INTRODUCTION
Rice field integrated farming, which combines aquaculture and rice cultivation, effectively stabilizes food security and maintains sustainable agricultural development.1 Integrated farming ecosystems are critical in stabilizing food synergy, improving soil fertility, and decreasing the incidence of insect pests and pesticide use.2 As a typical integrated agricultural-aquaculture system, rice field farming activities include rice tilapia, rice carp, rice crayfish, and rice-turtle.1,3,4 However, the risk assessment indicated that the exposure to toxic levels of pesticides for aquaculture is significant, at least shortly after pesticide application.5 Less research has been done in rice field habitats, particularly in rice-crayfish culture systems without artificial diets for crayfish.
Cypermethrin (CYP), the α-cyano-3-phenoxybenzyl ester of 2,2dimethyl- 3-(2,2-dichlorovinyl)2-2-dimethyl cyclopropane carboxylate, has been widely used to control of pests as a public health agent, often enters wastewater effluent because of its use in agriculture. CYP residues have been detected in water and sediment samples from streams and rivers draining major agricultural districts.6 According to previous reports, the concentration of CYP in surface water was <1 μg/L in most cases, but it can be as high as 2.8 μg/L.7 Chemical exposure may cause oxidative stress and reactive oxygen species (ROS) generation in fish.8,9 Under normal physiological conditions, ROS is detoxified by antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT).9,10 According to previous studies, CYP is more toxic to crustaceans11 than to fish species.12 Further, its toxicological effects were found to be species-specific.13
Emamectin benzoate (EMB) is a neurotoxic insecticide of the avermectin group. The careless use of EMB could cause environmental pollution and serious harm to other, non-target organisms. Research has shown that juvenile lobsters exposed to the highest dosages of EMB with longer exposure increased mortality, with LC50 values decreasing with increasing test material concentration.14 In addition, other reports have shown tubule degeneration in the kidney, mild glycogen vacuolation in the liver, loss of absorptive vacuoles, inflammation and disintegration of the epithelial layer in the intestine of Nile tilapia fed the 1X EB-diet.15 Although toxicity to some natural enemies and non-target arthropods was reported, this insecticide was considered less harmful to beneficial arthropods than broad-spectrum compounds. The insecticide primarily acts through ingestion, causing paralysis in the insect by activating the glutamate-gated chloride channels in the synapses allosterically. Because EMB is widely used as an insecticide for controlling rice leaf roller on paddy in China and applied to agricultural products just before harvest, the development of a simple, accurate detection method for this insecticide in paddy is highly desirable.
Imidacloprid [IMI; 1-[(6-chloro-3-pyridinyl) methyl]-N-nitro-2imidazolidinimine] is the most widely used neonicotinoid insecticide, and its use continues to increase.16 The residues of IMI may enter the food chain, making it important to investigate the potential adverse effects of imidacloprid exposure. This neurotoxic insecticide of the neonicotinoid group is an acetylcholine receptor agonist with a broad spectrum and highly systemic activity, acting by ingestion and direct contact and causing neuronal hyperexcitation at the level of synapses.17 IMI exhibited high toxicity to Mysid shrimp and aquatic insect midges, black flies, and mosquito larvae.18–20 Given IMI’s widespread environmental persistence and high solubility in water, there is a significant need for research on the aquatic toxicity of mixtures of IMI with other pesticides. In the Wu et al.21 study, pesticide mixtures of triazophos (TRI) and IMI synergistically affected zebrafish embryos. Activities of carboxylesterase (Car-E) and catalase (CAT) were significantly changed in most of the individual and joint exposures to pesticides compared to the control group.
Aquatic organisms in natural environments are commonly exposed to chemical mixtures rather than individual compounds.22 The behavior of chemical mixtures may not correspond to that predicted from data for individual compounds.23 Therefore, the evaluation of mixture toxicity, a more realistic approach to evaluating the toxicity of chemicals in the ecosystem, is crucial for risk assessment.24,25
The synergistic effects indicated that it is important to incorporate joint toxicity studies, especially at low concentrations, when assessing the risk of pesticides. In this study, the toxicity of CYP, EMB, IMI, and their mixtures to crawfish was explored by immersion, and the LC50 of the five pesticides was calculated. It can provide data to support future toxicological repeated drug administration experiments and provide some data references for pest control in aquatic environments of the aquaculture industry. In addition, determining the toxicity value can also provide some data reference for clinical medicine toxicity and side reaction detection, and the LC50 value can also provide a basis for formulating relevant water quality standards in water pollution prevention and control.
METHODS AND MATERIALS
TEST ORGANISMS AND CHEMICAL
All crayfish handling followed the guidelines for the care and use of animals for scientific purposes established by the Institutional Animal Care and Use Committee (IACUC) of Yancheng Institute of Technology, China. Red swamp crayfish were obtained from Jiangsu Jinfeng Agricultural Technology Co., Ltd. A commercial feed (Tongwei (Dafeng) Feed Co., Ltd., Jiangsu, China) was used to feed the crayfish twice daily (7:00 and 19:00), and they were temporarily cultured to acclimate to the experimental conditions for two weeks (average body weight: (4.04 ± 1.13) g). The conditions for temporary culture were temperature ((25 ± 1) °C), pH 7.60–8.40, NH3 and H2S less than 0.04 mg/L and 0.03 mg/L, respectively, and dissolved oxygen kept more than 6.0 mg/L throughout the experiment.
A pure cypermethrin (4.5%) was supplied from Zhejiang Well-done Chemical Co., Ltd., China. Purified grade of imidacloprid (10%) was supplied by Yancheng Limin Agrochemical Co., Ltd., Jiangsu, China. A purified grade of emamectin benzoate (5%) was supplied by Henan Yongguan Agricultural Technology Co., Ltd., China. Pesticide mixtures of CYP and IMI were supplied by Jiangsu Gushun Agrochemical Co., Ltd., China. Pesticide mixtures of CYP and EMB (4.2%) were supplied by Qingdao Haina Biotechnology Co., Ltd., Shandong, China. Pesticide mixtures of CYP and IMI, pesticide mixtures of CYP and EMB are emulsifiable oils, and EMB and IMI are both wettable powders. The above reagents were used as mother liquor and diluted according to the experimental concentration.
TOXICITY TESTS
To investigate the five pesticides’ toxicity on crayfish, a 28-day exposure experiment in a semi-static manner was carried out under laboratory conditions. The five pesticides were arranged into five concentration gradients (CYP mother liquor was 0.060, 0.105, 0.135, 0.180, 0.270μg/ml; EMB mother liquor was 40, 60, 70, 80, 100μg/ml; IMI mother liquor was 14, 17, 20, 25, 36 μg/ml, pesticide mixtures of CYP and EMB mother liquor 0.01, 0.03, 0.05, 0.08, 0.1μg/ml, the ratio was 37:3, pesticide mixtures of CYP and IMI mother liquor were 0.06, 0.12, 0.18, 0.24, 0.30μg/ml, the ratio was 1:1 (Table 1), 30 crayfish were selected for each concentration to set up three groups of parallel experiments. Then 30 crayfish without drug impregnation were selected to set up three groups and cultured in aerated water as the control group, and the toxicity was determined by the liquid immersion method. The soaking process involved immersing the crayfish in pesticide for approximately 10 minutes. After the soaking, the extra pesticide on the body surface of the crayfish was wiped off with absorbent paper, and then the crayfish was put into the aerated water for cultivation, and the crayfish was fed regularly. In the process of culture, the survival status of crayfish was checked at 12, 24 and 36 h after treatment, and the dead crayfish were picked out in time. The dead crayfish was assessed based on the criterion that there was no reaction to the crayfish when it was touched gently. Regression equations, χ2, LC50 values, and 95% confidence intervals were calculated and the mortality of crayfish toxicity of the five pesticides.
The color changes and shapes of each organ and tissue of crayfish were observed, and photographed, compared with the clear water.
STATISTICAL ANALYSIS
The experiment data was arranged using Excel 2016, and IBM SPSS Statistics24 performed the analysis.
RESULTS
TOXICITY OF CYP, EMB, AND IMI ON CRAYFISH
As shown in (Table 1), the experimental results displayed that the LC50 of CYP, EMB, and IMI after exposure for 24h, 48h, and 96h were 0.141, 0.137, 0.135, 75.813, 72.345, 70.568, 34.581, 27.930, 22.298 μg/ml, respectively, the death rate of crayfish in parallel with concentration and the extension of poisoning time. The experimental results show that the LC50 of EMB was the highest among the three pesticides, and its toxicity was the lowest. The LC50 of EMB was about 700 times higher than that of CYP, so the toxicity of CYP was significantly greater than that of EMB. The toxicity of IMI was between CYP and EMB.
JOINT TOXICITY OF CYP WITH EMB AND IMI ON CRAYFISH
To evaluate the interaction of CYP and EMB against crayfish, we determined the LC50 values of pesticide mixtures after 24h, 48h, 96h exposure. From Table 1, the experimental results show that the LC50 of the mixture of CYP and EMB was 0.053, 0.050, and 0.048 μg/ml for 24, 48 and 72h, respectively. The LC50 increased when the exposure time was extended as shown in Figure 1, indicating that the joint toxicity was positively correlated with exposure time. Interestingly, the mortality of crayfish exposed to the mixture of CYP and IMI for 48h and 72h was identical, resulting in LC50 of 0.160 μg/mL. In addition, when CYP was mixed with IMI, the toxicity of the pesticide was lower than that of CYP alone, contrary to the results of changes in CYP toxicity after mixing with EMB.
MANIFESTATIONS OF CRAYFISH POISONING
The behavior of crayfish to exposure was observed every 12 h during the toxicity tests. Upon exposure to all pesticides in the initial period, most crayfish showed transient excitement phenomena, including enhanced activity and quick swimming. The changes in behavioral responses initiated half an hour following dosing in all exposure groups. With the prolonged time and concentration, the movements of the crayfish slowed down and they tended to congregate in the corners of the aquarium. Some crayfish attempted to climb the vertical walls, whilst others settled in the middle of the aquaria. Fighting incidents among crayfish increased. Before death, crayfish lost equilibrium and presented themselves in a knock-down position. Then crayfish flipped over and turned upside-down on their backs.
ANATOMY AND OBSERVATION
As shown in Figure 1, the gut of crayfish in all experimental groups changed from dark brown to blue, others became empty, and some sections lost elasticity. The size of the gills did not change, but they turned black in color. The color of the hepatopancreas did not change significantly, but gradually changed from solid to liquid with the increase of concentration, and obvious lesions occurred. In contrast to the other groups, the color of the gastric pouch changed from dark brown to white, and obvious lesions occurred in the mixture CYP and EMB group (Figure 1e K, L), while the color of the gastric pouch changed to dark cyan in the other groups. Nonetheless, no CYP, EMB, and IMI response to heart and muscle tissue was observed in our study.
DISCUSSION
Various types of pesticides are widely used in China for a wide range of purposes. CYP is extensively used to control moths and pests in cotton, fruits, rice, vegetables and horticulture, as well as kill miscellaneous aquatic biota in ponds.26 However, excessive use of pesticides can lead to the destruction of the ecological environment and death of aquatic organisms, as documented in numerous species of aquatic organisms.27,28 CYP has been reported for a novel biodegradation pathway with Bacillus sp. strain SG2.29 Meanwhile, EMB has been experimentally confirmed as a pesticide that is readily biodegradable.30 In a study on the extensive use of imidacloprid in rice ecosystems, the total soil microbial biomass carbon content was reduced following imidacloprid application, concluding that IMI had transient negative effects on soil microbes.31 Waseem Hayat et al.32 pointed out that adding tert-butyl alcohol to nanoscale zero-valent iron (n-ZVI)/sodium persulfate (SPS) system decreased degradation, suggesting OH primarily degraded IMI. Previous studies have shown that CYP, EMB, and IMI can be efficiently degraded, but whether they can be degraded after combining them requires more research in the future. In this study, the 24, 48, and 72h acute LC50 values (with 95% confidence limits) of CYP for crayfish were 0.141, 0.137, and 0.135 μg/ml, respectively, the results were lower than CYP on juvenile crayfish in Yuan’s experiment (1.305, T0.424, 0.287 μg/ml).33 The cause of this experiment’s LC50 being smaller may be attributed to different age-related crayfish immunity, but more work is needed to explain the cause of the phenomenon. Pesticide contamination was often found as a mixture of different pesticides in water bodies than individual compounds.34 This study demonstrated, for the first time, that the joint action of CYP with EMB and IMI was highly toxic to crayfish. Mixtures of CYP and IMI exerted a strong synergistic effect on the acute toxicity to crayfish; the LC50 was 0.180, 0.160, and 0.160 μg/ml, indicating the higher toxicity of mixtures compared with individual pesticides, the synergistic effect of CYP and EMB was more obvious, the LC50 was 0.053, 0.050, 0.048 μg/ml. Lakshmi et al.35 supported the cypermethrin’s effectiveness of synergism; the cypermethrin and Croton bonplandianum Baill, the synergistic effect was found to provide better larvicidal activity. In Wu et al.21 study, pesticide mixtures of triazophos (TRI) and IMI displayed synergistic response on zebrafish embryos. The activities of carboxylesterase (Car-E) and catalase (CAT) were significantly altered in most individual and joint pesticide exposures compared with the control group. Therefore, we should consider the toxic effects of both individual pesticides and their mixtures in the risk management of pesticides.
In an experiment on acute toxicity testing of crayfish,36 the behavioral responses of crayfish to various pesticides were consistent, ranging from initial transient excitement to eventual slow action until death. This study did not conduct in-depth research on the sectioning of the heart and muscles, so it was impossible to determine whether pesticide exposure could have harmed the multinucleated and branched myocardial cells or if the muscles exhibited myofiber fracture and lysis.37
Since the natural aquatic environment encounters pesticide complexity, it is necessary to assess the joint effects of pesticides. Based on the abovementioned results, the mixed pesticide elicits a more significant and pronounced lethal effect on crayfish, providing a more comprehensive insight into joint toxic effects at the physiological level. Furthermore, the results also provided theoretical and scientific significance for the safety application of CYP, EMB, and IMI to farmers and promote the healthy development of the modern eco-agriculture model.
ACKNOWLEDGMENTS
This study was funded by research grants from the National Natural Science Foundation of China (32201874), the Northern Jiangsu Science and Technology Special Project (SZ-YC202123), and Funding for school-level research projects of the Yancheng Institute of Technology (XJR2021035, XJR2021024). A fellowship from the China Scholarship Council supports Yanming Sui.
AUTHOR CONTRIBUTION - CRediT
Conceptualization: Kaixin Zhang (Lead). Funding acquisition: Kaixin Zhang (Lead). Investigation: Kaixin Zhang (Lead). Validation: Kaixin Zhang (Lead). Writing – original draft: Kaixin Zhang (Equal), Jiyi Chen (Equal). Data curation: Jiyi Chen (Lead). Formal Analysis: Jiyi Chen (Lead). Methodology: Shuxin Zhang (Lead). Project administration: Xinxin Chen (Lead). Visualization: Xinxin Chen (Lead). Software: Ke Sun (Lead). Supervision: Pujie Liu (Lead). Resources: Hongshan Li (Lead). Writing – review & editing: Yanming Sui (Lead).
Institutional Animal Care and Use Committee (IACUC)
All crayfish handling followed the guidelines for the care and use of animals for scientific purposes established by the Institutional Animal Care and Use Committee (IACUC) of Yancheng Institute of Technology, China.