# Evaluation of Histopathological and Hematological Effects of Neonicotinoid (Acetamiprid 20% SP) on Grass Carp (Ctenopharyngodon idella)

Authors: Dariush Azadikhah; Mozhgan Velayati Baghdari; Mahnaz Dadras; Sokaina Issa Kadhim; Ali Kamil Kareem; Hussein Ali Hussein
Journal: Aquaculture Research
 (2023)

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## Abstract

Pesticides are usually used as an effective tool to control pests in agriculture; however, these chemicals may be a threat to nontarget organisms, especially aquatic organisms, because aquatic environments are the last station of pollutants. The present study evaluated the toxicity of a commercial formulation of neonicotinoid (acetamiprid 20% SP) as a systematic pesticide on the survival, hematology, and histology of the grass carp (Ctenopharyngodon idella). For these purposes, 105 fingerlings of the grass carp with an average body weight of 30 ± 2 g were exposed to 0, 50, 100, 150, and 200 mg·L−1 of acetamiprid for 96 h. According to the data, a clear mortality was observed as the concentration of acetamiprid elevated (P<0.01). The LC50 96 h of acetamiprid was 121.146 mg·L−1. The hematological parameters in fish changed, following exposure to acetamiprid (P<0.05). Also, a strong correlation was found between acetamiprid concentrations and stress bioindicators such as glucose, total protein, albumin, and cholesterol (P<0.01). There was not significant tissue damage in the control group (0 mg·L−1 of acetamiprid); however, acetamiprid led to tissues lesions such as hypertrophy, hyperplasia, uplifting of gill filaments, necrosis of gill epithelial cells, pyknosis and karyorrhexis of liver cells, and hemorrhage and necrosis of liver cell. Finally, acetamiprid-exposed fish exhibited some clinical signs including unbalanced swimming near the water surface, increasing operculum movement, and deaths with open-mouthed. The results of the present study clearly showed the survival-reducing effects of acetamiprid in C. Idella, which may return to tissue damage and stress induced by the pesticide. The results of the present study can be used as a base for future studies and environmental management.


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## Body

## 1. Introduction

Pesticides are widely used in the world to control plant pests in agriculture [1–3]. According to previous studies, only 1% of pesticides act on target pests, while the remainders (99%) are discharged into the environment, where they may be toxic for nontarget organisms [4]. This matter is so important for aquatic life, since aquatic environments are the last station of pollutants [5]. Although biological methods are developing as environmentally friendly ways to control pests, the use of chemicals is still unavoidable due to the extent of agricultural lands throughout the world [6]. Pesticides improve and stabilize agriculture by controlling of weeds, fungi, and harmful insects. Nevertheless, it is suggested that farmers use at least pesticides with less toxicity and half-life, such as systematic pesticides [7, 8].Acetamiprid as a systematic pesticide is a neonicotinoid insecticide which used to control a variety of pests such as rice stem borer (Chilo suppressalis). Acetamiprid is a chloropyridinyl neonicotinoid that is distinct from the nitro-guanidine neonicotinoid [9]. Acetamiprid is a member of a new class of insecticides, developed in the late 1980s [10, 11]. Acetamiprid is an α-chloro-N-heteroaromatic chemical compound. It has 6-chloro-3-pyridine methyl as other neonicotinoids such as imidacloprid, nitenpyram, and thiacloprid. However, their differences are in the nitroguanidine, nitromethane, or cyanoamidine substituent on an acyclic or cyclic moiety [12]. According to the United States Environmental Protection Agency reports [13], the lowest and highest toxicity of acetamiprid is for insects and mammals, respectively. Kimura–Kuroda et al. [14] stated that bioconcentration potentials of acetamiprid is little for aquatic organisms and can be an ideal systemic pesticide with a selective toxicity effect. However, very little work has been conducted to evaluate its acute toxicity among freshwater fish [15, 16].Toxicological studies can provide important information about the toxicity and possible effects of chemicals and their threats on nontarget organisms [17]. The acute toxicity test (the LC50 96 h test) is a base for toxicological studies [18]. This test can determine lethal concentrations of chemicals for different organisms; however, a static test has some limitations. This method cannot give clear results on the effect of sublethal concentrations, because it is limited to survival results only [3, 19]. Also, toxicity of pollutants alters depending on environmental conditions, species, age, size, and nutrition of organism, which restrict this method (as a static test) to calculate the toxicity in a static environment such as laboratory condition, resulting in different results in comparison with natural conditions [20].Fishes are one of the main sources of protein for human and also play an important role in food chain of water bodies [5, 21]. For example, the grass carp (Ctenopharyngodon idella) has been introduced for the biological control of aquatic plants in many countries. Also, the grass carp is an important freshwater fish for aquaculture in Iran [22]. Grass carp have a spindle-shaped body with large scales, gray back, and light gray belly. In the larval stage, grass carp fed on zooplankton and after that it relays mainly on plants, especially plants with soft tissues [23]. Grass carp farms are usually located near agriculture farms and also there is a polyculture of plant and fish as well [24]. Due to the different sensitivity of the organism to pollutants, toxicological tests are usually performed for different organisms [25, 26].The histopathological and hematological assays can use as a quick method to detect the direct effects of chemicals and stimulants (especially sublethal concentration) in target organs of fish on a laboratory scale. These methods can provide information about the side effects of pollutants with fish health [5, 27, 28]. Blood parameters such as red blood cell count (RBC), white blood cell count (WBC), hematocrit (HCT), glucose, cholesterol, albumin, and total protein concentrations, and histology of target tissues are usually evaluated in toxicological studies [17, 29]. Owning to the importance of grass carp for consumers in northern Iran and the proximity of the fish farms to the consumption hubs of acetamiprid (rice farms), this study was conducted to evaluate the histopathological and hematological effects of neonicotinoid (acetamiprid 20% SP) on grass carp (C. idella).


## 2. Methods and Materials

### 2.1. Preparation

One hundred and fifty grass carp (Ctenopharyngodon idella) with an average body weight of 30 ± 2 g were supplied from a local fish farm (Talesh County, Guilan province, Iran) and moved to a laboratory (Tehran, Iran). Fish were kept in 4 fiberglass tanks (500 Liters dewatering volume) for adaptation to laboratory conditions for 14 days. During the adaptation step, fish were fed twice a day about 1.5% of their weight (FFC, Faradaneh Co., Shahrekord, Iran). Water physicochemical parameters were checked every day. Average concentrations and levels of these parameters were as follow: total hardness 186 ± 4 mg·L−1 CaCO3, Oxygen concentration 8 ± 1 mg·L−1, temperature 27 ± 2°C, pH 7.8 ± 0.4, and NH3 less than 0.01 mg·L−1. Finally, the photoperiod of 16 h light to 8 h dark was employed.


### 2.2. Acute Toxicity Test (The LC50 96 h Test)

The grass carp were acclimated with methods are used for acute toxicity tests as suggested for fish, macroinvertebrates, and amphibians [30].After the adaption period, 105 fingerlings of the grass carp were randomly selected and transferred to the test tanks. The number of test tanks was 15 aquariums (100 × 50 × 50 cm) containing 200 l water. Fish were divided into 5 treatments with 3 replicates. Nominal concentrations of neonicotinoid (acetamiprid 20% SP-Hisun Chemical Co., Ltd., Zhejiang, China) were 0, 50, 100, 150, and 200 mg·L−1 at the present test. The test time was 96 h and the fish mortality was recorded at 24, 48, 72, and 96 h post-acetamiprid exposure. All water physicochemical parameters were the same as the adaptation period and those checked every day. During the test, 50% of the water volume of tanks were replaced daily with fresh water that had the same pesticide concentrations. Fish were transferred to the test tanks 24 h before beginning of the acute toxicity test (the LC50 96 h) and did not feed during the test. To make normal distribution of the pesticide and improve dissolved oxygen content in the test tanks, water was circulated by an internal water pump (No. 118, Guangzhou Ample Technology co. ltd., Guangzhou, China) during the test.


### 2.3. Haematological Assays

Fish (3 samples from each group) were randomly taken after 96 h and anesthetized with 200 mg·L−l ground clove (Syzygium aromaticum) to reduce stress during the sampling [31]. Blood was taken from the caudal vessels of live fish 96 h after exposure to acetamiprid by heparinized syringe and transferred to heparinize 1.5 mL Eppendorf tubes and kept on ice. The whole blood was suspended in the diluent for red and white blood cell examinations using a hemocytometer [32]. Blood plasma was separated by centrifuging of blood in 4800 ×g for 10 min. Plasma was kept at −20°C until biochemical analysis. Mean red blood cell count (RBC) and mean white blood cell count (WBC) were calculated using the hemocytometer according to a Houston study [33].Haematocrit (HCT) was determined by centrifuging whole blood in heparinized microhematocrit capillary tubes at 3500 ×g for 10 min (Deltalab, Barcelona, Spain). Hemoglobin concentration (Hb) was measured by the cyanmethemoglobin method using a commercial kit (Pars Azmun Co., Tehran, Iran) [34, 35]. Also, glucose, total protein, triglyceride, cholesterol, and albumin concentrations were calculated by auto-analyzer (Roche Cobas Mira, Basel, Switzerland) using commercial kits (Pars Azmun Co., Tehran, Iran). Total serum protein was measured by the Biuret method. The Biuret test involves the reaction of Cu ions with peptide bonds in an alkaline solution, which eventually forms a purple-blue complex; this complex stability is about 60 min. The color intensity at 520 to 560 nm is proportional to the concentration of total protein [36]. The concentrations of cholesterol, triglyceride, and glucose in serum were measured upon an enzymatic method by the calorimetric test (CHOD-PAP). Triglyceride, cholesterol, and glucose in combination with a group of enzymes produced a red solution, where the red intensity is directly related to their concentrations [37]. Finally, the albumin was measured using the bromocresol green method (BCG method), and the color change of suspension (from yellow-green to green-blue) was determined at 640 nm [38].


### 2.4. Histopathological Observations

The tissue sample was taken after blood sampling. For this purpose, the abdomen of the fish was cut from under the gills to the anus. The samples were taken from the second left gill arch and the middle part of the liver. The samples were fixed in Bouin’s fluid for 48 h and then transferred to the buffer solution and kept for 12 h. After this step, tissues were submerged in paraffin. Slides were prepared in thickness of 5–7μm using a microtome [29] and were examined under a light microscope and photographed by microscope tablet (scopepad-LX116V1, Labex, London, UK) after staining by hematoxylin and eosin (H&E).


### 2.5. Data Analyses

The lethal concentration of acetamiprid in intervals of 24, 48, 72, and 96 h (LC50 24 h, 48 h, 72 h, and 96 h of acetamiprid) was calculated by the probit test with a 95% confidence. Also, significance between the means of blood parameters in independent groups were determined by One-Way ANOVA with a 95% confidence. The authors used the Spearman test to find the correlation between nominal concentrations of neonicotinoid (Acetamiprid 20% SP) with the mortality rate, tissue damage, and change of hematological parameters (2-tail). The video data were analyzed by Adobe After Effects software (AAE CS6). The clinical signs of fish were evaluated through direct observation of recorded videos, counting the average movement of the gill operculum in 1 min, and comparing the color of the object (fish) during a period.


## 2.1. Preparation

One hundred and fifty grass carp (Ctenopharyngodon idella) with an average body weight of 30 ± 2 g were supplied from a local fish farm (Talesh County, Guilan province, Iran) and moved to a laboratory (Tehran, Iran). Fish were kept in 4 fiberglass tanks (500 Liters dewatering volume) for adaptation to laboratory conditions for 14 days. During the adaptation step, fish were fed twice a day about 1.5% of their weight (FFC, Faradaneh Co., Shahrekord, Iran). Water physicochemical parameters were checked every day. Average concentrations and levels of these parameters were as follow: total hardness 186 ± 4 mg·L−1 CaCO3, Oxygen concentration 8 ± 1 mg·L−1, temperature 27 ± 2°C, pH 7.8 ± 0.4, and NH3 less than 0.01 mg·L−1. Finally, the photoperiod of 16 h light to 8 h dark was employed.


## 2.2. Acute Toxicity Test (The LC50 96 h Test)

The grass carp were acclimated with methods are used for acute toxicity tests as suggested for fish, macroinvertebrates, and amphibians [30].After the adaption period, 105 fingerlings of the grass carp were randomly selected and transferred to the test tanks. The number of test tanks was 15 aquariums (100 × 50 × 50 cm) containing 200 l water. Fish were divided into 5 treatments with 3 replicates. Nominal concentrations of neonicotinoid (acetamiprid 20% SP-Hisun Chemical Co., Ltd., Zhejiang, China) were 0, 50, 100, 150, and 200 mg·L−1 at the present test. The test time was 96 h and the fish mortality was recorded at 24, 48, 72, and 96 h post-acetamiprid exposure. All water physicochemical parameters were the same as the adaptation period and those checked every day. During the test, 50% of the water volume of tanks were replaced daily with fresh water that had the same pesticide concentrations. Fish were transferred to the test tanks 24 h before beginning of the acute toxicity test (the LC50 96 h) and did not feed during the test. To make normal distribution of the pesticide and improve dissolved oxygen content in the test tanks, water was circulated by an internal water pump (No. 118, Guangzhou Ample Technology co. ltd., Guangzhou, China) during the test.


## 2.3. Haematological Assays

Fish (3 samples from each group) were randomly taken after 96 h and anesthetized with 200 mg·L−l ground clove (Syzygium aromaticum) to reduce stress during the sampling [31]. Blood was taken from the caudal vessels of live fish 96 h after exposure to acetamiprid by heparinized syringe and transferred to heparinize 1.5 mL Eppendorf tubes and kept on ice. The whole blood was suspended in the diluent for red and white blood cell examinations using a hemocytometer [32]. Blood plasma was separated by centrifuging of blood in 4800 ×g for 10 min. Plasma was kept at −20°C until biochemical analysis. Mean red blood cell count (RBC) and mean white blood cell count (WBC) were calculated using the hemocytometer according to a Houston study [33].Haematocrit (HCT) was determined by centrifuging whole blood in heparinized microhematocrit capillary tubes at 3500 ×g for 10 min (Deltalab, Barcelona, Spain). Hemoglobin concentration (Hb) was measured by the cyanmethemoglobin method using a commercial kit (Pars Azmun Co., Tehran, Iran) [34, 35]. Also, glucose, total protein, triglyceride, cholesterol, and albumin concentrations were calculated by auto-analyzer (Roche Cobas Mira, Basel, Switzerland) using commercial kits (Pars Azmun Co., Tehran, Iran). Total serum protein was measured by the Biuret method. The Biuret test involves the reaction of Cu ions with peptide bonds in an alkaline solution, which eventually forms a purple-blue complex; this complex stability is about 60 min. The color intensity at 520 to 560 nm is proportional to the concentration of total protein [36]. The concentrations of cholesterol, triglyceride, and glucose in serum were measured upon an enzymatic method by the calorimetric test (CHOD-PAP). Triglyceride, cholesterol, and glucose in combination with a group of enzymes produced a red solution, where the red intensity is directly related to their concentrations [37]. Finally, the albumin was measured using the bromocresol green method (BCG method), and the color change of suspension (from yellow-green to green-blue) was determined at 640 nm [38].


## 2.4. Histopathological Observations

The tissue sample was taken after blood sampling. For this purpose, the abdomen of the fish was cut from under the gills to the anus. The samples were taken from the second left gill arch and the middle part of the liver. The samples were fixed in Bouin’s fluid for 48 h and then transferred to the buffer solution and kept for 12 h. After this step, tissues were submerged in paraffin. Slides were prepared in thickness of 5–7μm using a microtome [29] and were examined under a light microscope and photographed by microscope tablet (scopepad-LX116V1, Labex, London, UK) after staining by hematoxylin and eosin (H&E).


## 2.5. Data Analyses

The lethal concentration of acetamiprid in intervals of 24, 48, 72, and 96 h (LC50 24 h, 48 h, 72 h, and 96 h of acetamiprid) was calculated by the probit test with a 95% confidence. Also, significance between the means of blood parameters in independent groups were determined by One-Way ANOVA with a 95% confidence. The authors used the Spearman test to find the correlation between nominal concentrations of neonicotinoid (Acetamiprid 20% SP) with the mortality rate, tissue damage, and change of hematological parameters (2-tail). The video data were analyzed by Adobe After Effects software (AAE CS6). The clinical signs of fish were evaluated through direct observation of recorded videos, counting the average movement of the gill operculum in 1 min, and comparing the color of the object (fish) during a period.


## 3. Results

### 3.1. The LC50 Test Results

The acetamiprid-exposed fingerlings of grass carp (Ctenopharyngodon idella) revealed some clinical signs such as increasing of operculum movement, fast swimming near the water surface, and death with open-mouthed. There was no mortality in the control group during the test. Also, there was a significant correlation between the pesticide concentration and the fish mortality (Figure 1), as the fish survival decreased significantly with increase of acetamiprid concentrations (P<0.01). Also, the LC50 9 6h of acetamiprid was 121.146 mg·L−1 in the present study (Table 1).Figure 1
Correlation between the nominal concentration of neonicotinoid (acetamiprid 20% SP) and the number of deaths of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure (P<0.01).Table 1
Lethal concentrations of neonicotinoid (acetamiprid 20% SP-Hisun Chemical Co., Ltd., Zhejiang, China) to fingerling grass carp (Ctenopharyngodon idella).
PointConcentration (mg·L−1)∗24 h48 h72 h96 hLC1087.13561.53144.71016.722LC20115.74188.29678.07952.568LC30136.368107.596102.14178.416LC40153.993124.087122.702100.503LC50170.466139.500141.919121.146LC60186.940154.914161.136141.789LC70204.564171.405181.696163.875LC80225.191190.704205.758189.723LC90253.797217.470239.138225.570LC95277.420264.450266.685288.491Note.∗All concentrations are nominal concentrations.


### 3.2. Haematological Assays

There was a significant correlation between the concentration of acetamiprid with blood parameters (Figure2, P<0.01). Red blood cell count (RBC), white blood cell count (WBC), hematocrit (HCT), and hemoglobin (Hb) concentrations significantly were different over 96 h exposure to acetamiprid (P<0.05). Also, the concentrations of glucose, cholesterol, albumin, and total protein in the serum were significantly different (Table 2).Figure 2
Correlation between blood parameters of fingerling grass carp (Ctenopharyngodon idella) and the nominal concentration of neonicotinoid (acetamiprid 20% SP) 96 h after exposure (P<0.01); (a) The nominal concentration of acetamiprid (con); red blood cell count (RBC); white blood cell count (WBC); haematocrit (HCT); the hemoglobin concentration (Hb); (b) glucose concentration (Glu); The total protein (TP); triglyceride concentration (Trig); cholesterol concentration (Cho); albumin concentration (Alb).
(a)(b)Table 2
Results of blood biochemical tests (mean ± SD) of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure to neonicotinoid (acetamiprid 20% SP).
Blood parametersConcentration of acetamiprid (mg·L−1)050100150200RBC (106 μL)1.41 ± 0.03d1.65 ± 0.06c1.89 ± 0.08b1.91 ± 0.08b1.94 ± 0.01aWBC (103 μL)95.43 ± 0.38d109.83 ± 0.3c123.83 ± 0.3b124.93 ± 0.6b126.60 ± 0.4aHCT (%)33.57 ± 0.42d37.10 ± 0.56c39.87 ± 0.72b40.92 ± 0.73b42.17 ± 0.15aHb (g/l)55.10 ± 0.61d65.13 ± 0.94c73.87 ± 0.77b74.18 ± 0.35b76.77 ± 0.25aGlucose (mg/dL)112.67 ± 0.43d132.54 ± 0.35c137.77 ± 0.53b142.67 ± 0.23a142.97 ± 0.51aTotal protein (g/dL)4.73 ± 0.05e5.82 ± 0.04d6.62 ± 0.07c7.12 ± 0.03b7.16 ± 0.05aTriglyceride (mg/dL)135.82 ± 0.11d143.42 ± 0.25c145.33 ± 0.15b145.62 ± 0.25b148.72 ± 0.13aCholesterol (mg/dL)118.48 ± 0.35c125.12 ± 0.09b129.92 ± 0.15a129.62 ± 0.93a129.98 ± 0.65aAlbumin (mg/dL)0.31 ± 0.02e0.42 ± 0.04d0.58 ± 0.05c0.67 ± 0.03b0.75 ± 0.05aNote; RBC: red blood cell count; WBC: white blood cell count; HCT: hematocrit; Hb: hemoglobin. Different letters (a–d) in the same rows indicate significant differences (P<0.05).


### 3.3. Pathological Assays

There were significant correlations between the concentrations of acetamiprid with the levels of liver and gill lesions (Tables3 and 4). Acetamiprid caused hypertrophy, hyperplasia, fusing of filaments, uplifting of gill filaments, and necrosis of epithelial cells over 96 h exposure (Figure 3); The dilation of sinusoids, revealing cytoplasmic vacuolation, congestion of erythrocytes, aggregation of erythrocytes within hepatic blood vessels, and necrosis of liver cells were recognized in treatments after 96 h exposure to acetamiprid (Figure 4). There were not defining significant tissue lesions in the control group.Table 3
Gills damages of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure to different levels of neonicotinoid (acetamiprid 20% SP).
Nominal concentrations (ml·L−1)Tissue damages050100150200Basal hyperplasia−−++++++++Epithelial hypertrophy−+++++++++Uplifting of gill filaments−+++++++Fusing of filaments−−+++++++Necrosis of epithelial cells−−+++++++(−) The tissue damages were not seen; (+) there was tissue slight damages; (++) there was tissue moderate damages; (+++) there was tissue intense damages; (++++) there was tissue very intense damages in the samples.Table 4
The aggregation of erythrocytes within hepatic blood vessels (AHBVs), congestion of erythrocytes (CE), dilation of sinusoids (DSSs), revealing cytoplasmic vacuolation (CV), and necrosis (N) of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure to different levels of neonicotinoid (acetamiprid 20% SP).
Nominal concentrations (ml·L−1)Liver damages050100150200AHBV−+++++++++CE−++++++++CV−++++++++++DSS−−++++++N−−++++++Note. (−) the tissue damages were not seen; (+) there was tissue slight damages; (++) there was tissue moderate damages; (+++) there was tissue intense damages; (++++) there was tissue very intense damages in the samples.Figure 3
Gills lesions of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure to lethal and sublethal concentration of neonicotinoid (acetamiprid 20% SP); (a) control group (acetamiprid was 0 mg·L−1); (b) hyperplasia and uplifting of gill filaments (black rectangle) along with hypertrophy (black rows) and necrosis of epithelial cells (red rectangle); (c) necrosis of epithelial cells (red stars) and fusing of filaments (black rows); (d) necrosis of epithelial cells (black rows); (e) hypertrophy of epithelia cells (black row) and fusing of filaments (black rectangle); (f) necrosis of epithelial cells (black rectangle), hyperplasia (black star), and hypertrophy of epithelia cells (black rows). All pictures were magnified ×400.
(a)(b)(c)(d)(e)(f)Figure 4
Liver lesions of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure to lethal and sub-lethal concentration of neonicotinoid (acetamiprid 20% SP); (a) control group (acetamiprid was 0 mg·L−1); (b) dilation of sinusoids; (c) revealing cytoplasmic vacuolation; (d) congestion of erythrocytes; (e) necrosis; (f) The aggregation of erythrocytes within hepatic blood vessels. All pictures were magnified ×400.
(a)(b)(c)(d)(e)(f)


## 3.1. The LC50 Test Results

The acetamiprid-exposed fingerlings of grass carp (Ctenopharyngodon idella) revealed some clinical signs such as increasing of operculum movement, fast swimming near the water surface, and death with open-mouthed. There was no mortality in the control group during the test. Also, there was a significant correlation between the pesticide concentration and the fish mortality (Figure 1), as the fish survival decreased significantly with increase of acetamiprid concentrations (P<0.01). Also, the LC50 9 6h of acetamiprid was 121.146 mg·L−1 in the present study (Table 1).Figure 1
Correlation between the nominal concentration of neonicotinoid (acetamiprid 20% SP) and the number of deaths of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure (P<0.01).Table 1
Lethal concentrations of neonicotinoid (acetamiprid 20% SP-Hisun Chemical Co., Ltd., Zhejiang, China) to fingerling grass carp (Ctenopharyngodon idella).
PointConcentration (mg·L−1)∗24 h48 h72 h96 hLC1087.13561.53144.71016.722LC20115.74188.29678.07952.568LC30136.368107.596102.14178.416LC40153.993124.087122.702100.503LC50170.466139.500141.919121.146LC60186.940154.914161.136141.789LC70204.564171.405181.696163.875LC80225.191190.704205.758189.723LC90253.797217.470239.138225.570LC95277.420264.450266.685288.491Note.∗All concentrations are nominal concentrations.


## 3.2. Haematological Assays

There was a significant correlation between the concentration of acetamiprid with blood parameters (Figure2, P<0.01). Red blood cell count (RBC), white blood cell count (WBC), hematocrit (HCT), and hemoglobin (Hb) concentrations significantly were different over 96 h exposure to acetamiprid (P<0.05). Also, the concentrations of glucose, cholesterol, albumin, and total protein in the serum were significantly different (Table 2).Figure 2
Correlation between blood parameters of fingerling grass carp (Ctenopharyngodon idella) and the nominal concentration of neonicotinoid (acetamiprid 20% SP) 96 h after exposure (P<0.01); (a) The nominal concentration of acetamiprid (con); red blood cell count (RBC); white blood cell count (WBC); haematocrit (HCT); the hemoglobin concentration (Hb); (b) glucose concentration (Glu); The total protein (TP); triglyceride concentration (Trig); cholesterol concentration (Cho); albumin concentration (Alb).
(a)(b)Table 2
Results of blood biochemical tests (mean ± SD) of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure to neonicotinoid (acetamiprid 20% SP).
Blood parametersConcentration of acetamiprid (mg·L−1)050100150200RBC (106 μL)1.41 ± 0.03d1.65 ± 0.06c1.89 ± 0.08b1.91 ± 0.08b1.94 ± 0.01aWBC (103 μL)95.43 ± 0.38d109.83 ± 0.3c123.83 ± 0.3b124.93 ± 0.6b126.60 ± 0.4aHCT (%)33.57 ± 0.42d37.10 ± 0.56c39.87 ± 0.72b40.92 ± 0.73b42.17 ± 0.15aHb (g/l)55.10 ± 0.61d65.13 ± 0.94c73.87 ± 0.77b74.18 ± 0.35b76.77 ± 0.25aGlucose (mg/dL)112.67 ± 0.43d132.54 ± 0.35c137.77 ± 0.53b142.67 ± 0.23a142.97 ± 0.51aTotal protein (g/dL)4.73 ± 0.05e5.82 ± 0.04d6.62 ± 0.07c7.12 ± 0.03b7.16 ± 0.05aTriglyceride (mg/dL)135.82 ± 0.11d143.42 ± 0.25c145.33 ± 0.15b145.62 ± 0.25b148.72 ± 0.13aCholesterol (mg/dL)118.48 ± 0.35c125.12 ± 0.09b129.92 ± 0.15a129.62 ± 0.93a129.98 ± 0.65aAlbumin (mg/dL)0.31 ± 0.02e0.42 ± 0.04d0.58 ± 0.05c0.67 ± 0.03b0.75 ± 0.05aNote; RBC: red blood cell count; WBC: white blood cell count; HCT: hematocrit; Hb: hemoglobin. Different letters (a–d) in the same rows indicate significant differences (P<0.05).


## 3.3. Pathological Assays

There were significant correlations between the concentrations of acetamiprid with the levels of liver and gill lesions (Tables3 and 4). Acetamiprid caused hypertrophy, hyperplasia, fusing of filaments, uplifting of gill filaments, and necrosis of epithelial cells over 96 h exposure (Figure 3); The dilation of sinusoids, revealing cytoplasmic vacuolation, congestion of erythrocytes, aggregation of erythrocytes within hepatic blood vessels, and necrosis of liver cells were recognized in treatments after 96 h exposure to acetamiprid (Figure 4). There were not defining significant tissue lesions in the control group.Table 3
Gills damages of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure to different levels of neonicotinoid (acetamiprid 20% SP).
Nominal concentrations (ml·L−1)Tissue damages050100150200Basal hyperplasia−−++++++++Epithelial hypertrophy−+++++++++Uplifting of gill filaments−+++++++Fusing of filaments−−+++++++Necrosis of epithelial cells−−+++++++(−) The tissue damages were not seen; (+) there was tissue slight damages; (++) there was tissue moderate damages; (+++) there was tissue intense damages; (++++) there was tissue very intense damages in the samples.Table 4
The aggregation of erythrocytes within hepatic blood vessels (AHBVs), congestion of erythrocytes (CE), dilation of sinusoids (DSSs), revealing cytoplasmic vacuolation (CV), and necrosis (N) of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure to different levels of neonicotinoid (acetamiprid 20% SP).
Nominal concentrations (ml·L−1)Liver damages050100150200AHBV−+++++++++CE−++++++++CV−++++++++++DSS−−++++++N−−++++++Note. (−) the tissue damages were not seen; (+) there was tissue slight damages; (++) there was tissue moderate damages; (+++) there was tissue intense damages; (++++) there was tissue very intense damages in the samples.Figure 3
Gills lesions of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure to lethal and sublethal concentration of neonicotinoid (acetamiprid 20% SP); (a) control group (acetamiprid was 0 mg·L−1); (b) hyperplasia and uplifting of gill filaments (black rectangle) along with hypertrophy (black rows) and necrosis of epithelial cells (red rectangle); (c) necrosis of epithelial cells (red stars) and fusing of filaments (black rows); (d) necrosis of epithelial cells (black rows); (e) hypertrophy of epithelia cells (black row) and fusing of filaments (black rectangle); (f) necrosis of epithelial cells (black rectangle), hyperplasia (black star), and hypertrophy of epithelia cells (black rows). All pictures were magnified ×400.
(a)(b)(c)(d)(e)(f)Figure 4
Liver lesions of fingerling grass carp (Ctenopharyngodon idella) 96 h after exposure to lethal and sub-lethal concentration of neonicotinoid (acetamiprid 20% SP); (a) control group (acetamiprid was 0 mg·L−1); (b) dilation of sinusoids; (c) revealing cytoplasmic vacuolation; (d) congestion of erythrocytes; (e) necrosis; (f) The aggregation of erythrocytes within hepatic blood vessels. All pictures were magnified ×400.
(a)(b)(c)(d)(e)(f)


## 4. Discussion

Acetamiprid is a new member of neonicotinoid insecticides, widely used in agriculture. It is accumulated in the water, thus threatening the life of nontarget organisms [39]. The results of the present study showed the survival reducing effects of acetamiprid, besides some disrupting impacts behavioral patterns and the fish health status. The swimming behavior of fish in acetamiprid exposed was significantly different from the control group, where showed clinical signs such as anxiety, fast swimming, and swimming near the surface. Yalsuyi et al. [5] attributed these clinical signs with stressful conditions.Houndji et al. [40] reported a 96 h LC50 of 265.5 mg·L−1 for acetamiprid in the African catfish (Clarias gariepinus). The LC50 96 h of acetamiprid was 121.146 mg·L−1 in the present was different from those reported by Houndji et al. [40]; which may be related to differences in environmental conditions, size, age, and fish species [3].Hematological assays are a useful method for evaluating pollutant effects on aquatic organisms [41], because blood parameters respond to low doses of pollutants [42]. In our finding, RBC, WBC, HCT, and Hb concentrations of treatments (50–200 mg·L−1 of acetamiprid) were significantly different upon 96 h exposure to acetamiprid. The results of previous studies showed the increasing concentration of biochemicals in serum (such as glucose, cholesterol, and total protein) in exposure to pollutants is a common response of the organism to deal with stressful conditions and oxidative stress [29, 43, 44]. In the present study, the concentrations of glucose, cholesterol, albumin, and total protein of serum as the stress biomarkers were significantly different in the treatments and were higher than in the control group (0 mg·L−1 of acetamiprid). These results were similar to Vali et al. [29] and Parrin et al. [44] studies.Ghayyur et al. [45] studied the toxicity of acetamiprid on hemato-biochemistry and tissue histology of the freshwater fish, Cirrhinus mrigala. The results of their study showed changes in blood parameters and gills and liver tissue in acetamiprid-exposed fish. Parrino et al. [46, 47] reported the toxicity effects of pesticides, their accumulations in aquatic environments and their threats for nontarget organisms, especially human. In the present study, the analyzing of liver and gills of groups (50–200 mg·L−1 of acetamiprid) revealed various tissue damages upon 96 h exposure to acetamiprid. These lesions included hypertrophy, hyperplasia, fusing of filaments, uplifting of gill filaments, the dilation of sinusoids, revealing cytoplasmic vacuolation, congestion of erythrocytes, the aggregation of erythrocytes within hepatic blood vessels, and necrosis of liver cells. In addition, there was a significant correlation between tissue lesions and acetamiprid concentrations. The highest level of tissue damages was related to the 200 mg·L−1 of acetamiprid.Liver is an important organ for controlling many functions of the body such as homeostasis, detoxification, enzyme production, and metabolism. The results of previous studies reported that liver damages can significantly reduce the survival of the organisms and these damages may lead to disruption of enzyme functions and body hemostasis, because there is a significant correlation between liver damage and increased oxidative stress [48–50]. Superoxide dismutase (SOD), catalase, glutathione peroxidases (GPXs), and transferases are the most important enzymes of the body’s antioxidant defense system [51]. Di Giulio and Meyer [52] observed a significant correlation between liver damages and the activity of these enzymes and oxidative stress. Raibeemol and Chitra [53] observed morphological changes in gills, such as the uplifting of gill filaments and hyperplasia, followed by the dilation of blood vessels associated with changes in the space between the exterior medium and blood flow. Dilate of blood vessels can lead to disturbance of ion exchange and following disruption of homeostasis and death of organism [54]. In the present study, there were significant correlations between tissues damages, the fish mortality, and acetamiprid concentrations, which were similar to the results of previous studies.


## 5. Conclusion

The present studies clearly showed the adverse effects of neonicotinoids on the survival rate, histology, and blood parameters of the grass carp as an important commercial and biological freshwater fish species. The results of the present study can be used for future studies and environmental management. Also, the studied parameters have the effective potentials to use as biomarkers of the stressful condition and toxicity of pesticides.



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