Josip Barišić

Hematological and biochemical changes in organically farmed sheep naturally infected with Fasciola hepatica.

A naturally occurring outbreak of fasciolosis in a group of 20 Merinolandschaf (German Merino) sheep was studied. Hematological and blood biochemical values in sheep spontaneously infected with liver fluke Fasciola hepatica were compared with equivalent values in 20 parasite-free sheep from organically farmed flock. Investigated animals were kept in outdoor system, on pastures covered with swamps, which remain flooded after rainy season. Significantly lower red blood cell (RBC) count, lymphocytes, hemoglobin (Hb), packed cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine, and albumin were recorded in sheep from the infected herd, whereas white blood cell (WBC) count, eosinophil, segmented and band neutrophil count, mean corpuscular volume (MCV), gamma-glutamyl transferase (GGT), concentrations of glucose, and globulins were significantly higher than in the parasite free herd. No significant correlation between the investigated blood parameters and the number of F. hepatica eggs in the feces was detected. This study shows that hematological and biochemical values can be useful in early diagnosis and prognosis of sheep fasciolosis.

Evaluation of histopathological alterations in the gills of Vardar chub (Squalius vardarensis Karaman) as an indicator of river pollution

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.

Impact of treated wastewater on organismic biosensors at various levels of biological organization

Relating the treated wastewater quality and its impact on organismic biosensors (Prussian carp, Carassius gibelio and earthworm, Eisenia fetida) was the main objective of the study. The impact on health status of fish living downstream, microbiological contamination and antimicrobial resistance, fish tissue structure, blood biochemistry, oxidative stress, genotoxic effects, as well as multixenobiotic resistance mechanism (MXR) was assessed. Treated wastewater discharged from the WWTP modified the environmental parameters and xenobiotic concentrations of the receiving surface waters. Potential bacterial pathogens from fish and respective waters were found in relatively low numbers, although they comprised aeromonads with a zoonotic potential. High resistance profiles were determined towards the tested antimicrobial compounds, mostly sulfamethoxazole and erythromycin. Histopathology primarily revealed gill lamellar fusion and reduction of interlamellar spaces of effluent fish. A significant increase in plasma values of urea, total proteins, albumins and triglycerides and a significant decrease in the activity of plasma superoxide dismutase were noted in carp from the effluent-receiving canal. Micronucleus test did not reveal significant differences between the examined groups, but a higher frequency of erythrocyte nuclear abnormalities was found in fish sampled from the effluent-receiving canal. Earthworms indicated to the presence of MXR inhibitors in water and sludge samples, thus proving as a sensitive sentinel organism for environmental pollutants. The integrative approach of this study could serve as a guiding principle in conducting evaluations of the aquatic habitat health in complex bio-monitoring studies.

Multilevel ecotoxicity assessment of environmentally relevant bisphenol A concentrations using the soil invertebrate Eisenia fetida.

Bisphenol A (BPA) presents a serious threat to soil ecosystems, yet its effects on soil-inhabiting organisms are mostly unexplored. Therefore, the impact of environmentally relevant BPA concentrations on a terrestrial model organism, the earthworm Eisenia fetida, was assessed. Animals were cutaneously exposed to 100nM and 10μM BPA up to 10days (10-d). Next, a battery of biomarkers was used for ecotoxicological evaluation on a cellular, tissue and behavioural level. HPLC analysis showed that after a 10-d exposure, BPA accumulation reached a maximum of 2.50μg BPA per g of wet tissue weight. On the cellular level, up to 3-d BPA exposure caused increased lipid oxidation indicating oxidative stress. Histopathological assessment of cell wall and ovaries after 7- and 10-d BPA exposure showed multiple abnormalities, i.e. hyperplasia of epidermis, increased body wall thickness and ovarian atrophy. Detection of these changes was facilitated by a newly proposed semi-quantitative scoring system. Finally, behavioural changes were detected after only 3days of exposure to 100nM BPA. Altogether, the presented multilevel toxicity evaluation indicates high sensitivity of earthworms to low BPA doses.

Detection and diversity of aeromonads from treated wastewater and fish inhabiting effluent and downstream waters

A two-season investigation of the wastewater treatment plant (WWTP) effluent, of related waters, sludge and fish across a wide area and 11 stations, with emphasis on Aeromonas spp. was conducted. Aeromonas veronii was the prevailing aeromonad isolated by MALDI TOF MS in the summer period. A rise of Aeromonas hydrophila was observed in summer in raw sewage, treated wastewater and effluent-carrying canal. The ratio of aeromonad species retrieved from fish tissues did not correspond with the water and sludge findings, as in spring in the effluent-carrying canal fish carried Aeromonas salmonicida ssp. salmonicida and Aeromonas bestiarum, while in summer mainly A. veronii and Acinetobacter johnsonii were isolated from fish tissues in the same location. No correlation was established between fecal coliforms/enterococci and aeromonad occurrence. All retrieved Aeromonas species demonstrated a distinct spectral pattern, with peaks showing unique mass distribution ranging from 4000 to 10,000Da. Hierarchical clustering separated aeromonads of all isolated species and clustered closely related strains together. Resistance was determined towards amoxicillin, and frequently towards sulfamethoxazole and erythromycin. In summer, a high proportion of water and sludge Aeromonas species demonstrated multiple resistance patterns towards five or more antimicrobials. The quinolone resistance of water aeromonads was mostly related to A. veronii. There are potential health concerns regarding aeromonad exposure amongst recreational fishermen who come into contact with fish inhabiting waters downstream from the WWTP, and WWTP workers who are occupationally exposed to wastewaters and their aerosols.

The effect of artificial feed on blood biochemistry profile and liver histology of wild saddled bream, Oblada melanura (Sparidae)

Floating fish farms attract a great number of wild fish species, changing their behaviour and physiology. The saddled bream, Oblada melanura, sampled from populations aggregated around the Adriatic fish farm and from natural/control populations, were analysed for differences in eleven blood biochemistry parameters and liver histomorphology. The levels of lactate dehydrogenase (LDH), alkaline phosphatase (ALKP) and urea (URE) in cage-associated saddled bream (428.00±SD 321.56 U/L, 86.13±SD 39.87 U/L and 0.05±SD 0.16 mmol/L, respectively) were significantly lower than those observed in the control specimens (1047.06±SD 505.56 U/L, 125.75±SD 34.70 U/L and 1.99±SD 0.73 mmol/L, respectively). In contrast to that, concentrations of aspartate aminotransferase (AST) in cage-associated fish (87.63±SD 132.34 U/L) were higher than values noted for the control population (6.55±SD 5.90 U/L). URE and AST presented the main variables contributing to the discrimination between two analysed populations. One-way ANOSIM based on the blood parameters showed significant difference between saddled bream that fed around cages and those from the remote waters (R=0.697; P < 0.01). Hepatocytes of cage-associated fish contained large cytoplasmatic clear spaces indicating excessive accumulation of fat in the hepatocyte cytoplasm. All observed differences can be attributed to contrasting feeding behaviour of sampled populations but basic nutritional differences between them should be quantified in the future. Moreover, further research is necessary to detect their impact on the health status of the fish.

Embryotoxic and genotoxic effects of sewage effluents in zebrafish embryo using multiple endpoint testing

Wastewater treatment plant (WWTP) effluents are often complex mixtures of various organic and inorganic substances. Quality control of wastewaters and sludges has been regulated with measuring several physico-chemical parameters and sometimes using biological methods with non-specific responses, while synergistic action mechanisms of contaminants in such complex mixtures is still unknown. Toxic effects of wastewaters within and downstream of the WWTP in City of Virovitica, Croatia, were tested on zebrafish Danio rerio using a set of biomarkers that enabled an insight in wastewaters toxic potential on embryos at the cellular, tissue and the whole organism level during an early ontogenesis (24 and 48 hpf). Exposure of embryos to the wastewater samples from WWTP Virovitica increased mortality and abnormality rate. Heart rate, spontaneous movements and pigmentation formation were also markedly affected. Biochemical markers confirmed the presence of MXR inhibitors in all tested wastewater samples, indicating the increase of pollutant accumulation in the cell/organism. Also, a tendency of DNA damage decrease measured with Comet assay was evident in wastewater samples downstream from WWTP although control levels were not reached in any environmental sample. Histopathological analysis showed that exposure to tested samples resulted in impaired muscle organization, notochord malformation and retardation in eye and brain development at embryos 48 hpf. Furthermore, semi-quantitative histopathology assessment indicated increased percentage of embryo defects in river water sampled several kilometers downstream from the WWTP, confirming toxic potential of WWTP effluents. Extension of the zebrafish embryotoxicity test (ZET) with biochemical and histopathological biomarkers could serve as a guiding principle in biomonitoring of wastewater contamination.

Indication of metal homeostasis disturbance in earthworm Eisenia fetida after exposure to semi-solid depot sludge.

Treated sewage sludge is commonly used in agriculture as fertilizer. It is, therefore, necessary to determine possible detrimental influences of sludge application on soil contamination and accumulation of contaminants in tissues of terrestrial animals, which in the long run could also have undesirable effects on humans. With that aim, the study was performed using earthworm Eisenia fetida as test organism and semi-solid depot sludge from a wastewater treatment plant as exposure media. The concentrations of 26 metals/metalloids were determined in depot sludge, and their bioaccumulation was estimated in whole tissue of E. fetida, and for the first time in the soluble tissue fraction, which represents metal fraction available for metabolic requirements and toxic effects. Obtained results have revealed acceptable levels of several elements (Cd, Cr, Cu, Ni, Pb, Zn) in depot sludge, when compared to currently valid regulations, and only moderate accumulation of some elements (e.g. As, Ba, Cd, Co, Fe, Tl, V, and Zn) in earthworms, as a consequence of exposure to depot sludge. However, a concentration increase after exposure to depot sludge was observed in E. fetida for several elements (Cd, Mo, and Zn), which were present in lower concentrations in the exposure mixtures than in soil. Contrary, a concentration decrease was observed for Cs, Mn, and Rb, although they were present in higher concentrations in depot sludge than in soil. It was an indication of disturbance in metal homeostasis in earthworms, possibly caused by exposure to complex mixture of contaminants present in depot sludge. The cumulative effect of exposure to a number of various contaminants (inorganic, organic, microbiological and pharmaceutical), even if each of them was not present in very high concentrations, could have caused distress in earthworms exposed to depot sludge.

PREDICTION OF LISTERIA MONOCYTOGENES GROWTH AS A FUNCTION OF ENVIRONMENTAL FACTORS

Listeria monocytogenes is a bacterium widespread in the environment, which has a capacity to survive and grow under various conditions. The bacterial growth results from interactions when subjected to various temperatures, pH levels, and NaCl concentrations were examined by measurements and predictive modelling. Good correlation across the range of growth conditions was shown among observed and predicted growth values, having similar trends and minimal deflections for pH levels 5.0 and 6.0. The growth condition in the 8% NaCl concentration (pH 7.0, temperature 4 °C) resulted with a growth curve of 1 log interval greater than the fitted curve for all the measurements. In all of the cases, there were consistent increases in the rates and decreases in the lag time when the growth temperature increased. Higher incubation temperatures provided higher growth rates as 30 °C and 35 °C yielded double increase of the fitted rate. Fitted and measured growth rates for salinity conditions were significantly different ...

Assessment of river sediment toxicity: Combining empirical zebrafish embryotoxicity testing with in silico toxicity characterization

Quantitative chemical analyses of 428 organic contaminants (OCs) indicated the presence of 313 OCs in the sediment extracts from Sava River, Croatia. Pharmaceuticals were present in higher concentrations than pesticides thus confirming their increasing threat to freshwater ecosystems. Toxicity evaluation of the sediment extracts from four locations (Jesenice, Rugvica, Galdovo and Lukavec) using zebrafish embryotoxicity test (ZET) accompanied with semi-quantitative histopathological analyses exhibited correlation with cumulative number and concentrations of OCs at the investigated sites (10.05, 15.22, 1.25, and 9.13 μg/g respectively). Toxicity of sediment extracts and sediment was predicted using toxic unit (TU) approach and persistence, bioaccumulation and toxicity (PBT) ranking. Additionally, influential OCs and genes were identified by graph mining of the prior knowledge informed, site-specific chemical-gene interaction models. Predicted toxicity of sediment extracts (TUext) was similar to the results obtained by ZET and associated histopathology with Rugvica sediment being the most toxic, followed by Jesenice, Lukavec and Galdovo. Sediment TU (TUsed) favoured OCs with low octanol-water partition coefficients like herbicide glyphosate and antibiotics ciprofloxacin and sulfamethazine thus indicating locations containing higher concentrations of these OCs (Galdovo and Rugvica) as the most toxic. Results suggest that comprehensive in silico sediment toxicity predictions advocate providing equal attention to organic contaminants with either very low or very high log Kow.