Elsevier

Aquatic Toxicology

Volume 196, March 2018, Pages 132-145
Aquatic Toxicology

Toxicity assessment of pyriproxyfen in vertebrate model zebrafish embryos (Danio rerio): A multi biomarker study

https://doi.org/10.1016/j.aquatox.2018.01.010Get rights and content

Highlights

  • PPF toxicity in zebrafish embryos was studied using multiple biomarker endpoints.

  • PPF causes severe developmental abnormalities in 1.66 μg/mL concentration.

  • PPF alters biochemical and antioxidant responses and inhibits AChE.

  • PPF causes DNA damage and alters histology at 1.66 μg/mL concentration.

Abstract

Pyriproxyfen (2-[1-methyl-2-(4-phenoxyphenoxy) ethoxy] pyridine) (PPF), a pyridine-based pesticide widely used to control agricultural insect pests and mosquitoes in drinking water sources. However, its ecotoxicological data is limited in aquatic vertebrates particularly in fish. Hence, the present study aimed to evaluate the adverse effect of PPF in zebrafish embryo development (Danio rerio). In order to investigate the impact of PPF, embryos were exposed to 0.16, 0.33 and 1.66 μg/mL (0.52, 1.04 and 5.2 μM, respectively) for 96 hpf and various biomarker indices such as developmental toxicity (edema formation, hyperemia, heart size and scoliosis), oxidative stress (reactive oxygen species (ROS), lipid peroxidation (LPO) and nitric oxide (NO)), antioxidant responses (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx) and reduced glutathione (GSH)), biochemical (lactate dehydrogenase (LDH) and acid phosphatase (AP)), neurotoxicity (acetylcholinesterase (AChE)), genotoxicity (apoptosis and DNA damage) and histopathological changes were determined. The results showed that severe developmental deformities and changes in heart rate were observed in embryos treated with highest (1.66 μg/mL) concentration than the control (P < 0.05). Heart size measurement showed that, significant change in heart size (P < 0.01) was observed in embryos of 96 hpf only at 1.66 μg/mL PPF exposure. The oxidative stress was apparent at highest test concentration (1.66 μg/mL) as reflected by the elevated ROS, LPO and NO and changes in antioxidant enzyme activities including SOD, CAT, GST and GPx (P < 0.05). Besides, GSH level and AChE activity were significantly lowered in 1.66 μg/mL PPF exposed group than the control. After 96 hpf of PPF exposure, no significant changes were found in AP activity whereas, a biphasic response was observed in the LDH activity. There was no genotoxic effect in embryos exposed to PPF at 0.16 and 0.33 μg/mL, while significant (P < 0.05) DNA damage and apoptosis were found in 1.66 μg/mL treated group. Histopathological analysis revealed that exposure to PPF at 1.66 μg/mL resulted in thinning of heart muscles, pericardial edema and hyperemia while there was no obvious changes were observed in other treatment groups. Hence, the results of the present study demonstrate that PPF could cause adverse effect on early developmental stages of zebrafish at higher concentration.

Introduction

Recently, Brazil and other parts of America facing a significant outbreak of the Zika virus which is transmitted by the Aedes mosquitoes and an associated increase in the microcephalic condition in the new born babies (Truong et al., 2016). Microcephaly, the neurological condition in which the head size of new born is significantly reduced due to developmental defects (Dzieciolowska et al., 2017). Since there was no proven evidence for the cause of microcephaly at the time of Zika outbreak, the anonymous concept has been raised that the pesticide pyriproxyfen used in the drinking water sources to control the mosquito population by the Brazilian government might be the reason (Truong et al., 2016). CDC (2016) reported that Zika virus is the real cause of microcephaly and there was no correlation between pyriproxyfen and microcephaly (Albuquerque et al., 2016). However, neurotoxic effect was reported at 120 hpf of zebrafish embryos exposed to PPF from low to mid micromolar concentration (Truong et al., 2016). Moreover, Dzieciolowska et al. (2017) concluded that PPF alone was unlikely to cause neurodevelopmental effects and microcephaly.

Pyriproxyfen (2-[1-methyl-2-(4-phenoxyphenoxy) ethoxy] pyridine) (PPF), the pyridine based insecticide used in household, agricultural and horticultural applications to control many insect species (Ginjupalli and Baldwin, 2013). PPF mimics the juvenile hormone, suppresses the embryogenesis process and adult formation of the whiteflies (Ishaaya and Horowitz, 1995). It is a registered pesticide for the agricultural use against whitefly, bollworm, jassids, aphids, and cutworms, and also widely used on citrus fruit in Israel, South Africa, Spain and Italy (WHO, 2008). In India, PPF have been used as vector control measures, against Culex quinquefasciatus and Anopheles stephensi (Jambulingam et al., 2008). Also, they reported that PPF (0.5% GR) has relatively longer residual effect at low dosage level. PPF is relatively stable aromatic compound with the half-life range between 16 and 21 days under aerobic lake water sediment system (WHO, 2008). The guideline value for acceptable daily intake of PPF is 100 mg per kg of body-weight per day for a lifetime and the recommended use of PPF in the drinking water sources at maximum final concentration of 0.01 mg/L (WHO, 2007; Truong et al., 2016; Dzieciolowska et al., 2017). However, PPF is not recommended to be generally applied to surface waters (Sullivan and Goh, 2008). Report shows that PPF can cause mortality in some non-target aquatic invertebrates and small fish at the levels used in mosquito control (Meir and Dhadialla, 2012; Caixeta et al., 2016; Lawler, 2017). Moreover, Belenguer et al. (2014) found PPF in the river water samples with the mean value of 89.66 ng/L (banned by EU since 22/9/2010) and also detected traceable amount in fish at one of the study site of Jucar River, Spain.

The median lethal concentration (LC 50) of PPF has been reported in various fish species, such as Pacific Blue Eye (Pseudomugil signifer) (Brown and Thomas, 1998), Rainbow trout (Oncorhynchus mykiss) and Bluegill sunfish (Lepomis macrochirus) (WHO, 2006). The lethal dose (LD 50) values were also evaluated in bird species like, Bobwhite quail and Mallard duck (WHO, 2006 and references therein). The toxic potency of PPF was determined as half maximal activity concentration (AC 50) of 26.13 μM in zebrafish embryos (Padilla et al., 2012). The report published recently determined the effective concentration (EC 50) of PPF in the zebrafish embryos was 5.2 μM (Truong et al., 2016). Further, Truong et al. (2016) reported that PPF exposure at 6.4 μM and 64 μM to zebrafish embryos causes mortality and sublethal effects.

Zebrafish (Danio rerio) is a well-established vertebrate model for assessing developmental toxicity of exposure to toxicant during early development in the aquatic environment (Deng et al., 2009). Short life cycle, rapid development (within 72 h), cost effective maintenance, high fecundity, transparency of embryos and sensitive to toxicants are the advantages of using zebrafish embryos as model system in pharmacology and toxicology (Schmidt et al., 2015). Further, its genetic and physiological similarity (70% gene homology) with human and the presence of approximately 84% of gene associated with human diseases (Howe et al., 2013) are the added advantage to extrapolate the data with mammalian system (Truong et al., 2014).

It is well known that the embryonic stages of development are the most susceptible to environmental toxins (Yang et al., 2009). Usually, the pesticides used in agriculture often affect the early life stages of aquatic organisms (Pašková et al., 2011). To investigate the toxicity of chemicals, early developmental stage assays have been increasingly used because of its great potential to study the wide range of endpoints (Scholz et al., 2008) including developmental and biochemical parameters (Oliveira et al., 2009). Generally, there is a balance between ROS production and the antioxidant enzyme activity under normal physiological condition (Hilscherova et al., 2003). The exposure to contaminants results in the increased production of reactive oxygen species (ROS) in the organisms (Livingstone, 2001; Lushchak, 2011) and leads to oxidative stress (Adeyemi et al., 2015). It results in damage to various cellular organelles by the oxidation of biomolecules like DNA, protein and lipids (Sabatini et al., 2009). Lipid peroxidation (LPO), has been used as a biomarker of oxidative stress by measuring malondialdehyde (MDA) content in the organism exposed to various contaminants (Valavanidis et al., 2006; Adeyemi et al., 2015). To counteract the damage caused by oxidative stress, the enzymatic antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferse (GST) and glutathione peroxidase (GPx) (Zhao et al., 2013) and the non-enzymatic antioxidants like reduced glutathione (GSH) is produced which are the potential biomarkers to assess the different environmental stress impact (Tabrez and Ahmad, 2009; Wu et al., 2011). Lactate dehydrogenase (LDH), a metabolic enzyme involved in the anaerobic pathway of carbohydrate metabolism has been used as biomarker of stress in fish (Diamantino et al., 2001; Vieira et al., 2008). ROS can also affect the phosphatases, which involved in various transphosphorylation reactions and have been measured by the enzyme, acid phosphatase (AP) (Aoyama et al., 2003; Clemente et al., 2014). Nitric Oxide (NO) plays a vital role in the physiology and pathology of diverse effects including cell apoptosis and used as biomarker. NO an intercellular messenger compete with superoxide dismutase for superoxide radical and forms highly reactive species, peroxynitrite (Beckmann and Koppenol, 1996) which supports oxidative damage in cells.

Acetylcholinesterase (AChE) is an important biomarker for neurotoxicity study in several environmental contaminants and it has been determined in zebrafish (Rico et al., 2006; Richetti et al., 2011). AChE, a neurotransmitter enzyme which hydrolyzes the acetylcholine in the synaptic cleft has been involved in normal functioning of nervous system. AChE is very essential for neuronal and muscular development (Behra et al., 2002) and its inhibition lead to accumulation of neurotransmitter in the synapse, hence hyperstimulation of receptors occurs. As a result, it affects various functions like respiration, feeding, behavior (Cunha et al., 2007), blood pressure and heart rate (Lazartigues et al., 1998; Lin et al., 2007). It has been measured both in situ and spectrophotometrically to assess the toxicity in the zebrafish embryos (Jacobson et al., 2010). Recently, the neurotoxic potential of PPF was studied by neurobehavioral assays suggested that at low to mid micromolar concentration, PPF was developmentally neurotoxic to zebrafish (Truong et al., 2016). In contrast, another study by Dzieciolowska et al. (2017) concluded that PPF does not affect central nervous system development in zebrafish. However, the effect of PPF on neurotransmitters is limited.

A strong relation have been exists between oxidative stress and genotoxicity in the vertebrates (Adeyemi et al., 2015). Comet Assay is widely used to assess the genotoxic potential of pollutants in zebrafish embryos (Kosmehl et al., 2006; Osterauer et al., 2011). JMPR concluded that PPF was not carcinogenic or genotoxic (WHO, 2008). However, the genotoxic potential of PPF was rarely reported in aquatic organisms. Apoptosis is an important toxicological event, occurs due to oxidative damage and mitochondrial injury (Simmons et al., 2009; Yu et al., 2011). The production of ROS in fish in response to contaminant exposure is closely associated with apoptotic cell death (Deng et al., 2009).

In summary, the toxicity of PPF has been extensively studied in invertebrate species (Ishaaya and Horowitz, 1995; WHO, 2006; Jambulingam et al., 2008; Ginjupalli and Baldwin, 2013; Caixeta et al., 2016; Jordão et al., 2016; Kakaley et al., 2017) whereas, in vertebrate model it is limited to few studies (WHO, 2006; Padilla et al., 2012; Truong et al., 2016; Dzieciolowska et al., 2017). There is no report of PPF toxicity on the physiological, biochemical and genotoxic aspect, particularly in aquatic vertebrates. Hence, in this study we aimed to assess the adverse effect of PPF on aquatic vertebrate zebrafish embryos. Multiple end points like, developmental toxicity, heart rate, and heart size, biochemical and antioxidant responses, neurotoxic, genotoxic and histopathological changes were investigated to reveal new insights into the molecular mechanisms underlying the PPF toxicity.

Section snippets

Test compound

Pyriproxyfen with IUPAC name of 4-phenoxyphenyl (RS)-2-(2-pyridyloxy) propyl ether was purchased from Sigma Aldrich (≥98.0%) purity (CAS No.95737-68-1; Cat No. 34174). The compound was dissolved in 100% DMSO at the concentration of 10 mg/mL as stock.

Zebrafish housing and embryo collection

Wild type zebrafish (AB strain) were procured from Zaman aquarium, Chennai, Tamil Nadu and housed in clean well aerated glass tanks. Fishes were fed with commercial feed (Taiyo max) twice daily. Healthy adult male and female fishes (2:1 ratio,

Quantification of PPF in exposure medium

The nominal exposure concentrations of PPF (0.16, 0.33 and 1.66 μg/mL) were measured to ensure the concentrations at during exposure time (T0) and before renewal period (T24). The result shows that at T0, the measured concentration of PPF in the exposure medium was slightly lower than the nominal concentrations (Table 1). The measured values at before renewal (T24) period were decreased than T0 with the deviation of 25.78, 36.90 and 35.03%, respectively.

Effect of PPF on development, heart rate and heart size of zebrafish embryo

In this study, different concentrations

Discussion

The present study was aimed to explore the adverse effect of PPF in vertebrate model zebrafish embryo using multiple biomarker end points. The results obtained in this study shows that at higher concentration of PPF could cause developmental deformities, oxidative stress, alterations in antioxidant response, cause genotoxic effect and histological changes in the embryonic stages of zebrafish.

The nominal exposure concentrations of PPF in the exposure medium of all the treatment group were

Conclusion

In summary, the ecotoxicological information of PPF was lacking in the aquatic vertebrates. Therefore, we aimed to study the effects of PPF in embryonic zebrafish by using various biomarkers like developmental toxicity, heart size, biochemical and antioxidant responses, neurotoxicity, genotoxicity and histopathological studies. We found that PPF does not cause major effects in zebrafish embryos in lowest tested concentration, whereas at highest concentration shown adverse effect in all the end

Conflict of interest statement

The authors declare no conflicts of interest.

Acknowledgement

Authors thank to Defense Research and Development Organization (DRDO), Government of India for the financial support through Phase II project in DRDO-BU CLS, Coimbatore, India, Dr. Babu Rajendran, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, India for the timely help by providing GC–MS facility and Dr. Ram Pratap Singh and Mr. K. Nambirajan for their valuable suggestions.

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