Cumulative effects of mutation accumulation on mitochondrial function and fitness.

The impact of mutations on the mitochondria deserves specific interest due to the crucial role played by these organelles on numerous cellular functions. This study examines the effects of repeated bottlenecks on mitochondrial function and fitness. Daphnia pulex mutation accumulation lines (MA) lines were maintained for over 120 generations under copper and no copper conditions. Following the MA propagation, Daphnia from MA lines were raised under optimal and high temperatures for two generations before assessing mitochondrial and phenotypic traits. Spontaneous mutation accumulation under copper led to a later age at maturity and lowered fecundity in the MA lines. Mitochondrial respiration was found to be 10% lower in all mutation accumulation (MA) lines as compared to the non-MA control. MtDNA copy number was elevated in MA lines compared to the control under optimal temperature suggesting a compensatory mechanism. Three MA lines propagated under low copper had very low mtDNA copy number and fitness, suggesting mutations might have affected genes involved in mtDNA replication or mitochondrial biogenesis. Overall, our study suggests that mutation accumulation had an impact on life history traits, mtDNA copy number, and mitochondrial respiration. Some phenotypic effects were magnified under high temperatures. MtDNA copy number appears to be an important mitigation factor to allow mitochondria to cope with mutation accumulation up to a certain level beyond which it can no longer compensate.

Developmental, behavioral, and biochemical effects of chronic exposure to sublethal concentrations of organic UV-filter compounds on a freshwater model species.

The prevalence of organic/chemical UV-filter compounds in aquatic ecosystems represents a growing environmental issue. The long-term toxicity risks of many UV-filters at environmentally relevant concentrations to aquatic biota are still less studied, especially in the case of invertebrates. This study was designed to evaluate the chronic toxicity of avobenzone (AVO), octocrylene (OCTO), and octinoxate (OCTI), three UV-filters which frequently occur in the aquatic environment, to the water flea (Daphnia magna) at an environmentally relevant concentration of 200 ng l-1 in a 21-day exposure. Potential alterations in the growth, reproduction, and heart rate were continuously monitored during the treatments. Filtration rate, swimming, and the state of the antioxidant- and metabolic functions were evaluated at the end of exposures. Avobenzone significantly increased the reproductive output, heart rate, and filtration rate, while evoked a significant decrease of swimming behavior, and inhibited the activity of catalase (CAT) and glutathione S-transferase (GST) enzymes. The body size, reproduction, heart rate, and superoxide dismutase (SOD) activity were significantly increased whereas the activity of GST and CAT was significantly reduced by OCTO. OCTI significantly increased reproduction, heart rate, CAT and SOD activity but significantly decreased the swimming behavior. Our results confirmed that chronic exposure to organic UV-filters even at environmentally relevant concentrations affect basic physiological traits and cellular defense pathways in D. magna. Highlighting, our observations revealed previously unknown physiological changes (e.g., altered heart rate, filtration rate, SOD activity) caused by the investigated UV-filter compounds. Future research is to be aimed at investigating the mixture effects of these compounds and at the understanding of the potential cellular and molecular mechanisms underlying the changes induced.

Differential susceptibility to hypoxia in hypoxia-inducible factor 1-alpha (HIF-1α)-targeted freshwater water flea Daphnia magna mutants.

The water flea, Daphnia magna, serves as a key model organism for investigating the response of aquatic organisms to environmental stressors, including hypoxia. Hypoxia-inducible factor 1-alpha (HIF-1α) is a central regulatory protein involved in the cellular response to hypoxic conditions. In this study, we used CRISPR/Cas9 gene editing to create D. magna mutant lines with targeted alterations in the HIF-1α gene. Mutants demonstrated decreased survival and reproductive output and down-regulated genes for the HIF-1α-mediated pathway in low-oxygen conditions. These findings suggest that the HIF-1α pathway is a critical component of resistance to hypoxia in D. magna. This study provides novel insights into the molecular basis of hypoxia tolerance of HIF-1α in D. magna and expands our understanding of how aquatic organisms can adapt to or be challenged by changing oxygen levels in the face of global environmental changes.

Biomonitoring emerging hazards of pharmaceuticals in river water using gut microbiome and behavioural Daphnia magna responses.

A cost-effective Daphnia magna testing framework was applied to identify emerging hazards such as neurological and cardiovascular defects as well as antibiotic resistant genes (ARGs), related to pharmaceuticals present in waste water treated (WWTP) effluent discharged into rivers. D. magna juveniles were exposed during 48 h to water samples from three rivers in the vicinity of Barcelona (NE Spain), Besós, Llobregat and Onyar, upstream and downstream of WWTP discharging points. The analyses included measuring levels of 80 pharmaceutical residues in water samples by HPLC-MS, determination of the loads of different clinically relevant antibiotic resistant genes (ARGs) in both water samples and exposed animals, and assessment of toxic effects in feeding, heartbeat responses, and behavioural indicators. ARG prevalence in water, but not in gut microbiomes, was associated with the presence of bactericides in water. These results suggest that their levels were high enough to put a selective pressure over river microbial populations, but that Daphnia guts were not easily populated by environmental bacteria. Toxic effects were found in 20 to 43% of water samples, depending on the river, and related to water quality parameters and to pollutant levels. For example, heartbeats were correlated with salinity, whereas feeding impairment did so with high loads of suspended solids. In contrast, behavioural alterations were associated to the concentration of neuroactive chemicals. Accordingly, we hypothesize that measured neuroactive chemicals have caused the observed effects. If this also applies to local invertebrate populations, the environmental consequences may be severe and unpredictable.

Effects of New Btk-Based Formulations BLB1 and Lip on Aquatic Non-Target Organisms.

Integrated pest management based on the use of biopesticides is largely applied. Experimental bioassays are critical to assess biopesticide biosafety at the ecotoxicological level. In this study, we investigated the effects of the new Bacillus thuringiensis subsp. kurstaki (Btk)-formulated-based biopesticides BLB1 and Lip, efficiently tested in field assays (IPM-4-CITRUS EC project no. 734921) on two aquatic non-target organisms, precisely the water flea Daphnia magna and the bioluminescent bacteria Aliivibrio fischeri. Acute toxicity studies, carried out in a comparative manner with Delfin® as the reference bioproduct and the lactose-based Blank formulation, show that no significant toxicity was observed up to 1 g/L. Our results indicated that BLB1- and Lip-formulated new bioproducts are far less toxic than the Delfin® reference bioproduct.

Possible Causes of Extreme Variation of Benzo[a]pyrene Acute Toxicity Test on Daphnia magna.

There are enormous differences in benzo[a]pyrene (BaP) acute toxicity tests on Daphnia magna, according to previous publications. The explanations of the reasons for this extreme variation are necessary. In this context, the acute toxicity tests of different experiment conditions (light/dark, culture medium, and solvent) were conducted on Daphnia magna with BaP as the toxicant of concern. Based on the experiments above, molecular dynamics (MD) simulations were employed to investigate the mechanisms of action. According to our results, the significant influence of light exposure on the acute toxicity test of BaP (p < 0.05) on D. magna was recorded. On the basis of the MD simulations, it was possible that BaP may not affect the normal operation of Superoxide Dismutase and Catalase directly, and it could be quickly transferred from the body through Glutathione S-transferase and Cytochromes P450. Therefore, when exposed to light, the oxidative stress process intensifies, causing damage to Daphnia magna. Apparently, the ecotoxicity tests based on inhibition for D. magna cannot adequately reflect the toxic effects of BaP.

Post-senescence reproductive rebound in Daphnia associated with reversal of age-related transcriptional changes.

A long-lived species of zooplankton microcrustaceans, Daphnia magna, sometimes exhibits late-life rebound of reproduction, briefly reversing reproductive senescence. Such events are often interpreted as terminal investments in anticipation of imminent mortality. We demonstrate that such post-senescence reproductive events (PSREs) neither cause nor anticipate increased mortality. We analyze an RNAseq experiment comparing young, old reproductively senescent, and old PSRE Daphnia females. We first show that overall age-related transcriptional changes are dominated by the increased transcription of guanidine monophosphate synthases and guanylate cyclases, as well as two groups of presumed transposon-encoded proteins, and by a drop in transcription of protein synthesis-related genes. We then focus on gene families and functional groups in which full or partial reversal of age-related transcriptional changes occur. This analysis reveals a reversal, in the PSRE individuals, of age-related up-regulation of apolipoproteins D, lysosomal lipases, and peptidases as well as several proteins related to mitochondrial and muscle functions. While it is not certain which of these changes enable reproductive rejuvenation, and which are by-products of processes that lead to it, we present some evidence that post-senescence reproductive events are associated with the reversal of age-related protein and lipid aggregates removal and apoptosis.

The Evolution of Extreme Genetic Variability in a Parasite-Resistance Complex.

Genomic regions that play a role in parasite defense are often found to be highly variable, with the major histocompatibility complex serving as an iconic example. Single nucleotide polymorphisms may represent only a small portion of this variability, with Indel polymorphisms and copy number variation further contributing. In extreme cases, haplotypes may no longer be recognized as orthologous. Understanding the evolution of such highly divergent regions is challenging because the most extreme variation is not visible using reference-assisted genomic approaches. Here we analyze the case of the Pasteuria Resistance Complex in the crustacean Daphnia magna, a defense complex in the host against the common and virulent bacterium Pasteuria ramosa. Two haplotypes of this region have been previously described, with parts of it being nonhomologous, and the region has been shown to be under balancing selection. Using pan-genome analysis and tree reconciliation methods to explore the evolution of the Pasteuria Resistance Complex and its characteristics within and between species of Daphnia and other Cladoceran species, our analysis revealed a remarkable diversity in this region even among host species, with many nonhomologous hyper-divergent haplotypes. The Pasteuria Resistance Complex is characterized by extensive duplication and losses of Fucosyltransferase (FuT) and Galactosyltransferase (GalT) genes that are believed to play a role in parasite defense. The Pasteuria Resistance Complex region can be traced back to common ancestors over 250 million years. The unique combination of an ancient resistance complex and a dynamic, hyper-divergent genomic environment presents a fascinating opportunity to investigate the role of such regions in the evolution and long-term maintenance of resistance polymorphisms. Our findings offer valuable insights into the evolutionary forces shaping disease resistance and adaptation, not only in the genus Daphnia, but potentially across the entire Cladocera class.

Elevated temperature increases the susceptibility of D. magna to environmental mixtures of carbamazepine, tramadol and citalopram.

The joint risks assessment of thermal stress and rising loads of pharmaceuticals (PhACs) in surface waters is a relevant topic in aquatic ecotoxicology. This study investigated the relevance of increased water temperature to alter the acute toxicity of environmentally relevant carbamazepine (CBZ), citalopram (CIT) and tramadol (TRA) concentrations as mixtures (ECs) and delayed outcomes in Daphnia magna. Responses of detoxification and antioxidant pathways in premature daphnids post an acute 24 h (pulsed) exposure to the PhACs mixtures and delayed responses as the reproductive output over 14 days recovery were investigated under 21- and 26 °C incubation. Biphasic modulation in glutathione S-transferase (GST) activity and significant inhibition of superoxide dismutase (SOD) activity were observed in both thermal regimes with significant shift in effective thresholds from 10-fold ECs at 21 °C to ECs at 26 °C incubation. Significant induction in catalase (CAT) activity and oxidative stress development were recorded at elevated temperatures from the 10-fold ECs dose onward. Pulsed exposures at 26 °C also led to significant decrease in the reproduction of daphnids above the 10-fold ECs of PhACs. The Integrated Biomarker Response scoring (IBRv2) approach outlined a 1.8-fold increase in alterations of daphnids exposed to 100-fold ECs of PhACs at 26 °C.

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.

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.

Combined toxic effects of polystyrene microplastic and benzophenone-4 on the bioaccumulation, feeding, growth, and reproduction of Daphniamagna.

The potential toxicity of microplastics (MPs) and UV filter Benzophenone-4 (BP4) to aquatic organisms has caused widespread concern among the public. However, the combined effects of MPs and BP4 on aquatic organisms are not well understood. This study sought to examine the combined impacts of 10 µg/L BP4, 1 mg/L Polystyrene (PS, 10 µm), and a mixture of both on the feeding, behavior, growth, and reproduction of Daphnia magna (D. magna) over a period of 21 days. The results showed that the combined exposure led to a reciprocal facilitation of bioaccumulation, along with a decrease in the second antenna beats frequency in D. magna. While the co-exposure did not change the body size or growth rate of D. magna, it did affect their feeding efficiency, leading to a decrease in Chlorella ingestion within a 24-h period. Furthermore, there was a high occurrence of malformations in two generations of D. magna exposed to BP4 and PS. The combined exposure also negatively affected reproductive parameters, such as the cumulative number of neonates and the days of first brood, suggesting a decline in overall reproductive success possibly due to feeding inhibition, with available energy potentially being redistributed between reproduction and growth in the daphnids. Co-exposure to BP4 and PS also led to elevated levels of Reactive Oxygen Species (ROS), Malonydialdehyde (MDA), and Glutathione (GSH) levels, as well as mRNA levels related to reproduction, growth, and detoxification in D. magna. Overall, this study delved into the consequences of BP4 and PS on bioaccumulation, feeding, behavior, growth, and reproduction, demonstrating that simultaneous exposure to BP4 and PS could pose a synergistic ecological hazard, potentially threatening aquatic organisms. These findings are critical and should be taken into account for accurate environmental risk assessments.

Intraspecific diversity is critical to population-level risk assessments.

Environmental risk assessment (ERA) is critical for protecting life by predicting population responses to contaminants. However, routine toxicity testing often examines only one genotype from surrogate species, potentially leading to inaccurate risk assessments, as natural populations typically consist of genetically diverse individuals. To evaluate the importance of intraspecific variation in translating toxicity testing to natural populations, we quantified the magnitude of phenotypic variation between 20 Daphnia magna clones exposed to two levels of microcystins, a cosmopolitan cyanobacterial toxin. We observed significant genetic variation in survival, growth, and reproduction, which increased under microcystins exposure. Simulations of survival showed that using a single genotype for toxicity tolerance estimates on average failed to produce accurate predictions within the 95% confidence interval over half of the time. Whole genome sequencing of the 20 clones tested for correlations between toxicological responses and genomic divergence, including candidate loci from prior gene expression studies. We found no overall correlations, indicating that clonal variation, rather than variation at candidate genes, predicts population-level responses to toxins. These results highlight the importance of incorporating broad intraspecific genetic variation, without focusing specifically on variation in candidate genes, into ERAs to more reliably predict how local populations will respond to contaminants.

Single and mixture toxicity of benzophenone-3 and its metabolites on Daphnia magna.

Benzophenone-3 (BP-3) is one of the organic ultraviolet (UV) filters widely used in personal care products, resulting in its ubiquitous occurrence in aquatic systems. This study discovered the potential risks of benzophenone-3 and its metabolites (BP-1 and BP-8) in aquatic environments. This study investigated the toxicity of three single BPs and their mixtures' effects on the survival of Daphnia magna. All three BP types were found to have toxic effects on D. magna, with median effective concentration (EC50) values of 22.55 mg/L for BP-1, 1.89 mg/L for BP-3, and 2.36 mg/L for BP-8, after 48 h of exposure. When the three BPs were binary and ternary mixtures, the EC50 values fell within 2.74-32.26 mg/L. Binary and tertiary mixtures of the three BPs indicated no strong synergistic or antagonistic effects. The mixture toxicity predictions using the classical mixture concept of concentration addition and measured toxicity data showed good predictability. The ecological risks of BPs were assessed using the maximum measured environmental concentrations of BPs collected from a river in Taiwan, divided by their respective predicted no-effect concentration (PNEC) values derived from the assessment factor (AF) method. The result showed a low ecological risk for the sum of three BPs. However, BP-3 had the highest potential risk, while BP-1 was the lowest among the three BPs. Therefore, BP-3 should pay attention to long-term environmental monitoring and management. This study provides valuable information for establishing scientifically-based water quality criteria for BPs and evaluating and managing the potential risk of BPs in the aquatic environment.

Combined environmental relevant exposure to perfluorooctanoic acid and zinc sulfate enhances apoptosis through binding with endogenous antioxidants in Daphnia magna.

Perfluorooctanoic acid (PFOA) is a long-chain legacy congener of the per- and polyfluoroalkyl substances (PFAS) family, notorious as a "forever chemical" owing to its environmental persistence and toxic nature. Essential elements such as zinc (Zn) can cause toxic effects when they change their metal speciation and become bioavailable, such as zinc sulfate (ZnSO4). Combined toxicity assessment is a realistic approach and a challenging task to evaluate chemical interactions and associated risks. Therefore, the present study aims to elucidate the acute mixture toxicity (12-48 h) of PFOA and ZnSO4 in Daphnia magna at environment-relevant concentrations (ERCs, low dose: PFOA 10 µg/L ZnSO4 20 µg/L; high dose: PFOA 20 µg/L ZnSO4 50 µg/L) in terms of developmental impact, apoptosis induction, and interaction with major endogenous antioxidants. Our results showed that deformity rates significantly increased (p < 0.05) with increasing exposure duration and exposure concentrations, compared to the control group. Further, lack of antenna, tale degeneration, and carapace alterations were the most commonly observed deformities following combined exposure to PFOA and ZnSO4, and these malformations were particularly pronounced after 48 h of exposure. Acridine orange (AO) staining was employed to examine apoptosis in D. magna, and apoptotic cells in terms of bright green fluorescence were detected in the abdominal claw carapace, heart, and post-abdominal area following exposure to a high dose of PFOA and ZnSO4. The molecular docking results revealed that both PFOA and ZnSO4 showed strong binding affinities with endogenous antioxidants CAT and GST, where PFOA was more strongly bound with CAT and GST with higher docking scores of -9.59 kcal/mol and -7.49 kcal/mol than those with ZnSO4 (-6.70 kcal/mol and -6.55 kcal/mol, respectively). In conclusion, the mixture exposure to PFOA and ZnSO4 at the environmental level induce developmental impacts and apoptosis through binding with major endogenous antioxidants in D. magna.

Cold snaps lead to a 5-fold increase or a 3-fold decrease in disease proliferation depending on the baseline temperature.

BACKGROUND: Climate change is driving increased extreme weather events that can impact ecology by moderating host-pathogen interactions. To date, few studies have explored how cold snaps affect disease prevalence and proliferation. Using the Daphnia magna-Ordospora colligata host-parasite system, a popular model system for environmentally transmitted diseases, the amplitude and duration of cold snaps were manipulated at four baseline temperatures, 10 days post-exposure, with O. colligata fitness recorded at the individual level. RESULTS: Cold snaps induced a fivefold increase or a threefold decrease in parasite burden relative to baseline temperature, with complex nuances and varied outcomes resulting from different treatment combinations. Both amplitude and duration can interact with the baseline temperature highlighting the complexity and baseline dependence of cold snaps. Furthermore, parasite fitness, i.e., infection prevalence and burden, were simultaneously altered in opposite directions in the same cold snap treatment. CONCLUSIONS: We found that cold snaps can yield complicated outcomes that are unique from other types of temperature variation (for example, heatwaves). These results underpin the challenges and complexity in understanding and predicting how climate and extreme weather may alter disease under global change.

Evaluation of Cardiotoxicity of Cancer Chemotherapeutics Using Daphnia magna as a Preclinical Model.

One of the major concerns following cancer treatment is cardiotoxicity. Therefore, it is important to predict potential cardiotoxicity of cancer chemotherapeutics at the preclinical phase. Current models of cardiotoxicity testing involve either cell culture models or rodent models. We developed a simple invertebrate animal model for rapid screening of cardiotoxicity of cancer chemotherapeutics. Daphnia magna (water flea, a crustacean) has a transparent body and a large myogenic heart that can be easily monitored under a microscope. Using this model, we have previously described comparative cardiotoxicity of several kinase inhibitors that were approved for the treatment of multiple cancers. In this article, we describe the step-wise protocols for evaluating the heart rate and survival of D. magna with relevant information on troubleshooting. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Culturing and maintenance of D. magna Basic Protocol 2: Experimental design for evaluating heart rate of Daphnia Basic Protocol 3: Long-term effect on Daphnia survival upon drug exposure.

In silico aquatic toxicity prediction of chemicals toward Daphnia magna and fathead minnow using Monte Carlo approaches.

The fast-increasing use of chemicals led to large numbers of chemical compounds entering the aquatic environment, raising concerns about their potential effects on ecosystems. Therefore, assessment of the ecotoxicological features of organic compounds on aquatic organisms is very important. Daphnia magna and Fathead minnow are two aquatic species that are commonly tested as standard test organisms for aquatic risk assessment and are typically chosen as the biological model for the ecotoxicology investigations of chemical pollutants. Herein, global quantitative structure-toxicity relationship (QSTR) models have been developed to predict the toxicity (pEC(LC)50) of a large dataset comprising 2106 chemicals toward Daphnia magna and Fathead minnow. The optimal descriptor of correlation weights (DCWs) is calculated using the notation of simplified molecular input line entry system (SMILES) and is used to construct QSTR models. Three target functions, TF1, TF2, and TF3 are utilized to generate 12 QSTR models from four splits, and their statistical characteristics are also compared. The designed QSTR models are validated using both internal and external validation criteria and are found to be reliable, robust, and excellently predictive. Among the models, those generated using the TF3 demonstrate the best statistical quality with R2 values ranging from 0.9467 to 0.9607, Q2 values ranging from 0.9462 to 0.9603 and RMSE values ranging from 0.3764 to 0.4413 for the validation set. The applicability domain and the mechanistic interpretations of generated models were also discussed.

Assessing the chronic toxicity of climbazole to Daphnia magna: Physiological, biochemical, molecular, and reproductive perspectives.

The widespread use of climbazole (CBZ) has led to its increased presence in aquatic environments, potentially threatening freshwater ecosystems. However, evidence regarding the harmful effects of CBZ on aquatic organisms remains limited. In this study, Daphnia magna was exposed to CBZ at concentrations of 0, 0.2, 20, and 200 µg/L for 21 days to evaluate its chronic toxicity through assessment of life-history traits, physiological parameters, biochemical analyses, and gene expression. The results indicated that CBZ exposure delayed the days to the first brood, reduced the frequency of molting per adult, decreased the offspring number at first brood, diminished the body length, and decreased both the total number of broods per female and the total number of offspring per female. Additionally, CBZ inhibited the swimming speed, filtration rate, and ingestion rate. Moreover, CBZ altered the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), while increasing malondialdehyde (MDA) levels. Gene expression analysis revealed varied responses in mRNA levels related to metabolic detoxification (cyp360a8, gst, and p-gp), digestive enzymes (α-amylase, α-esterase, and trypsin), energy (ak), oxygen transport (dhb), and reproduction (nvd, cyp314, ecr, vtg, and jhe) following CBZ exposure. These results indicate that the presence of CBZ in aquatic environments can induce toxicity by altering energy acquisition, supply, and metabolism; impairing metabolic detoxification pathways; eliciting oxidative stress; and causing reproductive toxicity in D. magna.

Ecotoxicological and metabolomic investigation of chronic exposure of Daphnia magna (Straus, 1820) to yttrium environmental concentrations.

In order to estimate the effects on aquatic organisms of long-term exposure to low doses of yttrium (Y) as a potential emerging contaminant, ecotoxicological and metabolomic data were collected on the model organism Daphnia magna, a keystone species in freshwater ecosystems. Following an initial acute toxicity assessment, a 21-day chronic exposure experiment was conducted using a sublethal concentration of 27 µg L⁻¹ of Y, corresponding to the effective concentrations inducing 10 % effect (EC10) value for mortality endpoint and simulating the environmental Y level in aquatic systems. Results from the 21-day two-factor experiment combining microcrustacean survival, growth and reproduction bioassays and targeted gas chromatography-mass spectrometry (GC-MS) metabolomics indicated significant adverse effects of chronic exposure to Y on D. magna. Daphnids exposed to Y exhibited a significantly lower survival at day 21, delayed the maturity stage, including their first breeding, and decreased clutch size. On the side of metabolomics, a clear and general increase over time of both the number and the level of detected metabolites in the hydroalcoholic extracts of the whole organisms was observed. However, emerging from this broad temporal pattern, several bioactive metabolites were identified (e.g., 2,4-di­tert­butylphenol, itaconic acid, 3-hydroxybutyric acid, and trehalose) whose levels in extracts are linked to the presence of Y. These results emphasize the necessity of considering low-dose, long-term exposure scenarios in environmental risk assessments of rare earth elements (REEs), which have often been overlooked in favour of higher concentration studies.