**1. Introduction**

Pesticides have been used successfully to improve agricultural productivity and meet the increasing demand for food, but they often end up in the natural environment where they may impact non-target organisms. Pesticides can act on organisms other than pest species. This is of particular concern in developing countries, where increasing intensification of agriculture leads to high pesticide use [1]. Pesticides cover a wide range of

**Citation:** Nazir, S.; Ali, M.N.; Tantray, J.A.; Baba, I.A.; Jan, A.; Popescu, S.M.; Paray, B.A.; Gulnaz, A. Study of Ultrastructural Abnormalities in the Renal Cells of *Cyprinus carpio* Induced by Toxicants. *Toxics* **2022**, *10*, 177. https://doi.org/10.3390/ toxics10040177

Academic Editors: François Gagné, Stefano Magni and Valerio Matozzo

Received: 4 February 2022 Accepted: 28 March 2022 Published: 2 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

substances including insecticides, acaricides, fungicides, molluscicides, herbicides, nematocides and rodenticides. Pesticides are widely used in agriculture to control insects, nematodes, fungi, etc. that affect food and other crops. These are easy to apply, being cost effective and, most importantly, they are readily available practical means of pest control. On the flip side, pesticides may have unwanted effects on the natural environment as they can enter through various routes including direct application, spray drift, atmospheric deposition and surface runoff. Among the different types of pesticides, insecticides are often used. Depending upon the mode of action, insecticides have been classified by the Insecticide Resistance Action committee (IRAC, 2017) into various categories, viz., botanicals (nicotine, rotenone, pyrethrum, etc.), organochlorines (e.g., DDT), organophosphates (Phorate, Dimethoate), carbamates, pyrethroids etc [2]. There are many pathways by which pesticides leave their sites of application and are distributed throughout the aquatic ecosystem. Different concentrations of the pesticides are present in many types of wastewater and several studies have revealed them to be toxic to aquatic organisms including fish [3,4]. Fish are sensitive to polluted water. Hence, fish have long being used to monitor the quality of the aquatic environment and fish histology is increasingly being used as an indicator of environmental stress [5,6].

Phorate is a systemic and broad spectrum organophosphorus (OP) insecticide, commonly used in agriculture to control sucking and chewing insecticides, leaf hoppers and mites. It is also used in pine forests and on root and field crops including corn, cotton, coffee and some ornamental plants and bulbs [7,8]. Phorate is primarily formulated as granules to be applied at planting in a band or directly to the seed furrow. In biota, it inhibits acetylcholinesterase activity by phosphorylating the serine hydroxyl group in the substrate binding domain, which results in the accumulation of acetylcholine and induces neurotoxicity. Though its use has been strictly restricted by the United States Environmental Protection Agency (USEPA), it is still being used in several countries such as India, China, Italy and Egypt [9].

Dimethoate is a systemic organophosphate insecticide used on a large variety of field grown agricultural crops, tree crops, and ornamentals. Dimethoate was first registered in 1962 in the US and later its use for non-agricultural practices (e.g., domestic purposes) was banned as of 2000. It is available as Rogor and like other organophosphates it acts as an acetylcholinesterase inhibitor and works as a nerve poison at synapses of neuromuscular junctions, which is evident by abnormal body movements and jerks [10–12]. Various studies on the toxicity of Phorate on aquatic organisms, especially fish, have been carried out, confirming its toxicity and genotoxic role [13–15]. Histopathological toxicity of brain of *Cyprinus carpio* exposed to Phorate was also reported by Lakshmaiah, (2017) [16]. Lakshmaiah (2016) reported acute toxicity of Phorate on succinate dehydrogenase enzyme activity, which is an important enzyme for Krebs Cycle [17]. Additionally, its genotoxicity potential was reported by Saquib et al., (2012) in male wistar rats and in human amniotic epithelial (WISH) cells [7]. Toxicity of dimethoate in fish fauna has also been reported by many researchers [12,18]. Demet and Canan (2011) reported toxic effects of Dimethoate on hematological, biochemical and behavioural patterns in *Oncorhynchus mykiss* exposed to sublethal concentrations of 0.0735, 0.3675, and 0.7350 mg/L for 5, 15, and 30 days [19]. Ganeshwade (2012) reported biochemical changes in the gills of freshwater fish *Puntius ticto* when exposed to lethal (5.012 ppm) and two sublethal (2.506 and 1.253 ppm) concentrations of Dimethoate for 96 h and 60 days, respectively [20]. Histopathological studies were reported in the kidney of *Cyprinus carpio* after exposure to dimethoate (EC 30%) by Singh (2012) [21]. Binukumari and Vasanthi (2013) observed the toxic effect of the Dimethoate 30% EC on protein metabolism of *Labeo rohita,* when exposed to a concentration of 0.398 ppm for 24, 48 and 72 h, respectively [22]. Singh (2013) exposed common carp, *Cyprinus* to dimethoate 0.40 mg/L for short-term exposure of 96 h and reported toxic effects on its liver [23]. Singh (2014) reported toxic effects of Dimethoate (EC 30%) on gill morphology, oxygen consumption and serum electrolyte levels of common carp, *Cyprinus Carpio.* The fish were exposed to a sub lethal concentration of 0.96 mg/L (60% of 96 h LC50) of dimethoate

at a 24, 48 and 96 h exposure duration [24]. Singh (2017) reported testicular toxicity of *Cyprinus carpio* when exposed to dimethoate at 0.96 mg/L and 0.48 mg/L, respectively in a short-term (96 h) and long-term study (36 days) [25].

As is evident, the toxic effects of these two organophosphates have been reported but no work has been reported on their potential role as cytotoxins, i.e., their role to cause ultrastructural abnormalities in fish cells. So, the present study has been designed to investigate the role of two organophosphate insecticides: Phorate and dimethoate, for their potential to damage cells in an in vivo system and contribute to acute toxicity. Hence, an investigation resulting from cellular events leading to cytotoxicity is proposed. Teleost fish have proved to be good models to evaluate the genotoxicity and effects of pollutants such as insecticides on animals, as their biochemical responses are similar to those of mammals and other vertebrates [14,26–28]. The advantage of using fish models includes the facility by which Teleostei, especially the small species, can be maintained and handled inside the laboratory under experimental conditions of toxic exposure [29]. Fish frequently respond to chemical exposure as superior vertebrates, which validate this model to study potential teratogenic and carcinogenic compounds in humans [30]. Common carp *Cyprinus carpio* is a robust fish which can tolerate a wide range of temperatures and is adaptable to varying environmental conditions. It is an economically important fish due to its nutritional value. Thus, it serves as an important vehicle for contamination.

The main objective of the present study was to observe Phorate and Dimethoate induced ultrastructural abnormality in the renal cells of common carp, *Cyprinus carpio* (as the kidney is not only the excretory organ but also functions as osmoregulatory organ of the fish). It will provide baseline data about the ultrastructural toxicity of these insecticides, which are widely used and finally reaches into our precious water bodies, thereby proving hazardous to both aquatic fauna (especially fish), as well as to its consumers. The present study was initiated to understand the acute toxicity of organophosphate insecticide exposure which could make a potential contribution towards the identification of the environmental toxicants. Such an acute toxicity testing would measure the adverse effects that occur within a short period of time. Thus, such information would be fruitful to serve as a basis for hazard classification and will provide information regarding the target tissue/organ toxicity by different doses of the toxicants.

## **2. Materials and Methods**

The fish species *Cyprinus carpio* L. (family: Cyprinidae) were chosen for the present study. Young specimens of *Cyprinus carpio communis* (age: <1 year; weight 30–40 g; length 10–12 cm) were used. The healthy young adult specimens of *Cyprinus carpio communis* were collected from the Dal Lake (34◦5 –34◦6 N latitude and 74◦8 –74◦9 E longitudes) using a net and hand-picking method, and then these collected fish were transported to a laboratory in a specially-designed container with oxygen supply. After collection, fish were acclimatized for 15 days in 60 L artificially aerated glass aquaria (5 fish each) containing dechlorinated tap water for 12/12 natural photoperiod (pH 7.6–8.4, temperature 25 ± 3). During acclimatization, fish were fed a commercial diet (Feed Royal®, Maa Agro Foods, Visakhapatnam, Andhra Pradesh, India). In order to avoid ammonia accumulation, the water in these aquariums were changed daily with dechlorinated tap water. During the test period, no feed was given to keep the insecticide concentrations constant throughout the test period of 72 h [18,19]. Water quality of the test solution was determined according to standard procedures [31]. The control fish were kept in experimental water without adding these insecticides, keeping all other conditions constant.
