Introduction

A major part of the total pesticide application in Assam state in the north eastern region of India is in its tea gardens, where, malathion [S-(1, 2-dicarboethoxyethyl) O, O-dimethyl phosphorodithioate], an organophosphate pesticide, is commonly used. Malathion residue is frequently detected in soil, water, human, animal and plant tissues11. In the present study, the Indian flying barb, Esomus danricus (Hamilton-Buchanan), a common teleost fish species of North India and economically important both as ornamental and food fish is used for finding both acute and chronic effects of malathion (EC50). Acute studies included deducing LC50 values and observing behavioural changes; while chronic studies included finding effects on growth in terms of changes in weight and somatic indices as well as rates of oxygen consumption. This fish species in the north eastern part of this country mostly inhabits floodplain wetlands, small streams and ponds adjoining tea plantations where they end up as non target victims of the pesticides that are being sprayed. Evaluations of growth, somatic indices and rates of oxygen consumption can serve as useful and sensitive biomarkers in fish due to pesticide stress and can be used to monitor toxicity18.

Material and Methods

Fishes of similar length (46.77 ± 4.30 mm) and weight (0.86 ± 0.16 g) were collected from unpolluted, freshwater ponds near Assam University campus, Barak valley, South Assam, India6. They were acclimatized under laboratory conditions seven days prior to experimentation and commercially available fish food was given ad libitum twice daily. Temperature, dissolved oxygen, hardness and pH under laboratory condition were 29°C, 5.5 mgl-1, 30 mgl-1 and 6.8 respectively. Stock solution of commercial grade Devimalt (malathion 50% E.C.) manufactured by Devidayal (Sales) limited, India, was prepared using double distilled water. Serial dilutions of stock solutions were prepared using chlorine free tap water.

Static-with-renewal acute toxicity tests were conducted with ten fish in each graded concentration. Fish were placed in five glass aquaria containing dechlorinated tap water. Thereafter, malathion was added as per the following concentrations: 0.0056, 0.01, 0.018, 0.032, and 0.056 mgl-1. The fish kept in chlorine free tap water served as a control. Food was withheld 24 hours prior to acute toxicity tests. The test solution was replaced and mortality monitored at 24, 48, 72 and 96 hours. Dead fish were removed as the test proceeded. The entire experiment was repeated thrice. The 24, 48, 72 and 96 h LC50 values of malathion was calculated by Probit method9 using SYSTAT 13 for Windows. The behavioural pattern of fish was monitored regularly under above treatment conditions upto 96 hours exposure.

For growth studies ten fish each were individually reared in three sublethal malathion concentrations (0.0017, 0.00017 and 0.000017 mgl-1) and in dechlorinated tap water that served as control. Test media were renewed every 24 hours and commercially available fish food given ad libitum twice daily. Test and control fish were weighed in an electrical balance at the beginning of the experiment and on 7, 14, 21 and 28 days. For calculating the different organo-somatic indices, 50 fish each were kept in control and the three test concentrations and ten fish sacrificed at the beginning of the experiment and on 7, 14, 21 and 28 days. Weights of the whole body as well as that of brain, liver and kidney were taken. The organo-somatic indices were calculated as weight of tissue divided by total weight of fish. Statistical significance of the differences in weight changes between control and exposed fish at different malathion concentrations were determined; and comparisons among the BSI, KSI and HSI values in control and the three test concentrations at the beginning of the experiment and on 7, 14, 21 and 28 days were also made by one-way ANOVA and Tukey Test using SYSTAT 13 software for Windows.

Three test chambers (each of 3 litre capacity) were marked A, B and C containing 0.0017, 0.00017 and 0.000017 mgl-1 of malathion respectively. Each test chamber contained ten fish. At the beginning (0 day), each fish of test chamber A was transferred to the respiratory chamber, which was also numbered in accordance with the test chamber and the experiment was run for a period of 1h. After the experiment, the fish was weighed and replaced in its respective test chamber. The same process was repeated for other fishes of the test chamber A (ten replicates) and for 7d, 14d, 21d and 28day. Controls were also run simultaneously in dechlorinated tap water to obtain information on the oxygen consumption of the fish in normal state. Similarly, the process was repeated for fishes in test chambers B and C. Respiratory measurements were made by the closed chamber method10 and the dissolved oxygen was estimated adopting Winkler’s method. Rate of oxygen consumption was measured in ml/hr/100g tissue. Statistical significance of the differences in oxygen consumption between control and exposed fish at different malathion concentrations were made by one-way ANOVA and Tukey Test using SYSTAT 13 software for Windows.

Results and Discussion

The fish in the control aquarium were healthy and no mortality was recorded in it. In malathion treated set no mortality was observed at 0.0056 mgl-1 concentrations after 96 h exposure. However, at 0.01, 0.018, 0.032 and 0.056 mgl-1, the percentage of mortality was found to be 30%, 50%, 70% and 100% respectively (Table 1). After 96 h treatment period, the LC50 was calculated and it was found to be 0.017 mgl-1 (Table 2). During acute toxicity studies, the normal colour was found to be fading in exposed fish along with copious secretion of mucous. Irregular, erratic and sometimes jerky movements were observable in fish exposed to pesticide. This abnormal behaviour set in after 30-36 hours of exposure. After 36 hours, the fish tried to jump out from medium. This type of escape behaviour was more pronounced in higher doses. Later, the fish resorted to erratic swimming.

Table 1: Acute toxicity of malathion (EC50) on Esomus danricus.

Table 2: Determination of LC50 values of malathion (EC50) on Esomus danricus.

Exposure to sublethal concentrations of 0.0017, 0.00017 and 0.000017 mgl-1 malathion revealed progressive decrease in body weight at weekly recordings with the mean decrease of 12.93, 10.1 and 8.35 per cent, respectively, as opposed to a 3.8 per cent mean increase in the weight of control fish, at the end of the 28 day exposure. Statistical comparisons reveal that the per cent changes in body weight of test fish at all exposure concentrations on every weekly measurement were significantly different from those of the control at p < 0.05. The lowest exposure concentration (0.000017 mgl-1) upto 14 days of exposure showed no significant changes when compared to changes in weight at initial exposure duration while other two higher concentrations showed significant decrease at each exposure duration (Table 3).

Table 3: Weekly changes in weight (g) of control and test Esomus danricus during a 28-day exposure to malathion (EC50).

‘-’ Indicates decrease; *Significantly different from Control at p < 0.05; ‘a’ indicates no significant difference and ‘b’ indicates significant differences at p < 0.05 amongst values.

BSI, KSI and HSI values at the beginning of the experiment (0 day) and in the four successive weekly measurements were not significantly different among one another in control. The three sublethal doses, in contrast, decreased BSI by 21.55, 14.28 and 6.76 per cent; and KSI by 24.94, 14.93 and 9.87 per cent; but increased HSI by 20.98, 12.87 and 5.23 per cent; respectively. Statistical analysis revealed that significant reduction in BSI was discernible from 7, 14 and 21 day onwards in 0.0017, 0.00017 and 0.000017 mgl-1 exposure respectively (p<0.05). HSI at 0.00017 and 0.000017 mgl-1 upto 7th and 14th day of exposure respectively, showed no significant difference with control; while the highest sub lethal concentration (0.0017 mg l-1) showed significant difference with control at all exposure intervals. KSI at 0.0017 and 0.00017 mgl-1 at 7, 14, 21 and 28 day of exposure showed significant difference with control while the lowest sub lethal concentration (0.000017mg l-1) upto the 7th day of exposure showed no significant difference but thereafter showed significant difference with the control upto 28th day of exposure (Table 4).

Table 4: Weekly changes in somatic indices (SI) of control and test Esomus danricus during 28 days of exposure to malathion (EC50).

‘-’ Indicates decrease; *significantly different from Control at p < 0.05; ‘a’ indicates no significant difference and ‘b’ indicates significant differences at p < 0.05 amongst values.

Percent decline in rate of oxygen consumption in 0.0017 mgl-1 malathion exposed fish for 7, 14, 21 and 28 days were 33.4, 48.7, 54.6 and 69.3 respectively; 0.00017 mgl-1 malathion for similar duration showed 28, 43, 50 and 57% decline respectively while 0.000017 mgl-1 malathion showed 10, 17, 31 and 41% decline respectively for the same exposure duration (Table 5). It was observed that with the increase in dose concentration, the oxygen consumption rate decreased. Again, with increased exposure duration, there was a corresponding decline in oxygen consumption.

Table 5: Effect of sublethal concentrations of malathion (EC50) on oxygen consumption of Esomus danricus.

*significantly different from Control at p < 0.05; ‘a’ indicates no significant difference and ‘b’ indicates significant difference at p < 0.05 amongst values.

In the present study, 96 h LC50 for E. danricus was found to be 0.017 mgl-1 which is lower than 96 h LC50 value of malathion reported in different species of fishes (0.091-22.9 mgl-1)21 which indicates that Indian flying barb is sensitive to this pesticide. Amongst behavioural anomaly, irregular, erratic and sometimes jerky movements were seen in the present study, indicating loss of equilibrium. Such behavioural changes caused by methyl parathion were reported for Brycon cephalus1. Organophosphates such as malathion is known to inhibit acetylcholine esterase, which in turn influence behavioural patterns as seen in Labeo rohita17. Besides, copious mucous secretions can act as an initial barrier between fish and toxicant while faded colour may be due to a decline in dispersed chromatophores and an increase in punctuate chromatophore in fish scale with exposure to stressors as noted by Das and Gupta7 in Esomus danricus.

Changes in growth rate of fish serve as an important biomarker in pesticide toxicity. In the present study, the exposed fish maintained their feeding regime; hence the loss of weight cannot be correlated with starvation but may be due to an indirect effect of toxicant on macromolecular syntheses which are secondary effects induced by physiological stress14. Organo-somatic indices are yet another simple biomarker of toxicity. In Seriola dumerilli malathion reduced brain AChE, subsequently leading to declined brain weight12. Present study revealed that brain was not affected with 0.000017 mgl-1 malathion upto 21 days.This was probably because the blood-brain barrier was able to prevent the entry of the pesticide up to a certain threshold value beyond which the brain tissue was affected. Pesticides are metabolized in the liver through cytochrome P450 system to hepatotoxic intermediates. Increase of liver weight and hepato somatic index has also been reported in animals exposed to pesticides by several researchers191513 while Figueiredo-Fernandes et al,8 recorded a corresponding decrease in hepatocytes nucleus per unit area of hepatic tissue. In accordance with this, the increase in weight of liver and HSI in Esomus danricus exposed to malathion in the present study was significant. On the contrary, reductions in HSI were also observed in Clarias gariepinus5. The observed differences probably reflect the nature of response of the liver to different toxicants. Along with liver, kidney is also involved in detoxification process. Altinok and Capkin2 reported that pesticide exposed rainbow trout had decreased amount of hematopoietic tissue in the trunk kidney, necrosis in hematopoietic tissue, glomerular and tubular cells, probably damaging and consequently decreasing the weight of kidney as seen in the present study. It is also possible that the variations in response are also governed, at least partly, by the concentration of the toxicant or duration of exposure.

Respiratory distress is one of the early symptoms of pesticide poisoning. The significant decrease in oxygen consumption is probably the result of alterations of energy metabolism16 or due to destruction of gills filaments resulting in asphyxia3. Pesticide like fenvalerate is known to increase oxygen consumption in Labeo rohita and Catla catla under sublethal concentrations20. This initial increase may be due to sudden response to the impeding toxicity and due to the acceleration of oxidative metabolism due to toxic stimulus. But prolonged exposure to toxicants, as seen in the present study, may depress oxygen consumption. In tilapia exposed to organophosphate, Folidol, an increase in oxygen consumption at lower doses (0.1, 0.5 and 1.0 mgl-1) were seen, however, exposure to higher doses (2.5, 5.0 and 10 mgl-1) decreased rates of oxygen consumption4. Here also, the highest dose had most severe effect compared to the lower doses.

Conclusion

Thus, short term (acute) as well as long term (chronic) studies with mortality, behaviour, growth, organo-somatic indices and oxygen consumption as end point appear to serve as useful yet simple biomarkers of malathion toxicity in E. danricus.