Aquatic Toxicity Tests Research Articles

Bioaccumulation and biochemical impact of polyethylene terephthalate microplastics in Cipangopaludina chinensis: Tissue-specific analysis and homeostasis disruption

Microplastics are a novel pollutant that adversely affect freshwater benthic organisms. However, few studies have investigated the mechanism underlying the bioaccumulation and the toxicity of microplastics. In this study, microplastics bioaccumulation of wild Cipangopaludina chinensis in the Songhua River were utilized, and a 28-day aquatic toxicity test was performed to determine the effects of exposure to polyethylene terephthalate (PET), the bioaccumulation of PET, and changes in multiple biomarkers in the muscle, gill, and kidney tissues. The concentration pattern of microplastics was as follows: kidney tissue > muscle tissue > gill tissue. Microplastic ingestion caused AChE inhibition led to significant increases in redox and energy metabolism indicators. Furthermore, the IBR analysis presented a "response-resistance-breakdown" process, indicating that Cipangopaludina chinensis possessed resistance with time (D14 and D21) and concentration (0.10 mg/L and 1.00 mg/L) thresholds. Tissue sensitivity to microplastics was ranked as gill > muscle > kidney, which was the opposite order of microplastic accumulation. These findings implied that less sensitive tissues stored a larger amount of pollutants, suggesting a reduction in tissue sensitivity to microplastics with higher microplastic occurrence rates. This study provides new insights into biological resistance to pollutant stress, warranting further investigation into the underlying mechanisms.

Modeling the Bioenergetics and Life History Traits of Chironomus riparius–Consequences of Food Limitation

Chironomids have a number of characteristics that make them a useful group for investigating the impact of environmental and chemical stressors on their life cycle stages. It is crucial to first understand sensitivities to environmental factors and provide a basis for interpreting the results of toxicity tests. We focused on Chironomus riparius–one of the most studied species in aquatic toxicity tests—to understand the changes during the larval stage under conditions of food abundance and limitation. We developed a model based on Dynamic Energy Budget (DEB) theory, a framework to capture the entire life cycle of an individual under varying food and temperature conditions. Available information from this study and the literature pointed out that the first three larval instars are immature and the fourth larval instar is mature, during which the organism saves, in two phases, energy for essential processes occurring during the subsequent non-feeding stages. The model can successfully predict the observed prolonged fourth instar duration under food limitation, the times of life history events (e.g., pupation and emergence), and egg production. This model has the potential to be integrated with toxicokinetic–toxicodynamic models to study the effects of toxicants on a variety of biological traits.

P21-71 Expanding fish invitrome-based methods as alternatives to animal use in aquatic toxicity testing

P21-71 Expanding fish invitrome-based methods as alternatives to animal use in aquatic toxicity testing

Comparison of Eco-Friendly Ionic Liquids and Commercial Bio-Derived Lubricant Additives in Terms of Tribological Performance and Aquatic Toxicity.

Approximately half of the lubricants sold globally find their way into the environment. The need for Environmentally Acceptable Lubricants (EALs) is gaining increased recognition. A lubricant is composed of a base oil and multiple functional additives. The literature has been focused on EAL base oils, with much less attention given to eco-friendly additives. This study presents the tribological performance and aquatic toxicity of four short-chain phosphonium-phosphate and ammonium-phosphate ionic liquids (ILs) as candidate anti-wear and friction-reducing additives for EALs. The results are benchmarked against those of four commercial bio-derived additives. The four ILs, at a mere 0.5 wt% concentration in a synthetic ester, demonstrated a 30-40% friction reduction and >99% wear reduction, superior to the commercial baselines. More impressively, all four ILs showed significantly lower toxicity than the bio-derived products. In an EPA-standard chronic aquatic toxicity test, the sensitive model organism, Ceriodaphnia dubia, had 90-100% survival when exposed to the ILs but 0% survival in exposure to the bio-derived products at the same concentration. This study offers scientific insights for the future development of eco-friendly ILs as lubricant additives.

Developing a Passive Dosing Method for Acute Aquatic Toxicity Tests of Cationic Surfactant Benzalkoniums (BACs).

Benzalkonium chlorides (BACs) have been of environmental concern due to their widespread use and potential harm. However, challenges arise in defining and controlling the exposure concentration (Cw) in aquatic toxicity tests involving BACs with a long alkyl chain (i.e., #C > 14). To address this, a novel passive dosing method was introduced in the 48 h-acute ecotoxicity test on Daphnia magna and compared to the conventional solvent-spiking method in terms of Cw stability and toxicity results. Among 13 sorbent materials tested for their sorption capacity, poly(ether sulfone) (PES) membrane was an optimal passive dosing reservoir, with equilibrium desorption of BACs to water achieved within 24 h. The Cw of BACs remained constant in both applied dosing methods during the test period. However, the Cw in solvent-spiking tests was lower than the nominal concentration for long-chain BACs, particularly at low exposure concentrations. Notably, the solvent-spiking tests indicated that the toxicity of BACs increased with alkyl chain length from C6 to 14, followed by a decline in toxicity from C14 to 18. In contrast, the passive dosing method displayed similar or slightly increasing toxicity levels of BACs from C14 to C18, indicating higher toxicity of C16 and C18-BACs than that inferred by the solvent spiking test. These findings emphasize the potential of applying this innovative passive dosing approach in aquatic toxicity tests to generate reliable and accurate toxicity data and support a comprehensive risk assessment of cationic surfactants.

Fish acute toxicity of nine nanomaterials: Need of pre-tests to ensure comparability and reuse of data

Fish acute toxicity tests are commonly used in aquatic environmental risk assessments, being required in different international substances regulations. A general trend in the toxicity testing of nanomaterials (NMs) has been to use standardized aquatic toxicity tests. However, as these tests were primarily developed for soluble chemical, issues regarding particle dissolution, agglomeration or sedimentation during the time of exposure are not considered when reporting the toxicity of NMs. The aim of this study was to characterize the NM behaviour throughout the fish acute test and to provide criteria to assay the toxicity of nine NMs based on TiO2, ZnO, SiO2, BaSO4, bentonite, and carbon nanotubes, on rainbow trout following OECD Test Guideline (TG) nº203. Our results showed the importance of conducting a preliminary test (without fish) when working with NMs. They provide valuable information on, sample monitoring, agglomeration, sedimentation, dissolution, actual concentrations of NMs, needed to design the test. Among the NMs tested, only bentonite nanoparticles were stable during the 96-h pre-test and test in aquarium water. In contrast, the remaining NMs exhibited considerable loss and sedimentation within the first 24 h. The high sedimentation observed for almost all NMs highlights the need of consistently measuring the concentrations throughout the entire duration of the fish acute toxicity test to make reliable concentration-response relationships. Notable differences emerged in LC50 values when using actual concentrations as nominal concentrations overestimated concentrations by up to 85.6%. Among all NMs tested, only ZnO NMs were toxic to rainbow trout. A flow chart was specifically developed for OECD TG 203, aiding users in making informed decisions regarding the selection of test systems and necessary modifications to ensure accurate, reliable, and reusable toxicity data. Our findings might contribute to the harmonization of TG 203 improving result reproducibility and interpretability and supporting the development of read-across and QSAR models.

First report on chemometric modeling of tilapia fish aquatic toxicity to organic chemicals: Toxicity data gap filling

The Toxic Substances Control Act (TSCA) mandates the Environmental Protection Agency (EPA) to document chemicals entering the US. Due to the vast range of toxicity endpoints, experimental toxicological study for all chemicals is impossible to conduct. To address this, in silico methods like QSAR and read-across are strategically used to prioritize testing for chemicals lacking ecotoxicity data. Aquatic toxicity is one of the most critical endpoints directly related to aquatic species, mainly fish, followed by direct to indirect effects on humans through drinking water and fish as food, respectively. Therefore, we have employed the ToxValDB database to curate acute LC50 toxicity data for three Tilapia species covering two different genera, an ideal species for aquatic toxicity testing. Employing the curated dataset, we have developed multiple robust and predictive QSAR and quantitative read-across structure-activity relationship (q-RASAR) models for Tilapia zillii, Oreochromis niloticus, and Oreochromis mossambicus which helped to understand the toxicological mode of action (MoA) of the modeled chemicals and predict the aquatic toxicity of new untested chemicals followed by toxicity data gap filling. The best three QSAR models showed encouraging statistical quality in terms of determination coefficient R2 (0.94, 0.74, and 0.77), cross-validated leave-one-out Q2 (0.90, 0.67 and 0.70), and predictive capability in terms of R2pred (0.95, 0.77, and 0.74) for T. zillii, O. niloticus, and O. mossambicus datasets, respectively. The developed best mathematical models were used for the prediction of aquatic toxicity in terms of pLC50 for 297 untested organic chemicals across three major Tilapia species ranging from 1.841 to 8.561 M in terms of environmental risk assessment.

Alternatives to Fish Acute Whole Effluent Toxicity (WET) Testing: Predictability of RTgill-W1 Cells and Fathead Minnow Embryos with Actual Wastewater Samples.

Toxicity assays using fish cells and embryos continue to gain momentum as a more ethical and informative alternative to fish acute toxicity testing. The goal of our study was to test the accuracy of RTgill-W1 cells and the fathead minnow (Pimephales promelas) embryos to predict actual whole effluent toxicity (WET) in the fathead minnow larvae. The three models were compared concurrently using samples of various origins and treatment types. Additionally, the toxicity of reference toxicants (Cd, Cu, NH3-N, 3,4-dichloraniline, and benzalkonium chloride) spiked into a nontoxic wastewater was compared. The toxicity of reference toxicants was tested in isosmotic and hypoosmotic exposure media in RTgill-W1 cells. Of the 28 wastewater samples, 14 induced a toxic response in fish larvae. Embryos predicted 11 of the 14 wastewater samples toxic to the larvae, whereas RTgill-W1 cells predicted the toxicity of all 14 toxic samples to the larvae. In addition, embryos and RTgill-W1 cells predicted toxicity in two and six additional samples, respectively, that were nontoxic to larvae. Exposures in hypoosmotic medium significantly increased sensitivity of RTgill-W1 cells to all reference toxicants, excluding benzalkonium chloride, compared to exposures in isosmotic medium and showed toxicity levels similar to that in larvae. Thus, hypoosmotic exposure medium should be considered for aquatic toxicity testing applications. Overall, both gill cell and embryo models predicted toxicity in the majority of wastewater samples toxic to larvae and demonstrated their applicability for regulatory WET testing.

Uncovering the Hidden Dangers of Microplastic Pollution in Lake Ecosystems: Effects of Ingestion on Talitrid Amphipods

Microplastic (MP) contamination is a globally recognised issue in aquatic environments, and recently, there has been an increase in investigations focusing on lake contamination, revealing significant amounts of dispersed MPs. However, our understanding of the ingestion and effects of MPs on organisms living in lake ecosystems remains limited. This study aims to develop an effective protocol for assessing the ingestion of MPs by the talitrid amphipod Cryptorchestia garbinii, with the goal of verifying and evaluating the biological effects following ingestion. Individuals sampled from the shores of Lake Albano were exposed to four different polymers, namely low-density polyethylene (PE), polyethylene terephthalate (PET), polyester (PES), and polypropylene (PP), under laboratory conditions. To deliver MPs through the diet, we decided to employ DECOTABs (DEcomposition and COnsumption TABlets) which have been successfully used as a food source in aquatic toxicity tests. At the end of the experiments, we employed the solvatochromic and fluorescent dye Nile red to detect and quantify the MPs present in the digestive tube contents of the animals. The results clearly demonstrate the ingestion of the supplied polymers through the tabs, validating this method of exposure as effective. Furthermore, the measurement of glucose, glycogen, and lipid levels reveals that within 24 h of ingestion, MPs had an impact on the macromolecules involved in the energy metabolism of C. garbinii. This research underscores the suitability of this species as a model organism for studying MP uptake and its effects.

Doping zinc oxide (ZnO) nanoparticles with molybdenum boosts photocatalytic degradation of Rhodamine b (RhB): Particle characterization, degradation kinetics and aquatic toxicity testing

Undoped and Mo-doped ZnO photocatalysts were synthesized by hydrothermal method and characterized by field emission scanning electron microscopy coupled to energy dispersive- X ray spectroscopy (FESEM- EDAX), X-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR), Brunauer–Emmett–Teller (BET) and thermogravimetric analysis (TGA) analyses. The photocatalytic activity of the photocatalysts and the kinetic of the reaction was assessed by degrading Rhodamine B under UV and sun light irradiation under variable conditions. Eventually the acute toxicity of undoped and doped nanophotocatalysts, RhB and the consequences of photocatalytic treatment were assessed using zebrafish (Danio rerio). The analytical results showed that Mo doping increased catalysts specific surface area and thermal stability. The results of photocatalytic degradation demonstrated that Mo doping has increased the photocatalytic activity under both UV and sunlight irradiation with 95 % RhB degradation efficiency and 41 % TOC removal efficiency over 30 min. The reusability of photocatalysts showed constant degradation efficiency over multiple cycles. The 96 h LC50 of undoped ZnO, Mo doped ZnO and RhB were 482.71 and 459.05 and 20.38 mg/L, respectively. Moreover, a lower toxicity was observed for the treated compared to untreated dye solution. This study indicates a promising potential of Mo-doped ZnO nanophotocatalysts for wastewater treatment and warrants further studies in this direction.

Recommendations for the advancement of oil-in-water media and source oil characterization in aquatic toxicity test studies

During toxicity testing, chemical analyses of oil and exposure media samples are needed to allow comparison of results between different tests as well as to assist with identification of the drivers and mechanisms for the toxic effects observed. However, to maximize the ability to compare results between different laboratories and biota, it has long been recognized that guidelines for standard protocols were needed. In 2005, the Chemical Response to Oil Spills: Ecological Effects Research Forum (CROSERF) protocol was developed with existing common analytical methods that described a standard method for reproducible preparation of exposure media as well as recommended specific analytical methods and analyte lists for comparative toxicity testing. At the time, the primary purpose for the data collected was to inform oil spill response and contingency planning. Since then, with improvements in both analytical equipment and methods, the use of toxicity data has expanded to include their integration into fate and effect models that aim to extend the applicability of lab-based study results to make predictions for field system-level impacts. This paper focuses on providing a summary of current chemical analyses for characterization of oil and exposure media used during aquatic toxicity testing and makes recommendations for the minimum analyses needed to allow for interpretation and modeling purposes.

Improving the design and conduct of aquatic toxicity studies with oils based on 20 years of CROSERF experience

Laboratory toxicity testing is a key tool used in oil spill science, spill effects assessment, and mitigation strategy decisions to minimize environmental impacts. A major consideration in oil toxicity testing is how to replicate real-world spill conditions, oil types, weathering states, receptor organisms, and modifying environmental factors under laboratory conditions. Oils and petroleum-derived products are comprised of thousands of compounds with different physicochemical and toxicological properties, and this leads to challenges in conducting and interpreting oil toxicity studies. Experimental methods used to mix oils with aqueous test media have been shown to influence the aqueous-phase hydrocarbon composition and concentrations, hydrocarbon phase distribution (i.e., dissolved phase versus in oil droplets), and the stability of oil:water solutions which, in turn, influence the bioavailability and toxicity of the oil containing media. Studies have shown that differences in experimental methods can lead to divergent test results. Therefore, it is imperative to standardize the methods used to prepare oil:water solutions in order to improve the realism and comparability of laboratory tests. The CROSERF methodology, originally published in 2005, was developed as a standardized method to prepare oil:water solutions for testing and evaluating dispersants and dispersed oil. However, it was found equally applicable for use in testing oil-derived petroleum substances. The goals of the current effort were to: (1) build upon two decades of experience to update existing CROSERF guidance for conducting aquatic toxicity tests and (2) to improve the design of laboratory toxicity studies for use in hazard evaluation and development of quantitative effects models that can then be applied in spill assessment. Key experimental design considerations discussed include species selection (standard vs field collected), test substance (single compound vs whole oil), exposure regime (static vs flow-through) and duration, exposure metrics, toxicity endpoints, and quality assurance and control.

Eco-toxicity assessment of industrial by-product-based alkali-activated binders using the sea urchin embryogenesis bioassay

New cement-based materials such as alkali-activated binders (AABs) or geopolymers allow the incorporation of waste or industrial by-products in their formulation, resulting an interesting valorization technique. Therefore, it is essential to inquire about the potential environmental and health impacts throughout their life cycle. In the European context, a minimum aquatic toxicity tests battery has been recommended for construction products, but their potential biological effects on marine ecosystems have not been considered. In this study, three industrial by-products, PAVAL® (PV) aluminum oxide, weathered bottom ash (WBA) resulting from incinerator bottom ash and glass cullet recycling waste (CSP), were evaluated as precursors in the AAB formulation from an environmental point of view. To determine the potential effects on marine environment caused by the leaching of contaminants from these materials into seawater, the leaching test EN-12457-2 and an ecotoxicity test using the model organism sea urchin Paracentrotus lividus were conducted. The percentage of abnormal larval development was selected as endpoint of the toxicity test. Based on the results obtained from the toxicity tests, AABs have less damaging impact (EC50 values: 49.2%–51.9%) on the marine environment in general than raw materials. The results highlight the need to stablish a specific battery of toxicity tests for the environmental assessment of construction products on marine ecosystem.

Advances to the CROSERF protocol to improve oil spill response decision making

The Chemical Response to Oil Spills: Ecological Effects Research Forum (CROSERF) created a standardized protocol for comparing the toxicity of physically dispersed oil versus chemically dispersed oil to address environmental concerns related to the proposed use of dispersants in the early 2000s. Since then, many revisions have been made to the original protocol to diversify the intended use of the data generated, incorporate emerging technologies, and to examine a wider range of oil types including non-conventional oils and fuels. Under the Multi-Partner Research Initiative (MPRI) for oil spill research under Canada's Oceans Protection Plan (OPP), a network of 45 participants from seven countries representing government, industry, non-profit, private, and academic sectors was established to identify the current state of the science and formulate a series of recommendations to modernize the oil toxicity testing framework. The participants formed a series of working groups, targeting specific aspects of oil toxicity testing, including: experimental conduct; media preparation; phototoxicity; analytical chemistry; reporting and communicating results; interpreting toxicity data; and appropriate integration of toxicity data to improve oil spill effects models. The network participants reached a consensus that a modernized protocol to assess the aquatic toxicity of oil should be sufficiently flexible to address a broad range of research questions in a ‘fit-for-purpose’ manner, where methods and approaches are driven by the need to generate scientifically-defensible data to address specific study objectives. Considering the many needs and varied objectives of aquatic toxicity tests currently being conducted to support and inform oil spill response decision making, it was also concluded that the development of a one size fits all approach would not be feasible.

A protocol for lixiviation of micronized plastics for aquatic toxicity testing

Plastics contain various types and amounts of additives that can leach into the water column when entering aquatic ecosystems. Some leached plastic additives are hazardous to marine biota at environmentally relevant concentrations. Disparate methodological approaches have been adopted for toxicity testing of plastic leachates, making comparison difficult. Here we propose a protocol to standardize the methodology to obtain leachates from microplastics (MPs) for aquatic toxicity testing. Literature reviewing and toxicity tests using marine model organisms and different types of MPs were conducted to define the main methodological aspects of the protocol. Acute exposure to leachates from the studied plastics caused negative effects on the early life stages of sea urchins and marine bacteria. We provide recommendations of key factors influencing lixiviation of MPs , such as particle size (<250 μm), solid-to-liquid ratio (1–10 g/L), mixing conditions (1–60 rpm), and lixiviation time (72 h). The proposed methodology was successful to determine the toxicity of leachates from different micronized plastics on marine biota. Our recommendations balance sensitivity, feasibility and environmental relevance, and their use would help ensure comparability amongst studies for a better assessment of the toxicity of plastic leachates on aquatic biota.

Ecotoxicological Effects of Four Commonly Used Organic Solvents on the Scleractinian Coral Montipora digitata.

Organic solvents are often used in aquatic toxicity tests to facilitate the testing of hydrophobic or poorly water-soluble substances such as ultraviolet (UV) filters, pesticides, or polycyclic aromatic hydrocarbons (PAHs). Knowledge of intrinsic effects (i.e., measured as standardized and non-standardized endpoints) of such carrier solvents in non-standardized organisms (i.e., corals), is critical to regulatory processes. Therefore, we exposed the reef-building coral Montipora digitata to the most commonly used carrier solvents ethanol, methanol, dimethyl sulfoxide, and dimethylformamide in the range of 10-100 µL L-1 for 16 days. The effects on mortality, photobiological, morphological, and oxidative stress markers were evaluated. In our study, all solvents resulted in significant morphological and/or oxidative stress responses, but not in mortality. Moreover, ethanol led to a rapid increase in turbidity, thus questioning its suitability as a carrier solvent in aquatic studies in general. Based on our observations, we could rank the solvent effects as follows: dimethylformamide < dimethyl sulfoxide ≈ methanol ≤ ethanol, with dimethylformamide showing the least and ethanol the most pronounced effects. We conclude that the use of solvents in toxicity studies with corals, particularly by examining non-standardized (e.g., morphological, physiological) endpoints, should be taken with caution and requires further elaboration.

The potential of Euglena species as a bioindicator for soil ecotoxicity assessment

Currently, there are no standard international test methods for assessing aquatic and soil toxicity, with aquatic toxicity tests based on limited Euglena species. Here, we proposed Euglena species as extended test species, especially as new soil test species for a paper-disc soil method, considering its ecologically important roles in providing highly bioavailable in-vivo nutrients to upper trophic level organisms. We conducted experiments to identify the optimal exposure duration for two Euglena species (Euglena viridis and Euglena geniculata). We demonstrated the toxic effects of nickel (model contaminant) on their photosynthetic parameters and growth in freshwater. The growth and photosynthetic activity of three Euglena species were significantly inhibited in nickel-contaminated soil during paper-disc soil tests, especially the test species adsorbed onto paper-disc soil. Euglena gracilis was more sensitive to nickel than E. viridis and E. geniculata in freshwater and soil. Thus, E. viridis and E. geniculata have potential as additional test species for improving test species diversity, while all three species have potential as new soil test species for soil toxicity assessment. Thus, results these species may be suitable for routine aquatic toxicity testing and new soil toxicity testing, addressing the current paucity of test species in freshwater and soil toxicity assessment.

Bridging the lab to field divide: Advancing oil spill biological effects models requires revisiting aquatic toxicity testing

Oil fate and exposure modeling addresses the complexities of oil composition, weathering, partitioning in the environment, and the distributions and behaviors of aquatic biota to estimate exposure histories, i.e., oil component concentrations and environmental conditions experienced over time. Several approaches with increasing levels of complexity (i.e., aquatic toxicity model tiers, corresponding to varying purposes and applications) have been and continue to be developed to predict adverse effects resulting from these exposures. At Tiers 1 and 2, toxicity-based screening thresholds for assumed representative oil component compositions are used to inform spill response and risk evaluations, requiring limited toxicity data, analytical oil characterizations, and computer resources. Concentration-response relationships are employed in Tier 3 to quantify effects of assumed oil component mixture compositions. Oil spill modeling capabilities presently allow predictions of spatial and temporal compositional changes during exposure, which support mixture-based modeling frameworks. Such approaches rely on summed effects of components using toxic units to enable more realistic analyses (Tier 4). This review provides guidance for toxicological studies to inform the development of, provide input to, and validate Tier 4 aquatic toxicity models for assessing oil spill effects on aquatic biota.Evaluation of organisms’ exposure histories using a toxic unit model reflects the current state-of the-science and provides an improved approach for quantifying effects of oil constituents on aquatic organisms. Since the mixture compositions in toxicity tests are not representative of field exposures, modelers rely on studies using single compounds to build toxicity models accounting for the additive effects of dynamic mixture exposures that occur after spills. Single compound toxicity data are needed to quantify the influence of exposure duration and modifying environmental factors (e.g., temperature, light) on observed effects for advancing use of this framework. Well-characterized whole oil bioassay data should be used to validate and refine these models.

Chemo-rheological, mechanical, morphology evolution and environmental impact of aged asphalt binder coupling thermal oxidation, ultraviolet radiation and water

Thermal oxidation, ultraviolet radiation and water significantly affect the aging of asphalt pavement, and the aging causes more serious emissions into aqueous environment. This study designed four aging methods to conduct coupling aging tests, and the dynamic shear oscillatory test, FT-IR spectrometer, gel permeation chromatography (GPC), binder bond strength test (BBS), fluorescent microscope, scanning electron microscope, and surface free energy (SFE), and zebrafish aquatic toxicity tests were used to explore the rheological, chemical, adhesive, surface evolution, and the environmental impact of asphalt binder. The aging degree increased under four aging methods, which led to a higher modulus, a lower phase angle, more oxygenated chemicals and asphaltenes, and higher water sensitivity. Thermal oxidation and ultraviolet radiation had the most significant aging acceleration effect. Although water greatly weakened the aging acceleration effect of UV radiation, through physical diffusion and chemical hydrolysis, water led to crack propagation, rougher surface and even holes coupling with high temperature, which increased the damage to the asphalt binder and the BBS failure mode changed from cohesion failure to adhesion failure. The results of the environmental impact of the organic matter released from the aged asphalt indicated that the asphalt leaching solution had certain biotoxicity. Moreover, the aged asphalt leaching solution was diluted 10 and 100 times, and the biotoxicity decreased significantly with the decrease of the concentration.

Applicability of OECD TG 201, 202, 203 for the aquatic toxicity testing and assessment of 2D Graphene material nanoforms to meet regulatory needs

Tests using algae and/or cyanobacteria, invertebrates (crustaceans) and fish form the basic elements of an ecotoxicological assessment in a number of regulations, in particular for classification of a substance as hazardous or not to the aquatic environment according to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS-CLP) (GHS, 2022) and the REACH regulation (Registration, Evaluation, Authorisation and Restriction of Chemicals, EC, 2006). Standardised test guidelines (TGs) of the Organisation for Economic Co-operation and Development (OECD) are available to address the regulatory relevant endpoints of growth inhibition in algae and cyanobacteria (TG 201), acute toxicity to invertebrates (TG 202), and acute toxicity in fish (TG 203). Applying these existing OECD TGs for testing two dimensional (2D) graphene nanoforms may require more attention, additional considerations and/or adaptations of the protocols, because graphene materials are often problematic to test due to their unique attributes. In this review a critical analysis of all existing studies and approaches to testing used has been performed in order to comment on the current state of the science on testing and the overall ecotoxicity of 2D graphene materials. Focusing on the specific tests and available guidance's, a complete evaluation of aquatic toxicity testing for hazard classification of 2D graphene materials, as well as the use of alternative tests in an integrated approach to testing and assessment, has been made. This information is essential to ensure future assessments generate meaningful data that will fulfil regulatory requirements for the safe use of this “wonder” material.