Jordan Crago

Benchmarking Organic Micropollutants in Wastewater, Recycled Water and Drinking Water with In Vitro Bioassays

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Pharmaceuticals and personal care products found in the Great Lakes above concentrations of environmental concern

The monitoring of pharmaceuticals and personal care products (PPCPs) has focused on the distribution in rivers and small lakes, but data regarding their occurrence and effects in large lake systems, such as the Great Lakes, are sparse. Wastewater treatment processes have not been optimized to remove influent PPCPs and are a major source of PPCPs in the environment. Furthermore, PPCPs are not currently regulated in wastewater effluent. In this experiment we evaluated the concentration, and corresponding risk, of PPCPs from a wastewater effluent source at varying distances in Lake Michigan. Fifty-four PPCPs and hormones were assessed on six different dates over a two-year period from surface water and sediment samples up to 3.2 km from a wastewater treatment plant and at two sites within a harbor. Thirty-two PPCPs were detected in Lake Michigan and 30 were detected in the sediment, with numerous PPCPs being detected up to 3.2 km away from the shoreline. The most frequently detected PPCPs in Lake Michigan were metformin, caffeine, sulfamethoxazole, and triclosan. To determine the ecological risk, the maximum measured environmental concentrations were compared to the predicted no-effect concentration and 14 PPCPs were found to be of medium or high ecological risk. The environmental risk of PPCPs in large lake systems, such as the Great Lakes, has been questioned due to high dilution; however, the concentrations found in this study, and their corresponding risk quotient, indicate a significant threat by PPCPs to the health of the Great Lakes, particularly near shore organisms.

Toxicity biomarker expression in daphnids exposed to manufactured nanoparticles: changes in toxicity with functionalization.

In previous work we have shown that the toxicity of nanomaterials to Daphnia spp. differs with the type of nanoparticle either due to the core of the particle or to the way in which a particle suspension is prepared. The purpose of this study was to investigate the toxicity and antioxidant response of Daphnia pulex in relation to a change in surface functionalization of nanomaterials with the same core material, nC60. Despite the lack of acute toxicity for various nC60 suspensions up to 100 ppm concentration, there was a significant production of the toxicity biomarkers glutathione-S-transferase and catalase, at lower concentrations indicating changes in reactive oxygen species. Nanoparticle functionalization significantly affected this response. Oxidative stress markers appear to be a good predictor of potential future toxicity of nanomaterials. Functionalization alters both toxicity and oxidative stress in whole organism assays.

Evaluation of a model for the removal of pharmaceuticals, personal care products, and hormones from wastewater

Current wastewater treatment processes are insufficient at removing many pharmaceutical and personal care products (PPCPs) from wastewater and it is necessary to identify the chemical characteristics that determine their fate. Models that predict the fate of various chemicals lack verification using in situ data, particularly for PPCPs. BIOWIN4 is a quantitative structure-activity relationship (QSAR) model that has been proposed to estimate the removal of PPCPs from wastewater, but data verifying the accuracy of its predictions is limited. In this study, the in situ soluble and suspended solid concentrations were assessed from raw influent, primary effluent, secondary effluent, and final effluent for 54 PPCPs and hormones over six dates. When assessing the removal efficiency across the different stages of the WWTP, the majority of the removal occurred across the secondary treatment process for the majority of the compounds. The primary treatment and disinfection process had limited impacts on the removal of most PPCPs. Sorption to solids was found to influence the removal for compounds with a log octanol-water partitioning coefficient greater than 4.5 across the secondary treatment process. For other compounds, the removal of PPCPs across the secondary treatment process was significantly correlated with the biodegradation predicted by BIOWIN4. Removal efficiencies across the aerobic secondary treatment process were predicted by integrating BIOWIN4 into pseudo-first order kinetics of PPCPs and these predicted values were compared to the in situ data. This study determines that under a certain set of operating conditions, two chemical characteristics - the expected hydrophobic interaction and the modeled biological degradation from BIOWIN4 - were found to predict the removal of highly degradable and recalcitrant PPCPs from a wastewater secondary treatment process.

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 worlds 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. Environ Toxicol Chem 2014;9999:1–6.

A mixture of an environmentally realistic concentration of a phthalate and herbicide reduces testosterone in male fathead minnow (Pimephales promelas) through a novel mechanism of action

Several chemicals that are used by humans, such as pesticides and plastics, are released into the aquatic environment through wastewater and runoff and have been shown to be potent disruptors of androgen synthesis at high concentrations. Although many of these chemicals have been studied in isolation, a large amount of uncertainty remains over how fish respond to low concentrations of anti-androgenic mixtures, which more accurately reflects how such chemicals are present in the aquatic environment. In this study male fathead minnows (FHM) (Pimephales promelas) were exposed to environmentally relevant concentrations of two anti-androgens, the herbicide linuron, and the plasticizer di(2-ethylhexyl) phthalate (DEHP) individually and as part of a mixture of the two for a 28-day period. At the end of this period there was a reduction in plasma testosterone (T) concentrations in male FHM exposed to the mixture, but not in FHM exposed individually to linuron or DEHP or the control FHM. There was also a significant reduction in 17β-estradiol (E2) in the DEHP-only and mixture exposed groups as compared to the control. Contrary to what has been previously published for these two chemicals in mammals, the lower plasma T concentrations in male FHM exposed to the mixture was not a result of the inhibition of genes involved in steroidogenesis; nor due to an increase in the expression of genes associated with peroxisome proliferation. Rather, an increase in relative transcript abundance for CYP3A4 in the liver and androgen- and estrogen-specific SULT2A1 and SULT1st2 in the testes provides evidence that the decrease in plasma T and E2 may be linked to increased steroid catabolism. Feedback from the pituitary is not repressed as the relative expression of follicle stimulating hormone β-subunit mRNA transcript levels in the brain was significantly higher in both DEHP and mixture exposed FHM. In addition, luteinizing hormone β-subunit mRNA transcript levels increased but were not significant in the mixture as compared to the control. Hormone receptor mRNA transcript levels in the liver and testes were not significantly different across all four exposure groups. This study highlights the importance of assessing environmentally relevant concentrations of mixtures when determining risk to aquatic organisms.

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.

Assessing bioavailability of DDT and metabolites in marine sediments using solid‐phase microextraction with performance reference compounds

Solid-phase microextraction (SPME) has often been used to estimate the freely dissolved concentration (Cfree ) of organic contaminants in sediments. A significant limitation in the application of SPME for Cfree measurement is the requirement for attaining equilibrium partition, which is often difficult for strongly hydrophobic compounds such as DDT. A method was developed using SPME with stable isotope-labeled analogues as performance reference compounds (PRCs) to measure Cfree of DDT and metabolites (DDTs) in marine sediments. Six (13) C-labeled or deuterated PRCs were impregnated into polydimethylsiloxane (PDMS) fiber before use. Desorption of PRCs from PDMS fibers and absorption of DDTs from sediment were isotropic in a range of sediments evaluated ex situ under well-mixed conditions. When applied to a historically contaminated marine sediment from a Superfund site, the PRC-SPME method yielded Cfree values identical to those found by using a conventional equilibrium SPME approach (Eq-SPME), whereas the time for mixing was reduced from 9 d to only 9 h. The PRC-SPME method was further evaluated against bioaccumulation of DDTs by Neanthes arenaceodentata in the contaminated sediment with or without amendment of activated carbon or sand. Strong correlations were consistently found between the derived equilibrium concentrations on the fiber and lipid-normalized tissue residues for DDTs in the worms. Results from the present study clearly demonstrated the feasibility of coupling PRCs with SPME sampling to greatly shorten sampling time, thus affording much improved flexibility in the use of SPME for bioavailability evaluation.

Linking biomarkers to reproductive success of caged fathead minnows in streams with increasing urbanization.

Reproductive and oxidative stress biomarkers have been recommended as tools to assess the health of aquatic organisms. Though validated in the laboratory, there are few studies that tie a change in gene expression to adverse reproductive or population outcomes in the field. This paper looked at 17 streams with varying degrees of urbanization to assess the use of biomarkers associated with reproduction or stress in predicting reproductive success of fathead minnows. In addition, the relationship between biomarkers and water quality measures in streams with varying degrees of urbanization was examined. Liver vitellogenin mRNA was correlated with reproduction within a period of 11d prior to sampling irrespective of habitat, but its correlation with egg output declined at 12d and beyond indicating its usefulness as a short-term biomarker but its limits as a biomarker of total reproductive output. Stress biomarkers such as glutathione S-transferase may be better correlated with factors affecting reproduction over a longer term. There was a significant correlation between GST mRNA and a variety of anthropogenic pollutants. There was also an inverse correlation between glutathione S-transferase and the amount of the watershed designated as wetland. Egg production over the 21-d was negatively correlated with the amount of urbanization and positively correlated to wetland habitats. This study supports the development of multiple biomarkers linking oxidative stress and other non-reproductive endpoints to changes in aquatic habitats will be useful for predicting the health of fish populations and identifying the environmental factors that may need mitigation for sustainable population management.

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.