BICTHEATABILITY STUDYAZilED-SIGNAL, INC,

COKE PLANT LAGOON AREAIROMTON, OHIO

PREPARED FOR:

ALLIED-SIGNAL, INC.MORRISTOWN, NFtf JERSEY

PREPARED BY:

IT CORPORATIONKNOXVILTjE, TENNESSEE

JANUARY 12, 199C

PROJECT NO. 303735

Table of Contents

List of Tables ii

List of Figures iii

1.0 Introduction 1

2.0 Methods 32.1 Experimental Design 32.2 Sample Handling 42.3 Respirometry 42.4 Inorganic Nutrients 52.5 pH 62.6 Microbial Enumerations 62.7 Total Carbon Analyses 72.8 Specific Organic Compound Analyses 7

3.0 Results3.1 Physical Observations 93.2 Inorganic Nutrients 93.3 Microbial Density 103.4 Oxygen and Carbon Consumption 103.5 Experimental Control Samples 123.6 Total Recoverable Petroleum Hydrocarbon 133.7 Target Compound Analysis 14

4.0 Summary 15

List of Tables

Table 1. Experimental design for assessing biodegradation inAllied-Signal Inc., coking plant waste from lagoon L2,Ironton, Ohio.

Table 2. Physical and chemical soil and water parameters beforeand after treatment.

Table 3. Microbial population size in soil and water from thelaboratory composite and following treatment.

Table 4. Naphthalene and phenanthrene-degrading bacteria in theinitial soil-water laboratory composite using a sprayed-plate technique.

Table 5. Total recoverable hydrocarbon analysis (TRPH-IR) of theuntreated soil and water and water/soil slurries.

Table 6. Quantitative analysis of priority pollutants from awater and soil derived from the laboratory sample compositeafter equilibration as a 10:1 (water:soil) slurry.

Table 7. Total dissolved organic carbon in filtered water fromthe laboratory composite and treated samples.

List of Figures

Figure 1. Cumulative oxygen consumption duringbiodegradation of coal-coking wastes.

Figure 2. Cumulative oxygen consumption in HgCl2 treatedcontrols.

Figure 3. Initial and final priority pollutant contentof Treatment 3, 50:1 (water:soil) slurry.

Figure 4. Initial and final priority pollutant contentof Treatments 4, 5, and 6, 10:1 (water:soil)slurries.

1.0 Introduction

International Technology Corporation (IT Corp.) was contracted byAllied-Signal, Inc. to investigate the potential forbioremediation of drained, inactive coal-coking waste lagoons atthe Allied-Signal coking plant in Ironton, Ohio. The laboratorytreatability study was performed at the IT Biotechnology Center,Knoxville, Tennessee from October to December, 1989. The purposeof the study was to determine the potential for successfulremediation of coal-coking waste lagoons using an in situbioremediation program.

The premise of in situ bioremediation is that the microbialpopulation, which exists at the site, has developed the capacityto biodegrade the target organic compounds given that otherconditions are conducive to microbial metabolism. This studyinvestigated if, in the presence of nutrients and oxygen, thecomplex coal-coking wastes deposited in lagoons could bebiodegraded by naturally-occurring microorganisms.

The type and concentration of organic compounds and fundamentalcharacteristics of the soil and groundwater in lagoon L2 sampleswere determined. Soil and groundwater composites from the sitewere supplemented with inorganic nutrients and monitored foraerobic biodegradation of the organic contaminants.Biodegradation was evaluated based on changes in theconcentration of organic carbon and specific coking by-productsduring the course of the laboratory study and on microbialactivity as determined by oxygen consumption and microbial cellgrowth.

The overall results of the treatability study indicated thatbioremediation is a viable means of remediating the wastelagoons. The site soil and water samples contained about 10

heterotrophic and hydrocarbon-degrading microorganisms permilliliter (mL). Respiratory activity and microbial growth onorganic carbon from site soil and water was observed. Totalorganic carbon and total petroleum hydrocarbons were reduced intreated samples and the concentrations of specific cokingby-products were reduced 100 to 10,000 fold during the study.These results demonstrated that the native microbial populationin the waste lagoon is capable of biodegrading coking wastesprovided that oxygen and nutrients were not limiting.

2.0 Methods

The treatability study invoked several technologies which wereused to evaluate the potential for bioremediation of waste lagoonL2. A description of the treatability study, the significance ofeach analytical determination, and a summary of the laboratoryprocedures follow.

2.1 Experimental DesignThe treatment scheme for the treatability study was designed todetermine the reduction of organic carbon and specific compoundsin nutrient and oxygen-amended samples when compared to abioticand untreated controls. An additional dilute treatment wasincluded to reveal any toxic effect that the lagoon solids mayhave on bacterial activity.

Analytical tests including total dissolved organic carbon, totalpetroleum hydrocarbon, volatile organic compounds, base/neutralsand acid extractables analyses were completed for untreatedsoil and water and for each sample at the end of the study todetermine the biodegradability of coking wastes. Additionalparameters such as oxygen consumption, bacterial density, pH, andnutrient content were monitored to support the conclusion ofbiodegradation in the active samples.

Soil-water slurries were sampled while the soil was suspended sothat the final results represented the entire sample. Theefficacy of this sampling procedure was determined by recoveringthe residual soil and measuring its dry weight. The differencebetween the initial dry weight of soil added and the amountremaining was the amount removed during sampling. Thisinformation was used to normalize the final analytical results sothat they were directly comparable to the initial data. In caseswhere sufficient soil remained in the respirometer vessels at the

end of the study, nitrogen, phosphate, and pH measurements veremade on the treated soil in addition to the slurries.

2.2 Sample HandlingTwenty liters (L) of water from well* C4 and 2 L of soil werecollected from lagoon site L2-1 and shipped on ice to IT Corp.Upon receipt, the soil and water samples were placed in a fivegallon carboy, mixed thoroughly, and allowed to equilibrate at4°C for 48 h. This ensured maximum homogeneity in the entiresample and the aliquots used for setting up the treatments.Without stirring the soil-water composite, water samples wereremoved for quantitative analysis, the respirometer study, andmicrobial enumerations. The remaining water was drained from thecarboy and soil samples were collected from the sediment forquantitative analysis, the respirometer study, and microbialenumerations. During all manipulations the samples were keptcold to avoid volatilization of organic compounds; materialtransfers were made quickly and without excess agitation.

2.3 RespirometryA computerized respirometer was used to assess the metabolicactivity of microorganisms by measuring the consumption ofoxygen. Since the primary carbon sources in the soil andgroundwater were the contaminants, oxygen consumption was anindication of microbial transformation of these compounds.

Table 1 summarizes the treatment scheme employed in therespirometer study. Treatment 1 evaluated biodegradation oforganic wastes in nutrient-supplemented ground water alone.Treatment 2 was a biologically-inhibited control for Treatment 1.Treatment 3 contained a 50:1 (water:soil) mixture. This dilutetreatment (compared to treatments 4, 5 and 6) was used todetermine if the relatively high concentration of aromaticcompounds in the lagoon solids adversely affected microbial

growth and metabolism. Treatment 4 examined the biodegradationof wastes in a 10:1 (water:soil) slurry. Treatment 5 was abiologically-inhibited control for Treatment 4. Treatment 6 wasan untreated control consisting of a 10:1 mixture of water andsoil respectively. This treatment was placed in a respirometervessel, sealed, and incubated at 25°C. No nutrients were added,and the contents were not stirred. This sample represented anychanges caused by experimental manipulation.

Water and soil were added to respirometer vesselsgravimetrically. The water content of the soil added to thereactors was 41%, i. e., the actual dry weight of soil added tothe reactors was 59% of the wet weight indicated in Table 1.Mercuric chloride was added to treatments 2 and 5 to aconcentration of 100 ppm. This level has been demonstrated inother studies to provide a biologically-inactive control inaqueous systems and soil-water slurries. Nutrients were addedaseptically to the treatments as a sterile solution of Restore375 brand nutrient (IT Corp.). All treatments were vigorouslystirred using a magnetic stirrer except Treatment 6, theuntreated control. All treatments were conducted in duplicateexcept 2 and 5, the biologically-inhibited controls.

2.4 Inorganic nutrientsNitrogen as ammonia and orthophosphate content of the initial(untreated) soil and water samples was measured. The data wereused to evaluate the need for nutrient amendment in thetreatments. Quantitation of nitrogen and phosphate at the end ofthe study revealed whether nutrients were likely to be limitingin the treatments. Generally, a ratio of 100:10:1carbon:nitrogen:phosphate is adequate to stimulate non-nutrientlimited growth and metabolism. This ratio was used toapproximate the need for additional nutrients based on nutrientconcentrations in the soil and water samples.

Ammonia analyses were performed on air-dried soil and aqueoussamples by the Nesslerization method (Standard Method I417B).Orthophosphate was determined by the ascorbic acid method(Standard Method #425F).

2.5 pHMicroorganisms from soil and ground water are typically mostactive in a pH range of 6 to 8. Measurements of pH wereconducted at the beginning and end of the study to determine ifdrastic changes in pH occurred during the study. Extreme changeswould affect biological activity. The pH of soils and waters wasdetermined with an Orion combination pH electrode.

2.6 Microbial EnumerationsChange in population size is an indirect indicator of microbialactivity. The initial densities of the heterotrophic microbialpopulation and the hydrocarbon-degrading bacteria were determinedfor the composite sample. These enumerations were compared withthe number of heterotrophic and hydrocarbon-degradingmicroorganisms recovered from each treatment at the end of thestudy.

Bacteria were enumerated using the standard plate counttechnique. Initial microbial enumerations were conducted on a10:1 (water:soil) sample. The final enumerations performed atthe end of the biodegradation study were conducted on each sampleusing both the slurry and sedimented soil collected from thebottom of selected treatment vessels.

Total heterotrophs were defined as those organisms capable offorming a colony on 0.23% nutrient agar after seven daysincubation at room temperature. Hydrocarbon-degraders weredefined as those organisms capable of forming colonies on mineral

salts agar incubated under diesel fuel vapors for one week atroom temperature.

Bacterial colonies from the enumeration of the starting microbialpopulation were examined for their ability to degrade naphthaleneand phenanthrene using the sprayed-plate technique (Kiyohara etal., Appl. Env. Microbiol 43:454-457. 1982.). After coloniesformed on the plates, they were sprayed with a solution of eithernaphthalene or phenanthrene dissolved in acetone so that a filmof the compound formed over the surface of the plate. Clearingaround individual colonies indicated those which were activelydegrading the added hydrocarbon.

2.7 Total Carbon AnalysesTwo independent analyses for total carbon content of initialsamples and experimental treatments were used to assess organiccarbon mineralization. Total recoverable petroleum hydrocarbon(TRPH-IR) analysis uses infrared spectroscopy to quantitate thecarbon-hydrogen bonding in a sample. Soil and water samples wereanalyzed at the beginning of the study and all treatments wereanalyzed at the end as described.

Total dissolved organic carbon analysis (TDOC) employs persulfateoxidation of reduced carbon compounds to carbon dioxide which isquantitated spectroscopically. Under UV irradiation all carbonis oxidized to carbon dioxide whereas in the absence of UVradiation only inorganic carbon is oxidized. The differencebetween total carbon and total inorganic carbon is totaldissolved organic carbon. Total dissolved organic carbon wasmeasured with a Dohrmann total carbon analyzer.

2.8 Specific Organic Compound AnalysesVolatile organic compounds analysis (USEPA SW-846 Method 8240),total recoverable petroleum hydrocarbons analysis (USEPA Method

418.1), and base neutrals and acid extractables analysis (USEPASW-846 Method 8270} were conducted at the beginning and end ofthe study. To compare starting concentrations with finalconcentrations, the amount of soil and ground water in eachtreatment was used to calculate the total mass of prioritypollutants present. The data from these analyses were used todemonstrate biodegradation of specific compounds.

3.0 Results

3.1 Physical ObservationsThe water sample received from well* C4 was slightly turbid withno odor. The soil sample from site L2-1 was black with fineblack particles and course grains. The sample contained nails,cinders, pieces of rubber, and droplets of tar-like substances.The soil had a strong naphthalene-like odor.

The pH data from the initial slurry and from each treatment atthe end of the study are presented in Table 2. The initial pH ofboth the soil and water was 7.1 and 7.6, respectively. The pHgenerally increased with the treatments, but remained withinreasonable limits. This increase was probably an artifact causedby inadvertant addition of sodium hydroxide to the reactionmixture when the treatments were disconnected from therespirometer.

3.2 Inorganic NutrientsThe initial ammonia content of the soil and water was 46 ppm and106 ppm, respectively (Table 2). The phosphate content was 71ppm in the soil and below detection (0.5 ppm) in the water. Thewater soluble nutrients supported some biological activity,however the phosphate level should limit extensivebiodegradation.

Nutrients were added to each active vessel on two occasionsduring the study. Neither addition caused a significant responsein bacterial activity in the sample containing only water andnutrients, indicating that nutrients were not limiting in thatsystem (Figure 1). There were, however, significant responses inthe water:soil slurries, indicating that nutrients were limitingthese systems after 100 hours of incubation. A total of

approximately 400 ppm of ammonia was added to the system duringthe study. On average approximately 50% of this mass wasconsumed during the study.

3.3 Microbial DensityThe initial heterotrophic microbial population density was 1.3 x10 colony-forming units (cfu) per mL slurry (Table 3). Thenumber of bacteria which were capable of using diesel fumes orbenzene-toluene-xylenes as their sole carbon and energy sourcesequaled the heterotrophic population. After the study wascompleted, the bacterial densities increased in all the water andsoil samples. The ground water-only samples exhibited netdecreases in the population, probably due to a lack of availablecarbon (treatment 1) or the presence of a biological inhibitor(treatment 2).

Table 4 provides information about the presence of nativebacteria which can degrade naphthalene and phenanthrene, twoprominent components in the soil sample. Using a sprayed-platetechnique on colonies growing on nutrient agar (uninduced forhydrocarbon degradation) and on diesel fumes (induced forhydrocarbon degradation), 7 and 13 percent of the nativemicrobial population assimilated naphthalene and phenanthrene,respectively. Up to 30 percent of the population could degradenaphthalene after induction by growing on diesel fumes. Theseresults indicate that the population is well-adapted to thecontaminants present in the lagoons.

3.4 Oxygen and Carbon ConsumptionRespirometric data on oxygen consumption by native bacteria insite ground water and slurries is shown in Figure 1. Treatmentsconsisting of ground water (Treatment 1), a 50:1 water:soilslurry (Treatment 3), and a 10:1 water:soil slurry (Treatment 4)are shown. Nutrients were added at the start of the experiment

10

and then again as indicated by the arrows in Figure 1 (thenutrient amendment schedule is also shown in Table 1). Thesedata indicated that respiration occurred in each of the threetreatments.

The available carbon source was quickly depleted in thegroundwater treatment (Treatment 1) as indicated by the quickrise and subsequent stabilization of oxygen consumption.Supporting evidence for this conclusion can also be seen in Table5 where the TDOC fell from 99 ppm to 5 ppm in Treatment 1. Datafrom TRPH-IR analyses shown in Table 6 are somewhat equivocal forTreatment 1 because the values are so near the detection limit of3.5 ppm. The observed trend for Treatment 1 does, however,suggest reduction of hydrocarbons during the study. This isfurther supported by the observation that the microbialpopulation diminished by the end of the study to a level lowerthan the starting population size (Table 3). This result wouldbe expected for a population that had depleted its carbon source.

The microorganisms in Treatment 3 (50:1 water:soil with nutrientsadded, Figure 1) actively consumed oxygen throughout the 500 hstudy. However, by 100 h this treatment had become nutrient-limited since the addition of extra nutrients at -200 h furtherstimulated oxygen consumption (Figure 1). The total dissolvedorganic carbon content of the water was reduced from 99 to 9 ppm(Table 5). TRPH-IR analysis indicated that the hydrocarboncontent fell from -11.5 ppm to less than the detection limit of 7ppm. Microbial activity was further indicated by the increase inthe total number of bacteria in the treatment (Table 3).

The microorganisms in Treatment 4 (10:1 water:soil with nutrientsadded, Figure 1) also actively consumed oxygen throughout thestudy, but at a greater rate than treatment 3. Observationsregarding nutrient limitation were similar to treatment 3.

11

TRPH-IR analysis indicated that the mass of hydrocarbon degradedwas similar to that degraded in treatment 3. TDOC analysis(Table 5) indicated a 66% reduction of the organic carbonconcentration. Microbial growth was also observed in thistreatment (Table 3).

3.5 Experimental Control SamplesThe biologically-inhibited control, treatment 2, containing waterand nutrients, was characterized by bacterial death (Table 3), noconsumption of oxygen (Figure 2) and no removal of hydrocarbons(Table 6). The reduction of total organic carbon in treatment 2(Table 5) was apparently due to adsorption of compounds to theinner surface of the sample vessel. Extraction and TRPH-IRanalysis of the inside surface of a respirometer vessel revealedthat 1 mg of hydrocarbon was adsorbed to the glass surface. Thisis a significant amount considering the relatively lowconcentration of hydrocarbon in the initial aqueous sample.Adsorption of organic compounds to surfaces in biologically-active treatments would not be as significant due to degradativeactivity against the compounds and solubilization of thecompounds by viable microorganisi

The second biologically-inhibited control, Treatment 5,containing 10:1 (water:soil) and nutrients, produced unexpectedresults. The soil appeared to sequester mercury so that a toxicconcentration was not achieved in the aqueous phase. (Theability of the 10:1 water:soil slurry to neutralize the toxiceffect of 100 ppm HgCl2 was also demonstrated in an unrelatedexperiment not reported here.) All the data indicated that thebacteria in this control were completely viable (see Tables 3, 5,and 6 and Figure 4). Oxygen consumption data also indicatedmetabolic activity (Figure 2). Two important features of theoxygen consumption curve are the 80 hour lag period prior tooxygen consumption and the relatively high rate of oxygen

12

utilization following the lag period. These observations suggestthat mercuric chloride affected the microbial population byselecting for a mercury-tolerant sub-population with a higherrespiratory rate, suffice it to say that this sample does notrepresent a true abiotic control.

Treatment 6 was an untreated control (10:1 water:soil);respirometric data was not collected, and no nutrients were addedbeyond the level in the initial samples. Temperature wasmaintained and oxygen was inadvertently supplied by the ambientatmosphere in 450 mL of head space in the reactor vessel.Approximately 120 mg of molecular oxygen was utilized in theactive samples. Since 640 mg of molecular oxygen is present in450 ml of ambient air, this sample is not a strict untreatedcontrol, but an oxygen-supplemented control.

This "oxygen-supplemented" control was valuable in assessing thenutrient requirement of the population. The final analysis ofthis treatment suggested that enough nutrients were available formicrobial growth (Table 3) and degradation of hydrocarbons(Tables 5 and 6, Figure 4), although the phosphate level wasprobably limiting at the end of the study (Table 2). Thisobservation suggests that some nutrients were available in thesystem and that oxygen was limiting the system.

3.6 Total Recoverable Petroleum HydrocarbonThe total recoverable petroleum hydrocarbon content of theinitial soil and water samples as well as the treated samples atthe completion of study are listed in Table 5. The initialconcentration* in the slurries were estimated to be 58 ppm in the10:1 (water:soil) samples, 11.5 ppa in the 50:1 samples and 4.3ppm in the water only samples. These concentrations werereduced by at least one-third in all samples. The water onlysample and the 50:1 diluted sample exhibited concentrations below

13

the detection limit. The concentration in the 10:1 samplesaveraged 37 ppm.

3.7 Target Compound AnalysisTable 6 lists the priority pollutants found in the soil andgroundwater from waste lagoon 2. Determination of the initialconcentrations of these compounds in groundwater and soil afterequilibration indicate that the compounds partition strongly tothe soil with relatively little solubilization into the aqueousphase. The absolute biodegradation of priority pollutants inTreatments 1 and 2 could not be determined because of the lowlevels of organic compounds in the water as shown in Tables 5 and6. The final concentration of all priority pollutants was belowthe detection limit in Treatments 1 and 2 with the exception ofbenzene and naphthalene which were detected only in Treatment 2(the biologically-inhibited control). Soil-containing treatmentshad higher levels of the priority pollutants.

The final concentrations of priority pollutants are given forTreatments 3, 4, 5, and 6 in Figures 3 and 4. Most base-neutralswere initially present between 1 and 10 mg. Individual compoundswere reduced to between .1 and 10 ug, a substantial decrease.The lighter compounds (benzene, ethylbenzene, and toluene) werepresent at much lower masses initially, (approximately 1 ug) andwere reduced to near the detection limit of 5 ng. All acid-extractable priority pollutants were below the detection limitsbefore and after treatment.

14

4.0 Sumary

The experimental observations provide substantive evidence thatbioremediation is a tenable approach to reducing the level ofhydrocarbons in the coking-waste lagoons at the Ironton Ohiosite.

o The calculated reduction of priority pollutant mass in thesoil-water slurries during the 500 h treatment periodindicated that reduction varied from 2-to-4 orders ofmagnitude depending on the compound. Many of thecompounds were below the detection limit at the end of thestudy.

o The microbial population at the site has adapted to theavailable organic carbon sources as indicated by thepresence of diesel, benzene-toluene-xylene, naphthalene,and phenanthrene degraders.

o Native bacteria are capable of degrading hydrocarbonsprovided that nutrients and oxygen are available.Quantitative analysis demonstrated reduction in theconcentration of hydrocarbons in general and diminution ofspecific hydrocarbons in the various treatments.Confirmation of biological activity lies in the measuredincrease in total and hydrocarbon-degrading bacteriaduring the study, stimulation of oxygen consumption by theaddition of nutrients, and the apparent lack of compoundremoval from the biologically-inhibited water-onlycontrol.

15

COLU_lCD

Table 1. Experimental design for assessing biodegradation in Allied-Signal Inc., cokingplant waste from lagoon L2, Ironton, Ohio.

Treatment3 Contents Inhibitor13Water Soil

1 -Water

2 -Water, abiotic

3-50 :ld

4-10:1

5-10:1, abiotic

6-10:1, untreated

(mL)

500

500

500

500

500

500

(g)

o —0 HgCl2

10 ——

50 ——

50 HgCl2

50 ——

Initial NutrientsConcc Time(ppm)

200

200

200

200

200

0

(h)

0

0

0

0

0

0

Nutrient AdditionCone Time Cone Time(ppm)

500

500

500

500

500

——

(h)

212

212

189

212

212

--—

(ppm)

500

500

500

500

500

——

(h)

336

336

336

336

336

——

aData represent the mean of duplicate samples in Treatments 1, 3, 4, and 6.

^Saturated HgCl- was added to treatments 2 and 5 to inhibit biological oxygen consumption.The final HgCl2 concentration was 100 ug'mL (pp»).

°A sterile solution of Restore brand nutrient was used to provide nutrient amendments.

Water:soil ratio

Table 2. Physical and chemical soil and water parameters beforeand after treatment.

Treatment3

Initial soilb

Initial water1-Water2 -Water, abiotic3-50:14-10:15-10:1, abiotic6-10:1, untreated

pH Ammonia Phosphateug'mL"1 (ppm) ug'mlT1 (ppm)

7.17.69.38.79.08.3 (6.3)d

7.36.7 (6.8)

46106231229216174 (55)154 (30)145 (63)

71<DLC

151144144124195<DL

(931)(302)(39)

aData represent the mean of duplicate samples in Treatments 1, 3,4, and 6.

Initial soil and water measurements were conducted afterequilibration as a 10:1 slurry.CDL, detection limit was 0.5 ppm for phosphate.dValues in parenthesis are for soil recovered from the treatmentsat the end of the study. For ammonia and phosphate the data aregiven as ug'g"1 dry wt. (ppm). Insufficient soil was availablefor analysis in those treatments where soil data are absent.

Table 3. Microbial population size in soil and water from thelaboratory composite and following treatment.

Treatment3 Carbon Utilization CFU*mL"lb Percent ChangeMode (xlO ) During Study

Initialcomposite

1 ( water )dwater

2 (water)Water, abiotic

3 (water)50:le

4 (water)10:1

4 (soil)10:1

5 (water)10:1, abiotic

5 (soil)10:1, abiotic

6 (water)10:1, untreated

6 (soil)10:1, untreated

HeterotrophsDiesel degradersBTX degraders

HeterotrophsDiesel degradersBTX degraders

HeterotrophsDiesel degradersBTX degraders

HeterotrophsDiesel degradersBTX degraders

HeterotrophsDiesel degradersBTX degraders

HeterotrophsDiesel degradersBTX degraders

HeterotrophsDiesel degradersBTX degraders

HeterotrophsDiesel degradersBTX degraders

HeterotrophsDiesel degradersBTX degraders

HeterotrophsDiesel degradersBTX degraders

1.31.41.2

0.730.320.24

000

1.510.03.9

8.78.32.0

167.53.3

6913017

412718

3.43.70.31

47104.4

NACNANA

-55-45-20

-100-100-100

+10+620+340

+550+500+170

+ 1180+540+290

+5200+9400+1500

+3100+2000+ 1600

+260+270-30

+3500+750+380

Continued

Table 3, continued.aData represent the mean of duplicate samples in Treatments 1, 3,4, and 6.bCFU, colony-forming units.CNA, not applicable.dMatrix evaluated (soil or water)eWater:soil ratio

Table 4. Naphthalene and phenanthrene-degrading bacteria in the

initial soil-water laboratory composite determined using a

sprayed-plate technique.

OriginalCarbonSource*

Nutrient Agar

Diesel

ChallengingCarbonSource*

Naphthalene

Phenanthrene

Naphthalene

Phenanthrene

DegraderjsCFU'mL"-1(XlO6)

0.093

0.18

0.40

0.17

Percent ofTotal

Population

7

13

30

13

aBacteria were cultured on the original carbon source untilcolonies were clearly visible.

Challenging carbon source was the compound used to spray theculture plates. Zones of clearing around individual colonieswere the indicator of biodegradation.

Table 5. Total dissolved organic carbon in filtered water from the laboratory

composite and treated samples.

Treatment* Total Dissolved Total Dissolved Total DissolvedCarbon Inorganic Carbon Organic Carbon

(ug'mlT1) (ug'mL"1)

Initial Composite1 -Water2 -Water, abiotic3-50:14-10:15-10:1, abiotic6-10:1, untreated

992713205610988

021510378175

995810192813

'Data represent the mean of duplicate samples in Treatments 1, 3, 4, and 6.

Table 6. Total recoverable hydrocarbon analysis (TRPH-IR) of theuntreated soil, water, and soil/water slurries.

Treatment* TRPH-IRb Calculated Initial Calculated(ppm)c Hydrocarbon Hydrocarbon

Initial waterInitial soil1 -Water2 -Water, abiotic3-50:14-10:15-10:1, abiotic6-10:1, untreated

<2.01920<3.54.3<3.57.99.86.1

Concentration^(ppm)

NAf

NA4.34.3

-11.5-58-58-58

Remaining8

(ppm)

NANA<3.54.3<7-47-29-37

aData represent the mean of duplicate samples in Treatments 1, 3,4, and 6.bTotal recoverable petroleum hydrocarbon analysis.cppm, ug/g for dry soils and mg/L for water samples. Watercontent of the soil was 41%.

Calculations of the initial concentration of petroleumhydrocarbons were performed based on a water content of 41% andthe efficiency of soil removal during preparation of the samplesfor analysis.

Calculations of the final concentration of petroleumhydrocarbons were performed using measurements of the efficiencyof soil removal during sample preparation for analysis.fNA, not applicable.

Table 7. Quantitative analysis of priority pollutants from waterand soil derived from the laboratory sample composite of vellf C4and site L2.

Method of Analysis Compound Initial ConcentrationWater Soilug'L"1 ug'kg"1

VOAa

VOAVOABNAC

BNABNABNABNABNABNABNABNABNABNABNABNABNABNA

BenzeneEthylbenzeneTolueneAcenaphtheneAcenaphthy 1 eneAnthraceneBenzo (a) anthraceneBenzo(b) fluorantheneBenzo (Jc) fluorantheneBenzo(a)pyreneBenzo (g, h, i) peryleneChryseneFluorantheneFluoreneIndeno (1,2, 3-cd) pyreneNapthalenePhenanthrenePyrene

<DLb

<DL<DL2213141412171410125733<DL397242

984681

9200018000028000035000024000034000030000066000290000990000270000170000600000120000077000

aVOA, volatile organic analysis.b<DL, detection limit for VOA, 5 ppb, for BNA, 20 ppb.CBNA, base neutrals and acid extractables. All other compoundswere below the detection limit.

FIGURES

100 200 300 400 500

T!m« (h)

Figure. 1. Ctnrulative oxygon consumption during biodagradation ofcoal-coJcing vastas. Arrows indicata points of addition of 500ppm Rasters to aaand th« nutrient content of the treatments.

700

~ 600

co

MCOO

XO

"a

500

400

300

Treatment 2-Water, mercury

Treatment 5-10:1, mercury

400 500

Tim* (h)

Figure 2. Cumulative oxygon consumption in HgCl- treatedcontrols. Arrows indlcmts points of addition of 500 ppm Restoreto amend the nutrient content of the treatments.

• Treatment 3, Initiai 50:1 gg Treatment 3, Final 50:1

PyrenePhenanthreneNaphttialen

lndeno(1,2,3-cd)pyreneRuorene

RuorantheneChrysene

Benzo(g,h,i)peryleneBenzo(a)pyrene

Benzo(k)fluorantfieneBenzo(b)fluoranthene

Benzo(a)anthraceneAnthracene

AcenaphthyleneAcenaphthene

TolueneEthylbenzene

Benzene

Mass (ng)

Figure 3. Initial and final priority pollutant content ofTraatmant 3, 50-to-l watar-to-soil slurry. Each valua raprasantstha maan of duplicate m«aaurra«nta. For compounds havino aaai ator balow th« dataction limit, th« dataction limit vaa usad tocalculata tha maan maaa. Tha amount of aach compound is givan asng in 510 mL, tha total voluma of tha traatmant.

• Initial Cone., 10:1Q TrMtrant S, FmaJ 10:1 HgCQ

Traatmarrt4, FmaJ 10:1Traatmant 6, Final untraatad

PyrenePhenanthreneNaphthalene

lndeno(1,2,3-cd)pyreneFluorene

Ruoranthene pBChrysene

Benzo(g,h,i)peryleneBenzo(a)pyrene B

Benzo(k)fluoranthene BBenzo(b)fluoranthene B

Benzo(a)anthracene BAnthracene B

Acenaphthyiene BAcenaphthene

TolueneEthyibenzene i

Benzene10° 101 10' 103 104 10'

Mass (ng)

10s 107 108

Figura 4. Initial and final priority pollutant contant ofTraatmants 4, 5, and 6, 10-to-l watar-to-soil alurriaa. Eachvalua rapraa«nta th« maan of duplicate maaaura««ita. Forcompounda having maas at or b«lov th« dataction limit, th«dataction limit was us*d to calculate the m«an ma««. Tha amountof «ach compound is given as ng in 550 mL, ths total volum* ofth« traatmant.