Effects of UV filter 4-methylbenzylidene camphor during early development of Solea senegalensis Kaup, 1858

https://doi.org/10.1016/j.scitotenv.2018.02.112Get rights and content

Highlights

  • 4MBC induced mortality, malformations and reduced growth in S. senegalensis larvae.

  • Swimming behaviour was affected by 4MBC at low concentrations.

  • 4MBC caused alterations in AChE and LDH biochemical markers.

  • Assessment of behaviour is recommended in ecotoxicological studies with sole larvae.

Abstract

The inclusion of organic UV filters in personal care products (PCPs) has increased in recent years. 4-Methylbenzylidene camphor (4MBC) is one of the most used UV filters, and thus it is commonly found in aquatic ecosystems, with proved negative effects on aquatic organisms. Effects on early life stages of marine vertebrates are largely unknown. Therefore, the main goal of this work was to evaluate 4MBC effects on Senegalese sole (Solea Senegalensis Kaup, 1858) larvae at different levels of biological organization. S. senegalensis were exposed to increasing concentrations of 4MBC from egg stage until 96 h. Mortality, growth, malformations, behaviour and biochemical responses, including enzymatic biomarkers were studied. The exposure to 4MBC until 96 h post-fertilization (hpf) induced mortality and malformations in a dose-response manner. Besides, reduced growth with increasing concentrations was observed. The exposure to 4MBC also caused alterations on behaviour, including overall lower swimming time during light and dark periods. Biomarker alterations caused by 4MBC included imbalance of neurotransmission related endpoints (increased acetylcholinesterase activity) and decreased activity of enzymes related to anaerobic metabolism (lower cellular lactate dehydrogenase activity) at the lower concentrations tested. Furthermore, our results suggest that 4MBC do not induce oxidative stress in S. senegalensis larvae, since catalase and lipid peroxidation levels were not significantly altered by 4MBC.

S. senegalensis revealed to be a good model species for vertebrate animal testing in the marine environment. Sub-lethal concentrations of 4MBC induced toxic effects at all organizational levels. Swimming behaviour was a sensitive endpoint and showed that exposure to 4MBC causes impairment on response to light stimulus which is possibly linked with the observed imbalances on cholinesterase activity in larvae. Conservation concerns along distribution range of S. senegalensis should consider that increasing levels of UV filters in marine environment might have impact on the ecology of the species.

Introduction

In last decades, the knowledge on harmful effects of ultraviolet (UV) radiation led to the increasing use of cosmetic products containing UV filters (Chisvert and Salvador, 2018). These personal care products (PCPs) include such compounds for dermal protection against deleterious UV radiation, but UV filters are also used as additives in plastics and household products for increasing shelf-life. However, UV filters exhibit some characteristics typical of organic priority pollutants, such as high stability and lipophilicity and low biotic degradation (USEPA, 2012). Besides, they tend to bioaccumulate and are reported to act as neurotoxicants and endocrine disruptors (Díaz-Cruz and Barceló, 2009; Gago-Ferrero et al., 2012; Krause et al., 2012; Ruszkiewicz et al., 2017).

Usually, more than one UV filter is combined on sunscreen formulations. 4-Methylbenzylidene camphor (4MBC) is one of the most common UV filters in Europe and Australia, with a maximum allowed concentration of 4% (Chisvert and Salvador, 2018; Krause et al., 2012). Bathing and swimming activities are one of the most common direct sources of this compound in aquatic ecosystems, particularly during warmer seasons when levels in water are prone to be high. Maximum reported concentrations are found at recreational coastal areas with concentrations up to 1.043 μg l−1 (Atlantic Gran Canaria) with peaks during daylight and warmer months (Balmer et al., 2005; Langford and Thomas, 2008; Sánchez-Rodríguez et al., 2015; Tovar-Sánchez et al., 2013). In addition, aquatic ecosystems can also receive 4MBC from wastewater treatment plants (WWTP), since this compound is not totally eliminated by typical treatment methods, reaching concentrations up to 2.7 μg l−1 (Badia-Fabregat et al., 2012; Balmer et al., 2005; Brausch and Rand, 2011; Fent et al., 2010b; Li et al., 2007; Liu et al., 2012). Besides, 4MBC has also been found in tap water (maximum concentration of 35 ng l−1) (Díaz-Cruz et al., 2012).

The ubiquity of 4MBC on coastal environments (Chisvert and Salvador, 2015) and high toxicity to marine organisms raises environmental concerns. Toxicity has already been described at concentrations below 10 ng l−1 to small organisms, such as protozoa (Gao et al., 2013), microalgae (Isochrysis galbana) or small crustaceans (Siriella armata) (Paredes et al., 2014). Besides, toxic effects have been detected in freshwater aquatic vertebrates, affecting development and reproduction (Inui et al., 2003; Kunz and Fent, 2006, Kunz and Fent, 2009; Martins et al., 2017; Schmitt et al., 2008; Schlumpf et al., 2004, Schlumpf et al., 2008). UV filters accumulate in fish tissues, and 4MBC have been detected in concentrations as high as 1.8 μg g−1 lipid in muscle tissue of Salmo trutta fario, brown trout (Balmer et al., 2005; Buser et al., 2006; Fent et al., 2010b; Gago-Ferrero et al., 2015; Zenker et al., 2008). However, effects of 4MBC at larval stages of marine fish are still very scarce and should be further assessed.

New alternative testing methods to animal experimentation are necessary for testing chemicals with toxicological risk (e.g. REACH regulations). Early life stages of aquatic vertebrates have been used as models for evaluating the effects of contaminants (Lammer et al., 2009; Scholz et al., 2008, Scholz et al., 2013). While several vertebrate freshwater species have been widely used and accepted by scientific community (e.g. Danio rerio, zebrafish; Pimephales promelas, fathead minnow; Xenopus laevis, African clawed frog; among others), the use of marine vertebrates as testing model species are still very scarce. Eggs of flatfish marine species from aquaculture facilities are becoming more frequently used for ecotoxicology studies (e.g. Foekema et al., 2008; Pavlaki et al., 2016).

Senegalese sole (Solea senegalensis Kaup, 1858) is a coastal flatfish species native to the Eastern Atlantic waters. This high economic important species is commercially exploited in Southwestern European aquaculture and its increasing scientific knowledge in different fields of biology has gained momentum in recent years (Imsland et al., 2003; Morais et al., 2014). Early development, metamorphosis and physiology of this species are well understood (Cañavate and Fernández-Diaz, 1999; Cañavate et al., 2006; Fernández-Díaz et al., 2001). Eggs can be obtained from several companies and national agencies throughout southern Europe. Besides, eggs and larvae are transparent and malformation screening is easily performed and endogenous energetic reserves last until between 80 and 100 h after fertilization, time when external feeding is in fact needed (Cañavate et al., 2006; Yúfera et al., 1999). Therefore, larval stages of this species arise as alternative animal models for toxicological evaluation of marine environmental contaminants, such as 4MBC.

Biochemical markers are sensitive and useful tools on the study of effects of chemical exposure at organisms' subcellular level. Specific biochemical responses can be associated with particular modes of action of chemicals. Neurotoxicity can be assessed through estimation of cholinergic activity (e.g. acetylcholinesterase, AChE). Exposure to specific chemicals can induce production of reactive oxygen species (ROS) exceeding basal cell levels. In such conditions, defence capacity of organism is surpassed and damage on DNA, peroxidation of membrane lipids and proteins, and cellular degenerative processes can occur (Oost et al., 2003; Storey, 1996). Therefore, oxidative stress can be detected through increased antioxidant enzymes activity (such as catalase, CAT) or measured through lipid peroxidation (LPO) levels (Antunes et al., 2010; Quintaneiro et al., 2008). Another relevant biomarker is lactate dehydrogenase (LDH), which catalyses the reversible reduction of pyruvate to lactate and may be related with low availability of oxygen and exposure to xenobiotics (Cohen et al., 2001; Guilhermino et al., 1994; Quintaneiro et al., 2006).

Non-invasive and non-lethal tools, are also becoming increasingly used as indicators of toxic effects in the assessment of chemicals. Behaviour is a sensitive and non-invasive tool for the evaluation of contaminants and the recent development of standardized methods and computer and video automation has been improving the analysis of behaviour in terms of reproducibility, reliability and turning it less time-consuming (Kane et al., 2005; Little et al., 1993; Melvin and Wilson, 2013; Scott and Sloman, 2004).

In this context, the main objective of this study was to understand the toxic effects of 4MBC to larval stages of S. senegalensis at different levels of biological organization using apical, biochemical and behavioural endpoints.

Section snippets

Chemicals

The 3-(4-methylbenzylidene) camphor (4MBC) (CAS Number 36861-47-9) used in bioassays was purchased from Sigma-Aldrich Co. LLC (St Louis, USA) and the ethanol used to prepare 4MBC stock solution and solvent control was supplied by Merck. All other chemicals used in biochemical marker analysis were of analytical grade quality (Sigma-Aldrich Co.).

In the chemical analysis, 4MBC and deuterated benzophenone (benzophenone-d10, Sigma–Aldrich) were used as analytical standard and surrogate,

Mortality, hatching and malformations

In the first experiment, there were no significant effects of 4MBC on hatching of S. senegalensis (p > 0.05). However, relatively lower hatching rates were observed at 24 hpf in sole embryos exposed to 0.447 and 0.599 mg l−1 4MBC (60%) when compared to hatching rates observed in solvent control and 0.935 mg l−1 4MBC treatment (100%). At 48 hpf all the fish in the different treatments have hatched.

There was no mortality of hatched larvae nor egg abortion in the treatments exposed to 4MBC at

Discussion and conclusions

In this study, toxicological effects of the organic UV-filter 4MBC on S. senegalensis larvae at different levels of biological organization were evaluated using apical behavioural and biochemical endpoints.

Despite the relatively low solubility of 4MBC, this compound can be frequently found in coastal ecosystems (Sánchez-Rodríguez et al., 2015; Tovar-Sánchez et al., 2013) and has high potential for adsorption to bed sediments and aquatic organism tissues, including fish (Balmer et al., 2005;

Acknowledgments

This work was supported by the Portuguese Foundation for Science and Technology (FCT) through CESAM (UID/AMB/50017/2013) and through the scholarships of MJA (SFRH/BD/52572/2014), RJMR (SFRH/BPD/99819/2014) and MSM (SFRH/BPD/100448/2014). The authors also acknowledge Sea8 for providing S. senegalensis eggs, MSc. Abel Ferreira and Dr. Ricardo Calado for all the technical support during this study.

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