Modernizing persistence-bioaccumulation-toxicity (PBT) assessment with high throughput animal-free methods.

The assessment of persistence (P), bioaccumulation (B), and toxicity (T) of a chemical is a crucial first step at ensuring chemical safety and is a cornerstone of the European Union's chemicals regulation REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals). Existing methods for PBT assessment are overly complex and cumbersome, have produced incorrect conclusions, and rely heavily on animal-intensive testing. We explore how new-approach methodologies (NAMs) can overcome the limitations of current PBT assessment. We propose two innovative hazard indicators, termed cumulative toxicity equivalents (CTE) and persistent toxicity equivalents (PTE). Together they are intended to replace existing PBT indicators and can also accommodate the emerging concept of PMT (where M stands for mobility). The proposed "toxicity equivalents" can be measured with high throughput in vitro bioassays. CTE refers to the toxic effects measured directly in any given sample, including single chemicals, substitution products, or mixtures. PTE is the equivalent measure of cumulative toxicity equivalents measured after simulated environmental degradation of the sample. With an appropriate panel of animal-free or alternative in vitro bioassays, CTE and PTE comprise key environmental and human health hazard indicators. CTE and PTE do not require analytical identification of transformation products and mixture components but instead prompt two key questions: is the chemical or mixture toxic, and is this toxicity persistent or can it be attenuated by environmental degradation? Taken together, the proposed hazard indicators CTE and PTE have the potential to integrate P, B/M and T assessment into one high-throughput experimental workflow that sidesteps the need for analytical measurements and will support the Chemicals Strategy for Sustainability of the European Union.

Molecular Responses of Daphnids to Chronic Exposures to Pharmaceuticals.

Pharmaceutical compounds are among several classes of contaminants of emerging concern, such as pesticides, heavy metals and personal care products, all of which are a major concern for aquatic ecosystems. The hazards posed by the presence of pharmaceutical is one which affects both freshwater organisms and human health-via non-target effects and by the contamination of drinking water sources. The molecular and phenotypic alterations of five pharmaceuticals which are commonly present in the aquatic environment were explored in daphnids under chronic exposures. Markers of physiology such as enzyme activities were combined with metabolic perturbations to assess the impact of metformin, diclofenac, gabapentin, carbamazepine and gemfibrozil on daphnids. Enzyme activity of markers of physiology included phosphatases, lipase, peptidase, ß-galactosidase, lactate dehydrogenase, glutathione-S-transferase and glutathione reductase activities. Furthermore, targeted LC-MS/MS analysis focusing on glycolysis, the pentose phosphate pathway and the TCA cycle intermediates was performed to assess metabolic alterations. Exposure to pharmaceuticals resulted in the changes in activity for several enzymes of metabolism and the detoxification enzyme glutathione-S-transferase. Metabolic perturbations on key pathways revealed distinct groups and metabolic fingerprints for the different exposures and their mixtures. Chronic exposure to pharmaceuticals at low concentrations revealed significant alterations of metabolic and physiological endpoints.

Chemical Pollution Levels in a River Explain Site-Specific Sensitivities to Micropollutants within a Genetically Homogeneous Population of Freshwater Amphipods.

Anthropogenic micropollutants alter chemical and ecological conditions of freshwater ecosystems and impact aquatic species that live along the pollution gradient of a river. Species sensitivity to micropollutants depends on the site-specific exposure; however, it remains unclear to what degree this sensitivity relates to the species' genetic structure. Here, we explored the relationship between the toxic sensitivity and genetic structure of the amphipod species Gammarus pulex (Linnaeus, 1758) along an organic micropollutant gradient in the Holtemme River in central Germany. We determined the river's site-specific micropollutant patterns and analyzed the genetic structure of G. pulex using nuclear and mitochondrial genetic markers. Furthermore, we examined the exposure sensitivities and bioaccumulation of the commonly detected insecticide imidacloprid in G. pulex from different sites. Our results show that throughout the Holtemme River, G. pulex forms a well-connected and homogeneous population with no observable pollution-related differences in the genetic structure. However, G. pulex from polluted sites responded more sensitively to imidacloprid; survival times for half of the amphipods were up to 54% shorter, the percentage of immobile individuals increased up to 65%, and the modeled imidacloprid depuration rate was lower in comparison to amphipods from non-polluted sites. Altogether, these results suggest that the level of sensitivity of G. pulex amphipods to micropollutants in the river depends on the degree of pollution: amphipods may thrive in food-rich but polluted habitats; yet, their sensitivity is increased when chronically exposed to organic micropollutants.

Can Environmentally Relevant Neuroactive Chemicals Specifically Be Detected with the Locomotor Response Test in Zebrafish Embryos?

Chemicals considered as neuroactive (such as certain pesticides, pharmaceuticals, and industrial chemicals) are among the largest groups of bioactive substances recently detected in European rivers. However, the determination of nervous-system-specific effects has been limited using in vitro tests or conventional end points including lethality. Thus, neurobehavioral tests using in vivo models (e.g., zebrafish embryo) have been proposed as complementary approaches. To investigate the specificity and sensitivity of a light-dark transition locomotor response (LMR) test in 4 to 5 days post fertilization zebrafish with respect to different modes of action (MoAs), we analyzed a set of 18 environmentally relevant compounds with various anticipated MoAs. We found that exposure-induced behavioral alterations were reproducible and dependent on concentration and time. Comparative and quantitative analyses of the obtained locomotor patterns revealed that behavioral effects were not restricted to compounds primarily known to target the nervous system. A clear distinction of MoAs based on locomotor patterns was not possible for most compounds. Furthermore, chemicals with an anticipated same MoA did not necessarily provoke similar behavioral phenotypes. Finally, we determined an increased sensitivity (≥10-fold) compared to observed mortality in the LMR assay for five of eight neuroactive chemicals as opposed to non-neuroactive compounds.

Uptake Kinetics and Subcellular Compartmentalization Explain Lethal but Not Sublethal Effects of Cadmium in Two Closely Related Amphipod Species.

Eulimnogammarus cyaneus and Eulimnogammarus verrucosus, closely related amphipod species endemic to Lake Baikal, differ with respect to body size (10- to 50-fold lower fresh weights of E. cyaneus) and cellular stress response (CSR) capacity, potentially causing species-related differences in uptake, internal sequestration, and toxic sensitivity to waterborne cadmium (Cd). We found that, compared to E. verrucosus, Cd uptake rates, related to a given exposure concentration, were higher, and lethal concentrations (50%; LC50) were 2.3-fold lower in E. cyaneus (4 weeks exposure; 6 °C). Upon exposures to species-specific subacutely toxic Cd concentrations (nominal LC1; E. cyaneus: 18 nM (2.0 µg L-1); E. verrucosus: 115 nM (12.9 µg L-1); 4 weeks exposure; 6 °C), Cd amounts in metal sensitive tissue fractions (MSF), in relation to fresh weight, were similar in both species (E. cyaneus: 0.25 ± 0.06 µg g-1; E. verrucosus: 0.26 ± 0.07 µg g-1), whereas relative Cd amounts in the biologically detoxified heat stable protein fraction were 35% higher in E. cyaneus. Despite different potencies in detoxifying Cd, body size appears to mainly explain species-related differences in Cd uptake and sensitivities. When exposed to Cd at LC1 over 4 weeks, only E. verrucosus continuously showed 15-36% reduced oxygen consumption rates indicating metabolic depression and pointing to particular sensitivity of E. verrucosus to persisting low-level toxicant pressure.

Use of a combined effect model approach for discriminating between ABCB1- and ABCC1-type efflux activities in native bivalve gill tissue.

Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds.

Time-Dependent Effects in Algae for Chemicals with Different Adverse Outcome Pathways: A Novel Approach.

Chemicals affect unicellular algae as a result of toxicokinetic and toxicodynamic processes. The internal concentration of chemicals in algae cells typically reaches equilibrium within minutes, while damage cumulatively increases over hours. The time gap between the steady state of internal exposure and damage development is thus suspected to span up to hours, mainly due to toxicodynamic processes. The quantification of rate-limited toxicodynamic processes, aggregated as a progressive effect from an initiating molecular event through biological key events toward the adverse outcome on algae growth inhibition, might discriminate between different adverse outcome pathways (AOPs). To support our hypothesis, we selected six chemicals according to different physicochemical properties and three distinctly dissimilar AOPs. The time courses of internal concentrations were linked to the observed affected Scenedesmus vacuolatus growth using toxicokinetic-toxicodynamic modeling. Effects on cell growth were explained by effect progression and not by the time to reach internal equilibrium concentration. Effect progression rates ranged over 6 orders of magnitude for all chemicals but varied by less than 1 order of magnitude within similar AOP (photosystem II inhibitors > reactive chemicals > lipid biosynthesis inhibitors), meaning that inhibitors of photosystem II advance an effect toward algae growth fastest compared to reactive chemicals and inhibitors of lipid biosynthesis.

Metabolic Effect Level Index Links Multivariate Metabolic Fingerprints to Ecotoxicological Effect Assessment.

A major goal of ecotoxicology is the prediction of adverse outcomes for populations from sensitive and early physiological responses. A snapshot of the physiological state of an organism can be provided by metabolic fingerprints. However, to inform chemical risk assessment, multivariate metabolic fingerprints need to be converted to readable end points suitable for effect estimation and comparison. The concentration- and time-dependent responsiveness of metabolic fingerprints to the PS-II inhibitor isoproturon was investigated by use of a Myriophyllum spicatum bioassay. Hydrophilic and lipophilic leaf extracts were analyzed with gas chromatography-mass spectrometry (GC-MS) and preprocessed with XCMS. Metabolic changes were aggregated in the quantitative metabolic effect level index (MELI), allowing effect estimation from Hill-based concentration-response models. Hereby, the most sensitive response on the concentration scale was revealed by the hydrophilic MELI, followed by photosynthetic efficiency and, 1 order of magnitude higher, by the lipophilic MELI and shoot length change. In the hydrophilic MELI, 50% change compares to 30% inhibition of photosynthetic efficiency and 10% inhibition of dry weight change, indicating effect development on different response levels. In conclusion, aggregated metabolic fingerprints provide quantitative estimates and span a broad response spectrum, potentially valuable for establishing adverse outcome pathways of chemicals in environmental risk assessment.

Identification and Characterization of Androgen-Responsive Genes in Zebrafish Embryos.

Responsive genes for fish embryos have been identified so far for some endocrine pathways but not for androgens. Using transcriptome analysis and multiple concentration-response modeling, we identified putative androgen-responsive genes in zebrafish embryos exposed to 0.05-5000 nM 11-ketotestosterone for 24 h. Four selected genes with sigmoidal concentration-dependent expression profiles (EC50 = 6.5-30.0 nM) were characterized in detail. The expression of cyp2k22 and slco1f4 was demonstrated in the pronephros; lipca was detected in the liver, and sult2st3 was found in the olfactory organs and choroid plexus. Their expression domains, the function of human orthologs, and a pathway analysis suggested a role of these genes in the metabolism of hormones. Hence, it was hypothesized that they were induced to compensate for elevated hormone levels. The induction of sult2st3 and cyp2k22 by 11-ketotestosterone was repressed by co-exposure to the androgen receptor antagonist nilutamide supporting a potential androgen receptor mediated regulation. Sensitivity (expressed as EC50 values) of sult2st3 and cyp2k22 gene expression induction after exposure to other steroidal hormones (11-ketotestosterone ∼ testosterone > progesterone > cortisol > ethinylestradiol) correlated with their known binding affinities to zebrafish androgen receptor. Hence, these genes might represent potential markers for screening of androgenic compounds in the zebrafish embryo.

Pollution-Induced Community Tolerance To Diagnose Hazardous Chemicals in Multiple Contaminated Aquatic Systems.

Aquatic ecosystems are often contaminated with large numbers of chemicals, which cannot be sufficiently addressed by chemical target analyses. Effect-directed analysis (EDA) enables the identification of toxicants in complex contaminated environmental samples. This study suggests pollution-induced community tolerance (PICT) as a confirmation tool for EDA to identify contaminants which actually impact on local communities. The effects of three phytotoxic compounds local periphyton communities, cultivated at a reference (R-site) and a polluted site (P-site), were assessed to confirm the findings of a former EDA study on sediments. The sensitivities of R- and P-communities to prometryn, tributyltin (TBT) and N-phenyl-2-naphthylamine (PNA) were quantified in short-term toxicity tests and exposure concentrations were determined. Prometryn and PNA concentrations were significantly higher at the P-site, whereas TBT concentrations were in the same range at both sites. Periphyton communities differed in biomass, but algal class composition and diatom diversity were similar. Community tolerance of P-communities was significantly enhanced for prometryn, but not for PNA and TBT, confirming site-specific effects on local periphyton for prometryn only. Thus, PICT enables in situ effect confirmation of phytotoxic compounds at the community level and seems to be suitable to support confirmation and enhance ecological realism of EDA.

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.

Curated mode-of-action data and effect concentrations for chemicals relevant for the aquatic environment.

Chemicals in the aquatic environment can be harmful to organisms and ecosystems. Knowledge on effect concentrations as well as on mechanisms and modes of interaction with biological molecules and signaling pathways is necessary to perform chemical risk assessment and identify toxic compounds. To this end, we developed criteria and a pipeline for harvesting and summarizing effect concentrations from the US ECOTOX database for the three aquatic species groups algae, crustaceans, and fish and researched the modes of action of more than 3,300 environmentally relevant chemicals in literature and databases. We provide a curated dataset ready to be used for risk assessment based on monitoring data and the first comprehensive collection and categorization of modes of action of environmental chemicals. Authorities, regulators, and scientists can use this data for the grouping of chemicals, the establishment of meaningful assessment groups, and the development of in vitro and in silico approaches for chemical testing and assessment.

Most oxidative stress response in water samples comes from unknown chemicals: the need for effect-based water quality trigger values.

The induction of adaptive stress response pathways is an early and sensitive indicator of the presence of chemical and non-chemical stressors in cells. An important stress response is the Nrf-2 mediated oxidative stress response pathway where electrophilic chemicals or chemicals that cause the formation of reactive oxygen species initiate the production of antioxidants and metabolic detoxification enzymes. The AREc32 cell line is sensitive to chemicals inducing oxidative stress and has been previously applied for water quality monitoring of organic micropollutants and disinfection byproducts. Here we propose an algorithm for the derivation of effect-based water quality trigger values for this end point that is based on the combined effects of mixtures of regulated chemicals. Mixture experiments agreed with predictions by the mixture toxicity concept of concentration addition. The responses in the AREc32 and the concentrations of 269 individual chemicals were quantified in nine environmental samples, ranging from treated effluent, recycled water, stormwater to drinking water. The effects of the detected chemicals could explain less than 0.1% of the observed induction of the oxidative stress response in the sample, affirming the need to use effect-based trigger values that account for all chemicals present.

The impact of amine and carboxyl functionalised microplastics on the physiology of daphnids.

Plastic waste is considered a major threat for terrestrial, marine and freshwater ecosystems. Ingestion of primary or secondary microparticles resulting from plastic degradation can lead to their trophic transfer raising serious health concerns. In this study, the effect of amine and carboxy functionalized polystyrene microparticles on the physiology of daphnids was investigated with a combination of phenotypic and metabolic endpoints. Carboxy functionalized microparticles showed higher toxicity in acute exposures compared to their amine counterparts. Accumulation of both microparticles in animal gut was confirmed by stereo-microscopy as well as fluorescent microscopy which showed no presence of particles in the rest of the animal. Fluorescence based quantification of microparticles extracted from animal lysates validated their concentration-dependent uptake. Additionally, exposure of daphnids to amine and carboxy functionalized microparticles resulted in increased activities of key enzymes related to metabolism and detoxification. Finally, significant metabolic perturbations were discovered following exposure to microplastics. These findings suggest that polystyrene microparticles can hinder organism performance of the freshwater species and highlight the importance of seeking for holistic and physiological endpoints for pollution assessment.

A data-derived reference mixture representative of European wastewater treatment plant effluents to complement mixture assessment.

Aquatic environments are polluted with a multitude of organic micropollutants, which challenges risk assessment due the complexity and diversity of pollutant mixtures. The recognition that certain source-specific background pollution occurs ubiquitously in the aquatic environment might be one way forward to approach mixture risk assessment. To investigate this hypothesis, we prepared one typical and representative WWTP effluent mixture of organic micropollutants (EWERBmix) comprised of 81 compounds selected according to their high frequency of occurrence and toxic potential. Toxicological relevant effects of this reference mixture were measured in eight organism- and cell-based bioassays and compared with predicted mixture effects, which were calculated based on effect data of single chemicals retrieved from literature or different databases, and via quantitative structure-activity relationships (QSARs). The results show that the EWERBmix supports the identification of substances which should be considered in future monitoring efforts. It provides measures to estimate wastewater background concentrations in rivers under consideration of respective dilution factors, and to assess the extent of mixture risks to be expected from European WWTP effluents. The EWERBmix presents a reasonable proxy for regulatory authorities to develop and implement assessment approaches and regulatory measures to address mixture risks. The highlighted data gaps should be considered for prioritization of effect testing of most prevalent and relevant individual organic micropollutants of WWTP effluent background pollution. The here provided approach and EWERBmix are available for authorities and scientists for further investigations. The approach presented can furthermore serve as a roadmap guiding the development of archetypic background mixtures for other sources, geographical settings and chemical compounds, e.g. inorganic pollutants.

Mixture toxicity revisited from a toxicogenomic perspective.

The advent of new genomic techniques has raised expectations that central questions of mixture toxicology such as for mechanisms of low dose interactions can now be answered. This review provides an overview on experimental studies from the past decade that address diagnostic and/or mechanistic questions regarding the combined effects of chemical mixtures using toxicogenomic techniques. From 2002 to 2011, 41 studies were published with a focus on mixture toxicity assessment. Primarily multiplexed quantification of gene transcripts was performed, though metabolomic and proteomic analysis of joint exposures have also been undertaken. It is now standard to explicitly state criteria for selecting concentrations and provide insight into data transformation and statistical treatment with respect to minimizing sources of undue variability. Bioinformatic analysis of toxicogenomic data, by contrast, is still a field with diverse and rapidly evolving tools. The reported combined effect assessments are discussed in the light of established toxicological dose-response and mixture toxicity models. Receptor-based assays seem to be the most advanced toward establishing quantitative relationships between exposure and biological responses. Often transcriptomic responses are discussed based on the presence or absence of signals, where the interpretation may remain ambiguous due to methodological problems. The majority of mixture studies design their studies to compare the recorded mixture outcome against responses for individual components only. This stands in stark contrast to our existing understanding of joint biological activity at the levels of chemical target interactions and apical combined effects. By joining established mixture effect models with toxicokinetic and -dynamic thinking, we suggest a conceptual framework that may help to overcome the current limitation of providing mainly anecdotal evidence on mixture effects. To achieve this we suggest (i) to design studies to establish quantitative relationships between dose and time dependency of responses and (ii) to adopt mixture toxicity models. Moreover, (iii) utilization of novel bioinformatic tools and (iv) stress response concepts could be productive to translate multiple responses into hypotheses on the relationships between general stress and specific toxicity reactions of organisms.

Concentration-response concept in ecotoxicoproteomics: effects of different phenanthrene concentrations to the zebrafish (Danio rerio) embryo proteome.

Concentration-response experiments, based on the testing of less replicates in favour of more exposure concentrations, represent the typical design of choice applied in toxicological and ecotoxicological effect assessment studies using traditional endpoints such as lethality. However, to our knowledge this concept has not found implementation in the increasingly applied OMICS techniques studying thousands of molecular endpoints at the same time. The present study is among the first applying the concentration-response concept for an ecotoxicoproteomics study. The effects of six different concentrations in the low effect range (

Photomotor response data analysis approach to assess chemical neurotoxicity with the zebrafish embryo.

The photomotor response (PMR) of zebrafish embryos, a light pulse-triggered undirected movement, is known to be altered by neuroactive chemicals. Here, we developed an approach for data analysis of the distribution of PMR movement activities along the time axis; differences between treatment and respective controls are expressed by an aggregated value integrating the time-resolved density of the movement parameter as a measure for a chemically elicited PMR effect. Logistic concentration-PMR effect relationships were modeled for neuroactive test compounds with different modes of action (acetylcholinesterase inhibition, activation and inhibition of voltage-gated sodium channels); 50% effect concentrations (EC50) were in the low to medium µM range (EC50 < 10 µM for flucythrinate, esfenvalerate, azinphos-methyl, propoxur; EC50 > 10 µM for tricaine). Modulation of movement activities in different phases of the PMR (i.e., "fingerprint") by neuroactive test compounds varied across concentrations, showing that mode of action-specific PMR fingerprints are also concentration-dependent. Above concentrations causing 10% lethality (LC10; 48 h), 3,4-dichlo­roaniline caused movement inhibition. This substance presumably is not neuroactive; its effect on the PMR therefore is considered a secondary toxic effect. Quantitative morphological examinations of chemically exposed embryos showed that malformations occurred only above PMR effect concentrations, indicating that changes in the PMR were not due to such indirect effects. The PMR assay will provide a useful measure in ecotoxicological risk assessment of neuroactive chemicals with zebrafish embryos and could potentially be used to infer acute fish toxicity levels from PMR effect concen­trations of neurotoxic compounds.

The Effects of Single and Combined Stressors on Daphnids-Enzyme Markers of Physiology and Metabolomics Validate the Impact of Pollution.

The continuous global increase in population and consumption of resources due to human activities has had a significant impact on the environment. Therefore, assessment of environmental exposure to toxic chemicals as well as their impact on biological systems is of significant importance. Freshwater systems are currently under threat and monitored; however, current methods for pollution assessment can neither provide mechanistic insight nor predict adverse effects from complex pollution. Using daphnids as a bioindicator, we assessed the impact in acute exposures of eight individual chemicals and specifically two metals, four pharmaceuticals, a pesticide and a stimulant, and their composite mixture combining phenotypic, biochemical and metabolic markers of physiology. Toxicity levels were in the same order of magnitude and significantly enhanced in the composite mixture. Results from individual chemicals showed distinct biochemical responses for key enzyme activities such as phosphatases, lipase, peptidase, ß-galactosidase and glutathione-S-transferase. Following this, a more realistic mixture scenario was assessed with the aforementioned enzyme markers and a metabolomic approach. A clear dose-dependent effect for the composite mixture was validated with enzyme markers of physiology, and the metabolomic analysis verified the effects observed, thus providing a sensitive metrics in metabolite perturbations. Our study highlights that sensitive enzyme markers can be used in advance on the design of metabolic and holistic assays to guide the selection of chemicals and the trajectory of the study, while providing mechanistic insight. In the future this could prove to become a useful tool for understanding and predicting freshwater pollution.