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Pesticide Biochemistry and Physiology 86 (2006) 157–161 www.elsevier.com/locate/ypest
Interactive eVect of monocrotophos and ammonium chloride on the freshwater Wsh Oreochromis mossambicus with reference to lactate/pyruvate ratio K. Vijayavel ¤, E.F. Rani, C. Anbuselvam, M.P. Balasubramanian Department of Pharmacology and Environmental Toxicology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600 113, Tamil Nadu, India Received 28 January 2006; accepted 3 March 2006 Available online 18 April 2006
Abstract Sublethal eVect of monocrotophos (pesticide) and ammonium chloride (fertilizer) was studied in the freshwater Wsh Oreochromis mossambicus, with reference to carbohydrate metabolism for a period of 96 h. The glycogen content was analysed in liver and muscle, while the lactate and pyruvate were assessed in blood along with liver and muscle. The results revealed that the glycogen content was found to be decreasing. In contrast increase in the tissues lactate and pyruvate level was found in the Wshes exposed to pesticide and fertilizer individually and in combinations. The combined eVects of these chemicals were more toxic to carbohydrate metabolism than the eVect produced by the individual chemicals. The results were tested to search for statistical signiWcance. The calculated lactate and pyruvate ratio (L/P) indicated that the Wshes were under chemical stress. © 2006 Elsevier Inc. All rights reserved. Keywords: Monocrotophos; Ammonium chloride; Glycogen; Lactate; Pyruvate; Lactate/pyruvate ratio; Oreochromis mossambicus
1. Introduction Pesticides and mineral fertilizers used extensively in modern agricultural practices pollute the nearby aquatic bodies due to runoV and aVect their Wsh populations. These chemicals applied simultaneously in agricultural operations have greater eVect on the metabolism and enzyme systems of non-target species than each chemical. Moreover, chemical fertilizers have greater synergistic eVects on the physiology of non-target species when combined with pesticides [1]. It has become increasingly apparent in recent years that combinations of chemicals result in greater toxicity than what would be expected on the basis of simple addition of their individual actions. Studies on the toxicity of mixtures are referred to as interaction studies. The interaction of
*
Corresponding author. E-mail address: [email protected] (K. Vijayavel).
0048-3575/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2006.03.002
chemicals may inXuence the toxicity induced by the individual chemicals [2]. Fishes especially Oreochromis mossambicus are often used as indicator organisms to investigate the biological eVects of pollutants in the aquatic environment. In recent years O. mossambicus serve as useful bioindicators and integrators of contaminants, because of various reasons, viz. their wide distribution in the freshwater environment, the fact of being free swimmers, their ability to respond against environmental pollution, and their importance as an economic food source for human beings [3]. Carbohydrates are considered to be Wrst among the organic nutrients degraded in response to stress conditions imposed on animals. Even though protein is the major source of energy in animals, stress or severe hypoxia causes depletion of stored carbohydrates (glycogen) in liver and muscle [4]. Studies on aquatic animals subjected to stress have revealed that lactic acid and pyruvic acid levels are altered following the changes in tissue
158
K. Vijayavel et al. / Pesticide Biochemistry and Physiology 86 (2006) 157–161
carbohydrates [5]. Moreover, the relative concentrations of lactate and pyruvate could also indicate the state of metabolism in vivo. Considerable studies have been recently carried out along these lines [6]. These studies revealed the importance of carbohydrates and its metabolites in pollution monitoring. A majority of studies have investigated the individual eVect of pesticides and fertilizers on various physiological responses in Wshes [7–10,3,11,12]. However, the inXuences of combination of pesticide–fertilizer on physiology of Wsh have received little attention [13,4,1,2]. Hence, it is of interest to study the changes in glycogen, lactic acid, and pyruvic acid levels in tissues of O. mossambicus with reference to interactive stress of pesticide and fertilizer.
Table 2 Percentage survival of O. mossambicus exposed to various concentrations of ammonium chloride for 96 h and its median lethal concentration (LC50 with 95% conWdence limits calculated by probit analysis) Concentration in ppm
24 h
48 h
72 h
96 h
400 425 450 475 500 525 550 575 600
100 100 100 100 100 100 70 50 0
100 100 100 100 90 60 30 0 0
100 100 90 80 50 20 0 0 0
100 80 70 50 30 10 0 0 0
96 h LC50 (mg ammonium chloride L¡1)
465.17 (431.17–475.3)
Juveniles of O. mossambicus weighing 2.9–3.2 g were collected from local pond, near Velacherry, Chennai and acclimated to the laboratory conditions with temperature 28 § 1 °C, and photoperiod of 12 L:12D, for one week. They were fed with chopped meat of goat liver pieces. The water in aquaria was renewed daily. One day prior to the experiment the Wshes were starved. In a preliminary experiment, acute toxicity study was carried out by following the standard guidelines of EPA/ROC [14] to determine the lethal (LC100), median lethal (LC50), and safe sublethal (LC0) level of monocrotophos and ammonium chloride to O. mossambicus. The 96 h LC50 value of the mortality in each exposure concentration was recorded and tested by probit analysis programme as described by Finney [15] (Tables 1 and 2). The results revealed that 4 mgL¡1 and 400 mgL¡1 were sublethal for monocrotophos and ammonium chloride respectively. The highest concentration of monocrotophos (4 mgL¡1) and ammonium chloride (400 mgL¡1), which showed 100% survival of Wsh, was considered as the sublethal level. For biochemical studies, O. mossambicus were divided into four groups of 10 specimens each. Fish of group I were reared in chemical-free water and treated as control. Fish belonging to groups II, III, and IV were exposed to suble-
thal concentrations of monocrotophos (4 mgL¡1), ammonium chloride (400 mgL¡1), and monocrotophos plus ammonium chloride (300 L¡1 + 1.5 L¡1) respectively. For each group, individuals were reared separately in plastic troughs containing 5 L of test medium. The exposure of Wshes to chemicals was continued up to 96 h. At the completion of 96 h, blood was collected from the Wsh by tail severance. Potassium oxalate (0.8%) mixed with ammonium oxalate (1.2%) was used as the anticoagulant and centrifuged at 2000 g for 10 min to separate serum. The Wshes were then sacriWced by severing the spinal cord behind the head and the tissues (liver and muscle) were separated from the control and test Wshes. The tissues were rinsed with distilled water to remove the adhering blood. One milliliter of serum and 100 mg of tissues were homogenized individually using 0.02 M Tris–HCl buVer. Homogenates were centrifuged at 5000 g for 10 min. The supernatant was collected and stored at ¡20 °C for the biochemical assay. Glycogen was estimated in liver and muscle by following the method of Caroll et al. [16] using glucose as standard. Lactic acid was estimated according to the method of Hohorst [17] in blood, liver, and muscle using lithium lactate as standard. By following the method of Landon et al. [18] pyruvic acid was estimated in blood, liver, and muscle using sodium pyruvate as standard. Each experiment was replicated three times and the data were subjected to Students t test by the method of Baily [19].
Table 1 Percentage survival of O. mossambicus exposed to various concentrations of monocrotophos for 96 h and its median lethal concentration (LC50 with 95% conWdence limits calculated by probit analysis)
3. Results and discussion
2. Materials and methods
Concentration in ppm
24 h
48 h
72 h
96 h
4 5 6 7 8 9 10 11 12 13 14
100 100 100 100 100 100 100 100 80 50 0
100 100 100 100 90 80 60 50 20 0 0
100 100 100 90 80 60 50 20 10 0 0
100 90 80 70 70 50 30 10 0 0 0
96 h LC50 (mg monocrotophos L¡1)
8.14 (7.35–9.08)
Carbohydrates in the tissues of aquatic animals exist as glycogen. It is well-known that the glycogen serves as energy reserve for various metabolic processes. The results of liver and muscle glycogen content of O. mossambicus exposed to pesticide and fertilizer individually and in combinations are presented in Table 3 and it reveals that the glycogen content reduced signiWcantly (P < 0.01, P < 0.001) when compared to control. The observed reduction in glycogen content indicates the utilization of stored glycogen possibly through anaerobic glycolysis to meet the extra energy requirement under hypoxia caused by pollutant stress [20]. Baskaran et al. [9] are of the
K. Vijayavel et al. / Pesticide Biochemistry and Physiology 86 (2006) 157–161 Table 3 Individual and combined eVect of monocrotophos and ammonium chloride on the glycogen content of liver and muscle of O. mossambicus Toxicants
Liver
Muscle
Control Monocrotophos Ammonium chloride Monocrotophos + ammonium chloride
5.070 § 1.703 2.246 § 0.306¤¤ 1.081 § 0.140¤¤ 0.98 § 0.03¤¤¤
0.511 § 0.164 0.223 § 0.058¤¤ 0.211 § 0.027¤¤ 0.187 § 0.027¤¤¤
Each value represents the average (X § SD) performance of 10 individuals for a period of 96 h. The results are expressed as (g mg¡1 wet tissue). Asterisks indicate values that are signiWcantly diVerent from controls. ¤¤ P < 0.01 ¤¤¤ P < 0.001
opinion that in any living animal, toxic inXuence exerts its eVects at the molecular and biochemical level. Thus, alteration in normal biochemical parameters serves as the earliest indicator of the toxic eVects on blood, liver, and muscle of aquatic animals [21]. The unequivocal reductions in stored glycogen content in liver and muscle of O. mossambicus exposed to sublethal concentrations of monocrotophos, ammonium chloride and their mixtures indicate the utilization of stored glycogen possibly through anaerobic glycolysis to meet the energy requirement under hypoxia [22] or may be due to chemical stress [23]. A similar reduction in the stored tissue glycogen content has been reported in Channa striatus exposed to demeton [24], C. striatus exposed to malathion [25], Sarotherodon mossambicus exposed to [23], and O. mossambicus exposed to urea [2]. Stressful situation in Wshes elicit neuroendocrine response, which in turn induce disturbance in carbohydrate metabolism. Both catecholamines and adrenocorticosteroids are secreted in increased amounts due to stress stimuli and elicit marked changes in carbohydrate energy reserves of Wshes [26]. Thus, the marked glycogenolysis in muscle and liver of Wshes exposed to chemicals may be due to the stress-induced increase of the circulating catecholamines. The observed reduction of glycogen in liver and muscle may be also due to the increased utilization of glycogen through the glycolytic pathway as suggested by [21]. On the other hand the observed decrease in the glycogen might have been due to the rapid turnover of glycogen synthe-
159
sized or due to the decreased rate of glycogenesis [27]. The amount of glycogen depleted may be due to the fact that energy reserves are used as substitutes during metabolic requirement, i.e., the rapid breakdown of glycogen to lactate and pyruvate and then released to meet the energy requirements during stress conditions. The lactic acid and pyruvic acid are the end products of glycolysis and their rate of production is considered as an index of physiological stress [28]. Hence, accumulation of lactic acid indicates that the metabolism is gearing toward a condition similar to anaerobiosis. This may be due to the reduction of the cellular oxidative process under the inXuence of pollutant [29]. The lactic acid level of blood, liver, and muscle of O. mossambicus exposed to sublethal concentrations of chemicals was found to be increasing when compared to control (Table 4). The lactic acid level in the blood of O. mossambicus reared in control medium, was 273.8 gmL¡1, which rose to 759.9, 853.3, and 879.9 gmL¡1 when reared in 4 mgL¡1 of monocrotophos, 400 mgL¡1 of ammonium chloride and mixture of monocrotophos and ammonium chloride (1.5 L¡1 + 300 L¡1) at the end 96 h exposure respectively. Similar increasing trend in the lactic acid level was observed in liver and muscle and the results were highly signiWcant (P < 0.001). The pyruvic acid levels of blood, liver, and muscle of O. mossambicus exposed to sublethal concentrations of chemicals were found to be increasing when compared to control (Table 4). The blood pyruvic acid level of O. mossambicus reared in control medium, was 200 gmL¡1 which raised to 460, 495, and 560 gmL¡1 when reared in 4 mgL¡1 of monocrotophos, 400 mgL¡1 of ammonium chloride and mixture of monocrotophos and ammonium chloride (1.5 L¡1 + 300 L¡1) at the end 96 h exposure respectively. Similar increasing trend in the pyruvic acid level was observed in liver and muscle and the results were highly signiWcant (P < 0.001). The above result revealed that the enhancement of blood, liver, and muscle lactic acid and pyruvic acid levels were higher in the Wshes reared in combination of monocrotophos and ammonium chloride than in the Wshes exposed to individual concentration of pesticide and fertilizer.
Table 4 Individual and combined eVect of monocrotophos and ammonium chloride on the lactate, pyruvate, and lactate/pyruvate ratio (L/P) of blood, liver, and muscle of O. mossambicus Toxicants
Control Monocrotophos Ammonium chloride Monocrotophos + ammoniumchloride
Lactic acid
Pyruvic acid
Lactate/pyruvate ratio (L/P)
Blood
Liver
Muscle
Blood
Liver
Muscle
Blood
Liver
Muscle
273.8 § 17.3 759.9 § 34.5¤¤ 853.3 § 54.4¤¤¤ 879.9 § 47.2¤¤¤
26.6 § 1.4 74.6 § 3.3¤¤ 81.33 § 4.02¤¤¤ 93.7 § 3.5¤¤¤
32.9 § 2.7 90.6 § 7.15¤¤¤ 105.3 § 9.3¤¤¤ 126.2 § 8.4¤¤¤
21.0 § 1.218 46.0 § 2.13¤¤¤ 49.5 § 3.12¤¤¤ 56.0 § 3.87¤¤¤
2.35 § 0.12 4.4 § 0.19¤¤ 4.72 § 0.28¤¤ 5.6 § 0.35¤¤¤
3.13 § 0.18 5.35 § 0.115¤¤ 6.65 § 0.32¤¤¤ 7.21 § 0.28¤¤¤
0.130 0.165 0.172 0.157
0.113 0.169 0.174 0.167
0.105 0.169 0.158 0.17
Each value represents the average (X § SD) performance of 10 individuals for a period of 96 h. Asterisks indicate values that are signiWcantly diVerent from controls. The results are expressed as g mL¡1 for blood and g mg¡1 for liver and muscle. ¤¤ P < 0.01 ¤¤¤ P < 0.001
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Hyperglycemia is a common phenomenon in Wshes following muscular excretion and both muscle and hepatic glycogen are the sources of the blood lactate [30]. Since catecholamines can cause an increase in the blood lactate level of Wsh directly [26], this response to chemicals might reXect a stress hormone-mediated response. Metabolically the lactic acid is formed in animal tissue as an anaerobic end product of glycolysis. On the contrary, the continued persistence of tissue lactic acid could not be cleared up immediately and this may be due to the lack of oxygen supply to tissues [5]. The following points are relevant to suggest for the cumulative increase of blood lactate caused by chemical poisoning viz: (1) lactate formed in tissues like muscles may have diVused into the blood, (2) lactate may be produced from the residual level of blood carbohydrates or other non-carbohydrate metabolites as the toxicants inhibit the oxygen binding capacity of hemoglobin content in blood cells, (3) circulatory failure inhibiting lactate removal and (4) lack or ineYciency of enzyme pathway involving ADH (alcohol dehydrogenase) genetic loci to convert the lactate into alcohol. In Wsh, such conversion of lactate into alcohol by ADH does not yield any energy [5]. It is an adaptation that could be related to the reduction of lactic acidemia, as the alcohol formed rapidly diVuses out through the gills. Increase of lactic acid in tissues is attributed to the inadequacy of oxygen supply in cells to cope up with complete breakdown of carbohydrates to carbondioxide and water. Hence, the persistence of tissue lactate in Wshes exposed to chemicals in the present study implies hypoxia. Moreover, the persistence of higher levels of lactic acid in both muscle and liver of chemical exposed Wshes may also be due to impairment in the diVusion from the tissues. Such impairment in the diVusion of tissue lactate into blood has been reported in stressed Wshes [6,31]. The increase in lactic acid content also suggests the reduced mobilization of pyruvic acid into citric acid [32]. Studies on Wshes subjected to physical stress have revealed that pyruvic acid contents are altered following the changes in tissue carbohydrates [32]. Above studies also suggest that pyruvate concentrations in tissues serve as good indices to assess the stress manifestations. The pyruvic acid content of the blood, liver, and muscle of O. mossambicus exposed to sublethal concentrations of monocrotophos, ammonium chloride, and mixtures of monocrotophos and ammonium chloride increased when compared to control. Similar observations were made in S. mossambicus exposed to DDT, malathion, and mercury [6] and Tilapia mossambicus exposed to lindane [33]. The tissue pyruvic acid increased following the depletion of glycogen. Increase in tissue pyruvic acid in Wshes subjected to stress conditions is not uncommon, however, the conversion of pyruvic acid and its further changes either through oxidation or reduction, depends on the expression of the diVerent isomeric forms of lactate dehydrogenase (LDH) [34]. The lactate/pyruvate ratio (L/P) increased when compared to control in the blood, liver, and muscle of O. mossambicus exposed to sublethal concentrations of
monocrotophos, ammonium chloride, and their mixtures, which is an indicator of stress. The results of the present study revealed that in O. mossambicus, the toxic stress by chemicals induced crisis in aerobic metabolism and a switch over to glycolytic potential. The higher concentrations of lactate and the L/P ratio exceeding unity than the control values emphasize the above change in carbohydrate metabolism and it is a good stress indicator for aquatic toxicology studies. Widespread contamination of aquatic ecosystems by a whole battery of pesticides and fertilizers has been responsible for the massive “Wsh kills” and recognized as a potential human health hazard as well. The combination of chemicals can result in toxicity that is simply additive or greater than or less than additive. Pesticides and fertilizers are a mixed blessing since they improve agricultural productivity, but can aVect the non-target organisms such as Wsh and aquatic animals. Hence, it can be concluded that pesticides and fertilizers applied in the agricultural operations probably have considerable joint action on non-target species such as Wshes. References [1] V. Palanivelu, K. Vijayavel, S. Ezhilarasibalasubramanianm, M.P. Balasubramanian, InXuence of pesticide–fertilizer combinations on food intake, growth and conversion eYciencies of Oreochromis mossambicus, Bullet. Environ. Contam. Toxicol. 69 (2002) 908–913. [2] V. Palanivelu, K. Vijayavel, S. Ezhilarasibalasubramanianm, M.P. Balasubramanian, Combined eVect of cartaphydrochloride and urea on biochemical constituents of freshwater Wsh Oreochromis mossambicus, J. Ecotoxicol. Environ. Monit. 14 (1) (2004) 23–29. [3] E.F. Rani, M. Elumalai, M.P. Balasubramanian, Toxic and sublethal eVects of ammonium chloride on a freshwater Wsh Oreochromis mossambicus, Water Air Soil Poll. 104 (1998) 1–8. [4] E.F. Rani, M. Elumalai, M.P. Balasubramanian, EVect of mixtures of pesticide and fertilizer on carbohydrates, protein, phosphomonoesterases and non-speciWc esterases in Oreochromis mossambicus, Nat. Acad. Sci. Lett. 22 (1999) 70–74. [5] K. Ramalingam, R. Kasinathadurai, Blood carbohydrates and phosalone poisoning in Rana tigrina (Daudin), Arch. Inter. Physiol. Biochem. 97 (1989) 369–374. [6] K. Ramalingam, Toxic eVects of DDT, malathion and mercury on the tissue carbohydrate metabolism of Sarotherodon mossambicus (Peters), Proc. Indian Acad. Sci. (Anim. Sci.). 97 (1988) 443–448. [7] S. Palanichamy, S. Arunachalam, M.P. Balasubramanian, Toxic and sub eVects of ammonium chloride on food consumption and growth in the air breathing Wsh Channa striatus, Warm Water Aqua. Hawaii 465 (1985) 480. [8] S. Palanichamy, S. Arunachalam, M.P. Balasubramanian, Toxic and sublethal eVects of diammonium phosphate on food consumption and growth in the Sarotherodon mossambicus, Hydrobiologia 128 (1985) 233–237. [9] P. Baskaran, S. Palanichamy, P. Visalakshi, M.P. Balasubramanian, EVects of mineral fertilizers on survival of the Wsh Oreochromis mossambicus, Environ. Ecol. 7 (1989) 463–465. [10] V. Palanivelu, M.P. Balasubramanian, Food consumption of Oreochromis mossambicus exposed to sublethal concentrations of cartap hydrochloride, Geobios 24 (1997) 51–54. [11] V. Palanivelu, K. Vijayavel, S. Ezhilarasibalasubramanianm, M.P. Balasubramanian, InXuence of insecticidal derivative (cartap hydrochloride) from the marine polychaete on certain enzyme systems of
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the freshwater Wsh Oreochromis mossambicus, J. Environ Biol. 26 (2) (2005) 191–196. V. Palanivelu, K. Vijayavel, S. Ezhilarasibalasubramanianm, M.P. Balasubramanian, Impact of fertilizer (urea) on oxygen consumption and feeding energetics in the freshwater Wsh Oreochromis mossambicus, Environ. Toxicol. Pharmacol. 19 (2) (2005) 351–352. E.F. Rani, M. Elumalai, M.P. Balasubramanian, The toxicity of mixtures of monocrotophos and ammonium chloride to a freshwater Wsh Oreochromis mossambicus, Biomed. Lett. 55 (1997) 193–198. EPA/ROC, Standard guide for conducting acute tests with Wshes. Static renewal test for common carp. NIEA B904.10B, Environmental Protection Administration of the Republic of China, Taipei, Taiwan, 1998. D.J. Finney, Probit Analysis, third ed., Cambridge University Press, London, 1971. W.V. Caroll, R.W. Longley, J.H. Rao, The determination of glycogen content in liver and muscle by the use of anthrogen reagent, J. Biol. Chem. 220 (1956) 583–593. H.J. Hohorst, L (+) Lactate: determination with lactate dehydrogenase and DPN+, in: H.U. Bergrneyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1965, pp. 266–270. J. Landon, J.K. Fawcett, V. Wynn, Blood pyruvate concentration measured by a speciWc method in control subjects, J. Clin. Path. 15 (1962) 579–584. N.T.J. Baily, Statistical Methods in Biology, The English University Press, London, 1959, p.200. A.G. Heath, Water Pollution and Fish Physiology. Chap. 8. Physiological Energetics, CRC Press, Florida, 1987, pp. 131–163. S. Omkar, Changes in acid and alkaline phosphatase activity of a freshwater prawn, Macrobrachium lamarrei exposed to aldrin, Ind. Health. 23 (1985) 155–157. D.M. Dhavale, V.B. Masurekar, Variations in the glucose and glycogen content in the tissues of Scylla serrata (Forskal) under the inXuence of cadmium toxicity, Geobios 13 (1986) 139–142.
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[23] M. Vasanthi, M. Ramasamy, A shift in metabolic pathway of Sarotherodon mossambicus (peters) exposed to thiodon (endosulfan), Proc. Indian. Acad. Sci. (Anim. Sci.). 91 (1987) 56–61. [24] G.M. Natarajan, Changes in the bimodal gas exchange and some blood parameters in the air-breathing Wsh Channa striatus (Bleeder) following lethal exposure to metasystor (Demeton), Curr. Sci. 51 (1) (1981) 40–41. [25] P.H. Shah, M.S. Dubale, Biochemical changes induced by malathion in the body organs of Channa punctatus, J. Anim. Morphol. Physiol. 30 (1983) 107–118. [26] A.L. Larsson, Metabolic eVects of epinephrine and norepinephrine in the eel Anguilla anguilla, Gen. Comp. Endocrinol. 20 (1973) 155–158. [27] V.J.S. Rani, P. Venkateshwaralu, C. Tanich, Changes in carbohydrate metabolism of Clarias batrachus (Linn) when exposed to organophosphorus insecticide, J. Environ. Biol. 10 (2) (1989) 197–204. [28] L. Lowejinde, A.J. Niimi, Short term and long term eVects of cadmium on glycogen reserves and liver size in rainbow trout (Salmo gairdneri Richardson), Arch. Environ. Contam. Toxicol. 13 (1984) 759–764. [29] B.V. Ravinder, B.R. Babu, G. Narajana, Decis toxicity study on lactate dehydrogenase isozymes of a freshwater Wsh catWsh Clarias batrachus (Linnaes), J. Environ. Biol. 10 (2) (1989) 217–225. [30] T.S. Gill, J. Pande, H. Tewari, EVects of endosulfan on blood and organ chemistry of freshwater Wsh Barius conchonius hamilton, Ecotoxicol. Environ. Saf. 21 (1) (1991) 80–91. [31] K. Ramalingam, K. Anbarasu, Lactate/Pyruavte ratio—a stress indicator of ecotoxicity, J. Ecotoxicol. Environ. Monit. 3 (3) (1993) 183–186. [32] A.T. Reddy, K. Yellamma, Perturbations in carbohydrate metabolism during cypermethrin toxicity in Wsh Tilapia mossambica, Biochem. Int. 23 (1991) 633–638. [33] M.D. Ferrando, E.A. Moliner, EVects of lindane on Wsh carbohydrate metabolism, Ecotoxicol. Environ. Saf. 22 (1) (1991) 17–23. [34] T.C. Diamantino, E. Almeida, M.V.M. Soares, L. Guilhermino, Lactate dehydrogenase as an eVect criterion in toxicity tests with Daphnia magna straus, Chemosphere 45 (2001) 553–560.
Interactive eVect of monocrotophos and ammonium chloride on the freshwater Wsh Oreochromis mossambicus with reference to lactate/pyruvate ratio K. Vijayavel ¤, E.F. Rani, C. Anbuselvam, M.P. Balasubramanian Department of Pharmacology and Environmental Toxicology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600 113, Tamil Nadu, India Received 28 January 2006; accepted 3 March 2006 Available online 18 April 2006
Abstract Sublethal eVect of monocrotophos (pesticide) and ammonium chloride (fertilizer) was studied in the freshwater Wsh Oreochromis mossambicus, with reference to carbohydrate metabolism for a period of 96 h. The glycogen content was analysed in liver and muscle, while the lactate and pyruvate were assessed in blood along with liver and muscle. The results revealed that the glycogen content was found to be decreasing. In contrast increase in the tissues lactate and pyruvate level was found in the Wshes exposed to pesticide and fertilizer individually and in combinations. The combined eVects of these chemicals were more toxic to carbohydrate metabolism than the eVect produced by the individual chemicals. The results were tested to search for statistical signiWcance. The calculated lactate and pyruvate ratio (L/P) indicated that the Wshes were under chemical stress. © 2006 Elsevier Inc. All rights reserved. Keywords: Monocrotophos; Ammonium chloride; Glycogen; Lactate; Pyruvate; Lactate/pyruvate ratio; Oreochromis mossambicus
1. Introduction Pesticides and mineral fertilizers used extensively in modern agricultural practices pollute the nearby aquatic bodies due to runoV and aVect their Wsh populations. These chemicals applied simultaneously in agricultural operations have greater eVect on the metabolism and enzyme systems of non-target species than each chemical. Moreover, chemical fertilizers have greater synergistic eVects on the physiology of non-target species when combined with pesticides [1]. It has become increasingly apparent in recent years that combinations of chemicals result in greater toxicity than what would be expected on the basis of simple addition of their individual actions. Studies on the toxicity of mixtures are referred to as interaction studies. The interaction of
*
Corresponding author. E-mail address: [email protected] (K. Vijayavel).
0048-3575/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2006.03.002
chemicals may inXuence the toxicity induced by the individual chemicals [2]. Fishes especially Oreochromis mossambicus are often used as indicator organisms to investigate the biological eVects of pollutants in the aquatic environment. In recent years O. mossambicus serve as useful bioindicators and integrators of contaminants, because of various reasons, viz. their wide distribution in the freshwater environment, the fact of being free swimmers, their ability to respond against environmental pollution, and their importance as an economic food source for human beings [3]. Carbohydrates are considered to be Wrst among the organic nutrients degraded in response to stress conditions imposed on animals. Even though protein is the major source of energy in animals, stress or severe hypoxia causes depletion of stored carbohydrates (glycogen) in liver and muscle [4]. Studies on aquatic animals subjected to stress have revealed that lactic acid and pyruvic acid levels are altered following the changes in tissue
158
K. Vijayavel et al. / Pesticide Biochemistry and Physiology 86 (2006) 157–161
carbohydrates [5]. Moreover, the relative concentrations of lactate and pyruvate could also indicate the state of metabolism in vivo. Considerable studies have been recently carried out along these lines [6]. These studies revealed the importance of carbohydrates and its metabolites in pollution monitoring. A majority of studies have investigated the individual eVect of pesticides and fertilizers on various physiological responses in Wshes [7–10,3,11,12]. However, the inXuences of combination of pesticide–fertilizer on physiology of Wsh have received little attention [13,4,1,2]. Hence, it is of interest to study the changes in glycogen, lactic acid, and pyruvic acid levels in tissues of O. mossambicus with reference to interactive stress of pesticide and fertilizer.
Table 2 Percentage survival of O. mossambicus exposed to various concentrations of ammonium chloride for 96 h and its median lethal concentration (LC50 with 95% conWdence limits calculated by probit analysis) Concentration in ppm
24 h
48 h
72 h
96 h
400 425 450 475 500 525 550 575 600
100 100 100 100 100 100 70 50 0
100 100 100 100 90 60 30 0 0
100 100 90 80 50 20 0 0 0
100 80 70 50 30 10 0 0 0
96 h LC50 (mg ammonium chloride L¡1)
465.17 (431.17–475.3)
Juveniles of O. mossambicus weighing 2.9–3.2 g were collected from local pond, near Velacherry, Chennai and acclimated to the laboratory conditions with temperature 28 § 1 °C, and photoperiod of 12 L:12D, for one week. They were fed with chopped meat of goat liver pieces. The water in aquaria was renewed daily. One day prior to the experiment the Wshes were starved. In a preliminary experiment, acute toxicity study was carried out by following the standard guidelines of EPA/ROC [14] to determine the lethal (LC100), median lethal (LC50), and safe sublethal (LC0) level of monocrotophos and ammonium chloride to O. mossambicus. The 96 h LC50 value of the mortality in each exposure concentration was recorded and tested by probit analysis programme as described by Finney [15] (Tables 1 and 2). The results revealed that 4 mgL¡1 and 400 mgL¡1 were sublethal for monocrotophos and ammonium chloride respectively. The highest concentration of monocrotophos (4 mgL¡1) and ammonium chloride (400 mgL¡1), which showed 100% survival of Wsh, was considered as the sublethal level. For biochemical studies, O. mossambicus were divided into four groups of 10 specimens each. Fish of group I were reared in chemical-free water and treated as control. Fish belonging to groups II, III, and IV were exposed to suble-
thal concentrations of monocrotophos (4 mgL¡1), ammonium chloride (400 mgL¡1), and monocrotophos plus ammonium chloride (300 L¡1 + 1.5 L¡1) respectively. For each group, individuals were reared separately in plastic troughs containing 5 L of test medium. The exposure of Wshes to chemicals was continued up to 96 h. At the completion of 96 h, blood was collected from the Wsh by tail severance. Potassium oxalate (0.8%) mixed with ammonium oxalate (1.2%) was used as the anticoagulant and centrifuged at 2000 g for 10 min to separate serum. The Wshes were then sacriWced by severing the spinal cord behind the head and the tissues (liver and muscle) were separated from the control and test Wshes. The tissues were rinsed with distilled water to remove the adhering blood. One milliliter of serum and 100 mg of tissues were homogenized individually using 0.02 M Tris–HCl buVer. Homogenates were centrifuged at 5000 g for 10 min. The supernatant was collected and stored at ¡20 °C for the biochemical assay. Glycogen was estimated in liver and muscle by following the method of Caroll et al. [16] using glucose as standard. Lactic acid was estimated according to the method of Hohorst [17] in blood, liver, and muscle using lithium lactate as standard. By following the method of Landon et al. [18] pyruvic acid was estimated in blood, liver, and muscle using sodium pyruvate as standard. Each experiment was replicated three times and the data were subjected to Students t test by the method of Baily [19].
Table 1 Percentage survival of O. mossambicus exposed to various concentrations of monocrotophos for 96 h and its median lethal concentration (LC50 with 95% conWdence limits calculated by probit analysis)
3. Results and discussion
2. Materials and methods
Concentration in ppm
24 h
48 h
72 h
96 h
4 5 6 7 8 9 10 11 12 13 14
100 100 100 100 100 100 100 100 80 50 0
100 100 100 100 90 80 60 50 20 0 0
100 100 100 90 80 60 50 20 10 0 0
100 90 80 70 70 50 30 10 0 0 0
96 h LC50 (mg monocrotophos L¡1)
8.14 (7.35–9.08)
Carbohydrates in the tissues of aquatic animals exist as glycogen. It is well-known that the glycogen serves as energy reserve for various metabolic processes. The results of liver and muscle glycogen content of O. mossambicus exposed to pesticide and fertilizer individually and in combinations are presented in Table 3 and it reveals that the glycogen content reduced signiWcantly (P < 0.01, P < 0.001) when compared to control. The observed reduction in glycogen content indicates the utilization of stored glycogen possibly through anaerobic glycolysis to meet the extra energy requirement under hypoxia caused by pollutant stress [20]. Baskaran et al. [9] are of the
K. Vijayavel et al. / Pesticide Biochemistry and Physiology 86 (2006) 157–161 Table 3 Individual and combined eVect of monocrotophos and ammonium chloride on the glycogen content of liver and muscle of O. mossambicus Toxicants
Liver
Muscle
Control Monocrotophos Ammonium chloride Monocrotophos + ammonium chloride
5.070 § 1.703 2.246 § 0.306¤¤ 1.081 § 0.140¤¤ 0.98 § 0.03¤¤¤
0.511 § 0.164 0.223 § 0.058¤¤ 0.211 § 0.027¤¤ 0.187 § 0.027¤¤¤
Each value represents the average (X § SD) performance of 10 individuals for a period of 96 h. The results are expressed as (g mg¡1 wet tissue). Asterisks indicate values that are signiWcantly diVerent from controls. ¤¤ P < 0.01 ¤¤¤ P < 0.001
opinion that in any living animal, toxic inXuence exerts its eVects at the molecular and biochemical level. Thus, alteration in normal biochemical parameters serves as the earliest indicator of the toxic eVects on blood, liver, and muscle of aquatic animals [21]. The unequivocal reductions in stored glycogen content in liver and muscle of O. mossambicus exposed to sublethal concentrations of monocrotophos, ammonium chloride and their mixtures indicate the utilization of stored glycogen possibly through anaerobic glycolysis to meet the energy requirement under hypoxia [22] or may be due to chemical stress [23]. A similar reduction in the stored tissue glycogen content has been reported in Channa striatus exposed to demeton [24], C. striatus exposed to malathion [25], Sarotherodon mossambicus exposed to [23], and O. mossambicus exposed to urea [2]. Stressful situation in Wshes elicit neuroendocrine response, which in turn induce disturbance in carbohydrate metabolism. Both catecholamines and adrenocorticosteroids are secreted in increased amounts due to stress stimuli and elicit marked changes in carbohydrate energy reserves of Wshes [26]. Thus, the marked glycogenolysis in muscle and liver of Wshes exposed to chemicals may be due to the stress-induced increase of the circulating catecholamines. The observed reduction of glycogen in liver and muscle may be also due to the increased utilization of glycogen through the glycolytic pathway as suggested by [21]. On the other hand the observed decrease in the glycogen might have been due to the rapid turnover of glycogen synthe-
159
sized or due to the decreased rate of glycogenesis [27]. The amount of glycogen depleted may be due to the fact that energy reserves are used as substitutes during metabolic requirement, i.e., the rapid breakdown of glycogen to lactate and pyruvate and then released to meet the energy requirements during stress conditions. The lactic acid and pyruvic acid are the end products of glycolysis and their rate of production is considered as an index of physiological stress [28]. Hence, accumulation of lactic acid indicates that the metabolism is gearing toward a condition similar to anaerobiosis. This may be due to the reduction of the cellular oxidative process under the inXuence of pollutant [29]. The lactic acid level of blood, liver, and muscle of O. mossambicus exposed to sublethal concentrations of chemicals was found to be increasing when compared to control (Table 4). The lactic acid level in the blood of O. mossambicus reared in control medium, was 273.8 gmL¡1, which rose to 759.9, 853.3, and 879.9 gmL¡1 when reared in 4 mgL¡1 of monocrotophos, 400 mgL¡1 of ammonium chloride and mixture of monocrotophos and ammonium chloride (1.5 L¡1 + 300 L¡1) at the end 96 h exposure respectively. Similar increasing trend in the lactic acid level was observed in liver and muscle and the results were highly signiWcant (P < 0.001). The pyruvic acid levels of blood, liver, and muscle of O. mossambicus exposed to sublethal concentrations of chemicals were found to be increasing when compared to control (Table 4). The blood pyruvic acid level of O. mossambicus reared in control medium, was 200 gmL¡1 which raised to 460, 495, and 560 gmL¡1 when reared in 4 mgL¡1 of monocrotophos, 400 mgL¡1 of ammonium chloride and mixture of monocrotophos and ammonium chloride (1.5 L¡1 + 300 L¡1) at the end 96 h exposure respectively. Similar increasing trend in the pyruvic acid level was observed in liver and muscle and the results were highly signiWcant (P < 0.001). The above result revealed that the enhancement of blood, liver, and muscle lactic acid and pyruvic acid levels were higher in the Wshes reared in combination of monocrotophos and ammonium chloride than in the Wshes exposed to individual concentration of pesticide and fertilizer.
Table 4 Individual and combined eVect of monocrotophos and ammonium chloride on the lactate, pyruvate, and lactate/pyruvate ratio (L/P) of blood, liver, and muscle of O. mossambicus Toxicants
Control Monocrotophos Ammonium chloride Monocrotophos + ammoniumchloride
Lactic acid
Pyruvic acid
Lactate/pyruvate ratio (L/P)
Blood
Liver
Muscle
Blood
Liver
Muscle
Blood
Liver
Muscle
273.8 § 17.3 759.9 § 34.5¤¤ 853.3 § 54.4¤¤¤ 879.9 § 47.2¤¤¤
26.6 § 1.4 74.6 § 3.3¤¤ 81.33 § 4.02¤¤¤ 93.7 § 3.5¤¤¤
32.9 § 2.7 90.6 § 7.15¤¤¤ 105.3 § 9.3¤¤¤ 126.2 § 8.4¤¤¤
21.0 § 1.218 46.0 § 2.13¤¤¤ 49.5 § 3.12¤¤¤ 56.0 § 3.87¤¤¤
2.35 § 0.12 4.4 § 0.19¤¤ 4.72 § 0.28¤¤ 5.6 § 0.35¤¤¤
3.13 § 0.18 5.35 § 0.115¤¤ 6.65 § 0.32¤¤¤ 7.21 § 0.28¤¤¤
0.130 0.165 0.172 0.157
0.113 0.169 0.174 0.167
0.105 0.169 0.158 0.17
Each value represents the average (X § SD) performance of 10 individuals for a period of 96 h. Asterisks indicate values that are signiWcantly diVerent from controls. The results are expressed as g mL¡1 for blood and g mg¡1 for liver and muscle. ¤¤ P < 0.01 ¤¤¤ P < 0.001
160
K. Vijayavel et al. / Pesticide Biochemistry and Physiology 86 (2006) 157–161
Hyperglycemia is a common phenomenon in Wshes following muscular excretion and both muscle and hepatic glycogen are the sources of the blood lactate [30]. Since catecholamines can cause an increase in the blood lactate level of Wsh directly [26], this response to chemicals might reXect a stress hormone-mediated response. Metabolically the lactic acid is formed in animal tissue as an anaerobic end product of glycolysis. On the contrary, the continued persistence of tissue lactic acid could not be cleared up immediately and this may be due to the lack of oxygen supply to tissues [5]. The following points are relevant to suggest for the cumulative increase of blood lactate caused by chemical poisoning viz: (1) lactate formed in tissues like muscles may have diVused into the blood, (2) lactate may be produced from the residual level of blood carbohydrates or other non-carbohydrate metabolites as the toxicants inhibit the oxygen binding capacity of hemoglobin content in blood cells, (3) circulatory failure inhibiting lactate removal and (4) lack or ineYciency of enzyme pathway involving ADH (alcohol dehydrogenase) genetic loci to convert the lactate into alcohol. In Wsh, such conversion of lactate into alcohol by ADH does not yield any energy [5]. It is an adaptation that could be related to the reduction of lactic acidemia, as the alcohol formed rapidly diVuses out through the gills. Increase of lactic acid in tissues is attributed to the inadequacy of oxygen supply in cells to cope up with complete breakdown of carbohydrates to carbondioxide and water. Hence, the persistence of tissue lactate in Wshes exposed to chemicals in the present study implies hypoxia. Moreover, the persistence of higher levels of lactic acid in both muscle and liver of chemical exposed Wshes may also be due to impairment in the diVusion from the tissues. Such impairment in the diVusion of tissue lactate into blood has been reported in stressed Wshes [6,31]. The increase in lactic acid content also suggests the reduced mobilization of pyruvic acid into citric acid [32]. Studies on Wshes subjected to physical stress have revealed that pyruvic acid contents are altered following the changes in tissue carbohydrates [32]. Above studies also suggest that pyruvate concentrations in tissues serve as good indices to assess the stress manifestations. The pyruvic acid content of the blood, liver, and muscle of O. mossambicus exposed to sublethal concentrations of monocrotophos, ammonium chloride, and mixtures of monocrotophos and ammonium chloride increased when compared to control. Similar observations were made in S. mossambicus exposed to DDT, malathion, and mercury [6] and Tilapia mossambicus exposed to lindane [33]. The tissue pyruvic acid increased following the depletion of glycogen. Increase in tissue pyruvic acid in Wshes subjected to stress conditions is not uncommon, however, the conversion of pyruvic acid and its further changes either through oxidation or reduction, depends on the expression of the diVerent isomeric forms of lactate dehydrogenase (LDH) [34]. The lactate/pyruvate ratio (L/P) increased when compared to control in the blood, liver, and muscle of O. mossambicus exposed to sublethal concentrations of
monocrotophos, ammonium chloride, and their mixtures, which is an indicator of stress. The results of the present study revealed that in O. mossambicus, the toxic stress by chemicals induced crisis in aerobic metabolism and a switch over to glycolytic potential. The higher concentrations of lactate and the L/P ratio exceeding unity than the control values emphasize the above change in carbohydrate metabolism and it is a good stress indicator for aquatic toxicology studies. Widespread contamination of aquatic ecosystems by a whole battery of pesticides and fertilizers has been responsible for the massive “Wsh kills” and recognized as a potential human health hazard as well. The combination of chemicals can result in toxicity that is simply additive or greater than or less than additive. Pesticides and fertilizers are a mixed blessing since they improve agricultural productivity, but can aVect the non-target organisms such as Wsh and aquatic animals. Hence, it can be concluded that pesticides and fertilizers applied in the agricultural operations probably have considerable joint action on non-target species such as Wshes. References [1] V. Palanivelu, K. Vijayavel, S. Ezhilarasibalasubramanianm, M.P. Balasubramanian, InXuence of pesticide–fertilizer combinations on food intake, growth and conversion eYciencies of Oreochromis mossambicus, Bullet. Environ. Contam. Toxicol. 69 (2002) 908–913. [2] V. Palanivelu, K. Vijayavel, S. Ezhilarasibalasubramanianm, M.P. Balasubramanian, Combined eVect of cartaphydrochloride and urea on biochemical constituents of freshwater Wsh Oreochromis mossambicus, J. Ecotoxicol. Environ. Monit. 14 (1) (2004) 23–29. [3] E.F. Rani, M. Elumalai, M.P. Balasubramanian, Toxic and sublethal eVects of ammonium chloride on a freshwater Wsh Oreochromis mossambicus, Water Air Soil Poll. 104 (1998) 1–8. [4] E.F. Rani, M. Elumalai, M.P. Balasubramanian, EVect of mixtures of pesticide and fertilizer on carbohydrates, protein, phosphomonoesterases and non-speciWc esterases in Oreochromis mossambicus, Nat. Acad. Sci. Lett. 22 (1999) 70–74. [5] K. Ramalingam, R. Kasinathadurai, Blood carbohydrates and phosalone poisoning in Rana tigrina (Daudin), Arch. Inter. Physiol. Biochem. 97 (1989) 369–374. [6] K. Ramalingam, Toxic eVects of DDT, malathion and mercury on the tissue carbohydrate metabolism of Sarotherodon mossambicus (Peters), Proc. Indian Acad. Sci. (Anim. Sci.). 97 (1988) 443–448. [7] S. Palanichamy, S. Arunachalam, M.P. Balasubramanian, Toxic and sub eVects of ammonium chloride on food consumption and growth in the air breathing Wsh Channa striatus, Warm Water Aqua. Hawaii 465 (1985) 480. [8] S. Palanichamy, S. Arunachalam, M.P. Balasubramanian, Toxic and sublethal eVects of diammonium phosphate on food consumption and growth in the Sarotherodon mossambicus, Hydrobiologia 128 (1985) 233–237. [9] P. Baskaran, S. Palanichamy, P. Visalakshi, M.P. Balasubramanian, EVects of mineral fertilizers on survival of the Wsh Oreochromis mossambicus, Environ. Ecol. 7 (1989) 463–465. [10] V. Palanivelu, M.P. Balasubramanian, Food consumption of Oreochromis mossambicus exposed to sublethal concentrations of cartap hydrochloride, Geobios 24 (1997) 51–54. [11] V. Palanivelu, K. Vijayavel, S. Ezhilarasibalasubramanianm, M.P. Balasubramanian, InXuence of insecticidal derivative (cartap hydrochloride) from the marine polychaete on certain enzyme systems of
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