Study on the Toxicity of Acetochlor to Loaches

Yan WANG Peng DONG Pei XU Yuxiang LIN Zexin CHEN Yihong HU Chenzhong JIN Yiding WANG Kaifa GUO Qianxia ZHU

Abstract ［Objectives］ This study was conducted to investigate the toxic effect of acetochlor in loaches.

［Methods］ Loaches were put into the acetochlor solution with concentrations of 0, 0.062 5, 0.125, 0.25, 0.5 and 1 μl/L, respectively. The effects on dismutase (SOD), peroxidase (POD), catalase (CAT) and glutathione (GSH) activity and malondialdehyde (MDA) content in the liver of loaches were analyzed after 12, 24, 48 and 72 h of exposure.

［Results］ The activity of SOD, POD, CAT and GSH increased with the stress time and concentration, reached their maximums at 48 h after acetochlor treatment, and then decreased. However, MDA content increased with the stress time and concentration.

［Conclusions］ Acetochlor had obvious oxidative stress on loaches, and the activity of SOD, POD, CAT and GSH showed an obvious induction-inhibition effect with the increase of acetochlor concentration and the extension of stress time.

Key words Acetochlor; Loach; Antioxidative enzyme system; Malondialdehyde

Received: February 26, 2020Accepted: April 19, 2020

Supported by the Hunan Provincial Science and Technology Innovation Plan Program (NO. 2019NK4170); the Important Project of Hunan Province Education Department (NO.19A259).

Yan WANG (1985-), female, P. R. China, lecturer, devoted to research about 4295248@qq.com.

*Corresponding author. E-mail: 532479626@qq.com.

The widespread use of pesticides has the potential to interfere with biological systems, and thus poses a certain threat to human and animal health. More and more pesticides are endocrine-disrupting chemicals that can cause thyroid dysfunction and oxidative stress in organisms［1-2］. Acetochlor is a selective amide pre-emergence herbicide. Because of its low and long-lasting efficacy and low toxicity to humans and animals, it application amount has increased year by year. It is currently one of the most widely used herbicides in China. The application amount has exceeded 1×107 kg［3］ every year in China. Due to the large amount of acetochlor used, it remains in the soil and enters the river basin with the erosion of rainwater, and accumulates in the environment, which has an impact on aquatic organisms, and even affects human health through indirect effects. In the farmland soil of northern Liaoning, the residual amount of acetochlor reaches 203.18 ng/g［4］. In addition, the residual amount of acetochlor in natural water of the Midwest of the United States is as high as 2.5 μg/L［5］. The widespread use and residues of acetochlor pose a great threat to the ecological environment. The US Environmental Protection Agency has classified acetochlor as a B-2 carcinogen for management［6］. Recent studies have also shown that the use of acetochlor has a certain correlation with the occurrence of lung cancer, colorectal cancer, melanoma and pancreatic cancer［7］. Acetochlor can also cause the death of zebrafish embryos and some non-fatal developmental malformations (such as yolk sac edema, heart cyst edema and egg coagulation) and induce the expression of innate immune phase genes［8］. Acetochlor can also interfere with Xenopus laevis gonad development and reproductive function, have estrogen activity and increase the proportion of females, so that the testicles have ovarian characteristics［9］. Acetochlor can also interfere with the animal餾 thyroid body, leading to the early appearance of tadpole forelimbs and distortion of the tail［10］. In summary, the data published so far indicates that acetochlor has a wide range of molecular and physiological effects on aquatic animals. Therefore, the impact of acetochlor on water pollution and animals is more likely to attract people餾 attention.

Loaches ( Misgurnus anguillucaudatus ) belong to Cobitidae in Cypriniformes of Pisce. It is a small freshwater fish with a certain anti-pollution ability that lives in still water at the bottom of sandy or silt or in a water body with a slow flow rate［11］. Loaches have very high nutritional value and strong vitality, and are highly sensitive to environmental pollution, especially sensitive to chemical mutagens and carcinogens. They are mainly distributed in natural water such as rivers, lakes and rice fields in China. They can be collected throughout the year and is easy to keep in the laboratory. They are one of the ideal experimental materials for toxicity studies and environmental testing［12］.

According to some studies, when aquatic animals are stressed by toxic substances, a variety of active oxygen free radicals that destroy biological function molecules will be produced in the body, and with the increase of active oxygen free radicals, the body will gradually form an antioxidant enzyme system that maintains the balance of oxygen free radical metabolism and protects against oxidative damage［13-14］. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and glutathione (GSH) are the key enzymes of the animal body餾 defense system against peroxidative damage. Their activity changes reflect the impact of toxic substances on organisms to a certain extent, and are more sensitive indicators of ecotoxicology［15］. At present, there have been many reports on the toxicity of acetochlor to fish and tadpoles［10,16］, but there are few reports about the toxicity of acetochlor to loaches. Therefore, in this study, with loaches as an experimental material, the effects of acetochlor on the activity of antioxidant enzymes and malondialdehyde content in livers of loaches were studied from a physiological and biochemical point of view, aiming to provide a theoretical basis for environmental pollution monitoring and the mechanism of toxic effects of acetochlor on loaches, and also to provide a scientific basis for the toxicity studies of other herbicides.

Materials and Methods

Test materials and instruments

The main test materials included test loaches (18-22 g, body length 11-15 cm) provided by Tangwan Farm in Loudi City , Hunan Province and 6 aquaria (50 cm×25 cm×20 cm) provided by Loudi Mingxing Aquarium. The main instruments included Sartorius electronic balance and 754 ultraviolet-visible spectrophotometer.

Reagents

Acetochlor was provided by Hunan Loudi Zhennong Company (purity 98%). Other chemical reagents were all of analytical grade.

Experimental methods

Acute toxicity test

First, a preliminary test was conducted to determine the maximum tolerance concentration (0.059 μl/L) and the minimum total lethal concentration (1.17 μl/L)［15］ to loaches, and then a formal test was conducted. One control check group (CK) and five concentration gradients were set between the maximum tolerance concentration and the minimum lethal concentration as follows: 0, 0.062 5, 0.125, 0.25, 0.5 and 1 μl/L. Each group was added with 16 tails of loaches, which were observed continuously for 72 h. The culture solution was replaced with equal volume of solution with an equal concentration every 24 h , and dead loaches were promptly removed to prevent water pollution.

Index measurement and method

Loach livers were taken out at 12, 24, 48, and 72 h after acetochlor treatment, placed in an ice box and accurately weighed. The livers and 0.9% saline were added to a mortar according to a weight-volume ratio of 1∶9 and ground until the tissue blocks were not visible to naked eyes in the homogenate. The homogenate was centrifuged at 12 000 r/min and 4 ℃ for 10 min. The resulting supernatant was the 10% tissue homogenate to be tested and to be used for the determination of antioxidant enzyme activity and propylene glycol content.

Protein, SOD activity, POD activity, CAT activity, GSH activity and MDA content were measured by Coomassie Brilliant Blue method, nitro blue tetrazolium photoreduction method, guaiacol method, spectrophotometry, DTNB developing process and thiobarbituric acid method, respectively.

Data analysis

The experimental data was represented as mean±standard error (SEM). SPSS 21.0 was used for data statistical processing. One-way ANOVA was used for Tukey test between groups. P <0.05 was considered as statistically significant.

Results and Analysis

Effect of acetochlor treatment on SOD activity in loach liver

As can be seen from Fig. 1, with the extension of the treatment time, the SOD activity showed a trend of increasing first and then decreasing in the 0.062 5, 0.125, 0.25, 0.5 and 1 μl/L acetochlor treatments. The induction of SOD activity was performed at 12 h after treatment on loaches, and the enzyme activity reached maximums in various concentration treatments at 48 h, which were 1.19, 1.63, 1.69, 2.31 and 2.13 times of the CK, respectively. At 12 and 24 h after acetochlor treatment, except for the SOD activity in the 0.062 5 and 0.125 μl/L treatments without a significant difference from the CK, the SOD activity in other treatment groups was significantly higher than that in the CK. At 48 and 72 h, the SOD activity of the acetochlor treatments at various concentrations was significantly higher than that of the CK. And at 72 h, the SOD activity of all concentrations decreased, which indicated that acetochlor had a significant activation-inhibition effect on the SOD activity of loaches.

Different letters indicate significant differences in the SOD activity in loach liver between different concentrations of acetochlor treatments. The same below.

Liver is the main organ of the body for metabolic detoxification, so it is susceptible to attack by exogenous toxic substances and is an important target organ for the body to undergo oxidative stress. When an organism is stressed by an exogenous toxic substance, the antioxidant capacity of the organism will be affected, reducing its ability to scavenge active oxygen in the body, resulting in the presence of excessive free radicals in the body, thus breaking the balance of the redox state, causing oxidative damage to cells and tissues［17］. However, SOD, POD, CAT, and GSH are important antioxidant enzymes to eliminate free radicals in the body, and the measurement of these indicators is very important for understanding the level of reactive oxygen species in organisms and the mechanism of toxic effects［18］. In the study on the effects of acetochlor on zebrafish liver antioxidant enzymes, it was found that the activity of liver SOD, CAT, POD and GSH increased with the treatment level, which showed a trend of first increasing and then decreasing, and the content of MDA increased with the increase of the treatment level［19］. In the study of the oxidative stress state in the liver of rats exposed to acetochlor, it was found that the activity of GSH decreased with the increase of the dose of acetochlor, which suggested that acetochlor reduced the body餾 antioxidant capacity, resulting in an imbalance of redox［2］. An important feature of antioxidant enzymes such as SOD and POD is that their active ingredients or contents can be changed due to the stress of toxic substances, which can indirectly reflect the degree of oxidative pollution in the environment, so they can be used as indicators of environmental pollution stress［20-21］. The results of this study found that in order to reduce the damage caused by acetochlor exposure, SOD, POD, CAT and GSH all showed a trend of increasing first and then decreasing with the concentration of the agent increasing and the extension of the exposure time, showing obvious activation-inhibition effect, which might be because that the low-concentration and short-time treatment increased the activity of antioxidant enzymes and accelerated free radical scavenging, but with the high-concentration and long-time exposure, the generated free radicals increased, which exceeded the body餾 scavenging ability and then broke down the dynamic balance of free radical generation and removal, and promoted the reduction of the activity or contents of SOD, POD, CAT and GSH. Existing studies have also shown that acetochlor and butachlor have obvious activation-inhibition effects on the SOD and CAT activity of earthworm liver, and as time goes on and the concentration of acetochlor increases, the inhibition effect also increases［22］, which is consistent with the research results in this study. Furthermore, butachlor also showed similar results on SOD and CAT activity in Pseudorasbora parva ［13］.

MDA is one of the important products of membrane lipid peroxidation. The change of its content can be used as an important indicator to measure the degree of lipid peroxidation, and can also reflect the degree of oxidative stress in the body［23］. The results of this study showed that with the increase of acetochlor concentration and the extension of treatment time, the content of MDA in loach liver increased, which meant that with the increase of the concentration of acetochlor and the extension of the exposure time, the lipid peroxidation in loach liver gradually increased, the accumulation of MDA increased, and the large amount of free radicals produced by acetochlor could not be removed, resulting in damage to liver tissue aggravating accordingly. Studies have shown that as the concentration of acetochlor increases, the MDA content in the liver of Bufo raddei Strauch also increases significantly, resulting in a greatly reduced survival rate of tadpoles［23］. In addition, other studies have shown that toxic substances such as copper ion stress can induce the production of a large amount of MDA in soldier fly larvae, and its MDA content is significantly positively correlated with copper ion concentration［24］.

In summary, acetochlor had certain oxidative stress and toxic effects on loaches, which changed with its concentration and treatment time. Based on this, the changes of SOD, POD, CAT and GSH activity and MDA content can be used to reflect the pollution of acetochlor in the environment, and can be used as indicators of oxidative stress and toxic effects on organisms. However, in order to further improve the study on the effects of acetochlor on the antioxidant enzyme system of loaches, such as the activity of acetylcholinesterase (AChE) and glutathione S-transferase (GSTs) and other enzymes, further research is needed.

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