Static Renewal Exposure Research Articles

Effective toxicity of pH to embryonic development of the banded lampeye killifish Aplocheilichthys spilauchen (Duméril, 1861)

The banded lampeye killifish, Aplocheilichthys spilauchen, feeds on larvae of the anopheles mosquito, which makes the fish a good candidate for biological control of malaria. Aquatic organisms require optimal hydrogen ion concentration (pH) for healthy aquatic life, but the impact of pH alteration on A. spilauchen reproductive potential is not known. This study investigated the toxic effect of pH on the embryonic development of A. spilauchen using a static renewal exposure method. Newly fertilised embryos were exposed to a range of pH treatments (5–6 acidic; 7 neutral; 8–9 alkaline) until hatching. All pH media involved triplicates and pH 7 was used as a control. The pH media were checked every 24 h for change and adjusted if necessary. Dissolved oxygen (DO), electrical conductivity (EC), and temperature were monitored throughout the experiment. The endpoints investigated were hatching success, hatchling length, and hatching period. Results indicated a mean hatching success of 95.83 % for pH 7 (neutral) group, while embryos exposed to acidic and basic media recorded lower hatching successes of 60.42 % and 83.34 %, respectively. Hatchling length and hatching period were longer in the control group than in groups of acidic and basic media. Effective toxic pH values of 4.5, 6 and 6.5 were estimated for the hatching period, hatching success, and hatchling length, respectively. These results suggest that sharp changes in the pH of aquatic environments from neutral may adversely impact the reproductive potential of A. spilauchen via embryonic development. This is the first time effective toxic pH values have been estimated for A. spilauchen embryonic development.

Novel Reproduction Toxicity Test Method for Hyalella azteca Using Sexually Mature Amphipods Provides More Robust Data Than Standard Methods in Exposures to Imidacloprid.

Hyalella azteca is an epibenthic crustacean used in ecotoxicology, but there are challenges associated with standard methods using reproduction as an endpoint. A novel, 28-day reproduction toxicity test method for H. azteca was created to address these issues by initiating tests with sexually mature amphipods to eliminate the confounding effects of growth, using a sex ratio of seven females to three males to reduce reproductive variability, and conducting tests in water-only conditions to make recovery of juveniles easier and expand testing capabilities to water-soluble compounds. In the present study, we evaluated the 28-day novel method by comparing it with the 42-day standard test method in duplicate and parallel water-only, static-renewal exposures to sublethal concentrations of imidacloprid (0.5-8 µg/L). Both methods showed similar effects on survival, with survival approaching 50% in the highest test concentration (8 µg/L). However, the 42-day median effect concentrations (EC50s) for growth were more sensitive in the standard method (1.5-3.2 µg/L) compared with the 28-day EC50s generated by the novel method (>8 µg/L). Reproduction endpoints (juveniles/female) produced similar EC50s between methods, but the data were less variable in novel tests (smaller coefficients of variation); therefore, fewer replicates would be required to detect effects on reproduction compared with the standard method. In addition, novel tests generated 28 days of reproduction data compared with 14 days using standard tests and allowed survival and growth of sexes to be assessed independently. Thus, the novel method shows promise to improve the use of reproduction as an endpoint in water-only toxicity tests with H. azteca. Environ Toxicol Chem 2024;43:723-735. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Interlaboratory Study on Zebrafish in Toxicology: Systematic Evaluation of the Application of Zebrafish in Toxicology's (SEAZIT's) Evaluation of Developmental Toxicity.

Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

Effects of aqueous fluoxetine exposure on gut microbiome of adult Pimephales promelas

The microbiome of the gut is vital for homeostasis of hosts with its ability to detoxify and activate toxicants, as well as signal to the immune and nervous systems. However, in the field of environmental toxicology, the gut microbiome has only recently been identified as a measurable indicator for exposure to environmental pollutants. Antidepressants found in effluents of wastewater treatment plants and surface waters have been shown to exhibit antibacterial-like properties in vitro, where some bacteria are known to express homologous proteins that bind antidepressants in vertebrates. Therefore, it has been hypothesized that exposure to antidepressant drugs might affect gut microbiota of aquatic organisms. In this study, the common antidepressant, fluoxetine, was investigated to determine whether it can modulate the gut microbiome of adult fathead minnows. A 28-day, sub-chronic, static renewal exposure was performed with nominal fluoxetine concentrations of 0.01, 10 or 100 μg/L. Using 16S rRNA amplicon sequencing, shifts among the gut-associated microbiota were observed in individuals exposed to the greatest concentration, with greater effects observed in females. These changes were associated with a decrease in relative proportions of commensal bacteria, which can be important for health of fish including bacteria essential for fatty acid oxidation, and an increase in relative proportions of pathogenic bacteria associated with inflammation. Results demonstrate, for the first time, how antidepressants found in some aquatic environments can influence gut microbiota of fishes.

Clothianidin alters leukocyte profiles and elevates measures of oxidative stress in tadpoles of the amphibian, Rana pipiens

Neonicotinoid pesticide use is widespread and highly debated, as evidenced by recent attention received from the public, academics and pesticide regulatory agencies. However, relatively little is known about the physiological effects of neonicotinoid insecticides on aquatic vertebrates. Amphibians (larval stages in particular) are excellent vertebrate bioindicators in aquatic systems due to their risk of exposure and sensitivity to environmental stressors. Previous work with wood frog (Rana sylvatica) tadpoles exposed to formulated products containing thiamethoxam or clothianidin in outdoor mesocosms found significant shifts in leukocyte profiles, suggesting the tadpoles were physiologically stressed. The main objective of the present study was to characterize this stress response further using complementary measures of stress after exposure to clothianidin on northern leopard frogs (Rana pipiens) during their aquatic larval stages. Laboratory static-renewal exposures were conducted over eight weeks with the technical product clothianidin at 0, 0.23, 1, 10 and 100 μg/L, and diquat dibromide at 532 μg/L was used as a positive control. We assessed tadpole leukocyte profiles and measures of oxidative stress as these sub-lethal alterations could affect amphibian fitness. We found changes in several types of leukocytes at 1 and 10 μg/L, suggesting that these tadpoles exhibited signs of mild physiological stress. Clothianidin also induced an oxidative stress response at 0.23, 1 and 100 μg/L. However, we found no differences in survival, growth, development time or hepatosomatic index in frogs exposed to clothianidin. Our study indicates that tadpoles chronically exposed to clothianidin have increased stress responses, but in the absence of concentration-response relationships and effects on whole-organism endpoints, the implications on the overall health and fitness of these changes are unclear.

Toxicological effects of micronized tire crumb rubber on mummichog (Fundulus heteroclitus) and fathead minnow (Pimephales promelas)

Recent studies on the distribution of microplastics in the Charleston Harbor, SC, USA revealed that a large part of the microplastic particles that are found in the intertidal sediments are tire wear particles. These particles originate from the wear of tire treads on roadways, and wash into the estuary during rain events. The abundance of these particles has raised questions about potential toxicity to aquatic organisms that ingest these particles. The synthetic rubber in car tires consists of a large variety of chemicals, which can vary between manufacturers, but usually contains styrene-butadiene rubber, carbon black and zinc. To investigate the potential toxicity of tire wear particles, both mummichogs (Fundulus heteroclitus) and fathead minnow (Pimephales promelas) were exposed to different concentrations of crumb rubber particles (38-355 µm) in a 7-day static renewal exposure. Dissection of the fish revealed that crumb rubber was ingested and accumulated in the intestinal tract. At the highest concentration tested (6 g/L) partial mortality was observed in the fathead minnow, which came close to the assumed LC50. To investigate if polynuclear aromatic hydrocarbons (PAHs) were leaching from the particles, bile fluorescence was measured, together with potential induction of cytochrome P450-1A through the ethoxyresorufin-O-deethylase (EROD) assay. Elevated levels of 2-, 4-, and 5-, ring structures representative of PAHs were detected in the bile of exposed animals. Bile fluorescence indicated that 4-ring PAH compounds were the most bioavailable from the crumb rubber particles. Induction of EROD activity was observed in exposed animals at environmentally relevant concentrations of the crumb rubber particles (<1-2 g/L), and this elevated EROD activity indicated that PAH compounds from the crumb rubber particles were being metabolized in both mummichogs and fathead minnow.

Effect of Louisiana sweet crude oil on a Pacific coral, Pocillopora damicornis

Recent oil spill responses such as the Deepwater Horizon event have underscored the need for crude oil ecotoxicological threshold data for shallow water corals to assist in natural resource damage assessments. We determined the toxicity of a mechanically agitated oil-seawater mixture (high-energy water-accommodated fraction, HEWAF) of a sweet crude oil on a branched stony coral, Pocillopora damicornis. We report the results of two experiments: a 96 h static renewal exposure experiment and a “pulse-chase” experiment of three short-term exposure durations followed by a recovery period in artificial seawater. Five endpoints were used to determine ecotoxicological values: 1) algal symbiont chlorophyll fluorescence, 2) a tissue regeneration assay and a visual health metric with three endpoints: 3) tissue integrity, 4) tissue color, and 5) polyp behavior. The sum of 50 entrained polycyclic aromatic hydrocarbons (tPAH50) was used as a proxy for oil exposure. For the 96 h exposure dose response experiment, dark-adapted maximum quantum yield (Fv/Fm) of the dinoflagellate symbionts was least affected by crude oil (EC50 = 913 μg/L tPAH50); light-adapted effective quantum yield (EQY) was more sensitive (EC50 = 428 μg/L tPAH50). In the health assessment, polyp behavior (EC50 = 27 μg/L tPAH50) was more sensitive than tissue integrity (EC50 = 806 μg/L tPAH50) or tissue color (EC50 = 926 μg/L tPAH50). Tissue regeneration proved to be a particularly sensitive measurement for toxicity effects (EC50 = 10 μg/L tPAH50). Short duration (6−24 h) exposures using 503 μg/L tPAH50 (average concentration) resulted in negative impacts to P. damicornis and its symbionts. Recovery of chlorophyll a fluorescence levels for 6−24 h oil exposures was observed in a few hours (Fv/Fm) to several days (EQY) following recovery in fresh seawater. The coral health assessments for tissue integrity and tissue color were not affected following short-term oil exposure durations, but the 96 h treatment duration resulted in significant decreases for both. A reduction in polyp behavior (extension) was observed for all treatment durations, with recovery observed for the short-term (6−24 h) exposures within 1–2 days following placement in fresh seawater. Wounded and intact fragments exposed to oil treatments were particularly sensitive, with significant delays observed in tissue regeneration. Estimating ecotoxicological values for P. damicornis exposed to crude oil HEWAFs provides a basis for natural resource damage assessments for oil spills in reef ecosystems. These data, when combined with ecotoxicological values for other coral reef species, will contribute to the development of species sensitivity models.

Chronic Toxicities of Neonicotinoids to Nymphs of the Common New Zealand Mayfly Deleatidium spp.

Neonicotinoid insecticides have been shown to have high chronic toxicity relative to acute toxicity, and therefore short-term toxicity tests ≤96 h in duration may underestimate their environmental risks. Among nontarget aquatic invertebrates, insects of the orders Diptera and Ephemeroptera have been found to be the most sensitive to neonicotinoids. To undertake a more accurate assessment of the risks posed by neonicotinoids to freshwater ecosystems, more data are needed from long-term tests employing the most sensitive taxa. Using nymphs of the common New Zealand mayfly genus Deleatidium spp., we performed 28-d static-renewal exposures with the widely used neonicotinoids imidacloprid, clothianidin, and thiamethoxam. We monitored survival, immobility, impairment, and mayfly moulting propensity at varying time points throughout the experiment. Imidacloprid and clothianidin exerted strong chronic toxicity effects on Deleatidium nymphs, with 28-d median lethal concentrations (LC50s) of 0.28 and 1.36 µg/L, respectively, whereas thiamethoxam was the least toxic, with a 28-d LC50 > 4 µg/L (highest concentration tested). Mayfly moulting propensity was also negatively affected by clothianidin (during 3 of 4 wk), imidacloprid (2 of 4 wk), and thiamethoxam (1 of 4 wk). Comparisons with published neonicotinoid chronic toxicity data for other mayfly taxa and larvae of the midge genus Chironomus showed similar sensitivities for mayflies and midges, suggesting that experiments using these taxa provide reliable assessments of the threats of neonicotinoids to the most vulnerable freshwater species. Environ Toxicol Chem 2019;38:2459-2471. © 2019 SETAC.

Comparative chronic toxicity of imidacloprid, clothianidin, and thiamethoxam to Chironomus dilutus and estimation of toxic equivalency factors.

Nontarget aquatic insects are susceptible to chronic neonicotinoid insecticide exposure during the early stages of development from repeated runoff events and prolonged persistence of these chemicals. Investigations on the chronic toxicity of neonicotinoids to aquatic invertebrates have been limited to a few species and under different laboratory conditions that often preclude direct comparisons of the relative toxicity of different compounds. In the present study, full life-cycle toxicity tests using Chironomus dilutus were performed to compare the toxicity of 3 commonly used neonicotinoids: imidacloprid, clothianidin, and thiamethoxam. Test conditions followed a static-renewal exposure protocol in which lethal and sublethal endpoints were assessed on days 14 and 40. Reduced emergence success, advanced emergence timing, and male-biased sex ratios were sensitive responses to low-level neonicotinoid exposure. The 14-d median lethal concentrations for imidacloprid, clothianidin, and thiamethoxam were 1.52 μg/L, 2.41 μg/L, and 23.60 μg/L, respectively. The 40-d median effect concentrations (emergence) for imidacloprid, clothianidin, and thiamethoxam were 0.39 μg/L, 0.28 μg/L, and 4.13 μg/L, respectively. Toxic equivalence relative to imidacloprid was estimated through a 3-point response average of equivalencies calculated at 20%, 50%, and 90% lethal and effect concentrations. Relative to imidacloprid (toxic equivalency factor [TEF] = 1.0), chronic (lethality) 14-d TEFs for clothianidin and thiamethoxam were 1.05 and 0.14, respectively, and chronic (emergence inhibition) 40-d TEFs were 1.62 and 0.11, respectively. These population-relevant endpoints and TEFs suggest that imidacloprid and clothianidin exert comparable chronic toxicity to C. dilutus, whereas thiamethoxam induced comparable effects only at concentrations an order of magnitude higher. However, the authors caution that under field conditions, thiamethoxam readily degrades to clothianidin, thereby likely enhancing toxicity. Environ Toxicol Chem 2017;36:372-382. © 2016 SETAC.

Experimental paradigm for in-laboratory proxy aquatic studies under conditions of static, non-flow-through chemical exposures.

Endocrine-disrupting chemicals (EDCs) such as 17α-ethynylestradiol, 17β-estradiol, estrone, and para-nonylphenol have been measured in wastewater-treatment plant effluents, surface waters, sediments, and sludge and have been shown to induce liver-specific vitellogenin (vtg) messenger RNA in male fathead minnows (Pimephales promelas). The purpose of the present study was to establish minimal concentrations of select EDCs necessary to induce transcription of vtg in 48-h static renewal exposures, as measured by quantitative real-time thermal cycle amplification. Adult males were exposed to 17α-ethynylestradiol, 17β-estradiol, estrone, and para-nonylphenol. Dose-dependent increases in vtg expression were significant with all chemicals tested. The lowest concentrations of these chemicals to induce measurable vtg expression, with significant difference from respective controls, were 17α-ethynylestradiol, 2.2 ng L(-1); para-nonylphenol, 13.9 μg L(-1); 17β-estradiol, 42.7 ng L(-1); and estrone, 46.7 ng L(-1), measured as 48-h average concentrations. The present experiments were designed to frame a commonly acceptable approach for investigators who conduct static, in-laboratory proxy environmental aquatic exposures. The present study highlights the need for investigators to report in peer-reviewed submissions the observed concentration values for minimal induction levels when measuring molecular responses to chemical exposures by means of real-time polymerase chain reaction, quantitative polymerase chain reaction, or other "omic" technologies.

Laboratory calibration of twelve pesticides using polar organic chemical integrative samplers

Passive sampling is an emerging tool for monitoring micropollutants in surface waters and enables the determination of time-weighted average (TWA) concentration over extended sampling periods. For the determination of TWA concentration, the sampling rates (Rs) for the compounds of interest are required. Laboratory calibration experiments were conducted in order to determine the sampling rates of 12 selected pesticides using Polar Organic Chemical Integrative Samplers (pest-POCIS). The experiments were performed on the basis of static renewal exposure of pest-POCIS under stirred conditions for different time intervals up to 28 days. The recoveries of target pesticides ranged between 71 % and 120 % with relative standard deviation (RSD) <18%. Detection limits (LODs) and quantification limits (LOQs) ranged between 0.5 and 6 ng L−1 and 1.6 and 19.7 ng L−1, respectively. The uptake in POCIS for the studied pesticides followed a linear pattern throughout the 28 days of exposure, except for fipronil. The calculated sampling rates ranged from 0.030 L day−1 to 0.223 L day−1. The pesticides pirimiphos methyl, clofentezine, cyprodinil, fipronil and kresoxim methyl have never been studied before in calibration experiments using pest-POCIS passive samplers. Using the described analytical procedure, trace levels (ng L−1) of the target pesticides could be determined in surface waters.

The effects of triclosan on spore germination and hyphal growth of the arbuscular mycorrhizal fungus Glomus intraradices

The effect of triclosan (5-chloro-2-[2,4-dichlorophenoxy]phenol; TCS), on spore germination, hyphal growth, and hyphal branching of the arbuscular mycorrhizal (AM) fungus, Glomus intraradices spores was evaluated at exposure concentrations of 0.4 and 4.0μg/L in a static renewal exposure system. To determine if potential effects were mycotoxic or a consequence of impaired signaling between a host plant and the fungal symbiont, spores were incubated with and without the addition of a root exudate. Exposed spores were harvested at days 7, 14, and 21. AM spore germination, hyphal growth, and hyphal branching were significantly lower in both TCS concentrations compared to controls in non-root exudate treatments suggesting direct mycotoxic effects of TCS on AM development. Greater hyphal growth and hyphal branching in controls and 0.4μg/L TCS treatments with root exudate compared to non-root exudate treatments demonstrated growth stimulation by signaling chemicals present in the root exudate. This stimulatory effect was absent in the 4.0μg/L TCS treatments indicating a direct effect on plant signaling compounds or plant signal response.

Copper toxicity to Lemna minor modelled using humic acid as a surrogate for the plant root

Humic acids are chemically analogous to plant root cell walls in that their surface sites are principally comprised of carboxylic and phenolic acids which bind both metals and protons. Based on this analogy, we developed a biotic-ligand type of model to predict Cu toxicity to Lemna minor, using particulate humic acid (HApart) of the Windermere Humic Aqueous Model (WHAM), and 7d static-renewal exposures with five surface waters and one nutrient media which varied in DOC (1–10mgL−1), pH (6.9–8.7), and water hardness (35–236mg equivalent CaCO3L−1). Although the range of waters tested resulted in a 36-fold variation in 50% inhibitory concentration (IC50) values, the calculated concentration of Cu bound to HApart using this framework was highly correlated with pooled percent net root elongation (%NRE) (R2=0.95). Ten and fifty percent IC values based on [Cu-HApart] were additionally within a factor of ±1.5 and ±1.4, respectively, inclusive of 95% confidence limits. This model construct, which defines the free metal ion and the first hydrolysis product (but not metal carbonate complexes) as being bioavailable, provides an alternative means of defining the binding surface in bioavailability models, whereby a heterogeneous mixture of ligands collectively influence root-metal sorption and toxicity.

Assessment of chromium toxicity in Cyprinus carpio through hematological and biochemical blood markers

Static renewal exposure of various sublethal concentrations of hexavalent chromium [Cr(VI)] produced a deleterious effect on some hematological and biochemical parameters of Cyprinus carpio with the increase in concentration of metal. C. carpio breeders (W = 500 ± 9.5 g; L = 25.60 ± 2.6 cm) were randomly divided into the control group (n = 33) and treated group (n = 198), with 11 breeders/tank (8.84 m3). The groups were distributed into tanks containing 1 of 7 sublethal concentrations of Cr(VI) (0, 25, 50, 75, 100, 125, and 150 mg/L), with 3 replicates per concentration for 6 months. Monthly Cr(VI) levels in the water were determined with an atomic absorption spectrophotometer. The Cr(VI) induced a significant increase (P < 0.05) in white blood cells, mean corpuscular volume, erythrocyte sedimentation rate, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and acid phosphatase, indicating anemia. However, red blood cells, hemoglobin, packed cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, glucose, protein, and cholesterol decreased significantly (P < 0.05) with the increase in Cr(VI) concentrations from 25 to 150 mg/L. The actual Cr(VI) concentration varied from 36 to 118 \mug/L in the tanks exposed to concentrations from 25 to 150 mg/L. The increasing trend was probably due to the toxic effects of the Cr(VI) and activation of the fish immune system to resist against metallic stress and liver damage. The decreasing trend reflected retarded and deteriorated fish health along with severe hemolysis.

Passive sampling of selected pesticides in aquatic environment using polar organic chemical integrative samplers

Polar chemical integrative samplers (POCIS) were examined for their sampling efficiency of 12 pesticides and one metabolite commonly detected in surface waters. Laboratory-based calibration experiments of POCISs were conducted. The determined passive sampling rates were applied for the monitoring of pesticides levels in Lake Amvrakia, Western Greece. Spot sampling was also performed for comparison purposes. Calibration experiments were performed on the basis of static renewal exposure of POCIS under stirred conditions for different time periods of up to 28days. The analytical procedures were based on the coupling of POCIS and solid phase extraction by Oasis HLB cartridges with gas chromatography-mass spectrometry. The recovery of the target pesticides from the POCIS was generally >79% with relative standard deviation (RSD) <16%. The calibration results revealed an integrative uptake of all pesticides for 28days and the calculated sampling rates ranged from 0.025 to 0.388 L day(-1) with RSD <29%. Low nanogram/liter levels of pesticides such as diazinon, alachlor, and s-metolachlor were detected during the monitoring campaign using both passive and spot sampling whereas higher concentrations were measured by spot sampling in most cases. Passive sampling by POCIS provides a useful tool for the monitoring of pesticides in aquatic systems since integrative sampling at rates sufficient for analytical quantitation of ambient levels was observed. Calibration data are in demand for a greater number of compounds in order to extend the use in environmental monitoring.

Acute effects of Fe 2O 3, TiO 2, ZnO and CuO nanomaterials on Xenopus laevis

Metal oxide nanomaterials have exhibited toxicity to a variety of aquatic organisms, especially microbes and invertebrates. To date, few studies have evaluated the toxicity of metal oxide nanomaterials on aquatic vertebrates. Therefore, this study examined effects of ZnO, TiO 2, Fe 2O 3, and CuO nanomaterials (20–100 nm) on amphibians utilizing the Frog Embryo Teratogenesis Assay Xenopus (FETAX) protocol, a 96 h exposure with daily solution exchanges. Nanomaterials were dispersed in reconstituted moderately hard test medium. These exposures did not increase mortality in static renewal exposures containing up to 1000 mg L −1 for TiO 2, Fe 2O 3, CuO, and ZnO, but did induce developmental abnormalities. Gastrointestinal, spinal, and other abnormalities were observed in CuO and ZnO nanomaterial exposures at concentrations as low as 3.16 mg L −1 (ZnO). An EC 50 of 10.3 mg L −1 ZnO was observed for total malformations. The minimum concentration to inhibit growth of tadpoles exposed to CuO or ZnO nanomaterials was 10 mg L −1. The results indicate that select nanomaterials can negatively affect amphibians during development. Evaluation of nanomaterial exposure on vertebrate organisms are imperative to responsible production and introduction of nanomaterials in everyday products to ensure human and environmental safety.

Toxicity of trinitrotoluene to sheepshead minnows in water exposures

Lethal effects of trinitrotoluene (TNT) to juvenile sheepshead minnows (JSHM) ( Cyprinodon variegatus) were assessed in ten-day water exposures. Ten-day median lethal concentrations (LC50s) were 2.3 and 2.5 mg L −1, the 10-d median lethal residue value (LR50) was 26.1 μmol kg −1 wet weight (ww), and bioconcentration factors (BCFs) ranged from 0.7 to 2.4 L kg −1. The lethal effects of TNT and its transformation products 2-aminodinitrotoluene (2-ADNT), 2,4-diaminonitrotoluene (2,4-DANT) and trinitrobenzene (TNB) to JSHM were compared in 5-d static-renewal exposures. Nitroreduction decreased the toxicity of TNT to SHM, as the 5-d LC50 for 2-ADNT was 8.6 mg L −1 and the lowest lethal concentration of 2,4-DANT was 50.3 mg L −1. TNB (5-d LC50=1.2 mg L −1) was more toxic than TNT to SHM. The 5-d LR50s were 4.3 mg kg −1 ww (20.4 μmol kg −1) for SumTNT (TNT exposure) and 54.2 mg kg −1 ww (275.3 μmol kg −1) for 2-ADNT and significant mortality occurred at 47.4 mg kg −1 ww (283.6 μmol kg −1). The range of BCF values was from 1.8 to 2.4, 5.6 to 8.0, and 0.6 to 0.9 L kg −1 for TNT, 2-ADNT, and 2,4-DANT, respectively.

A proteomics based approach to assessing the toxicity of bisphenol A and diallyl phthalate to the abalone ( Haliotis diversicolor supertexta)

The contamination of marine ecosystems by endocrine disrupting compounds (EDCs) is of great concern. Protein expression profile maybe a good method to help us understand the molecular mechanisms of EDCs-toxicity to aquatic organisms. In this study, the abalone ( Haliotis diversicolor supertexta), was selected as the target organism. Toxicological effects of two reference endocrine disruptors: diallyl phthalate (DAP, 50 μg L −1) and bisphenol-A (BPA, 100 μg L −1) were investigated after a three months static-renewal exposure on abalones using proteomics to analyze their hepatopancreas tissues. Some enzyme activity parameters of hepatopancreas extracts were also performed, including Na +–K +-ATPase, Ca 2+–Mg 2+-ATPase, peroxidase (POD) and malondialdehyde (MDA) production. After analyzing the proteomics profile of hepatopancreas by 2D gel electrophoresis, we found that 24 spots significantly increased or decreased at protein expression level (⩾2-fold difference) in the 2D-maps from the treatment groups. Eighteen out of 24 protein spots were successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI–TOF-MS). These proteins can be roughly categorized into diverse functional classes such as detoxification, oxidative stress, hormone regulating, cellular metabolism and innate immunity. In addition, the enzymatic results indicated that DAP/BPA exposure affected the oxidative stress status and the cellular homeostasis, which partly corroborated the proteomics’ results. Taken together, these data demonstrate that proteomics is a powerful tool to provide valuable insights into possible mechanisms of toxicity of EDCs contaminants in aquatic species. Additionally, the results highlight the potential of abalone as a valuable candidate for investigating EDCs impacts on marine ecosystems.

Real-time PCR array to study effects of chemicals on the Hypothalamic–Pituitary–Gonadal axis of the Japanese medaka

This paper describes the development and validation of a PCR array for studying chemical-induced effects on gene expression of selected endocrine pathways along the hypothalamic–pituitary–gonadal (HPG) axis of the small, oviparous fish, the Japanese medaka ( Oryzias latipes). The Japanese medaka HPG-PCR array combines the quantitative performance of SYBR® Green-based real-time PCR with the multiple gene profiling capabilities of a microarray to examine expression profiles of 36 genes associated with endocrine pathways in brain, liver and gonad. The performance of the Japanese medaka HPG-PCR array was evaluated by examining effects of two model compounds, the synthetic estrogen, 17α-ethinylestradiol (EE2) and the anabolic androgen, 17β-trenbolone (TRB) on the HPG axis of the Japanese medaka. Four-month-old medaka was exposed to three concentrations of EE2 (5, 50, 500 ng/L) or TRB (50, 500, 5000 ng/L) for 7 d in a static renewal exposure system. A pathway-based approach was implemented to analyze and visualize concentration-dependent mRNA expression in the HPG axis of Japanese medaka. The compensatory response to EE2 exposure included the down-regulation of male brain GnRH RI and testicular CYP17. The down-regulation of AR-α expression in brain of EE2-exposed males was associated with suppression of male sexual behavior. Compensatory responses to TRB in the female HPG axis included up-regulation of brain GnRH RII and ovary steroidogenic CYP19A. Overall, the results suggested that the Japanese medaka HPG-PCR array has potential not only as a screening tool of potential endocrine-disrupting chemicals but also in elucidating mechanisms of action.

In vivo assessment of the capacity of androstenedione to masculinize female mosquitofish (Gambusia affinis) exposed through dietary and static renewal methods

In this study, we investigated the capacity of androstenedione to masculinize female mosquitofish. Previous studies have identified androstenedione in the water and sediment of the Fenholloway River, a Florida, USA, coastal river that receives paper mill effluent and contains masculinized eastern mosquitofish (Gambusia holbrooki). Females of the closely related western mosquitofish, Gambusia affinis, were exposed to androstenedione through both dietary and static renewal treatments. Morphological masculinization of female mosquitofish is characterized by the development of a male secondary sexual trait: an elongated and modified anal fin (gonopodium). Dietary exposure to 0.7, 7, 70, and 700 microg of androstenedione per gram of food failed to induce gonopodial development at any concentration within the six-week exposure period. Static renewal treatments used androstenedione concentrations of 0.14, 1.4, 14, 140, and 350 nM. Significant anal fin ray elongation was observed in all but the lowest exposure group. Fish growth during the static renewal exposure experiment was negatively correlated with androstenedione concentration. No significant effects were observed for gonadosomatic index, vitellogenin expression, or ovarian area in fish exposed to androstenedione via either the dietary or static renewal methods. These results indicate that exposure to androstenedione via water can cause masculinization of adult female mosquitofish in a relatively short period of time and that acute dietary exposure to androstenedione at the concentrations used is not sufficient to induce masculinization.