Total Hydrocarbons, Polycyclic Aromatic ... - Science Direct

Marine Environmental Research 11 (1984) 163-181

Total Hydrocarbons, Polycyclic Aromatic Hydrocarbons and Synthetic Organic Compounds in the Hard Shell Clam, Mercenaria mercenaria, Purchased at Commercial Seafood Stores Richard J. Pruell, Eva J. Hoffman & James G. Quinn Graduate School of Oceanography, University of Rhode Island, Narragansett, Rhode Island 02882, USA (Received: 8 September, 1983)

ABSTRACT An analytical method for the determination of polycyclic aromatic hydrocarbons (PAHs) and substituted benzotriazoles in clams was developed using a combination of silica gel and Sephadex chromatography to isolate these compounds from large concentrations of polyolefinic hydrocarbons. The concentrations of total hydrocarbons, PAHs and substituted benzotriazoles were measured in hard shell clams (Mercenaria mercenaria) purchased from Rhode Island seafood stores. The levels of total hydrocarbons, PA Hs and substituted benzotriazoles in these clams were generally higher than the concentrations found in clams collected from a lower Narragansett Bay control location. The significance of these findings on the health of consumers is unknown at this time because human health standards or alert levels for these compounds in seafoods have not been established. However, this study illustrates that contaminants entering Narragansett Bay from a variety of sources may eventually make their way to commercial outlets.

INTRODUCTION In recent years, many studies have been conducted on organic contaminants in Narrangansett Bay. Considerable work has been done on total hydrocarbon concentrations in bay waters (Schultz & Quinn, 163 Marine Environ. Res. 0141-1136/84/$03.00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain

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Richard J. Pruell, Eva J. Hoffman, James G. Quinn

1977; Van Vleet & Quinn, 1977) and sediments (Van Vleet & Quinn, 1978; Hurtt & Quinn, 1979; Wade & Quinn, 1979). Polycyclic aromatic hydrocarbons (PAHs) in bay sediments were analyzed by Lake et al. (1979). In addition, recent studies have detected a wide variety of synthetic organic compounds in water, sediments and organisms from this estuary (Lopez-Avila & Hites, 1980; Lake et al., 1981). The interaction of marine filter-feeding bivalves with such man-made or man-mobilized substances has been a very active area of research in the last several years. Many studies have documented the uptake (Lee et al., 1972: Boehm & Quinn, 1976; Courtney & Denton, 1976; Neff et al., 1976; Clement et al., 1980; Riley et ai., 1981) and effects (Roberts, 1975; Gilfillan et al., 1977; Stekoll et al., 1980; Bayne et al., 1982) of hydrocarbons and other organic compounds in filter-feeding bivalves. Also, several studies have investigated the correspondence between bivalve tissue levels and contaminant concentrations in sediment and water from the same location (Courtney & Denton, 1976; Dunn & Stich, 1976; Teal & Farrington, 1977; Boehm & Quinn, 1978; Murray et al., 1981). M e r c e n a r i a m e r c e n a r i a (the hard shell clam, known locally as the quahog) is a commercially valuable species in Narragansett Bay providing a $4.3 million per annum industry in 1978 (Rhode Island Statewide Planning Program, 1979). Because of its commercial value in the bay, several studies have been conducted on the interaction of the hard shell clam with contaminants. Farrington & Quinn (1973) and Farrington & Medeiros (1975) reported hydrocarbon levels in clams from the bay. Also, Boehm & Quinn (1977) showed that clams exposed to chronic inputs of anthropogenic hydrocarbons in the Providence River area of upper Narragansett Bay, depurated these hydrocarbons only at a slow rate when transferred to tanks in a clean water laboratory system. Lake et al. (1981) reported that clams and a few other marine organisms from several locations in the bay contain organic compounds which have been detected in the wastewater of a chemical plant which discharges into a tributary of the bay (Lopez-Avila, 1979; Lopez-Avila & Hites, 1980). The present study was initiated to analyze clams purchased from local seafood stores for PAHs and substituted benzotriazoles. PAHs are widely distributed in the marine environment (Laflame & Hites, 1978) and many of them are toxic and/or carcinogenic (Harvey, 1982). Many studies have reported levels of PAHs in field collected shellfish (e.g. Fazio, 1971 ~Dunn & Stich, 1976; Pancirov & Brown, 1977). Also, Dunn & Fee (1979)

Hydrocarbons, PAlls and substituted benzotriazoles in M. mercenaria

165

reported benzo[a]pyrene in fish and shellfish from seafood stores. However, little information is available on other PAHs in commercial seafood. Substituted benzotriazoles are compounds used as ultraviolet light absorbers in plastics, paints and other materials. They are produced by a chemical company located on the Pawtuxet River, a tributary of the Providence River. Jungclaus et al. (I 978) first identified these compounds in Pawtuxet and Providence River water and sediments. Lopez-Avila & Hites (1980) extended this analysis to include Narragansett Bay sediments. Substituted benzotriazoles have not been previously reported in organisms. However, because of the amounts discharged and several of their properties (e.g. water solubility, n-octanol partition coefficient, environmental persistence), their occurrence in marine organisms was suspected. Acute toxicity studies with rats indicated that the substituted benzotriazoles were only toxic in extremely high doses (EPA, 1977); however, there is little information available on their chronic effects. METHODS Collection

On May 30, 1979, hard shell clams were purchased at nine seafood stores in Rhode Island. About two weeks later, clams were purchased from four more stores. Store personnel in nine of the 13 stores surveyed indicated that the clams were harvested from Narragansett Bay; and several did not know where their clams were harvested. Three seafood stores were sampled again on August 6, 1979 to determine if the higher values obtained at these establishments were representative of their usual stock. To serve as controls, clams were collected from a relatively unpolluted site in lower Narragansett Bay near Dutch Island (Boehm & Quinn, 1977). The clams from each store and the controls were divided into the following size categories: small (3-6 cm in length); medium (6-9 cm); and large (< 9 cm). For each location, clams from each size class were combined and then homogenized in a stainless steel blender and frozen at - 20 °C until analyzed. Extraction

About 100 g of clam homogenate was transferred to a round bottom flask

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Richard J. Prueil, Eva J. Hoffman, James G. Quinn

along with two internal standards (usually 51.6/zg of/'/-C22 and 4.75/zg of anthracene). Samples were then digested and homogenized using a method described by Boehm & Quinn (1977). Briefly, the homogenate was digested with 0-5 N KOH in methanol under reflux conditions for 2 h. The resultant solution was allowed to set overnight and the supernatant decanted into a separatory funnel through silanized glass wool. A saturated NaCI solution was added to inhibit emulsions, and the basic aqueous-methanol solution was extracted three times with petroleum ether. Combined extracts were then back washed twice with a 1:1 methanol-water mixture, and then finally once with distilled water. Extracts were reduced to a small volume by rotary evaporation and chromatographed on a silica gel column. The column consisted of a 0-5 cm (int. diam.) × 15 cm Pasteur pipette fitted with silanized glass wool and filled with 1.5 g of fully activated silica gel (Grace grade 922, 200-325 mesh size). The column was cleaned with 15 ml of CH2CI 2 followed by 15 ml of hexane. Samples were then charged to the column in I ml of petroleum ether and chromatographed using nitrogen pressure to produce a solvent flow rate ofabout 5 ml min- 1. The first fraction (fro)was eluted from the column with 15 ml of hexane and contained saturated hydrocarbons and hydrocarbons with one or two double bonds. The second fraction (t"2) was then eluted with 15ml of an 80:20 hexane-methylene chloride mixture. This fraction contained PAHs, polyolefinic hydrocarbons and substituted benzotriazoles. The f2 fraction was evaporated to near dryness at room temperature by rotary evaporation and 0.5 ml of methanol added to the residue. This solution was charged to a 1.0cm (int. diam.)× 50cm column of Sephadex LH-20 (Pharmacia Inc., Piscataway, N J, USA) in methanol. A solvent flow rate of approximately 1 - 2 m l m i n -~ was initiated using nitrogen pressure. The first 35 ml of eluate from the column contained the polyolefinic material and was discarded. The next 90 ml was collected as the PAH and substituted benzotriazole fraction. An equal volume of water was added to the methanol eluate and the sample extracted three times with petroleum ether. The fl eluate was evaporated to a small volume and injected on a Hewlett- Packard 5840A gas chromatograph equipped with a 30 m SE-52 glass capillary column and a flame ionization detector (FID). The temperature programming was from 45 to 270°C at 4°Cmin - 1 with a nitrogen carrier gas flow of ~ 1 ml rain- 1. Samples were quantified by comparing the area of the n-C22 internal standard to the area of the

Hydrocarbons, PAils and substituted benzotriazoles in M. mercenaria

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unresolved envelope, which was measured by planimetry. The Sephadex eluate was concentrated and then analyzed on a Hewlett-Packard 5711A gas chromatograph (FID) using a 20 m SE-52 or SE-30 glass capillary column, programmed from 40 to 270 °C with helium as the carrier gas. These samples were quantified using the anthracene internal standard and employing a Hewlett-Packard 3385A reporting integrator. Authentic standards were obtained for all compounds reported in this study. Relative retention times on glass capillary columns using two different supports (SE-30 and SE-52), were compared. Also, gas chromatography-mass spectrometry (GC-MS) analyses were conducted for selected samples. This instrumentation included a Varian 1400 gas chromatograph equipped with an SE-52 glass capillary column coupled to a Finnigan 1015 quadrupole mass spectrometer. Data was acquired and processed with a Finnigan INCOS 2300 data system. Blanks were usually 90 %. Results were not corrected for recoveries or instrumental response factors. Three aliquots of a clam homogenate were analyzed to determine the precision of the analyses. Total hydrocarbons showed a relative standard deviation (Rel. SD) of 6 % for the three analyses. For specific PAH compounds the precision ranged from 15 % Rel. SD for fluoranthene to 46% Rel. SD for benz[a]anthracene. Analysis for substituted benzotriazoles produced Rel. SD values of 9 % and 21% for C ~o-benzotriazole and chloro-benzotriazole respectively.

RESULTS The results of total hydrocarbon analyses of the clam homogenates are given in Table 1 and a compilation of statistical data is given in Table 2.

Richard J. Pruell, Eva J. Hoffman, James G. Quinn

168

TABLE 1 Total Hydrocarbon Concentrations in Clams Purchased from Rhode Island Seafood Stores

Store number

I 2 3 4 5 6 7 8 9 10 II 12 13 DI ~

Total hydrocarbons (gg g - ' wet weight) small

medium

13"5 32"2 22"3 24"2 40"1 (15"7)" 20"3 12.7 24-9 12.3 21.2 (13.2) 20.3 7.6 16.3 8'0

NA 14-0 30"9 NA 33'9 (12"6) 21'3 27.2 48.4 (28.7) 21.3 67.8 (17.9) 24.8 14.3 NA 11'6

large 15'0 NA NA 18'7 30'9 NA NA 20.9 NA 35.6 NA 9.6 4.7 9'8

(21.1)

NA = Not analysed. ° Numbers in parentheses are values obtained on a second visit. b DI = Dutch Island clams (lower Narragansett Bay) collected as controls.

TABLE 2 Mean Concentration ( + o n e standard deviation) of Total Hydrocarbons (ggg-~ wet weight) in Clams from Rhode Island Seafood Stores

Small clams Medium clams Large clams Mean of all samples ° Thirteen stores. b Three stores. c One analysis.

1st Visit a

2nd risit b

Dutch Island

20.6 + 8.8 30-4 _+ 16.6 19.3 +_ l l.0 23-6 _+ 12.9

14.5 _+ 1.8 19.7 _+8-2 21.V 18.2 + 6-0

8.0 11.6 9-8 9.8 +_ I-8

Hydrocarbons. PAils and substituted ben-otria:oles in M. mercenaria

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The mean concentration of hydrocarbons in clams purchased at Rhode Island seafood stores on the first visit was 23.6 +_ 12.9 ~ugg- ~ wet weight. Three stores (numbers 5, 8 and 10) whose clams showed higher than usual values (arbitrarily assigned as >_40 pg g - t ) w e r e sampled a second time. The hydrocarbon values of clams purchased on this visit averaged 18.2 _+ 6.0, which is similar to the mean concentration of clams purchased at all stores on the first visit. O.

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A typical chromatogram for the f~ fraction of clam tissue extract is given in Fig. 1. This fraction shows a large unresolved complex mixture of saturated hydrocarbons which is similar, although of a somewhat lower boiling range, to that found in Narragansett Bay sediments (Van Vleet & Quinn, 1978; Hurtt & Quinn, 1979). This phenomenon has previously been noted by Farrington & Quinn (1973), Teal & Farrington (1977) and Boehm & Quinn (1978). Peak I in Fig. 1 is the biogenic cycloalkene, HC344, which was reported in the ocean quahog, Arctica islandica, by Boehm & Quinn (1978). An f2-Sephadex eluate chromatogram of clam tissue extract is shown in Fig. 2. The insert in this figure (Fig. 2(b)) demonstrates the separation achieved for the substituted benzotriazole standards. Figure 3 shows mass spectra of authentic standards for the two substituted benzotriazoles, and spectra obtained from clam tissue extracts, The spectra

medium medium medium medium large medium large medium large

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phenanthrene 1.2 1.3 4.1 7.2 1.5 1.9 1.0 0.8 0.7 2-6 _+ 2.3

fluoranthene 1.2 I-3 2.7 6.6 !.6 1.5 1.6 0.6 0.3 2.4 +_ !.9

pyrene 0.4 0.3 0.4 0-8 0. I 0.2 0.3 0. I 0.3 0-4 _+0.2

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P A H concentrations (ng g - J wet weight)

° Analysis done on clams from the first seafood store visits. b Sufficient sample was not available for PAH analysis of the small size class. c Excluding DI clams.

Size class~

Store number"

TABLE 3 PAH Concentrations in Clams Purchased from Rhode Island Seafood Stores

3.6 3-4 8.3 17 3.9 4.3 3.5 ! .8 I-5 6.3 _+5.0

Total identified PAH

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174

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TABLE 4 Substituted Benzotriazole Concentrations in Clams Purchased from Rhode Island Seafood Stores Store number a

Size class b

3 5 7 8 l0 13 !3 DI DI

medium medium medium medium large medium large medium large

C~o-benzotria=ole c Chloro-ben:otriazole d (ngg TM wet weight) (ngg - t wet weight)

65 7 25 61 17 16 28 II Il

7-4 1-0 2-6 8.5 6. l 2-0 5-5 2.3 1-9

" Analysis done on clams from the first seafood store visits. Sufficient sample was not available for substituted benzotriazole analysis of the small size class. C~o-benzotriazole is 2-(2'-hydroxy-3',5'-di-t-amylphenyl)-2H-benzotriazole. d Chloro-benzotriazole is 2-(2'-hydroxy-Y,5'-di-t-butylphenyl)-5-chioro-2H-benzotriazole. obtained for authentic C~o-benzotriazole (Fig. 3(a)) and peak No. 6 (Fig. 2) in clam tissue extracts (Fig. 3(b)) are virtually identical. Also, the spectra o f peak No. 7 (Fig. 2) in clam tissue extract (Fig. 3(d)) is very similar to that o f authentic chloro-benzotriazole (Fig. 3(c)). The occurrence o f the extra peak at m/e 351 (Fig. 3(d)) probably results from overlap o f the C~ o-benzotriazole peaks but the appearance o f the peak at m/e 308 cannot be explained at this time. The results o f P A H analyses are compiled in Table 3. Figure 4 shows a comparison o f P A H s found in clams with the P A H distribution found in Narragansett Bay sediments. D a t a obtained on the substituted benzotriazoles are present in Table 4.

DISCUSSION Total hydrocarbons

The total h y d r o c a r b o n content o f clams, M e r c e n a r i a m e r c e n a r i a , purchased from R h o d e Island seafood stores varied from 4.7 to

Hydrocarbons, PAils and substituted benzotriazoles in M. mercenaria

175

TABLE 5 Total Hydrocarbon Concentrations in Mercenaria mercenaria Collected from Narragansett Bay by Various Investigators Date collected

Total hydrocarbons (Itgg- ' wet weight) Providence River

1970-1971

Wickford Dutch Island or GSO

14-16

4- l 0

1973

90

9-6

1976

42

1979

Reference

3

5.3-7.1 8.0- I 1.6

Farrington & Quinn, 1973 Farrington & Medeiros, 1975 Boehm & Quinn, 1977 This work

67.8 #g g- ~wet weight (Table 1) with an average of 23.6 _+ 12.9 #g g- ~wet weight (Table 2). There was no consistent trend in hydrocarbon content with size of the clams, with geographical location of the store, or with date of purchase. The absence of such relationships is to be expected since the most important variable influencing hydrocarbon content of the clams is probably the location where they were harvested. In this survey, the location of harvesting was not known to the investigators, as is the usual case for consumers, and in many cases the retail outlets. Table 5 compares the total hydrocarbon levels in clams found in this study with published values for Narragansett Bay clams reported by other investigators. As can be noted from these studies, the highest values for hydrocarbons are found in clams collected from the Providence River. The Providence River is an area receiving large anthropogenic hydrocarbon inputs from many sources including sewage eflquents (Van Vleet & Quinn, 1977) and urban runoff (Hoffman et al., 1983). Hydrocarbon levels in river sediments have been reported to reach 5 mg g- ~ dry weight (Van Vleet & Quinn, 1977). The Providence River has been closed to shellfishing for over 30 years due to unacceptable levels of fecal coliform bacteria in shellfish from this area. Of the 36 samples analyzed in this survey, three of the samples (from store numbers 5, 8 and 10) exhibited hydrocarbon levels (> 40/~g g- ~) in the range reported by Boehm & Quinn (1977) and Farrington & Medeiros (1975) for Providence River clams. This serves only as weak circumstantial evidence of the possible entry of clams from closed shellfishing areas

176

Richard J. Pruell, Eva J. Hoffman, James G. Quinn

into the market place. Also stores found to have higher hydrocarbon levels on the first visit had average levels on a subsequent visit, suggesting that no one store routinely purchased clams collected from contaminated areas.

Polynuclear aromatic hydrocarbons The concentrations of selected PAHs in clams purchased from Rhode Island seafood stores are given in Table 3. Values range from 0.1 to 7.2ngg -~ wet weight for specific compounds and from 1.5 to 17ngg -~ for total identified PAHs with an average of 6.3 +_5.0 ng gThese results are similar to those reported by Fazio (1971) for several PAHs in oysters collected from Galveston Bay, Texas. The pyrene concentrations are also similar to those reported by Pancirov & Brown (1977) for several shellfish species collected from locations along the east coast of the United States. Farrington & Davis (1978) reported levels (on a dry weight basis) of several aromatic hydrocarbons in mussels from Narragansett Bay. Using an average wet to dry weight ratio ,,-7.0 for Narragansett Bay mussels (R. J. Pruell, unpublished work) the calculated values of phenanthrene, fluoranthene and pyrene were similar to those determined for clams in this study. PAH levels reported for mussels from the coast of Norway (Knutzen & Sortland, 1982), and Scotland (Mackie et al., 1980), however, were higher than those determined for hard shell clams in this study. PAH concentrations in clams from the seafood stores were generally higher than those in clams from the lower bay control location. However, the levels detected were similar to those reported for several other foods (including cereals, vegetables, fruits, fish and cheeses) and lower than levels found in smoked food products (Howard & Fazio, 1969; Lo & Sandi, 1978: Howard & Fazio, 1980). The PAH distributions found in all clam samples, including the lower bay controls, were similar and independent of size. Figure 4(a) shows the PAH distribution detected in clams from the Dutch Island control site. Also included in this figure,(Fig. 4(b))iis the PAH distribution of a surface (0-2.5cm) sediment from North Jamestown, a nearby location in Narragansett Bay (Pruell & Quinn, unpublished work). Although the sediment and clam samples were analyzed using similar analytical methods, the PAH patterns detected were very different. Compared to the

Hydrocarbons, PAils and substituted benzotria:oles in M. mercenaria

177

PAHs in bay sediments, the clams showed a.much narrower distribution of compounds. Clam samples contained 3 or 4 ring compounds (phenanthrene to chrysene) and were depleted in the lower (e.g. naphthalenes) and higher molecular weight compounds (e.g. benzopyrenes) compared to the sediment. The sources of PAHs to Narragansett Bay have been investigated by Lake et al. (1979). They concluded that the dominant sources of PAHs were combustion related (produced during the combustion of fossil fuels) with contributing inputs of petroleum in the Providence River. The PAH distributions in Narragansett Bay sediments are similar to those observed by others in studies on Recent marine sediments throughout the world (Youngblood & Blumer, 1975; Laflamme & Hites, 1978: Gschwend & Hites, 1981). Substituted benzotriazoles The data presented in Table 4 represents the first report of substituted benzotriazoles in marine organisms. This data shows that C~o-benzotriazole, a compound produced since 1970, is present in higher concentrations in clams than chloro-benzotriazole, a compound which was produced from 1963 to 1972 (Lopez-Avila & Hites, 1980). Although the production of chloro-benzotriazole ended in 1972, trace levels of this compound can still be found in particulate material in the Pawtuxet River (R. J. Pruell, unpublished work, 1980). It is also found in surface sediment throughout Narragansett Bay (Pruell & Quinn, unpublished work). Reasons for this probably include the resuspension of contaminated sediment particles by both physical and biological processes and bioturbation in the sediments. The ratio of C~o-benzotriazole to chlorobenzotriazole in clams varies from 2.7 to 9-5. This is similar to the ratio in surface sediments of the bay which ranges from 2.0 to 7.6 (Pruell & Quinn, unpublished work). Statistical analyses A significant correlation (r --- 0.746, P > 95 o~) was found between total hydrocarbon and total PAH levels in the clam samples as well as among all of the PAHs quantified (P > 99 %). Also, a significant correlation existed between C~ o-benzotriazole and chloro-benzotriazole (r = 0.856,

178

Richard J. Pruell, Eva J. Hoffman, James G. Quinn

P > 99 %). Total hydrocarbons and total PAHs were not significantly correlated with the substituted benzotriazoles. Therefore, there was a strong correspondence between all of the hydrocarbon parameters measured as well as between the two substituted benzotriazoles. However, the trends for hydrocarbons and substituted benzotriazoles were not strongly correlated in this study. The substituted benzotriazoles have one major source in Narragansett Bay whereas the hydrocarbons have many sources and input locations. Both substituted benzotriazoles and hydrocarbons have their highest concentrations in the Providence River and decrease in concentration with distance down the bay (Pruell & Quinn, unpublished work). However, the rate of decrease for the substituted benzotriazoles is much faster than that for the hydrocarbons. This may account for the weak correspondence between these parameters. The hydrocarbon concentrations of the clams may also be influenced by local discharges in some areas. Alternatively, the uptake, retention or metabolism of hydrocarbons and benzotriazoles may be different and thus account for the observed distributions.

CONCLUSIONS Total hydrocarbons, PAHs and substituted benzotriazoles were measured in the hard shell clam, Mercenaria mercenaria, from Rhode Island seafood stores. Total hydrocarbon values in store samples were generally higher than in samples collected from a control location in lower Narragansett Bay. PAH concentrations were similar to levels which have been reported for other shellfish species. They are also within the range reported for other foods. Two substituted benzotriazoles were also detected in the clam extracts. There are no Rhode Island State Department of Health or US Food and Drug Administration standards or alert levels for any of these compounds in edible shellfish, and they are not routinely monitored. Therefore, the potential health significance of these findings is unknown. This study does illustrate, however, that contaminants which are discharged to Narragansett Bay by a variety of paths (e.g. sewage effluents, urban runoff and industrial discharges) may eventually make their way to commercial outlets.

Hydrocarbons, PAlls and substituted benzotriazoles in M. mercenaria

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ACKNOWLEDGEMENTS We wish to thank Evelyn Dyer (URI) for help with sample preparation, and Dr Nelson Frew and Dr John Farrington (WHOI) for assistance in G C - M S analysis.

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