Microplastics - Back to Reality: Impact of Pristine and Aged Microplastics in Soil on Earthworm Eisenia fetida under Environmentally Relevant Conditions.

Recently, studies have highlighted the potential danger for soil organisms posed by film-derived microplastics (MPs). However, the majority of those does not accurately reflect the field conditions and the degree of MP contamination that can be found in actual settings. To fill the gap between laboratory and field scenarios, the polyethylene (PE) plastic film was made into PE-MPs and aged. Toxicity and molecular mechanisms of pristine PE-MPs (PMPs) and aged PE-MPs (AMPs) with the concentration at 500 mg/kg of dry weight were determined after 14 days of exposure by measuring the oxidative stress, osmoregulation pressure, gut microbiota, and metabolic responses in earthworms under environmentally relevant conditions. Our research showed that, when compared to PMPs (13.13 ± 1.99 items/g), AMPs accumulated more (16.19 ± 8.47 items/g), caused more severe tissue lesions, and caused a higher increase of cell membrane osmotic pressure in earthworms' intestines. Furthermore, the proportion of probiotic bacteria Lactobacillus johnsonii in the gut bacterial communities was 24.26%, 23.26%, and 12.96%, while the proportion of pathogenic bacteria of the phylum Verrucomicrobia was 2.28%, 4.79%, and 10.39% in the control and PMP- and AMP-exposed earthworms, indicating that the decrease in number of probiotic bacteria and the increase in number of pathogenic bacteria were more pronounced in the gut of AMP- rather than PMP-exposed earthworms. Metabolomic analysis showed that AMP exposure reduced earthworm energy metabolites. Consequently, the constant need for energy may result in protein catabolism, which raises levels of some amino acids, disturbs normal cell homeostasis, causes changes of cell membrane osmolarity, and destroys the cell structure. Our studies showed that aged MPs, with the same characteristics as those found in the environment, have greater toxicity than pristine MPs. The results of this study broaden our understanding of the toxicological effects of MPs on soil organisms under environmentally relevant conditions.

A new set of microsatellite markers for Phoxinus lumaireul senso lato, Phoxinus marsilii and Phoxinus krkae for population and molecular taxonomic studies.

Minnows of the genus Phoxinus are common and an often highly abundant fish species in Palearctic freshwater habitats. Phoxinus species have a complex evolutionary history, phylogenetic relationships are not well understood and there are a number of unresolved taxonomic problems. There are currently 23 different mitochondrial genetic lineages identified in the genus Phoxinus, 13 of which are recognized as valid species. The taxonomic status of these lineages requires resolution, including the degree to which they can interbreed. Suitable nuclear molecular markers for studies of population divergence and interbreeding between morphotypes and mitochondrial lineages are lacking for Phoxinus species. Therefore, the authors developed a set of microsatellite markers using genomic information from Phoxinus lumaireul and tested their suitability for this and two related species, Phoxinus krkae and Phoxinus marsilii. Out of 16 microsatellite candidate loci isolated, 12 were found to be in Hardy-Weinberg equilibrium when tested on two P. lumaireul senso lato populations. Seven loci amplified across the three species, enabling the study of intraspecific genetic diversity and population structure within P. marsilii and P. krkae. The markers were able to clearly resolve differences among the three tested species, including the recently described P. krkae, and are therefore suitable for the detection of introgression and hybridization among populations consisting of mixtures of two or more of P. lumaireul s. l., P. marsilii and P. krkae.

Gold and silver nanoparticles effects to the earthworm Eisenia fetida - the importance of tissue over soil concentrations.

To address and to compare the respective impact of gold and silver nanoparticles (Au and Ag NPs) in soil invertebrate, the earthworm Eisenia fetida was exposed to soil containing 2, 10, and 50 mg/kg of Au and Ag in both nanoparticulate and ionic forms for 10 days. Both metal NPs were 2-15 times less bioavailable than their ionic forms, and displayed similar transfer coefficients from soil to earthworm tissues. Both metal NPs triggered the onset of an oxidative stress as illustrated by increased glutathione S-transferase levels, decreased catalase levels, and increased malondialdehyde concentrations. Protein carbonylation distinguished the nanoparticular from the ionic forms as its increase was observed only after exposure to the highest concentration of both metal NPs. Au and Ag NPs triggered DNA modifications even at the lowest concentration, and both repressed the expression of genes involved in the general defense and stress response at high concentrations as did their ionic counterparts. Despite the fact that both metal NPs were less bioavailable than their ionic forms, at equivalent concentrations accumulated within earthworms tissues they exerted equal or higher toxic potential than their ionic counterparts.Capsule: At equivalent concentrations accumulated within earthworm tissues Au and Ag NPs exert equal or higher toxic potential than their ionic forms.

Predictive Capability of QSAR Models Based on the CompTox Zebrafish Embryo Assays: An Imbalanced Classification Problem.

The CompTox Chemistry Dashboard (ToxCast) contains one of the largest public databases on Zebrafish (Danio rerio) developmental toxicity. The data consists of 19 toxicological endpoints on unique 1018 compounds measured in relatively low concentration ranges. The endpoints are related to developmental effects occurring in dechorionated zebrafish embryos for 120 hours post fertilization and monitored via gross malformations and mortality. We report the predictive capability of 209 quantitative structure-activity relationship (QSAR) models developed by machine learning methods using penalization techniques and diverse model quality metrics to cope with the imbalanced endpoints. All these QSAR models were generated to test how the imbalanced classification (toxic or non-toxic) endpoints could be predicted regardless which of three algorithms is used: logistic regression, multi-layer perceptron, or random forests. Additionally, QSAR toxicity models are developed starting from sets of classical molecular descriptors, structural fingerprints and their combinations. Only 8 out of 209 models passed the 0.20 Matthew's correlation coefficient value defined a priori as a threshold for acceptable model quality on the test sets. The best models were obtained for endpoints mortality (MORT), ActivityScore and JAW (deformation). The low predictability of the QSAR model developed from the zebrafish embryotoxicity data in the database is mainly due to a higher sensitivity of 19 measurements of endpoints carried out on dechorionated embryos at low concentrations.

New insights into the genetic diversity of the stone crayfish: taxonomic and conservation implications.

BACKGROUND: Austropotamobius torrentium is a freshwater crayfish species native to central and south-eastern Europe, with an intricate evolutionary history and the highest genetic diversity recorded in the northern-central Dinarides (NCD). Its populations are facing declines, both in number and size across its entire range. By extanding current knowledge on the genetic diversity of this species, we aim to assist conservation programmes. Multigene phylogenetic analyses were performed using different divergence time estimates based on mitochondrial and, for the first time, nuclear DNA markers on the largest data set analysed so far. In order to reassess taxonomic relationships within this species we applied several species delimitation methods and studied the meristic characters with the intention of finding features that would clearly separate stone crayfish belonging to different phylogroups. RESULTS: Our results confirmed the existence of high genetic diversity within A. torrentium, maintained in divergent phylogroups which have their own evolutionary dynamics. A new phylogroup in the Kordun region belonging to NCD has also been discovered. Due to the incongruence between implemented species delimitation approaches and the lack of any morphological characters conserved within lineages, we are of the opinion that phylogroups recovered on mitochondrial and nuclear DNA are cryptic subspecies and distinct evolutionary significant units. CONCLUSIONS: Geographically and genetically isolated phylogroups represent the evolutionary legacy of A. torrentium and are highly relevant for conservation due to their evolutionary distinctiveness and restricted distribution.

Description of a new species of Phoxinus from the upper Krka River (Adriatic Basin) in Croatia (Actinopterygii: Leuciscidae), first discovered as a molecular clade.

Using morphometric, meristic and qualitative characters, we confirmed a specific status of a clade discovered based on molecular data only. The newly described species Phoxinus krkae has a very local distribution in the upper Krka River but is easily morphologically distinguished from geographically neighbouring species with much wider ranges: Phoxinus lumaireul, Phoxinus marsilii and Phoxinus csikii. Phoxinus krkae is well differentiated in statistical analyses and differs from other species of Phoxinus in northern Adriatic Sea drainages and right-bank tributaries of the middle and lower River Danube by a combination of characters, none of them unique: caudal peduncle length 1.9-2.2 times minimum depth of caudal peduncle; upper lip not projecting beyond lower lip; rostral fold absent; total number of scales in lateral series 74-84; lateral line incomplete and interrupted, commonly 17-49 (< 60% of total number of scales in lateral series) with no or few canal segments on posterior body and caudal peduncle; scales on belly extending forward to over middle of distance between pelvic-fin base and pectoral-fin origin; patches of breast scales commonly connected by scattered scales or forming 1-3 regular rows of scales; total vertebrae commonly 38-39 (22 + 17, 22 + 16 or 21 + 17). The study reveals unrecognised morphological diversity within the group and provides the groundwork for future taxonomic study in Phoxinus in the Adriatic region and the Danube Basin.

Intestinal damage, neurotoxicity and biochemical responses caused by tris (2-chloroethyl) phosphate and tricresyl phosphate on earthworm.

Organophosphate esters (OPEs) draw growing concern about characterizing the potential risk on environmental health due to its wide usage and distribution. Two typical types of organophosphate esters (OPEs): tris (2-chloroethyl) phosphate (TCEP) and tricresyl phosphate (TCP) were selected to evaluate toxicity of OPEs to the soil organism like earthworm (Eisenia fetida). Histopathological examination (H&E), oxidative stress, DNA damage and RT-qPCR was used to identify the effects and potential mechanism of their toxicity. Hameatoxylin and eosin (H&E) demonstrated that intestinal cells suffered serious damage, and the observed up-regulation of chitinase and cathepsin L in mRNA levels confirmed it. Both TCEP and TCP significantly increased the DNA damage when the concentrations exceeded 1 mg/kg (p < 0.01), and a dose-response relationship was observed. In addition, TCEP and TCP also changed the acetylcholinesterase (AChE) activity and expression of genes associated with neurotoxic effects in earthworms even under exposure to low OPEs concentration (0.1 mg/kg). Moreover, genes associated with nicotinic acetylcholine receptors (nAChR) and carrier protein further demonstrated that highest concentration of TCEP (10 mg/kg) may have an overloading impact on the cholinergic system of E. fetida. Integrated Biological Response index (IBRv2) showed that TCEP exerted stronger toxicity than TCP under the same concentrations. We deduced that the observed intestinal damage, oxidative stress and neurotoxic effect might be the primary mechanisms of TCEP and TCP toxicity. This study provides insight into the toxicological effects of OPEs on earthworm model, and may be useful for risk assessment of OPEs on soil ecosystems.

Emerging human pathogen Acinetobacter baumannii in the natural aquatic environment: a public health risk?

Bacterium Acinetobacter baumannii is an emerging human pathogen whose presence in the aquatic environment raises the issue of public health risk. Fish colonization represents the potential route of pathogen transmission to humans. The aim was to examine the colonization of A. baumannii to freshwater fish Poecilia reticulata. An extensively drug-resistant A. baumannii was tested at three concentrations in natural spring water. Additionally, 70 fish from the Sava River (Croatia) were screened for the presence of A. baumannii, which was not found in gill swabs or analysed gut. The colonization potential of A. baumannii in freshwater fish is dependent upon its concentration in surrounding water. The low concentration of A. baumannii in natural waters represents low colonization potential of freshwater fish. The risk for public health exists in closed water bodies where there is constant inflow of water polluted by A. baumannii in concentrations above 3 log CFU mL-1.

Insights into the molecular phylogeny and historical biogeography of the white-clawed crayfish (Decapoda, Astacidae).

In this study, the evolutionary history of the white-clawed crayfish (WCC) was evaluated using large-scale datasets comprising >1350 specimens from the entire distribution range. Using species delimitation methods on mitochondrial DNA (mtDNA) sequences, we propose four primary species hypotheses for WCC. Sequences for several nuclear regions were screened but none showed significant variation within WCC. This result favours a single secondary species hypothesis and indicates the existence of a mito-nuclear discordance in WCC. Therefore, mtDNA groups were considered only as genetic units that carry information about ancient divergences within WCC and not as taxonomic units. The reconstruction of ancestral ranges and divergence time estimates were used to link the current genetic structure with paleogeographic processes. These results showed that the emergence of mtDNA groups in WCC could be related to the Messinian Salinity Crisis, the climate cooling during the Pliocene and Pleistocene, and (paleo)shifting of the Adriatic Sea coastline in the Padanovenezian Plain. The most recent common ancestor of the mtDNA groups most likely originated from Dalmatia (eastern Adriatic coast) as indicated by the reconstruction of ancestral ranges. This ecoregion, along with the Gulf of Venice Drainages, harbours a high genetic diversity and should be emphasised as an area of the highest conservation priority.

Prevalence of the pathogen Aphanomyces astaci in freshwater crayfish populations in Croatia.

The Oomycete Aphanomyces astaci is an obligate crayfish parasite that co-evolved with American crayfish species, and they therefore generally live in a balanced relationship. On the contrary, European native crayfish are highly susceptible to A. astaci, and infestation with it causes development of the lethal disease termed crayfish plague. Until now, 5 A. astaci strains have been described from the freshwater crayfish present in Europe. In this study we aimed to investigate the occurrence of the pathogen A. astaci in Croatian native and non-native crayfish populations, as well as to genotype established strains using microsatellite markers and obtain information on the pathogen's epidemiology. Our results showed that the pathogen is widespread in both native and non-native crayfish populations. Agent level, when positive, in non-native crayfish was generally low; in native species it was higher. Genotyping from microsatellites proved the presence of the B (Ps) strain in non-native species (Pacifastacus leniusculus), while the A (As) strain was detected from viable native species (Astacus astacus and Austropotamobius torrentium) that are distributed in areas lacking non-native crayfish. The genotype from A. torrentium differed from a typical A (As) by 1 allele. Strain B (Ps) was identified in native Astacus leptodactylus from the population that co-occurs with P. leniuscuslus. Interestingly, in 1 A. leptodactylus population both A (As) and B (Ps) strains were present.

Genomic and gene expression responses to genotoxic stress in PAC2 zebrafish embryonic cell line.

PAC2 cell line is, along most of the developed zebrafish cell lines, poorly characterized concerning its response to genotoxicants. To define the PAC2 cell line response to different forms of genotoxic stress, we exposed the cells to model genotoxic agents (benzo[a]pyrene, B[a]P, and ethyl methanesulfonate) and subsequently monitored DNA damage and alterations by using the battery of tests, including the Comet assay, quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism. The expression of several DNA repair (xpc, xpd, hr23b, rad51, msh2) and oxidative stress response (sod (Cu/Zn)) genes was monitored as well. To obtain an indication of the PAC2 cell line metabolizing capacity, the expression of genes belonging to cyp1, cyp2 and cyp3 families was assessed upon exposure to B[a]P. Genotoxic responses were observed in all the used methods, and quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism proved to be more sensitive by revealing DNA alterations even when the Comet assay indicated lack of significant damage. The PAC2 cell line demonstrated basal and B[a]P-induced expression of several cyp genes, suggesting its ability to metabolize indirect acting xenobiotics to a certain point. Based on these results, PAC2 cells seem to be sensitive zebrafish in vitro model in the genotoxicity assessment of the direct acting genotoxicant; however, they are less sensitive toward the indirect acting genotoxicant due to their limited metabolizing properties.

Zebrafish genome instability after exposure to model genotoxicants.

Sublethal exposure to environmental genotoxicants may impact genome integrity in affected organisms. It is therefore necessary to develop tools to measure the extent and longevity of genotoxicant-induced DNA damage, and choose appropriate model organisms for biomonitoring. To this end, markers of DNA damage were measured in zebrafish larvae and adults following exposure to model genotoxicants (benzo[a]pyrene and ethyl methanesulfonate). Specifically, we assessed primary DNA damage and the existence of potentially persistent genomic alterations through application of the comet assay, quantitative random amplified polymorphic DNA (qRAPD) and amplified fragment length polymorphism (AFLP) assays. Furthermore, expression of genes involved in DNA repair, oxidative stress response and xenobiotic metabolism was evaluated as well. Additionally, the AFLP method was applied to adult specimens 1 year after larval exposure to the genotoxicants to evaluate the longevity of the observed DNA alterations. Large numbers of DNA alterations were detected in larval DNA using the comet assay, qRAPD and AFLP, demonstrating that zebrafish larvae are a sensitive model for revealing genotoxic effects. Furthermore, some of these genomic alterations persisted into adulthood, indicating the formation of stable genomic modifications. qRAPD and AFLP methods proved to be highly sensitive to genotoxic effects, even in cases when the comet assay indicated a lack of significant damage. These results thus support the use of zebrafish larvae as a sensitive model for monitoring the impact of genotoxic insult and give evidence of the longevity of genomic modifications induced by genotoxic agents.

Investigating the toxic effects of ethoprophos on Eisenia fetida: Integrating light microscopy, scanning electron microscopy, and biochemical analysis.

This research investigated the ecological impact of exposing Eisenia fetida, an essential component of soil ecosystems, to the organophosphate pesticide ethoprophos, widely used in agriculture. With a focus on understanding the specific effects on earthworms, we employed three concentrations (7.5, 15, and 30 mg/kg) over 28 days, considering the pesticide's short half-life and existing data on environmental concentrations. We aimed to contribute to a broader understanding of how these pesticides affect soil health. Histological analysis, including staining with Hematoxylin-eosin, Mallory Trichrome, Periodic acid-Schiff, and Alcian blue methods, was conducted on control and treatment groups. The histological and histopathological results were evaluated using the light microscopy, revealing various degenerations in the epithelial and muscle layers. Scanning electron microscopy analysis detected concentration-related notable compaction of the body surface, asymmetry, and distortion in the body segments. In the exposed groups, especially those subjected to higher ethoprophos concentrations, the grid-like appearance of the clitellum was visibly disturbed. This disturbance in the grid-like pattern is indicative of structural changes and disruptions at the microscopic level. Furthermore, total protein, carbohydrate, lipid analyses, as well as acid phosphatase and alkaline phosphatase enzyme activities, were also evaluated for earthworms from each experimental group. The analyses showed a concentration-related decrease in all biochemical measurements, except acid phosphatase enzyme activity. In conclusion, our study reveals that the environmentally realistic concentrations of ethoprophos, an effective and widely used pesticide in pest control, have detrimental effects on the health and physiology of E. fetida. These effects are manifested through histological deformities, altered biochemical profiles, and observable physiological disturbances. These results shed light on the harmful effects of ethoprophos on earthworms, underlining the necessity to restrict its usage in agricultural practices and thereby support environmental sustainability.

Oxidative and genotoxic effects of 900 MHz electromagnetic fields in the earthworm Eisenia fetida.

Accumulating evidence suggests that exposure to radiofrequency electromagnetic field (RF-EMF) can have various biological effects. In this study the oxidative and genotoxic effects were investigated in earthworms Eisenia fetida exposed in vivo to RF-EMF at the mobile phone frequency (900 MHz). Earthworms were exposed to the homogeneous RF-EMF at field levels of 10, 23, 41 and 120 V m(-1) for a period of 2h using a Gigahertz Transversal Electromagnetic (GTEM) cell. At the field level of 23 V m(-1) the effect of longer exposure (4h) and field modulation (80% AM 1 kHz sinusoidal) was investigated as well. All exposure treatments induced significant genotoxic effect in earthworms coelomocytes detected by the Comet assay, demonstrating DNA damaging capacity of 900 MHz electromagnetic radiation. Field modulation additionally increased the genotoxic effect. Moreover, our results indicated the induction of antioxidant stress response in terms of enhanced catalase and glutathione reductase activity as a result of the RF-EMF exposure, and demonstrated the generation of lipid and protein oxidative damage. Antioxidant responses and the potential of RF-EMF to induce damage to lipids, proteins and DNA differed depending on the field level applied, modulation of the field and duration of E. fetida exposure to 900 MHz electromagnetic radiation. Nature of detected DNA lesions and oxidative stress as the mechanism of action for the induction of DNA damage are discussed.

Assessment of surface water in the vicinity of fertilizer factory using fish and plants.

The genotoxic and toxic potential of polluted surface water exposed to a fertilizer factory effluent was evaluated using assays with fish (Cyprinus carpio) and plant (Lemna minor) model organisms. Beside classical physicochemical parameters, the contents of fluorides, some heavy metals and polycyclic aromatic hydrocarbons were analyzed as well. Surface water caused inhibition of plant growth and decrease of photosynthetic pigment content. Regarding DNA damage and oxidative stress parameters, both fish and plants showed similar response to the surface water. In confirmation to biochemical markers, histopathological analysis of gill and liver tissues revealed a higher incidence of lesions in fish exposed to polluted surface water. Generally, results obtained by biological monitoring were mostly in agreement with chemical analysis of the surface water, although several discrepancies were observed which might be due to difference in sensitivity of model organisms or in experimental conditions (laboratory and field exposure). The results imply that conventional chemical analysis should be extended to genotoxicity/toxicity assays as measured biological effects and the potential health hazard cannot be predicted based on the physicochemical characteristics of water samples alone.

Assessing the persistence, mobility and toxicity of emerging organic contaminants in Croatian karst springs used for drinking water supply.

Emerging organic contaminants (EOCs) are a vast group of often (very)persistent, (very)mobile and toxic (PMT/vPvM) substances that are continuously released worldwide, posing environmental and human health risks. Research on occurrence and behavior of EOCs in karst is in its infancy, thus policy measures and legislative control of these compounds in groundwater are still lacking. The Dinaric karst aquifers are an essential source of drinking water for almost half of Croatia's territory. Intense karstification, complex heterogeneous characteristics, and high fracture-cavernous porosity result in rapid, far-reaching groundwater flow and large karst springs, but also high intrinsic vulnerability due to low contaminant attenuation. To prioritize future monitoring and establish appropriate thresholds for EOCs detected in Croatian karst drinking water resources, in silico tools based on quantitative structure-activity relationships were used in PBT (persistence, bioaccumulation, and toxicity) and PMT/vPvM analyzes, while toxicological assessment helped identify potential threats to human health. In 33 samples collected during two sampling campaigns in 2019 at 16 karst springs and one lake used for water supply, we detected 65 compounds (EOCs and some legacy chemicals), of which 7 were classified as potentially PBT or vPvB compounds (PFOS, PFHxS, PFHpA, PFOA, PFNA, boscalid, and azoxystrobin), while only 2 compounds were assessed as not PMT/vPvM. This finding underlines that most of detected EOCs potentially endanger karst (ground)water ecosystems and important drinking water sources in Croatia. Comparison of maximum concentrations with existing or derived drinking water guideline values revealed how 2 of 65 detected compounds represent a potential risk to human health at lifelong exposure (sulfadiazine and hydrochlorothiazide), while 5 chemicals warrant additional human health impacts studies and groundwater monitoring. Although most compounds do not individually pose a significant risk to human health at current environmental levels, their potential synergistic and long-term effects remain unknown.

Using a Battery of Bioassays to Assess the Toxicity of Wastewater Treatment Plant Effluents in Industrial Parks.

Bioassays, as an addition to physico-chemical water quality evaluation, can provide information on the toxic effects of pollutants present in the water. In this study, a broad evaluation of environmental health risks from industrial wastewater along the Yangtze River, China, was conducted using a battery of bioassays. Toxicity tests showed that the wastewater treatment processes were effective at lowering acetylcholinesterase (AChE) inhibition, HepG2 cells' cytotoxicity, the estrogenic effect in T47D-Kbluc cells, DNA damage of Euglena gracilis and the mutagenicity of Salmonella typhimurium in the analyzed wastewater samples. Polycyclic aromatic hydrocarbons (PAHs) were identified as potential major toxic chemicals of concern in the wastewater samples of W, J and T wastewater treatment plants; thus, the potential harm of PAHs to aquatic organisms has been investigated. Based on the health risk assessment model, the risk index of wastewater from the industrial parks along the Yangtze River was below one, indicating that the PAHs were less harmful to human health through skin contact or respiratory exposure. Overall, the biological toxicity tests used in this study provide a good basis for the health risk assessment of industrial wastewater and a scientific reference for the optimization and operation of the treatment process.

Estimating risk of cardiovascular pharmaceuticals in freshwaters using zebrafish embryotoxicity test - statins threat revealed.

Cardiovascular pharmaceuticals (CVPs) are globally present in inland waters and have also been found in the sediment and plasma of fish from the Sava River, Croatia. Based on the previous research, CVPs amiodarone (AMI), ramipril (RAM), simvastatin (SIM), and verapamil (VER) have been selected for this study. Their effect has been investigated, individually and in a mixture, on the development of the zebrafish embryo Danio rerio (Hamilton, 1822) within the first 96 h of development. Upon exposure to environmentally relevant concentrations of tested CVPs (0.1, 1, and 10 µg/L) zebrafish survival and development as apparent from observed morphological abnormalities, heartbeat rates and changes in behavior, hatching success, larval length and oxidative stress level were monitored. The CVP causing the highest mortality and pathological changes was SIM (1 and 10 µg/L), which corresponds well with the observed effects during zebrafish exposure to CVPs' mixtures (4 and 40 µg/L). All pharmaceuticals affected cardiac function and decreased heart rate. SIM (1 µg/L), VER and RAM (10 µg/L) decreased larval length, while induced oxidative stress was recorded in the SIM- and VER-exposed specimens. Behavioral alterations of zebrafish were observed only in AMI-treated group (10 µg/L). Our amino acid sequence comparison and structural and docking analysis showed a highly conserved binding site between human and zebrafish HMG-CoA reductase for SIM and its main metabolite simvastatin acid. Using these ecotoxicological bioassays on a zebrafish model with particular emphasis on sublethal endpoints, the risk of CVPs, especially statins, for fish in inland waters has been identified.

Data on occurrence and ecotoxicological risk of emerging contaminants in Dinaric karst catchment of Jadro and Zrnovnica springs.

Karst catchments are valuable drinking water sources and fragile habitats to many endemic species. This dataset presents initial insights into the occurrence and ecotoxicological risk of 21 emerging contaminants (ECs) (including 11 pharmaceuticals, 4 lifestyle products, 2 personal care products, 3 agricultural and 1 industrial compound) detected in Dinaric karst catchment of Jadro and Zrnovnica springs in Croatia. Contaminants concentrations were determined with UHD Q-TOF LC/MS and UHP LC/MS in samples from two springs (Jadro and Zrnovnica), one river (Cetina), and a deep borehole (Gizdavac). Persistence (P), bioaccumulation (B), mobility (M) and toxicity (T) of detected ECs were assessed based on in silico strategy for PBT assessment and recently developed REACH PMT/vPvM guidelines. Risk quotients were calculated from PNEC values and measured contaminants' concentrations. In addition, physicochemical properties (estimated and existing experimental values of solubility in water, log KOW, log KOC, and pKa) of detected substances and water (measured values of temperature and electrolytic conductivity) are provided. This dataset could be useful for setting up the regular monitoring and improvement of existing water-related legislative, water safety plans, for modelling contaminant transport and identification of potential sources, and lastly for comparison with other studies conducted in karst aquifers. The present dataset was interpreted and discussed in the article entitled "Ecotoxicological aspects related to the occurrence of emerging contaminants in the Dinaric karst aquifer of Jadro and Zrnovnica springs" by Selak et al. (2022).

Ecotoxicological aspects related to the occurrence of emerging contaminants in the Dinaric karst aquifer of Jadro and Zrnovnica springs.

Karst aquifers are globally important source of drinking water and harbor specific ecosystems that are vulnerable to anthropogenic contamination. This paper provides insights into the occurrence and ecotoxicological characterization of 21 emerging contaminants (ECs) detected in the karst catchment of Jadro and Zrnovnica springs (Dinarides, Croatia). Karst springs used for water supply, surface water, and groundwater were sampled during seven campaigns. The ECs concentration levels ranged from 0.3 ng/L (tramadol in Jadro spring) to 372 ng/L (1H-benzotriazole in Cetina River). DEET was the most frequently detected ECs with an average concentration of around 50 ng/L in both surface water and groundwater. To prioritise detected ECs, their persistence (P), bioaccumulation (B), mobility (M) and toxicity (T) were assessed based on in silico strategy for PBT assessment and recently developed REACH PMT guidelines. PBT scores ranging below the threshold of 0.5, indicated non-PBT compounds of expected low concern. However, only 4 out of 21 detected ECs were not assessed as PMT/vPvM. Concerningly, 20 ECs were categorised as very mobile. Karst springs exhibited larger proportions of ECs meeting PMT/vPvM criteria than surface water. To characterise the contamination extent and estimate the incidence of adverse effects of detected ECs, a preliminary environmental risk assessment (ERA) was conducted. Most ECs posed no environmental risk with RQ values predominantly below 0.01. The total risk quotient RQsite accentuated Cetina River as having the highest risk compared to other sampling sites. This is the first study on ECs in Croatian karst, contributing to a growing need to understand the impacts of emerging contaminants in karst aquifers, which are still largely unexplored.