Carbendazim exposure induces developmental, biochemical and behavioural disturbance in zebrafish embryos
Introduction
Carbendazim (methyl-1-H-benzimidazol-2-yl-carbamate) is one of the most widely used benzimidazole fungicides. It is highly toxic to target organisms, inhibiting the development of a wide variety of fungi even at low doses. It is used in agriculture, horticulture, forest and home gardening and as a preservative in paint, papermaking, textile, leather industry and fruits (Selmanoğlu et al., 2001). Carbendazim is a metabolite of benomyl and it is known to target the tubulin in cells, causing disruption of microtubule assembly and cell division (Davidse, 1986).
Low concentrations of carbendazim ranging from 0.2 to 200 μg/L have already been detected in surface waters near agriculture and forestry areas (Palma et al., 2004, Readman et al., 1997). Moreover, carbendazim has shown to be very persistent in the water with a half-life of 6–25 weeks (Cuppen et al., 2000a). Many studies have reported the adverse effects of carbendazim on mammals, mainly on reproductive organs (Farag et al., 2011, Ireland et al., 1979, Lim and Miller, 1997, Nakai et al., 2002, Urani et al., 1995), but unlike mammals, effects on aquatic organisms are poorly studied. The majority of studies available focus on zooplankton and macroinvertebrate communities in which chronic exposures to carbendazim decreased survival, reproduction and feeding rates (Cuppen et al., 2000b, Daam et al., 2010, Ferreira et al., 2008, Ribeiro et al., 2011, Van den Brink et al., 2000). To our knowledge, only two studies report carbendazim effects on fish early life stages. Ludwikowska et al. (2013) showed that carbendazim affects the survival and hatching success of Prussian carp embryos at concentrations above 0.036 mg/L and Jiang et al. (2014) demonstrated that embryonic exposure to carbendazim led to significant changes in the expression of genes related to apoptosis, immunotoxicity and endocrine disruption in zebrafish (Danio rerio). In this later study concentrations between 4 and 500 μg/L of carbendazim were tested.
Risk characterization is better achieved by studying chemical effects at several levels of biological organization. Recently, behavioural parameters such as locomotion (whose evaluation have been considered time consuming and lacking objectivity) have been increasingly used due to the development of technology for automated analysis. In the case of zebrafish, locomotion has been used as an endpoint to assess the neurotoxic effects of chemicals in early life stages (Irons et al., 2010, Padilla et al., 2011, Selderslaghs et al., 2010) and the sublethal toxicity of pollutants (Ulhaq et al., 2013). In fact, many contaminants disrupt fish behaviour at concentrations much lower than those causing mortality, e.g.: Klüver et al. (2015) recorded alteration of behaviour of fish embryos at concentrations 375-fold lower than the LC10. Thus, behaviour has proven to provide very sensitive measures of stress exposure; furthermore it has high ecological relevance as effects can be translated long term health and survival of populations (Scott and Sloman, 2004, Tierney, 2011).
Recently, the approval of the OECD test guideline 236 (fish embryo toxicity test) has consolidated the zebrafish embryo test as a true alternative for the acute fish toxicity test with adults (Braunbeck et al., 2014) in the European Union. This test has been increasingly used to assess the toxicity of chemicals and waste waters as reviewed by Scholz et al., 2008, Scholz et al., 2013. The low volume of test solutions needed (tests are deployed in 24 or 96-wells microplates) and the rapid development and transparency of embryos that allow the monitoring of the entire organogenesis are among the advantages of this test. Zebrafish embryos also comprise an excellent model for determining the effects of chemicals at biochemical level (Oliveira et al., 2009).
Thus, based on the hypothesis that carbendazim could have serious adverse effects in fish early life stages, the aim of this work was to assess the toxic effects of carbendazim in zebrafish embryos at several organizational levels, namely:
- i)
lethality,
- ii)
developmental (including embryo development delays and malformations),
- iii)
biochemical (including the measurement of the enzymes ChE- cholinesterase, GST-glutathione S-transferase, LDH-lactate dehydrogenase and CAT- catalase) and
- iv)
behavioural (by measuring locomotion of zebrafish eleutheroembryos expressed by distance moved and time spent moving)
The parameters selected to be evaluated at biochemical levels include not only parameters directly related to neurobehavioral action as it is the case of ChE but also enzymes representative of different metabolic pathways such as GST (involved in the phase II of the detoxification process), LDH (involved in the anaerobic way of energy production) and CAT (involved in the antioxidant defence).
Is this way an integrated analysis of carbendazim can be done contributing to understand the mechanisms of toxicity of this compound.
Section snippets
Zebrafish maintenance and embryo collection
All the embryos used in the present study were provided by the zebrafish facility established at the Department of Biology, University of Aveiro (Portugal). Adults were maintained in carbon-filtered water, complemented with 0.34 mg/L salt (“Instant Ocean Synthetic Sea Salt”, Spectrum Brands, USA) and automatically adjusted for pH and conductivity. Water temperature was kept at 26.0 ± 1 °C, conductivity at 750 ± 50 μS, pH at 7.5 ± 0.5 and dissolved oxygen equal or above 95% saturation. A 16:8 h
Stability of carbendazim in the exposure medium
Chemical analysis of the exposure media showed stable exposure concentrations and within 80–120% of the nominal concentrations in what refers the FET test (1.1–1.8 mg/L). However, analysis of the exposure media of the sublethal range of concentrations (0.16–500 μg/L) revealed some inconsistencies (Table S2, Supplementary data) probably because the tested concentrations were very close to the limit of quantification.
Effects on embryos development
The calculated LC50 and EC50 values for zebrafish embryos exposed to carbendazim
Discussion
Carbendazim acts by inhibiting the assembly of tubulin and the formation of microtubules in fungi, and also in mammals (Davidse, 1986, Ireland et al., 1979, Lim and Miller, 1997). Thus, carbendazim effects are prone to be more relevant in very early embryonic life stages where active cell division is ongoing. This may explain why in this work the lethal toxicity was not time dependent, being fully established at 48 h of exposure. Accordingly, in the clawed frog (Xenopus laevis) the lethal
Conclusion
By acting on tubulin assembly which directly affects many cellular processes including mitosis, carbendazim is particularly pernicious to early life stages of development where active cell division is ongoing. This mechanism of action is reflected in several developmental anomalies here recorded in the range of 1–2 mg/L of carbendazim, such as pericardial edemas, body and tail deformities, decreased heart beat rate and body length. At the biochemical level, the increase in the ChE activity (in
Acknowledgements
This study was supported by a PhD grant (SFRH/BD/74501/2010) attributed to Thayres Andrade and by the Post-Doc grant (SFRH/BPD/90521/2012) attributed to Inês Domingues by the Portuguese Science and Technology Foundation (FCT), funding by FEDER through COMPETE and Programa Operacional Factores de Competitividade and by National funding through FCT, within the research project Climatox—Impact of climatic changes on toxicity of pollutants (Ref. FCT PTDC/AAG-GLO/4059/2012).
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