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.     

First record of mosquitofish,Gambusia holbrooki,in Tasmania,Australia: Stock structure and reproductive biology

The first population of mosquitofish, Gambusia holbrooki, was discovered in a wetland area of the upper Tamar Estuary, northern Tasmania, Australia, in November 2000. We report aspects of the structure and reproductive biology of this population, based on 1567 fish collected between August 2001 and January 2002. Females dominated most catches. Maximum lengths were 48.9 and 23.6 mm standard length (SL) for females and males, respectively. Growth of the over‐wintered cohort was evident from October, when temperatures reached >19°C; growth rates in the young‐of‐season cohort from length frequency plots were 0.38 mm/ day for females and 0.19 mm/day for males. Estimated SL at 50% maturity for females (25.0 mm) and males (17.4 mm) were attained within 7 weeks of birth. Developing embryos were present from mid October and offspring from mid November; average number of stage III embryos was 55.5 (3–144). Given their rapid reproductive turnover (c. 34‐day gestation period) and resilience to broad environmental conditions, it is likely that this exotic pest will spread to other Tasmanian localities, as it has already been discovered in areas near the wetlands since the completion of this study.     

Effects of dietary exposure to the pyrethroid pesticide esfenvalerate on medaka (Oryzias latipes)

The pyrethroid insecticide esfenvalerate is widely used on orchard crops throughout California. In the aquatic environment, this compound is likely to accumulate in sediments, food particles and benthic organisms due to its lipophilicity and environmental persistence. This pilot project tested the hypothesis that esfenvalerate is toxic to medaka (Oryzias latipes) when taken up with the diet. For 7 days fish were fed diets, which contained esfenvalerate in three different concentrations (4, 21, 148 mg/kg, measured). Endpoints measured were mortality, fecundity, fertilization and hatching success of embryos, viability of larvae and cellular stress protein (hsp60, hsp70, hsp90) levels. The toxicity of aqueous exposure of medaka to esfenvalerate was also determined. Whereas the 96-h LC50 in the aqueous exposure was <9.4 microg/l, the dietary exposure did not cause mortality. Possible effects of dietary esfenvalerate were seen on fertilization and hatching success and the number of non-viable larvae. Expression of hsp60 and hsp90 showed a dose-dependent response pattern.     

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.     

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.     

Effects of prolonged exposure to 4-tert-octylphenol on toxicity and indices of oestrogenic exposure in the sand goby (Pomatoschistus minutus, Pallas)

Alkylphenolic compounds are present in estuarine and marine environments. They are moderately bioaccumulative, and oestrogenic to fish following three week, in vivo, exposures. The effects of prolonged exposure are, as yet, unclear. Sand goby (Pomatoschistus minutus) were exposed to measured concentrations of 4-tert-octylphenol (OP) in a 28-day, dose-ranging study, and a six month temporal response study. Following 28-days exposure to measured 31+/-6 or 101+/-47 microg l(-1) OP (mean+/-SD), immature male sand goby showed elevated vitellogenin (VTG) mRNA expression. Plasma alkali-labile phosphate concentrations were elevated in both sexes and were higher in females than males. Although measured OP concentrations were variable, they were significantly different for each treatment and a range of concentration-dependent effects were observed. The LC(50) for chronic OP exposure (8 weeks) was 29+/-6 microg l(-1), and both sexes demonstrated concentration and duration dependent increases in VTG mRNA expression. Exposure to 28+/-20 microg l(-1) OP (median+/-95% CI) for 6 months inhibited development of male nuptial colouration and sperm duct glands. These findings are discussed in relation to environmental concentrations of alkylphenols and ecological concerns.     

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.     

Effects of dietary and water-borne oil exposure on larval pacific herring (Clupea harengus pallasi)

Pacific herring (Clupea harengus pallasi) larvae were exposed either directly to the water-soluble fraction (WSF) of crude oil or indirectly via oil-contaminated prey (OCP) (Artemia salina) for <- 28 days, to determine the relative effects of diet and water as routes of contamination. Larvae were affected rapidly by 0·9 ppm WSF, which caused high larval mortality, reduced swimming ability and rapid reductions in feeding rates. Larval length was significantly reduced by 0·7 ppm WSF in 7 days and by 0·3 ppm WSF within 14 days. The WSF exposure also caused similar reductions in larval weights. Highly contaminated prey (6 ppm prey exposure) caused significant mortality, but surviving larvae appeared robust. The OCP did not affect swimming, feeding, or growth. Exposure of larvae was not significantly extended by OCP, which rapidly depurated WSF in clean water (98% in 1 day). Therefore, OCP is probably not an important source of low molecular weight petroleum contamination to larval fish in the marine environment.     

Metallothionein and heavy metal levels in rainbow trout (Salmo gairdneri) during exposure to cadmium in the water

Rainbow trout were exposed to 200 ppb cadmium in the water during four months at 5°C. The liver, kidney and gills were analyzed for cadmium, copper, zinc and metallothionein. Cadmium accumulated in all three organs and reached the highest concentration in the kidney. The zinc and copper concentrations were not altered during the experiment. The metallothionein concentration was significantly higher in liver of exposed fish than in control fish after three months. The kidneys reached significantly elevated levels of metallothionein in the exposed group after four months. These results demonstrate that metallothionein is induced by cadmium after exposure to the metal via water.     

Prey selection and dietary overlap of shortfinned (Anguilla australis) and longfinned (A. dieffenbachii) eels during summer in the Horokiwi Stream,New Zealand

Benthic macroinvertebrates were collected and the diets of shortfinned eels (Anguilla australis) and longfinned eels (Anguilla dieffenbachii) inhabiting a small coastal New Zealand stream were examined to investigate intraspecific and interspecific trophic partitioning and prey selection. Our results showed intraspecific segregation of preferred prey among three size classes of juvenile shortfinned eels, but there was significant overlap between different size classes of longfinned eels. All size classes of both species of eel fed nocturnally on similar prey, and so there was no indication of temporal or trophic segregation. Therefore, habitat separation is assumed to be the main mechanism to reduce interspecific competition in these two co‐occurring species of eels.     

Kinetics of Cd accumulation and elimination in tissues of juvenile rockfish (Sebastes schlegeli) exposed to dietary Cd

Cadmium (Cd) accumulation and elimination were investigated in the tissues (gill, intestine, kidney, lever and muscle) of juvenile rockfish, Sebastes schlegeli, after sub-chronic dietary Cd exposure (0, 0.5, 5, 25 and 125 mg/kg). No mortality occurred during the sub-chronic exposure to dietary Cd. Specific growth rates of the rockfish estimated by weight and length were significantly different from those of the control, and a significant inverse relationship was observed between weight gain and the exposure concentration of dietary Cd at 25, 125 mg/kg. Cd accumulation in the tissues increased with exposure periods and concentrations for the 60 days of dietary Cd exposure. Cd accumulation in the intestine of rockfish was higher than other tissues, and the order of Cd accumulation in tissues were intestine>kidney approximately liver>gill>muscle. Accumulation factors showed an increase with the exposure period and an inverse relationship between the accumulation factor and the exposure concentrations in the gill, intestine, liver and muscle, but not in kidney. Cd elimination in tissues of rockfish decreased during the 30 days of depuration except kidney and muscle. Intestine showed the fastest elimination rates of Cd at all concentrations compared with other tissues.     

Differential gene expression in the brain of Sebastiscus marmoratus in response to exposure to polychlorinated biphenyls (PCBs)

Effects of exposure to polychlorinated biphenyls (PCBs) on Sebastiscus marmoratus were investigated using a suppression subtractive hybridization method. A total of 108 gene sequences were identified as having the potential for being differentially expressed, and 45 could be identified with homologous database sequences. Functions with which they were associated included long-term potentiation and neurotransmitter release, neuroendocrine, mitosis and cell proliferation, energy-related metabolism, general metabolism, signal protein, hemopoiesis system, immune system, and structure. The expression of 17 of these genes was analyzed in the brain using real time fluorescent quantitative PCR. The present study provided a basis for studying the response of fish to PCB exposure and allowed the characterization of new potential neurotoxicol biomarkers of PCB contamination in seawater.     

Differential gene expression in the brain of Sebastiscus marmoratus in response to exposure to polychlorinated biphenyls (PCBs)

Effects of exposure to polychlorinated biphenyls (PCBs) on Sebastiscus marmoratus were investigated using a suppression subtractive hybridization method. A total of 108 gene sequences were identified as having the potential for being differentially expressed, and 45 could be identified with homologous database sequences. Functions with which they were associated included long-term potentiation and neurotransmitter release, neuroendocrine, mitosis and cell proliferation, energy-related metabolism, general metabolism, signal protein, hemopoiesis system, immune system, and structure. The expression of 17 of these genes was analyzed in the brain using real time fluorescent quantitative PCR. The present study provided a basis for studying the response of fish to PCB exposure and allowed the characterization of new potential neurotoxicol biomarkers of PCB contamination in seawater.     

A pockmark field in the Patras Gulf (Greece) and its activation during the 14/7/93 seismic event

During a recent oceanographical-geophysical survey carried out in the southeastern part of the Gulf of Patras in Western Greece for the construction of an outfall, an active pockmark field was found. The pockmark field was formed in soft layered Holocene silts. The pockmarks are associated with acoustic anomalies attributed to gas-charged sediments. The pockmarks vary in size and shape from 25 to 250 m in diameter and from 0.5 to 15 m in depth and are among the largest and deepest observed in the world.

On July 14th, 1993, during the survey, a major earthquake of magnitude 5.4 on the Richter scale occurred in the area. During the 24 hour period prior to the earthquake the bottom water temperature anomalously increased on three occasions, whilst for a few days after the earthquake it was noted that the majority of the pockmarks were venting gas bublles.

It is considered that the three abrupt sea-water temperature increases were probably the result of upward migrating high-temperature gas bubbles in the water column. It is further suggested that the earthquake was the triggering mechanism and that the gas expulsion was caused by the reduction in the pore volume in the sediments resulting from changes in the stress regime prior to the earthquake. Therefore, it can be suggested that in seismic areas adjacent to pockmark fields, earthquake prediction may be achieved by monitoring the water temperature and/or the rate of gas venting in the pockmark field.

Our analysis indicates that the pockmark field in the Patras Gulf has formed slowly during the Holocene by continuous gas venting, which is periodically being interrupted by short-duration events of enhanced gas seepage triggered by earthquakes.     

Sex-specific biochemical and histological differences in gonads of sea urchins (Psammechinus miliaris) and their response to phenanthrene exposure

Female and male individuals of the same species often differ with respect to their susceptibility to toxicant stress. In the present study, sea urchins (Psammechinus miliaris) of both sexes were exposed to high (150 μg L?1) and environmentally relevant (5 μg L?1) concentrations of phenanthrene over 10 days. While food intake was significantly decreased following exposure to 150 μg L?1 phenanthrene, histological indices (lipofuscin accumulation, fibrosis, oocyte atresia), energetic status (energy charge, sum adenylates, AMP/ATP ratio) as well as ascorbate levels in the gonads showed either little or no effect upon phenanthrene exposure. However, most parameters (vitamin C, energy charge, sum adenylates, AMP/ATP ratio, ATP and ADP concentrations, lipofuscin content, fibrosis) significantly differed between male and female animals. This study illustrates the difficulties to identify toxic injury in reproductive tissue as it may be superimposed by gametogenesis and spawning of gametes.