Applying Artificial Neural Networks to Oxidative Stress Biomarkers in Forager Honey Bees (Apis mellifera) for Ecological Assessment.

Insect pollinators provide an important ecosystem service that supports global biodiversity and environmental health. The study investigates the effects of the environmental matrix on six oxidative stress biomarkers in the honey bee Apis mellifera. Thirty-five apiaries located in urban, forested, and agricultural areas in Central Italy were sampled during the summer season. Enzyme activities in forager bees were analyzed using an artificial neural network, allowing the identification and representation of the apiary patterns in a Self-Organizing Map. The SOM nodes were correlated with the environmental parameters and tissue levels of eight heavy metals. The results indicated that the apiaries were not clustered according to their spatial distribution. Superoxide dismutase expressed a positive correlation with Cr and Mn concentrations; catalase with Zn, Mn, Fe, and daily maximum air temperature; glutathione S-transferase with Cr, Fe, and daily maximal air temperature; and glutathione reductase showed a negative correlation to Ni and Fe exposure. This study highlights the importance of exploring how environmental stressors affect these insects and the role of oxidative stress biomarkers. Artificial neural networks proved to be a powerful approach to untangle the complex relationships between the environment and oxidative stress biomarkers in honey bees. The application of SOM modeling offers a valuable means of assessing the potential effects of environmental pressures on honey bee populations.

Phylogenetic Diversity of the Red Swamp Crayfish Procambarus clarkii and Its Dispersal Pattern in Northern and Central Italy.

The red swamp crayfish Procambarus clarkii is one of the most threatening freshwater species in the world. The aim of this study is to provide a better understanding of the phylogeography and the invasion routes of P. clarkii populations in the Italian Peninsula through the analysis of mitochondrial phylogeny. Mitochondrial control region and cytochrome c oxidase subunit I (COI) sequences of 153 samples collected from six Italian basins were analyzed and compared to worldwide data. Except for the lakes Bolsena and Posta Fibreno, a high genetic variability was found in the other basins. The mitochondrial DNA pattern of P. clarkii from the lakes Candia and Massaciuccoli confirmed the hypothesis of double introduction events. Another entry point could be represented by Lake Trasimeno, which shows haplotypes originating from Louisiana and not shared with other Italian basins. Moreover, unique lineages were also found in the Stella River, thus enhancing the hypothesis that multiple introductions of P. clarkii occurred in northern and Central Italy and strengthening the idea that knowledge about the dispersion routes of this alien species can be useful to predict its invasiveness and elaborate control strategies to preserve biodiversity.

Sex and seasonal differences in metal accumulation of selected tissues in red swamp crayfish from Lake Trasimeno (Umbria, Italy).

Concentrations of essential metals (Cu, Co, Cr, Fe, Ni, Mn, and Zn) and non-essential and toxic metals (Ag, Cd, Hg, and Pb) were quantified in hepatopancreas and abdominal muscle for 73 specimens (37 males and 36 females) of Procambarus clarkii from Lake Trasimeno (Italy) in a year's campaign (July 2018 to June 2019). Elemental concentrations determined in crayfish differed in tissues, seasons, and gender. Both sexes showed lower concentrations in abdominal muscle than in hepatopancreas. Essential metals accumulated in higher concentrations in both tissues than non-essential elements, while associations between essential metals were stronger, especially for females; however, a strong correlation was also observed between Cu and Ag in both sexes. No significant differences in concentrations were recorded between sexes for all seasons; contrarily, significant differences among the total seasonal concentrations (males plus females) were observed only for essential metals and for Ag. Seasonal Toxic Contamination Index (TCI) for Cr, Ni, Cu, Zn, Cd, Pb, and Hg were calculated to assess the toxicity level of metals in the sediments. TCI higher than 1 was detected for Ni only in summer, confirming the close connection between the fluctuating hydrological levels of the shallow Lake Trasimeno, strongly dependent on precipitation, and the metal contamination levels of sediments.

Ecotoxicity of Copper(I) Chloride in Grooved Carpet Shell (Ruditapes decussatus).

Copper (Cu) is a ubiquitous trace element in the aquatic environment, and is usually found at low levels. Copper environmental concentrations can be altered as a result anthropogenic activities. Shellfish are useful bioindicators to ensure adequate environmental monitoring. Thus, the aim of the present study was as follows: (a) determine the LC50 of copper(I) chloride in grooved carpet shell (Ruditapes decussatus) collected in the Santa Gilla lagoon (Sardinia, Italy), and (b) analyze the antioxidant biomarkers in digestive gland and gills of same specimens exposed to different concentrations of the above-mentioned metal (0.045, 0.45, and 0.90 mg/L) for 96 h. A withdrawal period of 96 h was considered for the treated clam, carrying out the same biochemical analyses, superoxide dismutase (SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GPx), glutathione S-transferases (GSTs), and total glutathione (GSH+2GSSG) in the two tissues. Different time and dose responses of the antioxidant biomarkers were recorded in the digestive glands and gills. Oxidative stress biomarkers highlighted the ability of Cu to induce oxidative stress in R. decussatus. Clam, following the withdrawal period of 96 h, has not been able to achieve the control levels of all biochemical markers in the digestive gland and gills. R. decussatus can be a suitable model to assess the ecotoxicity of copper in aquatic ecosystems. These findings may advance knowledge on the role and the effects of copper on oxidative stress biomarkers in grooved carpet shell. The metal ecotoxicity response can be useful to perform accurate biomarker-based monitoring programs using this bivalve species.

Multi-Level System to Assess Toxicity in Water Distribution Plants.

The toxicity of water samples from water distribution plants needs to be investigated further. Indeed, studies on the pro-oxidant effects driven by tap water are very limited. In this study, the water quality, pro-oxidant effects, and potential health risks driven by exposure to groundwater samples from two water plants (sites A and B) located in Northwestern Italy were investigated in a multi-level system. Physicochemical parameters and the absence of pathogens, cyanotoxins, and endocrine active substances indicated a good water quality for both sites. The 25 metals analyzed were found under the limit of quantification or compliant with the maximum limits set by national legislation. Water samples were concentrated by the solid-phase extraction system in order to assess the aquatic toxicity on Epithelioma papulosum cyprini (EPC) cell line. Levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase were evaluated through the Integrated Biomarkers Response (IBRv2) index. EPC cell line was found a sensible model for assessing the antioxidant responses driven by both water concentrates. A similar antioxidant response was shown by plots and IBRv2 suggesting a muted risk for the two sampling sites.

Modulation of Antioxidant Defense in Farmed Rainbow Trout (Oncorhynchus mykiss) Fed with a Diet Supplemented by the Waste Derived from the Supercritical Fluid Extraction of Basil (Ocimum basilicum).

Phytotherapy is based on the use of plants to prevent or treat human and animal diseases. Recently, the use of essential oils and polyphenol-enriched extracts is also rapidly increasing in the aquaculture sector as a means of greater industrial and environmental sustainability. Previous studies assessed the antibacterial and antiparasitic effects of these bioactive compounds on fish. However, studies on the modulation of oxidative stress biomarkers are still scant to date. Thus, in this study, the modulation of antioxidant defense against oxidative stress exerted by fish diets supplemented with a basil supercritical extract (F1-BEO) was assessed in rainbow trout Oncorhynchus mykiss. The F1-BEO extracted with supercritical fluid extraction was added to the commercial feed flour (0.5, 1, 2, 3% w/w) and mixed with fish oil to obtain a suitable compound for pellet preparation. Fish were fed for 30 days. The levels of stress biomarkers such as superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, glyoxalase I, glyoxalase II, lactate dehydrogenase, glutathione and malondialdehyde showed a boost in the antioxidant pathway in fish fed with a 0.5% F1-BEO-supplemented diet. Higher F1-BEO supplementation led to a failure of activity of several enzymes and the depletion of glutathione levels. Malondialdehyde concentration suggests a sufficient oxidative stress defense against lipid peroxidation in all experimental groups, except for a 3% F1-BEO-supplemented diet (liver 168.87 ± 38.79 nmol/mg prot; kidney 146.86 ± 23.28 nmol/mg prot), compared to control (liver 127.76 ± 18.15 nmol/mg prot; kidney 98.68 ± 15.65 nmol/mg prot). Our results suggest supplementing F1-BEO in fish diets up to 0.5% to avoid potential oxidative pressure in farmed trout.

Ecotoxicity in Hyriopsis bialatus of copper and zinc biocides used in metal-based antifouling paints.

Biofouling is a costly burden for the shipping industry. Metal-based antifouling paints are widely used to protect submerged surfaces, but the release of metals from coatings and the recoating of hulls can leach large amounts of copper and zinc into aquatic environments, posing a risk for aquatic ecosystems and biodiversity. With this study, we studied the time-course metal accumulation and oxidative stress in the digestive gland and the gills of Hyriopsis bialatus, an Asian freshwater mussel, exposed to sublethal concentrations of cuprous chloride (50 and 5 µg/L) and zinc sulfate (1000 and 100 µg/L). Time-dependent accumulation was observed after exposure to copper, but zinc uptake was negligible. Integrated biomarker response (IBRv2) and statistical analysis of individual biomarker levels showed a greater biomarker response in the digestive gland and the gills after exposure to the higher concentration of CuCl and ZnSO4. Both compounds elicited a biochemical response, especially in the digestive gland. Glutathione peroxidase activity was increased after exposure to both metals at both concentrations, suggesting a powerful defense against lipid peroxidation. The biological impact of zinc was less than that of copper, suggesting mitigated ecological pressure.

Population Ecology and Genetic Diversity of the Invasive Alien Species Procambarus clarkii in Lake Trasimeno (Italy).

The deliberate or accidental introduction of invasive alien species (IAS) causes negative ecological and economic impacts altering ecosystem processes, imperiling native species and causing damage to human endeavors. A monthly monitoring program was performed in Lake Trasimeno (Central Italy) from July 2018 to July 2019 in order to provide an upgrade of the population ecology of Procambarus clarkii and to assess the genetic diversity by analyzing the relationships among mitochondrial DNA diversity. Our results confirmed that P. clarkii is well acclimatized in the lake, revealing a stable population structure favored by the resources and conditions typical of this ecosystem, which seem to be optimal for the maintenance of the species. Four distinct mitochondrial haplotypes were detected, but one of them was clearly overrepresented (76%), suggesting that a single predominant introduction event may have occurred in this area, likely followed by secondary events. The identification of the typical genetic variants provides a better understanding of the evolutionary scenarios of P. clarkii in this biotope and it can be helpful in management plans concerning the expanding populations of this invasive alien species.

First insights into oxidative stress and theoretical environmental risk of Bronopol and Detarox® AP, two biocides claimed to be ecofriendly for a sustainable aquaculture.

Bronopol and Detarox® AP are broad spectrum antimicrobial biocides of growing interest for the aquaculture sector. While their effectiveness against aquatic pathogens has been demonstrated, toxicity data on wild or farmed species are still lacking, as is information on their potential environmental risk for aquatic ecosystems. With this study, we assessed the acute and sublethal toxicity of Bronopol and Detarox® AP in the freshwater bivalve Sinanodonta woodiana and their theoretical risk for aquatic ecosystem. The 96-h median lethal concentration (LC50) was determined using the acute toxicity test, while for the sublethal toxicity test the bivalves were exposed to two concentrations for 14 days of Bronopol (2.5 and 50 mg/L) and Detarox® AP (1.11 and 22.26 mg/L) followed by a 14-day withdrawal period. Biocide-mediated oxidative processes were investigated via a panel of oxidative stress biomarkers (malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase). Theoretical environmental risk assessment of both biocides, with predicted concentration of no effect (PNEC), expected theoretical concentration (TEC) in the environment, and risk quotient (RQ) was performed. TEC was calculated using a model based on the size of the aquaculture facility and the receiving basin, the estimated quantity of biocide dissolved in water, and published data on biocide stability in water. Although the LC50 was higher for Bronopol (2440 mg/L) than for Detarox® AP (126 mg/L), fluctuations in oxidative stress biomarkers levels indicated that both biocides exert a slight oxidative pressure on S. woodiana. Theoretical environmental risk assessment suggested a muted risk with Detarox® AP and greater eco-sustainability compared to Bronopol.

Metal load and oxidative stress driven by organotin compounds on rainbow trout.

Tributyltin-based (TBT) antifouling paints, widely used for the treatment of flooded surfaces, have been banned in 2008 for their high environmental persistence and bioaccumulation in aquatic organisms. Although it is still present in aquatic ecosystems, oxidative stress driven by TBT has been still poorly investigated in fish. The aim of the study was to examine the time-course stress responses in liver of rainbow trout that received a single intraperitoneal injection of tributyltin chloride (TBTC) or tributyltin ethoxide (TBTE), both at a dose of 0.05 and 0.5 mg/kg. Levels of metallothioneins, total glutathione, malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase were evaluated at 3 and 6 days post-injection. Tin load was measured in the muscle of the same fish. Differences were observed in the time-course accumulation of tin with a clear dose-response relationship. Although individual oxidative stress biomarkers varied, the biomarker profile indicated different stress mechanisms caused by both TBTC and TBTE. The weak induction of metal-trapping metallothioneins and the changes of oxidative stress biomarkers suggested a stress-pressure in both TBT-treated trout, advising for an ecotoxicological risk for freshwater ecosystems.

Oxidative stress ecology on Pacific oyster Crassostrea gigas from lagoon and offshore Italian sites.

Crassostrea gigas is a sentinel species along the Italian coast. In mussels, the levels of oxidative stress biomarkers can be modulated by several environmental pollutants or pathogens and also fluctuate in response to reproductive stages and seasonal changes. In this study, adult Crassostrea gigas were sampled during summer and autumn from two lagoon and two offshore sites along the Adriatic coast of Italy in order to investigate the influence of seasonality on oxidative stress biomarkers. Trace elements load of Al, As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Se and Zn suggests low contamination for lagoon and offshore sites. Levels of total glutathione, superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase and glutathione S-transferase were analyzed in digestive gland and gills of the Pacific oysters in June, July, September and October. OsHV-1 and Vibrio aestuarianus were detected in lagoon sites, but both pathogens did not affect the biomarkers levels in both tissues. Although several biological responses were found different among the four sites in the same month, principal component analysis revealed similar trend in biomarkers levels between sites during the whole sampling period. On the other hand, a different biochemical pattern through the months emerged, suggesting that the level of oxidative stress biomarkers in both tissues may be related to seasonal progress and biological cycle of oysters sampled from the two lagoons and offshore sites along the Italian coasts of the Mediterranean Sea.

Oxidative stress ecology in brook trout (Salvelinus fontinalis) from a high-mountain lake (Cottian Alps).

High-mountain lakes are pristine ecosystems characterized by extreme environmental conditions. The atmospheric transport of pollutants from lowlands may add further stress to organisms inhabiting these environments. We investigated the environmental stress pressure on brook trout (Salvelinus fontinalis) from a high-mountain lake in the Cottian Alps (Piedmont, northwest Italy). To do this, males and females of brook trout were sampled from Balma Lake in summer (August) and autumn (October) 2017 in order to assess the influence of trace elements accumulation and environmental parameters (physicochemical parameters and nutrient characteristics of water) on oxidative stress biomarkers. Bioaccumulation of Al, As, Cd, Cr, Cu, Fe, Hg, Pb, Ni, Se, and Zn and metallothionein levels were measured in muscle tissue of males and females. Liver, gills, kidney, and spleen tissue samples were analyzed for superoxide dismutase, catalase, total glutathione peroxidase, selenium-dependent glutathione peroxidase, glutathione reductase, and glutathione S-transferase activity. Analysis of environmental parameters showed changes in biomarker levels with seasonal variations. Water temperature was significantly higher in summer than autumn (Wilcoxon test; p = .0078), while pH was significantly higher in autumn than in summer (Wilcoxon test; p = .0078). Sex-related differences in oxidative stress biomarkers in tissues were unremarkable, whereas seasonal variability of oxidative stress biomarkers was observed, with major differences occurred for liver in summer and for gills, kidney, spleen and muscle in autumn. Positive correlations between environmental parameters and biomarkers were noted. Major fluctuations in water temperature, pH, Cu, Pb and Hg produced changes in biomarker levels; however, increased food intake during the ice-free season was probably the main factor that influenced changes in oxidative stress biomarker levels in brook trout in this extreme ecosystem.

A comparative study on subacute toxicity of arsenic trioxide and dimethylarsinic acid on antioxidant status in Crandell Rees feline kidney (CRFK), human hepatocellular carcinoma (PLC/PRF/5), and epithelioma papulosum cyprini (EPC) cell lines.

Arsenic (As) is a global contaminant of terrestrial and aquatic environments posing concern for environmental and human health. The effects of subacute concentrations of arsenic trioxide (AsIII) and dimethylarsinic acid (DMAV) were examined using Crandell Rees feline kidney (CRFK), human hepatocellular carcinoma (PLC/PRF/5), and epithelioma papulosum cyprini (EPC). Whole monolayer with suffering cells (confluence 100%, pyknosis and refractive cells; value scale = 2) led to identification of subacute As concentrations for the three cell lines. The selected AsIII concentrations were 1.33 µM for CRFK and 33.37 µM for PLC/PRF/5 and EPC, at 48 hr time point. The selected DMAV concentrations were 0.67 mM for PLC/PRF/5, 1.33 mM for CRFK, and 2.67 mM for EPC for 48 hr. Unlike the AsIII test, the three cell lines did not exhibit marked susceptibility to DMAV-mediated toxicity. Several oxidative stress biomarker levels, directly or indirectly associated with reactive oxygen species (ROS) elimination including superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase, glutathione S-transferase, glyoxalase I, glyoxalase II, and total glutathione, were determined in the three cell lines at 24 and 48 hr. Antioxidant responses in metal-treated cells were significantly altered compared to controls, suggesting a perturbation of redox state. The weakening of antioxidant pathway in either healthy or tumoral cells was greater using AsIII than DMAV. Differences in level of several oxidative stress biomarkers suggest that the oxidative stress mechanism induced by AsIII is distinctly different from DMAV. Multifaceted mechanisms of action underlying ROS generation in tumor and nontumor cells versus AsIII and DMAV exposure are thus involved. Since As-mediated toxicity is quite complex, more data regarding both oxidant-enhancement and oxidant-lowering strategies may be useful to improve knowledge regarding the influence of As on human and animal cells.