LOCKHEED MARTIN J S^TE_poundL bdquo 4 ^

Lockheed Environmental Systems and Technologies Co Environmental Services Assistance Teams

^ ^ deg 19 Crosby Drive Suite 220 Bedford MA 01730Phone617-272-7868 Fax617-275-1373

Environmental Services Division US EPA - Region I 60 Westview street Lexington Massachusetts 02173

J u l v 2 4 1 9 9 5 SDMS DOCID 451894 ^ ^ ^ ^ 2 4 1995

L - 9 5 - 0 7 - J - 0 3

To Mr Anthony Palermo ESAT RPO

VIA Jack Berges ESAT Team Manager Region I

Subject TID No 01-9505-11 Surface Water and Sediment Sampling Surface WaterSediment Toxicity Testing Grain Size Analyses Cheshire Groundwater Site Cheshire CT

Dear Mr Palermo

c Environmental Services Assistance Team (ESAT) members Daniel Huber Amy Fox and David McDonald developed a surface water sediment sampling plan scuapled these two media and performed toxicity testing and grain size analyses as specified under this TID The task was requested by Peter Nolan EPA Task Monitor and was authorized under Technical Instruction Document (TID) No 01-9505-11 The requested start date was May 15 1995 The requested completion-date is July 22 1995

This TID encompassed the performance of several different tasks all to pursue the evaluation of the ecological issues likely to arise at the Cheshire groundwater Superfund site

I Preliminary Planning Phase

This task began with a preliminary planning element which included a conference call involving Jane Dolan (EPARPM) Dan Granz (EPA) Bill Andrade (EPA) Scott Clifford (EPA) Dan Huber (ESAT) and David McDonald (ESAT) During the conference call issues such as analyses to be performed tentative date of and time of sampling media to be sampled number of samplinglocations analyses are to performed and by whom were discussedFurther preliminary preparation in the ESD Biology Laboratoryincluded the inventory and subsequent ordering of additional toxicity test sampling vessels and sample containers for theactual sampling event

^ (shy rshy

^

Nbdquo

c

Mr Anthony Palermo July 24 1995 Page two

A step in preparation also included a site visit for reconnaissance purposes Participants in this visit included Jane Dolan Dan Granz and David McDonald During this visit tentative surface watersediment sampling locations in both site ponds and a nearby reference pond as well as soil sample locations both outside and beneath the facility were selected Svirface watersediment locations were examined as to their depth consistency and composition of sediment size and representativeness to the sampling area and to each other

An interim deliverable requested by the task monitor was to write a surface water and sediment sampling plan This plan was submitted to the task monitor for incorporation into the site field sampling plan This plan included the locations to be sampled the media to be sampled at each location the analytical and biological parameters to be sampled A table of approved sample containers and means of preservation minimxim amount of media required holding times and recommended detection limits was prepared

In addition a health and safety plan for the surface watersediment sampling event was also developed and approved

II Filed Activities

Details of the sampling event are as follows ESAT members Dan Huber Amy Fox and Dave McDonald accompanied by Tom Faber of the EPA left ESD on June 19th at approximately 0530 hours with all equipment needed for the seunpling event The team arrived at the site at approximately 0740 hours The team was accompanied by Tim Prior (USFWS) the Superfund Ecological Assistance Team (SEAT) lead for the site After some additional reconnaissance seunpling was initiated at the reference pond an unnamed pond located due west of the site approximately one quarter mile north of the intersection of Marion Rd and Interstate Rte 84 The pond was approximately one eight of a acre in size with an average water depth of 18 inches The bottom substrate consisted of a 6 inch siltymuck layer underlain by coarser sandy material The vegetation in the immediate area surrounding the pond consisted of mixed deciduous upland species a small area of bordering wetland species and emergent species within the pond itself Topographically the pond was situated at the base of a steep rising slope and was fed by at least one very small brook that was most likely an expression of groundwater

Mr Anthony Palermo July 24 1995 Page three

Surface water was sampled first Samples were taken by gently submersing the sample bottles approximately six inches below the surface Volatile organic carbon (VOC) samples were taken first followed by the other parameters The second media sampled was sediment The samples were taken with a petit Ponar dredge The sediment was placed in a stainless steel bowl VOCs were taken first then the sediment was homogenized and the other parameters were seunpled for beginning with the simultaneously extracted metalacid volatile sulfide sample Benthic invertebrate collection was performed last and consisted of three individual grabs that were sieved through an ASTM 30 sieve and then placed into separate sample containers

All samples were preserved as stated in the field sampling and analysis plan (FSAP) and placed on ice in coolers When the sampling at this station was complete all sampling equipment was decontaminated as stated in the FSAP

The team then moved to the larger of the two ponds located on site This pond was in the range of an acre in size with water depths from 3 - 5 feet in the sampling areas The bottom ^ substrate in this pond appeared to be made up of silt and muck with more of an organic matrix The pond is immediately bordered to the east by a band of scrubshrub wetland between the pond and the facility parking lot and to the south by West Johnson Rd There was very little rooted emergent vegetation evident The pond itself contained an extremely dense growth of milfoil The first location BG-l was located approximately 150 feet north of West Johnson Rd and the southern shore The second location BGshy2 was approximately 30 feet ENE of the northern end of the island The third location BG-3 was at the mouth of the cove into which the suspected drainage outfall pipe from the facility flows Surface water samples were taken by hand extending the bottle below the surface to a depth of approximately two feet All samples were taken and preserved in the same manner as was done in the reference pond with one exception The benthic invertebrate samples were not sieved on site Due to the extensive period of time taken to sieve these samples the benthic invertebrate samples were placed into 2 gallon ziploc bags and placed on ice to be sieved at the USFWS field office or at EPAESD the following day Field duplicate samples were taken at location BG-l except for VOCs which were taken at BG-3

Mr Anthony Palermo July 24 1995 Page four

The final sampling area was the smaller pond on site It was at this time after decontamination of equipment and prior to sampling the last location that rinseate blanks were taken The pond had a surface area of approximately one-half acre The water depth in this pond ranged up to ten feet in the middle The bottom substrate in this pond was more compact possibly due to leaf pack The immediate surroundings of this pond were a mixture of forested and scrtib shrub wetland This pond also had little rooted emergent vegetation The dense growth of rooted sxibmerged vegetation seen in the bigger pond was not evident in this pond The topography on the south side of the pond quickly steepened to meet the facility parking lot and delivery area

One sampling location was selected during the scoping phase as being adec[uate representation of this pond The location selected was approximately fifteen feet from the southeastern shore where a drainage swale carrying surface runoff from the site enters the pond

Once again surface water was sampled first in the same manner as the last area following the FSAP The sediment seunples were taken last Due to sediment composition the samplers had a more difficult time finding a sufficient amount of sediment in this pond having to attempt several locations before being able to provide enough sediment from one location Customarily surface water and sediment samples are tsdcen from the same location However at this location due to the sediment make-up the sediment saunples were taken from almost the center of the pond at a different location than the surface water

At each location dissolved oxygen temperature conductivity and pH were taken (See Table 1 for field chemistry results)

During this event chain of custody procedures were maintained Prior to the return trip fresh ice was added to each cooler and coolers were then chain of custody sealed The team left the site at 2030 hours and returned to the EPAESD laboratory at 2300 hours Sample coolers were stored in the Biology Section laboratory and then relinquished to EPA sample custody personnel at the laboratory the following day

Mr Anthony Palermo July 24 1995 Page five

III Laboratory Activities

ESAT Biology personnel were tasked with the responsibility of performing surface water sediment toxicity testing and grain size determination on the four site samples and site reference sample taken on June 19th in Cheshire CT

A seven day chronic surface water toxicity test using the freshwater cladoceran Ceriodaphnia dubia and minnow Pimephales promelas was initiated on Jine 20 1995 A ten day sediment toxicity test using the benthic invertebrates Chironomus tentans and Hyalella azteca began on June 29th Methodologies used were those found in EPA dociiments andor as modified within EPAESD Biology Section standard operating procedures (SOPs)

All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in the case of P^ promelas and 10 replicates containing 1 test organism in the case of C dubia

Sediment samples were homogenized and placed in 1 liter or 250 milliliter test chambers for Q^ tentans and IL azteca respectively with overlying water and allowed to aerate over night The following day inoculation of test vessels containing ogt site and reference samples and laboratory control sediments with early life stage test organisms was performed at a rate of 10 C tentans and 15 gj^ azteca

The following attachments provide separate detailed laboratory reports on both the surface water and sediment toxicity tests

Lastly during the week of July 17th grain size determination according to ASTM Method D 422-63 was performed on the four site and one site reference sediment samples The determination was limited to sieve manipulation Portions of the samples determined to have a grain size of less than 75 jim were reported as such Results of the grain size determination can be foxind in Table 2 of the following attachments

C

C Mr Anthony Palermo July 24 1995

Page six Should you have any questions or comments please contact

David McDonald 617860-4609 at the EPAESD Biology Section Lexington Mass

Very truly yours

Lockheed Environmental Sciences and Technologies Company

^y^nJ]^ V^gtML David F McDonald SrEnvironmental Scientist

dfm

Table 1 FIELD CHEMISTRY - CHESHIRE ASSOCIATION GROUNDWATER SUPERFUND SITE

JUNE 19 1995

C o n d u c t i v i t y D i s s o l v e d T e m p e r a t u r e pH (jumhocm) Oxygen (mgL) (deg C)

| R W - I 5 7 36 3 4 1 8 5

BG-l 6 2 49 5 8 1 5 5

BG-2 5 9 59 3 4 14

BG-3 6 8 39 7 0 1 3 8

LT-1 5 4 67 17 14

O)

Table 2

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o RW-1

G r a i n S i z e

gt 2 0 0 mm 0

850 xm - 2 0 0 mm 4 0

425 fim - 850 jim 1 6 3

250 nm - 425 Mm 2 1 7

125 nm - 250 xm 2 7 7

75 M^ - 125 jum 1 1 0

lt 75 ^m 1 9 0

Sample N o BG-l

G r a i n S i z e

gt 2 0 0 mm 0

850 fm - 2 0 0 mm 2 2

425 jum - 850 nm 1 5 7

250 xm - 425 xm 7 1

125 tm - 250 xm 8 5

75 xm - 125 xm 5 0

lt 75 xm 6 4 0

Sample N o BG-2

G r a i n S i z e

gt 2 0 0 xm 0

850 xm - 2 0 0 mm 0 6

425 xm- S50 xm 1 2 2

250 xm - 425 xm 1 1 2

125 xm - 250 xm 1 2 3

75 xm - 125 xm 56 1 lt 75 xm 593

Table 2 (contd) - ^

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 xm - 850 xm

250 nm - 425 xm

125 tm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 min

425 xm - 850 xm

250 xm - 425 tm

li25 tm - 250 xm

75 xm - 125 xm

lt 75 xm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 tm - 850 xm

250 xm - 425 tm

1 2 5 xm - 250 xm

75 tm - 125 xm bull mdash bull

lt 75 xm

0

10

7 9

8 5

1 3 3

1 1 0

5 9 4

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

1

)

Table 2 (contd)

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

1 850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 tm - 850 tm

250 xm - 425 tm

125 xm - 250 xm

75 xm - 125 xm

lt 75 tm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 xm - 125 xm

lt 75 xm

0

1 0

7 9

8 5

1 3 3

1 1 0

5 9 4

]

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

c

Mr Anthony Palermo July 24 1995 Page two

A step in preparation also included a site visit for reconnaissance purposes Participants in this visit included Jane Dolan Dan Granz and David McDonald During this visit tentative surface watersediment sampling locations in both site ponds and a nearby reference pond as well as soil sample locations both outside and beneath the facility were selected Svirface watersediment locations were examined as to their depth consistency and composition of sediment size and representativeness to the sampling area and to each other

An interim deliverable requested by the task monitor was to write a surface water and sediment sampling plan This plan was submitted to the task monitor for incorporation into the site field sampling plan This plan included the locations to be sampled the media to be sampled at each location the analytical and biological parameters to be sampled A table of approved sample containers and means of preservation minimxim amount of media required holding times and recommended detection limits was prepared

In addition a health and safety plan for the surface watersediment sampling event was also developed and approved

II Filed Activities

Details of the sampling event are as follows ESAT members Dan Huber Amy Fox and Dave McDonald accompanied by Tom Faber of the EPA left ESD on June 19th at approximately 0530 hours with all equipment needed for the seunpling event The team arrived at the site at approximately 0740 hours The team was accompanied by Tim Prior (USFWS) the Superfund Ecological Assistance Team (SEAT) lead for the site After some additional reconnaissance seunpling was initiated at the reference pond an unnamed pond located due west of the site approximately one quarter mile north of the intersection of Marion Rd and Interstate Rte 84 The pond was approximately one eight of a acre in size with an average water depth of 18 inches The bottom substrate consisted of a 6 inch siltymuck layer underlain by coarser sandy material The vegetation in the immediate area surrounding the pond consisted of mixed deciduous upland species a small area of bordering wetland species and emergent species within the pond itself Topographically the pond was situated at the base of a steep rising slope and was fed by at least one very small brook that was most likely an expression of groundwater

Mr Anthony Palermo July 24 1995 Page three

Surface water was sampled first Samples were taken by gently submersing the sample bottles approximately six inches below the surface Volatile organic carbon (VOC) samples were taken first followed by the other parameters The second media sampled was sediment The samples were taken with a petit Ponar dredge The sediment was placed in a stainless steel bowl VOCs were taken first then the sediment was homogenized and the other parameters were seunpled for beginning with the simultaneously extracted metalacid volatile sulfide sample Benthic invertebrate collection was performed last and consisted of three individual grabs that were sieved through an ASTM 30 sieve and then placed into separate sample containers

All samples were preserved as stated in the field sampling and analysis plan (FSAP) and placed on ice in coolers When the sampling at this station was complete all sampling equipment was decontaminated as stated in the FSAP

The team then moved to the larger of the two ponds located on site This pond was in the range of an acre in size with water depths from 3 - 5 feet in the sampling areas The bottom ^ substrate in this pond appeared to be made up of silt and muck with more of an organic matrix The pond is immediately bordered to the east by a band of scrubshrub wetland between the pond and the facility parking lot and to the south by West Johnson Rd There was very little rooted emergent vegetation evident The pond itself contained an extremely dense growth of milfoil The first location BG-l was located approximately 150 feet north of West Johnson Rd and the southern shore The second location BGshy2 was approximately 30 feet ENE of the northern end of the island The third location BG-3 was at the mouth of the cove into which the suspected drainage outfall pipe from the facility flows Surface water samples were taken by hand extending the bottle below the surface to a depth of approximately two feet All samples were taken and preserved in the same manner as was done in the reference pond with one exception The benthic invertebrate samples were not sieved on site Due to the extensive period of time taken to sieve these samples the benthic invertebrate samples were placed into 2 gallon ziploc bags and placed on ice to be sieved at the USFWS field office or at EPAESD the following day Field duplicate samples were taken at location BG-l except for VOCs which were taken at BG-3

Mr Anthony Palermo July 24 1995 Page four

The final sampling area was the smaller pond on site It was at this time after decontamination of equipment and prior to sampling the last location that rinseate blanks were taken The pond had a surface area of approximately one-half acre The water depth in this pond ranged up to ten feet in the middle The bottom substrate in this pond was more compact possibly due to leaf pack The immediate surroundings of this pond were a mixture of forested and scrtib shrub wetland This pond also had little rooted emergent vegetation The dense growth of rooted sxibmerged vegetation seen in the bigger pond was not evident in this pond The topography on the south side of the pond quickly steepened to meet the facility parking lot and delivery area

One sampling location was selected during the scoping phase as being adec[uate representation of this pond The location selected was approximately fifteen feet from the southeastern shore where a drainage swale carrying surface runoff from the site enters the pond

Once again surface water was sampled first in the same manner as the last area following the FSAP The sediment seunples were taken last Due to sediment composition the samplers had a more difficult time finding a sufficient amount of sediment in this pond having to attempt several locations before being able to provide enough sediment from one location Customarily surface water and sediment samples are tsdcen from the same location However at this location due to the sediment make-up the sediment saunples were taken from almost the center of the pond at a different location than the surface water

At each location dissolved oxygen temperature conductivity and pH were taken (See Table 1 for field chemistry results)

During this event chain of custody procedures were maintained Prior to the return trip fresh ice was added to each cooler and coolers were then chain of custody sealed The team left the site at 2030 hours and returned to the EPAESD laboratory at 2300 hours Sample coolers were stored in the Biology Section laboratory and then relinquished to EPA sample custody personnel at the laboratory the following day

Mr Anthony Palermo July 24 1995 Page five

III Laboratory Activities

ESAT Biology personnel were tasked with the responsibility of performing surface water sediment toxicity testing and grain size determination on the four site samples and site reference sample taken on June 19th in Cheshire CT

A seven day chronic surface water toxicity test using the freshwater cladoceran Ceriodaphnia dubia and minnow Pimephales promelas was initiated on Jine 20 1995 A ten day sediment toxicity test using the benthic invertebrates Chironomus tentans and Hyalella azteca began on June 29th Methodologies used were those found in EPA dociiments andor as modified within EPAESD Biology Section standard operating procedures (SOPs)

All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in the case of P^ promelas and 10 replicates containing 1 test organism in the case of C dubia

Sediment samples were homogenized and placed in 1 liter or 250 milliliter test chambers for Q^ tentans and IL azteca respectively with overlying water and allowed to aerate over night The following day inoculation of test vessels containing ogt site and reference samples and laboratory control sediments with early life stage test organisms was performed at a rate of 10 C tentans and 15 gj^ azteca

The following attachments provide separate detailed laboratory reports on both the surface water and sediment toxicity tests

Lastly during the week of July 17th grain size determination according to ASTM Method D 422-63 was performed on the four site and one site reference sediment samples The determination was limited to sieve manipulation Portions of the samples determined to have a grain size of less than 75 jim were reported as such Results of the grain size determination can be foxind in Table 2 of the following attachments

C

C Mr Anthony Palermo July 24 1995

Page six Should you have any questions or comments please contact

David McDonald 617860-4609 at the EPAESD Biology Section Lexington Mass

Very truly yours

Lockheed Environmental Sciences and Technologies Company

^y^nJ]^ V^gtML David F McDonald SrEnvironmental Scientist

dfm

Table 1 FIELD CHEMISTRY - CHESHIRE ASSOCIATION GROUNDWATER SUPERFUND SITE

JUNE 19 1995

C o n d u c t i v i t y D i s s o l v e d T e m p e r a t u r e pH (jumhocm) Oxygen (mgL) (deg C)

| R W - I 5 7 36 3 4 1 8 5

BG-l 6 2 49 5 8 1 5 5

BG-2 5 9 59 3 4 14

BG-3 6 8 39 7 0 1 3 8

LT-1 5 4 67 17 14

O)

Table 2

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o RW-1

G r a i n S i z e

gt 2 0 0 mm 0

850 xm - 2 0 0 mm 4 0

425 fim - 850 jim 1 6 3

250 nm - 425 Mm 2 1 7

125 nm - 250 xm 2 7 7

75 M^ - 125 jum 1 1 0

lt 75 ^m 1 9 0

Sample N o BG-l

G r a i n S i z e

gt 2 0 0 mm 0

850 fm - 2 0 0 mm 2 2

425 jum - 850 nm 1 5 7

250 xm - 425 xm 7 1

125 tm - 250 xm 8 5

75 xm - 125 xm 5 0

lt 75 xm 6 4 0

Sample N o BG-2

G r a i n S i z e

gt 2 0 0 xm 0

850 xm - 2 0 0 mm 0 6

425 xm- S50 xm 1 2 2

250 xm - 425 xm 1 1 2

125 xm - 250 xm 1 2 3

75 xm - 125 xm 56 1 lt 75 xm 593

Table 2 (contd) - ^

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 xm - 850 xm

250 nm - 425 xm

125 tm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 min

425 xm - 850 xm

250 xm - 425 tm

li25 tm - 250 xm

75 xm - 125 xm

lt 75 xm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 tm - 850 xm

250 xm - 425 tm

1 2 5 xm - 250 xm

75 tm - 125 xm bull mdash bull

lt 75 xm

0

10

7 9

8 5

1 3 3

1 1 0

5 9 4

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

1

)

Table 2 (contd)

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

1 850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 tm - 850 tm

250 xm - 425 tm

125 xm - 250 xm

75 xm - 125 xm

lt 75 tm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 xm - 125 xm

lt 75 xm

0

1 0

7 9

8 5

1 3 3

1 1 0

5 9 4

]

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

Mr Anthony Palermo July 24 1995 Page three

Surface water was sampled first Samples were taken by gently submersing the sample bottles approximately six inches below the surface Volatile organic carbon (VOC) samples were taken first followed by the other parameters The second media sampled was sediment The samples were taken with a petit Ponar dredge The sediment was placed in a stainless steel bowl VOCs were taken first then the sediment was homogenized and the other parameters were seunpled for beginning with the simultaneously extracted metalacid volatile sulfide sample Benthic invertebrate collection was performed last and consisted of three individual grabs that were sieved through an ASTM 30 sieve and then placed into separate sample containers

All samples were preserved as stated in the field sampling and analysis plan (FSAP) and placed on ice in coolers When the sampling at this station was complete all sampling equipment was decontaminated as stated in the FSAP

The team then moved to the larger of the two ponds located on site This pond was in the range of an acre in size with water depths from 3 - 5 feet in the sampling areas The bottom ^ substrate in this pond appeared to be made up of silt and muck with more of an organic matrix The pond is immediately bordered to the east by a band of scrubshrub wetland between the pond and the facility parking lot and to the south by West Johnson Rd There was very little rooted emergent vegetation evident The pond itself contained an extremely dense growth of milfoil The first location BG-l was located approximately 150 feet north of West Johnson Rd and the southern shore The second location BGshy2 was approximately 30 feet ENE of the northern end of the island The third location BG-3 was at the mouth of the cove into which the suspected drainage outfall pipe from the facility flows Surface water samples were taken by hand extending the bottle below the surface to a depth of approximately two feet All samples were taken and preserved in the same manner as was done in the reference pond with one exception The benthic invertebrate samples were not sieved on site Due to the extensive period of time taken to sieve these samples the benthic invertebrate samples were placed into 2 gallon ziploc bags and placed on ice to be sieved at the USFWS field office or at EPAESD the following day Field duplicate samples were taken at location BG-l except for VOCs which were taken at BG-3

Mr Anthony Palermo July 24 1995 Page four

The final sampling area was the smaller pond on site It was at this time after decontamination of equipment and prior to sampling the last location that rinseate blanks were taken The pond had a surface area of approximately one-half acre The water depth in this pond ranged up to ten feet in the middle The bottom substrate in this pond was more compact possibly due to leaf pack The immediate surroundings of this pond were a mixture of forested and scrtib shrub wetland This pond also had little rooted emergent vegetation The dense growth of rooted sxibmerged vegetation seen in the bigger pond was not evident in this pond The topography on the south side of the pond quickly steepened to meet the facility parking lot and delivery area

One sampling location was selected during the scoping phase as being adec[uate representation of this pond The location selected was approximately fifteen feet from the southeastern shore where a drainage swale carrying surface runoff from the site enters the pond

Once again surface water was sampled first in the same manner as the last area following the FSAP The sediment seunples were taken last Due to sediment composition the samplers had a more difficult time finding a sufficient amount of sediment in this pond having to attempt several locations before being able to provide enough sediment from one location Customarily surface water and sediment samples are tsdcen from the same location However at this location due to the sediment make-up the sediment saunples were taken from almost the center of the pond at a different location than the surface water

At each location dissolved oxygen temperature conductivity and pH were taken (See Table 1 for field chemistry results)

During this event chain of custody procedures were maintained Prior to the return trip fresh ice was added to each cooler and coolers were then chain of custody sealed The team left the site at 2030 hours and returned to the EPAESD laboratory at 2300 hours Sample coolers were stored in the Biology Section laboratory and then relinquished to EPA sample custody personnel at the laboratory the following day

Mr Anthony Palermo July 24 1995 Page five

III Laboratory Activities

ESAT Biology personnel were tasked with the responsibility of performing surface water sediment toxicity testing and grain size determination on the four site samples and site reference sample taken on June 19th in Cheshire CT

A seven day chronic surface water toxicity test using the freshwater cladoceran Ceriodaphnia dubia and minnow Pimephales promelas was initiated on Jine 20 1995 A ten day sediment toxicity test using the benthic invertebrates Chironomus tentans and Hyalella azteca began on June 29th Methodologies used were those found in EPA dociiments andor as modified within EPAESD Biology Section standard operating procedures (SOPs)

All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in the case of P^ promelas and 10 replicates containing 1 test organism in the case of C dubia

Sediment samples were homogenized and placed in 1 liter or 250 milliliter test chambers for Q^ tentans and IL azteca respectively with overlying water and allowed to aerate over night The following day inoculation of test vessels containing ogt site and reference samples and laboratory control sediments with early life stage test organisms was performed at a rate of 10 C tentans and 15 gj^ azteca

The following attachments provide separate detailed laboratory reports on both the surface water and sediment toxicity tests

Lastly during the week of July 17th grain size determination according to ASTM Method D 422-63 was performed on the four site and one site reference sediment samples The determination was limited to sieve manipulation Portions of the samples determined to have a grain size of less than 75 jim were reported as such Results of the grain size determination can be foxind in Table 2 of the following attachments

C

C Mr Anthony Palermo July 24 1995

Page six Should you have any questions or comments please contact

David McDonald 617860-4609 at the EPAESD Biology Section Lexington Mass

Very truly yours

Lockheed Environmental Sciences and Technologies Company

^y^nJ]^ V^gtML David F McDonald SrEnvironmental Scientist

dfm

Table 1 FIELD CHEMISTRY - CHESHIRE ASSOCIATION GROUNDWATER SUPERFUND SITE

JUNE 19 1995

C o n d u c t i v i t y D i s s o l v e d T e m p e r a t u r e pH (jumhocm) Oxygen (mgL) (deg C)

| R W - I 5 7 36 3 4 1 8 5

BG-l 6 2 49 5 8 1 5 5

BG-2 5 9 59 3 4 14

BG-3 6 8 39 7 0 1 3 8

LT-1 5 4 67 17 14

O)

Table 2

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o RW-1

G r a i n S i z e

gt 2 0 0 mm 0

850 xm - 2 0 0 mm 4 0

425 fim - 850 jim 1 6 3

250 nm - 425 Mm 2 1 7

125 nm - 250 xm 2 7 7

75 M^ - 125 jum 1 1 0

lt 75 ^m 1 9 0

Sample N o BG-l

G r a i n S i z e

gt 2 0 0 mm 0

850 fm - 2 0 0 mm 2 2

425 jum - 850 nm 1 5 7

250 xm - 425 xm 7 1

125 tm - 250 xm 8 5

75 xm - 125 xm 5 0

lt 75 xm 6 4 0

Sample N o BG-2

G r a i n S i z e

gt 2 0 0 xm 0

850 xm - 2 0 0 mm 0 6

425 xm- S50 xm 1 2 2

250 xm - 425 xm 1 1 2

125 xm - 250 xm 1 2 3

75 xm - 125 xm 56 1 lt 75 xm 593

Table 2 (contd) - ^

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 xm - 850 xm

250 nm - 425 xm

125 tm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 min

425 xm - 850 xm

250 xm - 425 tm

li25 tm - 250 xm

75 xm - 125 xm

lt 75 xm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 tm - 850 xm

250 xm - 425 tm

1 2 5 xm - 250 xm

75 tm - 125 xm bull mdash bull

lt 75 xm

0

10

7 9

8 5

1 3 3

1 1 0

5 9 4

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

1

)

Table 2 (contd)

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

1 850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 tm - 850 tm

250 xm - 425 tm

125 xm - 250 xm

75 xm - 125 xm

lt 75 tm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 xm - 125 xm

lt 75 xm

0

1 0

7 9

8 5

1 3 3

1 1 0

5 9 4

]

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

Mr Anthony Palermo July 24 1995 Page four

The final sampling area was the smaller pond on site It was at this time after decontamination of equipment and prior to sampling the last location that rinseate blanks were taken The pond had a surface area of approximately one-half acre The water depth in this pond ranged up to ten feet in the middle The bottom substrate in this pond was more compact possibly due to leaf pack The immediate surroundings of this pond were a mixture of forested and scrtib shrub wetland This pond also had little rooted emergent vegetation The dense growth of rooted sxibmerged vegetation seen in the bigger pond was not evident in this pond The topography on the south side of the pond quickly steepened to meet the facility parking lot and delivery area

One sampling location was selected during the scoping phase as being adec[uate representation of this pond The location selected was approximately fifteen feet from the southeastern shore where a drainage swale carrying surface runoff from the site enters the pond

Once again surface water was sampled first in the same manner as the last area following the FSAP The sediment seunples were taken last Due to sediment composition the samplers had a more difficult time finding a sufficient amount of sediment in this pond having to attempt several locations before being able to provide enough sediment from one location Customarily surface water and sediment samples are tsdcen from the same location However at this location due to the sediment make-up the sediment saunples were taken from almost the center of the pond at a different location than the surface water

At each location dissolved oxygen temperature conductivity and pH were taken (See Table 1 for field chemistry results)

During this event chain of custody procedures were maintained Prior to the return trip fresh ice was added to each cooler and coolers were then chain of custody sealed The team left the site at 2030 hours and returned to the EPAESD laboratory at 2300 hours Sample coolers were stored in the Biology Section laboratory and then relinquished to EPA sample custody personnel at the laboratory the following day

Mr Anthony Palermo July 24 1995 Page five

III Laboratory Activities

ESAT Biology personnel were tasked with the responsibility of performing surface water sediment toxicity testing and grain size determination on the four site samples and site reference sample taken on June 19th in Cheshire CT

A seven day chronic surface water toxicity test using the freshwater cladoceran Ceriodaphnia dubia and minnow Pimephales promelas was initiated on Jine 20 1995 A ten day sediment toxicity test using the benthic invertebrates Chironomus tentans and Hyalella azteca began on June 29th Methodologies used were those found in EPA dociiments andor as modified within EPAESD Biology Section standard operating procedures (SOPs)

All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in the case of P^ promelas and 10 replicates containing 1 test organism in the case of C dubia

Sediment samples were homogenized and placed in 1 liter or 250 milliliter test chambers for Q^ tentans and IL azteca respectively with overlying water and allowed to aerate over night The following day inoculation of test vessels containing ogt site and reference samples and laboratory control sediments with early life stage test organisms was performed at a rate of 10 C tentans and 15 gj^ azteca

The following attachments provide separate detailed laboratory reports on both the surface water and sediment toxicity tests

Lastly during the week of July 17th grain size determination according to ASTM Method D 422-63 was performed on the four site and one site reference sediment samples The determination was limited to sieve manipulation Portions of the samples determined to have a grain size of less than 75 jim were reported as such Results of the grain size determination can be foxind in Table 2 of the following attachments

C

C Mr Anthony Palermo July 24 1995

Page six Should you have any questions or comments please contact

David McDonald 617860-4609 at the EPAESD Biology Section Lexington Mass

Very truly yours

Lockheed Environmental Sciences and Technologies Company

^y^nJ]^ V^gtML David F McDonald SrEnvironmental Scientist

dfm

Table 1 FIELD CHEMISTRY - CHESHIRE ASSOCIATION GROUNDWATER SUPERFUND SITE

JUNE 19 1995

C o n d u c t i v i t y D i s s o l v e d T e m p e r a t u r e pH (jumhocm) Oxygen (mgL) (deg C)

| R W - I 5 7 36 3 4 1 8 5

BG-l 6 2 49 5 8 1 5 5

BG-2 5 9 59 3 4 14

BG-3 6 8 39 7 0 1 3 8

LT-1 5 4 67 17 14

O)

Table 2

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o RW-1

G r a i n S i z e

gt 2 0 0 mm 0

850 xm - 2 0 0 mm 4 0

425 fim - 850 jim 1 6 3

250 nm - 425 Mm 2 1 7

125 nm - 250 xm 2 7 7

75 M^ - 125 jum 1 1 0

lt 75 ^m 1 9 0

Sample N o BG-l

G r a i n S i z e

gt 2 0 0 mm 0

850 fm - 2 0 0 mm 2 2

425 jum - 850 nm 1 5 7

250 xm - 425 xm 7 1

125 tm - 250 xm 8 5

75 xm - 125 xm 5 0

lt 75 xm 6 4 0

Sample N o BG-2

G r a i n S i z e

gt 2 0 0 xm 0

850 xm - 2 0 0 mm 0 6

425 xm- S50 xm 1 2 2

250 xm - 425 xm 1 1 2

125 xm - 250 xm 1 2 3

75 xm - 125 xm 56 1 lt 75 xm 593

Table 2 (contd) - ^

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 xm - 850 xm

250 nm - 425 xm

125 tm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 min

425 xm - 850 xm

250 xm - 425 tm

li25 tm - 250 xm

75 xm - 125 xm

lt 75 xm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 tm - 850 xm

250 xm - 425 tm

1 2 5 xm - 250 xm

75 tm - 125 xm bull mdash bull

lt 75 xm

0

10

7 9

8 5

1 3 3

1 1 0

5 9 4

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

1

)

Table 2 (contd)

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

1 850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 tm - 850 tm

250 xm - 425 tm

125 xm - 250 xm

75 xm - 125 xm

lt 75 tm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 xm - 125 xm

lt 75 xm

0

1 0

7 9

8 5

1 3 3

1 1 0

5 9 4

]

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

Mr Anthony Palermo July 24 1995 Page five

III Laboratory Activities

ESAT Biology personnel were tasked with the responsibility of performing surface water sediment toxicity testing and grain size determination on the four site samples and site reference sample taken on June 19th in Cheshire CT

A seven day chronic surface water toxicity test using the freshwater cladoceran Ceriodaphnia dubia and minnow Pimephales promelas was initiated on Jine 20 1995 A ten day sediment toxicity test using the benthic invertebrates Chironomus tentans and Hyalella azteca began on June 29th Methodologies used were those found in EPA dociiments andor as modified within EPAESD Biology Section standard operating procedures (SOPs)

All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in the case of P^ promelas and 10 replicates containing 1 test organism in the case of C dubia

Sediment samples were homogenized and placed in 1 liter or 250 milliliter test chambers for Q^ tentans and IL azteca respectively with overlying water and allowed to aerate over night The following day inoculation of test vessels containing ogt site and reference samples and laboratory control sediments with early life stage test organisms was performed at a rate of 10 C tentans and 15 gj^ azteca

The following attachments provide separate detailed laboratory reports on both the surface water and sediment toxicity tests

Lastly during the week of July 17th grain size determination according to ASTM Method D 422-63 was performed on the four site and one site reference sediment samples The determination was limited to sieve manipulation Portions of the samples determined to have a grain size of less than 75 jim were reported as such Results of the grain size determination can be foxind in Table 2 of the following attachments

C

C Mr Anthony Palermo July 24 1995

Page six Should you have any questions or comments please contact

David McDonald 617860-4609 at the EPAESD Biology Section Lexington Mass

Very truly yours

Lockheed Environmental Sciences and Technologies Company

^y^nJ]^ V^gtML David F McDonald SrEnvironmental Scientist

dfm

Table 1 FIELD CHEMISTRY - CHESHIRE ASSOCIATION GROUNDWATER SUPERFUND SITE

JUNE 19 1995

C o n d u c t i v i t y D i s s o l v e d T e m p e r a t u r e pH (jumhocm) Oxygen (mgL) (deg C)

| R W - I 5 7 36 3 4 1 8 5

BG-l 6 2 49 5 8 1 5 5

BG-2 5 9 59 3 4 14

BG-3 6 8 39 7 0 1 3 8

LT-1 5 4 67 17 14

O)

Table 2

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o RW-1

G r a i n S i z e

gt 2 0 0 mm 0

850 xm - 2 0 0 mm 4 0

425 fim - 850 jim 1 6 3

250 nm - 425 Mm 2 1 7

125 nm - 250 xm 2 7 7

75 M^ - 125 jum 1 1 0

lt 75 ^m 1 9 0

Sample N o BG-l

G r a i n S i z e

gt 2 0 0 mm 0

850 fm - 2 0 0 mm 2 2

425 jum - 850 nm 1 5 7

250 xm - 425 xm 7 1

125 tm - 250 xm 8 5

75 xm - 125 xm 5 0

lt 75 xm 6 4 0

Sample N o BG-2

G r a i n S i z e

gt 2 0 0 xm 0

850 xm - 2 0 0 mm 0 6

425 xm- S50 xm 1 2 2

250 xm - 425 xm 1 1 2

125 xm - 250 xm 1 2 3

75 xm - 125 xm 56 1 lt 75 xm 593

Table 2 (contd) - ^

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 xm - 850 xm

250 nm - 425 xm

125 tm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 min

425 xm - 850 xm

250 xm - 425 tm

li25 tm - 250 xm

75 xm - 125 xm

lt 75 xm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 tm - 850 xm

250 xm - 425 tm

1 2 5 xm - 250 xm

75 tm - 125 xm bull mdash bull

lt 75 xm

0

10

7 9

8 5

1 3 3

1 1 0

5 9 4

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

1

)

Table 2 (contd)

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

1 850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 tm - 850 tm

250 xm - 425 tm

125 xm - 250 xm

75 xm - 125 xm

lt 75 tm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 xm - 125 xm

lt 75 xm

0

1 0

7 9

8 5

1 3 3

1 1 0

5 9 4

]

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

C

C Mr Anthony Palermo July 24 1995

Page six Should you have any questions or comments please contact

David McDonald 617860-4609 at the EPAESD Biology Section Lexington Mass

Very truly yours

Lockheed Environmental Sciences and Technologies Company

^y^nJ]^ V^gtML David F McDonald SrEnvironmental Scientist

dfm

Table 1 FIELD CHEMISTRY - CHESHIRE ASSOCIATION GROUNDWATER SUPERFUND SITE

JUNE 19 1995

C o n d u c t i v i t y D i s s o l v e d T e m p e r a t u r e pH (jumhocm) Oxygen (mgL) (deg C)

| R W - I 5 7 36 3 4 1 8 5

BG-l 6 2 49 5 8 1 5 5

BG-2 5 9 59 3 4 14

BG-3 6 8 39 7 0 1 3 8

LT-1 5 4 67 17 14

O)

Table 2

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o RW-1

G r a i n S i z e

gt 2 0 0 mm 0

850 xm - 2 0 0 mm 4 0

425 fim - 850 jim 1 6 3

250 nm - 425 Mm 2 1 7

125 nm - 250 xm 2 7 7

75 M^ - 125 jum 1 1 0

lt 75 ^m 1 9 0

Sample N o BG-l

G r a i n S i z e

gt 2 0 0 mm 0

850 fm - 2 0 0 mm 2 2

425 jum - 850 nm 1 5 7

250 xm - 425 xm 7 1

125 tm - 250 xm 8 5

75 xm - 125 xm 5 0

lt 75 xm 6 4 0

Sample N o BG-2

G r a i n S i z e

gt 2 0 0 xm 0

850 xm - 2 0 0 mm 0 6

425 xm- S50 xm 1 2 2

250 xm - 425 xm 1 1 2

125 xm - 250 xm 1 2 3

75 xm - 125 xm 56 1 lt 75 xm 593

Table 2 (contd) - ^

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 xm - 850 xm

250 nm - 425 xm

125 tm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 min

425 xm - 850 xm

250 xm - 425 tm

li25 tm - 250 xm

75 xm - 125 xm

lt 75 xm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 tm - 850 xm

250 xm - 425 tm

1 2 5 xm - 250 xm

75 tm - 125 xm bull mdash bull

lt 75 xm

0

10

7 9

8 5

1 3 3

1 1 0

5 9 4

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

1

)

Table 2 (contd)

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

1 850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 tm - 850 tm

250 xm - 425 tm

125 xm - 250 xm

75 xm - 125 xm

lt 75 tm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 xm - 125 xm

lt 75 xm

0

1 0

7 9

8 5

1 3 3

1 1 0

5 9 4

]

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

Table 1 FIELD CHEMISTRY - CHESHIRE ASSOCIATION GROUNDWATER SUPERFUND SITE

JUNE 19 1995

C o n d u c t i v i t y D i s s o l v e d T e m p e r a t u r e pH (jumhocm) Oxygen (mgL) (deg C)

| R W - I 5 7 36 3 4 1 8 5

BG-l 6 2 49 5 8 1 5 5

BG-2 5 9 59 3 4 14

BG-3 6 8 39 7 0 1 3 8

LT-1 5 4 67 17 14

O)

Table 2

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o RW-1

G r a i n S i z e

gt 2 0 0 mm 0

850 xm - 2 0 0 mm 4 0

425 fim - 850 jim 1 6 3

250 nm - 425 Mm 2 1 7

125 nm - 250 xm 2 7 7

75 M^ - 125 jum 1 1 0

lt 75 ^m 1 9 0

Sample N o BG-l

G r a i n S i z e

gt 2 0 0 mm 0

850 fm - 2 0 0 mm 2 2

425 jum - 850 nm 1 5 7

250 xm - 425 xm 7 1

125 tm - 250 xm 8 5

75 xm - 125 xm 5 0

lt 75 xm 6 4 0

Sample N o BG-2

G r a i n S i z e

gt 2 0 0 xm 0

850 xm - 2 0 0 mm 0 6

425 xm- S50 xm 1 2 2

250 xm - 425 xm 1 1 2

125 xm - 250 xm 1 2 3

75 xm - 125 xm 56 1 lt 75 xm 593

Table 2 (contd) - ^

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 xm - 850 xm

250 nm - 425 xm

125 tm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 min

425 xm - 850 xm

250 xm - 425 tm

li25 tm - 250 xm

75 xm - 125 xm

lt 75 xm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 tm - 850 xm

250 xm - 425 tm

1 2 5 xm - 250 xm

75 tm - 125 xm bull mdash bull

lt 75 xm

0

10

7 9

8 5

1 3 3

1 1 0

5 9 4

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

1

)

Table 2 (contd)

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

1 850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 tm - 850 tm

250 xm - 425 tm

125 xm - 250 xm

75 xm - 125 xm

lt 75 tm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 xm - 125 xm

lt 75 xm

0

1 0

7 9

8 5

1 3 3

1 1 0

5 9 4

]

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

Table 2

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o RW-1

G r a i n S i z e

gt 2 0 0 mm 0

850 xm - 2 0 0 mm 4 0

425 fim - 850 jim 1 6 3

250 nm - 425 Mm 2 1 7

125 nm - 250 xm 2 7 7

75 M^ - 125 jum 1 1 0

lt 75 ^m 1 9 0

Sample N o BG-l

G r a i n S i z e

gt 2 0 0 mm 0

850 fm - 2 0 0 mm 2 2

425 jum - 850 nm 1 5 7

250 xm - 425 xm 7 1

125 tm - 250 xm 8 5

75 xm - 125 xm 5 0

lt 75 xm 6 4 0

Sample N o BG-2

G r a i n S i z e

gt 2 0 0 xm 0

850 xm - 2 0 0 mm 0 6

425 xm- S50 xm 1 2 2

250 xm - 425 xm 1 1 2

125 xm - 250 xm 1 2 3

75 xm - 125 xm 56 1 lt 75 xm 593

Table 2 (contd) - ^

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 xm - 850 xm

250 nm - 425 xm

125 tm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 min

425 xm - 850 xm

250 xm - 425 tm

li25 tm - 250 xm

75 xm - 125 xm

lt 75 xm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 tm - 850 xm

250 xm - 425 tm

1 2 5 xm - 250 xm

75 tm - 125 xm bull mdash bull

lt 75 xm

0

10

7 9

8 5

1 3 3

1 1 0

5 9 4

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

1

)

Table 2 (contd)

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

1 850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 tm - 850 tm

250 xm - 425 tm

125 xm - 250 xm

75 xm - 125 xm

lt 75 tm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 xm - 125 xm

lt 75 xm

0

1 0

7 9

8 5

1 3 3

1 1 0

5 9 4

]

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

Table 2 (contd) - ^

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 xm - 850 xm

250 nm - 425 xm

125 tm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 min

425 xm - 850 xm

250 xm - 425 tm

li25 tm - 250 xm

75 xm - 125 xm

lt 75 xm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 tm - 850 xm

250 xm - 425 tm

1 2 5 xm - 250 xm

75 tm - 125 xm bull mdash bull

lt 75 xm

0

10

7 9

8 5

1 3 3

1 1 0

5 9 4

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

1

)

Table 2 (contd)

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

1 850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 tm - 850 tm

250 xm - 425 tm

125 xm - 250 xm

75 xm - 125 xm

lt 75 tm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 xm - 125 xm

lt 75 xm

0

1 0

7 9

8 5

1 3 3

1 1 0

5 9 4

]

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

Table 2 (contd)

GRAIN SIZE ANALYSIS - CHESHIRE SITE

Sample N o BG-3

G r a i n S i z e

gt 2 0 0 mm

1 850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 tm - 125 xm

lt 75 xm

Sample N o LT-1

G r a i n S i z e

gt 2 0 0 mm

850 xm - 2 0 0 mm

425 tm - 850 tm

250 xm - 425 tm

125 xm - 250 xm

75 xm - 125 xm

lt 75 tm

Sample N o Saw M i l l

G r a i n S i z e

gt 2 0 0 xm

850 xm - 2 0 0 mm

425 xm - 850 tm

250 xm - 425 xm

125 xm - 250 xm

75 xm - 125 xm

lt 75 xm

0

1 0

7 9

8 5

1 3 3

1 1 0

5 9 4

]

0

0 5

4 6

2 3 9

1 6 7

1 5 8

3 8 2

0

16

9 4

1 3 2

3 8 2

1 6 1

2 1 2

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-D1-1058

Cheshire Association Groundwater Site Sediment Toxicity Testing

Chironomus tentans Hvallela azteca

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

US EPA Region I 60 Westview St

Lexington Massachusetts 02173

Submitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

O^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

c

INTRODUCTION

As part of the site investigation at the Cheshire Association Groundwater Superfund site sediment toxicity testing was performed on four sediment samples from the site three from the larger pond and one from the small pond In addition one sediment sample was also taken from a reference pond located within close proximity to the site in the same watershed

The sediment samples were collected on Monday June 19 1995 and stored the Biology Section refrigerator at 4degC The test consisted of seven stations five site related and two laboratory controls of eight replicates per station for each species On June 291995 the sediments were homogenized and cleaned with 10 sieves For Hvalella azteca^ approximately 50 ml of sediment and 200 ml of overlying water were placed into each 250 ml test chamber Chironomus tentans were placed into the 1 liter test chambers 100 ml of sediment and 900 ml of overlying water were added The overlying water consisted of 25 Perrier75 80 mgL CaC03 culture water for Hyalella azteca and aerated tap water for Chironomus tentans The sediment and water were allowed to settle overnight before inoculation

On Friday and Saturday June 30th and July 1 each C tentans and JL azteca beaker was inoculated with 10 and 15 organisms respectively Initial hardness pH dissolved oxygen conductivity and temperature readings were taken On a daily basis pH conductivity DO and temperature were measured On Monday July 3rd and Friday July 7th a 50 renewal was performed for each test chamber

Young organisms ie less than seven day old Hi azteca and 2nd instar Cj tentans were exposed to sediment samples from four on-site sampling stations 1 site related reference station and two laboratory controls One sample was collected from a reference station Saw Mill Brook Pond in Concord MA and the other an artificial sediment made at the laboratory with a formulation of 77 sand 17 siltclay 5 organic matter and 1 CaCOj buffer The tests were performed to assess the sensitivity and response of these organisms to the sediments After organisms were recovered from each test chamber and tallied as live or dead the live organisms were dried at 75deg F over night and weighed Comparison of sediments from four on-site stations with the site reference station and the laboratory controls were examined statistically For g^ azteca and C tentans ^ survival and growth data were used to assess any significant effects

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

SAMPLE COLLECTION

On Monday June 29 1995 ESAT members Dan Huber Amy Fox and Dave McDonald and Tom Faber of the EPA met Tim Prior of the USFWS at the site There was a brief discussion of sampling procedures safety precautions and sampling locations At each sampling location sediment samples were extracted from depositional areas using a petit Ponar dredge sampler homogenized in a stainless steel bowl (with the exception of volatile organics) and then placed in appropriate seunple containers All sampling equipment was deconteuninated between sampling locations Seonples were labeled and sealed then placed in a cooler of ice for transport back to EPA New England Regional Laboratory for testing On site sample stations BG-l BG-2 and BG-3 from the larger pond and LT-1 from the smaller of the two along with RW-1 from the reference pond were designated for sediment toxicity testing SEMAVS TOC grain size and CLP TAL and TCL analyses and benthic invertebrate identification All samples were returned to the EPA New England Regional Laboratory for analysis

MATERIALS AND METHODS

A Toxic i ty t e s t procedures were followed according t o

bullInterim Modifications to ASTM Method as per EPA Tiered Sediment Testing Workshop September 16-18 1992 Washington DC

bullEPA Region I ESD Biology Section SOP 13 Sediment Toxicitv Testing Procedures for the preparation and analysis of sediments

bullThe statistical procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001

O)

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

c

MATERIALS AND METHODS Contd

B SAMPLE HANDLING

Sediment samples received at ESD were designated as follows BG-l BG-2 BG-3 RW-1 LT-1 laboratory reference station Saw Mill Brook Pond as SAW MILL and formulated sediment as ART SED There were 8 replicatesspecies for each seunple All test chambers were maintained at 25 degrees Celsius + - 1 degree and 168 hour lightdark cycle

Overlying water included aerated tap water for C^ tentans and a water mixture of 25 Perrier mineral water and 75 80 mgL as CaC03 hardness water for H^ azteca

C S T A T I S T I C S

Statistical analyses were performed on the accvimulated data Dunnetts Test Wilcoxon Rank Sum Test ind the T-test with the Bonferroni Adjustment were the statistical methods utilized to analyze the data The data for growth and survival were compared between the laboratory control ART SED the site reference and the on-site ponds to determine any statistical differences

D TEST FOOD

C tentans Tetramin shake 05 ml 4 gL deionized water blended and fed daily

H azteca Tetramin shake 02ml of 20 gL deionized water suspension daily

E HARDWARE

10 45 50 and 60 sieves plastic scoops 250 ml beedcers (test chambers) 1 liter beakers Ctest chambers) 1 ml plastic disposable pipets Parafilm Tygon tubing Nalgene trays

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

PROCEDURES ^^ o A I N I T I A L TEST SET-UP ^

Sediments were homogenized and cleaned using a standard 10 sieve Sediments followed by overlying water were added to the test vessels in the following quantities For the H azteca 50 ml of sediment was added to each 250 ml beaker with approximately 200 ml of overlying water per replicate For Si tentans 100 ml of sediment was added to each beedcer followed by 900 ml of overlying water Whenever water was added to a particular vessel either in a renewal or to begin a particular test a turbulence reducer was utilized to reduce the amount of sediment resuspension into the water column

J5 ORGANISM ORIGIN amp INTRODUCTION

The ESD Biology Laboratory cultures provided the C tentans and gjj azteca organisms for the sediment tests The C tentans were all in the age window from 7 to 10 days H gizteca fell within the 2nd and 3rd instar or 2-3 millimeters in length Prior to introduction of iLs azteca and C tentans the sediments were allowed to settle overnight Ten C tentans and fifteen iL azteca were introduced to the appropriate test chambers O) C FEEDING

The test organisms were fed according to the following schedule The C tentans were fed 05 ml of 4 gL Tetramin solution per day The Hj azteca were fed 02 ml of 20 gL Tetramin suspension daily

D TEST CHEMISTRY

Initial chemistry was performed on the overlying water and monitored every 24 hours All chemistry results were recorded in wet chemistry laboratory log book initial chemistry included the following pH conductivity hardness temperature and dissolved oxygen (DO) Daily chemistry monitoring included pH DO conductivity and temperature The environmental chamber temperature was also monitored on a daily basis

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

c

c

PROCEDURES contd

E TEST DESIGN

See appropriate Biology Section SOP for sediment toxicity testing unless stated below

Eight replicates per location per species were used in the test Tenchambers

Q^ tentans were placed into its appropriate test

RESULTS

Results of data on which statistical analyses were performed can be found in Tables 1 and 2 of this report

The Hyallela azteca survival and growth data were analyzed using the Shapiro Wilks test for normality and Bartletts test for homogeneity The survival data passed both of these tests However the growth data passed only the test for homogeneity The survival data were subsequently analyzed using the Dunnetts Test while growth data was evaluated using the Wilcoxon Rank Test w Bonferroni Adjustment These tests were used to examine the data for significant differences in survival and growth between each station and the laboratory reference sediment (formulated sediment-ART SED) as well as the site rieference pond sediment Statistical analyses of survival data indicate significant difference between sediment locations BG-2 and BG-3 and both the laboratory control and the site reference sediment Analyses of growth data indicates no significant difference between the site pond sediments and either the laboratory control or the site reference pond

The Chironomus tentans survival data were analyzed using the Shapiro Wilks test for normality and the Bartletts test for homogeneity The data passed for both of these tests As a consequence Dunnetts test was utilized to examine for significant difference of the on-site ponds from the laboratory control and site reference pond Results of this test indicate that there is no significant difference between on-sita sediments and either the laboratory control or the site reference

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

RESULTS contd O The growth data was subjected to a Chi-square test for

normality and a Hartley test for homogeneity These data passed both tests but due to unequal replicates were tested with a Bonferroni t-Test for significance between the on-site ponds and both the site reference and the laboratory control Results of these tests on growth data indicate a significant difference in growth between the laboratory control (ART SED) and RW-1 BG-l BG-2 and LT-1 However when comparing the site reference sediment to the on-site pond sediments there is no significant difference

Table 1 10-day SEDIMENT TOXICITY TEST

Chironomus tentans June 1995

1 bull

Location Percent Average weight per survival location (mg)

Art sediment 725 23

RW-1 588 12

BG-i 612 10 O) BG-2 662 07

BG-3 625 18

LT-1 638 09

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

w TABLE 2

10-day SEDIMENT TEST Hvallela azteca

June 1995

Concentration Percent Average weight per survival location (mg)

1 Art Sediment 719 009 1

1 RW-1 848 013

1 BG-l 792 013 1 B6-2 242 013 1

1 BG-3 383 011 1 LT-1 690 0 12

DISCUSSION

c

Results of analyses for the ILi azteca survival data indicate an impact on survival at location BG-2 and BG-3 These two locations were closest to the point where a suspected outfall may have entered the larger site pond However it should be noted that one of the laboratory controls Saw Mill Brook had only 117 survival Results of test chemistry would imply that the problem with survival did not come from a lack of dissolved oxygen or abrupt change in temperature The lower than expected survival in a control sample from an area regarded as pristine reduces the interpretation of the test data to point to site related impacts Growth data analyses indicate no difference between laboratory controls and on-site ponds or between the site reference pond and on-site ponds

The survival data analyses for C^ tentans show no significant difference between either the laboratory control and the on-site ponds or the site reference pond and on-site ponds implj^ng no adverse effects on survival Analyses of growth datar indicate a significant difference between the laboratory control and the site reference and all on-site locations except BG-3 However when a comparison between the site reference and on-site locations is made no significant difference is seen This may imply that- any statistically significant differences may be caused by natural physicalchemical characteristics rather than site related impacts

Vwshy

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

^ CONCLUSIONS

The percent survival for both species in the artificial sediment laboratory control used in statistical analyses was approximately 72 This percentage is slightly lower than the acceptable 80 currently in use at the laboratory In addition due to the unexplainable low survival of the Saw Mill Brook samples only the artificial sediment was used for statistical analyses In spite of these points a reasonable evaluation made on a comparative basis could still be made

Both test species are tolerant of a wide range in grain size which according to the grain size analyses should not have adversely effected the tests

Results from this testing are not definitive in defining a lack of toxicity Some statistically significant differences between laboratory controls and site related seunples were observed Whether they are biologically significant andor may be associated with the site is still a question Chemical analyses of these sediments may add to the weight of evidence and provide some clarity into defining potential causes of these differences

^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

r^

Cheshire Association Superfund Site Surface Water Toxicity Tests

Ceriodaphnia (a feja Pimephaleis promelas

TID No 01-9505-11

Submitted to

ESAT Regional Project Officer Environmental Services Division

U S EPA Region I 60 Westview St

Lexington Massachusetts 02173

^ Stibmitted by

ESAT Region I Lockheed Engineering and Sciences Company

19B Crosby Drive Bedford Massachusetts 01730

July 24 1995

Environmental Services Assistance Team US EPA REGION I

EPA Contract 68-Dl-1058

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

INTRODUCTION

The site is located in Cheshire Connecticut and comprised of two industrial facilities Previous sampling events revealed some contamination limited to VOCs in the nearby residential wells The two ponds one large and one small on the site are adjacent to the Airpax Corporation An additional reference pond was selected off of Marion Road The site ponds are ringed with a mixed deciduous type forest and abundant amounts of milfoil were evident The ponds were not deep averaging between 3 and 10 feet and a John boat was used to access both of these waterbodies Sediments and surface water were collected from all three of the ponds for toxicity testing AVSSEM TOC VOAs SVGAs PestPCBs and metals

Samples were collected on June 19 1995 The surface water toxicity tests were initiated on June 20 1995 A chronic toxicity test was run with both Ceriodaphnia dubia and Pimephales promelas Renewals were made daily for both organisms The six samples including the control were run at full strength to determine any effects on growth and mortality (P promelas) and mortality and reproduction fC dubia) All surface water samples were taken from refrigeration and brought to room temperature ie 25deg C Straight surface water samples and a laboratory control were set out in replicates of four containing 10 minnows in thecase of Pjs promelas and 10 replicates containing 1 test organism in the case of C dubia

SURFACE WATER COLLECTION

The surface samples were collected on June 19 1995 by ESAT EPA and USFWS personnel The sampling locations consisted of the reference pond RW-1 three large pond samples BG-l BG-2 BG-3 and the small pond LT-1 All bull samples were collected from a John boat except for the reference pond sample The samples were taken by submerging the sample containers about 2 feet under the surface Sample preservation was followed according to the FSAP All samples were then properly tagged and placed in an ice chest for shipment to the ESD facility

^

^ ^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

f^ MATERIALS AND METHODS

The procedures used followed those outlined in the EPA document Short-Term Methods For Estimating The Chronic Toxicitv Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA6004897001

Test P r o c e d u r e s

The tests was performed using piire seunple (100 sample) from each of the sampling locations There were no serial dilutions performed for these seunples The seunple locations are as follows BG-l BG-2 BG-3 LT-1 RW-1 and the control Sixty hardness culture water was used as a laboratory control solution Both tests were randomized according to the Short-Term Methods For Estimating The Chronic Toxicity Of Effluents and Receiving Waters To Freshwater Organisms 2nd Edition (Methods 10000 and 10020) EPA600489001 The organisms Ceriodaphnia dubia amp Pimephales promelas^ were then introduced into the test dilutions listed above

^

Test vessels were maintained at 25 degrees Celsius +- 1 degree in a 168 hour lightdark cycle Tests were monitored

every 24 hours and observations regarding the niomber of organisms dead (for Cj dubie and E^ promelas) and the number of neonates (S^ dubie^ only) were recorded on standard lab data sheets QC checks were performed on each cotint of organisms at the beginning of each test All test replicates were randomized throughout the test Chronic tests were renewed with new test solutions daily as indicated above

Initial and final test chemistry included determination of pH conductivity temperature dissolved oxygen (DO) on all test samples and hardness on the control and 100 solutions DO pH and temperature were also measured for the renewal waste every 24 hours Environmental chamber temperature was also monitored on a daily basis

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

MATERIALS AND METHODS contd

Tes t Food

a Cj dubia

-2 drops daily of Selenastrum capricornutum per tube

-2 drops daily of YAT (yeast alfalfa trout chow mixture) per tube

b Ei promelas

-2 drops of Artemia twice daily per vessel

Statistical Analyses

For the P^ promelas test the endpoints are determined for survival and growth Since there was no toxicity in the samples statistical analyses were not necessary However the growth data was analyzed with the Dunnetts Test

This is also the case for the data from the S dubia test As a result of the lack of significant differences insiorvival between the control and any concentration no attempt was made to perform statistical analysis Howeverstatistics were run on the reproduction data The BonferroniT-Test was used to analyze the data The results are notedin the following sections

^

^ ^ ^bullgt

)

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

c RESULTS

From both sampling events as noted above there was no significant mortality in either the chronic test for both C dubia and Pj promelas The data is provided in the tables below

Table 1 7-day chronic test with Pimephales promela^

June 1995

Concentration Percent Average weight per | survival concentration (mg)

Control 975 062

RW-1 875 058

1 BG-l 975 062

BG-2 925 059

BG-3 875 062

LT-1 100 072

V

TABLE 2 7-day chronic test with Ceriodaphnia^ dubia

June 1995 1

Concentration Percent Average number of survival young per

concentration

Control 100 462

RW-1 90 484

BG-l 90 283

BG-2 80 326

BG-3 80 321

LT-1 90 426

Indicates that a c dubia was lost during the renewals

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

DISCUSSION

The chronic test for poundi dubia displayed no significant mortality over the seven day period However there was a suppression in the mean value of neonates produced over 7 days ie iZ2 for the laboratory control 283 for sampling location BG-l (See Table 2) The statistical results showed a significant difference for BG-l in neonate production when compeired to the lab control When the reference pond (RW-1) is used as a control BG-l BG-2 amp BG-3 are statistically significant for the reproduction data analyzed The average number of neonates for the reference pond is 484 while the large pond samples BG-l is 283 BG-2 is 326 and BG-3 is 321

The large pond samples yielded smaller mean value brood size when compared to the brood size from the small pond The differences between the ponds are not readily apparent The wet chemistry data reveals no precipitous drops in dissolved oxygen or problems with pH Both ponds are similar in that they are fairly shallow and have a heavy milfoil growth Besides their size they are quite compatible The only obvious differences is the large ponds proximity toWest Johnson Road and the outfall from the site It is

^

possible that the pond is effected by surface water rtinoff although this seems unlikely due to the 25 buffer zonebetween the street and the pond

^

The chronic Pj promelas test displayed no toxicity in regards to survival over the seven day period In addition the average weight for each replicate was approximately that of the control No statistical analyses were necessary for this test

SUMMARY

From the statistical results and visual inspection of these two chronic tests there does not appear to be any toxicity as far as survival and growth are concerned However as noted in the previous section there was some statistical significance in the Cj dubia chronic neonate reproduction for BG-l (when compared to lab control water) and BG-l BG-2 amp BG-3 (when compared to the reference pond or RW-1)

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

i^cUha fi pto d^ft ^ i i J-i ^ c^frJi CHESHIRE SURFACE WATER C DUBIA REPRO File CTOXSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

C^ BONFERRONI T-TEST - TABLE 1 OF 2 HoControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 48400 48400 2 BG-l 28250 28250 2934

3 BG-2 32556 32556 2382 bullk

4 BG-3 32100 32100 2518

5 LT-1 42600 42600 0896

Bonferroni T table value = 233 (1 Tailed Value P=005 df=404)

SeeeeSeeeeeeeeeeeegesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue n ^eeeeeeeegeeeeSeeeeSeeeeSgeeeeeeeeeSegSeeeeeeampeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRWRPR Transform NO TRANSFORMATION

- BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 10 2 BG-l 8 15992 330 20150 3 BG-2 9 15491 320 15844 4 BG-3 10 15078 312 16300 5 LT-1 10 15078 312 5800

SeeeeeeeeeeeeeeeegeeSeSeeeeeeeeegeeeeeeSeeeeeeeSeeeeeE 0 Press any key t o continue or Esc t o r e tu rn to menu n elesecteeeeeeeeeeeuroeeeSeSeeeeeeeeeeeesecteeeSsecteeuroeeSeegegeeeeeyen

v^

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRPR Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 1 OF 2 HoControlltTreatment

TRANSFORMED MEAN CALCtJLATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 CONTROL 46200 46200 2 RW-1 48400 48400 - 0 3 5 6 3 BG-2 32556 32556 2 1 5 0 4 BG-3 32400 32400 2 2 3 4 5 LT-1 42600 42600 0 5 8 3 6 BG-l 28250 28250 2 7 4 0

Bonferroni T table value = 240 (1 Tailed Value P=005 df=505)

eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeE n Press any key to continue n ^eeeeeeeeSeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeyen

CHESHIRE SURFACE WATER C DUBIA REPRO File CT0XSTAT3CHESCDRPR Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 CONTROL 10 2 RW-1 10 14850 321 -2200 3 BG-2 9 15257 330 13644 4 BG-3 10 14850 321 13800 5 LT-1 10 14850 321 3600 6 BG-l 8 15751 341 17950

dee3eeeesecteeeeeeSeeeeeeeeeeeeeeeeSesectee^eeeeeeeeeeeeeeepound n Press any key to continue or Esc to return to menu n ^eeeeeeeeeeeeeesecteeeeeeeeeeeeeeeesecteeeeeeeeeeeeeeeeeeeeyen

o

CHESHIRE SURFACE WATER P PROMELAS GROWTH File CT0XSTAT3CHESPPGTH Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment -c- TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

1 CONTROL 0 618 0 618 2 RW-1 0 583 0 583 0 783 3 BG-2 0 593 0 593 0 559 4 BG-3 0 623 0 623 - 0 1 1 2 5 LT-1 0 718 0 718 - 2 2 3 6 6 BG-l 0 618 0 618 0 000

Dunnett table value = 241 (1 Tailed Value P=005 df=185)

^eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeE n Press any key to continue n aeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeyen

CHESHIRE SURFACE WATER P PROMELAS GROWTH File CT0XSTAT3CHESPPGTH Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 Ho ControKTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 CONTROL 4 2 RW-1 4 0108 175 0035 3 BG-2 4 0108 175 0025 4 BG-3 4 0108 175 -0005 5 LT-1 4 0108 175 -0100 6 BG-l 4 0108 175 0000

secteeeeeeeeeeeeeeeeeeeeeesecteeeeeeeeeeeeeeeeeeeeeeeeeeeeepound n Press any key to continue or Esc to return to menu a Seeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeyen

ffi-gt A- ]j^klt^^^C^ JU^O y U i gt ^ - X-^^ ^ArZ^y ^C^i^ Q

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

Ch( quare test for normality actual and expected frecjuencies

INTERVAL lt-15 -15 to lt-05 -05 to 05 gt05 to 15 gt15

EXPECTED 3149 11374 17954 11374 3149 OBSERVED 1 13 19 10 4

Calculated Chi-Sguare goodness of fit test statistic = 21559 Table Chi-Square value (alpha = 001) = 13277

Data PASS normality test Continue analysis

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

Shapiro Wilks test for normality

D = 18201

W raquo 0872

Critical W (P = 005) (n = 47) = 0946 Critical W (P = 001) (n = 47) = 0928

Data FAIL normality test Try another transformation

Warning - The two homogeneity tests are sensitive to non-normal data and should not be performed

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

Hartley test for homogeneity of variance

Calculated H statistic (max Varmin Var) = 942 Closest conservative Table H statistic = 184 (alpha = 001)

Used for Table H ==gt R ( groups) = 6 df ( reps-1) = 7 Actual values ==gt R ( groups) = 6 df ( avg reps-1) = 683

(average df used)

-c-Data PASS homogeneity test Continue analysis

NOTE This test requires equal replicate sizes If they are unequal

Bonferroni T table value = 242 (1 Tailed Value P=005 df=405)

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 ART SED 8 2 RW-1 7 0836 360 1111 3 BG-l 8 0808 347 1288 4 BG-2 8 0808 347 1575 5 BG-3 8 0808 347 0488 6 LT-1 8 0808 347 1275

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS ^

Between 5 13993 2799 6304

Within (Error) 41 18201 0444

Total 46 32194

Critical F value = 245 (005540) Since F gt Critical F REJECT HoAll groups equal

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 1 OF 2 HoControlltTreatment

TRANSFORMED MEAN CALCULATED IN GROUP TION MEAN ORIGINAL UNITS T STAT SIG

ART SED 2325 2325 2 RW-1 1214 1214 3221 3 BG-l 1038 1038 3864 4 BG-2 0750 0750 4727 5 BG-3 1838 1838 1463 6 LT-1 1050 1050 3827

Bonferroni T table value = 242 (1 Tailed Value P=005 df=405)

6 LT-1 1050 1050 3827

Bonferroni T table value = 242 (1 Tailed Value P=005 df=405)

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 2 OF 2 Ho ControKTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 bull SED 8 2 RW-1 7 0836 360 1111 3 BG-l 8 0808 347 1288 4 BG-2 8 0808 347 1575 5 BG-3 8 0808 347 0488 6 LT-1 8 0808 347 1275

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS F

Between 5 13993 2799 6304

Within (Error) 41 18201 0444

Total 46 32194

Critical F value = 245 (005540) Since F gt Critical F REJECT HoAll groups equal

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 1 OF 2 Ho ControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP LTION MEAN ORIGINAL UNITS T STAT SIG

mdash

1 ART SED 2325 2325 RW-1 1214 1214 3221

BG-l 1038 1038 3864

BG-2 0750 0750 4727 5 BG-3 1838 1838 1463 6 LT-1 1050 1050 3827

CHESHIRE SEDIMENT CT GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

Chi-slt3uare test for normality actual and expected frecjuencies

INTERVAL lt-15 -15 to lt-05 -05 to 05 gt05 to 15 gt15

slaquo

EXPECTED OBSERVED

2613 0

9438 12

14898 16

9438 7

2613 4

Calculated Chi-Scjuare goodness of fit test statistic =Table Chi-Stjuare value (alpha = 001) = 13277

47560

Data PASS normality test Continue analysis

CHESHIRE SEDIMENT CT GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

Hartley test for homogeneity of variance

Calculated H statistic (max Varmin Var) =Closest conservative Table H statistic =

Used for Table H ==gt R ( groups) = 5Actual values ==gt R ( groups) = 5

942 165 (alpha = 001)

df ( reps-1) = df ( avg reps-1) =

(average df used)

^ 680

^

Data PASS homogeneity test Continue analysis

NOTE This test retjuires ecjual replicate sizes If they are unecjual but do not differ greatly the Hartley test may still be used as an approximate test (average df are used)

Bonferroni T table value = 242 (1 Tailed Value P=005 df=405)

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 ART SED 8 2 RW-1 7 0836 360 1111 3 BG-l 8 0808 347 1288 4 BG-2 8 0808 347 1575 5 BG-3 8 0808 347 0488 6 LT-1 8 0808 347 1275

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS ^

Between 5 13993 2799 6304

Within (Error) 41 18201 0444

Total 46 32194

Critical F value = 245 (005540) Since F gt Critical F REJECT HoAll groups equal

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 1 OF 2 HoControlltTreatment

TRANSFORMED MEAN CALCULATED IN GROUP TION MEAN ORIGINAL UNITS T STAT SIG

ART SED 2325 2325 2 RW-1 1214 1214 3221 3 BG-l 1038 1038 3864 4 BG-2 0750 0750 4727 5 BG-3 1838 1838 1463 6 LT-1 1050 1050 3827

Bonferroni T table value = 242 (1 Tailed Value P=005 df=405)

6 LT-1 1050 1050 3827

Bonferroni T table value = 242 (1 Tailed Value P=005 df=405)

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 2 OF 2 Ho ControKTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 bull SED 8 2 RW-1 7 0836 360 1111 3 BG-l 8 0808 347 1288 4 BG-2 8 0808 347 1575 5 BG-3 8 0808 347 0488 6 LT-1 8 0808 347 1275

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS F

Between 5 13993 2799 6304

Within (Error) 41 18201 0444

Total 46 32194

Critical F value = 245 (005540) Since F gt Critical F REJECT HoAll groups equal

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 1 OF 2 Ho ControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP LTION MEAN ORIGINAL UNITS T STAT SIG

mdash

1 ART SED 2325 2325 RW-1 1214 1214 3221

BG-l 1038 1038 3864

BG-2 0750 0750 4727 5 BG-3 1838 1838 1463 6 LT-1 1050 1050 3827

CHESHIRE SEDIMENT CT GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

Chi-slt3uare test for normality actual and expected frecjuencies

INTERVAL lt-15 -15 to lt-05 -05 to 05 gt05 to 15 gt15

slaquo

EXPECTED OBSERVED

2613 0

9438 12

14898 16

9438 7

2613 4

Calculated Chi-Scjuare goodness of fit test statistic =Table Chi-Stjuare value (alpha = 001) = 13277

47560

Data PASS normality test Continue analysis

CHESHIRE SEDIMENT CT GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

Hartley test for homogeneity of variance

Calculated H statistic (max Varmin Var) =Closest conservative Table H statistic =

Used for Table H ==gt R ( groups) = 5Actual values ==gt R ( groups) = 5

942 165 (alpha = 001)

df ( reps-1) = df ( avg reps-1) =

(average df used)

^ 680

^

Data PASS homogeneity test Continue analysis

NOTE This test retjuires ecjual replicate sizes If they are unecjual but do not differ greatly the Hartley test may still be used as an approximate test (average df are used)

6 LT-1 1050 1050 3827

Bonferroni T table value = 242 (1 Tailed Value P=005 df=405)

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 2 OF 2 Ho ControKTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 bull SED 8 2 RW-1 7 0836 360 1111 3 BG-l 8 0808 347 1288 4 BG-2 8 0808 347 1575 5 BG-3 8 0808 347 0488 6 LT-1 8 0808 347 1275

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS F

Between 5 13993 2799 6304

Within (Error) 41 18201 0444

Total 46 32194

Critical F value = 245 (005540) Since F gt Critical F REJECT HoAll groups equal

CHESHIRE SEDIMENT CT GROWTH File CHESSEDASG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 1 OF 2 Ho ControKTreatment

TRANSFORMED MEAN CALCULATED IN GROUP LTION MEAN ORIGINAL UNITS T STAT SIG

mdash

1 ART SED 2325 2325 RW-1 1214 1214 3221

BG-l 1038 1038 3864

BG-2 0750 0750 4727 5 BG-3 1838 1838 1463 6 LT-1 1050 1050 3827

CHESHIRE SEDIMENT CT GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

Chi-slt3uare test for normality actual and expected frecjuencies

INTERVAL lt-15 -15 to lt-05 -05 to 05 gt05 to 15 gt15

slaquo

EXPECTED OBSERVED

2613 0

9438 12

14898 16

9438 7

2613 4

Calculated Chi-Scjuare goodness of fit test statistic =Table Chi-Stjuare value (alpha = 001) = 13277

47560

Data PASS normality test Continue analysis

CHESHIRE SEDIMENT CT GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

Hartley test for homogeneity of variance

Calculated H statistic (max Varmin Var) =Closest conservative Table H statistic =

Used for Table H ==gt R ( groups) = 5Actual values ==gt R ( groups) = 5

942 165 (alpha = 001)

df ( reps-1) = df ( avg reps-1) =

(average df used)

^ 680

^

Data PASS homogeneity test Continue analysis

NOTE This test retjuires ecjual replicate sizes If they are unecjual but do not differ greatly the Hartley test may still be used as an approximate test (average df are used)

CHESHIRE SEDIMENT CT GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

Chi-slt3uare test for normality actual and expected frecjuencies

INTERVAL lt-15 -15 to lt-05 -05 to 05 gt05 to 15 gt15

slaquo

EXPECTED OBSERVED

2613 0

9438 12

14898 16

9438 7

2613 4

Calculated Chi-Scjuare goodness of fit test statistic =Table Chi-Stjuare value (alpha = 001) = 13277

47560

Data PASS normality test Continue analysis

CHESHIRE SEDIMENT CT GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

Hartley test for homogeneity of variance

Calculated H statistic (max Varmin Var) =Closest conservative Table H statistic =

Used for Table H ==gt R ( groups) = 5Actual values ==gt R ( groups) = 5

942 165 (alpha = 001)

df ( reps-1) = df ( avg reps-1) =

(average df used)

^ 680

^

Data PASS homogeneity test Continue analysis

NOTE This test retjuires ecjual replicate sizes If they are unecjual but do not differ greatly the Hartley test may still be used as an approximate test (average df are used)

CHESHIRE SEDIMENT CT GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

ANOVA TABLE - ^

SOURCE DF SS MS F

Between 4 5243 1311 2676

Within (Error) 34 16666 0490

38 21909

Critical F value = 269 (005430) Since F lt Critical F FAIL TO REJECT HoAll groups equal

CHESHIRE SEDIMENT CT- GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 1 OF 2 HoControlltTreatment

GROUP IDENTIFICATION IN TRANSFORMED

MEAN MEAN CALCULATED IN

ORIGINAL UNITS T STAT SIG

4 5

RW-1 BG-l BG-2 BG-3 LT-1

1214 1038 0750 1838 1050

1214 1038 0750 1838 1050

0488 1282

-1720 0453

Bonferroni T table value = 235 (1 Tailed Value P=005 df=344)

CHESHIRE SEDIMENT CT GROWTH File CT0XSTAT3CHESSEDCTG Transform NO TRANSFORMATION

BONFERRONI T-TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 7 2 BG-l 8 0850 700 0177 3 BG-2 8 0850 700 0464 4 BG-3 8 0850 700 -0623 5 LT-1 8 0850 700 0164

^ i ici^^ C^^ W

CHESHIRE SEDIMENT CT SURVIVAL J ) File CHESSEDCT Transform NO TRANSFORMATION -

ShaJro Wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 708 Table Chi-square value = 1509 (alpha = 001) Table Chi-scjuare value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 Use for Chi-scjuare table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Shapiro Wilks test for normality

D = 158500

W = 0979

Critical W (p = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Da-^ PASS normality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL

2 BG-l 3 BG-2 4 BG-3 5 LT-1

Dunnett table value = 225

6125 6 1 2 5 -0261 6625 6 6 2 5 -0782 6250 6 2 5 0 -0391 6250 6 2 5 0 -0391

(1 Tailed Value P=005 df=304)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSITESEDCT Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

GROUP

1 2 3 4 5

IDENTIFICATION

RW-1 BG-l BG-2 BG-3 LT-1

NUM OFREPS

8 8 8 8 8

Minimum Sig Diff (IN ORIG UNITS)

2157 2157 2157 2157

of CONTROL

367 367 367 367

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS

Between 5 9167 1833

Within (Error) 42 158500 3774

Total 47 167667

Critical F value = 245 (005540) Since F lt Critical F FAIL TO REJECT HoAll groups equal

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

DIFFERENCE FROM CONTROL

-0250 -0750 -0375 -0375

F

0486

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

GROUP IDENTIFICATION

1 ARTIFICIAL SED 2 RW-1 3 BG-l 4 BG-2 5 BG-3

TRANSFORMED MEAN

MEAN CALCULATED ORIGINAL UNITS

IN T STAT SIG

mdash 7250 5875 6125 6625 6250

7250 5875 6125 6625 6250

1416 1158 0643 1030

C

6 LT-1 6375 6375 0901

Dunnett table value = 231 (1 Tailed Value P=005 df=405)

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoCentrelltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 ARTIFICIAL SED 8 2 RW-1 8 2244 309 1375 3 BG-l 8 2244 309 1125 4 BG-2 8 2244 309 0625 5 BG-3 8 2244 309 1000 6 LT-1 8 2244 309 0875

vbdquo

I

CHESHIRE SEDIMENT CT SURVIVAL ^ ^ File CT0XSTAT3CHESSITESED Transform NO TRANSFORMATION

Shajrtro Wilks test for normality

D = 128625

W = 0945

Critical W (P = 005) (n = 40) = 0940 Critical W (P = 001) (n = 40) = 0919

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSITESED Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 770 Table Chi-scjuare value = 1328 (alpha = 001) Table Chi-slt5uare value = 949 (alpha = 005)

AvL ige df used in calculation ==gt df (avg n - 1) = 700 UseH for Chi-s(3uare table value ==gt df (groups-l) = 4

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

( ^

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSITESED Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF ss MS F

Between 4 2350 0587 0160

Within (Error) 35 128625 3675

Total 39 130975

Critical F value = 269 (005430) Since F lt Critical F FAIL TO REJECT HoAll groups equal

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSITESED Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATIONN MEAN ORIGINAL UNITS T STAT SIG

1 RW-1 5875 5875 2 BG-l 6125 6125 -0261 3 BG-2 6625 6625 -0782 4 BG-3 6250 6250 -0391 5 LT-1 6250 6250 -0391

Dunnett table value = 225 (1 Tailed Value P=005 df=3 04)

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSITESED Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 8 2 BG-l 8 2157 367 -0250 3 BG-2 8 2157 367 -0750 4 BG-3 8 2157 367 -0375 5 LT-1 8 2157 367 -0375

y ^ f l i M t - y r -LAi ^t^ yi-- 1- ^- ilaquo-^ p y euro 9 - ^

CHESHIRE SED HA SURVIVAL - SITE REFERENCE File CHESSEDRWl Transform NO TRANSFORMATION

ANOVA TABLE C - SOURCE DF SS MS F

Between 4 514850 128712 12721

Within (Error) 35 354125 10118

Total 39 868975

Critical F value = 269 (005430) Since F gt Critical F REJECT HoAll groups equal

CHESHIRE SED HA SURVIVAL - SITE REFERENCE File CHESSEDRWl Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

RW-1 12500 12500 BG-l 11875 11875 0393

3 BG-2 3375 3375 5737 4 BG-3 5750 5750 4244 5 LT-1 10375 10375 1336

Dunnett table value = 225 (1 Tailed Value P=005 df=3 04)

CHESHIRE SED HA SURVIVAL - SITE REFERENCE File CHESSEDRWl Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 RW-1 8 2 BG-l 8 3578 286 0625 3 BG-2 8 3578 286 9125 4 BG-3 8 3578 286 6750 5 LT-1 8 3578 286 2125

CHESHIRE SED HA SURVIVAL - SITE REFERENCE File CHESSEDRWl Transform NO TRANSFORMATION

Shapiro Wilks test for normality

D laquo 354125

W = 0969

Critical W (P = 005) (n = 40) = 0940 Critical W (P = 001) (n = 40) = 0919

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SED HA SURVIVAL - SITE REFERENCE File CHESSEDRWl Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 379 Table Chi-square value = 1328 (alpha = 001) Table Chi-sltiuare value = 949 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 Used for Chi-square table value ==gt df (groups-l) = 4

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

CHESHIRE SED HA SURVIVAL File CHESSEDHAS Transform NO TRANSFORMATION

Shailaquo- ro Wilks test for normality

D = 369625

W = 0969

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SED HA SURVIVAL File CHESSEDHAS Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 832 Table Chi-scjuare value = 1509 (alpha = 001) Table Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 U s ^ for Chi-s(iuare table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

n

CHESHIRE SED HA SURVIVAL - SITE REFERENCE File CHESSEDRWl Transform NO TRANSFORMATION

Shapiro Wilks test for normality

D laquo 354125

W = 0969

Critical W (P = 005) (n = 40) = 0940 Critical W (P = 001) (n = 40) = 0919

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SED HA SURVIVAL - SITE REFERENCE File CHESSEDRWl Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 379 Table Chi-square value = 1328 (alpha = 001) Table Chi-sltiuare value = 949 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 Used for Chi-square table value ==gt df (groups-l) = 4

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

CHESHIRE SED HA SURVIVAL File CHESSEDHAS Transform NO TRANSFORMATION

Shailaquo- ro Wilks test for normality

D = 369625

W = 0969

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SED HA SURVIVAL File CHESSEDHAS Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 832 Table Chi-scjuare value = 1509 (alpha = 001) Table Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 U s ^ for Chi-s(iuare table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

n

CHESHIRE SED HA SURVIVAL File CHESSEDHAS Transform NO TRANSFORMATION

Shailaquo- ro Wilks test for normality

D = 369625

W = 0969

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SED HA SURVIVAL File CHESSEDHAS Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 832 Table Chi-scjuare value = 1509 (alpha = 001) Table Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 U s ^ for Chi-s(iuare table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

n

CHESHIRE SED HA SURVIVAL File CHESSEDHAS Transform NO TRANSFORMATION

ANOVA TABLE ^

SOURCE DF SS MS F

Between 5 540854 108171 12291

Within (Error) 42 369625 8801

Total 47 910479

Critical F value = 245 (005540) Since F gt Critical F REJECT HoAll groups etjual

CHESHIRE SED HA SURVIVAL File CHESSEDHAS Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

GROUP IDENTIFICATION TRANSFORMED

MEAN MEAN CALCULATED

ORIGINAL UNITS IN

mdash T STAT SIG

1 2 3 4 5 6

ARTFICIAL SED RW-1 BG-l BG-2 BG-3 LT-1

10750 12500 11875 3375 5750 10375

10750 12500 11875 3375 5750 10375

-1180 -0758 4972 3371 0253

Dunnett table value = 231 (1 Tailed Value P=005 df=405)

CHESHIRE SED HA SURVIVAL File CHESSEDHAS Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

GROUP IDENTIFICATION NUM OFREPS

Minimum Sig Diff (IN ORIG UNITS)

of CONTROL

DIFFERENCE FROM CONTROL

1 ARTFICIAL SED 8 2 RW-1 8 3426 319 -1750 3 BG-l 8 3426 319 -1125 4 BG-2 8 3426 319 7375 5 BG-3 8 3426 319 5000 6 LT-1 8 3426 319 0375

CHESHIRE SED HA GROWTH F i l e CHESSEDHAG T r a n s f o r m NO TRANSFORMATION

S h ^ i r o W i l k s t e s t f o r n o r m a l i t y

-

D = 8 9 9 1

W = 0 8 9 5 Critical W (P = 005) (n = 46) = 0945 Critical W (P = 001) (n = 46) = 0927

Data FAIL normality test Try another transformation

Warning - The two homogeneity tests are sensitive to non-normal data and should not be performed

CHESHIRE SED HA GROWTH File CHESSEDHAG Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 1263 Ta^e Chi-square value = 1509 (alpha = 001) Tar s Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 667 Used for Chi-square table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

0

CHESHIRE SED HA GROWTH File CHESSEDHAG Transform NO TRANSFORM

WILCOXON RANK SUM TEST W BONFERRONI ADJUSTMENT Ho ControKTreatment

TRANSFORMED RANK CRIT GROUP IDENTIFICATION MEAN SUM VALUE REPS SIG

1 2 3 4 5 6

ARTFICIAL SED RW-1 BG-l BG-2

LT-1

0988 1163 1288 1300 1071 1200

8300 7650 6550 6600 8000

4500 4500 3500 3500 4500

8 8 7 7 8

Critical values use k = 5 are 1 tailed and alpha =005

^

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform NO TRANSFORMATION

fiC ro Wilks test for normality

D = 0000

W = 0902

Critical w (p = 005) (n = 38) = 0938 Critical W (P = 001) (n = 38) = 0916

Data FAIL normality test Try another transformation

Warning - The two homogeneity tests are sensitive to non-normal data and should not be performed

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 584 Tablaquo Chi-square value = 1328 (alpha = 001) TaAy Chi-square value = 949 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 660 Used for Chi-square table value ==gt df (groups-l) = 4

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

r

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform ARC SINE(SQUARE ROOT(Y))

WILCOXON RANK SUM TEST W BONFERRONI ADJUSTMENT HoControlltTreatment

GROUP IDENTIFICATIOINN TRANSFORMED

MEAN RANK SUM

CRIT VALUE REPS SIG

1 2 3 4 5

RW-1 BG-l BG-2 BG-3 LT-1

0036 0035 0035 0031 0034

5950 5250 4900 6200

4600 3600 3600 4600

8 7 7 8

Critical values use k = 4 are 1 tailed and alpha = 005

o

o

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION -

Shaoiro wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS nomnality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 708 Table Chi-scjuare value = 1509 (alpha = 001) Table Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 Us- for Chi-square table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Shapiro Wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL

2 BG-l 3 BG-2 4 BG-3 5 LT-1

Dunnett table value 225

6125 6 1 2 5 -0261 6625 6 6 2 5 -0782 6250 6 2 5 0 -0391 6250 6 2 5 0 -0391

(1 Tailed Value P=005 df=304)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSITESEDCT Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

GROUP

1 2 3 4 5

IDENTIFICATION

RW-1 BG-l BG-2 BG-3 LT-1

NUM OFREPS

8 8 8 8 8

Minimum Sig Diff (IN ORIG UNITS)

2157 2157 2157 2157

of CONTROL

367 367 367 367

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS

Between 5 9167 1833

Within (Error) 42 158500 3774

Total 47 167667

Critical F value = 245 (005540) Since F lt Critical F FAIL TO REJECT HoAll groups ecjual

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

DIFFERENCE FROM CONTROL

-0250 -0750 -0375 -0375

_ ^o F

0486

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

GROUP IDENTIFICATION

1 ARTIFICIAL SED 2 RW-1 3 BG-l 4 BG-2 5 BG-3

TRANSFORMED MEAN

MEAN CALCULATED ORIGINAL UNITS

IN

mdash

7250 5875 6125 6625 6250

7250 5875 6125 6625 6250

T STAT SIG

1416 1158 0643 1030

^

bull

CHESHIRE SED HA GROWTH F i l e CHESSEDHAG T r a n s f o r m NO TRANSFORMATION

S h ^ i r o W i l k s t e s t f o r n o r m a l i t y

-

D = 8 9 9 1

W = 0 8 9 5 Critical W (P = 005) (n = 46) = 0945 Critical W (P = 001) (n = 46) = 0927

Data FAIL normality test Try another transformation

Warning - The two homogeneity tests are sensitive to non-normal data and should not be performed

CHESHIRE SED HA GROWTH File CHESSEDHAG Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 1263 Ta^e Chi-square value = 1509 (alpha = 001) Tar s Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 667 Used for Chi-square table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

0

CHESHIRE SED HA GROWTH File CHESSEDHAG Transform NO TRANSFORM

WILCOXON RANK SUM TEST W BONFERRONI ADJUSTMENT Ho ControKTreatment

TRANSFORMED RANK CRIT GROUP IDENTIFICATION MEAN SUM VALUE REPS SIG

1 2 3 4 5 6

ARTFICIAL SED RW-1 BG-l BG-2

LT-1

0988 1163 1288 1300 1071 1200

8300 7650 6550 6600 8000

4500 4500 3500 3500 4500

8 8 7 7 8

Critical values use k = 5 are 1 tailed and alpha =005

^

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform NO TRANSFORMATION

fiC ro Wilks test for normality

D = 0000

W = 0902

Critical w (p = 005) (n = 38) = 0938 Critical W (P = 001) (n = 38) = 0916

Data FAIL normality test Try another transformation

Warning - The two homogeneity tests are sensitive to non-normal data and should not be performed

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 584 Tablaquo Chi-square value = 1328 (alpha = 001) TaAy Chi-square value = 949 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 660 Used for Chi-square table value ==gt df (groups-l) = 4

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

r

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform ARC SINE(SQUARE ROOT(Y))

WILCOXON RANK SUM TEST W BONFERRONI ADJUSTMENT HoControlltTreatment

GROUP IDENTIFICATIOINN TRANSFORMED

MEAN RANK SUM

CRIT VALUE REPS SIG

1 2 3 4 5

RW-1 BG-l BG-2 BG-3 LT-1

0036 0035 0035 0031 0034

5950 5250 4900 6200

4600 3600 3600 4600

8 7 7 8

Critical values use k = 4 are 1 tailed and alpha = 005

o

o

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION -

Shaoiro wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS nomnality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 708 Table Chi-scjuare value = 1509 (alpha = 001) Table Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 Us- for Chi-square table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Shapiro Wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL

2 BG-l 3 BG-2 4 BG-3 5 LT-1

Dunnett table value 225

6125 6 1 2 5 -0261 6625 6 6 2 5 -0782 6250 6 2 5 0 -0391 6250 6 2 5 0 -0391

(1 Tailed Value P=005 df=304)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSITESEDCT Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

GROUP

1 2 3 4 5

IDENTIFICATION

RW-1 BG-l BG-2 BG-3 LT-1

NUM OFREPS

8 8 8 8 8

Minimum Sig Diff (IN ORIG UNITS)

2157 2157 2157 2157

of CONTROL

367 367 367 367

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS

Between 5 9167 1833

Within (Error) 42 158500 3774

Total 47 167667

Critical F value = 245 (005540) Since F lt Critical F FAIL TO REJECT HoAll groups ecjual

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

DIFFERENCE FROM CONTROL

-0250 -0750 -0375 -0375

_ ^o F

0486

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

GROUP IDENTIFICATION

1 ARTIFICIAL SED 2 RW-1 3 BG-l 4 BG-2 5 BG-3

TRANSFORMED MEAN

MEAN CALCULATED ORIGINAL UNITS

IN

mdash

7250 5875 6125 6625 6250

7250 5875 6125 6625 6250

T STAT SIG

1416 1158 0643 1030

^

bull

0

CHESHIRE SED HA GROWTH File CHESSEDHAG Transform NO TRANSFORM

WILCOXON RANK SUM TEST W BONFERRONI ADJUSTMENT Ho ControKTreatment

TRANSFORMED RANK CRIT GROUP IDENTIFICATION MEAN SUM VALUE REPS SIG

1 2 3 4 5 6

ARTFICIAL SED RW-1 BG-l BG-2

LT-1

0988 1163 1288 1300 1071 1200

8300 7650 6550 6600 8000

4500 4500 3500 3500 4500

8 8 7 7 8

Critical values use k = 5 are 1 tailed and alpha =005

^

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform NO TRANSFORMATION

fiC ro Wilks test for normality

D = 0000

W = 0902

Critical w (p = 005) (n = 38) = 0938 Critical W (P = 001) (n = 38) = 0916

Data FAIL normality test Try another transformation

Warning - The two homogeneity tests are sensitive to non-normal data and should not be performed

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 584 Tablaquo Chi-square value = 1328 (alpha = 001) TaAy Chi-square value = 949 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 660 Used for Chi-square table value ==gt df (groups-l) = 4

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

r

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform ARC SINE(SQUARE ROOT(Y))

WILCOXON RANK SUM TEST W BONFERRONI ADJUSTMENT HoControlltTreatment

GROUP IDENTIFICATIOINN TRANSFORMED

MEAN RANK SUM

CRIT VALUE REPS SIG

1 2 3 4 5

RW-1 BG-l BG-2 BG-3 LT-1

0036 0035 0035 0031 0034

5950 5250 4900 6200

4600 3600 3600 4600

8 7 7 8

Critical values use k = 4 are 1 tailed and alpha = 005

o

o

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION -

Shaoiro wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS nomnality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 708 Table Chi-scjuare value = 1509 (alpha = 001) Table Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 Us- for Chi-square table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Shapiro Wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL

2 BG-l 3 BG-2 4 BG-3 5 LT-1

Dunnett table value 225

6125 6 1 2 5 -0261 6625 6 6 2 5 -0782 6250 6 2 5 0 -0391 6250 6 2 5 0 -0391

(1 Tailed Value P=005 df=304)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSITESEDCT Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

GROUP

1 2 3 4 5

IDENTIFICATION

RW-1 BG-l BG-2 BG-3 LT-1

NUM OFREPS

8 8 8 8 8

Minimum Sig Diff (IN ORIG UNITS)

2157 2157 2157 2157

of CONTROL

367 367 367 367

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS

Between 5 9167 1833

Within (Error) 42 158500 3774

Total 47 167667

Critical F value = 245 (005540) Since F lt Critical F FAIL TO REJECT HoAll groups ecjual

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

DIFFERENCE FROM CONTROL

-0250 -0750 -0375 -0375

_ ^o F

0486

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

GROUP IDENTIFICATION

1 ARTIFICIAL SED 2 RW-1 3 BG-l 4 BG-2 5 BG-3

TRANSFORMED MEAN

MEAN CALCULATED ORIGINAL UNITS

IN

mdash

7250 5875 6125 6625 6250

7250 5875 6125 6625 6250

T STAT SIG

1416 1158 0643 1030

^

bull

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform NO TRANSFORMATION

fiC ro Wilks test for normality

D = 0000

W = 0902

Critical w (p = 005) (n = 38) = 0938 Critical W (P = 001) (n = 38) = 0916

Data FAIL normality test Try another transformation

Warning - The two homogeneity tests are sensitive to non-normal data and should not be performed

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 584 Tablaquo Chi-square value = 1328 (alpha = 001) TaAy Chi-square value = 949 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 660 Used for Chi-square table value ==gt df (groups-l) = 4

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

r

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform ARC SINE(SQUARE ROOT(Y))

WILCOXON RANK SUM TEST W BONFERRONI ADJUSTMENT HoControlltTreatment

GROUP IDENTIFICATIOINN TRANSFORMED

MEAN RANK SUM

CRIT VALUE REPS SIG

1 2 3 4 5

RW-1 BG-l BG-2 BG-3 LT-1

0036 0035 0035 0031 0034

5950 5250 4900 6200

4600 3600 3600 4600

8 7 7 8

Critical values use k = 4 are 1 tailed and alpha = 005

o

o

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION -

Shaoiro wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS nomnality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 708 Table Chi-scjuare value = 1509 (alpha = 001) Table Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 Us- for Chi-square table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Shapiro Wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL

2 BG-l 3 BG-2 4 BG-3 5 LT-1

Dunnett table value 225

6125 6 1 2 5 -0261 6625 6 6 2 5 -0782 6250 6 2 5 0 -0391 6250 6 2 5 0 -0391

(1 Tailed Value P=005 df=304)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSITESEDCT Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

GROUP

1 2 3 4 5

IDENTIFICATION

RW-1 BG-l BG-2 BG-3 LT-1

NUM OFREPS

8 8 8 8 8

Minimum Sig Diff (IN ORIG UNITS)

2157 2157 2157 2157

of CONTROL

367 367 367 367

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS

Between 5 9167 1833

Within (Error) 42 158500 3774

Total 47 167667

Critical F value = 245 (005540) Since F lt Critical F FAIL TO REJECT HoAll groups ecjual

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

DIFFERENCE FROM CONTROL

-0250 -0750 -0375 -0375

_ ^o F

0486

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

GROUP IDENTIFICATION

1 ARTIFICIAL SED 2 RW-1 3 BG-l 4 BG-2 5 BG-3

TRANSFORMED MEAN

MEAN CALCULATED ORIGINAL UNITS

IN

mdash

7250 5875 6125 6625 6250

7250 5875 6125 6625 6250

T STAT SIG

1416 1158 0643 1030

^

bull

CHESHIRE SED HA GROWTH File CHESHAGRWl Transform ARC SINE(SQUARE ROOT(Y))

WILCOXON RANK SUM TEST W BONFERRONI ADJUSTMENT HoControlltTreatment

GROUP IDENTIFICATIOINN TRANSFORMED

MEAN RANK SUM

CRIT VALUE REPS SIG

1 2 3 4 5

RW-1 BG-l BG-2 BG-3 LT-1

0036 0035 0035 0031 0034

5950 5250 4900 6200

4600 3600 3600 4600

8 7 7 8

Critical values use k = 4 are 1 tailed and alpha = 005

o

o

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION -

Shaoiro wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS nomnality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 708 Table Chi-scjuare value = 1509 (alpha = 001) Table Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 Us- for Chi-square table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Shapiro Wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL

2 BG-l 3 BG-2 4 BG-3 5 LT-1

Dunnett table value 225

6125 6 1 2 5 -0261 6625 6 6 2 5 -0782 6250 6 2 5 0 -0391 6250 6 2 5 0 -0391

(1 Tailed Value P=005 df=304)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSITESEDCT Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

GROUP

1 2 3 4 5

IDENTIFICATION

RW-1 BG-l BG-2 BG-3 LT-1

NUM OFREPS

8 8 8 8 8

Minimum Sig Diff (IN ORIG UNITS)

2157 2157 2157 2157

of CONTROL

367 367 367 367

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS

Between 5 9167 1833

Within (Error) 42 158500 3774

Total 47 167667

Critical F value = 245 (005540) Since F lt Critical F FAIL TO REJECT HoAll groups ecjual

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

DIFFERENCE FROM CONTROL

-0250 -0750 -0375 -0375

_ ^o F

0486

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

GROUP IDENTIFICATION

1 ARTIFICIAL SED 2 RW-1 3 BG-l 4 BG-2 5 BG-3

TRANSFORMED MEAN

MEAN CALCULATED ORIGINAL UNITS

IN

mdash

7250 5875 6125 6625 6250

7250 5875 6125 6625 6250

T STAT SIG

1416 1158 0643 1030

^

bull

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION -

Shaoiro wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS nomnality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Bartletts test for homogeneity of variance

Calculated B statistic = 708 Table Chi-scjuare value = 1509 (alpha = 001) Table Chi-square value = 1107 (alpha = 005)

Average df used in calculation ==gt df (avg n - 1) = 700 Us- for Chi-square table value ==gt df (groups-l) = 5

Data PASS homogeneity test at 001 level Continue analysis

NOTE If groups have unequal replicate sizes the average replicate size is used to calculate the B statistic (see above)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSEDCT Transform NO TRANSFORMATION

Shapiro Wilks test for normality

D = 158500

W = 0979

Critical W (P = 005) (n = 48) = 0947 Critical W (P = 001) (n = 48) = 0929

Data PASS normality test at P=001 level Continue analysis

CHESHIRE SEDIMENT CT SURVIVAL

2 BG-l 3 BG-2 4 BG-3 5 LT-1

Dunnett table value 225

6125 6 1 2 5 -0261 6625 6 6 2 5 -0782 6250 6 2 5 0 -0391 6250 6 2 5 0 -0391

(1 Tailed Value P=005 df=304)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSITESEDCT Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

GROUP

1 2 3 4 5

IDENTIFICATION

RW-1 BG-l BG-2 BG-3 LT-1

NUM OFREPS

8 8 8 8 8

Minimum Sig Diff (IN ORIG UNITS)

2157 2157 2157 2157

of CONTROL

367 367 367 367

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS

Between 5 9167 1833

Within (Error) 42 158500 3774

Total 47 167667

Critical F value = 245 (005540) Since F lt Critical F FAIL TO REJECT HoAll groups ecjual

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

DIFFERENCE FROM CONTROL

-0250 -0750 -0375 -0375

_ ^o F

0486

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

GROUP IDENTIFICATION

1 ARTIFICIAL SED 2 RW-1 3 BG-l 4 BG-2 5 BG-3

TRANSFORMED MEAN

MEAN CALCULATED ORIGINAL UNITS

IN

mdash

7250 5875 6125 6625 6250

7250 5875 6125 6625 6250

T STAT SIG

1416 1158 0643 1030

^

bull

2 BG-l 3 BG-2 4 BG-3 5 LT-1

Dunnett table value 225

6125 6 1 2 5 -0261 6625 6 6 2 5 -0782 6250 6 2 5 0 -0391 6250 6 2 5 0 -0391

(1 Tailed Value P=005 df=304)

CHESHIRE SEDIMENT CT SURVIVAL File CHESSITESEDCT Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

GROUP

1 2 3 4 5

IDENTIFICATION

RW-1 BG-l BG-2 BG-3 LT-1

NUM OFREPS

8 8 8 8 8

Minimum Sig Diff (IN ORIG UNITS)

2157 2157 2157 2157

of CONTROL

367 367 367 367

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

ANOVA TABLE

SOURCE DF SS MS

Between 5 9167 1833

Within (Error) 42 158500 3774

Total 47 167667

Critical F value = 245 (005540) Since F lt Critical F FAIL TO REJECT HoAll groups ecjual

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

DIFFERENCE FROM CONTROL

-0250 -0750 -0375 -0375

_ ^o F

0486

DUNNETTS TEST TABLE 1 OF 2 HoControlltTreatment

GROUP IDENTIFICATION

1 ARTIFICIAL SED 2 RW-1 3 BG-l 4 BG-2 5 BG-3

TRANSFORMED MEAN

MEAN CALCULATED ORIGINAL UNITS

IN

mdash

7250 5875 6125 6625 6250

7250 5875 6125 6625 6250

T STAT SIG

1416 1158 0643 1030

^

bull

6 LT-1 6375 6375 0901

Dunnett table value = 231 (1 Tailed Value P=005 df=405)

CHESHIRE SEDIMENT CT SURVIVAL File CT0XSTAT3CHESSED Transform NO TRANSFORMATION

DUNNETTS TEST TABLE 2 OF 2 HoControlltTreatment

NUM OF Minimum Sig Diff Of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG UNITS) CONTROL FROM CONTROL

1 ARTIFICIAL SED 8 2 RW-1 8 2244 309 1375 3 BG-l 8 2244 309 1125 4 BG-2 8 2244 309 0625 5 BG-3 8 2244 309 1000 6 LT-1 8 2244 309 0875

c