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The effect of salinity on the microbial mineralization of two polycyclic aromatic hydrocarbons in estuarine sediments
Marine Environmental Research 26 (1988) 18 l-198
The Effect of Salinity on the Microbial Mineralization of Two Polycyclic Aromatic Hydrocarbons in Estuarine Sediments Robert P. Kerr Cosper Environmental Services, Inc., PO Box 525, Northport, NY 11768, USA
& Douglas G. Capone* Center for Environmental and Estuarine Studies, Chesapeake Biological Laboratory. University of Maryland, Box 38, Solomons, MD 20688-0038, USA (Received 17 May 1988; revised version received 23 August 1988; accepted 29 August 1988)
ABSTRACT Sediments from the lower Hudson River estuary and two other coastal environments were examined experimentally for their ability to mineralize (convert to CO z) the polycyclic aromatic hydrocarbons ( PAHs) naphthalene and anthracene over a range of salinities. Routine assays employed 1:1 (vol fresh sed: vol water) sediment slurrys in order to overcome natural variability in mineralization rates among replicates. Mineralization rates were stimulated by about 2"5 fold, compared to unslurried controls, while the coefficient of variation fell from 13% to 3"5%. Rates of naphthalene mineralization in surface sediments from along the mainstem of the Hudson River (salinities from 2 to 27%0) ranged from 0.011 to 1.5nmolcm 3day x (pool turnover I T , ] f r o m 60 to 2040 days) with no discernible trends along the estuarine gradient. For two stations examined experimentally (mile point 5, salinity 23%0; mile point 26, salinity 5%0), microbial assemblages appeared acclimated to broad salinity variations as * To whom correspondence should be addressed. 181
Marine Environ. Res. 0141-1136/89/$03.50 ~ 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain
182
Robert P. Kerr, Douglas G. Capone imposed increases or decreases in salinity did not signi~'cantly alter rates g[" mineralization compared to controls. Sediments from two upstream marshes of the Hudson (mile points 36 and 45) showed rates of naphthalene mineralization from 0.007 to 0"15 nmol cm- 3 day 1 ( T , from 14 to 368 days), while sediments from a third marsh in freshwater ( mile point 76) had high rates (66 nmol cm- 3 day 1; 7", 40 days). For the two upstream marsh stations which rarely experienced salt intrusion, there was a substantial decrease in mineralization of naphthalene and anthracene with increasing salinity. Consistently high rates of naphthalene mineralization (780 to 1600 nmol cm 3day 1; 7", 5 to 6 days) were observed in petroleum contaminated sediments from Port Jefferson Harbor (PJH) on the north shore of Long Island. P J H has a relatively constant salinity regime (about 27%0) and imposed decreases in salinity effected decreases in rates of naphthalene and anthracene mineralization. Lowest rates of naphthalene mineralization 0"003 to 0"004 nmol cm - 3 day- 1; T, from 714 days to 833 days) were Jbund m sediments ,from two stations in the relatively pristine Carmans River estuao' on the south shore of Long Island, The abili O, oJ"increases or decreases in salinity to affect the rate o[model PA H mineralization appeared to be dependent on the natural variation in the salinity regime from which a sample was obtained. Data from all the environments studied indicated a strong positive correlation between PA H concentration and the rates of mineralization of naphthalene. Rates of PA H mineralization in all environments examined appear to be primarily controlled by the extent of pollutant loading and not by natural variations in the salinity regime.
INTRODUCTION Aquatic environments receive polycyclic aromatic hydrocarbons (PAHs) from many sources including direct discharge, sewage outfall, urban and agricultural run-off as well as groundwater flow (Van Vleet & Quinn, 1978; Bedding et al., 1982). Because of their relative insolubility, PAHs readily adsorb to particulate matter and become concentrated in sediments at levels many times those found in the water column (MacLeod et al., 1981; Mueller et al., 1982; Olsen et al., 1982). It has been shown that microbes in sediments from chronically polluted environments can metabolize PAHs at significant rates (Herbes & Schwall, 1978; Atlas, 1981; Saltzmann, 1982; Spain & Van Veld, 1983; Heitkamp et al. 1987; Bauer et al. 1988). Determining the rates at which these refractory compounds are removed by indigenous microbes is fundamental in assessing their persistence and effects on benthic communities. Numerous environmental factors (e.g. oxygen, nutrient availability,
Effect of salinity on mineralization of two poly~Tclic aromatic hydrocarbons
183
temperature and pH) are known to affect the activities of sediment microbes including their ability to degrade PAHs and other xenobiotics (Ward & Brock, 1976; Hambrick et al., 1980; Atlas, 1981). Estuaries are characterized by a variety of gradients which affect and define the nature of the microbiota within the estuary. Major inorganic ion composition, as well as the quality and quantity of organic matter, vary through the estuary in a systematic and microbiologically relevant way (Capone & Kiene, 1988). In the water column of estuaries, salinity, or some factor covarying with salinity, has been identified as an important factor regulating microbial activities, including the mineralization of xenobiotics. The degradation of mcresol, chlorobenzene and 1,2,4-trichlorobenzene was shown to be drastically inhibited as salinity increased through an estuary (Bartholomew & Pfaender, 1983). Degradation of" the synthetic detergent nitrilotriacetate (NTA) was inhibited in the water column of two estuaries at brackish and marine salinities (Bourquin & Przybyszewski, 1977; Hunter et al., 1986). Similarly, p-nitrophenol degradation in the water of sediment water microcosms was inhibited at marine salinities (Spain et al., 1980). While PAHs are known to be microbially mineralized (converted to CO2) in fresh water, brackish and marine sediments (Herbes & Schwall, 1978; Readman el al., 1982; Lee et al., 1982; Massie et al., 1985), the effect of an estuary's salinity gradient on PAH mineralization in sediments is not well defined. The objective of this study was to determine the ability of fresh, brackish and salt water sediments of the Hudson River Estuary and two other sites on Long Island to mineralize two low molecular weight PAHs (naphthalene and anthracene), and to examine experimentally the effect of imposed salinity variation on PAH mineralization at several of the sites.
MATERIALS AND METHODS All samples in the lower Hudson River were taken between June and October of 1984 and May and August of 1985. Site locations in 1984 spanned the estuary's entire salinity gradient. Sites included mile points (MP) (as defined by the US Army Corps of Engineers) 0 (southernmost tip of Manhattan), 5 (midtown Manhattan), 10 (George Washington Bridge), 26 (Tappan Zee Bridge), and 36 (Haverstraw Bay) (Fig. 1, Table 1). Samples were taken by boat from the subtidal banks along the edge of the mid channel of the river in waters of 5 to 15 m depth. Samples obtained in 1985 were from subtidal regions of marches adjacent to the Hudson. Marsh locations within the River are components of the Hudson River National Estuarine Sanctuary and include Piermont (adjacent to MP 36), Iona (MP 45) and Tivoli (MP 76) (Fig. 1, Table 2). Iona
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marsh is slightly brackish, while Tivoli marsh resides within the fresh water reach of the River. Other locations sampled during 1985 were: Port Jefferson H a r b o r (PJH), a small developed bay of relatively constant salinity with an oil fired power plant on the north shore of Long Island, New York; and Carmans River Estuary, a relatively pristine (with regard to petroleum contamination), eutrophic estuary on the south shore of Long Island. For the 1984 studies of the Hudson River, two gravity cores were taken at each site on each sampling date. Samples were kept in the dark and cool in a tall can with an ice-water mixture and returned to the Marine Sciences
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Research Center (SUNY, Stony Brook, NY) where they were placed in their container in a controlled environment room (20°C) overnight until experiments commenced the next day. Salinity of both the overlying water and surface sediment pore water (top 1 cm) was determined with a hand held refractometer (Atago Co., Ltd). Rarely did the two differ by more than 2%0. Sediment samples were taken by removing 1 cm 3 subsamples from the top 1 cm or to the bottom of the brown (aerobic) zone, whichever was less, from two cores from each site. A total of 30 subsamples from the cores were combined and amended with 30 ml of 0.45/~m filtered ambient bottom water from the corresponding sample site. The slurry was magnetically stirred for 3 min at an intermediate speed to minimize cell disturbance. Subsamples (2 ml) were removed from the slurry, placed in 20 ml scintillation vials and capped with butyl rubber stoppers. These 1 : 1 diluted samples were used to estimate the in situ rates of microbial mineralization. From the remaining slurry, subsamples (5-10ml) were taken and diluted 10:1 (vol :vol) with either ambient water from that site or artificial sea water (31 g NaC1, 10 g MgSO4.7H20, 9"04 g NaHCO3 in one liter distilled water, Strickland & Parsons, 1972) diluted to various salinities (0-30%o). From these diluted slurries, subsamples (2ml) were also removed, placed in scintillation vials and treated with either of the two radiolabeled PAHs. Experimental treatments were prepared in triplicate while killed controls (autoclaved for 20 minutes) were run in duplicate. All samples were incubated in the dark and at the in situ temperature. In situ temperatures ranged from 20°C (June, Oct) to 26°C (August). Rotary shaking during incubation ensured rapid equilibration between gas and liquid phases and dispersal of pollutants. On several occasions, in order to assess the effect of dilution on mineralization, comparisons were made among sediment diluted by various degrees with sediment which had been neither unslurried or diluted. [l,(4,5,8)-14C]-Naphthalene (5mCi/mmol) and E9(10)-14C] anthracene (5mCi/mmol), obtained from Amersham Co., were diluted with acetone to yield a working solution with a final concentration of 2000-300011M. Radiolabeled compounds were added to samples through the stopper using a microliter syringe (10pl injection). The final concentration of acetone ( < 0 ' 2 % vol:vol) did not interfere with several measures of sediment metabolism (Bauer & Capone, 1985a). The addition of naphthalene and anthracene added to sediments (about 200 ppb) was not inhibitory to either thymidine incorporation or glucose metabolism in pristine marine sediments (Bauer & Capone, 1985a). At selected intervals during experiments, the headspace of assay vials were flushed with compressed air, and newly evolved 14COz was trapped in vials with 10ml of Oxosol (National Diagnostics). Radioactivity was
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Robert P. Kerr, Douglas G. Capone
quantified on a Packard Tri-Carb 300C liquid scintillation counter. The same 14CO1 trapping vial was reused for sequential, cumulative trapping of the headspace of its respective assay vial for the duration of an experiment, counting each trapping vial after each trapping (Bauer & Capone, 1985b). Previous studies had shown that extended flushing of the headspace was effective in removing recently produced 14CO2 (Bauer & Capone, 1985b). Only small additional amounts of 14CO2 (< 5% of total) were obtained by acidification (Bauer & Capone, 1985b). Net ~4CO2 production for each time point was calculated by subtracting 14C flushed from control vials and trapped. This would include 14CO2 contamination in stocks as well as any ~4C-naphthalene volatilized into the flask headspace. Naphthalene volatilization in controls over the whole time course (9 days) never accounted for more than 1"3% of the total injected. Rates of mineralization were determined from linear portions of ~4CO2 production curves. Any lag period was recorded. Analysis of sediments for the presence of ambient naphthalene, phenanthrene and anthracene was conducted using a Waters' high-pressure liquid chromatograph (HPLC) equipped with an Adsorbosphere (Applied Science) C18 (5pm mesh size) column (15cm length) and detected on a Waters model 400 absorbance detector (254 nm). Sample preparation and flow conditions were identical to those of Bauer & Capone (1985b). Retention times for naphthalene, phenanthrene and anthracene were about 2.4, 4.2 and 4.7 min, respectively. The level of detection was about 100ng (gwetsed) 1 Turnover times of naphthalene and anthracene were calculated by using the inverse of the rate of 14CO2 evolution. This procedure was identical to that used by Readman e t al. (1982), which found 14CO2 measurements more reproducible than those based on residual hydrocarbon and polar metabolite measurements. Absolute rates (Va) of mineralization (nmol c m - 3 day-1) were calculated for all sites where ambient concentrations of the substrate were obtained. Absolute rates were calculated by using the relative rate (V,) of mineralization (fraction of label mineralized) divided by the fraction (F) of radiolabeled naphthalene added to the ambient concentration: V~ = V , / F = V, x
(ambient naphthalene) ([ 14C] naphthalene)
The effects of salinity on the rate of mineralization of PAHs within a specific environment were statistically analyzed by single class analysis of variance and unplanned mean comparison (Sokal & Rohlf, 1981). Organic carbon determinations were performed by ashing at 450°C. Bacterial numbers were enumerated by the method of McDaniel & Capone (1985).
E~fect of salinity on mineralization of two polycyclic aromatic hydrocarbons
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RESULTS
Naphthalene mineralization Sites used in this paper can be broadly categorized as either fresh water (Tivoli Bay and upper Carmans River), brackish (Iona and Piermont marshes, mile points 10, 26 and 36), or marine (Port Jefferson Harbor, lower Carmans River, MP 0 and 5). Ambient sediment pore water salinities for all sites are listed in Tables 1 and 2. Measurable levels of naphthalene were detected in sediments from all environments except Carmans River, Piermont and Iona (Tables 1 and 2). Highest concentrations of naphthalene were found in sediments from both MP 5 and Port Jefferson Harbor (PJH). Concentrations of naphthalene in sediments from the subtidal banks (1984 sites) of the Hudson River generally decreased up-river (away from midtown Manhattan). Samples for PAH analysis taken from the marshes along the Hudson did not show any consistent trend. Interestingly, phenanthrene, or a compound co-eluting with phenanthrene (such as retene) was the only PAH consistently detected at Piermont and Iona. For samples taken in 1984 along the edges of the main channel of the Hudson, there appeared to be no consistent trend in naphthalene mineralization rates along the estuarine gradient (Table 1). Turnover times (Tn) of naphthalene along the mainstem of the lower Hudson River varied substantially both among and within sites, ranging between 60 and 2040 days at the four upstream sites with an extreme value of 9100 days for the one sampling at the most seaward site. Absolute rates of naphthalene mineralization ranged from 0.011 to 1-5 nmol c m - 3 day- 1. Turnover times of naphthalene did not significantly correlate with salinity (r 2 = 0"40) or microbial numbers by direct counts (r 2 = 0" 12). For the six samples for which concentration of naphthalene, anthracene and phenanthrene were available, there was no apparent correlation. However, this included samples for three sites on one day (6 Sep.) on which naphthalene mineralization rates were unusually low and may not be representative (see Discussion). Turnover time was strongly correlated with organic content (r 2 = 0-72). Turnover times appeared less variable among and within sites along the subtidal marsh banks of the Hudson River, ranging from 14 to 368 days (Table 2). While absolute rates of mineralization were quite low (0.007 to 0.15 nmol cm -3 day -1) at the marshes at MP 36 and 45, naphthalene mineralization rates at Tivoli marsh (MP 76) were 66 nmol cm - 3 day- 1. For the 1985 data, turnover times (T,) of naphthalene were negatively correlated with the summed concentration of naphthalene, phenanthrene and anthracene (NPA) (Fig. 2) found at each site: that is, sediments containing
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higher concentrations of N P A had elevated rates of n a p h t h a l e n e mineralization (shorter T,). Anthracene mineralization was examined on three occasions and turnover times were of the same order as parallel naphthalene mineralization studies at each site (Table 2). The extent of any period of low or minimal rate (lag) before a higher steady rate of naphthalene mineralization varied a m o n g stations. For PJH sediments, high initial rates of mineralization without a lag period were observed on all three occasions. Similarly, for three out of four samplings at MP 5 (exception Sep. 6), there were high rates with no apparent lag period. For all sites, the extent of any lag period before rates of naphthalene mineralization became constant, appeared positively correlated with turnover time (Fig. 3). Several comparisons of the effect of dilution were made with sediments from Port Jefferson Harbor (Table 3). Slurring consistently stimulated mineralization rates while lowering the variation a m o n g replicates. The greatest stimulation occurred between unslurried samples and 1 : 1 dilution, with greater dilutions having lesser effect.
Effects of salinity change The effect of experimentally imposed salinity changes on the rate of mineralization of naphthalene and anthracene was examined in sediments from several of our sites using additions of artificial sea water adjusted to
Effect o f salinity on mineralization o f two polycyclic aromatic hydrocarbons
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specific salinities. For two sites along the mainstem of the tidally dominated lower H u d s o n River (MPs 5 and 26), there was no statistically significant effect (P < 0.05) of either increase or decreases of salinity on the rate of mineralization of naphthalene (Fig. 4). In both cases, sediments were subjected to salinities between 4 and 31%o and monitored for over two weeks. A second experiment at M P 5 (3 Oct., 27%0) revealed no difference for treatments from 13 to 36%0 with a 35% decrease in mineralization at the lowest salinity used (9%0). Sediments from the three most upstream sites in the H u d s o n (Tivoli Bay, Iona Marsh and M P 36) all responded to salinity changes in the same TABLE 3 Effect of Dilution on Mineralization of Naphthalene in Sediments from Port Jefferson Harbor. Values are Cumulative A m o u n t Mineralized, Normalized per cm 3 of the Undiluted Sediment
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manner, but by varying degrees. Sediments from Tivoli Bay (ambient salinity 0%0) were significantly (P < 0-01) inhibited in their ability to mineralize naphthalene by salinities of 15%0 (Fig. 5). Mineralization at this fresh water site completely ceased at 30%0. Naphthalene mineralization rates for sediments from Iona Marsh were also significantly reduced (P _<0-05) at a salinity of 15%o compared to treatments receiving 0%0 water (Fig. 5). Mineralization was further reduced at 30%0, but did not cease. Anthracene 1.4
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Effect of salinity on mineralization of two polycyclic aromatic hydrocarbons
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mineralization was also significantly reduced (P < 0.01) at 30%o, but not at 15%o. Sediments from the mainstem station at MP 36 during one experiment (ambient salinity 2-3%0) tolerated salinities of 10%o, but were also significantly inhibited (P<0.01) by salinities of 15%o (data not shown). Sediments from MP 36 used in a subsequent experiment (Oct. 1984), however, tolerated all salinities between 0 and 25%0 with no significant inhibition. On this date, pore water salinity had increased to 7%0 (Table 1). Port Jefferson Harbor, located adjacent to mid Long Island Sound, has a relatively constant salinity regime around 27 to 28%o. The effect of salinity on rates of microbial mineralization of naphthalene and anthracene for PJH sediments is shown in Fig. 6. In all cases, rates were greatest in sediments treated with artificial seawater of either 30 or 15%o (no significant difference). However, rates were significantly reduced (P < 0.01) by adjusting the sediment salinity to 0%0.
DISCUSSION The effect of large, instantaneous salinity changes on PAH mineralization in surficial sediments depended on the ambient salinity regime. In the lower reaches of the Hudson estuary (MP 5 and 26) and as far north as mile point 36, large salinity changes did not inhibit the mineralization of naphthalene by sediment microbes. This is probably a result of adaptation by downstream populations of sediment microbes to large daily changes in salinity, resulting from the large tidal oscillation occurring within the lower
194
Robert P. Kerr, Douglas G. Capone
reaches of the estuary. Hourly salinity measurements taken in the main channel near MP 10 between September and October 1980 (National Ocean Services, New York Harbor Circulatory Survey 1980-81, Rockville, MD) found that the salinity of surface and bottom waters normally varies diurnally between 16 and 24%o and can change by as much as 16%oin a single day as a result of passing storm fronts. Seasonal fluctuations in river flow accentuate salinity excursions even further. While sediments from PJH are exposed to roughly the same mean salinity as that for MP 5, they are not subjected to any significant change in salinity either during a tidal cycle or seasonally. The lack of large oscillations in salinity at PJH appears to account for a microbial population poorly adapted to lower salinities and inhibited in naphthalene and anthracene mineralization at salinities of 15 and 0%0. Sediments progressively further upstream in the Hudson River displayed maximum rates of naphthalene mineralization at lower salinities and also within a narrower range of salinities. Fresh water (Tivoli Bay) and slightly brackish (Iona and MP 36) sediments were significantly inhibited by increases in salinity to 15%o. This may reflect adaptation of the microbial population to lower ambient salinity and a narrower range of salinities experienced in these upstream stations. Sediments from MP 36, however, also displayed a tolerance to increases in salinity on one occasion (Sept. 1984, a characteristically low flow month; Mueller et al. 1982) when the sediment pore water salinity had increased to 7%0 from its earlier salinity of 2-3%o. This increase may be indicative of seasonal shifts in the salinity tolerance of indigenous microbial populations. Significant differences in rates of mineralization were observed in some experiments between samples incubated with artificial water compared to natural water at the ambient salinity. The low nutrient levels in the artificial sea water (0"5FtM NO 3 and 0"4#M NH~), and/or the absence of other required ions may explain why, as was most often the case, sediments incubated with artificial sea water had somewhat lower rates of PAH mineralization than sediments incubated with natural sea water at the same salinity. High in situ rates of naphthalene mineralization in the Hudson River estuary did not appear to be associated with any particular salinity zone, but generally occurred at stations having the greatest naphthalene and/or phenanthrene concentration (Fig. 2). Interestingly, sediments from Iona and Piermont both mineralized naphthalene quickly, yet were devoid of measurable naphthalene (Table 2). Both of these environments, however, contained substantial quantities of phenanthrene, indirectly supporting studies (Barnsley, 1983; Bauer & Capone, 1988) which showed that soil and sediment bacteria could catabolize both anthracene and naphthalene after
Effect of safinity on mineralization of two polycyclic aromatic hydrocarbons
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pre-exposure to phenanthrene. Alternatively, microbes from Iona and Piermont marshes may have been previously exposed to naphthalene. On one date (6 Sept. 1984), the turnover time of naphthalene in the lower Hudson River was unexpectedly low given the levels of naphthalene and phenanthrene detected. Turnover times of naphthalene in sediments from MP 0, 5, and 10 were approximately 9, 1.3 and 3 times longer, respectively, than for the pristine sediments from Carmans River (about 2 years). The very low rates of mineralization of naphthalene on this occasion might have been the result of several factors. Early September is the period of highest temperatures and lowest river flows, conditions which lend themselves to bottom water anoxia. Rates of naphthalene mineralization under anoxic conditions are extremely low (Bauer & Capone 1985b) and anoxia either at the time of sampling or during the immediately preceding period may have impaired the capacity of the sediment surface microbiota to mineralize naphthalene. Unfortunately, we did not collect water chemistry information during coring cruises. However, water chemistry data obtained from the NY Dept. of Environmental Protection for cruise on 4 Sept. indicated strong stratification and near bottom waters with about 45% of saturation 02 values (T. Brosnan, pers. comm.). Hence, it is possible that low O 2 conditions may have been developing and could have affected naphthalene mineralization rates for samples collected on 6 Sep. Alternatively, toxicological stress may have accounted for the lessened ability to utilize [14C]naphthalene. The summed concentration of NPA in sediments from the lower Hudson comprises only 10-40% of the total PAH pool and a yet smaller fraction of the persistent organic contaminant pool (MacLeod et al., 1981). The summed and possibly synergistic effect of part per million concentrations for each of the numerous PAHs present may inhibit microbial mineralization of naphthalene as well as other processes. However, turnover times at the same sites on other dates as well as in PJH sediments (an environment containing NPA at concentrations comparable to those of the lower Hudson) were often orders of magnitude shorter than on this occasion. The rate of mineralization of naphthalene in another chronically polluted aquatic environment having similar levels of naphthalene contamination (7/~g g dry sed -1) was 190nmol cm -3 day -1 (Herbes & Schwall, 1978). These findings agree well with those for the PJH station, and again are significantly greater than those of the lower Hudson. Sediments around oil drilling platforms in the North Sea (Massie et al., 1985; Saltzmann, 1982) which were contaminated with naphthalene by as much as 7/~g g dry sed- 1, produced rates of mineralization of naphthalene that varied between zero and 2"4 nmol c m - 3 d a y - 1. While in general, high rates appeared positively correlated with the degree of contamination (Saltzmann, 1982), it was also
196
Robert P. Kerr, Douglas G. Capone
noted that in some circumstances the lowest rates of mineralization were observed in sediments possessing the highest total PAH concentration. These findings are corroborative with the alternative hypothesis that sediment microbes in the lower Hudson River estuary could be stressed by PAH loading. If, in fact, a toxicological response does occur at some point, heavily contaminated sediments would become a proportionately larger sink for these contaminants. Most recently, Heitkamp et al. (1987) reported mineralization rates of from 1.7 to 2-7% day- 1 for a number of aquatic sediments which varied in their previous exposure to petroleum hydrocarbons. In summary, sediment microbial communities from each site within the Hudson River estuary and in Port Jefferson Harbor appeared adapted to the ambient salinity regime with respect to the mineralization of naphthalene and anthracene. Sediments from downstream areas exhibited the ability to mineralize these PAHs over a wider range of salinities than upstream sites. This is probably a result of the large, tidally mediated oscillation in salinity occurring in the lower reaches of the Hudson River. Rates of PAH mineralization appear to be primarily related to PAH concentrations but may be substantially modified by changes in salinity outside ranges normally experienced.
ACKNOWLEDGEMENTS At the the outset of the project, James Bauer provided generously of his time and experience and his contribution is acknowledged. Mark Bautista, Jennifer Slater, and Susan D u n h a m are also thanked for their assistance and comments. We are also indebted to Dr Bruce Deck for his help in the field. This work was supported by the Hudson River Foundation's grant 14-83B12 and 16-85B-6, Environmental Protection Agency Grant R809475011 and the Hudson River Foundation--National Estuarine Sanctuary Polgar Fellowship Program.
REFERENCES Atlas, R. M. (1981). Microbial degradation of petroleum hydrocarbons: An environmental perspective. Microbiol. Rev., 45, 180-209. Barnsley, E. A. (1983). Bacterial oxidation of naphthalene and phenanthrene. J. Bacteriol., 153, 1069-71. Bartholomew, G. W. & Pfaender, F. K. (1983)~ Influence of spatial variations on organic pollutant biodegradation rates in an estuarine environment. Appl. Environ. Microbiol., 45, 103-9.
Effect of salinity on mineralization of two poly
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Bauer, J. E. & Capone, D. G. (1985a). Effects of four aromatic organic pollutants on microbial glucose metabolism and thymidine incorporation in marine sediments. Appl. Environ. Microbiol., 49, 828-35. Bauer, J. E. & Capone, D. G. (1985b). Degradation and mineralization of the polycyclic aromatic hydrocarbons anthracene and naphthalene in intertidal marine sediments. Appl. Environ. Microbiol., 50, 81-90. Bauer, J. E. & Capone, D. G. (1988). Interactive effects of co-occurring aromatic hydrocarbons on the degradation of model polycyclic aromatic hydrocarbons in marine sediments. Appl. Environ. Microbiol., 54, 1649-55. Bauer, J., Kerr, R. P., Bautista, M. F., Decker, C. J. & Capone, D. G. (1988). Stimulation of microbial activities and polycyclic hydrocarbon degradation in marine sediments inhabited by Capitella capitata. Mar. Environ. Res., 25. 63-84. Bedding, N. D., McIntyre, A. E., Perry, R. & Lester, J. N. (1982). Organic contaminants in the aquatic environment I. Sources and occurrence. Sci. Tot. Environ., 32, 411-18. Bourquin, A. W. & Przybyszewski, V. A. (1977). Distribution of bacteria with nitrilotriacetate degrading potential in an estuarine environment. Appl. Environ. Microbiol., 34, 411 18. Capone, D. G. & Kiene, R. P. (1988). Comparison of microbial dynamics in freshwater and marine environments: Contrasts in anaerobic catabolism. Limnol. Oceanogr. Special Issue, 33, 725-49. Hambrick III, G. A., DeLaune, R. D. & Patrick Jr., W. H. (1980). Effect of estuarine sediment pH and oxidation-reduction potential on microbial hydrocarbon degradation. Appl. Environ. Microbiol., 40, 365-9. Heitkamp, M. A., Freeman, J. P. & Cerniglia, C. E. (1987). Naphthalene biodegradation in environmental microcosms: Estimates of degradation rates and characterization of metabolites. Appl. Environ. Microbiol, 53, 129-36. Herbes, S. E. & Schwall, L. R. (1978). Microbial transformation of polycyclic aromatic hydrocarbons in pristine and petroleum-contaminated sediments. Appl. Environ. Microbiol., 35, 306-16. Hunter, M., Stephenson, T., Kirk, P. W. W., Perry, R. & Lester, J. N. (1986). Effect of salinity gradients and heterotropic microbial activity on biodegradation of nitrilotriacetic acid in laboratory simulations of the estuarine environment. Appl. Environ. Microbiol., 51,919-25. Lee, R. F., Hinga, K. & Almquist, G. (1982). Fate of radiolabeled polycyclic aromatic hydrocarbons and pentachlorophenol in enclosed marine ecosystems. In Marine Mesocosms. ed. D. G. Grice & M. Reeve, Springer-Verlag, New York, NY, pp. 123-35. McDaniel, J. A. & Capone, D. G. (1985). A comparison of procedures for the separation of aquatic bacteria from sediments for subsequent direct enumeration. J. Microbiol. Meth., 3, 291-302. Massie, L. C., Ward, A. P. & Davies, J. M. (1985). The effects of oil exploration and production in the northern North Sea: Part 2--Microbial biodegradation of hydrocarbons in water and sediments, 1978-1981. Marine Environmental Research, 15, 235-62. MacLeod, W. D., Ramos, L. S., Friedman, A. J., Burrows, D. G., Prohaska, P. G., Fisher, D. L. & Brown, D. W. (1981). Analysis of residual chlorinated hydrocarbons, aromatic hydrocarbons and related compounds in selected
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sources, sinks and biota of the New York Bight. NOAA Technical Memorandum, OMPA-6. Boulder, Co. Mueller, J. A., Gerish, T. A. & Casey, M. C. (1982). Contaminant Inputs to the Hudson Raritan Estuary. NOAA Technical Memorandum OMPA-21. Boulder, Co. Olsen, S., Pilson C. Oviatt, M. E. Q. & Gearing, J. N. (1982). Ecological consequences of low, sustained concentrations of petroleum hydrocarbons in temperate estuaries. Marine Ecosystems Res. Lab. University of Rhode Island. 30 pp. Readman, J. W., Mantoura, R. F. C., Rhead, M. M. H. & Brown, L. (1982). Aquatic distribution and heterotrophic degradation of polycyclic aromatic hydrocarbons (PAH) in the Tamar Estuary. Estuarine, Coastal and Shelf Science., 14, 369-89. Saltzman, H. A. (1982). Biodegradation of aromatic hydrocarbons in marine sediments of three North Sea oil fields. Mar. Biol., 72, 17 26. Sokal, R. R. & Rohlf, F. J. (1981). Biometry. (2nd edn), W. H. Freeman and Co., San Francisco. Spain, J. C., Pritchard, P. H. & Bourquin, A. W. (1980). Effects of adaptation on biodegradation rates in sediment/water cores from estuarine and freshwater environments. Appl. Environ. MicrobioL, 49, 726-34. Spain, J. C. & Van Veld, P. A. (1983). Adaptation of natural microbial communities to degradation of xenobiotic compounds: Effects of concentration, exposure time, inoculum, and chemical structure. Appl. Environ. MicrobioL, 45, 428 35. Strickland, J. D. & Parsons, T. R. (1972). A Manual of Seawater Analysis. (2nd edn), Bull. Fish. Res. Bd. Can. p. 76. Van Vleet, E. S. & Quinn, J. G. (1978). Contribution of chronic petroleum inputs to Narragansett Bay and Rhode Island Sound sediments. J. Fish. Res. Board Can., 35, 536-43. Ward, D. M. & Brock, T. D. (1976). Environmental factors influencing the rate of hydrocarbon oxidation in temperate lakes. Appl. Environ. Microbiol., 31, 764-72.
The Effect of Salinity on the Microbial Mineralization of Two Polycyclic Aromatic Hydrocarbons in Estuarine Sediments Robert P. Kerr Cosper Environmental Services, Inc., PO Box 525, Northport, NY 11768, USA
& Douglas G. Capone* Center for Environmental and Estuarine Studies, Chesapeake Biological Laboratory. University of Maryland, Box 38, Solomons, MD 20688-0038, USA (Received 17 May 1988; revised version received 23 August 1988; accepted 29 August 1988)
ABSTRACT Sediments from the lower Hudson River estuary and two other coastal environments were examined experimentally for their ability to mineralize (convert to CO z) the polycyclic aromatic hydrocarbons ( PAHs) naphthalene and anthracene over a range of salinities. Routine assays employed 1:1 (vol fresh sed: vol water) sediment slurrys in order to overcome natural variability in mineralization rates among replicates. Mineralization rates were stimulated by about 2"5 fold, compared to unslurried controls, while the coefficient of variation fell from 13% to 3"5%. Rates of naphthalene mineralization in surface sediments from along the mainstem of the Hudson River (salinities from 2 to 27%0) ranged from 0.011 to 1.5nmolcm 3day x (pool turnover I T , ] f r o m 60 to 2040 days) with no discernible trends along the estuarine gradient. For two stations examined experimentally (mile point 5, salinity 23%0; mile point 26, salinity 5%0), microbial assemblages appeared acclimated to broad salinity variations as * To whom correspondence should be addressed. 181
Marine Environ. Res. 0141-1136/89/$03.50 ~ 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain
182
Robert P. Kerr, Douglas G. Capone imposed increases or decreases in salinity did not signi~'cantly alter rates g[" mineralization compared to controls. Sediments from two upstream marshes of the Hudson (mile points 36 and 45) showed rates of naphthalene mineralization from 0.007 to 0"15 nmol cm- 3 day 1 ( T , from 14 to 368 days), while sediments from a third marsh in freshwater ( mile point 76) had high rates (66 nmol cm- 3 day 1; 7", 40 days). For the two upstream marsh stations which rarely experienced salt intrusion, there was a substantial decrease in mineralization of naphthalene and anthracene with increasing salinity. Consistently high rates of naphthalene mineralization (780 to 1600 nmol cm 3day 1; 7", 5 to 6 days) were observed in petroleum contaminated sediments from Port Jefferson Harbor (PJH) on the north shore of Long Island. P J H has a relatively constant salinity regime (about 27%0) and imposed decreases in salinity effected decreases in rates of naphthalene and anthracene mineralization. Lowest rates of naphthalene mineralization 0"003 to 0"004 nmol cm - 3 day- 1; T, from 714 days to 833 days) were Jbund m sediments ,from two stations in the relatively pristine Carmans River estuao' on the south shore of Long Island, The abili O, oJ"increases or decreases in salinity to affect the rate o[model PA H mineralization appeared to be dependent on the natural variation in the salinity regime from which a sample was obtained. Data from all the environments studied indicated a strong positive correlation between PA H concentration and the rates of mineralization of naphthalene. Rates of PA H mineralization in all environments examined appear to be primarily controlled by the extent of pollutant loading and not by natural variations in the salinity regime.
INTRODUCTION Aquatic environments receive polycyclic aromatic hydrocarbons (PAHs) from many sources including direct discharge, sewage outfall, urban and agricultural run-off as well as groundwater flow (Van Vleet & Quinn, 1978; Bedding et al., 1982). Because of their relative insolubility, PAHs readily adsorb to particulate matter and become concentrated in sediments at levels many times those found in the water column (MacLeod et al., 1981; Mueller et al., 1982; Olsen et al., 1982). It has been shown that microbes in sediments from chronically polluted environments can metabolize PAHs at significant rates (Herbes & Schwall, 1978; Atlas, 1981; Saltzmann, 1982; Spain & Van Veld, 1983; Heitkamp et al. 1987; Bauer et al. 1988). Determining the rates at which these refractory compounds are removed by indigenous microbes is fundamental in assessing their persistence and effects on benthic communities. Numerous environmental factors (e.g. oxygen, nutrient availability,
Effect of salinity on mineralization of two poly~Tclic aromatic hydrocarbons
183
temperature and pH) are known to affect the activities of sediment microbes including their ability to degrade PAHs and other xenobiotics (Ward & Brock, 1976; Hambrick et al., 1980; Atlas, 1981). Estuaries are characterized by a variety of gradients which affect and define the nature of the microbiota within the estuary. Major inorganic ion composition, as well as the quality and quantity of organic matter, vary through the estuary in a systematic and microbiologically relevant way (Capone & Kiene, 1988). In the water column of estuaries, salinity, or some factor covarying with salinity, has been identified as an important factor regulating microbial activities, including the mineralization of xenobiotics. The degradation of mcresol, chlorobenzene and 1,2,4-trichlorobenzene was shown to be drastically inhibited as salinity increased through an estuary (Bartholomew & Pfaender, 1983). Degradation of" the synthetic detergent nitrilotriacetate (NTA) was inhibited in the water column of two estuaries at brackish and marine salinities (Bourquin & Przybyszewski, 1977; Hunter et al., 1986). Similarly, p-nitrophenol degradation in the water of sediment water microcosms was inhibited at marine salinities (Spain et al., 1980). While PAHs are known to be microbially mineralized (converted to CO2) in fresh water, brackish and marine sediments (Herbes & Schwall, 1978; Readman el al., 1982; Lee et al., 1982; Massie et al., 1985), the effect of an estuary's salinity gradient on PAH mineralization in sediments is not well defined. The objective of this study was to determine the ability of fresh, brackish and salt water sediments of the Hudson River Estuary and two other sites on Long Island to mineralize two low molecular weight PAHs (naphthalene and anthracene), and to examine experimentally the effect of imposed salinity variation on PAH mineralization at several of the sites.
MATERIALS AND METHODS All samples in the lower Hudson River were taken between June and October of 1984 and May and August of 1985. Site locations in 1984 spanned the estuary's entire salinity gradient. Sites included mile points (MP) (as defined by the US Army Corps of Engineers) 0 (southernmost tip of Manhattan), 5 (midtown Manhattan), 10 (George Washington Bridge), 26 (Tappan Zee Bridge), and 36 (Haverstraw Bay) (Fig. 1, Table 1). Samples were taken by boat from the subtidal banks along the edge of the mid channel of the river in waters of 5 to 15 m depth. Samples obtained in 1985 were from subtidal regions of marches adjacent to the Hudson. Marsh locations within the River are components of the Hudson River National Estuarine Sanctuary and include Piermont (adjacent to MP 36), Iona (MP 45) and Tivoli (MP 76) (Fig. 1, Table 2). Iona
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marsh is slightly brackish, while Tivoli marsh resides within the fresh water reach of the River. Other locations sampled during 1985 were: Port Jefferson H a r b o r (PJH), a small developed bay of relatively constant salinity with an oil fired power plant on the north shore of Long Island, New York; and Carmans River Estuary, a relatively pristine (with regard to petroleum contamination), eutrophic estuary on the south shore of Long Island. For the 1984 studies of the Hudson River, two gravity cores were taken at each site on each sampling date. Samples were kept in the dark and cool in a tall can with an ice-water mixture and returned to the Marine Sciences
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Research Center (SUNY, Stony Brook, NY) where they were placed in their container in a controlled environment room (20°C) overnight until experiments commenced the next day. Salinity of both the overlying water and surface sediment pore water (top 1 cm) was determined with a hand held refractometer (Atago Co., Ltd). Rarely did the two differ by more than 2%0. Sediment samples were taken by removing 1 cm 3 subsamples from the top 1 cm or to the bottom of the brown (aerobic) zone, whichever was less, from two cores from each site. A total of 30 subsamples from the cores were combined and amended with 30 ml of 0.45/~m filtered ambient bottom water from the corresponding sample site. The slurry was magnetically stirred for 3 min at an intermediate speed to minimize cell disturbance. Subsamples (2 ml) were removed from the slurry, placed in 20 ml scintillation vials and capped with butyl rubber stoppers. These 1 : 1 diluted samples were used to estimate the in situ rates of microbial mineralization. From the remaining slurry, subsamples (5-10ml) were taken and diluted 10:1 (vol :vol) with either ambient water from that site or artificial sea water (31 g NaC1, 10 g MgSO4.7H20, 9"04 g NaHCO3 in one liter distilled water, Strickland & Parsons, 1972) diluted to various salinities (0-30%o). From these diluted slurries, subsamples (2ml) were also removed, placed in scintillation vials and treated with either of the two radiolabeled PAHs. Experimental treatments were prepared in triplicate while killed controls (autoclaved for 20 minutes) were run in duplicate. All samples were incubated in the dark and at the in situ temperature. In situ temperatures ranged from 20°C (June, Oct) to 26°C (August). Rotary shaking during incubation ensured rapid equilibration between gas and liquid phases and dispersal of pollutants. On several occasions, in order to assess the effect of dilution on mineralization, comparisons were made among sediment diluted by various degrees with sediment which had been neither unslurried or diluted. [l,(4,5,8)-14C]-Naphthalene (5mCi/mmol) and E9(10)-14C] anthracene (5mCi/mmol), obtained from Amersham Co., were diluted with acetone to yield a working solution with a final concentration of 2000-300011M. Radiolabeled compounds were added to samples through the stopper using a microliter syringe (10pl injection). The final concentration of acetone ( < 0 ' 2 % vol:vol) did not interfere with several measures of sediment metabolism (Bauer & Capone, 1985a). The addition of naphthalene and anthracene added to sediments (about 200 ppb) was not inhibitory to either thymidine incorporation or glucose metabolism in pristine marine sediments (Bauer & Capone, 1985a). At selected intervals during experiments, the headspace of assay vials were flushed with compressed air, and newly evolved 14COz was trapped in vials with 10ml of Oxosol (National Diagnostics). Radioactivity was
188
Robert P. Kerr, Douglas G. Capone
quantified on a Packard Tri-Carb 300C liquid scintillation counter. The same 14CO1 trapping vial was reused for sequential, cumulative trapping of the headspace of its respective assay vial for the duration of an experiment, counting each trapping vial after each trapping (Bauer & Capone, 1985b). Previous studies had shown that extended flushing of the headspace was effective in removing recently produced 14CO2 (Bauer & Capone, 1985b). Only small additional amounts of 14CO2 (< 5% of total) were obtained by acidification (Bauer & Capone, 1985b). Net ~4CO2 production for each time point was calculated by subtracting 14C flushed from control vials and trapped. This would include 14CO2 contamination in stocks as well as any ~4C-naphthalene volatilized into the flask headspace. Naphthalene volatilization in controls over the whole time course (9 days) never accounted for more than 1"3% of the total injected. Rates of mineralization were determined from linear portions of ~4CO2 production curves. Any lag period was recorded. Analysis of sediments for the presence of ambient naphthalene, phenanthrene and anthracene was conducted using a Waters' high-pressure liquid chromatograph (HPLC) equipped with an Adsorbosphere (Applied Science) C18 (5pm mesh size) column (15cm length) and detected on a Waters model 400 absorbance detector (254 nm). Sample preparation and flow conditions were identical to those of Bauer & Capone (1985b). Retention times for naphthalene, phenanthrene and anthracene were about 2.4, 4.2 and 4.7 min, respectively. The level of detection was about 100ng (gwetsed) 1 Turnover times of naphthalene and anthracene were calculated by using the inverse of the rate of 14CO2 evolution. This procedure was identical to that used by Readman e t al. (1982), which found 14CO2 measurements more reproducible than those based on residual hydrocarbon and polar metabolite measurements. Absolute rates (Va) of mineralization (nmol c m - 3 day-1) were calculated for all sites where ambient concentrations of the substrate were obtained. Absolute rates were calculated by using the relative rate (V,) of mineralization (fraction of label mineralized) divided by the fraction (F) of radiolabeled naphthalene added to the ambient concentration: V~ = V , / F = V, x
(ambient naphthalene) ([ 14C] naphthalene)
The effects of salinity on the rate of mineralization of PAHs within a specific environment were statistically analyzed by single class analysis of variance and unplanned mean comparison (Sokal & Rohlf, 1981). Organic carbon determinations were performed by ashing at 450°C. Bacterial numbers were enumerated by the method of McDaniel & Capone (1985).
E~fect of salinity on mineralization of two polycyclic aromatic hydrocarbons
189
RESULTS
Naphthalene mineralization Sites used in this paper can be broadly categorized as either fresh water (Tivoli Bay and upper Carmans River), brackish (Iona and Piermont marshes, mile points 10, 26 and 36), or marine (Port Jefferson Harbor, lower Carmans River, MP 0 and 5). Ambient sediment pore water salinities for all sites are listed in Tables 1 and 2. Measurable levels of naphthalene were detected in sediments from all environments except Carmans River, Piermont and Iona (Tables 1 and 2). Highest concentrations of naphthalene were found in sediments from both MP 5 and Port Jefferson Harbor (PJH). Concentrations of naphthalene in sediments from the subtidal banks (1984 sites) of the Hudson River generally decreased up-river (away from midtown Manhattan). Samples for PAH analysis taken from the marshes along the Hudson did not show any consistent trend. Interestingly, phenanthrene, or a compound co-eluting with phenanthrene (such as retene) was the only PAH consistently detected at Piermont and Iona. For samples taken in 1984 along the edges of the main channel of the Hudson, there appeared to be no consistent trend in naphthalene mineralization rates along the estuarine gradient (Table 1). Turnover times (Tn) of naphthalene along the mainstem of the lower Hudson River varied substantially both among and within sites, ranging between 60 and 2040 days at the four upstream sites with an extreme value of 9100 days for the one sampling at the most seaward site. Absolute rates of naphthalene mineralization ranged from 0.011 to 1-5 nmol c m - 3 day- 1. Turnover times of naphthalene did not significantly correlate with salinity (r 2 = 0"40) or microbial numbers by direct counts (r 2 = 0" 12). For the six samples for which concentration of naphthalene, anthracene and phenanthrene were available, there was no apparent correlation. However, this included samples for three sites on one day (6 Sep.) on which naphthalene mineralization rates were unusually low and may not be representative (see Discussion). Turnover time was strongly correlated with organic content (r 2 = 0-72). Turnover times appeared less variable among and within sites along the subtidal marsh banks of the Hudson River, ranging from 14 to 368 days (Table 2). While absolute rates of mineralization were quite low (0.007 to 0.15 nmol cm -3 day -1) at the marshes at MP 36 and 45, naphthalene mineralization rates at Tivoli marsh (MP 76) were 66 nmol cm - 3 day- 1. For the 1985 data, turnover times (T,) of naphthalene were negatively correlated with the summed concentration of naphthalene, phenanthrene and anthracene (NPA) (Fig. 2) found at each site: that is, sediments containing
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higher concentrations of N P A had elevated rates of n a p h t h a l e n e mineralization (shorter T,). Anthracene mineralization was examined on three occasions and turnover times were of the same order as parallel naphthalene mineralization studies at each site (Table 2). The extent of any period of low or minimal rate (lag) before a higher steady rate of naphthalene mineralization varied a m o n g stations. For PJH sediments, high initial rates of mineralization without a lag period were observed on all three occasions. Similarly, for three out of four samplings at MP 5 (exception Sep. 6), there were high rates with no apparent lag period. For all sites, the extent of any lag period before rates of naphthalene mineralization became constant, appeared positively correlated with turnover time (Fig. 3). Several comparisons of the effect of dilution were made with sediments from Port Jefferson Harbor (Table 3). Slurring consistently stimulated mineralization rates while lowering the variation a m o n g replicates. The greatest stimulation occurred between unslurried samples and 1 : 1 dilution, with greater dilutions having lesser effect.
Effects of salinity change The effect of experimentally imposed salinity changes on the rate of mineralization of naphthalene and anthracene was examined in sediments from several of our sites using additions of artificial sea water adjusted to
Effect o f salinity on mineralization o f two polycyclic aromatic hydrocarbons
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specific salinities. For two sites along the mainstem of the tidally dominated lower H u d s o n River (MPs 5 and 26), there was no statistically significant effect (P < 0.05) of either increase or decreases of salinity on the rate of mineralization of naphthalene (Fig. 4). In both cases, sediments were subjected to salinities between 4 and 31%o and monitored for over two weeks. A second experiment at M P 5 (3 Oct., 27%0) revealed no difference for treatments from 13 to 36%0 with a 35% decrease in mineralization at the lowest salinity used (9%0). Sediments from the three most upstream sites in the H u d s o n (Tivoli Bay, Iona Marsh and M P 36) all responded to salinity changes in the same TABLE 3 Effect of Dilution on Mineralization of Naphthalene in Sediments from Port Jefferson Harbor. Values are Cumulative A m o u n t Mineralized, Normalized per cm 3 of the Undiluted Sediment
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manner, but by varying degrees. Sediments from Tivoli Bay (ambient salinity 0%0) were significantly (P < 0-01) inhibited in their ability to mineralize naphthalene by salinities of 15%0 (Fig. 5). Mineralization at this fresh water site completely ceased at 30%0. Naphthalene mineralization rates for sediments from Iona Marsh were also significantly reduced (P _<0-05) at a salinity of 15%o compared to treatments receiving 0%0 water (Fig. 5). Mineralization was further reduced at 30%0, but did not cease. Anthracene 1.4
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mineralization was also significantly reduced (P < 0.01) at 30%o, but not at 15%o. Sediments from the mainstem station at MP 36 during one experiment (ambient salinity 2-3%0) tolerated salinities of 10%o, but were also significantly inhibited (P<0.01) by salinities of 15%o (data not shown). Sediments from MP 36 used in a subsequent experiment (Oct. 1984), however, tolerated all salinities between 0 and 25%0 with no significant inhibition. On this date, pore water salinity had increased to 7%0 (Table 1). Port Jefferson Harbor, located adjacent to mid Long Island Sound, has a relatively constant salinity regime around 27 to 28%o. The effect of salinity on rates of microbial mineralization of naphthalene and anthracene for PJH sediments is shown in Fig. 6. In all cases, rates were greatest in sediments treated with artificial seawater of either 30 or 15%o (no significant difference). However, rates were significantly reduced (P < 0.01) by adjusting the sediment salinity to 0%0.
DISCUSSION The effect of large, instantaneous salinity changes on PAH mineralization in surficial sediments depended on the ambient salinity regime. In the lower reaches of the Hudson estuary (MP 5 and 26) and as far north as mile point 36, large salinity changes did not inhibit the mineralization of naphthalene by sediment microbes. This is probably a result of adaptation by downstream populations of sediment microbes to large daily changes in salinity, resulting from the large tidal oscillation occurring within the lower
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reaches of the estuary. Hourly salinity measurements taken in the main channel near MP 10 between September and October 1980 (National Ocean Services, New York Harbor Circulatory Survey 1980-81, Rockville, MD) found that the salinity of surface and bottom waters normally varies diurnally between 16 and 24%o and can change by as much as 16%oin a single day as a result of passing storm fronts. Seasonal fluctuations in river flow accentuate salinity excursions even further. While sediments from PJH are exposed to roughly the same mean salinity as that for MP 5, they are not subjected to any significant change in salinity either during a tidal cycle or seasonally. The lack of large oscillations in salinity at PJH appears to account for a microbial population poorly adapted to lower salinities and inhibited in naphthalene and anthracene mineralization at salinities of 15 and 0%0. Sediments progressively further upstream in the Hudson River displayed maximum rates of naphthalene mineralization at lower salinities and also within a narrower range of salinities. Fresh water (Tivoli Bay) and slightly brackish (Iona and MP 36) sediments were significantly inhibited by increases in salinity to 15%o. This may reflect adaptation of the microbial population to lower ambient salinity and a narrower range of salinities experienced in these upstream stations. Sediments from MP 36, however, also displayed a tolerance to increases in salinity on one occasion (Sept. 1984, a characteristically low flow month; Mueller et al. 1982) when the sediment pore water salinity had increased to 7%0 from its earlier salinity of 2-3%o. This increase may be indicative of seasonal shifts in the salinity tolerance of indigenous microbial populations. Significant differences in rates of mineralization were observed in some experiments between samples incubated with artificial water compared to natural water at the ambient salinity. The low nutrient levels in the artificial sea water (0"5FtM NO 3 and 0"4#M NH~), and/or the absence of other required ions may explain why, as was most often the case, sediments incubated with artificial sea water had somewhat lower rates of PAH mineralization than sediments incubated with natural sea water at the same salinity. High in situ rates of naphthalene mineralization in the Hudson River estuary did not appear to be associated with any particular salinity zone, but generally occurred at stations having the greatest naphthalene and/or phenanthrene concentration (Fig. 2). Interestingly, sediments from Iona and Piermont both mineralized naphthalene quickly, yet were devoid of measurable naphthalene (Table 2). Both of these environments, however, contained substantial quantities of phenanthrene, indirectly supporting studies (Barnsley, 1983; Bauer & Capone, 1988) which showed that soil and sediment bacteria could catabolize both anthracene and naphthalene after
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pre-exposure to phenanthrene. Alternatively, microbes from Iona and Piermont marshes may have been previously exposed to naphthalene. On one date (6 Sept. 1984), the turnover time of naphthalene in the lower Hudson River was unexpectedly low given the levels of naphthalene and phenanthrene detected. Turnover times of naphthalene in sediments from MP 0, 5, and 10 were approximately 9, 1.3 and 3 times longer, respectively, than for the pristine sediments from Carmans River (about 2 years). The very low rates of mineralization of naphthalene on this occasion might have been the result of several factors. Early September is the period of highest temperatures and lowest river flows, conditions which lend themselves to bottom water anoxia. Rates of naphthalene mineralization under anoxic conditions are extremely low (Bauer & Capone 1985b) and anoxia either at the time of sampling or during the immediately preceding period may have impaired the capacity of the sediment surface microbiota to mineralize naphthalene. Unfortunately, we did not collect water chemistry information during coring cruises. However, water chemistry data obtained from the NY Dept. of Environmental Protection for cruise on 4 Sept. indicated strong stratification and near bottom waters with about 45% of saturation 02 values (T. Brosnan, pers. comm.). Hence, it is possible that low O 2 conditions may have been developing and could have affected naphthalene mineralization rates for samples collected on 6 Sep. Alternatively, toxicological stress may have accounted for the lessened ability to utilize [14C]naphthalene. The summed concentration of NPA in sediments from the lower Hudson comprises only 10-40% of the total PAH pool and a yet smaller fraction of the persistent organic contaminant pool (MacLeod et al., 1981). The summed and possibly synergistic effect of part per million concentrations for each of the numerous PAHs present may inhibit microbial mineralization of naphthalene as well as other processes. However, turnover times at the same sites on other dates as well as in PJH sediments (an environment containing NPA at concentrations comparable to those of the lower Hudson) were often orders of magnitude shorter than on this occasion. The rate of mineralization of naphthalene in another chronically polluted aquatic environment having similar levels of naphthalene contamination (7/~g g dry sed -1) was 190nmol cm -3 day -1 (Herbes & Schwall, 1978). These findings agree well with those for the PJH station, and again are significantly greater than those of the lower Hudson. Sediments around oil drilling platforms in the North Sea (Massie et al., 1985; Saltzmann, 1982) which were contaminated with naphthalene by as much as 7/~g g dry sed- 1, produced rates of mineralization of naphthalene that varied between zero and 2"4 nmol c m - 3 d a y - 1. While in general, high rates appeared positively correlated with the degree of contamination (Saltzmann, 1982), it was also
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noted that in some circumstances the lowest rates of mineralization were observed in sediments possessing the highest total PAH concentration. These findings are corroborative with the alternative hypothesis that sediment microbes in the lower Hudson River estuary could be stressed by PAH loading. If, in fact, a toxicological response does occur at some point, heavily contaminated sediments would become a proportionately larger sink for these contaminants. Most recently, Heitkamp et al. (1987) reported mineralization rates of from 1.7 to 2-7% day- 1 for a number of aquatic sediments which varied in their previous exposure to petroleum hydrocarbons. In summary, sediment microbial communities from each site within the Hudson River estuary and in Port Jefferson Harbor appeared adapted to the ambient salinity regime with respect to the mineralization of naphthalene and anthracene. Sediments from downstream areas exhibited the ability to mineralize these PAHs over a wider range of salinities than upstream sites. This is probably a result of the large, tidally mediated oscillation in salinity occurring in the lower reaches of the Hudson River. Rates of PAH mineralization appear to be primarily related to PAH concentrations but may be substantially modified by changes in salinity outside ranges normally experienced.
ACKNOWLEDGEMENTS At the the outset of the project, James Bauer provided generously of his time and experience and his contribution is acknowledged. Mark Bautista, Jennifer Slater, and Susan D u n h a m are also thanked for their assistance and comments. We are also indebted to Dr Bruce Deck for his help in the field. This work was supported by the Hudson River Foundation's grant 14-83B12 and 16-85B-6, Environmental Protection Agency Grant R809475011 and the Hudson River Foundation--National Estuarine Sanctuary Polgar Fellowship Program.
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Bauer, J. E. & Capone, D. G. (1985a). Effects of four aromatic organic pollutants on microbial glucose metabolism and thymidine incorporation in marine sediments. Appl. Environ. Microbiol., 49, 828-35. Bauer, J. E. & Capone, D. G. (1985b). Degradation and mineralization of the polycyclic aromatic hydrocarbons anthracene and naphthalene in intertidal marine sediments. Appl. Environ. Microbiol., 50, 81-90. Bauer, J. E. & Capone, D. G. (1988). Interactive effects of co-occurring aromatic hydrocarbons on the degradation of model polycyclic aromatic hydrocarbons in marine sediments. Appl. Environ. Microbiol., 54, 1649-55. Bauer, J., Kerr, R. P., Bautista, M. F., Decker, C. J. & Capone, D. G. (1988). Stimulation of microbial activities and polycyclic hydrocarbon degradation in marine sediments inhabited by Capitella capitata. Mar. Environ. Res., 25. 63-84. Bedding, N. D., McIntyre, A. E., Perry, R. & Lester, J. N. (1982). Organic contaminants in the aquatic environment I. Sources and occurrence. Sci. Tot. Environ., 32, 411-18. Bourquin, A. W. & Przybyszewski, V. A. (1977). Distribution of bacteria with nitrilotriacetate degrading potential in an estuarine environment. Appl. Environ. Microbiol., 34, 411 18. Capone, D. G. & Kiene, R. P. (1988). Comparison of microbial dynamics in freshwater and marine environments: Contrasts in anaerobic catabolism. Limnol. Oceanogr. Special Issue, 33, 725-49. Hambrick III, G. A., DeLaune, R. D. & Patrick Jr., W. H. (1980). Effect of estuarine sediment pH and oxidation-reduction potential on microbial hydrocarbon degradation. Appl. Environ. Microbiol., 40, 365-9. Heitkamp, M. A., Freeman, J. P. & Cerniglia, C. E. (1987). Naphthalene biodegradation in environmental microcosms: Estimates of degradation rates and characterization of metabolites. Appl. Environ. Microbiol, 53, 129-36. Herbes, S. E. & Schwall, L. R. (1978). Microbial transformation of polycyclic aromatic hydrocarbons in pristine and petroleum-contaminated sediments. Appl. Environ. Microbiol., 35, 306-16. Hunter, M., Stephenson, T., Kirk, P. W. W., Perry, R. & Lester, J. N. (1986). Effect of salinity gradients and heterotropic microbial activity on biodegradation of nitrilotriacetic acid in laboratory simulations of the estuarine environment. Appl. Environ. Microbiol., 51,919-25. Lee, R. F., Hinga, K. & Almquist, G. (1982). Fate of radiolabeled polycyclic aromatic hydrocarbons and pentachlorophenol in enclosed marine ecosystems. In Marine Mesocosms. ed. D. G. Grice & M. Reeve, Springer-Verlag, New York, NY, pp. 123-35. McDaniel, J. A. & Capone, D. G. (1985). A comparison of procedures for the separation of aquatic bacteria from sediments for subsequent direct enumeration. J. Microbiol. Meth., 3, 291-302. Massie, L. C., Ward, A. P. & Davies, J. M. (1985). The effects of oil exploration and production in the northern North Sea: Part 2--Microbial biodegradation of hydrocarbons in water and sediments, 1978-1981. Marine Environmental Research, 15, 235-62. MacLeod, W. D., Ramos, L. S., Friedman, A. J., Burrows, D. G., Prohaska, P. G., Fisher, D. L. & Brown, D. W. (1981). Analysis of residual chlorinated hydrocarbons, aromatic hydrocarbons and related compounds in selected
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