Marlo K. Sellin Jeffries

The anti-estrogenic activity of sediments from agriculturally intense watersheds: assessment using in vivo and in vitro assays.

The goal of the current study was to determine whether sediments from agriculturally intense watersheds can act as a potential source of anti-estrogenic endocrine-disrupting compounds. The specific objectives of the current study were to determine (1) whether female fathead minnows (Pimephales promelas) experience alterations in endocrine function when exposed to sediments collected from agriculturally intense watersheds and (2) if these sediments display anti-estrogenic activity in an in vitro assay. In addition, sediment samples were analyzed for the presence of steroid hormones and pesticides associated with local agricultural practices. To accomplish this, sediments and water were collected from three sites within two agriculturally intense Nebraska watersheds (Bow Creek and the Elkhorn River). In 2009, minnows were exposed to sediment and/or water collected from the two Bow Creek sites (East Bow Creek and the Confluence) in the laboratory, while in 2010, minnows were exposed to sediment and/or water from East Bow Creek, the Confluence and the Elkhorn River. Following the 7-day exposure period, the hepatic mRNA expression of two-estrogen responsive genes, estrogen receptor α (ERα) and vitellogenin (Vtg) was determined. In 2009, females exposed to Confluence sediments, in the presence of laboratory water or Confluence water, experienced significant reductions in ERα expression relative to unexposed and Confluence water-exposed females. The defeminization of these females suggests the presence of a biologically available anti-estrogenic compound in sediments collected from this site. In 2010, sediments were assessed for anti-estrogenic activity on days 0 and 7 of the exposure period using a 4-h yeast estrogen screen. Lipophilic extracts (LEs) of day 0 sediments collected from the Confluence and the Elkhorn River induced significant reductions in the estrogenic reporter activity of treated yeast cultures suggesting the presence of a lipophilic anti-estrogenic compound in these extracts. Chemical analysis revealed the presence of a variety of steroid hormones, including those associated with the production of beef cattle (i.e. β-trenbolone, α-zearalanol and α-zearalenol), in sediments indicating that compounds utilized by local beef cattle operations are capable of entering nearby watersheds. Overall, the results of this study indicate that an environmentally relevant anti-estrogenic compound is present in sediments from agriculturally intense watersheds and that this compound is bioavailable to fish. Furthermore, the presence of steroid hormones in sediments from these watersheds provides evidence indicating that steroids are capable of sorbing to sediments.

Quantitative risk model for polycyclic aromatic hydrocarbon photoinduced toxicity in Pacific herring following the Exxon Valdez oil spill.

Phototoxicity occurs when exposure to ultraviolet radiation increases the toxicity of certain contaminants, including polycyclic aromatic hydrocarbons (PAHs). This study aimed to (1) develop a quantitative model to predict the risk of PAH phototoxicity to fish, (2) assess the predictive value of the model, and (3) estimate the risk of PAH phototoxicity to larval and young of year Pacific herring (Clupea pallasi) following the Exxon Valdez oil spill (EVOS) in Prince William Sound, Alaska. The model, in which median lethal times (LT50 values) are estimated from whole-body phototoxic PAH concentrations and ultraviolet A (UVA) exposure, was constructed from previously reported PAH phototoxicity data. The predictive value of this model was confirmed by the overlap of model-predicted and experimentally derived LT50 values. The model, along with UVA characterization data, was used to generate estimates for depths of de minimiz risk for PAH phototoxicity in young herring in 2003/2004 and immediately following the 1989 EVOS, assuming average and worst case conditions. Depths of de minimiz risk were estimated to be between 0 and 2 m deep when worst case UVA and PAH conditions were considered. A post hoc assessment determined that <1% of the young herring population would have been present at depths associated with significant risk of PAH phototoxicity in 2003/2004 and 1989.

A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples

BackgroundThis study compared the performance of five commercially available kits in extracting total RNA from small eukaryotic tissue samples (<15 mg). Total RNA was isolated from fathead minnow (Pimephales promelas) tissues (spleen, blood, kidney, embryo, and larvae) using the Qiagen RNeasy® Plus Mini, Qiagen RNeasy® Plus Universal, Promega Maxwell® 16 LEV simplyRNA, Ambion MagMAX™-96 and Promega SimplyRNA HT kits. Kit performance was evaluated via measures of RNA quantity (e.g., total RNA amount) and quality (e.g., ratio of absorbance at 260 and 280 nm, RNA integrity number (RIN), presence of gDNA).ResultsWith the exception of embryos, each kit generally extracted ≥5 μg of total RNA from each sample. With regard to RNA quality, the RINs of RNA samples isolated via the Plus Mini and Maxwell® 16 kits were consistently higher than those of samples extracted via the remaining three kits and for all tissues, these kits produced intact RNA with average RIN values ≥7. The Plus Universal and SimplyRNA HT kits produced moderately degraded (RIN values <7, but ≥5), while the RNA recovered via the MagMAX™ kit tended to exhibit a high degree of degradation (RIN values <5).ConclusionsEach kit was generally capable of extracting the amount of RNA required for most downstream gene expression applications suggesting that RNA yield is unlikely to be a limiting factor for any of the kits evaluated. However, differences in the quality of RNA extracted via each of the kits indicate that these kits may differ in their ability to yield RNA acceptable for some applications. Overall, the findings of this study demonstrate that there are practical differences between commercially available RNA extraction kits that should be taken into account when selecting extraction methods to be used for isolating RNA designated for gene expression analysis.

Occurrence and endocrine effects of agrichemicals in a small Nebraska, USA, watershed.

The Bow Creek watershed (Nebraska, USA) is dominated by the production of beef cattle and row crops; therefore, surface waters are likely to receive runoff containing steroid hormones and pesticides. The goal of the present study was to determine the occurrence and endocrine effects of agrichemicals in this watershed. To accomplish this, four sites within the watershed-Pearl, Bow, and East Bow Creeks and a site at the confluence with the Missouri River-were selected. In June of 2008, polar organic chemical integrative samplers (POCIS) were deployed at each site, whereas in June of 2009, water and sediment samples were collected. Caged fathead minnows (Pimephales promelas) were deployed at all of the selected sites in both years. Analysis of these samples revealed that steroid hormones were not present; however, pesticides were present in POCIS extracts and water samples. In general, the amount of pesticides was higher in POCIS retrieved from Pearl and Bow Creeks than in POCIS from East Bow Creek and the confluence. This variation between sites appeared to be related to row crop density, as row crop land cover surrounding the Pearl and Bow Creek sites was higher than that surrounding the East Bow and confluence sites. To determine the endocrine effects of agrichemicals within this watershed, the hepatic mRNA expression of vitellogenin and estrogen receptor α (ERα), as well as the gonadal expression of P450 aromatase A, was determined for the caged minnows. Females deployed at East Bow Creek and the confluence experienced decreases in the expression of ERα, suggesting that these females had been defeminized; however, this defeminization could not be attributed to any of the pesticides detected at these sites.

The fish embryo toxicity test as a replacement for the larval growth and survival test: A comparison of test sensitivity and identification of alternative endpoints in zebrafish and fathead minnows

The fish embryo toxicity (FET) test has been proposed as an alternative to the larval growth and survival (LGS) test. The objectives of the present study were to evaluate the sensitivity of the FET and LGS tests in fathead minnows (Pimephales promelas) and zebrafish (Danio rerio) and to determine if the inclusion of sublethal metrics as test endpoints could enhance test utility. In both species, LGS and FET tests were conducted using 2 simulated effluents. A comparison of median lethal concentrations determined via each test revealed significant differences between test types; however, it could not be determined which test was the least and/or most sensitive. At the conclusion of each test, developmental abnormalities and the expression of genes related to growth and toxicity were evaluated. Fathead minnows and zebrafish exposed to mock municipal wastewater-treatment plant effluent in a FET test experienced an increased incidence of pericardial edema and significant alterations in the expression of genes including insulin-like growth factors 1 and 2, heat shock protein 70, and cytochrome P4501A, suggesting that the inclusion of these endpoints could enhance test utility. The results not only show the utility of the fathead minnow FET test as a replacement for the LGS test but also provide evidence that inclusion of additional endpoints could improve the predictive power of the FET test.

Alternative methods for toxicity assessments in fish: Comparison of the fish embryo toxicity and the larval growth and survival tests in zebrafish and fathead minnows

An increased demand for chemical toxicity evaluations has resulted in the need for alternative testing strategies that address animal welfare concerns. The fish embryo toxicity (FET) test developed for zebrafish (Danio rerio) is one such alternative, and the application of the FET test to other species such as the fathead minnow (Pimephales promelas) has been proposed. In the present study, the performances of the FET test and the larval growth and survival (LGS; a standard toxicity testing method) test in zebrafish and fathead minnows were evaluated. This required that testing methods for the fathead minnow FET and zebrafish LGS tests be harmonized with existing test methods and that the performance of these testing strategies be evaluated by comparing the median lethal concentrations of 2 reference toxicants, 3,4-dicholoraniline and ammonia, obtained via each of the test types. The results showed that procedures for the zebrafish FET test can be adapted and applied to the fathead minnow. Differences in test sensitivity were observed for 3,4-dicholoraniline but not ammonia; therefore, conclusions regarding which test types offer the least or most sensitivity could not be made. Overall, these results show that the fathead minnow FET test has potential as an alternative toxicity testing strategy and that further analysis with other toxicants is warranted in an effort to better characterize the sensitivity and feasibility of this testing strategy.

The endocrine effects of dietary brominated diphenyl ether-47 exposure, measured across multiple levels of biological organization, in breeding fathead minnows

The goal of the present study was to evaluate the reproductive function of fathead minnows (Pimephales promelas) exposed to brominated diphenyl ether-47 (BDE-47) at doses lower than those used in previous studies. This was accomplished by evaluating the impacts of BDE-47 exposures across multiple levels of biological organization. Breeding pairs were exposed to BDE-47 via diet for 21 d, during which reproductive success was monitored. At the conclusion of the exposure, fish were euthanized to assess the effects of BDE-47 on sex steroid-related and thyroid-related transcripts, plasma androgen levels, gonadosomatic index (GSI), and secondary sexual characteristics. Several alterations in gene expression were noted including a >2.1-fold decrease in hepatic estrogen receptor α (erα) and a 2.9-fold decrease in ovarian aromatase (arom). In addition, BDE-47-exposed males experienced increases in deiodinase 2 (dio2) expression in brain tissue (∼1.5-fold) and decreases in hepatic transthyretin (ttr) expression (∼1.4-fold). Together, these gene expression alterations suggest the potential for BDE-47 to disrupt endocrine signaling. There were no significant differences in plasma hormone levels, GSI, secondary sexual characteristics, or reproductive success. Overall, the present study demonstrates that exposure to BDE-47 is capable of altering both sex steroid-related and thyroid-related transcripts but that these observed alterations do not necessarily manifest themselves at higher levels of biological organization for the endpoints selected. Environ Toxicol Chem 2016;35:2048-2057.

Enhancing the fathead minnow fish embryo toxicity test: Optimizing embryo production and assessing the utility of additional test endpoints

The fathead minnow fish embryo toxicity (FET) test has been identified as a potential alternative to toxicity test methods that utilize older fish. However, several challenges have been identified with the fathead minnow FET test, including: 1) difficulties in obtaining appropriately-staged embryos for FET test initiation, 2) a paucity of data comparing fathead minnow FET test performance to the fathead minnow larval growth and survival (LGS) test and 3) a lack of sublethal endpoints that could be used to estimate chronic toxicity and/or predict adverse effects. These challenges were addressed through three study objectives. The first objective was to optimize embryo production by assessing the effect of breeding group composition (number of males and females) on egg production. Results showed that groups containing one male and four females produced the largest clutches, enhancing the likelihood of procuring sufficient numbers of embryos for FET test initiation. The second study objective was to compare the performance of the FET test to that of the fathead minnow LGS test using three reference toxicants. The FET and LGS tests were similar in their ability to predict the acute toxicity of sodium chloride and ethanol, but the FET test was found to be more sensitive than the LGS test for sodium dodecyl sulfate. The last objective of the study was to evaluate the utility and practicality of several sublethal metrics (i.e., growth, developmental abnormalities and growth- and stress-related gene expression) as FET test endpoints. Developmental abnormalities, including pericardial edema and hatch success, were found to offer the most promise as additional FET test endpoints, given their responsiveness, potential for predicting adverse effects, ease of assessment and low cost of measurement.

Embryo-larval BDE-47 exposure causes decreased pathogen resistance in adult male fathead minnows (Pimephales promelas)

&NA; Exposures to polybrominated diphenyl ethers (PBDEs) have been shown to alter immune function in adult organisms across a variety of taxa. However, few if any studies have investigated the long‐term consequences of early life stage PBDE exposures on immune function in fish. This study sought to determine the effects of early life stage BDE‐47 exposure on pathogen resistance in the fathead minnow (Pimephales promelas) following an extended depuration period (≥180 d). Minnows were exposed to BDE‐47 via a combination of maternal transfer and diet through 34 days post fertilization (dpf), raised to adulthood (>215 dpf) on a clean diet, then subjected to pathogen resistance trials. Early life stage exposures to BDE‐47 did not affect the ability of females to survive from Yersinia ruckeri infection. However, the survival of BDE‐47 exposed males was significantly reduced relative to controls, indicating that developmental exposures to BDE‐47 altered male immunity. Because BDE‐47 is a known thyroid hormone disruptor and thyroid hormone disruptors have the potential to adversely impact immune development and function, metrics indicative of thyroid disruption were evaluated, as were immune parameters known to be altered in response to thyroid disruption. BDE‐47 exposed minnows exhibited signs of thyroid disruption (i.e, reduced growth); however, no alterations were observed in immune parameters known to be influenced by thyroid hormones (i.e, thymus size, expression of genes associated with lymphoid development) suggesting that the observed alterations in immunocompetence may occur through alternative mechanisms. Regardless of the mechanisms responsible, the results of this study demonstrate the potential for early life stage PBDE exposures to adversely impact immunity and illustrate that the immunological consequences of PBDE exposures are sex dependent. HighlightsEarly life stage BDE‐47 exposures reduced the pathogen resistance of adult minnows.The effects of developmental BDE‐47 exposure on pathogen resistance were sex specific.Males were less immunocompetent than females, regardless of exposure history.Mechanisms underlying BDE‐47 induced alterations in immunity remain elusive.Results suggest that fathead minnows may be a promising immunotoxicity model.

Characterization of basic immune function parameters in the fathead minnow (Pimephales promelas), a common model in environmental toxicity testing

Abstract The fathead minnow (Pimephales promelas) is an environmental sentinel species, commonly used in toxicity testing. However, there is a lack of data regarding basic immune function in this species. To improve the usefulness of the fathead minnow as a model for basic immune function and immunotoxicity, this study sought to 1) compare the differential expression of immune function genes in naïve fathead minnows and 2) determine the effects of pathogen exposure on immune gene expression and spleen index. To accomplish this, kidney, spleen and liver tissue were collected three days post injection (dpi) from adult male fathead minnows from each of the following groups: 1) uninjected control 2) sham‐injected (Hanks balanced salt solution) and 3) pathogen‐injected (Yersinia ruckeri). Spleen tissue was also collected at seven and 14 dpi. Differential tissue expression of immune function genes was evaluated in naïve minnows and expression patterns were similar to those found in other fish species, with liver tissue generally having the highest amount of expression. Following pathogen injection, the expression of complement component 3 (c3) (4.4‐fold, kidney; 2.5‐fold, liver), interleukin 11 (il11) (4.8‐fold, kidney; 15.2‐fold, liver) and interleukin 1&bgr; (il1&bgr;) (8.2‐fold, kidney; 17.2‐fold, spleen; 2.6‐fold, liver) were significantly upregulated. Elastase 2 (elas2) was significantly downregulated (5.8‐fold) in liver tissue. A significant increase in spleen index at seven dpi was also observed in pathogen‐injected minnows. This study has identified endpoints that are part of the normal response to pathogen in fathead minnows, an essential step toward the development of the fathead minnow as a model for immunotoxicity evaluations. HighlightsHepatic tissue had the highest expression of immune‐related genes in naïve fish.Pathogen injection resulted in alterations in expression of immune‐related genes.Spleen index was significantly increased seven days following pathogen injection.Immune‐sensitive endpoints can be utilized in future studies using this species.