A comparative study of the differential blood cell counts of winter flounder (Pseudopleuronectes americanus) collected in New England

Major alterations in the distribution of peripheral blood cells were observed among winter flounder (Pseudopleuronectes americanus) from Martha's Vineyard (MV), East Cape Cod Bay (ECCB), Fox Island (FI) (Narragansett Bay), Central Long Island Sound (CLIS), Gaspee Point (GP) (Narragansett Bay), Black Rock Harbor (BRH) (Bridgeport, CT), New Bedford Harbor (NBH) and Quincy Bay (QB) (Boston Harbor). Collection locations ranged from relatively uncontaminated offshore (MV) and nearshore (ECCB) sites to the more contaminated estuarine (FI, CLIS) and urban embayments (GP, BRH, NBH, QB). Differential diagnosis demonstrated a shift in lymphocyte counts from 28% (MV) to 54% (QB) that increased with the incidence of liver lesions in relation to known and suspected levels of sediment chemical contamination. A significant change in the ratio of circulating immature to mature erythrocytes was observed in flounder collected from Boston Harbor. Immature erythrocytes comprised 18% of the total erythrocyte population of fish from QB in comparison to 6% observed in flounder from ECCB.     

Comparative histopathological effects of chemically contaminated sediment on marine organisms

Comparative pathological effects in three species of fish and six species of invertebrates were investigated using a chemically contaminated sediment from Black Rock Harbor (BRH), Connecticut, USA. Substances contained in BRH sediment are known to be genotoxic, carcinogenic, co-carcinogenic and tumor-promoting. Proliferative lesions were observed in winter flounder (Pseudopleuronectes americanus) external, oral and esophageal epithelial surfaces, renal vascular and nephroblastic elements, and the pancreatic islets. Islet lesions included cystic adenomas and a diffuse islet proliferative condition, nesidioblastosis. In addition, pathology was enhanced when winter flounder were fed mussels (Mytilus edulis) previously exposed to BRH sediment. Neoplastic lesions developed in kidney tubule epithelia, gills, some regions of the gastrointestinal tract, neural elements and heart of oysters (Crassostrea virginica) exposed to BRH sediment for 30 days in the laboratory. In addition, renal carcinomas in three oysters had metastasized to the viseral ganglion. Lesions were also found in oysters exposed in situ in BRH and Long Island Sound for 30 or 36 days. Digestive diverticula, gills and kidneys appeared to be the prime organs affected in mussels, soft-shelled clams (Mya arenaria) and oysters exposed to BRH sediment. Mucous cell activity increased greatly in corals (Astrangia densa) while no microscopic lesions were detectable in lobster (Homarus americanus) exposed in the laboratory. Myxomas were also found in the hearts of < 5% of the mussels exposed in the laboratory.     

Nuclear changes in blood, early liver anomalies and hepatocellular cancers in flounder (Platichthys flesus L.) as prognostic indicator for a higher cancer risk?

Nuclear changes (micronuclei) in blood are a well-established biomarker of toxic injury and related genotoxicity in mussel, fish and humans, as well. Yet, the relationship between the frequencies of micronuclei in blood and nuclear changes in liver cells itself and the higher potential risk to develop liver cancer is not yet fully implemented. We analysed and diagnosed macroscopic liver changes and H&E and Feulgen stained histological liver sections (2 microm of thickness) together with blood smears (stained with Sytox Green) from same individuals in flounder caught along a well-known contaminant gradient in the North Sea. Results showed an increase in micronuclei frequencies in blood at the more contaminated sites and higher frequencies of macroscopically visible neoplastic liver changes. In livers of the same individuals, nuclear changes were diagnosed in early to degenerative liver lesion, extra-focal tissue surrounding neoplastic lesion and various stages during cancer progression from early neoplastic foci, benign adenomas and malignant carcinomas. The aim of this approach is to expand diagnostic criteria, well-established in the frame of BEQUALM to nuclear changes which might help diagnosis of malignancies in doubtful cases.     

Identification of a c-Ki-ras oncogene in a neoplasm isolated from winter flounder

It has been shown that a high incidence of hepatomas are present in winter flounder (Pseudopleuronectes americanus) obtained from Boston Harbor. It has been suggested that this may be a consequence of locally high levels of polycyclic aromatic hydrocarbons found in the sediment. The purpose of this study was to determine whether transforming DNA sequences (oncogenes) could be identified in liver neoplasms isolated from feral fish and to study their relationship to their corresponding proto-oncogenes. The ultimate aim of this study is to characterize novel mutations in oncogenes derived from these hepatomas to correlate these genetic changes with chemical exposure history. Genomic DNA was isolated from liver neoplasms and transfected into NIH3T3 mouse fibroblasts to assay for the formation of transformed foci. DNA was prepared from transformed foci and analyzed by Southern blot hybridization to viral DNA probes specific for c-Ki-ras and c-Ha-ras DNA sequences. A c-Ki-ras oncogene was identified in a transformant derived from one of the two tumors assayed. Comparison of c-Ki-ras DNA sequences of tumor and tumor-derived transformants indicate that the activated oncogene in the transformant is of flounder origin. We are currently analyzing the flounder oncogene for activating point mutations by primer-directed enzymatic amplification and direct sequence analysis.     

In-vitro metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by liver and intestinal mucosa homogenates from the winter flounder (Pseudopleuronectes americanus)

Freshly prepared homogenates were used to assess the relative ability of winter flounder (Pseudopleuronectes americanus) liver and intestinal mucosal cells to metabolize the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) and its proximate carcinogenic metabolite, BaP-7,8-dihydrodiol (7,8-Diol). Data obtained from homogenates prepared from fish previously fed β-naphthoflavone (BNF) indicated that both tissues had similar abilities to metabolize either BaP or 7,8-Diol on a per gram of protein basis. Metabolite profiles produced indicate that water-soluble metabolite formation is favored at low doses. These findings support the hypothesis that the intestine plays an important role in first-pass metabolism of dietary carcinogens in the winter flounder.     

Endogenously-mediated, pretranslational suppression of cytochrome P4501A in PCB-contaminated flounder

Gonadally mature fish display strong sex-related differences in the content and activity of P4501A, the major polynuclear aromatic hydrocarbon-inducible P450 form in teleosts. Such differences appear related to plasma levels of the female sex steroid, estradiol (E₂); however, neither the mechanism of estradiol suppression of P4501A nor the capacity for hormonal regulation to overcome P4501A induction by high concentrations of potent inducers are known. Gonadally mature flounder (Pseudopleuronectes americanus) were collected from Fox Island (FI), Rhode Island, a reference site, and New Bedford Harbor (NB), Massachusetts, a site highly contaminated with polychlorinated biphenyls (PCBs). Differences in flounder P4501A expression were determined at the level P4501A catalytic activity (measured as ethoxyresorufin-O-deethylase, EROD), P4501A protein content (immunoquantitated), and P4501A mRNA content (by Northern blot) as they relate to sex, reproductive status, and hepatic PCB content. Our results confirm that suppression of P4501A in gonadally mature female fish is probably due, at least in part, to elevated E₂ titers, and demonstrate that such suppression occurs at a pretranslational level and, further, that endogenous regulation of P4501A expression can ‘override’ exogenous regulation by even high concentrations of P4501A inducers.     

Long-term monitoring using resident and caged mussels in Boston Harbor yield similar spatial and temporal trends in chemical contamination

Measurements of chemical contaminants in caged (transplanted) and resident mussel populations have become a routine tool for monitoring and assessing the status and trends of coastal water quality. However, few long-term data sets are available to assess the comparability and efficacy of these two monitoring approaches. Three long-term independent data sets exist for Boston Harbor: the National Mussel Watch program has analyzed resident blue mussels (Mytilus edulis) from the Boston Harbor/Massachusetts Bay region for over twenty years, the Massachusetts Water Resources Authority has annually deployed caged (transplanted) mussels (M. edulis) to assess bioaccumulation potential of sewage effluent discharged under its NPDES permit for over fourteen years, and the GulfWatch program has analyzed resident blue mussel populations for over twelve years. Together, these data provide consistent and comparable information on temporal and spatial changes in chemical contamination in Boston Harbor as steps were taken to reduce contaminant loading. The data also demonstrate the complementary nature of resident and caged (transplanted) mussels for assessing contaminant trends even when the basic approaches and sampling frequency differ. These fifteen-year data sets demonstrate contaminant concentrations in mussels from Boston Harbor are similar and with few exceptions have significantly decreased since the early 1990s. The observed trends also demonstrate broad scale improvements to the quality of Boston Harbor and expand understanding of the response of coastal systems to interventions that reduce the load of chemicals to the ocean.     

Liver histopathology in Baltic eelpout (Zoarces viviparus) – A baseline study for use in marine environmental monitoring

The use of liver histopathology in various fish species as a biomarker in marine monitoring programmes to assess the biological effect of exposure to anthropogenic contaminants is well established. However, although the eelpout (Zoarces viviparus) has repeatedly been recommended as an appropriate bioindicator for monitoring purposes in the Baltic Sea, information on histopathological liver alterations of this species is scarce. In order to obtain an overview, a total of 1070 eelpout, collected in the period between spring 2001 and winter 2010 from 34 sampling sites distributed over large parts of the Baltic Sea, were histologically analyzed. Liver lesions were recorded and classified according to established liver lesion categories. A high prevalence of non-specific and early toxicopathic non-neoplastic hepatic lesions was recorded. In contrast, pre-neoplastic lesions were observed only in rare cases and neoplastic changes were absent. A comparison with published data on other fish species revealed that the observed alterations may indicate effects of environmental stressors including contaminants. The results of the present study, thus, support the application of eelpout liver histopathology in environmental monitoring.     

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) in tissues of aquatic animals utilized in toxicity bioassays

Cell proliferation is an important component of toxic and carcinogenic mechanisms. A recently developed immunohistochemical assay for proliferating cell nuclear antigen (PCNA) identifies an endogenous marker for cell proliferation in routinely-fixed and paraffin-embedded specimens. This assay was adapted to identify proliferating cells in tissues of several fish species used in toxicity bioassays: medaka (Oryzias latipes), guppy (Poeata), and western mosquitofish (Gambusia affinis). The present studies show that this PCNA technique can identify proliferating cells in aquatic animals if attention is given to primary fixative, primary antibody, protein blocking reagent and antigen retrieval processing. PCNA immunoreactivity was demonstrated in tissues with normally high cell turnover such as gill, gastrointestinal tract and testis. Proliferation was also demonstrated in carcinogen-induced hepatic neoplasms.     

Quantitation of CYP1A expression in two flatfish species showing different prevalences of contaminant-induced hepatic disease

English sole (Pleuronectes vetulus) and starry flounder (Platichthys stellatus) are two sympatric flatfish species which show markedly different responses to chemical contaminant exposure. Whereas English sole develop hepatic neoplasms, the prevalences of which are strongly linked to exposure to polycyclic aromatic hydrocarbons (PAHs), evidence of neoplasia is virtually nonexistent in starry flounder, even those residing in areas with very high levels of PAH in the sediments. Because PAHs are activated to genotoxic forms by the action of cytochrome P450 1A (CYP1A) in teleosts, we are examining the hypothesis that variation in the hepatic expression of this major inducible cytochrome P450 isozyme may contribute to the differential contaminant response. These two species were captured in the Duwamish Waterway, a contaminated area of Puget Sound, Washington. Catalytic activity of CYP1A [determined as aryl hydrocarbon hydroxylase (AHH) activity] was measured in the liver to quantitatively assess the relative induction of CYP1A in these two species, and AHH activity was significantly higher in English sole than in starry flounder (p = 0.015). Cellular expression of CYP1A was determined by immunohistochemical localization of tissues, using an avidin-biotin peroxidase complex method, with polyclonal rabbit anti-cod CYP1A. The results showed a markedly reduced expression of CYP1A in hepatocytes of starry flounder, which is consistent with the apparent resistance of this species to the development of hepatocellular neoplasia. This reduced expression of CYP1A in hepatocytes of contaminant-exposed fish was previously seen in oyster toadfish from the Elizabeth River, Virginia, and this species is also apparently resistant to hepatocellular neoplasia.     

Kinetics of vitellogenin protein and mRNA induction and depuration in fish following laboratory and environmental exposure to oestrogens

Plaice, flounder and sand goby were exposed to ethynylestradiol (EE2) for 21 days and then followed for up to 31 days after removal of the oestrogen. Plasma vitellogenin (VTG) and hepatic VTG mRNA were determined in groups of fish sampled during the induction and post-exposure phases. VTG mRNA increased slightly earlier than plasma protein, but both reached maxima by 21 days. In contrast, VTG mRNA decayed much more rapidly than protein after EE2 exposure was terminated (typical values t(1/2) mRNA 3 days, protein 15-30 days). Vitellogenin and VTG mRNA thus measure different temporal events and this is illustrated by field data of male flounder in which both parameters have been determined. Few fish show co-ordinate increased VTG mRNA and vitellogenin but rather more fish have increased vitellogenin. Low level increases of VTG mRNA (5 X) is observed in some fish without increased vitellogenin and this may represent polymorphic differences between individual fish.     

Localisation of a tumor-associated phenotype of benz-aldehyde dehydrogenase in liver carcinogenesis of flounder by quantitative histochemistry

Aldehyde dehydrogenases (ALDHs) oxidize endogeneous reactive aldehydes including lipid peroxidation products (propionaldehyde, malondialdehyde) and are also inducible by xenobiotics like DDT and TCDD. ALDHs are found in injured livers and cancers of contaminant-exposed flounder (Platichthys flesus L.) and showed identical characteristics as in hepatocellular carcinomas of rats by its cytosolic localisation, the preferential oxidation of aromatic compounds (benzaldehyde), dependency on NADP as coenzyme and sensitivity to the specific inhibitor disulfiram. Image analysis showed increased activity of NADP-dependent benzaldehyde dehydrogenase localised in a few cells of early eosinophilic foci and in basophilic adenomas, carcinomas and their invasively growing protrusions (satellites). Disulfiram sensitivity to differentiate between the xenobiotic-inducible isozyme and the tumor-specific isozyme in rats was highest in the early stages of carcinogenesis in the liver of fish. Moderate disulfiram inhibition was observed in non-neoplastic and extrafocal tissue. In neoplastic livers of flounder from contaminated areas (Elbe estuary), hepatocytes in early stages of carcinogenesis were best protected against accumulation of toxic aldehydes by increased activity of the specific tumor-associated isozyme.     

Pure compound and sediment elutriate embryo larval assay of winter flounder from the Hudson/Raritan Estuary and Long Island Sound

Winter flounder (Pseudopleuronectes americanus), a demersal egg layer, is at risk from dioxins and metals in the sediments of the Hudson/Raritan Estuary (the Estuary). The effects of tetrachlorodibenzo-p-dioxin (TCDD), mercuric chloride and sediment elutriates on the embryos of winter flounder from four sites in the Estuary, and a reference site: Long Island Sound (LI) were examined. Embryos were exposed from 2 days after fertilization until 2 days post hatch. TCDD absorption by embryos/larvae ranged from 13 to 44%, with an average 23.4%. One experiment, with the LI population showed a LD₅₀ of 1.8 pg/larva (2.6 ng/g) and LD₁₀ of 0.9 pg/larva (1.4 ng/g). Major observed lesions included pericardial edema (PE), and spinal lesions (SL). Background lesion occurrence in controls was 0.31 lesions/individual, and 0.27 major lesions/individual. NOELs were observed for PE 0.3–0.5 pg/larva, and SL 1.4–9.0 pg/larva. PE and SL are good indicators of low and high dose effects of TCDD in winter flounder, respectively. With mercuric chloride all populations showed 50% mortality at 100 μg/l, and 50% lesions at 25–50 μg/l. Three of seven sediment elutriates collected from the Estuary were acutely toxic. Site-specific variability in sediment toxicity and breeding stock sensitivity to known contaminants was observed. Future studies should incorporate metamorphosis from larva to juvenile. (USEPA:CR825437, NIEHS:ES07148).     

Natural modulation of hepatic metallothionein and cytochrome P4501A in flounder, Platichthys flesus L.

The use of biomarkers such as cytochrome P4501A (CYP1A) or metallothionein (MT) for pollution monitoring is based on the assumption that an increased activity or concentration is primarily caused by exposure to contaminants. Previous studies have shown that the response of biomarkers may be affected by factors such as season, temperature, gender, nutritional status or size. The objective of the present study was to identify natural factors that affect the hepatic activity of CYP1A and hepatic concentration of MT in flounder (Platichthys flesus L.). Thirty flounder were sampled at each of two sites in the Hvaler archipelago, southern Norway, monthly through one year. A set of variables were recorded for each sampling and individual including water temperature, salinity, external and internal lesions, intestinal content, sex, size and organ weights. Hepatic CYP1A activity (EROD), MT and metal concentrations were determined for each individual flounder. The influence of environmental and endogenous factors on the response of these two biomarkers was assessed in multiple regression models. For both biomarkers, 50–60% of the total variability could be explained from factors directly related to season, gender and maturation. Season contributed significantly in the model for EROD, as did external lesions. Size, condition and diet did not contribute greatly when the above factors were included. The results confirm previous findings that season, gender and maturation must be taken into account in biomarker monitoring, but also indicate that other factors such as external lesions should be considered.     

Availability of cadmium and zinc from sewage sludge to the flounder, Platichthys flesus, via a marine food chain

This paper examines the potential availability to a demersal fish of cadmium and zinc associated with digested sewage sludge, via a food chain as well as directly from the sludge, and the tissue distribution and possible excretion of any accumulated cadmium and zinc. Radioactive tracer techniques were used in order to follow the food chain transfer of the metals. Flounder (Platichthys flesus) accumulated cadmium both directly from sludge (delivered in newly ingested unassimilated gutfuls in the amphipods) and in assimilated form from the tissues of the amphipod crustacean Corophium volutator that had been feeding on a sludge–sediment mixture. Cadmium from both sources could be excreted by the fish. The concentration of cadmium within the whole fish increased as the input of cadmium in the diet increased. Zinc, similarly, is available to the flounder both directly from sludge and from zinc accumulated in C. volutator. Only a limited increase in net accumulation of zinc by the flounder was observed upon increased inputs of zinc through the diet, perhaps indicating some regulation of body zinc concentration by the flounder.     

Organochlorine residues in common terns and associated estuarine organisms,Massachusetts, USA, 1971–81

Organochlorine residues were measured in eggs of common terns Sterna hirundo at nine stations and in Atlantic silversides Menidia menidia at 11 stations in coastal Massachusetts, 1973–74. Tern eggs were sampled at one station from 1971 to 1981 and at another from 1973 to 1978. Mussels Mytilus edulis and various fish were sampled in 1975–76. PCBs were the predominant residues in all samples. DDE, TDE, DDT and HEOD were detected in most samples of fish. HE, Alpha-chlordane, oxychlordane and HCB were also detected in most samples of tern eggs. Concentrations of DDE, TDE and PCBs in tern eggs were highly correlated with those in fish at the same stations. Concentrations of PCBs in tern lipids were about 10 times those in the lipids of their prey. Except for HCB, concentrations of organochlorines were highest in urban areas (Boston Harbor and Buzzards Bay) and lowest in remote areas (outer Cape Cod). In Buzzards Bay, levels of DDE, HEOD, HCB and PCBs declined significantly between 1971 and 1981. However, on outer Cape Cod, only PCBs clearly declined after 1973, and by 1976 most of the urban-rural differences in levels of other organochlorines had disappeared. The geographical patterns in pesticide residue levels were associated both with urban uses and with specific agricultural uses. The highest levels of PCBs were associated with industrial effluents at New Bedford and Boston. Hatching failure in common terns was associated with elevated residues of DDE in one sample, but population effects of DDE were only marginal after 1971.     

鱼类甲状腺轴对胚胎发育、仔鱼变态及性别分化的调控作用研究进展

鱼类甲状腺轴由下丘脑、垂体、甲状腺组成,下丘脑通过分泌促甲状腺激素释放激素、促肾上腺皮质激素释放激素调节垂体促甲状腺激素的合成和分泌,促甲状腺激素促进甲状腺滤泡合成并释放甲状腺激素(主要为甲状腺素—T4),T4运输至肝脏等外周组织中,经过脱碘转化形成生物活性更强的三碘甲腺原氨酸(T3),T3主要通过同核受体结合发挥生物学功能。甲状腺激素对鱼类胚胎发育、仔鱼变态及性别分化等早期生长发育过程具有重要调控作用:甲状腺激素为鱼类胚胎发育期所必须,外加适量甲状腺激素能够提高胚胎或仔鱼存活率;仔鱼变态过程中,某些外部形态变化及相关组织分化依赖甲状腺激素的调控,牙鲆等仔鱼变态期体内甲状腺激素水平及其受体表达量会显著升高;性腺发育期,甲状腺激素对调控鱼类性别分化方向可能具有一定作用。     

Hematological analyses of some fish species in the Gulf of Riga

The objective of this work was to detect and compare blood parameters of European flounder (Platichthys flesus), herring (Clupea harertgus membras), eelpout (Zoarces viviparous) and perch (Perca fluviatilis) at the Eastern and Western coast of the Gulf of Riga. The number of erythrocytes in herring of the Gulf of Riga ranges from 1.45 to 2.57 × 10¹²/L. At the same time no statistically significant difference in red blood cells (RBC) count between herring of both coasts was detected. The most common white blood cells in GoR herring blood smear were lymphocytes ranging from 73 to 94%. The number of lymphoblasts was very small (0?C4%), indicating that herring of the GoR is not exposed to chronic stress. The number of erythrocytes in flounder ranged from 0.8 to 2.65 × 10¹²/L, but hemoglobin??from 4.7 to 16.5 g/dL. RBC count and hemoglobin level in European flounder did not differ between coasts however hematocrit was significantly higher at the Eastern coast. White blood cell count in flounder near the Western and Eastern coast was almost equal. Blood indices in eelpouts were slightly higher at the Eastern cost. Slightly higher number of red blood cells and significantly higher hemoglobin level has been observed in perch feeding near the Eastern coast, indicating physiological disturbances of fish. Although hematological analysis pointed at slightly worse living conditions of fish at the Eastern coast, in general hematological picture did not give evidence of fish welfare decline in the Gulf of Riga.     

Plasma steroid levels in female flounder (Platichthys flesus) after chronic dietary exposure to single polycyclic aromatic hydrocarbons

The chronic effects of polycyclic aromatic hydrocarbons (PAHs) on ovary development, total hepatic lipids and plasma sex- and corticosteroid levels in female flounder (Platichthys flesus) were examined. Sexually mature feral female flounder were exposed via the diet to phenanthrene (0.5, 2.5 or 12.5 nmol/g food) or chrysene (0.4 nmol/g food) for 12 weeks, during the previtellogenic phase of the annual reproductive cycle. PAH exposure did not directly affect germ cell development since no structural and/or developmental differences were observed between control and exposed fish. On the contrary, all treatments resulted in altered plasma steroid levels. The most pronounced effect was the significant decrease in plasma 17β-estradiol to 19±11%, 27±7%, 63±20% and 61±12% in relation to control fish, respectively, in flounders exposed to 12.5, 2.5 or 0.5 nmol phenanthrene/g food and 0.4 nmol chrysene/g food. Impaired ovarian growth was not observed, most likely because experiments were ended before the period of vitellogenesis, even though a non-significant general decline in total hepatic lipids could be observed. Moreover, all exposed flounders, except fish fed with the highest amount of phenanthrene, showed a negative correlation between plasma 17β-estradiol and 17α-hydroxyprogesterone levels (r=−0.46). One possible explanation is that PAH action may be mediated by a specific inhibition of steroidogenic enzymes. These findings provide evidence that selected PAHs are antiestrogenic xenobiotics with the capability to impair female teleost reproductive function.     

Availability of cadmium and zinc from sewage sludge to the flounder, Platichthys flesus, via a marine food chain

This paper examines the potential availability to a demersal fish of cadmium and zinc associated with digested sewage sludge, via a food chain as well as directly from the sludge, and the tissue distribution and possible excretion of any accumulated cadmium and zinc. Radioactive tracer techniques were used in order to follow the food chain transfer of the metals. Flounder (Platichthys flesus) accumulated cadmium both directly from sludge (delivered in newly ingested unassimilated gutfuls in the amphipods) and in assimilated form from the tissues of the amphipod crustacean Corophium volutator that had been feeding on a sludge-sediment mixture. Cadmium from both sources could be excreted by the fish. The concentration of cadmium within the whole fish increased as the input of cadmium in the diet increased. Zinc, similarly, is available to the flounder both directly from sludge and from zinc accumulated in C. volutator. Only a limited increase in net accumulation of zinc by the flounder was observed upon increased inputs of zinc through the diet, perhaps indicating some regulation of body zinc concentration by the flounder.