Ecotoxicological Studies Research Articles

Hidden impacts of environmental stressors on freshwater communities could be revealed at lower concentrations by correlation of abundances network analyses: An example with herbicides glyphosate, 2,4‐D, and their mixtures

AbstractMultiple stressors affect freshwater systems worldwide. Ecotoxicological studies are often focused on the abundance analysis of their communities. In this study, I discuss the possibility of incorporating correlation networks to earlier bring to light the possible hidden effects of environmental stressors on community interactions. A re‐analysis of the published data from an experiment where the herbicides glyphosate, 2,4‐D and their mixtures were applied in microcosms revealed that correlation networks can detect impacts on the community interactions at herbicide concentrations lower than those in which abundances were drastically modified. I propose the correlation of abundances network analysis (CANA) as a new approach for ecotoxicological freshwater community studies.

Histological Alteration of Green Mussel Perna viridis Organs Exposed to Microplastics

Microplastic in the oceans might interfere the health of marine organisms, including the green mussels (Perna viridis). This is due to microplastic accumulation in mussels organs, such as gills, hepatopancreas, and gonads. Therefore, tissue alteration is a good indicator for ecological risk analysis and other ecotoxicological study activities. Green mussels with shells 4.1 to 5.0 cm in length were collected from Mandalle waters, Pangkep Regency (Pangkajene Islands), Indonesia. Green mussels were exposed for seven days to microplastic with concentrations of 0.05 (A), 0.5 (B), and 5 (C) g/L. The results showed that the higher the concentration of microplastics exposed to the green mussel, the higher the accumulation of microplastics in the body of the mussel, within the tested concentrations. The increased concentration of microplastics increased the level of tissue alteration in the gills, hepatopancreas, and gonads, with the most sensitive organ being the hepatopancreas. Overall, the study confirmed that the histological assay of mussel organs could be used as a biomarker in ecotoxicological studies.

Waste brick dust as a prospective eco-friendly alternative component of artificial soils for ecotoxicological studies

Current artificial soils for ecotoxicological studies contain non-renewable materials that must be mined, and their production and processing consume a lot of energy and generate a significant amount of carbon dioxide (CO2). In this paper, waste brick dust is proposed as an alternative to calcium carbonate (CaCO3), which is used for pH adjustment of the Organization for Economic Co-operation and Development (OECD) soils. The artificial soils containing brick dust are contaminated with boric acid as a reference substance in the concentration range of 100-500mg/kg and studied in the tests with enchytraeids (E. crypticus), springtails (F. candida), and plants (L. sativa and B. napus). Experimental results shows the suitability of replacing calcium carbonate with waste brick dust, as neither toxicity nor ability of model organisms to inhabit the analyzed soil is found. A comparison with the standard OECD soil does not reveal any substantial differences between the parameters (survival, reproduction, and root elongation) of the applied ecotoxicological tests. The brick dust as waste material is found to have a lower carbon footprint than CaCO3, while a similar amount of water is necessary for the adjustment of tests with both kinds of artificial soil. The waste brick dust can be considered as asuitable eco-friendly alternative to CaCO3 in artificial soils for ecotoxicological studies.

Seasonal composition and activity of the intertidal macrobenthic community of Caleta Valdés (Patagonia, Argentina) applying in situ and ex situ experimental protocols

Caleta Valdés (CV) is a coastal lagoon of the Patagonian Atlantic coast located in the Península de Valdés declared as Humanity Mundial Patrimony due to its remarkable biodiversity, ecosystemic richness, and pristine state. Marine mammal populations are well documented in this area but few studies have been carried out on the local macrobenthic communities. The goals of this study were (a) to evaluate for the first time the seasonal variation of the structure and activity (i.e., sediment reworking) of the intertidal macrobenthic communities of CV, and (b) to validate an ex situ experimental protocol for future ecotoxicological studies. To do so, sedimentary (granulometry, water content, and organic matter) and biological (macrobenthic assemblages, biodiffusive-like and advective sediment reworking components) parameters were analyzed using a combination of in situ and ex situ measurements. Overall, polychaete and crustacean dominated the macrobenthic community. The highest abundances were found in spring–summer along with a predominance of biodiffusors versus conveyors. Ex situ and in situ measurements demonstrated similar results, thus allowing validation of an ex situ experimental procedure for macrobenthic community and functioning studies. In addition, these results provide a first baseline of benthic information on CV that will be helpful to monitor the effects of potential pollution in Patagonian coastal systems.

Comparative genome analysis of the monogonont marine rotifer Brachionus manjavacas Australian strain: Potential application for ecotoxicology and environmental genomics

This is the first study to analyze the whole-genome sequence of B. manjavacas Australian (Aus.) strain through combination of Oxford Nanopore long-read seq, resulting in a total length of 108.1 Mb and 75 contigs. Genome-wide detoxification related gene families in B. manjavacas Aus. strain were comparatively analyzed with those previously identified in other Brachionus spp., including B. manjavacas German (Ger.) strain. Most of the subfamilies in detoxification related families (CYPs, GSTs, and ABCs) were highly conserved and confirmed orthologous relationship with Brachionus spp., and with accumulation of genome data, clear differences between genomic repertoires were demonstrated the marine and the freshwater species. Furthermore, strain-specific genetic variations were present between the Aus. and Ger. strains of B. manjavacas. This whole-genome analysis provides in-depth review on the genomic structural differences for detoxification-related gene families and further provides useful information for comparative ecotoxicological studies and evolution of detoxification mechanisms in Brachionus spp.

No effect of dual exposure to sulfoxaflor and a trypanosome parasite on bumblebee olfactory learning

Bees are important pollinators in wild and agricultural ecosystems, and understanding the factors driving their global declines is key to maintaining these pollination services. Learning, which has been a focus of previous ecotoxicological studies in bees, may play a key role in driving colony fitness. Here we move beyond the standard single-stressor approach to ask how multiple stressors, an agrochemical (sulfoxaflor, a relatively new insecticide) and a parasite (Crithidia bombi, a prevalent gut parasite of bumblebees), impact learning in the bumblebee Bombus terrestris. We developed a modified version of the classic proboscis extension reflex assay to assess the combined effects of acute oral sulfoxaflor exposure and infection by C. bombi on olfactory learning of bumblebee workers. We found no evidence that either sulfoxaflor, C. bombi, or their combination had any significant effect on bumblebee olfactory learning, despite their known negative impacts on other aspects of bumblebee health. This suggests that losses in cognitive ability, as measured here, are unlikely to explain the impacts of sulfoxaflor and its interactions with other stressors on bumblebees. Our novel methodology provides a model system within which to test interactive effects of other key stressors on bee health.

Nickel and cadmium tissue bioaccumulation and blood parameters in Chelon auratus and Mugil cephalus from Anzali free zone in the south Caspian Sea (Iran) and Faro Lake (Italy): A comparative analysis

BackgroundAnalysis of heavy metal concentrations in fish blood is a valuable tool in environmental pollution monitoring. Among different type of fish, detritivorous fish are a very good indicator for monitoring pollution and environmental stress, along with hematological studies, which are an important indicator in eco-toxicological and biological studies. AimThe aim of this study was to evaluate the influence of environment on bioaccumulation of Ni and Cd and on blood parameters in Chelon auratus and Mugil cephalus (a detritivorous fish which is widely distributed in the world) captured in Caspian Sea and Faro Lake. MethodsFor the research blood and tissues samples were collected from 40 mullets (20 Mugil cephalus from Italy and 20 Chelon auratus from Iran) in 2019. The hematological (white blood cell, WBC; red blood cell, RBC; thrombocyte count, TC; hematocrit, Hct; hemoglobin concentration, Hb; mean corpuscular volume, MCV; mean corpuscular hemoglobin, MCH and mean corpuscular hemoglobin concentration, MCHC) and biochemical parameters (aspartate aminotransferase, AST; alanine aminotransferase, ALT; alkaline phosphatase, ALP; lactate dehydrogenase, LDH and creatine phosphokinase, CPK were assessed. ResultsAlthough the concentration of Ni and Cd in the muscle of fish have no significant health risks and were low in both regions, the most elevated concentration was found in the liver of Caspian Sea mullet. In all cases, the results obtained for all biochemical and most hematological parameters of individual Faro Lake, were considerably lower than the Caspian Sea, demonstrating that habitats and environmental conditions affect the blood metabolites. ConclusionsThe results of this study show that these measurements can be used as criteria for the quantitative evaluation of fish /health and provide information on the extent of potential poisoning and the risks posed to the populations and fisheries.

Application of land snail Helix lucorum for evaluation of genotoxicity of soil pollution

Application of native species as sentinels allows environmental scientists to determine real genotoxic impact of environmental pollutants. The present study aims at investigating the DNA damage in the land snail Helix lucorum as a biomarker of soil pollution. For this reason, the genotoxic impact of contaminated soil on H. lucorum, collected from different polluted areas, was investigated using the comet assay in haemocytes and digestive gland cells. An increase in DNA damage was found in the snails sampled from polluted sites compared with the reference one. Strong correlations between DNA damage in haemocytes and digestive gland cells with the level of contamination indicate pollution-induced genotoxic effects in both tissues. At the same time, the digestive gland was more sensitive towards pollutants compared with haemolymph. A direct relationship between concentrations of Cu, As and Mo in soil and the number of damaged cells for hаemolymph and digestive gland tissue was found. However, the data obtained reflect the total genotoxicity of all pollutants in the studied areas. Significant correlations between the DNA damage measured by the comet assay and metal contents in soil indicate that it is a suitable biomarker in ecotoxicological studies. Our results indicate the effectiveness of H. lucorum in biomonitoring of environmental pollution.

Comparative analysis of novel and common reference genes in adult tissues of the mussel Mytilus galloprovincialis.

BackgroundReal-time quantitative PCR is a widely used method for gene expression analyses in various organisms. Its accuracy mainly relies on the correct selection of reference genes. Any experimental plan involving real-time PCR needs to evaluate the characteristics of the samples to be examined and the relative stability of reference genes. Most studies in mollusks rely on reference genes commonly used in vertebrates.ResultsIn this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1α, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues.ConclusionMytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-022-08553-1.

Effects of trace elements contaminations on the larval development of Paracentrotus lividus using an innovative experimental approach.

Several experiments were performed using larvae of Paracentrotus lividus (Lamarck, 1816) in order to determine the consequences of different chronic contamination with mixtures of (i) fifteen trace elements from concentrations measured in the world ocean seawater, and (ii) seven trace elements from contamination resulting from mining. To predict the impact of increased marine pollution, higher concentrations were also used. These bioassays were conducted using spawners collected from Calvi (reference site, Corsica), and Albo (mining area, Corsica). The effects of trace elements have been studied on the entire larval development. The results show wider arms and delayed development as the number and concentration of trace elements increases. Therefore, the synergy between the different trace elements is of paramount importance with regard to the impact on organisms. Probably due to a hormesis phenomenon, larvae contaminated with seven trace elements at average concentrations developed more quickly. This work also highlighted the importance of the origin of spawners in ecotoxicological studies. To our knowledge, this is the first study to investigate the effects of such a broad combination of trace elements for chronic contamination on the entire larval stage of Paracentrotus lividus.

A mini-review of toxicokinetics and toxicity of heavy metals in marine and freshwater fish

The aquatic environment continues to be polluted by anthropogenic activities that cause heavy metals contamination of fish. These trace metals contaminants are present in abundance in the aquatic environment because their inputs also originate from several natural processes. In addition, they are bioaccumulative, persistent and non-biodegradable over time. Therefore the present mini-review accounts for to assesses the bio-kinetics and known effects of the heavy metals and their toxicity in fish. Among the most toxic metals are arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg), and they have been the primary focus of many aquatic ecotoxicological studies in recent years. According to the previous studies analyzed; heavy metals bioaccumulate in the body of fish, whose kinetic activity depends on the metal and also varies from one fish species to another, and can cause irregular and sometimes devastating effects in different organs and systems of the body, acute or chronic, depending on the duration of exposure and the dose of metal assimilated through water or food. Metal uptake can affect all life stages of fish, and these effects are a function of the concentration of the metal in the surrounding environment, its chemical form and also the type of the water in which it lives; fresh or marine.

Assessment of RNA extraction protocols from cladocerans.

The usage of cladocerans as non-model organisms in ecotoxicological and risk assessment studies has intensified in recent years due to their ecological importance in aquatic ecosystems. The molecular assessment such as gene expression analysis has been introduced in ecotoxicological and risk assessment to link the expression of specific genes to a biological process in the cladocerans. The validity and accuracy of gene expression analysis depends on the quantity, quality and integrity of extracted ribonucleic acid (RNA) of the sample. However, the standard methods of RNA extraction from the cladocerans are still lacking. This study evaluates the extraction of RNA from tropical freshwater cladocerans Moina micrura using two methods: the phenol-chloroform extraction method (QIAzol) and a column-based kit (Qiagen Micro Kit). Glycogen was introduced in both approaches to enhance the recovery of extracted RNA and the extracted RNA was characterised using spectrophotometric analysis (NanoDrop), capillary electrophoresis (Bioanalyzer). Then, the extracted RNA was analysed with reverse transcription polymerase chain reaction (RT-PCR) to validate the RNA extraction method towards downstream gene expression analysis. The results indicate that the column-based kit is most suitable for the extraction of RNA from M. micrura, with the quantity (RNA concentration = 26.90 ± 6.89 ng/μl), quality (A260:230 = 1.95 ± 0.15, A280:230 = 1.85 ± 0.09) and integrity (RNA integrity number, RIN = 7.20 ± 0.16). The RT-PCR analysis shows that the method successfully amplified both alpha tubulin and actin gene at 33-35 cycles (i.e. Ct = 32.64 to 33.48). The results demonstrate that the addition of glycogen is only suitable for the phenol-chloroform extraction method. RNA extraction with high and comprehensive quality control assessment will increase the accuracy and reliability of downstream gene expression, thus providing more ecotoxicological data at the molecular biological level on other freshwater zooplankton species.

NUTRITIONAL AND TOXICOLOGICAL STUDIES ON Periophthalmus koelreuteri FROM OKPOAMA-BRASS, BAYELSA STATE

Mudskippers have been used extensively in ecotoxicological studies due to their tendency to bioaccumulate heavy metals and other environmental toxicants. The present study investigated the proximate, fatty acids, minerals and heavy metals composition of Periophthalmus koelreuteri (mudskipper) from Okpoama-Brass, Bayelsa State. Proximate composition was determined by AOAC methods, fatty acids composition was determined by gas chromatography and mineral/heavy metals were determined by atomic absorption spectrophotometry. Health risks associated with consumption of mudskippers (estimated daily intake-EDI, target hazard quotient-THQ and carcinogenic risk-CR) were determined by calculation. Proximate composition of samples were as follows: 52.50±0.20% crude protein, 7.50±0.02% crude fat, 0.29±0.01% carbohydrates, 15.71±0.015% crude fibre, 18.00±1.00% ash and 6.00±0.04% moisture. The following fatty acids were detected: lauric acid, palmitic acid, margaric acid, stearic acid, oleic acid, linoleic acid and arachidonic acid. Minerals detected includes Na, K, Ca, Mg, Fe, Zn, Cu, Co and Mo and heavy metals (Cd>Cr>Pb). Health risk assessment showed the following: EDI for Cd was 0.001 mg/100g-1bw/day; EDI for Cr in the samples was 0.000628mg/100g-1bw/day. THQ value for Cd was >1 hence consumption of mudskipper (with respect to Cd) is not advised as it may pose health threats to the consumers. THQ for Cr and Pb were <1 hence there are no health risks associated with respect to these metals. CR values for all heavy metals (Cd, Cr and Pb) in the sample were within the permissible limits. Mudskipper is a good source of protein, fatty acids and minerals however it accumulates toxic heavy metals from the environment.

LipidTOX: A fatty acid-based index efficient for ecotoxicological studies with marine model diatoms exposed to legacy and emerging contaminants

Contaminants, when present above certain thresholds, can induce physiological constraints to organisms, namely diatoms, a model group representative of marine phytoplankton, triggering feedback mechanisms, such as changes in cell’s fatty acid profiles, that can be used as biomarkers towards xenobiotic exposure. Having this in mind and considering the ecological relevance of diatom fatty acid profiles as well as their recognized potential as biomarkers of contaminant exposure, the present work aims to develop and test the accuracy of an integrative multi-biomarker response index based on the fatty acid profiles of marine diatoms (using Phaeodactylum tricornutum as model diatom) exposed to several emerging contaminants.In terms of the impacts at the individual fatty acid level, it was possible to observe changes transversal to different contaminants, such as the reduction of C14:0 and C16:0 fatty acids, with increasing xenobiotic concentration, as observed, for example, under propranolol and fluoxetine exposure. Enhancement of C16:2n-7 and C16:3n-4 concentrations as well as complete disruption of the basal fatty acid profile was observed in diatoms exposed to copper nanoparticles. These individual diverse and intrinsically connected alterations in fatty acid concentrations depended on the type and dose of the xenobiotic applied, highlighting the need to address these profiles as a whole. The evaluation of the diatom cells’ fatty acids using a multivariate approach revealed a high degree of sensitivity of these biochemical traits to disclose the type of xenobiotic applied to the diatoms, as well as the exogenous concentration used. These biochemical profiles were later incorporated into a unifying numerical index (LipidTOX) using an integrated biomarker response approach. The LipidTOX index showed strong correlations with both the exogenous xenobiotic concentration applied as well as with the growth features assessed for the exposed cultures, revealing a very high efficiency in translating growth impairments imposed by each of the xenobiotics tested at the different test concentrations. The LipidTOX index proved to be an efficient tool for ecotoxicological assays with marine model diatoms and evidenced a high degree of reliability for classifying the exposure of the cells to emerging contaminants. The results and benefits of the LipidTOX index application can be easily communicated to non-expert audiences such as stakeholders, policymakers and environmental managers so that this approach can be used in future toxicological evaluations of the impacts of classical and emerging xenobiotics in marine primary producers.

Ecotoxicological assessment of waste and soil cover of anthropogenic polluted territories using biotest systems

The possibility of using various methods of biotesting together with chemical-analytical methods for the ecotoxicological assessment of waste from the oil industry (drilling sludge) and contaminated soil cover of objects of accumulated environmental damage located in the north-west of Russia is considered. Ecotoxicological studies by biotesting methods have been carried out using test organisms from different systematic groups. Conventional and proprietary biotesting methods were used. The proposed set of biotest systems for the purposes of environmental control of such objects along with chemical and analytical methods may increase the reliability of the research results.

Commentary: Assessing the endocrine disrupting effects of chemicals on invertebrates in the European Union

Evidence from both laboratory and field studies has shown that currently used synthetic and naturally occurring chemical substances may potentially disrupt invertebrate endocrine systems, although the extent of this in field populations remains unclear. Translating concerns about potential endocrine disrupting chemicals (EDCs) into practical and effective regulatory action is currently hampered by the breadth of invertebrate endocrinology when compared to the better understood vertebrate systems, a lack of fundamental knowledge about the endocrinology of many invertebrate groups, and the resulting uncertainty when making regulatory decisions. This commentary (i) outlines the breadth of invertebrate endocrine pathways for which European Union regulation of potential EDCs may be relevant; (ii) reviews the extent to which current knowledge meets regulatory requirements for invertebrates, including an assessment of the suitability of current invertebrate test guidelines for detecting endocrine modes of action; and (iii) proposes a roadmap towards the regulation of potential EDCs with greater confidence, based on the Adverse Outcome Pathway (AOP) concept and a focus on identifying Molecular Initiating Events (MIEs) within AOPs. We conclude there are no validated tools to determine any invertebrate endocrine mode of action in vitro or in vivo. However, there are commonly used invertebrate toxicity tests which might capture adverse effects that could potentially result from an endocrine mode of action but would not identify the causal mechanisms. Therefore, EU regulatory requirements for the identification of EDCs cannot currently be satisfied for invertebrates, either in general or for the specific invertebrates used in standard ecotoxicological studies. We propose that the most important research need is compilation of a comprehensive list of endocrine-related MIEs across invertebrate taxa via use of high-throughput ‘omics in combination with bioinformatics reverse engineered analyses. Although tractable, such an approach would require significant resource investment for development and implementation.

The distinct toxicity effects between commercial and realistic polystyrene microplastics on microbiome and histopathology of gut in zebrafish

As a ubiquitous emerging pollutant, microplastics (MPs) have attracted widespread attention. At this stage, researchers mainly employed commercial MPs (CMPs) as the model particles to explore the toxic effects of MPs. But whether CMPs can reflect the effects of realistic MPs (RMPs) still remains unknown. Herein, the effects of commercial and realistic polystyrene MPs on gut microbiota of zebrafish were compared. Considering MPs co-exist with antibiotics in real environment, we further distinguished the effects of CMPs and RMPs when they co-existed with enrofloxacin (ENR). The results revealed that while both CMPs and RMPs significantly shifted the gut microbiota, CMPs exhibited stronger toxic effects and more severe damage to gut. Furthermore, ENR exhibited a distinct effect with both CMPs and RMPs on gut microbiota, while the addition of CMPs and RMPs significantly alleviated the toxicity of ENR. In addition, analysis via Kyoto Encyclopedia of Genes and Genomes pathway database revealed that seven major level 1 pathways associated with metabolism, information processing and diseases in the microbial community were affected. Taken together, this work is the first to report that CMPs could not represent RMPs in terms of toxicity and other behaviors, reminding people the limits of using CMPs in ecotoxicology studies.

Parasite infection influences the biomarker response and locomotor activity of Gammarus fossarum exposed to conventionally-treated wastewater

Modern wastewater treatment plants cannot completely remove pollutants. Often, effluents entering the aquatic environment still contain micropollutants such as pharmaceuticals or pesticides, which may impose adverse effects on aquatic biota. At the same time, a large proportion of free-living aquatic species are known to be infected with parasites, which raises the question of interactions between environmental stressors (such as micropollutants) and parasite infection. We chose the freshwater amphipod Gammarus fossarum (Koch, 1835) as a test organism to investigate potential pollutant-parasite interactions. This gammarid is frequently used in ecotoxicological tests and is also commonly infected with larvae of the acanthocephalan parasite species Polymorphus minutus (Zeder, 1800) Lühe, 1911. We exposed infected and uninfected specimens of G. fossarum to conventionally-treated wastewater and river water in a 22-day flow channel experiment. The test organisms’ response was measured as mortality rates, concentrations or activities of five biomarkers, and overall locomotor activity. No significant differences were found between mortality rates of different exposure conditions. Contrastingly, three biomarkers (phenoloxidase activity, glycogen, and lipid concentrations) showed a significant increase in infected gammarids, while the effect of the water type was insignificant. Infected gammarids also showed a significantly higher locomotor activity in both water types. Our results suggest that the response of G. fossarum during the exposure experiments was mainly driven by parasite infection. This implies that parasites may act as additional biotic stressors in multiple stressor scenarios, and therefore, might play an important role when measuring the response of organisms to chemical stressors. Future ecotoxicological studies and assessments thus should consider parasite infection as an additional test parameter.

A systematic review of the water treatment sludge toxicity to terrestrial and aquatic biota: state of the art and management challenges

Safe drinking water’ supply is an essential service and depends directly on the water treatment that produces water treatment sludge (WTS) as a product, whose final destination varies and remains a challenge. The ecotoxicity assessment of the WTS address the ecological implications of the WTS disposal but these information is still scarce in the literature. In this sense, we did a systematic review of the ecotoxicological studies on WTS using databases from six platforms. From the 785 papers recovered; 16 studies were eligible and showed the ecotoxicity assays’ applicability to evaluate the WTS. We discussed WTS ecotoxicity considering sample characterization; terrestrial and aquatic toxicity assays; and WTS challenges. WTS proved to be a highly heterogeneous matrix composed mainly of coagulant precipitates, including Al and Fe. Studies lack consensus concerning the most representative/sensitive species for evaluating WTS’ toxicity. Crustaceans were the most studied aquatic group, although algae species were more sensitive. Besides, soil ecotoxicity assessed only plant growth, and a single study used the earthworm. Even papers used bioassays to indicate the recycling WTS’ feasibility, there is a lack of specific legislation regarding the WTS reuse. Furthermore, are necessary a regulation for WTS management that involves an ecological risk assessment.

In silico modeling of the antagonistic effect of mercuric chloride and silver nanoparticles on the mortality rate of zebrafish (Danio rerio) based on response surface methodology.

In this study, in silico modeling was designed to examine the antagonistic effect of mercuric chloride (HgCl2) and silver nanoparticles (AgNPs) on the mortality rate of zebrafish (Danio rerio) based on response surface methodology (RSM). Adult zebrafish (Danio rerio) with an average weight of 0.75 ± 0.16g were used in this study. An interaction between HgCl2 and AgNPs was evaluated using DLS, TEM, and EDX mapping. In addition, RSM was applied to determine and predict the mortality rate of zebrafish induced by HgCl2 in the presence of non-lethal concentrations of AgNPs and to optimize dependent and independent variables. Following exposure to HgCl2, in vitro observations showed an increase in the hydrodynamic size of AgNPs and the formation of irregular nanoparticles. EDX mapping analysis also demonstrated the deposition of Hg ions on the surface of AgNPs, indicating the interaction between HgCl2 and AgNPs (i.e., the amalgamation of Hg and AgNPs). Moreover, in silico and in vivo findings illustrated that the mortality rate of zebrafish increased significantly in a concentration-dependent manner; however, the mortality rate reduced greatly in the presence of AgNPs during 96-h exposure. Statistically significant correlation and regression were also observed for the mortality rate between the actual and predicted values based on the ANOVA results, showing that the proposed model fits well. The most critical conditions of mortality rate were occurred by HgCl2 concentration of 0.23mg L-1 and AgNP concentration of 0.04mg L-1 that yielding maximum fish mortality rate of 96.541%. Additionally, the obtained value for model desirability was equal to 1.000 (i.e., the highest possible value). In conclusion, this statistical model could accurately describe the relationship between independent and dependent variables, and consequently boost substantially the experimental design of ecotoxicological studies by reducing the number of model organisms, toxic and chemical substances, time, and budget.