James M. Lazorchak

Collapse of a fish population after exposure to a synthetic estrogen

Municipal wastewaters are a complex mixture containing estrogens and estrogen mimics that are known to affect the reproductive health of wild fishes. Male fishes downstream of some wastewater outfalls produce vitellogenin (VTG) (a protein normally synthesized by females during oocyte maturation) and early-stage eggs in their testes, and this feminization has been attributed to the presence of estrogenic substances such as natural estrogens [estrone or 17β-estradiol (E2)], the synthetic estrogen used in birth-control pills [17α-ethynylestradiol (EE2)], or weaker estrogen mimics such as nonylphenol in the water. Despite widespread evidence that male fishes are being feminized, it is not known whether these low-level, chronic exposures adversely impact the sustainability of wild populations. We conducted a 7-year, whole-lake experiment at the Experimental Lakes Area (ELA) in northwestern Ontario, Canada, and showed that chronic exposure of fathead minnow (Pimephales promelas) to low concentrations (5–6 ng·L−1) of the potent 17α-ethynylestradiol led to feminization of males through the production of vitellogenin mRNA and protein, impacts on gonadal development as evidenced by intersex in males and altered oogenesis in females, and, ultimately, a near extinction of this species from the lake. Our observations demonstrate that the concentrations of estrogens and their mimics observed in freshwaters can impact the sustainability of wild fish populations.

Pharmaceuticals and Personal Care Products in the Environment: What Are the Big Questions?

Background: Over the past 10–15 years, a substantial amount of work has been done by the scientific, regulatory, and business communities to elucidate the effects and risks of pharmaceuticals and personal care products (PPCPs) in the environment. Objective: This review was undertaken to identify key outstanding issues regarding the effects of PPCPs on human and ecological health in order to ensure that future resources will be focused on the most important areas. Data sources: To better understand and manage the risks of PPCPs in the environment, we used the “key question” approach to identify the principle issues that need to be addressed. Initially, questions were solicited from academic, government, and business communities around the world. A list of 101 questions was then discussed at an international expert workshop, and a top-20 list was developed. Following the workshop, workshop attendees ranked the 20 questions by importance. Data synthesis: The top 20 priority questions fell into seven categories: a) prioritization of substances for assessment, b) pathways of exposure, c) bioavailability and uptake, d) effects characterization, e) risk and relative risk, f ) antibiotic resistance, and g) risk management. Conclusions: A large body of information is now available on PPCPs in the environment. This exercise prioritized the most critical questions to aid in development of future research programs on the topic.

Management options for reducing the release of antibiotics and antibiotic resistance genes to the environment

Background: There is growing concern worldwide about the role of polluted soil and water environments in the development and dissemination of antibiotic resistance. Objective: Our aim in this study was to identify management options for reducing the spread of antibiotics and antibiotic-resistance determinants via environmental pathways, with the ultimate goal of extending the useful life span of antibiotics. We also examined incentives and disincentives for action. Methods: We focused on management options with respect to limiting agricultural sources; treatment of domestic, hospital, and industrial wastewater; and aquaculture. Discussion: We identified several options, such as nutrient management, runoff control, and infrastructure upgrades. Where appropriate, a cross-section of examples from various regions of the world is provided. The importance of monitoring and validating effectiveness of management strategies is also highlighted. Finally, we describe a case study in Sweden that illustrates the critical role of communication to engage stakeholders and promote action. Conclusions: Environmental releases of antibiotics and antibiotic-resistant bacteria can in many cases be reduced at little or no cost. Some management options are synergistic with existing policies and goals. The anticipated benefit is an extended useful life span for current and future antibiotics. Although risk reductions are often difficult to quantify, the severity of accelerating worldwide morbidity and mortality rates associated with antibiotic resistance strongly indicate the need for action.

Endocrine disrupting chemicals in fish: Developing exposure indicators and predictive models of effects based on mechanism of action

Knowledge of possible toxic mechanisms (or modes) of action (MOA) of chemicals can provide valuable insights as to appropriate methods for assessing exposure and effects, thereby reducing uncertainties related to extrapolation across species, endpoints and chemical structure. However, MOA-based testing seldom has been used for assessing the ecological risk of chemicals. This is in part because past regulatory mandates have focused more on adverse effects of chemicals (reductions in survival, growth or reproduction) than the pathways through which these effects are elicited. A recent departure from this involves endocrine-disrupting chemicals (EDCs), where there is a need to understand both MOA and adverse outcomes. To achieve this understanding, advances in predictive approaches are required whereby mechanistic changes caused by chemicals at the molecular level can be translated into apical responses meaningful to ecological risk assessment. In this paper we provide an overview and illustrative results from a large, integrated project that assesses the effects of EDCs on two small fish models, the fathead minnow (Pimephales promelas) and zebrafish (Danio rerio). For this work a systems-based approach is being used to delineate toxicity pathways for 12 model EDCs with different known or hypothesized toxic MOA. The studies employ a combination of state-of-the-art genomic (transcriptomic, proteomic, metabolomic), bioinformatic and modeling approaches, in conjunction with whole animal testing, to develop response linkages across biological levels of organization. This understanding forms the basis for predictive approaches for species, endpoint and chemical extrapolation. Although our project is focused specifically on EDCs in fish, we believe that the basic conceptual approach has utility for systematically assessing exposure and effects of chemicals with other MOA across a variety of biological systems.

Analysis of Ecologically Relevant Pharmaceuticals in Wastewater and Surface Water Using Selective Solid-Phase Extraction and UPLC−MS/MS

A rapid and sensitive method has been developed for the analysis of 48 human prescription active pharmaceutical ingredients (APIs) and 6 metabolites of interest, utilizing selective solid-phase extraction (SPE) and ultraperformance liquid chromatography in combination with triple quadrupole mass spectrometry (UPLC-MS/MS). The single-cartridge extraction step was developed using a mixed mode reversed-phase/cation-exchange cartridge (Oasis MCX) and validated in both wastewater effluent and surface water. Recoveries for the majority of compounds ranged from 80% to 125%, with relative standard deviations generally below 15%. Analytes were quantified using a multiple injection analysis with four chromatographic runs, with a combined run time of 48 min and SPE-UPLC-MS/MS method detection limits ranging from 1.0 to 51 ng/L. The analysis of seven wastewater effluents and one surface water sample revealed at least one detection for 38 of the 54 compounds, with effluent concentrations ranging from 7 to 2950 ng/L and surface water concentrations ranging from 10 to 140 ng/L. This initial data demonstrates that a significant number of the selected target analytes are present in wastewater treatment plant discharges.

Differential Gene Expression in Daphnia magna Suggests Distinct Modes of Action and Bioavailability for ZnO Nanoparticles and Zn Ions

Zinc oxide nanoparticles (ZnO NPs) are being rapidly developed for use in consumer products, wastewater treatment, and chemotherapy providing several possible routes for ZnO NP exposure to humans and aquatic organisms. Recent studies have shown that ZnO NPs undergo rapid dissolution to Zn(2+), but the relative contribution of Zn(2+) to ZnO NP bioavailability and toxicity is not clear. We show that a fraction of the ZnO NPs in suspension dissolves, and this fraction cannot account for the toxicity of the ZnO NP suspensions to Daphnia magna. Gene expression profiling of D. magna exposed to ZnO NPs or ZnSO(4) at sublethal concentrations revealed distinct modes of toxicity. There was also little overlap in gene expression between ZnO NPs and SiO(x) NPs, suggesting specificity for the ZnO NP expression profile. ZnO NPs effected expression of genes involved in cytoskeletal transport, cellular respiration, and reproduction. A specific pattern of differential expression of three biomarker genes including a multicystatin, ferritin, and C1q containing gene were confirmed for ZnO NP exposure and provide a suite of biomarkers for identifying environmental exposure to ZnO NPs and differentiating between NP and ionic exposure.

Identification of metabolites of trenbolone acetate in androgenic runoff from a beef feedlot.

Little is known concerning the potential ecological effects of hormonally active substances associated with discharges from animal feeding operations. Trenbolone acetate is a synthetic anabolic steroid that is widely used in the United States to promote growth of beef cattle. Metabolites of trenbolone acetate include the stereoisomers 17α- and 17β-trenbolone, both of which are stable in animal wastes and are relatively potent androgens in fish and mammals. Our purpose in this study was to evaluate the occurrence of 17α- and 17β-trenbolone in a beef cattle feedlot discharge and in river water upstream and downstream from the discharge. In conjunction with that effort, we measured in vitro androgenic activity of the discharge using CV-1 cells that had been transiently cotransfected with human androgen receptor and reporter gene constructs. Samples were collected on nine different occasions during 2002 and 2003. Whole-water samples from the discharge caused a significant androgenic response in the CV-1 cells and contained detectable concentrations of 17α- and 17β-trenbolone. Further work is needed to ascertain the degree to which synthetic androgens such as trenbolone contribute to androgenic activity of feedlot discharges.

Toxicogenomic responses of nanotoxicity in Daphnia magna exposed to silver nitrate and coated silver nanoparticles.

Applications for silver nanomaterials in consumer products are rapidly expanding, creating an urgent need for toxicological examination of the exposure potential and ecological effects of silver nanoparticles (AgNPs). The integration of genomic techniques into environmental toxicology has presented new avenues to develop exposure biomarkers and investigate the mode of toxicity of novel chemicals. In the present study we used a 15k oligonucleotide microarray for Daphnia magna, a freshwater crustacean and common indicator species for toxicity, to differentiate between particle specific and ionic silver toxicity and to develop exposure biomarkers for citrate-coated and PVP-coated AgNPs. Gene expression profiles revealed that AgNO(3) and AgNPs have distinct expression profiles suggesting different modes of toxicity. Major biological processes disrupted by the AgNPs include protein metabolism and signal transduction. In contrast, AgNO(3) caused a downregulation of developmental processes, particularly in sensory development. Metal responsive and DNA damage repair genes were induced by the PVP AgNPs, but not the other treatments. In addition, two specific biomarkers were developed for the environmental detection of PVP AgNPs; although further verification under different environmental conditions is needed.

Effects from filtration, capping agents, and presence/absence of food on the toxicity of silver nanoparticles to Daphnia magna

Relatively little is known about the behavior and toxicity of nanoparticles in the environment. Objectives of work presented here include establishing the toxicity of a variety of silver nanoparticles (AgNPs) to Daphnia magna neonates, assessing the applicability of a commonly used bioassay for testing AgNPs, and determining the advantages and disadvantages of multiple characterization techniques for AgNPs in simple aquatic systems. Daphnia magna were exposed to a silver nitrate solution and AgNPs suspensions including commercially available AgNPs (uncoated and coated), and laboratory-synthesized AgNPs (coated with coffee or citrate). The nanoparticle suspensions were analyzed for silver concentration (microwave acid digestions), size (dynamic light scattering and electron microscopy), shape (electron microscopy), surface charge (zeta potentiometer), and chemical speciation (X-ray absorption spectroscopy, X-ray diffraction). Toxicities of filtered (100 nm) versus unfiltered suspensions were compared. Additionally, effects from addition of food were examined. Stock suspensions were prepared by adding AgNPs to moderately hard reconstituted water, which were then diluted and used straight or after filtration with 100-nm filters. All nanoparticle exposure suspensions, at every time interval, were digested via microwave digester and analyzed by inductively coupled argon plasma-optical emission spectroscopy or graphite furnace-atomic absorption spectroscopy. Dose-response curves were generated and median lethal concentration (LC50) values calculated. The LC50 values for the unfiltered particles were (in µg/L): 1.1 ± 0.1-AgNO(3) ; 1.0 ± 0.1-coffee coated; 1.1 ± 0.2-citrate coated; 16.7 ± 2.4 Sigma Aldrich Ag-nanoparticles (SA) uncoated; 31.5 ± 8.1 SA coated. LC50 values for the filtered particles were (in µg/L): 0.7 ± 0.1-AgNO(3) ; 1.4 ± 0.1-SA uncoated; 4.4 ± 1.4-SA coated. The LC50 resulting from the addition of food was 176.4 ± 25.5-SA coated. Recommendations presented in this study include AgNP handling methods, effects from sample preparation, and advantages/disadvantages of different nanoparticle characterization techniques.

Perfluorinated compounds in whole fish homogenates from the Ohio, Missouri, and Upper Mississippi Rivers, USA

A method for the analysis of 10 perfluorinated compounds (PFCs) in whole fish homogenate is presented and applied to 60 fish samples collected from the Ohio, Missouri, and upper Mississippi Rivers in 2005. Method accuracy ranged between 86 and 125% with limits of quantitation between 0.2 and 10 ng/g wet weight. Intra- and inter-batch precision was generally +/-20%. Perfluorooctane sulfonate (PFOS) was the predominant compound identified in these samples, contributing over 80% of total PFC composition in the fish from these rivers, with median PFOS concentrations of 24.4, 31.8, and 53.9 ng/g wet wt in the Missouri, Ohio, and Mississippi Rivers, respectively. Median PFOS levels were significantly (p=0.01) elevated in piscivorous fish (88.0 ng/g) when compared with non-piscivorous fish (15.9 ng/g). The 10 samples with PFOS concentrations above 200 ng/g were broadly scattered across all three rivers, providing evidence of the widespread presence of this compound in these US waterways.