Residues of C-4n-nonylphenol in mosquitofish (Gambusia holbrooki) oocytes and embryos during dietary exposure of mature females to this xenohormone

In order to assess in fish the maternal transfer of alkylphenolic compounds to the progeny, the identification and quantification of the labelled compounds present in oocytes and embryos was conducted after dietary exposure of mature female mosquitofish to ¹⁴C-4n-nonylphenol during vitellogenesis and embryogenesis respectively. Radioactivity found in bile and liver extracts accounted for 0.9–0.6 and 0.2–0.1% of ingested radioactivity for females exposed during vitellogenesis and embryogenesis respectively. The amount of extractable radioactivity present in oocytes and embryos was 0.19 and 0.07% of the ingested dose respectively. The radio-HPLC profiles obtained from bile, liver, oocyte and embryo extracts were similar. They showed the presence of 4n-NP-glucuronide as the major metabolite and traces of unchanged 4n-NP. The other metabolites corresponded to 8-hydroxynonylphenol, 9-(4-hydroxyphenyl)-nonanoic acid and para-hydroxybenzoic acid which is the final product of the alkyl chain oxidation. Our results indicate that exposure of ovoviviparous female fish to 4-NP during vitellogenesis and embryogenesis leads to the contamination of the progeny by 4-NP and its metabolites.     

Simultaneous exposure of English sole (Parophrys vetulus) to sediment-associated xenobiotics: Part 1—uptake and disposition of 14C-polychlorinated biphenyls and 3H-benzo[a]pyrene

English sole (Parophrys vetulus) were exposed to environmentally realistic levels of sediment-associated ³H-benzo[a]pyrene (3 μg BaP/g sediment, dry weight) and ¹⁴C-Aroclor 1254 (1 μg PCBs/g), separately and together, for up to 10 days. BaP and its metabolites in tissues reached steady-state concentrations by the first day of the exposure, whereas PCBs did not approach steady-state concentrations until the tenth day of exposure. Simultaneous exposure of sole to BaP and PCBs, relative to separate exposure to the xenobiotics, significantly increased the concentrations of BaP-derived radioactivity and decreased the concentrations of PCB-derived radioactivity in some tissues and bile. Accumulation of PCB-derived radioactivity, estimated as the burden in tissues, was significantly greater (4- to 13-fold) than that of BaP-derived radioactivity throughout the experiments and regardless of the type of exposure. The rank of the concentrations of PCB-derived radioactivity in tissues and bile was: bile ～ liver > brain > kidney ～ gill > skin ～ blood ～ muscle and for BaP-derived radioactivity the order was: bile > liver > gill > kidney > skin ～ blood > muscle > brain. BaP-derived radioactivity in liver and bile was present primary (85–99%) as metabolites, whereas PCB-derived radioactivity was present equally as parent compounds and metabolites in bile and primarily (98 %) as parent compounds in liver. Hydrolysis of bile from PCB/BaP-exposed sole with β-glucuronidase/arylsulfatase released 35 % of the BaP-derived radioactivity and 32 % of the PCB-derived radioactivity as primary metabolites. A much higher proportion of the BaP-derived radioactivity (64 %) than the PCB-derived radioactivity (13 %) in bile was unaffected by the enzyme treatment, indicating differences in the conjugation of the primary metabolites of PCBs and BaP. The results suggest that in contaminated environments the tissue to sediment concentration ratios for PCBs would exceed that for BaP and its metabolites; however, BaP would be continually absorbed and metabolized by sole to potentially carcinogenic and mutagenic compounds. In addition, the tissue levels of these toxic compounds may be increased by simultaneous exposure to PCBs.     

Disposition and metabolism of 2,3,7,8-tetrachlorodibenzofuran (TCDF) in rainbow trout

In order to elucidate the metabolic fate of 2,3,7,8-tetrachlorodibenzofuran (TCDF) in fish and to thereby facilitate the assessment of the risks posed by this environmental toxin, we determined the whole body half-life, tissue distribution and metabolism of [³H TCDF in rainbow trout (Onchorynchus mykiss), treated orally. A whole body biphasic elimination pattern resulted in the excretion of 60% of the administered chemical during the first 3 days, after which a much slower elimination-rate (half-life = 14 days) was observed. Significant amounts of water-soluble metabolites were found in both bile and liver. Of the TCDF-derived radioactivity in bile, approximately 50% represented glucuronide conjugates, predominantly 4-hydroxy-2,3,7,8-TCDF: substantial amounts of the sulfate conjugate of this same metabolite were also present. Except at early time points, muscle contained the predominant fraction of TCDF-derived radioactivity, amounting to 25–65% of the total radioactivity present in the fish. More than 95% of the radioactivity present in muscle represented unmetabolized TCDF.     

Effect of reproductive toxicants on lipovitellin in female blue crabs, Callinectes sapidus

Changes in the concentration of lipovitellin, a lipoprotein that is the major protein in mature oocytes of crustaceans, were determined during ovarian development in female blue crabs, Callinectes sapidus. To test the effects of reproductive toxins on vitellogenesis, crabs were fed food containing cadmium or injected with the juvenile hormone antagonist, precocene II. Monoclonal antibodies prepared against one of the lipovitellin peptides (peptide A—107 kDa) were used in an enzyme linked immunosorbent assay to determine lipovitellin concentrations. Recently molted female crabs fed food containing cadmium (0.02 mg/g) for 18 days had oocytes with the same lipovitellin concentration as controls, but after 24 days the oocytes in cadmium treated crabs had significantly lower lipoprotein contents than controls (30 ± 4 ng/oocyte—cadmium treated; 45 ± 4 ng/oocyte—control). Precocene II-treated crabs had significantiy lower lipovitellin concentrations after 18 and 24 days. Diameters of oocytes were the same in both treated and control crabs. The results suggest that some pollutants may affect reproduction by acting during secondary vitellogenesis i.e., period when there is a large increase in lipovitellin synthesis. Since lipovitellins serve many of the nutritional needs of recently emerged crab embryos, poor larval survival may occur as a result of lower lipovitellin content.     

Evidence for exposure of fish to oil spilled into the Columbia river

On March 19, 1984, more than 170000 gallons of oil were spilled into the Columbia River. We had recently developed analytical methods for estimating the exposure of fish to aromatic compounds by measuring the concentrations of metabolites of these contaminants in fish bile. The oil spill provided an opportunity to field test our methods in assessing the exposure of fish to petroleum aromatic compounds from the spilled oil. Our findings indicated that, within 5 days after the spill, mean concentrations of metabolites of aromatic compounds in the bile of white sturgeon (Acipenser transmontanus) captured 57 miles downstream from the spill were significantly higher than those of sturgeon caught upriver.     

Ethoxyresorufin-O-deethylase activity and fixed wavelength fluorescence detection of PAHs metabolites in bile in turbot (Scophthalmus maximus L.) Exposed to a dispersed topped crude oil in a Continuous Flow System

Long term effects of sublethal concentrations of oil on the marine environment have become of general concern. Cytochrome P4501A activity (EROD) in liver and fixed wavelength fluorescence detection of PAHs metabolites (FF) have in this study been used as biomarkers for dispersed oil exposure on a long term period of juvenile turbot (Scophthalmus maximus L.). A Continuous Flow System was used to carry out the study. The fish were continuously exposed to 0.125, 0.5 or 2.0 mg litre⁻¹ dispersed topped crude oil for 6, 15, 24 h, 4 and 21 days followed by a 9 days recovery period in clean seawater. No induction of the cytochrome P4501A was measured. A maximum level in bile metabolites (4- to 5-fold) was recorded after 24 h of exposure revealing thereby a detoxification process, but a decline occurred from day 4 to day 21. This study demonstrated that FF detection of PAHs metabolites in bile could be a more sensitive biomarker than EROD activity in a long term exposure to sublethal concentration of oil.     

Bioconcentration and distribution of 4-tert-octylphenol residues in tissues of the rainbow trout (Oncorhynchus mykiss)

Branched chain alkylphenols are weak oestrogen mimics which are present in the aquatic environment and have been implicated in the feminisation of fish. This study reports the biotransformation, bioconcentration and tissue distribution of the xenoestrogen 4-tert-octylphenol (t-OP) in juvenile rainbow trout. Fish were exposed for 10 days to a concentration of 4 micrograms/l of [14C] t-OP in a flow-through system and were sampled after 1, 4, 7 and 10 days of exposure. t-OP residues were extracted from all tissues and analysed by radio-high-performance liquid chromatography. After 1 day of exposure radioactive residues were detected in all tissues and reached steady state conditions in the whole fish after 4 days of exposure. The concentration of t-OP residues were highest in bile, followed by faeces, pyloric caeca, liver and intestine. In these tissues the majority of alkylphenol was in the form of two metabolites which were identified by gas chromatography-mass spectroscopy as the glucuronide conjugates of t-OP and t-octylcatechol. t-OP accumulated as the parent compound in fat with a bioconcentration factor (BCF) of 1190, and in brain, muscle, skin, bone, gills, and eye with BCFs of between 100 and 260. This study suggests that exposure to water-borne alkylphenols results in rapid conjugation and elimination of the chemical via the liver/bile route, but that high amounts of the parent xenoestrogen can accumulate in a variety of other fish tissues.     

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 17 beta-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 17 beta-estradiol and 17 alpha-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.     

Metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar).

Nonylphenol (NP) is a breakdown product of alkylphenol polyethoxylates (APEs), an important class of non-ionic surfactants that are widely used in many detergent formulations and plastic products for industrial and domestic use. A complex microbial degradation pattern, characterized by the formation of several metabolic products that are more toxic than the parent compound, has been established for APEs. We have studied the in vivo metabolism and organ distribution of NP in juvenile salmon. Fish were exposed to a single oral dose of [3H]-4-n-NP (1295 KBq, 25 micrograms) and sampled at 24, 48 and 72 h after exposure. Metabolites were separated by radio-high-performance liquid chromatography and tentatively identified by cochromatography with standards characterized by mass spectrometry. Our results show that 4-n-NP was mainly metabolized in vivo to its corresponding glucuronide conjugate and to a lesser extent to various hydroxylated and oxidated compounds. Biliary excretion at 72 h after dosing amounted to 2.83 +/- 0.75% of the administered radioactivity. Kinetic analysis shows that NP-glucuronide accounted for 83, 95 and 81% of total radioactivity in the HPLC-injected bile sample at 24, 48 and 72 h, respectively, after exposure. The half-life of residues in carcass and muscle was between 24 and 48 h after exposure.     

B[a]P-DNA binding in early life-stages of Atlantic tomcod: population differences and chromium modulation

Atlantic tomcod (Microgadus tomcod) from the Hudson River (HR) are resistant at the molecular and organismic levels to the effects of exposure to dioxin-like aromatic hydrocarbon (AH) compounds, but much less so to benzo[a]pyrene (B[a]P). The aims of this study were to determine in early life-stages of tomcod exposed to B[a]P: (1) if DNA binding levels differed between fish from the HR and Miramichi River (MR), and (2) if co-exposure to chromium could modulate this genotoxic effect. After exposure to [(3)H]B[a]P alone, DNA-bound radioactivity was 5-10-fold higher in embryos and larvae of MR than HR descent. Co-exposure to chromium modulated DNA binding levels in offspring of both populations. In MR embryos, co-exposure to chromium inhibited B[a]P uptake. These results demonstrated resistance to the genotoxic effects of B[a]P in early life stages of HR tomcod at an ecologically important endpoint and suggest the ability of chromium to modulate AH-induced genotoxicity.     

Metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar)

Nonylphenol (NP) is a breakdown product of alkylphenol polyethoxylates (APEs), an important class of non-ionic surfactants that are widely used in many detergent formulations and plastic products for industrial and domestic use. A complex microbial degradation pattern, characterized by the formation of several metabolic products that are more toxic than the parent compound, has been established for APEs. We have studied the in vivo metabolism and organ distribution of NP in juvenile salmon. Fish were exposed to a single oral dose of [³H]-4-n-NP (1295 KBq, 25 μg) and sampled at 24, 48 and 72 h after exposure. Metabolites were separated by radio-high-performance liquid chromatography and tentatively identified by co-chromatography with standards characterized by mass spectrometry. Our results show that 4-n-NP was mainly metabolized in vivo to its corresponding glucuronide conjugate and to a lesser extent to various hydroxylated and oxidated compounds. Biliary excretion at 72 h after dosing amounted to 2.83±0.75% of the administered radioactivity. Kinetic analysis shows that NP-glucuronide accounted for 83, 95 and 81% of total radioactivity in the HPLC-injected bile sample at 24, 48 and 72 h, respectively, after exposure. The half-life of residues in carcass and muscle was between 24 and 48 h after exposure.     

Toxicopathic liver lesions in English sole and chemical contaminant exposure in Vancouver Harbour,Canada

The prevalence of toxicopathic liver lesions in English sole (Pleuronectes vetulus) was determined along a presumed gradient of chemical contamination in Vancouver Harbour, Canada. Fish were captured from five sites in or near Vancouver Harbour, British Columbia, Canada. No toxicopathic lesions were observed in fish examined at the reference site (Howe Sound outside Vancouver Harbour), or at the outer harbour site. In contrast, 20-23% of the fish from three sites located in the central harbour, Indian Arm and Port Moody Arm had one or more types of toxicopathic lesions. Likewise, aromatic hydrocarbon (AH) metabolites measured in bile exhibited a gradient in levels from lower concentrations at the reference site to significantly higher levels in fish from Indian Arm and Port Moody Arm harbour sites. The occurrence of toxicopathic liver lesions was statistically associated with concentrations of AHs measured in sediment and AH metabolite levels measured in bile.     

Isolation and characterisation of pristane metabolites formed in vivo by rainbow trout

Pristane (2,6,10,14 -tetramethylpentadecane) occurs ubiquitously in the marine environment. This hydrocarbon may be of biogenic or petrogenic origin.¹ Recently it has been shown that residual amounts of this branched alkane increased in marine organisms after an oil spill.^2,3 The lack of data on the fate of pristane in fish, added with the fact that this compound was considered by some authors as a non-metabolisable substance in vertebrates, including man,⁴ led us to investigate the capability of fish to metabolise pristane. In this study, urinary and fecal excretion, tissue distribution and metabolism of ³H-pristane were analysed in Salmo gairdneri R. after a single intragastric dose. In addition to unchanged hydrocarbon, various labelled compounds have been isolated and identified in liver, bile, faeces, urine and surrounding water, demonstrating that pristane was first oxidised to alcohols (pristanol and pristane-diol) and to acid (pristanic acid). The elimination of these compounds occurred in the form of conjugated products (primarily glucuronides) as well as free metabolites.     

High matrix interference affecting detection of PAH metabolites in bile of Atlantic hagfish (Myxine glutinosa) used for biomonitoring of deep-water oil production

The characteristic biology and wide distribution of hagfish species makes them relevant for use in pollution biomonitoring at great water depths, particularly in regions where deep-water oil production may take place. The exposure of fish to petrogenic contaminants can normally be detected from the level of polycyclic aromatic hydrocarbon (PAH) metabolites in bile fluid. Some of these metabolites are strong fluorophores, allowing analytical detection by means of simple fluorometric techniques such as fixed wavelength fluorescence (FF) and synchronous fluorescence scanning (SFS). In the present study bile from Atlantic hagfish (Myxine glutinosa) collected in pristine areas (Barents Sea and southwestern Norway) displayed strong bile fluorescence levels, suggesting the presence of PAH contaminants. However, gas-chromatography-mass spectrometry (GC-MS) analyses ruled out PAHs as the origin for this fluorescence signal. Rather, the bile of Myxine contains components resulting in unusually strong background fluorescence interfering at the wavelength pairs used for detection of PAH metabolites. Possible background for the observed matrix interference and implications for detection of PAH metabolites in hagfish is discussed.     

Reproductive life history stages sensitive to oil and naphthalene in Atlantic croaker

The reproductive effects of chronic exposure to water-soluble fractions (WSF) of diesel fuel oil and naphthalene in female Atlantic croaker during critical periods of their reproductive life history were investigated. Fish were repeatedly exposed to WSF of oil (2.5 or 5%) or naphthalene (0.5 or 1.0 ppm) for 5 or 8 weeks. Both pollutants blocked sexual maturation in some fish and impaired ovarian recrudescence in others. The majority of the oocytes in exposed fish were undeveloped and widespread oocyte atresia was evident at higher concentrations of these polycyclic aromatic hydrocarbons (PAH). The disruption of ovarian growth and oocyte development in croaker after PAH exposure was associated with alterations in reproductive endocrine function at multiple sites on the hypothalamus-pituitarygonadal axis. A third period of increased sensitivity to xenobiotics was observed during final oocyte maturation.     

DNA strand breaks (comet assay) and embryo development effects in grass shrimp (Palaemonetes pugio) embryos after exposure to genotoxicants.

Grass shrimp embryos develop in egg sacs (stages 1-10) attached to the female for 14-20 days after which they 'hatch' from the egg sacs into a swimming zoea stage (stage 11). Until they emerge from the egg sacs, embryos depend on lipids and lipovitellin stored within the egg. The percent of embryos which hatch after exposure to toxicants relative to controls was the basis of an embryo development assay. Exposure of embryos to chromium(III) chloride, sodium chromate, mercuric chloride, and 2-methyl-1,2-naphthoquinone (MNQ) resulted in a reduced hatching rate. In addition to effects on embryo development, DNA strand damage tests were carried out on contaminant-exposed embryos, using the single-cell electrophoresis method often referred to as comet assay. Development of stage 4 embryos was more affected by MNQ exposure than stage 7 embryos. The hatching rates of stages 4 and 7 embryos exposed to MNQ (172 micrograms/l) were 0 and 90%, respectively. DNA strand damage, measured as DNA tail moments, were 3.4 and 4.4, respectively. Thus, exposure of an early embryo stage to MNQ prevented full embryo development while development of later embryo stages was not affected. It may be that the DNA repair systems are more efficient in later embryo stages than in early stages and thus DNA damaged in the early stages affects development.     

B[a]P–DNA binding in early life-stages of Atlantic tomcod: population differences and chromium modulation

Atlantic tomcod (Microgadus tomcod) from the Hudson River (HR) are resistant at the molecular and organismic levels to the effects of exposure to dioxin-like aromatic hydrocarbon (AH) compounds, but much less so to benzo[a]pyrene (B[a]P). The aims of this study were to determine in early life-stages of tomcod exposed to B[a]P: (1) if DNA binding levels differed between fish from the HR and Miramichi River (MR), and (2) if co-exposure to chromium could modulate this genotoxic effect. After exposure to [³H]B[a]P alone, DNA-bound radioactivity was 5–10-fold higher in embryos and larvae of MR than HR descent. Co-exposure to chromium modulated DNA binding levels in offspring of both populations. In MR embryos, co-exposure to chromium inhibited B[a]P uptake. These results demonstrated resistance to the genotoxic effects of B[a]P in early life stages of HR tomcod at an ecologically important endpoint and suggest the ability of chromium to modulate AH-induced genotoxicity.     

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。     

三氯异氰尿酸对剑尾鱼(Xiphophorus helleri)毒性及其抗氧化酶影响

采用生物毒性测试与评价方法对常用消毒剂药物三氯异氰尿酸（Trichloroisocyanuric acid，TCCA）对剑尾鱼的急性毒性及其Ⅰ、Ⅱ相代谢酶活性影响进行了研究。结果表明，TCCA对剑尾鱼的96hLG0为2．15mg／L。TCCA对剑尾鱼肝脏还原型谷光苷肽（GSH）、谷胱苷肽硫转酶（GST）和7。乙氧基异吩恶唑酮-脱乙基酶（EROD）都存在诱导作用。雌雄个体在GST和EROD的诱导响应时间有显著差异性，其中雄性个体GST对TCCA暴露响应比雌性个体敏感。     

PAH in fish bile detected by fixed wavelength fluorescence

Fixed wavelength fluorescence (FF) for detection of polyaromatic hydrocarbon (PAH) metabolites in fish bile has been studied, and is presented through a laboratory and a field experiment. The method, which can be regarded as a biomarker of exposure to PAH, has proved to be a simple, rapid and cost effective method for monitoring PAH contamination.