# Ambient lead (Pb) concentration, bioaccumulation and its effects on leaf epidermal structures of Bougainvillea spectabilis.

IntroductionLead (Pb) the widely used element of the transition metals, had played various roles in the development of human civilization. The major source of Pb in the environment is the automobile exhaust fumes because of the addition of the lead in the form of tetra ethyl lead or tetra methyl lead to the gasoline as an antiknock substance in the last decades. Thus road traffic plays a major role in polluting the air of urban environment. In spite of all regulatory measures and total ban on the use of lead antiknock additives, it continues to be most serious global environmental problem as a major pollutant for both terrestrial and aquatic ecosystems. Excess lead causes a number of toxicity symptoms in plants as stunted growth, chlorosis, blackening of root system. Harmful effects of polluted air on plant as alterations in leaf epidermal structures have been studied by various workers (Salgare and Achereker, 1991; Rai and Kulshreshth, 2006). Some biochemical and ultra-structural changes have been reported in plant foliage exposed to auto-exhaust pollution by Verma and Singh, (2006). Malviya and Wagela (2001) had also estimated the Pb content in plant vegetation. Sharma and Dubey (2005) reviewed the morphological, physiological and biochemical effects of Pb toxicity on plants. Ji Tao et al. (2006) assessed the bioavailability, phytotoxicity and bioaccumulation of Pb in ryegrass and millet. Bougainvillea spectabilis is also a wild and ornamental shrub, which is widely used in green belt and social forestry to enhance the beauty and purity of roadside environment. Thus this paper also aims:

1.to estimate the ambient Pb concentration. 2. to estimate bioaccumulation of Pb by leaves of B. spectabilis. 3. to investigate the leaf epidermal structure of B. spectabilis and 4. to correlate with Pb present in air and leaves.

Materials and Methods

Air quality monitoring of ten selected sites (Sirmaur Chowk, Civil Lines, A.P.S. University campus (contorl site), Stadium Chowk, Dhobia Tanki area, Jaistambh chowk, PTS chowk, Nagar Nigam Building area, Prakash chowk, Saman chowk) have been carried out to assess the ambient Pb concentration along the major roadsides and national highways and crossings through the Rewa city. Air samples for Pb analysis were collected by Respirable Dust Sampler (Envirotech model APM 460-BL/APM 411) for eight hours in a day as per the standards of Central Pollution Control Board (CPCB), India. Leaf samples were also taken from the selected eleven sites.

Quantitative estimation of lead ([Pb.sup.++]): Quantification of [Pb.sup.++] in the air was done by the digestion of dust collected in the filter paper (GFA-3). To determine suspended metal ([Pb.sup.++]) the filter paper was digested with the help of nitric acid- perchloric acid. A little amount was taken for metal quantification. The concentration was analyzed by Atomic Absorption Spectrophotometer, Model PERKIN ELMER, MLA MLS 300 (1998-99) as per standards of CPCB, India.

Calculation: Concentration of heavy metals in the sample was calculated by comparing the sample minus blank reading of the calculation curve. [micro]g/[m.sup.3] heavy metal = CV/T, Where, C=mg/L Heavy metal in sample V=Sample Volume in ml, T=Total volume of air passed through the cyclone (m3).

Quantitative estimation of lead ([Pb.sup.++]) in leaves: Leaves are oven dried at 70 [degrees]C for 46 hours. 2-3 gm of finely grounded leaf material was taken in 100 ml flask and added the mixture of 25 ml nitric acid and 5 ml perchloric acid, digested by slow heating initially then fast heating until acid refluxes down the side of the flask. After cooling, transferred it to 50 ml. calibrated flask and make up volume with distilled water.

Calculation: mg/gm heavy metal=CV/w, Where, C=mg/L heavy metal in sample, V=Sample volume in ml, W=Weight of leaf sample in gm.

Microscopy or Anatomical Study: Leaf surface characteristics were studied with light microscope and Motic images camera. The leaf epidermal peel slides were made by the method of lasting Impressions. Leaf impression was examined under at least 400x magnification by light microscope and 400x, 1000x magnification by digital microscope. Number of stomata and epidermal cell were counted per square millimeter area and the stomatal frequency and stomatal index were calculated by using the following formulae. Stomatal frequency (S.F.) = S / E x 100, Stomatal Index (S.I.) = S /E + S x 100, Where S=Average no. of stomata, E=Average no. of Epidermal cells.

Cell size measurement: Size (Length & breadth) of epidermal cells and guard cells were measured with ocular micrometer under high power magnifications with the help of "stage-ocular micro-metery" and "Motic Images plus 2.0 ML" software.

Statistical Analysis were done by the "Graph Pad In stat 3" software, student's t-test and coefficient of correlation were applied for interpretation.

Results and Discussion

Lead contents in particulate matters of ambient air

Seasonal [Pb.sup.++] concentrations in ambient air at selected roadsides are shown in Table I. The highest Pb content was recorded at Sirmaur Chowk during winter months (0.4367 [micro]g/[m.sup.3]) and rainy (0.3758 [micro]g/[m.sup.3]) months, but in summer months at Jai Stambh Chowk (0.1386 [micro]g/[m.sup.3]). The lowest Pb content in the ambient air was recorded at UTD during winter (0.0542 [micro]g/[m.sup.3]), rainy (0.0324 [micro]g/[m.sup.3]) and summer (0.0295 [micro]g/[m.sup.3]) months. Seasonal average lead contents of particulate matters in the ambient air of Rewa city were computed from the basic data (Figure I). Results revealed higher lead contents during winter months (0.175 [micro]g/[m.sup.3]) followed by rainy (0.103 [micro]g/[m.sup.3]) and summer (0.073 [micro]g/[m.sup.3]) months. It is obvious from the results that Pb levels were higher during winter months followed by rainy and summer months. The similar trend of variation was also observed by Sayed et al., 2002 and Pradhan et al. (2004). Comparatively higher Pb contents estimated from the ambient air can be attributed to the traffic density of different types of auto-vehicles in operation. Wagela et al. (1998) and Malviya and Wagela (2001) have also reported similar findings in areas adjacent to the roadsides.

Automobile exhaust fumes results emission of lead in ambient air which ultimately settles down and gets mingled with roadside dust (Wagela, 1998). This study also follows the same trend having high lead concentration in the area where automobile pollution was high. Despite abatement on the use of Pb additives in the petrol fuels the presence of considerable amount of Pb concentration in the air suggests either of two concluding options. The first one is that the ban on the use of lead additives in petrol is not practiced honestly on ground level of execution and the second one is the recycling of Pb particulates settled down on the ground and arrived on the ground from other sources like battery discharging processes, continuous demolition of older buildings containing leaded paints (Montgomery and Mather, 2005; Farfel et al., 2005), which might be the another reason for the major source of inventory of Pb particulate emissions.

[FIGURE I OMITTED]

Various recent studies also support the second option of lead recycling by soil containing Pb in heavy amount, as the major source of inventory (Banat et al., 2005; Li, 2006; Nabulo et al., 2006).

Bioaccumulation of Lead (Pb): Table II shows the seasonal Pb content in the leaves of B. spectabilis growing at selected roadsides. Maximum accumulation of [Pb.sup.++] by the leaves of B. spectabilis was recorded at Sirmaur Chowk during winter (12.84 mg/g), rainy (10.99 mg/g) and summer (9.446 mg/g) seasons while the minimum was recorded for leaves at UTD campus during winter (2.034 mg/g), rainy (1.07 mg/g) and summer (1.001 mg/g). Seasonal average Pb contents of B. spectabilis leaves revealed highest Pb in the leaves during winter (5.013 mg/g) followed by rainy (3.636 mg/g) and summer (2.801 mg/g) months (Figure II). The correlation was calculated between these seasonal average values of leaf Pb and seasonal average Pb in ambient air which shows very strong positive correlation (r=0.9540). This study indicates the accumulation of substantial amount of lead in the leaves of B. spectabilis growing along roadsides. Presence of lead in the leaves of plant of urban areas is also conformed by various recent studies (Jaradat and Momani, 1999; Indukumari et al., 2005 Tomasevic et al, 2005; Swaileh et al, 2006; Oliva and Mingorance, 2006; Nabulo et al., 2006). Plant leaves have shown greater accumulation of Pb at sites with greater particulate contents in the ambient air (r=0.9540). The higher Pb was accumulated by the leaves of plants of high traffic area than those of low traffic area and control site. This increase could be due to prolonged exposure of plants to roadside exhaust of high traffic area and recycled lead dust (Indukumari et al., 2005). Swhaileh et al (2006) have reported Pb accumulation almost 3-4 times more in old leaves than new ones. This is perhaps one of the reasons for higher Pb contents in the leaves during winter months.

[FIGURE II OMITTED]

Effects on Bougainvillea spectabilis: Figure. III depicts the healthy as well as damaged epidermal and guard cells of Bougainvillea spectabilis at control and polluted zones, respectively.

Guard cells: At ventral surface no significant changes have been observed in guard cell lengths during winter months. However a significant increase was observed in width at most of the sites. The stomatal number, stomatal frequency and stomatal index showed decreasing pattern. A significant increase was observed in cell length and width at most of sites during summer and rainy months. Decrease in number of stomata has been observed during summer while increased number of stomata was noticed in rainy months. Stomatal frequency and stomatal index were notably increased than control site during both the seasons (Table-III and Table-IV).

Epidermal cells: Table-V and Table-VI presents the alterations in the cell length and width of epidermal cells. At dorsal surface the epidermal cell length exhibited significant decrease for some sites during winter months. There was significant decrease in epidermal cell width at most of sampling sites as compared to control plants during winter month and rainy months. However significant increase in cell width was noticed for most of sites during summer months. Leaves of sampling sites registered increased number of epidermal cells at all sites except two during winter months. The epidermal cell numbers were found to be increased on dorsal surface. In rainy season the number of epidermal cells was increased in all sites except three sampling sites as compared to control. Although, reduction in epidermal cell size was not observed in all seasons at dorsal and ventral surfaces some of the individual cells were in highly ruptured, degenerated and deteriorated conditions in all samples. Correlation was established between Pb contents and anatomical features of leaves with respect to three seasons (Table VII). Results revealed increase in length and width of guard cells of ventral surface (winter samples) and decreased width of rainy samples were negatively correlated with leaf Pb contents. Similarly, there was negative correlation between lead contents and epidermal cells size of both the surface of leaves, except on dorsal surface of summer samples and ventral surface of monsoon samples. The number of stomata and epidermal cells were also negatively correlated with Pb contents during summer months (Table VIII). These negative correlations indicate the negative effect of lead contents on cell size. Raina and Sharma (2006) have also observed marked alterations in the size and number of foliar epidermal and guard cells, stomatal frequency and stomatal index in both abaxial and adaxial surfaces of leaves of B.spectabilis as air pollution effects. Verma and Singh (2006) have reported the similar results regarding effect of urban air pollution on leaf epidermal parameters.

[FIGURE III OMITTED]

The significant reduction observed in cell size of B. spectabilis may be attributed to Pb toxicity, similarly reduced number of stomata per square millimeter, stomata frequency and stomata index could be attributed to Pb toxicity which is supported by Wehyszko-Chmielwska et al. (2005). However the significant increase in cell size may be attributed to the physiological and anatomical adaptive measures adopted by the plants naturally, if the no. of stomata will reduce then the guard cell size will increase at same surface. Whether the number of stomata reduction at dorsal surface leads the increased no. of stomata, stomata frequency and stomata index at ventral surface (Hethrington and Woodward, 2003).

Conclusion and Recommendations

From the present study it may be concluded that ambient Pb concentration is in considerable amount at roadside air. Pb in less than the permissible limit may cause toxicity in the plants and human by prolonged exposure. Despite the higher accumulation of [Pb.sup.++] by leaves of B. spectabilis. The plant shows much resistant nature through the less reduction observed in leaf epidermal structures due to [Pb.sup.++] toxicity. So it can be recommended for continuous use for roadside plantation as pollution indicators and for phytoremediation of Pb. Further investigation is also needed regarding other physiological effects (especially photosynthesis) of the plant as cell deterioration and alterations are found to be significant in the study.

References

[1] Banat K.M.,.Howari F.M and Al-Hamad A. A. (2005). Heavy metals in urban soils of central Jordan: Should we worry about their environmental risks? Environmental Research. 97:258-273

[2] Farfel Mark R., Orlava Anna O., Lees Peter S. J., Rohde C., Ashley Peter J. and Julian Chisolm Jr. J. (2005). A study of urban housing demolition as a source of lead in ambient dust on sidewalks, streets and alleys. Environmental Research. 99: 204-213.

[3] Hetherington A. M. and Woodward F. I. (2003). The role of stomata in sensing and driving environmental change. Nature 424, 901-908

[4] Indu kumari S, Usha rani P and Suresh Ch. (2005). Absorption of automobile pollutants by leaf surfaces of various road side plants and their effect on plant biochemical constituents. Poll Res. 24(3):509-512

[5] Jaradat Q. M. and Momani K. A. (1999). Contamination of road side soil, plants and air with heavy metals in Jordan, A comparative study. Turk J Chem 23,209-220.

[6] Ji-tao SI, Bao-guo TIAN, Hong-tao WANG Basta N., Schroder J. and Casillas M. (2006). Assessing availability, phytotoxicity and bioaccumulation of lead to ryegrass and millet based on 0.1 mol/L Ca[(N[O.sub.3]).sub.2] extraction. J.Envir. Res. 18, 5, 958-963

[7] Li Loretta Y. (2006). Retention Capacity and Environmental mobility of Pb in Soil along highway corridor. Water, Air and Soil Pollution. 170 (1-4) 211-227

[8] Malviya R. and Wagela D.K. (2001). Studies on lead concentration in ambient air, roadside dust and its influence on the healthy traffic police personnel at Indore city. Poll Res. 20(4); 635-638

[9] Montgomery M. and Mathee A. (2005). A preliminary study of residential paint lead concentrations in Johannesburg. Environmental Research. 98: 279283

[10] Nabulo G., Oryem-Origa H. and Diamond M. (2006). Assessment of lead, cadmium and Zinc contamination of roadside soils, surface films, and vegetables in Kampala City, Uganda. Environmental Research 101, 42-52

[11] Oliva S. R. and Mingorance M.D. (2006). Assessment of airborne heavy metal pollution by aboveground plant parts. Chemosphere 65, 177-182

[12] Pradhan A., Waseem M., Dogra S., Khanna A.K. and Kaw J.L. (2004). Trends of metals in the Respirable Particulates: A Comparative seasonal study in Lucknow city. Poll Res. 23(3):445-450

[13] Rai A. and Kulshreshtha K. (2006). Effect of particulates generated from automobile emission on some common plants. Journal of food, agriculture & Environment. 4(1): 253-259

[14] Raina A.K. and Sharma A. (2006). Assessment of air pollution and its impact on the leaves of some plant species. Poll Res. 25(3):543-547

[15] Salgare S.A. and Acharekar, C. (1991). Effect of ambient air on the leaf anatomy of some wild plants -1, J.Environ.Biol.,12(4):347-352

[16] Sayed, A.M., Al-Azmi D. and Khuraibet N.A. (2002). Measurement of 210Pb concentrations in Airborne in Kuwait. Environ. Mon. Assess. 79: 47-55

[17] Sharma P. and. Dubey R. S. (2005). Lead Toxicity in Plants. Braz. J. Plant Physiol. 17(1) 1677

[18] Swaileh K. M., Matani M. and Hussein R.M. (2006). Heavy metals in urban road side plants from Amman, Jordan. Bull.Environ.Contam. Toxicol. 77:445-450

[19] Tomasevic M., Z. Vukmirovic, Rajsic S., Tasic M. and Stevanovic B. (2005). Characterisation of trace metal particles deposited on some deciduous tree leaves in urban area. Chemosphere. 61(6): 753-760

[20] Verma A. and Singh S.N. (2006). Biochemical and ultra-structural changes in plant foliage exposed to auto-pollution. Env. Mon. Assess. 120 (1-3), 585-602.

[21] Wagela D.K., Joshi, O.P., and Pawar K. (1998). Urban air pollution effects on two species of CASSIA. Poll. Res. 16 (1):1-3.

[22] Weryszko-Chmielewska E. and Miroslawa C. (2005). Lead-induced histological and ultra structural changes in the leaves of Soyabean (Glycine max(L) Merr.). SoilSci PlantNutr. 51(2): 203-212.

Mani Singh * and R. M. Mishra

School of Environmental Biology, Awdhesh Pratap Singh University, Rewa (M.P.) India

* E-mail: smani801@gmail.com

Table I: Pb content in Ambient Air of Rewa City ([micro]g/[m.sup.3]). Winter Summer Rainy Stadium Chowk 0.1723 0.0528 0.1166 Sirmaur chowk 0.4367 0.1063 0.3758 UTD 0.0542 0.0295 0.0324 Saman chowk 0.1924 0.1013 0.1190 PTS chowk 0.0695 0.0513 0.0360 Dhobia Tanki 0.1783 0.0633 0.0565 Civil Lines 0.0761 0.0439 0.0531 Jaistambh chowk 0.3688 0.1386 0.1417 Nagar Nigam 0.1025 0.0769 0.0417 Prakash chowk 0.0992 0.0635 0.0599 Table II: Pb content in the leaves of B. spectabilis (mg/g). Winrer Rainy Summer Stadium Chowk 4.001 2.205 1.066 Sirmaur chowk 12.846 10.991 9.446 UTD 2.034 1.078 1.001 Saman chowk 4.552 3.020 1.999 PTS chowk 2.878 1.894 1.031 Dhobia Tanki 4.471 3.121 2.543 Civil Lines 3.097 2.795 1.545 Jaistambh chowk 11.083 9.637 8.771 Nagar Nigam 3.894 1.843 1.252 Prakash chowk 3.551 1.830 1.077 Transport Nagar 2.744 1.588 1.082 Table III: Average length and width of Guard cells, number of stomata, stomatal frequency and stomatal index of Bougainvillea spectabilis growing at different sites of Rewa city. SITES Stadium Sirmaur SEASONS Chowk chowk WINTER DORSAL L 66.85 65.46 [+ or -] 12.531 [+ or -] 6.954 W 15.67 17.25 [+ or -] 3.525 [+ or -] 3.577 NFS 10 30 SF 1.818 3.030 SI 1.785 2.941 VENTRAL L 66.37 61.86 [+ or -] 8.520 [+ or -] 9.997 W 18.25 15.27 [+ or -] 4.259 [+ or -] 3.084 NFS 30 170 SF 3.659 15.740 SI 3.529 13.60 SUMMER DORSAL L 64.51 67.242 [+ or -] 7.512 [+ or -] 9.222 W 12.67 13.48 [+ or -] 3.1320 [+ or -] 3.916 NFS 25 30 SF 4.464 25 SI 4.273 20 VENTRAL L 67.23 69.15 [+ or -] 6.452 [+ or -] 4.46 W 13.00 13.95 [+ or -] 3.001 [+ or -] 3.121 NFS 145 160 SF 19.333 22.222 SI 16.201 18.182 RAINY DORSAL L 59.867 67.86 [+ or -] 11.273 [+ or -] 12.480 W 13.97 12.21 [+ or -] 4.269 [+ or -] 4.837 NFS 25 40 SF 2.631 2.564 SI 2.564 2.00 VENTRAL L 75.09 83.58 [+ or -] 9.574 [+ or -] 13.294 W 14.37 18.2 [+ or -] 3.007 [+ or -] 3.282 NFS 135 140 SF 1.157 10.935 SI 10.037 9859 SITES Saman PTS SEASONS chowk Chowk WINTER DORSAL L 6589 58.02 [+ or -] 11.24 [+ or -] 6.451 W 14.5 12.22 [+ or -] 3.546 [+ or -] 5.123 NFS 20 20 SF 4 2.903 SI 3.846 2.821 VENTRAL L 6332 62.15 [+ or -] 7.55 [+ or -] 8456 W 1794 18.67 [+ or -] 4.894 [+ or -] 4.568 NFS 60 110 SF 7.407 14.102 SI 6.896 12.359 SUMMER DORSAL L 62.03 48.24 [+ or -] 7.571 [+ or -] 6.879 W 1298 11.89 [+ or -] 3.193 [+ or -] 2787 NFS 30 47 SF 2.727 5.341 SI 2.655 5.070 VENTRAL L 6828 63.456 [+ or -] 7.727 [+ or -] 7.46 W 13.78 10.33 [+ or -] 3.002 [+ or -] 3.521 NFS 150 160 SF 21.127 18.823 SI 17.442 15.841 RAINY DORSAL L 60.48 59.65 [+ or -] 6.789 [+ or -] 5.48 W 14.78 13.50 [+ or -] 5.456 [+ or -] 4.234 NFS 29 20 SF 3.258 2.705 SI 3.156 2.062 VENTRAL L 7789 50.49 [+ or -] 8.486 [+ or -] 7.685 W 1489 12.37 [+ or -] 4.56 [+ or -] 3.481 NFS 145 132 SF 11.837 10.688 SI 10.584 9.656 SITES Dhoba Tanki Civil SEASONS Lines WINTER DORSAL L 66.54 61.15 [+ or -] 13.604 [+ or -] 12.465 W 13.07 14.54 [+ or -] 1.871 [+ or -] 2.154 NFS 40 55 SF 5.00 6.548 SI 4.762 6.145 VENTRAL L 65.84 65.46 [+ or -] 14.157 [+ or -] 8.02 W 13.05 15.95 [+ or -] 3.345 [+ or -] 13.14 NFS 130 135 SF 13.131 15.517 SI 11.607 13.433 SUMMER DORSAL L 65.35 53.46 [+ or -] 5.781 [+ or -] 6.452 W 13.00 12.02 [+ or -] 4.632 [+ or -] 3.156 NFS 30 50 SF 3.158 5.464 SI 3.061 5.181 VENTRAL L 67.75 60.35 [+ or -] 6.482 [+ or -] 4.453 W 12.38 11.26 [+ or -] 3.45 [+ or -] 5.461 NFS 155 156 SF 19.645 15.757 SI 16.419 13.612 RAINY DORSAL L 59.56 61.45 [+ or -] 6.15 [+ or -] 9.79 W 13.08 15.53 [+ or -] 4.153 [+ or -] 3.478 NFS 30 35 SF 3.030 4.242 SI 2.941 4.070 VENTRAL L 79.86 58.78 [+ or -] 8.795 [+ or -] 9.78 W 14.89 14.9 [+ or -] 2.451 [+ or -] 3.846 NFS 138 134 SF 11.500 11.260 SI 10.314 10.121 SITES Jaistambh Nagar Nigam SEASONS chowk WINTER DORSAL L 63.38 76.245 [+ or -] 11.928 [+ or -] 10.488 W 14.37 14.89 [+ or -] 3.441 [+ or -] 3.480 NFS 40 45 SF 4.933 4.438 SI 4.706 4.249 VENTRAL L 63.53 62.45 [+ or -] 11.05 [+ or -] 10.46 W 18.44 16.45 [+ or -] 4.551 [+ or -] 4.346 NFS 140 115 SF 14.286 11.386 SI 12.50 10.222 SUMMER DORSAL L 67.542 63.468 [+ or -] 7.251 [+ or -] 5.780 W 13.23 12.45 [+ or -] 2.96 [+ or -] 2.461 NFS 30 43 SF 2.5 4.3 SI 2.439 4.122 VENTRAL L 68.46 66.57 [+ or -] 5.467 [+ or -] 5.461 W 13.85 13.01 [+ or -] 3.67 [+ or -] 4.43 NFS 140 148 SF 19.774 17.011 SI 17.327 14.538 RAINY DORSAL L 58.58 61.46 [+ or -] 9.051 [+ or -] 8.184 W 11.27 14.95 [+ or -] 2.326 [+ or -] 3.485 NFS 20 27 SF 2 2.903 SI 1.961 2.821 VENTRAL L 67.67 69.764 [+ or -] 10.855 [+ or -] 12.463 W 14.58 14.46 [+ or -] 2.508 [+ or -] 4.45 NFS 170 140 SF 20 14.141 SI 16.667 12.389 SITES Prakash Transport SEASONS chowk Nagar WINTER DORSAL L 73.12 55.36 [+ or -] 10.928 [+ or -] 9.656 W 16.78 12.56 [+ or -] 3.729 [+ or -] 4.098 NFS 50 40 SF 4.167 3.448 SI 4.000 3.333 VENTRAL L 6697 67.01 [+ or -] 8.941 [+ or -] 8.622 W 14 19.84 [+ or -] 3.383 [+ or -] 5.169 NFS 80 130 SF 7.692 17.105 SI 7.1429 14.607 SUMMER DORSAL L 62.00 48.89 [+ or -] 4.245 [+ or -] 7.546 W 12.06 11.23 [+ or -] 3.156 [+ or -] 3.15 NFS 24 18 SF 1.846 1.488 SI 1.813 1.464 VENTRAL L 62.48 61.45 [+ or -] 7.462 [+ or -] 5.451 W 12.48 11.46 [+ or -] 3.123 [+ or -] 3.124 NFS 156 165 SF 17.528 19.527 SI 14.914 16.337 RAINY DORSAL L 61.489 60 [+ or -] 10.79 [+ or -] 14.561 W 1521 13.84 [+ or -] 5.51 [+ or -] 3.542 NFS 30 24 SF 3.261 2.667 SI 3.158 2.597 VENTRAL L 67.75 51.01 [+ or -] 11.46 [+ or -] 13.592 W 13.46 12.46 [+ or -] 3.48 [+ or -] 4.453 NFS 135 133 SF 13.500 11.083 SI 11.894 9.977 SITES UTD SEASONS WINTER DORSAL L 59.17 [+ or -] 11.047 W 12.8 [+ or -] 2.06 NFS 75 SF 10.949 SI 9.868 VENTRAL L 64.02 [+ or -] 5.160 W 13.02 [+ or -] 3.625 NFS 170 SF 20 SI 16.667 SUMMER DORSAL L 50.24 [+ or -] 8.967 W 10.64 [+ or -] 1.449 NFS 70 SF 7.778 SI 7.216 VENTRAL L 57.6 [+ or -] 5.06 W 9.7 [+ or -] 1.703 NFS 185 SF 15.417 SI 13.357 RAINY DORSAL L 63.57 [+ or -] 8.949 W 16.74 [+ or -] 2.748 NFS 40 SF 5 SI 4.762 VENTRAL L 54 [+ or -] 8.540 W 14.3 [+ or -] 3.293 NFS 130 SF 11.077 SI 9.367 Table IV: Values of 't' test between size of guard cells of Bougainvillea spectabilis leaves of polluted and control sites. SITES Stadium Sirmaur Saman PTS Chowk Chowk Chowk Chowk SEASONS WINTER DORSAL LGC 1.454 1.524 1.348 0.284 P=0.1632 P=0.1449 P=0.1943 P=0.7794 WGC 2.223 * 3.409 ** 1.311 0.332 P=0.0393 P=0.0031 P=0.2064 P=0.7436 VENTRAL LGC 0.746 0.607 0.242 0.597 P=0.4653 P=0.5513 P=0.8115 P=0.5580 WGC 2.957 ** 1.495 2.555 3.064 ** P=0.0084 P=0.1522 0.0199 P=0.0067 SUMMER DORSAL LGC 3.858 ** 4.180 *** 3.177 ** 0.560 P=0.0012 P=0.0006 P=0.0052 P=0.5826 WGC 1.860 * 2.151 * 2.110 * 1.258 P=0.0793 P=0.0453 P=0.0491 P=0.2243 VENTRAL LGC 3.714 ** 5 415 *** 3.657 ** 2.054 * P=0.0016 P<0.0001 P=0.0018 P=0.0548 WGC 3.024 ** 3.780 ** 3.738 ** 0.509 P=0.0073 P=0.0014 P=0.0015 P=0.6167 RAINY DORSAL LGC 0.816 0.883 0.870 1.181 P=0.4265 P=0.8834 P=0.3958 P=0.2529 WGC 1.725 2.575 * 1.015 2.030 * P=0.1016 P=0.0191 P=0.3237 P=0.0574 VENTRAL LGC 5 198 *** 5.920 *** 6.275 *** 0.9661 P<0.0001 P<0.0001 P<0.0001 P=0.3468 WGC 0.050 2.653 * 0.332 1.274 P=0.9610 P=0.0127 P=0.7439 P=0.2190 SITES Dhobia Civil Jaistambh Tanki Lines Chowk SEASONS WINTER DORSAL LGC 1.330 0.3759 0.819 P=0.2002 P=0.7114 P=0.4236 WGC 0.307 1.846 * 1.238 P=0.7625 P=0.0814 P=0.2316 VENTRAL LGC 0.382 0.809 0.127 P=0.7070 P=0.4290 P=0.9003 WGC 0.192 0.678 2.946 ** P=0.9849 P=0.6797 P=0.0086 SUMMER DORSAL LGC 4.479 *** 0.922 4.745 *** P=0.0003 P=0.3688 P=0.0002 WGC 1.538 1.257 2.485 * P=0.1415 P=1.421 P=0.0230 VENTRAL LGC 3.903 ** 1.290 4.610 *** P=0.0010 P=0.2133 P=0.0002 WGC 2.203 * 0.862 3.244 ** P=0.0409 P=0.3998 P=0.0045 RAINY DORSAL LGC 1.168 0.505 1.240 P=0.2581 P=0.6194 P=0.2310 WGC 2.324 0.863 4.805 P=0.0320 P=.3994 P=0.0001 VENTRAL LGC 6.671 *** 1.164 3.130 ** P<0.0001 P=0.2595 P=0.0058 WGC 0.454 0.3747 0.2139 P=0.6549 P=0.7122 P=0.8330 SITES Nagar Prakash Transport Nigam Chowk Nagar SEASONS WINTER DORSAL LGC 3.545 ** 2.839 * 0.821 P = 0.0023 P=0.0109 P=0.4223 WGC 1.634 2.954 ** 0.165 P = 0.1196 P=0.0085 P=0.8704 VENTRAL LGC 0.426 0.904 0.941 P = 0.6754 P=0.3781 P=0.3592 WGC 1.917 * 0.625 3.416 ** P = 0.0713 P=0.5398 P=0.0031 SUMMER DORSAL LGC 3.921 ** 3.748 ** 0.364 P = 0.0010 P=0.0015 P=0.7199 WGC 2.004 * 1.293 0.538 P = 0.0603 P=0.2123 P=0.5971 VENTRAL LGC 3.810 ** 1.712 1.637 P = 0.0013 P=0.1041 P=0.1190 WGC 2.205 * 2.471 * 1.564 P = 0.0407 P=0.0237 P=0.1352 RAINY DORSAL LGC 0.550 0.469 0.660 P=5889 P=0.6444 P=0.5173 WGC 1.275 0.786 2.046 * P = 0.2184 P=0.4422 P=0.0557 VENTRAL LGC 3.300 ** 3.042 ** 0.589 P = 0.0040 P=0.0070 P=0.5632 WGC 0.09140 0.554 1.051 P = 0.9282 P=0.5861 P=0.3073 LGC = Length of guard cells WGC = Width of guard cell *** = extremely significant ** = Very significant * = Significant t' value at 18 d.f. on 0.05% level is 1.734 Table V: Average length ([micro]m), width ([micro]m) and number of epidermal cells of Bougainvillea spectabilis growing at different sites of Rewa city. SITES Stadium Sirmaur Chauk chauk SEASONS WINTER DORSAL L 87.78 81.76 [+ or -] 2.589 [+ or -] 13.064 W 58.75 51.48 [+ or -] 18.142 [+ or -] 9.644 NEC 550 990 VENTRAL L 65.6 76.03 [+ or -] 12.624 [+ or -] 17.354 W 45.64 45.73 [+ or -] 8.813 [+ or -] 7.330 NEC 820 1080 SUMMER DORSAL L 94.61 95.6 [+ or -] 11.632 [+ or -] 13.133 W 71.34 60.83 [+ or -] 6.554 [+ or -] 7.667 NEC 560 120 VENTRAL L 85.55 78.13 [+ or -] 10.48 [+ or -] 8.179 W 42.15 40.001 [+ or -] 6.157 [+ or -] 6.546 NEC 750 720 RAINY DORSAL L 100.99 94.55 [+ or -] 17.503 [+ or -] 20.960 W 79.36 58.28 [+ or -] 15.292 [+ or -] 7.109 NEC 950 1560 VENTRAL L 70.56 95.94 [+ or -] 20.421 [+ or -] 12.435 W 48.59 50.94 [+ or -] 11.247 [+ or -] 8.523 NEC 1210 1280 SITES Saman chauk PTS chauk SEASONS WINTER DORSAL L 84.55 90.01 [+ or -] 6.846 [+ or -] 15.32 W 56.2 61.42 [+ or -] 12.54 [+ or -] 10.654 NEC 500 689 VENTRAL L 64.49 75.12 [+ or -] 14.54 [+ or -] 12.512 W 44.68 48.68 [+ or -] 8.243 [+ or -] 8.117 NEC 810 780 SUMMER DORSAL L 96.61 88.45 [+ or -] 14.117 [+ or -] 8.46 W 72.23 60.31 [+ or -] 14.063 [+ or -] 5.452 NEC 1100 880 VENTRAL L 87.86 74.64 [+ or -] 14.015 [+ or -] 7.89 W 40.29 46.546 [+ or -] 7.401 [+ or -] 7.85 NEC 710 850 RAINY DORSAL L 99.79 97.46 [+ or -] 16.45 W [+ or -] 14.89 60.476 78.94 [+ or -] 14.5 NEC [+ or -] 6.486 950 890 VENTRAL L 71.59 68.987 [+ or -] 15.49 [+ or -] 16.458 W 47.46 48.08 [+ or -] 7.164 [+ or -] 8.146 NEC 1225 1235 SITES Dhoba Tanki Civil Lines SEASONS WINTER DORSAL L 98.35 98.01 [+ or -] 23.343 [+ or -] 20.45 W 61.77 61.544 [+ or -] 11.007 [+ or -] 12.346 NEC 800 840 VENTRAL L 91.77 87.56 [+ or -] 17.629 [+ or -] 12.004 W 56.92 55.413 [+ or -] 10.517 [+ or -] 9.153 NEC 990 870 SUMMER DORSAL L 95.34 88.79 [+ or -] 11.53 [+ or -] 13.96 W 65.12 59.06 [+ or -] 7.54 [+ or -] 7.24 NEC 950 915 VENTRAL L 79.456 83.79 [+ or -] 11.45 [+ or -] 4.56 W 42.5 50.45 [+ or -] 5.489 [+ or -] 5.79 NEC 789 990 RAINY DORSAL L 99.59 98.578 [+ or -] 4.95 [+ or -] 12.456 W 70.99 75.48 [+ or -] 15.78 [+ or -] 11.46 NEC 990 825 VENTRAL L 70.998 82.499 [+ or -] 14.879 [+ or -] 13.48 W 47.89 50.48 [+ or -] 6.789 [+ or -] 8.978 NEC 1200 1190 SITES Jastambh Nagar Ngam chauk SEASONS WINTER DORSAL L 88.61 85.94 [+ or -] 20.73 [+ or -] 12.548 W 68.53 60.132 [+ or -] 9.972 [+ or -] 8.456 NEC 810 1014 VENTRAL L 79.42 80.59 [+ or -] 13.993 [+ or -] 14.243 W 46.65 46.02 [+ or -] 10.286 [+ or -] 6.487 NEC 980 1010 SUMMER DORSAL L 96.61 93.25 [+ or -] 3.342 [+ or -] 5.642 W 73.68 71.34 [+ or -] 6.45 [+ or -] 4.58 NEC 1200 1000 VENTRAL L 78.53 80.67 [+ or -] 7.458 [+ or -] 5.468 W 39.97 44.56 [+ or -] 8.486 [+ or -] 4.792 NEC 708 870 RAINY DORSAL L 100.75 99.09 [+ or -] 12.542 [+ or -] 13.489 W 63.1 79.86 [+ or -] 16.081 [+ or -] 12.245 NEC 1000 930 VENTRAL L 80.17 80.45 [+ or -] 9.988 [+ or -] 12.489 W 51.36 52.4536 [+ or -] 12.257 [+ or -] 7.798 NEC 850 990 SITES Prakash Transport chauk nagar SEASONS WINTER DORSAL L 80.97 91.28 [+ or -] 12.209 [+ or -] 19.689 W 56.17 61.6 [+ or -] 12.851 [+ or -] 12.382 NEC 1200 1160 VENTRAL L 78.85 73.95 [+ or -] 12.091 [+ or -] 12.205 W 46.65 50.17 [+ or -] 10.286 [+ or -] 8.503 NEC 1040 760 SUMMER DORSAL L 92.12 90.45 [+ or -] 7.46 [+ or -] 9.43 W 70.76 59.89 [+ or -] 8.67 [+ or -] 6.43 NEC 1300 1210 VENTRAL L 79.97 81.48 [+ or -] 4.751 [+ or -] 9.465 W 48.45 45.54 [+ or -] 10.46 [+ or -] 6.481 NEC 890 845 RAINY DORSAL L 98.89 99.458 [+ or -] 11.463 [+ or -] 13.457 W 79.89 64.45 [+ or -] 15.45 [+ or -] 8.49 NEC 920 900 VENTRAL L 81.49 69.987 [+ or -] 10.468 [+ or -] 11.484 W 52.576 49.99 [+ or -] 10.479 [+ or -] 11.121 NEC 1000 1200 SITES UTD SEASONS WINTER DORSAL L 98.01 [+ or -] 19.622 W 73.43 [+ or -] 13.463 NEC 685 VENTRAL L 85.17 [+ or -] 34.703 W 56.42 [+ or -] 14.987 NEC 850 SUMMER DORSAL L 84.89 [+ or -] 10.534 W 57.46 [+ or -] 8.017 NEC 900 VENTRAL L 82.64 [+ or -] 10.45 W 56.87 [+ or -] 6.492 NEC 1200 RAINY DORSAL L 97.229 [+ or -] 22.720 W 80.83 [+ or -] 27.124 NEC 800 VENTRAL L 86.9 [+ or -] 13.087 W 58.53 [+ or -] 6.889 NEC 1180 Table VI: Values of 't' test between size of epidermal cells of Bougainvillea spectabilis leaves of polluted and control sites. SITES Stadium Sirmaur Saman Chowk Chowk Chowk SEASONS WINTER DORSAL LEC 1.634 2.180 * 2.048 * P=0.1195 P=0.0428 P=2.048 WEC 2.055 * 4.191 *** 2.961 ** P=0.0547 P=0.0005 P=0.0084 VENTRAL LEC 1.676 0. 7449 1.738 * P=0.1110 P=0.4659 P=0.0993 WEC 1.961 * 2. 026 * 2.171 * P=0.0656 P=0.0578 P=0.0436 SUMMER DORSAL LEC 1.959 2.012 * 2.104 * P=0.0658 P=0.0595 P=0.0497 WEC 4.239 *** 0.961 2.885 ** P=0.0005 P=0.3494 P=0. 0099 VENTRAL LEC 0.622 1.075 0.944 P=0.5419 P=0.2967 P=0.3576 WEC 5.203 *** 5.786 *** 5.326 *** P<0.0001 P<0.0001 P<0.0001 RAINY DORSAL LEC 0.415 0.274 0.298 P=0.6833 P=0.7872 P=7690 WEC 0.149 2.543 * 0.214 P=0.8830 P=0.0204 P=0. 8327 VENTRAL LEC 2.130 * 1.584 2.388 * P=0.0472 P=0.1307 P=0.0281 WEC 2. 383 * 2. 190 * 3.522 ** P=0.0284 P=0.0419 P=0.0024 SITES PTS Chowk Dhobia Tanki Civil Lines SEASONS WINTER DORSAL LEC 1.016 0.035 0.024 P=0.3230 P=0.9723 P=0.9807 WEC 2.212 * 2.120 * 1.019 P=0.0401 P=0.0481 P=0.3217 VENTRAL LEC 0. 865 0.536 0. 2058 P=0.4003 P=0.5984 P=0.8392 WEC 1.436 0.086 0.1813 P=0.1681 P=0.9321 P=0.85 81 SUMMER DORSAL LEC 0.833 2.116 * 0.705 P=0.4156 P=0.0485 P=0.4897 WEC 0. 929 2.201 * 0.468 P=0.3649 P=0.0410 P=0.6451 VENTRAL LEC 1.932 * 0.649 0.319 P=0.0693 P=0.5242 P=0.7534 WEC 3.205 ** 5 345 *** 2.334 * P=0.0049 P<0.0001 P=0.0314 RAINY DORSAL LEC 0. 026 0.321 0.165 P=0.9795 P=0.7518 P=0.8711 WEC 2.093 * 0.992 0.575 P=0.0508 P=0.3345 P=0.5727 VENTRAL LEC 2.694 * 2.538 * 0.741 P=0.0148 P=0. 0206 P=0.4684 WEC 3.098 ** 3 479 ** 2. 249 * P=0.0062 P=0.0027 P=0.0372 SITES Jaistambh Nagar Prakash Chowk Nigam Chowk SEASONS WINTER DORSAL LEC 1.041 1.639 2.332 * P=0.3115 P=0.1186 P=0.0315 WEC 0.925 2.645 * 2.933 ** P=0.3673 P=0.0165 P=0.0089 VENTRAL LEC 0.4859 0.386 0.544 P=0.6329 P=0.7040 P=0.5932 WEC 1.700 2.014 * 1.700 P=0.1064 P=0.0592 P=0.1064 SUMMER DORSAL LEC 3.354 ** 2.212 * 1.771 P=0.0035 P=0.0401 P=0.0935 WEC 4.985 *** 4.754 *** 3.562 ** P<0.0001 P=0.0002 P=0.0022 VENTRAL LEC 1.012 0.528 0.735 P=0.3248 P=0.6038 P=0.4715 WEC 5.002 *** 4.824 *** 2.163 * P<0.0001 P<0.0001 P=0.0443 RAINY DORSAL LEC 0.429 0.223 0.206 P=0.6730 P=0.8263 P=0.8388 WEC 1.778 * 0.1031 0.095 P=0.0923 P=0.9215 P=0.9252 VENTRAL LEC 1.293 1.128 1.021 P=0.2125 P=0.2743 P=0.3209 WEC 1.613 1.847 * 1.501 P=0.1242 P=0.0811 P=0.1506 SITES Transport Nagar SEASONS WINTER DORSAL LEC 0.766 P=0.4538 WEC 2.045 * P=0.0557 VENTRAL LEC 0.964 P=0.3476 WEC 1.147 P=1.147 SUMMER DORSAL LEC 1.224 P=0.2296 WEC 0.748 P=0.4643 VENTRAL LEC 0.260 P=0.7977 WEC 3.906 ** P=0.0010 RAINY DORSAL LEC 0.267 P=0.7926 WEC 1.822 * P=0.085 VENTRAL LEC 3.072 ** P=0.0066 WEC 2.064 * P=0.0537 LEC=Length of epidermal cells WEC=Width of epidermal cell *** = extremely significant ** = Very significant * = Significant t' value at 18 d.f. on 0.05% level is 1.734 Table VII: Values of Correlation coefficient (r) between Pb content and numbers of stomata and epidermal cells of Bougainvillea spectabilis (Willd.) leaves. Stomata Epidermal cells Dorsal Ventral Dorsal Ventral Winter -0.064 0.512 0.0384 0.584 Summer -0.131 -0.224 -0.422 -0.588 Rainy 0.294 0.655 * 0.783 * -0.161 Table VIII: Values of Correlation coefficient (r) between Pb content and size of guard cells and epidermal cells of Bougainvillea spectabilis (Willd.) leaves. Guard cells Epidermal cells Dorsal surface Ventral surface Dorsal surface Length Width Length Width Length Width Winter 0.04791 0.4723 0.4921 -0.0944 -0.3604 -0.1781 Summer 0.5623 0.7162 0.5920 0.6176 0.5602 0.05328 Rainy 0.5106 -0.7810 ** 0.4457 0.7335 * -0.4125 -0.5928 Epidermal cells Ventral surface Length Width Winter -0.0024 -0.3278 Summer -0.3311 -0.6265 * Rainy 0.6938 * 0.2382 * Significant at 0.05 % (8 d. f.).