Ecotoxicological Studies Research Articles

Validating an In-House Method for Assessing Effluent Discharge Toxicity Using Acartia tonsa in the Black Sea

The marine copepod Acartia tonsa is an important bioindicator in ecotoxicological studies due to its sensitivity to pollutants and ecological importance. This research evaluates the effects of reference toxicant on the survival of A. tonsa, aiming to validate an in-house method that assesses the sensitivity and reliability of the species for testing in the Black Sea. A. tonsa individuals were exposed in controlled laboratory conditions to varying 3,5-dichlorophenol (reference toxicant) concentrations, with their survival rates monitored over 48 h. Survival data were analyzed to determine the lethal concentration (LC50), a metric for assessing toxicity. The derived LC50 values offer important benchmarks for assessing the environmental impact of different effluent discharges in the Black Sea region. The outcomes will contribute to improving the accuracy and consistency of environmental monitoring and risk assessment practices in the Black Sea region, ensuring that variations in toxicity data are attributable to environmental factors rather than methodological discrepancies.

The influence of cultivation conditions on the testate amoeba Arcella gibbosa: a perspective model for ecotoxicological studies

The influence of cultivation conditions on the testate amoeba Arcella gibbosa: a perspective model for ecotoxicological studies

Insights into nanoparticle toxicity against aquatic organisms using multivariate regression, read-across, and ML algorithms: Predictive models for Daphnia magna and Danio rerio

The production of nanoparticles (NPs) has recently become more prevalent owing to their numerous applications in the fast-growing nanotechnology industry. Although nanoparticles have growing applications, there is a significant concern over their environmental impact due to their inevitable release into the environment. With the increasing risk to aquatic organisms, D. magna and zebrafish (Danio rerio) have been preferred as important freshwater model organisms for risk assessment and ecotoxicological studies on metal oxide-based nanoparticles (MeOxNPs) in aquatic environments. It is unfeasible to assess the risks associated with every single NP through in vivo or in vitro experiments. As an alternative, in silico approaches are employed to evaluate the NP toxicity. To evaluate such performance, we have collected data from databases and literature reviews to develop models based on multivariate regression, read-across approach (RA), and machine learning (ML) algorithms following the principles of OECD (Organization for Economic Cooperation and Development) for QSAR modeling. This work has aimed to investigate which features are important drivers of nanotoxicity in D. magna and Danio rerio using simple periodic table-derived descriptors. Further, we have examined the effectiveness of read-across-derived similarity measures compared to traditional QSAR models. The results obtained from model 1 infers that nanoparticles' size, the number of metals, the core environment of the metal present in the metal oxide, and the oxidation number of the metal play a key role in the final expression of toxicity of nanoparticles to D. magna. On the other hand, the presence of higher molecular weight, the core of the metal, and the presence of oxygen influence the enzyme inhibition activity. The enzyme inhibition is correlated with the ability of zebrafish embryos to hatch, and therefore, the inhibition of ZHE1 seems to be the factor driving hatch delay. The study emphasized the importance of developing transferable, reproducible, and easily interpretable models for the early identification of nanoparticle features contributing to aquatic toxicity.

The optimization of sample preparation on zebrafish larvae in vibrational spectroscopy imaging

The zebrafish (Danio rerio) larvae are widely used in biomedical, pharmaceutical, and ecotoxicological studies. Their transparency and translational potential make them particularly valuable for fluorescence imaging. In addition to fluorescence imaging, microspectroscopy, which combines vibrational spectroscopy: Raman or Fourier transform infrared (FT-IR) with microscopy, allows the collection of spatially resolved, label-free information. According to available literature, it was the first application of FT-IR imaging in zebrafish larvae.This study aims to compare different fixation methods for 10-day post-fertilization (dpf) zebrafish larvae using vibrational spectroscopy imaging. Paraformaldehyde (PFA), glutaraldehyde (GA), low temperature, and embedding in gelatin and agarose were investigated. Amides, lipids, and phosphates distribution were more informative in embedded samples but with challenging handling of the sample due to stiffness at −20 °C. FT-IR and Raman mapping revealed that frozen samples had better-preserved tissue structure than chemical fixation. PFA showed uniform amide distribution, while GA treatment exhibited tissue disruptions and denser protein networks in both. Handling of embedded samples is challenging for an operator, but provides more reliable results in developmental biology or disease modeling, compared to chemical treatment.

Histological Based Environmental Monitoring of the Lower Nun River: Using the Histo-morphometry of Kidney, Gills, Liver, and Testes of Coptodon zillii

It is an ecotoxicological study aimed at using chemical and bio-monitoring methods to pollution status of the Lower Nun River (LNR) basin using the histo-morphometry of the Liver, Kidney, Gills, and the Testes of Coptodon zillii. Based on literature review on the environmental burdens of the LNR, the following Target Chemicals (TC) were chosen for the study: Cadmium (Cd), Chromium (Cr), Copper (Cu), Nickel (Ni) and Lead (Pb). The study involved the sampling of water, sediment and feral fish from two stations along the LNR, Peremabiri (PER) and Igbomotoru(IGB) – Experimental sites.Water and sediment samples were analyzed to ascertain the physico-chemical concentrations of potential contaminants, while the harvest fish organs of liver, kidney, gills and testes were subjected to histological study to ascertain the exposure effect. Control fishes were harvested from African Aqua-culture Centre (ARAC) – Reference Site. Analyzed sediment and water values were used to evaluate Environmental Water Quality Index (EWQI) and sediment quality, while the fish organs were analyzes and used for pollution certification and stratification. Study results showed that EWQI of PER was poor (41.9); sediment quality showed that Cr, CU and Pb were above the Maximum Allowable Toxicant Concentration (MATC) for fresh water body. Fish from IGB showed worse lesion prevalence and organ histopathologic Index (Iorg) when compared to PER, with ARAC having the best outcome. The environmental pollution status (EPS) LNR was normal at PER and was slightly polluted at IGB. The study gave credence to the use of histology as a biomarker to assess sublethal effects of environmental stressors, and in determination of the pollution status.

The phenotypic and demographic response to the combination of copper and thermal stressors strongly varies within the ciliate species, Tetrahymena thermophila.

Copper pollution can alter biological and trophic functions. Organisms can utilise different tolerance strategies, including accumulation mechanisms (intracellular vacuoles, external chelation, etc.) to maintain themselves in copper-polluted environments. Accumulation mechanisms can influence the expression of other phenotypic traits, allowing organisms to deal with copper stress. Whether copper effects on accumulation strategies interact with other environmental stressors such as temperature and how this may differ within species are still unsolved questions. Here, we tested experimentally whether the combined effect of copper and temperature modulates traits linked to demography, morphology, movement and accumulation in six strains of the ciliate Tetrahymena thermophila. We also explored whether copper accumulation might modulate environmental copper concentration effects on phenotypic and demographic traits. Results showed high intraspecific variability in the phenotypic and demographic response to copper, with interactive effects between temperature and copper. In addition, they suggested an attenuation effect of copper accumulation on the sensitivity of traits to copper, but with great variation between strains, temperatures and copper concentrations. Diversity of responses among strains and their thermal dependencies pleads for the integration of intraspecific variability and multiple stressors approaches in ecotoxicological studies, thus improving the reliability of assessments of the effects of pollutants on biodiversity.

Facilitating microplastic ingestion in aquatic models: A verified protocol for daphnia magna as a trojan horse vector

Microplastic pollution poses a significant environmental threat due to its persistence, widespread distribution, and inherent toxic potential. Despite the increasing number of publications in this field, a standardized protocol for the laboratory intake of microplastics by Daphnia magna has yet to be established. In this study, we introduce a verified protocol designed to facilitate the ingestion of microplastic particles (MPs) by D. magna, ranging in size from 5–55 µm. This protocol can be further applied to evaluate the toxicity of MPs on D. magna, a crucial organism model in ecotoxicology. Furthermore, this protocol can be used to assess toxicity of MPs in other aquatic species, such as fish, by using daphnids as a vehicle for ensuring the ingestion of these particles. Consequently, this protocol can be applied to study also one of the most pressing concerns regarding exposure to MPs, the transfer of MPs through different trophic levels, which has a great potential for ecotoxicological studies.•The influence of MPs concentration, duration and exposure dynamics and D. magna age/size in MPs intake were tested.•We have determined the optimal conditions for promoting microplastic ingestion by D. magna.

Natural modulation of redox status throughout the ontogeny of Amazon frog Physalaemus ephippifer (Anura, Leptodactylidae)

During their development, amphibians undergo various physiological processes that may affect their susceptibility to environmental pollutants. Naturally occurring fluctuations caused by developmental events are often overlooked in ecotoxicological studies. Our aim is to investigate how biomarkers of oxidative stress are modulated at different stages of larval development in the Amazonian amphibian species, Physalaemus ephippifer. The premetamorphosis, prometamorphosis and metamorphic climax stages were used to analyze total antioxidant capacity (ACAP), glutathione S-transferase (GST) activity, lipid peroxidation (LPO) levels and the expression of genes nrf2, gst, gsr (glutathione reductase) and gclc (glycine-cysteine ligase, catalytic subunit). Although there was no difference in ACAP and the genes expression among the studied stages, individuals from the premetamorphosis and prometamorphosis showed higher GST activity than ones under the climax. LPO levels were highest in individuals from the metamorphic climax. The present study suggests that the oxidative status changes during ontogeny of P. ephippifer tadpoles, especially during the metamorphic climax, the most demanding developmental phase. Variations in the redox balance at different developmental stages may lead to a divergent response to pollution. Therefore, we recommend that studies using anuran larvae as biomonitors consider possible physiological differences during ontogeny in their respective analyses.

Assessing ecological responses of exposure to the pyrethroid insecticide lambda-cyhalothrin in sub-tropical freshwater ecosystems

Pyrethroid insecticides are widely detected in aquatic ecosystems due to their extensive use in agriculture and horticulture, which could pose a potential risk to aquatic non-target organisms. While previous ecotoxicological studies have been conducted mainly with standard tests and local species under temperate conditions, scarce information is available on the effects of pyrethroid insecticides on communities and ecosystems under (sub-)tropical conditions. A single application of lambda-cyhalothrin at concentrations of 0, 9, 30, and 100 ng/L was evaluated in outdoor mesocosms under sub-tropical conditions. Lambda-cyhalothrin was found to dissipate rapidly in the water column, with only 11 % and 7 % of the remaining dose measured at 1 and 3 days after application, respectively. Lambda-cyhalothrin concentrations disappeared considerably faster from the water compartment compared to temperate conditions. Consistent decreases in abundance were observed for Lecane lunaris at the medium and higher treatments (NOEC = 9 ng/L) and at the highest treatment (NOEC = 30 ng/L) for Keratella tropica. On the contrary, two taxa belonging to Cladocera (i.e., Ceriodaphnia sp. and Diaphanosoma sp.) showed the most prominent increase in abundance related to the lambda-cyhalothrin treatments. At the community level, a consistent no observed effect concentrations (NOECs) of 9 ng/L could be calculated for the zooplankton community. A marginal significant overall treatment related effect was observed for the macroinvertebrate community. The results of species sensitivity distribution (SSD) analysis based on results of acute toxicity experiments conducted alongside the mesocosm experiment and obtained from the literature indicated that macroinvertebrates from temperate regions may be generally more sensitive than their counterparts in (sub-)tropical regions. Overall, these findings suggest that environmentally relevant concentrations of the pyrethroid insecticide lambda-cyhalothrin may lead to different ecological outcomes in freshwater ecosystems in the (sub-)tropics relative to temperate regions.

Interactive impact of residual pyrethroid compounds used in the Chilean salmon farming industry and coastal acidification conditions on the feeding performance of farmed mussels in northern Patagonia

The use of pyrethroids in aquaculture has been an important component of achieving a thriving salmon farming industry in Chile. While the residual presence of such substances is known to depend on environmental conditions, most ecotoxicological studies to date have not considered environmental context. Here, we conducted oceanographic monitoring combined with experiments aiming to estimate the effects of two pyrethroids on the feeding rates of larvae of farmed mussels, Mytilus chilensis. In additional experiments, mussel spats were exposed to both pyrethroids, but under contrasting temperature/pH so as to mimic winter and summer conditions. Experiments mimicking spring conditions revealed that both pyrethroid substances affected the feeding of mussel larvae as a function of concentration. Conversely, significant impact of pyrethroids on adults were not observed with regard to temperature and pH, but a significant impact of low temperature/low pH condition on ingestion rates was confirmed. Given the current status of increasing ocean acidification, the results of this study are expected to provide useful information with regard to achieving sustainable mussel aquaculture, especially considering both activities occur in similar geographic areas, and the expansion of salmon farming areas is ongoing in Chile.

Health status biomarkers and hemato-biochemical indices in Nile tilápia

Considering the aquatic environment, fish are notorious bioaccumulators of persistent pollutants, and that Oreochromis niloticus (Nile tilapia) is a species of great economic interest and an excellent model for ecotoxicological experimental studies. Because of this, establishing baseline reference parameters for this healthy animal is extremely valuable for comparison. Thus, our aim is to establish reference values for Nile tilapia for comparative purposes for: (i) functional activation of the alternative pathway of the complement system (ACH50), (ii) some serum biochemical parameters of liver, kidney and muscle tissue integrity, and (iii) hematological profile of Nile tilapia under controlled conditions. Juvenile specimens of O. niloticus were obtained from the fish farming and acclimatized for 15 days. Blood samples were collected without and with anticoagulants (heparin and EDTA) and submitted to hematological assays for leukogram and erythrogram evaluation. Serum was used for the alternative complement and biochemical assays. A mean of 137.6 U/mL was found for ACH50, 52.3 U/mL for alanine aminotransferase, 81.7 U/mL for aspartate aminotransferase, 30.3 U/mL for alkaline phosphatase, 4.3 g/dL for albumin, 8.4 mg/dL for urea, 1.9 mg/dL for uric acid, 3654 U/L for total creatine kinase, 1329.9 U/L for creatine kinase MB fraction, 260.2 U/L for lactic dehydrogenase, 227.2 mg/dL for glucose, 5.6 g/dL for total protein, 252.7 mg/dL for triglycerides, 169.5 md/dL for cholesterol, 19.1 mg/dL for high-density lipoprotein, and 12.5 mg/dL for low-density lipoprotein. For all hematological parameters we found a difference between the lack and presence of the anticoagulants, being the lack and heparin the conditions that presented more stability of the cell numbers along the time. Thus, in this study we establish baseline health parameters for Nile tilapia and it will be applied as an essential and comparative tool for monitoring fish health and physiological fitness at various experimental levels, such as bioassays and environmental biomonitoring.

Ecotoxicological study of seven pharmaceutically active compounds: Mixture effects and environmental risk assessment

Pharmaceutically active compounds (PhACs) have been detected in several aquatic compartments, which has been of environmental concern since PhACs can cause adverse effects on the aquatic ecosystem at low concentrations. Despite the variety of PhACs detected in surface water, ecotoxicological studies are non-existent for many of them, mainly regarding their mixture. In addition, water bodies can continuously receive the discharge of raw or treated wastewater with micropollutants. Thus, PhACs are subject to mixture and interactions, potentiating or reducing their toxicity. Therefore, the present study evaluated the toxicity on Aliivibrio fischeri of seven PhACs, which still needs to be explored in the literature. The effects were evaluated for the PhACs individually and for their binary and tertiary mixture. Also, the experimental effects were compared with the concentration addition (CA) and independent action (IA) models. Finally, an environmental risk assessment was carried out. Fenofibrate (FEN), loratadine (LOR), and ketoprofen (KET) were the most toxic, with EC50 of 0.32 mg L-1, 6.15 mg L-1 and 36.8 mg L-1, respectively. Synergistic effects were observed for FEN + LOR, KET + LOR, and KET + FEN + LOR, showing that the CA and IA may underestimate the toxicity. Environmental risks for KET concerning algae, and LOR e 17α-ethynylestradiol (EE2) for crustaceans and fish were high for several locations. Besides, high removals by wastewater treatment technologies are required to achieve the concentrations necessary for reducing KET and LOR risk quotients. Thus, this study contributed to a better understanding of the toxic interactions and environmental risks of PhACs.

Structural and functional alterations under stress conditions by contamination: A multi-species study in a non-forced multi-compartmented mesocosm

Despite the existing connectivity and heterogeneity of aquatic habitats, the concept of interconnected landscapes has been frequently overlooked in ecotoxicological risk assessment studies. In this study, a novel mesocosm system, the HeMHAS (Heterogeneous Multi-Habitat Assay System), was constructed with the potential to assess structural and functional changes in a community resulting from exposure to contaminants, while also considering the complex ecological scenarios. Fish (Sparus aurata), shrimp (Palaemon varians) and three species of marine microalgae (Isochrysis galbana, Nannochloropsis gaditana and Tetraselmis chuii) were used as test organisms. Other species, such as Artemia sp. and macroalgae were also introduced into the system as environmental enrichment. All the species were distributed in five interconnected mesocosm compartments containing a copper gradient (0, 1, 10, 100 and 250 μg/L). The mobile fish avoided the copper contaminants from 1 μg/L (24 h-AC50: 4.88 μg/L), while the shrimp avoided from 50 μg/L (24 h-AC50: 136.58 μg/L). This finding suggests interspecies interactions influence habitat selection in contaminated environments, potentially jeopardizing population persistence. Among the non-motile organisms, the growth and chlorophyll content of the microalgae were concentration dependent. The growth of I. galbana was more sensitive (growth inhibition of 50 % at the highest concentration) in contrast to N. gaditana (30 % inhibition at the highest concentration) and T. chuii (25 % inhibition at the last two highest concentrations). In summary, the mesocosm HeMHAS showed how contamination-driven responses can be studied at landscape scales, enhancing the ecological relevance of ecotoxicological research.

Establishment of a Flow-Through System for the Macrophyte Growth Inhibition Test (OECD 239) Including Photosynthetic Activity Measurement to Determine Early Effects.

Maintaining constant exposure concentrations during ecotoxicological studies while testing rapidly degradable substances is a challenge. To achieve stable concentrations during exposure, flow-through systems are used. To assess the impact of substances on higher aquatic plants, the 14-day macrophyte water-sediment Myriophyllum spicatum growth inhibition test (Organisation for Economic Co-operation and Development [OECD, 2014a] test guideline 239) only includes a static or a semistatic test design. The main aim of our study was to investigate the applicability of a flow-through system for M. spicatum. The standard OECD test design was miniaturized, and a flow-through system with spill-over was developed to achieve stable exposure concentrations of a rapidly degrading substance. The main endpoints were total shoot length and fresh and dry weight. Photosynthetic activity was used as an endpoint for the identification of early effects using the noninvasive Image-Producing Pulse Amplitude Modulation (IMAGING-PAM) procedure. Atorvastatin (AV; fast degrading) and bentazone (BT; photosynthesis inhibitor) were used as model substances to observe differences of the effect concentration depending on the test design. At higher exposure levels of AV, stronger necrosis combined with lower effect concentrations was observed in the flow-through test compared with the semistatic test, indicating the applicability of the flow-through test for evaluating degradable substances. The test with BT demonstrated a concentration-dependent decrease in the photosynthetic yield (Y(II)) from day 3 onward even before macroscopically visible changes occurred. Our results show that the flow-through system in the macrophyte growth inhibition test (OECD test guideline 239; 2014a) is a suitable alternative when one is testing rapidly degradable substances such as AV. In addition, we showed that photosynthetic yield can serve as a supplementary endpoint, when one is testing substances with photosynthesis inhibition as a mode of action. Environ Toxicol Chem 2024;00:1-12. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Genome-wide identification of 769 G protein–coupled receptor (GPCR) genes from the marine medaka Oryzias melastigma

The marine medaka Oryzias melastigma is a useful fish model for marine and estuarine ecotoxicology studies and can be applied to field-based population genomics because of its distribution in Asian estuaries and other coastal areas. We identified 769 full-length G protein–coupled receptor genes in the O. melastigma genome and classified them into five distinct classes. A phylogenetic comparison of GPCR genes in O. melastigma to humans and two other small fish species revealed a high-level orthological relationship. Purinergic and chemokine receptors were highly differentiated in humans whereas significant differentiation of chemosensory receptors was evident in fish species. Our results suggest that the GPCR gene families among the species used in this study exhibit evidence of sporadic evolutionary processes. These results may help improve our understanding of the advanced repertoires of GPCR and expand our knowledge of physiological mechanisms of fish in response to various environmental stimuli.

Advancing ecotoxicological studies: Utilizing new approach methodologies to enable cross-species extrapolation and reduce avian testing

Ecological risk assessments of agrochemicals have traditionally depended on in vivo guideline tests using northern bobwhite and mallard to provide relevant endpoints for avian species. However, these studies have limitations, including animal welfare concerns, the time and cost involved, limited potential for extrapolation to more realistic exposure conditions, and the lack of mechanistic understanding. The proof-of-concept work presented a case study for thiamethoxam in three avian species, demonstrating the potential of physiologically based kinetic (PBK) modeling to enable dosimetry extrapolations that inform hazard characterization in risk assessment, and reduce the use of avian testing. The model structure for northern bobwhite and mallard contained ten compartments, while an additional ovulation model was included for chicken in the physiological state of egg-laying. The model was first parameterized and evaluated for chicken and northern bobwhite using in vitro kinetic measurements and in vivo toxicokinetic (TK) data. The chicken model was then extrapolated to mallard based on allometric scaling. The models were then used to map the TK profiles across species by simulating internal dose metrics in different avian toxicology studies. These metrics, including peak blood concentrations (Cmax) and area under the curve (AUC) for blood concentration, were determined for acute, subacute, or chronic toxicity endpoints for mallard and northern bobwhite, enabling a quantitative cross-species and cross-route comparison of dosimetry. The results suggested that the chronic toxicological response of birds exposed to thiamethoxam is highly dependent on internal exposure, while mallard appeared to be more dynamically sensitive to thiamethoxam on an acute oral exposure basis. The case study increases the confidence in using new approach methodologies (NAMs) for interpreting avian toxicity studies and facilitating in vitro-in silico-based ecological risk assessments of agrochemicals.

Acute and chronic toxicity of rare earth elements-enriched sediments from a prospective mining area: Effects on life history traits, behavioural and physiological responses of Gammarus fossarum (Crustacea Amphipoda)

Rare earth elements (REE) have an essential role and growing importance in the world's economy. They are attracting interest from society, policymakers, and scientists. The rapidly growing global demand for REE in several strategic industrial and agricultural sectors led many countries to consider the (re)-opening of mining activities for REE extraction. Hence, their increasing use led to the disruption of their biogeochemical cycles with anthropic abnormalities already observed in aquatic ecosystems. Nonetheless, REE remain less studied, and their mechanisms of toxicity actions are not fully understood. As amphipods, Gammarus fossarum represent an important part of the aquatic macroinvertebrate assemblage and are generally used in ecotoxicological studies for their high ecological relevance. However, their use for the study of REE effects has been rather limited so far. The current study aims to assess the potential effects of two naturally REE-enriched sediments (N2 and B4) on G. fossarum. Effects on life history traits, behavioural and physiological responses have been evaluated. Exposing G. fossarum males for 72h to sediments N2 and B4 led to a decrease in haemolymph osmolality and locomotion while an increase in ventilatory activity was observed. Exposing G. fossarum pre-copula pairs with females at the same reproductive stage to the naturally REE-enriched sediments, for one moult cycle duration (∼30 days) showed that sediment B4 led to i) a significant uptake of REE, ii) a significant decrease in the proportion of females with oocytes and iii) a significant reduction in the total number of juveniles. The physicochemical analyses of sediments showed that B4 contains the highest amount of REE with a higher proportion of light REE. The present study gives the first insights into the potential toxicity of REE on G. fossarum as they may have deleterious effects on G. fossarum population's dynamics, which may alter the functioning of aquatic ecosystems.

The sediment of a river having ‘’good ecological status” turned out to be toxic to midge Chironomus riparius larvae: Implication for environmental monitoring?

Globally, monitoring of the surface waters is largely limited to the physico-chemical analysis of water in rivers and lakes. Sediment state in the aquatic systems including sediment chemical content or the structure and diversity of benthic communities or ecotoxicological studies with natural sediments remains largely overlooked by the monitoring programs. Hence we assessed the potential toxicity of three riverine sediments on the life-cycle traits (emergence and reproduction) of midge Chironomus riparius via an ecotoxicological testing method over two generations (according to OECD test 233 guidelines). In addition, the riverine sediments were spiked with polyamide (nylon) microplastic particles (1 g kg−1) to analyze an additive effect of microplastic on the sediment toxicity. As model river systems, three rivers (Karchaghbyur, Gavaraget, Argichi) in the Lake Sevan basin (Armenia) were selected. Results of ecotoxicity testing were compared with the indices of water quality (derived from the physico-chemical analysis) and the indices of the ecological status of the rivers (derived from the analysis of benthic communities). The results of testing demonstrated an unexpectedly low emergence of midges after the first generation exposed to the sediment of the river having ‘’good ecological status’’ - the Argichi. Sediments of the Karchaghbyur and Gavaraget rivers impeded the emergence and reproduction of midges after the second generation. An addition of polyamide particles to the sediments did not significantly affect the life-cycle traits of C. riparius indicating the primary effect of the sediments’ condition. The discrepancy of biotesting result with that of the other two methods (which indicated ‘’average water quality’’ and “good ecological status”) underlies the importance of designing more comprehensive monitoring programs for better assessment and protection of aquatic systems and resources.

Designing and applying a methodology to assess sperm cell viability and DNA damage in a model amphipod

Sperm quality is defined as the sperm cell ability to successfully fertilize eggs and allow normal embryo development⁠. Few studies explore sperm quality using aquatic invertebrates. Parhyale hawaiensis is a marine amphipod with a circumtropical distribution and considered a model for evolution, development, and ecotoxicological studies. We aimed to develop a methodology to collect sperm cells of P. hawaiensis and evaluate their viability and DNA damage (comet assay). We directly exposed the sperm cells to different mutagenic agents to optimize/develop the protocols. Then, as a proof of concept, we exposed the males to mutagenic compounds (EMS, benzo[a]pyrene (BaP), azo and anthraquinone dyes) at non-lethal concentrations verified by the proposed viability test and analyzed their sperm cells for DNA damage (comet assay). Organisms exposed to EMS presented a clear concentration response in the DNA damage response. We also showed that BaP was able to induce a statistically significant increase in DNA damage of the sperm cells. For the two dyes, although DNA damage increased, statistically differences were not observed. We believe we successfully developed a test to detect genotoxicity of chemicals in sperm cells using an invertebrate model. The protocol for sperm cell viability needs to be further explored with different chemicals to verify its utility as a toxicity endpoint. The developed genotoxicity test has the advantages to employ organisms that are easily cultivated in reduced space, use simple laboratory resources and reduced amount of material and reagents. Positive responses with this model could be used to disclose new germ cell mutagen candidates which could be further confirmed in vertebrates' systems.

Effects of Non-steroidal Anti-inflammatory Drugs (Ibuprofen and Diclofenac) and Their Mixtures on the Growth of Three Green Algae

Ibuprofen (IBU) and diclofenac (DFN) are the most widely consumed non-steroidal anti-inflammatory (NSAIDs) worldwide. Since they are not completely metabolized in the human body, the unaltered active ingredients can reach aquatic ecosystems through domestic effluents. The aim of this study was to analyse the toxic effects of IBU and DFN, individually and in mixtures, on three freshwater green algae strains, the international standard strain Raphidocelis subcapitata and two native strains, Ankistrodesmus fusiformis and Tetradesmus obliquus. Bioassays were carried out according to the 72-h algal growth inhibition test. The concentration-response curve of the mixture was compared to predicted effects based on both the concentration addition (CA) and the independent action (IA) models. The two drugs tested individually were toxic to all three algal strains, with EC50 values <100 mg L-1. According to these values, the most sensitive strain was R. subcapitata (IBU = 20 mg L-1 and DFN = 8 mg L-1). The most resistant strain was T. obliquus (IBU = 74.1 mg L-1 and DFN = 92.7 mg L-1) and the A. fusiformis strain showed intermediate sensitivity (IBU = 26.7 mg L-1 and DFN = 14.6 mg L-1). The mixtures showed a synergistic effect on R. subcapitata, an additive effect on A. fusiformis, and an antagonistic effect on T. obliquus. Neither the CA nor the IA model was appropriate for predicting the toxicity of the mixtures. In conclusion, IBU and DFN showed toxic effects on the algae growth at the concentrations tested. Individual and mixed toxicity was different according to the species. These differences could be species-specific, showing the importance of including native strains in ecotoxicological studies to evaluate the potential effects of pollutants in regional aquatic environments.