Thijs Bosker

Challenges in current adult fish laboratory reproductive tests: Suggestions for refinement using a mummichog (Fundulus heteroclitus) case study

Concerns about screening endocrine-active contaminants have led to the development of a number of short-term fish reproductive tests. A review conducted of 62 published adult fish reproductive papers using various fish species found low samples sizes (mean of 5.7 replicates with a median of 5 replicates) and high variance (an average coefficient of variance of 43.8%). The high variances and low sample sizes allow only relatively large differences to be detected with the current protocols; the average significant difference detected was a 68.7% reduction in egg production, while only differences above 50% were detected with confidence. This result indicates low power to detect more subtle differences and a high probability of type II errors in interpretation. The present study identifies several ways to increase the power of the adult fish reproductive test in the mummichog (Fundulus heteroclitus). By identifying the peak timing of egg production (before and after the new moon), extending the duration of the experiment (increased from 7 to 14 d), and determining that a sample size of eight replicate tanks per treatment accurately predicts variance in the sample population (based on pre-exposure variation calculations of replicate tanks), the power of the test has been significantly increased. The present study demonstrates that weaknesses in the current adult fish reproductive tests can easily be addressed by focusing on improved understanding of the reproductive behavior of the test species and developing study designs that include calculating desired variability levels and increasing replicates.

Challenges and opportunities with the use of biomarkers to predict reproductive impairment in fishes exposed to endocrine disrupting substances.

Biomarkers are commonly used as signposts to evaluate the potential of contaminants to disrupt the endocrine system. However, the relationship between responses in these biomarkers and whole organism endpoints that directly affect population status is not clearly understood. In this study, the relationship between egg production (a whole-organism endpoint which has been directly linked to population-level responses) and biomarkers (sex steroids, vitellogenin (VTG) and gonad size) is examined. Data were collected from short-term reproductive tests in which a wide variety of fish species were exposed to a suite of contaminants with known or unknown modes/mechanisms of action (MOA). The potential to use biomarkers as signposts was evaluated by determining the occurrence of false negatives (i.e., an effect in egg production was not accompanied by a biomarker response) and false positives (i.e., an effect in biomarkers was not followed by an effect in egg production). The quantitative relationships between biomarkers and egg production, and the ability to use these quantitative relationships to predict population-level responses based on modeling was also assessed. A suite of female biomarkers resulted in a relatively low occurrence of both false positives and negatives, indicating the potential for their use as signposts for reproductive effects via endocrine disruption. Egg production in short-term adult fish reproductive tests showed significant relationships to 17β-estradiol (E2), changes in female VTG levels, and relative female gonad size (gonadosomatic index; GSI). Weaker significant relationships were found between egg production and both VTG levels and GSI in males. However, use of these quantitative relationships to predict population-level effects are cautioned because of high levels of uncertainty. This study demonstrates that there are qualitative and quantitative relationships among biomarkers, regardless of fish species used or the MOA of contaminants and concludes that a suite of female reproductive biomarkers can be used as effective signposts to screen chemicals and assess waste streams for endocrine disrupting substances with different MOA.

The effects of 17-α-ethinylestradiol (EE2) on molecular signaling cascades in mummichog (Fundulus heteroclitus).

Exposures to ≤10 ng/L of 17-α-ethinylestradiol (EE2) will reduce or shut down egg production in freshwater fish models, while mummichog (Fundulus heteroclitus), an estuarine species, are able to produce eggs at EE2 concentrations >3000 ng/L. The objective of this study was to gain mechanistic insight into how mummichog are able to produce eggs during exposures to high EE2. Mummichog were exposed to 0, 50 or 250 ng/L of EE2 for 14 d. There were no changes in gonadosomatic index, liversomatic index, gonad development, or plasma estradiol levels after exposure to EE2. However, testosterone significantly decreased with EE2 exposures (50, 250 ng/L). Microarray analysis in the liver revealed that cell processes associated with lipids were affected by EE2 at the transcriptome level. Based on the transcriptomics data, we hypothesize that mummichog are able to maintain lipid transport and uptake into the ovary and this may be associated with apolipoproteins, facilitating normal oocyte development. Novel gene regulatory networks for protein modification targets were also constructed to learn more about the potential roles of estrogens in the teleost liver. Although post-translational modifications (PTMs) are important regulatory mechanisms, the roles of PTMs in protein regulation in fish and the susceptibility of PTMs to aquatic pollutants are largely unexplored and may offer novel insight into mechanisms of endocrine disruption.

Transcriptomics profiling and steroid production in mummichog (Fundulus heteroclitus) testes after treatment with 5α-dihydrotestosterone

5α-Dihydrotestosterone (DHT) is a potent androgen in mammals with multiple roles; however the physiological actions of DHT in male fishes are not well known. To address this knowledge gap, male mummichog (Fundulus heteroclitus) were continuously exposed to 0, 5, and 50 μg/L DHT for 21 days. Following exposure, testes were separated for histology, ex vivo incubation to measure steroidogenic capacity, and gene expression analyses (real-time PCR and microarray). DHT significantly decreased ex vivo 11-ketotestosterone (11KT) production in males exposed to 50 μg/L DHT but not 5 μg/L DHT, and DHT exposure did not affect ex vivo testosterone production. Histological examination revealed that the amount of interlobular and connective tissue present in the testes was increased in the 50 μg/L DHT treatment. Despite reductions in the production of 11KT, DHT did not affect the expression of targeted genes in the steroidogenic pathway such as steroidogenic acute regulatory protein (star), P450 side chain cleavage (cyp11a1) and 11β-hydroxysteroid dehydrogenase (hsd11b3). Microarray analysis in the testes of individuals from control and 50 μg/L DHT revealed that males exposed to 50 μg/L DHT showed regulated transcriptional sub-networks that were related to immunity, regulation of blood flow, lipids and xenobiotic clearance, suggesting that DHT may be involved in the physiological regulation of these processes in the fish testes. A second objective of this study was to determine the feasibility of measuring mRNA levels in tissues used for ex vivo steroid production by comparing RNA integrity and transcript levels in testes of both immediately flash frozen tissue and incubated tissue. There was no significant difference in RNA quality between the two time points, indicating RNA integrity can remain intact for at least 18 h in ex vivo assays, thereby providing a viable option for researchers assessing multi-level biological reproductive endpoints when limited tissue is available. While the gene expression levels of actb, efla, rps12, rps18, star, and hsd11b3 remained unchanged, esr2a (esrba), esr2b (esrbb) and cyp11a1 were significantly lower in incubated tissue compared to flash frozen tissue. Therefore caution must be used as the steady-state levels of select genes may change over time. This study improves our understanding of DHT action in the teleostean testis and generates new hypotheses regarding cell processes that are regulated by this underexplored and potent androgen.

Detectable effect size and bioassay power of mummichog (Fundulus heteroclitus) and fathead minnow (pimephales promelas) adult reproductive tests.

Although multiple reproductive tests have been developed in small-bodied fish to determine the effects of endocrine-disrupting substances, few direct comparisons have been made among the available tests. Side-by-side reproductive tests with mummichog (Fundulus heteroclitus) and fathead minnow (FHM; Pimephales promelas) were conducted with 0, 3, 10, and 30% effluent from a bleached kraft pulp mill in Saint John, New Brunswick, Canada. Egg production was significantly increased in mummichog exposed to 3% combined mill effluent, but no difference was observed in FHM. No differences were found in whole-body testosterone or estradiol levels in mummichog, and whole-body 11-ketotestosterone levels in males were increased in 3% exposed fish compared to those in 10% effluent. Male FHM exposed to 30% effluent had increased whole-body testosterone levels, and female FHM in 30% effluent had decreased testosterone. No differences in estradiol or 11-ketotestosterone were observed in FHM. Relatively limited response occurred in other endpoints. A comparison of the results of the present study to other published studies suggests that current reproductive bioassays are only sensitive for detecting magnitudes of change of greater than 50% and that differences exist in the sensitivities of fish. Future research should address methods of reducing variability within test populations and focus on understanding the comparative responses among species commonly used for endocrine-disrupting substance testing.

The influence of feeding strategy on growth and rejection of herbage around dung pats and their decomposition

SUMMARY Fresh cattle dung from four farms with different feeding strategies was used to create artificial dung pats in a continuously grazed pasture in order to compare the rejection of herbage growing around the pats, the effect on undisturbed herbage growth under cages and pat decomposition. The first farm was an extensive organic farm (ORGE) with young steers grazing on a biodiverse sward. The second was an intensive organic farm (ORGI) with dairy cattle grazing on a grass/clover sward during the day and fed low-protein forages indoors. The third dung used was from an integrated farm (INT), where the feeding strategy was aiming for high dung quality by including straw in the diet. The fourth examined dung was from a conventional farm (CONV) aiming for a high milk production per cow, where fertilized grazed grass was the main component of the diet. A human smell test was performed to rank the odour of the four dungs. After 6 weeks of continuous grazing with dairy cattle, herbage yield around INT pats tended to be lowest, whilst undisturbed herbage yield in and around caged INT pats was highest (P<0 . 05). Therefore, it could be concluded that rejection was lowest for INT. The CONV pats gave highest rejection (P<0 . 05). However, herbage yield around the dung pats under grazing showed no significant correlation with both the human smell test and the contents of total-N and sugar in the rejected herbage. The feeding strategy had a significant effect on the decomposition of dung pats under the cages. After 6 weeks, the most liquid and least fibrous dung (CONV) showed highest decomposition (P<0 . 05), whilst decomposition of the most solid and fibrous dung (ORGE) tended to be lowest. However, no relationship was found between the decomposition of dung and the rejection of herbage around the dung pats. When combining a number of parameters determined in the experiment and comparing them using index figures for dung quality in terms of rejection, herbage growth and decomposition, the index figures of ORGI (102) and especially INT (113) were above average (100), while those of ORGE (94) and CONV (90) were below average. The difference between ORGI and INT might be explained by the addition of straw to the diet in the latter. The study showed that there are possibilities to improve dung quality by altering feeding strategy.

Effects of 17α-ethinylestradiol (EE2) on reproductive endocrine status in mummichog (Fundulus heteroclitus) under differing salinity and temperature conditions.

Exposure to 17α-ethinylestradiol (EE₂), a synthetic estrogen, has previously been shown to decrease reproductive endocrine status and egg production in northern mummichog (Fundulus heteroclitus macrolepidotus). The objective of this study was to evaluate if variations in salinity or temperature conditions of EE₂-exposed mummichog modify the effect on whole organism reproductive endocrine status and gonadal steroidogenesis. Mummichog were exposed in vivo for 14 days to 0, 50 and 250 ng/L EE₂ in 0, 16 and 32 ppt salinity at 18 °C and to 0 and 250 ng/L EE₂ at 10, 18 and 26 °C at 16 ppt. There was a little overall effect of salinity on measured endpoints. In the salinity exposure, 250 ng/L EE₂-exposed females had significantly reduced 17β-estradiol (E₂) levels. Increased temperature triggered gonadal growth in both sexes and increased plasma E₂ and E₂ production and decreased 11-KT (11-ketotestosterone) production. EE₂ counteracted the effect of temperature by depressing gonadal growth in males. In both exposures, EE₂ effects on testosterone (T) production were variable. The use of steroidogenic precursors (25-OH-cholesterol, and/or pregnenolone and/or testosterone) in the in vitro gonadal incubations indicated decreased E₂ production in females and 11-KT production in males were predominately due to suppression of the terminal conversion step between T and E₂ or 11-KT. Ovarian aromatase A (cyp19a) gene expression at 16 ppt and 18 °C was not affected by 250 ng/L EE₂ (the only treatment combinations tested). Overall, temperature is a factor regulating northern mummichog reproduction; EE₂ overrides its effects and disrupts the terminal step of steroidogenesis. Our results should be considered in designing future estuarine fish bioassays and in understanding effects of estrogenic endocrine disruptors in estuaries.

Sustained high temperature increases the vitellogenin response to 17α-ethynylestradiol in mummichog (Fundulus heteroclitus)

Mummichog (Fundulus heteroclitus), an estuarine fish of the western Atlantic, were acclimated to three salinities (0, 16 or 32 ppt) or three temperatures (10, 20 or 26 °C) and exposed to nominal 50 or 250 ng/L 17α-ethynylestradiol (EE2) for 14 days. In a separate experiment, fish were exposed to the same levels of EE2 and were subjected to a 1h heat shock (20-30 °C) on the 14th day and allowed to recover for 20 h. We were interested in whether or not susceptibility to EE2 exposure, as indicated by increases in vitellogenin (vtg) gene expression would change with high and low salinity, warm or cold temperature acclimation or acute heat shock. We also investigated the potential role of heat shock proteins (HSPs) under these conditions. Liver vtg1 mRNA was significantly induced in male mummichog exposed to 50 and 250 ng/L EE2, but salinity acclimation or acute heat shock did not further affect this induction. Males acclimated to 26 °C and exposed to 250 ng/L EE2 induced 3.5-fold more vtg1 mRNA than EE2 exposed males acclimated to 10 °C. HSP90 and HSP70 protein were largely unaffected by EE2 exposure. Our findings suggest that mummichog are more susceptible to EE2 under sustained temperature increases that may occur seasonally or with warming of coastal waters.

Validation of a refined short-term adult fish reproductive test with improved power for mummichog (Fundulus heteroclitus) to test complex effluents

Short-term adult fish reproductive tests are widely used to assess the toxicity of chemicals and waste streams. However, these tests often have low power to detect differences in egg production among treatments, due to high variance and small sample sizes, limiting their effectiveness for informing regulatory decisions. A protocol for a fish reproductive test using mummichog (Fundulus heteroclitus) was refined to increase statistical power. Three studies using the original protocol were compared with three studies using the refined protocol. Tank pre-selection and sample size increased the a priori power from 11.2% to 85.7%. After exposure, average power levels were 62.0%, a more than five-fold increase compared to studies that used the original protocol (power of 15.0%). There was a high level of consistency compared to the original protocol; differences >33% in female and male gonad size and egg production could be detected among treatments. This study demonstrates that a refinement process can address shortcomings in short-term adult fish reproductive protocols, creating a solid foundation for further standardization and possible regulatory use.

Statistical reporting deficiencies in environmental toxicology.

Null hypothesis significance testing is one of the most widely used forms of statistical testing in environmental toxicology. In this short communication, the authors show that the reporting of statistical information when using null hypothesis significance testing is frequently inadequate in environmental toxicology research. The authors demonstrate this by analyzing the statistical information reported for papers employing t tests or analyses of variance in the Environmental Toxicology section of Environmental Toxicology and Chemistry in 2010, which comprised 68% of papers published by this journal in that year. Of these papers, 60% fail to report exact p values, 85% fail to provide degrees of freedom, and 90% fail to report critical effect sizes. Statistical power was reported in only <2% of the published papers. The insufficient provision of statistical information makes interpretation of study results by reviewers and readers difficult. Consistently reporting exact p values with degrees of freedom, considering and explicitly stating biologically relevant critical effect sizes, and reporting statistical power associated with nonsignificant results would be easy to implement and would promote scientific progress in environmental toxicology through increased statistical transparency.