Predictive modelling reveals Australian continental risk hotspots for marine debris interactions with key threatened species.

Anthropogenic debris is a global threat that impacts threatened species through various lethal and sub-lethal consequences, as well as overall ecosystem health. This study used a database of over 24,000 beach surveys of marine debris collated by the Australian Marine Debris Initiative from 2012 to 2021, with two key objectives: (1) identify variables that most influence the occurrence of debris hotspots on a continental scale and (2) use these findings to identify likely hotspots of interaction between threatened species and marine debris. The number of particles found in each beach survey was modelled alongside fifteen biological, social, and physical spatial variables including land use, physical oceanography, population, rainfall, distance to waste facilities, ports, and mangroves to identify the significant drivers of debris deposition. The model of best fit for predicting debris particle abundance was calculated using a generalized additive model. Overall, debris was more abundant at sites near catchments with high annual rainfall (mm), intensive land use (km2), and that were nearer to ports (km) and mangroves (km). These results support previous studies which state that mangroves are a significant sink for marine debris, and that large ports and urbanized catchments are significant sources for marine debris. We illustrate the applicability of these models by quantifying significant overlap between debris hotspots and the distributions for four internationally listed threatened species that exhibit debris interactions; green turtle (26,868 km2), dugong (16,164 km2), Australian sea lion (2903 km2) and Flesh-footed Shearwater (2413 km2). This equates to less than 1% (Flesh-footed Shearwater, Australian sea lion), over 2% (green sea turtle) and over 5% (dugong) of their habitat being identified as areas of high risk for marine debris interactions. The results of this study hold practical value, informing decision-making processes, managing debris pollution at continental scales, as well as identifying gaps in species monitoring.

Non-targeted proteomics reveals altered immune response in geographically distinct populations of green sea turtles (Chelonia mydas).

All seven species of sea turtle are facing increasing pressures from human activities that are impacting their health. Changes in circulating blood proteins of an individual, or all members of a population, can provide an early indicator of adverse health outcomes. Non-targeted measurement of all detectable proteins in a blood sample can indicate physiological changes. In the context of wildlife toxicology, this technique can provide a powerful tool for discovering biomarkers of chemical exposure and effect. This study presents a non-targeted examination of the protein abundance in sea turtle plasma obtained from three geographically distinct foraging populations of green turtles (Chelonia mydas) on the Queensland coast. Relative changes in protein expression between sites were compared, and potential markers of contaminant exposure were investigated. Blood plasma protein profiles were distinct between populations, with 85 out of the 116 identified proteins differentially expressed (p < 0.001). The most strongly dysregulated proteins were predominantly acute phase proteins, suggestive of differing immune status between the populations. The highest upregulation of known markers of immunotoxicity, such as pentraxin fusion and complement factor h, was observed in the Moreton Bay turtles. Forty-five different organohalogens were also measured in green turtle plasma samples as exposure to some organohalogens (e.g., polychlorinated biphenyls) has previously been identified as a cause for immune dysregulation in marine animals. The few detected organohalogens were at very low (pg/mL) concentrations in turtles from all sites, and are unlikely to be the cause of the proteome differences observed. However, the changes in protein expression may be indicative of exposure to other chemicals or environmental stressors. The results of this study provide important information about differences in protein expression between different populations of turtles, and guide future toxicological and health studies on east-Australian green sea turtles.

Interactions among multiple stressors vary with exposure duration and biological response.

Coastal ecosystems are exposed to multiple anthropogenic stressors. Effective management actions would be better informed from generalized predictions of the individual, combined and interactive effects of multiple stressors; however, few generalities are shared across different meta-analyses. Using an experimental study, we present an approach for analysing regression-based designs with generalized additive models that allowed us to capture nonlinear effects of exposure duration and stressor intensity and access interactions among stressors. We tested the approach on a globally distributed marine diatom, using 72 h photosynthesis and growth assays to quantify the individual and combined effects of three common water quality stressors; photosystem II-inhibiting herbicide exposure, dissolved inorganic nitrogen (DIN) enrichment and reduced light (due to excess suspended sediment). Exposure to DIN and reduced light generally resulted in additivity, while exposure to diuron and reduced light resulted in additive, antagonistic or synergistic interactions, depending on the stressor intensity, exposure period and biological response. We thus find the context of experimental studies to be a primary driver of interactions. The experimental and modelling approaches used here bridge the gap between two-way designs and regression-based studies, which provides a way forward to identify generalities in multiple stressor interactions.

Individual and combined effects of diuron and light reduction on marine microalgae.

Coastal ecosystems such as those in the Great Barrier Reef (GBR) lagoon, are exposed to stressors in flood plumes including low light (caused by increased turbidity) and agricultural pesticides. Photosystem II (PSII)-inhibiting herbicides are the most frequently detected pesticides in the GBR lagoon, but it is not clear how their toxicity to phototrophic species depends on light availability. This study investigated the individual and combined effects of PSII-inhibiting herbicide, diuron, and reduced light intensity (as a proxy for increased turbidity) on the marine diatom, Phaeodactylum tricornutum. Effective quantum yield (EQY) and cell density were measured to calculate responses relative to the controls over 72-h, in tests with varying stressor intensities. Individually, diuron concentrations (0.1-3 µg l-1) were not high enough to significantly reduce growth (cell density), but led to decreased EQY; while, low light generally led to increased EQY, but only reduced growth at the lowest tested light intensity (5 µmol photons m-2 s-1) after 48-hours. P. tricornutum was less affected by diuron when combined with low light scenarios, with increased EQY (up to 163% of the controls) that was likely due to increased electron transport per photon, despite lesser available photons at this low light intensity. In contrast, growth was completely inhibited relative to the controls when algae were simultaneously exposed to the highest stressor levels (3 µg l-1 diuron and 5 µmol photons m-2 s-1). This study highlights the importance of measuring more than one biological response variable to capture the combined effects of multiple stressors. Management of water quality stressors should consider combined impacts rather than just the impacts of individual stressors alone. Reducing suspended sediment and diuron concentrations in marine waters can decrease harmful effects and bring synergistic benefits to water quality.

The Karyotype of Blainville's Beaked Whale, Mesoplodon densirostris.

The karyotype of the Odontocete whale, Mesoplodon densirostris, has not been previously reported. The chromosome number is determined to be 2n = 42, and the karyotype is presented using G-, C-, and nucleolar organizer region (NOR) banding. The findings include NOR regions on 2 chromosomes, regions of heterochromatic variation, a large block of heterochromatin on the X chromosome, and a relatively large Y chromosome. The karyotype is compared to published karyograms of 2 other species of Mesoplodon.

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.

Adaption and application of cell-based bioassays to whole-water samples.

The increasing presence of contaminants of emerging concern in wastewater and their potential environmental risks require improved monitoring and analysis methods. Direct toxicity assessment (DTA) using bioassays can complement chemical analysis of wastewater discharge, but traditional in vivo tests have ethical considerations and are expensive, low-throughput, and limited to apical endpoints (mortality, reproduction, development, and growth). In vitro bioassays offer an alternative approach that is cheaper, faster, and more ethical, and can provide higher sensitivity for some environmentally relevant endpoints. This study explores the potential benefits of using whole water samples of wastewater and environmental surface water instead of traditional solid phase extraction (SPE) methods for in vitro bioassays testing. Whole water samples produced a stronger response in most bioassays, likely due to the loss or alteration of contaminants during SPE sample extraction. In addition, there was no notable difference in results for most bioassays after freezing whole water samples, which allows for increased flexibility in testing timelines and cost savings. These findings highlight the potential advantages of using whole water samples in DTA and provide a framework for future research in this area.

Elucidating the performance of UV-based photochemical processes for the removal of trace organic contaminants: Degradation and toxicity evaluation.

In this study, the performance of standalone ultraviolet (UV) photolysis and UV-based advanced oxidation processes (AOPs), namely, UV/hydrogen peroxide, UV/chlorine, UV/persulphate, and UV/permonosulphate, were investigated for the degradation of 31 trace organic contaminants (TrOCs). Under the tested conditions, standalone UV photolysis did not achieve effective removal of TrOCs. To improve the degradation efficiency of UV photolysis, four different oxidants were added individually to the test solution. The effect of these oxidants in the absence of UV irradiation was also explored and only chlorine showed promising degradation of some contaminants. During the chlorination of 31 investigated TrOCs, only six demonstrated greater than 50% degradation. The combined UV-based AOPs demonstrated much improved degradation (ranging from 65 to 100%) depending on TrOC-structure and oxidant concentration. The UV/hydrogen peroxide process showed similar degradation of TrOCs, irrespective of the functional groups (i.e., electron withdrawing groups, EWGs and electron donating groups, EDGs) present in their structures. Conversely, the UV/sulphate and UV/chlorine based processes achieved better degradation of the TrOCs with EDGs in their structures. TrOCs degradation improved up to 40% when oxidants concentrations were increased from 0.1 to 1 mM, and further increasing the concentration to 2 mM did not improve degradation. Toxicity evaluation using bioluminescence test (BLT assay) demonstrated that except for UV/hydrogen peroxide, all UV-based AOPs increased the toxicity of the treated effluent, indicating generation of toxic by-products. This study elucidates the performance of four different UV-based AOPs for the removal of commonly detected diverse TrOCs for the first time.

Correction: Trace element concentrations in forage seagrass species of Chelonia mydas along the Great Barrier Reef.

[This corrects the article DOI: 10.1371/journal.pone.0269806.].

Green Turtle (Chelonia mydas) Blood and Scute Trace Element Concentrations in the Northern Great Barrier Reef.

Marine turtles face numerous anthropogenic threats, including that of chemical contaminant exposure. The ecotoxicological impact of toxic metals is a global issue facing Chelonia mydas in coastal sites. Local investigation of C. mydas short-term blood metal profiles is an emerging field, while little research has been conducted on scute metal loads as potential indicators of long-term exposure. The aim of the present study was to investigate and describe C. mydas blood and scute metal profiles in coastal and offshore populations of the Great Barrier Reef. This was achieved by analyzing blood and scute material sampled from local C. mydas populations in five field sites, for a suite of ecologically relevant metals. By applying principal component analysis and comparing coastal sample data with those of reference intervals derived from the control site, insight was gleaned on local metal profiles of each population. Blood metal concentrations in turtles from coastal sites were typically elevated when compared with levels recorded in the offshore control population (Howick Island Group). Scute metal profiles were similar in Cockle Bay, Upstart Bay, and Edgecumbe Bay, all of which were distinct from that of Toolakea. Some elements were reported at similar concentrations in blood and scutes, but most were higher in scute samples, indicative of temporal accumulation. Coastal C. mydas populations may be at risk of toxic effects from metals such as Co, which was consistently found to be at concentrations magnitudes above region-specific reference intervals. Environ Toxicol Chem 2023;42:2375-2388. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Dose metric evaluation of a cell-based bioassay for assessing the toxicity of metals to Dugong dugon: Effect of metal-media interactions on exposure concentrations.

Cell-based toxicity testing has emerged as a useful tool in (eco)toxicological research, allowing the ethical assessment of the effects of contaminants such as trace metals on marine megafauna. However, metal interactions with various dissolved ligands in the microplate environment may influence the effective exposure concentrations. Hence, the cells are not exposed to the nominal concentrations within the test system. This study aimed to establish and evaluate the effectiveness of cell-based bioassays for investigating the toxicity of selected metals in dugongs through the following objectives: (1) measure the cytotoxic potential of cadmium (Cd2+), and chromium (Cr6+) to dugong skin cell cultures, (2) investigate the interactions between media constituents and selected trace metals in cell-based bioassays, and (3) evaluate the risk to a free-ranging population of dugong based on effect values. Chromium was the most toxic of the metals tested (EC50 = 1.14 µM), followed by Cd (EC50 = 6.35 µM). Assessment of ultrafiltered (< 3 kDa) exposure media showed that 1% and 92.5% of Cr and Cd were associated with larger organic components of the media. Further, the binding of Cd to media constituents was calculated to underestimate Cd toxicity in cell-based assays by an order of magnitude. This understanding of metal partitioning in cell-based bioassays provides a more accurate method for assessing toxicity in cell-based bioassays. In addition, this study illustrated that dugong cells are more sensitive to Cr and Cd than other marine wildlife species. The chemical risk assessment found the dugong population in Moreton Bay to be at high risk from Cd exposure.

Influence of chemical dose and exposure duration on protein synthesis in green sea turtle primary cells.

Understanding the impacts of chemical exposure in marine wildlife is challenging, due to practical and ethical constraints that preclude traditional toxicology research on these animals. This study addressed some of these limitations by presenting an ethical and high throughput cell-based approach to elucidate molecular-level effects of contaminants on sea turtles. The experimental design addressed basic questions of cell-based toxicology, including chemical dose and exposure time. Primary green turtle skin cells were exposed to polychlorinated biphenyl (PCB) 153 and perfluorononanoic acid (PFNA) for 24 and 48 h, at three sub-lethal, environmentally relevant concentrations (1, 10 and 100 µg/L). Sequential window acquisition of all theoretical mass spectra (SWATH-MS) identified over 1000 differentially abundant proteins within the 1% false discovery rate (FDR) threshold. The 24 h exposure resulted in a greater number of differentially abundant proteins, compared to 48 h exposure, for both contaminants. However, there were no statistically significant dose-response relationships for the number of differentially synthesised proteins, nor differences in the proportion of increased vs decreased proteins between or within exposure times. Known in vivo markers of contaminant exposure, superoxide dismutase and glutathione S-transferase, were differentially abundant following exposure to PCB153 and PFNA. SIGNIFICANCE: Cell-based (in vitro) proteomics provides an ethical and high throughput approach to understanding the impacts of chemical contamination on sea turtles. Through investigating effects of chemical dose and exposure duration on unique protein abundance in vitro, this study provides an optimised framework for conducting cell-based studies in wildlife proteomics, and highlights that proteins detected in vitro could act as biomarkers of chemical exposure and effect in vivo.

Understanding contaminant exposure risks in nesting Loggerhead sea turtle populations.

Queensland loggerhead turtle nest numbers at Mon Repos (MR) indicate population recovery that doesn't occur at Wreck Island (WI). Previous research illustrated that MR and WI turtles forage in different locations, potentially indicating risks differences. Blood, scute, and egg were collected from turtles nesting at MR and WI, with known foraging sites (from concurrent studies). Trace element and organic contaminants were assessed via acid digestion and in vitro cytotoxicity bioassays, respectively. WI turtles had significantly higher scute uranium and blood molybdenum compared to MR turtles, and arsenic was higher in WI turtles foraging north and MR turtles foraging south. Egg and blood titanium, manganese, cadmium, barium, lead, and molybdenum, and scute and egg selenium and mercury significantly correlated. Blood (75 %) extracts produced significant toxicity in vitro in turtle fibroblast cells. In conclusion, reducing chemical exposure at higher risk foraging sites would likely benefit sea turtles and their offspring.

Trace element concentrations in forage seagrass species of Chelonia mydas along the Great Barrier Reef.

Toxic metal exposure is a threat to green sea turtles (Chelonia mydas) inhabiting and foraging in coastal seagrass meadows and are of particular concern in local bays of the Great Barrier Reef (GBR), as numerous sources of metal contaminants are located within the region. Seagrass species tend to bioaccumulate metals at concentrations greater than that detected in the surrounding environment. Little is known regarding ecotoxicological impacts of environmental metal loads on seagrass or Chelonia mydas (C. mydas), and thus this study aimed to investigate and describe seagrass metal loads in three central GBR coastal sites and one offshore site located in the northern GBR. Primary seagrass forage of C. mydas was identified, and samples collected from foraging sites before and after the 2018/2019 wet season, and multivariate differences in metal profiles investigated between sites and sampling events. Most metals investigated were higher at one or more coastal sites, relative to data obtained from the offshore site, and cadmium (Cd), cobalt (Co), iron (Fe) and manganese (Mn) were found to be higher at all coastal sites. Principle Component Analysis (PCA) found that metal profiles in the coastal sites were similar, but all were distinctly different from that of the offshore data. Coastal foraging sites are influenced by land-based contaminants that can enter the coastal zone via river discharge during periods of heavy rainfall, and impact sites closest to sources. Bioavailability of metal elements are determined by complex interactions and processes that are largely unknown, but association between elevated metal loads and turtle disease warrants further investigation to better understand the impact of environmental contaminants on ecologically important seagrass and associated macrograzers.

Temporal changes in chemical contamination of green turtles (Chelonia mydas) foraging in a heavily industrialised seaport.

Port Curtis, a major shipping port, has undergone significant expansion in the last decade, with plans for further development into the future. These activities may result in an increase of contaminant concentrations, threatening local wildlife including sea turtles. This study used a species-specific in vitro bioassay to examine spatial and temporal differences in exposure to, and effects of, organic contaminants in green sea turtles foraging in Port Curtis. Blood was collected from 134 green sea turtles (Chelonia mydas) from five locations in the port over four years. Organic contaminants were extracted from blood, and the cytotoxicity of the extracts to primary green sea turtle cells was assessed. Results indicated spatially similar chemical contamination throughout Port Curtis, at levels significant to sea turtle health, and with signs that chemical contamination may be increasing over time. These results can provide valuable information on the health of green turtles as further development occurs.

Pelagic and coastal green turtles (Chelonia mydas) experience differences in chemical exposure and effect.

Green turtles foraging in coastal areas are exposed to land-based chemical pollutants that accumulate in the habitats to which they show high site fidelity. However, prior to coastal recruitment, they may be exposed to a different range of chemical threats. The recent development of species-specific in vitro bioassays for marine turtles allows for an effect-based assessment of toxicological endpoints. Blood was collected from green turtles of two life-stages, 'recent recruits' and 'coastal residents', in Hervey Bay and Moreton Bay. Organic contaminants were extracted from blood using the QuEChERS method, and cytotoxicity of the extracts measured in green turtle skin cells. Although not statistically significant, extracts from 'coastal residents' exhibited greater mean toxicity compared to 'recent recruits', possibly indicative of increased chemical accumulation from coastal habitat exposure. The bioassay results also indicated that turtles foraging in Hervey Bay are at greater risk of chemical exposure than those foraging in Moreton Bay.

Optimisation of an automated high-throughput micronucleus (HiTMiN) assay to measure genotoxicity of environmental contaminants.

Anthropogenic contaminants can have a variety of adverse effects on exposed organisms, including genotoxicity in the form of DNA damage. One of the most commonly used methods to evaluate genotoxicity in exposed organisms is the micronucleus (MN) assay. It provides an efficient assessment of chromosomal impairment due to either chromosomal rupture or mis-segregation during mitosis. However, evaluating chromosomal damage in the MN assay through manual microscopy is a highly time-consuming and somewhat subjective process. High-throughput evaluation with automated image analysis could reduce subjectivity and increase accuracy and throughput. In this study, we optimised and streamlined the HiTMiN assay, adapting the MN assay to a miniaturised, 96-well plate format with reduced steps, and applied it to both primary cells from green turtle fibroblasts (GT12s-p) and a freshwater fish hepatoma cell line (PLHC-1). Image analysis using both commercial (Columbus) and freely available (CellProfiler) software automated the scoring of MN, with improved precision and drastically reduced time compared to manual scoring and other available protocols. The assay was validated through exposure to two inorganic (chromium and cobalt) and one organic (the herbicide metolachlor) compounds, which are genotoxicants of concern in the marine environment. All compounds tested induced MN formation below cytotoxic concentrations. The HiTMiN assay presented here greatly increases the suitability of the MN assay as a quick, affordable, sensitive and accurate assay to measure genotoxicity of environmental samples in different cell lines.

Field-scale monitoring of green sea turtles (Chelonia mydas): Influence of site characteristics and capture technique on the blood metabolome.

Given their threatened status, there is considerable interest in establishing monitoring techniques that can be used to evaluate the health of sea turtles in the wild. The present study represents a methodological contribution towards field-scale metabolomic assessment of sea turtles, by exploring differences in blood biochemistry associated with site characteristics and capture technique. We compared the metabolome of blood from animals at three locations (two coastal and one reefal), collected from turtles that were either resting or active, and sampled across multiple seasons at one location. Our results show clear differences in the metabolome of turtles from the three locations, some of which are likely attributable to differences in diet or forage quality and others which may reflect differences in other factors (e.g., occurrence of land-based contaminants or other biotic and/or abiotic stressors) between coastal and reefal sites. Our analysis also revealed the influence of capture technique on metabolite profiles, with numerous markers of physical exertion in animals captured while active that were absent in turtles sampled while resting. We observed a modest potential for temporal differences in the metabolome, but controlling for sampling time did not change the overall conclusions of our study. This suggests that temporal differences in the metabolome warrant consideration when designing studies to evaluate the status of sea turtles in the wild, but that site characteristics and capture technique are bigger drivers. However, sample size for this comparison was relatively small and further investigation of seasonal differences in the metabolome are warranted. Research exploring each of these factors more closely will further contribute towards achieving robust metabolomics analysis of sea turtles across large spatial and temporal scales.

Review of ecologically relevant in vitro bioassays to supplement current in vivo tests for whole effluent toxicity testing - Part 1: Apical endpoints.

Whole effluent toxicity (WET) testing is commonly used to ensure that wastewater discharges do not pose an unacceptable risk to receiving environments. Traditional WET testing involves exposing animals to (waste)water samples to assess four major ecologically relevant apical endpoints: mortality, growth, development, and reproduction. Recently, with the widespread implementation of the 3Rs to replace, reduce and refine the use of animals in research and testing, there has been a global shift away from in vivo testing towards in vitro alternatives. However, prior to the inclusion of in vitro bioassays in regulatory frameworks, it is critical to establish their ecological relevance and technical suitability. This is part 1 of a two-part review that aims to identify in vitro bioassays that can be used in WET testing and relate them to ecologically relevant endpoints through toxicity pathways, providing the reader with a high-level overview of current capabilities. Part 1 of this review focuses on four apical endpoints currently included in WET testing: mortality, growth, development, and reproduction. For each endpoint, the link between responses at the molecular or cellular level, that can be measured in vitro, and the adverse outcome at the organism level were established through simplified toxicity pathways. Additionally, literature from 2015 to 2020 on the use of in vitro bioassays for water quality assessments was reviewed to identify a list of suitable bioassays for each endpoint. This review will enable the prioritization of relevant endpoints and bioassays for incorporation into WET testing.

Review of ecologically relevant in vitro bioassays to supplement current in vivo tests for whole effluent toxicity testing - Part 2: Non-apical endpoints.

Whole effluent toxicity (WET) testing uses whole animal exposures to assess the toxicity of complex mixtures, like wastewater. These assessments typically include four apical endpoints: mortality, growth, development, and reproduction. In the last decade, there has been a shift to alternative methods that align with the 3Rs to replace, reduce, and refine the use of animals in research. In vitro bioassays can provide a cost-effective, high-throughput, ethical alternative to in vivo assays. In addition, they can potentially include additional, more sensitive, environmentally relevant endpoints than traditional toxicity tests. However, the ecological relevance of these endpoints must be established before they are adopted into regulatory frameworks. This is Part 2 of a two-part review that aims to identify in vitro bioassays that are linked to ecologically relevant endpoints that could be included in WET testing. Part 2 of this review focuses on non-apical endpoints that should be incorporated into WET testing. In addition to the four apical endpoints addressed in Part 1, this review identified seven additional toxic outcomes: endocrine disruption, xenobiotic metabolism, carcinogenicity, oxidative stress, inflammation, immunotoxicity and neurotoxicity. For each, the response at the molecular or cellular level measured in vitro was linked to the response at the organism level through a toxicity pathway. Literature from 2015 to 2020 was used to identify suitable bioassays that could be incorporated into WET testing.