Detection of Wastewater Plumes from the 15

N Isotopic Composition of

Groundwater, Algae and Bivalves in West Falmouth Harbor

Kara Marie Annoni

University of Minnesota Duluth

Duluth, MN

Advisor: Dr. Kenneth Foreman

The Ecosystems Center: The Marine Biological Laboratory

Semester in Environmental Science (SES)

Woods Hole, MA

2012

Abstract:

Nutrient loading to coastal and estuarine waters poses a threat to the

structure and function of biotic communities within the ecosystem. It is important

to locate and diagnose the sources of excess nitrogen input in order to mitigate

chronic detrimental responses. In this study, we aim to locate sources of nitrogen

inputs via organismal uptake of the 15

N isotope tracer. We focus on δ15

N and

nitrate concentrations within groundwater, Mya arenaria and Ulva lactuca at 8

different sites along the West Falmouth Harbor shoreline. Specifically, we

attempt to support the hypothesis suggesting that a main source of nutrient

loading is due to wastewater contamination of groundwater within the watershed

via septic systems and wastewater treatment facilities. 5 Mya arenaria were

collected at each shoreline site and combined to create 1 composite sample per

site. Ulva lactuca was also collected and compared to potential nitrogen source

signals. Nutrient and isotopic analyses revealed increasing trends of nitrogen

signals at sites 1-3. Results from this study suggest that excess nutrients from

wastewater may affect the biotic factors of an ecosystem. δ15

N revealed

correlations with groundwater samples that had higher nitrate concentrations

suggesting that the excess nutrients may be coming from wastewater. Data also

suggests that groundwater that is affected by wastewater inputs, can be localized

and detected within Mya arenaria.

Introduction:

In southeastern Massachusetts, West Falmouth Harbor (WFH) is

experiencing excessive nitrogen loading through groundwater inputs (Bowles

et.al., 2007). As a response, eutrophication of coastal environments can occur in

addition to macro-algae blooms (Costello & Kenworthy, 2011), which affects the

biological function and environmental quality of an ecosystem. Short and long

term anoxia may increase due to excessive decomposition of overly produced

organic matter, which in turn results from nitrogen loading. Widespread losses

of biota due to physiological stress can occur (Holmer & Laursen, 2002), causing

changes in abundance of consumers and producers within the environment.

Furthermore, nitrogen loading contributes to the loss of tourism, cleanliness,

recreational and commercial uses of West Falmouth Harbor.

Many anthropogenic sources of nitrogen have been identified using N

stable isotope ratios, which include fertilizers, atmospheric deposition, and

wastewater from septic systems and wastewater treatment facilities (McClelland

& Valiela, 1997). West Falmouth Harbor is susceptible to nitrogen loading

primarily because of its sandy unconsolidated aquifers (McClelland & Valiela,

1997). Because of this characteristic, nitrogen leaches into the watershed,

contaminating the groundwater, which eventually contributes excess nitrogen

input to the seep.

In 2007, the largest source of nitrogen loading into West Falmouth Harbor

originated from wastewater treatment facilities (60%) and septic systems (20%);

both caused by anthropogenic activity (Bowles et al., 2007). Because of its

location within the watershed of West Falmouth Harbor, the Falmouth

Wastewater Treatment Plant (FWTP) was suspect to be a major source of nitrogen

loading into the estuary. In 2006, the FWTP switched management from primary

to tertiary systems, which decreased the concentration of nitrogen within

wastewater from 35mg/L to 5mg/L. The wastewater is then released into a

leaching field. Before the switch, treated wastewater was disposed of by spray

irrigation (Jordan, Knute, & Fry, 1997). The spray methods lead to higher

concentrations of nitrogen leaching into the watershed, eventually seeping into

West Falmouth Harbor. It is important to note that the rate at which groundwater

travels through the WFH watershed, originating from the wastewater treatment

facility, is approximately 1 foot per year (Jordan, Knute, & Fry, 1997). The

amount of time needed for groundwater to reach the seep from the wastewater

treatment facility is approximately 10 years (Thoms, Giblin & Foreman, 2003).

Therefore, the present nitrogen loading in West Falmouth Harbor originates from

nitrogen sources in 2002.

In order to monitor or diagnose the current condition of the ecosystem, it

is important to determine the localized areas of nitrogen input, and the sources of

nitrogen loading responsible for the increase or decrease of biota. Therefore, we

aim to detect spatial variation of wastewater inputs to WFH by measuring

organismal uptake of the isotope tracer 15

N.

To assess the spatial variation and localized areas of enriched groundwater

input, we will utilize Ulva lactuca and Mya arenaria ability to assimilate

nitrogen. Ulva lactuca is a macro-algae in which studies suggest may be used as a

“non-discriminatory bio-integrator” of nitrogen (Cohen and Fong, 2005). Mya

arenaria is a filter-feeding soft-bodied clam. Through the use of isotopic

analysis, we know that fractionation in consumers discriminate against lighter

isotopes when the element of 15

N is passed to higher trophic levels, increasing the

15

N value. Therefore, organisms that are heavy in 15

N will become heavier;

essentially you are what you eat (McClelland & Valiela, 1997). The thought is

that if either organism exhibits an increased 15

N value, it may be due to nitrogen

loading. If the organisms resemble the same 15

N value as wastewater, it may

suggest that the organism is receiving nutrients from groundwater contaminated

by wastewater or resources influenced by wastewater. The wastewater 15

N value

of Falmouth Wastewater Treatment Plant ranged between +8 to +11 parts per

thousand in the winter. In the summer, del 15

N values were as high as +40 parts

per thousand but an average of +13 to +19 parts per thousand due to increased

human activity. These numbers were calculated from the year that the wastewater

was incorporated into the groundwater within the WFH watershed (Jordan, Knute

& Fry, 1997). Because the nitrogen loading to WFH was originally sourced in

2002, these values deem as a reference until 2016 (when the management of the

wastewater treatment facility was upgraded in 2006). Additionally, we will assess

wastewater inputs in regards to dissolved inorganic nitrogen, ammonium and

nitrate, to provide insight to the natural recycling of nitrogen.

Field Methods

Groundwater Sites (G1, G2, … G9)

We examined the nitrogen isotope signature of groundwater, ulva and

mussels at 8 sites along the eastern shoreline of West Falmouth Harbor during the

month of November 2012 (Figure 4). At each site, we collected ulva (preferably

attached to a substrate), 5 mussels, and 250 mL of groundwater. Groundwater

was drawn from wells at different depths, using a hydro-lab pump. For each well

collection, the groundwater was pumped 5 times the well casing volume to purge

the well, ensuring a representative sample was collected. The sample was then

filtered through a swinnex filter holder and 25mm GF/F ashed filter to remove

particulates. Samples of wastewater were collected from the Falmouth

Wastewater Treatment Plant.

Harbor Sites (H1, H2, … H7)

Seven harbor sites were chosen to examine the ability to trace wastewater

contamination through organismal isotopic composition (Figure 4). Ulva was

collected from Waquoit Bay and incubated for one week with running seawater,

provided by the Marine Biological Laboratory. At each site, we launched 1

mooring with 5 samples of incubated Ulva as a bioassay for nitrogen. Each

mooring was comprised of 1 cinderblock, rope with a length to accommodate tide

cycles, 1 buoy, and 5 mesh bags encasing the ulva. Wet weight measurements

were taken before the ulva was introduced to West Falmouth Harbor, and a

composite control sample was prepared for isotopic analysis. Overall, there were

7 deployed moorings and 35 mesh bags containing ulva (5 per mooring). The

moorings were collected after one week.

Laboratory Methods

Nutrient Analyses

Nutrient analyses (NO3-,NH4

+, PO4

3-) were carried out for each groundwater

collection, and analyzed separately. Protocols used for nitrate, ammonium, and

phosphate analysis were adapted from Wood et. al. 1967, Solarzano 1969, and

Murphy et. al. 1962 respectively.

Each groundwater sample tested for nitrate required the use of a Lachat

Flow Injection Analyzer (FIA). Nitrate standards and samples were incubated for

2 hours at room temperature in darkness after reagent additions. Samples and

standards tested for ammonium were treated with phenol, sodium nitroprusside

and oxidizing reagents respectively, vortexing the test tube between each addition.

Samples and standards tested for phosphate were treated with the PO43-

mixed

reagent, vortexed and incubated for at least one hour. Both phosphate and

ammonium samples were analyzed with a Shimadzu 1601 spectrophometer with

wavelength settings at 885nm, and 640 nm respectively.

Ulva and Mussel Samples

Ulva samples were rinsed carefully and quickly (as to not lyse cells) with

deionized water to reduce contamination. Ulva at the ground sites were dried and

analyzed for 15

N separately by site. The 5 ulva samples attached to each mooring

were combined by site, totaling 7 composite samples. Wet weights were

measured before drying.

Each mussel sample (5 per site) was scrubbed with deionized water to

reduce contamination. Within the mussel, the adductor muscle was an “easy to

locate” body tissue in which I prepared for isotopic analysis by dissecting using a

scalpel and tweezers, cleaning using 10% HCL, and rinsing with deionized water.

The five mussel samples collected at each ground site were combined to create

one composite sample per site. The mussel and ulva samples were dried at 60

degrees Fahrenheit.

Ulva samples were homogenized using the Wig-L-Bug and mussel

samples were coarsely ground in a glass vial using a glass rod. Dr. Marshall Otter

carried out isotopic analysis at the MBL Starr Stable Isotope Laboratory. For

more information concerning the stable isotope methods please visit the following

website: http://dryas.mbl.edu/silab/

Diffusion Protocol

Groundwater samples were analyzed separately by site and well. To

prepare groundwater samples for isotopic analysis, I used diffusion protocols

adapted from Holmes, R.M., et al. 1998 and Sigman, et al. 1997.

Thirty-seven filter packs were made using 10 mm GF/D ashed filters, and

25mm teflon filter tape. Each 10mm GF/D filter was placed upon the teflon filter

tape and 25 μL of 2M H2SO4 was pipetted onto the filter. The tape was then

folded over the filter making a “teflon tape sandwich” and an empty plastic scint

vial was centered over the filter and pressed down to seal the edges of the teflon

tape creating a visibly thinner ring around the GF/D filter. This enabled the filter

pack to float atop the groundwater sample during incubation without being

contaminated by the sample. Filter packs were immediately transferred to a clean,

closed, storage bottle until ready to incubate.

Because we wanted to examine the isotopic values of both nitrate and

ammonium, we did not boil the sample with MgO to remove ammonia. In order

to collect 10μM of DIN per filter, the amount of sample to analyze was

determined based on previous nitrate concentrations collected at the same sites in

October of 2012. The total amount of sample plus deionized water equaled 150

mL. Using a 250 mL square nalgene HDPE bottle, materials were added in the

respective order: DI water, 7g ashed NaCl, 1g ashed MgO, filter pack, sample,

and 0.3g Devardas Alloy. The bottles were capped tightly and quickly to reduce

the loss of nitrate/ammonium. Two blanks were made (150 mL of DI) and three

standards (100μM, 50μM, and 25μM) were made using 100 uM KNO3 stock

solution. The 37 diffusion bottles were then placed in a shaker table held at a

constant temperature of 38 degrees Celsius. After 7 days of incubation, the filter

packs were removed and dried in a desiccator for 2 days before isotopic analysis.

Results

Nitrate concentrations within the groundwater ranged from 53 (μM) to

274.33 (μM). This was a much larger scale compared to ammonium and

phosphate concentrations (Figure 5). The largest concentration of nitrate was

found in groundwater sites 2-4, which correlates with analyzed nitrate

concentrations in 2006 (Figure 13). Suspended ulva also exhibited high

concentrations of nitrate within harbor sites 1,2,3, and 4 (Figure 6). The N:P ratio

of groundwater exhibited increased values at sites 2 through 4 (Figure 7). All

values of N:P ratio exceeded the Redfield Ratio of 16:1. Likewise, the δ15

N of

groundwater displayed similar trends with increased values at groundwater sites 1

through 3 (Figure 8). Ribbed mussels were found to have δ15

N values ranging

from 10.4 to 12.4 and ulva 7.1 – 10.8. Groundwater δ15

N values in comparison

with δ15

N of ulva and ribbed mussels exhibited slightly higher values at sites 1

and 2 (Figure 9). Ulva suspended in the harbor sites increased in δ15

N values

compared to the control sample (5.3 o/oo), although the values did not exhibit

critical observational differences between sites (Figure 10). As in Figure 11, the

δ15

N and δ

13C

are compared in terms of ulva and ribbed mussel, in which ulva

indicates lower values of the δ15

N and δ

13C

than the ribbed mussel.

Compared to

spartina (-13.8, 3.9) and plankton (-20.5, 8.5) values of δ13

C and δ

15N of ribbed

mussels within West Falmouth Harbor displayed higher values of δ15

N and

differed in δ13

C (Figure 12). Sites 1 and 2 revealed the highest value of δ15

N.

Discussion

There was no general pattern in ammonium and phosphate concentrations

within groundwater. This was expected due to the general recycling

characteristics of nitrogen. Sites 1 through 4 exhibited the highest concentration

of groundwater input in reference to thermal imaging (Figure 4). Because these

sites exhibited high concentration of nitrate, it is likely that the excess nitrogen is

introduced into the system by groundwater. It is important to note that high

concentrations of nitrate correlate with high values of δ15

N of groundwater

(10.16-13.4 o/oo) at the same groundwater sites; suggestive that excess nutrients

is due to groundwater that is contaminated with wastewater. Overall, this

suggests that the localized groundwater is contaminated with excess nitrogen,

possibly in the form of nitrate; wastewater contains nitrate as it is leached into the

watershed (Jordan et al, 1997). The trend of high nitrogen concentrations was

also exhibited by the C:N ratio of the ulva suspended in the harbor at harbor sites

1,2, and 3 which were similarly located near ground sites 1,2,3 and 4 (Figure 6).

The N:P ratio revealed similar trends at ground sites 2,3 and 4 furthering

evidence that the groundwater input is enriched in nitrogen in those areas (Figure

7). All values of the N:P ratio exceeded the Redfield ratio of 16:1. Most coastal

systems are nitrogen limited and the presence of high N:P ratios exceeding the

Redfield ratio may suggest that the excess nitrogen is creating a phosphorous

limited system.

Comparison of data found within this study and data collected from

previous experiments show correlation in location of increased nitrate

concentration and high values of δ15

N (Figures 8 & 13), furthering the support of

groundwater contaminated with wastewater. Likely, the mussel and ulva would

exhibit similarly increased isotopic values because the data suggests that

wastewater is present within the seep; essentially assimilation of nitrogen within

each organism is influenced by the excess nutrients and will possess an isotopic

value resembling the material taken in. Again, at sites 1,2, and 3 mussel and ulva

samples from the ground sites showed an increased δ15

N value suggesting that as

groundwater δ15

N increases, so does the δ15

N in ribbed mussel and ulva (Figure

9). The suspended algae at the harbor sites had increased δ15

N values compared

to the control algae, yet there was no variation between sites, which could be due

to the placement of the mooring (not close enough to the shoreline to exhibit full

effects of groundwater), although there was evidence of excess nitrogen due to the

C:N ratios – which did exhibit increases of nitrogen at sites 1,2, and 3.

The δ13

C and δ15

N of mussels and ulva both exhibit high values of δ15

N

but different values of δ13

C. This data can support the assumption that additional

nutrients added to the estuary affects organisms within different food webs

(Figure 11). Figure 12 describes the relationship between δ15

N values of mussels

and isotope values of which mussels would acquire their usual nitrogen content

(Fry & Peterson, 1985). According to a study carried out in the Great Sippiwisset

Marsh (GSM), nitrogen can increase with a dieting shift from spartina to

plankton, but in this case there are higher δ15

N values from samples taken in the

Mashapaquit Creek marsh sites, with similar abiotic and biotic factors as the GSM

(ground sites 1,2 and 3), suggesting that their excess nutrients is coming from a

different source rather than diet. This source, according to previous data and data

collected for this study, may be coming from groundwater inputs that have been

contaminated with wastewater leeching into the watershed from septic and the

wastewater treatment plant.

Overall, the data collected in this experiment provides supporting evidence

that excess nutrients from wastewater may affect the whole ecosystem due to the

evidence of elevated δ15

N values. The characteristics of West Falmouth Harbor

and the geological attributes of the watershed suggest that a large source of excess

nutrients may originate from the wastewater treatment plant and seep into the

estuary. Data suggests that groundwater, which is affected by wastewater inputs,

can be localized and detected within ribbed mussels.

Acknowledgements

I would like to thank, my advisor Dr. Kenneth Foreman, Richard

McHorney, and Carrie Harries for the many hours spent helping me to perfect

protocols, collecting and analyzing data. I would also like to thank Alice Carter

for the time spent in West Falmouth Harbor on very cold days deploying

moorings and collecting and analyzing data. Thank you to Marshall Otter of the

MBL Starr Stable Isotope Laboratory who contributed his expertise enabling me

to analyze my data. Thank you to Suzanne Thomas for lending a helping hand

with the diffusion protocol. The Semester in Environmental Science at the

Marine Biological Laboratory was one of the best experiences of my life, and I

want to thank all the people who made the experience worthwhile. This project

was funded by the MBL Semester in Environmental Science Program.

Figure 1: Display of the groundwater watershed leading to the West Falmouth

Harbor, Massachusetts. An orange shape, east of the harbor, represents the

Falmouth Wastewater Treatment Plant.

Figure 2: The Massachusetts Department of Environmental Protection measured

the amount of nitrogen loading within the West Falmouth Harbor in 2007. Most

nitrogen input is due to wastewater treatment facilities (WWTP) and septic

systems. The wastewater treatment facilities account for approximately 60% of

nitrogen being loaded into the seep.

70

50

30

10

Figure 3: Records of the total dissolved nitrogen, total nitrogen, and flow of

wastewater through the Falmouth Wastewater Treatment Plant. In 2006, the

treatment plant changed management from primary to tertiary hence the decreased

concentration of total nitrogen.

Figure 4: Thermal image of West Falmouth Harbor. Dark areas represent

groundwater input and experimental sites are displayed. Orange marks indicate

groundwater sites from which groundwater, ribbed mussel and ulva samples were

collected. The blue marks indicate harbor sites where moorings were deployed; 5

samples of incubated ulva were attached to each mooring in order to act as a

bioassay of nitrogen.

Figure 5: Nutrient values were taken from groundwater during November 2012.

Wells were located along the eastern shoreline of West Falmouth Harbor,

Massachusetts. Concentrations of nitrate show increasing trends at sites 2,3, and

4.

Co

nce

ntr

ati

on

(μ

M)

Groundwater Sites

0.0

0.5

1.0

1.5

2.0

0 2 4 6 8 10

PO

43

-

-2

0

2

4

6

NH

4+

0

100

200

300

400

NO

3-

Figure 6: Carbon to nitrogen ratio of ulva suspended within the harbor. Ulva was

incubated for one week within the MBL laboratories before introduced to West

Falmouth Harbor to act as bioassay of nitrogen. More nitrogen per carbon was

present at sites 1-3.

R² = 0.867

0

2

4

6

8

10

12

14

16

18

20

0 1 2 3 4 5 6 7

C:N

Ra

tio

(μ

M)

Harbor Sites

Figure 7: Nitrogen per phosphorous was greater at sites 1-4. All values exceed

the Redfield Ratio. Data was collected from West Falmouth Harbor in

Massachusetts during November of 2012.

Figure 8: Groundwater samples were collected from the eastern shoreline of West

Falmouth Harbor in Massachusetts. The isotopic composition of the groundwater

samples exhibit increased trends of δ15

N at sites 1-3. Each site corresponds to the

groundwater sites depicted in Figure 4.

10

100

1000

10000

0 1 2 3 4 5 6 7 8 9

N:P

Ra

tio

(u

M)

Sites

N:P Groundwater

Redfield Ratio 16:1

0

4

8

12

16

20

0 2 4 6 8 10

δN

15

(o

/oo

vs.

AIR

)

Sites

Figure 9: δ

15N concentrations were taken of ulva (Ulva lactuca) and ribbed

mussels (Mya arenaria) collected from groundwater sites within West Falmouth

Harbor in Massachusetts. At sites 1-3, both mussels and ulva exhibited higher

δ15

N values.

Figure 10: The δ15

N of incubated Ulva lactuca increased from 5.3 o/oo to an

average of 8.1 o/oo. Ulva was originally collected from Waquoit Bay Estuaries

and incubated for one week with running seawater from MBL facilities. Samples

4

5

6

7

8

9

10

11

12

13

4 6 8 10 12 14

δ1

5N

(o

/oo

vs.

AIR

)

Groundwater δ15N (o/oo vs. AIR)

Ulva Ribbed Mussel

0

2

4

6

8

10

0 1 2 3 4 5 6 7

d1

5N

(o

/o

o v

s. A

IR)

Harbor Sites

Suspended Algae d15N vs. sites

Suspended Ulva

Control

were attached to moorings and deployed in the West Falmouth Harbor for one

week during November of 2012.

Figure 11: δ

15N and δ

13C values of ribbed mussels and ulva collected at the

groundwater sites display increased values of δ15

N and differences in δ13

C values.

Samples were collected in November of 2012 from West Falmouth Harbor in

Massachusetts.

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

-25.0-20.0-15.0-10.0

δ1

5N

(o/o

o v

s. A

IR)

δ13C (o/oo vs. PDB)

Ulva

Mussel

Figure 12: δ

15N and δ

13C of ribbed mussel collected from groundwater sites in

West Falmouth Harbor (WFH) are compared to the δ15

N and δ13

C of spartina and

plankton collected from the Great Sippiwisset Marsh (Fry & Peterson 1985).

Note that the ribbed mussel values do not resemble that of either spartina or

plankton at groundwater site 1 and 2. Sites 1 and 2 within WFH exhibit similar

abiotic and biotic factors as the Great Swippiwisset Marsh.

G1&2 G3

G4 G5

G7

G8 G9

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

-21.0-19.0-17.0-15.0-13.0

d1

5N

(o

/oo

vs.

AIR

)

d13C (o/oo vs. PDB)

Mussel WFH

Spartina SIP

Plankton SIP

Figure 13: Cross section of West Falmouth Harbor well sites along the eastern

shore. The colored graph indicates the sites in relation to nitrate concentration

and depth. The bottom graph indicates groundwater sites in relation to δ15

N and

distance between sites. According to Foreman and McHorney, sites 1,2,3 and 4

have the highest amount of nitrate, and sites 3, 2 and 1 are most influenced by

wastewater input.

G4

G3

G5

G7

G8

G2 G1

G9

G9 G8 G7 G5 G4 G3 G2 G1

-5

-10

-15

0 1 2 3 4 5 6 7 8 9 10 11 12

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