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Responses of benthic fish exposed to contaminants in outdoor microcosms--examining the ecological relevance of previous laboratory toxicity tests.

Previous laboratory studies indicate that coal combustion wastes (a mixture composed of fly ash and other lower volume wastes such as bottom ash; hereafter collectively referred to as ash) adversely affect the health of benthic fish (Erimyzon sucetta; lake chubsucker), but fish in these studies were provided with ample uncontaminated food resources. Because aquatic disposal of ash can also adversely affect food resources for benthic fish, we hypothesized that changes in resources might exacerbate the effects of ash on fish observed in laboratory studies. We exposed juvenile E. sucetta in outdoor microcosms to water, sediment, and benthic resources from an ash-contaminated site or a reference site for 45 days and compared our findings to previous laboratory studies. Benthic invertebrate biomass was nearly three times greater in controls compared to ash microcosms. Total organic content of control sediment (41%) was also greater than in ash sediments (17%), suggesting that additional benthic resources may have also been limited in ash microcosms. Benthic invertebrates isolated from the ash microcosms had trace element concentrations (As, Cd, Co, Cr, Cs, Se, Sr, and V) up to 18 times higher than in weathered ash used in laboratory studies. The concentrations of trace elements accumulated by fish reflected the high dietary concentrations encountered in the ash microcosms and were associated with reduced growth (final mass = 0.07 g) and survival (25%) compared to controls (0.37 g and 67%, respectively). Accumulation of trace elements, as well as reductions in growth and survival, were more pronounced than in previous laboratory studies, suggesting that resource conditions may be important in mediating ash toxicity. Taken together, our studies suggest that ash discharge into aquatic systems is a more serious threat to the health of benthic fish than previously predicted based upon laboratory toxicity tests.

Analysis of Variance↗

Molecular cloning and expression analysis of phospholipase Cdelta from mud loach, Misgurnus mizolepis.

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCdelta, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCdelta was isolated by screening the cDNA library of mud loach liver and using the 5'-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCdelta gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCdelta isozymes (PH domain, EF-hands, X-Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCdelta shares relatively high sequence identity with mammalian PLCdelta1 (51-52%) and catfish PLCdelta (64%). The recombinant ML-PLCdelta protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni(2+)-NTA affinity chromatography. The recombinant ML-PLCdelta showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP(2)) and its activity was Ca(2+)-dependent, which was similar to mammalian PLCdelta isozymes.

Amino Acid Sequence↗

Vitellogenic effects of 17beta-estradiol in male Chinese loach (Misgurnus anguillicaudatus).

Male Chinese loaches (Misgurnus anguillicaudatus) were exposed to 17beta-estradiol (E2) in the ambient water at nominal concentrations of 0.5, 1, 5 and 10 mug/L continuously for 8 weeks using a semi-static system. The gonadosomatic index (GSI), total plasma protein, total plasma calcium, magnesium and zinc, and plasma vitellogenin (Vtg) were determined as test endpoints. Our results indicate no significant changes in the GSI between exposed and control groups (P<0.05). All exposure concentrations of E2 dramatically induced the production of Vtg within 7 days. The vitellogenic response of male loach was time- and dose-dependent. At higher concentrations of E2 (5 and 10 microg/L), total plasma protein, total plasma calcium, and total plasma magnesium presented a significant time- and dose-dependent increase, and the three parameters were significantly correlated with plasma Vtg. We conclude that Chinese loaches are sensitive to estrogenic compounds and may be chosen as potential sentinel species in field and laboratory studies. In addition, total plasma protein, total plasma calcium, and total plasma magnesium can be used as indirect indicators to predict Vtg levels in estrogenized Chinese loach.

Animals↗

Intersex and other reproductive disruption of fish in wastewater effluent dominated Colorado streams.

Intersex white suckers (Catostomus commersoni) were collected in Boulder Creek and the South Platte River downstream of wastewater treatment plant (WWTP) effluent but not at reference sites. Eighty-three percent of the 60 white suckers collected downstream of the Boulder WWTP in spring and fall were female compared to 45% upstream (n=33). Only female (n=16) and intersex white suckers (n=4) were collected in the South Platte River downstream of the Denver WWTP in the spring. Gonad deformities and delayed follicular maturation were noted in some white suckers in Boulder Creek and the South Platte River downstream of the WWTP effluents. Asynchronous ovarian development was found in some female white suckers downstream of the WWTP effluents, but not upstream. The types and extent of sexual disruption differed in each river studied.

Animals↗

Oral exposure to Microcystis increases activity-augmented antioxidant enzymes in the liver of loach (Misgurnus mizolepis) and has no effect on lipid peroxidation.

Recently, eutrophication has induced severe cyanobacterial blooms in the Naktong River, the second largest river of Korea. In the present study, lipid peroxidation and the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, were evaluated in the liver of loach (Misgurnus mizolepis) that were orally exposed to a low dose of Microcystis through dietary supplementation with bloom scum. Loach received 75 mg of dry cells/kg body weight mass (equal to 10 microg microcystin-RR/kg body mass), for 28 days under controlled conditions. Antioxidant enzymatic activity and lipid peroxidation were measured after termination of exposure. The activities of antioxidant enzyme were significantly increased in the livers of toxin-exposed loach after 28 days of exposure, as compared to control fish. However, lipid peroxidation remained stable in both groups. These results suggest that antioxidant enzymes were able to eliminate oxidative stress induced by low concentrations of microcystins and to prevent increased lipid peroxidation in the liver of loach.

Animals↗

Mercury body burdens in Gambusia holbrooki and Erimyzon sucetta in a wetland mesocosm amended with sulfate.

This study used an experimental model of a constructed wetland to evaluate the risk of mercury methylation when the soil is amended with sulfate. The model was planted with Schoenoplectus californicus and designed to reduce copper, mercury, and metal-related toxicity in a wastestream. The sediments of the model were varied during construction to provide a control and two levels of sulfate treatment, thus allowing characterization of sulfate's effect on mercury methylation and bioaccumulation in periphyton and two species of fish--eastern mosquitofish (Gambusia holbrooki) and lake chubsucker (Erimyzon sucetta). After one year in the experimental model, mean dry-weight normalized total mercury concentrations in mosquitofish from the non-sulfate treated controls (374+/-77 ng/g) and the reference location (233+/-17 ng/g) were significantly lower than those from the low and high sulfate treatments (520+/-73 and 613+/-80 ng/g, respectively). For lake chubsucker, mean total mercury concentration in fish from the high sulfate treatment (276+/-63 ng/g) was significantly elevated over that observed in the control (109+/-47 ng/g), the low sulfate treatment (122+/-42 ng/g), and the reference population (41+/-2 ng/g). Mercury in periphyton was mostly inorganic as methylmercury ranged from 6.6 ng/g (dry weight) in the control to 9.8 ng/g in the high sulfate treatment, while total mercury concentrations ranged from 1147 ng/g in the control to a high of 1297 ng/g in the low sulfate treatment. Fish methylmercury bioaccumulation factors from sediment ranged from 52 to 390 and from 495 to 3059 for water. These results suggest that sulfate treatments add a factor of risk due to elevated production of methylmercury in sediment and porewater which biomagnified into small fish, and may potentially increase through the food web.

Analysis of Variance↗

Selenium impacts on razorback sucker, Colorado: Colorado River III. Larvae.

Razorback sucker (Xyrauchen texanus) larvae from adults exposed to selenium at three sites near Grand Junction, Colorado, for 9 months were used in a 30-day waterborne and dietary selenium study. Selenium concentrations in water averaged <1.6 microg/L from 24-Road, 0.9 microg/L from Horsethief, 5.5 microg/L from Adobe Creek, and 10.7 microg/L from the North Pond. Selenium in dietary items averaged 2.7 microg/g in brine shrimp, 5.6 microg/g in zooplankton from Horsethief east wetland, 20 microg/g in zooplankton from Adobe Creek, and 39 microg/g in zooplankton from North Pond. The lowest survival occurred in larvae fed zooplankton rather than brine shrimp. Survival of larvae at Adobe Creek and North Pond was lower in site water than in reference water. Survival of brood stock larvae was higher than Horsethief larvae even though they received the same water and dietary treatments. Arsenic concentrations in brine shrimp may have resulted in an antagonistic interaction with selenium and reduced adverse effects in larvae. Deformities in larvae from North Pond were similar to those reported for selenium-induced teratogenic deformities in other fish species. Selenium concentrations of 4.6 microg/g in food resulted in rapid mortality of larvae from Horsethief, Adobe Creek, and North Pond, and suggested that selenium toxicity in the Colorado River could limit recovery of this endangered fish.

Abnormalities, Drug-Induced↗

Reduced growth and survival of larval razorback sucker fed selenium-laden zooplankton.

Four groups of larval razorback sucker, an endangered fish, were exposed to selenium-laden zooplankton and survival, growth, and whole-body residues were measured. Studies were conducted with 5, 10, 24, and 28-day-old larvae fed zooplankton collected from six sites adjacent to the Green River, Utah. Water where zooplankton were collected had selenium concentrations ranging from <0.4 to 78 microg/L, and concentrations in zooplankton ranged from 2.3 to 91 microg/g dry weight. Static renewal tests were conducted for 20 to 25 days using reference water with selenium concentrations of <1.1 microg/L. In all studies, 80-100% mortality occurred in 15-20 days. In the 28-day-old larvae, fish weight was significantly reduced 25% in larvae fed zooplankton containing 12 microg/g selenium. Whole-body concentrations of selenium ranged from 3.7 to 14.3 microg/g in fish fed zooplankton from the reference site (Sheppard Bottom pond 1) up to 94 microg/g in fish fed zooplankton from North Roadside Pond. Limited information prior to the studies suggested that the Sheppard pond 1 site was relatively clean and suitable as a reference treatment; however, the nearly complete mortality of larvae and elevated concentrations of selenium in larvae and selenium and other elements in zooplankton indicated that this site was contaminated with selenium and other elements. Selenium concentrations in whole-body larvae and in zooplankton from all sites were close to or greater than toxic thresholds where adverse effects occur in fish. Delayed mortality occurred in larvae fed the two highest selenium concentrations in zooplankton and was thought due to an interaction with other elements.

Animals↗

Commentary: selenium study on endangered razorback sucker is flawed.

The razorback sucker (Xyrauchen texanus) is listed as federally endangered throughout its range. A massive recovery effort by the Recovery Implementation Program for Endangered Fish Species in the Upper Colorado River Basin has focused its efforts in the upper Colorado River. The upper Colorado River basin also has two locations that have been identified by the National Irrigation Water Quality Program as having substantial selenium contamination. Selenium is toxic to fishes, affecting reproductive success. Thus, there is concern about potential effects of selenium on the endangered razorback sucker. Two sets of studies have investigated the effects of selenium on razorback suckers, but study results are conflicting. This commentary evaluates studies that claim selenium is not a problem for razorback sucker. We find that study bias was so pervasive that purported conclusions were unwarranted. Contaminated control water, older life stages of fish tested, lack of methodology for analysis of selenium in water, diet, or fish, use of rotifer food, low feeding rates, low growth rates of fish, and improper storage of site waters resulted in an apparent erroneous linkage of high selenium in whole-body residues with no adverse effects.

Age Factors↗

Development and validation of an enzyme-linked immunosorbent assay for vitellogenin in Chinese loach (Misgurnus angaillicaudatus).

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of vitellogenin (Vtg) in the plasma of Chinese loach (Misgurnus angaillicaudatus). Vtg was isolated by anion exchange membrane chromatography from the plasma of 17beta-estradiol (E(2))-treated loach. Polyclonal antibody against Vtg was raised in rabbits, and the specificity of the antibody was assessed by Western blotting analysis. No cross-reactivity was observed with plasma from male loach. Plasma samples from vitellogenic females and E(2)-treated males were diluted parallel with the purified Vtg standard curve in the ELISA. The detection limit of the assay was 5.7 ng ml(-) (1) and a working range of the standard curve was between 15.6 and 1000 ng ml(-) (1). The intra- and inter-assay coefficients of variations were 6.9% and 10.4%, respectively. The recovery was 104.4%. The assay was applied for measuring vitellogenin concentration in the plasma from 24 wild Chinese loach from Lianyungang, Jiangsu Province, in October 2003. It was found that, in addition to females (plasma Vtg concentration ranging from 518.4 microg ml(-) (1) to 1922.3 microg ml(-) (1)), male loach showed low plasma Vtg--from undetectable (two fish) to 2.9+/-0.7 microg ml(-) (1) (mean+/-S.D.). The results indicate that this method provides a valuable tool for the study of estrogenic effect in Chinese loach.

Animals↗

Cytochrome P4501A induction in brown trout exposed to small streams of an urbanised area: results of a five-year-study.

This case study examines the ability of the cytochrome P4501A (CYP1A) biomarker to distinguish the pollution status of two small streams, Krähenbach and Körsch, receiving different levels of urban and agricultural impact, with low to moderate contamination by arylhydrocarbon receptor (AhR)-binding PAHs and PCBs. Brown trout, Salmo trutta, exposed in enclosure restrictions, showed significant between-stream differences of hepatic CYP1A levels. EROD activities were the better discriminator than CYP1A protein levels. The CYP1A response was consistent and repeatable over the 5-year observation period from 1995 to 1999. In contrast to brown trout, hepatic CYP1A of stone loach, Barbatula barbatula, did not clearly distinguish the streams. The findings of this long-term study lend support to the use of CYP1A as a biomarker of degraded environmental conditions, provided that sufficiently long observation periods are used to average out confounding factors, that sufficiently sensitive detection methods are used, and that a responsive monitoring species is chosen.

Agriculture↗

Perchlorate in fish from a contaminated site in east-central Texas.

Perchlorate, a known thyroid endocrine disruptor, contaminates surface waters near military instillations where solid fuel rocket motors are manufactured or assembled. To assess potential perchlorate exposure to fish and the human population which may feed on them, fish were collected around the Naval Weapons Industrial Reserve Plant in McLennan County, TX, and analyzed for the presence of the perchlorate anion. The sampling sites included Lake Waco and Belton Lake, and several streams and rivers within their watersheds. The general tendency was that perchlorate was only found in a few species sampled, and perchlorate was not detected in every individual within these species. When detected in the fish, perchlorate tissue concentrations were greater than that in the water. This may be due to highly variable perchlorate concentrations in the water coupled with individual-level variation in elimination from the body, or to routes of exposure other than water.

Animals↗

Serum acute phase response (APR)-related proteome of loach to trauma.

Proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2-DE PAGE) together with mass spectrometry was applied to screen acute phase response (APR)-related proteins in serum from loach following injury. Six APR-related proteins were identified, in which apolipoprotein, cathepsin, C-reactive protein (CRP) were known APP, while signal recognition protein (SRP), gastrin 71 and parvalbumin were new APR-related proteins.

Acute-Phase Proteins↗

Identification of 24 genes and two pseudogenes coding for olfactory receptors in Japanese loach, classified into four subfamilies: a putative evolutionary process for fish olfactory receptor genes by comprehensive phylogenetic analysis.

Twenty-four olfactory receptor (OR) genes and two pseudogenes have been identified in the genome of Japanese loach (Misgurnus anguillicaudatus). The genes were classified into four subfamilies according to the similarity of the amino acid sequences. In each subfamily, members showed high sequence similarity not only to each other but also to orthologues of other fish species. The number of members in each OR subfamily was roughly estimated to be from 3 to 10 by genomic Southern blot analysis. The genes of all four OR subfamilies were shown to express on olfactory neurons of the olfactory epithelium by in situ hybridization analysis. Two major features of fish OR genes were found by comprehensive and comparative analyses on OR genes of Japanese loach and other fish species including catfish, zebrafish and pufferfish. First, the phylogenetic tree comprising of representative subfamily members suggests the existence of several prototype genes common to the genomes of many fish species. Second, when all members of orthologous subfamilies identified in each clade of the tree are integrated, the members of a single species comprise a monophyletic group. This means that 'intraspecies' sequence homology, that is, homology among paralogous genes of the same subfamily in a species, is higher than 'interspecies' homology, that is, homology between orthologous genes of different species. This suggests that the subfamily members of a species have evolved recently. Taken together, fish OR genes have evolved from a limited number of prototype genes common to most fish species, and several genes in a subfamily have diversely evolved in each species from each prototype.

Amino Acid Sequence↗

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis.

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.

Animals↗

Apoptosis induction on HL-60 cells of a novel polysaccharide from the mucus of the loach, Misgurnus anguillicaudatus.

This study was designed to determine the anti-proliferative, apoptotic properties of a novel polysaccharide from the loach, Misgurnus anguillicaudatus (MAP), using a human promyelocytic leukemia line (HL-60) as a model system. HL-60 cells were cultured in the presence of MAP at various concentrations (50-800 mg/l) for 5 days and the percentage of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results showed that MAP inhibited the cells viability in time and concentration-dependent characteristics. We found that anti-proliferative effect of MAP was associated with apoptosis on HL-60 cells by determinations of morphological changes and oligonucleosomal DNA fragments. In addition, the content of nitric oxide (NO) and activity of lactate dehydrogenase (LDH) release increased when the cells incubated with MAP at various concentrations and times. These investigations suggest that the polysaccharide from loach has the function of anti-proliferation and induction of apoptosis in tumor cells in vitro.

Animals↗

Electron microscopic examination of the viromatrix of Rana grylio virus in a fish cell line.

Rana grylio virus (RGV), a Ranavirus belonging to the family Iridoviridae, assembles in the viromatrix which is a factory for viral genome replication and particle assembly. Ultrastructural studies of the viromatrix will clarify the pathway of assembly. The viromatrix and quantitative changes in RGV infected epithelipma papulosum cyprini (EPC) cells, one of fish cell lines, were studied by electron microscopy. It was shown that viromatrices were adjacent to the nucleus, and the electron density was lower than that of the surrounding cytoplasm. The viromatrix contained virus particles with different forms, electron-dense materials and amorphous structures which included tubules and membranous materials. Tubules were often observed in direct continuity with empty capsids. Several bundles of intermediate filaments were seen alongside the viromatrix and crystalline aggregates. Large clusters of mitochondria occurred in proximity to viromatrix. A total of 990 cells profiles were examined. The results showed that 394 cells contained viromatrix: 89.3% contained one, and 10.7% contained two to four viromatrices. The number of viromatrices increased gradually and reached a peak at 16 h p.i. The viromatrix area at 24 h p.i. increased up to 7.4 +/- 0.69 microm(2) which was three-times lower than that at 6 h p.i. The number of empty capsids within viromatrix was generally more than that of "full" particles at different time points, and there was a strong positive correlation between them.

Animals↗

Environmental contaminants and biomarker responses in fish from the Columbia River and its tributaries: spatial and temporal trends.

Fish were collected from 16 sites on rivers in the Columbia River Basin (CRB) from September 1997 to April 1998 to document temporal and spatial trends in the concentrations of accumulative contaminants and to assess contaminant effects on the fish. Sites were located on the mainstem of the Columbia River and on the Snake, Willamette, Yakima, Salmon, and Flathead Rivers. Common carp (Cyprinus carpio), black bass (Micropterus sp.), and largescale sucker (Catostomus macrocheilus) were the targeted species. Fish were field-examined for external and internal lesions, selected organs were weighed to compute somatic indices, and tissue and fluid samples were preserved for fish health and reproductive biomarker analyses. Composite samples of whole fish, grouped by species and gender, from each site were analyzed for organochlorine and elemental contaminants using instrumental methods and for 2,3,7,8-tetrachloro dibenzo-p-dioxin-like activity (TCDD-EQ) using the H4IIE rat hepatoma cell bioassay. Overall, pesticide concentrations were greatest in fish from lower CRB sites and elemental concentrations were greatest in fish from upper CRB sites. These patterns reflected land uses. Lead (Pb) concentrations in fish from the Columbia River at Northport and Grand Coulee, Washington (WA) exceeded fish and wildlife toxicity thresholds (>0.4 microg/g). Selenium (Se) concentrations in fish from the Salmon River at Riggins, Idaho (ID), the Columbia River at Vernita Bridge, WA, and the Yakima River at Granger, WA exceeded toxicity thresholds for piscivorous wildlife (>0.6 microg/g). Mercury (Hg) concentrations in fish were elevated throughout the basin but were greatest (>0.4 microg/g) in predatory fish from the Salmon River at Riggins, ID, the Yakima River at Granger, WA, and the Columbia River at Warrendale, Oregon (OR). Residues of p,p'-DDE were greatest (>0.8 microg/g) in fish from agricultural areas of the Snake, Yakima, and Columbia River basins but were not detected in upper CRB fish. Other organochlorine pesticides did not exceed toxicity thresholds in fish or were detected infrequently. Total polychlorinated biphenyls (PCBs; >0.11 microg/g) and TCDD-EQs (>5 pg/g) exceeded wildlife guidelines in fish from the middle and lower CRB, and ethoxyresorufin O-deethylase (EROD) activity was also elevated at many of the same sites. Temporal trend analysis indicated decreasing or stable concentrations of Pb, Se, Hg, p,p'-DDE, and PCBs at most sites where historical data were available. Altered biomarkers were noted in fish throughout the CRB. Fish from some stations had responded to chronic contaminant exposure as indicated by fish health and reproductive biomarker results. Although most fish from some sites had grossly visible external or internal lesions, histopathological analysis determined these to be inflammatory responses associated with helminth or myxosporidian parasites. Many largescale sucker from the Columbia River at Northport and Grand Coulee, WA had external lesions and enlarged spleens, which were likely associated with infections. Intersex male smallmouth bass (Micropterus dolomieu) were found in the Snake River at Lewiston, ID and the Columbia River at Warrendale, OR. Male bass, carp, and largescale sucker containing low concentrations of vitellogenin were common in the CRB, and comparatively high concentrations (>0.3 mg/mL) were measured in male fish from the Flathead River at Creston, Montana, the Snake River at Ice Harbor Dam, WA, and the Columbia River at Vernita Bridge, WA and Warrendale, OR. Results from our study and other investigations indicate that continued monitoring in the CRB is warranted to identify consistently degraded sites and those with emerging problems.

Animals↗