Vol. 41, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1981, p. 20-280099-2240/81/010020-09$02.00/0

Rates of Microbial Transformation of Polycyclic AromaticHydrocarbons in Water and Sediments in the Vicinity of a

Coal-Coking Wastewater DischargetSTEPHEN E. HERBES

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

To facilitate predictions of the transport and fate of contaminants at futurecoal conversion facilities, rates of microbial transformation of polycyclic aromatichydrocarbons were measured in stream water and sediment samples collected inthe vicinity of a coal-coking treated wastewater discharge from November 1977through August 1979. Six radiolabeled polycyclic aromatic hydrocarbons wereincubated with sediment and water samples; '4CO2, cell-bound 14C, and polartransformation products were isolated and quantified. Whereas 14CO2 and bound14C were major transformation products in sediment assays, soluble polar '4Cdominated transformation in water samples. Mean rate constants (measured at20°0) in sediments collected downstream from the effluent outfall were 7.8 x10-2 h'1 (naphthalene), 1.6 x 10-2 h-1 (anthracene), and 3.3 x 10-3 h-1[benz(a)anthracene], which corresponded to turnover times of 13, 62, and 300 h,respectively. No unequivocal evidence for transformation of benzo(a)pyrene ordibenz(a,h)anthracene was obtained. Only naphthalene and anthracene transfor-mations were observed in water samples; rate constants were consistently 5- and20-fold lower, respectively, than in the corresponding sediment samples. Themeasured rate constants for anthracene transformation in July 1978 sedimentsamples were not related to total heterotroph numbers. In late July 1978, theeffluent was diverted from the primary study area; however, no differences wereobserved either in transformation rate constants or in the downstream/upstreamsediment rate constant ratio. These results are consistent with the hypothesisthat continuous inputs of polycyclic aromatic hydrocarbons result in an increasedability within a microbial community to utilize certain polycyclic aromatic hydro-carbons. However, because transformation rates remained elevated for more than1 year after removal of the polycycic aromatic hydrocarbon source, microbialcommunities may shift only slowly in response to changes in polycyclic aromatichydrocarbon concentrations.

Commercial production of synthetic fuelsfrom coal has been proposed as one means ofalleviating the present shortage of liquid hydro-carbon fuels in the United States. A large-scalecoal conversion facility would almost certainlycause some degree of contamination of localsurface waters due to site runoff, product spil-lage, or discharge of treated effluents. One classof contaminants produced during coal conver-sion which is anticipated to be of major concernis the polycyclic aromatic hydrocarbons (PAH)(9). Several PAH are potent carcinogens (4), andtheir continuous presence at trace levels in sur-face waters may constitute a chronic humanhealth hazard. PAH are found in effluents fromsuch high-temperature industrial processes ascoal coking and petroleum refining (1) and have

t Publication from the Environmental Sciences Division,Oak Ridge National Laboratory.

20

been detected at microgram per liter concentra-tions in condensate samples from several pilot-scale coal conversion facilities (15, 21).Any assessment of the potential hazards to

humans from waterborne PAH resulting fromcoal conversion facilities requires knowledgeconcerning the transport and fate of PAH insurface waters downstream from such facilities.An environmental process which may be of im-portance in determining the fate of PAH is mi-crobial transformation, both in sediments and inthe water column. The ability of some microor-ganisms to degrade PAH has been known for 30years (22), and there have been reports whichhave quantitated degradation of PAH by en-riched cultures (23, 24). However, few measure-ments ofPAH transformation rates in sedimentsor natural waters have been reported. Moreover,PAH transformation rates in a relatively highlyPAH-contaminated environment may differ

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substantially from rates measured in less con-taminated systems (7).Because untreated coal-coking wastewater is

qualitatively and quantitatively similar to un-treated coal conversion wastewater (17) and be-cause PAH have been found in elevated concen-trations in sediments downstream from coal-cok-ing facilities (2), a coke plant was selected asbeing the most likely existing surrogate for afuture coal conversion facility. Therefore, theobjectives of this study were (i) to measure ratesof microbial transformation of representativePAH in water and sediments in the environmentreceiving effluents from a coal-coking plant, and(ii) to assess correlations of PAH levels withrates of PAH transformation.

MATERIALS AND METHODSSite description. More than 70 by-product coke

plants were evaluated to find a suitable surrogate fora coal conversion facility; criteria included effluentvolume, efficacy of wastewater treatment, length ofcontinuous discharge, and hydrological simplicity andwater quality of the receiving river (6). The site se-lected was the Bethlehem Steel Corp. coke plant inBethlehem, Pa., which is one of the five largest cokeplants in the United States (14). Approximately 9 x105 liters of highly contaminated wastewater per dayis treated by an activated sludge unit which removesphenols and cyanide before discharge. This unit wasone of the first of its kind and it has been operatingcontinuously with high efficiency since the early 1960s(12).

Until late July 1978, the treated coke effluent wasdiluted and discharged at a rate of 0.25 m3/s intoSaucon Creek, a shallow, rapidly flowing stream about15 m wide and 30 cm deep (average flow rate, approx-imately 2.5 m3/s) which originates as groundwater 15km upstream. A transient outfall of the BethlehemMunicipal Wastewater Treatment Plant is located 1.2km below the coke outfall. A blast furnace slag pileapproximately 50 m high lines the eastern bank 50 to100 m from the stream. With the exception of a smalltown (Hellertown) 3 km upstream from the outfall,the region upstream is rural. Water quality in SauconCreek above the effluent discharge is generally high,although zinc concentrations are elevated as a resultof zinc mine drainage 15 km upstream (3). Sedimentsare predominantly gravel and coarse sand, with pock-ets of silt.On 28 July 1978, a diversion pipeline parallel to

Saucon Creek was completed by the Bethlehem SteelCorp., and the entire flow of the coking effluent wasdiverted from the creek and discharged directly intoLehigh River 10 m east of the Saucon Creek-LehighRiver confluence.Sample collection. Sediment samples were col-

lected on the following seven dates: 10 October 1977,30 January 1978,25 April 1978, 18 July 1978,29 August1978, 28 November 1978, and 31 July 1979. Watersamples were obtained on the last five dates. Sampleswere collected at an upstream control site (Fig. 1, site1) and at three downstream locations (sites 4, 6, and

0 0.5 kmSCiSCALE

COKE PLANT

FIG. 1. Cokingplant study area in Bethlehem, Pa.,showing sampling locations I through 7.

7). Sediments were collected by scraping the upper 2cm into acetone- and water-washed, 200-ml, glass,wide-mouthed bottles. Each sample was sieved toremove objects larger than 6.25 mm and was held at4°C. Water samples were collected directly in 200-mlbottles. Samples were shipped in a refrigerated con-tainer to Oak Ridge National Laboratory by air ex-press and were processed within 48 h of collection.Additional sediment samples (200 g) were frozen(-78°C) for later PAH analysis; 3.8- or 7.6-liter watersamples for PAH measurements were sterilized byadding chloroform (40 ml) and were held at 4°C duringshipment to Oak Ridge National Laboratory.PAH analysis. The procedures used for PAH anal-

ysis have been described in detail elsewhere (5). Sed-iment portions (40 g) were thawed, spiked with 0.05.g of '4C-labeled benzo(a)pyrene, and extracted for 48h with acetone in a Soxhlet apparatus. Acetone wasreplaced with cyclohexane under nitrogen, and theextract was purified by florasil and alumina columnchromatography. Concentrations of PAH were deter-mined by gas chromatography, using a Perkin-Elmer3920 instrument equipped with a flame ionization de-tector and a 3% Dexsil 400 column (3 m; 80/100-meshHP Chromosorb G support; 110 to 320°C; 2°C/min).Compounds were identified by cochromatography andgas chromatography-mass spectrometry. Water sam-ples were spiked with 104 dpm of [14C]benzo(a)pyreneand were extracted twice with 200-mi portions ofmethylene chloride, which were reduced in volumeand purified similarly by column chromatography;PAH were quantitated by gas chromatography. Meas-ured PAH concentrations were corrected for recoveryof [14C]benzo(a)pyrene, which generally exceeded 80%in sediments and 60% in water samples.

Microbial transformation rate assays. The ra-diolabeled PAH used were obtained commercially

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(American Radiochemical and Amersham Corp.) andincluded [1-"C]naphthalene (specific activity, 2.00 x108 Bq/mmol), [9-"C]anthracene (1.25 x 109 Bq/mmol), [5,6-"C]benz(a)anthracene (BA) (2.22 x 109Bq/mmol), [7,10-"C]benzo(a)pyrene (BP) (9.8 x 10"Bq/mmol), [7-"C]dibenz(a,h)anthracene (DBA) (1.06x 109 Bq/mmol), and [12-14C]-7,12-dimethyl-benz(a)anthracene (DMBA) (8.2 x 10W Bq/mmol).Each PAH was dissolved in several milliliters of ace-

tone; each stock solution (except ["4C]naphthalene)was purified at intervals of several months by thin-layer chromatography on silica gel, using toluene as

the eluant. All work was performed under gold flu-orescent lighting (cutoff, 450 nm) to reduce photolyticdecomposition of the PAH.Sediment samples were assayed for PAH transfor-

mation rates as described previously (19). Each sedi-ment sample was mixed well with a glass rod; portions(0.5 ± 0.05 g) were weighed and placed into acetone-and water-rinsed 20-ml glass scintillation vials. Se-lected vials were autoclaved at 1220C for 30 min toserve as controls. Sufficient radiolabeled PAH dis-solved in acetone were added to result in total activi-ties of approximately 5 x 104 dpm per vial (final PAHconcentrations, approximately 1 ,ug/g of sediment); no

more than 2 pl of acetone solution per vial was re-

quired. Glass cups containing KOH were added, andvials were tightly sealed and held in a constant tem-perature incubator. The incubation temperature was

21 ± 10C for all samples except those collected in April1978, which were incubated at 150C.The kinetics of transformation of anthracene and

BA were measured in October 1977 sediment samplesfrom sites 1, 2, and 6 by incubation of replicate vialsfor 1, 2, 3, 5, and 24 h; replicate sterilized control vialswere incubated for 1, 2, and 24 h. In all other studiestwo samples and two sterilized control vials were sac-rificed after incubations of 4 h (naphthalene), 24 h(anthracene), or 72 or 96 h (BA, BP, DBA, andDMBA). Incubation times were selected from prelim-inary studies so that they resulted in transformationof 5 to 30% of the "4C-labeled PAH added.At each termination time, microbial activity was

halted by adding 5 ml of acetone; 14CO2 was liberatedby adding HNO3, trapped in KOH, and quantitated byliquid scintillation counting. Each sediment samplewas extracted with acetone in a micro-Soxhlet unit;bound 14C in sediments was measured by combustionof the sediment residues (Packard Tri-Carb SampleOxidizer) after extraction. Extracts were concentratedunder nitrogen and applied to silica gel thin-layerplates, which were developed in toluene; fractions atthe origin and solvent front were isolated by scraping,and "4C was quantitated by liquid scintillation. Prelim-inary studies (19) demonstrated that essentially all ofthe "4C-labeled transformation products remained atthe origin, whereas "4C-labeled PAH eluted near thesolvent front. For each fraction ("4CO2, bound 14C,polar 14C, and unaltered PAH), the amount of 14C wasexpressed as a percentage of the amount of 14C initiallyadded as "4C-labeled PAH.To test the efficacy of thin-layer chromatograms in

separation of "4C-labeled BP transformation products,acetone extracts of sterile and nonsterile sedimentsfrom site 4 (July 1978) were reduced in volume undernitrogen, and acetone was replaced with methylene

chloride. The radiolabeled compounds in the extractswere separated by reverse-phase high-performanceliquid chromatography, using established techniques(20); fractions of column eluant were collected, and "Clevels were determined by liquid scintillation counting.The procedures for measuring transformation rates

in water samples were similar to those used in earlierstudies with batch cultures (8). Portions (5 ml) of awater sample (or sterilized control) were added toacetone- and distilled water-rinsed scintillation vials.Approximately 5 x 10' dpm of "C-labeled PAH stocksolution was added to each vial with gentle mixing, aKOH trap was added, and the vial was sealed tightly.After 24 h (naphthalene), 48 h (anthracene), or 96 h(all other PAH) microbial activity was halted by add-ing 0.1 ml of 1 N HNO3 to two nonsterile and twosterile vials, which were then capped tightly for 1 hbefore KOH traps were removed. Each water layerwas filtered (Reeve-Angel 984 H glass fiber filter;nominal particle retention diameter, 0.4 ,um); the fil-trate was extracted twice with ethyl acetate, and the14C remaining in the water was quantified by liquidscintillation. Each filter was rinsed twice with ethanol;the 14C bound in cells was measured by direct liquidscintillation counting of the filter in a dioxane-basedscintillation cocktail. Ethanol and ethyl acetate ex-tracts were combined and replaced with heptane,which was reduced to 2 ml under nitrogen; the amountof 14C in a 0.4-ml portion was determined by liquidscintillation counting. The remainder was applied toa silica gel thin-layer plate, which was developed intoluene, and the 14C at the origin and the solvent frontwas quantitated as described above for the sedimentrate assays. Bound "4C, 14C02, water-soluble 14C, andthin-layer chromatography-separable polar "4C frac-tions were expressed as percentages of the 14C addedinitially as PAH. Tests with sterile sediment and watersamples amended with "4C-labeled PAH andNaH"4C03 demonstrated the completeness of the sol-vent extraction and 14CO2 trapping procedures, respec-tively.

Microbial transformations of ["C]anthracene weremeasured at all sites samples. Transformations of allother PAH compounds were measured only in site 4samples.Enumeration of microbial populations. Sedi-

ment samples were also collected by G. S. Sayler(Department of Microbiology, University of Tennes-see) by scraping to a 2-cm depth at three locations ona transect at each of sites 1, 3, and 7 on 18 July 1978.The number of total viable colony-forming units wasdetermined by plating serial dilutions of a sedimentsuspension on yeast extract peptone glucose agar andcounting colonies after 14 to 28 days of incubation at250C. Phenanthrene-utilizing microorganisms wereenumerated similarly after serial'dilutions on silica gelplates containing 10% reagent-grade phenanthrene(18). Plates containing between 30 and 300 colonieswere counted; at least three plates were counted foreach transect location.

RESULTSPAH concentrations in water and sedi-

ments. Table 1 shows the levels of representa-tive PAH in sediment and water samples col-

22 HERBES

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lected during July 1978 from several sites inSaucon Creek. Similar concentrations were ob-served in April 1978 sediment and water sam-ples. No PAH were detected in water samplesfrom sites 1, 4, and 7 in either August 1978 orNovember 1978 (detection limit, approximately0.03 jig/liter). Low PAH concentrations in site 1sediment were attributable to background levels(possibly from atmospheric deposition), whereasconcentrations at sites 4, 6, and 7 were elevated,apparently due to the coke plant wastewaterdischarge. Sediment concentrations at all siteswere somewhat lower in August and Novembersamples, but variability among samples waslarge, possibly due to differing sediment organiccarbon contents and particle size distributions.In July 1978, concentrations in sediments down-stream from the effluent outfall exceeded water-borne concentrations by factors of 104 to 105.Transformations of added "4C-labeled

PAH in sediments. Mean recoveries of added4C ranged from 88% (naphthalene) to 103%(DBA) in sediment assays. Mean recoveries forsterile controls did not differ significantly fromrecoveries for nonsterile samples.

Nonextractable bound 14C was the majortransformation product of anthracene in sedi-ments, averaging 90% of the total transformedanthracene in 18 assays. The quantities of "4CO2and bound 14C were approximately equal in mostassays of naphthalene and BA. In the assayswhere transformation products were detected inBP, DBA, and DMBA, bound 14C usually ex-ceeded 14CO2 evolution.As observed previously (19), the accumulation

of "4C-labeled polar metabolites separable bythin-layer chromatography was not quantita-tively significant in anthracene or BA assays.Polar transformation products of BP andDMBA varied widely and sometimes were quan-titatively less in nonsterile sediments than insterile controls, apparently due to PAH oxida-tion on the thin-layer plates (10). Separations ofextracts of sterile and nonsterile sediments in-cubated with BP by using high-performance liq-uid chromatography confirmed the absence ofpolar metabolites produced during incubation,although several polar compound peaks presentin the chromatogram of the sterile control ex-tract appeared to be reduced in size in the chro-matogram of the nonsterile sample (Fig. 2).Thus, calculated sediment transformation rateconstants were based on formation of "4CO2 andbound 14C only.As observed previously in petroleum-contam-

inated sediments (19), the appearance of trans-formation products in October 1977 sedimentsfrom sites 1, 2, and 6 occurred without a lagperiod, and the accumulation of these products

continued at a linear rate for 24 h (Fig. 3).Transformation rate constants were calculatedfor all sampling periods with the assumptionthat degradation proceeded as a first-order proc-ess without a lag period.

Correlations among relative quantities ofbound "4C, polar "4C, and "4CO2 were tested foreach compound for all sediments assayed.Bound 14C and "4CO2 were significantly corre-lated for naphthalene (r = 0.803, n = 6) and BA(r = 0.905, n = 7), but not for anthracene (r =0.382, n = 23; critical ro.05 = 0.404). Polar "4C didnot correlate significantly with either bound 14Cor "0CO2 for any compound.The sediment rate constants for site 4 are

shown in Fig. 4. The mean rate constants cal-culated before and after the diversion did notdiffer significantly for any of the four com-pounds. The geometric means of the rate con-

TABLE 1. Mean concentrations of replicatedeterminations of several PAH in water and

sediment samples collected from Saucon Creek inJuly 1978

Concn in:

PAH Water (ug/liter) Sediment (ug/g, dry

Site Sites 4, 6, Site 1 Sites 4, 6,1 and 7 and 7

Anthracene BDa BD 0.14 (0.15)b 3.4 (2.1)Fluoranthene BD 0.58 (0.09) 3.0 (3.6) 19 (17)Pyrene BD 0.39 (0.08) 2.3 (2.9) 12 (11)BA BD 0.11 (0.08) 1.4 (1.5) 6.9 (7.2)BP BD 0.04 (0.02) 0.33 (0.23) 4.8 (5.7)Dibenzanthra- BD BD BD 12 (10)

cenes

Below detection limit (0.03 pg/liter of water or 0.03 jig/kgof sediment).

b Numbers in parentheses are standard deviations.

60 1 1 1 1 1 1 1

40 (0)

t20 -

20

20

0 20 40 60 go 4 00 4 20 4 40 460 480 200FRACTION NUMER

FIG. 2. High-performance liquid chromatographicelution profiles of extracts of "C-labeled BP incu-bated for 96 h with nonsterile sediment (a) and sterilesediment (b) (site 4). The majorpeak at fractions 170to 180 corresponds to unaltered BP.

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24 HERBES

stants for all sample periods were as follows:naphthalene, 7.8 x 10-2 h-'; anthracene, 1.6 x10-2 h-1; BA, 3.3 x 10-3 h-1; BP, 3.4 x 10-4 h-1.The calculated mean turnover times (1/rate con-

25

a 20

,, 15

-04

L, S0

STERILE CONTROL STERILE CONTROL

I go I I 10. . . . - 3~~~-A 0

0 6 42 48 24 0 6 12 t8 24TIME (hr4 TIME (h4

u

FIG. 3. Appearance of 14CO2 (O and 0) and bound"4C (O and *) from ["4C]anthracene incubated withsterile and nonsterile sediments from site 1 (a) andsite 2 (b) (October 1977) at 21°C.

2

j0-1

lIIl lIN5

2T

a 5j:0

2

5

lo-4 -I' lI

i OCT i JAN I APR I JUL I OCT i JAN i APR I JUL IOCT77 78 78 78 78 79 79 79 79

FIG. 4. Sediment rate constants (ksED) for trans-formation of "4C-labeled PAH in sediments collecteddownstream from the coking outfall. All sedimentswere from site 4, except the October 1977 and January1978 samples, which were from site 6. Vertical barsrepresent the standard errors of replicate determi-nations. A, Anthracene; N, naphthalene.

APPL. ENVIRON. MICROBIOL.

stant) for the four compounds were as follows:naphthalene, 13 h; anthracene, 62 h; BA, 300 h;BP, 2,900 h (approximately 120 days). Of thefour rate constants measured forDBA (January,April, August, and November 1978), only one(1.2 x 1O-4 h-1, August 1978) differed signifi-cantly from zero; the turnover time for that rateconstant was 5,800 h (240 days). The onlyDMBA rate constant measured (July 1978) cor-responded to a turnover time of 3,600 h (150days). Although the rate constant patterns atsite 4 appeared to be similar (Fig. 4) for naph-thalene, anthracene, and BA, only naphthaleneand anthracene were significantly correlated (r= 0.900, n = 6, P < 0.01).The rate constants for anthracene transfor-

mation at sites 1, 4, 6, and 7 for all sampleperiods are shown in Fig. 5. To examine theeffect of the outfall diversion in late July 1978on the rate of anthracene degradation in sedi-ments, the rate data were subdivided into pre-diversion (October 1977 and January, April, andJuly 1978) values and postdiversion (August andNovember 1978 and August 1979) values. Down-stream sites 4, 6, and 7 were assumed to bereplicates at each sample time to permit esti-mation of within-site variance. The within-sitevariance was assumed to be independent of theseason and the presence or absence of the outfalland was assumed to be equal at all sites. Afterlogarithmic transformation of the rate constantsto equalize variances, the within-site variancewas estimated as the pooled mean of theweighted variances for sites 4, 6, and 7 for theApril 1978, July 1978, August 1978, and August1979 sampling periods (S2 = 0.0270). Two-sidedt tests with 5 degrees of freedom were then usedto compare the rate constants from site 1 withthe means of the rate constants from sites 4, 6,and 7 before and after diversion. Three compar-isons were tested, with the following results. (i)Upstream and downstream rate constants dif-fered significantly before diversion (t = 5.09, P< 0.01). (ii) Upstream and downstream rate con-stants differed significantly after diversion (t =4.76, P < 0.01). And (iii) The difference betweenupstream and downstream rate constants beforediversion was statistically the same as after di-version (t = 0.28). Mean downstream values ofthe rate constant for anthracene were 4.1 and3.7 times greater than the upstream values ofthis rate constant before and after diversion,respectively.Transformation of added "4C-labeled

PAH in water samples. Recoveries of 14C werelower for each compound in water samples thanthey were in sediment assays; mean recoveriesranged from 52 to 87%. The large difference inmean recoveries of naphthalene between sam-ples (52%) and sterile controls (69%) suggested

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Ci j1O3)40 r-

01 467 1 467 1467 1 467 1 467 1467 (SITES)OCT 77 APR78 JULY 78 AUG78 NOV78 AUG 79

FIG. 5. Sediment rate constants (ksED) for ['4CJan-thracene transformation in Saucon Creek sediments.The vertical bars represent the standard errors ofreplicate determinations. Where bars are not drawn,no rate constants were determined.

that incomplete recovery of transformationproducts may have occurred in addition to vol-atilization (and perhaps sorption) losses.

In contrast to sediment assays, polar 14C wasthe dominant anthracene transformation prod-uct fraction in water samples and averaged 82%of the total transfonned "IC. Despite the rela-tively large variability in controls, the quantityofpolar "C-labeled metabolites was significantlygreater than zero in 10 anthracene assays; how-ever, neither the quantity of polar 14C nor thequantity of total transformed 14C was differentfrom zero in any BA, BP, DBA, orDMBA assay.

All three transformation product fractions(polar 14C was not measured due to the volatilityof "4C from thin-layer plates) in the five naph-thalene assays were significantly correlated; thecorrelation coefficients for filter-bound "4C and14CO2, filter-bound "4C and water-soluble 14C,and water-soluble 14C and '4CO2 were 0.964,0.954, and 0.995, respectively. In anthracene as-says only water-soluble '4C and thin-layer-sepa-rable polar 14C were significantly correlated (r= 0.631, n = 20).

In water samples, transfornation of naphtha-lene was observed at all sampling times at site 4;the mean of log-transformed rate constants was3.2 x 10-3 h-1, resulting in a turnover time of 310h (13 days). However, because some losses of

transformation products apparently occurred,the calculated rate constants for naphthalenemay have underestimated true values. At notime did the transformation rate constants foranthracene in water samples from site 1 differsignificantly from zero. The log-transformedmean for sites 4, 6, and 7 over all samplingperiods was 2.0 x 10-3 h-1, corresponding to aturnover time of 500 h (21 days). Transforma-tions of BA, BP, DBA, and DMBA were notdetectable.Before the effluent diversion, similar patterns

of anthracene transformation activity in SauconCreek water samples were observed in April andJuly 1978 (Fig. 6). Rate constants were essen-tially zero both immediately upstream anddownstream from the effluent, reaching maxima1.2 to 1.5 km below the outfall. Rate constantsmeasured in July 1978 were two- to sixfoldgreater at each site than in April 1978. After thediversion, only five downstream (sites 4, 6, and7) rate constants for anthracene transformationwere measured; two of these were significantlynonzero, compared with five of six nonzero rateconstants before the diversion. The means oflog-transforned rate constants for sites 4, 6, and7 before and after diversion did not differ signif-icantly (t = 1.73, df = 9). Similarly, log-trans-formed mean rate constants for naphthalenedegradation at site 4 did not differ significantlybefore and after diversion (t = 0.24, df = 3).The rate constants for naphthalene and an-

thracene in site 4 water samples were stronglycorrelated (r = 0.999, df = 8, P < 0.01). If waterrate constants for naphthalene were in fact un-derestimated by a constant factor, the correla-tion between naphthalene and anthracene wouldremain the same.

400/'O*

80 t / \_

0-2 /JULY

20KW

APRIL4

c~~~~~~

0 0.5 40 4 5DISTANCE FROM EFFLUENT DiSCmARGE Xk,n)

FIG. 6. Rate constants (k) for ['4C]anthracenetransformation in Saucon Creek water samples col-lected in April and July 1978. The vertical barsrepresent the standard errors of replicate determi-nations.

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Interrelationships between water andsediment rate constants. Rate constants fornaphthalene in water and sediment assays werepaired by sampling date; the correlation wassignificant (r = 0.868, P< 0.05). A similar pairingof anthracene rate constants by sampling dateand location (sites 1, 4, 6, and 7) produced asimilar relationship (r = 0.840, P < 0.01).Microbial population densities in sedi-

ment samples. Means and standard deviations(in parentheses) of triplicate samples for thelogarithm of the total viable colony-formingunits per gram (dry weight) of sediment duringthe July 1978 sample period were as follows: site1, 7.37 (0.43); site 3, 6.22 (0.28); and site 7, 6.31(0.38). The corresponding values for phenan-threne-utilizing colony-forming units per gram(dry weight) of sediment were as follows: site 1,5.60 (0.13); site 3, 5.60 (0.25); and site 7, 5.78(0.67). One-way analyses of variance were usedto test for significant differences among log totalviable colony-forming unit values and among logphenanthrene-utilizing colony-forming unit val-ues. The F2,6 values for these analyses were 9.87and 0.20, respectively; the former is significant(P < 0.05), whereas the latter is not. To deter-mine whether upstream and downstream valuesfor log total viable colony-forming units differedsignificantly, the mean of sites 3 and 7 wascompared with the log total viable colony-form-ing unit value at site 1. The upstream and down-stream values differed significantly (t = 4.26, df= 6, P < 0.01).

DISCUSSIONThe relationship between PAH molecular size

and transformation rates in both water and sed-iment was similar to the relationship observedpreviously in petroleum-contaminated sediment(7); there was a consistent decrease in the rateconstant with increasing molecular size. In thisprevious work, Herbes and Schwall speculatedthat contamination of sediments with certainPAH may favor development of microorganismpopulations capable of utilizing those com-pounds at more rapid rates. The sediment con-centrations of the larger PAH were consistentlyhigher downstream from the coke effluent in thepresent study than in the petroleum-contami-nated sediments analyzed previously (7); trans-formation rate constants for anthracene and BAwere also greater by more than 10-fold. Simi-larly, the anthracene concentration at site 4 inthe July 1978 sample exceeded that at site 1 by1 order of magnitude; the transformation rateconstant at site 4 was more than fivefold higher.Therefore, the anthracene and BA concentra-tion and transformation rate data for the July1978 sediment and the previously analyzed pe-

troleum-contaminated sediments are consistentwith the hypothesis that increased levels ofPAHcontamination in sediments cause enrichment ofthe microbial community with strains which arecapable of transforming the PAH at a relativelyrapid rate.

In contrast to PAH levels, which appear to berelated to sediment transformation rates, thelimited amount of data suggests that sedimentmicrobial population estimates are not relatedto transformation rates. Whereas the [14C]an-thracene transformation rate constants in theJuly 1978 sediment samples were sevenfoldhigher at the two downstream locations (sites 4and 6) than at site 1, phenanthrene-utilizingpopulations (which presumably utilize anthra-cene also) were the same size both above andbelow the outfall. At the same time, total het-erotroph numbers were more than 1 order ofmagnitude greater above the outfall than at thedownstream sites. More rapid downstreamtransformation rates may be due to elevatednutrient levels downstream from the effluent,which contains relatively high concentrations ofphosphorus and ammonia; hydrocarbon degra-dation in lake water has been shown previouslyto be increased 5- to 20-fold by nutrient addition(25).Transformation of the five-ring PAH in sedi-

ments did not relate directly to sediment con-centrations, in contrast to transformation of an-thracene and BA. Although 14CO2 and bound 14Cwere formed during site 4 sediment incubationswith BP in the present study, the disappearanceof '4C-labeled impurities observed in the high-performance liquid chromatogram (Fig. 2) sug-gests that the impurities, rather than BP, weredegraded. Thus, no unambiguous evidence existsfor BP transformation in the site 4 sediment,although the BP concentration in July 1978exceeded the concentration in the petroleum-contaminated sediment analyzed previously by100-fold. Similarly, virtually no transformationofDBA was observed, although in the July 1978site 4 sediment the dibenzanthracene concentra-tion (albeit mixed isomers) was similar to theanthracene and BA concentrations. McKennaand Heath (13) have reported cometabolism ofthese compounds in the presence of naphtha-lene, which suggests that there are bacteriawhich can transform them. Khesina et al. (11)found degradation ofup to 50% of the BP in soilsover a 3-month period, with the most extensivedegradation in highly contaminated soil from arefinery. Because in the latter study the BPconcentration of the refinery soil was about 10-fold higher than the site 4 July 1978 sedimentBP concentration in the present study, the pos-sibility exists that higher concentrations are re-

26 HERBES

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RATES OF TRANSFORMATION IN COKING WASTEWATER 27

quired to induce degradation of BP. An alter-native explanation for the lack of BP or DBAtransformation in sediment is that the five-ringPAH partition onto sediment particles to suchan extent that only an extremely small fractionis present in the interstitial water, and thusvirtually none is available for uptake and trans-formation by microorganisms (26, 27).Although sedimentary and waterborne PAH

concentrations downstream from the coking out-fall appeared to decrease substantially after theeffluent diversion, little effect on microbial PAHtransformation rate constants was observed.Perhaps the lack of observable effects was dueto slowness of population shifts; although Pierceet al. (16) observed a rapid increase in naphtha-lene-degrading ability upon exposure of bacteriato naphthalenic hydrocarbons, more than 1 yearwas required for the proportion of naphthaleneutilizers to decline to pre-contamination levels,even though the concentration of aromatics de-clined by 75% within 8 days. Alternatively, thelack of effect of the diversion may indicate thatresidual PAH concentrations were sufficientlyhigh to maintain degradative enzyme activitiesat prediversion levels.

ACKNOWLEDGMENTSC. P. Allen provided excellent technical assistance in PAH

transformation rate determinations. W. H. Griest, R. Reagan,and S. Watson (Analytical Chemistry Division, Oak RidgeNational Laboratory) performed PAH analyses on water andsediment samples. J. Beauchamp (Mathematics Division, OakRidge National Laboratory) and J. Solomon provided assist-ance with statistical tests. J. Selkirk (Biology Division, OakRidge National Laboratory) performed high-performance liq-uid chromatographic separations of extracts of sediment sam-ples after incubation with "4C-labeled BP. G. S. Sayler (Micro-biology Department, University of Tennessee-Knoxville) gen-erously furnished unpublished data on microbial populationmeasurements. E. Coppenhaver and N. George (PennsylvaniaDepartment of Environmental Resources, Bureau of WaterQuality) assisted in sample collection. W. Grim (BethlehemMunicipal Wastewater Treatment Plant) furnished buildingspace for sample pretreatment and equipment storage. E. A.Bondietti and R. F. Strayer provided technical commentsconcerning the manuscript.

This research was sponsored by the Office of Health andEnvironmental Research, U. S. Department of Energy, undercontract W-7405-eng-26 with Union Carbide Corp.

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