Addressing chemical pollution in biodiversity research.

Climate change, biodiversity loss, and chemical pollution are planetary-scale emergencies requiring urgent mitigation actions. As these "triple crises" are deeply interlinked, they need to be tackled in an integrative manner. However, while climate change and biodiversity are often studied together, chemical pollution as a global change factor contributing to worldwide biodiversity loss has received much less attention in biodiversity research so far. Here, we review evidence showing that the multifaceted effects of anthropogenic chemicals in the environment are posing a growing threat to biodiversity and ecosystems. Therefore, failure to account for pollution effects may significantly undermine the success of biodiversity protection efforts. We argue that progress in understanding and counteracting the negative impact of chemical pollution on biodiversity requires collective efforts of scientists from different disciplines, including but not limited to ecology, ecotoxicology, and environmental chemistry. Importantly, recent developments in these fields have now enabled comprehensive studies that could efficiently address the manifold interactions between chemicals and ecosystems. Based on their experience with intricate studies of biodiversity, ecologists are well equipped to embrace the additional challenge of chemical complexity through interdisciplinary collaborations. This offers a unique opportunity to jointly advance a seminal frontier in pollution ecology and facilitate the development of innovative solutions for environmental protection.

Systematic evidence on migrating and extractable food contact chemicals: Most chemicals detected in food contact materials are not listed for use.

Food packaging is important for today's globalized food system, but food contact materials (FCMs) can also be a source of hazardous chemicals migrating into foodstuffs. Assessing the impacts of FCMs on human health requires a comprehensive identification of the chemicals they contain, the food contact chemicals (FCCs). We systematically compiled the "database on migrating and extractable food contact chemicals" (FCCmigex) using information from 1210 studies. We found that to date 2881 FCCs have been detected, in a total of six FCM groups (Plastics, Paper & Board, Metal, Multi-materials, Glass & Ceramic, and Other FCMs). 65% of these detected FCCs were previously not known to be used in FCMs. Conversely, of the more than 12'000 FCCs known to be used, only 1013 are included in the FCCmigex database. Plastic is the most studied FCM with 1975 FCCs detected. Our findings expand the universe of known FCCs to 14,153 chemicals. This knowledge contributes to developing non-hazardous FCMs that lead to safer food and support a circular economy.

Conflicts of Interest in the Assessment of Chemicals, Waste, and Pollution.

Pollution by chemicals and waste impacts human and ecosystem health on regional, national, and global scales, resulting, together with climate change and biodiversity loss, in a triple planetary crisis. Consequently, in 2022, countries agreed to establish an intergovernmental science-policy panel (SPP) on chemicals, waste, and pollution prevention, complementary to the existing intergovernmental science-policy bodies on climate change and biodiversity. To ensure the SPP's success, it is imperative to protect it from conflicts of interest (COI). Here, we (i) define and review the implications of COI, and its relevance for the management of chemicals, waste, and pollution; (ii) summarize established tactics to manufacture doubt in favor of vested interests, i.e., to counter scientific evidence and/or to promote misleading narratives favorable to financial interests; and (iii) illustrate these with selected examples. This analysis leads to a review of arguments for and against chemical industry representation in the SPP's work. We further (iv) rebut an assertion voiced by some that the chemical industry should be directly involved in the panel's work because it possesses data on chemicals essential for the panel's activities. Finally, (v) we present steps that should be taken to prevent the detrimental impacts of COI in the work of the SPP. In particular, we propose to include an independent auditor's role in the SPP to ensure that participation and processes follow clear COI rules. Among others, the auditor should evaluate the content of the assessments produced to ensure unbiased representation of information that underpins the SPP's activities.

Zebrafish early life stages as alternative model to study 'designer drugs': Concordance with mammals in response to opioids.

The number of new psychoactive substances (NPS) on the illicit drug market increases fast, posing a need to urgently understand their toxicity and behavioural effects. However, with currently available rodent models, NPS assessment is limited to a few substances per year. Therefore, zebrafish (Danio rerio) embryos and larvae have been suggested as an alternative model that would require less time and resources to perform an initial assessment and could help to prioritize substances for subsequent evaluation in rodents. To validate this model, more information on the concordance of zebrafish larvae and mammalian responses to specific classes of NPS is needed. Here, we studied toxicity and behavioural effects of opioids in zebrafish early life stages. Synthetic opioids are a class of NPS that are often used in pain medication but also frequently abused, having caused multiple intoxications and fatalities recently. Our data shows that fentanyl derivatives were the most toxic among the tested opioids, with toxicity in the zebrafish embryo toxicity test decreasing in the following order: butyrfentanyl>3-methylfentanyl>fentanyl>tramadol> O-desmethyltramadol>morphine. Similar to rodents, tramadol as well as fentanyl and its derivatives led to hypoactive behaviour in zebrafish larvae, with 3-methylfentanyl being the most potent. Physico-chemical properties-based predictions of chemicals' uptake into zebrafish embryos and larvae correlated well with the effects observed. Further, the biotransformation pattern of butyrfentanyl in zebrafish larvae was reminiscent of that in humans. Comparison of toxicity and behavioural responses to opioids in zebrafish and rodents supports zebrafish as a suitable alternative model for rapidly testing synthetic opioids.

Role of epidemiology in risk assessment: a case study of five ortho-phthalates.

BACKGROUND: The association between environmental chemical exposures and chronic diseases is of increasing concern. Chemical risk assessment relies heavily on pre-market toxicity testing to identify safe levels of exposure, often known as reference doses (RfD), expected to be protective of human health. Although some RfDs have been reassessed in light of new hazard information, it is not a common practice. Continuous surveillance of animal and human data, both in terms of exposures and associated health outcomes, could provide valuable information to risk assessors and regulators. Using ortho-phthalates as case study, we asked whether RfDs deduced from male reproductive toxicity studies and set by traditional regulatory toxicology approaches sufficiently protect the population for other health outcomes. METHODS: We searched for epidemiological studies on benzyl butyl phthalate (BBP), diisobutyl phthalate (DIBP), dibutyl phthalate (DBP), dicyclohexyl phthalate (DCHP), and bis(2-ethylhexyl) phthalate (DEHP). Data were extracted from studies where any of the five chemicals or their metabolites were measured and showed a statistically significant association with a health outcome; 38 studies met the criteria. We estimated intake for each phthalate from urinary metabolite concentration and compared estimated intake ranges associated with health endpoints to each phthalate's RfD. RESULT: For DBP, DIBP, and BBP, the estimated intake ranges significantly associated with health endpoints were all below their individual RfDs. For DEHP, the intake range included associations at levels both below and above its RfD. For DCHP, no relevant studies could be identified. The significantly affected endpoints revealed by our analysis include metabolic, neurodevelopmental and behavioral disorders, obesity, and changes in hormone levels. Most of these conditions are not routinely evaluated in animal testing employed in regulatory toxicology. CONCLUSION: We conclude that for DBP, DIBP, BBP, and DEHP current RfDs estimated based on male reproductive toxicity may not be sufficiently protective of other health effects. Thus, a new approach is needed where post-market exposures, epidemiological and clinical data are systematically reviewed to ensure adequate health protection.

Characterization of the Mercapturic Acid Pathway, an Important Phase II Biotransformation Route, in a Zebrafish Embryo Cell Line.

In view of the steadily increasing number of chemical compounds used in various products and applications, high-throughput toxicity screening techniques can help meeting the needs of 21st century risk assessment. Zebrafish (Danio rerio), especially its early life stages, are increasingly used in such screening efforts. In contrast, cell lines derived from this model organism have received less attention so far. A conceivable reason is the limited knowledge about their overall capacity to biotransform chemicals and the spectrum of expressed biotransformation pathways. One important biotransformation route is the mercapturic acid pathway, which protects organisms from harmful electrophilic compounds. The fully functional pathway involves a succession of several enzymatic reactions. To investigate the mercapturic acid pathway performance in the zebrafish embryonic cell line, PAC2, we analyzed the biotransformation products of the reactions comprising this pathway in the cells exposed to a nontoxic concentration of the reference substrate, 1-chloro-2,4-dinitrobenzene (CDNB). Additionally, we used targeted proteomics to measure the expression of cytosolic glutathione S-transferases (GSTs), the enzyme family catalyzing the first reaction in this pathway. Our results reveal that the PAC2 cell line expresses a fully functional mercapturic acid pathway. All but one of the intermediate CDNB biotransformation products were identified. The presence of the active mercapturic acid pathway in this cell line was further supported by the expression of a large palette of GST enzyme classes. Although the enzymes of the class alpha, one of the dominant GST classes in the zebrafish embryo, were not detected, this did not seem to affect the capacity of the PAC2 cells to biotransform CDNB. Our data provide an important contribution toward using zebrafish cell lines, specifically PAC2, for animal-free high- throughput screening in toxicology and chemical hazard assessment.

Impacts of food contact chemicals on human health: a consensus statement.

Food packaging is of high societal value because it conserves and protects food, makes food transportable and conveys information to consumers. It is also relevant for marketing, which is of economic significance. Other types of food contact articles, such as storage containers, processing equipment and filling lines, are also important for food production and food supply. Food contact articles are made up of one or multiple different food contact materials and consist of food contact chemicals. However, food contact chemicals transfer from all types of food contact materials and articles into food and, consequently, are taken up by humans. Here we highlight topics of concern based on scientific findings showing that food contact materials and articles are a relevant exposure pathway for known hazardous substances as well as for a plethora of toxicologically uncharacterized chemicals, both intentionally and non-intentionally added. We describe areas of certainty, like the fact that chemicals migrate from food contact articles into food, and uncertainty, for example unidentified chemicals migrating into food. Current safety assessment of food contact chemicals is ineffective at protecting human health. In addition, society is striving for waste reduction with a focus on food packaging. As a result, solutions are being developed toward reuse, recycling or alternative (non-plastic) materials. However, the critical aspect of chemical safety is often ignored. Developing solutions for improving the safety of food contact chemicals and for tackling the circular economy must include current scientific knowledge. This cannot be done in isolation but must include all relevant experts and stakeholders. Therefore, we provide an overview of areas of concern and related activities that will improve the safety of food contact articles and support a circular economy. Our aim is to initiate a broader discussion involving scientists with relevant expertise but not currently working on food contact materials, and decision makers and influencers addressing single-use food packaging due to environmental concerns. Ultimately, we aim to support science-based decision making in the interest of improving public health. Notably, reducing exposure to hazardous food contact chemicals contributes to the prevention of associated chronic diseases in the human population.

Clobetasol propionate causes immunosuppression in zebrafish (Danio rerio) at environmentally relevant concentrations.

Synthetic glucocorticoids (GCs) are potential endocrine disrupting compounds that have been detected in the aquatic environment around the world in the low ng/L (nanomolar) range. GCs are used as immunosuppressants in medicine. It is of high interest whether clobetasol propionate (CP), a highly potent GC, suppresses the inflammatory response in fish after exposure to environmentally relevant concentrations. Bacterial lipopolysaccharide (LPS) challenge was used to induce inflammation and thus mimic pathogen infection. Zebrafish embryos were exposed to ≤1000nM CP from ~1h post fertilization (hpf) to 96 hpf, and CP uptake, survival after LPS challenge, and expression of inflammation-related genes were examined. Our initial experiments were carried out using 0.001% DMSO as a solvent vehicle, but we observed that DMSO interfered with the LPS challenge assay, and thus masked the effects of CP. Therefore, DMSO was not used in the subsequent experiments. The internal CP concentration was quantifiable after exposure to ≥10nM CP for 96h. The bioconcentration factor (BCF) of CP was determined to be between 16 and 33 in zebrafish embryos. CP-exposed embryos showed a significantly higher survival rate in the LPS challenge assay after exposure to ≥0.1nM in a dose dependent manner. This effect is an indication of immunosuppression. Furthermore, the regulation pattern of several genes related to LPS challenge in mammals supported our results, providing evidence that LPS-mediated inflammatory pathways are conserved from mammals to teleost fish. Anxa1b, a GC-action related anti-inflammatory gene, was significantly down-regulated after exposure to ≥0.05nM CP. Our results show for the first time that synthetic GCs can suppress the innate immune system of fish at environmentally relevant concentrations. This may reduce the chances of fish to survive in the environment, as their defense against pathogens is weakened.

In Vitro Toxicity Testing of Food Contact Materials: State-of-the-Art and Future Challenges.

Currently, toxicological testing of food contact materials (FCMs) is focused on single substances and their genotoxicity. However, people are exposed to mixtures of chemicals migrating from food contact articles (FCAs) into food, and toxic effects other than genotoxic damage may also be relevant. Since FCMs can be made of more than 8 thousand substances, assessing them one-by-one is very resource-consuming. Moreover, finished FCAs usually contain non-intentionally added substances (NIAS). NIAS toxicity can only be tested if a substance's chemical identity is known and if it is available as a pure chemical. Often, this is not the case. Nonetheless, regulations require safety assessments for all substances migrating from FCAs, including NIAS, hence new approaches to meet this legal obligation are needed. Testing the overall migrate or extract from an FCM/FCA is an option. Ideally, such an assessment would be performed by means of in vitro bioassays, as they are rapid and cost-effective. Here, we review the studies using in vitro bioassays to test toxicity of FCMs/FCAs. Three main categories of in vitro assays that have been applied include assays for cytotoxicity, genotoxicity, and endocrine disruption potential. In addition, we reviewed studies with small multicellular animal-based bioassays. Our overview shows that in vitro testing of FCMs is in principle feasible. We discuss future research needs and FCM-specific challenges. Sample preparation procedures need to be optimized and standardized. Further, the array of in vitro tests should be expanded to include those of highest relevance for the most prevalent human diseases of concern.

Endocrine disrupting compounds affecting corticosteroid signaling pathways in Czech and Swiss waters: potential impact on fish.

This study investigated the occurrence of corticosteroid signaling disruptors in wastewaters and rivers in the Czech Republic and in Switzerland. 36 target compounds were detected using HPLC-MS/MS, with up to 6.4 µg/L for azole antifungals that indirectly affect corticosteroid signaling. Glucocorticoid receptor (GR)-mediated activity was determined using the GR-CALUX bioassay with dexamethasone equivalent concentrations ranging from

LC-MS/MS determination of potential endocrine disruptors of cortico signalling in rivers and wastewaters.

A targeted analytical method was established to determine a large number of chemicals known to interfere with the gluco- and mineralocorticoid signalling pathway. The analytes comprise 30 glucocorticoids and 9 mineralocorticoids. Ten out of these corticosteroids were primary metabolites. Additionally, 14 nonsteroids were included. These analytes represent a broader range of possible adverse modes of action than previously reported. For the simultaneous determination of these structurally diverse compounds, a single-step multimode solid-phase extraction and pre-concentration was applied. Extracts were separated by a short linear HPLC gradient (20 min) on a core shell RP column (2.7 µm particle size) and compounds identified and quantified by LC-MS/MS. The method provided excellent retention time reproducibility and detection limits in the low nanograms per litre range. Untreated hospital wastewater, wastewater treatment plant influent, treated effluent and river waters were analysed to demonstrate the applicability of the method. The results show that not all compounds were sufficiently eliminated by the wastewater treatment, resulting in the presence of several steroids (∼20 ng/L) and nonsteroids in the final effluent, some of them at high concentrations up to 200 ng/L. Most of the detected mono-hydroxylated steroidal transformation products were found at significantly higher concentrations than their parent compounds. We therefore recommend to include these potentially bioactive metabolites in environmental toxicity assessment.

Mass spectrometry in environmental toxicology.

In environmental toxicology, mass spectrometry can be applied to evaluate both exposure to chemicals as well as their effects in organisms. Various ultra-trace techniques are employed today to measure pollutants in different environmental compartments. Increasingly, effect-directed analysis is being applied to focus chemical monitoring on sites of ecotoxicological concern. Mass spectrometry is also very instrumental for studying the interactions of chemicals with organisms on the molecular and cellular level, providing new insights into mechanisms of toxicity. In the future, diverse mass spectrometry-based techniques are expected to become even more widely used in this field, contributing to the refinement of currently used environmental risk assessment strategies.

Chemical Aspects of Nanoparticle Ecotoxicology.

Nanoecotoxicology strives to understand the processes and mechanisms by which engineered nanoparticles (ENP) may exert toxic effects on aquatic organisms. Detailed knowledge of the chemical reactions of nanoparticles in the media and of their interactions with organisms is required to understand these effects. The processes of agglomeration of nanoparticles, of dissolution and release of toxic metal ions, and of production of reactive oxygen species (ROS) are considered in this article. Important questions concern the role of uptake of nanoparticles in various organisms, in contrast to uptake of ions released from nanoparticles and to nanoparticle attachment to organism surfaces. These interactions are illustrated for effects of silver nanoparticles (AgNP), cerium oxide (CeO2 NP) and titanium dioxide (TiO2 NP), on aquatic organisms, including algae, biofilms, fish cells and fish embryos.

Exploring BPA alternatives - Environmental levels and toxicity review.

Bisphenol A alternatives are manufactured as potentially less harmful substitutes of bisphenol A (BPA) that offer similar functionality. These alternatives are already in the market, entering the environment and thus raising ecological concerns. However, it can be expected that levels of BPA alternatives will dominate in the future, they are limited information on their environmental safety. The EU PARC project highlights BPA alternatives as priority chemicals and consolidates information on BPA alternatives, with a focus on environmental relevance and on the identification of the research gaps. The review highlighted aspects and future perspectives. In brief, an extension of environmental monitoring is crucial, extending it to cover BPA alternatives to track their levels and facilitate the timely implementation of mitigation measures. The biological activity has been studied for BPA alternatives, but in a non-systematic way and prioritized a limited number of chemicals. For several BPA alternatives, the data has already provided substantial evidence regarding their potential harm to the environment. We stress the importance of conducting more comprehensive assessments that go beyond the traditional reproductive studies and focus on overlooked relevant endpoints. Future research should also consider mixture effects, realistic environmental concentrations, and the long-term consequences on biota and ecosystems.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics.

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.