Does short-term exposure to a Neonicotinoid insecticide trigger Biochemical and Physiological responses in Juvenile catfish?

doi:10.21203/rs.3.rs-2193834/v1

Imidacloprid is among the most widely used insecticides and may contaminate surface waters, yet there is limited information available on their effects on fish. In the present study, juvenile catfish (Clarias gariepinus) were exposed to 100, 130, 160, and 190 mg/L of imidacloprid for 96 hours, and the effects on mortality, behavior, hepatic and endocrine functions, oxidative stress, and tumor and inflammatory responses were investigated. The 96 hours LC₅₀ was 166.60 (143.38-193.59 95% confidence intervals) mg/L with mortality recorded at concentrations ≥ 130 mg/L. Exposure to imidacloprid induced behavioral alterations and clinical symptoms including gulping, hypoactivity, abnormal surface distribution, loss of buoyancy, and excessive mucus secretion. Furthermore, amylase, lipase, conjugate bilirubin, carbohydrate antigen 19 − 9 (CA 19 − 9), thyroid stimulating hormone (TSH), and glutathione peroxidase (GPx) activity were significantly increased, while significant reductions were observed in triiodothyronine (T3) and superoxide dismutase (SOD) activity. Non-significant changes were observed in aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, 17β- estradiol (E2), thyroxine (T4), C-reactive protein (CRP), glutathione S-transferases (GST), total antioxidant capacity (TAC) and catalase (CAT) activity. Our results demonstrate that imidacloprid causes behavioral, hepatic, and endocrine toxicity in catfish as well as oxidative stress and tumor marker elevation. Our findings show that short-term exposure to imidacloprid triggers biochemical and physiological responses in juvenile catfish.

Toxicology

Behavioral Ecology

Animal Physiology

behavioral markers

imidacloprid

oxidative stress

thyroid hormones

reproductive hormones

hepatic markers

tumor marker

inflammatory marker

Pollution has taken the headlines in recent stories across the globe as more contaminants with varying magnitudes of toxicity are being introduced into aquatic and terrestrial environments. Man's attempt to improve the quality of life subjects the ecological system to enormous stress and deteriorating modifications.¹ These activities affect the health and survival of individual organisms, as well as their performance, and alter the function and structure of natural ecosystems, especially the aquatic ecosystem.² Commercial, industrial, and agricultural chemicals can cause several harmful effects on aquatic organisms when they enter the aquatic environment. Fish accumulate these chemicals, including pesticides, directly from the polluted water and indirectly from the food chain.^3,4

Over a thousand varieties of pesticides are used in the world today, with different properties and exploring diverse modes of action, with more than 80% of farmers relying on pesticides to secure their produce.⁵ Some pesticides like DDT and lindane have been proven to be very persistent in soil and water for many years after application.⁶ Most pesticides deviate from their main objective of the action, causing a variety of toxic effects in non-target species.⁷ As pesticides ultimately flow into the surface water, it is expected that aquatic environments are more affected than terrestrial environments.^8,9 Aquatic ecosystems are threatened by the pollution of these pesticides, thus the development and usage of pesticides with low ecological impact and toxicity to non-target organisms are pertinent for the preservation of these ecosystems and biodiversity.¹⁰

Great efforts have been made over the last three decades to produce pesticides that are specific in action, highly degradable, and have selective qualities that work on certain chemical and physiological aspects of insects, reducing the risk of non-target species. This tremendous advance in knowledge paved the way for a more effective group of insecticides, the neonicotinoids, of which imidacloprid is a widely used example. Neonicotinoids are a class of nitroguanidine systemic insecticides often used to treat soil and seeds before planting crops to prevent pest infestations.¹¹ Owing to their effectiveness against a wide range of insects and the breadth of their uses, as well as their functioning at low dosages with relatively low volatility and high solubility in water.^12,13,14 The idea that neonicotinoids function as nicotinic acetylcholine receptor (nAChR) agonists, which are thought to have little impact on vertebrate species and limited cross-resistance with other pesticides, is the basis for their appeal. Also, neonicotinoids have systemic activity, which allows them to spread more widely throughout plants than conventional pesticides to control the targeted pest species.¹⁰ However, neonicotinoids' high water solubility also makes them very mobile and, as a result, easily transferred into aquatic habitats, potentially causing harm to aquatic organisms that are not intended targets. Seven neonicotinoid pesticides are currently in use, including acetamiprid, dinotefuran, clothianidin, nitenpyram, imidacloprid, thiamethoxam, and thiacloprid.

Imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitro-imidazolidin-2-ylideneamine] is a neonicotinoid synthetic insecticide that forms an agonistic relationship with the postsynaptic nicotinic acetylcholine receptors^15,16 causing impairment of normal nerve function. It was the first neonicotinoid to be introduced in 1991, and it has since become a key product in several pest control operations.^17,18 Products containing imidacloprid dominate the insecticide industry and are authorized for use in more than 120 countries on more than 140 different types of crops.¹⁹ Its continuously increasing popularity not just in the control of household pets’ pests but especially in agricultural use can be attributed to its excellent effectiveness in the control of sucking, soil, and chewing insects.²⁰

Recently, numerous studies have focused on the toxicity of imidacloprid in the aquatic environment due to the negative effects of neonicotinoids on non-target beneficial invertebrates. Although there is little information available about its toxicity on aquatic vertebrates including fish, several studies have assessed the toxic effects of imidacloprid on several endpoints of different species of fish in various parts of the world.^14,21,22 Imidacloprid has been reported to cause DNA damage and oxidative stress¹⁴ decreased hatching rate of fertilized eggs²² and histopathological alterations in tissues²³ of different fish species. Nonetheless, no study has conducted a broad range of assessments of behavioral and physiological responses and biomarkers for a fish species. This is necessary to understand the underlying mechanism of action for imidacloprid toxicity in fish and other vertebrates.

Considering these knowledge gaps, this study aimed to assess the impacts of imidacloprid on Clarias gariepinus behavior, hepatic and endocrine functions, oxidative stress, and tumor and inflammatory responses. To achieve this objective, catfish were exposed to acute concentrations of imidacloprid for 96 hours. C. gariepinus was chosen for this study due to its wide distribution in tropical freshwater systems, adaptation to laboratory conditions, and stress resistance.²⁴

Fish Maintenance. Juvenile Clarias gariepinus (mean length = 10.73 ± 1.34 cm; mean weight = 12.34 ± 3.44 g) were purchased from Farm Project, Faculty of Agriculture, University of Benin, Nigeria and maintained in a 160 L plastic tank for two weeks according to OECD²⁵ at the Laboratory for Ecotoxicology and Environmental Forensics at the University of Benin, Nigeria.

Chemical Exposures. Chemical exposure was conducted through a static renewal system for 96 hours, following OECD guidelines for fish acute toxicity testing²⁵. The treatment groups consisted of four concentrations of imidacloprid: 100, 130, 160, and 190 mg/L using a commercially available insecticide, Imi Force® (Jubaili™), containing an imidacloprid concentration of 200 g/L, and a control group. The treatment concentrations were achieved by mixing 10, 13, 16, and 19 mL of the Imi force® insecticide in 20 L water to get the required imidacloprid concentrations of 100, 130, 160, and 190 mg/L respectively. No insecticide or chemical was added to the control group. The concentrations and duration were selected based on previous studies and a range-finding test. Each group was comprised of two replicates (n = 6). Mortalities were counted daily for LC₅₀ determination.

Behavioral Alterations and Mortality. The fish were inspected multiple times daily during the exposure period to record behavioral changes and clinical diagnostic signs through direct observation, according to OECD²⁵. The toxicological endpoint of this study was mortality. Mortality was recorded daily when the fish failed to respond to gentle prodding. The dead fish were removed immediately.

Sampling. All fish were euthanized after 96 hours of exposure to a lethal dose of benzocaine (0.5 g L^− 1; Sigma-Aldrich). Samples were selected randomly from each aquarium (n = 2), homogenized, and used for the assays.

Biomarker Analysis. The samples were used in the assay of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities according to Reitman and Frankel²⁶; total and conjugate bilirubin levels according to Jendrassik and Grof²⁷; amylase activity according to Bern²⁸; lipase activity according to Bier²⁹; 17β-estradiol (E2) and testosterone levels using an Estradiol EIA Kit (Cayman Chemical, USA) according to the manufacturer’s instructions; glutathione S-transferases (GST) activity according to Habig and Jakoby³⁰; carbohydrate antigen (CA-19-9) level according to Minamide et al.³¹; C-reactive protein (CRP) level according to Lowry et al.³²; total antioxidant capacity (TAC) activity according to Teimouri et al.³³; catalase (CAT) activity according to Beer and Sizer³⁴; glutathione peroxidase-1 (GPx) activity according to Hafemann et al.³⁵; superoxide dismutase (SOD) according to McCord and Fridovich³⁶; triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) using ELISA kit (Millipore-Sigma, USA) according to the manufacturer’s instructions.

Statistical Analysis. LC₅₀ was calculated with probit analysis using Microsoft Excel 2016. Statistical analyses were conducted with GraphPad Prism (version 5.01). All data were analyzed using a single factor one-way analysis of variance (ANOVA), followed by Dunnett’s posthoc test. Statistical significance was determined if P < 0.05.

Fish are ideal sentinels for behavioral and health assays of various stressors and toxic chemical exposure due to their constant direct contact with the aquatic environment, where chemical exposure occurs over the entire body surface. Imidacloprid is a promising and rapidly expanding pesticide, known for its selective toxicity to insects. Its detection in food and water prompts the verification of its possible negative effects on non-target organisms. To identify its effect on Clarias gariepinus, various biomarkers were evaluated along with mortality and behavioral changes. This present study contributes to the ongoing efforts to unravel the adverse effects and mode of action of imidacloprid and other neonicotinoid pesticides on non-target species. This study is the first to report behavioral alterations and endocrine disruption in Clarias gariepinus after imidacloprid exposure, as well as the effects of imidacloprid on Ca 19-9 in fish. The results of this study show that exposure to different imidacloprid concentrations adversely impacted C. gariepinus.

Imidacloprid LC₅₀, Mortalities, Fish Behavior, and Clinical Symptoms. The water temperature and pH in the aquariums throughout the toxicity testing were 26.64 ± 0.09 ℃ and 6.74 ± 0.05 respectively. The survival rate of fish in the control was 100%, indicating that the toxicity data were acceptable.

The mortality rate was time- and concentration-dependent, with % mortality peaking at 66.67% after 96 hours of exposure. Mortality was observed at concentrations ≥130 mg/L. (Table 1). The 96-h LC₅₀ of imidacloprid was 166.60 mg/L (95% confidence intervals: 143.38-193.59 mg/L). This quantity is substantially higher than the environmental values already measured and is unlikely to occur in aquatic systems. The value obtained in this study is considerably higher than the previously reported 96-h LC₅₀ (10.16 µg/L) for C. gariepinus.²¹ However, similar values have been reported for imidacloprid in other fish species, including Danio rerio (143.7 mg/L)³⁷ and Misgurnus anguillicaudatus (145.8 mg/L)³⁸.

Table 1. Cumulative mortality of Clarias gariepinus exposed to various concentrations of imidacloprid

Imidacloprid concentration (mg/L)	Number of exposed fish	Cumulative mortality of fish per time interval (h)				Mortality %
Imidacloprid concentration (mg/L)	Number of exposed fish	24	48	72	96	Mortality %
0	12	0	0	0	0	0
100	12	0	0	0	0	0
130	12	0	0	1	3	25
160	12	1	1	3	5	42
190	12	3	6	7	8	67

Fish behavior varied with concentration and time of exposure. All exposed fish showed noticeable distress symptoms, represented by under-reactivity to stimulus, hypoventilation, gulping, abnormal surface distribution, and poor swimming movement (Table 2). Loss of buoyancy was also observed in fish, a few hours before mortality. Fish exposed to ≥ 130 mg/L imidacloprid were severely hypoventilated, exhibiting almost no opercular movement, while fish in ≥ 160 mg/L constantly gasped for air at the water's surface. This could be a result of imidacloprid affecting fish gills directly, which may affect fish respiration.³⁹ Although fish activity was largely reduced in all exposed fish compared to the control, fish activity in the 100 mg/L groups showed improvement on the final day of observation. Furthermore, mucus secretion in all exposed fish peaked after 48 hours of exposure but declined after 72 hours. These observations of fish activity and mucus secretion suggest that catfish may recover from acute behavioral effects caused by imidacloprid after a prolonged time of exposure. Literature on the effects of imidacloprid on fish behavior is scarce, however, imidacloprid has been reported to cause similar behavioral alterations by Qadir et al.⁴⁰ in Labeo rohita exposed to 120 mg/L imidacloprid for 8 days. Qadir et al.⁴⁰ also observed that as exposure time increased, fish tended to recover from the disturbing condition and the occurrence of abnormal behavior declined, similar to our findings. The behavioral changes observed in our study could be attributed to the effect of imidacloprid on the fish nervous system via impairment of brain activity and acetylcholinesterase level alteration, resulting in the impediment of many physiological functions.^41,42

Table 2. Behavioral abnormalities of C. gariepinus due to the exposure to imidacloprid within the 96-h observation period

Concentration (mg/L)	Hypo activity	Abnormal surface distribution	Under-reactivity to stimuli	Hypoventilation	Gulping	Mucus secretion
24h
0	-	-	-	-	-	-
100	+++	+++	++	+++	++	++
130	+++	+++	+++	+++	++	++
160	+++	+++	+++	+++	+++	+++
190	+++	+++	+++	+++	+++	+++

48h
0	-	-	-	-	-	-
100	+++	+++	++	+++	++	++
130	+++	+++	++	+++	+++	++
160	+++	+++	+++	+++	+++	+++
190	+++	+++	+++	+++	+++	+++

72h
0	-	-	-	-	-	-
100	+++	+++	++	+++	++	+
130	+++	+++	+++	+++	+++	+
160	+++	+++	+++	+++	+++	++
190	+++	+++	+++	+++	+++	++

96h
0	-	-	-	-	-	-
100	++	++	++	+++	++	-
130	+++	+++	+++	+++	+++	-
160	+++	+++	+++	+++	+++	-
190	+++	+++	+++	+++	+++	-

'-' indicates no behavioral abnormality; '+' indicates the number of individuals showing behavioral abnormalities; '+'= Mild (around 30 to 40% of individuals showing behavioral abnormalities); '++' = Moderate (around 45 to 65% of individuals showing the behavioral abnormalities); ‘+++’ = Severe (≥ 70% of individuals showing the behavioral abnormalities).

Hepatic Biomarkers. Concentrations of ALT and AST were increased in fish exposed to imidacloprid, however, these changes were not significantly different from the control. ALT levels increased with increasing concentrations of imidacloprid until 160 mg/L peaking at a 176% difference from the control. AST levels increased considerably in fish exposed to imidacloprid, with a difference of more than 17 folds observed at 130 mg/L (Table 3). The increased enzymatic activities could result from elevated enzyme production to counter the harm caused by the insecticide or a disturbance in the cell membrane integrity, preventing the enzymes from moving freely.^43,44,45 Increases in ALT and AST levels have previously been reported in C. gariepinus and other fish species exposed to neonicotinoids.^40,46,47 Desai and Parikh⁴⁶ studied the biochemical effects of imidacloprid on Labeo rohita and Oreochromis mossambicus. Fish exposure to imidacloprid for 21 days elevated AST and ALT levels in several tissues of Oreochromis mossambicus and Labeo rohita.⁴⁶ Increased activities of ALT and AST could be due to amplified transamination processes that occur as a result of amino acid input into the tricarboxylic acid cycle to manage energy crises during toxicant-based stress.^48,49 The likelihood of the elevation of the enzymes due to an energy crisis is supported by our observations on amylase and lipase activity in exposed groups.

Amylase activity in catfish increased with increasing concentrations of imidacloprid. A significant increase in amylase activity was observed in catfish exposed to 190 mg/L imidacloprid. Similarly, a concentration-dependent increase was observed in lipase activity. A significant difference from the control was observed in fish exposed to ≥ 130 mg/L with the confidence intervals increasing with each observed concentration of imidacloprid (Table 3). Amylase and lipase are important enzymes that are directly related to carbohydrate and lipid metabolism. Amylase is secreted by the exocrine region of the pancreas. The elevated activity could be the result of amylase secretory cell damage or pancreatitis.⁴⁵ Alteration in serum amylase indicates an abnormality of the digestive process.⁵⁰ In biological systems, lipase is crucial for maintaining the integrity of membranes and cell signaling.⁵¹ Increased lipase activity could be due to tissue damage, the development of a stress situation, or the effective use of lipid molecules for digestion to match the high energy demand during the imidacloprid-induced stress condition.⁵² Literature available on the effects of imidacloprid on lipase and amylase activities in fish is limited, however, increases in their activities in several fish species due to other pesticides have been reported.^50,51,52,53 Our study indicates that imidacloprid can impact fish metabolic activities, forcing increased energy production through carbohydrate and lipid metabolism.

No significant difference was observed between total bilirubin levels in control and catfish exposed to imidacloprid. The total bilirubin levels in exposed catfish were generally higher than the control, with a drop below the control level observed only in the 190 mg/L groups. In contrast, a significant difference from the control was observed in conjugate bilirubin levels of the fish exposed to 130 mg/L. However, similar to the patterns observed in total bilirubin levels, conjugate bilirubin levels of catfish exposed to imidacloprid were generally higher than that of the control, with a drop below control levels observed only in the 190 mg/L groups (Table 3). Bilirubin is one of the main bile pigments found in the fish circulatory system and is derived from hemoglobin disruption.⁵⁴ The increased bilirubin production could result from imidacloprid inducing increased red blood cell breakdown or reduced hepatic uptake of bilirubin due to the decreased transport protein-albumin level⁵⁵, severe damage to hepatocytes, and hindrance of the bile duct.⁵⁶ Increase in total and conjugate bilirubin levels have also been linked to liver and biliary tract disease.⁵⁷ Experimental studies have shown that exposure of C. gariepinus and other fish species to neonicotinoids, including imidacloprid, causes an increase in bilirubin levels^40,58 which among others has been attributed to acute hepatic necrosis.⁵⁸

Table 3. Effects on hepatic functions of C. gariepinus after 96 hours of exposure to imidacloprid

Imidacloprid (mg/L)	ALT (U/l)	AST (U/l)	Amylase (U/l)	Lipase (U/l)	Total bilirubin (µmol/l)	Conjugate bilirubin (µmol/l)
0	3.38 ± 1.24	4.45 ± 0.15	3.70 ± 1.60	0.15 ± 0.03	0.31 ± 0.04	0.06 ± 0.02
100	7.51 ± 3.22	11.75 ± 1.05	7.95 ± 0.55	0.44 ± 0.15	0.37 ± 0.04	0.14 ± 0.01
130	8.58 ± 2.15	78.22 ± 18.22	10.05 ± 4.75	0.55 ± 0.03*	0.58 ± 0.31	0.28 ± 0.07*
160	9.35 ± 2.92	67.50 ± 20.40	17.45 ± 4.75	0.70 ± 0.03**	0.37 ± 0.14	0.18 ± 0.01
190	6.43 ± 0.00	66.45 ± 21.45	23.80 ± 1.60*	1.13 ± 0.01***	0.12 ± 0.01	0.06 ± 0.01
P value	0.4537	0.0541	0.0340	0.0013	0.4383	0.0189

The values are mean ± standard error of two fish replicates. *, ** and *** indicate significant difference between exposure and control group at p < 0.05, 0.01 and 0.001 respectively.

Endocrine Disrupting Biomarkers. Catfish 17β-estradiol (E2) levels increased with increasing concentrations of imidacloprid, having an 85% increase from control levels in the group exposed to 190 mg/L. However, these increments were not significantly different from the control. Testosterone levels in catfish fluctuated minimally with increasing concentrations of imidacloprid, with a maximum 5% difference from the levels observed in the control fish. These changes were not significantly different from the control as well (Table 4). The primary internal regulators of gonadal differentiation and its activities are sex steroid hormones.⁵⁹ They influence how the brain is organized during the formative stages and sex-dependently coordinate a range of behaviors.⁶⁰ They also have an impact on the neurological, skeletal, and cardiovascular systems, as well as stress and immunological responses.^61,62 E2 is a major sex hormone that controls female fish maturity, and high E2 levels are a reliable sign of endocrine disturbance.⁶³ Elevated E2 concentrations are especially worrisome because they can upset the endocrine system's delicate balance, impairing reproductive function and resulting in lower fecundity and fertility, repressed sexual behavior, and altered sexual differentiation.⁶³ Fish immune systems and numerous leukocyte functions are regulated by estrogens.⁶⁴ Acidophilic granulocytes, which have been linked to tissue damage⁶⁵, migrate from the head kidney as a result of an increase in E2 levels following an inflammatory reaction.⁶⁶ This is the first study on the effects of imidacloprid on catfish sex steroid hormones. Experimental studies have shown that Clarias and several fish species' exposure to pesticides and other stressors cause elevations in E2 levels.^{63,67,68,69,70} Additional research is necessary to identify whether imidacloprid affects E2 levels directly or indirectly through the conversion of testosterone to E2, slowed sex steroid hormone degradation mechanisms, or at the hypothalamic-pituitary-gonad axis. The findings indicate that additional research is required to fully understand the negative effects of imidacloprid on the reproductive endocrine axis, regardless of the mode of action that led to the elevated E2 reported in this study.

Significant differences were observed between T3 levels in the control and catfish exposed to 190 mg/L. T3 and T4 levels were generally reduced with increasing concentrations of imidacloprid. However, no significant difference was observed between T4 levels in control and fish exposed to imidacloprid. In contrast, TSH levels of fish increased on exposure to imidacloprid, with a significant difference observed between the control and 190 mg/L groups (Table 4). Thyroid hormones in fish regulate growth, development, metabolism, skin pigmentation, behavior, metamorphosis, osmoregulation, and lipid and protein metabolism, often in association with growth hormone and cortisol.^71,72,73,74 Production of T3 is largely by peripheral enzymatic monodeiodination of T4 majorly in the liver and other tissues.⁷⁵ Typically, a decrease in T4 synthesis and secretion is what causes T3 levels to decrease.⁷¹ In the present study, a decrease in the level of thyroid hormones might be indicative of metabolic energy redirecting away from the anabolic process, which is essential to carry through life, thereby triggering thyroid endocrine disruption.⁷⁶ Thyroid hormone levels are regulated by the negative feedback that thyroid hormones have on their upstream regulators. Reductions in T3 or T4 enhance the production of TSH by the pituitary gland, hence our findings could be a reflection of this mechanism. Ghayyur et al.⁷⁷ reported similar findings in their 30-day exposure of Cirrhinus mrigala to the neonicotinoid acetamiprid. They observed significant increases in thyroid hormones and a significant reduction in TSH levels in their experiment after fish exposure to acetamiprid, chlorfenapyr, and dimethoate. Saha et al.⁷⁸ observed a reduction in thyroid hormone levels in Clarias batrachus after chronic exposure to diazinon and suggested these reductions could be due to pesticide effects on thyroid hormone signaling and metabolism in the hypothalamic-pituitary-thyroid axis.^78,79 Our study highlights the disruptive endocrine properties of imidacloprid, confirming its potential to adversely affect normal ecological functions and the survival of fish.

Table 4. Effects on endocrine functions of C. gariepinus after 96 hours of exposure to imidacloprid

Imidacloprid (mg/L)	17β-estradiol (ng/ml)	Testosterone (ng/ml)	T3 (ng/ml)	T4 (µg/dL)	TSH (µlm/mL)
0	68.60 ± 4.00	0.80 ± 0.01	1.05 ± 0.05	7.40 ± 1.30	0.70 ± 0.00
100	106.30 ± 15.95	0.80 ± 0.01	1.00 ± 0.00	6.50 ± 0.80	0.80 ± 0.00
130	108.70 ± 16.20	0.84 ± 0.01	1.05 ± 0.05	5.45 ± 0.25	0.80 ± 0.00
160	111.50 ± 5.65	0.81 ± 0.02	0.90 ± 0.00	5.35 ± 0.75	0.80 ± 0.00
190	126.60 ± 17.40	0.78 ± 0.01	0.85 ± 0.05*	4.35 ± 0.35	0.95 ± 0.05**
P value	0.1561	0.0551	0.0448	0.2019	0.0047

The values are mean ± standard error of two fish replicates. * and ** indicate a significant difference between the exposure and control group at p < 0.05 and 0.01 respectively.

Oxidative Biomarkers. Significant reductions were observed between SOD activity in control and exposed fish at different concentrations of imidacloprid. GPx activity of fish increased in groups exposed to ≥ 130 mg/L imidacloprid. A significant difference between GPx activity in control and exposed fish was observed in 130 mg/L imidacloprid concentration. A non-significant increase in CAT, TAC, and GST activity of the exposed groups compared to the control group was observed (Table 5). Pesticide exposure has the potential to cause oxidative damage.⁸⁰ To deal with oxidative stress and prevent excessive reactive oxygen species (ROS) formation, which can cause oxidative damage to nucleic acids, lipids, and proteins, fish produce a variety of anti-oxidative defenses including GPx, CAT, and SOD.^21,41,81,82 The role of GST is to catalyze the detoxification of harmful compounds via the conjugation of glutathione.⁸³ The current study demonstrated reduced SOD, and elevated GPx, CAT, GST, and TAC in C. gariepinus exposed to imidacloprid. An increase in CAT, GPx, and TAC activity could be an indication that imidacloprid caused an overproduction of ROS in exposed fish.¹⁴ The reduced SOD activity may be due to the elevated oxygen free radicals in catfish, rendering SOD inactive through oxidation.⁸⁴ Additionally, elevated quantities of H₂O₂ and superoxide anions may be the cause of the rise in CAT activity.⁸⁵ The increase in H₂O₂ levels is supported by Svensson et al.⁸⁶ who reported that the sulfhydryl of microsomal GST’s 49th cysteine could specifically combine with H₂O₂, thus enhancing GST activity. Nnadi et al.²¹ reported significant SOD activity reductions and CAT activity elevations in C. gariepinus exposed to imidacloprid, similar to our findings. However, contrary to our results, they found that imidacloprid induced a significant reduction in GPx activity in exposed catfish. Several studies have reported oxidative stress induced by imidacloprid in various fish species such as Danio rerio⁸⁷, Oncorhynchus mykiss⁴¹, Oreochromis niloticus⁸⁸, and Prochilodus lineatus¹⁴. Our study further affirms the potential of imidacloprid to induce oxidative stress in fish.

Table 5. Effects on oxidative stress of C. gariepinus after 96 hours of exposure to imidacloprid

Imidacloprid (mg/L)	SOD (U/ml)	GPx (Unit/ml)	CAT (mmol/min/ml)	TAC (mmol/L)	GST (mmol/mi/min)
0	1.81 ± 0.07	11.20 ± 2.80	22.30 ± 3.10	1.59 ± 0.16	0.55 ± 0.05
100	1.44 ± 0.14	11.20 ± 0.00	44.60 ± 12.80	1.92 ± 0.01	1.40 ± 0.40
130	1.26 ± 0.08*	36.45 ± 5.65*	36.95 ± 6.85	1.80 ± 0.04	1.20 ± 0.10
160	1.35 ± 0.08*	28.00 ± 2.80	40.15 ± 4.55	1.81 ± 0.05	3.60 ± 2.60
190	1.58 ± 0.00	33.65 ± 8.45	40.35 ± 3.15	1.81 ± 0.13	3.15 ± 2.05
P value	0.0244	0.0353	0.3369	0.3080	0.5814

The values are mean ± standard error of two fish replicates. * indicates a significant difference between the exposure and control group at p < 0.05.

Tumor and Inflammatory Biomarkers. Significant increases (p = 0.0270) between CA 19-9 levels in control and fish exposed to imidacloprid were observed at imidacloprid concentrations ≥ 160mg/L (Figure 1A). Increases in serum CA 19-9 are helpful in the diagnosis of upper gastrointestinal tract adenocarcinoma and the monitoring of colonic carcinoma, but their greatest sensitivity has been found in the detection of pancreatic adenocarcinoma, according to various studies.^89,90 This is the first study assessing pesticide effects on fish CA 19-9 levels. Literature on the effects of toxicants on CA 19-9 in animal studies is incredibly scarce, however, Omnia et al.⁹¹ observed a significant increase in CA 19-9 levels in rats exposed to 1,2-dimethylhydrazine, similar to our results. Our findings indicate that imidacloprid may pose a high risk of malignant and pancreaticobiliary diseases in fish. However, Kim et al.⁹² found CA-19-9 elevation in patients with pulmonary, gynecologic, endocrine, and splenic diseases who did not have a malignant and pancreaticobiliary disease.

There was no significant difference (p = 0.0807) between CRP levels in the control and imidacloprid-exposed groups. Furthermore, the CRP levels fluctuated, and the comparison of these two groups did not yield a consistent pattern over time, having only minor deviations from the control group levels except in the group exposed to 190 mg/L (Figure 1B). As an initial line of defense against environmental contaminants, toxic chemicals cause fish to produce more non-antibody proteins like CRP.⁹³ CRP is mostly produced in the liver and is responsible for several immunological responses.⁹⁴ Our studies reveal that although imidacloprid has a mild effect on CRP levels at concentrations below the 96-h LC₅₀, its effects become more noticeable at higher concentrations, where a 32% increase in CRP levels was observed in catfish exposed to 190 mg/L imidacloprid. Currently, there is no comprehensive data available on CRP levels due to imidacloprid and other neonicotinoid exposure in fish with which to compare this data. However, elevated CRP levels have been observed in Clarias species on exposure to various stressors.^95,96 Chatterjee⁹⁶ exposed C. gariepinus to endosulfan for 96 hours and observed a steady rise in CRP levels. Furthermore, pesticides, as well as other physiological and chemical stressors, have been reported to increase CRP levels in several fish species.^93,97,98,99 This elevated level of CRP in the acute phase response indicates that fish exposed to imidacloprid may develop inflammatory responses.

In conclusion, short-term exposure to imidacloprid, a neonicotinoid insecticide, now largely used worldwide as a replacement for more toxic and persistent pesticides triggered biochemical and physiological responses in juvenile catfish (Clarias gariepinus). The present study clearly showed that imidacloprid has the potential to disrupt various physiological and behavioral functions in fish, which could lower the probability of survival. While extensive research has focused on the effects of imidacloprid on fish behavior, transaminases, and oxidative stress, our work highlights the need for further examination of its effects on hepatic and endocrine functions, as well as tumor and inflammatory responses. Whether or not the environmentally relevant concentrations of imidacloprid will cause these same effects in catfish remains yet to be known. Controlled use of imidacloprid-based pesticides, especially close to aquatic ecosystems, is therefore essential.

Ethics approval and consent to participate

Ethical approval for this research was given by the Research Ethics committee of the College of Medicine of the University of Benin. CMS/REC/2020/34.

Consent for publication

Not applicable.

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest

The authors declare no conflict of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

Nosakhare O. Erhunmwunse: Conceived and designed the experiments. Timeyin J. Pajiah, Junior V. Ogodo performed the experiments. Nosakhare O. Erhunmwunse, Timeyin J. Pajiah, Junior V. Ogodo: Analyzed and interpreted the data. Nosakhare O. Erhunmwunse, Timeyin J. Pajiah, Junior V. Ogodo, Endurance Ewere: Materials, analysis tools, and ideas. All authors have read and approved the manuscripts.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

We appreciate the diligence, and relentless efforts of Mr. Christopher who helped us all through the work.

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Graphical Abstract

Does short-term exposure to a Neonicotinoid insecticide trigger Biochemical and Physiological responses in Juvenile catfish?

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Abstract

Figures

Introduction

Materials And Methods

Results And Discussion

Declarations

References

Supplementary Files

Status:

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