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.

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.

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.

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.

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.

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.

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).

COVID-19 Lockdowns-Effect on Concentration of Pharmaceuticals and Illicit Drugs in Two Major Croatian Rivers.

In order to prevent the spread of COVID-19, contingency measures in the form of lockdowns were implemented all over the world, including in Croatia. The aim of this study was to detect if those severe, imposed restrictions of social interactions reflected on the water quality of rivers receiving wastewaters from urban areas. A total of 18 different pharmaceuticals (PhACs) and illicit drugs (IDrgs), as well as their metabolites, were measured for 16 months (January 2020-April 2021) in 12 different locations at in the Sava and Drava Rivers, Croatia, using UHPLC coupled to LCMS. This period encompassed two major Covid lockdowns (March-May 2020 and October 2020-March 2021). Several PhACs more than halved in river water mass flow during the lockdowns. The results of this study confirm that Covid lockdowns caused lower cumulative concentrations and mass flow of measured PhACs/IDrgs in the Sava and Drava Rivers. This was not influenced by the increased use of drugs for the treatment of the COVID-19, like antibiotics and steroidal anti-inflammatory drugs. The decreases in measured PhACs/IDrgs concentrations and mass flows were more pronounced during the first lockdown, which was stricter than the second.

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.

Combining short-term bioassays using fish and crustacean model organisms with ToxCast in vitro data and broad-spectrum chemical analysis for environmental risk assessment of the river water (Sava, Croatia).

This study focused on the short-term whole organism bioassays (WOBs) on fish (Danio rerio) and crustaceans (Gammarus fossarum and Daphnia magna) to assess the negative biological effects of water from the major European River Sava and the comparison of the obtained results with in vitro toxicity data (ToxCast database) and Risk Quotient (RQ) methodology. Pollution profiles of five sampling sites along the River Sava were assessed by simultaneous chemical analysis of 562 organic contaminants (OCs) of which 476 were detected. At each sampling site, pharmaceuticals/illicit drugs category was mostly represented by their cumulative concentration, followed by categories industrial chemicals, pesticides and hormones. An exposure-activity ratio (EAR) approach based on ToxCast data highlighted steroidal anti-inflammatory drugs, antibiotics, antiepileptics/neuroleptics, industrial chemicals and hormones as compounds with the highest biological potential. Summed EAR-based prediction of toxicity showed a good correlation with the estimated toxicity of assessed sampling sites using WOBs. WOBs did not exhibit increased mortality but caused various sub-lethal biological responses that were dependant relative to the sampling site pollution intensity as well as species sensitivity. Exposure of G. fossarum and D. magna to river water-induced lower feeding rates increased GST activity and TBARS levels. Zebrafish D. rerio embryo exhibited a significant decrease in heartbeat rate, failure in pigmentation formation, as well as inhibition of ABC transporters. Nuclear receptor activation was indicated as the biological target of greatest concern based on the EAR approach. A combined approach of short-term WOBs, with a special emphasis on sub-lethal endpoints, and chemical characterization of water samples compared against in vitro toxicity data from the ToxCast database and RQs can provide a comprehensive insight into the negative effect of pollutants on aquatic organisms.

Genomic characterization and assessment of pathogenic potential of Legionella spp. isolates from environmental monitoring.

Several species in the genus Legionella are known to cause an acute pneumonia when the aerosols containing the bacteria from man-made water systems are inhaled. The disease is usually caused by Legionella pneumophila, but other species have been implicated in the infection. The disease is frequently manifested as an outbreak, which means several people are affected when exposed to the common source of Legionella contamination. Therefor environmental surveillance which includes isolation and identification of Legionella is performed routinely. However, usually no molecular or genome-based methods are employed in further characterization of the isolates during routine environmental monitoring. During several years of such monitoring, isolates from different geographical locations were collected and 39 of them were sequenced by hybrid de novo approach utilizing short and long sequencing reads. In addition, the isolates were typed by standard culture and MALDI-TOF method. The sequencing reads were assembled and annotated to produce high-quality genomes. By employing discriminatory genome typing, four potential new species in the Legionella genus were identified, which are yet to be biochemically and morphologically characterized. Moreover, functional annotations concerning virulence and antimicrobial resistance were performed on the sequenced genomes. The study contributes to the knowledge on little-known non-pneumophila species present in man-made water systems and establishes support for future genetic relatedness studies as well as understanding of their pathogenic potential.

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.

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.

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.

Toxicity prediction and effect characterization of 90 pharmaceuticals and illicit drugs measured in plasma of fish from a major European river (Sava, Croatia).

Chemical analysis of plasma samples of wild fish from the Sava River (Croatia) revealed the presence of 90 different pharmaceuticals/illicit drugs and their metabolites (PhACs/IDrgs). The concentrations of these PhACs/IDrgs in plasma were 10 to 1000 times higher than their concentrations in river water. Antibiotics, allergy/cold medications and analgesics were categories with the highest plasma concentrations. Fifty PhACs/IDrgs were identified as chemicals of concern based on the fish plasma model (FPM) effect ratios (ER) and their potential to activate evolutionary conserved biological targets. Chemicals of concern were also prioritized by calculating exposure-activity ratios (EARs) where plasma concentrations of chemicals were compared to their bioactivities in comprehensive ToxCast suite of in vitro assays. Overall, the applied prioritization methods indicated stimulants (nicotine, cotinine) and allergy/cold medications (prednisolone, dexamethasone) as having the highest potential biological impact on fish. The FPM model pointed to psychoactive substances (hallucinogens/stimulants and opioids) and psychotropic substances in the cannabinoids category (i.e. CBD and THC). EAR confirmed above and singled out additional chemicals of concern - anticholesteremic simvastatin and antiepileptic haloperidol. Present study demonstrates how the use of a combination of chemical analyses, and bio-effects based risk predictions with multiple criteria can help identify priority contaminants in freshwaters. The results reveal a widespread exposure of fish to complex mixtures of PhACs/IDrgs, which may target common molecular targets. While many of the prioritized chemicals occurred at low concentrations, their adverse effect on aquatic communities, due to continuous chronic exposure and additive effects, should not be neglected.

Toxicological effects of polystyrene microplastics on earthworm (Eisenia fetida).

Microplastics are plastic fragments of particle sizes less than 5 mm, which are widely distributed in marine and terrestrial environments. In this study, earthworms Eisenia fetida were exposed to 100 and 1000 µg of 100 nm and 1300 nm fluorescent polystyrene microplastics (PS-MPs) per kg of artificial soil for 14 days. Uptake or accumulation of PS-MPs in earthworm intestines, histopathological changes, oxidative stress, and DNA damage were assessed to determine the toxicological effects of PS-MPs on E. fetida. The results showed that the average accumulated concentrations in the earthworm intestines were higher for 1300 nm PS-MPs (0.084 ± 0.005 and 0.094 ± 0.003 µg/mg for 100 and 1000 µg/kg, respectively) than for 100 nm PS-MPs (0.015 ± 0.001 and 0.033 ± 0.002 µg/mg for 100 and 1000 µg/kg, respectively). In addition, histopathological analysis indicated that the intestinal cells were damaged after exposure to PS-MPs. Furthermore, PS-MPs significantly changed glutathione (GSH) level and superoxide dismutase (SOD) activity. The GSH levels were 86.991 ± 7.723, 165.436 ± 4.256-167.767 ± 18.642, and 93.590 ± 4.279-173.980 ± 15.523 µmol/L in the control, 100 nm, and 1300 nm PS-MPs treatment groups. In addition, the SOD activities were 10.566 ± 0.621, 9.039 ± 0.787-9.408 ± 0.493, and 7.959 ± 0.422-9.195 ± 0.327 U/mg protein for the control, 100 nm, and 1300 nm PS-MPs treatment groups, respectively, indicating that oxidative stress was induced after PS-MPs exposure. Furthermore, the comet assay suggested that exposure to PS-MPs induced DNA damage in earthworms. Overall, 1300 nm PS-MPs showed more toxic effect than 100 nm PS-MPs on earthworms. These findings provide new insights regarding the toxicological effects of low concentrations of microplastics on earthworms, and on the ecological risks of microplastics to soil animals.

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.

To evaluate the toxicity of atrazine on the freshwater microalgae Chlorella sp. using sensitive indices indicated by photosynthetic parameters.

Atrazine is a widely-applied herbicide used primarily to control weeds, which can persist in the ecosystem and exert potential toxicity to phytoplankton in the aquatic environment. In this study, acute toxicity of atrazine on microalgae Chlorella sp. was investigated with different initial cell densities (1 × 105 and 1 × 106 cells mL-1) and exposure periods (4 d and 8 d). Both growth rate and photosynthetic parameters of the microalgae in response of atrazine stress were determined to find out the sensitive indices and toxicological mechanisms. Because of the independence of initial cell density as well as the high sensitivity and reliability, the performance index PIABS was verified as the most convincing photosynthetic parameter for indicating IC50 of atrazine on Chlorella sp., being superior to the traditional parameters of growth rate and FV/FM. The IP amplitude (ΔFIP, fluorescence amplitude of the I-to-P-rise in the OJIP curve) was another sensitive biomarker to reflect atrazine stress. Results from chlorophyll fluorescence transient revealed that atrazine damaged the photosystem II (PS II) reaction center, suppressed the electron transport at the donor and receptor sides, and acted on the absorption, transfer, and utilization of light energy. Our results provide confirmatory references for understanding the toxicity and mechanisms of atrazine on freshwater microalgae.

Biomarker response of Mediterranean mussels Mytilus galloprovincialis regarding environmental conditions, pollution impact and seasonal effects.

The complexity of seasonally and spatially variable environments, coupled with complex biological interactions, makes it difficult to pinpoint biological responses to specific environmental stressors, including chemical pollution. To disentangle causative factors and reveal biomarker responses, we applied biomarker-based multivariate approaches to 15 native populations of Mediterranean mussel Mytilus galloprovincialis in spring and autumn. In addition, we used a subset of these populations in transplant experiments between clean and polluted environments in nature and in lab mesocosms. The extent of biomarker responses in native populations is affected by season, and significantly lower variability across seasons was observed among mussels from clean than from polluted sites. Results of paired block designed transplant experiment demonstrated both regional and pollution effect, with mussels uniformly exhibiting higher responses on more impacted sites in each of the Adriatic regions. Biomarker status of mussels varied among Adriatic regions in dependence on the set of environmental variables, and between clean and polluted sites in dependence on measured concentrations of metals in mussels' tissue. Results of the mesocosm experiment revealed distinctive biomarker responses of two populations of different origin when exposed to common conditions. Multivariate description of biomarker activity and application of specific experiments allowed us to link environmental condition, exposure to pollution and seasonality to mussels' biomarker responses.

Ecotoxicity and genotoxicity of polystyrene microplastics on higher plant Vicia faba.

Nano- and microplastics have been widely spread in environmental matrices, especially in marine and terrestrial systems. In this study, higher plant Vicia faba root tips were exposed to 5 µm and 100 nm with 10, 50 and 100 mg/L polystyrene fluorescent microplastics (PS-MPs) for 48 h. Root length, weight, oxidative stress and genotoxicity of V. faba were assessed to investigate toxic effects of PS-MPs. The results showed that the biomass and catalase (CAT) enzymes activity of V. faba roots decreased under 5 µm PS-MPs whereas superoxide dismutase (SOD) and peroxidase (POD) enzymes activity significantly increased. Under the 100 nm PS-MPs exposure a significant decrease of growth was observed only at the highest concentration (100 mg/L). However, micronucleus (MN) test and antioxidative enzymes activities showed that 100 nm PS-MPs induce higher genotoxic and oxidative damage to V. faba than 5 µm PS-MPs. Furthermore, the laser confocal scanning microscopy (LCSM) demonstrated that 100 nm PS-MPs can accumulate in V. faba root and most probably block cell connections or cell wall pores for transport of nutrients. These findings provide a new insight into the toxic effects of microplastics on V. faba, and further apply to the ecological risk assessment of microplastics on higher plants.