Chronic toxicity of hexavalent chromium to the fathead minnow (Pimephales promelas)

The chronic effects of hexavalent chromium on the fathead minnow (Pimephales promelas) were investigated. Survival was affected only at the high test concentration of 3.95 mg Cr/L. All chromium concentrations, including 0.018 mg/L, the lowest tested, retarded the early growth of first-generation fish, but this effect was only temporary. Growth of second-generation fish was not affected at concentrations of 1.0 mg/L or lower. Reproduction and hatchability of eggs were not affected at any chromium concentration tested.The maximum acceptable toxicant concentration (MATC) for fathead minnows in hard water (209 mg/L as CaCO₃ at pH 7.7) was based on survival and lies between 1.0 and 3.95 mg Cr/L, respectively. The application factor (MATC/96-hr LC50) is between 0.03 and 0.11.     

Effects of the synthetic estrogen 17 alpha-ethinylestradiol on the life-cycle of the fathead minnow (Pimephales promelas)

A fish full life-cycle (FFLC) study was conducted for 17 alpha-ethinylestradiol (EE2) using the fathead minnow, Pimephales promelas. Newly fertilized embryos (< 24 h old) were exposed to five concentrations of EE2 (0.2, 1.0, 4.0, 16, and 64 ng/L nominal) in continuous flow-through conditions for 305 d at 25 +/- 1 degrees C. Exposure concentrations were verified by 14C-EE2 radiochemistry, supported by radioimmunoassay, and mean measured values were > or = 70% of nominal. For the F0 adult phase until 301 d posthatch, the no-observed-effect concentrations (NOECs) for growth, survival, and reproduction (as egg production) were all > or = 1.0 ng/L. The NOEC values for F1 embryo hatching success and larval survival (at 28 d posthatch) were both > or = 1.0 ng/L. While statistically detectable changes in F1 growth were evident at 0.2 ng/L, these were not considered to be biologically significant when compared with historical control data. Male fish exposed to EE2 at 4.0 ng/L failed to develop normal secondary sexual characteristics; on the other hand, assumed females exposed to this level of EE2 were able to breed when paired with males that had not been exposed to EE2. Histology of F0 control, 0.2-, and 1-ng/L exposed fish at 56 d posthatch indicated an approximate female-to-male (F:M) sex ratio of 50:50 (with no ovatestes observed in the control), while fish exposed to EE2 at 4.0 ng/L for 56 d posthatch had a F:M sex ratio of 84:5 (with ovatestes in 11% of fish). After 172 d posthatch, no testicular tissue was observed in any fish exposed to EE2 at 4.0 ng/L. At the same time point, plasma vitellogenin levels were significantly higher in fish exposed to EE2 at 16 ng/L. A lack of sexual differentiation occurred in males at concentrations > or = 4.0 ng/L. Taking into account these data, the overall no-observed-adverse-effect concentration was considered to be 1.0 ng/L.     

Acute toxicity of ten chlorinated aliphatic hydrocarbons to the fathead minnow (Pimephales promelas)

Ninety-six-hour LC50 values were determined for 10 chlorinated aliphatic hydrocarbons in freshwater flow-through toxicity tests on fathead minnows (Pimephales promelas). The 96-hr measured LC50 values in mg/L from combined duplicate tanks were: tetrachloroethylene 13.4; 1,1′,2,2′-tetrachloroethane 20.4; pentachloroethane 7.34; 1,1′,2-trichloroethane 81.6; hexachlorobutadiene 0.10; 1,1′2-trichloroethylene 45.0; 1,2-dichloropropane 140; 1,2-dichloroethane 116; hexachloroethane 1.51; 1,3-dichloropropane 131.     

Toxicity and bioaccumulation of 2,4,6-trinitrotoluene in fathead minnow (Pimephales promelas)

Few studies have determined the toxicity and bioaccumulation potential of explosive compounds in freshwater fish. In the present study, fathead minnow (Pimephales promelas) were exposed to a range of 2,4,6-trinitrotoluene (TNT) concentrations (0.44-44 micromol/L [0.1-10 mg/L] and 4.4-22.0 micromol/L [1.0-5.0 mg/L] in 4- and 10-d experiments, respectively). Median lethal concentrations of 11.93 micromol/L (2.7 mg/L; 95% confidence limit [CL], 10.29-13.83 micromol/L) and 9.68 micromol/L (2.20 mg/L; 95% CL, 9.17-10.22 micromol/L) were calculated in the 4- and 10-d experiments, respectively, and median lethal body residue of 101.0 micromol/kg (95% CL, 86.0-118.7 micromol/kg) was calculated in 4-d experiments. To study bioaccumulation, fish were exposed to 4.4 micromol/L (1 mg/L) of TNT for 12 h. Rapid bioaccumulation of TNT occurred within the first 10 min of exposure (ku = 30.4 L/kg/ h). Elimination of sigmaTNT (molar sum of TNT and degradation products 2- and 4-aminodinitrotoluenes) was fast, with an elimination rate (ke) of 2.24/h and a short half-life (0.31 h). The bioconcentration factors determined using 6-h mean tissue and water concentrations of sigmaTNT were 8.40 and 4.68 L/kg for the uptake experiment and the uptake portion of the elimination experiments, respectively. To determine the target organ for TNT in fish, juvenile fathead minnow were exposed to 2.2 micromol/L (0.5 mg/L) of [14C]TNT for 10 d. Radiolabeled compounds primarily bioaccumulated in the visceral tissues and spleen in comparison to gill, brain, muscle, and remainder tissue groups. The present study demonstrates the low bioaccumulation potential and rapid uptake of TNT in the fathead minnow.     

Effect of toluene on fathead minnow (Pimephales promelas Rafinesque) development

The environmental levels of aromatic hydrocarbons present in freshwater systems have been poorly characterized. This study examines the effect of toluene, a major water-soluble component of petroleum fuels on teleost development under controlled conditions. The fathead minnowPimephales promelas Rafinesque, was chosen as a model teleost for this study. Live embryos as well as serial sections of paraffin-embedded embryos were examined to determine effects of the toxicant. The toluene concentrations utilized ranged from 30 to 45 mg/L. Abnormalities noted under these conditions included distorted embryonic axis, abnormal heart and circulatory system development, hydration and swelling of the pericardial coelom, hemorrhaging, an overall stunted appearance, microphthalmia, and a unique migration of the ventrally located yolk syncytial layer and its associated nuclei.     

Effects of 17alpha-ethinylestradiol in a fathead minnow (Pimephales promelas) gonadal recrudescence assay

To contribute to the development and evaluation of a practical and cost-effective in vivo testing system for endocrine disruption (specifically environmental estrogens), the effects of 17alpha-ethinylestradiol (EE2) were assessed in a gonadal recrudescence assay with the fathead minnow (Pimephales promelas). Mature male and female fathead minnows were kept first at 15 degrees C on a 8 h light/16 h dark regime and then transferred to 25 degrees C and a 16 h light/8 h dark regime to induce gonadal recrudescence. They were then exposed to various nominal concentrations of the synthetic estrogen EE2 (0, 0.1, 1, 3, 10, 100 ng/L). After 3 weeks of chemical exposure, effects on plasma vitellogenin (VTG), secondary sex characteristics, gonad growth (gonadosomatic index; GSI), and condition factor were assessed. Additionally, the effects on liver and gonad tissue morphology were investigated by means of light (LM) and electron microscopy (EM). Reproductive output (egg production) and fertilization rate were measured during a subsequent 3-week period in breeding adults maintained in clean water. Exposure to EE2 resulted in a significant decrease in GSI, condition factor, and number of batches of eggs and their fertilization rate at EE2 exposure concentrations between 10 and 100 ng/L. A reduction in the extent of parenchymatic areas in ovaries and ultrastructural changes in the livers of females could be detected at EE2 concentrations > or =3 ng/L. The lowest observed effective concentration (LOEC) of EE2 for plasma VTG induction in both sexes and for ultrastructural changes in the testes and livers was 1 ng/L. A significant increase in the mean number of eggs spawned per pair occurred at EE2 exposure doses of 0.1 and 1 ng/L. However, at higher EE2 concentrations, a dose-dependent decrease in the mean number of eggs per pair was apparent. Therefore, the LOEC for a biological effect of EE2 in the fathead minnow using the selected endpoints in the recrudescence assay was 1 ng/L for biomarkers such as plasma VTG and number of tubercles, and 0.1 ng/L for an increased number of eggs spawned per pair.     

Chronic toxicity of Dursban (chlorpyrifos) to the fathead minnow (Pimephales promelas) and the resultant acetylcholinesterase inhibition

Fathead minnows (Pimephales promelas) were exposed to Dursban during a chronic toxicity test for 200 days including a reproductive period of their life cycle. The fish concentrated Dursban approximately 1700 times. Survival of first-generation fish was adversely affected at 2.68 micrograms/liter within 60 days. A significant increase in deformities occurred at 2.68 micrograms/liter within 30 days. Growth was significantly reduced at 2.68 micrograms/liter within 30 days and at 1.21 micrograms/liter by 60 days. Maturation of the first-generation fish was reduced at all Dursban exposures and reproduction was significantly reduced at 0.63 micrograms/liter and above. Growth and estimated biomass of 30-day-old second-generation fish were significantly reduced at 0.12 micrograms/liter, the lowest concentration tested. Brain acetylcholinesterase (AChE) activity was significantly inhibited at 0.27 micrograms/liter and above. AChE inhibition ranged from near 10% in fish exposed at 0.12 micrograms/liter to 89% for those exposed at 2.68 micrograms/liter. Inhibition results are compared to other results demonstrated during the chronic study. The use of exposure units (exposure concentration X exposure duration) is discussed as a tool for determining the effects of organophosphate pesticides on the environment.     

Response spectrum of fluoranthene and pentachlorobenzene for the fathead minnow (Pimephales promelas)

Internal body residue has been recognized as a potential dose metric for toxicological assessments. This relationship between body residue and biological effects, including both lethal and sublethal effects, is critically important for determining environmental quality in risk assessments. The present study identified the toxic equivalent body residues for fluoranthene (FLU) and pentachlorobenzene (PCBz) associated with mortality, reduced growth, and decreased hatchability in the fathead minnow. The toxic equivalent body residue was defined as the total of the parent compound and the organically extractable metabolites for FLU and of the parent compound only for PCBz, because no biotransformation was measurable. The lethal body residues corresponding to 50% mortality were 0.80 and 1.26 micromol/g wet weight for FLU and PCBz, respectively. As expected, residues associated with sublethal effects generally are 2- to 40-fold lower than the lethal residues for FLU and PCBz. Juvenile fish growth was the most sensitive endpoint examined for both compounds. The maximum allowable toxicant residues were 0.02 and 0.43 micromol/g wet weight for FLU and PCBz, respectively. The information collected from the present study will permit a greater understanding of residue-response relationships, which will be useful in risk assessments.     

Evaluation of a commercial kit for measuring vitellogenin in the fathead minnow (Pimephales promelas)

Vitellogenin (vtg) concentrations in oviparous animals such as fish represent an integrated indicator of the status of the reproductive endocrine system. As such, vtg is a common measurement endpoint in tests designed to detect certain classes of endocrine-disrupting chemicals (EDCs). The most common approach to measuring vtg is via enzyme-linked immunosorbent assays (ELISAs). However, because labs testing EDCs in fish often use slightly different ELISAs (e.g., in terms of antibodies, binding antigens, standards), results among studies are not always comparable. One approach to obviating this would be for researchers to use standardized ELISA kits from a common source(s). The fathead minnow (Pimephales promelas) is a small fish model commonly used for EDC testing. The purpose of this study was to evaluate a recently developed commercial ELISA kit for measuring vtg in the fathead minnow. The commercial ELISA, based on a monoclonal antibody to fathead minnow vtg, was compared to an ELISA that utilizes a fathead minnow polyclonal antibody, which has been used extensively in our lab and others for several years. Plasma samples for this comparison came from three studies in which fathead minnows had been exposed to different model EDCs, including an androgen (17beta-trenbolone), an anti-androgen (flutamide), and two CYP19 (aromatase) inhibitors (prochloraz, fadrozole). Results obtained using the two different ELISA methods were consistently similar.     

Development and validation of a 2,000-gene microarray for the fathead minnow (Pimephales promelas)

Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000-gene oligonucleotide microarray for the fathead minnow Pimephales promelas, a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17beta-estradiol, a well-characterized estrogen receptor (ER) agonist, were examined. For these experiments, adult male fathead minnows were exposed for 24 h to waterborne 17beta-estradiol (40 or 100 ng/L) in a flow-through system, and gene expression in liver samples was characterized. Seventy-one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiol's expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species.     

The accumulation and disposition of benz(a)acridine in the fathead minnow,Pimephales promelas

The bioconcentration and metabolism of benz(a)acridine in fathead minnows (Pimephales promelas) was investigated using¹⁴C-labelled benz(a)acridine. The rates of uptake, elimination, and metabolic transformation of benz(a)acridine were estimated in the fish. The equilibrium concentration factor [ratio of benz(a)acridine concentration in fish (wet weight) to benz(a)acridine concentration in water] was estimated at 106±17. The observed bioconcentration factor was about one tenth of that predicted by octanol-water partitioning models. Metabolic alteration was estimated to reduce the degree of bioconcentration 50 to 90% from the hypothetical case in which metabolic transformation did not occur. Benz(a)acridine metabolites attained concentrations in the fish considerably in excess of benz(a)acridine itself.Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with Union Carbide Corp.Publication No. 1598, Environmental Sciences Division, ORNL.