BIOAVAILABILITY OF PETROLEUM HYDROCARBONS FROM WATER, SEDIMENTS, AND DETRITUS TO THE MARINE , ARENACEODENTATA

Steven S. Rossi Marine Biology Research Division Scripps Institution of Oceanography Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1977/1/621/2349813/2169-3358-1977-1-621.pdf by guest on 29 September 2021 La Jolla, California 92093

ABSTRACT Methods

Uptake, retention, metabolism, and depuration of diaromatic hydrocar- All experiments were performed with immature young adult bons by the , Neanthes arenaceodentata, were examined in from a laboratory population of Neanthes arenaceodentata which was experiments utilizing seawater solutions and sediments contaminated with originally cultured by D. J. Reish, California State University, Long Beach. either No. 2 fuel oil water-soluble fractions or radio-labelled naphthalenes. Equal numbers of males and females were used, and all work was conducted Polychaetes rapidly accumulate 14C'-naphthalene (magnification factor at 21 ±2°C, with artificial seawater (Instant Ocean), adjusted to a salinity of = 40X) from solution during short-term exposure (24 hr). Worms slowly 32 ppt (7oo). released hydrocarbons accumulated during acute exposure down to unde- In experiments on the uptake, release, and metabolism of PHCs in tectable levels (<0.05ppm) within 300 hours after return to clean seawater. solution, male-female pairs were exposed for 24 hours to seawater contain- x 4 14C-naphthalene accumulated from solution was metabolized by ing 0.15 ppm C-naphthalene (specific activity = 1.72 mCi/mM) in air-free polychaetes, and associated microflora apparently play no role in uptake, containers. Following exposure, worms were transferred to hydrocarbon release, or metabolism. Analyses of worms held for 28 days in clay-silt (radioactivity) free seawater in aerated 1 liter bowls. At selected intervals sediments artificially contaminated with No. 2 fuel oil (9 ßg total during the latter period (depuration), pairs of worms were placed in air-free naphthalenes Ig wet sediment) indicate that naphthalenes were not accumu- containers for 24 hours to monitor the release of radioactivity from exposed lated by worms at tissue concentrations above 0.1 ppm. Polychaetes into seawater. Tissue samples were taken periodically throughout likewise failed to accumulate ^C-methylnaphthalene from ingestion of exposure and depuration for analysis of hydrocarbon content. Tissue and contaminated detritus (10-15 μ-g ^^C-methylnaphthalenelg dry detritus) for seawater samples were extracted with a known volume of high grade 16 consecutive days. These data suggest that petroleum hydrocarbons hexane, and radioactivity in the hexane and water extracts was determined in bound to sediment particles or paniculate organic matter are less available Aquasol-2 (New England Nuclear) on a Packard Tricarb liquid scintillation to marine worms than those in solution. spectrometer. Radioactivity extracted by hexane was assumed to be 14C- naphthalene, while that remaining in aqueous phases presumably rep- resented 14C-naphthalene-polar derivatives (metabolites). All data were corrected for background interference and quenching. The above experi- ments were performed both in the presence and absence of antibiotic agents (300 mg Penicillin G + 200 mg streptomycin sulphate + 50 mg Chloram- INTRODUCTION phenicol per liter of seawater). This mixture of antibiotic agents completely inhibits bacterial and fungal metabolism,4 permitting an assessment of the Members of several marine phyla concentrate petroleum hydrocarbons role which symbiotic microbes might play in the accumulation, metabolism, (PHCs) in their tissues when acutely exposed to seawater contaminated with and release of naphthalene by Neanthes. oil or specific hydrocarbons.3'27 In most cases, a majority of the accumulated PHC transfer from sediments to infauna (Neanthes) was studied by compounds are rapidly released when exposed animals are returned to clean exposing worms to sediment which had been previously contaminated seawater. Molluscs appear to be an exception to this trend, since depuration (artificially) by No. 2 fuel oil water-soluble fraction, prepared as described of PHCs from mussels and oysters occurs somewhat more slowly than in by Anderson et al.2 At selected times during exposure, worms and sediment other marine .10'31 Some marine crustaceans8'1217 and fish19'23 can were analyzed for diaromatic hydrocarbons (naphthalenes) by ultraviolet biochemically alter (metabolize) accumulated PHCs, which may play a role spectrophotometry.22 Polychaetes were exposed in 1-liter culture dishes in their depuration from, and possibly detoxification with, body tissues. containing 600 ml flowing (15 ml/min) seawater and 80 g contaminated 918 Molluscs apparently lack the ability to metabolize PHCs. Dynamics of sediment (3 parts clay-silt mud: 1 part powdered alfalfa) for 28 days. soluble PHC uptake and release by polychaetous , which are ecolog- Neanthes burrowed within and actively ingested sediment throughout the ically important in most bottom communities, have not been studied. In exposure period. The initial concentration of naphthalenes in sediments was addition, the role of PHC metabolism in members of this important marine approximately 9 ppm (9 μ-g total naphthalenes/ g fresh weight sediment). 16 phylum has only recently been investigated. To examine the feasibility of PHC contamination from ingestion of Crustacean zooplankters1115 andbenthic decapods1217 accumulate, depu- contaminated detritus (particulate organic matter), young adult Neanthes rate, and metabolize a broad variety of PHCs originally introduced through were offered detritus (powdered alfalfa) previously contaminated with 10-15 the diet. Studies on the bioavailability of PHCs in the diet of other important ppm * 4C-2-methylnaphthalene (specific activity = 7.98 mCi/mM), for 16 marine organisms have not been made. While some work has concerned days in succession. Worms were offered 5 mg of contaminated alfalfa daily itself with the transfer of pesticides in sediments to benthic (bottom- in individual culture dishes containing 50 ml seawater. After each 24-hour dwelling) species,21'25 no such work has been performed using PHCs. Thus, feeding period, some worms were transferred to clean feeding chambers for an examination of dynamics describing PHC contamination of marine or- continued feeding, others were sacrificed for analysis of hydrocarbon con- ganisms from food or sediments, particularly among burrowing species (e.g. tent (radioactivity), while others were fed powdered Tetramin for 24 hours polychaetes), merited further attention. before analysis of hydrocarbon content. After feeding on Tetramin for 24

621 622 1977 OIL SPILL CONFERENCE hours, worms had voided their guts of all alfalfa detritus, as determined by Table 1. Radioactivity in seawater containing pairs of polychaetes visual examination. Culture dish seawater, fecal pellets, and alfalfa were for selected 24 hour intervals during depuration, following acute ex- also monitored for radioactivity, both before and after daily feeding. posure to seawater solutions of l^C-naphthalene; total radioactivity Radioactivity was extracted with hexane, and determined as described equals that amount extracted by hexane and aqueous radioactivity earlier. not ex tractable with hexane (metabolized radioactivity); mean values for 8 samples are given; standard deviations were generally ± 20 cpm/ml Results Metabolized 14 Total radioactivity Kinetics of C-naphthalene uptake and release by both control and Depuration interval radioactivity released released treated (with antibiotic mixture—ABM) Neanthes are seen in Figure 1. Both (hours) groups accumulated and released 14C-naphthalene in nearly identical fash- (cpm/ml) (% of total) ion, and the quantitative role of metabolism appeared similar in both groups. Control ABM Control ABM

14 Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1977/1/621/2349813/2169-3358-1977-1-621.pdf by guest on 29 September 2021 Uptake from solution was rapid, with close to maximal (i.e., 6 /xg C- 2 2 naphthalene/g tissue) accumulation observed after only three hours of expo- 0-24 6 X 10 4.8 X 10 60 69 sure. There was no trend toward a net decrease in tissue concentrations 24-48 5.5 X 102 4 X 102 78 71 during the latter stages of exposure, as has been observed in work with other 1 1 marine organisms.3 Neanthes did not metabolize significant quantities of 72-96 2 X 10 1.5 X 10 80 82 14 C-naphthalene during exposure. 168-192 40 25 100 100 When returned to clean seawater, both groups slowly released accumu- lated 14C-naphthalene, and depuration of native x 4C-naphthalene was com- plete (<0.05 ppm in tissues) within 300 hours. Polychaetes treated with ABM depurated 14C-naphthalene in much the same manner as did untreated (control) worms. Thus, bacteria or fungi do not appear to play any part in the uptake, metabolism, and depuration of 14C-naphthalene by Neanthes. Data from the analysis of radioactivity released by worms during depuration are presented in Table 1. Both groups released radioactivity in the un- metabolized (native), as well as metabolized, form. Approximately one- / third of the radioactivity released by both groups during their first 24 hours in SEDIMENTS clean seawater was in the native form. The relative percentages of radioac- tivity released during depuration closely paralleled those which simulta- neously occurred within tissues. Specifically, concentrations of hexane- b o.e extractable radioactivity in tissues approached undetected levels at the same o: 0.6 time detectable levels were no longer found in external media. The presence of significant levels of metabolized radioactivity in worm tissues sampled \- UJ. 504 hours (21 days) after exposure suggests the possibility of complex O 0.2 14 2 incorporation of C-naphthalene (or its derivatives) into stable biochemical O pathways.

Concentrations of total naphthalenes in sediment used to expose Neanthes TISSUE for 28 days are graphically presented in Figure 2. Naphthalenes initially 0 \ present in quantities near 9 ppm declined to concentrations less than 3 ppm < ·' — ×··×· X ×· · during the exposure period. Decreases were presumably due to combina- tions of microbial degradation, photo-oxidation, and volatilization of TIME (DAYS)

Figure 2. Concentration of total naphthalenes (naphthalene + methyl- naphthalenes + dimethylnaphthalenes) in aritificially-contaminated Á,Δ ABM-TREATED sediment used to expose polychaetes for 28 days; mean values for 4 •,Ο CONTROL samples ± S.D. (vertical bars) are presented; total naphthalenes in M worms were below the level of detection (0.1 ppm) throughout the .* 4 + experiment

naphthalenes bound to sediment particles.29 Periodic water sampling failed to detect measurable concentrations (>0.01 ppm total naphthalenes) of diaromatic PHCs. Analyses of more than 20 replicate samples of Neanthes tissues, taken periodically throughout the exposure period, showed that polychaetes contained less than 0.1 ppm total naphthalenes at all times. Results concerned with the feasibility of PHC transfer from food to Neanthes are given in Table 2 and Figure 3. As can be seen in Table 2 (row 2), polychaetes contained no radioactivity (following gut clearance) after 50 70 100 200 ingestion of contaminated food for 16 consecutive days. Radioactivity DEPURATION present in worms prior to gut clearance represented that amount still bound to TIME (HOURS) alfalfa within the digestive tract of animals analyzed after 192 and 384 hours of feeding (row 1). Hexane-extractable radioactivity in fecal pellets gradu- ally increased in direct relation to total (accumulative) feeding time. Figure 1. Radioactivity in ABM-treated and untreated polychaetes Throughout the experiment, all of the alfalfa introduced daily had been during, and after placement in clean seawater following acute expo- consumed by the end of each 24-hour period. These data indicate a corres- sure to seawater containing 14C-naphthalene; open symbols depict ponding decrease in the efficiency of digestion, which was expected as the hexane-extractable radioactivity, dark symbols indicate total extract- nutritional state of starved worms improved with increasing cumulative able radioactivity; each symbol represents the mean value of 4 sam- feeding time. Consequently, fecal pellets sampled later on in the experiment ples ± S.D. (vertical bars); samples were composed of one male and represented alfalfa which had not been as thoroughly digested as those one female worm amounts consumed earlier (immediately following starvation). OIL SPILL BEHAVIOR AND EFFECTS 623

Figure 3 characterizes the fate of radioactivity within feeding chambers 4 for selected intervals during the experiment. Approximately 10 total counts 100 were introduced to chambers, in the form of * 4C-2-methylnaphthalene bound to powdered alfalfa. A large majority of this radioactivity (>90%) 80 was found in chamber seawater after each 24-hour interval. As the nutri- tional condition of Neanthes improved with continued feeding, radioactivity in expelled fecal pellets, as well as digestive tracts, increased. Increased 60 L recovery of 14C-2-methylnaphthalene in fecal pellet and tissue (gut) frac- tions was paralleled by decreasing concentrations in seawater. Subsequent analyses demonstrated that >85% ofthat radioactivity recovered in seawa- 40 L ter was in the hexane extractable (unmetabolized) form. These results offer o further evidence that polychaetes may not effectively accumulate diaromatic LU PHCs from contaminated sediments. O O O Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1977/1/621/2349813/2169-3358-1977-1-621.pdf by guest on 29 September 2021 oc 20 L Discussion

The abundance of naphthalene and its alkyl derivatives in all crude and < refined oils is justification enough for studying its biological effects and fate.2,24 These water soluble diaromatic hydrocarbons (fused benzene rings; MW — 150) are toxic to most marine organisms in the low (0.1 - 1) ppm >- range.3'28'32 As such, they are believed to account for a majority of the > toxicity elicited by a diverse range of petroleum products. Furthermore, I- Armstrong (personal communication) has monitored changes in populations of benthic invertebrates (including several species of polychaetes) actually I inhabiting sediments in Trinity Bay, Texas containing 1 - 5 ppm TN (total

Table 1. Results of experiments on bioavailability of detritus-bound l^C-methylnaphthalene; starved Neanthes arenaceodentata were offered daily rations of 3-5 mg alfalfa pre-loaded with l^C-2-methyl- naphthalene (2 X 10^ counts/g =10 ppm), in individual feeding chambers containing 50 ml Millipore- filtered seawater; after selected 24 hr feeding periods, worm tissues, fecal pellets, and chamber seawater were analyzed for radioactivity; alfalfa in worm guts was voided by offering animalsTetramin for 24 hr.

Cumulative feeding times 24 H 48 H 96 H 192 H 384 H

Radioactivity in worm tissues 7 X 103 IX 104 before voiding (counts/g tissue) Radioactivity in worm tissues after voiding (counts/g tissue) Radioactivity in fecal pellets 2 X 103 6 X 104 IX 105 3 X 105 (counts/g feces) Radioactivity in seawater 1.8 XIO^ 1.9 X 102 1.8 X 102 1.5 X 102 1.1 X 102 (counts/ml) 624 1977 OIL SPILL CONFERENCE

Uptake of heavy metals from sediments is apparently a species-specific 2. Anderson, J. S., J. M. Neff, B. A. Cox, H. E. Tatem, and G. M. function among polychaetes. The unselective deposit feeder Arenicola Hightower, 1974. Characteristics of dispersions and water-soluble marina does not accumulate heavy metals upon ingestion of contaminated extracts of crude and refined oils and their toxicity to estuarine sediments.1 Nereis diversicolor on the other hand, does accumulate a broad crustaceans and fish. Marine Biology. v27, pp75-88 variety of heavy metals during prolonged inhabitation of contaminated 3. Anderson, J. W., J. M. Neff, B. A. Cox, H. E. Tatem, and G. M. sediments.5'6'7 It has not been clearly demonstrated whether the major mode Hightower, 1974. The effects of oil on estuarine animals: toxicity, of metal contamination in N. diversicolor occurs by ingestion of loaded uptake and depuration, respiration, in Pollution and Physiology of sediments or adsorption from metallic ion-rich interstitial water. If signifi- Marine Organisms. F. J. Vernberg and W. B. Vernberg (eds). cant concentrations of naphthalenes were present in interstitial water of Academic Press, New York. pp285-310 PHC-contaminated sediments studied here, they were not accumulated by 4. Anderson, J.W. and Stephens, G. C., 1969. Uptake of organic material polychaetes in concentrations greater than 0.1 ppm. by aquatic invertebrates VI. Role of epiflora in apparent uptake of Marine crustaceans can accumulate naphthalenes and other PHCs from glycine by marine crustaceans. Marine Biology. v4, pp243-249 artificially contaminated food.1217 Corner et al.11 indicated that PHCs 5. Bryan, G. W., and L. G. Hummerstone, 1971. Adaptation of the

introduced through the diet may be more difficult to depurate than those polychaete Nereis diversicolor to estuarine sediments containing Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1977/1/621/2349813/2169-3358-1977-1-621.pdf by guest on 29 September 2021 accumulated from solution. On the other, Lee etal.,17 found that blue crabs, high concentrations of heavy metals-I. General observations and Callinectes sapidus, rapidly depurate PHCs accumulated by ingestion of adaptation to copper. J. Mar. Biol. Assoc. U.K. v51, pp845-863 tainted oysters. The question of relative persistence of diet-introduced PHCs 6. Bryan, G. W. and L. G. Hummers tone, 1973. Adaptation of the within Neanthes is of little consequence since this polychaetous annelid polychaete Nereis diversicolor to estuarine sediments containing failed to accumulate methylnaphthalene by ingestion of contaminated de- high concentrations of zinc and cadmium. J. Mar. Biol. Assoc. U.K. tritus (Table 2). Clearly much more work is needed in this area. Specifically, v53, pp839-857 feasibility of contamination from tainted food among representatives of all 7. Bryan, G. W. and L. G. Hummerstone, 1973. Adaptation of the marine phyla (especially fish) should be investigated. Work with prolonged polychaete Nereis diversicolor to manganese in estuarine sediments. exposure periods (>1 month), utilizing the presumably more persistent J. Mar. Biol. Assoc. U.K. v53, pp839-872 polycyclic aromatic hydrocarbons (e.g., benzo(a)pyrene, fluoranthene, or 8. Burns, K. A., 1976. Hydrocarbon metabolism in the intertidal fiddler chrysene), merits particular attention. crab Uca pugnax. Marine Biology.v36, pp5-12 Metabolism of PHCs typically involves the formation of phenolic and/or 9. Carlson, G.P., 1972. Detoxification of foreign organic compounds by conjugated hydrocarbon derivatives, via hydroxylation and binding with the quahog, Mercenaria mercenaria. Comp Biochem. other organic molecules (glutathione and simple sugars, respectively).10 In Physiol. v43B, pp295-302 both instances, the hydrocarbon is converted into a more polar form and is 10. Corner, E. D. S., 1975. The fate of fossil fuel hydrocarbons in marine thus more water soluble (less lipophilic). Consequently, metabolized aroma- animals. Proc. Roy. Soc. Lond. B. vl88, pp391-413 tic hydrocarbons are presumably more mobile within organisms, and 11. Corner, E. D. S., R. P. Harris, C. C. Kilvington, andS. C. M. O'Hara, thereby easier to depurate than their parent compound. The present study 1976. Petroleum compounds in the marine food web: short-term offers evidence that marine annelids can metabolize diaromatic hydrocar- experiments on the fate of naphthalenes in Calanus. J. mar. biol. bons (naphthalene). The presence of non-hexane-extractable radioactivity Assoc. U.K. (in press) within its tissues and depuration seawater indicated that Neanthes had 12. Corner, E. D. S., C. C. Kilvington, and S. C. M. O'Hara, 1973. converted 14C-naphthalene into more polar forms (Figure 1). Associated Qualitative studies on the metabolism of naphthalene in Maia microflora (bacteria) were eliminated as a possible contributing factor by squinado (Herbst). J. mar. biol. Assoc. U.K. v53, pp819-832 experiments performed in the presence of antimicrobial agents. Many 13. Cox, B. A., 1974. Responses of the marine crustaceans Mysidopsis marine bacteria are vigorous degraders (metabolizers) of PHCs.33'34 Thus, almyra Bowman Penaeus aztecus Ives and P. setiferus (Linn.) to investigation of their contribution to the apparent metabolism of naphthalene petroleum hydrocarbons. Ph.D. dissertation. Texas A&M Univer- by Neanthes was essential. sity, College Station, Texas. 167pp Non-hexane-extractable radioactivity in tissue homogenates may not 14. Farrington, J. W. and J. G. Quinn, 1973. Petroleum hydrocarbons of necessarily represent metabolized 14C-naphthalene. This radioactivity may Narragansett Bay. I. Survey of hydrocarbons in sediments and clams have represented naphthalene so strongly associated with lipid fractions that (Mercenaria mercenaria). Est. and Coastal Mar. Sei. vi, pp71-79 its extraction into hexane was not possible, using the methods described 15. Lee, R. F., 1975. Fate of petroleum hydrocarbons in marine zoo- earlier. However, the presence of polar radioactivity in seawater holding plankton. 1975. in Proceedings of 1975 Conference on Prevention contaminated Neanthes offers fairly conclusive evidence that these worms, and Control of Oil Pollution. American Petroleum Institute, in some way, metabolize this diaromatic hydrocarbon prior to or during its Washington, D.C. pp549-553 excretion (efflux). A majority of tissue radioactivity not soluble in hexane 16. Lee, R. F., E. Furlong, S. Singer, 1976. Detoxification systems in conceivably represents 14C-naphthalene which has been conjugated with marine invertebrates. Aryl hydrocarbon hydroxylase from the tissues various water soluble molecular species (proteins, mucopolysaccharides, of the blue crab, Callinectes sapidus, and the polychaete worm, simple sugars). Conjugation with such molecules by linkage through cys- Nereis sp. in I.D.O.E. Biological Effects Program Workshop, May teine (amino acid) is a major metabolic fate of naphthalene in the spider crab, 16-19. Texas A&M University, College Station, Texas Maia squinado.12 Much of the same could occur in Neanthes. In a recent 17. Lee, R. F., C. Ryan, and M. L. Neuhauser, 1976. Fate of petroleum study, Lee et al.16 found significant aryl hydrocarbon hydroxylase (AHH) hydrocarbons taken up from food and water by the blue crab, Cal- activity in microsomes from the polychaete Nereis sp. The presence of an linectes sapidus. Marine Biology, (in press) active PHC-metabolizing enzyme such as AHH in a closely related worm 18. Lee, R. F., R. Sauerherber, and A. A. Benson, 1972. 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