Name: Chihaka, A.I. Home Country: Zimbabwe Research Country: Zimbabwe Project period: 1995-1998 Title The agrobotany and use of the plant molluscicide Phytolacca dodecandra in rural areas: towards community based schistosomiasis control in Zimbabwe Abstract Schistosomiasis is officially recognised as a disease of public health importance in Zimbabwe and control strategies including the control of intermediate host snails have not been sustained because of the high cost of niclosamide, the only available molluscicide endorsed by WHO. Schistosomiasis control is also given low priority by the Government, compared to the control of more acute illnesses like malaria. The situation is compounded by the fact that some affected communities accept living with schistosomiasis because the problem is only obvious and more serious in chronic infections. Plant molluscicides have generated a lot of interest in both the scientific community and national disease control agencies in schistosomiasis endemic areas because they are simple to use and are available locally. Phytolacca dodecandra, commonly known as Endod (or Gopo in Zimbabwe), is by far the most studied plant molluscicide with toxicity data available. Agrobotanical studies of wild strains of P. dodecandra, based on evaluation of berry yield, molluscicidal potency and resistance towards drought and predators led to the selection of cultivar E44 as the best candidate for domestication. Acute mammalian toxicity studies have demonstrated that Endod is either slightly toxic or non-toxic but it is piscicidal and toxic to aquatic invertebrates such as Daphnia magna. However, despite a long history of experimental work on P. dodecandra, no assessment has been made of the feasibility of involving the affected communities in the domestication and use of the plant molluscicide as a self-help schistosomiasis control project. There is need for more information on growth and productivity of P. dodecandra under varying nutrient and watering conditions. This information would be valuable in making recommendations for cultivation of this plant in endemic areas with diverse rainfall patterns and edaphic conditions. There is also not enough information on the fate of saponins in the environment. The potential of bio-accumulation of the molluscicidal compounds needs to be systematically evaluated in view of possible damage to non-target organisms at higher trophic levels. While natural compounds which are soluble in water would be expected to be biodegradable, this has not been demonstrated under standardised conditions. This study was aimed at assessing the feasibility and effectiveness of community involvement in schistosomiasis control using locally produced P. dodecandra berries and at evaluating the fate of saponins in water after mollusciciding. A comparison was made of village and school community groups to determine the better strategy for promoting mass cultivation of P. dodecandra at a sustainable level. The effect on nutrient type and watering on plant growth and productivity was studied in order to form recommendations on simple but effective cultivation methods for rural communities. The fate of saponins in an aquatic environment was evaluated in standardised tests to assess the possible environmental impact of the molluscicide. Potted P. dodecandra E44 plants were introduced to six community groups for planting, harvesting berries and treating their streams with the plant molluscicide as a self-help project. The research team and a local Environmental Health Technician gave technical advice when required and monitored growth performance of systematically selected plants at each plot. Monthly snail population surveys were conducted by trained field orderlies to assess the effectiveness of mollusciciding. The effects of nutrient types and watering on plant growth and productivity were assessed at an experimental station in Harare. The biodegradability and the persistence in aqueous solutions of saponins from the berries of P. dodecandra type E44 was evaluated. The biodegradability of P. dodecandra E44 saponins was evaluated under OECD standardised conditions. The persistence of saponins was evaluated by determination of the saponin concentration in aqueous solutions periodically over 30 days. For the determination of saponin concentration a quantitative HPLC method was used and compared to a semiquantitative haemolytic assay, which is considered a potential field method. Bioassays were simultaneously conducted using Biomphalaria glabrata snails to assess the molluscicidal potency OVI time. All experiments were carried out in two sets parallel to each other. Chemically balanced water to sustain snails was used in one set, while in the other set the medium was a mixture of snail water and river water. Finally an evaluation was made of the proportion of berries to water that would give optimal extraction of saponins before mollusciciding. Domestication was generally successful at all plots but poor drainage at some sites led to high plant losses when the plants became waterlogged. This and the poor timing of care activities such as weeding and harvesting resulted in insufficient quantities of berries being available for effective mollusciciding Mwembezi river, the river that runs close to homesteads of village community groups, was successful cleared of snail intermediate hosts for five months after mollusciciding. Mollusciciding was not very effective in the other river, Mupinge, which was treated with berry extracts from the school group. Plant growth management conditions considered alone explain the significant variation in the quantity of saponins in berries from village and school community plots (F=13.8, p<0.001). Optimum extraction of saponins from berries occurred when 1 g fine crushed berries was soaked in 10 ml cold water to give an extract concentration of 1,000 ppm. It appears from the best growth performance an highest berry yields of appr. 200 g/plant observed in plants treated with manure, that this is the most suitable nutrient type for P. dodecandra cultivation. Plants grown with cattle manure as single treatment at planting had better growth performance than those treated with synthetic fertiliser: Nutrient types added at planting had a significant effect on molluscicidal saponin content of berries (p<0.001) but the effect of watering was insignificant (p>0.05). Saponin concentrations in water treated with an aqueous extract of P. dodecandra E44 were stable during the first two days, then rapidly declined during the third and fourth days. Only traces of the saponin mixture could be detected later than five days after application of the molluscicide. From bio-assays conducted simultaneously in the same aquaria the lethal concentrations were LC50 = 9.6 mg/l in tanks containing water that was chemically balanced to sustain snails during breeding (snail water) and LC50= 6.8 mg/l in the aquaria whose medium was a mixture of snail water and river water Comparison of a haemolytic field assay and an HPLC method in determining saponin concentrations in water samples showed no significant difference in mean saponin concentrations determined (t = 0.3: p>0.05). Differences between the mean values varied between -2.67 and 3.64 mg/l at 95% CI. The correlation (r2) of mean saponin concentrations determined by the two methods was 0.84. An evaluation of the biodegradability of an aqueous extract of P. dodecandra E44 berries under standardised conditions showed that the compound is biodegraded after a lag phase of 30.7 h. Calculation of the best fit of the degradation data using the Richards model gave a half-life t1/2 = 15.8 h. Since complete biodegradation occurred within a 10 day window, the extract is considered to be readily biodegradable in most environments under aerobic conditions. It has been evident from the pilot study in Guruve district that community involvement in the domestication of P. dodecandra is feasible but achievement of berry productivity levels sufficient for effective control of intermediate host snails, is heavily influenced by management conditions. Village community groups should be targeted for mass cultivation programmes because this activity can be incorporated into their normal farming activities. The use of cattle manure has been shown to be very effective and should therefore be encouraged especially since manure is more readily available and cheaper than synthetic fertilisers. The study of pruning strategies has not been conclusive in determining effect on synchronisation of flowering but the lack of productivity when all male plants were waterlogged at Dambatsoko plot highlights the need to make a provision for always having some male plants in flower when female plants are inflorescent. This can be done by planting male P dodecandra along the perimeter of each plot thus serving the dual purpose of pollination and security against damage of plants by free grazing domestic animals. The haemolytic assay has been shown to be an effective tool for determining saponin concentration in water under field conditions but further developments are necessary before it can be used as a "field kit" at primary health care level. The biodegradability demonstrated in this study also means that P dodecandra extracts can be used in the natural environment without fear of bio-accumulation. Involved research institutions Blair Research Laboratory, Harare, Zimbabwe Royal Danish School of Pharmacy Institute for Health Research and Development Supervisor(s) J. Ndamba and N. Nyazema, Blair Research Laboratory; P. Mølgaard and E. Lenunich, Royal Danish School of Pharmacy; P. Furu, DBL Correspondence