International Journal of Fisheries and Aquaculture Vol. 5(1), pp. 1-6, January 2013 Available online at http://www.academicjournals.org/IJFA DOI: 10.5897/IJFA12.050 ISSN 2006-9839 ©2013 Academic Journals

Full Length Research Paper

Toxic effect of potassium permanganate on Oreochromis niloticus based on hematological parameters and biomarkers of oxidative stress

Jakeline G. França1*, Maria J. T. Ranzani-Paiva2, Julio V. Lombardi2, Solange de Carvalho1, Francisco Filipak-Neto3 and Ciro A. Oliveira-Ribeiro3

1Aquaculture Center, UNESP, Via de Acesso Prof. Paulo Donato Castellane, 14884-900 Jaboticabal, SP, Brazil. 2Fisheries Institute/APTA/SAA SP, Avenida Francisco Matarazzo, 05001-900 São Paulo, SP, Brazil. 3Departament of Celular Biology, University Federal do Paraná, 81.531-990 Curitiba, PR, Brazil.

Accepted 21 December, 2012

Potassium permanganate (KMnO4) is an oxidizing agent used in aquaculture for many years. Despite its known efficacy for the treatment and prevention of fish diseases, information about their potential toxicity in non-target organisms is still very limited. The objective of this study was to evaluate the sublethal effects of potassium permanganate (1.0 and 4.0 mg L-1) on Nile tilapia, Oreochromis niloticus, using hematological analysis and oxidative stress (reduced glutathione concentration, glutathione S- transferase activity, catalase and lipid peroxidation) as biomarkers. Fish exposed to a concentration at 1.0 mg L-1 had no significant variations in blood parameters. At 4.0 mg L-1, the changes in hematological parameters of the exposed animals indicated hemolysis as a result of the oxidizing action of potassium permanganate. Regarding the oxidative stress analyses, only the reduced glutathione presented significant increase in fish exposed to potassium permanganate, indicating adaptive and protection responses against the oxidative stress. This study demonstrated that concentrations of potassium permanganate (1.0 to 4.0 mg L−1) usually recommended for the treatment of fish diseases, can be toxic to Nile tilapia (O. niloticus).

Key words: Potassium permanganate, Nile tilapia, hematology, oxidative stress.

INTRODUCTION

Potassium permanganate is an oxidizing agent used for approved by the U.S. Food and Drug Administration as a many years in aquaculture for the treatment or prevention therapeutic agent in aquaculture (FDA, 2007). of diseases in fish, removal of parasites, disinfection of Potassium permanganate is potentially toxic to humans fish tanks and aquariums, reduction of products toxic to and other organisms (Kegley et al., 2010), including fish as rotenone and antimycin, and for the control of several fish species (Marking and Bills, 1975; Cruz and fungi and algae for managing problems when temporary Tamse, 1989; Bills et al., 1993; Straus, 2004; Silva et al., reduction of oxygen in tanks occurs (Lay, 1971; Duncan, 2006) but is not included in regulatory categories. 1974). Despite its widespread use for the treatment of Therefore, there is no pattern or established limit to this diseases in cultivated fish, this chemical compound is not compound and it may be used freely and without restriction in aquaculture. According to the FDA (2007), the inclusion of this compound in a regulatory class still depends on further studies. Several studies on the imple- *Corresponding author. E-mail: [email protected]. Tel: +55 mentation and effectiveness of potassium permanganate 11 38717563. in aquaculture are available in the literature, but information

2 Int. J. Fish. Aquaculture.

about its toxic potential in organs and tissues in fish is still were cleaned up at every operation of water replacement. scanty. The assay was carried out for 30 days, with sampling of 10 The impact of contaminants on the aquatic ecosystem individuals per group for the hematological analyses and biochemical after 7, 15 and 30 days of exposure time. can be measured through a variety of parameters, from For hematological analyses, the animals were anesthetized with low levels of biological organization (molecular and benzocaine (3%); blood was drawn by caudal puncture, with the biochemical responses) to high organization levels help of a needle and syringe previously heparinized. The blood (population and community response) (Au, 2004). In this specimens were assayed for: number of erythrocytes (RBC), context, the use of biomarkers at cellular and molecular counted in a Neubauer chamber; hematocrit (Ht) by the microhematocrit technique; and hemoglobin level (Hb) by the levels is extremely important as a sensitive tool to cyanomethemoglobin method. After these procedures, the following measure the biological effects during the assessment of RBC indices were calculated: mean corpuscular volume (MCV), environmental quality (Sarkar et al., 2006), since they are mean corpuscular hemoglobin (MCH) and mean corpuscular more specific, sensitive, reproducible and easy to hemoglobin concentration (MCHC). Smears were made using the determine (Au, 2004). same blood aliquots, and the slides were later stained with May- The Nile tilapia, O. niloticus (Linnaeus, 1758) was Grunwald-Giemsa, which were utilized for total leukocyte number (WBC) and differential count for each type of leukocyte selected as the test organism in this study for its great (lymphocytes, neutrophils, basophils, eosinophils and monocytes) aquaculture and commercial value. Tilapia is the third and total thrombocytes. most important cultured fish group in the world, after The biochemical analyses were performed at the Laboratory of carps and salmonids. Their hardiness and adaptability to Cell Toxicology, in the Federal University of Parana (UFPR), a wide range of culture systems has led to the Curitiba, Parana State, Brazil. Fish were sacrificed by deep commercialization of tilapia production in more than 100 sedation with benzocaine. Portions of the liver were sampled and maintained at −80°C freezer before performing analysis. Then, countries. Nile tilapia represents approximately 84% of samples were homogenized in phosphate buffered saline (PBS, pH total global tilapia production. In 2008, Nile tilapia culture 7.4) and frozen. Further, aliquots were separated from samples to alone was ranked fifth among the most cultured species the determinations of total protein concentration, glutathione S- in the world, with a total aquaculture production of 2.3 transferase (GST) and catalase (CAT) activities, as well as the million tonnes (FAO, 2009). In 2010, it is anticipated that concentrations of reduced glutathione (GSH) and lipid peroxidation (LPO). Total protein concentration was determined by the Bradford global Nile tilapia production will reach nearly 2.5 million method (Bradford, 1976). The activity of glutathione S-transferase tonnes (FAO, 2010). was monitored by the reaction of the substrate 1-chloro-2,4- The objective of this study was to evaluate the sub- dinitrobenzene with GSH, generating a thioether that absorbs light lethal effects of potassium permanganate for tilapia, O. at 340 nm (Habig et al., 1974; Keen et al., 1976). The concentration niloticus, using the hematological analyses and oxidative of GSH was determined according to Sedlak and Lindsay (1968) stress as biomarkers. after protein precipitation with 10% of trichloroacetic acid, and the activity of CAT was determined according to Aebi (1984). Lipid peroxidation was determined by the FOX method (Ferrous Oxidation/ Xylenol Orange Method) according to Jiang et al. (1991, MATERIALS AND METHODS 1992). In order to assure that the probable toxic effects observed were This study was carried out at the Laboratory of Pathology of Aquatic attributed to potassium permanganate, the following physical and Organisms (Fisheries Institute, Sao Paulo State, Brazil) under a chemical variables of water were measured at the beginning of the controlled environment. experiment and then at every 24 h: temperature (°C) and dissolved The fish used as test organisms were juveniles of tilapia, O. oxygen (mg L-1 and % saturation) were measured using the niloticus, purchased in commercial fish farming, with mean weight oximeter YSI - Mod.55; pH values were obtained using of 44.52 ± 11.07 g and mean length of 13.87 ± 1.14 cm. Fish were potentiometer and the electrical conductivity (μScm-1) using previously acclimated for 15 days in the same environmental conductivimeter. The analysis of hardness was done by the titration -1 conditions of testing containers, that is, they were randomly method with EDTA, alkalinity (mg CaCO3 L ) by titration. The distributed into three 300 L tanks equipped with artificial aeration following analyses were performed weekly: the hardness, alkalinity -1 -1 and biological filters previously matured, at the density of 70 fish (mg CaCO3 L ) and total ammonia (mg NH4L ) according to per tank (7.6 g L-1). The water quality variables were measured standardized methodology in APHA et al. (2005). Non-ionized -1 daily during the period of acclimation and they were appropriate for ammonia (NH3, mg L ) was calculated according to Bower and the specie in the initial conditions: temperature 22 ± 0.7°C; pH 7.7 ± Bidwell (1978). 0.2; dissolved oxygen 6.9 ± 0.3 mg L-1 and total ammonia between For data analyses we used the statistical program TOXSTAT 3.5 -1 0.33 and 0.40 mg NH4 L . Test solutions were set up by dissolving (West and Gulley, 1996). Data normality was tested by Shapiro TM potassium permanganate (KMnO4) Merck in dechlorinated tap Wilk’s test and homoscedasticity of variances by the Bartlett's test. water. Nominal test concentrations of potassium permanganate (1.0 Then, data was analyzed using the statistical test ANOVA. and 4.0 mg L-1) were used in the assays, and it was chosen based Alterations were considered significant when P < 0.05. on minimum and maximum doses of this compound reported in the literature to treat diseases in fish (Francis-Floyd and Klinger, 1997). A control group with no addition of potassium permanganate was RESULTS also carried out in the same conditions of testing containers. Test solutions were partially replaced every three days at the proportion of 1/3 of total volume (renewal test system). Fish were Survival rate of 100% was observed in experiments and fed with commercial ration 32% crude protein, and food residues all fish appeared healthy. Physical and chemical variables

França et al. 3

Table 1. Mean values and standard error of the mean of red blood series of Oreochromis niloticus exposed to potassium permanganate.

Treatments RBC (106 µL-1) Ht (%) Hb (g dL-1) MCH (pg) MCV (fL) MCHC (%) 7 days Control 2.03 ± 0.05a 30.10 ± 1.10a 6.43 ± 0.25a 32.01 ± 1.83a 149.92 ± 08.33a 21.39 ± 0.48a 1 mg L-1 2.11 ± 0.07a 27.20 ± 0.36a 5.92 ± 0.36a 28.47 ± 2.13a 130.29 ± 07.75a 21.86 ± 1.04a 4 mg L-1 1.52 ± 0.06b 26.85 ± 1.82a 6.41 ± 0.54a 42.19 ± 3.36a 177.78 ± 12.55a 23.60 ± 0.82a

15 days Control 1.98 ± 0.06a 28.00 ± 1.08a 6.67 ± 0.36a 33.58 ± 1.39a 141.39 ± 08.33a 24.09 ± 1.42a 1 mg L-1 1.86 ± 0.06ab 29.80 ± 0.26a 5.92 ± 0.36a 39.49 ± 1.48a 161.15 ± 07.75a 24.69 ± 0.96a 4 mg L-1 1.71 ± 0.06bc 26.45 ± 0.78a 6.41 ± 0.54a 35.20 ± 2.44a 156.03 ± 12.55a 22.70 ± 1.45a

30 days Control 1.79 ± 0.13a 28.25 ± 1.07a 6.58 ± 0.32a 37.58 ± 2.07a 162.08 ± 08.53a 23.32 ± 0.83a 1 mg L-1 1.48 ± 0.09a 26.50 ± 0.92ab 6.19 ± 0.37a 42.62 ± 2.48a 183.71 ± 09.16a 23.27 ± 0.95a 4 mg L-1 1.64 ± 0.11a 24.15 ± 0.87bc 6.09 ± 0.18a 38.18 ± 2.03a 152.68 ± 11.56a 25.42 ± 0.92a

abc Different letters indicate significant different (P < 0.05) mean values between treatments. RBC, Number of erythrocytes; Ht: hematocrit; Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration.

of water used in the bioassays were similar among potassium permanganate (Figure 1A). No significant different treatments (P=0.05). The mean data recorded change occurred in the activity of GST, CAT and LPO were: temperature (°C): 22.54 ± 1.30; pH: 7.77 levels in fish exposed to potassium permanganate ± 0.10; dissolved oxygen (% saturation): 80.67 ± 1.39; (Figure 1B to D). dissolved oxygen (mg L-1): 7.00 ± 0.41; electrical conductivity (µs cm-1): 248.16 ± 31.11; alkalinity (mg -1 -1 CaCO3 L ): 76.56 ± 13.51; total ammonia (mg NH4 L ): DISCUSSION -1 0.40 ± 0.20 and un-ionized ammonia (mg NH3 L ): 0.009 -1 ± 0.006. Fish kept at a concentration of 1 mg L KMnO4 The reduced values of hematimetric values found in this did not present significant variations in blood variables present study suggest the induction of anemia caused by herein analyzed (RBC, Ht and Hb) and in hematimetric the toxicity of potassium permanganate. The anemia may indices (MCV, MCH and MCHC) (Table 1). be due to the inhibition of erythropoiesis and hemo- The animals exposed to a concentration at 4 mg L-1 had synthesis and to an increase in the rate of erythrocyte a significant reduction (P<0.05) in red blood cells on the destruction in hemopoietic organs, resulting in a reduced seventh and 15th day after collection, and a reduction of RBC (Adhikari et al., 2004). Other xenobiotics also hematocrit on the 30th day (Table 1). On the seventh day induce changes that give evidence of decreased of collection, a significant increase of MCH (P<0.05) was hematopoiesis followed by anemia induction in fish, for detected in fish exposed to a concentration of 4 mg L-1. example, changes in erythrocyte profile induced by Thrombocytes were quite numerous in fish exposed to sublethal exposure to cypermethrin and carbofuran in potassium permanganate on the seventh collection day. Labeo rohita (Adhikari et al., 2004), diazinon in Cyprinus However, only at 1 mg L-1 a significant difference was carpio (Banaee et al., 2008), mercuric chloride in O. registered (Table 2). The total count of leukocytes and niloticus (Ishikawa et al., 2007). deltamethrin in Cyprinus -1 lymphocytes in tilapia exposed to 4.0 mg L KMnO4 was carpio (Svoboda et al., 2003) and chromium in Labeo significantly lower on the seventh day of sampling (Table rohita (Vutukuru, 2005). 2). On the 30th day, a significant decrease was observed Another observed response was the significant in the total count of leukocytes and lymphocytes in both increase of MHC in fish exposed to potassium treatments with potassium permanganate (1.0 and 4.0 permanganate, indicating the presence of cells with mg L-1). Eosinophils were not verified in any treatment. larger volume and greater amount of hemoglobin. Even But basophils were found in two samples of the control with the inverse variation of MHC in relation to the -1 and one sample of the treatment at 1 mg L . number of erythrocytes, the level of hematocrit, hemoglobin On the 15th day of collection, significant increase of and MCHC had no significant alteration (P > 0.05), with GSH was observed in the two tested concentrations of values close to control (Table 1). Moreover, the mean value

4 Int. J. Fish. Aquaculture.

Table 2. Mean values and standard error of the mean of leukocytes and thrombocytes of O. niloticus exposed to potassium permanganate.

Treatments Lc (104 µL-1) Lf (104 µL-1) Nt (104 µL-1) Mn (104 µL-1) Tr (104 µL-1) 7 days Control 4.45 ± 0.66a 4.27 ± 0.64a 0.07 ± 0.01a 0.08 ± 0.03a 0.44 ± 0.14a 1 mg L-1 4.86 ± 0.67a 4.65 ± 0.69a 0.12 ± 0.05a 0.07 ± 0.02a 1.68 ± 0.30b 4 mg L-1 2.78 ± 0.38b 2.65 ± 0.39b 0.08 ± 0.03a 0.04 ± 0.01a 1.22 ± 0.38a

15 days Control 4.24 ± 0.45a 4.13 ± 0.45a 0.05 ± 0.01a 0.06 ± 0.01a 0.65 ± 0.19a 1 mg L-1 2.84 ± 0.44a 2.75 ± 0.43a 0.05 ± 0.02a 0.03 ± 0.01a 1.05 ± 0.25a 4 mg L-1 2.84 ± 0.51a 2.77 ± 0.51a 0.03 ± 0.01a 0.05 ± 0.01a 0.94 ± 0.23a

30 days Control 3.70 ± 0.22a 3.53 ± 0.20a 0.07 ± 0.02a 0.07 ± 0.01a 0.49 ± 0.14a 1 mg L-1 2.00 ± 0.25b 1.92 ± 0.25b 0.03 ± 0.01a 0.04 ± 0.01a 0.76 ± 0.22a 4 mg L-1 2.06 ± 0.31b 1.99 ± 0.31b 0.02 ± 0.01a 0.04 ± 0.01a 0.51 ± 0.12a

ab Different letters indicate significant different (P < 0.05) mean values between treatments. Lc, Leukocytes; Lf, lymphocytes; Nt, neutrophils; Mn, monocytes; Tr, thrombocytes.

Figure 1. Concentration of GSH (A); activity of GST (B); activity of CAT (C); Lipid peroxidation (D), in liver of O. niloticus, exposed to potassium permanganate. Data are reported as mean ± standard error (n=10). *, Significant difference from control group (P < 0.05).

França et al. 5

of MCV was higher than other groups, but without significant response to avoid the increase of reactive oxygen difference this indicates that the same amount of species (ROS) and oxidative damage. Similar results hemoglobin has occupied a larger volume in the cell. were reported by Ventura-Lima et al. (2009), where the These results suggested the presence of a great amount of absence of changes in the activity of GST, CAT and LPO old and large erythrocytes, probably owing the prolonged levels in fish exposed to arsenic was due to the mechanism or failure to replace these cells (Oliveira-Ribeiro effectiveness of the increased levels of GSH for et al., 2006), inducing a macrocytic anemia. Changes in preventing additional oxidative disturbances. Thus, the the differential count of leukocytes provide an evidence results of this present study confirm that GSH is often the for a decrease in non-specific immunity in fish after first defense against oxidative stress. exposure to toxic substances (Svoboda et al., 2003; Oliveira-Ribeiro et al., 2006; El-Sayed et al., 2007). Thus, the leukopenia observed in fish exposed to potassium Conclusion permanganate is primarily assigned to the reduction in the number of lymphocytes probably due to a reduced Information on the physiological changes in fish by acute production and rapid destruction, leading to an increased and subchronic exposure to potassium permanganate is rate of removal of these cells. Svoboda et al. (2001) and limited. Study performed by França et al. (2011) in O. Banaee et al. (2008) observed a decrease in the niloticus, Kori-Siakpere (2008) in Clarias gariepinus and percentage of lymphocytes and monocytes in Cyprinus Silva et al. (2006) in Colossoma macropomum, revealed carpio after exposure to diazinon, indicating a decrease that the compost has high toxicity to fish in a short term in immunity, in response to stress caused by the toxic exposure. substance, corroborating the results of this present study. The results obtained in this present study have The increased number of thrombocytes registered in demonstrated that the prolonged use of concentrations stressed fish after being exposed to pollutants is related considered safe by literature for prophylaxis and to damage to organs and tissues (Mazon et al., 2002; treatment of diseases in fish farming (1.0 and 4.0 mg L-1 Gabriel et al., 2007). According to Francis-Floyd and KMnO4) may cause considerable changes to health of Klinger (1997), the potassium permanganate is a exposed fish, evidencing the toxic potential of potassium compound capable of reacting indiscriminately with any permanganate in non-target organisms. Such alterations, organic material in the water, including fish tissues like especially in hematological variables, have pointed out skin and gills. Since it is a strong oxidizing agent, the high that potassium permanganate induced the destruction of values of thrombocytes found in fish of both treatments red blood cells in a short period of exposure to the -1 (1.0 and 4.0 mg L KMnO4) can be associated with chemical, indicating the direct action of its toxicity. injuries in tissues such as skin and gills, once these Therefore, the use of different biomarkers become organs are directly exposed to this chemical in the water. important as they can reflect more accurately the toxicity According to Zhang et al. (2004), under severe stress, of contaminant substances studied and their effects suppression in the levels of GSH may occur, due to the impacting the aquatic ecosystem. reduction capacity of synthesis, regeneration or oxidation of GSH. Moreover, the increase in GSH observed in this study reflects the stimulation of the detoxification ACKNOWLEDGMENT metabolism, suggesting an adaptive response and a protection response of this biomolecule against a rapid The authors would like to thank the São Paulo Research oxidative stress, induced by contaminants (Pandey et al., Foundation (FAPESP) for financial support (Process 2003; Zhang et al., 2004; Farombi et al., 2007). 0756481-6R). As observed in this study, Ventura-Lima et al. (2009) found no significant change in the levels of GST in Danio rerio exposed to arsenic, indicating that the tested REFERENCES concentrations were not deleterious enough to interfere in Adhikari S, Sarkar B, Chatterjee A, Mahapatra CT Ayyappan S (2004). the activity of this enzyme. Sanchez et al. 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