Comparative toxicokinetics and toxicity of PFOA and its replacement GenX in the early stages of zebrafish.

PER: and poly-fluoroalkyl substances (PFAS) are receiving attention due to their persistence, and potential adverse effects on environmental and human health. Efforts to reduce long-chained PFAS (≥C8) compounds were implemented in 2006 as a part of "PFOA Stewardship Program Initiative" (PFOA-perfluorooctanoic acid). Short-chained PFAS (

Species- and Tissue-Specific Chronic Toxicity Values for Northern Bobwhite Quail (Colinus virginianus) Exposed to Perfluorohexane Sulfonic Acid and a Binary Mixture of Perfluorooctane Sulfonic Acid and Perfluorohexane Sulfonic Acid.

Per- and polyfluoroalkyl substances (PFAS) are globally distributed and present in nearly every environmental compartment. Characterizing the chronic toxicity of individual PFAS compounds and mixtures is necessary because many have been reported to cause adverse health effects. To derive toxicity reference values (TRVs) and conduct ecotoxicological risk assessments (ERAs) of PFAS-contaminated ecosystems for wildlife, species-specific PFAS chronic toxicity values (CTVs) are needed. The present study quantified PFAS residues from liver and eggs of birds chronically exposed to perfluorohexanoic acid (PFHxA) or a mixture of perfluorooctane sulfonate (PFOS) and PFHxA that produced a no-observable-adverse-effect level (NOAEL) and/or a lowest-observable-adverse-effectlevel (LOAEL). The CTVs we present are lower than those previously reported for birds and should be considered in future regulatory evaluations. From the estimated species- and tissue-specific PFAS CTVs, we found that PFOS and perfluorohexane sulfonate (PFHxS) were more bioaccumulative than PFHxA in avian tissues, but PFHxA was more toxic to reproducing birds than either PFOS or a PFOS:PFHxS mixture. We further determined that avian toxicity was not necessarily additive with respect to PFAS mixtures, which could have implications for PFAS ERAs. The PFAS LOAEL CTVs can be used to predict reproductive and possible population-level adverse health effects in wild avian receptors. Environ Toxicol Chem 2022;41:219-229. © 2021 SETAC.

Chronic Reproductive Toxicity Thresholds for Northern Bobwhite Quail (Colinus virginianus) Exposed to Perfluorohexanoic Acid (PFHxA) and a Mixture of Perfluorooctane Sulfonic Acid (PFOS) and PFHxA.

Terrestrial toxicology data are limited for comprehensive ecotoxicological risk assessment of ecosystems contaminated by per- and polyfluoroalkyl substances (PFAS) partly because of their existence as mixtures in the environment. This complicates logistical dose-response modeling and establishment of a threshold value characterizing the chronic toxicity of PFAS to ecological receptors. We examined reproduction, growth, and survival endpoints using a combination of hypothesis testing and logistical dose-response modeling of northern bobwhite quail (Colinus virginianus) exposed to perfluorohexanoic acid (PFHxA) alone and to PFHxA in a binary mixture with perfluorooctane sulfonic acid (PFOS) via the drinking water. The exposure concentration chronic toxicity value (CTV) representative of the lowest-observable-adverse effect level (LOAEL) threshold for chronic oral PFAS toxicity (based on reduced offspring weight and growth rate) was 0.10 ng/mL for PFHxA and 0.06 ng/mL for a PFOS:PFHxA (2.7:1) mixture. These estimates corresponded to an adult LOAEL average daily intake CTV of 0.0149 and 0.0082 µg × kg body weight-1 × d-1 , respectively. Neither no-observable-adverse effect level threshold and representative CTVs nor dose-response and predicted effective concentration values could be established for these 2 response variables. The findings indicate that a reaction(s) occurs among the individual PFAS components present in the mixture to alter the potential toxicity, demonstrating that mixture affects avian PFAS toxicity. Thus, chronic oral PFAS toxicity to avian receptors represented as the sum of the individual compound toxicities may not necessarily be the best method for assessing chronic mixture exposure risk at PFAS-contaminated sites. Environ Toxicol Chem 2021;40:2601-2614. © 2021 SETAC.

Toxicity Testing of Effluent-Dominated Stream Using Predictive Molecular-Level Toxicity Signatures Based on High-Resolution Mass Spectrometry: A Case Study of the Lubbock Canyon Lake System.

Current aquatic toxicity assessments usually focus on targeted analyses coupled with toxicity testing to determine the impacts of complex mixtures on aquatic organisms. However, based on this approach alone, it is sometimes difficult to explain observed toxicity from the selected chemical analytes. Recent analytical advances such as high-resolution mass spectrometry (HRMS) can improve the characterizations of the chemical composition of complex mixtures, but the intensive labor required to produce confident identifications limits its utility in high-throughput screening. In the present study, we evaluated a rapid workflow to predict potential toxicity signatures of complex water samples based on high-throughput, tentative HRMS identifications derived from database matching, followed by identification of chemical-ligand interactions and pathway identification. We tested the workflow with water samples from the effluent-dominated Lubbock Canyon Lake System (LCLS). Results across all sites showed that predicted toxicity signatures had little variation when correcting for HRMS false-positive rates. The most common pathways across sites were gonadotropin-releasing hormone receptor and α-adrenergic receptor signaling. Alterations to the predicted pathways were successfully observed in larval zebrafish exposures to LCLS water samples. These results may allow researchers to better utilize rapid assessments of HRMS data for the assessment of adverse impacts on aquatic organisms.

Transgenerational effects of developmental exposure to chlorpyrifos-oxon in zebrafish (DANIO RERIO).

The organophosphate chlorpyrifos, and its active metabolite chlorpyrifos-oxon (CPO), have been attributed to a number of neurodevelopmental disorders. It is unclear if the adverse effects associated with developmental exposure to the active CPO persist into adulthood and future generations. The goal of this study was to investigate whether CPO-associated changes in embryo-larval zebrafish (ZF) behavior at the F0 5 dpf were manifest throughout the life of the exposed F0, and are inherited by subsequent generations. For this study, embryos were exposed to chlorpyrifos-oxon at the environmentally relevant concentration of 0.01 µg/L and a high concentration of 50 µg/L starting at 4 hpf to 5 dpf, and then raised to F2. There was a significant decrease in distance traveled with 5 dpf F0 ZF exposed to the 50 µg/L CPO, with alterations in noncholinergic genes CFOS and LINGO, and alterations in global DNA methylation. CPO-related behavioral effects were ameliorated by day 21 through the F1 generation. This trend changed with hyperactive behavior, increase acetylcholine concentration in F2 zebrafish that were exposed to 50 µg/L CPO during the F0 development. There was also an increase in AChE activity and hypermethylation in F2 0.01 µg/L exposure larvae, indicating that even low dose exposures can have transgenerational effects. Results from this study demonstrate that early life stage exposures to CPO can lead to epigenetic changes in neurological activity, which may lead to alterations in response to CPO in future generations. ABSTRACT SUMMARY: This study identified a correlation between CPO exposure during F0 development and significant differences in F2 behavioral, AChE activity and neurotransmitter concentration.

In vivo effects on the immune function of fathead minnow (Pimephales promelas) following ingestion and intraperitoneal injection of polystyrene nanoplastics.

Nanoplastics (1-100 nm) are potentially the most hazardous litter in the environment. Recent scientific studies have documented their toxic effects at the cellular and molecular levels, but knowledge underlying mechanisms of their toxicity is still scarce. Nanoplastics are known for their ability to induce immune and inflammatory responses as well as generating reactive oxygen species. While some studies have addressed the immunotoxicity of nanoplastics in vitro and on in vivo in fish after intraperitoneal injection, no information is available on adult fish after ingestion of a contaminated prey. The present study is the first to attempt to address the immunotoxicity of nanoplastics in adult fish after trophic transfer. Pimephales promelas is a well-established bioindicator species to study the immunotoxicity of nanoparticles and the innate immune responses of fish. This study aims to assess the in vivo innate immune response of adult P. promelas following exposure to polystyrene nanoplastics by measuring the gene expression of ncf, nox2, mst1 and c3; these genes are related with the immune function of neutrophils, macrophages and complement in fish. Two target organs (liver and head kidney) and two routes of exposure (IP- injection and ingestion) were analyzed. After 48 h of exposure, polystyrene nanoplastics were encountered in the liver and kidney of both IP-injection and ingestion exposed fish, and significantly affected the innate immune system of P. promelas by downregulating the gene expression ncf, mst1, and c3 in liver and kidney. Significant difference between treatments was only observed for the gene expression of nfc in liver. Results of this study indicate that polystyrene nanoplastics can exhibit immunotoxicity in fish through an environmentally relevant route of exposure, interfering with the synthesis and function of neutrophils, macrophages, and complement of P. promelas in their principal hematopoietic tissues, which may potentially compromise its ability to survive in nature.

Translocation, trophic transfer, accumulation and depuration of polystyrene microplastics in Daphnia magna and Pimephales promelas.

In recent years, reports of plastic debris in the gastrointestinal (GI) tract of fish have been well documented in the scientific literature. This, in turn, increased concerns regarding human health exposure to microplastics through the consumption of contaminated fish. Most of the available research regarding microplastic toxicity has focused on marine organisms through direct feeding or waterborne exposures at the individual level. However, little is known about the trophic transfer of microplastics through the aquatic food chain. Freshwater zooplankton Daphnia magna (hereafter Daphnia), and the fathead minnow Pimephales promelas (FHM), are well-known model species used in standard toxicological studies and ecological risk assessments that provide a simple model for trophic transfer. The aim of this study was to assess the tissue translocation, trophic transfer, and depuration of two concentrations (20 and 2000-part ml-1) of 6 µm polystyrene (PS) microplastics particles between Daphnia and FHM. Bioconcentration factors (BCF) and bioaccumulation factors (BAF) were determined. Fluorescent microscopy was used to determine the number of particles in the water media and within the organs of both species. Throughout the five days of exposure, PS particles were only found within the GI tract of both species. The BCF for Daphnia was 0.034 ± 0.005 for the low concentration and 0.026 ± 0.006 for the high concentration. The BAF for FHM was 0.094 ± 0.037 for the low concentration and 0.205 ± 0.051 for the high concentration. Between 72 and 96 h after exposure all microplastic particles were depurated from both species. The presence of food had a significant effect on the depuration of microplastic particles from Daphnia but not for FHM. Based on the low BCF and BAF values for both species, rapid depuration rates, and null translocation of microplastic particles to organs and tissues from the GI tract, there is a low probability that microplastics will bioconcentrate and bioaccumulate under environmental conditions.

Comparative analyses of the neurobehavioral, molecular, and enzymatic effects of organophosphates on embryo-larval zebrafish (Danio rerio).

Organophosphates insecticides (OPs) are common surface water contaminants in both urban and agricultural landscapes. Neurobehavioral effects on larval fish are known to occur at concentrations higher than those reported in the environment. The aim of this study was to perform a comparative analysis of neurobehavioral, molecular, and biochemical responses of four OPs (diazinon, dichlorvos, malathion, methyl-parathion) via the following endpoint measurements: distance traveled, velocity, gene expression (AChE, c-Fos, LINGO-1B, GRIN-1B), enzymatic acetylcholinesterase (AChE) activity, and carboxylesterase (CES) activity. OP exposures (5 hpf - 120 dpf) on embryo-larval zebrafish (Danio rerio) were assessed using a larval zebrafish behavior assay at concentrations: 0.01, 0.1, 10, and 100 µg/L. Individual OPs had varying degrees of neurotoxicity. Significant hypoactivity was observed in the 100 µg/L treatments for diazinon and malathion (p < 0.05) as compared to the controls. Diazinon-exposed larvae exhibited a 26% locomotor decrease, and hypoactivity was observed in malathion-exposed larvae at a reduction of 22% and 29% for distance traveled and velocity, respectively. Gene regulation and enzymatic activity changes were measured for both 0.1 and 100 µg/L exposures across OP treatments. Increased CES activity was observed for the 0.1 µg/L treatments of diazinon and methyl-parathion as well as the 100 µg/L treatment of dichlorvos; meanwhile, decreased CES activity was observed for 100 µg/L treatments of diazinon and malathion. Relative enzymatic activity of AChE was inhibited as compared to the control for the 0.1 µg/L diazinon. No other treatment group exhibited a significant effect on biochemical AChE activity; however, AChE upregulation was observed in the 0.1 µg/L exposure for diazinon, dichlorvos, and malathion. Methyl-parathion was observed to downregulate c-Fos at 0.1 µg/L exposure. Malathion upregulated LINGO-1B at 100 µg/L, a gene associated with neuronal regeneration; meanwhile, downregulation of LINGO-1B was observed for 0.1 µg/L exposure of methyl-parathion. Additional downregulation was observed for GRIN-1B in the 100 µg/L diazinon, 100 µg/L dichlorvos, and 0.1 µg/L methyl-parathion treatments. Exposure of ZF embryos to independent concentrations of 100 µg/L concentrations of diazinon and malathion resulted in hypoactivity and decreased CES activity at 5 dfp. No changes in swimming behavior were observed for either the 0.1 µg/L or 100 µg/L dichlorvos or methyl-parathion treatments. Observations from this study indicate that AChE inhibition may not be the most sensitive biomarker of OP pesticide exposure in zebrafish. Rather, the enzyme CES demonstrated higher sensitivity as a biomarker of OP toxicity.

Behavioral, molecular and physiological responses of embryo-larval zebrafish exposed to types I and II pyrethroids.

Pyrethroids are potent neurotoxicants that may elicit multiple pathways of toxicity in non-target organisms. Comparative studies on the mechanistic and developmental effects of types I and II pyrethroids against non-target aquatic species are limited. This study assessed the effects of the two pyrethroid types against embryo-larval zebrafish (Danio rerio) at environmentally relevant and laboratory concentrations. Zebrafish embryos were exposed to type-I (permethrin, bifenthrin) and type-II (deltamethrin, λ-cyhalothrin, fenvalerate, esfenvalerate) pyrethroids at 1000, 10, 0.1, 0.01, 0.0 µg/L, starting at 5-h post-fertilization (hpf) through 5-d post-fertilization (dpf) under static exposure conditions. Swimming behavior (distance traveled and velocity) was assessed at 5-dpf. The relative expression of Nrf2a, GST, Casp-9 and p53 mRNA transcripts, carboxyl esterase (CES) activity and total reactive oxygen species (ROS) were measured. The stability of the pyrethroids across 5 days was analyzed. Bifenthrin-(10 µg/L) and esfenvalerate-(1000 µg/L) significantly (p < 0.05) reduced total distance traveled by larvae while 1000 µg/L deltamethrin and λ-cyhalothrin were lethal causing body axis curvature and pericardial edema. At environmentally relevant concentrations-(µg/L) compared to control, permethrin-(0.122) upregulated Nrf2a and Casp-9 expressions while λ-cyhalothrin-(0.053) downregulated Nrf2a and fenvalerate-0.037 downregulated GST. At laboratory concentrations-(µg/L), permethrin-(1000) upregulated Nrf2a, Casp-9 and p53 expressions, bifenthrin-(10) upregulated Casp-9 while fenvalerate-(0.1) and esfenvalerate-(1000) downregulated GST. There was concentration dependent increase in CES activity which correlated positively with total ROS. Pyrethroid concentrations decreased significantly by day 5. This study showed disparity in the mechanistic effects across the pyrethroids types and their instability in aqueous media may underestimate toxicity against non-target aquatic species when exposed in their natural environment.

Place-based screening of mixtures of dominant emerging contaminants measured in Lake Michigan using zebrafish embryo gene expression assay.

Determining impacts of emerging contaminants is difficult due to the different concentrations of mixtures of these chemicals over a landscape. Assessment approaches need to account for absorption, distribution, metabolism and excretion of the chemicals in an organism, and potential crosstalk between molecular pathways. The goal of this study was to assess the utility of employing a modified zebrafish embryo toxicity (ZFET) assay that assesses morphological alterations and measurements of estrogen-associated mRNA transcripts, to exposure of a mixtures of chemicals at concentrations measured in several locations in Lake Michigan. The 5 pharmaceuticals in this study were carbamazepine, diltiazem, fluoxetine, gemfibrozil and metformin. Exposures consisted of 4 concentrations of each individual chemical, mixture concentrations measured at seven locations in Lake Michigan, or 17ß-estradiol. The relative expression of Estrogen Receptor-alpha, brain aromatase (CYP19A2), and gonadotropin releasing hormone 3 mRNA were measured at the end the 6-d exposure to determine estrogenicity of the individual chemical or mixture. In this study, there was significant induction of CYP19A2 in individual exposures of diltiazem, fluoxetine, gemfibrozil and metformin at concentrations measured in Lake Michigan. Exposure to 5 of the 7 chemical mixtures altered the expression of one of the three biomarkers. Transcripts varied across mixtures, indicating that biological screening of whole water samples for potential estrogenicity may need to include alternative molecular pathways other than just steroid receptor binding. This research demonstrates that pairing chemical measurements with a modified ZFET assay, twhich incorporates molecular biomarkers and morphological endpoints, could provide location and mixture specific toxic profiling.

Trophic Transfer and Accumulation of Multiwalled Carbon Nanotubes in the Presence of Copper Ions in Daphnia magna and Fathead Minnow (Pimephales promelas).

The increase in use of nanomaterials such as multiwalled carbon nanotubes (MWCNTs) presents a need to study their interactions with the environment. Trophic transfer was measured between Daphnia magna and Pimephales promelas (fathead minnow, FHM) exposed to MWCNTs with different outer diameter (OD) sizes (MWCNT1 = 8-15 nm OD and MWCNT2 = 20-30 nm OD) in the presence and absence of copper. Pristine FHM were fed D. magna, previously exposed for 3 d to MWCNT1 or MWCNT2 (0.1 mg/L) and copper (0.01 mg/L), for 7 d. D. magna bioaccumulated less MWCNT1 (0.02 µg/g) than MWCNT2 (0.06 µg/g), whereas FHM accumulated more MWCNT1 (0.81 µg/g) than MWCNT2 (0.04 µg/g). In the presence of copper, MWCNT bioaccumulation showed an opposite trend. Mostly MWCNT1 (0.03 µg/g) bioaccumulated in D. magna, however less MWCNT1 (0.21 µg/g) than MWCNT2 (0.32 µg/g) bioaccumulated in FHM. Bioaccumulation factors were higher for MWCNT1s than MWCNT2. However, an opposite trend was observed when copper was added. Plasma metallothionein-2 was measured among treatments; however concentrations were not statistically different from the control. This study demonstrates that trophic transfer of MWCNTs is possible in the aquatic environment and further exploration with mixtures can strengthen the understanding of MWCNT environmental behavior.

Effects of alkylphenols on the biotransformation of diuron and enzymes involved in the synthesis and clearance of sex steroids in juvenile male tilapia (Oreochromus mossambica).

Previous studies using in vivo bioassay guided fractionation indicated that the herbicide diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and alkylphenol (AP)-containing surfactants were detected in fractions of extracts that induced the estrogenic biomarker, vitellogenin in fish exposed to surface water extracts from the United States. However, when the compounds were evaluated individually using in vivo estrogenic assays or in vitro estrogen receptor assays, estrogenic activity was not observed. Since APs have been shown to alter activity and content of cytochrome P450s (CYP) which convert diuron to potential estrogenic metabolites, the hepatic biotransformation of diuron was measured with and without a 7day pretreatment of p-Octylphenol (OP) and p-Nonylphenol (NP) at low (OP 13ng/L+NP 91ng/L), and high concentrations (OP 65ng/L+NP 455ng/L) in juvenile male Nile tilapia (Oreochromus niloticus). Pre-treatment with the OP/NP (AP) mixture caused elevated levels of NADPH-catalyzed formation of 3,4-dichlorophenyl-N-methylurea (DCPMU) but not 3,4-dichlorophenylurea (DCPU). Fish were also treated with nominal concentrations of low (40ng/L) and high (200ng/L) diuron and each of its three degradates/metabolites: DCPMU, DCPU and 3,4-dichloroaniline (DCA). Additional treatments were conducted with APs and Diuron as a mixture at the low concentrations which mimicked concentrations observed in surface waters. Hepatic vitellogenin (Vtg) mRNA was induced by exposure to the high concentrations of Diuron, as well as DCPMU and DCPU in both concentrations. Brain cytochrome P450 aromatase activity was generally diminished by diuron, its metabolites, and the AP/diuron mixtures. 17ß-Hydroxysteroid dehydrogenase (17ßHSD) levels were also reduced by DCPMU and DCA in the lower concentrations, but not by higher concentrations. While the AP mixture reduced 17ßHSD, the AP/diuron mixture induced testosterone (T) biosynthesis at the single concentration tested. Although CYP3A expression was induced by all diuron metabolites, it was unchanged by the AP mixture. These data indicate that mixtures of AP and diuron enhanced the formation of the metabolite (DCPMU) which induced vitellogenin, and reduced T biosynthetic enzymes (17ßHSD inhibition). Overall, these data showed that APs may have induced the biotransformation of diuron to at least one metabolite, that may disrupt androgen biosynthesis and potentially alter steroid feedback pathways in the central nervous system.

Trophic transfer and effects of DDT in male hornyhead turbot (Pleuronichthys verticalis) from Palos Verdes Superfund site, CA (USA) and comparisons to field monitoring.

High concentrations of DDT and metabolites (ΣDDT) have been detected in sediment and the demersal flatfish hornyhead turbot (Pleuronichtys verticalis) collected from Palos Verdes (PV), California, USA, a site contaminated with over 100 metric tons of DDT throughout 1960s-70s. This study was conducted to assess the transfer of ΣDDT from PV-sediment into polychaetes (Neanthes arenaceodentata) and hornyhead turbot, and to investigate if the responses in turbots from two different laboratory exposures mimic those in turbots caught in PV (PV-turbot). Turbot fed PV-sediment-contaminated polychaete for 7 days had liver concentrations of ΣDDT similar to PV-turbot. After 28 days, ΣDDT also accumulated in livers of turbot gavaged with a ΣDDT mixture. In vitro cell bioassays indicated significant increases of 17ß-estradiol equivalents (EEQ) in turbot bile extracts as compared to the control in the 7-day study. These responses corresponded to those measured in PV-fish. Glucocorticoid receptor (GR), anti-androgen receptor (anti-AR), estrogen receptor (ER) or aryl hydrocarbon receptor (AhR) activities were also observed in extracts of PV-sediment, and PV-sediment-exposed worm. Anti-AR, AhR and GR activities were significantly higher in PV-sediment than reference sediment (San Diego, SD). Higher transcripts of hepatic VTG, ERα and ERß were found in PV-turbot than SD-turbot, but were unaltered in fish exposed to sediment-contaminated worms for the 7-day study. In contrast, liver extracts from the 28-day treatment of ΣDDT showed lower EEQ but similar hepatic VTG and ERß transcripts relative to those of PV-turbot. These data indicated that trophic transfer of sediment-associated DDT in 7-day exposures corresponded to field measurements of DDT residues and in vitro ER bioactivities, but failed to mimic in vivo biological effects observed in field fish. In contrast, treatment with ΣDDT alone for 28 days mimicked in vivo biological effects of DDTs in PV fish, but did not correspond to liver concentrations or in vitro bioactivities.

Developmental alterations and endocrine-disruptive responses in farmed Nile crocodiles (Crocodylus niloticus) exposed to contaminants from the Crocodile River, South Africa.

In the present study, the developmental (including fertility) and endocrine-disruptive effects in relation to chemical burden in male and female Nile crocodiles (Crocodylus niloticus), from a commercial crocodile farm in the Brits district, South Africa, exposed to various anthropogenic aquatic contaminants from the natural environment was investigated. Hepatic transcript levels for vitellogenin (Vtg), zona pellucida (ZP) and ERα (also in gonads) were analyzed using real-time PCR. Plasma estradiol-17ß (E2), testosterone (T) and 11-ketotestosterone (11-KT) were analyzed using enzyme immunoassay. Gonadal aromatase and hepatic testosterone metabolism (6ß-hydroxylase (6ß-OHase)) were analyzed using biochemical methods. Overall, there is high and abnormal number (%) of infertile and banded eggs during the studied reproductive seasons, showing up to 57 and 34% of infertile eggs in the 2009/2010 and 2013/2014 seasons, respectively. In addition, the percentage of banded eggs ranged between 10 and 19% during the period of 2009-2014 seasons. While hepatic ERα, Vtg, ZP mRNA and testosterone 6ß-OHase, were equally expressed in female and male crocodiles, gonadal ERα mRNA and aromatase activity were significantly higher in females compared to male crocodiles. On the other hand, plasma T and 11-KT levels were significantly higher in males, compared to female crocodiles. Principal component analysis (PCA) produced significant grouping that revealed correlative relationships between reproductive/endocrine-disruptive variables and liver contaminant burden, that further relates to measured contaminants in the natural environment. The overall results suggest that these captive pre-slaughter farm crocodiles exhibited responses to anthropogenic aquatic contaminants with potentially relevant consequences on key reproductive and endocrine pathways and these responses may be established as relevant species endocrine disruptor biomarkers of exposure and effects in this threatened species.

Age-dependent effects in fathead minnows from the anti-diabetic drug metformin.

The anti-diabetic drug metformin is thought to be the pharmaceutical most deposited into the aquatic environment by mass at up to 6tons per year from individual WWTPs in urban areas. Recent studies have shown that exposure to 40ug/L of metformin increased the relative expression of the egg yolk precursor protein vitellogenin in adult male fathead minnows (Pimephales promelas) (FHM). For this study, the expression of several other genes involved in estrogen biosynthesis, clearance and downstream effects were assessed in FHM after treatment to three concentrations of metformin, to better understand the estrogenic effects of metformin on FHM. In contrast to the previous study, although upward trends were observed, metformin failed to significantly alter the expression of VTG, ERα, GnRH3, and CYP3A126 in adult male FHM. However, a concentration-dependent response to metformin was observed in younger 80-90day juvenile FHM. A 17.7-, 22-, and 22-fold increase in the relative expression of VTG mRNA in juvenile FHM exposed to 1, 10, and 100µg/L as compared to the control was observed. There was also a 3.3-, 4.7-, and 5.5-fold increase in GnRH3 in juvenile FHM exposed to 1, 10, and 100µg/L as compared to the control. Similarly, a 14-, 16-, and 24-fold increase in the relative expression of CYP3A126 mRNA was measured in juvenile FHM exposed to 1, 10 and 100µg/L metformin as compared to the control. These results indicate that juvenile FHM were more susceptible to the estrogenic effects of metformin during a 7-d exposure than older, sexually mature male FHM.

Interlaboratory comparison of in vitro bioassays for screening of endocrine active chemicals in recycled water.

In vitro bioassays have shown promise as water quality monitoring tools. In this study, four commercially available in vitro bioassays (GeneBLAzer(®) androgen receptor (AR), estrogen receptor-alpha (ER), glucocorticoid receptor (GR) and progesterone receptor (PR) assays) were adapted to screen for endocrine active chemicals in samples from two recycled water plants. The standardized protocols were used in an interlaboratory comparison exercise to evaluate the reproducibility of in vitro bioassay results. Key performance criteria were successfully achieved, including low background response, standardized calibration parameters and high intra-laboratory precision. Only two datasets were excluded due to poor calibration performance. Good interlaboratory reproducibility was observed for GR bioassay, with 16-26% variability among the laboratories. ER and PR bioactivity was measured near the bioassay limit of detection and showed more variability (21-54%), although interlaboratory agreement remained comparable to that of conventional analytical methods. AR bioassay showed no activity for any of the samples analyzed. Our results indicate that ER, GR and PR, were capable of screening for different water quality, i.e., the highest bioactivity was observed in the plant influent, which also contained the highest concentrations of endocrine active chemicals measured by LC-MS/MS. After advanced treatment (e.g., reverse osmosis), bioactivity and target chemical concentrations were both below limits of detection. Comparison of bioassay and chemical equivalent concentrations revealed that targeted chemicals accounted for ≤5% of bioassay activity, suggesting that detection limits by LC-MS/MS for some chemicals were insufficient and/or other bioactive compounds were present in these samples. Our study demonstrated that in vitro bioassays responses were reproducible, and can provide information to complement conventional analytical methods for a more comprehensive water quality assessment.

The effect of bifenthrin on the dopaminergic pathway in juvenile rainbow trout (Oncorhynchus mykiss).

Bifenthrin is a type I pyrethroid pesticide, which has been shown to increase plasma estrogen concentrations in several fish models. The mechanism of action by which bifenthrin alters 17ß-estradiol (E2) is unclear. E2 biosynthesis is regulated through pituitary follicle stimulating hormone, which is directly controlled by hypothalamic gonadotropin releasing hormone (GnRH2). Since dopaminergic signaling significantly influences GnRH2 release in fish, the goal of the study was to determine the effect of a 96 h and 2 weeks exposure to bifenthrin on dopaminergic signaling in juvenile rainbow trout (Oncorhynchus mykiss) (RT). Our results indicated that a decrease in dopamine receptor 2A (DR2A) expression was associated with a trend toward an increase in plasma E2 following exposure at 96 h and 2 weeks, and a significant increase in the relative expression of vitellogenin mRNA at 2 weeks. DR2A mRNA expression decreased 426-fold at 96 h and 269-fold at 2 weeks in the brains of 1.5 ppb (3.55 pM) bifenthrin treated RT. There was an increase in tyrosine hydroxylase transcript levels at 96 h, which is indicative of dopamine production in the brains of the 1.5 ppb (3.55 pM) bifenthrin treated RT. A significant increase in the relative expression of GnRH2 was observed at 96 h but a significant decrease was noted after 2 weeks exposure indicating potential feedback loop activation. These results indicate that the estrogenic-effects of bifenthrin may result in part from changes in signaling within the dopaminergic pathway, but that other feedback pathways may also be involved.

Metformin exposure at environmentally relevant concentrations causes potential endocrine disruption in adult male fish.

Pharmaceuticals and personal care products (PPCPs) are emerging contaminants that have been found ubiquitously in wastewater and surface waters around the world. A major source of these compounds is incomplete metabolism in humans and subsequent excretion in human waste, resulting in discharge into surface waters by wastewater treatment plant (WWTP) effluent. One pharmaceutical found in particularly high abundance in recent WWTP effluent and surface water studies is metformin, one of the world's most widely prescribed antidiabetic drugs. Interactions between insulin signaling and steroidogenesis suggest potential endocrine-disrupting effects of metformin found in the aquatic environment. Adult fathead minnows (Pimephales promelas) were chronically exposed to metformin for 4 wk, at 40 µg/L, a level similar to the average found in WWTP effluent in Milwaukee, Wisconsin, USA. Genetic endpoints related to metabolism and endocrine function as well as reproduction-related endpoints were examined. Metformin treatment induced significant up-regulation of messenger ribonucleic acid (mRNA) encoding the egg-protein vitellogenin in male fish, an indication of endocrine disruption. The present study, the first to study the effects of environmentally relevant metformin exposure in fathead minnows, demonstrates the need for further study of the endocrine-disrupting effects of metformin in aquatic organisms.

Exploring the impacts of two separate mixtures of pesticide and surfactants on estrogenic activity in male fathead minnows and rainbow trout.

In this study, male fathead minnows (FHM) (Pimephales promelas) and juvenile rainbow trout (RT; Oncorhynchus mykiss) were exposed to two different surfactant mixtures of analytical-grade nonlyphenol, 4-tert octyphenol, octylphenol ethoxylates, nonylphenol ethoxylates, and the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). After a 7-days exposure to environmentally relevant concentrations of these compounds, there was no difference in the relative mRNA expression of vitellogenin (VTG) in male juvenile RT exposed to individual compounds or the 2,4-D-surfactant mixture compared with the control. In male FHM, there was a significant increase in VTG mRNA expression in the high individual treatments of 2,4-D and the surfactants but not the 2,4-D-surfactant mixtures compared with the control. These results were compared with another study exposing male FHM to individual and a mixture of alkylphenols and alkylphenol ethoxylates in two different combinations with the herbicide diuron and the insecticide bifenthrin. There were no differences in the relative expression of VTG mRNA amongst individual exposures and the control. Interestingly, when the ethoxylate mixture was combined with diuron, there was a significant decrease in the relative mRNA expression of VTG compared with the control. However, when the ethoxylate mixture was combined with both diuron and bifenthrin, there was a significant increase in the relative mRNA expression of VTG in male compared with all other groups in the multichemical mixture. The results of this study highlight differences between species and measurements of VTG in assessing the risk of mixtures to aquatic organisms.

Use of isotope dilution method to predict bioavailability of organic pollutants in historically contaminated sediments.

Many cases of severe environmental contamination arise from historical episodes, where recalcitrant contaminants have resided in the environment for a prolonged time, leading to potentially decreased bioavailability. Use of bioavailable concentrations over bulk chemical levels improves risk assessment and may play a critical role in determining the need for remediation or assessing the effectiveness of risk mitigation operations. In this study, we applied the principle of isotope dilution to quantify bioaccessibility of legacy contaminants DDT and PCBs in marine sediments from a Superfund site. After addition of 13C or deuterated analogues to a sediment sample, the isotope dilution reached a steady state within 24 h of mixing. At the steady state, the accessible fraction (E) derived by the isotope dilution method (IDM) ranged from 0.28 to 0.89 and was substantially smaller than 1 for most compounds, indicating reduced availability of the extensively aged residues. A strong linear relationship (R2=0.86) was found between E and the sum of rapid (Fr) and slow (Fs) desorption fractions determined by sequential Tenax desorption. The IDM-derived accessible concentration (Ce) was further shown to correlate closely with tissue residue in the marine benthic polychaete Neanthes arenaceodentata exposed in the same sediments. As shown in this study, the IDM approach involves only a few simple steps and may be readily adopted in laboratories equipped with mass spectrometers. This novel method is expected to be especially useful for historically contaminated sediments or soils, for which contaminant bioavailability may have changed significantly due to aging and other sequestration processes.