Acute Toxicity of an Emerging Insecticide Pymetrozine to Procambarus clarkii Associated with Rice-Crayfish Culture (RCIS)

This study aims to evaluate the acute toxicity of pymetrozine to juvenile Procambarus clarkii. Two 96-h toxicity tests were conducted to assess the lethal concentration 50 (LC50) values, behaviors, and histopathology (at 50% of the 96 h LC50) after pymetrozine exposure. The results showed high toxicity of pymetrozine to juvenile P. clarkii in a dose and time dependent manner, with a decreasing LC50 from 1.034 mg/L at 24 h to 0.479 mg/L at 96 h. The maximum allowable concentration (MAC) of pymetrozine for P. clarkii was 0.106 mg/L. Behavioral abnormalities were observed in pymetrozine-treated crayfish, such as incunabular hyperexcitability, subsequent disequilibrium, lethargy, and increased defecation. Significant lesions were observed in all pymetrozine-treated tissues, including: (1) in gill, hemocytic infiltration and 33.27% of epithelial cells lesions; (2) in perigastric organs, 64.37%, 29.06%, and 13.99% of tubules with lumen atrophy, vacuolation, and cell lysis, respectively; (3) in heart, 2.5%, 8.55% and 7.74% of hemocytic infiltration, vacuolization, and hyperplasia, respectively; (4) in stomach, 80.82%, 17.77%, 6.98%, 5.24% of cuticula swelling, vacuolization, muscle fragmentation, hemocytic infiltration, respectively; (5) in midgut, 7.45%, 10.98%, 6.74%, and 13.6% of hyperplasia, tissue lysis and vacuolation, hemocytic infiltration, muscle fracture; and (6) in abdominal muscle, 14.09% of myofiber fracture and lysis. This research demonstrates that pymetrozine is highly toxic to juvenile P. clarkii, with significant effects on mortality, behavior and histopathology at concentrations of ≤1.1 mg/L, while the estimated practical concentration of pymetrozine in rice-crayfish culture water was around 20 times lower than the calculated MAC.


Introduction
The red swamp crayfish, Procambarus clarkii, is a widely-distributed freshwater crayfish and key species in many water bodies, and has great influences on the ecosystems, such as water quality, sediment, food web, and biodiversity [1][2][3]. P. clarkii has been widely used in water quality and pollution determination as a model organism [4][5][6].
P. clarkii has also become a globally important cultured species in crayfish industry, with an annual yield of~9 × 10 5 t and the highest share in global freshwater crayfish [7,8]. In major culturing regions, such as China, the USA and Portugal, crayfish culture is commonly combined with rice planting  [21] In this study, two 96 h toxicity tests were performed to assess the lethal (mortality) and sublethal effects (behavior and histopathology in gill, perigastric organ, heart, stomach, midgut, and abdominal muscle) of pymetrozine on juvenile P. clarkii. The LC 50 values at 24, 48, 72 and 96 h were obtained from the first 96 h experiment, along with the behavioral changes and maximum allowable concentration (MAC), and the histopathological alterations were determined at a sublethal concentration of pymetrozine in the second 96 h experiment. Furthermore, a safety evaluation of pymetrozine was conducted to estimate the ecological risk of applying pymetrozine in RCIS. The results will provide a better understanding of the toxicity of pymetrozine to aquatic animals, and a guideline for pymetrozine application in RCIS.

Ethical Statement
All procedures performed in studies involving animals were in accordance with ethical standards in Laboratory animal-Guideline for Ethical Review of Animal Welfare (The National Standard of the People's Republic of China GB/T 35892-2018). All dissections were performed under MS-222 anesthesia. In addition, all efforts were made to minimize suffering.

Test Organisms and Chemical
Juvenile P. clarkii were supplied by a crayfish breed cooperative in Qianjiang City, Hubei Province, China. Crayfish were transported to the laboratory of Institute of Hydrobiology, Chinese Academy of Sciences, and acclimated for 14 days, according to Yu et al. [6]. Mortality during acclimation was below 5%. Healthy intermolt-staged crayfish (mean weight of 0.27 ± 0.05 g) with complete appendages were selected.
Technical-grade pymetrozine (purity 99.10%, Beijing JSYH Chemical Technology Research Institute, Beijing, China) was dissolved in double-distilled water as a 0.1 g/L stock solution.

Test Conditions
Tap water was used in exposure tank after a 48-h aeration for chlorine elimination and ultraviolet sterilization. Water temperature and photoperiod were maintained at 20 • C and a 16:8 h light:dark cycle, respectively. Water quality were daily measured, and the dissolved oxygen, hardness (CaCO 3 ), ammonia, water temperature, and pH were 5.7 ± 0.8, 127 ± 9, <0.1 mg/L, 7.47-7.86, and 20 ± 0.6 • C, respectively.

Acute Toxicity Tests
A 96-h semi-static bioassay was conducted with a daily renewal of pymetrozine solution to maintain the test concentrations. No feeding was conducted during the 96-h exposure, and artificial Elodea nuttallii and PVC pipes were provided in the exposure tanks to minimize aggression and cannibalism of the crayfish. A preliminary range-finding test (from 0.01 to 100 mg/L) was conducted and then six concentrations (0.1, 0.3, 0.5, 0.7, 0.9, 1.1 mg/L at nominal) were chosen for the following exposure. Ten crayfish per triplicated tank were exposed to 10 L pymetrozine solution of each concentration or control water (N = 21). Behavioral changes and mortality of the crayfish were recorded at 1, 12, 24, 48, 72 and 96 h after the introduction. Death was defined as lack of any movement of a crayfish within 5 min when probed gently with a glass rod, and dead crayfish were removed from the tanks [6].

Histopathology Test
Ten crayfish per triplicated tank were exposed to nominal 0.24 mg/L pymetrozine (50% of the 96 h LC 50 ) or control water (N = 6). At 96 h, gills, perigastric organs, hearts, stomachs, midguts and abdominal muscles of 12 survivors were freshly dissected out, separately. The tissues were fixed in Bouin's Solution, dehydrated in a graded series of ethanol, cleared in xylene, and then embedded in paraffin wax. Sections of 4 µm were prepared and stained with hematoxylin-eosin (H&E) [22]. An OLYMPUS BX53 microscope (Olympus Corporation, Tokyo, Japan) was used to examine the sections, and the histological impacts were quantified by measuring the percentages of different lesions' area, number or length in three sections as repeats (N = 3) [6].

Statistical Analysis
The mortalities at 24, 48, 72 and 96 h were used to determine the 24, 48, 72 and 96 h LC 50 and 95% confidence with Probit analysis [16] in SPSS 13.0 (IBM, Armonk City, NY, USA). The MAC of pymetrozine in water was calculated by Reed-Muench method [23]. Mortalities in the regressions and percentages of lesions were given as mean ± standard error (Mean ± SE). Analysis of the quantitative histology was performed in SPSS 13.0, and normality of percentages or transformed percentages of the lesions were tested. Data on hemocytic infiltration in stomach were analyzed using Mann-Whitney U test and independent-sample t-test for the rest. A probability of p < 0.05 was considered to be significant.

Mortality, LC 50 Values and MAC
The mortalities of crayfish increased with time and increasing pymetrozine concentrations ( Figure 1). No mortality was observed in control tanks. The 24, 48, 72 and 96 h LC 50 were 1.034, 0.724, 0.551 and 0.479 mg/L, respectively ( Table 2). The calculated MAC of pymetrozine in water was 0.106 mg/L. pymetrozine in water was calculated by Reed-Muench method [23]. Mortalities in the regressions and percentages of lesions were given as mean ± standard error (Mean ± SE). Analysis of the quantitative histology was performed in SPSS 13.0, and normality of percentages or transformed percentages of the lesions were tested. Data on hemocytic infiltration in stomach were analyzed using Mann-Whitney U test and independent-sample t-test for the rest. A probability of p < 0.05 was considered to be significant.

Mortality, LC50 Values and MAC
The mortalities of crayfish increased with time and increasing pymetrozine concentrations ( Figure 1). No mortality was observed in control tanks. The 24, 48, 72 and 96 h LC50 were 1.034, 0.724, 0.551 and 0.479 mg/L, respectively ( Table 2). The calculated MAC of pymetrozine in water was 0.106 mg/L.  In the regression equation, R 2 , P and C are the regression coefficient, probability unit of mortality, and logarithm of pymetrozine concentration, respectively.

Behavioral Responses
Behavioral abnormalities were only found in the pymetrozine-exposed crayfish. The initial response to pymetrozine was hyperexcitability, such as fast movement, climbing the chamber wall, or increased agonism. Then, some individuals showed body jerk or belly arch, and then slow movement, equilibrium loss, sank to the bottom, and lethargy. Increased defecation was noted in pymetrozine-exposed crayfish compared with controls.  In the regression equation, R 2 , P and C are the regression coefficient, probability unit of mortality, and logarithm of pymetrozine concentration, respectively.

Behavioral Responses
Behavioral abnormalities were only found in the pymetrozine-exposed crayfish. The initial response to pymetrozine was hyperexcitability, such as fast movement, climbing the chamber wall, or increased agonism. Then, some individuals showed body jerk or belly arch, and then slow movement, equilibrium loss, sank to the bottom, and lethargy. Increased defecation was noted in pymetrozine-exposed crayfish compared with controls.

Gills
Gills of P. clarkii is composed of branching gill filaments (lamellae) (Figure 2A), which is covered by a thick cuticula underlain with a single epithelial layer ( Figure 2B). The control gills showed uniform arrangements of lamellae and intralamellar spaces, clear cuticula ( Figure 2A-C), and closely and uniformly located epithelial cells ( Figure 2B,C). A significantly higher percentage of gill cuticula vagueness and degeneration (44.29 ± 2.21%) was observed after the pymetrozine exposure than in controls (6.55 ± 1.09%) ( Figure 2D,E; p < 0.001, Figure 3A). A significantly higher percentage of gill epithelial cells exhibited lesions (e.g., cell disorganization and detachment from the cuticula) in pymetrozine-treated groups (33.27 ± 6.65%) than the control group (4.58 ± 1.34%) ( Figure 2E; p < 0.05, Figure 3A). Hemocytic infiltration in the intralamellar space was also found in the pymetrozine-exposed gills ( Figure 2D).  covered by a thick cuticula underlain with a single epithelial layer ( Figure 2B). The control gills showed uniform arrangements of lamellae and intralamellar spaces, clear cuticula (Figure 2A-C), and closely and uniformly located epithelial cells ( Figure 2B,C). A significantly higher percentage of gill cuticula vagueness and degeneration (44.29 ± 2.21%) was observed after the pymetrozine exposure than in controls (6.55 ± 1.09%) ( Figure 2D,E; p < 0.001, Figure 3A). A significantly higher percentage of gill epithelial cells exhibited lesions (e.g., cell disorganization and detachment from the cuticula) in pymetrozine-treated groups (33.27 ± 6.65%) than the control group (4.58 ± 1.34%) ( Figure 2E; p < 0.05, Figure 3A). Hemocytic infiltration in the intralamellar space was also found in the pymetrozine-exposed gills ( Figure 2D).  , heart (C), stomach (D), midgut (E) and abdominal muscle (F) of the pymetrozine-exposeed and control P. clarkii (Mean ± SE). All of the rounded and elliptical gill crosscuts, and tubules of the perigastric organs were quantified by counting the total number and those with different lesions; whole the stomach cuticula and the cuticula with swelling were measured on length, and other lesions in stomach, all the lesions in hearts, midguts and abdominal muscles were quantified by measuring areas of the total samples and parts with different lesions, separately. Percentage of hemocytic infiltration in stomach was analyzed using Mann-Whitney U test, and others were analyzed using independent-sample t-test. Asterisks indicate significant differences between the control and treatment groups (* p < 0.05, ** p < 0.01).
stomach cuticula and the cuticula with swelling were measured on length, and other lesions in stomach, all the lesions in hearts, midguts and abdominal muscles were quantified by measuring areas of the total samples and parts with different lesions, separately. Percentage of hemocytic infiltration in stomach was analyzed using Mann-Whitney U test, and others were analyzed using independent-sample t-test. Asterisks indicate significant differences between the control and treatment groups (* p < 0.05, ** p < 0.01).

Hearts
A normal myocardium of P. clarkii is composed of multinucleated and branched myocardial cells and an adventitia (epicardium) ( Figure 5A). The adventitia consists of several layers of varisized and shapeless epithelial cells, which had no cytoplasm and eccentrically located nuclei, and these give the adventitia a net-like structure ( Figure 5A). Following the 96-h exposure to pymetrozine, hemocytic infiltration ( Figure 5B), vacuolization and hyperplasia ( Figure 5C) appeared, with percentages of 2.58 ± 2.36%, 8.55 ± 1.02% and 7.74 ± 1.33%, respectively, which were

Hearts
A normal myocardium of P. clarkii is composed of multinucleated and branched myocardial cells and an adventitia (epicardium) ( Figure 5A). The adventitia consists of several layers of varisized and shapeless epithelial cells, which had no cytoplasm and eccentrically located nuclei, and these give the adventitia a net-like structure ( Figure 5A). Following the 96-h exposure to pymetrozine, hemocytic infiltration ( Figure 5B), vacuolization and hyperplasia ( Figure 5C) appeared, with percentages of 2.58 ± 2.36%, 8.55 ± 1.02% and 7.74 ± 1.33%, respectively, which were significantly higher than those in the control group (p-value = 0.002, 0.002 and 0.004, respectively, Figure 3C). Some myocardial fibers showed swelling, fracture and lysis as well ( Figure 5B).

Midguts
The midgut of P. clarkii contains several longitudinal ridges which are composed of simple columnar epithelia and subepithelial connective tissues ( Figure 7A). Cytoplasm of the epithelial cells is fibrous, and the connective tissues consist of longitudinal muscles and numerous bladder  Figure 3C). Some myocardial fibers showed swelling, fracture and lysis as well ( Figure 5B).

Midguts
The midgut of P. clarkii contains several longitudinal ridges which are composed of simple columnar epithelia and subepithelial connective tissues ( Figure 7A). Cytoplasm of the epithelial cells is fibrous, and the connective tissues consist of longitudinal muscles and numerous bladder

Abdominal Muscles
The structures of the control abdominal muscles were generally compact and damage-free ( Figure 8A). 14.09 ± 2.46% of the pymetrozine-exposed muscles exhibited myofiber fracture and lysis ( Figure 8B), which was significantly higher than that of the control group (p < 0.05, Figure 3F).

Abdominal Muscles
The structures of the control abdominal muscles were generally compact and damage-free ( Figure 8A). 14.09 ± 2.46% of the pymetrozine-exposed muscles exhibited myofiber fracture and lysis ( Figure 8B), which was significantly higher than that of the control group (p < 0.05, Figure 3F).

Lethal Effects of Pymetrozine
The estimated LC 50 of pymetrozine was 0.48 mg/L, indicating a high toxicity (0.1-1 mg/L [24]) to juvenile P. clarkii. Unexpectedly, pymetrozine exhibited a much lower LC 50 to P. clarkii than other organisms in Table 1, suggesting that P. clarkii is much more sensitive and vulnerable to pymetrozine than many other species. This may be due to the certain physiological similarities between crustacea and insects [16]. Pymetrozine acts on insects in a unique way interfering in the neuroregulation or nerve-muscle interaction of feeding behavior (blockage of stylet penetration), resulting in starvation to death [13,25]. However, the mechanisms of pymetrozine on sucking insects are still largely unknown.

Behavioral Effects of Pymetrozine
When an organism is exposed to a contaminant, it either remains unaware of the environmental change, or chemosensory perception initiates a suite of behavioral responses, such as avoidance, locomotion, feeding and mating [26]. Irritation at the start of the exposure are consistent with the universal behavioral responses of crayfish to ethion [3], thiamethoxam [16], 2,4-D [27], and etofenprox [28]. Hyperactivity may accelerate crayfish to death by enhancing agonism [16] and oxygen consumption [3,29]. The subsequent changes, such as slow movement, equilibrium loss and lethargy, could increase the vulnerability to predation in the environment, and affect the survival rate, feeding and growth of the survivals [1,30].

Gill
Gill has been demonstrated to be a major target of water-borne contaminants and the first organ showing histological changes [3]. For example, gill was identified as the most severely affected and the first organ to show pathology in pentachlorophenol-treated Palaemonetes pugio [31]. At a concentration of 0.24 mg/L, pymetrozine caused significant degeneration in cuticula and epithelial cells, and hemocytic infiltration, which are similar to the alterations in crustacean gills caused by trichlor, chlorpyrifos, ethion, and etofenprox [3,23,28,32]. Gill is the major organ for respiration, osmotic and ionic regulation, so pymetrozine may disrupt respiratory and osmoregulatory functions of crayfish, ultimately resulting in mortality. This has been exampled by studies on the effects of fenitrothion and trichlorfon in crustaceans [32,33].

Perigastric Organ
As a major gland for digestion, absorption, secretion, excretion and detoxification in crustacea, perigastric organ is sensitive to pesticides and other water-borne contaminants [34,35]. Tubule lumen atrophy, lumen dilatation, vacuolation, and epithelial cell lysis in perigastric organ of crayfish in this study were consistent with other studies [3,5,6,23]. These pathological changes could, due to accumulation of pymetrozine or its degradation products in perigastric organs, lead to increased activity of the lysosomal enzymes which damages the cell organelles [3].

Heart
Cardiotoxicity of P. clarkii has been previously reported after mixture of bensulfuron-methyl and acetochlor, chlorpyrifos, and recombinant VP28 protein exposure [6,23,36], including the lesions of cardiac muscle fiber, epithelial hyperplasia, hemocytic infiltration, vacuolization, and myocardial edema. Except for myocardial edema, other symptoms were also detected in the present study. Structural abnormality of heart could lead to cardiac malfunctions. For example, toxicant-induced heart malformations and reduction in heartbeat rate were observed in different fish species [37][38][39][40].

Abdominal Muscle
Muscle is usually not a target organ and accumulates a relatively low amount of toxicants, due to its low-fat content [43,44]. Carapace of crustaceans may also help to prevent abdominal muscle to be damaged, which is suggested by higher content of fluoride, ethion and its degradation products in carapace than in abdominal muscle [3,45]. However, when detoxification systems are saturated, abdominal muscle may still suffer damages [44]. The pymetrozine-exposed muscles exhibited distinct myofiber fracture and lysis, which were also detected in P. clarkii treated with chlorpyrifos, and a mixture of bensulfuron-methyl and acetochlor [3,6].

Pymetrozine Safety Evaluation for RCIS
According to the estimation by Yu et al. [6], the water volume of a typical RCIS in China is over 10 8 L/ha. Given the recommended dose of pymetrozine on rice is under 600 g a.i./ha [6,21], the practical concentration of pymetrozine in RCIS water is estimated less than 6 µg/L, which is far lower than the calculated MAC (0.106 mg/L). In this sense, a proper application of pymetrozine in RCIS is unlikely to induce mortality of crayfish. However, the MAC was calculated based on only mortality data from acute tests and the potential impact of chronic exposure is still unknown. In addition, the actual environmental concentrations of pymetrozine in RCIS could be elevated or even higher than the recommended dose due to the possible misconduct (over-dosing) of pymetrozine application, long-term application of pymetrozine, accumulation of pymetrozine in particles and sediment, shallowed water in RCIS etc., implying the concerns and uncertainties in the safety of pymetrozine application in RCIS.

Conclusions
The present study revealed that pymetrozine was highly toxic to juvenile P. clarkii, with the 96 h LC 50 and maximum allowable concentration of 0.479 and 0.106 mg/L, respectively. Pymetrozine caused behavioral abnormalities at lethal and sublethal concentrations (0.01-1.1 mg/L), and significant pathological changes in gills, perigastric organs, hearts, stomachs, midguts and abdominal muscles at 0.24 mg/L. The results, to our knowledge, are the first to provide a comprehensive toxicity dataset incorporating both lethal and sublethal measurements to better understand the impact of pymetrozine to crayfish, and call for further long-term investigations on the environmental fate (e.g., degradation, bioaccumulation, and transformation) and chronic effects of pymetrozine.