# Toxic Effects of Copper Sulfate and Trichlorfon on the Intestines of Zebrafish (Danio rerio)

Authors: Qing Li; Yangyang Cao; Yuting Wang; Xiaodong Long; Wenbo Sun; Hui Yang; Yingying Zhang
Journal: Aquaculture Research
 (2023)

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## Abstract

Copper sulfate and trichlorfon are commonly used in aquaculture. They are often used alone or in combination to kill pathogens and parasites. However, there are few studies evaluating the toxic effects of copper sulfate and trichlorfon on fish. The intestine is an important digestive and immune tissue for fish, which is essential for the growth and development. In this study, zebrafish were used as the research model to detect the toxic effects of copper sulfate (0.5 mg/L) and trichlorfon (0.5 mg/L) at common production concentrations on the fish intestine. The results showed that copper sulfate and trichlorfon significantly induced the activities of intestinal catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), and alkaline phosphatase (AKP), but had no significant effects on intestinal histology after short-term exposure for 7 days. Following 21 days of long-term exposure, copper sulfate and trichlorfon could significantly induce intestinal mucosal damage, and inhibit the activity of antioxidant enzymes, and the expression of tight junction protein genes tight junction protein 1 (ZO1), claudin 1 (Cldn1), and occludin (Oclna) in the zebrafish intestine. These results suggested that copper sulfate and trichlorfon probably induced intestinal oxidative stress injury by inhibiting the activity of antioxidant enzymes, or induced intestinal mucosal damage by inhibiting the expression of intestinal tight junction protein genes. Moreover, the combined exposure of copper sulfate and trichlorfon was more toxic than the single exposure, which suggest the toxicity of copper sulfate and trichlorfon had a synergistic effect. The present results suggest that copper sulfate and trichlorfon should be used with caution in aquaculture.


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## Body

## 1. Introduction

Copper is an essential trace element that plays an important role in the growth and development of organisms [1]. Copper sulfate is a commonly used drug in aquaculture to prevent fish diseases caused by bacteria and parasites [2]. Copper ion can combine with protein to form protein salt precipitation, which has a strong ability to kill pathogens. At the same time, copper sulfate also has a good control effect on moss, blue-green algae, and snails in the water body [3]. However, when the concentration of copper ions in water is too high, the gill epithelium, hematopoietic tissue, kidney, spleen, and liver of fish will be damaged [4]. Moreover, studies have shown that copper in water can be absorbed by fish through the gastrointestinal tract, and mainly accumulate in metabolically active tissues [5, 6]. It has been found that copper sulfate can damage various aquatic animals such as Cyprinus carpio, Daphnia magna, Chironomus tentans, and Pimephales promelas [7, 8]. Trichlorfon is an antiparasitic drug that can be used to control parasites, leeches, and aquatic insects in aquaculture. But at the same time, trichlorfon can enter the fish through the gills, skin, and digestive tract of aquatic economic animals and cause toxic effects on fish [9, 10]. Previous studies have shown that trichlorfon could inhibit the activities of acetylcholine (ACh), glutamic oxalacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) in the heterotrophic silver crucian carp, resulting in neurotoxic symptoms [11]. It can also affect the glutathione (GSH) level and catalase (CAT) activity in Chinese sea bass [12]. Previous studies also showed that high concentrations of trichlorfon could inhibit the activity of crustacean cholinesterase, resulting in accumulation of acetylcholine, disturbing the nervous system, and finally leading to death [13, 14].In aquaculture, copper sulfate and trichlorfon are often used alone or in combination to control fish diseases, but there are few studies on their toxicity to fish. Further research is urgently needed. Zebrafish (Danio rerio), as a small teleost fish, is an excellent research model with small size, low feeding cost, short growth, and reproduction cycle. It has been widely used in the fields of toxicology, developmental biology, pharmacology, and neuroscience. In pathological conditions, the intestine is a conduit for bacteria and toxins to invade. Referring to the safe concentrations of copper sulfate and trichlorfon, and the actual concentrations of these two drugs used in aquaculture, 0.5 mg/L trichlorfon and 0.5 mg/L copper sulfate were selected to treat zebrafish for 7 days and 21 days. The intestinal histology, antioxidant enzyme and alkaline phosphatase activities, and the expression of tight junction-related genes were detected to investigate whether copper sulfate and trichlorfon would affect the zebrafish intestine within the safe concentrations. The results of the present study will provide guidance for the safe use of fishery drugs in aquaculture.


## 2. Materials and Methods

### 2.1. Animals and Drug Exposure

The 2-month-old wild-type AB line zebrafish juveniles used in this experiment were bred in our laboratory and raised in a zebrafish recirculating aquaculture system. Healthy and uninjured zebrafish (0.31 ± 0.16 g) were selected and randomly assigned to the experimental glass tank (about 1 L water/g fish). Fish were raised in dechlorinated tap water (pHat 7.2 ± 0.5, dissolved oxygen at 6.0–7.5 mg·L−1, temperature at 25 ± 0.5°C) with a 14 h/10 h light/dark cycle and fed with chironomid larvae twice a day. Four experimental groups were divided: control group (Con), copper sulfate group (Cu, Sinopharm, 0.5 mg/L), trichlorfon group (Dip, Jiangshan, 90% purity, 0.5 mg/L), copper sulfate + trichlorfon group (Cu, 0.5 mg/L + Dip, 0.5 mg/L), and three replicates were set for each treatment. The concentration of copper sulfate and trichlorfon was selected based on the safe concentrations of copper sulfate and trichlorfon, and the actual concentrations of these two drugs were used in the production. Half of the water in each tank was replaced every two days with fresh dechlorinated tap water dosed with the appropriate amount of copper sulfate and trichlorfon to keep the drug concentration constant.


### 2.2. Sampling and Morphometry

Fish were sacrificed following 7 and 21 days of exposure, respectively. At each time point, fish were euthanized with MS-222 (500 mg/L buffered with 200 mg/L NaHCO3) and drained on the filter paper, and the body weight and length were measured using sensitive balance and caliper, respectively. For each treatment, six intestine tissues were taken from each treatment (two intestines per tank in triplicate), and fixed with Bouin’s solution for paraffin section analysis. For each treatment, nine intestines for enzymatic activity (three intestines per tank mixed in a sample in triplicate) and six intestines for mRNA expression analysis (two intestines per tank in triplicate) were frozen using liquid nitrogen and kept individually at −80°C until use. 168 fish in total were used in the present study.


### 2.3. Intestinal Histological Examination

The intestine was dissected from the fish and fixed in Bonn solution for 48 h at 4°C. The intestines were wrapped in gauze, washed with tap water for 24 hours, dehydrated by ethanol gradient, and treated with methyl salicylate for 12–24 hours until the tissue was transparent, and then embedded in paraffin. Sections with a thickness of 6μm were made using a rotary microtome (Leica). Sections were stained using hematoxylin eosin (HE) method. Microscopic examination was carried out using CHC binocular microscope (Olympus).


### 2.4. Enzyme Activity Assay

For CAT, superoxide dismutase (SOD), glutathione peroxidase (GPX), and alkaline phosphatase (AKP) activity assay, 9 times volume of precooled phosphate buffer solution (PBS) (1 g of tissue: 9 mL PBS) was added to tube with intestines, and the intestines were mechanically homogenized in ice water bath, and then centrifuged at a speed of 4000 rpm for 10 minutes at 4°C. The supernatant was isolated for enzyme activity analysis according to the manufacturer’s protocol of the relevant kit (Nanjing Jiancheng). Concentrations of final activities of enzymes were normalized to protein concentrations of the corresponding samples. The total protein concentrations of the samples were determined with protein quantitation kit (Beyotime).


### 2.5. Gene Expression Analysis

mRNA expression of genes related to cell junction including tight junction protein 1 (ZO1), occludin (Oclna), and claudin 1 (Cldn1) was investigated in the intestine following 7 and 21 days of exposure. The intestines of two fish in each replicate were mixed into one sample, and each treatment group had three replicates. A total of 48 fish were used for this test. Before dissection, the body surface water was blotted with absorbent paper, and the body length and body weight were measured. Total RNAs of these tissues were isolated using the TRIzol one-step method. RNA integrity was checked by analyzing 28S and 18S rRNA ratios with 1% agarose gel electrophoresis. The cDNAs were synthesized from total RNA with M-MLV reverse transcriptase with gDNA wiper (Vazyme Biotech Co., Ltd, China) and oligo (dT) 18 primer. qPCR was applied to evaluate mRNA expression profiles of these genes. Beta actin (actb) and eukaryotic translation elongation factor 1 alpha (ef1a) were selected as the reference genes in the present study. The arithmetic mean of the two Cq values of reference genes to calculate the relative transcript changes. The primer sequences are listed in Table 1. The quantitative real-time PCR (qPCR) efficiency of these genes were all between 90% and 110%. The relative transcript changes were calculated using the 2−ΔΔCq method [15].Table 1
The primers for qRT-PCR.
GeneSequencesTmAmplification efficiency (%)GenBank accession noactbF: AGA​TGG​GAA​CCG​CTG​CCT​CTT63.8893.4NM_131031.2R: GTG​GTC​TCG​TGG​ATA​CCG​CAA62.42ef1aF: ACA​GCC​AGA​AGC​GTT​ATG​AGG60.4196.2NM_200009.2R: GTC​AGT​TGG​ACG​AGT​TGG​AGG60.61ZO1F: TGC​TCA​GTG​GTA​TCC​CAT​CGT​A60.69103.1XM_021477863.1R: GAA​TGG​TGT​CCT​TCA​GTG​CTC​C61.20Cldn1F: AGT​AAA​GTG​GCT​CTG​GCT​GG59.6794.1NM_131770.1R: ATA​CAG​CAG​AGC​AGG​GAA​CC59.46OclnaF: CAG​CGA​TTA​CGA​CCG​CAG​AT60.60105.7NM_212832.2R: GAG​GCA​CAT​TAG​GAA​CAA​GCA​A59.18


### 2.6. Statistical Analysis

All the statistical data were expressed as mean ± standard deviation (SD). Before analysis, data were tested for normality of distribution (Kolmogorov–Smirnov test) and homogeneity of variance (Levene’s test). Then, one-way analysis of variance (ANOVA) was performed on the data sets with significant differences. The data with significant difference carried out a Tukey post hoc test.P<0.05 was considered statistically significant. Data analysis was performed with SPSS 18.0.


## 2.1. Animals and Drug Exposure

The 2-month-old wild-type AB line zebrafish juveniles used in this experiment were bred in our laboratory and raised in a zebrafish recirculating aquaculture system. Healthy and uninjured zebrafish (0.31 ± 0.16 g) were selected and randomly assigned to the experimental glass tank (about 1 L water/g fish). Fish were raised in dechlorinated tap water (pHat 7.2 ± 0.5, dissolved oxygen at 6.0–7.5 mg·L−1, temperature at 25 ± 0.5°C) with a 14 h/10 h light/dark cycle and fed with chironomid larvae twice a day. Four experimental groups were divided: control group (Con), copper sulfate group (Cu, Sinopharm, 0.5 mg/L), trichlorfon group (Dip, Jiangshan, 90% purity, 0.5 mg/L), copper sulfate + trichlorfon group (Cu, 0.5 mg/L + Dip, 0.5 mg/L), and three replicates were set for each treatment. The concentration of copper sulfate and trichlorfon was selected based on the safe concentrations of copper sulfate and trichlorfon, and the actual concentrations of these two drugs were used in the production. Half of the water in each tank was replaced every two days with fresh dechlorinated tap water dosed with the appropriate amount of copper sulfate and trichlorfon to keep the drug concentration constant.


## 2.2. Sampling and Morphometry

Fish were sacrificed following 7 and 21 days of exposure, respectively. At each time point, fish were euthanized with MS-222 (500 mg/L buffered with 200 mg/L NaHCO3) and drained on the filter paper, and the body weight and length were measured using sensitive balance and caliper, respectively. For each treatment, six intestine tissues were taken from each treatment (two intestines per tank in triplicate), and fixed with Bouin’s solution for paraffin section analysis. For each treatment, nine intestines for enzymatic activity (three intestines per tank mixed in a sample in triplicate) and six intestines for mRNA expression analysis (two intestines per tank in triplicate) were frozen using liquid nitrogen and kept individually at −80°C until use. 168 fish in total were used in the present study.


## 2.3. Intestinal Histological Examination

The intestine was dissected from the fish and fixed in Bonn solution for 48 h at 4°C. The intestines were wrapped in gauze, washed with tap water for 24 hours, dehydrated by ethanol gradient, and treated with methyl salicylate for 12–24 hours until the tissue was transparent, and then embedded in paraffin. Sections with a thickness of 6μm were made using a rotary microtome (Leica). Sections were stained using hematoxylin eosin (HE) method. Microscopic examination was carried out using CHC binocular microscope (Olympus).


## 2.4. Enzyme Activity Assay

For CAT, superoxide dismutase (SOD), glutathione peroxidase (GPX), and alkaline phosphatase (AKP) activity assay, 9 times volume of precooled phosphate buffer solution (PBS) (1 g of tissue: 9 mL PBS) was added to tube with intestines, and the intestines were mechanically homogenized in ice water bath, and then centrifuged at a speed of 4000 rpm for 10 minutes at 4°C. The supernatant was isolated for enzyme activity analysis according to the manufacturer’s protocol of the relevant kit (Nanjing Jiancheng). Concentrations of final activities of enzymes were normalized to protein concentrations of the corresponding samples. The total protein concentrations of the samples were determined with protein quantitation kit (Beyotime).


## 2.5. Gene Expression Analysis

mRNA expression of genes related to cell junction including tight junction protein 1 (ZO1), occludin (Oclna), and claudin 1 (Cldn1) was investigated in the intestine following 7 and 21 days of exposure. The intestines of two fish in each replicate were mixed into one sample, and each treatment group had three replicates. A total of 48 fish were used for this test. Before dissection, the body surface water was blotted with absorbent paper, and the body length and body weight were measured. Total RNAs of these tissues were isolated using the TRIzol one-step method. RNA integrity was checked by analyzing 28S and 18S rRNA ratios with 1% agarose gel electrophoresis. The cDNAs were synthesized from total RNA with M-MLV reverse transcriptase with gDNA wiper (Vazyme Biotech Co., Ltd, China) and oligo (dT) 18 primer. qPCR was applied to evaluate mRNA expression profiles of these genes. Beta actin (actb) and eukaryotic translation elongation factor 1 alpha (ef1a) were selected as the reference genes in the present study. The arithmetic mean of the two Cq values of reference genes to calculate the relative transcript changes. The primer sequences are listed in Table 1. The quantitative real-time PCR (qPCR) efficiency of these genes were all between 90% and 110%. The relative transcript changes were calculated using the 2−ΔΔCq method [15].Table 1
The primers for qRT-PCR.
GeneSequencesTmAmplification efficiency (%)GenBank accession noactbF: AGA​TGG​GAA​CCG​CTG​CCT​CTT63.8893.4NM_131031.2R: GTG​GTC​TCG​TGG​ATA​CCG​CAA62.42ef1aF: ACA​GCC​AGA​AGC​GTT​ATG​AGG60.4196.2NM_200009.2R: GTC​AGT​TGG​ACG​AGT​TGG​AGG60.61ZO1F: TGC​TCA​GTG​GTA​TCC​CAT​CGT​A60.69103.1XM_021477863.1R: GAA​TGG​TGT​CCT​TCA​GTG​CTC​C61.20Cldn1F: AGT​AAA​GTG​GCT​CTG​GCT​GG59.6794.1NM_131770.1R: ATA​CAG​CAG​AGC​AGG​GAA​CC59.46OclnaF: CAG​CGA​TTA​CGA​CCG​CAG​AT60.60105.7NM_212832.2R: GAG​GCA​CAT​TAG​GAA​CAA​GCA​A59.18


## 2.6. Statistical Analysis

All the statistical data were expressed as mean ± standard deviation (SD). Before analysis, data were tested for normality of distribution (Kolmogorov–Smirnov test) and homogeneity of variance (Levene’s test). Then, one-way analysis of variance (ANOVA) was performed on the data sets with significant differences. The data with significant difference carried out a Tukey post hoc test.P<0.05 was considered statistically significant. Data analysis was performed with SPSS 18.0.


## 3. Results

### 3.1. Effects of Copper Sulfate and Trichlorfon on the Growth of Zebrafish

Following 7 days and 21 days of exposure, copper sulfate and trichlorfon alone or in combination had no significant effect on the body length of zebrafish (Figure1(a)). After 21 days of exposure, the body weight of zebrafish was significantly suppressed by the combined effect of copper sulfate and trichlorfon (Figure 1(b)).Figure 1
Effects of copper sulfate and trichlorfon on the growth of zebrafish; (a) body length and (b) body weight.∗represents significant difference compared with the control group (P<0.05).
(a)(b)


### 3.2. Effects of Copper Sulfate and Trichlorfon on the Intestinal Histology in Zebrafish

Following 7 days of exposure, there were no significant changes in zebrafish intestinal histology in all treatment groups (data are not shown). Following 21 days of exposure, the intestines villi were morphologically intact, arranged regularly, and had no obvious pathological damage in the Con group (Figure2(a)). Compared with the Con group, the intestinal mucosa was damaged in all the drug treatment groups, and there was local hemorrhage, and the intestinal mucosal damage in the Cu + Dip group was the most serious (Figures 2(b)–2(d)).Figure 2
Effects of copper sulfate and trichlorfon on intestinal histology of zebrafish; (a) Con group, (b) Cu group, (c) Dip group, and (d) Cu + Dip group; arrows indicate intestinal villi and triangles indicate local bleeding.


### 3.3. Effects of Copper Sulfate and Trichlorfon on the Activity of Antioxidant Enzymes in the Zebrafish Intestine

Following 7 days of exposure, the intestinal CAT activity of the Cu group and Dip group was significant higher than that of the Con group (P<0.05). Following 21 days of exposure, the CAT activities of the three treatment groups were significantly lower than those of the Con group (Figure 3(a)). Following 7 days of exposure, the SOD and GPX activities in gut of the three treatment groups were significantly higher than those of the Con group (P<0.05), while after 21 days of exposure, the SOD and GPX activities of three experimental groups were significantly lower than those of the control group (P<0.05) (Figures 3(b) and 3(c)). In conclusion, after Cu, Dip, and Cu + Dip stress, the CAT, SOD, and GXP activities in the intestine increased in a short time, but with the extension of exposure duration, the activities of these enzymes decreased significantly. Moreover, the activity of these enzymes decreased more in the Cu + Dip group than in the Cu group or Dip group.Figure 3
Effects of copper sulfate and trichlorfon intestinal antioxidant enzyme activities of zebrafish; (a) CAT, (b) SOD, and (c) GPX;∗represents significant difference compared with the control group (P<0.05).
(a)(b)(c)


### 3.4. Effects of Copper Sulfate and Trichlorfon on the Intestinal AKP Activity in Zebrafish

At 7 days of exposure, the intestinal AKP activity significantly increased in all the treatment groups, and the combined effect of copper sulfate and trichlorfon had a stronger inductive effect. At 21 days of exposure, the intestinal AKP activity significantly decreased in all the treatment groups, and the combined effect of copper sulfate and trichlorfon showed a stronger inhibiting effect (Figure4).Figure 4
Effects of copper sulfate and trichlorfon on intestinal AKP activity of zebrafish;∗represents significant difference compared with the control group (P<0.05).


### 3.5. Effects of Copper Sulfate and Trichlorfon on Intestinal Tight Junction-Related Genes

After 7 days of exposure, the gene expression ofZO1 significantly decreased in the Cu + Dip group, the gene expression of Cldnl significantly increased in the Cu group and the Dip group, while the expression of Oclna had no significant change (Figure 5(a)). At 21 days of exposure, the gene expressions of ZO1, Cldn1, and Oclna significantly decreased in all the treatment groups, and the downregulation was greater in the Cu + Dip group.Figure 5
Effects of copper sulfate and trichlorfon on intestinal tight junction-related genes; (a)ZO1, (b) Oclna, and (c) Cldn1; ∗represents significant difference compared with the control group (P<0.05).
(a)(b)(c)


## 3.1. Effects of Copper Sulfate and Trichlorfon on the Growth of Zebrafish

Following 7 days and 21 days of exposure, copper sulfate and trichlorfon alone or in combination had no significant effect on the body length of zebrafish (Figure1(a)). After 21 days of exposure, the body weight of zebrafish was significantly suppressed by the combined effect of copper sulfate and trichlorfon (Figure 1(b)).Figure 1
Effects of copper sulfate and trichlorfon on the growth of zebrafish; (a) body length and (b) body weight.∗represents significant difference compared with the control group (P<0.05).
(a)(b)


## 3.2. Effects of Copper Sulfate and Trichlorfon on the Intestinal Histology in Zebrafish

Following 7 days of exposure, there were no significant changes in zebrafish intestinal histology in all treatment groups (data are not shown). Following 21 days of exposure, the intestines villi were morphologically intact, arranged regularly, and had no obvious pathological damage in the Con group (Figure2(a)). Compared with the Con group, the intestinal mucosa was damaged in all the drug treatment groups, and there was local hemorrhage, and the intestinal mucosal damage in the Cu + Dip group was the most serious (Figures 2(b)–2(d)).Figure 2
Effects of copper sulfate and trichlorfon on intestinal histology of zebrafish; (a) Con group, (b) Cu group, (c) Dip group, and (d) Cu + Dip group; arrows indicate intestinal villi and triangles indicate local bleeding.


## 3.3. Effects of Copper Sulfate and Trichlorfon on the Activity of Antioxidant Enzymes in the Zebrafish Intestine

Following 7 days of exposure, the intestinal CAT activity of the Cu group and Dip group was significant higher than that of the Con group (P<0.05). Following 21 days of exposure, the CAT activities of the three treatment groups were significantly lower than those of the Con group (Figure 3(a)). Following 7 days of exposure, the SOD and GPX activities in gut of the three treatment groups were significantly higher than those of the Con group (P<0.05), while after 21 days of exposure, the SOD and GPX activities of three experimental groups were significantly lower than those of the control group (P<0.05) (Figures 3(b) and 3(c)). In conclusion, after Cu, Dip, and Cu + Dip stress, the CAT, SOD, and GXP activities in the intestine increased in a short time, but with the extension of exposure duration, the activities of these enzymes decreased significantly. Moreover, the activity of these enzymes decreased more in the Cu + Dip group than in the Cu group or Dip group.Figure 3
Effects of copper sulfate and trichlorfon intestinal antioxidant enzyme activities of zebrafish; (a) CAT, (b) SOD, and (c) GPX;∗represents significant difference compared with the control group (P<0.05).
(a)(b)(c)


## 3.4. Effects of Copper Sulfate and Trichlorfon on the Intestinal AKP Activity in Zebrafish

At 7 days of exposure, the intestinal AKP activity significantly increased in all the treatment groups, and the combined effect of copper sulfate and trichlorfon had a stronger inductive effect. At 21 days of exposure, the intestinal AKP activity significantly decreased in all the treatment groups, and the combined effect of copper sulfate and trichlorfon showed a stronger inhibiting effect (Figure4).Figure 4
Effects of copper sulfate and trichlorfon on intestinal AKP activity of zebrafish;∗represents significant difference compared with the control group (P<0.05).


## 3.5. Effects of Copper Sulfate and Trichlorfon on Intestinal Tight Junction-Related Genes

After 7 days of exposure, the gene expression ofZO1 significantly decreased in the Cu + Dip group, the gene expression of Cldnl significantly increased in the Cu group and the Dip group, while the expression of Oclna had no significant change (Figure 5(a)). At 21 days of exposure, the gene expressions of ZO1, Cldn1, and Oclna significantly decreased in all the treatment groups, and the downregulation was greater in the Cu + Dip group.Figure 5
Effects of copper sulfate and trichlorfon on intestinal tight junction-related genes; (a)ZO1, (b) Oclna, and (c) Cldn1; ∗represents significant difference compared with the control group (P<0.05).
(a)(b)(c)


## 4. Discussion

Copper is an essential trace element for both human and animal, which plays an important role in maintaining the functions of red blood cells, nerve cells, and the immune system. However, when the copper content exceeds the normal level required for the growth and development of the organism, it will accumulate in the organism, causing irreversible damage [1]. In aquaculture, copper has been widely used as a bactericidal and antiparasitic drug [16]. In the present study, we found that short-term (7 days) exposure to production-related concentrations of copper sulfate stimulated the upregulation of intestinal antioxidant enzymes in zebrafish while there was no significant change in intestinal histology. Long-term exposure to copper sulfate could significantly decrease the activity of intestinal antioxidant enzymes and cause intestinal mucosal damage. Similar to the results of the present study, exposure of juvenile grouper to 20 or 200 μg/L copper sulfate for 25 days dramatically inhibited the activity of intestinal antioxidant enzymes and induced apoptosis of intestinal wall cells [17]. Exposure of zebrafish embryos to 0.375 or 0.50 mg/L copper sulfate led to thinner intestinal wall and shorter intestinal villi of larval fish [18]. Trichlorfon, an organophosphorus ester insecticide, is widely used as an insecticide in aquaculture because of its high efficiency, low toxicity, and low residue. Exposure to high concentrations of trichlorfon could cause hyperemia in the gill filament and fin, decreased respiration, dysmotility, and even death in fish [19, 20]. Few studies focused on the effects of production-related concentrations of trichlorfon on the intestine of fish. In this study, we found that the effect of trichlorfon exposure alone on the zebrafish intestine was similar to those of copper sulfate exposure alone. Long-term exposure would lead to a significant decrease in the activity of antioxidant enzymes in zebrafish intestinal.The SOD-CAT system provided the first defense against antioxidant toxicity under ambient stress. SOD catalyzed the dismutation of the superoxide anion radical to water and H2O2, and H2O2 was subsequently detoxified by CAT [21]. GPX also plays an important role in scavenging reactive oxygen species (ROS), which can remove 80%–90% of intracellular H2O2 [22, 23]. Oxidative stress was caused by the imbalance between the increase of the ROS level and the decrease of the antioxidant enzyme activity [24]. In the present study, following long time (21 days) exposure, the activities of several antioxidant enzymes including SOD, CAT, and GPX were significantly decreased regardless of the exposure of copper sulfate and trichlorfon alone or in combination. These results suggested that the ROS level in the intestine of zebrafish may increase, resulting in oxidative stress, which might be one of the important reasons for intestinal mucosal damage. AKP is sensitive to environmental pollutants and is often used as a biomarker of water pollutants [25]. In the present study, we detected the changes of the intestinal tissue AKP level. The activity of AKP was significantly increased under the short-term exposure to copper sulfate and trichlorfon, while it was significantly decreased under long-term exposure. AKP is widely distributed in various tissues of the organism, and intestinal AKP exists in the apical microvilli of the brush border of the intestine, which plays an important role in maintaining the integrity of intestinal mucosa [26]. As an important part of the intestinal mucosal barrier, the intestinal epithelial tight junction protein plays an important role, while AKP plays a role in maintaining the tight junction between cells. AKP deletion in mice resulted in a significant decrease in intestinal tight junction protein ZO1 and Oclna levels, while AKP overexpression stimulated an increase in the mRNA level of tight junction protein ZO1. Therefore, it has been confirmed that AKP can affect intestinal mucosal integrity by regulating tight junction proteins [27]. Similar to previous studies, the expression trend of intestinal tight junction-related genes ZO1, Cldn1, and Oclna in zebrafish exposed to copper sulfate and trichlorfon was consistent with the changes of intestinal AKP activity in this study. These results suggested that prolonged exposure to copper sulfate and trichlorfon can damage intestinal mucosa by inhibiting AKP activity.In the present study, the body weight, intestinal enzyme activity, tight junction-related genes expression, and intestinal histological results of zebrafish exposed to copper sulfate and trichlorfon for a long time all indicated that the combined toxicity of copper sulfate and trichlorfon to zebrafish was synergistic. Similar phenomena have been found in other studies, in which the combination of chromium and acaricide thiophosphate increased testicular damage in rats by increasing oxidative stress, reducing antioxidant capacity, and changing androgen levels [28]. The combination of cadmium (Cd) and chlorpyrifos (CPF) can improve its toxicity to the liver by increasing the accumulation of intracellular cholesterol and triglyceride [29, 30]. The combination of nickel (Ni) and chlorpyrifos can induce more abnormal gene expression in purple mussels [31]. The combination of Cd and diazinon can cause a significant decrease in the sperm production capacity of rats [32]. Some studies demonstrated that synergistic effects between pesticides and heavy metals can only occur at high concentrations [33]. The concentrations of copper sulfate and trichlorfon selected in this study are both production-related and much higher than their concentrations in natural water, which belong to relatively high concentrations, consistent with the above speculation. Studies have shown that the mechanism of synergistic effect between heavy metals and pesticides may be because that they can form complexes and promote their accumulation in the body [29]. Some studies have also shown that pesticides may promote the absorption of heavy metals and increase the toxicity of heavy metals [34]. The specific mechanism of the synergistic effect between copper sulfate and trichlorfon in this study is still unknown, and additional investigation is needed. However, it can be confirmed that their long-term combined effects will enhance the damage effect on the intestine of zebrafish, which is an important digestive and immune tissue of fish, and intestinal damage will affect the normal growth and development of fish. In this study, the weight of zebrafish decreased significantly after 21 days of combined exposure of copper sulfate and trichlorfon. But the synergistic mechanism of copper sulfate and trichlorfon is still unclear, and further research is needed to clarify.


## 5. Conclusions

In the present study, the toxic effects of copper sulfate and trichlorfon on the intestine of zebrafish were investigated. The results showed that copper sulfate and trichlorfon stimulated the activity of antioxidant enzymes in zebrafish after short exposure, but had no significant effect on intestinal histology. Copper sulfate and trichlorfon can cause intestinal mucosal damage after long-term exposure, which may be caused by inhibiting antioxidant enzyme activity and inducing intestinal oxidative stress injury, or damaging intestinal mucosal barrier by inhibiting intestinal tight junction protein gene expression. And the toxicity of copper sulfate and trichlorfon has a synergistic effect, and the combined use of the two will bring greater damage to the intestines of fish. The present results suggest that copper sulfate and trichlorfon should be used with caution in aquaculture, and attention should be paid to the duration and combination of the drug used.



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*Source: 8360334-2023-02-06.xml*