Determining residence patterns of rainbow trout using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis of selenium in otoliths.

Toxicological studies are often hampered by concerns of fish residency in the industrial effluent being evaluated. Contaminants in muscle or visceral tissue are useful indicators of recent exposure, but depuration, metabolic transformation, and tissue recompartmentalization of contaminants makes their use as temporal markers tenuous. Otoliths are metabolically stable and can provide temporal resolution for exposure to some elements that are incorporated into their calcified structure, including the divalent cations Sr, Zn, and Mn. Here we provide the first determinations of selenium, an anion in biological systems, in the otoliths of rainbow trout captured from a site receiving runoff with elevated selenium from a coal mine operation. Concentrations of selenium in annual growth zones of otoliths suggest that fish from the mine-impacted system are recent immigrants from nearby reference streams not receiving selenium-bearing effluent.

Collapse of a fish population after exposure to a synthetic estrogen.

Municipal wastewaters are a complex mixture containing estrogens and estrogen mimics that are known to affect the reproductive health of wild fishes. Male fishes downstream of some wastewater outfalls produce vitellogenin (VTG) (a protein normally synthesized by females during oocyte maturation) and early-stage eggs in their testes, and this feminization has been attributed to the presence of estrogenic substances such as natural estrogens [estrone or 17beta-estradiol (E2)], the synthetic estrogen used in birth-control pills [17 alpha-ethynylestradiol (EE2)], or weaker estrogen mimics such as nonylphenol in the water. Despite widespread evidence that male fishes are being feminized, it is not known whether these low-level, chronic exposures adversely impact the sustainability of wild populations. We conducted a 7-year, whole-lake experiment at the Experimental Lakes Area (ELA) in northwestern Ontario, Canada, and showed that chronic exposure of fathead minnow (Pimephales promelas) to low concentrations (5-6 ng x L(-1)) of the potent 17 alpha-ethynylestradiol led to feminization of males through the production of vitellogenin mRNA and protein, impacts on gonadal development as evidenced by intersex in males and altered oogenesis in females, and, ultimately, a near extinction of this species from the lake. Our observations demonstrate that the concentrations of estrogens and their mimics observed in freshwaters can impact the sustainability of wild fish populations.

Biochemical and histopathological effects in pearl dace (Margariscus margarita) chronically exposed to a synthetic estrogen in a whole lake experiment.

Potential effects of exposure to the synthetic estrogen 17alpha-ethynylestradiol (EE2) were examined in several species of fish from a lake experimentally treated with environmentally relevant concentrations of the contaminant. Ethynylestradiol was added to Lake 260, a small Precambrian shield lake at the Experimental Lakes Area in northwestern Ontario, Canada, from May to October of 2001, 2002, and 2003. Mean concentrations of EE2 in epilimnetic waters ranged between 4.5 and 8.1 ng/L during the three years, with overall means of 6.1 (+/- 2.8), 5.0 (+/- 1.8), and 4.8 (+/- 1.0) ng/L for the three years, respectively. Male and female pearl dace (Margariscus margarita) captured after EE2 additions began contained up to 4,000-fold higher concentrations of the egg yolk precursor vitellogenin than fish captured from the same lake before the EE2 additions or when compared to fish from reference lakes. Edema in the ovaries, inhibited development of testicular tissue, intersex, and histopathological kidney lesions were all evident in fish exposed to EE2. Some indications that EE2 exposure affected in vitro steroidogenic capacity of the ovaries and the testes existed, although results were not always consistent between years. Pearl dace abundance was similar in the lake treated with EE2 and the reference lake. A trend exists toward a reduced overall population of pearl dace from the treated and reference lakes, as do indications that young-of-the-year size classes are less abundant in the EE2-treated lake. Biochemical and histopathological impacts observed in fish exposed to EE2 in this study have not yet been linked to clear population level impacts in pearl dace. Monitoring of these populations is ongoing.

Metabolism of selenomethionine by rainbow trout (Oncorhynchus mykiss) embryos can generate oxidative stress.

Although selenium is required by vertebrates, toxicity can arise at concentrations only slightly greater than those they require. The toxicity of Se is thought to arise from its ability to substitute for sulfur during the assembly of proteins. However, recent studies also indicate that some forms of selenium are capable of generating oxidative stress in an in vitro test system that includes glutathione. L-Selenomethionine, the predominant form of selenium in the eggs of oviparous vertebrates, does not generate oxidative radicals in this system, but lesions consistent with oxidative stress have been identified in fish and birds with high concentrations of Se. Here we report on the ability of rainbow trout embryos to transform L-Selenomethionine to a form capable of producing a superoxide radical. Oxidative stress appears to be generated by methioninase enzyme activity in the embryos that liberates methylselenol from l-Selenomethionine. Methylselenol redox cycles in the presence of glutathione producing superoxide and likely accounts for oxidative lesions present in fish and birds environmentally exposed to excessive loads of selenomethionine.

Altered thyroid status in lake trout (Salvelinus namaycush) exposed to co-planar 3,3',4,4',5-pentachlorobiphenyl.

Recent studies indicate that co-planar 3,3',4,4',5-pentachlorobiphenyl (PCB) congeners or their metabolites may disrupt thyroid function in fishes. Although co-planar PCB have been detected at microgram per kilogram levels in fish from contaminated areas, few studies have examined mechanisms whereby, co-planar PCBs may alter thyroid function in fish. We treated immature lake trout by intraperitoneal (i.p.)-injection or dietary gavage with vehicle containing 0, 0.7, 1.2, 25 or 40 microg 3,3',4,4',5-pentachlorobiphenyl (PCB 126) per kgBW. Blood and tissue samples were collected at various times up to 61 weeks following exposure. The treatments produced sustained dose-dependent elevations of tissue (PCB 126) concentrations. Thyroid epithelial cell height (TECH), plasma thyroxine (T4) and 3,3',5-triiodo-l-thyronine (T3) concentrations, hepatic 5'-monodeiodinase, hepatic glucuronidation of T4 and T3, as well as plasma T4 kinetics and fish growth were analyzed. Exposure to the highest doses of PCB 126 caused increased TECH, plasma T4 dynamics and T4-glucuronidation (T4-G). PCB 126 did not affect 5'-monodeiodinase and T3-glucuronidation (T3-G) and there were no effects on fish growth or condition. Because T3 status and growth were unaffected, the thyroid system was able to compensate for the alterations caused by the PCB 126 exposure. It is clear that concentrations of co-planar PCBs similar to those found in predatory fish from contaminated areas in the Great Lakes are capable of enhancing metabolism of T4. These changes may be of significance when T4 requirements are high for other reasons (e.g. periods of rapid growth, warm temperatures, metamorphosis, and parr-smolt transformation).

Waterborne ethynylestradiol induces vitellogenin and alters metallothionein expression in lake trout (Salvelinus namaycush).

Estrogenic contaminants isolated from waters receiving sewage treatment plant effluents are known to induce the egg yolk precursor vitellogenin (VTG) in male fish. Levels of the metal binding protein metallothionein (MT) have also been shown to be affected by estrogens in fish. It has been postulated that MT declines in estrogen exposed fish to facilitate transfer of the essential metal Zn to cellular components required for VTG synthesis. To examine the changes in MT and VTG concentrations in fish exposed to an estrogen contaminant, lake trout (Salvelinus namaycush) were exposed to waterborne ethynylestradiol at 0, 4, 40 or 400 ng/l(-1) for 21 days. Blood and tissues were collected after 21 days of exposure to measure circulating levels of VTG as well as MT concentrations in liver and kidney. VTG increased in male and female fish from all three exposure groups compared to control fish. MT in liver significantly decreased in males and females compared to the controls, in the two highest exposures. MT in kidney was significantly higher in both sexes of fish exposed to the two highest concentrations of ethynylestradiol. These data are supportive of a relationship between estrogen exposure and the regulation of MT. Further studies to examine the specific links between estrogen exposure, VTG induction and regulation of essential metals like Zn are required.

Dietary accumulation and biochemical responses of juvenile rainbow trout (Oncorhynchus mykiss) to 3,3',4,4',5-pentachlorobiphenyl (PCB 126).

Juvenile rainbow trout (Oncorhynchus mykiss) (initial weights 2-5 g) were exposed to three dietary concentrations (0, 12.4 and 126 ng g(-1), wet weight) of a 14C-labelled 3,3',4,4',5-pentachlorobiphenyl (PCB 126) for 30 days followed by 160 days of clean food. We assessed bioaccumulation, histology (liver and thyroid) and biochemical responses (liver ethoxyresorufin-O-deethylase (EROD), liver vitamins (retinoids and tocopherol) and muscle thyroid hormone levels) along with growth and survival. The half-life of PCB 126 in the rainbow trout ranged from 82 to 180 days while biomagnification factors (BMF) ranged from 2.5 to 4.1 providing further evidence that PCB 126 is among the most bioaccumulative PCB congeners. Toluene extractable 14C declined with time in the trout suggesting the possibility of some biotransformation and/or covalent bonding with biological macromolecules. The threshold for liver EROD induction by PCB 126 was approximately 0.1 ng g(-1) (wet weight). EROD activities in the low- and high treatments were 9 and 44 times greater than control, respectively, and remained elevated throughout the experiment. EROD activity was correlated with whole body concentrations of PCB 126 although there was evidence of EROD activity suppression in the highly exposed fish. Liver didehydroretinoids and tocopherol concentrations were depressed by the high PCB 126 dose after 30 days exposure. Initially, muscle concentrations of thyroxine (T4) and triiodo-L-thyronine (T3) declined as the fish grew during the experiment, and exposure to PCB 126 accelerated the growth related decline. More information is needed to assess the functional significance of the reduced muscular stores of thyroid hormones. Despite the changes in liver EROD, liver vitamins and muscle thyroid hormones, liver and thyroid histology in trout examined after 30 days exposure and growth parameters were unaffected by PCB 126. This indicates that the functional competences of the physiological factors associated with growth were maintained under the experimental conditions.