Alan M. Vajda

Antidepressant Pharmaceuticals in Two U.S. Effluent-Impacted Streams: Occurrence and Fate in Water and Sediment, and Selective Uptake in Fish Neural Tissue

Antidepressant pharmaceuticals are widely prescribed in the United States; release of municipal wastewater effluent is a primary route introducing them to aquatic environments, where little is known about their distribution and fate. Water, bed sediment, and brain tissue from native white suckers (Catostomus commersoni) were collected upstream and at points progressively downstream from outfalls discharging to two effluent-impacted streams, Boulder Creek (Colorado) and Fourmile Creek (Iowa). A liquid chromatography/tandem mass spectrometry method was used to quantify antidepressants, including fluoxetine, norfluoxetine (degradate), sertraline, norsertraline (degradate), paroxetine, citalopram, fluvoxamine, duloxetine, venlafaxine, and bupropion in all three sample matrices. Antidepressants were not present above the limit of quantitation in water samples upstream from the effluent outfalls but were present at points downstream at ng/L concentrations, even at the farthest downstream sampling site 8.4 km downstream from the outfall. The antidepressants with the highest measured concentrations in both streams were venlafaxine, bupropion, and citalopram and typically were observed at concentrations of at least an order of magnitude greater than the more commonly investigated antidepressants fluoxetine and sertraline. Concentrations of antidepressants in bed sediment were measured at ng/g levels; venlafaxine and fluoxetine were the predominant chemicals observed. Fluoxetine, sertraline, and their degradates were the principal antidepressants observed in fish brain tissue, typically at low ng/g concentrations. A qualitatively different antidepressant profile was observed in brain tissue compared to streamwater samples. This study documents that wastewater effluent can be a point source of antidepressants to stream ecosystems and that the qualitative composition of antidepressants in brain tissue from exposed fish differs substantially from the compositions observed in streamwater and sediment, suggesting selective uptake.

Antidepressants at environmentally relevant concentrations affect predator avoidance behavior of larval fathead minnows (Pimephales promelas).

The effects of embryonic and larval exposure to environmentally relevant (ng/L) concentrations of common antidepressants, fluoxetine, sertraline, venlafaxine, and bupropion (singularly and in mixture) on C-start escape behavior were evaluated in fathead minnows (Pimephales promelas). Embryos (postfertilization until hatching) were exposed for 5 d and, after hatching, were allowed to grow in control well water until 12 d old. Similarly, posthatch fathead minnows were exposed for 12 d to these compounds. High-speed (1,000 frames/s) video recordings of escape behavior were collected and transferred to National Institutes of Health Image for frame-by-frame analysis of latency periods, escape velocities, and total escape response (combination of latency period and escape velocity). When tested 12 d posthatch, fluoxetine and venlafaxine adversely affected C-start performance of larvae exposed as embryos. Conversely, larvae exposed for 12 d posthatch did not exhibit altered escape responses when exposed to fluoxetine but were affected by venlafaxine and bupropion exposure. Mixtures of these four antidepressant pharmaceuticals slowed predator avoidance behaviors in larval fathead minnows regardless of the exposure window. The direct impact of reduced C-start performance on survival and, ultimately, reproductive fitness provides an avenue to assess the ecological relevance of exposure in an assay of relatively short duration.

Demasculinization of male fish by wastewater treatment plant effluent

Adult male fathead minnows (Pimephales promelas) were exposed to effluent from the City of Boulder, Colorado wastewater treatment plant (WWTP) under controlled conditions in the field to determine if the effluent induced reproductive disruption in fish. Gonadal intersex and other evidence of reproductive disruption were previously identified in white suckers (Catostomus commersoni) in Boulder Creek downstream from this WWTP effluent outfall. Fish were exposed within a mobile flow-through exposure laboratory in July 2005 and August 2006 to WWTP effluent (EFF), Boulder Creek water (REF), or mixtures of EFF and REF for up to 28 days. Primary (sperm abundance) and secondary (nuptial tubercles and dorsal fat pads) sex characteristics were demasculinized within 14 days of exposure to 50% and 100% EFF. Vitellogenin was maximally elevated in both 50% and 100% EFF treatments within 7 days and significantly elevated by 25% EFF within 14 days. The steroidal estrogens 17β-estradiol, estrone, estriol, and 17α-ethynylestradiol, as well as estrogenic alkylphenols and bisphenol A were identified within the EFF treatments and not in the REF treatment. These results support the hypothesis that the reproductive disruption observed in this watershed is due to endocrine-active chemicals in the WWTP effluent.

Predator avoidance performance of larval fathead minnows (Pimephales promelas) following short-term exposure to estrogen mixtures

Aquatic organisms exposed to endocrine disrupting compounds (EDCs) at early life-stages may have reduced reproductive fitness via disruption of reproductive and non-reproductive behavioral and physiological pathways. Survival to reproductive age relies upon optimal non-reproductive trait expression, such as adequate predator avoidance responses, which may be impacted through EDC exposure. During a predator-prey confrontation, larval fish use an innate C-start escape behavior to rapidly move away from an approaching threat. We tested the hypotheses that (1) larval fathead minnows exposed to estrogens, a primary class of EDCs, singularly or in mixture, suffer a reduced ability to perform an innate C-start behavior when faced with a threat stimulus; (2) additive effects will cause greater reductions in C-start behavior; and (3) effects will differ among developmental stages. In this study, embryos (post-fertilization until hatching) were exposed for 5 days to environmentally relevant concentrations of estrone (E1), 17beta-estradiol (E2), and 17alpha-ethinylestradiol (EE2) singularly and in mixture. Exposed embryos were allowed to hatch and grow in control well water until 12 days old. Similarly, post-hatch fathead minnows were exposed for 12 days to these compounds. High-speed (1000frames/s) video recordings of escape behavior were collected and transferred to National Institutes of Health Image for frame-by-frame analysis of latency period, escape velocity, and total escape response (combination of latency period and escape velocity). When tested 12 days post-hatch, only E1 adversely affected C-start performance of larvae exposed as embryos. Conversely, larvae exposed for 12 days post-hatch did not exhibit altered escape responses when exposed to E1, while adverse responses were seen in E2 and the estrogen mixture. Ethinylestradiol exposure did not elicit changes in escape behaviors at either developmental stage. The direct impact of reduced C-start performance on survival, and ultimately, reproductive fitness provides an avenue to assess the ecological relevance of exposure in an assay of relatively short duration.

Fish Endocrine Disruption Responses to a Major Wastewater Treatment Facility Upgrade

The urban-water cycle modifies natural stream hydrology, and domestic and commercial activities increase the burden of endocrine-disrupting chemicals, such as steroidal hormones and 4-nonylphenol, that can disrupt endocrine system function in aquatic organisms. This paper presents a series of integrated chemical and biological investigations into the occurrence, fate, and effects of endocrine-disrupting chemicals in the City of Boulder Colorados WWTF and Boulder Creek, the receiving stream. Results are presented showing the effects of a full-scale upgrade of the WWTF (that treats 0.6 m(3) s(-1) of sewage) from a trickling filter/solids contact process to an activated sludge process on the removal of endocrine-disrupting compounds and other contaminants (including nutrients, boron, bismuth, gadolinium, and ethylenediaminetetraacetic acid) through each major treatment unit. Corresponding impacts of pre- and postupgrade effluent chemistry on fish reproductive end points were evaluated using on-site, continuous-flow experiments, in which male fathead minnows (Pimephales promelas) were exposed for 28 days to upstream Boulder Creek water and WWTF effluent under controlled conditions. The upgrade of the WWTF resulted in improved removal efficiency for many endocrine-disrupting chemicals, particularly 17β-estradiol and estrone, and fish exposed to the postupgrade effluent indicated reduction in endocrine disruption relative to preupgrade conditions.

An environmental oestrogen disrupts fish population dynamics through direct and transgenerational effects on survival and fecundity

Summary 1. Increased need for water and projected declines in precipitation due to climate change could leave waterways increasingly dominated by wastewater effluent. Understanding how components of wastewater influence fish populations is necessary for effective conservation and management. Despite research demonstrating effects of oestrogens, such as 17a-ethynylestradiol (EE2), on fish physiology and population failure, the generality of population responses is uncertain and the underlying mechanisms affecting population declines are unknown. EE2 is the steroid oestrogen in human contraceptive pills and has been measured up to 11 ng L 1 in the environment. 2. We identify disrupted population dynamics due to direct and transgenerational effects on survival and fecundity. We conducted a year-long study on three generations of fathead minnows Pimephales promelas Rafinesque in aquatic mesocosms and laboratory aquaria. We added environmentally relevant concentrations of EE2 daily using a static renewal, which approximates a pulsed exposure that fish experience in natural systems. 3. EE2 (3 2n g L 1 ) reduced F0 male survival to 17% (48% lower than controls) and juvenile production by 40% compared to controls. F1 fish continuously exposed to EE2 failed to reproduce, and reproduction of the F1 transferred to clean water was 70–99% less than controls. 4. F2 larval survival, exposed only as germ cells in their parents, was reduced by 51–97% compared to controls. The indirect effect on F2 survival suggests the possibility of transgenerational effects of EE2. 5. Synthesis and applications. Our results suggest that fish populations exposed to environmentally relevant 17a-ethynylestradiol (EE2) concentrations may not recover from exposure. Management of short-lived highly fecund fishes should be prioritized to protect fish from the embryo through gonadal differentiation. Reducing effluent will not be possible in many situations; hence, conservation of breeding and rearing habitat in unpolluted tributaries or reaches is needed. Additionally, resource managers could enhance habitat connectivity in rivers to facilitate immigration. Finally, investment in advanced wastewater processing technology should improve removal of bioactive chemicals such as EE2. Our results provide a baseline for regulatory agencies to consider when assessing the ecological effects of environmental oestrogens, and our approach to evaluating population-level effects could be widely applied to other contaminants.

Perturbation and restoration of the fathead minnow gut microbiome after low-level triclosan exposure.

BackgroundTriclosan is a widely used antimicrobial compound and emerging environmental contaminant. Although the role of the gut microbiome in health and disease is increasingly well established, the interaction between environmental contaminants and host microbiome is largely unexplored, with unknown consequences for host health. This study examined the effects of low, environmentally relevant levels of triclosan exposure on the fish gut microbiome. Developing fathead minnows (Pimephales promelas) were exposed to two low levels of triclosan over a 7-day exposure. Fish gastrointestinal tracts from exposed and control fish were harvested at four time points: immediately preceding and following the 7-day exposure and after 1 and 2 weeks of depuration.ResultsA total of 103 fish gut bacterial communities were characterized by high-throughput sequencing and analysis of the V3-V4 region of the 16S rRNA gene. By measures of both alpha and beta diversity, gut microbial communities were significantly differentiated by exposure history immediately following triclosan exposure. After 2 weeks of depuration, these differences disappear. Independent of exposure history, communities were also significantly structured by time. This first detailed census of the fathead minnow gut microbiome shows a bacterial community that is similar in composition to those of zebrafish and other freshwater fish. Among the triclosan-resilient members of this host-associated community are taxa associated with denitrification in wastewater treatment, taxa potentially able to degrade triclosan, and taxa from an unstudied host-associated candidate division.ConclusionsThe fathead minnow gut microbiome is rapidly and significantly altered by exposure to low, environmentally relevant levels of triclosan, yet largely recovers from this short-term perturbation over an equivalently brief time span. These results suggest that even low-level environmental exposure to a common antimicrobial compound can induce significant short-term changes to the gut microbiome, followed by restoration, demonstrating both the sensitivity and resilience of the gut flora to challenges by environmental toxicants. This short-term disruption in a developing organism may have important long-term consequences for host health. The identification of multiple taxa not often reported in the fish gut suggests that microbial nitrogen metabolism in the fish gut may be more complex than previously appreciated.

Chronic Toxicity of Ammonia to Early Life Stage Rainbow Trout

Abstract A 90-d ammonia toxicity test for early life stage rainbow trout Oncorhynchus mykiss was conducted using newly fertilized eggs from a wild strain of fish. The toxicity test was conducted at a pH of 7.75 and temperature of 11.4°C. Hatch success and survival of sac fry were not affected by ammonia exposure. Survival, growth, and biomass of swim-up fry were significantly reduced at an ammonia-nitrogen (NH3-N) concentration of 16.8 mg NH3-N/L of water but were unaffected by exposures to 7.44 mg NH3-N/L or lower concentrations. The chronic toxicity value was 11.2 mg NH3-N/L, and the EC20 (concentration estimated to cause a 20% reduction in organism performance compared with the control) based on biomass at test termination was 7.72 mg NH3-N/L. Development of sac fry to the swim-up stage was retarded by ammonia, but fry exposed to 7.44 mg NH3-N/L or lower concentrations appeared to recover by the end of the test. Histological analysis of the gills of exposed fry did not detect any gill pathology. U.S. E...

Reproductive failure of the red shiner (Cyprinella lutrensis) after exposure to an exogenous estrogen

Endocrine disrupting chemicals (EDCs) have been detected in surface waters worldwide and can lead to devel- opmental and reproductive disruption in exposed fishes. In the US Great Plains, EDCs are impacting streams and rivers and may be causing adverse reproductive effects. To examine how estrogenic EDCs might affect reproductive success of plains fishes, we experimentally exposed male red shiners (Cyprinella lutrensis) to exogenous 17b-estradiol. We character- ized the effects of estradiol on male gonadal histology and secondary sexual characteristics, determined whether exposure reduced reproductive success, and examined the effects of depuration. Adults were exposed to a mean concentration of 70 ngL -1 estradiol, a solvent control, or a water control for at least 83 days. Male exposure to estradiol resulted in ele- vated plasma vitellogenin concentrations, changes in spermatogenesis, reduced mating coloration and tubercles, altered mating behaviors, and reduced reproductive success with no viable progeny produced. Reproductive endpoints improved upon depuration (28 days). Exposure to estradiol had significant adverse effects on red shiners, indicating that wild popula- tions may face developmental and reproductive difficulties if they are chronically exposed to estradiol.

Integrated assessment of wastewater treatment plant effluent estrogenicity in the Upper Murray River, Australia, using the native Murray rainbowfish (Melanotaenia fluviatilis)

The contamination of major continental river systems by endocrine-active chemicals (EACs) derived from the discharge of wastewater treatment plant (WWTP) effluents can affect human and ecosystem health. As part of a long-term effort to develop a native fish model organism for assessment of endocrine disruption in Australias largest watershed, the Murray-Darling River Basin, the present study evaluated endocrine disruption in adult males of the native Australian Murray rainbowfish (Melanotaenia fluviatilis) exposed to effluent from an activated sludge WWTP and water from the Murray River during a 28-d, continuous-flow, on-site experiment. Analysis of the WWTP effluent and river water detected estrone and 17β-estradiol at concentrations up to approximately 25 ng L(-1) . Anti-estrogenicity of effluent samples was detected in vitro using yeast-based bioassays (yeast estrogen screen) throughout the experiment, but estrogenicity was limited to the first week of the experiment. Histological evaluation of the testes indicated significant suppression of spermatogenesis by WWTP effluent after 28 d of exposure. Plasma vitellogenin concentrations and expression of vitellogenin messenger RNA in liver were not significantly affected by exposure to WWTP effluent. The combination of low contaminant concentrations in the WWTP effluent, limited endocrine disrupting effects in the Murray rainbowfish, and high in-stream dilution factors (>99%) suggest minimal endocrine disruption impacts on native Australian fish in the Murray River downstream from the WWTP outfall.