Investigating endocrine-disrupting properties of chemicals in fish and amphibians: Opportunities to apply the 3Rs.

Many regulations are beginning to explicitly require investigation of a chemical's endocrine-disrupting properties as a part of the safety assessment process for substances already on or about to be placed on the market. Different jurisdictions are applying distinct approaches. However, all share a common theme requiring testing for endocrine activity and adverse effects, typically involving in vitro and in vivo assays on selected endocrine pathways. For ecotoxicological evaluation, in vivo assays can be performed across various animal species, including mammals, amphibians, and fish. Results indicating activity (i.e., that a test substance may interact with the endocrine system) from in vivo screens usually trigger further higher-tier in vivo assays. Higher-tier assays provide data on adverse effects on relevant endpoints over more extensive parts of the organism's life cycle. Both in vivo screening and higher-tier assays are animal- and resource-intensive and can be technically challenging to conduct. Testing large numbers of chemicals will inevitably result in the use of large numbers of animals, contradicting stipulations set out within many regulatory frameworks that animal studies be conducted as a last resort. Improved strategies are urgently required. In February 2020, the UK's National Centre for the 3Rs and the Health and Environmental Sciences Institute hosted a workshop ("Investigating Endocrine Disrupting Properties in Fish and Amphibians: Opportunities to Apply the 3Rs"). Over 50 delegates attended from North America and Europe, across academia, laboratories, and consultancies, regulatory agencies, and industry. Challenges and opportunities in applying refinement and reduction approaches within the current animal test guidelines were discussed, and utilization of replacement and/or new approach methodologies, including in silico, in vitro, and embryo models, was explored. Efforts and activities needed to enable application of 3Rs approaches in practice were also identified. This article provides an overview of the workshop discussions and sets priority areas for follow-up. Integr Environ Assess Manag 2022;18:442-458. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009.

This Guidance describes how to perform hazard identification for endocrine-disrupting properties by following the scientific criteria which are outlined in Commission Delegated Regulation (EU) 2017/2100 and Commission Regulation (EU) 2018/605 for biocidal products and plant protection products, respectively.

Development of the first certified reference materials for several brominated flame retardants in polymers.

The first reference materials certified for several polybrominated flame retardants in polymers were developed. Commercially available polyethylene and polypropylene were fortified with technical mixtures of Pentabrominated diphenylether (Penta-BDE), Octa-BDE, Deca-BDE, and Decabrominated biphenyl (BB) (where the capitalized forms refer to the technical mixtures). Homogeneity was tested on 20 units of each material, and between-unit variation was confirmed to be below 4% for all congeners. Stability was assessed after storage of samples for 1 year at 4, 18, and 60 degrees C. Uncertainty of degradation during transport was found negligible for all congeners, whereas uncertainty of degradation for storage of 24 months at 4 degrees C was estimated between 2% and 11%. A characterization intercomparison involving 16 laboratories was organized. After exclusion of technically doubtful results, between-laboratory standard deviations ranged from 3% to 12%, making this intercomparison the best for this field of analysis so far. Statistical analysis revealed that the use of isotopically labeled internal standards did not improve analytical precision in this study. The good comparability, together with the independent confirmation of the assigned mass fractions via the total bromine content as well as by using non-GC/MS-based methods, allowed for the first time the certification of polymer materials for several brominated flame retardants.

Analysis of short-chain chlorinated paraffins: a discussion paper.

Short-chain chlorinated paraffins are a class of organic compounds widely used in many industrial applications, extensively diffused into the environment, persistent, bioaccumulative, and toxic towards aquatic organisms. However, their study and monitoring in the environment are still limited. Because of the enormous number of positional isomers that characterise their mixtures, the analysis of this class of pollutants is very difficult to perform. Beside this, the lack of certified reference materials poses a problem for the assessment of the quality assurance/quality control of any analytical procedure. At present, the scientific community does not agree on any analytical reference method, although the monitoring of short-chain chlorinated paraffins has already started in order to comply with the Water Framework Directive of the European Union on water quality. In this paper the regulatory framework, in which chlorinated paraffins are included, and the status concerning their determination are summarized. The main analytical difficulties still existing are discussed, and the definition of a method-defined parameter as well as the development of a standardised method are suggested as a way to obtain comparable monitoring data.

Organochlorine compounds (polychlorinated biphenyls and pesticides) and polycyclic aromatic hydrocarbons in populations of Hexaplex trunculus affected by imposex in the lagoon of Venice, Italy.

Concentrations of polychlorinated biphenyls (PCBs), organochlorine pesticides, and polycyclic aromatic hydrocarbons (PAHs) were measured in gastropods from the Lagoon of Venice, Italy. The visceral coil and the rest of the soft body of organisms (Hexaplex trunculus) sampled at two stations inside the lagoon and three stations on the seaward side were analyzed to evaluate their contamination levels. Preferential accumulation of PCBs and pesticides in the visceral coil (>80%) compared with the rest of the soft body was observed, whereas on average, PAHs showed no preferential partitioning. Differences between levels of organochlorine contaminants in the gastropods highlighted a gradient of pollution from the stations inside the lagoon (PCBs, 45-363 ng/g; pesticides, 4-51 ng/g) to the sea (PCBs, 13-131 ng/g; pesticides, 2-29 ng/g). The possible role of the three classes of contaminants, in addition to that of organotin compounds (OTCs), previously analyzed in the same samples, in causing one of the anatomic modifications because of imposex in this gastropod also was studied. A modeling approach by partial least squares (PLS) in latent variables was applied to explain the penis length of imposex-affected females with concentrations of organic pollutants. The synergistic role of PCBs, pesticides, and OTCs was evidenced, whereas the contribution of PAHs appeared to be very low.

The changing state of contamination in the Lagoon of Venice. Part 2: heavy metals.

In order to verify whether pollution is increasing or decreasing, in 25 locations uniformly distributed in the central part of the Lagoon of Venice, a transitional environment suffering from man's urban and industrial activities, the same sampling scheme was repeated three times (in 1987, 1993 and 1998) over a 12-year period during which the lagoonal environment underwent substantial changes. Superficial sediments were sampled and analysed for heavy metals and total organic carbon contents, grain size and density. In general heavy metal contents were found to be correlated, with concentrations above the background level, e.g., for Hg, a concentration factor of 24 was observed in 1987. A temporal decrease in concentrations was observed for most of the metals. Detailed analysis on a smaller spatial scale showed that contamination significantly decreases from the inner border of the lagoon seawards, as highlighted in contour maps. The role of the Porto Marghera industrial zone as a source of pollutants at the border of the Lagoon was confirmed. The decrease in contamination could not be attributed only to a decrease in the intensity of sources, but also to erosion processes, worsened by intensive harvesting of clams with hydraulic dredges.

The changing state of contamination in the Lagoon of Venice. Part 1: organic pollutants.

The Lagoon of Venice is a transitional environment suffering from industrial, urban and agricultural impact. Recently the mechanical clam fishing, preventing a regular deposition of sediments has made sampling of cores suitable for radiodating very problematic. Therefore, in this study, in order to assess temporal and spatial trends of contamination, the same sampling scheme was repeated three times over a 12-year period. The concentrations of PCBs, PAHs and organochlorine pesticides were measured in the uppermost 5 cm of sediments collected in 25 sites in the central portion of the lagoon during three campaigns conducted in 1987, 1993 and 1998. Analytical results, validated statistically, indicated that, while PCB and pesticide concentrations tend to decrease during the considered period of time, PAH do not, showing that the input rate of these compounds to the lagoon environment is still important. A spatial trend of pollution was also found in the lagoon for PCBs and pesticides, which decrease from the sources, namely the industrial zone and the mouths of rivers, to areas closer to the sea: a reduction up to 80% and 90%, respectively, was monitored. PAH contamination, originating chiefly from incomplete combustion of fossil fuels, as inferred from ratios of congeners, was mainly located around the historical center of Venice, where most of the boat traffic is concentrated and has an increasing trend.

Concentrations of organotin compounds and imposex in the gastropod Hexaplex trunculus from the Lagoon of Venice.

In four stations located close to the channels connecting the Lagoon of Venice with the Adriatic Sea (two inside the lagoon and two outside it), individuals of Hexaplex trunculus were collected in order to assess their contamination. Concentrations of some organotin compounds, i.e. tributyltin and triphenyltin with their di- and mono- substituted metabolites, were measured and endocrine disruption such as imposex (superimposition of male sexual characteristics on females of gonochoristic gastropods) was observed. Vas deferens sequence indexes (VDSI) of 4.1-4.9 were found in organisms from stations inside the lagoon, and 3.6-4.2 in the sea stations. Organotin derivatives were measured in both the visceral coil and the rest of the soft body of the organisms. Total concentrations of butyltin compounds ranged from 102 +/- 17 to 432 +/- 27 ng Sn g(-1) d.w. in the visceral coil, and from 96 +/- 24 to 297 +/- 107 ng Sn g(-1) d.w. in the rest of the soft body. Phenyltins were found at far lower concentrations, ranging from 8 +/- 1 to 41 +/- 3 ng Sn g(-1) d.w. (visceral coil) and from 0.25 to 32 +/- 22 ng Sn g(-1) d.w. (rest of soft body). The degree of imposex in female gastropods, evaluated from VDSI and penis lengths, was related to organotin contents in the soft body. In particular, female penis lengths were significantly correlated (r = 0.917 and r = 0.982, P<0.05) to tributyltin (TBT) contents and the sum of organotin compounds in organisms.