Int. J. Aquat. Biol. (2018) 6(5): 254-257 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2018 Iranian Society of Ichthyology Short Communication Effects of Dimethoate and Bacilar fertilizer on biochemical and immunological parameters in common carp, Cyprinus carpio Fatemh Mohseni1, Mahdi Banaee*1, Parvaneh Shokat1, Mohammad Mohiseni2 1Department of Aquaculture, Faculty of Natural Resources and the Environment, Behbahan Khatam Alanbia University of Technology, Behbahan, Iran. 2Department of Fisheries Science and Engineering, Faculty of Agriculture and Natural Resources, Lorestan University, Khorramabad, Iran. Article history: Received 19 May 2018 Accepted 23 October 2018 Available online 2 5 October 2018 Keywords: Agrochemicals Carp Immunotoxicity Abstract: Fish exposure to agrochemicals can suppress their immune system and survival. Therefore, this study investigates adverse effects of sublethal concentrations of Dimethoate alone or combined with Bacilar (an organophosphorus pesticide and bio-fertilizer) on the innate immune parameters of common carp, Cyprinus carpio, within 14 days. No significant changes were found in levels of total protein, immunoglobulin, the activity of lysozyme and complement C3 in fish exposed to Bacilar alone; however, globulin and complement C4 level indicated a significant reduction. Fish exposure to Dimethoate alone or combined with Bacilar resulted in a decrease in the activity of ACH50, lysozyme, complement C3, C4 and levels of total protein, globulin, and immunoglobulin in compared with the control group. In conclusion, the results of this study showed that innate immune parameters decreased in fish exposed to dimethoate and/or Bacilar. As consequences: Dimethoate or/and Bacilar have the immunosuppressive effect on fish. Introduction The presence of different pollutants, such as pesticides and chemical or organic fertilizers in aquatic ecosystems near agricultural farms can be a serious threat to aquatic organisms (Fazilat et al., 2017). Dimethoate, with the chemical formula of O, O- dimethyl S-methyl carbamoyl methyl phosphoro- dithioate and the organophosphorus pesticide – Bacilar – which is the biological product of Bacillus subtilis, along with some micronutrients are used in agriculture. Therefore, the introduction of these compounds into surface waters can bring about alterations in biochemical parameters of blood (Fazilat et al., 2017), cause oxidative stress (Dogan et al., 2011) and behavioral disorders in fish. Changes in the activity of innate immune parameters (Ahmadi et al., 2014), alterations in the gene expression of cytokines IL-6, IL-8, IL-10 and TNF-α (Chen et al., 2014), and an increase in expression of gene atrophies LC3-II, dynein (Chen et al., 2015) and Hsp60, Hsp70, and Hsp90 genes (Xing et al., 2015) in the head kidney and spleen, alterations *Correspondence: Mahdi Banaee DOI: https://doi.org/10.22034/ijab.v6i5.404 E-mail: mahdibanaee@yahoo.com in expression of immunoglobulin M (IgM), complement C3, and lysozyme (LYZ) (Ma and Li, 2015) are a number of damages incurred to the immune system of fish exposed to different pesticides and agrochemical compounds. These changes can increase fish sensitivity to biological and environmental infections. Thus, this study evaluates changes in the immune parameters of common carp treated with varied concentrations of either Dimethoate or Dimethoate and Bacilar. Materials and Methods This study is conducted according to the Ethics of Animal Experimentation in Iran. Common carp, Cyprinus carpio, were divided into 9 groups and tested for 14 days under different conditions: Fish under standard environmental conditions, or the control group (Group I); fish treated with 16 and 32 g L-1 of Dimethoate (respectively Groups II and III); Fish treated with 0.1 and 0.2 ml L-1 of Bacilar bio-fertilizer (respectively Groups IV and V); fish treated with 16 g L-1 of Dimethoate + 0.1 ml L-1 of Bacilar (Group 255 Int. J. Aquat. Biol. (2018) 6(5): 254-257 VI); fish exposed to 32 g L-1 of Dimethoate + 0.1 ml L-1 of Bacilar (Group VII); and fish exposed to 32 g L-1 of Dimethoate + 0.2 ml L-1 of Bacilar (Group IX). Blood samples were collected from all groups. The following methods measured immune parameters of plasma: total immunoglobulin by Amar and colleagues’ method (Amar et al., 2000), alternative complement pathway activity (ACH50) by Yano’s method (Yano, 1992) and based on hemolysis of sheep red blood cells (ShRBC) (Baharafshan Research Company), lysozyme activity in blood plasma via Micrococcus luteus (Actinobacteria: Micrococcaceae) (Lange et al., 2001), the activity of complements C3 and C4 via immunoturbidimetry (Abdollahi et aal., 2016), and total protein, albumin and globulin (Johnson et al., 1999) by Pars Azmun Kits. After checking the normality of data via Shapiro-Wilk, data analysis was done by one-way ANOVA, using SPSS 22. The means were compared by Duncan Test at a 99% confidence level (𝛼 = 0.01%). Results No mortality was observed in fish during the experiment; however, an increase in mucus secretions, changes in color and behavior, such as unbalanced swimming, swimming on the surface, and neurological reactions are among the significant behavioral changes in fish exposed to Dimethoate alone or combined with Bacilar on the final days of the experiment. Biochemical parameters of Common carp exposed to Dimethoate alone or combined with Bacilar for 14 days are presented in Table 1. The results of this study indicated that Bacilar alone had no significant effects on levels of total protein, immunoglobulin, lysozyme activity, and complement C3 in fish. However, levels of these parameters in fish treated with either Dimethoate or Dimethoate and Bacilar were significantly reduced. Fish exposure to Bacilar and Dimethoate decreased globulin and complement C4 levels. There was a significant increase in albumin levels in fish treated with Bacilar and Dimethoate compared to that of the control. No significant changes were found in ACH50 activity in fish treated with 0.2 ml L-1 of Bacilar; however, ACH50 activity was significantly reduced in other experimental groups. Discussion The immunotoxicity of agrochemicals on fish has been widely investigated (Marchand et al., 2017; Chen et al., 2015; Ma and Li, 2015). In the present study, immunological parameters in plasma of fish exposed Table 1. Biochemical parameters of Cyprinus carpio exposed to Dimethoate alone or combined with Bacilar for a period of 14 days. Treatments Total protein (g/dl) Albumin (g/dl) Globulins (g/dl) Immunoglobulins (mg/ml) Lysozyme (U/ml) ACH50 (U/ml) Complement C3 (mg/dl) Complement C4 (mg/dl) Control 4.8±0.2c 2.1±0.6a 2.7±0.5b 15.6±3.3c 23.3±2.4b 92.0±8.5e 15.5±2.9c 16.3±0.4c 16 µg/l Dimethoate 4.0±0.1ab 3.0±0.5bc 0.9±0.5a 2.7±1.1a 13.5±3.0a 81.2±3.6cd 10.9±1.8a 10.2±0.6a 32 µg/l Dimethoate 3.9±0.4ab 2.7±0.3b 1.13±0.4a 8.0±4.0b 14.0±3.6a 70.6±2.0b 8.4±1.2a 8.9±1.9a 0.1 ml/l Bacilar 4.6±0.7c 3.0±0.3bc 1.6±0.8a 15.8±5.6c 22.3±4.5b 83.0±3.7cd 15.7±4.7c 14.1±0.9b 0.2 ml/l Bacilar 4.4±0.3bc 3.5±0.4c 0.9±0.5a 17.8±4.7c 19.9±0.5b 90.6±5.9e 14.1±2.8bc 13.6±0.6b 16 µg/l Dimethoate & 0.1 ml/l Bacilar 4.1±0.7ab 3.1±0.7bc 1.0±0.8a 6.3±3.6ab 12.9±2.2a 84.8±5.0d 10.8±1.9a 13.8±0.4b 16 µg/l Dimethoate & 0.2 ml/l Bacilar 4.0±0.3ab 3.1±0.6bc 0.9±0.6a 7.3±3.7ab 15.7±3.2a 78.0±1.8c 11.4±1.1ab 14.0±1.5b 32 µg/l Dimethoate & 0.1 ml/l Bacilar 4.1±0.4ab 3.2±0.4bc 0.9±0.4a 6.5±3.7ab 12.2±0.9a 61.4±2.0a 10.4±1.4a 10.5±2.0a 32 µg/l Dimethoate & 0.2 ml/l Bacilar 3.7±0.2a 2.8±0.3b 0.9±0.4a 4.0±1.9ab 12.2±0.8a 62.6±2.1a 9.9±1.8a 10.4±2.3a Data are presented as mean±S.D. Significant differences between values when compared with control group were characterized by alphabetical symbol (P<0.01). 256 Mohseni et al./ Effects of Dimethoate and Bacilar fertilizer on common carp to Dimethoate and/or Bacilar fertilizer are evaluated. A significant decrease in levels of total protein may indicate liver necrosis or disturbance in the physiological performance of fish treated with Dimethoate or Dimethoate and Bacilar. Dimethoate can reduce levels of total protein in plasma, especially globulins, by reducing appetite, causing a disturbance in the absorption of amino acids in intestines, increased activity of proteolytic enzymes, the increased rate of proteins break down in the liver and preventing protein synthesis in the liver (Narra, 2017). Liver failure, malnutrition as well as a biochemical reaction between agrochemicals and the amino acid sequences of proteins found in blood may account for lower plasma total protein. Similar results are reported in fish exposed to different pesticides (Ahmadi et al., 2014). An increase in albumin can be attributed to its role in distributing the pesticide in the blood (Tarhoni et al., 2008). Total immunoglobulin is a main element of blood adaptive immunity in bony fish and a well-known biomarker in evaluating the immunotoxicity of fish exposed to environmental pollutants (Li et al., 2013). In this study, a decrease in total immunoglobulin levels is due to disturbance in the adaptive immune system in fish exposed to Dimethoate alone or both Dimethoate and Bacilar. A reduction of total immunoglobulin levels in the blood may be due to an insufficient synthesis of immunoglobulins, or changes in the gene expression involved in the biosynthesis of Ig’s subunits (Ghazy et al., 2017). Similar effects were observed in other species exposed to different agrochemical compounds (Narra, 2017). Lysozyme, a vital element of the innate immune system of bony fish, is very sensitive to pollution- induced stress (Ahmadi et al., 2014). Lysozyme is expressed to a great extent in hematopoietic cells, granulocytes, monocytes, and macrophages (Merlini and Bellotti, 2005). Therefore, a reduction in the activity of lysozyme in fish exposed to Dimethoate alone or Dimethoate and Bacilar indicates the influence of this pollutant on lysozyme biosynthesis and thus disturbance in the innate immune system of common carp. Furthermore, the significant decrease in lysozyme activity in plasma of fish exposed to Dimethoate and Bacilar may indicate debility of defense mechanisms against bacterial agents. The activity of lysozyme is reduced in fish treated with different pesticides (Li et al., 2013). The complement system, as the first element of the innate immune system, plays a vital role in the immune system and affects the adaptive immune system by stimulating the reproduction of B cells (Li et al., 2013; Pushpa et al., 2014). Both C3 and C4 are glycoproteins that are produced by liver cells, macrophages, and monocytes. New molecular and cellular findings indicate that complement proteins are synthesized in different parts (Løvoll et al., 2007). Therefore, the reduced activity of ACH50, C3, and C4 in fish exposed to Dimethoate or Dimethoate and Bacilar may disturb the biosynthesis of these elements in hepatocytes, macrophages, and monocytes. A reduction in total complement and its elements in fish exposed to different pollutants demonstrate a deficiency in the innate immune system (Ahmadi et al., 2014). In general, our results indicate that exposure to agrochemicals, such as Dimethoate and Bacilar can suppress the innate immune system in common carp. 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(2018) 6(5): 254-257 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2018 Iranian Society of Ichthyology چکیده فارسی Cyprinusهای بیوشیمیایی و ایمنی در ماهی کپور معمولی )اثرات دایمیتوات و کود باسیالر بر فراسنجه carpio) 2، محمد محیسنی1، پروانه شوکت1*، مهدی بنایی1فاطمه محسنی نبیا ص( ببببا،، ایرا، گروه شیالت، دانشکده منابع طبیعی و محیط زیست، دانشگاه صنعتی خاتم اال1 ، خرم آباد، ایرا، روه علوم و مبندسی شیالت، دانشکده کشاورزی و منابع طبیعی، دانشگاه لرستا،گ2 چکیده: تواند موجب سرکوب سیستم ایمنی و کاهش بقای آنبا گردد بنابراین در قرار گرفتن ماهیا، در معرض مواد شیمیایی مورد استفاده در کشاورزی می یستی کش فسفره آلی و یک کود زهای زیرکشنده دایمیتوات به تنبایی و توام با کود باسیالر صیک آفتامطلوب غلظتاین مطالعه به بررسی اثرات ن داری در سطح پروتئین تام، ایمنوگلبولین و فعالیت روز، پرداخته شده است تغییر معنی 14های ایمنی ذاتی ماهی کپور معمولی در طی بر فراسنجه داری کاهش طور معنیبه C4با این حال، سطح گلبولین و کمپلما، در ماهیا، در معرض کود باسیالر به تنبایی یافت نشد C3لیزوزیم و کمپلما، ، لیزوزیم، کمپلما، ACH50نشا، داد قرار گرفتن ماهیا، در معرض دایمیتوات به تنبایی و یا توام با کود باسیالر موجب کاهش فعالیت کمپلما، تام C3 ،C4 پروتئین تام، گلبولین و ایمنوگلبولین تام در مقایسه با ماهیا، گروه کنترل گردید نتایج این مطالعه نشا، داد که پارامترهای ایمنی و سطح هیا، اذاتی در ماهیا، در معرض دایمیتوات و یا باسیالر کاهش یافت در نتیجه، دایمیتوات و باسیالر دارای اثر مبار کنندگی بر سیستم ایمنی م هستند مواد شیمیایی کشاورزی، ماهی کپور، مسمومیت ایمنی :کلمات کلیدی