CHAPTER V Seed Germination Bioassay Seed germination, root elongation and seedling growth are rapid and widely used acute phytotoxicity test with several advantages: sensitivity, simplicity, low cost and suitability for chemicals or environmental samples (Munzuroglu and Geckil, 2002; Wang et al., 2001). Germination is normally known as a physiological process beginning with water imbibition by seeds and culminating in the emergence of the rootlet (Kordan, 1992). However, there are different definitions of seed germination according to its root length: emergence of root, >1 mm or >5 mm (Kordan, 1992; Moore et al., 1999; Munzuroglu and Geckil, 2002; Murata et al., 2003; Ren et al., 1996). Seed coat plays a very important role in protecting the embryo from harmful external factors. Seed coats can have selective permeability (Wierzbicka and Obidzin ska, 1998). Pollutants, though having obviously inhibitory effect on root growth, may not affect germination if they cannot pass through seed coats. Recent reports have considered phytotoxicity test to be useful in assessing environmental (soils, sediments) and anthropogenic matrix toxicity (Czerniawska-Kusza et al., 2006). It is well documented that germination process is highly disturbed by metal stress. Seed germination is more sensitive to metal pollution because of a lack of some defense mechanisms (Liu et al., 2005; Xiong and Wang, 2005). The purpose of the seed germination bioassay was to assess the discriminatory ability of this test between contaminated sediment and control sediments or less contaminated sediment. Therefore, in the present study, seed germination bioassay was employed with 138
sediment and sediment elutriates to determine the toxic effect of the contaminants. The primary end point used were the survival (viability) and growth (root and shoot growth). Results Seed Germination Bioassay of Sediment Seed germination was determined by directly exposing the seeds with whole sediment taken in petridishes and also by placing the seed on the sediment layer filled in plates used for seedling growth (Materials and Methods Page-51). Effect of sediment exposure on viability of seed. Sediment toxicity was determined in samples from three stations from upstream area (Bithoor) and two stations from downstream area (Jajmau) and the results are presented in (Table 34). In the case of controls 100% seeds showed viability. Exposure to sediment from three station of upstream area (Bithoor) reduced the viability to 62.5% at all the three stations. Exposure to sediment from downstream area (Jajmau) showed the viability from 50-87.5 % with average 62.25±22.98 % close to that observed with upstream sediment (Table 34). This showed the viability of seed was reduced to almost similar level by up and downstream sediment. Effect of sediment exposure on seed germination. Seed germination was examined on exposure to the sediment samples collected from three stations of upstream area (Bithoor) along with controls where no exposure was given. Controls had average root length 7.87±3.56, ranging from 4.0 to 16.0 cms, and average shoot length 15.12±4.19, ranging from 8.0 to 20.0 cms. 139
Table. 34. Viability of seed exposed to sediment from the two sampling locations Sampling Station Germination (%) Bithoor Jajmau Control Station-1 62.50 50.0 100 Station-2 62.50 82.5 100 Station-3 62.50-100 Average 62.5 66.25 100 SD ±0.00 ±22.98 - Range 62.50-65.50 50.00-82.50-140
The seeds germinated in the sediment from Station 1 had the average root length 9.37±1.14 cms (ranging from 8.0 to 12.0 cms) and shoot length was 16.2±3.21 cms (ranging from 12.0 to 20.0 cms). Germination in sediment from Station 2 had the average root length 11.75±2.25 cms (ranging from 10.0 to 16.0 cms) and shoot length was 19.87±3.87 cms (ranging from 15.0 to 22.0 cms), and exposure to sediment from Station 3 germinated seed had the average root length 8.25±2.31 cms (ranging from 10.0 to 16.0 cms) and shoot length 16.62±5.51 cms (ranging from 5.0 to 24.0 cms) (Table 35). Sediment from two stations of downstream area (Jajmau) were tested for seed germination toxicity test. Seed exposed to sediment from Station 1 had the average root length 6.25±1.49 cms (ranging from 5.0 to 8.0 cms) and shoot length was 7.62±1.60 cms (ranging from 5.0 to 16.0 cms) and from Station 2 average root length 6.25±1.75 cms (ranging from 4.0 to 8.0 cms) and shoot length was 6.62±1.60 cms (ranging from 5.0 to 10.0 cms) (Table 36 and 37). Seed Germination Toxicity Test with Sediment Elutriate. Seed Germination test was done in sediment elutriate from both the sampling locations at four different dilutions, results are presented below. In the case of controls the average root length was 5.54±2.31, ranging from 3.0 to 8.0 cms, while control test had average shoot length 8.0±1.86, ranging from 5.0 to 12.0 cms. 141
Table. 35. Seed Germination Toxicity Test Sediment Sample from Upstream (Bithoor-one week) Legnth in Centimeters SRNo. Station-1 Station-2 Station-3 Control Root Shoot Root Shoot Root Shoot Root Shoot 1 10 15 16 22 10 24 16 20 2 12 20 10 15 9 16 8 16 3 10 17 14 26 3 5 8 16 4 8 12 10 22 8 18 8 17 8 9 16 12 20 10 15 6 12 6 8 13 10 16 8 16 6 20 7 10 20 10 16 8 19 4 12 8 8 20 12 22 10 20 7 8 Average 9.37 16.62 11.75 19.87 8.25 16.62 7.87 15.12 SD ±1.14 ±3.21 ±2.25 ±3.87 ±2.31 ±5.51 ±3.56 ±4.19 Range 8.0-12.0 12.0-20 10.0-16.0 15.0-22.0 10.0-16.0 5.0-24.0 4.0-16.0 8.0-20 142
Table. 36. Seed Germination Toxicity Test Sediment Sample from Downstream (Jajmau-one week) Legnth in Centimeters SRNo. Station-1 Station-2 Control Root Shoot Root Shoot Root Shoot 1 4 6 4 6 16 20 2 8 8 5 8 8 16 3 5 6 8 6 8 16 4 7 5 8 5 8 17 8 7 6 7 6 6 12 6 5 6 4 6 6 20 7 8 16 8 10 4 12 8 6 8 6 6 7 8 Average 6.25 7.62 6.25 6.62 7.87 15.12 SD ±1.49 ±1.60 ±1.75 ±1.60 ±3.56 ±4.19 Range 5.0-8.0 5.0-16.0 4.0-8.0 5.0-10.0 4.0-16.0 8.0-20 143
Table. 37. Summary Table of Seed Germination Toxicity Test in Sediment Samples from Upstream (Bithoor) and Downstream (Jajmau) Legnth in Centimeters Station Bithoor Jajmau Control Average SD Average SD Average SD Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot 1 9.37 16.62 1.14 3.21 6.25 7.62 1.49 1.60 7.87 15.12 3.56 4.19 2 11.75 19.87 2.25 3.87 6.25 6.62 1.75 1.60 7.87 15.12 3.56 4.19 3 8.25 16.62 2.31 5.51 - - - - 7.87 15.12 3.56 4.19 Average 9.79 17.70 1.9 4.19 6.25 7.12 1.62 1.60 7.87 15.12 3.56 4.9 144
Seed exposed to Elutriates of sediment from upstream Station 1 with 100 % elutriate concentration had the average root length 3.8±1.32 cms, ranging from 1.5 to 6.0 cms, and shoot length was 2.82±0.91 cms, ranging from 1.5 to 5.0 cms. Exposure of seeds at 50 % elutriate concentration resulted in average root length 4.8±1.13 cms (ranging from 3.0 to 7.0 cms) and shoot length 5.58±1.48 cms (ranging from 4.0 to 8.0 cms), at 25 % elutriate concentration the average root length was 6.27±1.39 cms (ranging from 4.0 to 9.0 cms) and shoot length 6.52±1.04 cms (ranging from 4.5 to 7.5 cms) and further dilution to 12.5 % elutriate concentration the average root length found was 6.75±2.83 cms (ranging from 2.0 to 8.5 cms) and the shoot length was 5.67±2.01 cms (ranging from 1.5 to 9.0 cms) (Table 38). Seed exposure to elutriate from upstream Station 2 at 100 % elutriate concentration resulted in the average root length 9.45±1.88 cms (ranging from 5.0 to 12.5 cms) and shoot length was 6.3±0.99 cms (ranging from 4.5 to 8.5 cms), at 50 % elutriate concentration the average root length was 10.77±4.4 cms (ranging from 3.0 to 17.5 cms) and shoot length was 6.45±2.14 cms (ranging from 2.5 to 11.0 cms), at 25 % elutriate concentration the average root length was 9.77±3.21 cms (ranging from 3.0 to 14.5 cms) and shoot length was 6.2±1.76 cms (ranging from 3.0 to 10.0 cms), at 12.5 % elutriate concentration the average root length was 9.02±2.61 cms (ranging from 3.0 to 13.0 cms) and shoot length was 6.1±1.35 cms (ranging from 3.0 to 9.0 cms) (Table 39). Exposure of seed to Elutriate of sediment from upstream Station 3 at 100 % exposure concentration resulted in the average root length 6.92±2.43 cms (rangin 145
Table. 38. Seed Germination Toxicity Test with Sediment Elutriate Station-1 (Bithoor-one week)length in Centimeters SRNo. 100% 50% 25% 12.50% Control Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot 1 6.5 4.0 6.0 5.0 7.5 7.5 8.5 7.5 8.0 9.0 2 5.0 3.0 5.0 8.0 7.5 7.5 1.5 7.0 5.0 7.0 3 3.0 1.5 5.0 4.0 4.5 7.0 10.5 7.5 2.0 4.0 4 3.0 2.0 4.5 5.0 7.5 7.0 10.5 9.0 3.0 7.0 5 3.0 2.5 3.0 4.0 7.5 7.5 7.0 7.0 3.0 5.0 6 4.5 3.0 7.0 8.0 8.0 8.0 8.5 7.0 6.0 8.0 7 2.5 2.5 6.5 5.0 6.5 6.0 10.0 7.5 6.0 9.0 8 4.0 2.5 3.0 4.0 4.0 7.5 4.0 4.5 6.0 7.0 9 3.0 3.0 5.0 6.0 7.0 7.0 7.5 5.5 3.0 8.0 10 5.0 5.0 4.5 7.0 3.5 4.5 7.5 6.5 3.0 8.0 11 6.0 4.5 4.5 5.5 6.5 7.0 8.5 7.5 3.0 6.0 12 4.5 3.0 4.5 8.0 6.0 6.5 5.5 5.5 5.0 8.0 13 4.5 1.5 4.5 7.0 6.0 6.5 7.0 6.0 5.0 8.0 14 4.0 3.0 4.5 4.5 6.0 7.0 5.0 5.0 3.0 7.0 15 2.0 3.0 4.5 5.0 5.0 5.0 3.0 4.5 6.0 10.0 16 4.5 3.0 5.5 6.5 5.5 5.0 7.5 6.0 9.0 11.0 17 3.5 2.5 3.0 4.0 5.5 4.5 5.0 3.5 8.0 8.0 18 4.0 3.0 6.0 4.0 7.0 6.0 3.0 2.0 9.0 12.0 19 2.0 1.5 3.0 0.0 5.5 6.5 2.0 3.0 8.0 9.0 20 1.5 2.5 0.0 0.0 9.0 7.0 3.0 1.5 8.0 9.0 Average 3.8 2.82 4.80 5.58 6.27 6.52 6.75 5.67 5.45 8.0 SD ±1.32 ±0.91 ±1.13 ±1.48 ±1.39 ±1.04 ±2.83 ±2.01 ±2.31 ±1.86 Range 1.5-6.0 1.5-5.0 3.00-7.0 4.0-8.0 4.0-9.0 4.5-7.50 2.0-8.5 1.5-9.0 3.0-8.0 5.0-12.0 146
Table. 39. Seed Germination Toxicity Test with Sediment Elutriate Station-2 (Bithoorone week) Length in Centimeters SRNo. 100% 50% 25% 12.50% Control Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot 1 9.5 6.0 12.0 7.5 10.0 6.5 10.0 6.0 8.0 9.0 2 12.0 8.5 17.5 11.0 13.0 10.0 11.0 9.0 5.0 7.0 3 10.0 7.5 14.0 9.0 14.0 5.0 5.0 7.0 2.0 4.0 4 9.0 7.0 15.0 8.0 11.0 7.0 11.0 7.0 3.0 7.0 5 10.0 6.0 13.0 6.0 13.0 6.0 9.0 6.0 3.0 5.0 6 10.0 7.0 12.0 6.0 10.0 8.0 9.0 6.0 6.0 8.0 7 5.0 4.5 13.0 8.0 10.5 6.5 10.0 6.5 6.0 9.0 8 10.0 6.5 13.0 5.0 3.0 8..0 10.5 6.5 6.0 7.0 9 10.0 7.0 12.0 8.0 11.0 8.5 10.0 6.5 3.0 8.0 10 10.0 7.0 5.0 8.0 11.0 5.5 13.0 7.0 3.0 8.0 11 8.0 5.0 15.0 8.0 8.0 7.0 8.0 5.0 3.0 6.0 12 11.0 6.0 3.0 5.0 14.5 6.0 9.0 6.0 5.0 8.0 13 9.0 6.0 12.0 6.0 10.0 7.0 3.0 5.0 5.0 8.0 14 12.5 6.0 11.0 7.0 12.0 4.0 9.0 7.0 3.0 7.0 15 9.0 6.0 15.0 7.0 5.0 6.0 13.0 7.0 6.0 10.0 16 5.0 4.5 12.0 3.0 9.0 7.0 8.0 4.0 9.0 11.0 17 11.0 7.0 3.0 5.0 11.0 5.0 10.0 7.0 8.0 8.0 18 10.0 7.0 7.0 3.0 8.0 5.0 5.0 3.0 9.0 12.0 19 8.0 5.5 3.0 2.5 8.5 3.0 6.0 4.0 8.0 9.0 20 10.0 6.0 8.0 6.0 3.0 3.0 11.0 6.5 8.0 9.0 Average 9.45 6.3 10.77 6.45 9.77 6.2 9.02 6.1 5.45 8.0 SD ±1.88 ±0.99 ±4.4 ±2.14 ±3.21 ±1.76 ±2.61 ±1.35 ±2.31 ±1.86 Range 5.0-12.5 4.5-8.5 3.0-17.5 2.5-11.0 3.0-14.5 3.0-10.0 3.0-13.0 3.0-9.0 3.0-8.0 5.0-12.0 147
from 2.0 to 9.5 cms) and shoot length was 5.15±1.94 cms (ranging from 1.0 to 9.0 cms) at 50 % elutriate concentration the average root length was 7.87±2.77 cms (ranging from 0.0 to 11.5 cms) and shoot length 5.55±1.81 cms (ranging from 0.0 to 8.0 cms), at 25 % elutriate concentration the average root length was 4.72±3.1 cms (ranging from 0.0 to 9.0 cms) and shoot length 3.77±3.32 cms (ranging from 0.0 to 7.0 cms), at 12.5 % elutriate concentration the average root length 7.35±3.71 cms (ranging from 1.0 to 11.5 cms) and shoot length was 5.72±2.14 cms (ranging from 1.5 to 8.0 cms) (Table 40). Similarly the exposure of seeds to elutriates of sediment from downstream Station 1 at 100 % elutriate concentration resulted in the average root length 5.7±2.21 cms (ranging from 1.5 to 9.0 cms) and shoot length was 4.0±1.85 cms (ranging from 1.0 to 8.0 cms), at 50 % elutriate concentration the average root length was 8.0±2.64 cms (ranging from 2.5 to 11.0 cms) and shoot length was 5.77±1.65 cms (ranging from 1.5 to 9.0 cms), at 25 % elutriate concentration the average root length 4.32±2.23 cms (ranging from 0.0 to 7.0 cms) and shoot length was 4.05±1.89 cms (ranging from 0.0 to 7.5 cms), at 12.5 % elutriate concentration the average root length was 4.92±2.59 cms (ranging from 0.0 to 8.5 cms) and shoot length was 4.05±2.13 cms, ranging from 0.0 to 7.0 cms (Table 41). Elutriate from downstream Station 2 at 100 % exposure concentration influenced the seed germination and the average root length was 3.15±1.49 cms (ranging from 1.0 to 6.0 cms) and shoot length was 2.4±0.0.75 cms (ranging from 1.0 to 3.5 cms), at 50 % elutriate concentration the average root length was 2.4±0.73 cms 148
Table. 40. Seed Germination Toxicity Test with Sediment Elutriate Station-3 (Bithoorone week) Length in Centimeters SRNo. 100% 50% 25% 12.50% Control Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot 1 9.0 7.0 9.0 6.0 8.0 6.5 8.0 6.0 8.0 9.0 2 7.0 6.5 8.0 7.0 7.0 6.0 11.0 8.0 5.0 7.0 3 7.0 5.0 10.0 7.0 6.0 7.0 5.5 4.0 2.0 4.0 4 8.0 6.0 5.0 7.0 7.0 6.0 2.5 4.0 3.0 7.0 5 7.0 5.0 9.0 6.0 6.0 4.5 6.0 4.0 3.0 5.0 6 7.0 5.5 9.0 7.5 7.0 4.5 11.0 6.5 6.0 8.0 7 9.5 7.0 10.5 6.5 8.0 5.0 9.5 6.5 6.0 9.0 8 8.0 6.0 5.5 4.5 9.0 5.5 3.0 6.0 6.0 7.0 9 7.0 2.5 4.5 4.0 7.0 4.0 9.0 3.5 3.0 8.0 10 9.0 9.0 8.0 4.0 5.0 4.5 12.0 7.5 3.0 8.0 11 8.5 7.5 6.0 5.5 5.0 3.5 7.0 4.0 3.0 6.0 12 7.5 5.5 8.0 6.0 2.0 4.0 11.5 9.0 5.0 8.0 13 9.5 5.5 11.5 8.0 6.0 5.0 8.5 7.5 5.0 8.0 14 8.5 5.5 10.0 6.0 7.0 5.5 8.0 5.5 3.0 7.0 15 8.0.0 5.5 11.0 6.0 1.0 1.5 7.0 5.5 6.0 10.0 16 8.0 4.5 11.0 6.0 3.5 2.5 11.0 7.0 9.0 11.0 17 2.0 3.0 8.0 6.0 0.0 0.0 1.5 2.0 8.0 8.0 18 3.0 2.5 7.0 5.0 0.0 0.0 12.0 8.0 9.0 12.0 19 3.5 3.0 6.5 3.0 0.0 0.0 2.0 8.5 8.0 9.0 20 1.5 1.0 0.0 0.0 0.0 0.0 1.0 1.5 8.0 9.0 Average 6.92 5.15 7.87 5.55 4.72 3.77 7.35 5.72 5.45 8.0 SD ±2.43 ±1.94 ±2.77 ±1.81 ±3.1 ±3.32 ±3.71 ±2.14 ±2.31 ±1.86 Range 2.0-9.5 1.0-9.0 0.0-11.5 0.0-8.0 0.0-9.0 0.0-7.0 1.0-11.5 1.5-8.0 3.0-8.0 5.0-12.0 149
Table. 41. Seed Germination Toxicity Test with Sediment Elutriate Station-1 (Jajmauone week) Length in Centimeters SRNo. 100% 50% 25% 12.50% Control Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot 1 5.0 4.5 9.0 8.5 5.0 4.5 7.0 6.0 8.0 9.0 2 7.0 5.0 9.0 6.5 4.0 4.5 8.5 6.5 5.0 7.0 3 9.0 8.0 9.0 7.0 7.0 7.5 6.0 5.0 2.0 4.0 4 4.5 4.0 11.0 9.0 5.0 6.0 6.0 3.5 3.0 7.0 5 10.5 6.5 10.0 6.0 5.0 3.0 8.0 6.0 3.0 5.0 6 4.5 3.5 9.0 6.0 8.0 7.0 5.0 7.0 6.0 8.0 7 5.5 3.0 9.5 6.0 5.0 4.0 5.0 4.0 6.0 9.0 8 6.5 4.5 1.0 1.5 3.0 3.0 9.5 5.0 6.0 7.0 9 8.0 5.5 8.0 6.5 5.0 4.5 5.0 4.5 3.0 8.0 10 7.0 5.0 8.0 5.0 7.0 5.5 2.5 1.5 3.0 8.0 11 7.0 6.0 10.0 5.5 4.5 3.0 3.0 3.5 3.0 6.0 12 4.5 3.0 8.0 6.0 4.0 4.0 4.0 2.5 5.0 8.0 13 6.0 6.0 8.0 7.0 2.0 4.0 3.5 2.5 5.0 8.0 14 5.5 3.0 8.0 4.5 7.5 5.0 4.0 4.5 3.0 7.0 15 4.0 2.5 8.0 5.0 6.0 5.5 7.5 6.5 6.0 10.0 16 8.0 3.5 8.5 5.0 3.0 3.5 3.0 5.0 9.0 11.0 17 3.0 2.5 10.0 6.0 3.0 3 4.0 1.5 8.0 8.0 18 3.5 1.0 9.5 6.0 2.5 3.5 7.0 6.0 9.0 12.0 19 3.5 1.0 4.0 5.5 0.0 0.0 0.0 0.0 8.0 9.0 20 1.5 2.0 2.5 3.0 0.0 0.0 0.0 0.0 8.0 9.0 Average 5.7 4.0 8.0 5.77 4.32 4.05 4.92 4.05 5.45 8.0 SD ±2.21 ±1.85 ±2.64 ±1.65 ±2.23 ±1.89 ±2.59 ±2.13 ±2.31 ±1.86 Range 1.5-9.0 1.0-8.0 2.5-11.0 1.5-9.0 0.0-7.0 0.0-7.5 0.0-8.5 0.0-7.0 3.0-8.0 5.0-12.0 150
(ranging from 1.0 to 3.0 cms) and shoot length was 1.6±0.57 cms (ranging from 1.0 to 2.5 cms), at 25 % elutriate concentration the average root length was 8.0±3.2 cms (ranging from 1.0 to 12.0 cms) and shoot length was 6.27±1.87 cms (ranging from 1.5 to 9.0 cms), at 12.5 % elutriate concentration the average root length was 3.0±1.11 cms (ranging from 1.0 to 4.0 cms) and shoot length was 2.5±0.93 cms (ranging from 1.0 to 4.0 cms) (Table 42). Seeds exposed to elutriate from downstream Station 3 at 100 % elutriate concentration resulted in the average root length 2.76±0.78 cms (ranging from 1.0 to 4.0 cms) and shoot length 1.77±0.47 cms (ranging from 1.0 to 3.0 cms), at 50 % elutriate concentration the average root length was 5.62±1.27 cms (ranging from 4.0 to 9.0 cms) and shoot length was 4.15±1.02 cms (ranging from 3.0 to 6.5 cms), at 25 % elutriate concentration the average root length 4.45±2.47 cms (ranging from 1.0 to 7.0 cms) and shoot length was 3.37±1.15 cms (ranging from 1.0 to 5.0 cms), at 12.5 % elutriate concentration the average root length was 2.65±0.96 cms (ranging from 1.0 to 4.0 cms) and shoot length was 2.15±0.71 cms, ranging from 1.0 to 3.0 cms. This suggested that elutriate from downstream stations exerted effect on the root growth and also on shoot growth thus resulting in stunted seedlings (Table 43). 151
Table. 42. Seed Germination Toxicity Test with Sediment Elutriate Station-2 (Jajmau-one week) Length in Centimeters SRNo. 100% 50% 25% 12.50% Control Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot 1 3.0 2.0 2.0 3.0 6.0 6.0 4.0 3.0 8.0 9.0 2 3.0 2.5 3.0 1.5 10.0 8.0 2.0 4.0 5.0 7.0 3 4.5 3.0 2.0 1.0 12.0 8.0 3.0 2.0 2.0 4.0 4 4.0 2.5 3.0 2.0 9.0 9.0 4.0 3.0 3.0 7.0 5 1.0 1.5 2.0 1.5 9.0 8.0 2.0 3.0 3.0 5.0 6 1.5 3.5 2.0 1.5 12.0 6.0 1.0 1.0 6.0 8.0 7 4.5 3.5 1.5 1.0 8.0 5.0 4.0 3.0 6.0 9.0 8 6.0 3.0 1.5 1.0 7.0 6.0 4.0 5.0 6.0 7.0 9 4.5 3.0 3.0 2.5 9.0 5.0 3.0 2.0 3.0 8.0 10 2.0 1.5 2.5 2.0 8.0 6.0 3.0 3.0 3.0 8.0 11 3.5 2.5 4.0 1.0 9.0 6.0 5.0 3.0 3.0 6.0 12 1.5 1.0 2.5 1.5 8.0 8.0 4.0 2.0 5.0 8.0 13 2.0 2.5 3.0 1.5 7.0 5.0 3.0 2.0 5.0 8.0 14 4.0 2.0 3.0 1.5 7.0 7.0 1.0 1.0 3.0 7.0 15 3.0 2.0 3.0 2.0 12.0 3.0 3.0 2.0 6.0 10.0 16 1.0 1.5 2.5 1.5 1.5 8.0 1.5 2.0 9.0 11.0 17 4.0 2.5 3.0 2.5 10.5 5.0 3.5 2.0 8.0 8.0 18 5.0 3.0 1.0 1.0 3.0 8.0 4.0 2.5 9.0 12.0 19 4.0 3.5 2.0 1.5 11.0 7.0 2.0 2.0 8.0 9.0 20 1.0 1.5 1.5 1.0 1.0 1.5 3.0 2.5 8.0 9.0 Average 3.15 2.4 2.4 1.6 8.0 6.27 3.0 2.5 5.45 8.0 SD ±1.49 ±0.75 ±0.73 ±0.57 ±3.2 ±1.87 ±1.11 ±0.93 ±2.31 ±1.86 Range 1.0-6.0 1.0-3.5 1.0-3.0 1.0-2.5 1.0-12.0 1.5-9.0 1.0-4.0 1.0-4.0 3.0-8.0 5.0-12.0 152
Table. 43. Seed Germination Toxicity Test with Sediment Elutriate Station-3 (Jajmau-one week) Length in Centimeters SRNo. 100% 50% 25% 12.50% Control Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot 1 4.0 3.0 6.0 4.5 7.0 4.0 2.0 1.0 8.0 9.0 2 3.5 2.5 6.5 5.0 6.0 4.0 1.5 1.0 5.0 7.0 3 3.0 2.0 4.5 3.5 5.5 5.0 1.0 3.5 2.0 4.0 4 3.0 1.5 6.5 6.5 1.0 2.0 3.0 2.5 3.0 7.0 5 3.0 1.5 5.0 4.0 1.0 1.0 2.0 2.0 3.0 5.0 6 2.0 1.5 6.0 4.5 1.0 1.5 3.0 1.5 6.0 8.0 7 3.0 1.5 5.0 3.0 2.0 4.0 2.0 1.5 6.0 9.0 8 2.5 1.5 7.0 4.0 2.0 3.0 3.0 1.5 6.0 7.0 9 3.0 2.0 6.0 5.0 1.5 2.0 3.0 1.5 3.0 8.0 10 1.5 1.0 6.0 5.0 7.0 4.0 2.0 2.0 3.0 8.0 11 2.0 1.5 3.0 3.5 6.0 4.5 3.0 3.0 3.0 6.0 12 2.0 1.5 9.0 6.0 5.5 3.0 4.0 3.0 5.0 8.0 13 1.0 1.5 6.5 3.0 4.0 3.0 3.5 2.5 5.0 8.0 14 3.0 2.0 5.5 3.0 4.0 2.5 4.0 3.0 3.0 7.0 15 2.0 1.5 6.0 3.5 6.0 3.0 3.5 2.5 6.0 10.0 16 2.0 1.5 6.0 5.0 4.0 4.5 4.0 2.0 9.0 11.0 17 3.0 2.0 4.5 4.0 6.0 4.5 3.0 3.0 8.0 8.0 18 3.0 2.0 4.5 3.0 7.0 5.0 1.0 2.0 9.0 12.0 19 3.0 1.5 5.0 4.0 7.0 4.0 1.5 2.0 8.0 9.0 20 4.0 2.5 4.0 3.0 5.5 3.0 3.0 2.0 8.0 9.0 Average 2.76 1.77 5.62 4.15 4.45 3.37 2.65 2.15 5.45 8.0 SD ±0.78 ±0.47 ±1.27 ±1.02 ±2.47 ±1.15 ±0.96 ±0.71 ±2.31 ±1.86 Range 1.0-4.0 1.0-3.0 4.0-9.0 3.0-6.5 1.0-7.0 1.0-5.0 1.0-4.0 1.0-3.0 3.0-8.0 5.0-12.0 153
Discussion Seed germination test was conducted with both sediment sample and elutriates to see the toxic effect on direct contact of seed with sediment matrix and the effect of aqueous elutable toxicant from the contaminated sediment. The sediment samples from upstream and downstream areas reduced the viability of seeds from 62.5-66% compared to 100% viability observed in control soil. This shows that irrespective of location there were some toxicants present in Ganga sediments that caused a reduction in seed viability. Moreover, the seed germinated have shown similar root and shoot growth in upstream sediment compared to controls, whereas the sediment from downstream area showed negligible effect on the growth of root but shoot growth was stunted(table 44&45). It was interesting to note that exposure of seeds to elutriate of downstream sediment samples inhibited both root and shoot growth (Fig. 13&14). A decrease of lower magnitude was also occurred in the growth of roots germinated in elutriates from upstream sediment samples whereas shoot growth was not influenced. Since toxic trace metals were poorly eluted and their concentration was very low, the inhibitory effect on the germination of seeds and specially the shoot growth may be attributed to other chemical contaminants possibly present in the sediment. It has been shown in the earlier studies that Ganga soil from the same sampling area contained pesticide contamination and the crops grown on the dry beds of Ganga accumulated higher concentration of pesticides (Hans et al. 1999). Bioaccumulation of trace metals in the seedlings was not done in this study; however, risk of the accumulation of some 154
Table. 44. Summary Table for Seed Germination Toxicity Test with Sediment Elutriate from Bithoor Station Bithoor Average 100% Average 50% Average 25% Average 12.5% Average Control Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot 1 3.8 2.28 4.80 5.58 6.27 6.52 6.75 6.67 5.45 8.00 2 9.45 6.30 10.77 6.45 9.77 6.20 9.02 6.1 5.45 8.00 3 6.92 5.15 7.87 5.55 4.72 3.77 7.35 5.72 5.45 8.00 Average 6.72 4.58 7.81 5.86 6.92 5.50 7.71 6.16 5.45 8.00 155
Table. 45. Summary Table for Seed Germination Toxicity Test with Sediment Elutriate from Jajmau Station Jajmau Average 100% Average 50% Average 25% Average 12.5% Average Control Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot 1 5.70 4.00 8.00 5.77 4.32 4.05 4.92 4.05 5.45 8.00 2 3.15 2.40 2.40 1.60 8.00 6.27 3.00 2.50 5.45 8.00 3 2.76 1.77 5.62 4.15 4.45 3.37 2.65 2.15 5.45 8.00 Average 3.87 2.72 5.34 3.84 5.59 4.56 3.52 2.90 5.45 8.00 156
Bulk Sediment Root Shoot 25 20 Length in cms 15 10 5 0 Control Upstream Downstream Sampling Area Figure 13. Seed Germination Bioassay of Bulk Sediment 157
Sediment elutriate Root Shoot 12 10 8 Length in cms 6 4 2 0 Control Upstream Downstream Sampling Area Figure 14. Seed Germination Bioassay of Sediment Elutriate 158
toxic metals especially Cr is possible if any crop is grown in dried beds near the study area as reported earlier. In the present study some of the trace metals were found in higher concentrations in downstream sediment, like Cr, Cu, Fe, Mn and Zn therefore, these metals alone or in combination may exert toxic effects on the seedling growth causing inhibition in root and shoot growth. Among the various trace metals chromium (Cr) concentration was sufficiently high in downstream sediment possible due to discharge from leather tanning, textile and electroplating industries. Several studies have shown inhibition of seed growth due to Cr (Valeria Scoccianti et al., 2006). In plants, macroscopic effects of Cr toxicity include alteration in the germination process as well as in growth and development, which may affect total dry matter production and yield (Shanker et al., 2005). Copper (Cu) is an essential micronutrient for plants, but it can be toxic to the plants at higher concentrations (Xiong and Wang, 2005). In plants, copper interacts with a wide range of physiological and biochemical processes in cells, and elevated copper concentration inhibits the normal growth and development (Caspi et al., 1999, Nagib et al., 2007). Copper was other trace metal that was found higher than sediment quality guidelines (SQG) and is known to exert toxic effects on plants (Wang, 1994; Wang and Zhou, 2005; Xiong and Wang, 2005; An et al., 2006). In downstream sediment manganese (Mn) was found about 2-fold higher than upstream sediment. Human practices have raised Mn content and availability in many soils. Mine tailings and metal smelters increase soil Mn concentration and availability with subsequent effects on vegetation structure and composition (Zheljazkov and Nielsen, 1996; Wong et al., 1983). Long-term and heavy dose 159
applications of sewage-sludge (biosolids) or other organic amendments to agricultural soils and soil anaerobic conditions such as waterlogging or poor drainage may also lead to an increase in the content or availability of Mn and other heavy metals (Ramachandran and D Souza, 1997). The availability of Mn in soils is largely controlled by soil ph (Smith and Paterson, 1990). However, the ph of the rhizosphere is more crucial for determining Mn availability to plants (Reisenaur, 1988). In general, nutrient uptake, especially in relation to elements entering the roots by diffusion, may be hampered by Mn due to Mn inhibition of root hair production and reduction of stomata dimensions (Lidon, 2002). High substrate Mn may thus reduce plant growth due to other nutrient deficiencies instead of Mn toxicity (Langheinrich et al., 1992). In fact, one of the first symptoms associated with Mn toxicity is related to both Ca and Mg deficiencies (Marschner, 1995). Iron toxicity is related to iron solubility that is itself dependent on the solution composition. Iron solubility increases with decreasing ph and the presence of sulfate ions, which are commonly encountered in the environment surrounding mines, sulfide mine wastes and other sulfide deposits. Iron in the form of Fe 3+ is more suitable for phytotoxicity studies because it is spontaneously oxidized from Fe 2+ to Fe 3+. To prevent iron precipitation, the determination of iron phytotoxic effects requires a low ph to obtain a wider iron concentration range, but the selection of ph is limited by the plant tolerance to acidity (Banášová and Suchá, 1998). Iron concentrations inhibiting plant growth (other than lettuce) have been determined at ph values above 4 (Samantaray et al., 1998; Wheeler et al., 1993; Wheeler and Power, 1995), where iron solubility is very low. The tests on the inhibition of root elongation by iron (Lepidium 160
sativum and Brassica rapa) included a low ph of 2.7 (Devare and Bahadir, 1994). However, the iron content in the waste solution was expressed in relative units, without any determination of the inhibiting concentrations (EC50 or any other). The determination of iron phytotoxicity requires the exclusion of interferring effects. If the conditions of iron solubility and the ionic strength phytotoxic effect are not taken into account, iron toxicity is either undetectable or the results obtained do not include changes in iron concentration and specification appearing during toxicity testing. All of the processes are dependent on the ph and the presence of accompanying substances in the tested solutions. All ph values lower than 3 also had a strong inhibitory effect on other plants (Banášová and Suchá, 1998; Evans et al., 1982). Zinc phytotoxicity is the most extensive microelement phytotoxicity. Natural vegetation has been hampered by the high concentration of Zn in the surface mineral soil. Zn toxicity to grasses has been reported be Chaney et al., (1989). Compared with shoot growth, much less is known about the rooting characteristics of plants grown on high-zn soils. Likewise, little is known of how the root and shoot growths of different species are affected by high-zn soil. The depth and area of rooting are major factors in the long term survival of plants grown on contaminated soils covered with a clean soil or organic material. Brar and Plazzo (1995) showed that some grasses root more deeply than others and hypothesized that deeper-rooting plants tolerant of Zn will be more likely to be successful in long term survival following a one time surface amendment. Another requirement for survival on surface-amended soil is plants ability to root through a change in substrate. Inhibition of root growth from a soil amendment layer into a toxic metal substrate below could cause plant injury 161
(Johnson et al., 1977). It is considered that seedling growth is extremely sensitive to metal pollution because of a lack of some defense mechanisms (Liu et al., 2005; Xiong and Wang, 2005). It is known that metal sensitivity and toxicity to plants are influenced by not only the concentration and the toxicant types, but are also dependent to several developmental stages of the plants (Liu et al., 2005). Studies have shown that absorption and accumulation of heavy metals in plant tissue depend upon many factors, which include temperature, moisture, organic matter, ph, and nutrient availability. Soil properties influencing heavy metal availability also vary between the sites. The alkaline range of soil (>8.0) is known to restrict the mobilization of heavy metals, thus reducing their uptake. The ph values of sediment observed in the present study were also in the range of 8. Therefore, it is quite possible that the trace metals were not bioavailable to cause toxic effects on shoot system but root on emergence came in contact to receive direct inhibitory effects. The inhibition in shoot growth observed in downstream sediment may be due to some other factors. Thus, seed germination bioassay could indicate the difference in the toxicity of upstream (less contaminated sediment) and downstream (highly contaminated sediment). Also seed germination could provide indication of the presence of factors other than trace elements present in the downstream sediment due to differential response observed in shoot growth of seedlings exposed to up- and downstream sediments making the test sensitive assay. The seed germination bioassay is fairly inexpensive requiring simple laboratory facilities, and can be conducted by the manpower after minimal training. 162