Oxidative stress and DNA alteration on the earthworm Eisenia fetida exposed to four commercial pesticides.

Modern agriculture is mainly based on the use of pesticides to protect crops but their efficiency is very low, in fact, most of them reach water or soil ecosystems causing pollution and health hazards to non-target organisms. Fungicide triazoles and strobilurins based are the most widely used and require a specific effort to investigate toxicological effects on non-target species. This study evaluates the toxic effects of four commercial fungicides Prosaro® (tebuconazole and prothioconazole), Amistar®Xtra (azoxystrobin and cyproconazole), Mirador® (azoxystrobin) and Icarus® (Tebuconazole) on Eisenia fetida using several biomarkers: lipid peroxidation (LPO), catalase activity (CAT), glutathione S-transferase (GST), total glutathione (GSHt), DNA fragmentation (comet assay) and lysozyme activity tested for the first time in E. fetida. The exposure to Mirador® and AmistarXtra® caused an imbalance of ROS species, leading to the inhibition of the immune system. AmistarXtra® and Prosaro®, composed of two active ingredients, induced significant DNA alteration, indicating genotoxic effects. This study broadened our knowledge of the effects of pesticide product formulations on earthworms and showed the need for improvement in the evaluation of toxicological risk deriving from the changing of physicochemical and toxicological properties that occur when a commercial formulation contains more than one active ingredient and several unknown co-formulants.

Assessing the effects of a commercial fungicide and an herbicide, alone and in combination, on Apis mellifera: Insights from biomarkers and cognitive analysis.

Agrochemicals play a vital role in protecting crops and enhancing agricultural production by reducing threats from pests, pathogens and weeds. The toxicological status of honey bees can be influenced by a number of factors, including pesticides. While extensive research has focused on the lethal and sublethal effects of insecticides on individual bees and colonies, it is important to recognise that fungicides and herbicides can also affect bees' health. Unfortunately, in the field, honey bees are exposed to mixtures of compounds rather than single substances. This study aimed to evaluate the effects of a commercial fungicide and a commercial herbicide, both individually and in combination, on honey bees. Mortality assays, biomarkers and learning and memory tests were performed, and the results were integrated to assess the toxicological status of honey bees. Neurotoxicity (acetylcholinesterase and carboxylesterase activities), detoxification and metabolic processes (glutathione S-transferase and alkaline phosphatase activities), immune system function (lysozyme activity and haemocytes count) and genotoxicity biomarkers (Nuclear Abnormalities assay) were assessed. The fungicide Sakura® was found to activate detoxification enzymes and affect alkaline phosphatase activity. The herbicide Elegant 2FD and the combination of both pesticides showed neurotoxic effects and induced detoxification processes. Exposure to the herbicide/fungicide mixture impaired learning and memory in honey bees. This study represents a significant advance in understanding the toxicological effects of commonly used commercial pesticides in agriculture and contributes to the development of effective strategies to mitigate their adverse effects on non-target insects.

Oceanographic and anthropogenic variables driving marine litter distribution in Mediterranean protected areas: Extensive field data supported by forecasting modelling.

Marine litter concentration in the Mediterranean Sea is strongly influenced both by anthropogenic pressures and hydrodynamic factors that locally characterise the basin. Within the Plastic Busters MPAs (Marine Protected Areas) Interreg Mediterranean Project, a comprehensive assessment of floating macro- and microlitter in the Pelagos Sanctuary and the Tuscan Archipelago National Park was performed. An innovative multilevel experimental design has been planned ad-hoc according to a litter provisional distribution model, harmonising and implementing the current sampling methodologies. The simultaneous presence of floating macro- and microlitter items and the potential influences of environmental and anthropogenic factors affecting litter distribution have been evaluated to identify hotspot accumulation areas representing a major hazard for marine species. A total of 273 monitoring transects of floating macrolitter and 141 manta trawl samples were collected in the study areas to evaluate the abundance and composition of marine litter. High mean concentrations of floating macrolitter (399 items/km2) and microplastics (259,490 items/km2) have been found in the facing waters of the Gulf of La Spezia and Tuscan Archipelago National Park as well in the Genova canyon and Janua seamount area. Accordingly, strong litter inputs were identified to originate from the mainland and accumulate in coastal waters within 10-15 nautical miles. Harbours and riverine outfalls contribute significantly to plastic pollution representing the main sources of contamination as well as areas with warmer waters and weak oceanographic features that could facilitate its accumulation. The results achieved may indicate a potentially threatening trend of litter accumulation that may pose a serious risk to the Pelagos Sanctuary biodiversity and provide further indications for dealing with plastic pollution in protected areas, facilitating future management recommendations and mitigation actions in these fragile marines and coastal environments.

State of the art, gaps and future perspectives on common kestrel ecotoxicology.

Anthropogenic activities have caused a steady decline of common kestrel (Falco tinnunculus) since the 1980 s. Effects, especially sublethal effects of contaminants, need to be investigated to ensure the conservation of this species. Data about countries, biological material, contaminants classes, and methodological approaches were collected from scientific publications to highlight gaps on common kestrel toxicology and ecotoxicology. We found that most studies have been conducted in Europe and in the field, underlining a lack of in vitro studies. The studies investigated mainly contaminant levels, while sublethal effects, evaluation of emerging contaminants and use of non-invasive or low-invasive samples were scarce. This work shows important gaps on toxicological status of the common kestrel, highlighting the importance of developing a non-lethal approach that combines responses at different levels of biological organization, as well as data on chemical contamination and on the environment in which the different populations inhabit.

The Culturable Mycobiota of Sediments and Associated Microplastics: From a Harbor to a Marine Protected Area, a Comparative Study.

Fungi are an essential component of marine ecosystems, although little is known about their global distribution and underwater diversity, especially in sediments. Microplastics (MPs) are widespread contaminants worldwide and threaten the organisms present in the oceans. In this study, we investigated the fungal abundance and diversity in sediments, as well as the MPs, of three sites with different anthropogenic impacts in the Mediterranean Sea: the harbor of Livorno, the marine protected area "Secche della Meloria"; and an intermediate point, respectively. A total of 1526 isolates were cultured and identified using a polyphasic approach. For many of the fungal species this is the first record in a marine environment. A comparison with the mycobiota associated with the sediments and MPs underlined a "substrate specificity", highlighting the complexity of MP-associated fungal assemblages, potentially leading to altered microbial activities and hence changes in ecosystem functions. A further driving force that acts on the fungal communities associated with sediments and MPs is sampling sites with different anthropogenic impacts.

Integrated biomarker responses in European seabass Dicentrarchus labrax (Linnaeus, 1758) chronically exposed to PVC microplastics.

Few studies evaluated long-term effects of polyvinyl chloride (PVC) microplastics (MPs) ingestion in fish. The present study aimed to investigate the integrated biomarker responses in the liver and blood of 162 European seabass, Dicentrarchus labrax, exposed for 90 days to control, virgin and marine incubated PVC enriched diets (0.1 % w/w) under controlled laboratory condition. Enzymatic and tissue alterations, oxidative stress, gene expression alterations and genotoxicity were examined. Additives and environmental contaminants levels in PVC-MPs, control feed matrices and in seabass muscles were also detected. The results showed that the chronic exposure at environmentally realistic PVC-MPs concentrations in seabass, cause early warning signs of toxicological harm in liver by induction of oxidative stress, the histopathological alterations and also by the modulation of the Peroxisome proliferator-activated receptors (PPARs) and Estrogen receptor alpha (ER-α) genes expression. A trend of increase of DNA alterations and the observation of some neoformations attributable to lipomas suggest also genotoxic and cancerogenic effects of PVC. This investigation provides important data to understand the regulatory biological processes affected by PVC-MPs ingestion in marine organisms and may also support the interpretation of results provided by studies on wild species.

First application of an Integrated Biological Response index to assess the ecotoxicological status of honeybees from rural and urban areas.

Understanding the effects of environmental contaminants on honeybees is essential to minimize their impacts on these important pollinating insects. The aim of this study was to assess the ecotoxicological status of honeybees in environments undergoing different anthropic pressure: a wood (reference site), an orchard, an agricultural area, and an urban site, using a multi-biomarker approach. To synthetically represent the ecotoxicological status of the honeybees, the responses of the single biomarkers were integrated by the Integrated Biological Response (IBRv2) index. Overall, the strongest alteration of the ecotoxicological status (IBRv2 = 7.52) was detected in the bees from the orchard due to the alteration of metabolic and genotoxicity biomarkers indicating the presence of pesticides, metals, and lipophilic compounds. Honeybees from the cultivated area (IBRv2 = 7.18) revealed an alteration especially in neurotoxicity, metabolic, and genotoxicity biomarkers probably related to the presence of pesticides, especially fungicides. Finally, in the urban area (IBRv2 = 6.60), the biomarker results (GST, lysozyme, and hemocytes) indicated immunosuppression in the honeybees and the effects of the presence of lipophilic compounds and metals in the environment.

Review on Sublethal Effects of Environmental Contaminants in Honey Bees (Apis mellifera), Knowledge Gaps and Future Perspectives.

Honey bees and the pollination services they provide are fundamental for agriculture and biodiversity. Agrochemical products and other classes of contaminants, such as trace elements and polycyclic aromatic hydrocarbons, contribute to the general decline of bees' populations. For this reason, effects, and particularly sublethal effects of contaminants need to be investigated. We conducted a review of the existing literature regarding the type of effects evaluated in Apis mellifera, collecting information about regions, methodological approaches, the type of contaminants, and honey bees' life stages. Europe and North America are the regions in which A. mellifera biological responses were mostly studied and the most investigated compounds are insecticides. A. mellifera was studied more in the laboratory than in field conditions. Through the observation of the different responses examined, we found that there were several knowledge gaps that should be addressed, particularly within enzymatic and molecular responses, such as those regarding the immune system and genotoxicity. The importance of developing an integrated approach that combines responses at different levels, from molecular to organism and population, needs to be highlighted in order to evaluate the impact of anthropogenic contamination on this pollinator species.

Multi-biomarker approach and IBR index to evaluate the effects of different contaminants on the ecotoxicological status of Apis mellifera.

The honeybee, Apis mellifera L. (Hymenoptera: Apidae), a keystone pollinator of wild plant species and agricultural crops, is disappearing globally due to parasites and diseases, habitat loss, genetic constraints, beekeeper management issues and to the widespread use of pesticides. Besides insecticides, widely studied in this species, honeybees are also exposed to herbicides and fungicides and heavy metals whose lethal and sublethal effects need to be investigated. In this context, our study aimed to evaluate the effects of fungicides and of heavy metals on honeybees and to develop and apply a multi-biomarker approach that include an Integrated Biological Index (IBRv2) to assess the toxicological status of this species. Biomarkers of neurotoxicity (AChE and CaE), metabolic alteration (ALP, and GST) and immune system (LYS, granulocytes) were measured, following honeybees' exposure to cadmium or to a crop fungicide, using the genotoxic compound EMS as positive control. A biomarker of genotoxicity (NA assay) was developed and applied for the first time in honeybees. At the doses tested, all the contaminants showed sublethal toxicity to the bees, highlighting in particular genotoxic effects. The data collected were analyzed by an IBRv2 index, which integrated the seven biomarkers used in this study. IBRv2 index increased with increasing cadmium or fungicide concentrations. The IBRv2 represents a simple tool for a general description of honeybees ecotoxicological health status. Results highlight the need for more in-depth investigations on the effects of fungicides on non-target organisms, such as honeybees, using sensitive methods for the determination of sublethal effects. This study contributes to the development of a multi-biomarker approach to be used for a more accurate ecotoxicological environmental monitoring of these animals.

Abundance and characterization of microplastics in the coastal waters of Tuscany (Italy): The application of the MSFD monitoring protocol in the Mediterranean Sea.

Monitoring efforts are required to understand the sources, distribution and abundance of microplastic pollution. To verify the abundance of microplastics along the Tuscan coastal waters (Italy), water-column and surface samples were collected in two seasons across four transects at different distances to the coast (0.5, 5, 10 and 20 km), within the implementation of the European Marine Strategy Framework Directive. The results show an average concentration of 0.26 items/m3 in the water-column samples and 41.1 g/km2 and 69,161.3 items/km2 of floating microplastics, with an increase with the distance to the coast The seasonality and the sampling area do not affect the abundance of microplastics. The most abundant size class is 1-2.5 mm as fragments and sheets suggesting that fragmentation of larger polyethylene and polypropylene items could be the main source of microplastics. These data represent the application of a harmonized protocol to make the data on microplastics comparable and reliable.

First ecotoxicological assessment of Caretta caretta (Linnaeus, 1758) in the Mediterranean Sea using an integrated nondestructive protocol.

The loggerhead sea turtle (Caretta caretta) is a long-lived carnivorous reptile included in the IUCN Red List. The IUCN assessment for this species underlines the lack of information regarding pollution and pathogens and indicates as a priority effort to investigate and reduce the impacts of these threats. In this contest, the aim of our study was to conduct the first ecotoxicological assessment of this species in the Mediterranean Sea using a nondestructive integrated methodology. We set up and applied a monitoring protocol which includes endpoints, such as CYP1A, LPO, ENA assay, B esterases, never investigated before in this species. Seventy-five loggerhead turtles were sampled in a nondestructive way in Italian Sea Turtle Rescue Centers or free-ranging along the Spanish coasts. Blood, skin and carapace samples were used to test biomarker responses (CYP1A, VTG, LPO, Comet and ENA assay, BChE, GGT) and contaminant (OCs, PAHs, Pb, Cd, Hg) levels. Elaboration of experimental results was carried out taking also into consideration different age classes of the specimens. Among the main results obtained we should underline the statistically significant correlation between carcinogenic PAHs in blood and DNA fragmentation as well as between Cd in carapace and GGT in plasma. We also evidenced as youngest animals showed significantly higher DNA fragmentations, BChE inhibition and increase of GGT. Older specimens showed the highest levels of erythrocyte nuclear abnormalities which may indicate a long term toxicological stress. This study contributed to expand the knowledge about the ecotoxicology of C. caretta in the Mediterranean. The nondestructive protocol could also be applied to other marine ecosystems and other sea turtle species.

Loggerhead sea turtles (Caretta caretta): A target species for monitoring litter ingested by marine organisms in the Mediterranean Sea.

Marine litter is any persistent, manufactured or processed solid material discarded, disposed of or abandoned in the marine and coastal environment. Ingestion of marine litter can have lethal and sub-lethal effects on wildlife that accidentally ingests it, and sea turtles are particularly susceptible to this threat. The European Commission drafted the 2008/56/EC Marine Strategy Framework Directive with the aim to achieve a Good Environmental Status (GES), and the loggerhead sea turtle (Caretta caretta, Linnaeus 1758) was selected for monitoring the amount and composition of litter ingested by marine animals. An analogous decision has been made under the UNEP/MAP Barcelona Convention for the protection of the Mediterranean Sea, following the Ecosystem Approach. This work provides for the first time, two possible scenarios for the Marine Strategy Framework Directive GES, both related to "Trends in the amount and composition of litter ingested by marine animals" in the Mediterranean Sea. The study validates the use of the loggerhead turtle as target indicator for monitoring the impact of litter on marine biota and calls for immediate use of this protocol throughout the Mediterranean basin and European Region. Both GES scenarios are relevant worldwide, where sea turtles and marine litter are present, for measuring the impact of ingested plastics and developing policy strategies to reduce it. In the period between 2011 and 2014, 150 loggerhead sea turtles, found dead, were collected from the Italian Coast, West Mediterranean Sea Sub-Region. The presence of marine litter was investigated using a standardized protocol for necropsies and lab analysis. The collected items were subdivided into 4 main categories, namely, IND-Industrial plastic, USE-User plastic, RUB-Non plastic rubbish, POL-Pollutants and 14 sub-categories, to detect local diversity. Eighty-five percent of the individuals considered (n = 120) were found to have ingested an average of 1.3 ± 0.2 g of litter (dry mass) or 16 ± 3 items.

Impact of Raw and Bioaugmented Olive-Mill Wastewater and Olive-Mill Solid Waste on the Content of Photosynthetic Molecules in Tobacco Plants.

Disposal and reuse of olive-mill wastes are both an economic and environmental problem, especially in countries where the cultivation of olive trees is extensive. Microorganism-based bioaugmentation can be used to reduce the pollutant capacity of wastes. In this work, bioaugmentation was used to reduce the polyphenolic content of both liquid and solid wastes. After processing, bioaugmented wastes were tested on the root development of maize seeds and on photosynthesis-related molecules of tobacco plants. In maize, we found that bioaugmentation made olive-mill wastes harmless for seed germination. In tobacco, we analyzed the content of RuBisCO (ribulose-1,5-bisphosphate carboxylase oxygenase) and of the photosynthetic pigments lutein, chlorophylls, and ß-carotene. Levels of RuBisCO were negatively affected by untreated wastewater but increased if plants were treated with bioaugmented wastewater. On the contrary, levels of RuBisCO increased in the case of plants treated with raw olive-mill solid waste. Pigment levels showed dissimilar behavior because their concentration increased if plants were irrigated with raw wastewater or treated with raw olive-mill solid waste. Treatment with bioaugmented wastes restored pigment content. Findings show that untreated wastes are potentially toxic at the commencement of treatment, but plants can eventually adapt after an initial stress period. Bioaugmented wastes do not induce immediate damages, and plants rapidly recover optimal levels of photosynthetic molecules.

Amount and distribution of neustonic micro-plastic off the western Sardinian coast (Central-Western Mediterranean Sea).

A plethora of different sampling methodologies has been used to document the presence of micro-plastic fragments in sea water. European Marine Strategy suggests to improve standard techniques to make future data comparable. We use Manta Trawl sampling technique to quantify abundance and distribution of micro-plastic fragments in Sardinian Sea (Western Mediterranean), and their relation with phthalates and organoclorine in the neustonic habitat. Our results highlight a quite high average plastic abundance value (0.15 items/m(3)), comparable to the levels detected in other areas of the Mediterranean. "Site" is the only factor that significantly explains the differences observed in micro-plastic densities. Contaminant levels show high spatial and temporal variation. In every station, HCB is the contaminant with the lowest concentration while PCBs shows the highest levels. This work, in line with Marine Strategy directives, represents a preliminary study for the analysis of plastic impact on marine environment of Sardinia.

First application of comet assay in blood cells of Mediterranean loggerhead sea turtle (Caretta caretta).

The aim of this study was to validate the comet assay in erythrocytes of Caretta caretta, a species never investigated for genotoxicity. We studied 31 loggerhead sea turtles from three Italian marine rescue centres. Peripheral blood samples were collected from all the animals and the comet assay applied. All comet cells were analysed using two methods: visual scoring and computer image analysis. The % DNA in tail mean value ± SD and Damage Index were 21.56 ± 15.41 and 134.83 ± 94.12, respectively. A strong and statistically significant statistically correlation between the two analytical methods was observed (r = 0.95; p < 0.05). These results demonstrate that the comet assay is a useful method to detect the possible effects of genotoxic agents in loggerhead sea turtle and to increase the knowledge about the ecotoxicological health status of this threatened species.

Presence of plastic debris in loggerhead turtle stranded along the Tuscany coasts of the Pelagos Sanctuary for Mediterranean Marine Mammals (Italy).

This work evaluated the presence and the frequency of occurrence of marine litter in the gastrointestinal tract of 31 Caretta caretta found stranded or accidentally bycaught in the North Tyrrhenian Sea. Marine debris were present in 71% of specimens and were subdivided in different categories according to Fulmar Protocol (OSPAR 2008). The main type of marine debris found was user plastic, with the main occurrence of sheetlike user plastic. The small juveniles showed a mean±SD of marine debris items of 19.00±23.84, while the adult specimens showed higher values of marine litter if compared with the juveniles (26.87±35.85). The occurrence of marine debris observed in this work confirms the high impact of marine debris in the Mediterranean Sea in respect to other seas and oceans, and highlights the importance of Caretta caretta as good indicator for marine litter in the Marine Strategy Framework Directive (MSFD) of European Union.