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Produced water is the largest effluent discharge from oil and gas/condensate production facilities in the North Sea. There is concern that contaminants originating from the reservoir and chemicals used in the production process may affect marine organisms. Developmental toxicity of extractable organic compounds in produced water effluents from oil and gas/condensate production platforms in the Norwegian sector of the North Sea was assessed in a temporal and spatial manner using zebrafish (Danio rerio) embryos. Large-scale solid-phase extraction (SPE) and on-column fractionation of water-soluble fraction (WSF) and an oil/particulate fraction was used in a rapid screening bioassay for embryotoxicity. Exposure to produced water extracts increased rate of mortality and reduced pigmentation and heart rate, as well as delaying time to hatch. The oil/particulate fraction was 10-fold less toxic than WSF, indicating that toxicity was predominantly produced by moderately polar and bioavailable compounds. Large spatial and temporal variation in produced water toxicity was observed, displaying considerable variability in the reservoir, oil well, and effluent composition over time. The noted toxicity did not correlate well with either reported produced water composition or parameters such as total hydrocarbons, thus challenging chemical measurements as a reliable source of information for predicting complex effects. Although embryotoxicity was observed following exposure to the extracts, dilution and transformation of produced water in the recipient are expected to rapidly reduce the concentrations of compounds in the effluents to levels below the thresholds of observed effects.

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Brominated dioxins have recently been detected in Baltic Sea biota. Due to their similarities to the highly toxic chlorinated dioxins, concern has been raised about their potential biological effects. The present study investigated retention and effects of brominated dioxins in adult zebrafish, as well as maternal transfer and effects on offspring. We exposed adult zebrafish for nine weeks via feed to 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) or to a mixture of brominated dioxins (Baltic Sea mixture), which was designed to reflect relative concentrations found in Baltic Sea biota. We studied spawning success, gonad morphology, hepatic vitellogenin gene expression, and offspring early life-stage development to investigate effects on zebrafish reproduction. Hepatic ethoxyresorufin-O-deethylase (EROD) activity and hepatic expression of a number of aryl hydrocarbon receptor (AHR)-regulated genes were studied to investigate if the brominated dioxins can activate gene transcription through the AHR pathway in zebrafish. In addition, glutathione reductase activity and expression of genes involved in adaptive responses to intracellular stress were studied to investigate potential stress effects of brominated dioxins. After nine weeks of exposure, all brominated dioxins spiked to the feed were detected in female fish and transferred to eggs. Exposure to the Baltic Sea mixture and TBDD clearly induced AHR-regulated genes and EROD activity. Exposure to TBDD reduced spawning success, altered ovarian morphology and reduced hepatic vitellogenin gene expression, which implies that TBDD has a similar effect pattern as the chlorinated analogue. Overall, our results show that dietary exposure to sublethal concentrations of brominated dioxins may impair reproductive physiology in fish and induce AHR-regulated genes.

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Proteomic effect screening in zebrafish liver was performed to generate hypotheses following exposure (21 days) to a structurally diverse mixture of brominated flame retardants (BFRs). Fish were exposed to two doses (10 and 100 nmol/g feed). Two-dimensional gel-electrophoresis, image analysis and MALDI-TOF mass-spectrometry revealed 13 and 19 significant responses in males and females, respectively. Effects on proteins related to cellular maintenance and stress were observed in both genders. Regulated proteins were gender-specific, but functionally indicated common protective responses (peroxiredoxin 6 and Zgc:92891 in males and transketolase in females) suggesting oxidative stress. Betaine homocysteine methyltransferase (BHMT) was induced in both genders. In addition a female-specific downregulation of ironhomeostatic proteins (iron-regulatory protein 1 and transferrin) were observed. Our proteomic approach revealed novel responses that suggest important gender-specific sensitivity to BFRs that should be considered when interpreting adverse effects of BFRs.

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The phthalate esters are a group of industrial chemicals considered to have endocrine-disrupting properties. The most common tonnage product among these, di-2-ethylhexyl phthalate (DEHP), is widely spread in the environment. The objectives with the present work were to study uptake and metabolism of orally administered DEHP and its major metabolite mono-2-ethyl hexyl phthalate (MEHP) and to evaluate the impact of early life exposure on sex differentiation in Atlantic salmon. The feeding with contaminated diet started immediately after yolk sac resorption and continued for 4 weeks. Nominal concentrations of DEHP in the diet were 400 (measured 359), 800 (measured 827), and 1500 (measured 1648) mg DEHP/kg and a control group was fed food mixed with solvent. After the exposure period, fish were fed non-contaminated diet until final sampling 4 months post-exposure. There were no effects on growth or survival of the fish and no late effects on hepatosomatic index or sex ratio. However, the histological examination of gonads from fish exposed to 1500 mg DEHP/kg revealed a small but significant incidence (3%) of intersex fish (ovo-testis). Chemical residues of DEHP and MEHP were analyzed weekly during the first 3 months of the post-exposure period. Both DEHP and MEHP were rapidly eliminated to near background levels within one week post exposure. The study indicates that exposure of Atlantic salmon to relatively high concentrations of DEHP during a sensitive part of the life cycle may interfere with gonad differentiation.

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The pharmaceutical estrogen 17alpha-ethinylestradiol (EE2) and the anabolic androgen 17beta-trenbolone (Tb) can interfere with the endocrine and reproductive systems of fish. The potency of these chemicals in zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) was assessed using the core end points vitellogenin (Vtg) concentration at 38 days post-hatch and sex ratio and gonad morphology at 60 days post-hatch. Vtg concentrations were measured in fish whole-body homogenate samples using enzyme-linked immunosorbent assay. Increased Vtg concentration and feminization of fish after exposure to 10 ng/L EE2, as well as masculinization after exposure to 50 ng/L of Tb, were observed in zebrafish. Intersex was observed in medaka exposed to EE2. A decrease in Vtg production after Tb exposure (50 ng/L) was measured in both zebrafish and medaka. Analyses of gonad morphology revealed increased testicular area and sperm percentage in Tb-exposed zebrafish, whereas increased sperm percentage was measured in Tb-exposed medaka. The higher sensitivity of zebrafish compared with medaka to both EE2 and Tb was revealed in the study.

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Synthetic musk substances have been found in a number of environmental samples. Some of these chemicals have been detected in concentrations above 1 microg/L in water, which raises concern about possible effects on aquatic life. The toxicity of four synthetic musks, 4-tert-butyl-2,6-dimethyl-3,5-dinitrophenylethanone (musk ketone), 1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene (musk xylene), 1-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethyl-2-naphthalenyl)ethanone (AHTN, tonalide) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-[g]-2-benzopyrane (HHCB, galaxolide), were studied in zebrafish by the use of two different early life-stage methods. In the first method, specific developmental characteristics during the first 48 hours were studied. In the second method, hatching and survival times were studied on eggs and larvae. The results on heart rate in the first test gave the following LOECs: musk ketone 10 microg/L, musk xylene and AHTN 33 microg/L, and HHCB showed no effect up to 1000 microg/L. In the study of survival time, LOEC for musk ketone was 100 microg/L, for musk xylene 33 microg/L, and AHTN gave no effect on survival time up to 100 microg/L. The LOECs for musk ketone, musk xylene, and AHTN in this study are in the range of what has been measured in sewage effluents and recipients, and consequently these substances may have adverse impact on wild fish.

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Rainbow trout, Oncorhynchus mykiss (Walbaum), sea trout, Salmo trutta L., and Atlantic salmon, Salmo salar L., were experimentally infected with Flavobacterium psychrophilum in order to evaluate any species differences in susceptibility to the bacterium. Furthermore, differences in pathological changes and distribution of the bacteria in internal organs were studied. The bacteria were injected intraperitoneally in two doses, high dose (Hd) 1 x 10(7) colony forming units (CFU) fish(-1) and low dose (Ld) 1 x 10(6) CFU fish(-1). The mortalities in the Ld groups varied between 0 and 7.5% and in the Hd groups between 55-70%. No significant differences in mortality between the species were recorded. Clinical signs and pathological findings were similar in the three species and in accordance with those of rainbow trout fry syndrome. Rainbow trout showed more pronounced lesions in the spleen compared with the other species. Necrosis of renal tubular epithelium and haematopoietic tissue was most prominent in rainbow trout and Atlantic salmon. Intracellular eosinophilic droplets in the kidney tubular epithelium were a prominent finding in rainbow trout and sea trout surviving the infection. The distribution of the bacteria in internal organs was similar in the three species, as studied with immunohistochemistry.

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The uptake and elimination of 20 structurally diverse tetra- to heptachlorinated biphenyls were studied in zebrafish (Danio rerio), three-spined stickleback (Gasterosteus aculeatus), and Arctic char (Salvelinus alpinus). The polychlorinated biphenyls (PCBs) were administered to the fish through food, intraperitoneal injection of peanut oil, or intraperitoneal implantation of silicone capsules. The retention of the PCBs in fish exposed through their diet was related with the substitution patterns of the compounds. Ortho-substituted congeners with no unsubstituted meta-para positions had high biomagnification potential. PCBs with low biomagnification all had adjacent vicinal hydrogens, indicating that congeners with this feature may have been metabolically eliminated. The retention characteristics of the PCBs in the diet-exposed and the injected zebrafish were similar. The pattern of congeners in Arctic char indicates that they have a lower capacity to metabolize PCBs compared to three-spined sticklebacks and zebrafish. The levels in the fish exposed to the PCBs through a silastic implant were negatively correlated with the hydrophobicity of the congeners. Most probably congener-specific release rates of the PCBs from the implants mask their retention characteristics. It is suggested that food, mimicking the natural intake route, should be used in PCB exposure studies to validate extrapolations to natural situations.

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Sea-run Baltic salmon (Salmo salar) populations have been affected by the M74 syndrome since 1974 causing high yolk-sac fry losses in Swedish compensatory rearing plants. M74 has been shown to be a maternally transmitted thiamine (vitamin B(1)) deficiency. The aim of this study was to investigate possible relationships between thiamine and hepatic activities of the thiamine-dependent enzymes transketolase (TK) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) in addition to glucose-6-phosphate dehydrogenase (G6PDH) and cytochrome P4501A (CYP1A), measured as 7-ethoxyresorufin O-deethylase (EROD), in Baltic salmon yolk-sac fry after treatment with thiamine. Thiamine concentrations and activities of TK, alpha-KGDH and EROD were significantly lower (P<0.05) in M74 groups compared to controls (not developing M74) and family groups of thiamine injected females. In M74-developing groups the thiamine immersions reduced the mortality from 86 to 13% and restored thiamine concentrations and activities of TK, alpha-KGDH and EROD to levels slightly lower than the immersed controls. An interesting fact was that the controls showed significantly elevated (P<0.05) TK and alpha-KGDH-activities after immersions in thiamine, indicating that they also may have a stressed thiamine metabolism. The TK and alpha-KGDH-activities of unimmersed groups correlated significantly (P<0.05) with the thiamine content. We suggest that the low activities of TK and alpha-KGDH in M74 groups may be an integrative part in the pathogenesis of M74 development.

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Zambia is a country with an extensive mining industry with the majority of mines located in the Copperbelt province. Through this region of the country, the Kafue River drains and receives effluent water from mining activities as well as from other industrial point sources. In addition, production of agricultural products and pest control requires use of different pesticides in the area. Information on industrial and agricultural pollution has not been clearly identified in Zambia, and little attention has been paid to pollution control and possible impact of metals, pesticides, and other persistent compounds in the environment. The objective of this study was to introduce and to evaluate a few methodologies based on in situ bioassays for environmental assessment to promote sustainable and environmentally sound water resource management of the Kafue River. The results show that caged threespot tilapia exposed downstream of industrial points sources rapidly bioaccumulate several trace elements, i.e., Cd, Co, Cu, Cr, Ni. These elements also occurred in much higher concentrations in water samples downstream of the industrial area compared with a locality upstream. Furthermore, the use of a semipermeable membrane device (SPMD) for passive absorption of lipophilic pollutants in the water showed relatively high concentration of several pesticides, i.e., DDT with major metabolites, PCB, and dieldrin. The present study shows that only 2 weeks of in situ studies in waters contaminated by pollutants affects in situ exposed fish and that the correlation between water and tissue concentrations was relatively good. Both trace elements and persistent organic pollutants occurred in such high concentrations that they must be considered from ecotoxicological aspects and may affect aquatic animal health.

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Juvenile Baltic salmon, Salmo salar, were fed commercial salmon food contaminated with different concentrations of polychlorinated naphthalenes (PCNs; 0.1, 1, 2, or 10 microg PCN/g food). Among other effects, possible estrogenic impact caused by PCNs were evaluated. Fish were therefore fed a diet contaminated with 17beta-estradiol (E2; 0.94 or 9.4 microg E2/g food), as a positive control. After 8, 13, 17, and 41 weeks, sampling took place. Growth, liver somatic index (LSI), EROD activity, and vitellogenin content in blood plasma were measured along with morphological studies of gonads and chemical analyses to determine the effects caused by PCNs. Exposure to PCNs did not seem to have any effects on body weight gain, since the group fed the high dose followed the growth in the control group during the entirely experimental period. After 41 weeks of exposure the groups fed 2 and 10 microg PCN/g food had significantly lower LSIs compared with the control, indicating liver toxic effects of PCNs. Furthermore, a dose-dependent induction of EROD activity was found. At week 41, the control group had an activity of 4.9 +/- 4.8 pmol/mg prot/min, whereas it was between 69 +/- 21 and 720 +/- 370 pmol/mg prot/min in the exposed groups, respectively. Examination of gonadal morphology showed that PCNs also had negative effects on ovaries in Baltic salmon, including delayed development. The distribution between females and males, gonadal morphology, and vitellogenin content in blood plasma did, however, indicate that PCNs are not capable of causing effects similar to E2 or xenoestrogens. Exposure to both of the concentrations of E2 resulted in decreased body weight gain, increased LSI, and feminization of the gonads. E2 did, however, not induce any EROD activity.

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Nitro musks are a group of nitrated benzenes mainly used by the fragrance industry as a substitute for natural musk. Two of the most common nitro musks, musk xylene and musk ketone, have been detected in water, fish, human adipose tissue, as well as in human breast milk. In this study, female zebrafish were dietary-exposed to musk ketone, prior to spawning with unexposed males. The fish were exposed for 8 weeks at two different dose levels. Accumulation of the compound in the fish as well as reproductive success were studied. Exposed females had reduced body weight and length, as well as reduced liver- and gonad somatic index. The results from the reproduction study showed a dose-dependent reduction in fecundity. Early life-stage mortality was increased and the median survival time was reduced. In addition, an embryo/larvae toxicity test was performed, using newly fertilized zebrafish eggs from unexposed parental fish. Eggs were exposed to a series of different concentrations of musk ketone via the surrounding water. A NOEC value of 10 micrograms/l and a LOEC value of 33 micrograms/l was determined. Conclusively, the present study clearly shows that musk ketone negatively affects reproduction and early life-stage survival in zebrafish.

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Chlorinated naphthalenes are planar halogenated aromatic compounds, which are widespread in the environment. Knowledge of their biochemical and toxicological actions in aquatic biota is, however, limited. The objective of this study was to assess the toxicity of highly chlorinated naphthalene congeners found in the aquatic environment on fish sac fry and to study their effects on xenobiotic metabolizing enzymes (CYP) using a short-term primary culture of fish hepatocytes and liver microsomes. A few days after hatching, rainbow trout sac fry were administered either Hallovax 1014, a mixture of 1,2,3,4,6,7-hexachloronaphthalene and 1,2,3,5,6, 7-hexachloronaphthalene (HxCN-mix), or 1,2,3,4,5,6, 7-heptachloronaphthalene (HpCN) (0.08, 0.8, and 4 microg/sac fry injected into the yolk sac). The exposure was terminated 2 weeks later. The naphthalene preparations did not cause any clinical signs of toxicity or difference in mortality rates between the control and treated groups. Immunohistochemical analysis of CYP1A expression in the treated sac fry revealed that staining was most pronounced in the hepatocytes and thereafter in kidney tubular epithelial cells. Moderate CYP1A staining was also seen in the mucosal epithelium of pyloric caecae and mild staining in the epithelium of olfactory organ. Staining in control sac fry was weak or absent. Exposure of the primary cell culture of trout hepatocytes to a low doses (

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In the present study, four structurally diverse polychlorinated biphenyls (PCBs) were chosen from a set of 20 PCBs selected to represent the 154 tetra- through hepta-chlorinated biphenyls. The purpose was to determine estrogenic activities of the chosen PCBs and five of their hydroxylated derivatives (OH-PCBs). A human breast cancer cell line (MCF-7) and primary cultures of rainbow trout (Oncorhyncus mykiss) hepatocytes were used to determine estrogenic effects. The PCBs 2,2',4,6,6'-pentachlorobiphenyl (104) and 2,2',3, 4', 5,6,6'-heptachlorobiphenyl (188), and the hydroxylated PCBs 2,2', 4',6'-tetrachloro-4-biphenylol (4'-50), 2',4', 6'-trichloro-4-biphenylol (4'-30), 2',3,5, 5'-tetrachloro-4-biphenylol (4'-72), 2',3,3',5', 6'-pentachloro-4-biphenylol (4'-112), and 2',3,4',5, 6'-pentachloro-4-biphenylol (4'-121) significantly increased MCF-7 cell proliferation. The coaddition of hydroxytamoxifen, an estrogen antagonist, inhibited increased cell proliferation. The activity of the hydroxylated PCBs 4'-50 and 4'-30 was significantly higher at all nominal concentrations tested as compared to the corresponding PCB, viz., PCB 104. The hydroxylated PCBs 4'-50, 4'-30, 4'-72 and 4'-112 induced vitellogenin synthesis in rainbow trout hepatocytes. Significant differences were found in the MCF-7 system between the parent PCB and its hydroxylated derivative, viz., for 4'-50/4'-30 and 104, and in the rainbow trout hepatocyte assay between 4'-112 and 112, respectively. No activity was observed for PCB 58 in any of the two assays in the present study. Even though cells from two different species (human and fish) are used in the present study, the results obtained by the two methods agree fairly well. In both studies the hydroxylated PCBs were more active than the PCBs, and 4'-30 was the most active compound second only to 17beta-estradiol. http://link.springer-ny. com/link/service/journals/00244/bibs/37n2p145.html

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Zebrafish (Danio rerio) were orally exposed to a mixture of 20 PCBs in three different dose levels (0.008, 0.08, and 0.4 microg of each congener per gram of freeze-dried chironomids). Generally, the PCBs accumulated in a dose-related manner. After 13 weeks of exposure body, liver, and ovary weights, as well as the liver and ovary somatic index, were significantly lower in exposed groups. In addition, the PCB mixture was an effective inducer of hepatic EROD activity. The reproduction study performed with exposed females and unexposed males after 9 weeks revealed that median survival time for larvae was only 7.7 days in the high-dose group as compared with 14 days in controls. Furthermore, egg production was reduced in all three groups exposed. No differences in hatching frequency or median hatching time were recorded. Histologically, females in both the intermediate and high-dose groups contained a reduced number of mature oocytes. The present study demonstrates that the potency of the mixture of selected PCBs induces hepatic EROD activity and has a clearly negative effect on zebrafish reproduction.

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A human breast cancer cell line (MCF-7) was used to develop an in vitro screening assay for the detection of xenoestrogenic environmental pollutants. MCF-7 cells were cultured in DMEM containing 5% fetal bovine serum (FBS). An estrogenic response was defined as an increase in the frequency of proliferating MCF-7 cells, and was measured using a thymidine analog, bromodeoxyuridine, and flow cytometry. Di-2-ethylhexyl phthalate (DEHP) and 4-n-nonylphenol (4-n-NP) were used as model chemicals. The proliferation rate of S-phase cells after 24 h of exposure to various concentrations of 17beta-estradiol and to model compounds was compared with a positive and a negative control, containing 1 nM 17beta-estradiol and 0.1% ethanol, respectively. DEHP and 4-n-NP increased the frequency of proliferating MCF-7 cells in a dose-dependent manner. The lowest concentration that significantly increased the proliferation of MCF-7 cells was 10 microM for DEHP and 1 microM for 4-n-NP. The results showed that the assay is accurate and quick to perform. It may prove a valuable tool for screening potential estrogen-mimicking environmental pollutants.

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The four arsenic-containing chemical warfare agents (CWA) Adamsite (technical, 10-chloro-9-10-dihydrophenarsazine), Clark 1 (Diphenyl arsine chloride), Clark 2 (Diphenyl arsine cyanide), and Lewisite (2-chloro-ethenyl dichloro arsine) were evaluated toxicologically using an in vitro and an in vivo model. The CWA were tested in vitro, using human leucocytes, in order to evaluate their effects on cell proliferation and cell cycle kinetics assayed by flow cytometry. The concentration for total inhibition of cell proliferation for the CWAs ranged from 0.3 micrograms/mL (Lewisite) to 15 micrograms/mL (Clark 1). The results showed no differences in cell proliferation between the different stages of the S-phase either at no-effect or at effect concentrations when samples treated with the CWAs were compared with untreated controls. As2O5 was used as a reference, and the arsenic concentration required for total inhibition of the cell proliferation was 1.7 mg/mL. A lower arsenic concentration, 0.83 micrograms/mL, showed a decreasing proliferation ration in the different parts of the S-phase with Searly and Slate having the highest and lowest ratios, respectively. In addition, there was a positive correlation between the unlabeled S-phase cell ratio and the arsenic concentration, indicating cessation of DNA-synthesis. Conclusively, the examined CWAs exerts a toxicity more potent than arsenic, with respect to cell proliferation. This might indicate that the toxicity of these arsenic-containing CWAs only to a minor extent can be explained by their arsenic content. Because of their relatively good water solubility and ability to easily degrade to arsenic acid, organo-arsenics, e.g., Lewisite, pose less of an environmental threat than organo-arsenics that are sparingly soluble and chemically persistent, like Adamsite. The four CWAs were also tested in vivo in a dietary exposure study, using juvenile three-spined stickleback (Gasterosteus aculeatus L.) as test organism, with measurement of EROD-activity and studies of liver morphology. The results from the in vivo study indicated small effects, suggesting that a 10-week period of dietary exposure at tested dose levels (1 and 100 ng/ml) affects juvenile three-spined stickleback only to a minor extent.

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The distribution of lead in lactating mice and suckling offspring was studied with whole body autoradiography at 4 and 24 h after a single intravenous injection of 203Pb (50 nmol Pb/kg) to the dams. In the lactating mice on day 14 of lactation, the highest uptake of radioactivity at 4 h after administration was recorded in renal cortex, skeleton and liver. A high uptake was also evident in the mammary gland. At 24 h after administration, the radioactivity had decreased in most organs except in the skeleton. In the suckling pups, exposed to lead only via dams' milk for 24 h, the highest level of radioactivity was present in the intestinal mucosa and a much lower level of radioactivity was present in the skeleton. The mammary glands from mice given three daily intravenous injections of 240 mumol Pb/kg were examined with X-ray microanalysis. At 4 h after the last injection, lead was found associated with casein micelles both inside the alveolar cell and in the milk lumen, indicating that lead is excreted into the milk, bound to casein, via the Golgi secretory system.

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Investigation of 12 Irish setter puppies from six litters with severe recurrent infections, neutrophilia and low body weight revealed a leucocyte adhesion protein deficiency with a total lack of CD11b and CD18. Their neutrophil function was severely impaired with a totally absent capacity to ingest C3b-opsonized particles, a significantly impaired capacity to ingest IgG-opsonized particles and significantly diminished adherence to nylon wool when compared with neutrophils from healthy control dogs. The chemiluminescence of patient neutrophils activated by C3b-opsonized particles was, consequently, significantly decreased compared with that of control neutrophils, while the respiratory burst assayed by phorbolmyristate acid (PMA) stimulated nitroblue tetrazolium (NBT)-reduction was normal in the patient group. Random migration and chemotactic responses of patient and control neutrophils, were similar. The etiology, pathogenesis and clinical manifestations of the Irish setter leucocyte adhesion deficiency were similar to that of the leucocyte adhesion deficiency in humans.

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