Toxicity of Carbon Dioxide to Freshwater Fishes: Implications for Aquatic Invasive Species Management.

Carbon dioxide (CO2 ) has been approved by the US Environmental Protection Agency as a new aquatic pesticide to control invasive Asian carps and other aquatic nuisance species in the United States. However, limited CO2 toxicity data could make it challenging for resource managers to characterize the potential risk to nontarget species during CO2 applications. The present study quantified the toxicity of CO2 to 2 native riverine fishes, bluegill (Lepomis macrochirus) and fathead minnow (Pimephales promelas), using 12-h continuous flow-through CO2 exposure at 5, 15, and 25 °C water temperatures. Resulting survival indicated that bluegill (median lethal concentration [LC50] range 91-140 mg/L CO2 ) were more sensitive to CO2 than fathead minnow (LC50 range 235-306 mg/L CO2 ) across all water temperatures. Bluegill were also more sensitive to CO2 at 5 °C (LC50 91 mg/L CO2 , 95% CI 85-96 mg/L CO2 ) than at 25 °C (LC50 140 mg/L CO2 , 95% CI 135-146 mg/L CO2 ). Fathead minnow showed an opposite response and were less sensitive at 5 °C (LC50 306 mg/L CO2 , 95% CI 286-327 mg/L CO2 ) relative to 25 °C (LC50 235 mg/L CO2 , 95% CI 224-246 mg/L CO2 ). Our results show that CO2 toxicity can differ by species and water temperature. Data from the present study may inform decisions related to the use of CO2 as a control tool. Environ Toxicol Chem 2020;39:2247-2255. Published 2020. This article is a U.S. government work and is in the public domain in the USA.

Contaminants of emerging concern presence and adverse effects in fish: A case study in the Laurentian Great Lakes.

The Laurentian Great Lakes are a valuable natural resource that is affected by contaminants of emerging concern (CECs), including sex steroid hormones, personal care products, pharmaceuticals, industrial chemicals, and new generation pesticides. However, little is known about the fate and biological effects of CECs in tributaries to the Great Lakes. In the current study, 16 sites on three rivers in the Great Lakes basin (Fox, Cuyahoga, and Raquette Rivers) were assessed for CEC presence using polar organic chemical integrative samplers (POCIS) and grab water samplers. Biological activity was assessed through a combination of in vitro bioassays (focused on estrogenic activity) and in vivo assays with larval fathead minnows. In addition, resident sunfish, largemouth bass, and white suckers were assessed for changes in biological endpoints associated with CEC exposure. CECs were present in all water samples and POCIS extracts. A total of 111 and 97 chemicals were detected in at least one water sample and POCIS extract, respectively. Known estrogenic chemicals were detected in water samples at all 16 sites and in POCIS extracts at 13 sites. Most sites elicited estrogenic activity in bioassays. Ranking sites and rivers based on water chemistry, POCIS chemistry, or total in vitro estrogenicity produced comparable patterns with the Cuyahoga River ranking as most and the Raquette River as least affected by CECs. Changes in biological responses grouped according to physiological processes, and differed between species but not sex. The Fox and Cuyahoga Rivers often had significantly different patterns in biological response Our study supports the need for multiple lines of evidence and provides a framework to assess CEC presence and effects in fish in the Laurentian Great Lakes basin.

Contaminants of emerging concern in tributaries to the Laurentian Great Lakes: II. Biological consequences of exposure.

The Laurentian Great Lakes contain one fifth of the world's surface freshwater and have been impacted by human activity since the Industrial Revolution. In addition to legacy contaminants, nitrification and invasive species, this aquatic ecosystem is also the recipient of Contaminants of Emerging Concern (CECs) with poorly understood biological consequences. In the current study, we documented the presence, concentrations, and biological effects of CECs across 27 field sites in six Great Lakes tributaries by examining over 2250 resident and caged sunfish (Lepomis ssp.) for a variety of morphological and physiological endpoints and related these results to CEC occurrence. CEC were ubiquitous across studies sites and their presence and concentrations in water and sediment were highest in effluent dominated rivers and downstream of municipal wastewater treatment plant discharges. However, even putative upstream reference sites were not free of CEC presence and fish at these sites exhibited biological effects consistent with CEC exposure. Only the Fox River exhibited consistent adverse biological effects, including increased relative liver size, greater prominence of hepatocyte vacuoles and increased plasma glucose concentrations. Canonical Redundancy Analysis revealed consistent patterns of biological consequences of CEC exposure across all six tributaries. Increasing plasma glucose concentrations, likely as a result of pollutant-induced metabolic stress, were associated with increased relative liver size and greater prominence of hepatocyte vacuoles. These indicators of pollutant exposure were inversely correlated with indicators of reproductive potential including smaller gonad size and less mature gametes. The current study highlights the need for greater integration of chemical and biological studies and suggests that CECs in the Laurentian Great Lakes Basin may adversely affect the reproductive potential of exposed fish populations.

An integrated approach for identifying priority contaminant in the Great Lakes Basin - Investigations in the Lower Green Bay/Fox River and Milwaukee Estuary areas of concern.

Environmental assessment of complex mixtures typically requires integration of chemical and biological measurements. This study demonstrates the use of a combination of instrumental chemical analyses, effects-based monitoring, and bio-effects prediction approaches to help identify potential hazards and priority contaminants in two Great Lakes Areas of Concern (AOCs), the Lower Green Bay/Fox River located near Green Bay, WI, USA and the Milwaukee Estuary, located near Milwaukee, WI, USA. Fathead minnows were caged at four sites within each AOC (eight sites total). Following 4d of in situ exposure, tissues and biofluids were sampled and used for targeted biological effects analyses. Additionally, 4d composite water samples were collected concurrently at each caged fish site and analyzed for 132 analytes as well as evaluated for total estrogenic and androgenic activity using cell-based bioassays. Of the analytes examined, 75 were detected in composite samples from at least one site. Based on multiple analyses, one site in the East River and another site near a paper mill discharge in the Lower Green Bay/Fox River AOC, were prioritized due to their estrogenic and androgenic activity, respectively. The water samples from other sites generally did not exhibit significant estrogenic or androgenic activity, nor was there evidence for endocrine disruption in the fish exposed at these sites as indicated by the lack of alterations in ex vivo steroid production, circulating steroid concentrations, or vitellogenin mRNA expression in males. Induction of hepatic cyp1a mRNA expression was detected at several sites, suggesting the presence of chemicals that activate the aryl hydrocarbon receptor. To expand the scope beyond targeted investigation of endpoints selected a priori, several bio-effects prediction approaches were employed to identify other potentially disturbed biological pathways and related chemical constituents that may warrant future monitoring at these sites. For example, several chemicals such as diethylphthalate and naphthalene, and genes and related pathways, such as cholinergic receptor muscarinic 3 (CHRM3), estrogen receptor alpha1 (esr1), chemokine ligand 10 protein (CXCL10), tumor protein p53 (p53), and monoamine oxidase B (Maob), were identified as candidates for future assessments at these AOCs. Overall, this study demonstrates that a better prioritization of contaminants and associated hazards can be achieved through integrated evaluation of multiple lines of evidence. Such prioritization can guide more comprehensive follow-up risk assessment efforts.

Integrated assessment of runoff from livestock farming operations: Analytical chemistry, in vitro bioassays, and in vivo fish exposures.

Animal waste from livestock farming operations can contain varying levels of natural and synthetic androgens and/or estrogens, which can contaminate surrounding waterways. In the present study, surface stream water was collected from 6 basins containing livestock farming operations. Aqueous concentrations of 12 hormones were determined via chemical analyses. Relative androgenic and estrogenic activity was measured using in vitro cell assays (MDA-kb2 and T47D-Kbluc assays, respectively). In parallel, 48-h static-renewal in vivo exposures were conducted to examine potential endocrine-disrupting effects in fathead minnows. Mature fish were exposed to surface water dilutions (0%, 25%, 50%, and 100%) and 10-ng/L of 17α-ethynylestradiol or 50-ng/L of 17ß-trenbolone as positive controls. Hepatic expression of vitellogenin and estrogen receptor α mRNA, gonadal ex vivo testosterone and 17ß-estradiol production, and plasma vitellogenin concentrations were examined. Potentially estrogenic and androgenic steroids were detected at low nanogram per liter concentrations. In vitro estrogenic activity was detected in all samples, whereas androgenic activity was detected in only 1 sample. In vivo exposures to the surface water had no significant dose-dependent effect on any of the biological endpoints, with the exception of increased male testosterone production in 1 exposure. The present study, which combines analytical chemistry measurements, in vitro bioassays, and in vivo fish exposures, highlights the integrated value and future use of a combination of techniques to obtain a comprehensive characterization of an environmental chemical mixture.

Developing predictive approaches to characterize adaptive responses of the reproductive endocrine axis to aromatase inhibition: I. Data generation in a small fish model.

Adaptive or compensatory responses to chemical exposure can significantly influence in vivo concentration-duration-response relationships. This study provided data to support development of a computational dynamic model of the hypothalamic-pituitary-gonadal axis of a model vertebrate and its response to aromatase inhibitors as a class of endocrine active chemicals. Fathead minnows (Pimephales promelas) were either exposed to the aromatase inhibitor fadrozole (0.5 or 30 µg/l) continuously for 1, 8, 12, 16, 20, 24, or 28 days or exposed for 8 days and then held in control water (no fadrozole) for an additional 4, 8, 12, 16, or 20 days. The time course of effects on ovarian steroid production, circulating 17ß-estradiol (E2) and vitellogenin (VTG) concentrations, and expression of steroidogenesis-related genes in the ovary was measured. Exposure to 30 µg fadrozole/l significantly reduced plasma E2 and VTG concentrations after just 1 day and those effects persisted throughout 28 days of exposure. In contrast, ex vivo E2 production was similar to that of controls on day 8-28 of exposure, whereas transcripts coding for aromatase and follicle-stimulating hormone receptor were elevated, suggesting a compensatory response. Following cessation of fadrozole exposure, ex vivo E2 and plasma E2 concentrations exceeded and then recovered to control levels, but plasma VTG concentrations did not, even after 20 days of depuration. Collectively these data provide several new insights into the nature and time course of adaptive responses to an aromatase inhibitor that support development of a computational model (see companion article).

A time-course analysis of effects of the steroidogenesis inhibitor ketoconazole on components of the hypothalamic-pituitary-gonadal axis of fathead minnows.

The objective of this study was to evaluate temporal effects of the model steroidogenesis inhibitor ketoconazole (KTC) on aspects of reproductive endocrine function controlled by the hypothalamic-pituitary-gonadal (HPG) axis in the fathead minnow (Pimephales promelas). Ketoconazole inhibits the activity of two cytochrome P450s (CYPs) key to sex steroid production in vertebrates, CYP11a (cholesterol side chain cleavage) and CYP17 (c17α-hydroxylase/17, 20-lyase). Sexually mature fish were exposed to water-borne KTC (30 or 300 µg/L) in a flow-through system for up to 8d, following which animals were allowed to recover in clean water. Fish were sampled after 1, 4 and 8d of exposure, and after 1, 8 and 16d of recovery. A shorter-term time-course experiment also was conducted in which females were sampled on seven occasions during a 12h KTC exposure. Ketoconazole consistently depressed ex vivo gonadal synthesis of testosterone (T) in both sexes, and 17ß-estradiol (E2) in females during both exposure and recovery phases of the time-course studies. Effects on ex vivo steroidogenesis in females occurred within as little as 1h of exposure. Plasma concentrations of T in males and E2 in females also were depressed by exposure to KTC, but these decreases did not persist to the same degree as observed for the ex vivo effects. In females, after decreases within 12h, plasma E2 concentrations were similar to (or greater than) controls at 24h of exposure, while in males, plasma T returned to levels comparable to controls within 1d of cessation of KTC exposure. The discrepancy between the ex vivo and in vivo data at later stages in the test is consistent with some type of compensatory response to KTC in fish. However, we were unable to ascertain the mechanistic basis for such a response. For example, although a number of genes related to steroid synthesis (e.g., cyp11a, cyp17) were up-regulated in the gonads of both males and females during the exposure and early recovery phases of the experiment, this did not seem to account for the resurgence in plasma steroid concentrations in KTC-exposed fish. Further studies focused on metabolism and clearance of steroids might lend insights as to the effects of KTC on plasma steroid concentrations. Overall, our results demonstrate the complex, temporally dynamic nature of the vertebrate HPG system in response to chemical stressors.

Ecotoxicogenomics to support ecological risk assessment: a case study with bisphenol A in fish.

Effects of bisphenol A (BPA) on ovarian transcript profiles as well as targeted end points with endocrine/reproductive relevance were examined in two fish species, fathead minnow (Pimephales promelas) and zebrafish (Danio rerio), exposed in parallel using matched experimental designs. Four days of waterborne exposure to 10 µg BPA/L caused significant vitellogenin induction in both species. However, zebrafish were less sensitive to effects on hepatic gene expression and steroid production than fathead minnow and the magnitude of vitellogenin induction was more modest (i.e., 3-fold compared to 13,000-fold in fathead minnow). The concentration-response at the ovarian transcriptome level was nonmonotonic and violated assumptions that underlie proposed methods for estimating hazard thresholds from transcriptomic results. However, the nonmonotonic profile was consistent among species and there were nominal similarities in the functions associated with the differentially expressed genes, suggesting potential activation of common pathway perturbation motifs in both species. Overall, the results provide an effective case study for considering the potential application of ecotoxicogenomics to ecological risk assessments and provide novel comparative data regarding effects of BPA in fish.

Effects of a glucocorticoid receptor agonist, dexamethasone, on fathead minnow reproduction, growth, and development.

Synthetic glucocorticoids are pharmaceutical compounds prescribed in human and veterinary medicine as anti-inflammatory agents and have the potential to contaminate natural watersheds via inputs from wastewater treatment facilities and confined animal-feeding operations. Despite this, few studies have examined the effects of this class of chemicals on aquatic vertebrates. To generate data to assess potential risk to the aquatic environment, we used fathead minnow 21-d reproduction and 29-d embryo-larvae assays to determine reproductive toxicity and early-life-stage effects of dexamethasone. Exposure to 500 µg dexamethasone/L in the 21-d test caused reductions in fathead minnow fecundity and female plasma estradiol concentrations and increased the occurrence of abnormally hatched fry. Female fish exposed to 500 µg dexamethasone/L also displayed a significant increase in plasma vitellogenin protein levels, possibly because of decreased spawning. A decrease in vitellogenin messenger ribonucleic acid (mRNA) expression in liver tissue from females exposed to the high dexamethasone concentration lends support to this hypothesis. Histological results indicate that a 29-d embryo-larval exposure to 500 µg dexamethasone/L caused a significant increase in deformed gill opercula. Fry exposed to 500 µg dexamethasone/L for 29 d also exhibited a significant reduction in weight and length compared with control fry. Taken together, these results indicate that nonlethal concentrations of a model glucocorticoid receptor agonist can impair fish reproduction, growth, and development.