Resource and animal use implications of the proposed REACH information requirements for endocrine disruptor assessment.

Revised information requirements for endocrine disruptor (ED) assessment of chemicals under the European Union's Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation have been proposed. Implementation will substantially increase demands for new data to inform ED assessment. This article evaluates the potential animal use and financial resource associated with two proposed ED policy options, and highlights areas where further clarification is warranted. This evaluation demonstrates that studies potentially conducted to meet the proposed requirements could use tens of millions of animals, and that the approach is unlikely to be feasible in practice. Given the challenges with implementing either policy option and the need to minimise the reliance on animal testing, further consideration and clarification is needed on several aspects prior to implementation of the requirements. This includes how testing will be prioritised in a proportionate approach; how to harness new approach methodologies to waive higher-tier animal testing; and need for provision of clear guidance particularly in applying weight-of-evidence approaches. There is now a clear opportunity for the European Commission to lead the way in developing a robust and transparent ED assessment process for industrial chemicals which fully implements replacement, refinement, and reduction of the use of animals (the 3Rs).

Comprehensive Two-Dimensional Gas Chromatography with Peak Tracking for Screening of Constituent Biodegradation in Petroleum UVCB Substances.

Petroleum substances, as archetypical UVCBs (substances of unknown or variable composition, complex reaction products, or biological substances), pose a challenge for chemical risk assessment as they contain hundreds to thousands of individual constituents. It is particularly challenging to determine the biodegradability of petroleum substances since each constituent behaves differently. Testing the whole substance provides an average biodegradation, but it would be effectively impossible to obtain all constituents and test them individually. To overcome this challenge, comprehensive two-dimensional gas chromatography (GC × GC) in combination with advanced data-handling algorithms was applied to track and calculate degradation half-times (DT50s) of individual constituents in two dispersed middle distillate gas oils in seawater. By tracking >1000 peaks (representing ∼53-54% of the total mass across the entire chromatographic area), known biodegradation patterns of oil constituents were confirmed and extended to include many hundreds not currently investigated by traditional one-dimensional GC methods. Approximately 95% of the total tracked peak mass biodegraded after 64 days. By tracking the microbial community evolution, a correlation between the presence of functional microbial communities and the observed progression of DT50s between chemical classes was demonstrated. This approach could be used to screen the persistence of GC × GC-amenable constituents of petroleum substance UVCBs.

Increasing the reliability and reproducibility of aquatic ecotoxicology: Learn lessons from aquaculture research.

Regulatory ecotoxicology highly relies on aquatic toxicity studies carried out under controlled conditions. Researchers recently expressed increasing concern about their possible lack of repeatability/reproducibility in many cases. Poor experimental designs, inappropriate statistics and lack of accurate reporting are often pointed out. However, I believe that there is also insufficient attention paid to the various experimental conditions under which fish studies are conducted. These conditions encompass numerous factors (temperature, photoperiod, food, stressors…) which modulate fish response to chemicals. Their effects are poorly studied in ecotoxicology but have been investigated for decades in aquaculture research. It is therefore proposed herein to consider experimental ecotoxicology from an aquaculture perspective. An overview of modulating factors and plausible associated experimental flaws is presented, with emphasis to fish health, growth and reproduction which are the most common regulatory endpoints. Photoperiod and temperature mainly determine growth/reproductive status for which fish also have species and stage-specific nutritional requirements. Stressors, sex ratio, density, water quality and factorial interactions may induce experimental bias. Modulating factors can strongly limit findings applicability and might explain the lack of reproducibility in some cases. Aquaculture knowledge/experience can already allow avoiding some experimental flaws (e.g., stress) while further research is warranted for some other aspects (e.g., nutrition). Detailed reporting of fish husbandry and experimental conditions is of utmost importance for study quality assessment.

Mobility in the context of exposure-based assessment of chemicals for drinking water resource protection.

In order to protect European Union (EU) drinking water resources from chemical contamination, criteria for identifying persistent, mobile, and toxic (PMT) chemicals and very persistent and very mobile (vPvM) chemicals under the EU REACH Regulation were proposed by the German Environment Agency (Umweltbundesamt-UBA). Additionally, new hazard classes for PMT and vPvM substances in the revised EU classification, labeling, and packaging (CLP Regulation) are intended. Therefore, a reliable approach in the identification of potential drinking water resource contaminants is needed. The scientific basis of the property-based PMT/vPvM criteria, focusing on mobility, which dictates the migration of chemical drinking water sources, was evaluated, and a critical analysis of the deviation of sorption metrics from simple behavior was carried out. Based on our evaluation, a Koc may be used for nonionic substances on a screening level only, requiring a higher tier assessment. It is considered inappropriate for hydrophilic and ionizable chemicals, particularly for soils with low organic carbon contents. The nonextractable residue formation is complex and not well understood but remains significant in limiting the mobility of chemicals through soils and sediments. In order to inform the EU commission's work on the introduction of new hazard classes for PMT and vPvM substances into the European legislation, the derivation of a tiered approach is proposed, which utilizes the weight of evidence available, with adoption of appropriate higher tier models commensurate with the nature of the substance and the data available. Integr Environ Assess Manag 2023;19:775-791. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Scientific concepts and methods for moving persistence assessments into the 21st century.

The evaluation of a chemical substance's persistence is key to understanding its environmental fate, exposure concentration, and, ultimately, environmental risk. Traditional biodegradation test methods were developed many years ago for soluble, nonvolatile, single-constituent test substances, which do not represent the wide range of manufactured chemical substances. In addition, the Organisation for Economic Co-operation and Development (OECD) screening and simulation test methods do not fully reflect the environmental conditions into which substances are released and, therefore, estimates of chemical degradation half-lives can be very uncertain and may misrepresent real environmental processes. In this paper, we address the challenges and limitations facing current test methods and the scientific advances that are helping to both understand and provide solutions to them. Some of these advancements include the following: (1) robust methods that provide a deeper understanding of microbial composition, diversity, and abundance to ensure consistency and/or interpret variability between tests; (2) benchmarking tools and reference substances that aid in persistence evaluations through comparison against substances with well-quantified degradation profiles; (3) analytical methods that allow quantification for parent and metabolites at environmentally relevant concentrations, and inform on test substance bioavailability, biochemical pathways, rates of primary versus overall degradation, and rates of metabolite formation and decay; (4) modeling tools that predict the likelihood of microbial biotransformation, as well as biochemical pathways; and (5) modeling approaches that allow for derivation of more generally applicable biotransformation rate constants, by accounting for physical and/or chemical processes and test system design when evaluating test data. We also identify that, while such advancements could improve the certainty and accuracy of persistence assessments, the mechanisms and processes by which they are translated into regulatory practice and development of new OECD test guidelines need improving and accelerating. Where uncertainty remains, holistic weight of evidence approaches may be required to accurately assess the persistence of chemicals. Integr Environ Assess Manag 2022;18:1454-1487. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Moving persistence assessments into the 21st century: A role for weight-of-evidence and overall persistence.

Assessing the persistence of chemicals in the environment is a key element in existing regulatory frameworks to protect human health and ecosystems. Persistence in the environment depends on many fate processes, including abiotic and biotic transformations and physical partitioning, which depend on substances' physicochemical properties and environmental conditions. A main challenge in persistence assessment is that existing frameworks rely on simplistic and reductionist evaluation schemes that may lead substances to be falsely assessed as persistent or the other way around-to be falsely assessed as nonpersistent. Those evaluation schemes typically assess persistence against degradation half-lives determined in single-compartment simulation tests or against degradation levels measured in stringent screening tests. Most of the available test methods, however, do not apply to all types of substances, especially substances that are poorly soluble, complex in composition, highly sorptive, or volatile. In addition, the currently applied half-life criteria are derived mainly from a few legacy persistent organic pollutants, which do not represent the large diversity of substances entering the environment. Persistence assessment would undoubtedly benefit from the development of more flexible and holistic evaluation schemes including new concepts and methods. A weight-of-evidence (WoE) approach incorporating multiple influencing factors is needed to account for chemical fate and transformation in the whole environment so as to assess overall persistence. The present paper's aim is to begin to develop an integrated assessment framework that combines multimedia approaches to organize and interpret data using a clear WoE approach to allow for a more consistent, transparent, and thorough assessment of persistence. Integr Environ Assess Manag 2022;18:868-887. © 2021 ExxonMobil Biomedical Sciences, Inc. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch, Perca fluviatilis.

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated. Plasma cortisol and glucose levels increased markedly 1h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1-72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses. Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.

Is the Arrhenius-correction of biodegradation rates, as recommended through REACH guidance, fit for environmentally relevant conditions? An example from petroleum biodegradation in environmental systems.

Biodegradation is a major determinant of chemical persistence in the environment and an important consideration for PBT and environmental risk assessments. It is influenced by several environmental factors including temperature and microbial community structure. According to REACH guidance, a temperature correction based on the Arrhenius equation is recommended for chemical persistence data not performed at the recommended EU mean surface water temperature. Such corrections, however, can lead to overly conservative P/vP assessments. In this paper, the relevance of this temperature correction is assessed for petroleum hydrocarbons, using measured surface water (marine and freshwater) degradation half-time (DT50) and degradation half-life (HL) data compiled from relevant literature. Stringent screening criteria were used to specifically select data from biodegradation tests containing indigenous microbes and conducted at temperatures close to their ambient sampling temperature. As a result, ten independent studies were identified, with 993 data points covering 326 hydrocarbon constituents. These data were derived from tests conducted with natural seawater, or freshwater, at temperatures ranging from 5 to 21 °C. Regressions were performed on the full hydrocarbon dataset and on several individual hydrocarbons. The results were compared to the trend as predicted by the Arrhenius equation and using the activation energy (Ea) as recommend in the REACH Guidance. The comparison shows that the correction recommended in REACH Guidance over predicts the effect of temperature on hydrocarbon biodegradation. These results contrast with temperature manipulated inocula where the test temperature is different from the ambient sampling temperature. In these manipulated systems, the effect of temperature follows the Arrhenius equation more closely. In addition, a more striking effect of temperature on the lag phase was observed with longer lag phases more apparent at lower temperatures. This indicates that the effect of temperature may indeed be even lower when considering hydrocarbon biodegradation without the initial lag phase.

Physiological and proteomic responses to corticosteroid treatments in Eurasian perch, Perca fluviatilis: Investigation of immune-related parameters.

The comparative effects of cortisol and 11-deoxycorticosterone (DOC), two major corticosteroids in fish, have yet received little attention in teleosts. We evaluated the proteomic and immune responses of Eurasian perch to chronic corticosteroid treatments. We implanted immature perch with cortisol (80mg/kg) or DOC (4mg/kg) and measured the proportions of blood leucocytes, immune indices in the plasma, spleen and liver (complement and lysozyme activity, total immunoglobulin and immune gene expression in the tissues) and differential proteome expression (corticosteroid versus control) in the liver and the spleen on days 2, 4 and 14 post-treatment. Implantation of cortisol decreased the ratio of blood leucocytes and depressed Ig levels in both organs while DOC modulated the proportion of leucocyte sub-populations (increase in lymphocytes and decrease in granulocytes). In contrast, the innate humoral immunity was not strongly influenced by any of corticosteroid implants. The only immune parameter that was significantly affected was lysozyme, after DOC treatment. A number of proteins were differentially regulated by these hormones and some were identified in the liver (21 for cortisol and 8 for DOC) and in the spleen (10 for cortisol and 10 for DOC). None of the proteins was directly linked to immunity, except the natural killer enhancing factor, which was repressed by cortisol in the spleen. Our results also confirm that the proteins involved in energetic and glucose metabolism are affected by corticosteroids. Furthermore, these corticosteroids differently regulate immune status in Eurasian perch and they primarily impact leucocytes, as opposed to innate immune function.

A multi-biomarker approach to assess the impact of farming systems on black tiger shrimp (Penaeus monodon).

This study examined the advantages of the use of biomarkers as an early warning system by applying it to different shrimp farming systems in Soctrang and Camau provinces, main shrimp producers in Mekong River Delta, Vietnam. Shrimp were collected at 15 different farms divided into four different farming systems: three farms were converted from originally rice paddies into intensive shrimp farming systems (IS1, IS2, IS3); three farms were rice-shrimp integrated farming systems (RS4, RS5, RS6); three farms were intensive farming systems (IS7, IS8, IS9); six farms were extensive shrimp farming systems (From ES1 to ES6). Lipid peroxidation (LPO) and total glutathione (GSH) were measured as well as catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST) and acetylcholinesterase activities (ACHE). Organ specificity was observed between gills and hepatopancreas with generally higher activity of GST in gills (GSTG) whereas the contrary was observed for LPO level in gills (LPOG). Hierarchical clustering and principal component analysis clearly indicated that shrimp reared in extensive culture system formed a distinct group from those reared in intensive or rice-shrimp integrated systems. CAT in gills (CATG), GPX in gills (GPXG) and hepatopancreas (GPXHP) and ACHE in muscle (ACHEM) of shrimp collected in extensive farms showed a general higher level than those in intensively farmed shrimp. On the contrary, we observed clear high levels of GSTG and GST in hepatopancreas (GSTHP) and LPOG and hepatopancreas (LPOHP) of shrimp sampled in intensive and rice-shrimp integrated systems. Thus, we propose that LPO and CAT, GPX, GST and ACHE can be used as a set of biomarkers for the assessment of health condition and can discriminate between shrimp cultivated in different farming systems. These findings provide the usefulness of integrating a set of biomarkers to define the health status of shrimp in different shrimp culture systems.

Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon hypophthalmus, exposed to trichlorfon.

Trichlorfon (TRC) is the most common organophosphorous insecticide used in aquaculture practices in Southeast Asian countries. Indiscriminate use of TRC can either damage or alter the enzymatic and hormonal activities in the living organisms. In this present study, therefore, toxicogenomic analyses using real time PCR was used to characterize expression levels of various genes in Pangasianodon hypophthalmus after exposure to three concentrations, the 96 h 1/100LC(50) (0.01 mg/L), the 96 h 110LC(50) (0.1 mg/L) and the 96 h 12LC(50) (0.5 mg/L) of TRC for 6 h, 24 h, 96 h, 7 days, 14 days, 28 days and 56 days respectively. The expression kinetics of stress and other cellular toxicity representative genes such as heat shock protein70 (HSP70), growth hormone, acetylcholinesterase (AChE), trypsinogen, cytochrome P4501B (CYP1B) and cytochrome oxidase subunit 1 (COI) were investigated in liver and gills. TRC at a level of 0.1 mg/L and 0.5 mg/L induced a time and dose-dependent increase in the expression of the HSP70, COI and CYPIB while the transcript level of AChE, growth hormone and trypsinogen were significantly down-regulated. These results could permit to develop a "molecular biomarker system" which can be applied as a first-tier method of identifying contaminant exposure before effects at population level occur.

Adverse effects of enrofloxacin when associated with environmental stress in Tra catfish (Pangasianodon hypophthalmus).

The aim of this study was to assess the adverse effects of enrofloxacin (EF) on Tra catfish, Pangasianodon hypophthalmus, in relation with density stress. Fish were held at 40, 80 or 120 fish m(-3) and fed with pellets containing either 1 g kg(-1) EF or no EF. Antibiotic exposure lasted 7d and all fish were fed without EF for another 7-d recovery period. Fish were sampled at 3, 7, 8, 10 and 14 d after the beginning of EF exposure. Lipid peroxidation (LPO) and total glutathione (GSH) levels, catalase (CAT), glutathione-s-transferase (GST) and acetylcholine-esterase (AChE) activities were assessed in gill, brain, liver and muscle. At day 7, LPO levels in gills of EF-fish reared at low or high density were significantly more than 5-fold higher than their respective control. On the contrary, LPO in gills of EF-fish reared at medium density was significantly 3-fold lower than the control fish. Similarly, CAT activities in gills of EF-fish reared under low or high density were higher than in their control groups, while this activity was lower in EF-fish of the medium density group. AChE activities in muscles of EF-fish reared at low or high density were lower than controls at days 3 and 7, respectively. These results suggest that EF exposure may lead to disorders like lipid peroxidation and neural dysfunction in fish. However, when reared under lower stress condition (medium density), they may cope better with EF-induced stress than chronically stressed fish (low or high density).