An acute exposure study of mancozeb (MAZ) fungicide was applied on Oreochromis niloticus for 96-h duration. Three hundred fish (20.50 ± 1.60 g) were assigned into six groups (50 fish/ group; 10 fish/replicate) and exposed to different six concentrations (0, 4, 8, 12, 16, and 20 mg L−1) of MAZ for 96-h. The Probit analysis program was used to compute the 96-h lethal concentration 50 (96-h LC50) of MAZ. During the exposure duration, the fish's behavior, clinical symptoms, and mortalities were recorded daily. After the exposure period was ended, the hematological, biochemical, immunological, and oxidant/antioxidant parameters were evaluated. The results of this study recorded the 96-h LC50 of MAZ for O. niloticus to be 11.49 mg L−1. Acute MAZ exposure badly affected the fish's behavior in the form of increased the breath gasping and swimming activity with aggressive mode. The exposed fish showed excessive body hemorrhages and fin rot. The survival rate of the exposed fish to MAZ was 100, 80, 66, 50, 38, and 30% in 0, 4, 8, 12, 16, and 20 mg L−1 MAZ, respectively. The hematological indices (red blood cell count, hemoglobin, packed cell volume%, and white blood cell count) were significantly decreased by increasing the MAZ exposure concentration (8–20 mg L−1). The acetylcholine esterase activity and immune indices (lysozyme, nitric oxide, immunoglobulin M, complement 3) were decreased by MAZ exposure (4–20 mg L−1). Acute MAZ exposure induced hepato-renal dysfunction and elevated stress-related parameter (cortisol) by increasing the MAZ concentration. A significant reduction in the antioxidant parameters (total antioxidant activity, catalase, and superoxide dismutase) with increasing the lipid peroxidation marker (malondialdehyde) was noticed by acute MAZ exposure (4 –20 mg L−1) in O. niloticus. Based on these outcomes, the MAZ exposure induced toxicity to the fish evident in changes in fish behavior, neurological activity, hepato-renal functioning, and immune-antioxidant responses which suggest physiological disruption.

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.

As an essential trace metal, copper (Cu) regulation, distribution and detoxification among different cellular organelles remain much unknown. In the current study, bioimaging tool was used in visualizing the locations of Cu among different organelles in fish fin cells isolated from rabbitfish Siganus fuscescens. Exposure concentration of Cu directly affected the Cu bioaccumulation and toxicity. When the exposure dosage of Cu reached 100 µM, it began to damage the cells and affect the cell viability after 10 min of exposure. Remarkably, while various Cu concentrations (50∼150 µM) initially reduced the cell viability, they did not lead to a further loss in viability over extended exposure period. Upon entry to the cells, Cu was mainly targeted to the mitochondria whose number, size and network responded immediately to the incoming Cu. However, Cu toxicity did not increase time-dependently, strongly indicating that these mitochondria damaged by Cu could be removed and its cytotoxicity could be relieved. Bioimaging results showed that lysosomes interacted with the mitochondria, which were subsequently digested within a few minutes. Meanwhile the lysosomal number increased, and the size and pH of lysosomes decreased. These reactions were in line with the observed mitophagy, suggesting that mitochondrial Cu could be detoxified, and the damaged mitochondria were removed by lysosome via mitophagy. By further purifying the cellular organelles, the mitochondrial and lysosomal Cu amounts were quantified and found to be in line with the imaging results. The present study suggested that excessive mitochondrial Cu could be removed via mitophagy to relieve the Cu toxicity.

The toxicity of ZnO nanoparticles (ZnO NPs) in aquatic organisms has been extensively studied, but little information is available on the effects associated with their interaction with other contaminants. In this context, the in vitro effects of co-exposure of chlorpyrifos (CPF) and ZnO NPs on fish-derived cells were investigated. A selection of concentrations was tested in single and binary exposures: CPF (0.312 - 75 mg/L) and ZnO NPs (10 - 100 mg/L). Cytotoxicity was measured using commonly used cellular endpoints: Alamar Blue/CFDA-AM for viability and plasma membrane integrity, NRU for lysosomal disruption and MTT for mitochondrial function. In addition, specific mechanisms of toxicity for CPF and ZnO NPs were tested: acetylcholinesterase (AChE) activity and ROS generation, respectively. AChE was by far the most sensitive assay for single exposure to CPF. There was no concentration-response relationship for ROS after single exposure to ZnO NPs, but 10 mg/L produced significant effects only for this cellular endpoint. Co-exposure of CPF with 10 m/L of ZnO NPs produced significant effects in almost all endpoints tested, which were enhanced by co-exposure with 100 mg/L of ZnO NPs. AChE testing of additional co-exposures with bulk ZnO, together with the application of the Independent Action (IA) prediction model, which allowed us to draw more in-depth conclusions on the toxicological behavior of the mixture. Synergism was observed at 0.625 mg/L CPF concentration and antagonism at 5 mg/L CPF in mixtures containing 100 mg/L of both ZnO NPs and bulk ZnO. However, more cases of synergism between CPF and ZnO NPs occurred at intermediate CPF concentrations, demonstrating that nano-sized particles have a more toxic interaction with CPF than bulk ZnO. Therefore it can be argued that in vitro assays allow the identification of interaction profiles of NP-containing mixtures by achieving multiple endpoints with a large number of concentration combinations.

The potential toxicity of microplastics (MPs) and hydrophilic pharmaceuticals to aquatic organisms has recently raised great public concern, yet their combined effects on aquatic organisms remain largely unknown. Herein, the combined effects of MPs and the commonly prescribed amitriptyline hydrochloride (AMI) on the intestinal tissue and gut microbiota of zebrafish (Danio rerio) were investigated. Adult zebrafish were exposed to microplastics (polystyrene, PS, 440 µg/L), AMI (2.5 µg/L), PS+AMI (440 µg/L PS + 2.5 µg/L AMI), and dechlorinated tap water (control) for 21 days, respectively. Our results showed that zebrafish rapidly ingested PS beads and accumulated them in the gut. Exposure to PS+AMI significantly enhanced the SOD and CAT activities compared to the control group, suggesting that combined exposure might increase ROS production in the zebrafish gut. Exposure to PS+AMI led to severe gut injuries, including cilia defects, partial absence and cracking of intestinal villi. Exposure to PS+AMI caused shifts in the gut bacterial communities, increasing the abundance of Proteobacteria and Actinobacteriota, and decreasing the abundance of Firmicutes, Bacteroidota and beneficial bacteria Cetobacterium, which caused dysbiosis in the gut microbiota, and subsequently may induce intestinal inflammation. Furthermore, exposure to PS+AMI disordered the predicted metabolic functions of gut microbiota, but functional changes in the PS+AMI group at KEGG level 1 and level 2 were not significantly different from those in the PS group. The results of this study extend our knowledge of the combined effects of MPs and AMI on the health of aquatic organisms, and will be helpful in assessing the combined effects of MPs and tricyclic antidepressants on aquatic organisms.

Dreissena polymorpha is a sentinel freshwater mussel providing key functional ecosystemic services like nutrient recycling and suspended matter filtration. Global warming and especially extreme events imply rapid fluctuations of environmental parameters that sessile organisms could not escape. The increase occurrence of heat waves and the subsequent expansion of hypoxic areas could challenge the survival of mussels. This study provided a deeper knowledge of energy management and cellular function during thermal (+15 °C) or hypoxic (30% of dissolved oxygen saturation) stress for 7 days. A potential metabolic rate depression was highlighted in D. polymorpha under hypoxia through a decline in the mitochondrial activity and a constant AMP content over time. A contrasted pattern of response was observed in thermal-stressed mussels between 24 h and 7 days of exposure. A global increase of metabolic activity was noticed in mussels after 24 h while a return to control level was noticed at the end of the experiment. Although D. polymorpha is considered as a temperature tolerant species, a significant increase of ADP:ATP ratio, related to a decrease of mitochondrial activity and density, suggested an overwhelming of organisms. This study pointed to the importance of considering time of exposure to natural factor variations in tolerance window of organisms in a long-term changing environment. The apparent short-term tolerance of D. polymorpha could hide much more deleterious consequences, i.e. mortality, if abiotic stresses persist, as suggested by climate change models.

Microplastics, plastic particles and fragments smaller than 5 mm are ubiquitous in various aquatic environments, but the hazards of microplastics with different particle sizes, concentrations and materials are not well understood. This study investigated the toxicity of polyethylene microplastics (PE-MPs) with different concentrations and particle sizes or polystyrene microplastics (PS-MPs) on freshwater algae Chlorella vulgaris Beij (C. vulgaris) for 11 days. Results indicated that the growth, colony formation, photosynthetic pigment contents and soluble intracellular polysaccharides were unaffected, whereas the photosynthetic efficiency and the total soluble protein (TSP) contents were remarkably decreased at 11 d with the increased concentration of PE-MP exposure. The growth, photosynthetic efficiency, soluble intracellular polysaccharides and TSP contents were unaffected after exposure to PE-MPs with different particle sizes or PS-MPs. By contrast, the colony formation and photosynthetic pigment contents were remarkably decreased after exposure to PS-MPs compared with the control or PE-MPs with the same particle size. The C. vulgaris colonization on microplastics was proven by scanning electron microscopy, indicating that the adsorption effects were the main harmful pathways of different microplastics to algal. Our results suggested that microplastics have limited harmful effects on algae, mainly in adsorption and shading.

Hypoxia causes irreversible damage to aquatic animals. However, few reports have explored the effect of hypoxia stress and reoxygenation on intestinal homeostatic imbalance and consequent hepatopancreas-intestine axis health in crustacean. Herein, 180 Chinese mitten crabs (Eriocheir sinensis) were equally divided into control (DO 7.0 ± 0.2 mg/L) and treatment groups. The treatment group was exposed with continuous hypoxic stress (DO 3.0 ± 0.1 mg/L) for 96 h and then reoxygenated (DO 6.9 ± 0.1 mg/L) for 96 h. The effects on intestines and hepatopancreas of Chinese mitten crab were investigated, and the role of gut microbiota in hypoxia induced damages was explored. Hypoxia impaired intestinal tissue structure, and decreased swelling and the number of goblet cells, which are features that did not significantly improve after reoxygenation. With prolonged hypoxic stress, the activities of antioxidant enzymes (LDH, SOD and CAT) and MDA content in intestine were significantly elevated. Moreover, the level of oxidative stress increased, which led to upregulated apoptosis rate and expression of apoptosis-related genes (Caspase 3, Caspase 8 and BAX). In addition, the expression of immune related genes (MyD88, ALF1, Relish and Crustin) in hepatopancreas and intestine was both significantly induced under hypoxia, which activated the immune defense mechanism of Chinese mitten crab to adapt to the hypoxic environment. Furthermore, diversity and relative abundance of gut microbiota decreased noticeably during hypoxic stress; the number of beneficial bacteria downregulated. Finally, KEGG pathway analysis revealed that nine pathways were significantly enriched in intestinal microorganisms, including autoimmune disease and environmental adaptation. Collectively, these results suggested that hypoxia negatively affected E. sinensis health by triggering oxidative stress, altering the composition of the gut microbiota and inhibiting the immune response.

Aquatic herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D) formulations, are commonly used for invasive species management throughout the United States. Ecologically relevant concentrations of 2,4-D can impair essential behaviors, reduce survival, and act as an endocrine disruptor; however, there is limited knowledge of its effects on the health of non-target organisms. Here, we investigate the acute and chronic exposure impacts of 2,4-D on adult male and female fathead minnow (Pimephales promelas) innate immune function. We exposed both adult male and female fathead minnows to three different ecologically relevant concentrations of 2,4-D (0.00, 0.40, and 4.00 mg/L) and took blood samples at three acute time points (6, 24, and 96 h) and one chronic time point (30 days). We found that male fatheads had higher total white blood cell concentrations when exposed to 2,4-D at the acute time points. For the females, only proportions of specific cell types were altered when exposed to 2,4-D at the acute time points. However, we did not observe any significant impacts of chronic exposure to 2,4-D on any innate immune responses for either males or females. Overall, this study is the first step in answering an important question for game fisheries and management agencies while providing insight to future studies that investigate the impacts of herbicide exposure to freshwater fish health and immunity.

Manganese (Mn) is an essential metal for organisms, but high levels can induce serious toxicity. To date, the toxic mechanism of Mn to marine fish is still poorly understood. In the present study, Oryzias melastigma embryos were exposed to different concentrations of MnCl2 (0–152.00 mg/L) to investigate its effect on early development. The results showed that exposure to MnCl2 caused developmental toxicity to embryos, including increased heart rate, delayed hatching time, decreased hatching rate and increased malformation rate. MnCl2 exposure could induce oxidative stress in O. melastigma embryos, as indicated by increased the contents of malondialdehyde (MDA) and the activities of the antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)). The heart might be an important target organ for MnCl2 because of cardiac malformations and disruption in the expression of cardiac development-related genes (ATPase, epo, fg8g, cox1, cox2, bmp4 and gata4). In addition, the expression levels of stress- (omTERT and p53) and inflammation-related genes (TNFα and il1β) were significantly up-regulated, suggesting that MnCl2 can trigger stress and inflammatory response in O. melastigma embryos. In conclusion, this study demonstrated that MnCl2 exposure can induce developmental toxicity, oxidative stress and inflammatory response in O. melastigma embryos, providing insights into the toxic mechanism of Mn to the early development of marine fish.

Contaminated wastewater (WW) can cause severe hazards to numerous delicate ecosystems and associated life forms. In addition, human health is negatively impacted by the presence of microorganisms in water. Multiple pathogenic microorganisms in contaminated water, including bacteria, fungi, yeast, and viruses, are vectors for several contagious diseases. To avoid the negative impact of these pathogens, WW must be free from pathogens before being released into stream water or used for other reasons. In this review article, we have focused on pathogenic bacteria in WW and summarized the impact of the different types of pathogenic bacteria on marine organisms. Moreover, we presented a variety of physical and chemical techniques that have been developed to provide a pathogen-free aquatic environment. Among the techniques, membrane-based techniques for trapping hazardous biological contaminants are gaining popularity around the world. Besides, novel and recent advancements in nanotechnological science and engineering suggest that many waterborne pathogens could be inactivated using nano catalysts, bioactive nanoparticles, nanostructured catalytic membranes, nanosized photocatalytic structures, and electrospun nanofibers and processes have been thoroughly examined.

Plastics are the most abundant marine litter in the world's oceans and can be degraded into micro-plastics. These emerging pollutants negatively affect marine organisms, but little is known on the effects on macroalgae. In this study, we investigated the effects of micro-plastics on two species of red algae: Grateloupia turuturu and Chondrus sp. Grateloupia turuturu has a slippery surface while Chondrus sp. has a rough surface. Different surface characteristics of these macroalgae may affect adherence of micro-plastics. Both species were exposed to 5 different concentrations (0, 20, 200, 2000 and 20,000 ng/L) of polystyrene microspheres. Adherence capacity, accumulating micro-plastics on the surface was higher for Chondrus sp. than G. turuturu. Chondrus sp. at 20,000 ng/L only showed a decrease in growth rate and photosynthesis activity, and an increase of reactive oxygen species (ROS). However, G. turuturu was not significantly affected by micro-plastics at all tested concentrations. Shaded light and inhibition of gas flow by adhered micro-plastics may be a reason for the reduction of growth and photosynthesis and production of ROS. Based on this result, the toxic effects of micro-plastics appear to be species specific, dependent on the adherence capacity of macroalgae.

Oxytetracycline (OTC), a commonly used tetracycline antibiotic in aquaculture, has been found to cause significant damage to the liver of largemouth bass (Micropterus salmoides). This study revealed that OTC can lead to severe histopathological damage, structural changes at the cellular level, and increased levels of reactive oxygen species (ROS) in M. salmoides. Meanwhile, OTC impairs the activities of antioxidant enzyme (such as T-SOD, CAT, GST, GR) by suppressing the activation of MAPK/Nrf2 pathway. OTC disrupts mitochondrial dynamics and mitophagy through via PINK1/Parkin pathway. The accumulation of damaged mitochondria, combined with the inhibition of the antioxidant enzyme system, contributes to elevated ROS levels and oxidative liver damage in M. salmoides. Further investigations demonstrated that an enzyme-treated soy protein (ETSP) dietary supplement can help maintain mitochondrial dynamic balance by inhibiting the PINK1/Parkin pathway and activate the MAPK/Nrf2 pathway to counteract oxidative damage. In summary, these findings highlight that exposure to OTC disrupts mitochondrial dynamics and inhibits the antioxidant enzyme system, ultimately exacerbating oxidative liver damage in M. salmoides. We propose the use of a dietary supplement as a preventive measure against OTC-related side effects, providing valuable insights into the mechanisms of antibiotic toxicity in aquatic environments.

Due to the high production volume and persistence in the environment of bisphenol A (BPA) and its substitutes, realistic exposure scenarii were proposed in some species to better understand the relationship between external and internal concentrations. For example, a recent PBTK model has been developed and adapted to BPA ADME (Absorption, Distribution, Metabolization, and Excretion) processes in three-spined stickleback. These substances have an impact on organism physiology including reproductive and immune functions. In this context, physiologically-based toxicokinetic models coupled with toxicodynamics (PBTK-TD) have proven to be valuable tools to fill the knowledge gap between external exposure and effect dynamics. The aim of the current work was to explain the impact of BPA on the immune response by determining its temporality. In addition, the relationship between BPA dose and these responses was investigated using a PBTK-TD model. Two experiments were performed on stickleback to characterize their biomarker responses, (i) a short exposure (14 days) at 0, 10 and 100 µg/L, including a depuration phase (7 days), and (ii) a long exposure (21 days) at 100 µg/L to measure the immunomarker dynamic over a long period. The fish spleens were sampled to analyze immune responses of stickleback at various times of exposure and depuration: leucocyte distribution, phagocytic capacity and efficiency, lysosomal presence and leucocyte respiratory burst index. At the same date, blood, muscle, and liver were sampled to quantify BPA and their metabolites (BPA monoglucuronide and BPA monosulfate). All these data enabled the development of the indirect pharmacodynamic models (PBTK-TD) by implementing the responses of biomarkers in the existing BPA PBTK of stickleback. The results shown a high induction of phagocytosis activity by BPA in the two exposure conditions. Furthermore, the immunomarkers exhibit very different temporal dynamics. This study demonstrates the need of a thorough characterization of biomarker response for a further use in Environmental Biomonitoring.

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.

Enrofloxacin (ENR) is a broad-spectrum antibiotic widely used due to its efficacy against pathogens. Microplastics (MPs) may bind to ENR and reduce its efficiency, whereas there would be an increase in its toxicity, bioavailability, and bio-accumulation rates. Therefore, the hypothesis is that the interaction between MPs and ENR can alter their toxicity and bioavailability. The subjective of this study is to examine the toxicity of various concentrations of ENR (0, 1.35, and 2.7 ml Kg−1 diet) and MPs (0, 1000, and 2000 mg Kg−1 diet) alone and in combination for 21 days. The rainbow trout (Oncorhynchus mykiss) is an economic aquaculture species used as an experimental model in ecotoxicology studies. Blood biochemical analytes indicated that ENR and MPs combination led to increasing enzymatic activity of each biomarker, except for gamma-glutamyl-transferase (GGT). Alterations related to triglycerides, cholesterol, glucose, urea, creatinine, total protein, and albumin blood contents were observed. An elevation in the levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glucose 6-phosphate dehydrogenase (G6PDH) was found in the liver. In contrast, catalase (CAT) and glutathione peroxidase (GPx) levels decreased. Furthermore, a decline was observed in the cellular total antioxidant (ANT) levels. These findings suggested that ENR and MPs could affect fish health both independently and together. Consequently, the study determined that when both ENR and MPs were present in high concentrations, the toxicity of ENR was amplified, providing further evidence of the synergistic impact of MPs on ENR toxicity.

Progestins are widely used and detected in surface waters, and can affect gonad development and sexual differentiation in fish. However, the toxicological mechanisms of sexual differentiation induced by progestins are not well understood. Here, we investigated the effects of norethindrone (NET) and androgen receptor (AR) antagonist flutamide (FLU) on gonadal differentiation in zebrafish from 21 dpf (days post-fertilization) to 49 dpf. The results showed that NET caused male bias, while FLU resulted in female bias at 49 dpf. The NET and FLU mixtures significantly decreased the percentage of males compared to the NET single exposure. Molecular docking analysis showed that FLU and NET had similar docking pocket and docking posture with AR resulting in competitively forming the hydrogen bond with Thr334 of AR. These results suggested that binding to AR was the molecular initiating event of sex differentiation induced by NET. Moreover, NET strongly decreased transcription of biomarker genes (dnd1, ddx4, dazl, piwil1 and nanos1) involved in germ cell development, while FLU significantly increased transcription of these target genes. There was an increase in the number of juvenile oocytes, which was consistent with the female bias in the combined groups. The bliss independence model analysis further showed that NET and FLU had antagonistic effect on transcription and histology during gonadal differentiation. Thus, NET suppressed the germ cell development via AR, resulting in male bias. Understanding the molecular initiation of sex differentiation in progestins is essential to provide a comprehensive biological basis for ecological risk assessment.

Environmental micro(nano)plastics have become a significant global pollution problem due to the widespread use of plastic products. In this review, we summarized the latest research advances on micro(nano)plastics in the environment, including their distribution, health risks, challenges, and future prospect. Micro(nano)plastics have been found in a variety of environmental media, such as the atmosphere, water bodies, sediment, and especially marine systems, even in remote places like Antarctica, mountain tops, and the deep sea. The accumulation of micro(nano)plastics in organisms or humans through ingestion or other passive ways poses a series of negative impacts on metabolism, immune function, and health. Moreover, due to their large specific surface area, micro(nano)plastics can also adsorb other pollutants, causing even more serious effects on animal and human health. Despite the significant health risks posed by micro(nano)plastics, there are limitations in the methods used to measure their dispersion in the environment and their potential health risks to organisms. Therefore, further research is needed to fully understand these risks and their impacts on the environment and human health. Taken together, the challenges of micro(nano)plastics analysis in the environment and organisms must be addressed, and future research prospects need to be identified. Governments and individuals must take action to reduce plastic waste and minimize the negative impact of micro(nano)plastics on the environment and human health.

Copper ions (Cu) are one of the most frequent trace-contaminants found in Brazilian waters and, although considered as an essential element, in high concentrations can accumulate and induce toxicity. Biomarkers are important tools that can be used to assess these impacts, but to be considered trustworthy, they have to be previously tested in target organisms through laboratory studies under controlled conditions. However, many of these experiments are conducted using only males, as it is believed that the hormonal variation of females can bias the results, increasing data variability. Notwithstanding, few studies have actually tested this hypothesis, highlighting the importance of considering and measuring the role of sex in ecotoxicological studies. The aim this study was to evaluate the influence of sex on biomarkers classically used in environmental monitoring programs using the fish Poecilia vivipara as model. For this, females and males were exposed for 96 h to two Cu concentrations (9 and 20 μg/L) and a control group. In liver and gills, Cu accumulation, total antioxidant capacity (TAC) and lipid peroxidation (LPO) were evaluated. In addition, samples of peripheral blood were used for neutrophil to lymphocyte ratio determination, a measure of the onset of secondary stress. Results show that Cu hepatic accumulation did not differ between females and males, but higher levels of this metal were observed in exposed animals compared to control fish. Additionally, interactive effects were observed for hepatic LPO, as males showed elevated oxidative damage in comparison to females. Moreover, Cu exposure elevated hepatic LPO relative to control only in males, but this increase in oxidative damage was not accompanied by changes in liver TAC. On the other hand, differences in branchial Cu accumulation and LPO were not observed. Conversely, control females showed elevated TAC in comparison to control males, but Cu exposure eliminated this difference. Cu exposure also induced an increase in the N:L ratio, indicating the presence of a secondary stress response unrelated to sex. Ultimately, the findings of this study demonstrate that sex can influence the response of biomarkers that are typically used in ecotoxicological investigations in a multifaceted manner. As a result, using animals from a singular sex in such studies may result in consequential outcomes, potentially leading to underestimation or overestimation of results.

Ammonia nitrogen is a long-lasting pollutant along the Chinese coast. In our previous studies, the clam Ruditapes philippinarum exhibited several toxic responses to environmental concentrations of ammonia nitrogen. To elucidate the underlying mechanism of ammonia nitrogen toxicity in clams at the post-transcriptional level, microRNA (miRNA) expression profiles were investigated by high-throughput sequencing after the clams were exposed to 0.1 mg/L ammonia nitrogen for 30 days. A total of 238 miRNAs were identified, including 49 conserved miRNAs and 189 novel miRNAs. After comparative analysis, six miRNAs were significantly expressed after 1 day of exposure, with three up-regulated and three down-regulated miRNAs. In addition, 35 miRNAs were significantly expressed after 30 days of exposure, of which 16 were up-regulated and 19 were down-regulated. Furthermore, the target genes of each differentially expressed miRNA were predicted, followed by Gene Ontology (GO) category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The target genes were predicted to be involved in the immune response, protein processing and transport, DNA damage repair, cellular communication, neural signaling, redox homeostasis, lipid metabolism, and biotransformation. A biological phagocytosis assay proved the speculation that ammonia nitrogen regulated the immunity of clams with the aid of a novel miRNA (novel_29). These findings support further research on miRNA levels in R. philippinarum exposed to ammonia nitrogen.