Fish cell lines as screening tools to predict acute toxicity to fish of biocidal active substances and their relevant environmental metabolites
Introduction
Biocides comprise a heterogeneous group of chemical agents used in treatments against a wide range of target organisms, having potential applications in hospital hygiene maintenance, agriculture, disinfection and quality preservation. However, they also suppose a risk for a great variety of non-target organisms, meaning they may have undesirable side effects harming human health and/or the environment. According to the Biocidal Products Regulation (BPR) (Regulation(EU)_528/2012), the assessment of the toxic potential of these compounds to non-target organisms in an aquatic environment is required for the approval of new biocidal active substances. Furthermore, the BPR focuses not just on parent compounds but also introduces the concept of “ecotoxicologically relevant metabolite”, and defines it as “any minor or major metabolite which e.g. poses a comparable or higher hazard than the active substance” (ECHA, 2018).
Traditionally, the environmental hazard on vertebrates in aquatic systems is evaluated by performing acute and chronic toxicity assays in fish. In practical terms, the most widely performed test is the fish acute toxicity test as it is one of the core data set required by BPR to evaluate the toxicity of biocides to aquatic species (Regulation(EU)_528/2012). This test should be done according to the OECD Test Guideline (TG) 203 (OECD, 2019). Because the toxicity of a substance can show important variations amongst different fish species and water conditions (Coppock and Nation, 2018), the TG recommends some fish species representative of a number of environmental scenarios. The species used to perform the experiments should be selected taking into account the foreseen use of the substance.
The BPR, in its Annex IV, refers to the validity of in vitro data for the risk assessment of biocides. Moreover, the need for in vitro test validation is coming from all the different international regulatory agencies, in an attempt to fulfil the 3R's (Refinement, Reduction and Replacement) principle and animal welfare regulation (e.g. Directive 2010/63/EU on the protection of animals used for scientific purposes). Efforts have been made over the past three decades to find out whether in vitro cytotoxicity assays can be used for aquatic hazard assessment as an alternative to in vivo tests (Knauer et al., 2007). In 2007, Knauer et al. (2007) examined the acute toxicity to PLHC-1 and to juvenile rainbow trout of 18 plant protection products. Authors found that the effective concentration causing a 50% of inhibition of the neutral red uptake with respect to the negative controls (EC50) overestimated the corresponding acute toxicity value (LC50, lethal concentration for 50% of the fish) obtained in vivo using OECD TG 203. They reported a weak correlation between both values. Their suggestion was to use a set of in vitro systems not only to address the toxicity of the compound but also to study specific mechanisms of toxic action. A more recent study (Taju et al., 2012) evaluated the potential use of the cell lines IEG (gills), IEE (eye) and IEK (kidney) of green chromide (Etroplus suratensis) for their potential use as screening tools for the ecotoxicological assessment of a tannery effluent. They applied different cytotoxicity assays obtaining highly significant linear correlations between in vitro cytotoxicity and in vivo acute toxicity data. Several authors have compared cytotoxicity data from RTgill-W1 cells and toxicity data from fish exposed to the same chemicals (Tanneberger et al., 2013; Natsch et al., 2018; Fischer et al., al.,2019), and proposed these cells as a true alternative to an animal in vivo test. Indeed, the OECD TG 249 has been recently published (OECD, 2021), detailing the usefulness of the RTgill-W1 to predict fish acute toxicity in product testing or perform a pre-screening before conducting a full fish acute or other fish-based toxicity test. However, it recognizes some exceptions depending on the compound assayed. For instance, for specific compounds such as neurotoxic chemicals or compounds that need to be transformed to more toxic metabolites, the RTgill-W1 is less sensitive than fish. This fact should be taken into account when selecting the cell lines or interpreting results.
The present study was performed in the framework of the EU funded LIFE-COMBASE Project (LIFE-COMBASE, 2019) aiming at the development of alternative methodologies for the aquatic hazard assessment of biocides and their environmentally relevant metabolites. The project focussed on a battery of organisms representative of the freshwater environment (algae, invertebrates, vertebrates) and sewage-treatment plants (STP) (microorganisms). Different in silico approaches were developed to predict the acute toxicity of these compounds to the four aquatic taxonomic groups (Blázquez et al., 2021). In addition, we collected information of acute toxicity to microorganisms, algae, invertebrates and fish for 196 biocidal substances and 206 environmental metabolites (Hernández-Moreno et al., 2019). More than 80% of the biocides were toxic to fish with ∼50% presenting a high toxicity (LC50 < 1 mg/L). Metabolites were mainly less toxic than the parent compounds, although 34% presented the same toxicity and 4% were more toxic. The high percentage of toxic metabolites and the absence of data for 46% of the selected 206 metabolites indicated the need to further study their impact in the aquatic compartment.
The objective of the present work was to generate new data on the acute toxicity of biocidal active substances and of environmentally relevant metabolites to fish and to establish a correlation between in vitro cytotoxicity and in vivo fish acute toxicity data, with the overarching target of assessing in vitro methods as potential screening tools in acute aquatic toxicity assessment. To reach this aim, we performed acute toxicity studies with 2 biocides and 5 metabolites in rainbow trout (Oncorhynchus mykiss) and in vitro toxicity tests in four different fish cell lines (PLHC-1 and RTH-149, RTG-2 and RTgill-W1) for 8 biocides and 8 metabolites. The number of the in vivo experiments could be reduced because LC50s were reported for some of the 16 substances selected. Complementarily, we increased the number of biocides and metabolites for the in vitro-in vivo correlations study with information from previously developed databases (LIFE-COMBASE, 2019; Hernández-Moreno et al., 2019). To the best of our knowledge, no previous studies describing the correlation between in vitro cytotoxicity and in vivo fish acute toxicity data for biocidal active substances and their environmentally relevant metabolites are currently available.
Section snippets
Selection of substances for the experimental assays
The biocidal substances and metabolites used in the in vitro and in vivo studies were selected from the LIFE-COMBASE database. The criteria followed for the selection were: 1) the molecule should be likely to appear in the aquatic compartment; 2) there should be an important scarcity or even absence of toxicity data for fish (specially for O. mykiss) and fish cell lines; 3) the parent compound should not be readily degradable; 4) the biocides and metabolites should be representative of
In vivo toxicity assays
Table 3 shows the acute toxicity in O. mykiss for the 7 compounds assayed (2 biocides and 5 metabolites). The metabolite 3-phenoxybenzyl alcohol showed medium toxicity whereas the other substances were non-toxic or exerted a low toxicity.
In vitro toxicity assays
Three cytotoxicity assays were performed with 16 compounds in hepatoma fish cell lines representative of cold and tropical water fish (RTH-149 and PLHC-1, respectively). In addition, the seven compounds assayed in fish were also tested in two additional rainbow
Discussion
During the last decades, the amount of chemicals on the market has increased considerably and, therefore, their possibility to reach the environment. It is becoming necessary to check the potential toxicity of these chemicals for humans and for the environment. The consequence will be an increase in the number of animals that should be used to conduct the different experiments. However, several European Agencies (e.g. EFSA, ECHA) have reported the need to fulfil the requirement of the Directive
Conclusions
We have generated a battery of results for fish acute toxicity corresponding to biocidal active substances and their environmentally relevant metabolites, including newly generated and previously existing in vivo and in vitro data. Based on the findings of the present study, the proposed categorization of hazard data into four different toxic-effect groups may be a valid approach to allow the use of in vitro data as a predictive tool for in vivo effects. There is not a clear pattern of
Funding
This work was supported by LIFE-COMBASE project (LIFE15 ENV/ES/000416).
CRediT authorship contribution statement
D Hernández-Moreno: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. M Blázquez: Funding acquisition, Project administration, Conceptualization, Formal analysis, Methodology, Writing – original draft, Writing – review & editing. JM Navas: Writing – review & editing. ML Fernández-Cruz: Funding acquisition, Project administration, Conceptualization, Formal analysis, Methodology, Writing – original draft, Writing – review &
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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