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Field assessment of biomarkers for winter flounder
Volume 22/Number 2/February 1991 Hong, W.-H., Meier, P. G. & Deininger, R. A. (1987). A microcomputer-interfaced continuous flow toxicity test system. Wat. Res. 21, 1249-1257. Mount, D. I. & Brungs, W. A. (1967). A simplified dosing apparatus for fish toxicology studies. Wat. Res. 1, 21-29. Mount, D. I. & Warner, R. E. (1965). A Serial Dilution Apparatus fi~r
Rand, G. M. & Petrocelli, S. R. (1985). Fundamentals of Aquatic 7oxicology.Hemisphere Publishing Corp., Washington, D.C. Singer, M. M., Smalheer, D. L. & Tjeerdema, R. S. (1990a). A simple continuous-flow toxicity test system for microscopic life stages of aquatic organisms. Wat. Res. 24,899-903. Singer, M. M., Smalheer, D. L., Tjeerdema, R. S. & Martin, M. (1990b). Continuous Delivery of Various Concentrations of Material in Water. Toxicity of an oil dispersant to the early life stages of four California PHS Publication No. 999-WP-23, Washington, D.C. marine species. Environ. ToxicoL Chem. 9, 1389-1397. N RC (National Research Council) (1989). Using Oil Spill Dispersants Singer, M. M., Smalheer, D. L., Tjeerdema, R. S. & Martin, M. (1990c). on lheSea. National Academy Press, Washington, D.C. Effects of spiked-exposure to an oil dispersant on the early life stages of four marine species. Environ. Toxicol. Chem. (in press).
Marine I'ollutiotl B,//etin. Volume22, N~ 2, pp 61 liT. 1991.
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Field Assessment of Biomarkers for Winter Flounder ROBERT SCOTT CARR*, ROBERT E. HILLMAN and JERRY M. NEFF+
Battelle, Ocean Sciences, 397 Washington Street, Duxbury, MA 02332, USA Present addresses: *US Fish and Wildlife Service, NFCR Field Research Station, Corpus Christi State University, Campus Box 315, 63(1(I Ocean Drive, Corpus Christi, TX 78412, USA +Arthur D. 1,iltlc Inc., Acorn Park. Cambridge, MA 02140, USA
Winter flounder, Pseudopleuronectes americanus, from Boston Harbor near the Deer Island sewage outfall and from a nearby reference population near Plymouth Beach, Massachusetts, USA, were collected on several occasions and analysed for a variety of biochemical variables and histopathological conditions. A number of biochemical variables including hepatic and pectoral fin ascorbic acid concentrations, hepatic glycogen and lipid levels, plasma glucose concentrations, brain serotonin and norepinephrine concentrations, and the concentration and ratio of various free amino acids in muscle tissue were significantly different between the two populations. The apparent apoptotic hepatic lesions were found in the majority of the winter flounder collected from Boston Harbor and were rarely observed in animals from the reference site. Low tissue concentrations of ascorbic acid and hepatic glycogen were found to have significant statistical associations with the presence and degree of severity of these hepatic lesions,
As part of a large, multidisciplinary programme designed to develop diagnostic biochemical indices of early pollutant stress in marine animals, a field study was conducted to evaluate the feasibility of using biochemical parameters to assess the relative health of winter flounder, Pseudopleuronectes americanus, collected near the discharge of a sewage treatment plant as
compared to a population from a nearby reference site. Recent results from the National Status and Trends Program of the National Oceanic and Atmospheric Administration indicate that sediment concentrations of target polycyclic aromatic hydrocarbons (PAHs) and total polychlorinated biphenyls (PCBs) are higher at the Boston Harbor site than at any of the other 49 coastal sites examined in this national survey (NOAA, 1987). The primary objectives of the investigation were 1. to assess whether significant differences in biochemistry and histopathology exist between fish from the two sites, and 2. to determine whether any correlations exist between observed histological abnormalities and the biochemical variables. Biochemical parameters investigated in this study were hepatic and pectoral fin ascorbic acid concentrations, lipid and glycogen concentrations, plasma glucose and cholesterol levels, muscle free amino acid concentrations, and brain indoleamine and catecholamine concentrations. These parameters have been evaluated previously in several laboratory and field investigations and have been found to be useful for diagnosing pollutant stress in marine teleosts (Thomas et al., 1980, 1981a, 1981b, 1982a, 1982b; Haensly etal., 1982; Neff& Haensly, 1982). Methods
Field Sampling Winter flounder, Pseudopleuronectes americanus, were coll6cted by otter trawl off Plymouth Beach and near the Deer Island sewage outfall in Boston Harbor 61
Marine Pollution Bulletin
(Fig. 1) during the summer on two consecutive years, Three stations at different distances from the outfall were sampled in Boston Harbor during the first collection. Liver and pectoral fins were excised and frozen on dry ice. Prior to freezing, a small portion of the eyedside lobe of the liver was removed and preserved in Helly's fixative for histological examination, The trawls for the second year's sampling lasted for 30-60 s of bottom time. Only the animals that could be bled within 10 rain of the start of the trawl were included in the collection because previous studies have shown that elevated plasma glucose levels resulting from collection and handling stress are not evident within 10 min for this species (Carr, unpublished). First, blood samples were obtained from the branchial sinus, and heparinized to prevent clotting. The whole body weight was measured with a spring scale to the nearest 10 g and the standard length recorded. Liver and brain tissues were then quickly excised and stored in liquid nitrogen for later biochemical analysis. A sample from the eyed-side lobe of the liver was taken
and preserved in Helly's fixative for later histological examination. The heparinized blood samples were kept on ice and centrifuged within 60 rain of collection. The plasma was collected and stored refrigerated (4°C). A scale sample from between the eyed-side pectoral fin and the lateral line was obtained from each fish. Growth rings on the scale samples were examined microscopically to obtain age estimates for each animal. Males were differentiated from females by the presence of rough scales on the blind-side of the caudal peduncle. Surface and bottom water temperature, salinity, and dissolved oxygen concentrations were measured at both stations withtheaid of a Hydrolab.
BiochemicalAnalyses Plasma glucose and cholesterol were measured with a Beckman glucose/cholesterol analyser within 48 h of collection. Ascorbic acid concentrations in liver tissues were determined by the method of Carr & Neff (1980) using liquid chromatography with electrochemical detection. This method has been used to quantify the
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Volume 22/Number 2/February 1991
ascorbic acid content of a wide variety of animal tissues (Carr et aL, 1983). Liver glycogen was quantified by the method of Carr & Neff (1983). Total lipid concentrations in liver tissues were determined in triplicate by the method of Holland & Gabbott (1971). Concentrations of free amino acids in muscle were determined by gradient high performance liquid chromatography (HPLC) with post-column derivatization (o-pthaldehyde) and fluorometric detection, Tissue samples were homogenized in 7% trichloroacetic acid (TCA) as described by Roesijadi el al. (1976). TCA supernatants were extracted three times with diethyl ether, then lyophilized. Lyophilized samples were redissoved in 0.1 N HC1 just prior to analysis. A Waters Associates gradient liquid chromatograph with a WISP autosampler, system controller, data module, and fluorometric detector was employed. The chromatographic conditions have been described previously (Pfeiffer et al., 1983). Norleucine was used as the internal standard, Catecholamines and indoleamines were determined by ion-pairing HPLC with electrochemical detection, Frozen brains were homogenized in 250 mM HCIO 4. The homogenate was centrifuged at 27 000 g for 3() rain and the supernatant analysed without further extraction or cleanup. An isocratic Waters Associates system was employed with an amperometric detector (Bioanalytical Systems, LC-4) using a thin-layer glassy carbon electrode (+800 mV vs. Ag/AgC1). A Partisil SAX guard column in conjunction with a Resolve C m column provided excellent separation and resolution of the components of interest. The mobile phase was 0.1 M KH2PO,:methanol, 85:15 (pH 3.76) with heptane sulphonic acid (Waters Assoc.) added as the ion-pairing reagent. Serotonin, the last component of interest, eluted at a flow rate of 0.5 ml rain -~ approximately 15 rain after injection. Dihydroxybenzylamine (DHBA) was used as the internal standard, Histology
Liver tissues were excised immediately after blood collection and individually placed in labelled plastic cassettes with a code number so that the evaluator did not know from which population the tissues were derived. The samples were fixed in Helly's fixative for 24 h and then rinsed in running water for 16-24 h to remove excess fixative. The samples were stored in 70% ethanol until preparation for final processing. The tissues were impregnated with Paraplast Plus in an American Optical TP8000 tissue processor and sectioned at 4 ~tm. Sections were stained with hematoxylin and eosin or Whipf's polychrome stain and mounted in Histoclad.
analysed by Spearman correlation analysis with the aid of the Statistical Analysis System (SAS Institute, Inc. 1982).
Results FirstColleetion
The average weight (±SD) and age (as determined by scale growth bands) of flounder from the Boston Harbor and Plymouth Beach sites were 325 + 54 g, 3, years, and 447+ 90 g, 4 years, respectively. Liver glycogen and ascorbic acid were the only biochemical measurements made for the first collection. The mean concentration of ascorbic acid in livers and blind-side pectoral fins, and mean hepatic glycogen concentrations were significantly lower in the Boston Harbor animals than in animals from the reference site (Fig. 2). The primary histological difference between the winter flounder collected from Boston Harbor in the first sampling and those collected during the same period off Plymouth Beach (Table 1) was the high prevalence of apparent apoptotic hepatic parenchymal cells (AAPHC, Fig. 3)in the Boston Harbor fish. Second Collection
The standard length of the winter flounder collected ranged 20-31 cm. The average weight (±SD) and age of the animals was 270 + 76 g, 2.6 years and 373 + 126 g, 3.2 years for Boston Harbor and Plymouth Beach animals, respectively. The bottom water temperature, dissolved oxygen concentration, and salinity were 16.5°C, 9.4 mg 1-~, and 32%0 for Boston Harbor and I4.5°C, 10.2 mgl-l, and 32%ofor Plymouth Beach. Biochemical analyses performed on liver tissue from the second collection included ascorbic acid, glycogen, and lipid concentrations (Table 2). As in the first collection, the mean hepatic ascorbic acid concentration was lower in the Boston Harbor animals than in animals from the reference site, but the variability was too great for the difference to be statistically significant. Hepatic glycogen and lipid concentrations of winter flounder from Boston Harbor were significantly lower than levels in animals from the reference site. Plasma glucose levels of the Boston Harbor animals were significantly lower than the levels of the reference animals (Table 3). Neurotransmitters measured in brain tissues in greatest concentration were norepinephrine and serotonin (5-hydroxytryptamine). The level of both of these neurotransmitters was significantly lower in the brains of winter flounder from Boston Harbor than in animals from Plymouth Beach (Table 3). In general, the muscle concentration of free amino acids was higher in the Boston Harbor animals (Table TABLE1
Statistical Analyses
Significant differences between population means for the various parameters were determined by the Student's t-test where applicable, and by the MannWhitney U-test where the data did not fulfill the requirements for parametric analysis. The descriptive information, biochemical, and histopathological data for each individual from the two populations were
Prevalence of apparent apoptotic hepatic parenchymal cells (AAHPC) in winter flounder (Pseudopleuronectes americanus) collected from Plymouth Beachand BostonHarbor.
iv First collection Second collection
13 20
Percentage of fish with AAHPC Plymouth Beach N BostonHarbor
8%
14
64%
0%
20
50%
63
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Fig. 2 Mean ( ± S E M , n >- 13) pectoral fin ascorbic acid and liver ascorbic acid and glycogen for winter flounder from the first collection. The difference between the two populations was highly significant (ct < 0.005, U-Test) for all parameters except eyed-side pectoral fin. TABLE 2
Mean ( _+SEM) hepatic ascorbic acid, glutathione, glycogen and lipid concentrations for winter flounder.
Site
N
Ascorbic Acid (~g g-~)
Glutathione (~tgg ])
Glycogen (mgg -~)
Total lipids (mgg -~)
Boston Harbor Plymouth Beach
20 20
11.2 _+2.5 13.9 _+2.2
20.1 _+ 1.9 20.5 _+2.2
28.1 ± 3.7* 45.8 _+4.2
17.5 -+ 0.25f 26.4 _+ 1.17
*~ < 0.001 (U-test)
+c~< 0.005 (U-test)
N = Number of animals analysed.
TABLE 3 Mean (± SEM) plasma glucose and cholesterol and brain norepinephrine and serotonin concentrations for winter flounder.
Site
N
Glucose (mgdl-')
N
Cholesterol (mgdl-')
N
Boston Harbor Plymouth Beach
20 20
23.3 -+ 1.5" 31.8 _+ 1.6
17 20
234.9 _+ 18.1 2(t8.0 _+ 13.0
20 19
*c~ < (I.001 (U-test)
tct< 0.025 (U-test)
"a < 0.005 (U-test)
4). This elevation was statistically significant for only glycine and serine. The serine+ threonine index and the taurine/glycine ratio were both significantly different between the two populations, The high prevalence of AAHPC of fish collected in Boston Harbor during the second year was again the major histological difference between the two populations (Table 1). Fifty percent of the fish collected from Boston Harbor during the second sampling period had aggregates of these apoptotic cells, while none of these cells were found in flounder collected off Plymouth Beach. 64
Norepinephrine (~tgg ~) 0.40 _+0.1 (1~ 1.03 +_0.22
N
Serotonin (~,gg ')
20 20
0.48 _+0.09~ (I.86 _+0,06
N = Number of animals analysed.
CorrelationAnalyses The results of the Spearman correlation analysis of the biochemical histological, and descriptive data are shown in Table 5. A number of significant correlations were observed when the data from both populations were analysed together. The most interesting correlations were those showing strong associations between levels of liver glycogen and tissue ascorbic acid, and the presence of AAHPC. Plasma cholesterol levels and hepatic glycogen concentrations were significantly associated with the weight and age of the animals. This relationship may account for some of the differences
Volume 22/Number 2/February 1991
Fig. 3 Apparent apoptotic hepatic parenchymalcells (arrows) found in majority of winter flounder collected from Boston Harbor. H&E x 100. observed between the two populations for these variables, because the animals from the reference site tended to be older and larger than those from Boston Harbor. There were no significant associations between the sex or hepatic P-450 levels (Stegeman et al., 1987) and any of the descriptive, biochemical, or histological data, and there was no difference in condition factor (length/weight) between the populations, Discussion Significant differences exist for a variety of biochemical parameters and the occurrence and severity of A A H P C between the two populations investigated. Results of a 10-year tagging study showed that the movement of winter flounder from these two sites was localized (Howe & Coates, 1975) and it is known that
winter flounder return to the same area to spawn each year (Saila, 1961). Because these populations are believed to be relatively discreet (Howe & Coates, 1975) and the water quality parameters (temperature, salinity, dissolved oxygen) essentially identical between the two sites, one might speculate that the biochemical and histological differences observed between the two populations are a result of conditions arising from the discharge of primary effluent and sewage sludge at the Boston Harbor site. The high incidence of disease reported for winter flounder from Boston Harbor (Murchelano & Wolke, 1985; NOAA, 1987) further supports this contention. In the present study, both the hepatic glycogen and lipid concentrations of the Boston Harbor animals were significantly lower than the levels in animals from the reference site. When winter flounder are stressed, their 65
Marine Pollution Bulletin TABLE 4
TABLE 5
Mean (±SEM) free amino acid (and ammonia) concentrations in muscle tissue (~tg mg -h) and amino acid indices for winter flounder.
Probability values for significant associations as determined by Spearman correlation analysis for data from winter flounder collected from Boston Harbor and Plymouth Beach combined.
Taurine Aspartic Acid Threonine Serine Glutamine Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Ammonia Arginine Taurine/Glycine Serine _+ Threonine
N
Boston Harbor
N
Plymouth Beach
19 19 19 19 19 19 19 19 14 19 19 17 19 19 19 19 6 19 19
3885 ± 211.8 106.8 ± 52.7 264.0 +- 30.4 311.9+46.2" 145.5 ± 4 2 . 7 2886"+ 552.9t 792.5 "+ 281.0 60.2 ± 10.9 31.9 ± 9.3 43.3 + 10.5 98.4 +_36.7 136.1 ± 9 7 . 7 6 1 . 7 ± 12.7 349.4± 29.1 136.9 ± 15.3 1730 ± 75.5 357.8 ± 251.8 1.99 ± 0.31" 576.0 ± 65.9*
20 20 20 20 20 20 20 20 10 20 20 11 20 20 20 20 13 20 20
3890 ± 174.4 49.7 _+7.6 147.7 ± 13.7 147.7+21.4 115.9"+13.7 779.6 ± 100.1 379.3 ± 27.5 64.7 ± 10.2 16.7 ± 1.5 48.9 ± 10.6 74.3 ± 13.4 26.6"+6.8 4 2 . 7 ± 5.5 431.9±23.0 203.7 ± 22.4 1826 ± 135.2 110.4"+12.6 5.88 ± 0.46 295.5 _+ 33.6
• ¢~<0.005(U-test). tot < 0.05 (U-test). N = Number of animals analysed,
adaptive reproductive strategy is to sacrifice egg production for somatic growth and homeostatic maintenance (Tyler & Dunn, 1976). Reduced carbohydrate and lipid reserves are also indicative of lowered energy reserves. The significant decrease in brain norepinephrine and serotonin in Boston Harbor flounder also suggests that the neurological and endocrine systems were depressed in these animals. Brain levels of epinephrine and serotonin decreased in mullet Mugil cephalus exposed to sublethal concentrations of dibenzofuran (Thomas etal., 1981b). The two amino acid indices, the taurine/glycine ratio and the serine + threonine index, showed a significant difference between the two populations. The taurine/ glycine ratio, which has been used most often as a stress index with molluscs and crustaceans, usually increases in stressed animals due to a decrease in glycine, with taurine levels remaining relatively constant (Jeffries, 1972; Bayne et aL, 1976). The increased glycine concentration in muscle tissue of the Boston Harbor animals resulted in a taurine/glycine ratio that was 34% of the ratio observed for the Plymouth Beach animals, As with invertebrates, the taurine levels remained unchanged. Glycine has been shown to function as a conjugate in Phase II biotransformation reactions in flounder (Lech & Vodicnik, 1984), which suggests t h a t the increased glycine levels observed in tissues of Boston Harbor winter flounder may have been induced
in response to contaminant exposure, The serine+ threonine index, which previously has been used almost exclusively with bivalves, normally decreases during stress (Livingstone, 1985). The sum of serine+threonine was almost twice as great in the muscle tissue of Boston Harbor animals as in the reference population. Although the changes in these indices are the opposite of what has been observed in invertebrates, the fact that highly significant differences were observed between the two populations for both indices suggests that these 66
Liver ascorbic acid Blind-side pectoral fin ascorbic acid AAHPC~
Blind-side pectoral fin ascorbic acid
Liver glycogen
Plasma cholesterol
0.0001 *
Whole body weight
0.0093*$
0.0052 *+ 0.0034§ 0.0006* 0.0001§
0.0001§
*Data from first sampling period. ]-Apparent apoptotic hepatic parenchymal cells. SNegative association. §Data from second sampling period.
parameters may be sensitive sublethal indicators of stress in teleosts as well.
The most conspicuous histological difference between the two populations was the occurrence of AAHPC observed in the majority of the livers of flounder collected in Boston Harbor. These AAHPC were observed in 64% and 50% of the animals collected in Boston Harbor during the first and second collections, respectively, whereas only 3% of the animals collected from the reference site exhibited this condition. The most interesting result of the correlation analysis werethehighlysignificantassociationsbetween the presence of the AAHPC and decreased concentrations of hepatic glycogen and tissue ascorbic acid. The results of this study suggest that, for these two populations of winter flounder, the presence of AAHPC was as sensitive a biomarker of contaminantinduced stress as any of the other biochemical indices examined.
We thank John Williams, Paul Perra, and Phillip Nimeskern for their assistance during the collection and processing of samples. Rosanna Buhl and Joanne Lahey provided experl technical assistance. We are grateful for the proofreading and editorial suggestions of Marcia B. Carr and Eleanore D. Scavotto. The comments of several anonymous reviewers helped improve the focus of the manuscript significantly. This study was supported in part by Grant No. O C E 81-11949 from the National Science Foundation to Jerry M. Neff as part of the Pollutant Responses in Marine Animals (PRIMA) program.
Bayne, B. L., Livingstone, D. R., Moore, M. N. & Widdows, J. (1976). A cytochemical and a biochemical index of stress in Mytilus edulis. Mar. Pollut. Bull. 7 , 2 2 1 - 2 2 4 . Cart, R. S., Bally, M. B., Thomas, P. & Neff, J. M. (1983). Comparison of methods for determination of ascorbic acid in animal tissues. Anal. Chem. 55, 1229-1232. Cart, R. S. & Neff, J. M. (198(I). Determination of ascorbic acid in tissues of marine animals by liquid chromatography with electrochemical detection. Anal. Chem, 52, 2428-2430. Cart, R. S. & Neff, J. M. (1983). Quantitative semi-automated enzymatic assay for tissue glycogen. Cornp. Biochem. Physiol. 77B, 447-449. Haensly, W. E., Neff, J. M., Sharp, 3. R., Morris, A. C., Bedgood, M. F. & Boehm, P. D. (1982). Histopathology of Pleuronectes platessa from Aber Wrac'h and Abet Benoit. Brittany, France: Long-term effects of the Amoco Cadiz crude oil spill. J. l%'h Diseases 5, 365391.
Volume 22/Number 2/February 1991 Holland, D. L. & Gabbott, P. A. (1971). A micro-analytical scheme for the determination of protein, carbohydrate, lipid and RNA levels of marine invertebrate larvae. J. Mar. Biol. Assoc. U.K. 51,659-668. Howe, A. B. & Coates, P. G. (1975). Winter flounder movements, growth and mortality off Massachusetts. Trans. Amer. Fish. Soc. 104, 13-29. Jeffries, H. P. (1972). A stress syndrome in the hard clam, Mercenaria mercenaria. J. Invert. Path. 20,242-287. Lech, J. J. & Vodicnik, M. J. (1984). Biotransformation of chemicals by fish: an overview. In Use of Small Fish Species in Carcinogenicity Testing, pp. 355-357. National Cancer Institute Monograph 65. NIH Publication No. 84-2653, Bethesda, Maryland. Livingstone, D. R. (1985). Biochemical measurements. In The Effects of Stress and Pollution on Marine Animals (B. L. Bayne, ed.), pp. 81132. Praeger Publishers, New York. Murchelano, R. A. & Wolke, R. E. (1985). Epizootic carcinoma in the winter flounder, Pseudopleuronectes americanus. Science 228, 587589. Neff, J. M. & Haensly, W. E. (1982). Long-term impact of the Amoco Cadiz crude oil spill on oysters Crassostrea gigas and plaice Pleuronectes platessa from Abet Benoit and Aber Wrac'h, Brittany, France. I. Oyster histopathology. II. Petroleum contamination and biochemical indices of stress in oysters and plaice. In EcologicalStudy of the Amoco Cadiz Oil Spill pp. 269-328. Report of the NOAACNEXO Joint Commission. U.S.D.C./NOAA, Washington. NOAA (National Oceanic and Atmospheric Administration) (1987). The National Status and Trends Program for marine environmental quality. Progress report and preliminary assessment of the findings of the 1984 benthic surveillance project. NOAA, Ocean Assessment Division, Office of Oceanography and Marine Assessment, National Ocean Service, Rockville, Maryland. Pfeiffer, R., Karol, R., Korpi, J., Burgoyne, R. & McCourt, D. (1983). Practical application of HPLC to amino acid analysis. Amer. Lab. 15, 78-87. Roesijadi, G., Anderson, J. W. & Giam, C. S. (1976). Osmoregulation
of the grass shrimp Palaemonetes pugio exposed to polychlorinated biphenyls (PCBs). II. Effect on free amino acids of muscle tissue. Mar. Biol. 38,357-363. Saila, S. B. (1961). A study of winter flounder movements. Limnol. Oceanogr.6, 292-298. SAS Institute, Inc. (1982). SAS user's guide: Basics, 1982 Edition. SAS Institute, Cary, North Carolina. Stegeman, J. J., Teng, F. Y. & Snowberger, E. A. (1987). Induced cytochrome P450 in winter flounder Pseudopleuronectes americanus from coastal Massachusetts evaluated by catalytic assay and monoclonal antibody probes. Can. J. Fish. Aquat. Sci. 44, 1270-1277. Thomas, R, Bally, M. & Neff, J. M. (1982a). Ascorbic acid status of mullet (Mugil cephalus Linn.) exposed to cadmium. J. Fish Biol. 20, 183-196. Thomas, P., Carr, R. S, & Neff, J. M. (1981a). Biochemical stress responses of mullet Mugil cephalus and polychaete worms Neanthes virens to pentachlorophenol. In Biological Monitoring of Marine Pollutants" (F. J. Vernberg, A. Calabrese, F. P. Thurberg & W. B. Vernberg, eds), pp. 73-103. Academic Press, New York. Thomas, R, Wofford, H. W. & Neff, J. M. (1981b). Biochemical stress responses of striped mullet (Mugil cephalus L.) to fluorene analogs. Aquat. Toxicol. 1,329-343. Thomas, R, Wofford, H. W. & Neff, J. M. (1982b). Effect of cadmium on glutathione content of mullet (Mugil cephalus) tissues. In Physiological Mechanisms of Marine Pollutant Toxicity (F. J. Vernberg, A. Calabrese, E P. Thurberg & W. B. Vernberg, eds), pp. 109-125. Academic Press, New York. Thomas, P., Woodin, B. & Neff, J. M. (1980). Biochemical responses of the striped mullet Mugil cephalus to oil exposure. I. Short-term responses--alteration of plasma cortisol, glucose, and cholesterol titers. Mar. Biol. 59, 141-149. Tyler, A. V. & Dunn, R. S. (1976). Ration, growth, and measures of somatic and organ condition in relation to meal frequency in winter flounder, Pseudopleuronectes americanus, with hypothesis regarding population homeostasis. J. Fish. Res. Board ('an. 33, 63-75.
MarinePollutionBulletin,Volume22. No. 2. pp. 67 72. 1991. Primedin GrealBritain.
0025 326X/91S3.00+(H)0 © 19g] PergamonPressplc
Trace Metal Levels in Sharks from British and Atlantic Waters PHILIP VAS D e p a r t m e n t o f B i o l o g i c a l Sciences, University o f Salford, Salford, M 5 4 W T , U K
Tissue samples from 46 sharks representing ten species and four locations were analysed for seven trace metals ( C u , M n , Fe, Cd, N i , P b , Z n ) using atomic absorption spectrophotometry. Observed concentration ranges were: C u 0 . 0 2 - 6 8 I~g g - t ; M n 0 . 0 2 - 9 lag g - t ; Fe 0 . 2 >200
og g-t; Cd <0.02-2.6
lag g - l ; N i 0 , 0 2 - 1 1 . 5
lag
g-i; Pb 0 . 1 - 1 3 og g-t; and Zn < 0 . 1 - 9 0 I~g g-t. The highest metal concentrations were observed in 'inshore demersal species', lowest were in 'offshore pelagic species'. Cu was of greater importance to demersai sharks, while a similar relationship was observed for Ni in pelagic sharks. These differences were believed to be a function of diet. Suggestions for future work are made.
M a n y p o l l u t a n t s s u c h as D D T ( M e t c a l f , 1 9 7 7 ) a n d H g ( R a t k o w s k y et al., 1 9 7 5 ; M a r c o v e c c h i o et al., 1986) h a v e b e e n o b s e r v e d to a c c u m u l a t e w i t h i n t h e h i g h e s t levels o f m a r i n e f o o d chains. T r a c e m e t a l s o t h e r t h a n H g m a y s i m i l a r l y a c c u m u l a t e in s u c h o r g a n i s m s ( E i s l e r , 1984), p a r t i c u l a r l y in l o n g - l i v e d s p e c i e s s u c h as t u n a a n d m a r l i n ( F 6 r s t n e r & W i t t m a n , 1981, p. 310). S h a r k s , as p r e d a t o r s at t h e t o p o f m a n y s u c h f o o d chains, m a y t h u s r e p r e s e n t sites f o r t h e b i o l o g i c a l a c c u m u l a t i o n o f
such pollutants (De Metrio et al., 1982). Because the c o m m e r c i a l i m p o r t a n c e o f s h a r k s is c u r r e n t l y i n c r e a s i n g ( F A O Y e a r b o o k s , 1 9 7 7 - 8 8 ) a n d b e c a u s e s u c h poll u t a n t s r e p r e s e n t a p o t e n t i a l t h r e a t to h u m a n h e a l t h ( F A O / W H O , 1 9 7 2 ; F l i c k et al., 1971), it is i m p o r t a n t to 67
Rand, G. M. & Petrocelli, S. R. (1985). Fundamentals of Aquatic 7oxicology.Hemisphere Publishing Corp., Washington, D.C. Singer, M. M., Smalheer, D. L. & Tjeerdema, R. S. (1990a). A simple continuous-flow toxicity test system for microscopic life stages of aquatic organisms. Wat. Res. 24,899-903. Singer, M. M., Smalheer, D. L., Tjeerdema, R. S. & Martin, M. (1990b). Continuous Delivery of Various Concentrations of Material in Water. Toxicity of an oil dispersant to the early life stages of four California PHS Publication No. 999-WP-23, Washington, D.C. marine species. Environ. ToxicoL Chem. 9, 1389-1397. N RC (National Research Council) (1989). Using Oil Spill Dispersants Singer, M. M., Smalheer, D. L., Tjeerdema, R. S. & Martin, M. (1990c). on lheSea. National Academy Press, Washington, D.C. Effects of spiked-exposure to an oil dispersant on the early life stages of four marine species. Environ. Toxicol. Chem. (in press).
Marine I'ollutiotl B,//etin. Volume22, N~ 2, pp 61 liT. 1991.
326X 91 $3.00+O.0() © 19~)1PergamnnPressplc
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Field Assessment of Biomarkers for Winter Flounder ROBERT SCOTT CARR*, ROBERT E. HILLMAN and JERRY M. NEFF+
Battelle, Ocean Sciences, 397 Washington Street, Duxbury, MA 02332, USA Present addresses: *US Fish and Wildlife Service, NFCR Field Research Station, Corpus Christi State University, Campus Box 315, 63(1(I Ocean Drive, Corpus Christi, TX 78412, USA +Arthur D. 1,iltlc Inc., Acorn Park. Cambridge, MA 02140, USA
Winter flounder, Pseudopleuronectes americanus, from Boston Harbor near the Deer Island sewage outfall and from a nearby reference population near Plymouth Beach, Massachusetts, USA, were collected on several occasions and analysed for a variety of biochemical variables and histopathological conditions. A number of biochemical variables including hepatic and pectoral fin ascorbic acid concentrations, hepatic glycogen and lipid levels, plasma glucose concentrations, brain serotonin and norepinephrine concentrations, and the concentration and ratio of various free amino acids in muscle tissue were significantly different between the two populations. The apparent apoptotic hepatic lesions were found in the majority of the winter flounder collected from Boston Harbor and were rarely observed in animals from the reference site. Low tissue concentrations of ascorbic acid and hepatic glycogen were found to have significant statistical associations with the presence and degree of severity of these hepatic lesions,
As part of a large, multidisciplinary programme designed to develop diagnostic biochemical indices of early pollutant stress in marine animals, a field study was conducted to evaluate the feasibility of using biochemical parameters to assess the relative health of winter flounder, Pseudopleuronectes americanus, collected near the discharge of a sewage treatment plant as
compared to a population from a nearby reference site. Recent results from the National Status and Trends Program of the National Oceanic and Atmospheric Administration indicate that sediment concentrations of target polycyclic aromatic hydrocarbons (PAHs) and total polychlorinated biphenyls (PCBs) are higher at the Boston Harbor site than at any of the other 49 coastal sites examined in this national survey (NOAA, 1987). The primary objectives of the investigation were 1. to assess whether significant differences in biochemistry and histopathology exist between fish from the two sites, and 2. to determine whether any correlations exist between observed histological abnormalities and the biochemical variables. Biochemical parameters investigated in this study were hepatic and pectoral fin ascorbic acid concentrations, lipid and glycogen concentrations, plasma glucose and cholesterol levels, muscle free amino acid concentrations, and brain indoleamine and catecholamine concentrations. These parameters have been evaluated previously in several laboratory and field investigations and have been found to be useful for diagnosing pollutant stress in marine teleosts (Thomas et al., 1980, 1981a, 1981b, 1982a, 1982b; Haensly etal., 1982; Neff& Haensly, 1982). Methods
Field Sampling Winter flounder, Pseudopleuronectes americanus, were coll6cted by otter trawl off Plymouth Beach and near the Deer Island sewage outfall in Boston Harbor 61
Marine Pollution Bulletin
(Fig. 1) during the summer on two consecutive years, Three stations at different distances from the outfall were sampled in Boston Harbor during the first collection. Liver and pectoral fins were excised and frozen on dry ice. Prior to freezing, a small portion of the eyedside lobe of the liver was removed and preserved in Helly's fixative for histological examination, The trawls for the second year's sampling lasted for 30-60 s of bottom time. Only the animals that could be bled within 10 rain of the start of the trawl were included in the collection because previous studies have shown that elevated plasma glucose levels resulting from collection and handling stress are not evident within 10 min for this species (Carr, unpublished). First, blood samples were obtained from the branchial sinus, and heparinized to prevent clotting. The whole body weight was measured with a spring scale to the nearest 10 g and the standard length recorded. Liver and brain tissues were then quickly excised and stored in liquid nitrogen for later biochemical analysis. A sample from the eyed-side lobe of the liver was taken
and preserved in Helly's fixative for later histological examination. The heparinized blood samples were kept on ice and centrifuged within 60 rain of collection. The plasma was collected and stored refrigerated (4°C). A scale sample from between the eyed-side pectoral fin and the lateral line was obtained from each fish. Growth rings on the scale samples were examined microscopically to obtain age estimates for each animal. Males were differentiated from females by the presence of rough scales on the blind-side of the caudal peduncle. Surface and bottom water temperature, salinity, and dissolved oxygen concentrations were measured at both stations withtheaid of a Hydrolab.
BiochemicalAnalyses Plasma glucose and cholesterol were measured with a Beckman glucose/cholesterol analyser within 48 h of collection. Ascorbic acid concentrations in liver tissues were determined by the method of Carr & Neff (1980) using liquid chromatography with electrochemical detection. This method has been used to quantify the
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Volume 22/Number 2/February 1991
ascorbic acid content of a wide variety of animal tissues (Carr et aL, 1983). Liver glycogen was quantified by the method of Carr & Neff (1983). Total lipid concentrations in liver tissues were determined in triplicate by the method of Holland & Gabbott (1971). Concentrations of free amino acids in muscle were determined by gradient high performance liquid chromatography (HPLC) with post-column derivatization (o-pthaldehyde) and fluorometric detection, Tissue samples were homogenized in 7% trichloroacetic acid (TCA) as described by Roesijadi el al. (1976). TCA supernatants were extracted three times with diethyl ether, then lyophilized. Lyophilized samples were redissoved in 0.1 N HC1 just prior to analysis. A Waters Associates gradient liquid chromatograph with a WISP autosampler, system controller, data module, and fluorometric detector was employed. The chromatographic conditions have been described previously (Pfeiffer et al., 1983). Norleucine was used as the internal standard, Catecholamines and indoleamines were determined by ion-pairing HPLC with electrochemical detection, Frozen brains were homogenized in 250 mM HCIO 4. The homogenate was centrifuged at 27 000 g for 3() rain and the supernatant analysed without further extraction or cleanup. An isocratic Waters Associates system was employed with an amperometric detector (Bioanalytical Systems, LC-4) using a thin-layer glassy carbon electrode (+800 mV vs. Ag/AgC1). A Partisil SAX guard column in conjunction with a Resolve C m column provided excellent separation and resolution of the components of interest. The mobile phase was 0.1 M KH2PO,:methanol, 85:15 (pH 3.76) with heptane sulphonic acid (Waters Assoc.) added as the ion-pairing reagent. Serotonin, the last component of interest, eluted at a flow rate of 0.5 ml rain -~ approximately 15 rain after injection. Dihydroxybenzylamine (DHBA) was used as the internal standard, Histology
Liver tissues were excised immediately after blood collection and individually placed in labelled plastic cassettes with a code number so that the evaluator did not know from which population the tissues were derived. The samples were fixed in Helly's fixative for 24 h and then rinsed in running water for 16-24 h to remove excess fixative. The samples were stored in 70% ethanol until preparation for final processing. The tissues were impregnated with Paraplast Plus in an American Optical TP8000 tissue processor and sectioned at 4 ~tm. Sections were stained with hematoxylin and eosin or Whipf's polychrome stain and mounted in Histoclad.
analysed by Spearman correlation analysis with the aid of the Statistical Analysis System (SAS Institute, Inc. 1982).
Results FirstColleetion
The average weight (±SD) and age (as determined by scale growth bands) of flounder from the Boston Harbor and Plymouth Beach sites were 325 + 54 g, 3, years, and 447+ 90 g, 4 years, respectively. Liver glycogen and ascorbic acid were the only biochemical measurements made for the first collection. The mean concentration of ascorbic acid in livers and blind-side pectoral fins, and mean hepatic glycogen concentrations were significantly lower in the Boston Harbor animals than in animals from the reference site (Fig. 2). The primary histological difference between the winter flounder collected from Boston Harbor in the first sampling and those collected during the same period off Plymouth Beach (Table 1) was the high prevalence of apparent apoptotic hepatic parenchymal cells (AAPHC, Fig. 3)in the Boston Harbor fish. Second Collection
The standard length of the winter flounder collected ranged 20-31 cm. The average weight (±SD) and age of the animals was 270 + 76 g, 2.6 years and 373 + 126 g, 3.2 years for Boston Harbor and Plymouth Beach animals, respectively. The bottom water temperature, dissolved oxygen concentration, and salinity were 16.5°C, 9.4 mg 1-~, and 32%0 for Boston Harbor and I4.5°C, 10.2 mgl-l, and 32%ofor Plymouth Beach. Biochemical analyses performed on liver tissue from the second collection included ascorbic acid, glycogen, and lipid concentrations (Table 2). As in the first collection, the mean hepatic ascorbic acid concentration was lower in the Boston Harbor animals than in animals from the reference site, but the variability was too great for the difference to be statistically significant. Hepatic glycogen and lipid concentrations of winter flounder from Boston Harbor were significantly lower than levels in animals from the reference site. Plasma glucose levels of the Boston Harbor animals were significantly lower than the levels of the reference animals (Table 3). Neurotransmitters measured in brain tissues in greatest concentration were norepinephrine and serotonin (5-hydroxytryptamine). The level of both of these neurotransmitters was significantly lower in the brains of winter flounder from Boston Harbor than in animals from Plymouth Beach (Table 3). In general, the muscle concentration of free amino acids was higher in the Boston Harbor animals (Table TABLE1
Statistical Analyses
Significant differences between population means for the various parameters were determined by the Student's t-test where applicable, and by the MannWhitney U-test where the data did not fulfill the requirements for parametric analysis. The descriptive information, biochemical, and histopathological data for each individual from the two populations were
Prevalence of apparent apoptotic hepatic parenchymal cells (AAHPC) in winter flounder (Pseudopleuronectes americanus) collected from Plymouth Beachand BostonHarbor.
iv First collection Second collection
13 20
Percentage of fish with AAHPC Plymouth Beach N BostonHarbor
8%
14
64%
0%
20
50%
63
Marine Pollution Bulletin
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0
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ASCORBIC ACID
GLYCOGEN
Fig. 2 Mean ( ± S E M , n >- 13) pectoral fin ascorbic acid and liver ascorbic acid and glycogen for winter flounder from the first collection. The difference between the two populations was highly significant (ct < 0.005, U-Test) for all parameters except eyed-side pectoral fin. TABLE 2
Mean ( _+SEM) hepatic ascorbic acid, glutathione, glycogen and lipid concentrations for winter flounder.
Site
N
Ascorbic Acid (~g g-~)
Glutathione (~tgg ])
Glycogen (mgg -~)
Total lipids (mgg -~)
Boston Harbor Plymouth Beach
20 20
11.2 _+2.5 13.9 _+2.2
20.1 _+ 1.9 20.5 _+2.2
28.1 ± 3.7* 45.8 _+4.2
17.5 -+ 0.25f 26.4 _+ 1.17
*~ < 0.001 (U-test)
+c~< 0.005 (U-test)
N = Number of animals analysed.
TABLE 3 Mean (± SEM) plasma glucose and cholesterol and brain norepinephrine and serotonin concentrations for winter flounder.
Site
N
Glucose (mgdl-')
N
Cholesterol (mgdl-')
N
Boston Harbor Plymouth Beach
20 20
23.3 -+ 1.5" 31.8 _+ 1.6
17 20
234.9 _+ 18.1 2(t8.0 _+ 13.0
20 19
*c~ < (I.001 (U-test)
tct< 0.025 (U-test)
"a < 0.005 (U-test)
4). This elevation was statistically significant for only glycine and serine. The serine+ threonine index and the taurine/glycine ratio were both significantly different between the two populations, The high prevalence of AAHPC of fish collected in Boston Harbor during the second year was again the major histological difference between the two populations (Table 1). Fifty percent of the fish collected from Boston Harbor during the second sampling period had aggregates of these apoptotic cells, while none of these cells were found in flounder collected off Plymouth Beach. 64
Norepinephrine (~tgg ~) 0.40 _+0.1 (1~ 1.03 +_0.22
N
Serotonin (~,gg ')
20 20
0.48 _+0.09~ (I.86 _+0,06
N = Number of animals analysed.
CorrelationAnalyses The results of the Spearman correlation analysis of the biochemical histological, and descriptive data are shown in Table 5. A number of significant correlations were observed when the data from both populations were analysed together. The most interesting correlations were those showing strong associations between levels of liver glycogen and tissue ascorbic acid, and the presence of AAHPC. Plasma cholesterol levels and hepatic glycogen concentrations were significantly associated with the weight and age of the animals. This relationship may account for some of the differences
Volume 22/Number 2/February 1991
Fig. 3 Apparent apoptotic hepatic parenchymalcells (arrows) found in majority of winter flounder collected from Boston Harbor. H&E x 100. observed between the two populations for these variables, because the animals from the reference site tended to be older and larger than those from Boston Harbor. There were no significant associations between the sex or hepatic P-450 levels (Stegeman et al., 1987) and any of the descriptive, biochemical, or histological data, and there was no difference in condition factor (length/weight) between the populations, Discussion Significant differences exist for a variety of biochemical parameters and the occurrence and severity of A A H P C between the two populations investigated. Results of a 10-year tagging study showed that the movement of winter flounder from these two sites was localized (Howe & Coates, 1975) and it is known that
winter flounder return to the same area to spawn each year (Saila, 1961). Because these populations are believed to be relatively discreet (Howe & Coates, 1975) and the water quality parameters (temperature, salinity, dissolved oxygen) essentially identical between the two sites, one might speculate that the biochemical and histological differences observed between the two populations are a result of conditions arising from the discharge of primary effluent and sewage sludge at the Boston Harbor site. The high incidence of disease reported for winter flounder from Boston Harbor (Murchelano & Wolke, 1985; NOAA, 1987) further supports this contention. In the present study, both the hepatic glycogen and lipid concentrations of the Boston Harbor animals were significantly lower than the levels in animals from the reference site. When winter flounder are stressed, their 65
Marine Pollution Bulletin TABLE 4
TABLE 5
Mean (±SEM) free amino acid (and ammonia) concentrations in muscle tissue (~tg mg -h) and amino acid indices for winter flounder.
Probability values for significant associations as determined by Spearman correlation analysis for data from winter flounder collected from Boston Harbor and Plymouth Beach combined.
Taurine Aspartic Acid Threonine Serine Glutamine Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Ammonia Arginine Taurine/Glycine Serine _+ Threonine
N
Boston Harbor
N
Plymouth Beach
19 19 19 19 19 19 19 19 14 19 19 17 19 19 19 19 6 19 19
3885 ± 211.8 106.8 ± 52.7 264.0 +- 30.4 311.9+46.2" 145.5 ± 4 2 . 7 2886"+ 552.9t 792.5 "+ 281.0 60.2 ± 10.9 31.9 ± 9.3 43.3 + 10.5 98.4 +_36.7 136.1 ± 9 7 . 7 6 1 . 7 ± 12.7 349.4± 29.1 136.9 ± 15.3 1730 ± 75.5 357.8 ± 251.8 1.99 ± 0.31" 576.0 ± 65.9*
20 20 20 20 20 20 20 20 10 20 20 11 20 20 20 20 13 20 20
3890 ± 174.4 49.7 _+7.6 147.7 ± 13.7 147.7+21.4 115.9"+13.7 779.6 ± 100.1 379.3 ± 27.5 64.7 ± 10.2 16.7 ± 1.5 48.9 ± 10.6 74.3 ± 13.4 26.6"+6.8 4 2 . 7 ± 5.5 431.9±23.0 203.7 ± 22.4 1826 ± 135.2 110.4"+12.6 5.88 ± 0.46 295.5 _+ 33.6
• ¢~<0.005(U-test). tot < 0.05 (U-test). N = Number of animals analysed,
adaptive reproductive strategy is to sacrifice egg production for somatic growth and homeostatic maintenance (Tyler & Dunn, 1976). Reduced carbohydrate and lipid reserves are also indicative of lowered energy reserves. The significant decrease in brain norepinephrine and serotonin in Boston Harbor flounder also suggests that the neurological and endocrine systems were depressed in these animals. Brain levels of epinephrine and serotonin decreased in mullet Mugil cephalus exposed to sublethal concentrations of dibenzofuran (Thomas etal., 1981b). The two amino acid indices, the taurine/glycine ratio and the serine + threonine index, showed a significant difference between the two populations. The taurine/ glycine ratio, which has been used most often as a stress index with molluscs and crustaceans, usually increases in stressed animals due to a decrease in glycine, with taurine levels remaining relatively constant (Jeffries, 1972; Bayne et aL, 1976). The increased glycine concentration in muscle tissue of the Boston Harbor animals resulted in a taurine/glycine ratio that was 34% of the ratio observed for the Plymouth Beach animals, As with invertebrates, the taurine levels remained unchanged. Glycine has been shown to function as a conjugate in Phase II biotransformation reactions in flounder (Lech & Vodicnik, 1984), which suggests t h a t the increased glycine levels observed in tissues of Boston Harbor winter flounder may have been induced
in response to contaminant exposure, The serine+ threonine index, which previously has been used almost exclusively with bivalves, normally decreases during stress (Livingstone, 1985). The sum of serine+threonine was almost twice as great in the muscle tissue of Boston Harbor animals as in the reference population. Although the changes in these indices are the opposite of what has been observed in invertebrates, the fact that highly significant differences were observed between the two populations for both indices suggests that these 66
Liver ascorbic acid Blind-side pectoral fin ascorbic acid AAHPC~
Blind-side pectoral fin ascorbic acid
Liver glycogen
Plasma cholesterol
0.0001 *
Whole body weight
0.0093*$
0.0052 *+ 0.0034§ 0.0006* 0.0001§
0.0001§
*Data from first sampling period. ]-Apparent apoptotic hepatic parenchymal cells. SNegative association. §Data from second sampling period.
parameters may be sensitive sublethal indicators of stress in teleosts as well.
The most conspicuous histological difference between the two populations was the occurrence of AAHPC observed in the majority of the livers of flounder collected in Boston Harbor. These AAHPC were observed in 64% and 50% of the animals collected in Boston Harbor during the first and second collections, respectively, whereas only 3% of the animals collected from the reference site exhibited this condition. The most interesting result of the correlation analysis werethehighlysignificantassociationsbetween the presence of the AAHPC and decreased concentrations of hepatic glycogen and tissue ascorbic acid. The results of this study suggest that, for these two populations of winter flounder, the presence of AAHPC was as sensitive a biomarker of contaminantinduced stress as any of the other biochemical indices examined.
We thank John Williams, Paul Perra, and Phillip Nimeskern for their assistance during the collection and processing of samples. Rosanna Buhl and Joanne Lahey provided experl technical assistance. We are grateful for the proofreading and editorial suggestions of Marcia B. Carr and Eleanore D. Scavotto. The comments of several anonymous reviewers helped improve the focus of the manuscript significantly. This study was supported in part by Grant No. O C E 81-11949 from the National Science Foundation to Jerry M. Neff as part of the Pollutant Responses in Marine Animals (PRIMA) program.
Bayne, B. L., Livingstone, D. R., Moore, M. N. & Widdows, J. (1976). A cytochemical and a biochemical index of stress in Mytilus edulis. Mar. Pollut. Bull. 7 , 2 2 1 - 2 2 4 . Cart, R. S., Bally, M. B., Thomas, P. & Neff, J. M. (1983). Comparison of methods for determination of ascorbic acid in animal tissues. Anal. Chem. 55, 1229-1232. Cart, R. S. & Neff, J. M. (198(I). Determination of ascorbic acid in tissues of marine animals by liquid chromatography with electrochemical detection. Anal. Chem, 52, 2428-2430. Cart, R. S. & Neff, J. M. (1983). Quantitative semi-automated enzymatic assay for tissue glycogen. Cornp. Biochem. Physiol. 77B, 447-449. Haensly, W. E., Neff, J. M., Sharp, 3. R., Morris, A. C., Bedgood, M. F. & Boehm, P. D. (1982). Histopathology of Pleuronectes platessa from Aber Wrac'h and Abet Benoit. Brittany, France: Long-term effects of the Amoco Cadiz crude oil spill. J. l%'h Diseases 5, 365391.
Volume 22/Number 2/February 1991 Holland, D. L. & Gabbott, P. A. (1971). A micro-analytical scheme for the determination of protein, carbohydrate, lipid and RNA levels of marine invertebrate larvae. J. Mar. Biol. Assoc. U.K. 51,659-668. Howe, A. B. & Coates, P. G. (1975). Winter flounder movements, growth and mortality off Massachusetts. Trans. Amer. Fish. Soc. 104, 13-29. Jeffries, H. P. (1972). A stress syndrome in the hard clam, Mercenaria mercenaria. J. Invert. Path. 20,242-287. Lech, J. J. & Vodicnik, M. J. (1984). Biotransformation of chemicals by fish: an overview. In Use of Small Fish Species in Carcinogenicity Testing, pp. 355-357. National Cancer Institute Monograph 65. NIH Publication No. 84-2653, Bethesda, Maryland. Livingstone, D. R. (1985). Biochemical measurements. In The Effects of Stress and Pollution on Marine Animals (B. L. Bayne, ed.), pp. 81132. Praeger Publishers, New York. Murchelano, R. A. & Wolke, R. E. (1985). Epizootic carcinoma in the winter flounder, Pseudopleuronectes americanus. Science 228, 587589. Neff, J. M. & Haensly, W. E. (1982). Long-term impact of the Amoco Cadiz crude oil spill on oysters Crassostrea gigas and plaice Pleuronectes platessa from Abet Benoit and Aber Wrac'h, Brittany, France. I. Oyster histopathology. II. Petroleum contamination and biochemical indices of stress in oysters and plaice. In EcologicalStudy of the Amoco Cadiz Oil Spill pp. 269-328. Report of the NOAACNEXO Joint Commission. U.S.D.C./NOAA, Washington. NOAA (National Oceanic and Atmospheric Administration) (1987). The National Status and Trends Program for marine environmental quality. Progress report and preliminary assessment of the findings of the 1984 benthic surveillance project. NOAA, Ocean Assessment Division, Office of Oceanography and Marine Assessment, National Ocean Service, Rockville, Maryland. Pfeiffer, R., Karol, R., Korpi, J., Burgoyne, R. & McCourt, D. (1983). Practical application of HPLC to amino acid analysis. Amer. Lab. 15, 78-87. Roesijadi, G., Anderson, J. W. & Giam, C. S. (1976). Osmoregulation
of the grass shrimp Palaemonetes pugio exposed to polychlorinated biphenyls (PCBs). II. Effect on free amino acids of muscle tissue. Mar. Biol. 38,357-363. Saila, S. B. (1961). A study of winter flounder movements. Limnol. Oceanogr.6, 292-298. SAS Institute, Inc. (1982). SAS user's guide: Basics, 1982 Edition. SAS Institute, Cary, North Carolina. Stegeman, J. J., Teng, F. Y. & Snowberger, E. A. (1987). Induced cytochrome P450 in winter flounder Pseudopleuronectes americanus from coastal Massachusetts evaluated by catalytic assay and monoclonal antibody probes. Can. J. Fish. Aquat. Sci. 44, 1270-1277. Thomas, R, Bally, M. & Neff, J. M. (1982a). Ascorbic acid status of mullet (Mugil cephalus Linn.) exposed to cadmium. J. Fish Biol. 20, 183-196. Thomas, P., Carr, R. S, & Neff, J. M. (1981a). Biochemical stress responses of mullet Mugil cephalus and polychaete worms Neanthes virens to pentachlorophenol. In Biological Monitoring of Marine Pollutants" (F. J. Vernberg, A. Calabrese, F. P. Thurberg & W. B. Vernberg, eds), pp. 73-103. Academic Press, New York. Thomas, R, Wofford, H. W. & Neff, J. M. (1981b). Biochemical stress responses of striped mullet (Mugil cephalus L.) to fluorene analogs. Aquat. Toxicol. 1,329-343. Thomas, R, Wofford, H. W. & Neff, J. M. (1982b). Effect of cadmium on glutathione content of mullet (Mugil cephalus) tissues. In Physiological Mechanisms of Marine Pollutant Toxicity (F. J. Vernberg, A. Calabrese, E P. Thurberg & W. B. Vernberg, eds), pp. 109-125. Academic Press, New York. Thomas, P., Woodin, B. & Neff, J. M. (1980). Biochemical responses of the striped mullet Mugil cephalus to oil exposure. I. Short-term responses--alteration of plasma cortisol, glucose, and cholesterol titers. Mar. Biol. 59, 141-149. Tyler, A. V. & Dunn, R. S. (1976). Ration, growth, and measures of somatic and organ condition in relation to meal frequency in winter flounder, Pseudopleuronectes americanus, with hypothesis regarding population homeostasis. J. Fish. Res. Board ('an. 33, 63-75.
MarinePollutionBulletin,Volume22. No. 2. pp. 67 72. 1991. Primedin GrealBritain.
0025 326X/91S3.00+(H)0 © 19g] PergamonPressplc
Trace Metal Levels in Sharks from British and Atlantic Waters PHILIP VAS D e p a r t m e n t o f B i o l o g i c a l Sciences, University o f Salford, Salford, M 5 4 W T , U K
Tissue samples from 46 sharks representing ten species and four locations were analysed for seven trace metals ( C u , M n , Fe, Cd, N i , P b , Z n ) using atomic absorption spectrophotometry. Observed concentration ranges were: C u 0 . 0 2 - 6 8 I~g g - t ; M n 0 . 0 2 - 9 lag g - t ; Fe 0 . 2 >200
og g-t; Cd <0.02-2.6
lag g - l ; N i 0 , 0 2 - 1 1 . 5
lag
g-i; Pb 0 . 1 - 1 3 og g-t; and Zn < 0 . 1 - 9 0 I~g g-t. The highest metal concentrations were observed in 'inshore demersal species', lowest were in 'offshore pelagic species'. Cu was of greater importance to demersai sharks, while a similar relationship was observed for Ni in pelagic sharks. These differences were believed to be a function of diet. Suggestions for future work are made.
M a n y p o l l u t a n t s s u c h as D D T ( M e t c a l f , 1 9 7 7 ) a n d H g ( R a t k o w s k y et al., 1 9 7 5 ; M a r c o v e c c h i o et al., 1986) h a v e b e e n o b s e r v e d to a c c u m u l a t e w i t h i n t h e h i g h e s t levels o f m a r i n e f o o d chains. T r a c e m e t a l s o t h e r t h a n H g m a y s i m i l a r l y a c c u m u l a t e in s u c h o r g a n i s m s ( E i s l e r , 1984), p a r t i c u l a r l y in l o n g - l i v e d s p e c i e s s u c h as t u n a a n d m a r l i n ( F 6 r s t n e r & W i t t m a n , 1981, p. 310). S h a r k s , as p r e d a t o r s at t h e t o p o f m a n y s u c h f o o d chains, m a y t h u s r e p r e s e n t sites f o r t h e b i o l o g i c a l a c c u m u l a t i o n o f
such pollutants (De Metrio et al., 1982). Because the c o m m e r c i a l i m p o r t a n c e o f s h a r k s is c u r r e n t l y i n c r e a s i n g ( F A O Y e a r b o o k s , 1 9 7 7 - 8 8 ) a n d b e c a u s e s u c h poll u t a n t s r e p r e s e n t a p o t e n t i a l t h r e a t to h u m a n h e a l t h ( F A O / W H O , 1 9 7 2 ; F l i c k et al., 1971), it is i m p o r t a n t to 67