Reviewing the use of zebrafish for the detection of neurotoxicity induced by chemical mixtures through the analysis of behaviour.

The knowledge and assessment of mixtures of chemical pollutants in the aquatic environment is a complex issue that is often challenging to address. In this review, we focused on the use of zebrafish (Danio rerio), a vertebrate widely used in biomedical research, as a model for detecting the effects of chemical mixtures with a focus on behaviour. Our aim was to summarize the current status of the ecotoxicological research in this sector. Specifically, we limited our research to the period between January 2012 and September 2023, including only those works aimed at detecting neurotoxicity through behavioural endpoints, utilizing zebrafish at one or more developmental stages, from egg to adult. Additionally, we gathered the findings for every group of chemicals involved and summarised data from all the works we included. At the end of the screening process 101 papers were considered eligible for inclusion. Results show a growing interest in zebrafish at all life stages for this kind of research in the last decade. Also, a wide variety of different assays, involving different senses, was used in the works we surveyed, with exposures ranging from acute to chronic. In conclusion, the results of this study show the versatility of zebrafish as a model for the detection of mixture toxicity although, for what concerns behavioural analysis, the lack of standardisation of methods and endpoints might still be limiting.

The role of ecotoxicology in the health impact assessment: an innovative ecosystem approach for the protection of human health in Italy.

BACKGROUND: The Health Impact Assessment (HIA) is a procedure with the aim to protect the populations exposed to the impacts deriving from the establishment or upgrading of large industrial enterprises, i.e. large combustion plants (>300 MWth). In Italy a guideline for the HIA procedure has been published in compliance with the 2014/52/EU Directive on the Environmental Impact Assessment (EIA) requirements. Italian HIA procedure. An ecotoxicological approach has been included for the first time in the HIA procedure with the aim to detect toxic effects caused by unknown not-monitored contaminants or mixtures in the ecosystem components affected by the potential emissions, discharges and releases of large industrial enterprises. Ecotoxicology plays an important bridge role between environment and human health in the scoping and monitoring step of the HIA procedure with a key function of early warning system and screening. The aim of this paper is to present the Italian experience in the first three years of the application of the new approach, proposing recommendations on specific case studies. Conclusion and future perspective. 80% of enterprises, that applied HIA, have delivered a robust, integrated and detailed documentation in relation to the ecotoxicological assessment, this positive feedback will generate environmental and human health benefits to the areas where the plants are established.

Mercury (Hg) and methylmercury (MeHg) in sediment and biota: A case study in a lagoon in Central Italy.

A quantification of total mercury (Hgtot) and methylmercury (MeHg) concentrations in sediment and mussels was carried out in the east basin of the Orbetello lagoon in order to assess their bioaccumulation potential. The sediment was sampled in four macroareas, mussels were transplanted in the same sites and collected after seven weeks. The results show that Hgtot concentrations in sediments exceeded (0.21-16.9 mg/kg dry weight (dw)) the environmental quality standard of the Italian legislation (0.3 mg/kg dw). The Hgtot concentration in mussels (0.050-0.324 mg/kg wet weight (ww)) does not exceed the limit values (0.5 mg/kg ww) of the European food legislation. The biota-sediment accumulation factors (BSAFs) derived for MeHg (80-306.7) and a biomagnification factor (BMF) greater than 1 for Hgtot demonstrate that in the lagoon, these compounds can be transferred in the upper levels of the trophic chain and pose a risk to human health.

An integrated approach for chemical water quality assessment of an urban river stretch through Effect-Based Methods and emerging pollutants analysis with a focus on genotoxicity.

The impact of emerging chemical pollutants, on both status and functionality of aquatic ecosystems is worldwide recognized as a relevant issue of concern that should be assessed and managed by researchers, policymakers, and all relevant stakeholders. In Europe, the Reach Regulation has registered more than 100.000 chemical substances daily released in the environment. Furthermore, the effects related to the mixture of substances present in aquatic ecosystems may not be predictable on the basis of chemical analyses alone. This evidence, coupled with the dramatic effects of climate changes on water resources through water scarcity and flooding, makes urgent the application of innovative, fast and reliable monitoring methods. In this context, Effect-Based Methods (EBMs) have been applied in the urban stretch of the Tiber River (Central Italy) with the aim of understanding if detrimental pressures affect aquatic environmental health. In particular, different eco-genotoxicological assays have been used in order to detect genotoxic activity of chemicals present in the river, concurrently characterized by chemical analysis. Teratogenicity and embryo-toxicity have been studied in order to cover additional endpoints. The EBMs have highlighted the presence of diffuse chemical pollution and ecotoxicological effects in the three sampling stations, genotoxicological effects have been also detected through the use of different tests and organisms. The chemical analyses confirmed that in the aquatic ecosystems there is a diffuse presence, even at low concentrations, of emerging contaminants such as pharmaceuticals, not routinely monitored pesticides, personal care products, PFAS. The results of this study can help to identify an appropriate battery of EBMs for future studies and the application of more appropriate measures in order to monitor, mitigate or eliminate chemical contamination and remediate its adverse/detrimental effects on the ecosystem health.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Need for a sustainable use of medicinal products: environmental impacts of ivermectin.

It is worldwide recognized that the use of pharmaceuticals for human and veterinary purposes could lead to unsustainable effects on the environment. A strategy to reduce the impact of pharmaceuticals on the environment has been recently established at European level, where guidelines to evaluate the impacts of veterinary drugs used to treat animal diseases are in place. The aim of this article is to focus on the worldwide used antiparasitic drug ivermectin (IVM) and its potential impact on the environment. A specific section is related to the IVM resistance that the massive use of this drug could generate enhancing the risk scenarios also for human health. The application of stringent measures for the veterinary use of this substance, in line with the recommendations provided by International frameworks such as One Health and EcoHealth, is recommended.

Application of effect-based methods (EBMs) in a river basin: a preliminary study in Central Italy.

INTRODUCTION: Effect-based methods (EBMs), i.e. in vitro and in vivo bioassays, represent innovative tools for the effect detection of environmental chemical pollutants on living organisms. The aim of this study was to evaluate the water quality of a river ecosystem implementing two in vivo bioassays on target freshwater animal species: the crustacean Daphnia magna and the small fish Danio rerio, also known as zebrafish. MATERIALS AND METHODS: The methods applied in this study, i.e. the Daphnia sp. Acute Immobilisation assay and the Fish Embryo Acute Toxicity (FET) test, are commonly used in water quality research and their application in short-term ecotoxicity detection is suggested by recent European projects. Two sampling sites were chosen in the urban part of the Tiber River in Rome, while a third one was chosen as a reference site in the Farfa River, a tributary upstream of the city. The sites in the Tiber River are potentially affected by different pollution sources, including urban and industrial wastewater discharges, the pesticide release, livestock waste products, and waste dumps. RESULTS AND DISCUSSION: The results of the study showed wide differences between the two applied bioassays. The FET test was generally more sensitive in detecting even low effects in all the water samples, but the strongest statistically results were observed with the D. magna Acute Immobilisation test. The results of this research confirm the effectiveness of EBMs in investigating and monitoring water chemical pollution, and stress the need for performing further studies, e.g. chemical analyses and other bioassays, to improve the knowledge of the health status of the Tiber River basin. CONCLUSIONS: Further results will aim to support the local authorities in adopting measures to reduce and to eliminate the sources of chemical pollution in the study area.

Tigriopus fulvus: The interlaboratory comparison of the acute toxicity test.

The paper reports the results of an interlaboratory comparison involving 11 laboratories, with the objectives of apply and validate a new standardized ecotoxicological method on marine crustacean Tigriopus fulvus. Copper was chosen as reference toxicant as indicated in the official method. The results of two independent tests performed by all the participants, demonstrated that the new method is simple, fast and easy to learn. This is confirmed even by the values of z-score index calculated for each laboratory and the relative coefficient of variation (CV) which are 6.32% after 24h, 6.56 after 48h and 35.3% after 96h, mentioned in the ISO standards for the precision of interlaboratory assays. Therefore its use could be recommended in environmental studies and monitoring.

Standardized methods for acute and semichronic toxicity tests with the copepod Acartia tonsa.

The availability of standardized protocols for both organism culture and bioassay with ecologically relevant species is of great concern in ecotoxicology. Acartia tonsa represents an important, often dominant, member of zooplankton communities and meets all the practical criteria suggested for model species. New standardized procedures for laboratory culturing of the copepod A. tonsa and standardized methods for acute (24- and 48-h) and semichronic (7-d, static-renewal) toxicity tests with the nauplius stage are described. In both cases, eggs are the starting stage, and nauplius immobilization is the endpoint. The methods were the object of an intercomparison test involving nine laboratories, and nickel was the reference toxicant. Relative reproducibility was 24, 25, and 34% for 24-h, 48-h, and 7-d tests, respectively.

Airborne microorganisms associated with waste management and recovery: biomonitoring methodologies.

This paper presents preliminary results from a year-long indoor bioaerosol monitoring performed in three working environments of a municipal composting facility treating green and organic waste. Composting, whereby organic matter is stabilized through aerobic decomposition, requires aeration, causing the dispersion of microbial particles (microorganisms and associated toxins). Waste can, therefore, become a potential source of biological hazard. Bioaerosol samples were collected on a monthly basis. Through a comparison of results obtained using two samplers - the Surface Air System DUO SAS 360 and the BioSampler - the study aimed at assessing the presence of biological pollutants, and at contributing to the definition of standard sampling methods for bioaerosols leading, eventually, to the establishment of exposure limits for these occupational pollutants.

Microbial characterization of water and biofilms in drinking water distribution systems at sport facilities.

The present study investigated the in situ microbial indicators' occurrence in water and biofilm in drinking water distribution systems of sport facilities such as gyms. The presence of Legionella, such as a potential pathogen, was also verified. Water and biofilm were contemporaneously collected and microbiologically analyzed. Few colonies of coliforms were detected in biofilm but not in the corresponding water samples. Conversely, some colonies of heterotrophs were counted at 22 degrees C in both the 80% biofilm samples and the 53% water samples. Legionella was detected in 29% of the hot water systems, and, in particular, L. pneumophila in 21% and L. non pneumophila in 9% of the samples. Higher counts were found in biofilm samples. As it was recommended for swimming pools, it is to be hoped that hygiene risk assessment criteria and safety plans could be also developed for gyms to reduce potential hazards to health for all attendants.