Introduction

Agrochemical is a substance or mixture of substances that is use for preventing, abolishing, resisting or mitigating any pest for enhancement in crop yield. Agrochemicals are frequently applied to agricultural commodities in order to fulfill the increasing food demand and for protection of crops from pests, pathogen and weeds (Gupta et al. 2013). Along with beneficial effects these agrochemicals have some disadvantages such as odor and change in taste of water and lethality of non-target organisms in aquatic environment (Sathyamoorthi et al. 2019). Pesticides enter in aquatic environment through runoff from place of application and pose a serious threat to aquatic life (Al-Otaibi et al. 2019). Aquatic organisms face significant problems similar to terrestrial species analysis of fish and water samples from different aquatic environments shows 90% to have one or more than one toxicant (Hussain et al. 2020). On the basis of chemical nature and mode of action agrochemical are categorized into five major categories namely; oraganochlorine, organophosphate, carbamates, pyrethroids and neonicotinoids (Xiao et al. 2017).

Profenofos is broad spectrum organophosphate highly effective for controlling chewing and sucking insects and mites mostly present on cotton plants (Reddy and Rao 2008). Deltamethrin is a synthetic pyrethroid., Natural pyrethrins are from Chrysanthemum cinerariaefolium (Bradberry et al. 2005). It is used to control aphids, white flies, lice, tsetse flies, fleas, ticks, spiders, bees, cockroaches, ants, weevils, beetles and bedbugs (Costa 2015). Endosulfan is an organochlorine is preferred for pests management programs such as mosquitoes and tsetse fly public health control programs (Guo et al. 2008).

As fish are in direct contact with water, they are exposed to the toxic chemical and accumulate raising concern about their biomagnification potential in the food webs (Zhang et al. 2020). Agrochemicals can disturb physiological functions and biochemical processes of organisms, so biochemical and endocrine parameters can be used as a biomarkers of stressor impacts (Isaac et al. 2017). Potential genotoxicity of pesticides are usually first monitored by using the micronucleus assay; a reliable, simple, easily performed bioassay (Talapatra and Banerjee 2007; Hemalatha et al. 2020). Induction of micronuclei in peripheral erythrocytes can be used as a biomarker for ecotoxicology, which can lead to damaging effects like retardation of growth and abnormal development in fish (Caliani et al. 2019). Common carp (Cyprinus carpio L.) is the third most frequently introduced important aquaculture species, and is source of 71.9% fresh water production, and a well-studied aquatic model organism in ecotoxicology when OECD standard bioassays are used (Rahman 2015). The aim of current study was to evaluate the comparative adverse effects of endosulfan, profenofos and deltamethrin on health status of the common carp during 96-h acute exposure.

Materials and Methods

Fish collection and acclimatization

Total one forty widespread freshwater omnivorous fish, Common carp (Cyprinus carpio) (50 ± 7.45g and 15 ± 5.86 cm) were collected from fish farm service road Kamra, Punjab Pakistan and brought to laboratory of Zoology department at Government Postgraduate College Haripur. Fish were allowed to acclimatize to lab condition for ten days prior to experiment, during this time period fish were fed with pelleted fish food and 70% of water was renewed on alternate days in order to prevent accumulation of toxic substances. The pH and temperature of laboratory water were maintained at 7.6-7.8 and 20-23°C, respectively.

Selected chemicals for exposure

Mmarketable commercial formulations of profenofos (40 EC), deltamethrin (60 EC) and endosulfan (37 EC) were selected for the study and purchased from local agrochemicals market of Peshawar Khyber Pakhtunkhwa, Pakistan.

Experimental setup

To investigate the possible toxic effect of Profenofos, Deltamethrin and Endosulfan, test solution of 4 ppb was prepared for each chemical from their commercial formulation. On the basis of fish mortality response and agrochemical concentration, the LC50 of profenofos, deltamethrin and endosulfan by Probit analysis(Finney 1980) was found to be 8.34 µg/L, 11.92 µg/L and 9.58 respectively for 96 h. Four glass aquaria of 60 L capacity were filled with 40 L of water and labeled as E0, E1, E2 and E3. E0 was control group having no chemical while E1 experiment one, E2 experiment two, E3 experiment three. Each aquarium contains 25 fish. Fish were exposed to the chemicals for 96 h (96-h acute bioassay).

Biochemical and endocrine analyses

After exposure of fish to agrochemicals for 96 h fish from each aquarium were collected and anesthetized with clove oil Blood samples were collected from the caudal vein of fish by using sterile syringes and immediately transmitted to a tube, allowed to clot. Collected blood samples were spun at 3000 rpm for 5 min to obtain blood serum. Sera were used for determination of biochemical parameters i.e., serum protein, serum glucose, triglyceride, cholesterol, creatinine, serum amylase, serum lipase and alkaline phosphatase, Na, P, Mg and endocrine parameters i.e., thyroid stimulating hormone (TSH), triiodothyronine (T3), tetraiodothyronine (T4), cortisol and insulin. Fully automated biochemical analyzer COBAS e 411 of Roche with Electro-Chemi-luminescence technology & CE approved 4^th generation immunodiagnostic kits was used for the analyses.

Genotoxicity (Micronucleus assay)

For MN assay blood samples were taken from the caudal fin of fish by using disposable heparinized syringes and then transferred into EDTA tubes after that two drops of blood were applied immediately on clean grease free slides for making peripheral blood smears, two for each fish specimen. These slides were air dried for 24 h and then dipped into cold absolute methanol for 15 min and then air dried for one hour. Slides were stained with Giemsa dye in phosphate buffer for 30 min then slides were washed and dried. Prepared slides were observed under 100x oil emersion lens. Then total magnification becomes 10 × 100 = 1000) and frequency for MN were counted by using following formula:

Statistical analysis

The data for biochemical parameters, endocrine parameters and micronuclei frequency was statistically analyzed using SPSS^® statistics software (version 24.0. Intergroup comparison was made by using one-way ANOVA while t-test was used for comparison between control and experimental group. The critical significance was set at 0.05.

Results

Physico-chemical parameters of water

The physico-chemical properties of laboratory water and experimental condition were regularly examined during the experiment. The reported values were as Mean ± SD for different parameters and are shown in Table 1.

Behavioral changes in C. carpio

Table 1: Physico-chemical parameters of laboratory water

Parameters	Data	Parameters	Data
Temperature	21± 1.19˚C	Calcium hardness	44 ± 1.55 mg/L
PH	7.5 ± 0.23	Total alkalinity	129±4.89 mg/L
Dissolve oxygen	6.4 ± 0.53	Conductivity	819±14.9 ms/L
Total dissolved solids	366 ± 10.81 mg/L	Chloride	18.5±2.1 mg/L

Fig. 1: A-J Serum protein, glucose, triglycerides, creatinine, serum amylase, serum lipase, ALP, Na, Mg and P levels of C. carpio after 96-h exposure to profenofos, deltamethrin and endosulfan. Sign (**) indicates significant difference (P < 0.05) to each other

Several changes in the behavior of treated group of C. carpio were observed throughout the experiment which varies from mild to severe depending upon the toxicity of pesticide. Various behavioral abnormalities shown by fish were, loss of body equilibrium, sinking to bottom, swimming at lateral side, darting swimming movement, hyper-excitability, increase in movement of operculum, mucus secretions and due to stress some fishes tried to jump out of aquarium on the other hand no such type of alterations were noticed in control group of fish.

Table 2: MNi frequency in control and treated groups of C. carpio

Parameter	Control group E0	Profenofos (4 ppb)	Deltamethrin (4 ppb)	Endosulfan (4 ppb)
MN frequency (%)	0.19 ± 0.10	2.14 ± 0.51**^Δ	3.16±0.56**^Δ	4.22 ± 0.51**^Δ

Fig. 2: (A-E) T3, T4, TSH, cortisol and insulin level of C. carpio against profenofos, deltamethrin and endosulfan exposure. Sign (**) indicates significant difference (P < 0.05) to each other

Fig. 3: MNi induction in erythrocytes of control, E0, (A) profenofos (B), deltamethrin (C) and endosulfan (D) treated groups

Fig. 4: Frequency (%) of MNi in erythroctes of C. carpio against Profenofos, Deltamethrin and Endosulfan exposure. Sign (**) indicates significant difference (P < 0.05) to each other

Biochemical analyses

In the present study C. carpio exposed to profenofos, deltamethrin and endosulfan showed alterations in biochemical parameters in response to agrochemical exposure as presented in Fig. 1 A–J. The values of serum protein and triglycerides decreased significantly in all treatment groups, while values of serum lipase and magnesium insignificantly decreased in group E1 exposed to profenofos, while significantly decreased in group E2 and E3 exposed to deltamethrin and endosulfan respectively. The values of glucose, serum amylase and alkaline phosphatase increased significantly in all the treated groups while Na and creatinine increased slightly in treated group I and significantly in group II and III, P level increased slightly in treated group I and II while significantly in group III compared to control group. The order of toxicities was endosulfan > deltamethrin > profenofos.

Endocrine parameters

In the present study endocrine variations in C. carpio was assessed on exposure to Profenofos, Deltamethrin and Endosulfan for 96-h as presented in Fig. 2 A–E. Values of T3 decreased significantly (P < 0.05) in all the treated groups while T4 decreased slightly in group I exposed to profenofos while significantly in group II and III exposed to deltamethrin and endosulfan respectively. TSH and cortisol values increased significantly (P < 0.05) in all the treated groups while values of insulin decrease slightly in group I and significantly in group II and III. No such type of alteration was recorded in endocrine parameters of fish belonging to control group. The order of toxicity of agrochemical was Endosulfan > Deltamethrin > Profenofos.

Estimation of genotoxic effect through MN assay

After agrochemical exposure micronuclei induction was observed in all the treated groups as compared to control group (Fig. 3). The highest micronuclei frequency was recorded against treated group III (4.22%) exposed to endosulfan while lowest MN frequency was calculated against group I (2.14%) exposed to profenofos (Fig. 4), which shows that endosulfan is more toxic amongst all three selected agrochemicals. The Micronuclei frequency increased in order of Endosulfan > Deltamethrin> Profenofos as represented in Table 2.

Discussion

Current study was conducted to assess the biochemical, endocrine and genotoxic effect of selected agrochemicals on C. carpio as an experimental model. To our knowledge, the present study provides the first ecotoxicological assessment for selected agrochemicals by using a multi-biomarker approach in a C. carpio. Our results indicated that a low acute sublethal exposure to agrochemicals in C. carpio has significant effects on the fish physiology and behavior. During experiment fish in E0, control group, showed normal behavior while various behavioral abnormalities were shown by treated group of fish such as; loss of body equilibrium, sinking to bottom, swimming at lateral side, darting swimming movement, hyper-excitability, increase in movement of operculum, mucus secretions and due to stress some fishes tried to jump out of aquarium. Similar changes were noticed by (Ghelichpour et al. 2020) by exposing C. carpio to lufenuron and by (Ghayyur et al. 2020) in C. mrigala against chlorfenapyr, dimethoate and acetamiprid exposure.

Biochemical study plays a vital role in monitoring fish health and assessing toxic effect of pollutants on aquatic organism (Poopal et al. 2017). The findings of present study showed a significant decrease in serum protein and triglycerides of agrochemical exposed fish as compare to control group. This result was in agreement with that of (Khan et al. 2019) in C. carpio treated with endosulfan and O. orientalis exposed to Cypermethrin (Shruti and Tantarpale 2014). The hypoproteinemia in present investigation might be due to boosted proteolysis, proteolysis seems to offer a physiological mechanism in a bid to provide energy to deal with stressful condition created by toxicant exposure. Triglyceride are major energy reserve in fish, the decline in triglyceride level in present investigation represent liver dysfunction (Prakash and Verma 2020). Similar results were obtained by (Qadir et al. 2014) when they exposed L. rohita to imidacloprid. Glucose is the major source of energy and its higher level in plasma act as a stress indicator in present study significant increase in glucose level observed. Our results was in line with findings of (Khan et al. 2018) and (Ghayyur et al. 2019). Changes in creatinine level shows kidney dysfunction, in present study level of creatinine increased in fish exposed to agrochemical in comparison to control group which may be due to oxidative damage. Similar consequences were reported by (Prusty et al. 2011). Our result also show similarity with the findings of (Amin and Hashem 2012) they noticed an increase in creatinine level in C. gariepinus against deltamethrin exposure.

When toxicants enter different tissues are injured and damaged cells release specific enzymes, in this investigation treated fish revealed elevation in alkaline phosphatase. Increase in ALP might be due to destruction of cell membrane of liver cells (Rahman et al. 2019). Similar increase in ALP was recorded by (Rahman et al. 2020) in C. carpio, by (Ghayyur et al. 2020) in Cirrhinus mrigala and by (Sancho et al. 2017) in Anguilla anguilla. Alteration in serum amylase indicates abnormality of digestive process in present study serum amylase increased in treated groups of fish, similar trend have been reported by (Lundstedt et al. 2004) and (Khan et al. 2019). Declined level of lipase due to agrochemical exposure may also be justified by the increased protein catabolism and reduced protein anabolism due to stress or may be due to decreased lipid levels. Similar reduction was reported in C. carpio exposed to malathion, chlorpyrifos and dimethoate (Rani et al. 2017).

Blood electrolytes are bio-indicator of physiology and health Status of fish. In present investigation values of sodium and phosphorus increased while magnesium level decreased. Increase in sodium and phosphorus was also reported by (Borges et al. 2007) when fish was exposed to Cypermethrin. Similar to our findings increase in sodium was reported by (Akhtar et al. 2019) in Schizothorax esocinus exposed to Cypermethrin. Our result shows disagreement with findings of (Atamanalp et al. 2002) who reported decrease in phosphorus after exposure of fish to cypermethrin.

Many fishes respond to stressors by showing endocrine shifts. In present study C. carpio showed decline in plasma T3 and T4 while significant increase in plasma TSH level. This result was in agreement with that reported by (Thangavel et al. 2010) in S. mossambicus exposed to endosulfan and by (Ghayyur et al. 2020) in Cirrhinus mrigala. Reduction in plasma T3 occur possibly because of diminution of T4 secretion or production (Li et al. 2008). Cortisol is an adrenocortical hormone involves in ion regulation and energy metabolism and used as a primary indicator of stress; it showed a significant increase in present study. Our results are in line with findings of other researchers e.g. C. carpio exposed to trichlorfon (Woo and Chung 2020), C. carpio exposed to cyfluthrin (Sepici-Dinçel et al. 2009) and Salmo salar exposed to atrazine (Waring and Moore 2004). In present study decline in level of insulin was recorded, the toxicant may have injured the islets of Langerhans which reduce the insulin secretion from β-cells (Thoker 2015). Similar to our outcomes, decline in insulin level was reported in Danio rerio (Ahmad et al. 2018), due to inhibition of β-cells by toxicant. Our result shows dissimilarity with the findings of (Shah et al. 2019) in which they reported an increase in insulin level of fish against dimethoate exposure.

MN assay is mostly used to detect biological impacts of aquatic pollutants on aquatic organisms, micronuclei induction in peripheral erythrocytes is due to genotoxic effect of pollutants (Tripathy 2020). Present study revealed genotoxicity of agrochemical in the peripheral erythrocytes of C. carpio as indicated by MN assay. Genetic damage increased in treated groups of fish as compare to control group, represented by formation of MN in erythrocytes. Our results show agreement with the findings of (Naqvi et al. 2016) when they treated O. mossambicus with different pesticides and (D’Costa et al. 2018) in Danio rerio exposed to monocrotophos. Similar genotoxic effects were also reported in erythrocytes of Grass carp on exposure to chromium, lead and copper (Shah et al. 2020). Likewise thiamethoxam administration also result in significant increase in micronuclei frequency in L. rohita (Ghaffar et al. 2020). Similar increase in micronucleus frequency was also reported in O. mykiss treated with fipronil (Ucar et al. 2020).

Conclusion

Present investigation clearly confirmed the harmful impacts of selected agrochemicals on biochemical, endocrine and genetic profile of C. carpio which showed that genetic, biochemical and endocrine parameters can be used as efficient biomarker for ecotoxicology. On the basis of current investigation, we can conclude that agrochemical pose serious threat to aquatic life in order to minimize poisonous effects of agrochemicals its use should be minimized and environment friendly agrochemical should be formulated having fast degrading ability and more target specificity.

Acknowledgments

This study was supported by the Higher Education Commission of Pakistan (10108).

Author Contributions

Sanaa: Perform experiment, Shehzad Ghayyur: Designed the study, Sadia Tabassum & Shumaila Noreen: Review the article, Sajid Mahmood: Analyzed the data, Mujadad Ur Rehman & Bashir Ahmad: Help in sample collection, Muhammad Kabir & Muhammad Sajid: Write the article, Muhammad Fiaz Khan: Supervised the study

Conflict of Interest

The auther have no conflict of interest.

Data Availability

Data presented in this study are available on fair request to the corresponding author.

Ethics Approval

All the experimental work was conducted in accordance with the ethical guideline approved by the ethical committee of Hazara University, Mansehra, Pakistan.

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