Name: Aditya Parmar Date: 15th November 2010Supervisor: Dr. Olga Calvo di AlegreInstitute: Institute of Landscape and Plant Ecology.Experiment: Lemna spp. (Duckweed) Growth Inhibition Test with 3,5-DCP as toxin (Inhibitor)IntroductionLemna plant was used to characterize the effect of different concentrations of 3,5-DCP on its growth. This aquatic plant was used due toits high representation in the ecosystem and its unique characteristics of being small in size, simple structure, asexual reproduction,genetic uniformity and short generation time. None the less, plant reduces the use of animal (for e.g. Fish) for ecotoxicological tests.PrincipleLemna shows a significant reduction in growth when exposed to different organic and inorganic toxins. The inhibited growth isdepicted in terms of lower frond number, lower frond area and lower quantity of pigments such as chlorophyll.MethodologyThe experiment was carried out in a time period of 1 week. Day 1, five concentrations (0, 1.25, 2.5, 5, 10,20mg/L) were prepared toexpose Lemna plant to the toxin (3, 5-DCP). Equal numbers of frond were added to all six samples. Day 3, frond area, frond numberand appearance was noted down in the data sheet. The nutrient solution was changed, samples were rotated and ph levels weremeasured again to compare them with Day 1 readings. Day 7, again frond number, frond area and appearance was noted down in thedatasheet. The ph was also checked, on Day 7 chlorophyll pigment levels were calculated with SPAD test for different concentrationsof toxin.ResultsFrond Numbers and Frond areas were plotted against different doses of the 3,5-DCP concentrations in point graphs as Figure 1 andFigure 2 respectively, showing variation for duration and concentration relationship to frond area and frond number. On the last daychlorophyll pigment was also measured and represented in a bar graph in Figure 3. Mean Frond Numbers vs Concentration- Figure 1 Area vs Concentration - Figure 2 30 4.00 3.50 25 3.00 Frond Ares ( cm2) Frond Numbers 20 2.50 15 20, 15 Fronds Day 3 2.00 20, 1.78 1.74 10 20, 12 Fronds Day 7 1.50 Day 3 1.00 Day 7 5 Fronds Day 1 0.50 0 0.00 0 5 10 15 20 25 0 5 10 15 20 25 (mg/L) 3,5-DCP (mg/L) 3,5-DCP SPAD Vs Concentration - Figure 3 NOTE: From the above results we can describe the reduced growth of 25 Lemna plant at high concentration of toxin. Figure 1 and 2 shows 20 15 that at more than10ml/L 3, 5-DCP the plant has shown almost no 10 growth in terms of increase in frond numbers and frond area after 5 the 3rd day of the experiment. 0 1 2 3 4 5 6 The plant has shown a steep decline in chlorophyll levels with increasing concentration of toxin. Figure 3 show how pigment Concentration Mean SPAD level decreased with the increasing concentrations of 3,5-DCP.Conclusion and DiscussionThe experiment showed the reduction in growth as an influence of toxin on plant as expected. Lemna plant responded to higherconcentration in a larger influence on its frond number and area. The increasing reduction in pigment levels were also depicted withincreasing concentrations of toxin. The test clearly gave an indication that Chlorinated Hydrocarbons (CHC) significantly reduces thegrowth and pigment levels at higher concentrations (more than 10ml/L). This test might not give accurate indication for very lowconcentrations of toxins, as at lower concentrations the significance was much less.Calculation of frond number and area could be a source of possible mistake if not done in a proper disciplined manner. During theexperiment some of the samples had difficulties in estimation of area as the pictures took were not of very good quality. However, testgave appropriate results in the end and users were satisfied with the estimates.