Quantification of metallothionein by differential pulse polarography overestimates concentrations in crustaceans.

If metallothionein concentrations in invertebrates are to be used as biomarkers for metal contamination in the aquatic environment, it is imperative thatthe methods used for quantitative analysis are reliable. A review of the literature concerned with quantification of crustacean metallothionein shows that utilization of differential pulse polarography generally results in higher concentrations than any other method. The obvious discrepancies were investigated by experimental comparison of three different methods (enzyme linked immuno sorbent assay (ELISA), a spectrophotometric assay, differential pulse polarography) for determination of metallothionein concentrations in the shore crab Carcinus maenas. Application of an ELISA to cytosolic tissue extracts of unexposed crabs gave basal metallothionein levels of approximately 180 and 80 microg g(-1) dw in midgut gland and gill, respectively; the levels increased 14-fold and 11-fold after exposure to 2 mg l(-1) Cd for 3 weeks. The spectrophotometric assay generally gave 2-fold higher results than the ELISA in unexposed crabs and similar results in Cd-exposed crabs. The determination of metallothionein by differential pulse polarography (successfully applied in vertebrate tissue) was found to be unsuitable for crustacean tissues due to unidentified interfering compounds which led to 5- to 20-fold overestimation of metallothionein levels. The method should not be used unless thoroughly validated in the group of organisms in question.

Orally administered bisphenol a in rainbow trout (Oncorhynchus mykiss): estrogenicity, metabolism, and retention.

The estrogenic effect of orally administered bisphenol A (BPA) was investigated in a rainbow trout (Oncorhynchus mykiss) test system. Bisphenol A was administered orally to sexually immature rainbow trout every second day for up to 12 d in doses between 1.8 and 258 mg/kg every second day (/2d). Plasma vitellogenin was measured before and during the exposures, and the concentrations of BPA in plasma, liver, and muscle and the plasma concentrations of BPA glucuronic acid (BPAGA) were determined at the end of the experiments. Increases in average plasma vitellogenin levels were seen at oral exposure to 24 mg BPA/kg/2d; the most sensitive fish responded to 9.3 mg/kg/2d. At day 12, the 10, 50, and 90% effective doses for increase in vitellogenin synthesis were 13, 19, and 25 mg/kg/2d, respectively. Bisphenol A could be detected in liver, muscle, and plasma at the end of the exposure, generally in increasing concentrations with increasing doses; liver concentrations generally were higher than muscle concentrations. Four to five hours after the last feeding of doses between 3.6 and 24 mg BPA/kg, plasma BPA concentrations ranged between 400 and 1,200 nM, whereas BPAGA concentrations were between 2- and 10-fold higher. The difference between BPA and BPAGA concentrations increased with increasing BPA dose. Bisphenol A showed little tendency to bioaccumulate in rainbow trout; less than 1% of the total amount of BPA administered orally at doses between 1.8 and 258 mg/ kg/2d over the 10- or 12-d experimental period was retained in muscle and liver at 5 or 24 h after the end of the experiments.

Estrogenic effect of dietary 4-tert-octylphenol in rainbow trout (Oncorhynchus mykiss).

The estrogenic effect of dietary 4-tert-octylphenol (octylphenol) in rainbow trout Oncorhynchus mykiss was investigated. Octylphenol was administered orally to sexually immature rainbow trout every second day for 11 days in doses between 0.4 and 50 mgkg(-1)2 d(-1). Plasma vitellogenin was measured at day 0, 6 and 11 and at the end of the experiments, the amounts of octylphenol retained in liver and muscle were determined. Increases in average plasma vitellogenin levels were seen at exposure to 40 mg octylphenol kg(-1) every second day; the most sensitive fish responded to 30 mgkg(-1). Doses below 20 mg octylphenol kg(-1)2 d(-1) had no effect. The ED(50) value for induction of vitellogenin synthesis was 35 mg octylphenol kg(-1)2 d(-1). Only 1 to 2 per thousand of the total amount of octylphenol administered orally over the 11 days experimental period was retained in muscle and liver at the end of the experiment. A clear dose-related increase was observed for concentrations of octylphenol in both liver and muscle of fish exposed to doses between 0.4 and 50 mgkg(-1)2 d(-1). A significant correlation was found between the concentrations of octylphenol in the liver and vitellogenin level in plasma.

Estrogenic effect of propylparaben (propylhydroxybenzoate) in rainbow trout Oncorhynchus mykiss after exposure via food and water.

The estrogenic effect of propylparaben was investigated in a rainbow trout Oncorhynchus mykiss test system. Propylparaben was administered orally to sexually immature rainbow trout every second day for up to 10 days in doses between 7 and 1830 mg kg(-1) 2 d(-1) and in the water at 50 and 225 microg l(-1) for 12 days. Plasma vitellogenin was measured before and during the exposures and the concentrations of propylparaben in liver and muscle were determined at the end of experiments. Increases in average plasma vitellogenin levels were seen at oral exposure to 33 mg propylparabenkg(-1) 2 d(-1); the most sensitive fish responded to 7 mg kg(-1). The ED(50) values for increase in vitellogenin synthesis were 35, 31 and 22 mg kg(-1) 2 d(-1) at day 3, 6 and 11, respectively. Exposure to 225 microg propylparabenl(-1) increased vitellogenin synthesis, but exposure to 50 microg l(-1) did not. Propylparaben showed little tendency to bioaccumulation in rainbow trout; less than 1 per thousand of the total amount of propylparaben administered orally at 1830 mg kg(-1) 2 d(-1) over the 10-d experimental period was retained in muscle and liver 24 h after the end of the experiment. Exposure to 225 microg propylparabenl(-1) for 12 d led to concentrations of 6700 and 870 microg propylparabenkg(-1) liver and muscle, respectively. Half lives for propylparaben were 8.6 h in liver and 1.5 h in muscle.

Effects of waterborne exposure of octylphenol and oestrogen on pregnant viviparous eelpout (Zoarces viviparus) and her embryos in ovario.

Exposure to oestrogenic chemicals (xeno-oestrogens) may have severe effects on embryonic development. The present study investigates whether the oestrogenic endocrine disruptor 4-tert-octylphenol (4-tOP) or 17beta-oestradiol (E(2)) is accumulated in the viviparous fish the eelpout (Zoarces viviparus) and transferred to the embryos in ovario and subsequently affects embryonic development, including gonadal differentiation. Pregnant eelpouts were exposed to nominal concentrations of 25 micro gl(-1) or 100 micro gl(-1) 4-tOP (OP25 or OP100, respectively) or 0.5 micro gl(-1) E(2) in water. During 4-tOP exposure, the compound accumulated in both plasma and ovarian fluid in a concentration-dependent manner. In the mother fish, the oestrogenic biomarkers, vitellogenin (Vtg) in plasma, Vtg mRNA in liver and oestrogen-binding activity in liver, were all induced by 4-tOP (and by E(2)) at an actual concentration of 14 micro gl(-1). E(2) and 4-tOP were examined for their potency to disturb the maternal-foetal trophic relationship by disturbing the physiology of the ovary and by changing the distribution of essential nutrients normally transported to embryos during pregnancy. After exposure to E(2) or 4-tOP, calcium was depleted from the ovarian fluid and the level of free amino acids available in maternal plasma was decreased. A marked overall effect on ovarian components, including the ovarian sac, ovarian fluid and embryonic mass, was evident. Embryonic growth was significantly decreased, which might in part be attributed to disturbances of the maternal-foetal trophic relationship. Marked inductions of Vtg mRNA and Vtg protein, determined by RT-PCR and immunohistochemistry, respectively, were found in embryos from the OP100 group - the only group to show considerable accumulation of an oestrogenic compound in the ovarian fluid. A different pattern of gonadal development was found in embryos from the OP100 group compared with embryos from the control, OP25 or E(2) groups, in which approximately 50% had normal ovaries and 50% had normal presumptive male gonads. In the OP100 group, 46% had normal ovaries but, in contrast to controls, only 22% had normal presumptive male gonads, whereas the remaining 32% had abnormal male gonads with structures resembling the endo-ovarian cavity of a female gonad. As oestrogen receptor (ER) expression was detected by in situ hybridisation in early differentiating gonads, these effects could be mediated by direct interaction of the xeno-oestrogens with gonadal ER. In conclusion, this study indicates that the xeno-oestrogen 4-tOP can be transferred from the water via the mother fish to the ovarian fluid and can subsequently disturb the maternal-foetal trophic relationship and cause severe effects on embryonic development, including gonadal differentiation in ovario.