Per- and polyfluoroalkyl substances (PFAS) are a large class of chemicals of concern for both human and environmental health because of their ubiquitous presence in the environment, persistence, and potential toxicological effects. Despite this, ecological hazard data are limited to a small number of PFAS although there are over 4,000 identified PFAS. Traditional toxicity testing will likely be inadequate to generate necessary hazard information for risk assessment. Therefore, this study investigated the utility of using transcriptomic points of departure (tPODs) for informing PFAS algal toxicity. Raphidocelis subcapitata, a freshwater green algal species, were exposed for 24 hr in 96-well microplates to multiple concentrations of 22 different PFAS. Following exposure, RNA was extracted, and the transcriptome was evaluated by RNA sequencing followed by concentration response modeling to determine a tPOD for each PFAS. Per- and polyfluoroalkyl substance tPODs, based on measured concentrations, ranged from 0.9 µg/L for perfluorotridecanoic acid to 1 mg/L for perfluorononanoic acid. These values derived from R. subcapitata exposures were compared with published hazard benchmarks from other taxa (larval fathead minnow and Daphnia magna) and in vitro data. Although R. subcapitata was generally more sensitive to the tested PFAS than previously tested taxa and in vitro assays, the algal tPODs were, on average, three orders magnitude greater than the maximum concentrations of PFAS detected in Great Lakes tributaries. This high throughput transcriptomics assay with algae is a promising new approach method for an ecologically relevant tiered hazard evaluation strategy.

Microalgae play important ecological roles and serve as useful models for assessing the impacts of environmental contaminants on lower trophic aquatic organisms. Sertraline, a commonly prescribed selective serotonin reuptake inhibitor (SSRI) found in urban waters, has been observed to induce chlorosis in Raphidocelis subcapitata close to environmentally relevant concentrations. This study estimated potentially hazardous concentrations of sertraline hydrochloride by deriving transcriptomic and metabolomic points of departure (tPODs and mPODs) below which chronic adverse effects would not be expected. Additionally, an Adverse Outcome Pathway describing the mechanism by which sertraline hydrochloride impacted R. subcapitata was proposed using a multiomics approach. The mPOD derived from nontargeted metabolomics was comparable to the tPOD (455 vs 690 μg/L), and annotated dysregulated metabolites along with reduced photosynthetic capability indicated that uncoupling of the cyclic electron flow required for photosynthesis led to a reactive oxygen species (ROS) burst. This probable ROS burst altered cell membrane composition and downregulated genes associated with protoporphyrinogen IX and heme biosynthesis pathways. This research highlights the molecular mechanisms underlying pharmaceutical-induced chlorosis in a model microalgae species and demonstrates the utility of integrating metabolomics and transcriptomics for assessing the potential ecological risks of SSRIs.

In 2019, the US EPA organized a federal government challenge aimed at identifying and evaluating low cost, high-throughput, RNA sequencing technologies that could support the aims of a new program in ecological high-throughput transcriptomics. Innovators worldwide were invited to demonstrate their solutions in an open competition. Each responding Solver was provided a set of nine pooled RNA samples from each of four species of aquatic organisms (n = 36 samples total). Five Solutions submitted by three Solver teams were evaluated according to a pre-defined scoring rubric that considered accuracy, precision, transcriptome coverage for each species, cost per sample, and throughput. A targeted approach (TempO-Seq) that employed sentinel gene sets representing 5-11 % of the whole transcriptome was ranked as the top solution. However, all were viable approaches and had specific strengths and weaknesses. In a follow up investigation, transcriptomic points of departure based on a sentinel gene set were generally found to fall within a factor of 10 or less of those based on whole transcriptome sequencing. Results support the conclusion that a wide range of sequencing technologies and approaches are suitable for the work. Detailed and transparent reporting of the approaches used will help support uptake in science-based decision-making.

As part of the Great Lakes Restoration Initiative, chemical monitoring and surveillance efforts have detected approximately 330 chemicals in surface water of Great Lakes tributaries. There were 140 chemicals for which no empirical toxicity data were available. The aim of this study was to generate transcriptomic points of departure (tPODs) for 10 of these compounds and demonstrate how they could be applied in a screening-level prioritization. Organisms representing three trophic levels of the aquatic food web (Pimephales promelas, Daphnia magna, and Raphidocelis subcapitata) were exposed for 24 hr to a half-log dilution series of nominal exposure concentrations typically ranging from 66.7-0.021 µM of each chemical. In addition to observations of apical effects (e.g., survival and morphology), whole body transcriptomic responses (tPODs) to each chemical were evaluated with targeted analysis using TempO-seq for P. promelas and D. magna and nontargeted RNA-seq for R. subcapitata. The tPODs ranged from 0.18-10.8 µM for P. promelas and 0.32-29 µM for D. magna, with the most potent of the chemicals tested being fipronil carboxamide for both species. For R. subcapitata, the tPODs ranged from 0.04-1.77 µM, with gabapentin as the most potent chemical tested. Empirically derived tPODs from these data-poor chemicals were compared with concentrations detected in the Great Lakes basin. Environmental concentrations were less than the tPODs except for R. subcapitata and 3,4-dichlorophenyl isocyanate. Similarly, tPODs from previously tested data-rich chemicals were compared with environmental concentrations, in which case tPODs from several chemicals overlapped environmental concentrations. This work demonstrates the potential utility of emerging ecological high-throughput transcriptomics assays to support screening and prioritization of data-poor environmental contaminants.

Generation of hazard information for chemicals already in production can be costly and time consuming. High-throughput techniques offer a method to define the hazard potential posed by chemicals and facilitate risk assessment activities in a rapid manner. Transcriptomic points of departure (tPODs), derived from the concentration at which global gene expression significantly differs from controls, are expected to precede concentrations that cause apical toxicity, suggesting tPODs could serve as an alternative point of departure for use in screening level hazard assessment. The present study extends previous work with a larval fathead minnow high-throughput transcriptomics assay but employs a targeted transcriptome rather than whole transcriptome approach to generate tPODs for 22 chemicals (pesticides and pharmaceuticals), expanding the diversity of chemical modes of action evaluated. One-day-post-hatch fathead minnows were exposed to eight concentrations of two piscicides, three herbicides, four insecticides, seven fungicides, two statins, and four PPAR agonists for 24 h. Following exposures, RNA from whole body homogenates were sequenced using TempO-Seq. Concentration-response modeling, using measured concentrations where feasible, was applied to all sequencing results to estimate benchmark concentrations and derive tPODs. To investigate the relationships of chemicals within the same mode of action (MoA), differentially expressed genes were visualized using Euler diagrams, sscMap, and exploratory principal component analysis using the sequencing counts for each chemical from the first concentration above the derived tPOD value was used. The targeted transcriptome approach produced results similar to what is expected from a whole transcriptome approach which suggests that if appropriate genes are selected, a targeted approach is sufficient to derive protective tPODs. In most cases, the derived tPODs were lower than concentrations associated with apical endpoints from fish data collected from the ECOTOX Knowledgebase. However, this was not the case when tPODS were compared to apical effects in other taxa (crustaceans, insects, algae). Our results showed that the individual genes impacted by chemical exposure rarely clustered within the same MoA. Overall, the results of this study provide additional data that can be used to examine the applicability of high-throughput assays for possible use in ecological hazard screening.

It is postulated that below a transcriptomic-based point of departure, adverse effects are unlikely to occur, thereby providing a chemical concentration to use in screening level hazard assessment. The present study extends previous work describing a high-throughput fathead minnow assay that can provide full transcriptomic data after exposure to a test chemical. One-day post-hatch fathead minnows were exposed to ten concentrations of three representatives of four chemical modes of action: organophosphates, ecdysone receptor agonists, plant photosystem II inhibitors, and estrogen receptor agonists for 24 h. Concentration response modeling was performed on whole body gene expression data from each exposure, using measured chemical concentrations when available. Transcriptomic points of departure in larval fathead minnow were lower than apical effect concentrations across fish species but not always lower than toxic effect concentrations in other aquatic taxa like crustaceans and insects. The point of departure was highly dependent on measured chemical concentration which were often lower than the nominal concentration. Differentially expressed genes between chemicals within modes of action were compared and often showed statistically significant overlap. In addition, reproducibility between identical exposures using a positive control chemical (CuSO4) and variability associated with the transcriptomic point of departure using in silico sampling were considered. Results extend a transcriptomic-compatible fathead minnow high-throughput assay for possible use in ecological hazard screening.

Traditional toxicity testing has been unable to keep pace with the introduction of new chemicals into commerce. Consequently, there are limited or no toxicity data for many chemicals to which fish and wildlife may be exposed. Per- and polyfluoroalkyl substances (PFAS) are emblematic of this issue in that ecological hazards of most PFAS remain uncharacterized. The present study employed a high-throughput assay to identify the concentration at which 20 PFAS, with diverse properties, elicited a concerted gene expression response (termed a transcriptomics-based point of departure [tPOD]) in larval fathead minnows (Pimephales promelas; 5-6 days postfertilization) exposed for 24 h. Based on a reduced transcriptome approach that measured whole-body expression of 1832 genes, the median tPOD for the 20 PFAS tested was 10 mu M. Longer-chain carboxylic acids (12-13 C-F); an eight-C-F dialcohol, N-alkyl sulfonamide; and telomer sulfonic acid were among the most potent PFAS, eliciting gene expression responses at concentrations <1 mu M. With a few exceptions, larval fathead minnow tPODs were concordant with those based on whole-transcriptome response in human cell lines. However, larval fathead minnow tPODs were often greater than those for Daphnia magna exposed to the same PFAS. The tPODs overlapped concentrations at which other sublethal effects have been reported in fish (available for 10 PFAS). Nonetheless, fathead minnow tPODs were orders of magnitude higher than aqueous PFAS concentrations detected in tributaries of the North American Great Lakes, suggesting a substantial margin of safety. Overall, results broadly support the use of a fathead minnow larval transcriptomics assay to derive screening-level potency estimates for use in ecological risk-based prioritization. (c) 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Per- and polyfluoroalkyl substances (PFAS) represent a large group of contaminants of concern based on their widespread use, environmental persistence, and potential toxicity. Many traditional models for estimating toxicity, bioaccumulation, and other toxicological properties are not well suited for PFAS. Consequently, there is a need to generate hazard information for PFAS in an efficient and cost-effective manner. In the present study, Daphnia magna were exposed to multiple concentrations of 22 different PFAS for 24 h in a 96-well plate format. Following exposure, whole-body RNA was extracted and extracts, each representing five exposed individuals, were subjected to RNA sequencing. Following analytical measurements to verify PFAS exposure concentrations and quality control on processed cDNA libraries for sequencing, concentration-response modeling was applied to the data sets for 18 of the tested compounds, and the concentration at which a concerted molecular response occurred (transcriptomic point of departure; tPOD) was calculated. The tPODs, based on measured concentrations of PFAS, generally ranged from 0.03 to 0.58 mu M (9.9-350 mu g/L; interquartile range). In most cases, these concentrations were two orders of magnitude lower than similarly calculated tPODs for human cell lines exposed to PFAS. They were also lower than apical effect concentrations reported for seven PFAS for which some crustacean or invertebrate toxicity data were available, although there were a few exceptions. Despite being lower than most other available hazard benchmarks, D. magna tPODs were, on average, four orders of magnitude greater than the maximum aqueous concentrations of PFAS measured in Great Lakes tributaries. Overall, this high-throughput transcriptomics assay with D. magna holds promise as a component of a tiered hazard evaluation strategy employing new approach methodologies. Environ Toxicol Chem 2024;00:1-16. (c) 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Concentrations at which global gene expression profiles in cells or animals exposed to a test substance start to differ significantly from those of controls have been proposed as an alternative point of departure for use in screening level hazard assessment. The present study describes pilot testing of a high throughput compatible transcriptomics assay with larval fathead minnows. One day post hatch fathead minnows were exposed to eleven different concentrations of three metals, three selective serotonin reuptake inhibitors, and four neonicotinoid-like compounds for 24 h and concentration response modeling was applied to whole body gene expression data. Transcriptomics-based points of departure (tPODs) were consistently lower than effect concentrations re-ported in apical endpoint studies in fish. However, larval fathead minnow-based tPODs were not always lower than concentrations reported to elicit apical toxicity in other aquatic organisms like crustaceans or insects. Random in silico subsampling of data from the pilot assays was used to evaluate various assay design and acceptance considerations such as transcriptome coverage, number of replicate individuals to sequence per treatment, and minimum number of differentially expressed genes to produce a reliable tPOD estimate. Results showed a strong association between the total number of genes for which a concentration response relationship could be derived and the overall variability in the resulting tPOD estimates. We conclude that, for our current assay design and analysis pipeline, tPODs based on fewer than 15 differentially expressed genes are likely to be unreliable for screening and that interindividual variability in gene expression profiles appears to be a more significant driver of tPOD variability than sample size alone. Results represent initial steps toward developing high throughput transcriptomics assays for use in ecological hazard screening.

New approach methods (NAMs) can reduce the need for chronic animal studies. Here, we apply benchmark dose (concentration) (BMD(C))–response modeling to transcriptomic changes in the liver of mice and in fathead minnow larvae after short-term exposures (7 days and 1 day, respectively) to several dose/concentrations of three organophosphate pesticides (OPPs): fenthion, methidathion, and parathion. The mouse liver transcriptional points of departure (TPODs) for fenthion, methidathion, and parathion were 0.009, 0.093, and 0.046 mg/Kg-bw/day, while the fathead minnow larva TPODs were 0.007, 0.115, and 0.046 mg/L, respectively. The TPODs were consistent across both species and reflected the relative potencies from traditional chronic toxicity studies with fenthion identified as the most potent. Moreover, the mouse liver TPODs were more sensitive than or within a 10-fold difference from the chronic apical points of departure (APODs) for mammals, while the fathead minnow larva TPODs were within an 18-fold difference from the chronic APODs for fish species. Short-term exposure to OPPs significantly impacted acetylcholinesterase mRNA abundance (FDR p-value <0.05, |fold change| ≥2) and canonical pathways (IPA, p-value <0.05) associated with organism death and neurological/immune dysfunctions, indicating the conservation of key events related to OPP toxicity. Together, these results build confidence in using short-term, molecular-based assays for the characterization of chemical toxicity and risk, thereby reducing reliance on chronic animal studies.

The US Environmental Protection Agency conducts ecological risk assessments with a battery of fish toxicity tests that include acute, early life stage, and reproduction tests. While endpoints in these tests (survival, growth and reproduction) are conceptually related, because they are measured in separate exposures, the quantitative relationships between them are difficult to determine and largely ignored. In the current test, fathead minnows (FHM) were exposed for 28 days to 1 mg/L or 2 mg/L carbaryl, a well-studied carbamate insecticide, in early life stages and then reared in clean water until adulthood, when reproduction was assessed. Also. weekly growth measurements were taken throughout the test to determine growth rates during and after exposure. Growth curves derived from these measurements were then compared to the reproductive output. The data indicate that carbaryl reduced growth rate only for a brief time early in the exposure. However, this brief effect impacted overall growth into adulthood and lowered the reproductive output of exposed FHM. The effect of a transient exposure early in life to carbaryl could have later population-level impacts by causing mortality, lowering growth rates, and reducing reproductive output.

Presentation to the EPA Board of Scientific Counselors (BOSC) Virtual Meeting February 2021

Environmental contamination can negatively impact fish populations. In addition to acute toxicity leading to death, toxicants can reduce fish growth and lower reproduction. The potential for adverse population level effects of environmental contaminants are estimated to conduct risk assessments from laboratory toxicity tests that most often measure apical endpoints related to growth, survival and reproduction. The relationships between these effect endpoints are being evaluated to predict shifts in fish population demography better after exposure to environmental toxicants. Environmental contaminants can also affect fish populations indirectly by reducing prey biomass. However, estimating the magnitude of the combined effects of prey reduction and direct toxicity is difficult and rarely attempted. Here we describe a toxicity test designed to estimate the effect on Japanese medaka of both reduced food and chronic exposure to diazinon, an acetylcholinesterase inhibiting organophosphate pesticide. Fish were reared with limited food ration and/or diazinon exposure through a full life cycle to assess possible interactions between the two stressors in their effects on growth and reproduction. Diazinon exposure (10 or 20 μg/L), reduced ration (50% and 25% of ad libitum), or combinations of both lowered growth rates and reproductive output of Japanese medaka. In addition, growth and reproduction alone were modeled, and then various relationships between the two stressors (diazinon and ration) and how they relate to growth and reproduction were modeled.

We describe designs for the temporal aspect of sampling in an experiment that estimates the population mean growth of an organism that can be modeled using a Weibull or von Bertalanffy growth curve. First, the properties of the Weibull growth curve are explained. Next, using the shape parameter ( v ), the Weibull growth curve can be categorized into two shape categories: sigmoid and saturation. D‐optimization and shape classification are used to determine the optimal sampling occasions and the effect of a sampling schedule on the precision of the size estimates. Finally, the results are used to recommend a generalized sampling design to best determine the shape and size of a growth curve.

The Medaka Extended One Generation Reproduction Test (MEOGRT) is a Tier 2 test within U.S. Environmental Protection Agency's (USEPA) Endocrine Disruptor Screening Program (EDSP), designed to characterize the potential adverse effects to fish of exposure to chemical that can cause disruption of the endocrine system. The MEOGRT focuses primarily on adverse effects to reproduction while collecting information regarding effects on growth, survival, and endocrine-related endpoints. However, the risk assessment process for fish, as mandated by legislation such as the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) or the Toxic Substances Control Act (TSCA), could benefit from a more detailed assessment of effects on growth. Typically, fish growth data in support of risk assessment are obtained from full life-cycle tests or early life stage tests using the fathead minnow. As an alternative to these tests, a modified MEOGRT was conducted to assess the effects of diazinon on the various parameters measured in the MEOGRT. Diazinon is an organophosphate insecticide that is detected in the environment, and whose efficacy is a result of inhibition of the acetylcholine esterase enzyme at neuromuscular junctions and synapses of the nervous system. Diazinon (2.9, 5.2, 10.3, 19.8, and 40.2 μg/L) was tested with the MEOGRT protocol, and the lowest observable effect concentrations of 2.9 μg/L for fecundity and 5.2 μg/L for growth were determined. Additional growth measurements were added to the MEOGRT protocol to more robustly define growth rates and to determine the impact size has on reproductive performance. Fish size starting at the first measurement day (i.e. 21 days post-fertilization), and continuing through the duration of the test was reduced with exposure to 5.2 μg/L and higher, and asymptotic size predicted from growth modeling was reduced at 10.3 μg/L and higher. By simply adding non-destructive growth measurements at two additional time points, the MEOGRT provided enough data for the parameterization of growth models, which could be used to characterize the reproductive implications of growth impairment.

Because of various Congressional mandates to protect the environment from endocrine disrupting chemicals (EDCs), the United States Environmental Protection Agency (USEPA) initiated the Endocrine Disruptor Screening Program. In the context of this framework, the Office of Research and Development within the USEPA developed the Medaka Extended One Generation Reproduction Test (MEOGRT) to characterize the endocrine action of a suspected EDC. One important endpoint of the MEOGRT is fecundity of medaka breeding pairs. Power analyses were conducted to determine the number of replicates needed in proposed test designs and to determine the effects that varying reproductive parameters (e.g. mean fecundity, variance, and days with no egg production) would have on the statistical power of the test. The MEOGRT Reproduction Power Analysis Tool (MRPAT) is a software tool developed to expedite these power analyses by both calculating estimates of the needed reproductive parameters (e.g. population mean and variance) and performing the power analysis under user specified scenarios. Example scenarios are detailed that highlight the importance of the reproductive parameters on statistical power. When control fecundity is increased from 21 to 38 eggs per pair per day and the variance decreased from 49 to 20, the gain in power is equivalent to increasing replication by 2.5 times. On the other hand, if 10% of the breeding pairs, including controls, do not spawn, the power to detect a 40% decrease in fecundity drops to 0.54 from nearly 0.98 when all pairs have some level of egg production. Perhaps most importantly, MRPAT was used to inform the decision making process that lead to the final recommendation of the MEOGRT to have 24 control breeding pairs and 12 breeding pairs in each exposure group.

The medaka extended one-generation test (MEOGRT) was developed as a multigenerational toxicity test for chemicals, particularly endocrine-disrupting chemicals. Briefly, 3 generations of Japanese medaka (Oryzias latipes) are exposed to a chemical over a 20-wk period: 3 wk in the parental generation (F0), 15 wk in the first generation (F1), and 2 wk in the second generation (F2). The present study reports the first MEOGRT results concerning branched isomer mixtures of 4-nonylphenol (NP). Adult F0 medaka exposed to NP at 5 actual concentrations (1.27, 2.95, 9.81, 27.8, 89.4 µg/L) were unaffected in terms of reproduction, although vitellogenin in the male liver was increased dose-dependently at concentration of 2.95 µg/L and higher. In F1, in contrast, total egg (fecundity), fertile egg, and fertility decreased as NP increased; lowest-observed-effect concentrations (LOECs) for total egg, fertile egg, and fertility were 1.27, 1.27, 27.8 µg/L, respectively. In F1, but not in F0, secondary sex characteristics (i.e., anal fin papillae in males) were suppressed at 27.8 µg/L NP. Vitellogenin induction in adult male fish was slightly weaker in F1 than it was in F0, however. Gonadal sex abnormality and sex reversal occurred at 27.8 and 89.4 µg/L NP in F1 subadults. At 89.4 µg/L NP, all genotypic F1 males in breeding pairs had female phenotype, and some even demonstrated spawning. Concentrations of NP lower than 89.4 µg/L did not affect F2 survival or hatching. The highest detected NP level in environmental freshwater in Japan was approximately a half of the LOEC (1.27 µg/L for F1 fecundity); in other countries, however, environmental concentrations above the LOEC are reported, suggesting that NP may be affecting fish populations. Environ Toxicol Chem 2017;36:3254-3266. © 2017 SETAC.

In response to various legislative mandates, the US Environmental Protection Agency (USEPA) formed its Endocrine Disruptor Screening Program (EDSP), which in turn, formed the basis of a tiered testing strategy to determine the potential of pesticides, commercial chemicals, and environmental contaminants to disrupt the endocrine system. The first tier of tests is intended to detect the potential for endocrine disruption mediated through estrogen, androgen, or thyroid pathways, whereas the second tier is intended to further characterize the effects on these pathways and to establish a dose-response relationship for adverse effects. One of these tier 2 tests, the Medaka Extended One Generation Reproduction Test (MEOGRT), was developed by the USEPA for the EDSP and, in collaboration with the Japanese Ministry of the Environment, for the Guidelines for the Testing of Chemicals of the Organisation for Economic Co-operation and Development (OECD). The MEOGRT protocol was iteratively modified based on knowledge gained after the successful completion of 9 tests with variations in test protocols. The present study describes both the final MEOGRT protocol that has been published by the USEPA and the OECD, and the iterations that provided valuable insights into nuances of the protocol. The various tests include exposure to 17β-estradiol, 4-t-octylphenol, o,p'- dichlorodiphenyltrichloroethane, 4-chloro-3-methylphenol, tamoxifen, 17β-trenbolone, vinclozolin, and prochloraz. Environ Toxicol Chem 2017;36:3387-3403. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

The focus of this session was on the proposed USEPA Endocrine Disruption Screening Program (EDSP) Tier 2 testing protocols. Tier 2 tests have been developed to evaluate the potential impacts of endocrine disrupting chemicals (EDCs) over the life cycle across organisms representing vertebrate and invertebrate classes. Key aspects of these Tier 2 testing protocols rely on selecting appropriate measurement end points to reveal differential sensitivity and adverse impacts across an organism’s life stages. To this end, certain Tier 2 tests utilize a multigenerational protocol, which detect both short- and long-term effects. However, multigenerational testing protocols can be time consuming and costly. As such, other testing protocols have also been considered, including partial life-cycle and extended one-generation tests. Regardless of the specifics of the multigenerational protocol, it is critical to identify key measurement end points that are responsive, reliable, and repeatable indicators of exposure to endocrine disrupting chemicals; these measures should also provide information to enable initial assessments of risk translated from individual to potential population level effects across a variety of living organisms. Presentations in Session three of the Society of Environmental Toxicology and Chemistry (SETAC) North America Focused Topic Meeting: Endocrine Disruption (February 4 – 6, 2014) focused on the current state of the science for EPA EDSP Tier 2 testing. Presentations in this session considered the strengths and weaknesses of the Tier 2 assays across several classes of organisms, and provided an industry perspective on Tier 2 testing. The interactive panel discussion provided an interesting perspective that balanced regulatory needs for reliable testing protocols that are highly repeatable and utilize consistent indices of exposure and adverse effect.

Various aquatic bioassays using one of several fish species have been developed or are in the process of being developed by organizations like the US Environmental Protection Agency and the Office of Economic Cooperation and Development for testing potential endocrine-disrupting chemicals (EDCs). Often, these involve assessment of the gonad phenotype of individuals as a key endpoint that is inputted into a risk or hazard assessment. Typically, gonad phenotype is determined histologically, which involves specialized and time-consuming techniques. The methods detailed here utilize an entirely different methodology, reverse-transcription quantitative polymerase chain reaction, to determine the relative expression levels of 4 genes after exposure to either 17-estradiol or 17-trenbolone and, by extension, the effects of EDCs on the phenotypic status of the gonad. The 4 genes quantified, Sox9b, protamine, Fig1, and ZPC1, are all involved in gonad development and maintenance in Japanese medaka (Oryzias latipes); these data were then inputted into a permutational multivariate analysis of variance to determine whether significant differences exist between treatment groups. This information in conjunction with the sexual genotype, which can be determined in medaka, can be used to determine adverse effects of exposure to EDCs in a similar fashion to the histologically determined gonad phenotype. Environ Toxicol Chem 2013;32:13441353. (c) 2013 SETAC