The Effects of Carbendazim on Acute Toxicity, Development, and Reproduction in Caenorhabditis elegans

Carbendazim, as a fungicide, was commonly used to control fungal diseases in agriculture, forestry, and veterinary medicines. In this study, the acute and reproductive toxicity of carbendazim was assessed using Caenorhabditis elegans (C. elegans) as a model in order to preliminarily evaluate the potential risks of this fungicide in agricultural production and application. *e results showed that the growth of C. elegans was inhibited by 0.01 μg/L carbendazim. *e treatment of 0.1 μg/L carbendazim caused a significant decrease in locomotion behavior and significant damage to the reproductive and antioxidant system, causing the lifespan of nematodes to be drastically shortened.*ese results provide a better understanding of the environmental risk of carbendazim and raise new concerns about safety.


Introduction
Pesticides, a kind of chemical or biological reagents, are widely used in agriculture to regulate plant growth and control diseases and insect pests, which can promote crop growth and improve crop yield [1]. However, the widespread use of pesticides will lead to different degrees of residues in crops or food and thus affect human health [2]. e problems of pesticide residues have not only attracted great attention of consumers but also become one of the key factors affecting food safety [3].
Carbendazim, as a broad-spectrum fungicide, has been used to control fungal diseases in agriculture, forestry, and veterinary medicines [4]. However, carbendazim is categorized in the hazardous category of chemicals by the World Health Organization [5] and has been classified in the priority list of endocrine-disrupting chemicals by the European Commission [6]. In recent years, it is obvious that the widespread use of carbendazim with over-range and overdose and the fact that carbendazim is hard to be degraded both lead to the problem of carbendazim residues in agriculture [7]. Although the toxic effect of carbendazim has been reported since the 1980s, the toxicity of carbendazim becomes a hot topic because of the increasing concern about environmental endocrine disruptors [4]. Carbendazim has been banned in several countries because of its negative impacts on the environment and health such as development and reproductive disturbances, toxicity, and mutagenicity [8].
e adverse effects of carbendazim on the biochemical, histopathological, and hematological parameters in the liver, kidney, and endocrine glands and their hormonal levels have been illustrated in rats [4]. Additionally, it needs further study on low concentration due to the residues of carbendazim.
Caenorhabditis elegans (C. elegans), as an important research model, is widely used to do some assessment. According to Amrit et al. [9], C. elegans has many advantages, such as small size, rapid generation time, easy of culturing on laboratory, and short adult lifespan. C. elegans was chosen in this study as the model organism to evaluate the toxicity of low concentration of carbendazim, which may be considered as a reference value for the application of carbendazim in agriculture.

Lethality.
Carbendazim original solutions (1 g/L) were diluted with S liquid medium (1.12 g K 2 HPO 4 , 5.92 g KH 2 PO 4 , and 5.85 g NaCl were diluted with 1 L water) to get the final carbendazim concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1 mg/L, which contained 0.1% DMF. S liquid medium with 0.1% DMF was the control group. 30 nematodes (L4) were tested in 96-well plates for each concentration. e survival of nematodes was counted under a microscope after culturing for 24 hours in the incubator. e process of the test is based on the method of Xiang et al. [11]. ree parallel experiments were needed.

Locomotion Behavior.
At least 10 C. elegans (L4) were picked randomly from each concentration to determine the locomotive behavior, which was recorded by both the headthrash frequency and the body bend times [12]. e number of head-thrash frequency was counted by changes in the direction of bending at the midbody of C. elegans in 1 min. Measurement of body bend was defined as the times of the direction changes of the part of the nematodes cultured in NGM without E. coli OP50 in the 30 s.

Growth and Development
Assays. C. elegans exposed to carbendazim for 24 h was analyzed. e body length of nematodes exposed to carbendazim was assessed by the Image J software. e offspring of each C. elegans from L4 larvae to day 1 was recorded at the L3 stage after individually transferring to a new plate every day until reproduction ceased [13]. At least three parallel tests were carried out.

Lifespan Analysis.
All of C. elegans tested for lifespan were cultured in the same condition at 20°C. e synchronized C. elegans were cultivated in NGM plates with different concentrations of carbendazim until day 4. e tested nematodes then would be transferred into new NGM plates every 2 days. Surviving and dead C. elegans were recorded daily (beginning on the first day of adulthood) until all nematodes for each concentration had died [11]. At least three parallel tests were carried out.

Determination of Oxidative Damage. Intracellular ROS
was measured with 2′,7′-dichlorodihydrofluoroscein diacetate (H 2 DCFH-DA), which is the most common and sensitive reactive oxygen detection probe by far. e wildtype N2 C. elegans were washed in M9 buffer and then ultrasonically disrupted. e supernatant was analyzed the ROS level following the instruction of the ROS kit. e final working concentration of H 2 DCHE-DA was 10 μΜ [11]. e excitation and emission absorbance wavelengths were 485 nm and 535 nm, respectively. At least three parallel tests were carried out.
Intracellular total superoxide dismutase (T-SOD) was determined according to the instruction of the T-SOD kit purchased from the Nanjing Jiancheng Bioengineering Institute. After washed with M9 three times, the examined nematodes were ultrasonically disrupted and reacted with a T-SOD kit. e absorbance wavelength was 550 nm. In addition, the supernatant was used to detect the level of protein for each concentration, in which absorbance wavelength was 595 nm. At least three parallel tests were carried out.

Data Analysis.
All data were given as mean ± standard error of the mean (SEM) by using one-way ANOVA. Graphs were presented using Origin 8.5 and GraphPad Primer 7, and statistical analysis was performed using the SPSS 19.0 software. e statistical significance level was conducted using * p < 0.05 and * * p < 0.01.

Determination of the Locomotion Behavior of C. elegans
after Carbendazim Acute Exposures. LC 50 C. elegans were exposed to carbendazim for 24 hours to assess its acute toxic effects. Data are represented as shown in Table 1, and the obtained linear fitting equation was y � 2.180x − 0.223 through data analysis. e obtained LC 50 is 0.867 mg/L. Next, we assayed the determination of the locomotive behavior of C. elegans after carbendazim acute exposures by analyzing the data about head-thrash frequency and bodybending times of nematodes (Figures 1(a) and 1(b)). Both of them showed significant decreases at the carbendazim concentrations ranging from 0.01 μg/L to 100 μg/L (p < 0.01). Additionally, the head thrashes of nematodes exposed to 100 μg/L decreased to 68.27%. For the body bends test, when the carbendazim concentrations were 10 μg/L and 100 μg/L, it had a significant inhibitory effect on the body bends of C. elegans by 36.77% and 35.48% compared with the control one, respectively.    lifespan of nematodes treated with 0.01 μg/L carbendazim was reduced by 20.00%. When the carbendazim exposure concentration was 100 μg/L, the lifespan of C. elegans was reduced the most by 45.83%.

Effects of Acute Exposure to Carbendazim on the Antioxidant System of C. elegans.
e ROS levels of control and treated C. elegans at carbendazim exposure of different concentrations are shown in Figure 4(a). It was indicated that the level of intracellular ROS was significantly increased (p < 0.01) at ranges from 0.01 μg/L to 100 μg/L carbendazim. Compared with the control one, the ROS level was increased at most by 70.60% with the treatment of 10 μg/L. According to the results of intracellular SOD levels, it had an increase at the treatment from 0.01 μg/L to 100 μg/L carbendazim (Figure 4(b)). e SOD level was increased by 10.70% at 0.1 μg/L carbendazim compared with the control group.

Discussion
Carbendazim, as the fungicides, is widely used in agriculture to inhibit the growth of fungus. Carbendazim has been prohibited to use in Australia, most of the European Union, and the USA because of its severe toxicology and persistent nature [14]. For this study, it was the first time to use C. elegans as the model organisms to evaluate the effects of carbendazim on locomotive behavior, growth and development, reproduction, lifespan, and antioxidant systems. Moreover, results showed that it had a negative influence on C. elegans.
According to the 24 h-LC 50 , the acute toxicity concentration of C. elegans exposed to different concentrations of carbendazim is 0.867 mg/L. e 96-h LC 50 of carbendazim in response to zebrafish has been illustrated as 1.75 mg/L [15]. e eggs of Prussian carp Carassius gibelio has shown the toxicity effects at the concentration of 0.036 mg/L [16,17]. Studies presented that the growth and development of Navicula sp. is inhibited by carbendazim with a 24 h-EC 50 value of 2.18 mg/L. ough the rate of algal growth is recovered after 72-h exposure, the chlorophyll-a content remains significantly decreased when the treatment of carbendazim was beyond 0.5 mg/L [18]. In this present, C. elegans exposed to a low concentration of carbendazim were selected to evaluate its effects depending on the actual concentration of human daily exposure. Low concentrations of carbendazim do not mean it is safe. Carbendazim shows negative biological impacts at much lower doses in some studies.
Locomotive behavior was evaluated to assess the neurotoxicity of C. elegans (L4 larva) after 24-h exposure to carbendazim. e results showed that carbendazim could have negative effects on locomotive behavior through the detection of head thrashing and body bending of C. elegans, which both were more sensitive at the higher exposure group. e locomotive behavior of zebrafish embryos exposed to carbendazim is sensitive [15]. Previous studies have shown that fish have an abnormal behavior when sublethal concentrations of carbendazim are 0.22-0.43 mg/L [19].
Developmental malformations could also be one reason for abnormal locomotion [20]. e growth and development of C. elegans were assayed in our study. Results showed that the body length and body area of C. elegans were significantly narrowed at the treatment exceeding 0.01 μg/L carbendazim. e normal growth of vertebrates is related to the metabolic thyroid hormone homeostasis [21,22]. Williams et al. [23] have indicated that carbendazim could cause sperm loss after implantation, fetal malformation, and slow growth and development. e reproductive toxicity of carbendazim has been demonstrated that carbendazim could inhibit the microtubule polymerization of fungal and mammalian cells, causing disruption of microtubule assembly by acting with Concentration (μg/L) * * * * * * * * * Figure 2: Effects of C. elegans on brood size exposed to carbendazim. Data (mean ± SEM) are shown as the percentage value compared with the control group. e asterisks present the significance between the each exposure group and control group ( * p < 0.05 and * * p < 0.01). β-tubulin, which results in impairing the segregation of chromosomes in the process of cell division [24]. e formation of microtubules by noncovalent bindings of α-and β-tubulin is responsible for chromosome segregation in the process of mitosis and meiosis [24]. e brood size of C. elegans significantly decreases at 0.1 μg/L carbendazim concentration. Carbendazim has been found to affect the reproduction systems in Japanese quails [25] and hamsters [23]. It was concluded that C. elegans lifespan was significantly decreased with carbendazim concentration of ≥0.01 μg/L based on our study. Studies have shown that carbendazim has led to infertility and developmental toxicity and manifests embryo toxicity, germ cell apoptosis, and teratogenesis in different mammalian species [17,18,24].
Apoptosis is a complex programmed cell death, which is a highly regulated phenomenon characterized by a series of cellular processes [26,27]. Many studies have presented that the production of ROS induced by oxidative stress is related to apoptotic cell death [28]. Our study found that carbendazim could induce a significant increase in the level of ROS values and a little increase in the level of SOD values. Oxidative stress caused by environmental pollution induces the increased expression of ROS and subsequently damages the antioxidant defense system [29]. SOD is responsible for the detoxification of toxic free radicals and their activities, which is used to evaluate the oxidative stress level and cellular antioxidant status [17,24]. Metalloenzyme SOD accelerates the transformation of endogenous cytotoxic superoxide radicals to H 2 O 2 , and the increase of SOD expression levels may contribute to improving the enzyme activities in order to eliminate the superoxide radicals induced by carbendazim and to prevent the occurrence of cellular dysfunction during exposure of carbendazim [24,29]. Higher exposure concentrations of carbendazim could cause severe oxidative stress, which subsequently destroys the balance of cell homeostasis and promote apoptosis [30]. However, carbendazim in low concentrations could still significantly damage the reproductive system according to our results.

Conclusion
As far as we know, the present study evaluated the safety of carbendazim exposed to C. elegans for the first time. It demonstrated that carbendazim could have a harmful effect on the locomotive behavior, development and growth, reproduction, lifespan, and antioxidant system of C. elegans. Hope that it needs to pay more attention to the application of carbendazim based on the results. In addition, the safety of carbendazim for use needs to evaluate further, especially the bioaccumulation toxicity and potential genotoxic effects.

Data Availability
All data generated or analyzed during this study are included in this article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.  Figure 4: Effects of C. elegans on antioxidative system exposed to carbendazim. (a) e ROS level of C. elegans after carbendazim exposure; (b) the SOD level of C. elegans after carbendazim exposure. Data (mean ± SEM) are shown as the percentage value compared with the control group. e asterisks present significance between the each exposure group and control group ( * p < 0.05 and * * p < 0.01).