Sub-lethal effects of organophosphates and synthetic pyrethroid insecticides on muscle tissue transaminases of Oreochromis niloticus in vivo

Abstract

Organophosphates and synthetic pyrethroid insecticides have been commonly used in public health and agriculture. The present study aimed to evaluate the sub-lethal effects of organophosphates and synthetic pyrethroid insecticides on transaminases: glutamate oxaloacetate/aspartate transaminase (AST) and glutamate pyruvate/alanine transaminase (ALT) in Oreochromis niloticus. Fish were exposed to malathion (OP), chlorpyrifos (OP) and λ-cyhalothrin (synthetic pyrethroid) at sub-lethal concentrations of 1.425, 0.125 and 0.0039 ppm, respectively for 24 and 48 h. AST and ALT activities were shown to be remarkably (p < 0.05) decreased and increased, respectively in O. niloticus treated with the insecticides. The highest and lowest inhibition in AST level were noted as -12.2% and -12.2% in chlorpyrifos and λ-cyhalothrin 24 h treated fish samples, respectively. The highest and lowest elevation in ALT level were recorded as + 313% and 237% in 48 h chlorpyrifos and 24 h malathion treated fish samples, respectively. This indicates that the insecticides used in this study did not result in death but in changes in AST and ALT enzyme activities. Therefore, organophosphates (malathion, chlorpyrifos) and synthetic pyrethroid (λ-cyhalothrin) insecticides are toxic to fishes and could affects their survival in their natural habitat.

Introduction

Organophosphate and synthetic pyrethroid insecticides have been used globally in agriculture, forests, wetlands and households because they are highly biodegradable and detrimental to insects and less harmful to mammals and birds [1, 2]. Organophosphate insecticides are phosphoric organic ester derivatives, usually amide or thiol analogues of phosphoric, thiophosphoric or phosphinic acid with extra-sided chains of cyanide, phenoxy and thiocyanate moiety and their approximate global usage reach to 50% [3–5]. Several organophosphate insecticides, including malathion and chlorpyrifos, have recently been identified. λ-cyhalothrin is a new type of a pyrethroid insecticide that has harmful effects on the aquatic ecosystem and human health [6, 7]. They are sometimes used with other types of insecticides such as the organochlorines and organophosphates, due to their hyper ingestion and touch toxicity abilities as well as their high efficacy on a wide variety of insects [8, 9]. Other relevant advantages of this group of insecticides have been observed including light stability, low-dose efficiency and low adverse effects on mammalian and avian groups [10].Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Also, kindly confirm the details in the metadata are correct.I have gone through the author names. Amendment was made to author 8 "Rania M. Waheed" was replaced with "Eida M. Alshammari". This was an error during submission.

According to El-Sayed et al. [11], insecticides have also an adverse impact on fish health and survival because they are often subjected to the stressful conditions of these insecticides. In the aquatic habitats, these chemicals may trigger diseases that causes ecosystem imbalances and affects fish immunity [12, 13]. Over a long time, organophosphates are the most widely used pesticides, example is the use of malathion since the 1950s. Hence, these pesticides occur in almost all aquatic reservoirs and ecosystems. These pesticides are taken up by aquatic organisms from the surrounding water and their particles are suspended in the water column [14]. Therefore, most current toxicological studies involves aquatic organisms. These organisms are used as model systems for assessing cellular damage, tissue injury and free radicals protection [15]. Aspartate transaminase (AST) and alanine amino transferase (ALT), also known as glutamic oxaloacetic transaminase (GOT) and glutamate pyruvate transaminase (GPT), respectively are biomarker enzymes for estimating organ injury or damage and they play a critical role in amino acid metabolism [16, 17]. Insecticides affect the activities of the transaminases (AST and ALT), which are vital biomarkers for detecting tissue injury in aquatic animals such as fish [18].Kindly check and confirm the inserted city name in affiliations.Affiliations are correct.

Moreover, pyrethroid toxicity in water is not negligible. For instance, LC₅₀ of some pyrethroids such as bifenthrin, tefluthrin and allethrin in trout was 150 (0.15 ppm), 0.06 (0.00006 ppm) and 19 (0.019 ppm) µg/L, respectively [19]. In literature, there were no studies on pyrethroid bioaccumulation in wild fish tissues. However, some researchers had investigated the bioaccumulation in treated fishes, these studies reported high bioaccumulation [20, 21]. Corcellas et al. [22] reported the bioaccumulation of pyrethroid in B. guiraonis and Salmo trutta as 68 (0.068 ppm) and 4934 (4.934 ppm) ng/g, respectively. Few studies were conducted on the bioaccumulation of pesticide residues in the muscles of Nile tilapia, a study reported wide difference in the number of detected pesticides between pond water samples and musculature tissue with 6 and 18 pesticides detected respectively [23]. The bio-accumulated pyrethroids found in samples of Nile tilapia collected from Egypt were cypermethrin (0.015 ppm) and lambda-cyhalothrin (0.005 ppm) [24].

Nile tilapia (Oreochromis niloticus) are valuable and abundant source of protein for humans worldwide. Nevertheless, the accumulation of insecticides in this fish leads to losses, it also has negative impact on human health on consumption. The bioaccumulation of insecticides by this fish could therefore harm humans and other animals [25].

The present study was conducted to evaluate and compare the effects of sub-lethal concentrations of organophosphates insecticide (malathion and chlorpyrifos) and synthetic pyrethroid (λ-cyhalothrin) on the activities of AST and ALT enzymes in the muscles tissue of exposed O. niloticus. The observations of the toxicological effects of these pesticides on O. niloticus will add to existing knowledge as well as provide information that will help in the management plan of these pesticides.

Materials and methods

Test chemicals

Three insecticides which include; malathion (OP) 57% EC, chlorpyrifos (OP) 40% EC, and λ-cyhalothrin 2.5% EC (synthetic pyrethroid were obtained from STEDEC Technology Commercialization Corporation Limited, Pakistan. Deionized water was used for the preparation of different concentrations of the insecticides, these concentrations were prepared using serial dilution formula C₁V₁ = C₂V₂. The structures of the organophosphate (malathion and chlorpyrifos) and synthetic pyrethroid (λ-cyhalothrin) insecticides used are shown in Fig. 1.

Fig. 1.

Structure of malathion, chlorpyrifos and λ-cyhalothrin

Sub-acute bioassays

Oreochromis niloticus (with mean ± SD total length 12.12 ± 0.6 cm) of Cichlidae family were obtained from the Chilia hatchery Thatta District of Sindh, Pakistan. Plastic bags with aerated water were used to transport the fish to the toxicology research laboratory at the Department of Zoology University of Karachi. Fish were housed in glass aquaria of 57 × 30 × 30 cm dimension, containing dechlorinated water, the fish were acclimatized for fifteen days under laboratory conditions. During acclimatization, the water of each aquarium was refilled every 24 h and aerated regularly by an electric aerators. Fish were fed once daily at fixed quantity with pellets of Oryza, containing 40% protein purchased from Pvt Limited. The water physicochemical properties, including temperature of 26 ± 0.7 °C, dissolved oxygen 5.5 ± 0.5 mg/l, ammonia (NH₃) 1.0 ± 0.1 mg/l, nitrate of 1.2 ± 0.4 mg/l, and pH 7.08 ± 0.2 were determined based on the method of APHA (2005) [26]. After 15 days of acclimation, the sub-acute semi-static test was conducted using OECD (1992) guideline [27]. Ten fish were added to each aquarium (25 × 25 × 25 cm) containing ten liter water. Separate aquarium was used for each chemical along with three control groups. After the preliminary trial, sub-lethal concentrations of each insecticide were selected for biochemical procedures. The test fish were exposed to sub-lethal concentrations of 1.425, 0.125 and 0.0039 ppm malathion, chlorpyrifos and λ-cyhalothrin, respectively for 24 and 48 h. The mortality and behavior of the fish were observed regularly.

Method of euthanasia used in killing of fish

Prior to dissection, experimental fish from the treated and control groups were removed. Concussion method of euthanasia was applied, which involves a blow on the back of the head. Death was confirmed by destruction of the brain.

Biochemical assays

Fish were dissected using hand dip nets after 24 and 48 h of exposure. Muscle tissue (1 g) of both control and treated groups was crushed in 5 ml deionized water by using mortar and pestle. The crushed muscle was then placed in a glass container and homogenized at 1000 rpm for 10 min using a Teflon coated tissue grinder (JJ-1 mechanical stirrer, No. 515069). Homogenates were centrifuged for 30 min at 4500 rpm using a centrifuge machine (Changzhou Guohua Electric Appliance Co, Ltd, Model 800 D). The supernatant was then transferred into quartz cuvettes (1 cm light path) and placed in spectrophotometer (Model: MOLEQULE-ON Ingenious) for estimating the activities of AST and ALT [28, 29]. The activities of AST and ALT were analyzed by IFCC method of Murray using kits (Spinreact S.A.U/SPINREACT, S.A. Ctra. Santa Coloma). Kits numbered LIQ298A and LIQ203A were used for AST and ALT, respectively [22, 23]. For AST, analysis, the substrate solution, L-aspartic acid (reagent, 500 µL; pH, 7.8) was added to a 10μL sample and incubated at 37 ºC for 1 min. For ALT analysis, buffered L-alanine, 2-oxoglutarate substrate (reagent, 500 µL; pH, 7.8) was added to a 10 µL sample and incubated at 37 ºC for 1 min. Initial initial absorbance reading was taken at 340 nm using spectrophotometer and the optical density (DO) reading was noted after 1, 2 and 3 min. The activity of AST and ALT was expressed as U/L.

$$\mathrm{Calculation}:\mathrm{U}/\mathrm{L}=\mathrm{\Delta }\mathrm{A}340\mathrm{nm}/\min \times 1750(\mathrm{Factor})$$

where ΔA 340 nm/min is directly proportional to the activity of AST and ALT in the sample.

Statistical analysis

The data obtained were subjected to descriptive statistics (mean and standard deviation). One way analysis of variance was performed and Tukey HSD test was used to test significant difference between control and treated groups. Analysis was performed using the Statistical Package for Social Sciences (SPSS version 20.0). Results at P < 0.05 were considered statistically significant. Enzymes percent enhancement (PE) and inhibition (PI) were calculated with the formula C ─ T/C × 100 of Ilahi et al. [30,

Results

Mortality and biochemical examination

Mortality and behavioral alterations were not observed in both control and treated groups. AST and ALT activities were significantly changed in the fish groups exposed to all insecticides used when compared to control group.

Glutamic oxaloacetic transaminase (GOT/AST) activity in fish exposed to insecticides

The AST mean activity in response to the three insecticides after 24 and 48 h is presented in Table 1. All fish groups exposed to the three insecticides showed consistency in enzyme inhibition at 24 and 48 h, except for λ-cyhalothrin treated group that showed no inhibition at 48 h. The highest (− 95.8%) and lowest (− 12.2%) inhibition in the AST activity was observed in chlorpyrifos and λ-cyhalothrin treated samples at 24 h, respectively. Figure 2 showed significant AST percent activity. The maximum and minimum percent activity of AST were recorded as 87.7% and 4.1% for λ-cyhalothrin and chlorpyrifos 24 h treated samples, respectively compared to the control one.

Table 1.

Effect of malathion, chlorpyrifos and λ-cyhalothrin on glutamic oxaloacetic transaminase (GOT/AST) in the muscle tissue of O. niloticus

Groups	Exposure time	GOT/AST activity	95% confidence interval		PI	p-value
Control	24 h	857.15a ± 2.05	859.47	854.84	–	p < 0.05
	48 h	682.26a ± 2.02	684.55	679.97	–
Malathion	24 h	468.03c ± 4.47	473.08	462.97	− 45.3
	48 h	405.61d ± 6.49	412.96	398.27	− 40.5
Chlorpyrifos	24 h	35.39e ± 7.07	43.39	27.40	− 95.8
	48 h	180.66f ± 6.42	187.93	173.40	− 73.5
λ-cyhalothrin	24 h	752.36g ± 7.35	760.68	744.04	− 12.2
	48 h	1055.66 h ± 5.07	1061.40	1049.93	–

Mean activity ± standard deviation, PI—enzymes percent inhibition, superscript letters indicate statistical significance at p < 0.05

Fig. 2.

Effect of malathion (1.425 ppm), chlorpyrifos (0.125 ppm) and lambda-cyhalothrin (0.0039 ppm) on the percent activities of AST in the muscle tissue of O. niloticus

Glutamate pyruvate transaminase (GPT/ALT) activity in fishes exposed to insecticides

ALT activity in fishes exposed to the three insecticides is presented in Table 2. The activity of these enzymes was remarkably increased in insecticide-treated fish compared to the control group. The highest (+ 313%) and lowest (+ 237%) ALT activity was recorded at 48 h for chlorpyrifos and at 24 h for malathion-treated groups, respectively. Figure 3 showed the percent activity of ALT in fishes exposed to insecticides. The maximum and minimum percent activity were 413% and 337% at 48 h for chlorpyrifos and 24 h for malathion-treated groups, respectively, compared to the control one.

Table 2.

Effect of malathion, chlorpyrifos and λ-cyhalothrin on glutamic pyruvic transaminase (GPT/ALT) in the muscle tissue of O. niloticus

Groups	Exposure time	GPT/ALT activity	95% confidence interval		PE	p-value
Control	24 h	98.24 ± 0.65a	98.97	97.51	–	p < 0.05
	48 h	91.63 ± 0.57a	92.27	90.99	–
Malathion	24 h	337.40 ± 0.62b	338.10	336.69	237
	48 h	355.13 ± 2.03c	357.43	352.84	282.5
Chlorpyrifos	24 h	376.92 ± 0.78d	377.81	376.04	283.6
	48 h	378.82 ± 21.31e	402.94	354.70	313
λ-cyhalothrin	24 h	331.40 ± 6.26f	338.49	324.32	237.3
	48 h	361.98 ± 0.53g	362.58	361.39	294

Mean activity ± standard deviation, (PE) percent enhancement, superscript letters indicate statistical significance

Fig. 3.

Effect of malathion (1.425 ppm), chlorpyrifos (0.125 ppm) and lambda-cyhalothrin (0.0039 ppm) on the percent activities of ALT in the muscle tissue of O. niloticus

Discussion

Transaminases are enzymes involved in the catabolism of amino acids, which convert the amino group from one amino acid to another keto acid for gluconeogenesis. These enzymes act as a link between carbohydrate and protein metabolism under altered physiological, pathological and stimulating environmental stress conditions. Changes in AST and ALT activity indicate tissue injury [16, 17, 31, 32]. AST and ALT are biomarker enzymes used to ascertain the level of organ damage in organisms after exposure to certain chemicals. De Smet and Blust [33], reported that both AST and ALT enzymes catabolise protein in animals exposed to pesticides for energy production. In addition, AST and ALT are used to diagnose disease and tissue injury induced by environmental pollution [34, 35]. To estimate the effect of the insecticides used in this study, AST and ALT activities in O. niloticus muscle tissues were estimated. From this study, remarkable alterations in AST and ALT enzyme activities were noticed in O. niloticus muscle tissue after exposure to the insecticides (malathion, chlorpyrifos and λ-cyhalothrin) for 24 and 48 h. Chlorpyrifos resulted in the highest inhibition (-95.8%) in AST activities followed by malathion (− 45.3%) and λ-cyhalothrin (− 12.2%). Also, chlorpyrifos resulted in the highest activity of ALT enzyme (+ 313%) followed by λ-cyhalothrin (+ 294%) and malathion (+ 282.5%). Our findings are consistent with other studies involving diazinon (OP) treated Cirrhinus mrigala [36], malathion treated Channa striatus [37], cadmium-treated Cyprinus carpio [32], monocrotophos-treated Channa punctatus [38], deltamethrin (synthetic pyrethroid)-treated O. niloticus and Cyprinus carpio [11, 39, 40], profenofos (OP)- and cypermethrin-treated O. niloticus [34, 41], λ-cyhalothrin-treated Macrobrachium rosenbergii [2], cypermethrin-treated Rhamdia quelen [42] and α-permethrin-treated Labeo rohita [43]. Inhibition in AST and ALT levels was observed in 2,4-diamin-treated Cyprinus carpio [44], in zinc oxide-treated Cyprinus carpio [45] and phosphamidon (OP)-treated Puntius conchonius [46]. Gill et al. [46], Oruc and Uner [44] and Lee et al. [45], and Velíšek et al [39, 40] reported a remarkable increase in AST levels and reduction in ALT activity in rainbow trout exposed to deltamethrin, which is in contrast to our study. The increase in ALT activity recorded may be attributed to increased enzyme synthesis to counteract organ injury due to chemicals, and inhibition or decrease in AST activity recorded may be due to reduced enzyme synthesis or cell permeability changes due to the leakage of certain enzymes from the cells [47]. El-Demerdash et al. [48] reported that the activities of AST and ALT in liver and testes of rabbits were increased; contrarily, the activities of AST and ALT in plasma were decreased due to Cypermethrin administration. This indicated that the same insecticide may show the different trend of their activity in different organs of the same organisms. Khan et al. [49] noticed inhibition in AST in organophosphates treated Calotes versicolor. Ramaswamy et al. [32] exposed Sarotherodon mossambicus to sub-lethal and lethal doses of carbaryl and noted increase in ALT level in muscles tissues. They concluded that aminotransferase enzymes in the tissues, thereby probably aiding gluconeogenesis through transamination of glucogenic amino acids to meet the energy demand under carbaryl toxicity. The AST inhibition observed in this study may be due to the tissue damage as investigated by Oluah [50]. Increased and decreased AST and ALT activity suggested tissue and organ injury and dysfunction^50–53.

In conclusion, the present study indicated that organophosphates (malathion, chlorpyrifos) and synthetic pyrethroid (λ-cyhalothrin) insecticides alters the activity ofAST and ALT enzymes in fishes. So insecticides may affect the fish's health, growth, habitat and survival. These parameters can be used as good biomarkers for early detection of insecticides pollution in aquatic ecosystem. Consequently, precautions should be taken when these chemicals are used near water supplies. In addition, the current findings indicate that O. niloticus is a good bioindicator of pollutants in aquatic systems.

Abbreviations

ALT

Alanine amino transferase

AST

Aspartate transaminase

GOT

Glutamic oxaloacetic transaminase

GPT

Glutamate pyruvate transaminase

h

Hours

LC

Lethal concentration

OD

Optical density

PE

Percent enhancement

PI

Percent inhibition

Author contributions

Conceptualization, MA, MY, MA, NA, MA and MI; methodology, MA, MY, MA, NA and MI; investigation, MA, MY, MA, NA and MI; writing—review and editing, MA, MY, MA, NA, MA, MI, AAAZ, CAY, EMA, YSB, GE-SB and IDB. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Declarations

Ethics approval and consent to participate

This study follows guidelines for the care and use of experimental animals established by the Animal Care and Use Committee, University of Karachi for the purpose of control and supervision of experiments on animals.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

The data sets in this study are available from the corresponding author on reasonable request.

Conflict of interest

The authors declare that they have no competing interests.