EFFECT OF SOME HEAVY METALS ON LAMPITO MAURITII KINBERG (ANNELIDA: OLIGO CHAETA) IN MUNICIPAL WASTES DISPOSAL SITE AND A RESERVE FOREST FLOOR SITE OF WEST BENGAL, INDIA

It is evident that earthworm can accumulate heavy metals from surrounding polluted soils and other media in their body (Gish and Christensen, 1973; van Hook, 1974; van Rhee, 1975; Ireland, 1979, 1983; Ash and Lee, 1980; Beyer, 1981; Beyer et 01. 1982; Kruse and Barrett, 1985). But no such work has been carried out in India. To fill up this lacuna the present study has been conducted with the following objectives:


INTRODUCTION
As a result of the increasing interest paid to the recycling of wastewater, municipal wastes and sewage sludge in agricultural practice, it becomes necessary to study the uptake of heavy metals in invertebrates in general and earthworm in particular.
It is evident that earthworm can accumulate heavy metals from surrounding polluted soils and other media in their body (Gish and Christensen, 1973;van Hook, 1974;van Rhee, 1975;Ireland, 1979Ireland, , 1983Ash and Lee, 1980;Beyer, 1981;Beyer et 01. 1982;Kruse and Barrett, 1985). But no such work has been carried out in India. To fill up this lacuna the present study has been conducted with the following objectives: To determine whether this dominant species of L. mauritii could be use to absorb the heavy metals in contaminated soil and to compare them with a less polluted controlled reserve forest floor.

MATERIALS AND METHODS
Earthworm samples were collected month wise at random by digging and hand sorting method. Collected samples were repeatedly washed in water and then kept in double distilled water for 72 hours to evacuate soil from its gut. After that period earthworm samples were preserved in 10% formalin. Preserved samples were washed in double distilled water and then oven dried at 65°C for 48 hours Dried samples were crushed, weighed on a microbalance and acid (Nitric and Perchloric) digested on a hot plate. KEYWORDS: Earthworm, Lampito mauritii, Heavy metals, Cadmium, Zinc, Lead, Copper, Wastes disposal site. 48-132 ppm., 17-41 ppm. and 10-42 ppm. respectively in the soil of Site-II (Tables 1 and 2). In the tissue of earthworm species, 10-25 ppm., 670-1045 ppm., 150-292 ppm., 60-200 ppm. of cadmium, zinc, lead and copper respectively was present at Site-I whereas in the same species when collected from Site-II it was 3-10 ppm., 66-122 ppm., 10-28 ppm. and 10-29 ppm. respectively (Tables 1 and 2).
It was observed that the accumulation of cadmium and zinc in the whole earthworm tissue of studied species were in higher level in comparison to the surrounding soil from the studied sites whereas a reverse results were found in case of the accumulation of heavy metals like copper and lead  Bioconcentration (E / S) factor of cadmium was 3-10 in polluted soil whereas in forest soil it was 2.16-15. Bioconcentration factor of zinc was 1.1-1.39 and 1.08-1.69, for lead it was 0.44-0.73 and 0.3-0.88, for copper it was 0.28-1.61 and 0.62-0.93 in polluted soil and in forest soil respectively. Only two examples for copper in forest soil was found where bioconcentration factor was one or more (Tables 1 and 2).
The level of concentration of heavy metals in soil and its accumulation in earthworm tissue varies from one month to another and as well as with in the studied year. In Site-I highest concentration of cadmium in soil was found in Sept. '02, Feb. '03 and in earthworm tissue accumulation of this metal was highest in Jan. '02, Dec. '02, March '03; highest concentration of zinc was found in March '03 in both soil and in earthworm tissue; for lead concentration in soil was high in Jan. '03 and in earthworm tissue it was in March '03; highest copper concentration in soil was found in Jan. '02, Dec. '02 and in earthworm tissue it was in Oct. '01 (Table-I).
In Site-II highest concentration of cadmium in soil was found in May '02 but in earthworm tissue accumulation of this metal was high in Jan. '03; highest concentration of zinc in soil was found in May '03, but for earthworm tissue it was in June '03, lead concentration in soil was high in March '03, but for earthworm tissue it was high in Nov. and Dec. '01; copper concentration in soil was high in May '03 and for earthworm tissue it was in July '02 (Table-2).
From figures 9-12 it has been found that the level of heavy metal accumulation in the earthworm tissue is directly proportional to the amount of heavy metals in soil.
Organic carbon, pH and electrical conductivity the soil of site-land II is given in Table 4.

STATISTICAL TREATMENT OF DATA
Data pertaining to the level of heavy metals in the soil and in earthworm tissue were subjected to statistical analysis. The application of linear correlation was undertaken in the present study involving the data of soil factors, heavy metal content of soil and of earthworm tissue for each site. Analysis was carried out by pulling together data for 24 months (as species not found in February '03) in Site-I (DP) and for 18 months (as species not found in January, April, May, '02 & February-May, '03) in Site-II (BRF).

LINEAR CORRELATION
From this analysis (Tables 3a and 3b), it is found that level of accumulation of Cd, Zn, Pb and Cu of earthworm tissue showed significant positive correlations with the level in soil of Site-II (BRF) but in Site-I (DP), level of accumulation of only Zn and Pb of earthworm tissue shows significant positive correlations with that of the level of Zn and Pb in soil.
In site-I (DP) significant negative correlations found between level of soil organic carbon and level of zinc and lead in soil; but in Site-II only lead content of soil shows significant negative correlations with soil organic carbon.

DISCUSSION
It is evident from the present study that heavy metal contents of soil and in earthworm tissue were much higher in Municipal wastes disposal site than that of the Reserve forest (Figs. 1-8).
There was a seasonal variation in the level of concentration of heavy metals in the soil as well as its accumulation in earthworm tissue (Tables 1 and 2). Higher accumulation of heavy metals in the tissue of earthworm was found mainly in the dry season i.e., in winter and summer; it may be due to the lower activity of earthworm at that time. Ireland (1975a) and Anderson (1979) was made similar observation regarding the variation of accumulation of heavy metals in earthworm tissue as well as in the soil. The absorption level of cadmium and zinc were maximum in earthworm than the soil in both studied area, on the other hand copper and lead concentration in earthworm tissues were low than the concentration of these heavy metals in surrounding soil . This might be due to an active regulation of Cu and Pb in the body of earthworm but not of Cd and Zn. This observation coincide with the findings of Carter et 01. (1980) in Lumbricus rubellus and AlloZobophora chZorotico, Pietz et oZ. (1984), Beyer et oZ. (1987) in A. tubercuZoto, Morgan et oZ. (1986) in L rubellus.
The present study reveals that this species has the capability to withstand toxicity concentration of Cd, Zn, Pb, and Cu upto 6.25 ppm, upto 750 ppm, upto 410 ppm and upto 300 ppm respectively in the soil. Moreover this species might have capable to increase absorption rate of Cd in its body from as low as 3 ppm to 25 ppm, Zn from 66 to 1045 ppm, Pb from 10 to 292 ppm and Cu from 10 to 200 ppm (Tables 1 and 2). But bioconcentration factor tends to fall for each metal in polluted site (Site-I) than least polluted environment (Site-II). Edwards and Bohlen (1996), after reviewing several literatures, opined that the bioconcentration factor for many heavy metals in earthworms tends to fall as soil concentrations rise.
Accumulation level of Cd, Zn, Pb and Cu in this species shows a significant positive correlation with that of the concentration of these metal in soil in least polluted forest site, but accumulation level of only Zn and Pb of earthworm tissue shows significant positive correlation with that of the concentration of these metals in polluted soil in wastes disposal site. Zietek and Pytasz (1979) also found a high correlation between concentrations in earthworm tissues and soils for both Zn and Pb. Marino et 01. (1998) showed significant interactions of Cu and Cd ions on Lumbricus rubellus.
It suggests that when soil Cd and Cu content is in the higher level, then accumulation of Cd and Cu in tissue either influenced by other factors or may be the leveling off of these metals in earthworm tissue. Carter et 01. (1980) opined that the leveling off of Cd levels in earthworm tissue without a concomitant increase in faecal levels might have been due to the high Cd levels in the soil having toxic effects. Ireland and Wooton (1976) found that Zn levels in tissue were not related to soil Zn levels where soil levels were high, on the contrary in this work concentration of Zn in tissue of this species shows significant positive correlation with the amount of Zn in soil in both these sites and bioconcentration factor of Zn is more than one in both cases.
The order of concentration factors of Cd, Zn, Pb and Cu in this species was Cd > Zn > Cu ~ Pb. Ma (1982) was made similar observations in populations of Aporrectodea caliginosa. Significant negative correlations existed between concentration factors and soil organic matter content for Cu only (Ma, 1982), but Van Rhee (1975) showed positive correlations between the copper and organic matter content. Present study indicates that soil organic carbon has significant negative correlations with zinc and lead content of soil in polluted site. Regarding the question of high densities of this species in such environments, where there is a great deal of heavy metals, it might be due to the sub-lethal effects of these heavy metal poisoning. The toxic effects of heavy metals are partly determined by soil pH and the content of organic matter, high values being protective (Peredney and Williams, 2000). Perhaps also others factors, not investigated here, had a protective effects on earthworm viability as, for example, bioavailibity of Zn, Pb and Cd is limited in the presence of phosphorus compounds (Maenpaa et 01. 2002).
From the foregoing discussion it is seen that the uptake pattern of heavy metals in earthworms are a complicated matter. There are differences in rates of uptake attributed to soil pH, to interactions in the environment and in the earthworms' internal chemistry between various combinations and relative concentrations of heavy metals that occur together, to the chemical form in which the heavy metals occur, to adsorption of heavy metals onto surfaces of clay or organic matter particles and to selection of food by the earthworms (Lee, 1985). Helmke et 01. (1979), Morgan (1986), Beyer et 01. (1987) stressed the value of using earthworms to assess heavy metal contamination.
This study indicates that analysis of tissue of this earthworm species could be proved to be a useful for lowering down the heavy metal pollution at any particular site.
With respect to the question of earthworms being a source of contamination for other animals it seems clear that earthworms are preyed on by various birds, amphibians, reptiles and mammals living near municipal waste disposal site will be subjected to ingestion of considerable amount of Cd, Zn and some amount of Pb and Cu from this species. The studies on this aspect are also in progress.

SUMMARY
Study has been conducted during the period from June, 2001 to August, 2003 (total 25 months) to know the level of accumulation of some heavy metals in the tissue of L. mauritii (Annelida: Oligochaeta) collected from the municipal wastes disposal site of Kolkata, as well as from a reserve forest floor and to determine whether this dominant species could be use as a tool for the absorption of heavy metal in a contaminated soil. Metal content was estimated from whole earthworm tissue and in the surrounding soil (per gm. dry weight). Besides soil features like organic carbon, pH and electrical conductivity were also analyzed. The concentration levels of studied metals were varied from the earthworm tissue and the surrounding soil in both the sites, as well as from one month to another. Accumulation of some heavy metals in earthworm tissue was in higher level in comparison to surrounding soil Detail analyses on all these aspects have been discussed. The earthworms being a very good source of food for other vertebrates, therefore the study on the effect of consumption of this contaminated species by those animals are also in progress.

ACKNOWLEDGMENTS
Authors are grateful to Dr. J.R.B. Alfred, Director, Zoological Survey of India for providing laboratory facilities. The authors are indebt to the Director, Indian Statistical Institute, Kolkata for helping in analyzing the statistical data. Thanks are also due to Dr. J.M. Julka, Emeritus scientist, Zoological Survey of India, Solan for constructive criticism and showing keen interest for this study, last but not the least to Prof. B.K. Senapati, Sambalpur University, Orissa for providing some valuable literatures to the authors.