Sample preparation and culture condition effects on MALDI-TOF MS identification of bacteria: A review.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an excellent tool for bacterial identification. It allows high throughput, sensitive and specific applications in clinical diagnostics and environmental research. Currently, there is no optimal standardized protocol for sample preparation and culture conditions to profile bacteria. The performance of MALDI-TOF MS is affected by several variables, such as sample preparation, culture media and culture conditions, incubation time/growth stage, incubation temperature, high salt content, blood in the culture media, and others. This review thus aims to clarify why a uniformed protocol is not plausible, to assess the effects these factors have on MALDI-TOF MS identification score, and discuss possible optimizations for its methodology, in relation to specific bacterial representatives and strain requirements.

Chemical Evaluation, Antioxidant, Antiproliferative, Anti-Inflammatory and Antibacterial Activities of Organic Extract and Semi-Purified Fractions of the Adriatic Sea Fan, Eunicella cavolini.

Due to sedentary lifestyle and harsh environmental conditions, gorgonian coral extracts are recognized as a rich source of novel compounds with various biological activities, of interest to the pharmaceutical and cosmetic industries. The presented study aimed to perform chemical screening of organic extracts and semi-purified fractions obtained from the common Adriatic gorgonian, sea fan, Eunicella cavolini (Koch, 1887) and explore its abilities to exert different biological effects in vitro. Qualitative chemical evaluation revealed the presence of several classes of secondary metabolites extended with mass spectrometry analysis and tentative dereplication by using Global Natural Product Social Molecular Networking online platform (GNPS). Furthermore, fractions F4 and F3 showed the highest phenolic (3.28 ± 0.04 mg GAE/g sample) and carotene (23.11 ± 2.48 mg ß-CA/g sample) content, respectively. The fraction F3 inhibited 50% of DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and ABTS (2,2'-azino-bis (3-ethylbenzthiazolin-6-yl) sulfonic acid) radicals at the concentrations of 767.09 ± 11.57 and 157.16 ± 10.83 µg/mL, respectively. The highest anti-inflammatory potential was exhibited by F2 (IC50 = 198.70 ± 28.77 µg/mL) regarding the inhibition of albumin denaturation and F1 (IC50 = 254.49 ± 49.17 µg/mL) in terms of soybean lipoxygenase inhibition. In addition, the most pronounced antiproliferative effects were observed for all samples (IC50 ranging from 0.82 ± 0.14-231.18 ± 46.13 µg/mL) against several carcinoma cell lines, but also towards non-transformed human fibroblasts pointing to a generally cytotoxic effect. In addition, the antibacterial activity was tested by broth microdilution assay against three human pathogenic bacteria: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The latter was the most affected by fractions F2 and F3. Finally, further purification, isolation and characterization of pure compounds from the most active fractions are under investigation.

Fish photobacteriosis-The importance of rapid and accurate identification of Photobacterium damselae subsp. piscicida.

MALDI-TOF MS was tested for the identification of Photobacterium damselae subsp. piscicida on isolates grown on two media, cultured at three incubation times and applied on the target plate by the direct sample spotting (DS), by the on-target extraction (OTE) and by the full extraction (FE) method, in triplicates. The identification of samples grown on blood agar (BA) outperformed identification on tryptic soya agar (TSA) by 0.64% for DS and OTE. The OTE gave the highest scores in both culture media, all incubation times and replicates. Reliable 24-hr species identification was 61.54%, 84.61% and 53.85% for samples grown on TSA and identified by DS, OTE and FE, respectively. For isolates grown on BA, they were 76.92%, 96.15% and 30.77%, respectively. When identified by OTE, the 48-hr identification was 93.58%, but for 72 hr declined to 71.79%. The reliable identification with the highest score from the first measurement was 100% only for OTE from BA (24 hr), whereas OTE from TSA gave 84.61% (24 hr), 76.92% (48 hr) and 84.61% (72 hr). The reliable MALDI-TOF MS identification of Ph. damselae subsp. piscicida is incubation time, media, target plate preparation and replicate-dependent.

Supplementation with imuno-2865® in gilthead sea bream (Sparus aurata Linnaeus, 1758): Effects on hematological and antioxidant parameters.

The aim of this study was to evaluate the effects of IMUNO-2865(®) on hematological and antioxidative parameters in sea bream. Total of 640 sea bream were fed with diets containing 0 (Group 1), 1 (Group 2), 10 (Group 3) and 25 (Group 4) g of IMUNO-2865(®) kg(-1) feed during 90 days. Samples were taken each month and three months after the supplementation. A significant heterophils increase was observed in group 4 compared to group 1 after two months, and an increase in monocytes number was observed in group 4 compared to the other groups after one month. Glutathione peroxidase (GSH-Px) and paraoxonase-1 (PON1) were significantly increased in groups 3 and 4 compared to the control group three months into the experiment. Superoxide dismutase (SOD) was increased in group 4 compared to the control group from day 60 until the end of the experiment, and in groups 2 and 3 compared to the control after three months. Based on the differences in the cellular immunity and oxidative stress parameters, with an overall absence of mortality, the results of this study suggest that the use of IMUNO-2865(®) in aquaculture is safe and possess a cumulative immunostimulatory effect on sea bream.

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.

Fish liver damage related to the wastewater treatment plant effluents.

Wastewater treatment plants (WWTPs) continuously release a complex mixture of municipal, hospital, industrial, and runoff chemicals into the aquatic environment. These contaminants are both legacy contaminants and emerging-concern contaminants, affecting all tissues in a fish body, particularly the liver. The fish liver is the principal detoxifying organ and effects of consistent pollutant exposure can be evident on its cellular and tissue level. The objective of this paper is thus to provide an in-depth analysis of the WWTP contaminants' impact on the fish liver structure, physiology, and metabolism. The paper also gives an overview of the fish liver biotransformation enzymes, antioxidant enzymes, and non-enzymatic antioxidants, their role in metabolizing xenobiotic compounds and coping with oxidative damage. Emphasis has been placed on highlighting the vulnerability of fish to xenobiotic compounds, and on biomonitoring of exposed fish, generally involving observation of biomarkers in caged or native fish. Furthermore, the paper systematically assesses the most common contaminants with the potential to affect fish liver tissue.

Effect of seasonal temperature shifts on body homeostasis, biochemical parameters and chemical composition of green ormer (Haliotis tuberculata) in the Northern Adriatic.

Seasonal responses of green ormer in terms of antioxidant capacity and lipid peroxidation, proximate and fatty acid tissue composition, trace and macro elements concentrations over the seasons were studied in relation to temperature shifts in the Northern Adriatic Sea. Overall antioxidative defenses (SOD, TBARS, TAS, LDH) varied significantly (p < 0.001) according to seasons (primarily spring and summer). The proportions of overall SFA were highest in summer. The proportions of MUFA increased in autumn, with significant differences between genders in spring and summer, and spring, summer and autumn for C18:1n7 and C20:4n6. The only fatty acid lacking significant variation between seasons was C22:5n3. Significant overall differences were observed in summer vs. winter samples for As, Ba, Co, Ni, Mn, Pb, Sb, and Se content in soft tissues, however, gender variations were not significant. The data obtained in the study are of utmost importance for the management of this under-investigated species.

Identification of environmental aquatic bacteria by mass spectrometry supported by biochemical differentiation.

In this study, the purposefulness of using the API20E biochemical identification system as a supportive tool for enhancing the discrimination of environmental bacteria by MALDI-TOF MS method was evaluated. The identification results of MALDI-TOF MS and API20E for 321 Gram-negative strains isolated from the riverine freshwater and its sediment, and from the tissues of fish from the same water body were compared. Of 190 isolates identified with probable to highly probable species-level identification, and secure genus to probable species identification, 14 isolates (7.37%) had identification score over 2.300, and from the same group 19 isolates (10%) had excellent or very good identification to the genus by API20E system. With regard to agreement at genus level, out of 231 strains with genus designation available by API20E at any level of identification reliability, MALDI-TOF MS genus identification agreed in 163 (70.6%) strains. Of these, 135 (82.8%) were Aeromonas species and the remaining isolates belonged to 7 different genera. Although API20E resulted in frequent misidentification due to a limited profile index, its individual biochemical reactions might contribute to overall characterization of isolates. For example, for all reliable A. hydrophila strain identifications with MALDI-TOF MS, ONPG, GLU and OX reactions were unarguably positive for all fish and water/sediment isolates, whereas only fish isolates yielded additional 100% positive TDA and VP reactions. Thus, after initial identification with MALDI-TOF MS, environmental isolates with lower identification scores should be further analyzed. Before commencing confirmatory testing with nucleic acid-based methods, biochemical API20E tests could be applied as a purposeful and inexpensive identification support in targeting better identification accuracy. In this study, this was particularly evident with A. hydrophila, Chryseobacterium sp. and Pseudomonas sp. This identification strategy could significantly resolve methodological and cost-related shortcomings frequently occurring with large number of environmental isolates.

Shewanella spp. from wastewater treatment plant-affected environment: isolation and characterization.

Bacteria from the genus Shewanella are inhabitants of marine and freshwater ecosystems, recognized fish spoilage bacteria, but less known as fish disease agents. Shewanella spp. isolated from fish living in waters close to effluents of a wastewater treatment plant (WWTP) were not previously characterized. We have tested Shewanella isolates from WWTP-affected waters and related fish. Genotypic characterization identified most strains as S. baltica and S. oneidensis. In order to investigate the sensibility and accuracy of their MALDI-TOF MS identification, they were grown on two culture media enriched by various NaCl concentrations, incubated at different temperatures and duration. We analyzed their antimicrobial susceptibility on a panel of antimicrobial drugs and capacity for biofilm production. With a view to demonstrate their capacity to produce fatty acids, we assessed the impact of different culture media on their lipid profile. We performed zebrafish embryotoxicity tests to simulate the environmental infection of the earliest life stages in S. baltica-contaminated waters. The best MALDI-TOF MS identification scores were for strains cultivated on TSA for 24 h at 22 °C and with supplementation of 1.5% NaCl. Less than 17% of isolates demonstrated antimicrobial resistance. Most isolates were weak biofilm producers. Strain-to-strain variation of MIC and MBC was low. The major fatty acids were C15:0, C16:0, C16:1, C17:1, and iC15:0. Exposure of Danio rerio to different S. baltica concentrations induced severe effects on zebrafish development: decreased heartbeat rate, locomotor activity, and melanin pigmentation. S. baltica passed through chorionic pores of zebrafish.

Evaluation of micronucleus and erythrocytic nuclear abnormalities in Balkan whip snake Hierophis gemonensis.

Over recent years, changes of erythrocytic nuclei have been increasingly used to evaluate genotoxic effects of different compounds such as polycyclic aromatic hydrocarbons (benzo-a-pyrene, naphthalene, ß-naphthoflavone), heavy metals (cadmium, mercury), textile mill effluent especially in aquatic ecosystem. However, in fish, both micronuclei and erythrocytic nuclear abnormalities also appear spontaneously and their frequency can be seasonally dependent. The aim of the study was to evaluate the frequency of micronuclei (MN), nuclear abnormalities (NA) including vacuolated nuclei (VN) and cytoplasmic vacuoles (CV) in erythrocytes of Balkan whip snake Hierophis gemonensis and establish the level of spontaneous appearance during the annual cycle. Average frequency of NA was 10.89 ± 4.72% while the MN (0.03 ± 0.03%) and VN (0.04 ± 0.08%) were seldom detected. NA significantly positively correlated with MN (r = 0.319; P < 0.05) and VN (r = 0.363; P < 0.05). Appearance of CV did not correlate with other measured parameters and average frequency was 11.06 ± 8.33%. Significant seasonal variation was found in NA appearance with the lowest value in spring and the highest in winter. VN increase was observed in autumn. MN and CV levels varied between seasons but not significantly. Considering the biological cycle, frequency of NA, VN, MN and CV recorded in pre-hibernation/hibernation increased compared to the active phase, but only NA elevation was significant. Although the obtained results showed differences according to sex, statistical analysis of measured parameters showed the same pattern of seasonal variation in both sexes.

Seasonal antioxidant and biochemical properties of the Northern Adriatic Pecten jacobaeus.

The present work is the first study of Mediterranean scallop (Pecten jacobaeus) biochemical properties, antioxidant defenses, and free radical scavengers during the yearly seasons in the Northern Adriatic, off Istria. Scallop nutrient reserves (glucose, triglyceride, and cholesterol) in four tissues under examination were positively correlated and were predominant in digestive gland and gonad. The muscle energy maxima were in correlation with the maximum fall gonosomatic index (GSI), when diatoms and coccolithophorids thrive. The decrease of GSI in summer might be related to the spawning or resorption of gametes. Summer also revealed elevated levels of glucose in gonad and digestive gland, while muscle glucose and cholesterol significantly varied in spring vs. winter samples. In relation to the diatom seasonal abundance, carotenoids, namely astaxanthin peaks were found in digestive gland, which, being stimulators of calcium transport over cell membranes, could have contributed to the high digestive gland levels of calcium in winter. In winter, total antioxidative status (TAS) of scallop tissues was 3-fold higher than in other seasons, particularly in digestive gland, having a significant correlation with magnesium, a regulatory tool in oxidative processes. The winter maxima of TAS and thiobarbituric acid reactive substances TBARS in relation to summer maxima of glutathione peroxidase and superoxide dismutase in digestive glands indicate to a decrease in antioxidant defense during cold months, and are related to the accumulation of lipid peroxidation products (such as malondialdehyde) in digestive gland of scallops. Although the increased susceptibility to oxidative stress could be attributed to winter temperature, other factors such as the gonad maturation, availability of food supply, and salinity might counteract that effect. The seawater alterations of salinity, temperature and water quality are in relation to the river Po influx, which is very likely to influence the physiological and biochemical responses of scallops in the Northern Adriatic.

Seasonality of nuclear abnormalities in gilthead sea bream Sparus aurata (L.) erythrocytes.

The frequencies of erythrocyte nuclear abnormalities, such as irregularly shaped (ISN), vacuolated nuclei (VN), binucleated cells (BN), including micronuclei (MN), were monitored in the peripheral blood of cultured gilthead sea bream (Sparus aurata L.) from the southern Adriatic Sea (Croatia) and assessed for a relation to seasonality. Fish were sampled in February, April, June, and September. The lowest frequencies of ISN and MN were measured in April, and the lowest frequencies of VN and BN were found in February. The highest values of ISN and BN were detected in September. The highest values of MN and the most frequent occurrence of VN occurred in June and April, respectively. The Spearman rank order correlation test revealed a significant positive correlation between the frequencies of ISN, BN and MN; VN did not correlate with any of the measured parameters. The ISN had significantly higher nuclear surface area values than normal nuclei (P < 0.001). Our results demonstrate that the measured parameters displayed a distinct seasonality. Consequently, we conclude that seasonal variations should be taken into account when nuclear abnormalities are used as biomarkers. This variation should be well understood in order to successfully separate true contamination effects from seasonal factors that have the potential to affect the frequency and occurrence of nuclear abnormalities.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream.

Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48-72 hours.

Aquatic bacterial contamination associated with sugarplant sewage outfalls as a microbial hazard for fish.

The aim of the study was to compare bacterial composition and load in waters and fish related to the wastewater treatment plant (WWTP), particularly waters and wild fish affected by sugarplant processing (sugar cane and sugar beet). Aeromonads were the most frequently isolated group from water and fish. A. hydrophila was a prevailing species in isolates from water, followed by A. veronii, Rheinheimera soli and Ochrobactrum anthropi. Of indicator bacteria for aquatic contamination from fish tissues, the most prominent were V. cholerae, Enterobacter cloacae and E. sakazakii. Sugar cane processing contributed to high viable cell counts at 37 °C while sugar beet processing contributed to high bacterial counts at 22 °C. Heterotrophs from gills of effluent fish were highest during sugar cane processing. Counts retrieved from fish skin were more uniform between effluent fish and fish from downstream waters. Antimicrobial resistance of bacteria isolated from water was high against amoxicillin, sulfamethoxazole, flumequine, norfloxacin and oxolinic acid in samples from the inflow of raw municipal wastewaters to WWTP, while resistance found in bacteria from the inflow of sugarplant mostly related to sulfamethoxazole and amoxicillin. The PCA analysis associated the occurrence of high heterotroph counts, P. aeruginosa, and intestinal enterococci on skin and gills with sugar cane, and yeasts and molds with sugar beet processing. Fish living in treated wastewaters and related water bodies could pose a microbial hazard if fished for human consumption, possibly causing infection when being handled and processed, as a risk of human pathogens penetrating fish tissues.

Observational study of hepatic cytochrome P-450 protein expression and activity in summer flounder (Paralichtys dentatus) after combination ormetoprim-sulfadimethoxine treatment.

The metabolism of aquaculture antibiotics on the piscine, hepatic cytochrome P-450 (CYP) system has not yet been defined. Fifty summer flounder, maintained at 20 degrees C, were fed ormetoprim-sulfadimethoxine (Romet-30(R)) at 1% body weight daily and were randomly sampled before treatment and on days 1, 6, 10 and 21 after treatment. Western blotting of hepatic microsomes included goat antirat CYP1A1 and rabbit antihuman CYP3A4 serum. Catalytic activities comprised: 3-cyano-7-ethoxycoumarin (CEC), 7-benzyloxy-4-trifluoromethylcoumarin (BFC), resorufin benzyl ether (BzRes). Treatment induced CYP1A1 and CYP3A4 expression. Dealkylation of CEC (CYP1A2) was increased after treatment. Romet-30 inhibited CYP3A4 activity measured by BFC, but induced BzRes CYP3A4. The usefulness of mammalian antibodies for piscine P-450 Western blotting was demonstrated. The hepatic P-450 1A2 and 3A4 metabolism was quantifiable by kits developed for mammalian microsomes.

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.

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.

Native Prussian carp (Carassius gibelio) health status, biochemical and histological responses to treated wastewaters.

The aim of this study was to assess the impact of treated wastewaters on native wild Prussian carp inhabiting effluent-receiving waters (ERC) receiving municipal and sugar plant treated wastewaters, further downstream waters (DW), and a detached canal unaffected by the WWTP activities. To that end, general fish health status was determined, including plasma biochemical, haematological, oxidative stress and tissue histopathological indices, over three seasons. The greatest tissue alterations were in fall in ERC during sugar beet processing, as hypertrophy of gill epithelial and interlamellar cells, necrosis and lymphocytic infiltration, hyperplasia and hypertrophy of renal tubules, distention of hepatic sinusoids. In fall the lowest leukocytes, lymphocytes and granulocytes (2467 ± 565, 1333 ± 264, 1133 ± 488 cells/µL respectively), as well as highest plasma ALP (52.7 ± 19.39 U/L) were measured. ERC in fall had the highest ammonium (20 mg/L), nitrite (1.48 mg/L), nitrate (13.4 mg/L), and lowest dissolved O2 (1.23 mg/L). Gill, kidney and liver alterations, and the highest plasma cholesterol (9.1 ± 1.98 mmol/L) were noted in DW fish in fall. Tissue morphology during sugar cane processing seems a consequence of cellular and structural tissue integrity loss. Structural heterogeneity of gills and spleen was enhanced with increasing concentrations of heavy metals and correlated with oxidative stress (SOD 392.5 ± 77.28 U/L). Monogenean infestation was moderate in ERC fish in all seasons compared with DW fish. Prussian carp biological responses to multiple stressors, measured by the effects of WWTP on blood and tissue parameters, reached far downstream and were not of localized nature. This study demonstrated that in aquatic environments impacted with complex contaminants acting synergistically, causal relationships between biological responses and environmental stressors should be interpreted. Integrated histopathological, haematological and biochemical findings are valuable biomarkers for native fish adaptive patterns and monitoring of water quality/pollution of freshwater ecosystems.

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.

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.