As a result of the efforts to introduce the principle of the 3Rs (replacement, reduction, and refinement) into animal testing, alternative in vitro skin corrosion test methods have been developed and standardized globally. However, alternative in vitro skin corrosion test methods have some limitations in terms of the use of humanely killed rats or commercial models and kits. The present study focused on the applicability of invertebrates as alternative in vivo skin models. Even though earthworm skin comprises the same biomolecules as human skin, the possibility of using earthworm skin as an alternative for skin testing remains unexplored. In this study, we developed a half-pipe tool for earthworm skin corrosion testing and optimized the test protocol. Subsequently, the applicability of the earthworm half-pipe assay for corrosion testing with six chemicals, including inorganic acids, organic acids, and alkalis, was investigated using stereomicroscopy and electron microscopy. It was observed that the specific concentrations for earthworm skin corrosion were lower than those for animal or in vitro tests. Therefore, the sensitivity of the earthworm half-pipe assay indicates that it could be useful as a screening tool before conducting in vivo animal tests or in vitro skin tests. This new method can contribute to research on alternative skin corrosion tests by reducing ethical issues, time, and cost while achieving effective results.

Robust antimicrobial capability is crucial for marine organisms survival in complex ocean environments. Although the detrimental impacts of emergent pollutants on cellular immune response of marine bivalve mollusks were increasingly documented, the effects of bisphenol A (BPA) and microplastics (MPs) on humoral immune response and hemocyte chemotactic activity remain unclear. Therefore, in this study, the toxicities of BPA and MPs, alone or in combination, to the antimicrobial ability, humoral immune response, and hemocyte chemotactic activity were investigated in the blood clam Tegillarca granosa. Our data demonstrated that exposure of blood clams to BPA, MPs, and BPA-MPs for 2 weeks lead to significant reductions in their survival rates upon pathogenic bacterial challenge, indicating evident impairment of antimicrobial ability. Compared to control, the plasma of pollutant-incubated blood clams exhibited significantly less antimicrobial activity against the growth of V. harveyi, suggesting significant reduction in humoral immune effectors including defensin, lysozyme (LZM), and lectin. Moreover, hemocytes migration across the polycarbonate membrane to the serum containing chamber was markedly arrested by 2-week exposure to BPA, MPs, and BPA-MPs, suggesting a hampered chemotactic activity. In addition, the intracellular contents of ROS and protein carbonyl in hemocytes were markedly induced whereas the expression levels of key genes from the MAPK and actin cytoskeleton regulation pathways were significantly suppressed upon exposure. In this study, it was also found that BPA-MP coexposure was significantly more toxic than single exposures. In summary, our findings revealed that exposure to the pollutants tested possibly impair the antimicrobial ability of blood clam through (1) reducing the inhibitory effect of plasma on bacterial growth, the contents of humoral immune effectors, and the chemotactic activity of hemocytes, (2) interrupting IL-17 activation of MAPK signal pathway, (3) inducing intracellular ROS, elevating protein carbonylation levels, and disrupting actin cytoskeleton regulation in hemocytes.

Plastics are produced and used in large quantities worldwide (e.g. as food packaging). In line with this, plastic particles are found throughout the ecosphere and in various foods. As a result, plastics are also present in energy-rich waste biomass derived from the food industry, supermarkets, restaurants, etc. These waste streams are a valuable source for biogas production but can also be used to feed insects that in turn upcycle it into new high-value biomass. In both applications, the remaining residue can be used as fertilizer. Due to the present plastic particles, these applications could pose a continued threat to the environment, and both human and animal health. Therefore, the need of determining the (micro)plastic content to assess the potential danger is rising. In this research, a closed-vessel microwave-assisted acid digestion method was developed to accurately determine meso- and microplastic contents in food (waste) matrices by solubilising this food matrix. Polyvinyl chloride (PVC) food packaging foil was used to develop the method, using a full factorial design with three parameters (nitric acid concentration (c(HNO₃)), temperature (T), and time (t)). According to this model, the best practical conditions were c(HNO₃) = 0.50 mol/L, T = 170 °C, and t = 5.00 min. Subsequently, the method was tested on five other plastics, namely high- and low-density polyethylene (HDPE and LDPE), polypropylene (PP), polystyrene (PS), and polyethylene terephthalate (PET), mixed with a food matrix, resulting in a mean plastic recovery of 102.2 ± 4.1%. Additionally, the polymers were not oxidised during the microwave digestion. For PVC and PS hardly any degradation was found, while HDPE, LDPE, and PP showed slight chain degradation, although without recovery loss. In conclusion, the method is an accurate approach to quantify the total meso- and microplastic content in food (waste) matrices with minimal change in their intrinsic characteristics.

Both amphibian metamorphosis assay (AMA) and larval amphibian growth and development assay (LAGDA) can detect thyroid-mediated modality and adversity on the basis of morphological changes during the thyroid hormone-dependent metamorphosis. They are used for identification of thyroid hormone system disrupting chemicals (TDCs) for non-target organisms or the environment. The EU Guidance recommends that the AMA and the LAGDA should be used to address sufficient investigation of the thyroid-mediated modality and adversity, respectively. In the EU discussions over identification of TDCs, the necessity of using LAGDA as a follow-up of positive results of the AMA has been questioned because of the overlap between the endpoints and the exposure of both tests. This study analyzed similarities, differences, and sensitivity of these two assays in detection of TDCs. For agonists and most of antagonists of the hypothalamic-pituitary-thyroid (HPT) axis, both AMA and LAGDA can detect the thyroid-mediated modality and adversity. The LAGDA, as a follow-up of the positive results of the AMA, may not be needed because the results of AMA are considered enough for identification of TDCs. For chemicals like inhibitors of iodotyrosine deiodinase, the LAGDA is considered necessary for identification of TDCs because the thyroid-mediated adversity cannot be detected until Nieuwkoop and Faber (NF) stage 62. Incorporation of mechanistic endpoints into existing test guidelines and the use of Xenopus Eleutheroembryo Thyroid Assay (XETA), extended amphibian metamorphosis assay (EAMA) and adverse outcome pathways (AOPs) for testing and identification of TDCs are further discussed.

Potential nuclear accidents propel serious environmental pollution, and the resultant radionuclide release devastates severely the environment severely and threatens aquatic organism survival. Likewise, ongoing climate change coupled with the gradual increase in global surface temperatures can also adversely impact the aquatic ecosystems. In the present study, we preconditioned zebrafish (Danio rerio) at three different temperatures (18 °C, 26 °C and 34 °C) to investigate the effects of a temperature profile on their radiosensitivity (exposure to 20 Gy of gamma rays) to identify the potential biochemical mechanism responsible for influencing radiosensitivity. We found that preconditioning of zebrafish at different temperatures moulded specific gut microbiota configurations and impacted hepatic glycometabolism and sensitivity to subsequent radiation. Following antibiotic treatment to reduce gut bacteria, these observed differences in the expression of hepatic glycometabolism-related genes and radiation-induced intestinal toxicity were minimal, supporting the hypothesis that the gut bacteria reshaped by different ambient temperatures might be the key modulators of hepatic functions and radiosensitivity in zebrafish. Together, our findings provide novel insights into the connection of radiation injuries with temperature alterations in fish, and suggest that maintaining the stability of gram-positive bacteria may be efficacious to protect aquatic organisms against short or long-term radioactive contamination in the context of global climate change.

Monitoring beach plastic contamination across space and time is necessary for understanding its sources and ecological effects, and for guiding mitigation. This is logistically and financially challenging, especially for microplastics. Citizen science represents an option for sampling accessible sites to support long term monitoring, but challenges persist around data validation. Here we test a simple citizen science methodology to monitor visible microplastic contamination on sandy beaches using a standard quadrat unit (50 cm × 50 cm x 5 cm depth) sieved to 1 mm, to support the analysis of microplastic on two islands within the marine protected area of the Galápagos Archipelago, Ecuador (San Cristóbal and Santa Cruz islands). High school and university students undertook supervised sampling of two beaches in 2019–2020 collecting over 7000 particles. A sub-sample of the suspected microplastics collected (n = 2,213, ∼30% total) were analysed using FTIR spectrometry, confirming 93% of particles >1 mm visually identified by students were microplastics or rubber, validating this method as a crowd-sourced indicator for microplastic contamination. These data provide important insights into the plastic contamination of Galápagos, revealing plastic abundances of 0–2524 particles m⁻² over the two beaches (the highest reported in Galápagos). Strong accumulation gradients were measured parallel to the waterline at Punta Pitt (San Cristobal island) and perpendicular to the waterline at Tortuga Bay (Santa Cruz island), where four-fold higher concentrations were recorded at the sea turtle nesting habitat on the back-beach dune. No significant seasonal trends were measured during one year. These results demonstrate the value of citizen science in filling spatiotemporal knowledge gaps of beach contamination to support intervention design and conservation.

In this study, the occurrence of pharmaceuticals, hormones and bacterial community structures was studied at a wastewater treatment plant in Finland having two different parallel treatment lines: conventional activated sludge (CAS) treatment with a sedimentation stage, and a membrane bioreactor (MBR). Influent and effluents were sampled seven times over a period of one year. The bacterial communities of the influent samples showed a high degree of similarity, except for the February sample which had substantially lower diversity. There was significant fluctuation in the species richness and diversity of the effluent samples, although both effluents showed a similar trend. A marked decrease in diversity was observed in effluents collected between August and November. The initiation of nitrogen removal as a result of an increase in temperature could explain the changes in microbial community structures. In overall terms, suspended solids, bacteria and total organic matter (COD and BOD) were removed to a greater extent using the MBR, while higher Tot-N, Tot-P and nitrate removal rates were achieved using the CAS treatment. Estrone (E1) concentrations were also consistently at a lower level in the MBR effluents (<0.1–0.68 ng/l) compared to the CAS effluents (1.1–12 ng/l). Due to the high variation in the concentrations of pharmaceuticals, no clear superiority of either process could be demonstrated with certainty. The study highlights the importance of long-term sampling campaigns to detect variations effectively.

Triclocarban (TCC), a bactericide widely used in personal care products, is frequently detected in soil and surface water, which may affect the environmental behavior of other environmental pollutants by changing the community structure of environmental microorganisms. This work evaluated the effects of TCC on the degradation and migration of seven herbicides and five fungicides in soil under co-occurrence conditions. TCC significantly increased the persistence of the pesticides in soil, and this effect increased with TCC concentration. For example, the half-life of metolachlor, atrazine, metribuzin, and metamitron increased 44%, 38%, 153%, and 33%, respectively, with 10 mg/kg TCC and increased 60%–640% with 100 mg/kg TCC. After 90 days, the residue of the pesticides in soil treated with TCC was significantly elevated relative to the control. TCC treatment could also increase the potential leaching risk of the herbicides in the soil, as indicated by an increased Groundwater Ubiquity Score (GUS) index. The reduced abundance of soil bacteria by TCC might be an essential reason for the impacts on the environmental behavior of the pesticides. This study confirmed that TCC could slow down the degradation of pesticides in soil, increase their persistence and even affect the leaching behavior, thus influencing the risks of the pesticides to the environment.

The development of saline-alkali lands has contributed to the increasing discharge of alkaline salt-laden wastewater, which poses a threat to aquatic organisms. However, the comprehensive effect of alkaline salt on Microcystis aeruginosa, a harmful cyanobacterium, remains unclear. In this study, the growth, physiology, cell ultrastructure and production of microcystin-LR (MC-LR) in Microcystis aeruginosa exposed to four levels of alkaline salt stress were evaluated. The growth of Microcystis aeruginosa was stimulated at an electrical conductivity (EC) of 2.5 mS/cm compared to the control, as supported by the increased cell density, photosynthetic pigment and protein contents. Microcystis aeruginosa could tolerate a certain level of alkaline salt (i.e., EC of 5 mS/cm) via increasing photosynthetic pigment contents to protect cells from alkaline salt stress, but the antioxidant defence system and cell ultrastructure were not affected. When EC increased to 7.5 mS/cm, alkaline salt caused oxidative stress and toxicity in Microcystis aeruginosa, as evidenced by analysis of the integrated biomarker response (IBR). Furthermore, the photosynthetic pigment and protein contents decreased, and cell apoptosis associated with ultrastructural changes was observed. Therefore, we propose that EC of 7.5 mS/cm is a threshold for growth of Microcystis aeruginosa. Additionally, the intracellular MC-LR content was stimulated by alkaline salt, and the highest value was observed at EC of 2.5 mS/cm. The extracellular MC-LR content increased with the increasing alkaline salt concentration. When EC was 7.5 mS/cm, the extracellular MC-LR content was significantly higher than in the control and was associated with the upregulated mcyH gene. This study recommends that more attention should be paid to the risk of Microcystis aeruginosa bloom and microcystin-LR pollution in lakes located in salinization regions.

Estuaries are one of the most valuable biomes on earth. Although humans are highly dependent on these ecosystems, anthropogenic activities have impacted estuaries worldwide, altering their ecological functions and ability to provide a variety of important ecosystem services. Many anthropogenic stressors combine to affect the soft sedimentary habitats that dominate estuarine ecosystems. Now, due to climate change, estuaries and other marine areas might be increasingly exposed to the emerging threat of megafires. Here, by sampling estuaries before and after a megafire, we describe impacts of wildfires on estuarine benthic habitats and justify why megafires are a new and concerning threat to coastal ecosystems. We (1) show that wildfires change the fundamental characteristics of estuarine benthic habitat, (2) identify the factors (burnt intensity and proximity to water's edge) that influence the consequences of fires on estuaries, and (3) identify relevant indicators of wildfire impact: metals, nutrients, and pyrogenic carbon. We then discuss how fires can impact estuaries globally, regardless of local variability and differences in catchment. In the first empirical assessment of the impact of wildfires on estuarine condition, our results highlight indicators that may assist waterway managers to empirically detect wildfire impacts in estuaries and identify catchment factors that should be included in fire risk assessments for estuaries. Overall, this study highlights the importance of considering fire threats in current and future estuarine and coastal management.