Effects of 17beta-estradiol on the reproduction of Java-medaka (Oryzias javanicus), a new test fish species

17β-estradiol (E2) is a female hormone which is known to be one of the strongest estrogenic chemicals in the environment. The present study investigated the effects of E2 on the reproduction of the estuarine fish, Java-medaka (Oryzias javanicus). Starting from the embryonic stages, Java-medaka was exposed for 6 months to 9.5, 16, 68, 159 and 243 ng/l of E2. The fecundity of Java-medaka exposed to E2 levels >16 ng/l was significantly lower than that of the control. The appearance of secondary sexual characteristics seemed to be inhibited by exposure to 159 and 243 ng/l E2. Vitellogenin concentrations in the liver of male fish exposed to 68, 159 and 243 ng/l were significantly higher than that of the control. Thirty-three and sixty percent of male fishes exposed to 159 and 243 ng/l, respectively, had testis-ova. These results suggest that reproduction in the Java-medaka, especially the male fish, could be affected by exposure to E2 concentrations greater than 16 ng/l.     

Gene Expression Analysis for Exposure to Estrogenic Substances

Zebrafish (Danio rerio) were exposed with 17β‐estradiol (E2) and nonylphenol (NP) in different concentrations. Gene expression analysis was carried out by two different approaches. First, RT‐PCR experiments were performed for the examination of expression levels of the two marker genes vitellogenin and aromatase. This approach showed a significant increase in the expression of the vitellogenin gene in exposed male fish (500 ng/L 17β‐estradiol and 250 μg/L nonylphenol). This egg yolk protein is usually not synthesized in male vertebrates. A slight decrease of expression of the aromatase gene was observed in exposed female zebrafish. Aromatase is known to catalyze the conversion of androgens to estrogens. Second, DNA microarray experiments were carried out, which allow the simultaneous examination of the expression levels of a great number of marker genes. The microarray experiments resulted in an up‐regulation of vitellogenin up to 850‐fold. In addition, several other genes were identified to be up‐regulated by estrogens, for example the high mobility group box protein ssrp1 (78‐fold) or the chaperonin containing t‐complex polypeptide 1, beta subunit cctb (22‐fold).     

Uptake and tissue distribution of C4-C7 alkylphenols in Atlantic cod (Gadus morhua): relevance for biomonitoring of produced water discharges from oil production

The sensitivity of different tissues for assessment of chronic low-dose environmental exposure of fish to alkylphenols (APs) was investigated. We exposed Atlantic cod (Gadus morhua) in the laboratory to tritium labelled 4-tert-butylphenol, 4n-pentylphenol, 4n-hexylphenol, and 4n-heptylphenol via seawater (8 ng/l) and via contaminated feed (5 μg/kg fish per day). Measurements of different fish tissues during eight days of exposure and eight subsequent days of recovery revealed that APs administered via spiked seawater were readily taken up whereas the uptake was far less efficient when APs were administered in spiked feed. AP residues were mainly located in the bile fluid whereas the concentrations in liver were very low, indicating a rapid excretion and the liver-bile axis to be the major route of elimination. The biological half-life of APs in the exposed cod was short, between 10 and 20 h. Our study shows that in connection with biomonitoring of AP exposure in fish, assessment of AP metabolites in bile fluid is a more sensitive tool than detection of parent AP levels in liver or other internal tissues.     

Biomarker responses in pelagic and benthic fish over 1 year following the Hebei Spirit oil spill (Taean, Korea)

After the Hebei Spirit oil spill incident (7th December, 2007) in the west coast of Korea, contamination of biliary PAH metabolite and hepatic biomarkers in a pelagic and a benthic fish was monitored for 1 year. Concentrations of 16 PAHs and alkylated PAHs in fish muscle were highest (22.0 ng/g d.w. for 16 PAHs and 284 ng/g d.w. for alkylated PAHs) at 5 days after the spill and then decreased rapidly to background levels at 11 months after the spill. Fish from the oiled site had elevated biliary PAH metabolite concentrations immediately after the spill; these declined steadily in both species, but were still above reference site concentrations 2 months after the spill. Oiled-site fish showed hepatic CYP 1A induction whose trend closely followed those of biliary PAH metabolite concentrations, implying continuous exposure to PAHs. Brain acetylcholinesterase activity was not related to oil exposure.     

A whole life cycle assessment on effects of waterborne PBDEs on gene expression profile along the brain-pituitary-gonad axis and in the liver of zebrafish

Polybrominated diphenyl ethers (PBDEs) are now found ubiquitously in the aquatic environment and biota, and there is a growing concern that PBDEs may disrupt endocrine systems, leading to reproductive impairments of aquatic animals. In our study, zebrafish (Danio rerio) were exposed to the 5 ng/L, 1 μg/L and 50 μg/L of DE-71 for the duration of the whole life cycle (120 days, from eggs to adults). The expression of selected genes along the brain-pituitary-gonadal (BPG) axis and liver, and the levels of plasma sex hormones were examined. In male fish, up-regulation of GnRH in brain, FSHβ and LHβ in pituitary, FSH-receptor, LH-receptor, and CYP19a in testis was clearly evident, while down-regulation of CYP11a and 3β-HSD was found in testis. In female fish, a 2.4-fold up-regulation of 3β-HSD was found in ovary upon exposure to 50 μg/L of DE-71. GnRH in brain, FSHβ and LHβ in pituitary were also up-regulated, while ERβ, TH and TPH in brain and GnRH-receptor in pituitary were significantly down-regulated. Hepatic ERα, AR and VTG in males were all down-regulated, while hepatic ERα and AR in female were up-regulated. Serum estradiol (E2) was reduced in both male and female upon exposure to DE-71, while significant increases in serum testosterone (T) and 11-keto-testosterone (11-KT) were only found in male but not female fish. The ratio of T/E2 as well as the ratio of 11-KT/E2 in male fish increased in a dose-dependent manner upon exposure to DE-71. Our overall results showed that whole life exposure of DE-71 altered the expression of regulatory genes and receptors at all three levels of the BPG axis in zebrafish, and the responses are sex dependent. The observed disruption of GnRH and GtHs can be further related to the subsequent disruption in both levels and balance sex steroid hormones.     

Estrogen in a karstic aquifer

Adverse impacts on the health of some fish populations, such as skewed sex distributions, have been noted in surface waters and in laboratory experiments with relatively low concentrations (above 25 ng/L) of natural estrogen (17 beta-estradiol--E2). Sources of E2 to surface and ground waters can include avian, human, and mammalian waste products. The Ozark Plateau Aquifer (OPA) is a karstic basin that receives a significant portion of its water through losing reaches of rivers. Thus, there is a direct connection between surface water and ground water. The OPA was targeted for an E2 study to assess the potential for adverse health effects to aquatic organisms living in the system. Eight springs, which drain the aquifer, were sampled quarterly. The concentrations of E2 in the OPA ranged from 13 to 80 ng/L. For any one sampling event, the concentrations of E2 at the spring waters were statistically similar; however, the concentrations of E2 at all springs varied throughout the year. At Maramec Spring, one of the larger springs, the E2 concentration, was correlated with discharge. Based on the correlation between discharge and E2 concentration, aquatic organisms living in the plateau or in its discharged waters, including the threatened southern cavefish T. subterraneus, are exposed to concentration of E2 above 25 ng/L approximately 60% of the time. This implies that organisms living in karst basins throughout the OPA are likely exposed to E2 concentrations that may adversely impact their reproductive success for a significant portion of each year.     

Sea lice treatments on salmon farms have no adverse effects on zooplankton communities: a case study

The long-term effects of the sea lice treatment products Excis and Slice on zooplankton communities in a Scottish sea loch were investigated at a commercially operating salmon farm over 31 months. Cypermethrin and emamectin benzoate are the active ingredients in Excis and Slice respectively, which are widely used to control ectoparasitic sea lice on farmed salmon. Excis and Slice treatments did not cause basin-wide effects on the zooplankton community. For both formulations, no adverse affects on zooplankton were detected, instead observed changes in zooplankton abundance and community composition displayed natural seasonal cycles of abundance. Water column concentrations of cypermethrin and emamectin benzoate following sea lice treatments at the fish farm were predicted using models. Cypermethrin concentrations of 3000 ng/l were predicted for short periods immediately after each cage treatment assuming no particle adhesion. The 3-h and 24-h Environmental Quality Standards (EQS) were exceeded for 10 h and 32 h respectively on the second day when five cages were treated. However, cypermethrin concentrations higher than 0.5 ng/l (24-h EQS) were predicted to occur over <2% of the total basin area on each treatment day. The 3-h EQS (16 ng/l) was exceeded in <0.3% of the basin on each treatment day. The concentration of soluble emamectin benzoate present in the water column was predicted from modelled deposition footprints and sediment concentrations to be of order 10(-3) ng/l. Predicted concentrations of both chemicals were generally lower than those causing toxicity to copepods in previous laboratory studies and further support the results of this field study that environmental concentrations of Excis and Slice do not adversely impact zooplankton communities.     

Perfluorinated compounds in estuarine and coastal areas of north Bohai Sea, China

Perfluorinated compounds (PFCs) in water, sediment, soil, and biota collected from estuarine and coastal areas of the north Bohai Sea, China, were determined by use of HPLC-MS/MS. Significant concentrations of PFCs were found in water (mean: 18.4 ng/l) and biologic samples (fish: 265 ng/g dw), while concentrations of PFCs in soils and sediments were less. The predominately detected compound was perfluorooctanesulfonate (PFOS), with a maximum concentration of 30.9 ng/l in water and 791 ng/g dw in fish. Concentrations of PFCs were significantly greater in the Liaohe River system than other locations, which suggests point sources in this urbanized and industrialized region. PFOS concentrations in water and biota were both less than the reported threshold concentrations. Detection of PFCs at relatively great concentrations in various environmental matrices from this region suggested that further studies characterizing concentrations of PFCs, their sources and potential risk to both humans and wildlife are needed.     

Acute toxicity of platinum to coho salmon (Oncorhynchus kisutch)

The effects of short-term exposure to tetravalent platinum on survival, opercular movement and post-treatment growth of coho salmon fry (Oncorhynchus kisutch) was investigated. Employing a static water acute toxicity bioassay with platinum as PtCl₄2HCl·6 H₂O, at 8.5±0.2°C, and a water hardness of 55.9±3.5 mg l.^?1 (as CaCo₃), the 24, 48, and 96-h LC₅₀ values were 15.5, 5.2, and 2.5 mg Pt⁴⁺ l.^?1 respectively.Rates of opercular movement for fish exposed to platinum increased with increasing concentrations to a level of 1.0 mg l.^?1. No further significant increases were evident above this level. Hypoactivity of fish exposed to 0.3 mg l.^?1 and higher was evident during the acute toxicity bioassay and much of the post-treatment study. Post-treatment rate of growth for fish exposed to sublethal concentration of platinum for 96 h was less than that of the controls.All organs examined histopathologically were within normal ranges with the exception of the gills and olfactory organ. Lesions in fish exposed to concentration of 0.3 mg l.^?1 and higher were characterized by branchial epithelial hypertrophy and hyperplasia, and necrosis of olfactory epithelial cells.     

Lethal and estrogenic effects of 4-nonylphenol in the cockle Cerastoderma glaucum

The lethal and sublethal effects of the xenoestrogen 4-nonylphenol (NP) were evaluated in the cockle Cerastoderma glaucum. In a 96-h lethality test, bivalves were exposed to 0, 0+ acetone, 0.19, 0.38, 0.75, 1.5 and 3.0 mg NP/l. The 96-h LC₅₀ value was 0.3 mg NP/l. No mortality was observed at 0.1 mg NP/l. The potential estrogenicity of NP was studied in both sexually undifferentiated (resting phase) and differentiated (pre-spawning phase) cockles, exposed for 7 and 14 days to 0, 0+ acetone, 0.0125, 0.025, 0.05, and 0.1 mg NP/l. Vitellogenin (Vg)-like protein levels were determined in both haemolymph and digestive gland by the alkali-labile phosphate (ALP) assay. In the resting phase, exposure for 7 days to 0.1 mg NP/l resulted in significant increases in ALP in both haemolymph and digestive gland, compared with controls. A significant increase was also observed in digestive gland of animals exposed to 0.0125 mg NP/l-exposed animals. After 14 days of exposure, haemolymph ALP levels were significantly increased in exposed animals at all NP concentrations tested, whereas no difference was recorded in digestive gland. In the pre-spawning phase, exposure for 7 days to NP significantly increased ALP levels in haemolymph from males exposed at all NP concentrations tested, whereas no significant variations were found in haemolymph from females. NP (0.05 and 0.1 mg/l) was also shown to increase ALP concentrations significantly in digestive gland of males, but not in those of females. Likewise, after 14 days’ exposure, ALP levels significantly increased in haemolymph from males only at 0.1 mg NP/l. Conversely, NP caused significant increases in ALP levels in digestive gland from both males (at all NP concentrations tested) and females (at 0.025 and 0.1 mg NP/l). These results demonstrate that NP induces Vg synthesis in C. glaucum. Interestingly, males were more responsive to NP than females.     

Retardation of Fin Regeneration in Freshwater Fish Oreochromis mossambicusby Zinc

Regeneration of partially amputated caudal fin was studied in freshwater fish Oreochromis mossambicus exposed to sublethal concentrations of zinc (5.0 mg L^—1 and 10.0 mg L^—1) under ambient laboratory conditions over a period of 20 days. Caudal fin regeneration was measured on 5th, 10th, 15th and 20th day of exposure and after amputation. Significant ( p < 0.05) retardation in fin regeneration was observed on day 5th and 10th in fish exposed to the nominal concentration of 5.0 mg L^—1 Zn, while retardation was found highly significant ( p < 0.01) at all the observations in 10.0 mg L^—1. The maximum inhibition in caudal fin regeneration (20.8 % and 24.3 %) was found during the initial observation at both of the concentrations (5.0 mg L^—1 and 10.0 mg L^—1) of zinc exposure. Later on the regeneration rate was almost as good as in the control group. Thus in this study fin regeneration was significantly inhibited at all time points following Zn exposure as a detrimental effect of Zn to fish. This study demonstrates that fish caudal fin regeneration is a simple assay, sensitive and easy to perform, and can serve as a model to determine the toxicity of pollutants in aquatic environment.     

Toxicity of Phenol,Pentachlorophenol and Sodium Pentachlorophenolate to a Freshwater Teleost Lebistes reticulatus (Peters)

Lebistes reticulatus (Peters) was exposed to various concentrations of phenol, pentachlorophenol and sodium pentachlorophenolate to determine their toxicity, lethal concentrations and slope functions by a static bioassay procedure. Simultaneous controls were also run. The 94h LC₅₀ values were 47.5mg/l (42.04 … 53.68) for phenol, 0.77 mg/l (0.616 … 0.963) for sodium pentachlorophenate and 0.97 mg/l (0.866 … 1.086) for pentachlorophenol. On this basis, the rank order is sodium pentachlorophenolate-pentachlorophenol-phenol. During the treatment, fish showed an abnormal swimming reflex, excessive mucus secretion, haemorrhage near lips, abdomen and basis of fins and loss of equilibrium followed by death.     

Chronic effects of tributyltin on the caprellid amphipod Caprella danilevskii

In order to examine the biological effects by TBT exposure, experiments involving the exposure of five levels of TBT concentrations (0, 10, 100, 1000 and 10,000 ngl(-1)) were conducted on the caprellid amphipod Caprella danilevskii, over a generation after hatching. Marked delays in growth and molting during the early developmental stage and maturation were found in both 100 and 1000 ng TBTCl l(-1) concentrations in spite of the sex. All specimens died in 10,000 ng TBTCl l(-1) within 4 days after hatching. Inhibition of maturation and reproduction such as delaying in the achievement of maturity and a decrease in the number of juveniles hatched was apparent in 10 and 100 ng TBTCl l(-1) concentrations. Furthermore, brood loss, and failure in egg formation and hatching were observed as the TBT concentration became higher. No significant changes in sex ratio were seen at any of the TBT exposure levels during hatching and maturation in the present study. This phenomenon was different from a previous study in which the proportion of females was increased with an increase in TBT concentrations in the embryonic exposure experiment. This suggests that sex disturbance is induced during the embryonic stage of the species. A drastic decrease in survival rate was observed at 10 ng TBTCl l(-1) (25%) which corresponds to the mean level in coastal waters. Therefore, the present encountering ambient TBT concentration may influence populations of C. danilevskii in the coastal environment.     

Polybrominated diphenyl ethers (PBDEs) in marine foodstuffs in Australia: residue levels and contamination status of PBDEs

Edible marine products (n=24) collected from the fish markets in Adelaide, Australia, were analysed for the presence of polybrominated diphenyl ethers (PBDEs). The total concentration of 11 PBDE congeners found in the marine products ranged from 1.01 to 45 ng/g fresh weight. Among the samples analysed, imported silver fish and prawns from Vietnam and Thailand respectively, recorded the highest ∑PBDEs concentrations (45.10 and 43.29 ng/g fresh weight) as against 13.08 and 12.57 ng/g fresh weight in Australian king prawns and Gar fish. BDE-209 was the most dominant congener in the marine products followed by BDE-47, 99 and 100. The calculated average daily intakes of ∑PBDEs per day for an adult male and female (19 or more years old) were 1026.8 and 1188.4 ng/kg per day, respectively. This study clearly shows that the dietary exposure to marine foods can contribute to PBDEs accumulation in human body.     

Changes in zooxanthellae density,morphology, and mitotic index in hermatypic corals and anemones exposed to cyanide

Sodium cyanide (NaCN) is widely used for the capture of reef fish throughout Southeast Asia and causes extensive fish mortality, but the effect of NaCN on reef corals remains debated. To document the impact of cyanide exposure on corals, the species Acropora millepora, Goniopora sp., Favites abdita, Trachyphyllia geoffrio, Plerogyra sp., Heliofungia actinformis, Euphyllia divisa, and Scarophyton sp., and the sea anemone Aiptasia pallida were exposed to varying concentrations of cyanide for varying time periods. Corals were exposed to 50, 100, 300, and 600 mg/l of cyanide ion (CN(-)) for 1-2 min (in seawater, the CN(-) forms hydrocyanic acid). These concentrations are much lower than those reportedly used by fish collectors. Exposed corals and anemones immediately retracted their tentacles and mesenterial filaments, and discharged copious amounts of mucus containing zooxanthellae. Gel electrophoreses techniques found changes in protein expression in both zooxanthellae and host tissue. Corals and anemones exposed to cyanide showed an immediate increase in mitotic cell division of their zooxenthellae, and a decrease in zooxanthellae density. In contrast, zooxanthellae cell division and density remained constant in controls. Histopathological changes included gastrodermal disruption, mesogleal degradation, and increased mucus in coral tissues. Zooxanthellae showed pigment loss, swelling, and deformation. Mortality occurred at all exposure levels. Exposed specimens experienced an increase in the ratio of gram-negative to gram-positive bacteria on the coral surface. The results demonstrate that exposure cyanide causes mortality to corals and anemones, even when applied at lower levels than that used by fish collectors. Even brief exposure to cyanide caused slow-acting and long-term damage to corals and their zooxanthellae.     

Biochemical and Physiological Responses in Atlantic Salmon (Salmo salar) Following Dietary Exposure to Copper and Cadmium

Three experiments were conducted with Atlantic salmon (Salmo salar) to assess the effects of dietary exposure to copper and cadmium. The results presented here provide an overview, details of each experiment will be published in full elsewhere. In the first experiment, salmon parr exposed for four weeks to 35 and 700 mg Cu kg⁻¹ diet had significantly elevated intestinal copper concentrations, cell proliferation (PCNA) and apoptosis rates compared to control fish. No differences were observed in gill or plasma copper concentrations among the groups. In contrast to the controls, the Cu exposed groups did not grow significantly during the exposure period. The second experiment (three months exposure) was conducted to assess the effects of dietary copper (control, 35, 500, 700, 900 or 1750 mg Cu kg⁻¹ diet) on growth and feed utilization in salmon fingerlings. Growth was significantly reduced after three months exposure to dietary Cu concentrations above 500 mg kg⁻¹. Similarly, copper body burdens were significantly higher in fish exposed to elevated dietary copper concentrations (above 35 mg Cu kg⁻¹ diet). In the third experiment, salmon parr were exposed to one of six dietary cadmium concentrations (0, 0.5, 5, 25, 125 or 250 mg Cd kg⁻¹ diet) for four months. Cadmium accumulated in the liver>intestine>gills of exposed fish. Rates of apoptosis and cell proliferation in the intestine increased following exposure to dietary cadmium. Exposure to elevated concentrations of dietary cadmium had no effect on growth in salmon parr. Results from these studies indicate that cellular biomarkers have potential as early warning signs of negative effects on the overall fitness of an organism.     

Immunotoxicity of the xenoestrogen 4-nonylphenol to the cockle Cerastoderma glaucum

The in vivo effects of 4-nonylphenol (NP) on functional responses of haemocytes from the cockle Cerastoderma glaucum were investigated after 7 days exposure to sublethal NP concentrations (0, 0+acetone, 0.0125, 0.025, 0.05 and 0.1 mg/l NP). Haemocytes from both controls and exposed cockles were collected, and the effects of NP on total haemocyte count (THC) and volume of circulating cells, intracellular superoxide anion () levels, acid phosphatase and lysozyme-like activities in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. Exposure of cockles to 0.1 mg/l NP significantly increased THC (p < 0.05) with respect to controls. Analysis of haemocyte size frequency distribution showed that the haemocyte fraction of about 7–8 μm in diameter and 250 femtolitres in volume increased markedly in cockles exposed to the highest NP concentration tested. Apoptosis resulting in cell volume reduction in NP-exposed animals cannot be excluded. No statistically significant variation in intracellular levels was observed. Conversely, significant increases (p < 0.05) in acid phosphatase activity were observed in CFH from 0.05 and 0.1 mg/l NP-exposed animals; no significant differences in enzyme activity were recorded in HL. Lysozyme-like activity also increased significantly in CFH from cockles exposed to 0.05 mg/l NP (p < 0.05) and 0.1 mg/l NP (p < 0.001). Instead, lysozyme-like activity decreased significantly (p < 0.05) in the HL of animals exposed to 0.05 mg/l NP. Our results suggest that NP induces variations in the functional responses of haemocytes of C. glaucum, mainly by reducing cell membrane stability and promoting cell degranulation.     

Seasonally Accumulation of Microcystins in the Various Tissues of an Endemic and Protected Fish Species (Rutilus panosi) with Different Sizes

The present study was undertaken to investigate the accumulation of microcystins (MCYST) in the various tissues of an endemic and protected fish species (Rutilus panosi). We also tried to identify any differences in sensitivity and accumulation of MCYST, for various specimens of the same fish species related to body length. MCYST concentrations in lake water and fish tissues were determined by a commercial Microcystin ELISA kit. Results showed that considerable amount of toxins were found to be accumulated in tissues of R. panosi throughout the year. Among them, liver had the highest MCYST concentrations (407.97 ± 32.43 ng/g). This study confirmed the accumulation of high MCYST concentrations in fish brain (308.75 ± 26.49 ng/g). Our results suggest, that MCYST accumulation in R. panosi tissues is size depended. High MCYST concentration in the muscle of the fish increases the risk of unsafe consumption, thus threatening human health.     

Development of a laboratory exposure system using marine fish to carry out realistic effect studies with produced water discharged from offshore oil production

A biotest system for environmentally realistic exposure of fish to produced water (PW) was developed and tested. Authentic PW was collected at an oil production platform in the North Sea and preserved by freezing in multiple aliquots a 25 L. After transport to the test laboratory onshore, daily PW aliquots were thawed, homogenised and administered to the test fish, Atlantic cod (Gadus morhua), in two diluted exposure concentrations, 0.1% and 0.5%, during a 15 d period, using a continuous flow-through exposure setup. Positive control groups were exposed to two crude oil treatments for comparison. Chemical analyses showed that alkylphenol (AP) and PAH concentrations in PW exposure waters were very low. Observations of significantly increased AP and PAH metabolite levels in PW exposed fish demonstrated the suitability of the biotest system for its use in biological exposure/effect studies of PW, and it also demonstrated the sensitivity of bile metabolites as PW exposure markers in fish. The relevance of the biotest system for PW effect studies and for validating modelled environmental risk estimates of PW dischargers from offshore oil production is discussed.