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Carbaryl induced alterations in the reproduction and metabolism of freshwater snail Lymnaea acuminata
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 79 (2004) 1–9 www.elsevier.com/locate/ypest
Carbaryl induced alterations in the reproduction and metabolism of freshwater snail Lymnaea acuminata Pankaj Kumar Tripathi and Ajay Singh* Natural Products Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur 273 009 (UP), India Received 22 July 2003; accepted 18 November 2003
Abstract When the freshwater snail Lymnaea acuminata was exposed to sub-lethal doses (2.0, 5.0, and 8.0 mg/L) of carbaryl, fecundity was significantly reduced and even stopped at higher sub-lethal doses and altered metabolic activity in the body tissue of the snail was observed. The change from aerobic to anaerobic metabolism results in lesser energy production in the body tissues of the snails, causing paralysis and finally death. This reduced fecundity and altered metabolism suggests that it would be better to avoid the use of carbaryl pesticides in the water bodies or fields adjoining the water bodies particularly in the rainy season. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Carbaryl; Lymnaea acuminata; Fecundity; Metabolism; Enzyme
1. Introduction Carbaryl pesticides are used for pest control in agricultural fields and then translocate in water bodies by several ways. The resulting pesticide pollution of the water becomes a great threat to the aquatic ecosystem. Carbaryl has been found to have significant inhibitory effect on the controlling mechanism of reproduction in fishes [1]. The survival of organisms is largely determined by the successful reproduction and good metabolism. Disruption in the reproduction will affect the abundance and distribution of the species [2].
* Corresponding author. Fax: +91-551-202-127. E-mail address: [email protected] (A. Singh).
Therefore, laboratory tests of long-term impact of sub-lethal pollutant concentrations on organisms are most instructive when done on reproductive success [3] and metabolism. The effect of pollutants on natural ecosystems can be partially understood by determining the effect of such pollutants on the reproductive process and metabolism in the key species of the ecosystem. Although carbamate pesticides persist as residue in the local environment for only a few days [4], there may be a cumulative effect on the reproduction of aquatic organisms because the electrophilic nature of carbaryl affects the various enzymes responsible for normal metabolic processes. The aim of this study was to study the effect of sub-lethal concentrations of carbaryl pesticides on the reproduction and metabolism of the
0048-3575/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2003.11.003
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freshwater snail Lymnaea acuminata, a cosmopolitan freshwater animal and an important link of detritus food chain, which shares habitat with the freshwater fishes.
into separate petri dishes for hatching under the same exposure conditions as above. The numbers of hatched snails were counted and their survival rate was recorded for 28 days after hatching. 2.2. Biochemical estimations
2. Materials and methods Adult freshwater snails (L. acuminata) of a uniform size range (42.5 3.6 mm shell height and 22.8 1.9 mm shell width) were collected from non-contaminated waters of Gorakhpur district of Uttar Pradesh, India and kept in glass aquaria containing 30 L of dechlorinated tap water for at least 96 h to acclimatize them to laboratory conditions. The water was changed every day and dead snails were removed as soon as possible to avoid water fouling. The snails were fed daily on washed and dried Nymphaea leaves during the whole acclimatization period. A technical grade of carbaryl (1-naphthyl-N-methylcarbamate) was used for the experimental study supplied by Phone-Poulenc Agrochem., Mumbai, India. For these snails, the LC50 value of carbaryl is 14.2 mg/ L for 96 h [5]. The sub-lethal concentrations of carbaryl used for the study were 2.0, 5.0, and 8.0 mg/L. 2.1. Fecundity experiments For the fecundity experiment the test amount of pesticide was added to each glass aquarium containing 10 L dechlorinated tap water. Thirty snails were placed in each aquarium. In the control groups the water was pesticide free. Each aquaria set had six replicates. Water temperature was kept at 23 1 °C during the whole experimental time. Nymphaea leaves were allowed to float at the top of the water surface for egg laying. The leaves were changed every day to avoid decay. No food was given to the snails during the entire experiment. Lymnaeid snails attached their egg masses, containing a large number of eggs, to the back surface of Nymphaea leaves when reproducing. The egg masses produced by the snails in the experiment were removed after every 24 h and the number of eggs counted under a compound microscope. All the egg masses for each group were transferred
For biochemical analysis six sets of aquaria were used, as with the fecundity experiment. Fifty snails were placed in each aquarium. No food was given to the snails during the whole experimental period. At the end of the allotted time the nervous, hepatopancreas, and ovotestis tissues of the treated and control groups of the snails were dissected out and various biochemical parameters were measured. Glycogen was measured according to Van der Vies [6]. The homogenate (50 mg/mL, w/v) was prepared in 5% trichloroacetic acid (TCA). Glycogen content was expressed as mg glycogen/g of tissue. The pyruvate level was measured according to Friedemann and Haugen [7]. The homogenate (50 mg/mL, w/v) was prepared in 10% TCA. The pyruvate content was expressed as lmol of pyruvate/g of tissue. Lactate was estimated according to Barker and Summerson [8] as modified by Huckabee [9]. The homogenate (50 mg/mL, w/ v) was prepared in 10% cold TCA. Lactate content was expressed as mg lactic acid/g of tissue. Total protein level was estimated according to the folin– phenol method of Lowry et al. [10]. The homogenate (50 mg/mL, w/v) was prepared in 10% TCA. Total protein content was expressed as lg protein/ mg of tissue. Total free amino acid level was measured according to Spies [11]. The homogenate (50 mg/mL, w/v) was prepared in 96%. Total free amino acid content was expressed as lg/mg of tissue. Nucleic acids (DNA and RNA) were estimated according to Schneider [12], using diphenylamine and orcinol reagents, respectively. The homogenates (100 mg/mL, w/v) were prepared in 5% TCA at 90 °C. Nucleic acids content was expressed as lg/mg of tissue. 2.3. Enzymological estimations Lactic dehydrogenase (LDH) activity was measured according to the method of Anon [13]. The homogenate (50 mg/mL, w/v) was prepared in
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0.1 M phosphate buffer (pH 7.5) for 5 min in an ice bath. Enzyme activity was expressed as nmol of pyruvate reduced/min/mg protein. Succinic dehydrogenase (SDH) activity was measured by the method of Arrigoni and Singer [14]. The homogenate (50 mg/mL, w/v) was prepared in 0.5 M potassium phosphate buffer (pH 7.6) in an ice bath and centrifuged at )4 °C. Enzyme activity is expressed as lmol dye reduced/min/mg protein. Cytochrome oxidase activity was measured according to the method of Cooperstein and Lazarow [15]. The homogenate (50 mg/mL, w/v) was prepared in 1.0 mL of 0.33 M phosphate buffer (pH 7.4) for 5 min in an ice bath. Enzyme activity was expressed in arbitrary units/min/mg of proteins (corresponding to the quantity of enzyme which catalyzed an O2 uptake by the oxidation of reduced cytochrome). Protease activity was measured according to the method of Moore and Stein [16]. The homogenate (50 mg/mL, w/v) was prepared in cold distilled water. The enzyme activity was expressed in lmol of tyrosine equivalent/mg protein/h. Aminotransaminases (glutamic oxalic transaminase and glutamic pyruvic transaminase) activities were measured according to Reitman and Frankel [17]. The homogenates (100 mg/mL, w/v) were prepared in phosphate buffer for 5 min and centrifuged at 1000g for 15 min and the supernatant kept for estimation of enzyme activity. Both
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the enzyme activities were expressed as lmol of pyruvate formed/mg protein/h. Acetylcholinesterase (AChE) activity was measured by the method of Ellman et al. [18]. The homogenate (50 mg/mL, w/v) was prepared in 0.1 M phosphate buffer (pH 8.0) in an ice bath and centrifuged at )4 °C and the supernatant was kept for enzyme assay. Enzyme activity is expressed as lM ÔSHÕ hydrolyzed/min/ mg wet tissue. Activities of phosphatases (acid and alkaline phosphatase) were measured according to the method of Andersch and Szcypinski [19] as modified by Bergmeyer [20]. The homogenates (50 mg/mL, w/v) were prepared in ice-cold 0.9% saline and centrifuged at 4 °C, supernatant was used as the enzyme source. Enzyme activities are expressed as the amount of p-nitrophenol formed/ 30 min/mg protein in supernatant. Each assay was replicated six times, values are expressed as means SE of six replicates. StudentÕs t test was applied to locate significant (P < 0:05) differences between treated and control groups.
3. Results and discussion 3.1. Fecundity experiment The results of the fecundity experiments on the freshwater snail L. acuminata are given in Table 1.
Table 1 Number SE (%) of eggs laid, egg masses, hatched eggs, and time to hatching (in days) of the freshwater snail Lymnaea acuminata after carbaryl exposure Control
Sub-lethal concentrations (mg/L) 2.0
5.0
8.0 —
Number of eggs after 96 h Number of egg masses after 24 h Number of hatched eggs Time to hatching in days
260 14.6 (100) 9 1.2 258 12.4 (100) 11–13
128 9.5 (49) 7 0.9 120 8.5 (47) 10–14
—
Survival rate 7 days after hatching 14 days after hatching 21 days after hatching 28 days after hatching
252 5.6 243 5.6 239 4.8 236 4.0
112 1.2 (93) 105 2.4 (88) 80 2.0 (67) 64 1.6 (53)
*
(98) (94) (93) (91)
4 0.4
2 0.4
—
—
—
—
—
—
—
—
—
—
—
—
Significant (P < 0:05), when StudentÕs t test was applied between control and treated groups. Significant (P < 0:05), when StudentÕs t test was applied between number of hatched eggs and survivability of hatchlings in corresponding treated groups. All the experiments were replicated six times. Values are means SE of six replicates. Values in parentheses are the percent of the corresponding value with control taken as 100%. —, Egg laying was stopped. Value in parentheses under survival rate are the percent of number of hatched eggs in corresponding treatment. **
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The cumulative number of eggs laid was highest in the control group. The number of eggs was significantly (P < 0:05) reduced at all the sub-lethal doses of carbaryl. Fecundity was reduced to 49% of control at 2.0 mg/L, while at 5.0 and 8.0 mg/L of carbaryl no egg was laid (Table 1). Some egg masses were laid without eggs at higher doses of carbaryl. No significant (P < 0:05) difference was found in the time of hatching between the control and treated groups but the survival rate of snail embryos exposed to carbaryl was significantly lower than the control value. The rate of survival was significantly reduced at 7 days after hatching and it was reduced to about 53% of the control after exposure to 2.0 mg/L of carbaryl after 28 days after hatching (Table 1).
3.2. Biochemical experiment Sub-lethal doses were enough to alter the biochemical parameters in the nervous, hepatopancreatic, and ovotestis tissues of the snail. Glycogen, pyruvate, total protein, and nucleic acid (DNA and RNA) levels were reduced after exposure, while lactate and free amino acid levels increased after exposure in nervous, hepatopancreatic, and ovotestis tissues of the freshwater snail L. acuminata after 96 h of exposure to carbaryl pesticide (Table 2). In the nervous, hepatopancreatic, and ovotestis tissues, respectively, of the snails exposed to 8.0 mg/L of carbaryl, glycogen content was reduced to 52, 40, and 56%, pyruvate level was reduced to 48, 31, and 26%, lactate content was
Table 2 Glycogen (GN), pyruvate (PV), lactate (LT), total protein (TP), free amino acid (FAA), nucleic acid (DNA and RNA) levels in nervous (NT), hepatopancreas (HP), and ovotestis (OT) tissues of the freshwater snail Lymnaea acuminata after 96 h exposure to carbaryl Control
Sub-lethal concentrations (mg/L) 2.0
5.0
8.0
GN
NT HP OT
2.19 0.29 (100) 2.64 0.38 (100) 2.54 0.38 (100)
2.05 0.62 (94) 2.15 0.65 (81) 2.18 0.54 (86)
1.56 0.28 (71) 1.91 0.23 (72) 2.08 0.28 (82)
1.14 0.26 (52) 1.06 0.28 (40) 1.41 0.22 (56)
PV
NT HP OT
1.05 0.28 (100) 1.09 0.23 (100) 1.28 0.36 (100)
0.87 0.16 (83) 0.82 0.14 (75) 0.79 0.12 (62)
0.67 0.10 (64) 0.65 0.11 (60) 0.46 0.12 (36)
0.50 0.11 (48) 0.34 0.08 (31) 0.33 0.10 (26)
LT
NT HP OT
2.42 0.15 (100) 2.59 0.19 (100) 2.32 0.13 (100)
2.79 0.34 (115) 2.84 0.28 (110) 2.92 0.35 (126)
3.19 0.24 (132) 3.30 0.27 (127) 3.27 0.37 (141)
4.74 0.38 (196) 5.02 0.29 (194) 5.17 0.32 (223)
TP
NT HP OT
47.64 2.11 (100) 55.44 3.20 (100) 70.06 5.21 (100)
44.46 2.29 (93) 50.29 3.37 (91) 49.36 4.87 (70)
39.54 1.36 (83) 40.31 2.21 (73) 37.83 3.89 (54)
36.68 1.17 (77) 33.26 2.71 (60) 28.02 2.54 (40)
FAA
NT HP OT
14.52 1.04 (100) 17.59 1.16 (100) 16.39 1.11 (100)
15.73 1.23 (108) 19.34 1.45 (110) 19.94 1.62 (122)
18.29 1.09 (126) 21.18 1.25 (120) 23.14 1.27 (141)
22.36 1.26 (154) 28.75 1.20 (163) 32.78 1.11 (200)
DNA
NT HP OT
42.23 1.94 (100) 46.81 0.89 (100) 52.96 0.72 (100)
36.43 3.02 (86) 36.83 3.29 (79) 41.28 4.17 (78)
29.56 1.84 (70) 29.49 0.47 (63) 34.36 0.44 (65)
22.80 1.42 (54) 20.13 0.28 (43) 16.95 0.29 (32)
RNA
NT HP OT
27.08 1.18 (100) 37.67 2.31 (100) 41.22 2.76 (100)
22.13 1.09 (82) 29.82 1.24 (79) 30.45 2.03 (74)
19.50 1.01 (72) 24.43 1.45 (65) 21.85 1.47 (53)
14.89 0.94 (55) 18.39 0.41 (49) 18.30 0.37 (44)
*
Significant (P < 0:05) when StudentÕs t test was applied between treated and control groups. Values are means SE of six replicates. Values given in the parentheses are percent values with control taken as 100%. Values are expressed as: lg/mg for TP, FAA, DNA, and RNA; mg/g for GN and LT; and lmol/g for PV.
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increased to 196, 194, and 223%, total protein level was reduced to 77, 60, and 40%, free amino acid content was increased to 154, 163, and 200%, DNA level was reduced to 54, 43, and 32%, and RNA level was reduced to 55, 49, and 44% (Table 2). Carbohydrates are the primary and immediate source of energy [21] in living creatures. The fall in glycogen content in the body tissues of L. acuminata indicates its rapid utilization by the respective tissues as a consequence of pesticide toxic stress. Under hypoxic conditions, animals derive their energy from anaerobic breakdown of glucose, which is available to the cells by increased glycogenolysis [22]. Nakano and Tomlinson [23] have suggested that catecholamine levels rise under stressful environmental conditions, enabling the increased utilization of glycogen for energy production. Glycogen levels appear to be related, at least to some extent, to the detoxification mechanisms, essential for metabolism or degradation and elimination of pesticides from the body [24]. The decrease in pyruvate levels is due to the higher energy demand during pesticidal exposure, and suggests the possibility of a shift towards anaerobic dependence due to a notable drop in the aerobic segment. The decrease in pyruvate could be due to its conversion to lactate, or its mobilization to form amino acids, lipids, triglycerides, and glycogen synthesis in addition to its role in detoxification [25]. The increase in lactate also suggests a shift towards anaerobiosis as a consequence of hypoxia created from pesticide toxic impact leading to respiratory distress [26]. Tissues receive less oxygen, leading to severe tissue hypoxia. Huckabee [27] reported that an upward trend in lactate in the tissues might be taken to indicate that oxygen supply to the tissues is not adequate for the normal metabolic functions. The appearance of such internal hypoxic conditions may be proximally responsible for the shift to the less efficient anaerobic metabolism, as evidenced by the change in lactate content observed during this study. Proteins are the most important and abundant macromolecules in living beings, playing a vital role in the architecture and physiology of the cell and in cellular metabolism [28]. The depletion of
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the protein fraction in the body tissues of the snails in this experiment may have been due to protein degradation for metabolic purposes. Under stress conditions, the dietary protein consumed by snails is not stored in the body tissue [29] and hence the treated snails met their extra energy requirements from body proteins which are mobilized to produce glucose, the instant energy of which is made available for the snail by the process of gluconeogenesis [30]. Thus, the decreased protein content may be attributed to the destruction/necrosis of cells and consequent impairment in protein synthesis machinery [31]. The increment in free amino acid level was also the result of the breakdown of proteins for energy use and the subsequent impaired incorporation of amino acids in protein synthesis [32]. It also attributed to the reduced use of amino acids in the maintenance of the acid–base balance [33]. Carbaryl pesticide appears as potential inhibitor of DNA synthesis, which might also result in reduction of the RNA level. It might also be noted that deprivation of food possibly caused some nutritional deficiency in the test snails, leading to lower concentration of nucleic acids and aberrations in DNA-directed RNA formation and protein synthesis, consequently limiting growth and adding to the metabolic stress of the snails. 3.3. Enzymological experiment Significant alteration in the enzyme levels was also observed in the body tissues of the freshwater snail L. acuminata exposed to different sub-lethal doses of carbaryl. The activities of succinic dehydrogenase (SDH), cytochrome oxidase, acetylcholinesterase (AChE), acid phosphatase, and alkaline phosphatase were inhibited, while the activities of lactic dehydrogenase (LDH), protease, glutamic oxalic transaminase (GOT), and glutamic pyruvic transaminase (GPT) were enhanced after the exposure to sub-lethal doses of the carbaryl pesticide (Table 3). In the nervous, hepatopancreatic, and ovotestis tissues, respectively, of snail exposed to 8.0 mg/L of carbaryl, lactic dehydrogenase activity was increased to 155, 175, and 198%, succinic dehydrogenase activity was inhibited to 46, 36, and 28%,
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Table 3 Lactic dehydrogenase (LDH), succinic dehydrogenase (SDH), cytochrome oxidase (CY), protease (PR), transaminases (GOT and GPT), acetylcholinesterase (AChE), and phosphatase (ACP and ALP) activities in nervous (NT), hepatopancreas (HP), and ovotestis (OT) tissues of the freshwater snail Lymnaea acuminata after 96 h exposure to carbaryl Control
Sub-lethal concentrations (mg/L) 2.0
5.0
8.0
LDH
NT HP OT
0.038 0.005 (100) 0.063 0.008 (100) 0.050 0.007 (100)
0.044 0.006 (116) 0.078 0.010 (124) 0.063 0.008 (126)
0.049 0.008 (129) 0.090 0.012 (143) 0.073 0.014 (146)
0.059 0.009 (155) 0.110 0.019 (175) 0.099 0.018 (198)
SDH
NT HP OT
50.34 5.17 (100) 53.83 4.18 (100) 45.95 5.08 (100)
43.25 4.09 (86) 40.27 4.54 (75) 32.18 3.37 (70)
35.74 3.54 (71) 33.37 3.54 (62) 23.89 2.73 (52)
23.16 3.42 (46) 19.38 3.29 (36) 12.87 2.54 (28)
CY
NT HP OT
56.71 5.09 (100) 74.98 5.18 (100) 68.84 5.17 (100)
50.24 4.45 (89) 66.12 5.65 (88) 56.34 5.18 (82)
47.07 4.69 (83) 61.87 4.53 (83) 46.46 4.13 (67)
34.03 4.69 (60) 41.73 3.74 (56) 37.08 3.19 (54)
PR
NT HP OT
0.316 0.044 (100) 0.320 0.058 (100) 0.299 0.099 (100)
0.337 0.042 (107) 0.354 0.076 (111) 0.376 0.092 (126)
0.369 0.064 (117) 0.392 0.037 (123) 0.443 0.067 (148)
0.429 0.074 (136) 0.464 0.037 (145) 0.454 0.066 (152)
GOT
NT HP OT
4.94 0.54 (100) 6.07 0.48 (100) 5.22 0.38 (100)
5.49 0.62 (111) 7.28 0.78 (120) 6.37 0.63 (122)
6.08 0.69 (123) 8.93 0.79 (147) 7.92 0.25 (152)
6.87 0.94 (139) 10.80 0.33 (178) 10.60 0.48 (203)
GPT
NT HP OT
6.62 0.84 (100) 7.71 0.45 (100) 6.61 0.65 (100)
7.45 0.92 (113) 10.84 1.12 (141) 8.57 0.85 (130)
9.00 1.17 (136) 17.22 1.31 (223) 11.44 1.10 (173)
12.31 1.46 (186) 19.33 1.69 (251) 12.79 1.17 (193)
AChE
NT HP OT
0.086 0.006 (100) 0.071 0.004 (100) 0.061 0.007 (100)
0.066 0.009 (77) 0.043 0.005 (61) 0.030 0.008 (49)
0.043 0.004 (50) 0.034 0.006 (48) 0.016 0.003 (26)
0.016 0.002 (19) 0.014 0.003 (20) 0.008 0.001 (14)
ACP
NT HP OT
0.191 0.022 (100) 0.157 0.017 (100) 0.124 0.014 (100)
0.143 0.026 (75) 0.129 0.019 (82) 0.084 0.015 (68)
0.115 0.016 (60) 0.085 0.009 (54) 0.059 0.009 (48)
0.057 0.007 (30) 0.050 0.008 (32) 0.036 0.006 (29)
ALP
NT HP OT
0.392 0.018 (100) 0.339 0.022 (100) 0.257 0.041 (100)
0.245 0.021 (63) 0.269 0.027 (79) 0.194 0.035 (75)
0.169 0.026 (43) 0.227 0.031 (67) 0.152 0.019 (59)
0.102 0.016 (26) 0.129 0.019 (38) 0.077 0.011 (30)
*
Significant (P < 0:05) when StudentÕs t test was applied between treated and control groups. Values are means SE of six replicates. Values given in the parentheses are percent values with control taken as 100%. Values are expressed as: LDH, lmol of pyruvate reduced/min/mg protein; SDH, lmol of dye reduced/min/mg protein; CY, arbitrary units/min/mg/proteins; PR, lmol of tyrosine equivalent/mg protein/h; GOT and GPT, lmol pyruvate formed/mg protein/h; AChE, lM ÔSHÕ hydrolyzed/min/mg protein; and ACP and ALP, p-nitrophenol formed/30 min/mg protein.
cytochrome oxidase activity was decreased to 60, 56, and 54%, protease activity was increased to 136, 145, and 152%, GOT activity was increased to 139, 178, and 203%, GPT activity was increased to 186, 251, and 193%, acetylcholinesterase activity was decreased to 19, 20, and 14%, acid phosphatase activity was decreased to 30, 32, and 29% while alkaline phosphatase activity was decreased to 26, 38, and 30% (Table 3).
The enzyme lactic dehydrogenase (LDH) forms the centre for a delicately balanced equilibrium between catabolism and anabolism of carbohydrates [34] and is associated with cellular metabolic activity [35]. LDH is present in most animal tissues and is involved in the interconversion of pyruvic acid and lactic acid. It serves as a pivotal enzyme between the glycolytic pathway and the tricarboxylic acid cycle. The action of pesticide in the
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respiratory chain is between FAD and co-enzyme Q, and SDH containing one molecule of covalently bound FAD is inhibited [36]. The exact mechanism of SDH inhibition is still unknown. A decrease in cytochrome oxidase activity might be either the result of reduced availability of oxygen, which in turns reduces the capacity of the ETS to produce ATP molecules, or could be due to the direct impact of the pesticides [37]. Stevans et al. [38] reported that anticholinesterase compounds are known to usually inhibit mitochondrial reactions such as the function of the cytochrome oxidase in the ETS. Pesticidal poisoning also effects KrebÕs cycle by diminishing the rate of the ETS and oxidative phosphorylation, resulting in less ATP synthesis. The enzyme protease functions in hydrolyzing proteins to free amino acids and small peptides, which is also called proteolysis. The increase in protease activity corroborates the enhancement of the free amino acid level and reduced protein level in the body tissues of the snails. Transaminases are released during cellular damage or lysis [39]. Stress conditions induce elevation in the transamination pathway [40]. The increase in transaminase values may be due to hepatopancreas enlargement indicating an increase in the functional load of the treated snails. The transamination reaction (transfer of an amino group from an amino acid to a keto acid with subsequent formation of different amino acid and keto acids) is the most important pathway in the metabolism of many amino acids [41]. Transamination not only serves as a pathway for the conversion of a-keto acids to L -amino acids, but also as an alternative means of replenishing the pyruvate pool [42]. Transamination represents the major mechanism leading towards the eventual deposition of nitrogen as waste products and also results in the production of carbon compounds, which may be metabolized for energy purposes. Christensen et al. [43] suggested that toxic chemical pollutants often affect the activity of enzymes to varying degrees, and hence enzymes are logical candidates to be used as biomonitors. The transaminases (GOT and GPT) are two key enzymes known for their role in utilization of protein and carbohydrates. The studies of Harper et al. [44] showed that transamination and
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transdeamination reactions are prominent under stress condition. GOT and GPT function at the junction between protein and carbohydrate metabolism by interconverting ketogluterate, oxalacetate, and pyruvate on one side and alanine, aspartate, and glutamate on the other side [44]. Acetylcholinesterase (AChE), is involved in the synaptic transmission of nerve impulses. Carbamates are well known inhibitor of AChE. Carbamate insecticides are a deadly nerve poison as they inhibit AChE. Carbamate compounds inhibit AChE reversibly by combining either at the active site or at a site specially removed from the active centre called the peripheric anionic site [45]. Inhibition of AChE results in the accumulation of acetylcholine (ACh), which causes twitching of muscle and leads to tetanus and eventual paralysis of the muscle. Paralysis of respiratory muscles may lead to death. Due to their neurotoxicant nature the physiology of several systems may be affected, resulting in disturbances of the metabolic system. At 20–70% inhibition of AChE, adverse effects become more subtle and can include reproduction problems [46]. It has also been shown that alkaline phosphate, through the process of phosphorylation of carbohydrates and fats, plays an important role in the active transport of chemicals across cell membranes [47]. Pilo et al. [48] demonstrated that alkaline phosphate is associated with protein synthesis, and it has also been shown that this enzyme is involved in the synthesis of certain proteinaceous digestive enzymes [49] and plays an important role in spermatogenesis [50]. Since the active sites of alkaline phosphatase [51] contain a serine residue, it is possible that the inhibition of alkaline phosphatase observed in this study could be due to the phosphorylation of the active sites of alkaline phosphatase. Acid phosphatase is a lysosomal enzyme and plays an important role in catabolism, pathological necrosis, autolysis, and phagocytosis [52]. A comparison of the differences in various enzymological constituents in the control and test animals suggests that exposure of L. acuminata to sub-lethal concentrations of carbaryl severely affects various physiological mechanisms. The toxic effects of the pesticide were not fully neutralized, and there was evidence of protein denaturation,
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disturbance of cellular metabolic activities and impairment in neural transmission. Therefore, pollution of the aquatic environment by carbaryl would adversely affect the metabolism of the snail, and have adverse effects on its reproduction, so the use of carbaryl pesticides in the water bodies or fields adjoining the water bodies should be undertaken only after careful consideration of less harmful alternatives. Acknowledgment The author extend his thanks to the Council of Science and Technology, Uttar Pradesh, India for financial support during this work. References [1] S. Arunachalam, K. Jayalakshmi, S. Aboobker, Toxic and sub-lethal effects of Carbaryl on a freshwater cat fish Mystus vittatus (Bloch), Arch. Environ. Contam. Toxicol. 9 (1980) 307–316. [2] P. Woin, C. Br€ onmark, Effects of DDT and MCPA (4chloro-2-methylphenoxyacetic acid) on reproduction of the pond snail, Lymnaea stagnalis L, Bull. Environ. Contam. Toxicol. 48 (1992) 7–13. [3] P.J. Sheehan, Effects on individuals and populations, in: P.J. Sheehan, D.R. Miller, G.C. Butler, P. Bourdeau (Eds.), Effects of Pollutants at the Ecosystem Level, SCOPE 22, Wiley, Chichester, UK, 1984, pp. 23–50. [4] A. Singh, D.K. Singh, R.A. Agarwal, Effect of cypermethrin, mexacarbate and phorate on the phospholipids and lipid peroxidation in the snail Lymnaea acuminata, Bull. Environ. Contam. Toxicol. 51 (1993) 68–71. [5] V.K. Srivastava, A. Singh, Toxicity of alphamethrin, dimethoate and carbaryl pesticides to the freshwater snails Lymnaea acuminata and Indoplanorbis exustus, Iberus 19 (2001) 1–5. [6] J. Van der Vies, Two methods for the determination of glycogen in liver, Biochem. J. 57 (1954) 410–416. [7] T.E. Friedemann, G.F. Haugen, Pyruvic acid. I. Collection of blood for the determination of pyruvic acid and lactic acid, J. Biol. Chem. 144 (1943) 67–77. [8] S.B. Barker, W.H. Summerson, The colorimetric determination of lactic acid in biological materials, J. Biol. Chem. 138 (1941) 535–542. [9] W.E. Huckabee, Blood analysis, determination of lactic acid, in: B.L. Oser (Ed.), HawkÕs Physiological Chemistry, Tata McGraw-Hill, New Delhi, 1961, p. 1103. [10] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.
[11] J.R. Spies, Colorimetric products for amino acids, in: S.P. Claowick, N.O. Kaplon (Eds.), Methods in Enzymology, Academic Press, New York, 1957, p. 468. [12] W.C. Schneider, Determination of nucleic acids in tissue by pentose analysis, in: S.P. Calowick, N.O. Kaplon (Eds.), Enzymology, Academic Press, New York, 1957, p. 680. [13] Anon, Sigma diagnostics TM, Lactic dehydrogenase (quantitative, colorimetric determination in serum, urine and cerebrospinal fluid) at 400–450 nm. Procedure No. 500, Sigma Chemical Company, St. Louis, USA, 1984. [14] O. Arrigoni, T.P. Singer, Limitations of the phenazine methosulphate assay for succinic and related dehydrogenase, Nature 193 (1962) 1256–1258. [15] S.J. Cooperstein, R. Lazarow, A microspectrophotometric method for the determination of cytochrome oxidase, J. Biol. Chem. 189 (1951) 665–670. [16] S. Moore, W.H. Stein, A modified ninhydrin reagent for the photometric determination of amino acids and related compounds, J. Biol. Chem. 211 (1954) 907–913. [17] S. Reitman, S. Frankel, A colorimetric method for the determination of glutamic-oxaloacetic and glutamic-pyruvic transaminases, Am. J. Clin. Pathol. 28 (1957) 56–63. [18] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone, A new and rapid colorometric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–95. [19] M.A. Andersch, A.J. Szcypinski, The colorimetric determination of phosphatases in biological materials, Am. J. Clin. Pathol. 17 (1947) 571–574. [20] U.H. Bergmeyer, Methods of Enzymatic Analysis, Academic Press, New York, 1967, p. 1129. [21] B.L. Umminger, Relation of whole blood sugar concentration in vertebrates to standard metabolic rate, Comp. Biochem. Physiol. 55 (1977) 457–460. [22] S. Vincent, T. Ambrose, L. Cyrill, M. Selvanaygam, Biochemical responses of the Indian major carp, Catla catla (Ham.) to chromium toxicity, Indian J. Environ. Health 37 (1995) 192–196. [23] T. Nakano, N. Tomlinson, Catecholamine and carbohydrate concentrations in rainbow trout (Salmo gairdneri) in relation to physical disturbance, J. Fish Res. Board Can. 24 (1967) 1707–1715. [24] R.K.R.S. Sambasiva, Pesticide Impact on Fish Metabolism, Discovery Publishing House, New Delhi, 1999, pp. 129–149. [25] P.K. Sathya, Studies on the toxic impact of lindane on tissue metabolic profiles in the freshwater fish, Tilapia mossambica (Peters) with emphasis on carbohydrate metabolism. Ph.D. thesis, S.V. University, Tirupati, India, 1983. [26] S. Domsche, W. Domsche, M. Classen, Zum mechanisms deri Leber Zeuschadigung durch Alkaylphosphate, Natururissenchaflan 58 (1971) 575–578. [27] W.E. Huckabee, Relationship of pyruvate and lactate during anaerobic metabolism. II. Exercise and formation of O2 -debt, J. Clin. Invest. 37 (1958) 255–263. [28] T.P. Mommsen, P.J. Walsh, Biochemical and environmental perspectives on nitrogen metabolism in fishes, Experientia 48 (1992) 583–593.
P.K. Tripathi, A. Singh / Pesticide Biochemistry and Physiology 79 (2004) 1–9 [29] P. Baskaran, S. Palanichamy, Impact of agricultural (ammonium chloride) fertilizer on physiology and biochemistry of fresh water teleost fish, Oreochromis mossambicus, J. Ecobiol. 2 (1990) 97–106. [30] R. Vasanthi, P. Baskaran, S. Palanichamy, Influence of carbofuran on growth and protein conversion efficiency in some fresh water fishes, J. Ecobiol. 2 (1990) 85–88. [31] S.P. Bradbury, J.R. Makim, J.R. Coats, Physiological responses of rainbow trout, Salmo gairdneri to acute fenvalerate intoxication, Pestic. Biochem. Physiol. 27 (1987) 275–285. [32] N.N. Singh, V.K. Das, S. Singh, Effect of Aldrin on carbohydrate, protein and ionic metabolism of a freshwater catfish Heteropneustes fossilis, Bull. Environ. Contam. Toxicol. 57 (1996) 204–210. [33] K.S. Moorthy, R.B. Kashi, K.S. Swamy, C.S. Chetty, Change in respiration and ionic content in the tissue of freshwater mussel exposed to methyl-parathion toxicity, Toxicol. Lett. 21 (1984) 287–291. [34] T. Everse, N.O. Kaplan, Lactate dehydrogenase: structure and function, in: A. Meister (Ed.), Advances in Enzymology, Wiley, New York, 1973, pp. 61–133. [35] P.A. Abston, J.D. Yarbrough, The in vitro effect of mirex on soluble hepatic enzymes in the rat, Pestic. Biochem. Physiol. 6 (1976) 192–199. [36] K. Kamalaveni, V. Gopal, U. Sampson, D. Aruna, Effect of pyrethroids on carbohydrate metabolic pathways in common carp, Cyprinus carpio, Pest. Manag. Sci. 57 (2001) 1151–1154. [37] J.R. Corbett, K. Wright, A.C. Bailee, Pesticides interfering with respiration, in: J.R. Corbett, K. Wright, A.C. Bailee (Eds.), The Biochemical Mode of Action of Pesticides, Academic Press, London, 1984, pp. 1–49. [38] J.T. Stevans, R.E. Stizel, J.J. Mc Phillips, Effects of anticholinesterase insecticides on hepatic microsomal metabolism, J. Pharmacol. Exp. Ther. 181 (1972) 576–580. [39] M. Pisam, Membrane system in the chloride cell of teleostean fish gill, their modifications in response to the salinity of the environment, Anat. Res. 200 (1981) 401– 414. [40] G.M. Natarajan, Inhibition of branchial enzymes in snake head fish (Channa striatus) by oxydementon—methyl, Pestic. Biochem. Physiol. 23 (1985) 41–46.
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[41] C. Hamen, Aminotransferase activities and the amino acid excretion of bivalve mollusks and brachiopods, Comp. Biochem. Physiol. 26 (1986) 697–705. [42] L. Manohar, P.V. Rao, K. Swami, Variations in aminotransferase activity and total free amino acid level in the body fluid of the snail Lymnaea luteola during different larval treamatodes infections, J. Invertebr. Pathol. 19 (1972) 36–41. [43] G.M. Christensen, D. Olson, B. Riedel, Chemical effects of the activity of eight enzymes: a review and a discussion relevant to environmental monitoring, Environ. Res. 29 (1982) 247–255. [44] H.A. Harper, V.W. Rodwell, P.A. Mayyer, Review of Physiological Chemistry, Lange Medical Publications, California, 1978, p. 328. [45] P. Taylor, S. Lappy, Interaction of fluorescent probes with acetylcholinesterase. The site and specificity of propodium binding, Biochemistry 14 (1975) 1989–1997. [46] D.W. Beyers, P.J. Sikoski, Acetylcholinesterase inhibition in federally endangered Colorado squawfish exposed to carbaryl and malathion, Environ. Toxicol. Chem. 13 (1994) 935–939. [47] J. Hugon, M. Bogers, Ultrastructural localization of alkaline phosphatase activity in the absorbing cells of the duodenum of mouse, J. Histochem. Cytochem. 14 (1966) 629–640. [48] B. Pilo, M.V. Ansari, R.V. Shah, Studies on wound healing and repairs in pigeon liver: III. Histochemical studies on acid and alkaline phosphatase during the process, J. Anim. Morphol. Physiol. 19 (1972) 205–212. [49] A.M. Ibrahim, M.G. Higazi, S.S. Demian, Histochemical localization of alkaline phosphatase activity in the alimentary tract of the snail, Marisa conuarietis (L.), Zool. Soc. Egypt Bull. 26 (1974) 94–105. [50] D. Pavlikova, S. Repas, Comparative histochemical studies of changes in spermatogenesis and inter-tubular tissue at male sterility, Biol. Bratisl. 30 (1975) 889–895. [51] L.S. Goodman, A.G. Gilman, T.W. Rall, F. Murad, The Pharmacological Basis of Therapeutics, MacMillan Publishing Company, New York, 1985, p. 427. [52] M.B. Abou-Donia, Increased acid phosphatase activity in hens following an oral dose of Leptophos, Toxicol. Lett. 2 (1978) 199–203.
Pesticide Biochemistry and Physiology 79 (2004) 1–9 www.elsevier.com/locate/ypest
Carbaryl induced alterations in the reproduction and metabolism of freshwater snail Lymnaea acuminata Pankaj Kumar Tripathi and Ajay Singh* Natural Products Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur 273 009 (UP), India Received 22 July 2003; accepted 18 November 2003
Abstract When the freshwater snail Lymnaea acuminata was exposed to sub-lethal doses (2.0, 5.0, and 8.0 mg/L) of carbaryl, fecundity was significantly reduced and even stopped at higher sub-lethal doses and altered metabolic activity in the body tissue of the snail was observed. The change from aerobic to anaerobic metabolism results in lesser energy production in the body tissues of the snails, causing paralysis and finally death. This reduced fecundity and altered metabolism suggests that it would be better to avoid the use of carbaryl pesticides in the water bodies or fields adjoining the water bodies particularly in the rainy season. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Carbaryl; Lymnaea acuminata; Fecundity; Metabolism; Enzyme
1. Introduction Carbaryl pesticides are used for pest control in agricultural fields and then translocate in water bodies by several ways. The resulting pesticide pollution of the water becomes a great threat to the aquatic ecosystem. Carbaryl has been found to have significant inhibitory effect on the controlling mechanism of reproduction in fishes [1]. The survival of organisms is largely determined by the successful reproduction and good metabolism. Disruption in the reproduction will affect the abundance and distribution of the species [2].
* Corresponding author. Fax: +91-551-202-127. E-mail address: [email protected] (A. Singh).
Therefore, laboratory tests of long-term impact of sub-lethal pollutant concentrations on organisms are most instructive when done on reproductive success [3] and metabolism. The effect of pollutants on natural ecosystems can be partially understood by determining the effect of such pollutants on the reproductive process and metabolism in the key species of the ecosystem. Although carbamate pesticides persist as residue in the local environment for only a few days [4], there may be a cumulative effect on the reproduction of aquatic organisms because the electrophilic nature of carbaryl affects the various enzymes responsible for normal metabolic processes. The aim of this study was to study the effect of sub-lethal concentrations of carbaryl pesticides on the reproduction and metabolism of the
0048-3575/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2003.11.003
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freshwater snail Lymnaea acuminata, a cosmopolitan freshwater animal and an important link of detritus food chain, which shares habitat with the freshwater fishes.
into separate petri dishes for hatching under the same exposure conditions as above. The numbers of hatched snails were counted and their survival rate was recorded for 28 days after hatching. 2.2. Biochemical estimations
2. Materials and methods Adult freshwater snails (L. acuminata) of a uniform size range (42.5 3.6 mm shell height and 22.8 1.9 mm shell width) were collected from non-contaminated waters of Gorakhpur district of Uttar Pradesh, India and kept in glass aquaria containing 30 L of dechlorinated tap water for at least 96 h to acclimatize them to laboratory conditions. The water was changed every day and dead snails were removed as soon as possible to avoid water fouling. The snails were fed daily on washed and dried Nymphaea leaves during the whole acclimatization period. A technical grade of carbaryl (1-naphthyl-N-methylcarbamate) was used for the experimental study supplied by Phone-Poulenc Agrochem., Mumbai, India. For these snails, the LC50 value of carbaryl is 14.2 mg/ L for 96 h [5]. The sub-lethal concentrations of carbaryl used for the study were 2.0, 5.0, and 8.0 mg/L. 2.1. Fecundity experiments For the fecundity experiment the test amount of pesticide was added to each glass aquarium containing 10 L dechlorinated tap water. Thirty snails were placed in each aquarium. In the control groups the water was pesticide free. Each aquaria set had six replicates. Water temperature was kept at 23 1 °C during the whole experimental time. Nymphaea leaves were allowed to float at the top of the water surface for egg laying. The leaves were changed every day to avoid decay. No food was given to the snails during the entire experiment. Lymnaeid snails attached their egg masses, containing a large number of eggs, to the back surface of Nymphaea leaves when reproducing. The egg masses produced by the snails in the experiment were removed after every 24 h and the number of eggs counted under a compound microscope. All the egg masses for each group were transferred
For biochemical analysis six sets of aquaria were used, as with the fecundity experiment. Fifty snails were placed in each aquarium. No food was given to the snails during the whole experimental period. At the end of the allotted time the nervous, hepatopancreas, and ovotestis tissues of the treated and control groups of the snails were dissected out and various biochemical parameters were measured. Glycogen was measured according to Van der Vies [6]. The homogenate (50 mg/mL, w/v) was prepared in 5% trichloroacetic acid (TCA). Glycogen content was expressed as mg glycogen/g of tissue. The pyruvate level was measured according to Friedemann and Haugen [7]. The homogenate (50 mg/mL, w/v) was prepared in 10% TCA. The pyruvate content was expressed as lmol of pyruvate/g of tissue. Lactate was estimated according to Barker and Summerson [8] as modified by Huckabee [9]. The homogenate (50 mg/mL, w/ v) was prepared in 10% cold TCA. Lactate content was expressed as mg lactic acid/g of tissue. Total protein level was estimated according to the folin– phenol method of Lowry et al. [10]. The homogenate (50 mg/mL, w/v) was prepared in 10% TCA. Total protein content was expressed as lg protein/ mg of tissue. Total free amino acid level was measured according to Spies [11]. The homogenate (50 mg/mL, w/v) was prepared in 96%. Total free amino acid content was expressed as lg/mg of tissue. Nucleic acids (DNA and RNA) were estimated according to Schneider [12], using diphenylamine and orcinol reagents, respectively. The homogenates (100 mg/mL, w/v) were prepared in 5% TCA at 90 °C. Nucleic acids content was expressed as lg/mg of tissue. 2.3. Enzymological estimations Lactic dehydrogenase (LDH) activity was measured according to the method of Anon [13]. The homogenate (50 mg/mL, w/v) was prepared in
P.K. Tripathi, A. Singh / Pesticide Biochemistry and Physiology 79 (2004) 1–9
0.1 M phosphate buffer (pH 7.5) for 5 min in an ice bath. Enzyme activity was expressed as nmol of pyruvate reduced/min/mg protein. Succinic dehydrogenase (SDH) activity was measured by the method of Arrigoni and Singer [14]. The homogenate (50 mg/mL, w/v) was prepared in 0.5 M potassium phosphate buffer (pH 7.6) in an ice bath and centrifuged at )4 °C. Enzyme activity is expressed as lmol dye reduced/min/mg protein. Cytochrome oxidase activity was measured according to the method of Cooperstein and Lazarow [15]. The homogenate (50 mg/mL, w/v) was prepared in 1.0 mL of 0.33 M phosphate buffer (pH 7.4) for 5 min in an ice bath. Enzyme activity was expressed in arbitrary units/min/mg of proteins (corresponding to the quantity of enzyme which catalyzed an O2 uptake by the oxidation of reduced cytochrome). Protease activity was measured according to the method of Moore and Stein [16]. The homogenate (50 mg/mL, w/v) was prepared in cold distilled water. The enzyme activity was expressed in lmol of tyrosine equivalent/mg protein/h. Aminotransaminases (glutamic oxalic transaminase and glutamic pyruvic transaminase) activities were measured according to Reitman and Frankel [17]. The homogenates (100 mg/mL, w/v) were prepared in phosphate buffer for 5 min and centrifuged at 1000g for 15 min and the supernatant kept for estimation of enzyme activity. Both
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the enzyme activities were expressed as lmol of pyruvate formed/mg protein/h. Acetylcholinesterase (AChE) activity was measured by the method of Ellman et al. [18]. The homogenate (50 mg/mL, w/v) was prepared in 0.1 M phosphate buffer (pH 8.0) in an ice bath and centrifuged at )4 °C and the supernatant was kept for enzyme assay. Enzyme activity is expressed as lM ÔSHÕ hydrolyzed/min/ mg wet tissue. Activities of phosphatases (acid and alkaline phosphatase) were measured according to the method of Andersch and Szcypinski [19] as modified by Bergmeyer [20]. The homogenates (50 mg/mL, w/v) were prepared in ice-cold 0.9% saline and centrifuged at 4 °C, supernatant was used as the enzyme source. Enzyme activities are expressed as the amount of p-nitrophenol formed/ 30 min/mg protein in supernatant. Each assay was replicated six times, values are expressed as means SE of six replicates. StudentÕs t test was applied to locate significant (P < 0:05) differences between treated and control groups.
3. Results and discussion 3.1. Fecundity experiment The results of the fecundity experiments on the freshwater snail L. acuminata are given in Table 1.
Table 1 Number SE (%) of eggs laid, egg masses, hatched eggs, and time to hatching (in days) of the freshwater snail Lymnaea acuminata after carbaryl exposure Control
Sub-lethal concentrations (mg/L) 2.0
5.0
8.0 —
Number of eggs after 96 h Number of egg masses after 24 h Number of hatched eggs Time to hatching in days
260 14.6 (100) 9 1.2 258 12.4 (100) 11–13
128 9.5 (49) 7 0.9 120 8.5 (47) 10–14
—
Survival rate 7 days after hatching 14 days after hatching 21 days after hatching 28 days after hatching
252 5.6 243 5.6 239 4.8 236 4.0
112 1.2 (93) 105 2.4 (88) 80 2.0 (67) 64 1.6 (53)
*
(98) (94) (93) (91)
4 0.4
2 0.4
—
—
—
—
—
—
—
—
—
—
—
—
Significant (P < 0:05), when StudentÕs t test was applied between control and treated groups. Significant (P < 0:05), when StudentÕs t test was applied between number of hatched eggs and survivability of hatchlings in corresponding treated groups. All the experiments were replicated six times. Values are means SE of six replicates. Values in parentheses are the percent of the corresponding value with control taken as 100%. —, Egg laying was stopped. Value in parentheses under survival rate are the percent of number of hatched eggs in corresponding treatment. **
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The cumulative number of eggs laid was highest in the control group. The number of eggs was significantly (P < 0:05) reduced at all the sub-lethal doses of carbaryl. Fecundity was reduced to 49% of control at 2.0 mg/L, while at 5.0 and 8.0 mg/L of carbaryl no egg was laid (Table 1). Some egg masses were laid without eggs at higher doses of carbaryl. No significant (P < 0:05) difference was found in the time of hatching between the control and treated groups but the survival rate of snail embryos exposed to carbaryl was significantly lower than the control value. The rate of survival was significantly reduced at 7 days after hatching and it was reduced to about 53% of the control after exposure to 2.0 mg/L of carbaryl after 28 days after hatching (Table 1).
3.2. Biochemical experiment Sub-lethal doses were enough to alter the biochemical parameters in the nervous, hepatopancreatic, and ovotestis tissues of the snail. Glycogen, pyruvate, total protein, and nucleic acid (DNA and RNA) levels were reduced after exposure, while lactate and free amino acid levels increased after exposure in nervous, hepatopancreatic, and ovotestis tissues of the freshwater snail L. acuminata after 96 h of exposure to carbaryl pesticide (Table 2). In the nervous, hepatopancreatic, and ovotestis tissues, respectively, of the snails exposed to 8.0 mg/L of carbaryl, glycogen content was reduced to 52, 40, and 56%, pyruvate level was reduced to 48, 31, and 26%, lactate content was
Table 2 Glycogen (GN), pyruvate (PV), lactate (LT), total protein (TP), free amino acid (FAA), nucleic acid (DNA and RNA) levels in nervous (NT), hepatopancreas (HP), and ovotestis (OT) tissues of the freshwater snail Lymnaea acuminata after 96 h exposure to carbaryl Control
Sub-lethal concentrations (mg/L) 2.0
5.0
8.0
GN
NT HP OT
2.19 0.29 (100) 2.64 0.38 (100) 2.54 0.38 (100)
2.05 0.62 (94) 2.15 0.65 (81) 2.18 0.54 (86)
1.56 0.28 (71) 1.91 0.23 (72) 2.08 0.28 (82)
1.14 0.26 (52) 1.06 0.28 (40) 1.41 0.22 (56)
PV
NT HP OT
1.05 0.28 (100) 1.09 0.23 (100) 1.28 0.36 (100)
0.87 0.16 (83) 0.82 0.14 (75) 0.79 0.12 (62)
0.67 0.10 (64) 0.65 0.11 (60) 0.46 0.12 (36)
0.50 0.11 (48) 0.34 0.08 (31) 0.33 0.10 (26)
LT
NT HP OT
2.42 0.15 (100) 2.59 0.19 (100) 2.32 0.13 (100)
2.79 0.34 (115) 2.84 0.28 (110) 2.92 0.35 (126)
3.19 0.24 (132) 3.30 0.27 (127) 3.27 0.37 (141)
4.74 0.38 (196) 5.02 0.29 (194) 5.17 0.32 (223)
TP
NT HP OT
47.64 2.11 (100) 55.44 3.20 (100) 70.06 5.21 (100)
44.46 2.29 (93) 50.29 3.37 (91) 49.36 4.87 (70)
39.54 1.36 (83) 40.31 2.21 (73) 37.83 3.89 (54)
36.68 1.17 (77) 33.26 2.71 (60) 28.02 2.54 (40)
FAA
NT HP OT
14.52 1.04 (100) 17.59 1.16 (100) 16.39 1.11 (100)
15.73 1.23 (108) 19.34 1.45 (110) 19.94 1.62 (122)
18.29 1.09 (126) 21.18 1.25 (120) 23.14 1.27 (141)
22.36 1.26 (154) 28.75 1.20 (163) 32.78 1.11 (200)
DNA
NT HP OT
42.23 1.94 (100) 46.81 0.89 (100) 52.96 0.72 (100)
36.43 3.02 (86) 36.83 3.29 (79) 41.28 4.17 (78)
29.56 1.84 (70) 29.49 0.47 (63) 34.36 0.44 (65)
22.80 1.42 (54) 20.13 0.28 (43) 16.95 0.29 (32)
RNA
NT HP OT
27.08 1.18 (100) 37.67 2.31 (100) 41.22 2.76 (100)
22.13 1.09 (82) 29.82 1.24 (79) 30.45 2.03 (74)
19.50 1.01 (72) 24.43 1.45 (65) 21.85 1.47 (53)
14.89 0.94 (55) 18.39 0.41 (49) 18.30 0.37 (44)
*
Significant (P < 0:05) when StudentÕs t test was applied between treated and control groups. Values are means SE of six replicates. Values given in the parentheses are percent values with control taken as 100%. Values are expressed as: lg/mg for TP, FAA, DNA, and RNA; mg/g for GN and LT; and lmol/g for PV.
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increased to 196, 194, and 223%, total protein level was reduced to 77, 60, and 40%, free amino acid content was increased to 154, 163, and 200%, DNA level was reduced to 54, 43, and 32%, and RNA level was reduced to 55, 49, and 44% (Table 2). Carbohydrates are the primary and immediate source of energy [21] in living creatures. The fall in glycogen content in the body tissues of L. acuminata indicates its rapid utilization by the respective tissues as a consequence of pesticide toxic stress. Under hypoxic conditions, animals derive their energy from anaerobic breakdown of glucose, which is available to the cells by increased glycogenolysis [22]. Nakano and Tomlinson [23] have suggested that catecholamine levels rise under stressful environmental conditions, enabling the increased utilization of glycogen for energy production. Glycogen levels appear to be related, at least to some extent, to the detoxification mechanisms, essential for metabolism or degradation and elimination of pesticides from the body [24]. The decrease in pyruvate levels is due to the higher energy demand during pesticidal exposure, and suggests the possibility of a shift towards anaerobic dependence due to a notable drop in the aerobic segment. The decrease in pyruvate could be due to its conversion to lactate, or its mobilization to form amino acids, lipids, triglycerides, and glycogen synthesis in addition to its role in detoxification [25]. The increase in lactate also suggests a shift towards anaerobiosis as a consequence of hypoxia created from pesticide toxic impact leading to respiratory distress [26]. Tissues receive less oxygen, leading to severe tissue hypoxia. Huckabee [27] reported that an upward trend in lactate in the tissues might be taken to indicate that oxygen supply to the tissues is not adequate for the normal metabolic functions. The appearance of such internal hypoxic conditions may be proximally responsible for the shift to the less efficient anaerobic metabolism, as evidenced by the change in lactate content observed during this study. Proteins are the most important and abundant macromolecules in living beings, playing a vital role in the architecture and physiology of the cell and in cellular metabolism [28]. The depletion of
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the protein fraction in the body tissues of the snails in this experiment may have been due to protein degradation for metabolic purposes. Under stress conditions, the dietary protein consumed by snails is not stored in the body tissue [29] and hence the treated snails met their extra energy requirements from body proteins which are mobilized to produce glucose, the instant energy of which is made available for the snail by the process of gluconeogenesis [30]. Thus, the decreased protein content may be attributed to the destruction/necrosis of cells and consequent impairment in protein synthesis machinery [31]. The increment in free amino acid level was also the result of the breakdown of proteins for energy use and the subsequent impaired incorporation of amino acids in protein synthesis [32]. It also attributed to the reduced use of amino acids in the maintenance of the acid–base balance [33]. Carbaryl pesticide appears as potential inhibitor of DNA synthesis, which might also result in reduction of the RNA level. It might also be noted that deprivation of food possibly caused some nutritional deficiency in the test snails, leading to lower concentration of nucleic acids and aberrations in DNA-directed RNA formation and protein synthesis, consequently limiting growth and adding to the metabolic stress of the snails. 3.3. Enzymological experiment Significant alteration in the enzyme levels was also observed in the body tissues of the freshwater snail L. acuminata exposed to different sub-lethal doses of carbaryl. The activities of succinic dehydrogenase (SDH), cytochrome oxidase, acetylcholinesterase (AChE), acid phosphatase, and alkaline phosphatase were inhibited, while the activities of lactic dehydrogenase (LDH), protease, glutamic oxalic transaminase (GOT), and glutamic pyruvic transaminase (GPT) were enhanced after the exposure to sub-lethal doses of the carbaryl pesticide (Table 3). In the nervous, hepatopancreatic, and ovotestis tissues, respectively, of snail exposed to 8.0 mg/L of carbaryl, lactic dehydrogenase activity was increased to 155, 175, and 198%, succinic dehydrogenase activity was inhibited to 46, 36, and 28%,
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Table 3 Lactic dehydrogenase (LDH), succinic dehydrogenase (SDH), cytochrome oxidase (CY), protease (PR), transaminases (GOT and GPT), acetylcholinesterase (AChE), and phosphatase (ACP and ALP) activities in nervous (NT), hepatopancreas (HP), and ovotestis (OT) tissues of the freshwater snail Lymnaea acuminata after 96 h exposure to carbaryl Control
Sub-lethal concentrations (mg/L) 2.0
5.0
8.0
LDH
NT HP OT
0.038 0.005 (100) 0.063 0.008 (100) 0.050 0.007 (100)
0.044 0.006 (116) 0.078 0.010 (124) 0.063 0.008 (126)
0.049 0.008 (129) 0.090 0.012 (143) 0.073 0.014 (146)
0.059 0.009 (155) 0.110 0.019 (175) 0.099 0.018 (198)
SDH
NT HP OT
50.34 5.17 (100) 53.83 4.18 (100) 45.95 5.08 (100)
43.25 4.09 (86) 40.27 4.54 (75) 32.18 3.37 (70)
35.74 3.54 (71) 33.37 3.54 (62) 23.89 2.73 (52)
23.16 3.42 (46) 19.38 3.29 (36) 12.87 2.54 (28)
CY
NT HP OT
56.71 5.09 (100) 74.98 5.18 (100) 68.84 5.17 (100)
50.24 4.45 (89) 66.12 5.65 (88) 56.34 5.18 (82)
47.07 4.69 (83) 61.87 4.53 (83) 46.46 4.13 (67)
34.03 4.69 (60) 41.73 3.74 (56) 37.08 3.19 (54)
PR
NT HP OT
0.316 0.044 (100) 0.320 0.058 (100) 0.299 0.099 (100)
0.337 0.042 (107) 0.354 0.076 (111) 0.376 0.092 (126)
0.369 0.064 (117) 0.392 0.037 (123) 0.443 0.067 (148)
0.429 0.074 (136) 0.464 0.037 (145) 0.454 0.066 (152)
GOT
NT HP OT
4.94 0.54 (100) 6.07 0.48 (100) 5.22 0.38 (100)
5.49 0.62 (111) 7.28 0.78 (120) 6.37 0.63 (122)
6.08 0.69 (123) 8.93 0.79 (147) 7.92 0.25 (152)
6.87 0.94 (139) 10.80 0.33 (178) 10.60 0.48 (203)
GPT
NT HP OT
6.62 0.84 (100) 7.71 0.45 (100) 6.61 0.65 (100)
7.45 0.92 (113) 10.84 1.12 (141) 8.57 0.85 (130)
9.00 1.17 (136) 17.22 1.31 (223) 11.44 1.10 (173)
12.31 1.46 (186) 19.33 1.69 (251) 12.79 1.17 (193)
AChE
NT HP OT
0.086 0.006 (100) 0.071 0.004 (100) 0.061 0.007 (100)
0.066 0.009 (77) 0.043 0.005 (61) 0.030 0.008 (49)
0.043 0.004 (50) 0.034 0.006 (48) 0.016 0.003 (26)
0.016 0.002 (19) 0.014 0.003 (20) 0.008 0.001 (14)
ACP
NT HP OT
0.191 0.022 (100) 0.157 0.017 (100) 0.124 0.014 (100)
0.143 0.026 (75) 0.129 0.019 (82) 0.084 0.015 (68)
0.115 0.016 (60) 0.085 0.009 (54) 0.059 0.009 (48)
0.057 0.007 (30) 0.050 0.008 (32) 0.036 0.006 (29)
ALP
NT HP OT
0.392 0.018 (100) 0.339 0.022 (100) 0.257 0.041 (100)
0.245 0.021 (63) 0.269 0.027 (79) 0.194 0.035 (75)
0.169 0.026 (43) 0.227 0.031 (67) 0.152 0.019 (59)
0.102 0.016 (26) 0.129 0.019 (38) 0.077 0.011 (30)
*
Significant (P < 0:05) when StudentÕs t test was applied between treated and control groups. Values are means SE of six replicates. Values given in the parentheses are percent values with control taken as 100%. Values are expressed as: LDH, lmol of pyruvate reduced/min/mg protein; SDH, lmol of dye reduced/min/mg protein; CY, arbitrary units/min/mg/proteins; PR, lmol of tyrosine equivalent/mg protein/h; GOT and GPT, lmol pyruvate formed/mg protein/h; AChE, lM ÔSHÕ hydrolyzed/min/mg protein; and ACP and ALP, p-nitrophenol formed/30 min/mg protein.
cytochrome oxidase activity was decreased to 60, 56, and 54%, protease activity was increased to 136, 145, and 152%, GOT activity was increased to 139, 178, and 203%, GPT activity was increased to 186, 251, and 193%, acetylcholinesterase activity was decreased to 19, 20, and 14%, acid phosphatase activity was decreased to 30, 32, and 29% while alkaline phosphatase activity was decreased to 26, 38, and 30% (Table 3).
The enzyme lactic dehydrogenase (LDH) forms the centre for a delicately balanced equilibrium between catabolism and anabolism of carbohydrates [34] and is associated with cellular metabolic activity [35]. LDH is present in most animal tissues and is involved in the interconversion of pyruvic acid and lactic acid. It serves as a pivotal enzyme between the glycolytic pathway and the tricarboxylic acid cycle. The action of pesticide in the
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respiratory chain is between FAD and co-enzyme Q, and SDH containing one molecule of covalently bound FAD is inhibited [36]. The exact mechanism of SDH inhibition is still unknown. A decrease in cytochrome oxidase activity might be either the result of reduced availability of oxygen, which in turns reduces the capacity of the ETS to produce ATP molecules, or could be due to the direct impact of the pesticides [37]. Stevans et al. [38] reported that anticholinesterase compounds are known to usually inhibit mitochondrial reactions such as the function of the cytochrome oxidase in the ETS. Pesticidal poisoning also effects KrebÕs cycle by diminishing the rate of the ETS and oxidative phosphorylation, resulting in less ATP synthesis. The enzyme protease functions in hydrolyzing proteins to free amino acids and small peptides, which is also called proteolysis. The increase in protease activity corroborates the enhancement of the free amino acid level and reduced protein level in the body tissues of the snails. Transaminases are released during cellular damage or lysis [39]. Stress conditions induce elevation in the transamination pathway [40]. The increase in transaminase values may be due to hepatopancreas enlargement indicating an increase in the functional load of the treated snails. The transamination reaction (transfer of an amino group from an amino acid to a keto acid with subsequent formation of different amino acid and keto acids) is the most important pathway in the metabolism of many amino acids [41]. Transamination not only serves as a pathway for the conversion of a-keto acids to L -amino acids, but also as an alternative means of replenishing the pyruvate pool [42]. Transamination represents the major mechanism leading towards the eventual deposition of nitrogen as waste products and also results in the production of carbon compounds, which may be metabolized for energy purposes. Christensen et al. [43] suggested that toxic chemical pollutants often affect the activity of enzymes to varying degrees, and hence enzymes are logical candidates to be used as biomonitors. The transaminases (GOT and GPT) are two key enzymes known for their role in utilization of protein and carbohydrates. The studies of Harper et al. [44] showed that transamination and
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transdeamination reactions are prominent under stress condition. GOT and GPT function at the junction between protein and carbohydrate metabolism by interconverting ketogluterate, oxalacetate, and pyruvate on one side and alanine, aspartate, and glutamate on the other side [44]. Acetylcholinesterase (AChE), is involved in the synaptic transmission of nerve impulses. Carbamates are well known inhibitor of AChE. Carbamate insecticides are a deadly nerve poison as they inhibit AChE. Carbamate compounds inhibit AChE reversibly by combining either at the active site or at a site specially removed from the active centre called the peripheric anionic site [45]. Inhibition of AChE results in the accumulation of acetylcholine (ACh), which causes twitching of muscle and leads to tetanus and eventual paralysis of the muscle. Paralysis of respiratory muscles may lead to death. Due to their neurotoxicant nature the physiology of several systems may be affected, resulting in disturbances of the metabolic system. At 20–70% inhibition of AChE, adverse effects become more subtle and can include reproduction problems [46]. It has also been shown that alkaline phosphate, through the process of phosphorylation of carbohydrates and fats, plays an important role in the active transport of chemicals across cell membranes [47]. Pilo et al. [48] demonstrated that alkaline phosphate is associated with protein synthesis, and it has also been shown that this enzyme is involved in the synthesis of certain proteinaceous digestive enzymes [49] and plays an important role in spermatogenesis [50]. Since the active sites of alkaline phosphatase [51] contain a serine residue, it is possible that the inhibition of alkaline phosphatase observed in this study could be due to the phosphorylation of the active sites of alkaline phosphatase. Acid phosphatase is a lysosomal enzyme and plays an important role in catabolism, pathological necrosis, autolysis, and phagocytosis [52]. A comparison of the differences in various enzymological constituents in the control and test animals suggests that exposure of L. acuminata to sub-lethal concentrations of carbaryl severely affects various physiological mechanisms. The toxic effects of the pesticide were not fully neutralized, and there was evidence of protein denaturation,
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disturbance of cellular metabolic activities and impairment in neural transmission. Therefore, pollution of the aquatic environment by carbaryl would adversely affect the metabolism of the snail, and have adverse effects on its reproduction, so the use of carbaryl pesticides in the water bodies or fields adjoining the water bodies should be undertaken only after careful consideration of less harmful alternatives. Acknowledgment The author extend his thanks to the Council of Science and Technology, Uttar Pradesh, India for financial support during this work. References [1] S. Arunachalam, K. Jayalakshmi, S. Aboobker, Toxic and sub-lethal effects of Carbaryl on a freshwater cat fish Mystus vittatus (Bloch), Arch. Environ. Contam. Toxicol. 9 (1980) 307–316. [2] P. Woin, C. Br€ onmark, Effects of DDT and MCPA (4chloro-2-methylphenoxyacetic acid) on reproduction of the pond snail, Lymnaea stagnalis L, Bull. Environ. Contam. Toxicol. 48 (1992) 7–13. [3] P.J. Sheehan, Effects on individuals and populations, in: P.J. Sheehan, D.R. Miller, G.C. Butler, P. Bourdeau (Eds.), Effects of Pollutants at the Ecosystem Level, SCOPE 22, Wiley, Chichester, UK, 1984, pp. 23–50. [4] A. Singh, D.K. Singh, R.A. Agarwal, Effect of cypermethrin, mexacarbate and phorate on the phospholipids and lipid peroxidation in the snail Lymnaea acuminata, Bull. Environ. Contam. Toxicol. 51 (1993) 68–71. [5] V.K. Srivastava, A. Singh, Toxicity of alphamethrin, dimethoate and carbaryl pesticides to the freshwater snails Lymnaea acuminata and Indoplanorbis exustus, Iberus 19 (2001) 1–5. [6] J. Van der Vies, Two methods for the determination of glycogen in liver, Biochem. J. 57 (1954) 410–416. [7] T.E. Friedemann, G.F. Haugen, Pyruvic acid. I. Collection of blood for the determination of pyruvic acid and lactic acid, J. Biol. Chem. 144 (1943) 67–77. [8] S.B. Barker, W.H. Summerson, The colorimetric determination of lactic acid in biological materials, J. Biol. Chem. 138 (1941) 535–542. [9] W.E. Huckabee, Blood analysis, determination of lactic acid, in: B.L. Oser (Ed.), HawkÕs Physiological Chemistry, Tata McGraw-Hill, New Delhi, 1961, p. 1103. [10] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.
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