Toxicological assessment and developmental abnormalities induced by butylparaben and ethylparaben exposure in zebrafish early-life stages

https://doi.org/10.1016/j.etap.2020.103504Get rights and content

Highlights

  • Ethylparaben and butylparaben exposure led to acute toxicity in zebrafish embryos.

  • Determination of BM dose values were performed using model averaging approach.

  • Developmental alterations were reported.

  • Zebrafish larvae showed behavioral changes.

Abstract

Toxicological effects of butylparaben (BuP) and ethylparaben (EtP) on zebrafish (Danio rerio) early-life stages are not well established. The present study evaluated, using zebrafish embryos and larvae, the toxicity of BuP and EtP through benchmark dose (BMD) approach. BuP was more toxic than EtP to zebrafish larvae. In fact, Lethal Concentration 50 (LC50) values at 96 h post-fertilization (hpf) for BuP and EtP were 2.34 mg/L and 20.86 mg/L, respectively. Indeed, BMD confidence interval (lower bound (BMDL) - upper bound (BMDU) was 0.91–1.92 mg/L for BuP and 10.8–17.4 mg/L for EtP. Zebrafish embryos exposed to 1 mg/L, 2.5 mg/L of BuP and 5 mg/L, 10 mg/L, 20 mg/L, 30 mg/L of EtP showed several developmental abnormalities and teratological effects compared to negative control. Exposed zebrafish developed reduced heartbeat, reduction in blood circulation, blood stasis, pericardial edema, deformed notochord and misshaped yolk sac. Embryos exposed to the highest concentrations of the chemicals (2.5 mg/L of BuP, 10 mg/L, 20 mg/L and 30 mg/L of EtP) showed the developmental abnormalities at 48 hpf while those treated with 1 mg/L of BuP and 10 mg/L of EtP reported behavioral changes at 72 hpf, including trembling of head, pectoral fins and spinal cord. This research identified the lethal and sublethal effects of BuP and EtP in zebrafish early-life stages and could be helpful to elucidate the developmental pathways of toxicity of parabens.

Introduction

Parabens are a group of synthetic chemicals used in cosmetics, food, and pharmaceuticals due to their broad-spectrum antimicrobial and antifungal properties (Nowak et al., 2018). In terms of chemical structure, they are esters of p-hydroxybenzoic acid with alkyl substituents ranging from methyl to butyl groups (Darbre et al., 2004). With the increase in the length of alkyl chain the value of octanol water-partition coefficient rises, resulting in decrease of their water solubility and in an increase of their lipophilicity (Artacho-Cordón et al., 2018). Antimicrobial activity of parabens is also directly proportional to the chain length of ester group increasing from methyl to n-butyl (Błędzka et al., 2014). In vivo studies showed as parabens are rapidly absorbed from the gastrointestinal tract and from blood, hydrolyzed to p-hydroxybenzoic acid, conjugated and then excreted in the urine. Parabens can also be absorbed quickly through intact skin and hydrolyzed by skin and subcutaneous fatty tissues carboxylesterases (Boberg et al., 2010). They are classified as endocrine disrupting chemicals (EDCs) due to their ability to bind to several nuclear receptors (Nowak et al., 2018). Indeed, their influence on adipose tissue and neurodevelopment is still less clear. The zebrafish is increasingly used as a model in human toxicological research and is highly suitable to assess developmental and teratogenic effects of EDCs (Staal et al., 2018). The early life stages of development are highly conserved between species and the observation of developmental abnormalities in zebrafish model is considered relevant to understand new modes of action and potential target tissues of EDCs in mammals (Zezza et al., 2019). Specific advantages of the zebrafish model are the relatively transparent body during the early life stages and, according to the new EU Directive 2010/63/EU, the fact that the earliest life-stages do not fall into the regulatory frameworks dealing with animal experimentation (Staal et al., 2018). Recently has been demonstrated that the sublethal exposure to propylparaben (PrP) interferes with lipid utilization in zebrafish larvae, in terms of decrease in neutral lipid mobilization from yolk and alteration of phospholipids metabolism. Moreover, PrP and methylparaben (MeP) exposure in zebrafish early life-stages led to acute toxicity and developmental abnormalities due to alterations in gene expression involved in cellular stress response, cell cycle and DNA damage, inflammation and fatty acid metabolism (Dambal et al., 2017; Bereketoglu and Pradhan, 2019). Indeed, available data on the effects of ethylparaben and butylparaben on zebrafish early life stages are still lacking. BuP is recognized to be the most potent paraben in terms of estrogenic activity and was found to be able to interfere with male reproductive functions (Routledge et al., 1998; Oishi, 2001). EtP is admitted as additive in food and it showed genotoxic activity related to telomere shortening in vitro studies (EFSA, 2004; Finot et al., 2017).

Data analysis methods for the assessment of zebrafish developmental toxicity end-points are under active evolution and include EC50/LC50 (half maximal effective concentration/median lethal concentration), LOAEL (lowest-observed-adverse-effect level) and NOAEL (no-observed-adverse-effect level) strategies (Hsieh et al., 2018). However, LOAEL and NOAEL approaches have some shortcomings including lack of consideration of concentration-response trend, limiting LOAE/NOAEL options to tested concentration and inability to provide uncertainty factors for reported potency (Davis et al., 2011). For these reasons EFSA Scientific Committee recommends using the BMD (Benchmark Dose) approach instead of NOAEL strategy, since it makes a more extended use of dose-response data and it allows for a quantification of the uncertainties in the dose-response data (EFSA, 2017). Moreover, the Scientific Committee suggests to always report the BMD confidence interval (lower bound, BMDL-upper bound, BMDU) rather than the value of the BMD (EFSA, 2017). The present study focuses on the evaluation of the acute toxicity and developmental abnormalities of butylparaben and ethylparaben on zebrafish early-life stages using the BMD approach as toxicological risk assessment.

Section snippets

Chemicals

Ethylparaben (CAS number 120−47-8, Pharmaceutical Secondary Standard; Certified Reference Material) and Butylparaben (CAS number 94−26-8, Pharmaceutical Secondary Standard; Certified Reference Material) were purchased from Sigma Aldrich, Milan, Italy. Dimethyl sulfoxide (DMSO) (> 99.9 % purity) and 3,4-dichloroaniline (> 98 % purity) were obtained from Sigma-Aldrich (Co. St. Louis, MO). Dilution water (DW) was prepared according to OECD TG 203, Annex 2 (OECD, 1992).

Zebrafish maintenance and eggs collection

Adults zebrafish (wild type

Fish embryo acute toxicity tests

FET test acceptance criteria were achieved, by following OECD n.236 since the mortality of negative control at 96 hpf was 0 %, and the mortality in the positive control group was 60 %. LC50 values at 96 hpf for BuP and EtP were 2.34 mg/L and 20.86 mg/L, respectively. BMD confidence intervals were 0.91–1.92 mg/L for BuP and 10.8–17.4 mg/L for EtP (Fig. 1). Coagulation of embryos and no heartbeat were the most common lethal endpoints recorded at 96 hpf. Lack of detachment of the tail-bud from the

Discussion

The present study demonstrated that BuP and EtP exposure in zebrafish early-life stages resulted in acute toxicity leading to developmental abnormalities and teratological effects. According to their physical and chemical properties, LC50 of BuP (2.34 mg/L) was lower than LC50 of EtP (20.86 mg/L). These results agree with data obtained from several acute studies on parabens toxicity towards fish as animal models. Medaka (Oryzias latipes) early life stages were used to assess the acute toxicity

Conclusion

The data of the present study showed that BuP and EtP exposure was able to induce acute toxicity in zebrafish early-life stages and that BuP was more toxic than EtP. LC50 values enriched data on toxicological properties of BuP and EtP in aquatic organism as there was deficient information available on acute toxicity of these parabens on zebrafish early-life stages. BMD confidence interval could be used as a starting point for risk assessment. Moreover, BuP and EtP exposure led to developmental

CRediT authorship contribution statement

C. Merola: Methodology, Validation, Investigation. O. Lai: Writing - original draft. A. Conte: Formal analysis, Software. G. Crescenzo: Writing - review & editing. T. Torelli: Methodology, Data curation. M. Alloro: Writing - original draft. M. Perugini: Resources, Supervision, Project administration.

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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