Materials and Methods:

Animals

    The freshwater vector snails Lymnaea acuminata (2.6±0.3 cm in total shell height) and Indoplanorbis exustus (0.85±0.03 cm in total shell height) and non-target fish Labeo rohita (5.15±0.5 cm total body length) were collected from the local freshwater bodies of Gorakhpur district (U.P.) in India. The collected animals were stored in glass aquaria containing de-chlorinated tap water for acclimatization to laboratory conditions. Dead animals were removed from the aquaria to avoid any contamination. Average sized adult animals were used for the experiments. Experimental conditions of water were determined in the beginning of the experiments by the methods of APHA, (2005).

Plant

    Plant Euphorbia hirta (Family: Euphorbiaceae) was collected from Botanical Garden of D.D.U. Gorakhpur University, Gorakhpur, Uttar Pradesh, India and identified by Prof. S.K. Singh, Plant taxonomist, Department of Botany, D.D.U. Gorakhpur University, Gorakhpur, Uttar Pradesh, India, where a voucher specimen is deposited.

Extraction of active moiety through different solvents

    The fresh leaves and bark was dried at 40°C over night and pulverized in a mortar and pestle. The dried powder of leaf and stem bark was extracted in soxhlet apparatus, using 200 mL of five different solvents viz. chloroform, carbon tetrachloride, acetone, diethyl ether and ethyl alcohol by the method of Jain, (1965). The extracted material was dried through vacuum pump. The extracted dried powder was stored in airtight desiccators and used for further toxicity studies. Different doses used for the toxicity experiments against freshwater vector snails Lymnaea acuminata and Indoplanorbis exustus, respectively (Table 1).

Toxicity experiments

    Toxicity experiments were performed by the method of Singh and Agarwal, 1988. Ten experimental animals Lymnaea acuminata and Indoplanorbis exustus were kept in glass aquaria containing 3 L de-chlorinated tap water for 24h, 48h, 72h or 96h. Control groups were kept in de-chlorinated tap water without any treatment. Each set of experiment was replicated six times. Mortality was recorded after every 24h during the observation period of 96h. The values were calculated by the probit log method of Robertson et al., 2007.

    For environmental toxicity, if any caused by the extracts of both the plant parts, the toxic effect of different solvent extracts of leaf and bark of Euphorbia hirta was also studies in mixed populations of fish and snails. In these experiments, group of 10 snail Lymnaea acuminata and 10 fish Labeo rohita were put together in 6 L de-chlorinated tap water. These mixed populations were exposed to previously determined LC90 (24h) of snail Lymnaea acuminata for 24h, (Table 4). The doses that can be used for killing the 90% population of snail Lymnaea acuminata are safe for non-target fish Labeo rohita, which shares the habitat with these vector snails.