Applying mixture toxicity modelling to predict bacterial bioluminescence inhibition by non-specifically acting pharmaceuticals and specifically acting antibiotics

Pharmaceuticals and antibiotics co-occur in the aquatic environment but mixture studies to date have mainly focused on pharmaceuticals alone or antibiotics alone, although differences in mode of action may lead to different effects in mixtures. In this study we used the Bacterial Luminescence Toxicity Screen (BLT-Screen) after acute (0.5 h) and chronic (16 h) exposure to evaluate how non-specifically acting pharmaceuticals and specifically acting antibiotics act together in mixtures. Three models were applied to predict mixture toxicity including concentration addition, independent action and the two-step prediction (TSP) model, which groups similarly acting chemicals together using concentration addition, followed by independent action to combine the two groups. All non-antibiotic pharmaceuticals had similar EC50 values at both 0.5 and 16 h, indicating together with a QSAR (Quantitative Structure-Activity Relationship) analysis that they act as baseline toxicants. In contrast, the antibiotics’ EC50 values decreased by up to three orders of magnitude after 16 h, which can be explained by their specific effect on bacteria. Equipotent mixtures of non-antibiotic pharmaceuticals only, antibiotics only and both non-antibiotic pharmaceuticals and antibiotics were prepared based on the single chemical results. The mixture toxicity models were all in close agreement with the experimental results, with predicted EC50 values within a factor of two of the experimental results. This suggests that concentration addition can be applied to bacterial assays to model the mixture effects of environmental samples containing both specifically and non-specifically acting chemicals

Exploring contaminants as a disruptor of temperature-dependent sex determination in sea turtle hatchlings

Sea turtle nesting beaches are experiencing increased sand temperatures as climate change progresses. In one major green turtle (Chelonia mydas) nesting beach in the northern Great Barrier Reef, over 99 percent of hatchlings are female. The effects of contaminants on sea turtle hatchling sex determination are not often explored. Liver samples were collected from green turtle hatchlings that were sacrificed for histological sex determination in a parallel study on the effects of sand cooling on sex ratios, and analysed for trace elements via acid digestion and organic contaminants via in vitro cytotoxicity bioassays. Chromium, antimony, barium, and cadmium have previously been demonstrated to be estrogenic, and concentrations of these elements were used to calculate three estrogenic indexes for each clutch: predicted relative estrogenic potency (PEEQA), the sum of percent trace elements above the median of all samples (TEOM), and the sum of percent estrogenic elements above the median of all samples (EstroEOM). Excluding an outlier clutch, cadmium, antimony, and EstroEOM had significant positive relationships with sex ratio deviation. Mean clutch cobalt, lead, antimony and barium, also had a significant positive relationship with clutch sex ratio. There was no relationship between in vitro cytotoxicity of liver extracts and sex ratio, however, 9% of hatchlings had organic contaminants high enough to suggest potential cellular damage. Contaminant effects on sex determination are likely to be caused by a mixture of contaminant interactions as well as temperature. Many trace elements detected in this study have also been linked to negative health effects on hatchlings in previous studies. Considering the risks of feminization due to climate change and potential contaminant effects on hatchling health and sex determination, future studies exploring contaminant effects on sea turtle hatchling sex determination are recommended

Estrogenic and androgenic potential of municipal sewage in Australia and New Zealand

Studies in Europe, Japan, and North America have reported that wild fish exposed to treated sewage effluents can exhibit significant physiological and reproductive abnormalities consistent with exposure to hormonally active chemicals. The main objective of this research project was to examine the estrogenic and androgenic activity in treated sewage to determine the risk associated with treated sewage discharges in Australia and New Zealand. Several bioassays, including a sheep estrogen receptor and a rainbow trout androgen receptor binding assay, were set up and validated with model compounds. The assays were then used to measure the estrogenic and androgenic activity in sewage samples from 15 municipal sewage treatment plants (STP) utilizing a variety of treatment technologies. Raw sewage samples contained high levels of both estrogenic and androgenic activity, up to 185 ng/L estradiol equivalents (EEq) and up to 9330 ng/L testosterone equivalents (TEq), respectively. Secondary treatment processes such as activated sludge had the greatest impact on removal of biological activity from the wastewater. The estrogenic and androgenic activity in final treated effluents were <1 to 4.2 ng/L EEq and <6.5 to 736 ng/L TEq, respectively. Based on lowest observable effective concentrations reported in the literature, these levels are unlikely to induce biological effects in exposed fish in the short term. To examine potential long-term effects, resident mosquitofish chronically exposed to undiluted treated sewage were sampled. Several morphological biomarkers indicative of endocrine disruption were measured and compared with mosquitofish captured at a reference site. Mosquitofish captured in a constructed wetland for tertiary treatment of secondary treated sewage exhibited morphological differences such as elongated anal fins consistent with exposure to androgenic chemicals, although this effect was not measurable in fish collected at sites further downstream or at any of the other sites. Based on these results, it is unlikely that mosquitofish populations would be significantly affected by exposure to final treated sewage. A reverse transcription real-time polymerase chain reaction (RT-PCR) method to measure the production of a female-specific protein (vitellogenin) mRNA in adult male mosquitofish was developed, and this could be used as a rapid test to detect early changes in individuals exposed to estrogenic activity

Assessing the role of different dissolved organic carbon and bromide concentrations for disinfection by-product formation using chemical analysis and bioanalysis

Concerns regarding disinfection by-product (DBP) formation during drinking water treatment have led water utilities to apply treatment processes to reduce the concentration of DBP precursor natural organic matter (NOM). However, these processes often do not remove bromide, leading to high bromide to dissolved organic carbon (DOC) ratios after treatment, which can increase the formation of more toxic brominated DBPs. In the current study, we investigated the formation and effect of DBPs in a matrix of synthetic water samples containing different concentrations of bromide and DOC after disinfection with chlorine. Trihalomethanes and haloacetic acids were analysed by chemical analysis, while effect was evaluated using in vitro bioassays indicative of the oxidative stress response and bacterial toxicity. While the addition of increasing bromide concentrations did not alter the sum molar concentration of DBPs formed, the speciation changed, with greater bromine incorporation with an increasing Br:DOC ratio. However, the observed effect did not correlate with the Br:DOC ratio, but instead, effect increased with increasing DOC concentration. Water samples with low DOC and high bromide did not exceed the available oxidative stress response effect-based trigger value (EBT), while all samples with high DOC, irrespective of the bromide concentration, exceeded the EBT. This suggests that treatment processes that remove NOM can improve drinking water quality, even if they are unable to remove bromide. Further, iceberg modelling showed that detected DBPs only explained a small fraction of the oxidative stress response, supporting the application of both chemical analysis and bioanalysis for monitoring DBP formation

Considerations when assessing antagonism in vitro: why standardizing the agonist concentration matters

There is increasing recognition of the importance of assessing both agonism and antagonism in parallel for environmental samples. Cell-based in vitro assays have the advantage over receptor binding assays as they are able to differentiate between agonist and antagonist activity, but at present there is no standardized approach to assess antagonism in vitro, and in particular the competing agonist concentration can vary in the literature anywhere from half maximal to maximal effect concentrations. In this study, we investigated the influence of changing agonist concentrations in the estrogen receptor alpha (ER alpha), progesterone receptor (PR) and glucocorticoid receptor (GR) assays run in antagonist mode. The antagonistic effect varied by over two orders of magnitude when using the range of agonist concentrations applied in the literature, clearly indicating the need for standardization. By comparing antagonist EC50 values with different background agonist concentrations, an EC80 background agonist concentration is recommended when assessing antagonism in vitro to optimise both assay sensitivity and reproducibility. (C) 2015 Elsevier Ltd. All rights reserved

Wastewater treatment plant effluent as a source of microplastics: review of the fate, chemical interactions and potential risks to aquatic organisms

Wastewater treatment plant (WWTP) effluent has been identified as a potential source of microplastics in the aquatic environment. Microplastics have recently been detected in wastewater effluent in Western Europe, Russia and the USA. As there are only a handful of studies on microplastics in wastewater, it is difficult to accurately determine the contribution of wastewater effluent as a source of microplastics. However, even the small amounts of microplastics detected in wastewater effluent may be a remarkable source given the large volumes of wastewater treatment effluent discharged to the aquatic environment annually. Further, there is strong evidence that microplastics can interact with wastewater-associated contaminants, which has the potential to transport chemicals to aquatic organisms after exposure to contaminated microplastics. In this review we apply lessons learned from the literature on microplastics in the aquatic environment and knowledge on current wastewater treatment technologies, with the aim of identifying the research gaps in terms of (i) the fate of microplastics in WWTPs, (ii) the potential interaction of wastewater-based microplastics with trace organic contaminants and metals, and (iii) the risk for aquatic organisms

Using fluorescence-parallel factor analysis for assessing disinfection by-product formation and natural organic matter removal efficiency in secondary treated synthetic drinking waters

Parallel factor (PARAFAC) analysis of fluorescence excitation-emission matrices (EEMs) was used to investigate the organic matter and DBP formation characteristics of untreated, primary treated (enhanced coagulation; EC) and secondary treated synthetic waters prepared using a Suwannee River natural organic matter (SR-NOM) isolate. The organic matter was characterised by four different fluorescence components; two humic acid-like (C1 and C2) and two protein-like (C3 and C4). Secondary treatment methods tested, following EC treatment, were; powdered activated carbon (PAC), granular activated carbon (GAC), 0.1% silver-impregnated activated carbon (SIAC), and MIEX® resin. Secondary treatments were more effective at removing natural organic matter (NOM) and fluorescent DBP-precursor components than EC alone. The formation of a suite of 17 DBPs including chlorinated, brominated and iodinated trihalomethanes (THMs), dihaloacetonitriles (DHANs), chloropropanones (CPs), chloral hydrate (CH) and trichloronitromethane (TCNM) was determined after chlorinating water sampled before and after each treatment step. Regression analysis was used to investigate the relationship between peak component fluorescence intensity (FMAX), DBP concentration and speciation, and more commonly used aggregate parameters such as DOC, UV254 and SUVA254. PARAFAC component 1 (C1) was in general a better predictor of DBP formation than other aggregate parameters, and was well correlated (R ≥ 0.80) with all detected DBPs except dibromochloromethane (DBCM) and dibromoacetonitrile (DBAN). These results indicate that the fluorescence-PARAFAC approach could provide a robust analytical tool for predicting DBP formation, and for evaluating the removal of NOM fractions relevant to DBP formation during water treatment

Effects of polyethylene microplastics on the acute toxicity of a synthetic pyrethroid to midge larvae (Chironomus tepperi) in synthetic and river water

Microplastics are ubiquitous pollutants in the aquatic environment. However, our understanding of the interaction of chemicals, particularly synthetic pyrethroids, with microplastics and the potential toxic effects of sorbed contaminants on aquatic organisms under realistic conditions is still extremely limited. In this study, we examined whether the presence of polyethylene (PE) microplastics can affect the acute toxicity of the synthetic pyrethroid bifenthrin to an invertebrate Chironomus tepperi in both synthetic and river water. Bifenthrin alone was, as expected, acutely toxic to exposed larvae (LC50 of 0.5 mu g/L after 48 h exposure). The addition of microplastics to synthetic water significantly reduced the toxicity of bifenthrin (apparent LC50 = 1.3 mu g/L), most likely because sorption of bifenthrin to microplastics reduced its bioavailability to the exposed larvae. A sorption capacity experiment showed that >92% of bifenthrin was sorbed to microplastics. In river water containing 9.6 mg/L organic carbon, bifenthrin alone was less toxic (LC50 = 1.3 mu g/L) than in synthetic water. Strikingly, the addition of microplastics to river water did not mitigate bifenthrin toxicity (apparent LC50 = 1.4 mu g/L), most likely due to greater interaction of bifenthrin with organic carbon than with microplastics. While PE microplastics reduced the negative effects of bifenthrin in synthetic water, the presence of organic carbon in river water without microplastics also reduced toxicity. This suggests that while sorption of contaminants to microplastics does occur, it may not be as relevant under environmentally realistic conditions with mg/L concentrations of organic matter. (C) 2019 Elsevier B.V. All rights reserved

Environmentally relevant concentrations of polyethylene microplastics negatively impact the survival, growth and emergence of sediment-dwelling invertebrates

Microplastics are a widespread environmental pollutant in aquatic ecosystems and have the potential to eventually sink to the sediment, where they may pose a risk to sediment-dwelling organisms. While the impacts of exposure to microplastics have been widely reported for marine biota, the effects of microplastics on freshwater organisms at environmentally realistic concentrations are largely unknown, especially for benthic organisms. Here we examined the effects of a realistic concentration of polyethylene microplastics in sediment on the growth and emergence of a freshwater organism Chironomus tepperi. We also assessed the influence of microplastic size by exposing C. tepperi larvae to four different size ranges of polyethylene microplastics (1-4, 10-27, 43-54 and 100-126 mu m). Exposure to an environmentally relevant concentration of microplastics, 500 particles/kg(sediment), negatively affected the survival, growth (i.e. body length and head capsule) and emergence of C tepperi. The observed effects were strongly dependent on microplastic size with exposure to particles in the size range of 10-27 mu m inducing more pronounced effects. While growth and survival of C tepperi were not affected by the larger microplastics (100-126 mu m), a significant reduction in the number of emerged adults was observed after exposure to the largest microplastics, with the delayed emergence attributed to exposure to a stressor. While scanning electron microscopy showed a significant reduction in the size of the head capsule and antenna of C. tepperi exposed to microplastics in the 10-27 mu m size range, no deformities to the external structure of the antenna and mouth parts in organisms exposed to the same size range of microplastics were observed. These results indicate that environmentally relevant concentrations of microplastics in sediment induce harmful effects on the development and emergence of C. tepperi, with effects greatly dependent on particle size. (C) 2018 Elsevier Ltd. All rights reserved

In vitro cytotoxicity and adaptive stress responses to selected haloacetic acid and halobenzoquinone water disinfection byproducts

The process of disinfecting drinking water inadvertently leads to the formation of numerous disinfection byproducts (DBPs). Some of these are mutagenic, genotoidc, teratogenic, and cytotoxic, as well as potentially carcinogenic both in vivo and in vitro. We investigated the in vitro biological activity of five DBPs: three monohaloacetic acids (monoHAAs) [chloroacetic acid (CAA), bromoacetic acid (BAA), and iodoacetic acid (IAA)] and two novel halobenzoquinones (HBQs) [2,6-dichloro-p-benzoquinone (DCBQ) and 2,6-dibromo-p-benzoquinone]. We focused particularly on cytotoxidty and induction of two adaptive stress response pathways: the oxidative stress responsive Nrf2/ARE and DNA-damage responsive p53 pathways. All five DBPs were cytotoidc to the Caco-2 cell line after a 4 h exposure, and all DBPs induced both of the adaptive stress response pathways, Nrf2/ARE and p53, in the micromolar range, as measured by two beta-lactamase-based reporter gene assays. The decreasing order of potency for all three endpoints for the five DBPs was IAA similar to BAA > DCBQ similar to DBBQ > CAA. Induction of oxidative stress was previously proposed to be the molecular initiating event (MIE) for both classes of DBPs. However, comparing the levels of activation of the two pathways uncovered that the Nrf2/ARE pathway was the more sensitive endpoint for HAAs, whereas the p53 pathway was more sensitive in the case of HBQs. Therefore, the DNA damage-responsive p53 pathway may be an important piece of information to fill in a gap in the adverse outcome pathway framework for the assessment of HBQs. Finally, we cautiously compared the potential risk of the two novel HBQs using a benchmarking approach to that of the well-studied CAA, which suggested that their relative risk may be lower than that of BAA and IAA