Bioenergetic Shifts in Humpback Whale Fibroblasts Upon Chemical Exposure.

Southern Hemisphere humpback whales accumulate persistent and toxic chemicals, which are transported to Antarctica through distant sources and in situ usage. The extreme seasonal migration-associated fast of humpback whales results in the remobilization of persistent and lipophilic environmental contaminants from liberated fat stores. Mitochondria play a key role in lipid metabolism, and any disruption to mitochondrial function is expected to influence whole-organism bioenergetics. It is therefore of interest to advance understanding of the impact of known contaminants of the Antarctic sea-ice ecosystem upon humpback whale cellular bioenergetics. Using cell line-based in vitro testing, this study employed the Seahorse Extracellular Flux Analyzer to study cellular metabolic activity in live humpback whale fibroblast cells. The assay, based on oxygen consumption rate, provides insights into the cause of cellular bioenergetic disruption. Immortalized skin fibroblasts were exposed to four priority environmental chemicals found in the Antarctic sea-ice ecosystem. Our findings reveal chemical-dependent functional alterations and varying bioenergetic profile responses. Chlorpyrifos was observed to decrease mitochondrial basal oxygen consumption; dieldrin increased basal oxygen consumption; trifluralin's impact was dose-specific, and endosulfan displayed no effect. Our results provide unique insights into environmental chemical mechanisms of action on cellular bioenergetics, generating much-needed taxa-specific chemical effect data in support of evidence-based conservation policy and management.

High-Throughput Transcriptomics of Water Extracts Detects Reductions in Biological Activity with Water Treatment Processes.

The presence of numerous chemical contaminants from industrial, agricultural, and pharmaceutical sources in water supplies poses a potential risk to human and ecological health. Current chemical analyses suffer from limitations, including chemical coverage and high cost, and broad-coverage in vitro assays such as transcriptomics may further improve water quality monitoring by assessing a large range of possible effects. Here, we used high-throughput transcriptomics to assess the activity induced by field-derived water extracts in MCF7 breast carcinoma cells. Wastewater and surface water extracts induced the largest changes in expression among cell proliferation-related genes and neurological, estrogenic, and antibiotic pathways, whereas drinking and reclaimed water extracts that underwent advanced treatment showed substantially reduced bioactivity on both gene and pathway levels. Importantly, reclaimed water extracts induced fewer changes in gene expression than laboratory blanks, which reinforces previous conclusions based on targeted assays and improves confidence in bioassay-based monitoring of water quality.

The "Microplastome" - A Holistic Perspective to Capture the Real-World Ecology of Microplastics.

Microplastic pollution, an emerging pollution issue, has become a significant environmental concern globally due to its ubiquitous, persistent, complex, toxic, and ever-increasing nature. As a multifaceted and diverse suite of small plastic particles with different physicochemical properties and associated matters such as absorbed chemicals and microbes, future research on microplastics will need to comprehensively consider their multidimensional attributes. Here, we introduce a novel, conceptual framework of the "microplastome", defined as the entirety of various plastic particles (<5 mm), and their associated matters such as chemicals and microbes, found within a sample and its overall environmental and toxicological impacts. As a novel concept, this paper aims to emphasize and call for a collective quantification and characterization of microplastics and for a more holistic understanding regarding the differences, connections, and effects of microplastics in different biotic and abiotic ecosystem compartments. Deriving from this lens, we present our insights and prospective trajectories for characterization, risk assessment, and source apportionment of microplastics. We hope this new paradigm can guide and propel microplastic research toward a more holistic era and contribute to an informed strategy for combating this globally important environmental pollution issue.

Removal of organic micropollutants in biologically active filters: A systematic quantitative review of key influencing factors.

Biofiltration utilizes natural mechanisms including biodegradation and biotransformation along with other physical processes for the removal of organic micropollutants (OMPs) such as pharmaceuticals, personal care products, pesticides and industrial compounds found in (waste)water. In this systematic review, a total of 120 biofiltration studies from 25 countries were analyzed, considering various biofilter configurations, source water types, biofilter media and scales of operation. The study also provides a bibliometric analysis to identify the emerging research trends in the field. The results show that granular activated carbon (GAC) either alone or in combination with another biofiltration media can remove a broad range of OMPs efficiently. The impact of pre-oxidation on biofilter performance was investigated, revealing that pre-oxidation significantly improved OMP removal and reduced the empty bed contact time (EBCT) needed to achieve a consistently high OMP. Biofiltration with pre-oxidation had median removals ranging between 65% and >90% for various OMPs at 10-45 min EBCT with data variability drastically reducing beyond 20 min EBCT. Biofiltration without pre-oxidation had lower median removals with greater variability. The results demonstrate that pre-oxidation greatly enhances the removal of adsorptive and poorly biodegradable OMPs, while its impact on other OMPs varies. Only 19% of studies we reviewed included toxicity testing of treated effluent, and even fewer measured transformation products. Several studies have previously reported an increase in effluent toxicity because of oxidation, although it was successfully abated by subsequent biofiltration in most cases. Therefore, the efficacy of biofiltration treatment should be assessed by integrating toxicity testing into the assessment of overall removal.

Effect-based monitoring of two rivers under urban and agricultural influence reveals a range of biological activities in sediment and water extracts.

Chemical contaminants, such as pesticides, pharmaceuticals and industrial compounds are ubiquitous in surface water and sediment in areas subject to human activity. While targeted chemical analysis is typically used for water and sediment quality monitoring, there is growing interest in applying effect-based methods with in vitro bioassays to capture the effects of all active contaminants in a sample. The current study evaluated the biological effects in surface water and sediment from two contrasting catchments in Aotearoa New Zealand, the highly urbanised Whau River catchment in Tamaki Makaurau (Auckland) and the urban and mixed agricultural Koreti (New River) Estuary catchment. Two complementary passive sampling devices, Chemcatcher for polar chemicals and polyethylene (PED) for non-polar chemicals, were applied to capture a wide range of contaminants in water, while composite sediment samples were collected at each sampling site. Bioassays indicative of induction of xenobiotic metabolism, receptor-mediated effects, genotoxicity, cytotoxicity and apical effects were applied to the water and sediment extracts. Most sediment extracts induced moderate to strong estrogenic and aryl hydrocarbon (AhR) activity, along with moderate toxicity to bacteria. The water extracts showed similar patterns to the sediment extracts, but with lower activity. Generally, the polar Chemcatcher extracts showed greater estrogenic activity, photosynthesis inhibition and algal growth inhibition than the non-polar PED extracts, though the PED extracts showed greater AhR activity. The observed effects in the water extracts were compared to available ecological effect-based trigger values (EBT) to evaluate the potential risk. For the polar extracts, most sites in both catchments exceeded the EBT for estrogenicity, with many sites exceeding the EBTs for AhR activity and photosynthesis inhibition. Of the wide range of endpoints considered, estrogenic activity, AhR activity and herbicidal activity appear to be the primary risk drivers in both the Whau and Koreti Estuary catchments.

Microplastics and Tire Wear Particles in Urban Stormwater: Abundance, Characteristics, and Potential Mitigation Strategies.

Stormwater has been identified as a pathway for microplastics (MPs), including tire wear particles (TWPs), into aquatic habitats. Our knowledge of the abundance of MPs in urban stormwater and potential strategies to control MPs in stormwater is still limited. In this study, stormwater samples were collected from microlitter capture devices (inlet and outlet) during rain events. Sediment samples were collected from the material captured in the device and from the inlet and outlet of a constructed stormwater wetland. MP (>25 µm) concentration in stormwater varied across different locations ranging from 3.8 to 59 MPs/L in raw and 1.8 to 32 MPs/L in treated stormwater, demonstrating a decrease after passage through the device (35-88% removal). TWPs comprised ∼95% of all particles, followed by polypropylene (PP) and poly(ethylene terephthalate) (PET). The concentration of TWPs ranged from 2.5 to 58 TWPs/L and 1450 to 4740 TWPs/kg in stormwater and sediment, respectively. A higher abundance of MPs was found in the sediment at the inlet of the constructed wetland compared to the outlet, indicating a potential role of wetlands in removing MPs from stormwater. These findings suggest that both constructed wetlands and microlitter capture devices can mitigate the transport of MPs from stormwater to the receiving waterways.

Novel Use of Cell Profiling Technology to Visualize Mitochondrial Responses of Humpback Whale Fibroblasts to Chemical Exposure.

Cetaceans are at elevated risk of accumulating persistent and lipophilic environmental contaminants due to their longevity and high proportion of body fat. Despite this, there is a paucity of taxa-specific chemical effect data, in part due to the ethical and logistical constraints in working with highly mobile aquatic species. Advances in cetacean cell culture have opened the door to the application of mainstream in vitro toxicological effect assessment approaches. Image-based cell profiling is a high-throughput, microscopy-based system commonly applied in drug development. It permits the analysis of the xenobiotic effect on multiple cell organelles simultaneously, hereby flagging its potential utility in the evaluation of chemical toxicodynamics. Here we exposed immortalized humpback whale skin fibroblasts (HuWaTERT) to six priority environmental contaminants known to accumulate in the Southern Ocean food web, in order to explore their subcellular organelle responses. Results revealed chemical-dependent modulation of mitochondrial texture, with the lowest observed effect concentrations for chlorpyrifos, dieldrin, trifluralin, and p,p'-dichlorodiphenyldichloroethane of 0.3, 4.1, 9.3, and 19.8 nM, respectively. By contrast, no significant changes were observed upon exposure to endosulfan and lindane. This study contributes the first fixed mitochondrial images of HuWaTERT and constitutes novel, taxa-specific chemical effect data in support of evidence-based conservation policy and management.

Application of Effect-Based Methods to Water Quality Monitoring: Answering Frequently Asked Questions by Water Quality Managers, Regulators, and Policy Makers.

Effect-based methods (EBM) have great potential for water quality monitoring as they can detect the mixture effects of all active known and unknown chemicals in a sample, which cannot be addressed by chemical analysis alone. To date, EBM have primarily been applied in a research context, with a lower level of uptake by the water sector and regulators. This is partly due to concerns regarding the reliability and interpretation of EBM. Using evidence from the peer-reviewed literature, this work aims to answer frequently asked questions about EBM. The questions were identified through consultation with the water industry and regulators and cover topics related to the basis for using EBM, practical considerations regarding reliability, sampling for EBM and quality control, and what to do with the information provided by EBM. The information provided in this work aims to give confidence to regulators and the water sector to stimulate the application of EBM for water quality monitoring.

ECHIDNA (Emerging CHemIcals Database for National Awareness): a framework to prioritise contaminants of emerging concern in water.

The widespread presence of contaminants of emerging concern (CEC) in surface waters, treated wastewater and drinking water is an ongoing issue for the water industry. The absence of regulatory guidance and limited occurrence, toxicity and removal data are defining criteria of CEC and make it difficult to prioritise which CEC pose the greatest risk. The online Emerging CHemIcals Database for National Awareness (ECHIDNA) aims to classify and prioritise CEC based on their potential risk, with the information presented in an easily accessible and intuitive manner. A candidate list of almost 1,800 potential CEC, including pesticides, pharmaceuticals and industrial compounds, was compiled using both Australian and international resources. These were ranked based on in silico assessment of their persistent, bioaccumulative and toxic (PBT) properties, as well as potential chronic toxicity hazard, yielding 247 CEC for further prioritisation. Risk Quotients (RQ) identified between 5 and 87 CEC posing a risk to human and ecosystem health, respectively, across drinking water, surface water, treated wastewater and raw wastewater. While the ability of the water industry to effectively prioritise CEC is limited by candidate identification and data availability, ECHIDNA can provide valuable information for better decision-making surrounding CEC management.

Airborne Microplastics in Indoor and Outdoor Environments of a Developing Country in South Asia: Abundance, Distribution, Morphology, and Possible Sources.

Airborne microplastics (AMPs) have been reported in indoor and outdoor air in high-income countries and are expected to be a significant contributor to daily microplastic (MP) exposure for human beings. To date, there are only a handful of studies in lower-middle-income countries. In this study, AMPs from 5000 to 50 µm were sampled across selected areas of Sri Lanka using an active sampling technique. Suspected AMPs were further characterized using Fourier transform infrared spectroscopy. MP concentrations were higher indoors compared to outdoor air (0.13-0.93, compared to 0.00-0.23 particles/m3, respectively). The types of indoor MPs were related to indoor-generating sources, and the occupant's lifestyles. The highest outdoor MP abundance was found near an industrial zone, followed by urban and inland locations in high-density areas. The dominant size range of MPs was 100-300 µm, and the only shapes observed indoors and outdoors were fibers (98%) and fragments. Polyethylene terephthalate was the most prominent MP type, followed by polyester, indicating that textile fibers could be the major source of these AMPs. This study provides the first report on AMPs in Sri Lanka. Considering population growth and industrialization, further research should evaluate possible trends and health risks upon inhalation.

Chiral Inversion of 2-Arylpropionic Acid Enantiomers under Anaerobic Conditions.

This work examined the chiral inversion of 2-arylpropionic acids (2-APAs) under anaerobic conditions and the associated microbial community. The anaerobic condition was simulated by two identical anaerobic digesters. Each digester was fed with the substrate containing 11 either pure (R)- or pure (S)-2-APA enantiomers. Chiral inversion was evidenced by the concentration increase of the other enantiomer in the digestate and the changes in the enantiomeric fraction between the two enantiomers. Both digesters showed similar and poor removal of 2-APAs (≤30%, except for naproxen) and diverse chiral inversion behaviors under anaerobic conditions. Four compounds exhibited (S → R) unidirectional inversion [flurbiprofen, ketoprofen, naproxen, and 2-(4-tert-butylphenyl)propionic acid], and the remaining seven compounds showed bidirectional inversion. Several aerobic and facultative anaerobic bacterial genera (Candidatus Microthrix, Rhodococcus, Mycobacterium, Gordonia, and Sphingobium) were identified in both digesters and predicted to harbor the 2-arylpropionyl-CoA epimerase (enzyme involved in chiral inversion) encoding gene. These genera presented at low abundances, <0.5% in the digester dosed with (R)-2-APAs and <0.2% in the digester dosed with (S)-2-APAs. The low abundances of these genera explain the limited extent of chiral inversion observed in this study.

Effect-based monitoring to integrate the mixture hazards of chemicals into water safety plans.

Water safety plans (WSPs) are intended to assure safe drinking water (DW). WSPs involve assessing and managing risks associated with microbial, chemical, physical and radiological hazards from the catchment to the consumer. Currently, chemical hazards in WSPs are assessed by targeted chemical analysis, but this approach fails to account for the mixture effects of the many chemicals potentially present in water supplies and omits the possible effects of non-targeted chemicals. Consequently, effect-based monitoring (EBM) using in vitro bioassays and well plate-based in vivo assays are proposed as a complementary tool to targeted chemical analysis to support risk analysis, risk management and water quality verification within the WSP framework. EBM is frequently applied to DW and surface water and can be utilised in all defined monitoring categories within the WSP framework (including 'system assessment', 'validation', 'operational' and 'verification'). Examples of how EBM can be applied within the different WSP modules are provided, along with guidance on where to apply EBM and how frequently. Since this is a new area, guidance documents, standard operating procedures (SOPs) and decision-making frameworks are required for both bioassay operators and WSP teams to facilitate the integration of EBM into WSPs, with these resources being developed currently.

Deriving safe short-term chemical exposure values (STEV) for drinking water.

Small and brief exceedances of chemicals above their guideline values in drinking water are unlikely to cause an appreciable increased risk to human health. As a result, short-term exposure values (STEV) can be derived to help decide whether drinking water can still be supplied to consumers without adverse health risks. In this study, three approaches were applied to calculate and compare STEV for pesticides. The three approaches included basing a STEV on the acute reference dose (ARfD) (Approach 1), removing conventional attribution rates and uncertainty factors from current guideline values (Approach 2) and extrapolating 1 d and 7 d no observed adverse effect levels (NOAEL) from existing toxicity data using a log-linear regression (Approach 3). Despite being very different methods, the three approaches produced comparable STEV generally within an order of magnitude, which often overlapped with other existing short-term exposure values such as short-term no adverse response levels (SNARL) and health advisories (HA). The results show that adjusting the current guideline value using standard extrapolation factors (Approach 2) often produced the most conservative values. Approach 2 was then applied to two other chemical classes, disinfection by-products (DBPs) and cyanotoxins, demonstrating the wider applicability of the approach.

Towards the development of standardised sea turtle primary cell cultures for toxicity testing.

Chemical contaminants are known to accumulate in marine megafauna globally, but little is known about how this impacts animal health. In vitro assays offer an ethical, reproducible and cost-effective alternative to live animal toxicity testing on large, long-lived or threatened species, such as sea turtles. However, using a cell culture from a single animal raise the question of whether the toxicity observed adequately represents the toxicity in that species. This study examined variation in the cytotoxic response of primary skin fibroblasts established from seven green (Chelonia mydas) and five loggerhead (Caretta caretta) sea turtles. Cell viability using resazurin dye was examined in response to exposure to five contaminants. The variation in cytotoxicity was generally low (within a factor of five) for both independent analyses of the same cell culture, and cell cultures from different individuals. This low within and between cell culture variation indicates that primary sea turtle cell cultures can provide a suitable approach to understanding toxicity in sea turtles. In addition, green and loggerhead turtle cells showed similar toxicity to the compounds tested, indicating that only subtle differences in chemical sensitivity may exist between sea turtle species. This study provides a framework for using species-specific cell cultures in future toxicological studies on sea turtles. Although in vivo studies are the gold standard for toxicological studies and species-specific risk assessments, the development of in vitro tools can provide important information when in vivo studies are not possible or practical. For large, endangered species such as sea turtles that are exposed to, and accumulate, a large number of contaminants, using validated cell cultures may facilitate the rapid assessment of chemical risk to these animals.

Impact of Microplastic Beads and Fibers on Waterflea (Ceriodaphnia dubia) Survival, Growth, and Reproduction: Implications of Single and Mixture Exposures.

There is limited knowledge regarding the adverse effects of wastewater-derived microplastics, particularly fibers, on aquatic biota. In this study, we examined the acute (48 h) and chronic (8 d) effects of microplastic polyester fibers and polyethylene (PE) beads on freshwater zooplankton Ceriodaphnia dubia. We also assessed the acute response of C. dubia to a binary mixture of microplastic beads and fibers for the first time. Acute exposure to fibers and PE beads both showed a dose-dependent effect on survival. An equitoxic binary mixture of beads and fibers resulted in a toxic unit of 1.85 indicating less than additive effects. Chronic exposure to lower concentrations did not significantly affect survival of C. dubia, but a dose-dependent effect on growth and reproduction was observed. Fibers showed greater adverse effects than PE beads. While ingestion of fibers was not observed, scanning electron microscopy showed carapace and antenna deformities after exposure to fibers, with no deformities observed after exposure to PE beads. While much of the current research has focused on microplastic beads, our study shows that microplastic fibers pose a greater risk to C. dubia, with reduced reproductive output observed at concentrations within an order of magnitude of reported environmental levels.

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.

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, genotoxic, 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 cytotoxicity and induction of two adaptive stress response pathways: the oxidative stress responsive Nrf2/ARE and DNA-damage responsive p53 pathways. All five DBPs were cytotoxic 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 ß-lactamase-based reporter gene assays. The decreasing order of potency for all three endpoints for the five DBPs was IAA ∼ BAA > DCBQ ∼ 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.

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.

A national survey of trace organic contaminants in Australian rivers.

Trace organic contaminant (TrOC) studies in Australia have, to date, focused on wastewater effluents, leaving a knowledge gap of their occurrence and risk in freshwater environments. This study measured 42 TrOCs including industrial compounds, pesticides, and pharmaceuticals and personal care products by liquid chromatography tandem mass spectrometry at 73 river sites across Australia quarterly for 1 yr. Trace organic contaminants were found in 92% of samples, with a median of three compounds detected per sample (maximum 18). The five most commonly detected TrOCs were the pharmaceuticals salicylic acid (82%, maximum = 1530 ng/L), paracetamol (also known as acetaminophen; 45%, maximum = 7150 ng/L), and carbamazepine (27%, maximum = 682 ng/L), caffeine (65%, maximum = 3770 ng/L), and the flame retardant (2-chloroethyl) phosphate (44%, maximum = 184 ng/L). Pesticides were detected in 28% of the samples. To determine the risk posed by the detected TrOCs to the aquatic environment, hazard quotients were calculated by dividing the maximum concentration detected for each compound by the predicted no-effect concentrations. Three of the 42 compounds monitored (the pharmaceuticals carbamazepine and sulfamethoxazole and the herbicide simazine) had a hazard quotient >1, suggesting that they may be causing adverse effects at the most polluted sites. A further 10 compounds had hazard quotients >0.1, indicating a potential risk; these included four pharmaceuticals, three personal care products, and three pesticides. Most compounds had hazard quotients significantly <0.1. The number of TrOCs measured in this study was limited and further investigations are required to fully assess the risk posed by complex mixtures of TrOCs on exposed biota.