Toxicity Test Methods Research Articles

Parameters Optimization for Improving Bioluminescence Inhibition Assay Using Vibrio fischeri Bacteria to Detect Lipopolysaccharide Toxicity in Aquatic Environments.

Bioluminescence inhibition of Vibrio fischeri is a widely used method for toxicity testing in aquatic environments. Certain complex biological contaminants, such as lipopolysaccharide (LPS), can interfere with metabolic pathways during toxicity assays. The standard 15-minute Vibrio fischeri bioluminescence assay has limitations when evaluating and screening water toxicity against complex and emerging chemicals like LPS. To accurately determine the effects of such substances, it is crucial to use a bioassay that encompasses a sufficient cell cycle period. This study tested LPS at varying incubation times (ranging from 60s to 60min) and concentrations (1-1*10- 12 mg/ml) to identify the appropriate incubation time for bioluminescence inhibition and toxicity testing. The results indicated that bioluminescence inhibition begins within 60s and reaches maximum inhibition at 60min. However, at 30 and 45min, the bacterial response to different concentrations of LPS varied, with some concentrations causing increased bioluminescence. The EC50 values at different times (60s, 15, 30, 45, and 60min) were found to be 0.0012, 0.0063, 4.07e + 54, 3.85e-8, and 3.34e-9mg/ml respectively. This study highlights the importance of considering incubation time when using bioluminescence inhibition to detect acute toxicity in aquatic ecosystems. A longer incubation time may enhance the method's sensitivity and improve its ability to detect low levels of toxins, such as LPS, in water resources.

Comparative QSAR and q-RASAR modeling for aquatic toxicity of organic chemicals to three trout species: O. Clarkii, S. Namaycush, and S. Fontinalis

Oncorhynchus clarkii, Salvelinus fontinalis, and Salvelinus namaycush are vital trout species in North America, crucial for maintaining ecological balance, economic stability, and human health. These species thrive in cold, unpolluted waters and are highly vulnerable to contaminants. Given the rapid proliferation of industrial organic chemicals, traditional in vivo toxicity testing methods are inadequate to ensure timely and comprehensive risk assessments. Therefore, we employed in silico tools, namely Quantitative Structure-Activity Relationship (QSAR) and Quantitative Read-Across Structure-Activity Relationship (q-RASAR), to efficiently predict the aquatic toxicity of chemicals. Utilizing acute median lethal concentration (LC50) data from the US EPA’s ToxValDB, we developed the first-ever species-specific QSAR and q-RASAR models. The q-RASAR models outperformed traditional QSAR models by achieving higher internal and external statistical quality for each species. Key toxicity-determining descriptors included electrotopological state indices, autocorrelation descriptors, and similarity-based RASAR descriptors. For O. clarkii, the presence of chlorine atoms and rotatable bonds significantly influenced toxicity. S. fontinalis toxicity was strongly affected by polarizability, and van der Waals volumes, while S. namaycush showed sensitivity to weak hydrogen bond acceptors and topological complexity. The models predicted the toxicity of 1172 external compounds, identifying the most and least toxic chemicals for each species. This study not only offers the first comprehensive q-RASAR models for predicting trout species-specific toxicity but also provides novel insights into species-specific toxicological modes of action. The results contribute significantly to chemical screening and prioritization in aquatic risk assessments, effectively filling critical data gaps and advancing predictive modeling techniques.

A new approach for risk assessment of pulmonary toxicants: a preliminary study using didecyldimethylammonium chloride.

Current human risk assessments often rely on animal toxicity data to establish point of departure (POD) values, followed by the application of uncertainty factors. Consequently, there is growing interest in alternative toxicity testing methods that reduce reliance on animal models. In this study, we propose a novel approach for inhalation toxicity risk assessment that integrates in silico and in vitro methods. Human primary alveolar epithelial cells were exposed to aerosolized didecyldimethylammonium chloride (DDAC) to assess cytotoxicity. This was followed by transcriptome analysis and biological pathway investigation, utilizing adverse outcome pathway (AOP), to calculate the POD. Additionally, human DDAC exposure was simulated using a multiple-path particle dosimetry (MPPD) model to predict exposure levels in the human alveolar region via inhalation. The results from in silico and in vitro studies were then compared, and a comprehensive risk assessment was performed. The POD for AOP 452 key events-oxidative stress, inflammation, epithelial-mesenchymal transition (EMT), apoptosis, and autophagy-was found to range between 19.0 and 23.89ng/cm2, according to benchmark dose calculation tools. The estimated human exposure to DDAC in the alveolar region under actual exposure conditions was 0.164ng/cm2/day, resulting in a margin of exposure (MOE) ranging from 121 to 145, suggesting caution regarding DDAC inhalation exposure. This study presents a novel risk assessment method that compares estimated human inhalation exposure values to in vitro results, applying the human equivalent concentration concept. Our findings demonstrate the potential for conducting human risk assessments using both in silico and in vitro methods as alternatives to traditional in vivo studies.

Uji Toksisitas Akut Ekstrak Etanol Daun Alpukat (Persea americana Mill.) dan Pengaruhnya Terhadap Hepar Mencit BALB/c

Avocado leaf ethanol extract as a traditional medicine has been proven to have various pharmacological activities, but the safety of its use is not yet known. This study aims to determine the range of LD50 values, toxic symptoms, changes in body weight, and SGPT SGOT levels of Balb/c mice. The research was carried out experimentally using a fixed dose toxicity test method with a randomized matched pre and post-test control group design. EEDA is made by 70% ethanol maceration. The acute toxicity test consists of 2 stages: preliminary and main tests. Preliminary tests were carried out with a dose of extract 300 mg/kg BW once administered. After 24 hours of observation, there were no toxic symptoms, the test dose was increased to 2000 mg/kg BW. The main test was carried out using 10 test animals which were divided into 2 groups, each given a dose of extract 2000 mg/kg BW once and CMC Na 1% (negative control). Observation of toxic symptoms and death was carried out for 14 days. The LD50 value is determined from the results of toxic symptoms and death of test animals. Toxic symptoms were analyzed descriptively, and changes in body weight and SGOT SGPT levels were analyzed statistically (95% confidence level). The research results show that avocado leaf ethanol extract has an LD50 value range of >2000 mg/kg BW. The test dose of 2000 mg/kg BW did not cause toxic symptoms and death. SGOT and SGPT levels were still within normal limits, however, changes in SGPT levels were significantly different from controls. Thus, an extract dose of 2000 mg/kg BW has an acute toxic effect on the liver of mice.

Acute Toxicity of 70% Ethanol Extract of Kenitu (Chrysophyllum cainito L.) Leaves of Wistar Rats (Rattus norvegicus)

The postmenopausal phase, which results in a lack of estrogen levels and is one of the triggers of menopausal symptoms, contributes to the elderly's cognitive function decline. This condition can also cause joint and bone pain, bladder and urinary tract disorders, and sexual problems. Based on several studies, it is known that kenitu (Chrysophyllum cainito L.) contains phytoestrogen compounds with a structure similar to the hormone estrogen, so kenitu has the potential as an alternative treatment for disease conditions with risk factors for estrogen deficiency. The study was conducted to determine the estimated lethal dose 50 (LD50) value based on the Globally Harmonized System (GHS) classification as well as macroscopic (physical, behavioral, and average weight gain per day) and microscopic (liver and kidney histology) values in Wistar rats against the administration of 70% ethanol extract of kenitu leaves at a dose of 2000 mg/kg body weight (BW). The Up-and-Down Procedure (UDP) acute toxicity test method was used for this study. The maximum dose was 2000 mg/kg BW of ethanol extract from 70% kenitu leaves. The study found that the 70% ethanol extract of kenitu leaves had more than 2000 mg/kg BW LD50 value against Wistar rats. Based on the classification of the GHS, it can be concluded that 70% ethanol extract of kenitu leaves against Wistar rats is classified as low toxicity.

An automatic measurement method for the response of Caenorhabditis elegans to chemicals.

Caenorhabditis elegans is a widely used model animal. Chemotaxis assay is one of the experiments that study the effects of different chemicals on nematodes. It is mainly used to study the effects of different chemicals on the perception behavior of nematodes. By conducting this experiment, not only can the neurotoxicity of chemicals be reflected, but also the impact of chemicals on physiological functions regulated by the nervous system, such as nematode feeding behavior and basic motor ability. The experiment of detecting the response of nematode to chemicals is also a common method of chemical toxicity testing based on nematode models. In the analysis of worm tendency behavior, manual operations are generally used. Manually processing a large number of worms under a microscope is very time-consuming and labor-intensive. The current quantitative methods for nematode chemotaxis experiments are not only time-consuming and labor-intensive, but also biased in experimental results due to differences in judgment standards among experimenters. The automatic and efficient quantification method for nematode chemotaxis experiments is a very important technical difficulty in the field of nematode experiments. Here, we have designed an automatic quantification method for nematode chemotaxis experiments by incorporating image acquisition and processing techniques into the nematode experiment. The experimental results show that the Pearson correlation coefficient between manual and automatic counting results is 0.978. This proves the effectiveness of our method. Applying the automatic measurement method to replace manual counting by the experimenter can improve work efficiency, and reduce errors in human counting operations.

Research Progress in Acute Oral Toxicity Testing Methods

Acute oral toxicity is the first phase of safety toxicological evaluation, with the median lethal dose (LD50) being the most commonly used assessment parameter. This paper aims to summarize and compare conventional methods for determining LD50 and alternative approaches, along with their respective advantages and disadvantages, to provide options for further toxicological studies. Alternative tests, which do not require the precise determination of LD50 values, minimize animal mortality to the greatest extent and reduce the waste of human and material resources, making them worthy of promotion. Additionally, the development of Quantitative Structure-Activity Relationship (QSAR) models for predicting in vivo acute toxicity and in vitro cell culture toxicity assays are also in progress.

Feeding Ration Impacts Larval Pimephales Promelas 7-Day Subchronic Growth Endpoint: Case Study with Perfluorooctanesulfonic Acid

The larval fathead minnow, Pimephales promelas, 7-day subchronic survival and growth standard toxicity test method is commonly used for research and regulatory testing of effluents and compounds, including emerging contaminants such as Perfluorooctanesulfonic Acid (PFOS). Existing feeding guidelines for testing are described in multiple methods but are open to interpretation. The current study sought to determine the impact of feeding ration on P. promelas survival and biomass during a subchronic exposure to PFOS. The study was conducted in two phases: (1) a control experiment to determine the most significant feeding ration factors that maximize biomass, with consideration to laboratory logistics, and (2) application of down-selected feeding rations in a PFOS exposure to determine toxicity reference values. The control optimization study supported that feeding ration and feeding frequency were significant factors in fish biomass. In the subsequent PFOS study, fish were fed a high or low ration of Artemia twice daily, while exposed to 0.3 to 3.4 mg/L PFOS. Fish fed a high ration of Artemia had significantly (p < 0.05) greater biomass than fish fed a low ration in all exposure concentrations except 3.4 mg/L, where survival was low in both treatments. The feeding ration was not a significant factor on the survival endpoint for either treatment, but the PFOS concentration was (p < 0.0001) (high ration LC50 = 2.44 mg/L; low ration LC50 = 2.25 mg/L). These findings contribute to a better understanding of the impact feeding ration has in toxicity assessments and downstream regulatory decisions.

Sediment Toxicity Tests: A Critical Review of Their use in Environmental Regulations.

Sediments are an integral component of aquatic systems, linking multiple water uses, functions, and services. Contamination of sediments by chemicals is a worldwide problem, with many jurisdictions trying to prevent future pollution (prospective) and manage existing contamination (retrospective). The present review assesses the implementation of sediment toxicity testing in environmental regulations globally. Currently, the incorporation of sediment toxicity testing in regulations is most common in the European Union (EU), North America, and Australasian regions, with some expansion in Asia and non-EU Europe. Employing sediment toxicity testing in prospective assessments (i.e., before chemicals are allowed on the market) is most advanced and harmonized with pesticides. In the retrospective assessment of environmental risks (i.e., chemicals already contaminating sediments), regulatory sediment toxicity testing practices are applied inconsistently on the global scale. International harmonization of sediment toxicity tests is considered an asset and has been successful through the widespread adoption and deployment of Organisation for Economic Co-operation and Development guidelines. On the other hand, retrospective sediment assessments benefit from incorporating regional species and protocols. Currently used toxicity testing species are diverse, with temperate species being applied most often, whereas test protocols are insufficiently flexible to appropriately address the range of environmental contaminants, including nanomaterials, highly hydrophobic contaminants, and ionized chemicals. The ever-increasing and -changing pressures placed on aquatic resources are a challenge for protection and management efforts, calling for continuous sediment toxicity test method improvement to insure effective use in regulatory frameworks. Future developments should focus on including more subtle and specific toxicity endpoints (e.g., incorporating bioavailability-based in vitro tests) and genomic techniques, extending sediment toxicity testing from single to multispecies approaches, and providing a better link with ecological protection goals. Environ Toxicol Chem 2024;43:1697-1716. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Exploring Alternatives for Marine Toxicity Testing: Initial Evaluation of Fish Embryo and Mysid Tests.

Current regulations require that toxicity assessments be performed using standardized toxicity testing methods, often using fish. Recent legislation in both the European Union and United States has mandated that toxicity testing alternatives implement the 3Rs of animal research (replacement, reduction, and refinement) whenever possible. There have been advances in the development of alternatives for freshwater assessments, but there is a lack of analogous developments for marine assessments. One potential alternative testing method is the fish embryo toxicity (FET) test, which uses fish embryos rather than older fish. In the present study, FET methods were applied to two marine model organisms, the sheepshead minnow and the inland silverside. Another potential alternative is the mysid shrimp survival and growth test, which uses an invertebrate model. The primary objective of the present study was to compare the sensitivity of these three potential alternative testing methods to two standardized fish-based tests using 3,4-dichloroaniline (DCA), a common reference toxicant. A secondary objective was to characterize the ontogeny of sheepshead minnows and inland silversides. This provided a temporal and visual guide that can be used to identify appropriately staged embryos for inclusion in FET tests and delineate key developmental events (e.g., somite development, eyespot formation, etc.). Comparison of the testing strategies for assessing DCA indicated that: (1) the standardized fish tests possessed comparable sensitivity to each other; (2) the mysid shrimp tests possessed comparable sensitivity to the standardized fish tests; (3) the sheepshead minnow and inland silverside FET tests were the least sensitive testing strategies employed; and (4) inclusion of sublethal endpoints (i.e., hatchability and pericardial edema) in the marine FETs increased their sensitivity. Environ Toxicol Chem 2024;43:1285-1299. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Intuitive toxicology in the 21st century-Bridging the perspectives of the public and risk assessors in Europe.

Three decades ago, several articles on the subjectivity in chemical risk judgments (i.e., labeled "intuitive toxicology") measured the divide between the public and toxicologists with different backgrounds regarding the validity of predicting health effects based on in vivo studies. Similar divides with impacts on societal discourse and chemical risk assessment practices might exist concerning alternative toxicity testing methods (i.e., in vitro and in silico). However, studies to date have focused either on the public's views of in vivo or stem cell testing or on experts' views of in vivo testing and potential alternatives (i.e., toxicologists and medical students), which do not allow for a direct investigation of potential divides. To fill this knowledge gap, we conducted two online surveys, involving members of the German-speaking public in Switzerland and European human health risk assessors, respectively. This article presents the results of these two surveys regarding the divide in the public's and risk assessors' perspectives on risk assessment based on in vivo, in vitro, and in silico testing. Particularly, the survey with the risk assessors highlights that, beyond scientific and regulatory barriers, alternatives to in vivo testing may encounter individual hurdles, such as higher uncertainty associated with them. Understanding and addressing these hurdles will be crucial to facilitate the integration of new approach methodologies into chemical risk assessment practices as well as a successful transition toward next-generation risk assessment, bringing us closer to a fit-for-purpose and more efficient regulatory landscape.

Effect of Extracellular Marennine produced by Haslea Ostrearia on the Blood Clams Tegillarca Granosa

Marennine is a blue-green pigment produced by diatoms of the Haslea genus, one of which is Haslea ostrearia. This marennine pigment is water-soluble and confirmed to contain polyphenols and glycosides. There are two forms of marennine pigment: the intracellular form of marennine (IMn) and the extracellular form of marennine (EMn). Marennine pigments exhibit various biological activities such as antibacterial, antiviral, antioxidant, allelopathic, and inhibiting the growth of other diatoms. With this biological activity, marennine can be used in various fields, one of which is aquaculture.This research aims to determine the level of toxicity and analyze the effect of exposure to the extracellular marennine produced by H. ostrearia on the juvenile blood clam Tegillarca granosa. By using the toxicity test method, Blue water which is supernatant of H. ostrearia (BW) was tested on juvenile of commercially important bivalve species, the blood clams Tegillarca granosa, with three treatments, namely control (0 mg/L), treatment A (0.25 mg/L) and treatment B (0.5 mg/L of BW). The observation parameters of this study were LC50-72 h with one-way ANOVA analysis. The results showed that the BW produced by H. ostrearia increased the survival of juvenile blood clams T. granosa up to 27.7% in treatment B (0.5 mg/L) compared to control within 72 h. The one-way ANOVA analysis revealed that the control and treatment B (0.5 mg/L) were significantly different. This study shows that there is potential to develop the use of marennine in shellfish aquaculture.

Southeast Asia's environmental challenges: emergence of new contaminants and advancements in testing methods.

Emerging contaminants, including pharmaceuticals, personal care products, microplastics, and per- and poly-fluoroalkyl substances, pose a major threat to both ecosystems and human health in Southeast Asia. As this region undergoes rapid industrialization and urbanization, the increasing presence of unconventional pollutants in water bodies, soil, and various organisms has become an alarming concern. This review comprehensively examines the environmental challenges posed by emerging contaminants in Southeast Asia and recent progress in toxicity testing methods. We discuss the diverse range of emerging contaminants found in Southeast Asia, shedding light on their causes and effects on ecosystems, and emphasize the need for robust toxicological testing methods. This review is a valuable resource for researchers, policymakers, and environmental practitioners working to mitigate the impacts of emerging contaminants and secure a sustainable future for Southeast Asia.

Novel Reproduction Toxicity Test Method for Hyalella azteca Using Sexually Mature Amphipods Provides More Robust Data Than Standard Methods in Exposures to Imidacloprid.

Hyalella azteca is an epibenthic crustacean used in ecotoxicology, but there are challenges associated with standard methods using reproduction as an endpoint. A novel, 28-day reproduction toxicity test method for H. azteca was created to address these issues by initiating tests with sexually mature amphipods to eliminate the confounding effects of growth, using a sex ratio of seven females to three males to reduce reproductive variability, and conducting tests in water-only conditions to make recovery of juveniles easier and expand testing capabilities to water-soluble compounds. In the present study, we evaluated the 28-day novel method by comparing it with the 42-day standard test method in duplicate and parallel water-only, static-renewal exposures to sublethal concentrations of imidacloprid (0.5-8 µg/L). Both methods showed similar effects on survival, with survival approaching 50% in the highest test concentration (8 µg/L). However, the 42-day median effect concentrations (EC50s) for growth were more sensitive in the standard method (1.5-3.2 µg/L) compared with the 28-day EC50s generated by the novel method (>8 µg/L). Reproduction endpoints (juveniles/female) produced similar EC50s between methods, but the data were less variable in novel tests (smaller coefficients of variation); therefore, fewer replicates would be required to detect effects on reproduction compared with the standard method. In addition, novel tests generated 28 days of reproduction data compared with 14 days using standard tests and allowed survival and growth of sexes to be assessed independently. Thus, the novel method shows promise to improve the use of reproduction as an endpoint in water-only toxicity tests with H. azteca. Environ Toxicol Chem 2024;43:723-735. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Validation of a new protocol for a zebrafish MEFL (malformation or embryo-fetal lethality) test method that conforms to the ICH S5 (R3) guideline.

Detecting the toxic effects of chemicals on reproduction and development without using mammalian animal models is crucial in the exploitation of pharmaceuticals for human use. Zebrafish are a promising animal model for investigating pharmacological effects and toxicity during vertebrate development. Several studies have suggested the use of zebrafish embryos for the assessment of malformations or embryo-fetal lethality (MEFL). However, a reproducible protocol as a standard for the zebrafish MEFL test method that fulfills global requests has not been established based on the International Council of Harmonisation (ICH) S5 (R3) guidelines. To establish such a toxicity test method, we developed a new and easy protocol to detect MEFL caused by chemicals, especially those with teratogenic potential, using fertilized zebrafish eggs (embryos) within 5 days of development. Our toxicity test trials using the same protocol in two to four different laboratories corroborated the high inter-laboratory reproducibility. Our test method enabled the detection of 18 out of 22 test compounds that induced rat MEFL. Thus, the prediction rate of our zebrafish test method for MEFL was almost 82% compared with that of rat MEFL. Collectively, our study proposes the establishment of an easy and reproducible protocol for the zebrafish MEFL test method for reproductive and developmental toxicity that meets ICH guideline S5 (R3), which can be further considered in combination with information from other sources for regulatory use.

Transforming Toxicity Assessment through Microphysiology, Bioprinting, and Computational Modeling

Background: Traditional toxicity testing emphasizes animal models with growing concerns regarding predictive capacity, throughput and ethics. Rapid innovation surrounding human cell platforms, bioengineered tissues, omics techniques and computational tools offers more modern alternatives aligned with expanding knowledge of chemical biological pathways. These disruptive approaches promise immense potential to transform next-generation chemical safety assessment and drug development pipelines. Purpose: This review provides clinical researchers an updated, comprehensive perspective across evolving areas of focus in new toxicity testing methods with analysis of latest advances and translational context. Main Body: We survey progress in two- and three-dimensional human cell cultures recapitulating tissue/organ complexity impossible in conventional assays. Complementing this, computational modeling integrates structure-activity relationships, physicochemical properties and physiological interactions to predict pharmacokinetics and toxicity in silico. Expanding model organisms add further dimensionality and demographic relevance. High-throughput omics and imaging technologies unravel mechanisms and illuminate biomarkers undetectable by standard measures. Specialized techniques show high promise addressing toxicodynamic intricacies within disease contexts like diabetes and NAFLD. Evaluating traditional medicines and expanding phytochemicals likewise represents an area of growth well-suited for contemporary platforms. Future outlook weighs remarkable potential advantages in reducing animal testing demands, enabling precision toxicology links to clinical medicine and overhauling core chemical risk assessment frameworks. Conclusion: This review intends to catalyze discourse on strategic optimization priorities and roadmaps towards fully unlocking the immense yet still emerging public health potential of these disruptive techniques poising transformation in toxicity sciences centered on human-focused models.

The benefits of validation of methods for toxicity testing outweigh its costs

The 4th Annual Forum on Endocrine Disrupters organized by the European Commission brought together the authors of this article around the topic: “From bench to validated test guidelines: (pre)val­idation of test methods”. Validation activities are meant to demonstrate the relevance and reliability of methods and approaches used in regulatory safety testing. These activities are essential to facil­itate regulatory use, still they are largely underfunded and unattractive to the scientific community. In the last decade, large amounts of funding have been invested in European research towards the development of approaches that can be used in regulatory decision-making, including for the identification of endocrine disrupters. There is a vast pool of candidate test methods for potential reg­ulatory applications, but most of them will not be used due to the absence of consideration of their relevance and reliability outside the method developer’s laboratory. This article explains the reasons why such a gap exists between the outputs of research projects and the uptake in a regulatory context. In parallel, there are also increasing expectations from the regulatory science community that validation becomes more efficient with respect to time and resources. This article shares some of the lessons learned and proposes paths forward for validation of new methods that are not intended as one-to-one replacements of animal studies. This includes submitting only mature methods for validation that were developed following good practices and good documentation, proposing a greater emphasis on well-documented transferability studies, and adopting a cost-sharing model among those who benefit from validated methods.

A review of toxicity assessment procedures of solar photovoltaic modules

Environmental management of solar photovoltaic (PV) modules is attracting attention as a growing number of field-operated PV modules approach end of life (EoL). PV modules may contain small amounts of toxic metals, and the procedures for assessing and regulating the toxic metal content and release of such materials at EoL differ widely across nations. This paper provides an overview of the metal composition of PV modules and common procedures for toxicity assessment through extensive research and review of technical literature and legislative documents. This review focuses on three primary aspects: first, it explores the distribution of toxic elements within current and emerging PV module designs, with a specific focus on obtaining representative samples for proportional toxicity testing within different module laminate areas. Second, it examines a sampling standard and the diverse toxicity testing methods and regulations employed in various regions, encompassing standards like the Environmental Protection Agency (EPA) Test Method 1311 (Toxicity Characteristic Leaching Procedure, TCLP) in the U.S., Restriction of Hazardous Substances (RoHS) in Europe, and the Waste Extraction Test (WET) in California. Third, the review examines the sources of variability in toxicity testing outcomes, including techniques for securing homogeneous samples from non-uniform PV modules, selecting particle sizes representative of landfill conditions in extracted samples, determining appropriate leachate characteristics such as leaching agents and pH levels, and considering factors like test duration and temperatures. In summary, this review summarizes relevant regulations and offers a comprehensive overview of the strengths and limitations associated with several toxicity assessment procedures currently in practice.

Acute Toxicity Assays with Adult Coral Fragments: A Method for Standardization.

Coral reefs are globally declining due to various anthropogenic stressors. Amongst those, chemical pollutants, such as pesticides from agricultural runoff, sewage or an overabundance of personal care products in coastal waters due to intense tourism, may be considered as a local stressor for reef-building corals. The extent to which such chemicals exhibit toxic effects towards corals at environmentally relevant concentrations is currently controversially discussed and existing studies are often based on varying and sometimes deficient test methods. To address this uncertainty, we adapted available methods into a reliable and comprehensive acute coral toxicity test method for the reef-building coral Montipora digitata. The toxicities of the four substances benzophenone-3 (BP-3), Diuron (DCMU), copper (Cu2+ as CuCl2, positive control) and dimethylformamide (DMF, solvent) were assessed in a 96 h semi-static test design. Endpoints such as maximum quantum yield, bleaching, tissue loss and mortality were evaluated with respect to their suitability for regulatory purposes. Overall, the endpoints bleaching and mortality yielded sensitive and robust results for the four tested substances. As the test method follows the principles of internationally standardized testing methods (ISO, OECD), it can be considered suitable for further validation and standardization. Once validated, a standardized test method will help to obtain reproducible toxicity results useful for marine hazard and risk assessment and regulatory decision making.

Optimizing Sex Ratios of Hyalella azteca to Reduce Variability in Reproduction and Improve Reproductive Toxicity Test Methods.

Standard toxicological test methods with Hyalella azteca incorporate both lethal and sublethal (growth, reproduction) endpoints, although lethal endpoints are often favored in routine testing. However, sublethal endpoints are important to consider because they are ecologically relevant and are often more sensitive than lethality. It is difficult to achieve robust data for reproduction in H. azteca because high biological variability is associated with reproductive yield, likely due to complex interactions in mate selection, which can add to the variability in brood sizes that females produce. In addition, effects on reproduction often co-occur with effects on growth, making it difficult to differentiate effects on growth from those on reproduction in standard tests initiated with juveniles. The present study characterized the reproductive capacity of H. azteca by investigating the role of sex ratios in reproductive yield. Experiments were initiated in the absence of toxicants with sexually mature (6-8-week-old) individuals that were placed in different female-to-male ratios (1:1, 2:3, 3:2, and 7:3). Reproduction was monitored during weekly static renewals for 7 weeks. The results indicated that a higher female:male ratio (seven females to three males) improved reproductive success and lowered biological variability in reproduction. In addition, the body lengths and weights of newly mature amphipods were used to create a size distribution to aid in the identification of small female amphipods. Based on the results of our study, a novel reproductive toxicity test method is proposed that begins with sexually mature amphipods in a ratio of seven females to three males to minimize reproductive variability in amphipods and to enable a more effective assessment of contaminants using reproduction as a highly sensitive endpoint. However, evaluation of this novel method with toxicants is needed to demonstrate that results are comparable to standard methods.Environ Toxicol Chem 2024;43:712-722. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.