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Red mud (RM) contains large quantities of microscale particles < 1 micrometer and high concentrations of potentially toxic elements. In this research, we have used two types of RM of similar chemical properties but containing different quantities of micro-particles, to test whether their size plays a role in the uptake of chemical elements by earthworm Eisenia fetida. Earthworms were exposed for seven days to artificial soils (prepared in the laboratory following a protocol) amended with increasing quantities of RM. Mortality of 86% occurred when earthworms were exposed to amended soil containing 46% of particles below 1 µm. Surprisingly, tissue analyses have shown decreased concentrations of metals instead of the expected toxic effect. SEM analysis revealed that micro-particles strongly adhere to the earthworm epidermis putting them under the large stress. Micro-particles in RM clog their minute dermal pores of 90 nm to 735 nm in diameter, which size depends on whether the earthworm’s body is contracted or stretched. Strong adhesion of micro-particles to earthworms’ epidermis and blockage of their microsize pores prevented normal dermal respiration and absorption of chemical elements through their epithelium resulting in a decrease of most measured metals, especially essential elements potassium, calcium and iron, followed by the lethal outcomes.

The presence of a novel adenovirus (AdV) was detected by PCR and sequencing, in the internal organs of a captive polar bear that had died in the Budapest zoo. The virus content of the samples proved to be high enough to allow for conventional Sanger sequencing on PCR-amplified genomic fragments. With this approach, the sequence of the entire genome of the putative polar bear adenovirus 1 (PBAdV-1) was obtained. Although the genome was found to be short, consisting of 27, 952 base pairs merely, with a relatively balanced G + C content of 46.3 %, its organisation corresponded largely to that of a typical mastadenovirus. Every genus-common gene could be identified except that of protein IX. The short E3 region of the PBAdV-1 consisted of two novel, supposedly type- specific ORFs only, whereas no homologue of any of the E3 genes, usually conserved in mastadenoviruses, such as for example that of the 12.5 K protein, were present. In the E4 region, only the highly conserved gene of the 34 K protein was found besides two novel ORFs showing no homology to any known E4 ORFs. In silico sequence analysis revealed putative splicing donor and acceptor sites in the genes of the E1A, IVa2, DNA-dependent DNA polymerase, pTP, 33 K proteins, and also of U exon protein, all being characteristic for mastadenoviruses. Phylogenetic calculations, based on various proteins, further supported that the newly- detected PBAdV is the representative of a new species within the genus Mastadenovirus, and may represent the evolutionary lineage of adenoviruses that coevolved with carnivorans.

International audience; Zeolites are materials widely used in many fields of human activities. Furthermore, new potential applications constantly emerge, so understanding their possible impact on the environment is necessary. Within this study, the potential toxicity of nanosized particles (140 and 600 nm) of a widely used zeolite beta was evaluated using zebrafish Danio rerio embryos. Embryotoxicity test, with an emphasis on sublethal changes, was performed on three concentrations of each nanosized zeolite sample (calcined and non-calcined). Toxicity of tetraethylammonium species (TEA) present in non-calcined zeolite samples was also investigated using experimental and computational approaches. The data suggest that non-calcined zeolites and tetraethylammonium hydroxide (TEAOH) itself caused hatching failure, but also initiated oxidative stress and apoptosis. Such observation confirmed certain TEA leaching from the zeolite framework, thus impacting embryonic development. Since molecular docking and molecular dynamics simulations did not show TEA inhibition of the hatching enzyme ZHE1 and the ROS formation was detected using fluorescence microscopy, it was concluded that oxidative stress is the major mechanism underlying the toxicity of non-calcined samples and TEAOH. Contrary to that, calcined zeolite nanoparticles, although having a strong interaction with the chorion and subsequently with the embryos, did not show a negative impact on zebrafish survival/development. Such a comprehensive study pinpointed zeolite nanoparticles as safe materials and opened the door for their application

Application of metallothionein (MT) as an early warning sign of metal exposure in aquatic organisms is common in biomonitoring, but there is a huge variability in MT concentrations among different studies. Present research aims to assess MT responses in freshwater fish brown trout (Salmo trutta Linnaeus, 1758) and gammarids(Gammarus balcanicus Schaferna, 1922 and Echinogammarus acarinatus Karaman, 1931) as indicators of metal exposure within the freshwater karst environment (Krka River, Croatia). Sampling was performed upstream (reference site) and downstream (anthropogenically impacted site) of the wastewater discharges in autumn and spring seasons. Brown trout intestine was applied as a bioindicator tissue due to its role in dietborne metal uptake while gammarids were chosen as fish food and potential metal uptake source. Moreover, there is a lack of data on intestinal MT levels, so our results on MT and metal/metalloid concentrations, measured as total and metabolically available cytosolic levels, represent the first data of this kind for the selected indicator species. The results indicated that the ecotoxicological impact of technological and municipal wastewaters on the biota of the karst Krka River was moderate, although higher metal levels at the affected site were evident in both, fish and gammarids. The modified Brdicka reaction applied in this study was confirmed as reliable electrochemical technique for MT quantification in both vertebrates and invertebrates, and it indicated higher MT levels in gammarids (1.9–4.1 mg g−1 w.w) than in fish intestine (0.5–2.8 mg g−1 w.w). Due to the lack of the data on MT concentrations in S. trutta and gammarid species G. balcanicus and E. acarinatus, presented results can serve as a preliminary data to establish MT background levels in intestine of wild freshwater fish and gammarids. Obtained MT levels showed species-, tissue- and method- specific differences, so comparison between MT levels should always involve the same species, tissue and measurement method.

In order to assess the extent of existing anthropogenic influence on biota of the vulnerable karst ecosystem of the Krka River, multi-biomarker approach was applied in the intestinal tissue of brown trout Salmo trutta Linnaeus, 1758. Biomarkers of the general stress (total cytosolic proteins), oxidative stress (malondialdehyde), antioxidant capacity (catalase activity, total glutathione) and of an exposure and effect of contaminants, especially metals (metallothionein) and organophosphorous pesticides and metals (acetylcholine esterase activity) were compared in the intestine of fish from the reference site (river source) and downstream of the technological and municipal wastewater impacted site (town of Knin) in two seasons, October 2015 and May 2016. Biological response was additionally evaluated by metal/metalloid concentrations in intestinal cytosol. Site-specific differences were observed as significantly higher As, Ca, Co, Cu, Se and Sr concentrations in intestinal cytosol of fish from the contaminated compared to the reference site. Significant seasonal differences existed for Ni, Cd, Mo, Cs and Na, with higher levels in autumn, following the trend of most of the dissolved metal levels in the river water. Impact of improperly treated wastewaters was also confirmed by significantly increased levels of glutathione, total proteins and Foulton condition indices, with 1.5, 1.13 and 1.12 times higher average values in fish from that site compared to the river source, respectively. The other biomarkers showed similar trend and pointed to specific biological changes regarding oxidative stress or metal exposure in fish from the anthropogenically impacted site, especially in autumn, but without significant differences. Thus, the anthropogenic impact still seems to be only moderate, although cytosolic metals and most of the biomarkers in fish intestine were confirmed as initial indicators of pollution impact, which pointed to the need of continuous monitoring of the Krka River in order to protect this natural karst world phenomenon.

Concentrations of 23 trace elements (TEs; essential: Co, Cu, Fe, Mn, Mo, Se, V, Zn; non-essential: Ag, Al, As, Ba, Cd, Cr, Cs, Li, Ni, Pb, Rb, Sr, Ti, Tl, U) in whole soft tissues of Noah's Ark shell (Arca noae) were determined monthly during one year (March 2013–February 2014) at two sampling sites in the central part of the Eastern Adriatic Sea. Our aim was to detect the influence of reproductive cycle and changes in the environmental factors on the variabilities of TEs' contents. Higher concentrations of Pb, Ba, V, Mo, Mn and Fe were found at potentially contaminated site in Pasman channel, whereas higher concentrations of Tl, Ni, Li, Cr, Cd, Ti and Se were found at reference site in Nature Park Telascica. Since several bioaccumulated TEs were associated to mean gonadal index, in TEs monitoring in A. noae, animal gonadal status has to be considered.

The environmental status of the marine environment in the NE Adriatic Sea was assessed, using as a bioindicator species the Mediterranean mussel Mytilus galloprovincialis Lamarck, 1819. Samples were collected seasonally from mariculture sites and from major Istrian ports between the years 2010 and 2013. The condition indices of mussels ranged from 13.3 to 20.9% at mariculture sites and from 14.3 to 23.3% at port locations. The seasonally δ13CDIC values of seawater varied between -10.9 to 0.7‰. Pollution by sewage sludge (based on δ15N values) was confirmed only in two ports. Tissue concentrations of Mn, Co, Ni, Cu, Zn, Se, Cd, and Pb were significantly higher in the tissue of the mussels collected from the ports (polluted sites). Arsenobetaine was the major As compound present in the samples and there was no significant difference in the levels of total As in mussel tissues from mariculture and port sites.

Quantification of histopathological alterations in the gills of Vardar chub (Squalius vardarensis Karaman) was performed in 2012 in rivers of north- eastern Macedonia, with the aim to examine the effects of water quality in the rivers (Zletovska and Kriva River - impacted by active Pb/Zn mines ; Bregalnica River - contaminated by agricultural waste). The biological alterations in chub were classified as: circulatory disturbances, regressive and progressive changes, but their severity differed. Altogether the mildest changes were observed in the gills of chub from the Bregalnica River, a less polluted river, whereas mining impacted rivers were characterized by more severe alterations. In the gills of chub from the Zletovska River, which is highly contaminated with numerous metals, sulphates and chlorides, the highest lesion indices were found for the regressive changes of both epithelium and supporting tissue, with typical lesions referring to atrophy, thinning and lifting of epithelial cells, necrosis of epithelium and chloride cells, as well as deformations of lamellar cartilaginous base. Gill damages of chub from the Kriva River were overall milder compared to the Zletovska River, in accordance with pollution status. In the gills of chub from that river, progressive changes were more pronounced, specifically severe hyperplasia of mucous cells and epithelium in the interlammellar space, leading to fusion of lamellae, as well as hypertrophy of chloride cells. The comparison between seasons indicated higher intensity of progressive changes in all three rivers in autumn, when water level was very low, and consequently, water contamination was more pronounced due to concentration effect. The pattern and severity of histopathological alterations in the chub gills reflected differences in contamination levels and type of contaminants in different rivers and sampling periods, and thus have been proven as a valuable indicator of water quality.

Croatian Adriatic coastal waters are systematically monitored within the Mediterranean Mussel Watch Project using mussels ( Mytilus galloprovincialis ) as a bio-indicator species. The study includes determination of naturally occurring ( 7 Be, 40 K, 232 Th, 226 Ra and 238 U), as well as anthropogenic 137 Cs radionuclides. Activity concentrations in dry weight of mussels' soft tissue along the Croatian Adriatic coast are presented, with spatial and seasonal variations given and discussed. Samples were collected in spring and autumn for the period between 2009 and 2013. Radionuclides were determined by gamma-ray spectrometry. Activity concentrations of 7 Be were the highest in spring periods, especially in the areas with significant fresh water discharges. Activity concentrations of 40 K did not vary significantly with season or location. 137 Cs activities were low, while 232 Th, 226 Ra and 238 U activities were mostly below the detection limit of performed gamma-spectrometric measurement.

A study was made of seasonal and spatial variability of metallothionein (MT) concentrations, determined spectrophotometrically in the soluble fraction of the digestive gland of mussels Mytilus galloprovincialis, collected between 1999 and 2001 from several coastal and estuarine locations along the central Eastern Adriatic coast (Croatia). The seasonal influence on the MT and metal concentrations (higher values in winter– spring season, than in summer– autumn season) is more pronounced than the local site-specific influence. Furthermore, within each season a significant site-specific dependence on the MT and trace metal variations can be detected. An inverse relationship of mussel condition index (CI) and temperature with MT and trace metals levels indicates the influence of food abundance and mussel annual reproductive cycle. Substantially higher concentrations of both MT and Cd were recorded in mussels inhabiting estuarine locations, possibly indicating a markedly higher Cd bioavailability at these locations. The positive correlations obtained between MT and Cd in all seasons except autumn support an argument for application of digestive gland MT as a biomarker of Cd exposure, providing evidence for assessing the most appropriate season for mussel sampling.

Two wild fish populations, Mullus surmuletus (striped red mullet), as a bottom and Liza aurata (golden grey mullet), as a pelagic species, were sampled in Kaštela Bay, Middle Dalmatia. Metallothionein (MT) and metal content (Cu, Zn, Cd) was determined in heat treated supernatants (S50) of fish tissues. MT levels were statistically different in liver and kidney of sampled species, with highest value in kidney of M. surmuletus (31.1&plusmn ; 7.96 &#956 ; g mg-1 proteins). Metal levels are statistically different between all tissues. Highest metal concentrations are in liver cytosols, especially of Cu in L. aurata (6.46&plusmn ; 3.45 &#956 ; g ml-1 S50). High positive correlation of MT and cytosolic metals is established with Zn in liver of M. surmuletus (0.95) and with Cu in liver of L. aurata (0.69). Total length, body weight and age are mostly correlated biometric parameters with MT and soluble metal concentrations. MT and metal levels increase with age in liver cytosol of both species.