Hepatic gene expression profiling using GeneChips in zebrafish exposed to 17alpha-methyldihydrotestosterone.

Concentration and time-dependent changes in hepatic gene expression were examined in adult, female zebrafish (Danio rerio) exposed to 0, 0.1, 0.7, 4.9 microg/L of a model androgen, 17alpha-methyldihydrotestosterone (MDHT). At 24 and 168 h, fish were sacrificed and liver was extracted for gene expression analysis using custom Affymetrix GeneChip Zebrafish Genome Microarrays. In an effort to link gene expression changes to higher levels of biological organization, blood was collected for measurement of plasma steroid hormones (17beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. Body and ovary weight were also measured. A significant reduction in E2 occurred at 24h (0.7 and 4.9 microg/L) and 168 h (4.9 microg/L) following MDHT exposure. In contrast, T was significantly increased at 24h (4.9 microg/L) and 168 h (0.1, 0.7, 4.9 microg/L). 171 and 575 genes were significantly affected in a concentration-dependent manner at either 24 or 168 h by MDHT exposure at p

Hepatic gene expression profiling using Genechips in zebrafish exposed to 17alpha-ethynylestradiol.

Genomic, proteomic, and metabolomic technologies continue to receive increasing interest from environmental toxicologists. This interest is due to the great potential of these technologies to identify detailed modes of action and to provide assistance in the evaluation of a contaminant's risk to aquatic organisms. Our experimental model is the zebrafish (Danio rerio) exposed to reference endocrine disrupting compounds in order to investigate compound-induced changes in gene transcript profiles. Adult, female zebrafish were exposed to 0, 15, 40, and 100ng/L of 17alpha-ethynylestradiol (EE2) and concentration and time-dependent changes in hepatic gene expression were examined using Affymetrix GeneChip Zebrafish Genome Microarrays. At 24, 48, and 168h, fish were sacrificed and liver mRNA was extracted for gene expression analysis (24 and 168h only). In an effort to link gene expression changes to effects on higher levels of biological organization, body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. EE2 exposure significantly affected gene expression, GSI, E2, T, and VTG. We observed 1622 genes that were significantly affected (p< or =0.001) in a concentration-dependent manner by EE2 exposure at either 24 or 168h. Gene ontology (GO) analysis revealed that EE2 exposure affected genes involved in hormone metabolism, vitamin A metabolism, steroid binding, sterol metabolism, and cell growth. Plasma VTG was significantly increased at 24, 48, and 168h (p< or =0.05) at 40 and 100ng/L and at 15ng/L at 168h. E2 and T were significantly reduced following EE2 exposure at 48 and 168h. GSI was decreased in a concentration-dependent manner at 168h. In this study, we identified genes involved in a variety of biological processes that have the potential to be used as markers of exposure to estrogenic substances. Future work will evaluate the use of these genes in zebrafish exposed to weak estrogens to determine if these genes are indicative of exposure to estrogens with varying potencies.

Bioconcentration and toxicity of dodecylbenzene sulfonate (C12LAS) to aquatic organisms exposed in experimental streams.

Fish, mollusks, and crustaceans were caged in the tail pool of streams during a C(12)LAS (dodecyl benzene sulfonate) model ecosystem experimental program. Bioconcentration of total C(12)LAS and individual isomers and acute and chronic toxicity were investigated during this study. Toxicity endpoints were based on water and tissue (i.e., body burden) concentrations at which adverse effects were observed. At 32 days, total C(12)LAS bioconcentration factors (BCFs) for the fathead minnow and three invertebrate species ranged from 9 to 116. In general, bioconcentration was affected by isomer position, exposure concentration, and species. BCF values tended to decrease as isomer position moved from external (e.g., 2-phenyl) to internal (e.g., 5,6-phenyl). BCFs also decreased as exposure concentration increased. Mean acute 4-d LC(50) values ranged from 1.5 to >3.0 mg/L for the six species tested. Lethal body burdens associated with 50% mortality (LBB(50)) varied from 0.21 to 0.60 mmole/kg (wet weight). During the 32-day chronic exposures, the EC(20) values were 0.27 (0.204-0.352), 0.95 (0.597-1.29), and approximately 1.0 mg/L for Corbicula (length), Hyalella (survival), and fathead minnow (survival), respectively. At these EC(20) values, C(12)LAS body burdens were 0.035, 0.23, and 0.19 mmoles/kg wet weight in Corbicula, Hyalella, and fathead minnow, respectively. Fish exposed to wastewater treatment plant effluent had total C(12)LAS tissue concentrations ranging from 0.0005 to 0.0039 mmoles/kg wet weight. These concentrations are approximately 45-360 times below the tissue concentration associated with subtle effects in the model ecosystem stream exposures. Total C(12)LAS body burdens in feral and caged Corbicula exposed to WWTP effluents were approximately 0.0013 mmoles/kg; approximately 25-fold below concentrations associated with effects in stream exposures.

The relationship between the interfacial properties of surfactants and their toxicity to aquatic organisms.

In previous work, the toxicity of several anionic and nonionic surfactants to rotifer (Brachionus calyciflorus) was shown to be highly correlated with interfacial activity. In this study, the relationship between interfacial properties of surfactants and their effects on aquatic organisms is extended to include the toxicity of the cationic surfactant class (homologues of alkyl trimethylammonium chloride and alkyl hydroxyethyl dimethylammonium chloride) to green algae (Selenastrum capricornutum) and the bioconcentration of linear alkylbenzene sulfonate (LAS) isomers and homologues by fish (Pimephales promelas and Ictalurus punctatus). In each case, the interfacial activity is expressed by the physicochemical parameter, delta G0ad/Amin, where delta G0ad is the standard free energy of adsorption of the surfactant at the air/solution interface and Amin is the minimum cross sectional area of the surfactant, or the analogous parameter, delta 1sG0ad/1sAmin, at the solid/liquid interface, where the solid is an immobilized artificial membrane (IAM) that mimics a biological cell membrane. The general nature of the relationship between interfacial activity of surfactants and their biological effects in aquatic systems indicates that sorption to biological membranes is a critical parameter for predicting and understanding environmental effects. While specific interactions probably occur once a surfactant has penetrated a membrane bilayer, nonspecific hydrophobic interactions appear to be driving the sorption process.

Aquatic toxicity testing of sparingly soluble, volatile, and unstable substances and interpretation and use of data. Task Force of the European Centre for Ecotoxicology and Toxicology of Chemicals.

Aquatic toxicity tests were originally designed for individual compounds that are soluble and stable in water. For sparingly soluble substances that are not toxic at the solubility limit, the issue is whether tests should be performed with insoluble test substance present. Based on a literature evaluation of the physiology of uptake, it was concluded that only the dissolved fraction is available for uptake and that the insoluble test substance may introduce artifacts that aggravate data interpretation. Therefore, toxicity tests should be conducted only up to the solubility limit. Testing of volatile, unstable, or adsorptive substances is complicated by the ability to keep exposure concentrations relatively constant. For these, appropriate test protocols including adequate design of the dosing systems must be employed. For test medium preparation, physical methods and, where necessary, use of low concentrations of certain solvents are recommended to support handling and speed of dissolution. However, recommendation is made against the use of dispersants. Water-accommodated fractions are recommended as one approach for dosing multicomponent substances. Interpretation of observed effects depends on appropriate test medium preparation, correct measurement and expression of exposure levels, and differentiation of true toxicity from indirect physical effects of the substance, or the toxicity of impurities.

Integrated approach for environmental assessment of new and existing substances.

To ensure the environmental safety of new and existing substances, the environmental fate and potential effects resulting from their release into the environment must be assessed. This requires the development of reasonable, consistent, and effective methods to conduct environmental risk assessments and to prioritize testing for these substances. This assessment must integrate fate and effects at the point-of-entry; it should also extend to an assessment of the potential to persist, and the consequences of increases in exposure concentrations, and to bioaccumulate. The conventional environmental risk assessment approach is used to assess the fate and effects of a substance at its point-of-entry into the environment. In this paper, an approach is presented for conducting quantitative environmental risk assessments of new and existing substances that builds on this conventional approach by including quantitative assessment of the potential for, and consequences of, persistence and bioaccumulation. The approach is described for aquatic, sediment, and terrestrial environments. For each environmental compartment, the approach includes (i) classification of the substance, based on environmental partitioning processes, to ensure that the appropriate data are collected and models used; (ii) a fate assessment to understand the ultimate fate of the substance after entry into the environment or "an environmental compartment" and to predict the exposure concentration of the substance at point-of-entry; (iii) a persistence assessment which determines the potential for increase in the exposure concentration as a result of repeated additions of the substance; (iv) effects assessment; (v) environmental risk assessment to examine the potential for adverse impact on ecosystems; and (vi) a bioaccumulation assessment to evaluate the potential for direct and indirect effects on the species of interest due to bioaccumulation. The assessment approach is illustrated using data for a hypothetical consumer product substance that is disposed down-the-drain.

Interspecies comparisons of A/D ratios: A/D ratios are not constant across species.

The hypothesis that the ratio of the adult (A) and developmental (D) toxicity of a chemical is constant across animal species has been proposed as the basis for identifying developmental hazards, both from traditional developmental toxicity screens using laboratory mammals and from alternative systems such as the coelenterate Hydra attenuata. The purpose of this study was to determine whether A/D ratios are constant across species. The developmental and adult toxicity of 14 chemicals was assessed in four phylogenetically different species. The chemicals tested were aminopterin, bromodeoxyuridine, cadmium chloride, caffeine, congo red, dinocap, dinoseb, diphenylhydantoin, epinephrine, ethylenethiourea, 2-methoxyethanol, mirex, all-trans-retinoic acid, and trypan blue. These chemicals are representative of a variety of toxic mechanisms and a range of potencies. Species used were the CD-1 mouse (Mus musculus), South African clawed frog (Xenopus laevis), fathead minnow (Pimephales promelas), and fruit fly (Drosophila melanogaster). The mouse is a commonly used model for developmental toxicity. The other species are known to be sensitive to mammalian toxicants and have well-studied embryologies. Mice were exposed to chemicals either po or by sc injection using a standard Segment II protocol in which pregnant mice are administered the test agent on a daily basis from Gestation Days 6 to 15, adult toxicity is evaluated during and after treatment, and developmental toxicity is evaluated in fetuses at term. The exposure duration spans the period of organ formation in the embryo. The other species were exposed to test agents for a developmentally comparable period. This was from blastulation (shortly after fertilization) to the free-swimming tadpole stage in Xenopus (4 days); from blastulation to the free-swimming fry stage in Pimephales (7 days); and for the entire larval period, the period of development of the imaginal discs, in Drosophila (6 days). Adults of each species were exposed to test agents for 4, 7, and 6 days, respectively. The route of exposure was via the water column in the two aquatic species and via the diet in Drosophila. Statistical lowest observed effect level (LOEL) and no observed effect level (NOEL) values were generated for adult and developmental toxicity in each species. A/D ratios were calculated using both LOEL and NOEL values.(ABSTRACT TRUNCATED AT 400 WORDS)

The histological and biochemical effects of cadmium exposure in the bluegill sunfish (Lepomis macrochirus).

Cadmium effects on the bluegill sunfish (Lepomis macrochirus) were assessed histologically and biochemically and the effects were compared with effects on the ecologically relevant parameters of growth and survival. Growth and survival were monitored and tissues were removed for histopathological assessment of toxicant effects in a 163-day chronic exposure. The biochemical effects of cadmium were determined in a 32-day subchronic exposure. Exposure of fish to cadmium in hard water (363 mg Cd/liter) caused significant reductions in growth at 3.9 and 12.7 mg Cd/liter. Mortality was significantly increased over controls at 12.7 mg Cd/liter. Histopathological lesions were observed in gill tissue from fish exposed to 3.9 and 12.7 mg Cd/liter at all times during the chronic exposure. No histopathological lesions were observed in any internal organ during this exposure. In a 32-day subchronic exposure, cadmium caused significant increases in serum acid phosphatase and N-acetyl-beta-d-glucosaminidase activities. Serum aspartate and alanine transaminase and lactate dehydrogenase activities were not increased by cadmium exposure. Liver lysosomal membranes were destabilized by cadmium exposure. This indicates an alteration in lysosome function. The utility of biochemical and histological procedures for estimating safe concentrations of environmental pollutants are discussed.