An international cross-laboratory survey on fish vitellogenin analysis: Methodological challenges and opportunities for best practice.

Vitellogenin (VTG) is a biomarker for possible endocrine activity of chemicals acting via the estrogen, androgen, or steroidogenesis pathways. VTG is assessed in standardised fish guideline studies conducted for regulatory safety assessment of chemicals. VTG data can be highly variable leading to concerns for potential equivocal, false positive and/or negative outcomes. Consequently, additional fish testing may be required to address uncertainties in the VTG response, and possibly erroneous/missed identification of endocrine activity. To better understand the technical challenges of VTG assessment and reporting for regulatory purposes, a survey was sent to 27 testing laboratories performing these analyses. The survey results from 16 respondents (6 from the UK, 3 from the USA, and 7 from the EU) were analysed and discussed in a follow-up webinar. High variability in background VTG concentrations was widely acknowledged and thought to be associated with fish batch, husbandry, laboratory practices, and several methodological aspects. These include sample collection and storage, VTG quantification, data handling, and the benchmarks used for data acceptability. Information gathered in the survey provides a basis for improving and harmonizing the measurement of VTG in fish, and an opportunity to reassess the suitability of current acceptability criteria in test guidelines.

Assessment of the bioaccumulation potential of four commonly used phenolic benzotriazoles based on in silico and experimental in vivo data.

Phenolic benzotriazoles (BTZs) are used globally as light stabilizers in various plastic products to protect them from photooxidative degradation. The same physical-chemical properties that confer their functionality, like a sufficient photostability and a high octanol-water partition coefficient, also raise concerns on their potential for environmental persistence and bioaccumulation based on in silico predictive tools. To evaluate their bioaccumulation potential in aquatic organisms, standardized fish bioaccumulation studies according to OECD TG 305 were conducted with four of the most commonly used BTZs: UV 234, UV 329, UV P, and UV 326. The resulting growth- and lipid-corrected BCF values revealed that UV 234, UV 329, and UV P were below the bioaccumulation threshold (BCF ≤ 2000), but UV 326 is considered very bioaccumulative (BCF ≥ 5000) with respect to the bioaccumulation criteria under REACH. Comparing these experimentally derived data with quantitative structure activity related or other calculated values using a logarithmic partitioning coefficient octanol-water (log Pow ) driven mathematical formula revealed significant discrepancies demonstrating the weakness of current in silico approaches for this group of substances. Furthermore, available environmental monitoring data demonstrate that these rudimentary in silico approaches can lead to unreliable bioaccumulation estimates for this chemical class due to considerable uncertainties in underlying assumptions (e.g., concentration and route of exposure). However, using more sophisticated in silico methods (i.e., CATALOGIC base-line model), the derived BCF values were better aligned with the experimentally derived ones.

Are changes in vitellogenin concentrations in fish reliable indicators of chemical-induced endocrine activity?

Vitellogenin (VTG), a biomarker for endocrine activity, is a mechanistic component of the regulatory assessment of potential endocrine-disrupting properties of chemicals. This review of VTG data is based on changes reported for 106 substances in standard fish species. High intra-study and inter-laboratory variability in VTG concentrations was confirmed, as well as discrepancies in interpretation of results based on large differences between fish in the dilution water versus solvent control, or due to the presence of outlier measurements. VTG responses in fish were ranked against predictions for estrogen receptor agonist activity and aromatase inhibition from bioactivity model output and ToxCast in vitro assay results, respectively. These endocrine mechanisms explained most of the VTG responses in the absence of systemic toxicity, the magnitude of the VTG response being proportional to the in vitro potency. Interpretation of the VTG data was sometimes confounded by an alternative endocrine mechanism of action. There was evidence for both false positive and negative responses for VTG synthesis, but overall, it was rare for substances without endocrine activity in vitro to cause a concentration-dependent VTG response in fish in the absence of systemic toxicity. To increase confidence in the VTG results, we recommend improvements in the VTG measurement methodologies and greater transparency in reporting of VTG data (including quality control criteria for assay performance). This review supports the application of New Approach Methodologies (NAMs) by demonstrating that endocrine activity in vitro from mammalian cell lines is predictive for in vivo VTG response in fish, suggesting that in vitro mechanistic data could be used more broadly in decision-making to help reduce animal testing.

Considerations of Environmentally Relevant Test Conditions for Improved Evaluation of Ecological Hazards of Engineered Nanomaterials.

Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.

Adapting OECD Aquatic Toxicity Tests for Use with Manufactured Nanomaterials: Key Issues and Consensus Recommendations.

The unique or enhanced properties of manufactured nanomaterials (MNs) suggest that their use in nanoenabled products will continue to increase. This will result in increased potential for human and environmental exposure to MNs during manufacturing, use, and disposal of nanoenabled products. Scientifically based risk assessment for MNs necessitates the development of reproducible, standardized hazard testing methods such as those provided by the Organisation of Economic Cooperation and Development (OECD). Currently, there is no comprehensive guidance on how best to address testing issues specific to MN particulate, fibrous, or colloidal properties. This paper summarizes the findings from an expert workshop convened to develop a guidance document that addresses the difficulties encountered when testing MNs using OECD aquatic and sediment test guidelines. Critical components were identified by workshop participants that require specific guidance for MN testing: preparation of dispersions, dose metrics, the importance and challenges associated with maintaining and monitoring exposure levels, and the need for reliable methods to quantify MNs in complex media. To facilitate a scientific advance in the consistency of nanoecotoxicology test results, we identify and discuss critical considerations where expert consensus recommendations were and were not achieved and provide specific research recommendations to resolve issues for which consensus was not reached. This process will enable the development of prescriptive testing guidance for MNs. Critically, we highlight the need to quantify and properly interpret and express exposure during the bioassays used to determine hazard values.

Endocrine disruption assessment in aquatic vertebrates - Identification of substance-induced thyroid-mediated effect patterns.

According to the World Health Organisation and European Commission definitions, substances shall be considered as having endocrine disrupting properties if they show adverse effects, have endocrine activity and the adverse effects are a consequence of the endocrine activity (using a weight-of-evidence approach based on biological plausibility), unless the adverse effects are not relevant to humans or non-target organisms at the (sub)population level. To date, there is no decision logic on how to establish endocrine disruption via the thyroid modality in non-mammalian vertebrates. This paper describes an evidence-based decision logic compliant with the integrated approach to testing and assessment (IATA) concept, to identify thyroid-mediated effect patterns in aquatic vertebrates using amphibians as relevant models for thyroid disruption assessment. The decision logic includes existing test guidelines and methods and proposes detailed considerations on how to select relevant assays and interpret the findings. If the mammalian dataset used as the starting point indicates no thyroid concern, the Xenopus Eleutheroembryonic Thyroid Assay allows checking out thyroid-mediated activity in non-mammalian vertebrates, whereas the Amphibian Metamorphosis Assay or its extended, fixed termination stage variant inform on both thyroid-mediated activity and potentially population-relevant adversity. In evaluating findings, the response patterns of all assay endpoints are considered, including the direction of changes. Thyroid-mediated effect patterns identified at the individual level in the amphibian tests are followed by mode-of-action and population relevance assessments. Finally, all data are considered in an overarching weight-of-evidence evaluation. The logic has been designed generically and can be adapted, e.g. to accommodate fish tests once available for thyroid disruption assessments. It also ensures that all scientifically relevant information is considered, and that animal testing is minimised. The proposed decision logic can be included in regulatory assessments to facilitate the conclusion on whether substances meet the endocrine disruptor definition for the thyroid modality in non-mammalian vertebrates.

Aquatic testing guidelines insufficiently control the influence of dilution water toc and hardness on cationic polymer toxicity - A proposal to improve standardized test procedures.

Cationic polymers (CPs) are widely used chemicals for wastewater treatment applications and in various "down-the-drain" household products. The aquatic toxicity of CPs results from an electrostatic interaction with negatively charged cell surfaces. These effects are greatly mitigated by the binding affinity of CPs to total organic carbon (TOC) in surface water. Consequently, baseline aquatic toxicity tests of CPs using clean lab water (TOC < 2 mg/L) typically overestimate toxicity and risk which is greatly mitigated at higher environmentally relevant OC levels. However, the point at which mitigation begins is not well defined and low-level TOC in lab water may influence the baseline toxicity outcome. Similarly, divalent cations, quantified as water hardness, may modulate the electrostatic binding between OC and CP. Although standard guidelines define limits for lab water hardness and TOC, the consequences of variability within those limits on test outcome is unknown. We investigated the impact of part-per-billion (ppb) additions of TOC to lab water at different hardness levels on CP acute toxicity to Daphnia magna and Raphidocelis subcapitata. In both species, the acute toxicities of CPs with different molecular weight and charge density varied by > 10-fold in response to slight changes in TOC and water hardness, although parameters were maintained within guideline limits. When determining the baseline aquatic toxicity of CPs, the lab water should be standardized at the lowest biologically tolerable hardness and TOC at a reliably measurable level (>1 - < 2 mg/L) to reduce variability and increase the reliability of the toxicity estimate.

Evaluation of the bioaccumulation of octocrylene after dietary and aqueous exposure.

Octocrylene is used as UV filter in personal care products with a high production volume and can be detected in surface water and biota. It is liquid at ambient temperature, highly lipophilic, has a high adsorption capacity to organic material and is considered as persistent in the environment. The very low water solubility complicates the evaluation of potential long-term effects in aquatic toxicity testing, since effect thresholds are often above the water solubility limit. Thus, the evaluation of the bioaccumulation potential becomes highly relevant for the assessment of long-term environmental effects. However, even the determination of the water solubility limit for a substance with such difficult properties is challenging. The following experiments are described, and results compared to available environmental monitoring data: A bioconcentration study with aqueous exposure (BCF) in zebrafish and a biomagnification study with dietary exposure (BMF) in rainbow trout, as well as supporting experiments to evaluate the water solubility. The growth and lipid corrected BCF determined by aqueous exposure was 858 L kg-1 while the corrected BMF was 0.0335. The model-based estimation of the BCF from BMF (152-1182 L kg-1) is in good agreement with the measured BCF value. Environmental monitoring data provide only limited information on the bioaccumulation potential of octocrylene, as only few investigations were made in biota and water in parallel and concentrations of octocrylene vary by several orders of magnitude during seasons. Based on the determined fish BCF data, we conclude that OCR is not bioaccumulative according to the criteria as laid down by ECHA, 2017. Furthermore, the low BMF value indicates no accumulation along the food chain.

Intrinsic Xenobiotic Metabolizing Enzyme Activities in Early Life Stages of Zebrafish (Danio rerio).

Early life stages of zebrafish (Danio rerio, zf) are gaining attention as an alternative invivo test system for drug discovery, early developmental toxicity screenings and chemical testing in ecotoxicological and toxicological testing strategies. Previous studies have demonstrated transcriptional evidence for xenobiotic metabolizing enzymes (XME) during early zf development. However, elaborate experiments on XME activities during development are incomplete. In this work, the intrinsic activities of representative phase I and II XME were monitored by transformation of putative zf model substrates analyzed using photometry and high pressure liquid chromatography techniques. Six different defined stages of zf development (between 2.5 h postfertilization (hpf) to 120 hpf) were investigated by preparing a subcellular fraction from whole organism homogenates. We demonstrated that zf embryos as early as 2.5 hpf possess intrinsic metabolic activities for esterase, Aldh, Gst, and Cyp1a above the methodological detection limit. The activities of the enzymes Cyp3a and Nat were measurable during later stages in development. Activities represent dynamic patterns during development. The role of XME activities revealed in this work is relevant for the assessing toxicity in this test system and therefore contributes to a valuable characterization of zf embryos as an alternative testing organism in toxicology.

Lignosulfonic acid blocks in vitro fertilization of macaque oocytes when sperm are treated either before or after capacitation.

Lignin-derived macromolecules (LDMs) are biologically active compounds that affect a variety of cell-to-cell interactions including the inhibition of fertilization and embryo development in a number of nonmammalian species. The effect of ligno-sulfonic acid (LSA), a highly sulfonated LDM, on cynomolgus macaque sperm-oocyte interaction was evaluated with a zona pellucida binding assay and by in vitro fertilization (IVF). Sperm were treated with LSA (1.5 mg/mL) either before washing or after capacitation. Capacitation included centrifugation through 80% Percoll followed by 2 consecutive washes with medium, overnight incubation, and activation with dibutyryl cyclic adenosine monophosphate and caffeine. The zona binding assay was performed using immature oocytes that had adhered to the center of glass "binding chambers." The number of capacitated sperm that attached to the zona over a 3-minute period was recorded. Sperm attachment was significantly inhibited by LSA as compared to controls whether treatment occurred after capacitation (92.5%; P <.001) or before washing (82.5%; P <.001). When sperm were treated similarly with fucoidin, a sulfated polysaccharide known to inhibit sperm-oocyte interaction, sperm-zona binding was significantly inhibited by postcapacitation treatment but not by prewash treatment. Treatment of sperm with LSA consistently blocked fertilization over 4 IVF cycles both before washing and after capacitation. Fertilization rate for controls was 65% +/- 17%. No LSA-treated sperm were observed on the surface of lightly rinsed oocytes after 4 hours of coincubation. Localization of biotinylated LSA showed labeling over the entire sperm surface with the greatest intensity observed over the head and midpiece. LSA treatment had no effect on the percentage of motile sperm or quality of sperm motility. Due to the antifertility properties of this nontoxic molecule, LSA appears to have potential as a vaginal contraceptive.

Aquatic toxicity tests with substances that are poorly soluble in water and consequences for environmental risk assessment.

Aquatic toxicity tests with substances that are poorly soluble in water have been conducted using different methods, and estimates of toxicity have varied accordingly. The present study illustrates differences in toxicity values resulting from variation in test designs and solution preparation methods, and offers guidance on the best way to conduct these tests. Consequences for environmental risk assessment and classification are also discussed. The present study mainly considers active ingredients of plant protection products, but is also considered relevant to other chemicals. It is recommended that toxicity tests be conducted only up to the saturation limit, dispersants avoided, and solvents used only if necessary to support handling and speed of dissolution. Analytical measurements of exposure concentrations should reflect what organisms are exposed to. If acute toxicity testing at the saturation limit yields no adverse effects, further testing should not normally be required; the toxicity value of the endpoints should be considered as the saturation limit and adverse classification should not be required. Chronic testing, if required, should then be conducted at the practical saturation limit as this is the most realistic worst-case exposure scenario. If no adverse effects occur, the risk should be acceptable because higher aqueous exposure cannot occur. This could be substantiated by testing additional species. Assessment factors on no observed effect concentration (NOEC) values at the saturation limit require careful consideration in the risk assessment to avoid unnecessarily low regulatory acceptable concentrations.