Endocrine, immune and renal toxicity in male largemouth bass after chronic exposure to glyphosate and Rodeo®.

Glyphosate is the most used herbicide worldwide, with no historical comparison. It is used for genetically modified crops, and particularly in Florida, it is used as a sugar cane ripener. An aquatic formulation (Rodeo®) is used to treat aquatic weeds in waterbodies and drainage canals. Because of its extended use, glyphosate can run off or be sprayed directly into waterbodies, and chronically expose aquatic wildlife. Exposure in animal models has been associated with kidney and liver damage and glyphosate has been suggested as an endocrine disruptor. We exposed adult male largemouth bass for 21 days to two doses of glyphosate and Rodeo® (chemically equivalent concentration of glyphosate) at 0.5 mg L-1 and 10 mg L-1 and to a clean water control (n=4 fish/tank in quadruplicate). Concentrations during the experiment were corroborated with UHPLC-MS/MS. Total RNA was isolated from the trunk kidney and head kidney. RNA-seq was performed for the high doses compared to controls. Transcripts were analyzed with fish and mammalian pathway analyses software. Transcripts mapped to Zebrafish metabolic pathways using PaintOmics showed steroid hormone biosynthesis in the trunk kidney as the most significantly enriched pathway. Steroid hormones were measured in plasma by UHPLC-MS/MS. Total androgens were significantly reduced at 0.5 mg L-1 of glyphosate and at equivalent concentrations in Rodeo® compared to controls. 11-ketotestosterone and estrone concentrations were significantly reduced in all doses. A gene involved in the conversion of testosterone to 11-ketotestosterone was down-regulated by glyphosate. Using the mammalian pathway analysis algorithm, cellular processes associated with T-cell activation/development and intracellular pH were significantly enriched in the trunk kidney by glyphosate and Rodeo® exposure. Endocrine disruption was corroborated at the hormone and gene expression levels. Rodeo® and glyphosate share gene expression pathways, however, Rodeo® had more pronounced effects in largemouth bass.

Impact of bisphenol-A and synthetic estradiol on brain, behavior, gonads and sex hormones in a sexually labile coral reef fish.

Endocrine disrupting chemicals, such as bisphenol A (BPA) and ethinylestradiol (EE2), are detected in the marine environment from plastic waste and wastewater effluent. However, their impact on reproduction in sexually labile coral reef fish is unknown. The objective of this study was to determine impacts of environmentally relevant concentrations of BPA and EE2 on behavior, brain gene expression, gonadal histology, sex hormone profile, and plasma vitellogenin (Vtg) levels in the anemonefish, Amphiprion ocellaris. A. ocellaris display post-maturational sex change from male to female in nature. Sexually immature, male fish were paired together and fed twice daily with normal food (control), food containing BPA (100 µg/kg), or EE2 (0.02 µg/kg) (n = 9 pairs/group). Aggression toward an intruder male was measured at 1, 3, and 6 months. Blood was collected at 3 and 6 months to measure estradiol (E2), 11-ketotestosterone (11-KT), and Vtg. At the end of the study, fish were euthanized to assess gonad morphology and to measure expression of known sexually dimorphic genes in the brain. Relative to control, BPA decreased aggression, altered brain transcript levels, increased non-vitellogenic and vitellogenic eggs in the gonad, reduced 11-KT, and increased plasma Vtg. In two BPA-treated pairs, both individuals had vitellogenic eggs, which does not naturally occur. EE2 reduced 11-KT in subordinate individuals and altered expression of one transcript in the brain toward the female profile. Results suggest BPA, and to a lesser extent EE2, pollution in coral reef ecosystems could interfere with normal reproductive physiology and behavior of the iconic sexually labile anemonefish.

Practical Euthanasia Method for Common Sea Stars (Asterias rubens) That Allows for High-Quality RNA Sampling.

Sea stars in research are often lethally sampled without available methodology to render them insensible prior to sampling due to concerns over sufficient sample quality for applied molecular techniques. The objectives of this study were to describe an inexpensive and effective two-step euthanasia method for adult common sea stars (Asterias rubens) and to demonstrate that high-quality RNA samples for further use in downstream molecular analyses can be obtained from pyloric ceca of MgCl2-immersed sea stars. Adult common sea stars (n = 15) were immersed in a 75 g/L magnesium chloride solution until they were no longer reactive to having their oral surface tapped with forceps (mean: 4 min, range 2-7 min), left immersed for an additional minute, and then sampled with sharp scissors. RNA from pyloric ceca (n = 10) was isolated using a liquid-liquid method, then samples were treated with DNase and analyzed for evaluation of RNA integrity number (RIN) for assessment of the quantity and purity of intact RNA. Aversive reactions to magnesium chloride solution were not observed and no sea stars regained spontaneous movement or reacted to sampling. The calculated RIN ranged from 7.3-9.8, demonstrating that the combination of animal welfare via the use of anesthesia and sampling for advanced molecular techniques is possible using this low-cost technique.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Acute and Chronic Toxicity Testing of Drinking Water Treatment Residuals in Freshwater Systems.

The beneficial use of drinking water treatment residuals (DWTRs) faces barriers due primarily to uncertainties and concerns about their potential environmental impacts. We used total and water leachable toxic metal concentrations and 2 benthic organism-based bioassays to identify suitable DWTR substrates for introduction to freshwater systems. Using total metal contents and the consensus probable effect concentration concept, 3 DWTRs were selected and used in elutriate and toxicity studies. The concentrations of water leachable Ag, As, Cd, Cu, Cr, Ni, Pb, and Zn were below the US Environmental Protection Agency's ambient water quality criteria. Using the long-term 65-d life cycle Chironomus tentans test and 4 different endpoints (survival, adult emergence, egg case production, and number of eggs produced per female), no statistical differences were found between the DWTR treatments and the controls. Similarly, results obtained using the 10-d Hyalella azteca test showed no toxicity. However, although both survival and growth were recorded in all bioassays, the results of the 10-d C. tentans and the 28-d H. azteca tests were ambiguous. For C. tentans, 2 of the 3 DWTRs resulted in significantly lower survival rates compared to the controls. For H. azteca, no significant growth differences were observed between controls and DWTR treatments, but 2 of the 3 DWTRs resulted in significantly lower survival rates than the controls. Overall, these results suggest that certain DWTR substrates could be suitable for introduction to aquatic systems. Environ Toxicol Chem 2021;40:2005-2014. © 2021 SETAC.

Chronic exposure to glyphosate in Florida manatee.

Florida manatees depend on freshwater environments as a source of drinking water and as warm-water refuges. These freshwater environments are in direct contact with human activities where glyphosate-based herbicides are being used. Glyphosate is the most used herbicide worldwide and it is intensively used in Florida as a sugarcane ripener and to control invasive aquatic plants. The objective of the present study was to determine the concentration of glyphosate and its breakdown product, aminomethylphosphonic acid (AMPA), in Florida manatee plasma and assess their exposure to manatees seeking a warm-water refuge in Crystal River (west central Florida), and in South Florida. We analyzed glyphosate's and AMPA's concentrations in Florida manatee plasma (n = 105) collected during 2009-2019 using HPLC-MS/MS. We sampled eight Florida water bodies between 2019 and 2020, three times a year: before, during and after the sugarcane harvest using grab samples and molecular imprinted passive Polar Organic Chemical Integrative Samplers (MIP-POCIS). Glyphosate was present in 55.8% of the sampled Florida manatees' plasma. The concentration of glyphosate has significantly increased in Florida manatee samples from 2009 until 2019. Glyphosate and AMPA were ubiquitous in water bodies. The concentration of glyphosate and AMPA was higher in South Florida than in Crystal River, particularly before and during the sugarcane harvest when Florida manatees depend on warm water refuges. Based on our results, Florida manatees were chronically exposed to glyphosate and AMPA, during and beyond the glyphosate applications to sugarcane, possibly associated with multiple uses of glyphosate-based herbicides for other crops or to control aquatic weeds. This chronic exposure in Florida water bodies may have consequences for Florida manatees' immune and renal systems which may further be compounded by other environmental exposures such as red tide or cold stress.

Novel effective mosquito larvicide DL-methionine: Lack of toxicity to non-target aquatic organisms.

Mosquito larvicides are an effective tool for reducing numbers of adult females that bite and potentially spread pathogenic organisms. Methionine, an essential amino acid in humans, has been previously demonstrated to be a highly effective larvicide against four (4) mosquito species in three (3) genera, Anopheles, Culex and Aedes. The aim of the present study was to determine the potential impact on non-target aquatic organisms of methionine applied as a mosquito larvicide. DL-methionine concentrations ranging from 0.06% to 1.00% were used; wherein the highest concentration of 1.00% would result in 100% mortality within 48 h in mosquitoes. Acute toxicity assays were conducted in accordance with the US Environmental Protection Agency (US EPA) guidelines for the water flea (Daphnia magna Straus; Cladocera: Daphniidae) and the fathead minnow (Pimephales promelas Rafinesque; Cypriniformes: Cyprinidae). Water fleas and fish were placed directly into the solutions in glass containers and tanks for 48-hours and 96-hours, respectively. When applied within the above-mentioned range of effective mosquito larvicide concentrations, DL-methionine meets US EPA criteria as a "practically non-toxic" pesticide for both species. These results suggest that methionine is a viable alternative to current mosquito larvicide options, which are typically classified as moderately to highly toxic and may be a valuable addition to a mosquito integrated pest management program.

Investigating an increase in Florida manatee mortalities using a proteomic approach.

Two large-scale Florida manatee (Trichechus manatus latirostris) mortality episodes were reported on separate coasts of Florida in 2013. The east coast mortality episode was associated with an unknown etiology in the Indian River Lagoon (IRL). The west coast mortality episode was attributed to a persistent Karenia brevis algal bloom or 'red tide' centered in Southwest Florida. Manatees from the IRL also had signs of cold stress. To investigate these two mortality episodes, two proteomic experiments were performed, using two-dimensional difference in gel electrophoresis (2D-DIGE) and isobaric tags for relative and absolute quantification (iTRAQ) LC-MS/MS. Manatees from the IRL displayed increased levels of several proteins in their serum samples compared to controls, including kininogen-1 isoform 1, alpha-1-microglobulin/bikunen precursor, histidine-rich glycoprotein, properdin, and complement C4-A isoform 1. In the red tide group, the following proteins were increased: ceruloplasmin, pyruvate kinase isozymes M1/M2 isoform 3, angiotensinogen, complement C4-A isoform 1, and complement C3. These proteins are associated with acute-phase response, amyloid formation and accumulation, copper and iron homeostasis, the complement cascade pathway, and other important cellular functions. The increased level of complement C4 protein observed in the red tide group was confirmed through the use of Western Blot.

Toxicity assessment of a novel oil dispersant based on silica nanoparticles using Fathead minnow.

Oil spill accidents are a major concern for aquatic organisms. In recent history, the Deepwater Horizon blowout spilled 500 million liters of crude oil into the Gulf of Mexico. Corexit 9500A was used to disperse the oil since it was the method approved at that time, despite safety concerns about its use. A better solution is necessary for dispersing oil from spills that reduces the toxicity to exposed aquatic organisms. To address this challenge, novel engineered nanoparticles were designed using silica cores grafted with hyperbranched poly(glycidol) branches. Because the silica core and polymers are known to be biocompatible, we hypothesized that these particles are nontoxic to fathead minnows (Pimephales promelas) and would decrease their exposure to oil polyaromatic hydrocarbons. Fathead minnow embryos, juveniles and adult stages were exposed to the particles alone or in combination with a water-accommodated fraction of oil. Acute toxicity of nanoparticles to fish was tested by measuring mortality. Sub-lethal effects were also measured including gene expression of cytochrome P450 1a (cyp1a) mRNA and heart rate in embryos. In addition, a mixture of particles plus the water-accommodated fraction was directly introduced to adult female fathead minnows by gavage. Three different nanoparticle concentrations were used (2, 10, and 50 mg/L) in either artificial fresh water or the water-accommodated fraction of the oil. In addition, nanoparticle-free controls were carried out in the two solutions. No significant mortality was observed for any age group or nanoparticle concentration, suggesting the safety of the nanoparticles. In the presence of the water-accommodated fraction alone, juvenile and adult fathead minnows responded by increasing expression of cyp1a. The addition of nanoparticles to the water-accommodated fraction reduced cyp1a gene expression in treatments. Heart rate was also restored to normal parameters in embryos co-exposed to nanoparticles and to the water-accommodated fraction. Measurement of polyaromatic hydrocarbons confirmed their presence in the tested solutions and the reduction of available PAH in WAF treated with the nanoparticles. Our findings suggest the engineered nanoparticles may be protecting the fish by sequestering polyaromatic hydrocarbons from oil, measured indirectly by the induction of cypa1 mRNAs. Furthermore, chemical analysis showed a reduction in PAH content in the water accommodated fraction with the presence of nanoparticles.

Transcriptome and physiological effects of toxaphene on the liver-gonad reproductive axis in male and female largemouth bass (Micropterus salmoides).

Toxaphene is an organochlorine pesticide and environmental contaminant that is concerning due to its atmospheric transport and persistence in soil. In Florida, toxaphene and other organochlorine pesticides were used heavily in agriculture on the north shore of Lake Apopka and they are still detectable in soil. Wild largemouth bass that inhabit the lake and the marshes along the north shore have been exposed to a variety of organochlorine pesticides including dieldrin, methoxychlor, and p,p'-DDE, among others. While these other organochlorine pesticides have been studied for their endocrine disrupting effects in largemouth bass, there is little information for toxaphene. In this study, male and female largemouth bass were given food containing 50 mg/kg toxaphene for almost 3 months, to achieve tissue levels similar to those found in fish at Lake Apopka. Sex-specific toxicity was then evaluated by measuring various reproductive endpoints and transcriptomic changes. In females, gonadosomatic index showed a trend towards reduction (p = 0.051) and plasma vitellogenin was reduced by ~40% relative to controls. However plasma levels of 17ß-estradiol and testosterone were not perturbed by toxaphene exposure. These data suggest that toxaphene does not act as a weak estrogen as many other organochlorine pesticides do, but rather appears to be acting as an antiestrogen in female fish. There were no obvious changes in the gonadosomatic index and plasma hormones in male bass. However, ex vivo explant experiments revealed that toxaphene prevented human chorionic gonadotropin-stimulated testosterone production in the testis. This suggested that toxaphene had anti-androgenic effects in males. Subsequent transcriptomic analyses of the testis revealed that androgen receptor/beta-2-microglobulin signaling was up-regulated while insulin-related pathways were suppressed with toxaphene, which could be interpreted as a compensatory response to androgen suppression. In the male liver, the transcriptome analysis revealed an overwhelming suppression in immune-related signaling cascades (e.g. lectin-like receptor and ITSM-Containing Receptor signaling, CD16/CD14 Proinflammatory Monocyte Activation, and CD38/CD3-JUN/FOS/NF-kB Signaling in T-cell Proliferation). Overall, this study showed that toxaphene induced sex-specific effects. The transcriptomic and physiological responses observed can contribute to the development of adverse outcome pathways for toxaphene exposure in fish.

Steroid hormones and estrogenic activity in the wastewater outfall and receiving waters of the Chascomús chained shallow lakes system (Argentina).

Natural and synthetic steroid hormones, excreted by humans and farmed animals, have been considered as important sources of environmental endocrine disruptors. A suite of estrogens, androgens and progestogens was measured in the wastewater treatment plant outfall (WWTPO) of Chascomús city (Buenos Aires province, Argentina), and receiving waters located downstream and upstream from the WWTPO, using solid phase extraction and high-performance liquid chromatography mass spectrometry. The following natural hormones were measured: 17ß-estradiol (E2), estrone (E1), estriol (E3), testosterone (T), 5α-dihydrotestosterone (DHT), progesterone (P), 17-hydroxyprogesterone (17OHP) and the synthetic estrogen 17α-ethinylestradiol (EE2). Also, in order to complement the analytical method, the estrogenic activity in these surface water samples was evaluated using the in vitro transactivation bioassay that measures the estrogen receptor (ER) activity using mammalian cells. All-natural steroid hormones measured, except 17OHP, were detected in all analyzed water samples. E3, E1, EE2 and DHT were the most abundant and frequently detected. Downstream of the WWTPO, the concentration levels of all compounds decreased reaching low levels at 4500 m from the WWTPO. Upstream, 1500 m from the WWTPO, six out of eight steroid hormones analyzed were detected: DHT, T, P, 17OHP, E3 and E2. Moreover, water samples from the WWTPO and 200 m downstream from it showed estrogenic activity exceeding that of the EC50 of the E2 standard curve. In sum, this work demonstrates the presence of sex steroid hormones and estrogenic activity, as measured by an in vitro assay, in superficial waters of the Pampas region. It also suggests the possibility of an unidentified source upstream of the wastewater outfall.

Quantification of steroid hormones in low volume plasma and tissue homogenates of fish using LC-MS/MS.

Quantification of steroid hormones in fish is an important step for toxicology and endocrinology studies. Among the hormone analysis techniques, liquid chromatography tandem mass spectrometry (LC-MS/MS) has widely been used for measuring hormones in various biological samples. Despite all improvements in the technique, detection of several hormones in a low volume of serum or plasma is still challenging. We developed a robust method for simultaneous quantification of 14 steroid hormones including corticosterone, cortisol, 11-ketotestosterone, progesterone, testosterone, 17OH-progesterone, aldosterone, dihydrotestosterone, estrone, 17ß-estradiol, estriol, ethinylestradiol, levonorgestrel and equilin from volumes as low as 10 µL serum or plasma in a short run by LC-MS/MS. The lowest limit of detection in 10 µL serum was 0.012 ng/mL measured for cortisol, progesterone, testosterone, 17OH-progesterone and estrone. Use of high (25 times more) serum volume improved detection limit of hormones by 2-40 times. The method was compared with the radioimmunoassay technique in which testosterone and 17ß-estradiol were highly correlated with R2 of 0.95 and 0.96, respectively. We validated the method by measuring four selected hormones, in low and high plasma volumes of largemouth bass (Micropterus salmoides). In addition, we developed a method to quantify hormones in whole body fish homogenates of small fish and compared the values to plasma concentrations, using fathead minnow (Pimephales promelas). Calculated concentrations of the hormones in plasma were consistent with those in the homogenate and 11-ketotestosterone and 17ß-estradiol were significantly different in males and females. The ability to measure hormones from whole body homogenates was further evaluated in two model small fish species, zebrafish (Danio rerio) and juvenile silverside (Menidia beryllina). These results suggest that whole tissue homogenate is a reliable alternative for hormone quantification when sufficient plasma is not available.

Steroidogenic acute regulatory protein transcription is regulated by estrogen receptor signaling in largemouth bass ovary.

Estrogenic contaminants in the environment are linked to the occurrence of reproductive abnormalities in many aquatic species, including largemouth bass (Micropterus salmoides; LMB). Previous work has shown that many different types of xenoestrogens regulate expression of the Steroidogenic Acute Regulatory protein (StAR), a cholesterol-transporting protein vital to steroid hormone biosynthesis; however, the regulatory mechanisms of StAR are incompletely characterized in fish. To learn more about endogenous expression patterns of StAR in the ovary, LMB were collected from the St. John's River (Florida, USA) over an entire breeding season to investigate StAR expression. Plasma 17ß-estradiol (E2) and StAR mRNA levels were positively correlated in females, and StAR mRNA levels displayed ~ 100-fold increase between primary oocyte growth stages and final maturation. To further study the regulation of StAR, female LMB in the laboratory were fed at ≃2% of their weight on a diet laden with 17α-ethinylestradiol (EE2, 70 or 200 ng EE2 per gram feed). Diets were designed to achieve a physiologically-relevant exposure to EE2, and StAR expression was assessed in vivo. We observed a dose-dependent suppression of StAR mRNA levels, however both diets led to high, pharmacological levels in the blood and do not represent normal physiological ranges of estrogens. In the 200 ng EE2/gm feed group, ovarian StAR mRNA levels were suppressed to approximately 5% of that of the LMB control group. These investigations suggest that LMB StAR increases in expression during oocyte maturation and that it is suppressed by E2 feedback when estrogen levels are high, through the HPG axis. A 2.9 kb segment of the LMB StAR promoter was examined for putative E2 response elements using in silico software, and a putative estrogen receptor binding element (ERE/-1745) was predicted in the promoter. The functionality of the ERE was examined using MA-10 mouse Leydig cells transfected with the LMB StAR promoter. Estrogen receptor (ER) interaction with ERE/-1745 was evaluated under basal and human chorionic gonadotropin (hCG)-treated conditions in the presence and absence of E2. Chromatin immunoprecipitation (ChIP) experiments revealed that ESR1 binding to the promoter was enriched under basal conditions and E2 exposure elicited an increase in enrichment (4-fold) above that observed under basal conditions. ESR2 was not strongly enriched at the ERE/-1745 site, suggesting that StAR may be preferentially regulated by LMB estrogen receptor 1 (esr1). Taken together, these different experiments provide evidence that LMB StAR is under the control of estrogens and that ESR1 binds directly to the LMB StAR promoter in an E2-responsive manner.

Comparative toxicity of three phenolic compounds on the embryo of fathead minnow, Pimephales promelas.

Phenols are classified as polar narcotics, which are thought to cause toxicity by non-specific mechanisms, possibly by disrupting membrane structure and function. Here we test three phenolic chemicals, phenol, 2,4-dichlorphenol and pentachlorophenol on embryo development, heartbeat rate and mitochondrial respiration in fathead minnow (Pimephales promelas). While these chemicals have been used on isolated mitochondria, they have not yet been used to verify respiration in intact embryos. Mitochondrial respiration in intact embryos was measured after optimizing the Seahorse XFe24 Extracellular Flux Analyzer. Heartbeat rate and mitochondrial respiration patterns of fathead minnow embryos at different developmental stages were also characterized. Exposures of embryos at developmental stage 20 occurred for 24 h with five concentrations of each phenolic compound ranging from 0.85 to 255 µM for phenol, 0.49 to 147 µM for 2,4-dichlorophenol and 0.3 to 90 µM for pentachlorophenol. Exposure to phenol at the concentrations tested had no effects on development, heartbeat or mitochondrial respiration. However, both 2,4-dichlorophenol and pentachlorophenol showed dose-dependent effects on development, heartbeat rate, and mitochondrial respiration, with the effects occurring at lower concentrations of pentachlorophenol, compared to 2,4-dichlorophenol, highlighting the higher toxicity of the more chlorinated phenols. Both 2,4-dichlorophenol and pentachlorophenol decreased basal mitochondrial respiration of embryos and ATP production. These results indicate that higher chlorinated phenolic chemicals cause developmental toxicity in fathead minnow embryos by decreasing mitochondrial respiration and heartbeat rate.

Toward an adverse outcome pathway for impaired growth: Mitochondrial dysfunction impairs growth in early life stages of the fathead minnow (Pimephales promelas).

Chemical contaminants present in the environment can affect mitochondrial bioenergetics in aquatic organisms and can have substantial effects on individual fitness. As early life stages of fish are particularly vulnerable to environmental contaminants, they are ideal models for examining the relationship between impaired mitochondrial bioenergetics (ATP-dependent respiration, basal oxidative respiration) and apical endpoints such as growth. Here, early life stages of the fathead minnow (Pimephales promelas), an ecologically relevant North American species, were used to investigate the relationship between mitochondrial bioenergetics and growth following perturbation with model mitochondrial toxicants 2,4-dinitrophenol and octylamine. Fathead minnows were exposed to 2,4-dinitrophenol and octylamine at 3 concentrations for 24 h and endpoints related to mitochondrial bioenergetics were measured with the Agilent Seahorse XFe24 Bioanalyzer. In order to link changes in mitochondrial bioenergetics to growth, fathead minnows were exposed to the same chemical contaminants for 7-14 days and growth was measured by measuring total length on a weekly basis. There was a significant correlation between decrease in average length at 14 days and basal respiration (r = 0.997, p = 0.050, n = 3), as well as maximal respiration (r = 0.998, p-value = 0.043, n = 3) for embryos exposed to 2,4 dinitrophenol. For octylamine, ATP production was highly correlated with average length at 7 days (p-value = 0.1) and spare respiratory capacity and average length at 14 days were highly correlated (p-value = 0.1). These data improve understanding of how mitochondrial toxicants impair growth in fish larvae and may be useful for developing an adverse outcome pathway for growth.

Activated carbon as a means of limiting bioaccumulation of organochlorine pesticides, triclosan, triclocarban, and fipronil from sediments rich in organic matter.

Addition of activated carbon to contaminated sediment is an established means of remediation but its applicability to sediments high in organic carbon is presently unknown. We evaluated the effects of adding either granular activated carbon (GAC) or pelletized fine-grained activated carbon (PfAC, containing ∼ 50% AC) to contaminated sediments from Lake Apopka featuring a very high total organic carbon content (∼39% w/w dry). Sediments showing background levels of legacy pesticides were spiked with a mixture of 5 chemicals (p,p'-DDE, dieldrin, triclosan, triclocarban, and fipronil) to a nominal concentration of 2 µg/g sediment for each chemical. Following incubation of spiked sediments with the addition of activated carbon for 30 days, we assessed the success on limiting bioaccumulation using Lumbriculus variegatus (blackworm). In contaminant-spiked sediments amended with PfAC, blackworm body burdens of triclosan, triclocarban, and fipronil decreased by >50% and those of p,p'-DDE and dieldrin decreased by <30%. GAC addition to spiked sediments was less impactful, and yielded notable benefits in worm body burden reduction only for fipronil (40%). Fipronil achieved high treatment efficiency within the 30 day amendment with both GAC and PfAC. This is the first study to examine AC treatment in artificially contaminated sediments intrinsically very rich in organic matter content. PfAC exhibited superior performance over GAC for mitigating the uptake of certain organochlorines by aquatic organisms. These results indicate that further studies focusing on additional types of sediments and a broader spectrum of hydrophobic pollutants are warranted.

Linking in vitro estrogenicity to adverse effects in the inland silverside (Menidia beryllina).

High-throughput cell assays that detect and integrate the response of multiple chemicals acting via a common mode of action have the potential to enhance current environmental monitoring practices. Establishing the linkage between in vitro and in vivo responses is key to demonstrating that in vitro cell assays can be predictive of ecologically relevant outcomes. The present study investigated the potency of 17ß-estradiol (E2), estrone (E1), nonylphenol (NP), and treated wastewater effluent using the readily available GeneBLAzer® estrogen receptor transactivation assay and 2 life stages of the inland silverside (Menidia beryllina). In vitro estrogenic potencies were ranked as follows: E2 > E1 >> NP. All 3 model estrogens induced vitellogenin and choriogenin expression in a dose-dependent manner in larvae and juveniles. However, apical effects were only found for E2 and E1 exposures of juveniles, which resulted in female-skewed sex ratios. Wastewater effluent samples exhibiting low in vitro estrogenicity (below the 10% effective concentration [EC10]), did not cause significant changes in M. beryllina. Significant induction of estrogen-responsive genes was observed at concentrations 6 to 26 times higher than in vitro responses. Gonadal feminization occurred at concentrations at least 19 to 26 times higher than the in vitro responses. These findings indicated that in vitro cell assays were more sensitive than the fish assays, making it possible to develop in vitro effect thresholds protective of aquatic organisms. Environ Toxicol Chem 2018;37:884-892. © 2017 SETAC.

Estrogen signaling through both membrane and nuclear receptors in the liver of fathead minnow.

Estradiol is a potent sex steroid hormone that controls reproduction and other cellular pathways in fish. It is known to regulate important proteins such as vitellogenin, the egg yolk precursor protein, and zona radiata proteins that form the eggshell for fish eggs. These proteins are made in the liver and transported out into the blood from where they are taken up into the ovary during oogenesis. Estradiol can exert its influence directly through soluble nuclear receptors (there are three in fish) or indirectly through membrane receptors and a phosphorylation cascade. Often there is coordination through both genomic and non-genomic pathways. We have used a toxicogenomics approach to determine the contribution of genomic and non-genomic regulation in the liver of fathead minnows exposed to 5ng ethinylestradiol per liter or to a mixture of 5ng ethinylestradiol and 100ng ZM189,154 (ZM) per liter. ZM has previously been shown to be a "perfect" antagonist for the fish nuclear estrogen receptors but has displayed agonistic activities for membrane receptors. We find that both nuclear and membrane receptors contribute to the biosynthesis of vitellogenin 1 and estrogen receptor one (Esr1), among others. In addition, lipid metabolism pathways appear to require both activities.

How consistent are we? Interlaboratory comparison study in fathead minnows using the model estrogen 17α-ethinylestradiol to develop recommendations for environmental transcriptomics.

Fundamental questions remain about the application of omics in environmental risk assessments, such as the consistency of data across laboratories. The objective of the present study was to determine the congruence of transcript data across 6 independent laboratories. Male fathead minnows were exposed to a measured concentration of 15.8 ng/L 17α-ethinylestradiol (EE2) for 96 h. Livers were divided equally and sent to the participating laboratories for transcriptomic analysis using the same fathead minnow microarray. Each laboratory was free to apply bioinformatics pipelines of its choice. There were 12 491 transcripts that were identified by one or more of the laboratories as responsive to EE2. Of these, 587 transcripts (4.7%) were detected by all laboratories. Mean overlap for differentially expressed genes among laboratories was approximately 50%, which improved to approximately 59.0% using a standardized analysis pipeline. The dynamic range of fold change estimates was variable between laboratories, but ranking transcripts by their relative fold difference resulted in a positive relationship for comparisons between any 2 laboratories (mean R2 > 0.9, p < 0.001). Ten estrogen-responsive genes encompassing a fold change range from dramatic (>20-fold; e.g., vitellogenin) to subtle (∼2-fold; i.e., block of proliferation 1) were identified as differentially expressed, suggesting that laboratories can consistently identify transcripts that are known a priori to be perturbed by a chemical stressor. Thus, attention should turn toward identifying core transcriptional networks using focused arrays for specific chemicals. In addition, agreed-on bioinformatics pipelines and the ranking of genes based on fold change (as opposed to p value) should be considered in environmental risk assessment. These recommendations are expected to improve comparisons across laboratories and advance the use of omics in regulations. Environ Toxicol Chem 2017;36:2593-2601. © 2017 SETAC.

Tissue distribution of organochlorine pesticides in largemouth bass (Micropterus salmoides) from laboratory exposure and a contaminated lake.

Tissue concentrations of persistent organochlorine pesticides in laboratory-exposed largemouth bass (Micropterus salmoides) and in bass collected from Lake Apopka, FL were determined by both total mass and lipid normalized mass to better understand the bioaccumulation pathways of contaminants. In the laboratory study, male bass were orally administered a single dose of a mixture of two pesticides (p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) and dieldrin) and then fed uncontaminated food for 28 days. Gastrointestinal tract, liver, brain, gonad, kidney, spleen, and muscle were collected for chemical analysis. Different profiles were observed by total contaminant mass in tissues compared to lipid normalized mass. On a lipid normalized basis, p,p'-DDE was highest in the gastrointestinal tract followed by the liver, gonad, spleen, muscle, kidney and then brain. Dieldrin, on the other hand, was highest in the gastrointestinal tract and spleen and then followed by the gonad, muscle, liver, kidney, and brain. Distribution of the chemicals among the organs differed by their log KOW values and generally followed the blood flow path after the gastrointestinal tract. The low contaminant levels found in kidney and brain suggest insufficient time for equilibration into these tissues, especially into the brain where the blood-brain barrier may be slow to traverse. In Lake Apopka fish, dichlorodiphenyltrichloroethanes (DDXs, sum of p,p'-DDE, p,p'-DDD, and p,p'-DDT), Drins (sum of aldrin, dieldrin, and endrin), and hexachlorocyclohexanes (HCHs) were found. For DDXs, the lipid normalized concentrations in each tissue were about the same, as predicted from theory. For Drins and HCHs, the lipid normalized concentrations were similar for kidney, spleen, brain, gonad and muscle, but much lower in the gastrointestinal tract and liver, probably because of metabolism occurring in those tissues.