Harmful effects of stone crushing dust pollution on pomegranate (Punica granatum L.) fruit plant

24 Bazai et al. Int. J. Biosci. 2020 RESEARCH PAPER OPEN ACCESS Harmful effects of stone crushing dust pollution on pomegranate ( Punica granatum L.) fruit plant Muhammad Nasir Bazai1, Saadullah Khan Leghari1*, Saeed-Ur-Rahman Kakar1, Saeed Ahmad Mughal1, Manzoor Iqbal Khatak2, Mahjabeen Tafuzal1 1Department of Botany University of Balochistan, Quetta, Pakistan 2Department of Chemistry University of Balochistan Quetta, Pakistan Key words: Stone dust, Punica granatum L., Physico-morphological contents. http://dx.doi.org/10.12692/ijb/17.1.24-34 Article published on July 17, 2020 Abstract This research was carried out on Punicagranitum L. grown near Abbaseen stone crushing plant at Kuchlak District Quetta of Balochistan, Pakistan. The investigation was aimed to understand the adverse effects of stone crushing dust pollution on morphological, physiological, growth, fruit quality and productivity of Punica granatum L.. The plants leaves and fruits were collected from three polluted sites with the distance of 200, 400 and 600 meters away from the stone crushing units respectively. Whereas a locality of 5000 meters away from stone crushing location comparatively clean air considered as a control sampling. Results showed that the dust load on the leaf surface was reported (1.16, 0.84, 0.62 and 0.13µg/cm2) at location 200, 400, 600 and 5000 m sites respectively. The site nearest (200 m) to the stone crushing units, contained highest dust amount showed significant destructive effects on the leaf physio-morphological characteristics, fruit yield and quality characteristics of Punica granatum L. The consequences of this study also revealed that a fine aerosol of stone dust is emitted from the crushing units has significantly effects on plant height (110.6-144 cm), Stem size (18.4224.06cm) and tree canopy (108.6-142.0 cm2). Petiole length, leaf length, breadth and Leaf area near to the stone crushing units was found; 0.3-0.6 cm, 5.3-6.6 cm, 1.9-2.6cm and 6.2-7.2cm2. Leaf relative water content (41.8375.23%) and Fruit Moisture Content (58.21-77.43%) was also noted significantly less at polluted site. More number of closed stomata/ cm2, decrease in photosynthetic rate and transpiration rate and stomatal conductance with an enhanced level of sub stomatal CO2 concentration was also reported near the crushing unit with respect to the control site. Results also showed less chlorophyll contents at polluted site. Number of fruits/tree and Fruit Size was also noted significantly low as compared to control site (5000 m). From the result it was concluded that the distance is directly proportional to the growth, yield, physiological and morphological characteristics of this plant. * Corresponding Author: Saadullah Khan Leghari  drsaadulla[email protected] International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 17, No. 1, p. 24-34, 2020 25 Bazai et al. Int. J. Biosci. 2020 Introduction An important constituent related to groundwork expansion is the usage of crushed rock (e.g., for building and road construction).Through screening and crushing processes, the consequential dust is a key environmental air impurity that can have significant effects on nearby vegetation and wildlife (Leghari et al., 2019a). Raina et al. (2008) reported that the Dust released from the crushing plants is hazardous to surrounding vegetation and by falling on the plant leaves cause injuries. The effects include a change in soil productivity and pH, decreased visibility in the neighboring areas, increased number of people with chronic respiratory illnesses and allergies, and degradation of natural habitats and resources, such as economic crops (Semban and Chandrasekhar, 2000; Das and Nandi, 2002; Mishra,2004; Sivacoumar et al., 2006). During the relatively simple stone crushing and screening process, largestones are broken into small pieces and sorted into different sizes which produce a large amount of dust that drifts over surrounding areas (Ravindran, 2013). This airborne dust falls onto the surrounding environment including plant leaves and people causing those adverse effects (Anon, 1999). Dust particulates accumulate on leaf surfaces causing a reduction in photosynthesis and stomatal activities and other foliar damages, all of which leads to a reduction in crop yield. A significant reduction on plant height, cover, number of leaves, and total chlorophyll content for Vitis vinifera L. was observed by Leghari et al., (2014) due to roadside dust accumulation. Wallenborn et al., (2009) found that stone crusher units add a significant amount of dust in the environment. Numerous investigations have revealed that the particulate matters settled down on the surface of the leaves of the plants due to gravitation and cause deterioration effects on the vegetation (Chatter, 1991). The dust is composed of homogenous mixtures as oxides of K, Ca, Al, N and S. (Raina et al., 2008). It is important to monitor the effects of dust on vegetation. (Saini et al., 2011). The accumulation of the dust on plants causes dangerous effects on the growth, yield, morphological and physiological features of the commercially valuable plants (Mughal et al., 2018). Significant reductions have been observed in the biomass of Apricots (Prunus armeniaca L.) fruits/tree, number of branches/tree and number of fruits/branch in stone dust pollution locations than those of the control sites. The results of this study were also revealed that the stones dust decreases the proximate and essential elements constituents at a considerable level (Leghari et al., 2018). Yield of plants is decreased due to dust fall on leaves which leads to decrease photosynthesis, respiration and transpiration (Raina et al., 2008; Saini et al., 2011). Leghari et al., (2019a) assess the effects of stone crushing dust pollution on three commonly cultivated fruit plant species (Vitis vinifera L., Morus alba L., and Prunus armeniaca L.) and on the health of workers working at crushing plants and found that the stone crushing units had high percentages of closed stomata both on the upper sides (Us) and lower sides (Ls) of leaves at 500-m distance from stone crushing installations. Results also showed that stone crushing workers suffered from symptoms of respiratory diseases (82.17%), allergies (72.13%), headaches (75.09%), coughing (78.36%), and tiredness (92.31%). Plants are an integral part of life in many indigenous communities. A great attention has been given in developed countries about the effects of dust and metal toxicities on germination, growth of plant and their productivities. Stone dust and metal toxicity is an important factor governing germination of seeds, growth and productivity of plants. Therefore, the main objective of the study was to determine the effect of stone dust onPomegranate (Punica granatum L.)Fruit plants locally grown nearby the stone crushing components in Kuchlakh district Quetta Balochistan, Pakistan. Materials and methods Study site and plant selection The study was conducted in Kuchlakharea district Quetta, Balochistan during 2019. Kuchlakh area is situated at latitude 30°19'31"N and longitude 66°56'33"E (Fig.1). It receives 212mm rain fall per annum with annual average temperature remain 15.8 26 Bazai et al. Int. J. Biosci. 2020 οC The altitude of the study area is about 1680 meters above sea level. Three sites (200 m, 400 m 600 m) near stone crushing units were selected for plant material and considered as polluted sites and comparatively less polluted site 5000 meter away from crushing unit was designated control site where the dust load was found zero. An orchard of Pomegranate (Punica granatum L.) fruit plants were selected to investigate the effects stone dust which were locally grown nearby the stone crushing componentsin Kuchlakh district Quetta Balochistan, Pakistan and these fruit plants (Pomegranate)were available at all four sites equally. In addition to that all the study sits had same physio-ecological conditions. Collection of plant material Mature areal parts (Leaf and fruits) of Punica granatum L.were collected from randomly selected 15 plants. All the plant samples were taken at 2-3m height from all four sides of the sampled trees at each study site as the methods used by Mughal et al., (2018); Leghari et al., (2018). Then these samples were brought to the laboratory in an ice can carefully for further analysis. Leaf morphological characteristics Leaf morphological characteristics as leaf length (cm) breadth (cm) and petiole length (cm) was determined by using ruler and leaf area (cm2) were determined byleaf Area Meter as described by Sahaand Padhy (2012); Leghari and Zaidi, (2013). Measurements of dust fall on leaf surface For the measurement of dust fall on the leaf surface of selected plant was measured from particular leaves marked at the lower branches. The leaf surface was cleaned nicely by wet cotton. After 15 days marked leaves were taken and each of them was kept in pre weighed (b) poly packs. The weight of the leaf along with pack were noted (a).Then leaves were nicely washed with tap water in the laboratory of Botany University of Balochistan Quetta and were dried at room temperature. Clean foliage were then weighted (c). Deposited dust on leaf surface were calculated with the help of formula [a-(b + c)]. After the interval of 15 days dust fall on leaves were measured during the whole study period then average were calculated and expressed as μg/cm2 and samples were taken three time during while study (Gamiand Parel, 2015); Prusty et al.,2005). Yield and quality determination Fruit plant yield was determined by fruit diameter and through counting the number of mature fruits at time of harvesting and sizes of fruits were measured by Vernier caliper as the method used by Olszyk, (1989); Mughal et al., (2018). Fruit quality was determined by measuring fruit size, fruit moisture content % (FMC), fruit grain size, fruit grain taste and fruit grain colours as method described by Leghari et al., (2019b); Prajapati and Tripathi, (2008) after some modifications. Growth parameters For the determination of growth parameters plant height (cm), stem diameter (cm) and Canopy (cm2) were measured. The plant height was noted by using Clinometer, stem diameter was measured by measuring tab and the canopy measured by using following formula: Tree canopy = A x B (Eq. 1) Where A is the east to west cover and B is the north to south cover and the cover was noted by the use of measuring tape as used by Mughal et al., (2018). Leaf and fruit moisture content Leaf and fruit moisture content was calculated by using the following formula as described by A.O.A.C (2005). (Eq. 2) Leaf stomatal study Leaves stomata was studied by method recommended by Salisbury, (1927) and reviewed by Radoglouand Jarvis, (1990). Imprints of upper (adaxial) and lower (abaxial) epidermis were isolated from central vein at 27 Bazai et al. Int. J. Biosci. 2020 “maximum leaf width. Colorless nail polish and adhesive transparent tape was used for impression. Replica imprints isolated from the leaf epidermis were observed by using a microscope (Ernst Leitz Gmbh Wetzlar, Type 20-446.023, Germany) at various magnifications (*50, *125 or *1250) for the studying of stomata. Minimum 5 microscopic regions was haphazardly chosen for each replica. Entire numbers of closed stomata/mm2 were counted under the microscope and mean %age of 20 observations from each species was calculated. Photosynthetic pigments The chlorophyll contents (Chl. “a”, “b” and Total Chl.) was determined through Ultra-violent spectrophotometer and concentration was calculated as method used by Arnon, (1949) and carotenoid contents was noted as noted by Davis, (1976). Gaseous exchange characteristics measurement Few parameters of gaseous exchange i.e. rate of photosynthesis (A) stomatal conductance” (gs), rate of transpiration (E) and sub-stomatal carbon dioxide concentration (Ci) were observed from younger and expanded foliage by utilizing LCA-4 ADC movable infra-red gas analyzer (IRGA). All the measurements were taken during 10:00 am - 2:00pm. Result and discussion Effects on growth characteristics Data depicted in the Table 1 revealed that the plant height (110.6, 120.4, 144 and 313 cm), Stem dimeter size (18.42, 20.19, 24.06 and 36 cm) and tree canopy (108.6, 119.2, 142.0 and 217 cm2) varied significant at all four locations 200 m 400 m600m and 5000 m. This variation is directly proportional to the distance from the crushing units. Similar observation was also reported by Leghari et al., (2019a) they found significant effects of stone crushing dust pollution on three fruit plant species (Vitis vinifera L., Morus alba L., and Prunus armeniaca L.) cultivated near the stone crushing units at three different distance sites (500, 1000, and 1500 m). Similarly Mughal et al., (2018) reported that the road side dust pollution causes reduction in plants height, stem size and tree canopy. A decline in tree cover of VitisveniferaL was also observed by Leghari et al., (2014) near road side plantation. Table 1. Summary of pair sample t-test at 95% Confidence level for the variation in Plant growth characteristics. Parameters Distance from stone crusher 200m 400m (600m) 5000 m (Control) Plant Height(cm) Mean T value Sig. (2 tailed) 110.6±3.2 24.32 0.001 120.4±8.9 16.22 0.002 144±11 4.3 0.012 313±1.5 Stem Size(cm) Mean T value Sig. (2 tailed) 18.42±1.04 18.22 0.002 20.19±1.67 9.33 0.003 24.06±2.06 3.2 0.005 36±2.1 Tree Canopy(cm2) Mean T value Sig. (2 tailed) 108.6±6.26 40.22 0.002 119.2±10.03 33.2 0.012 142.0±11.94 5.34 0.018 217±4.2 Leaf morphological characteristics and dust accumulation The leaf morphological characteristics are directly proportional to the distance from the crushing plant. Data illustrated in Table 2 exposed that the petiole length (0.3, 0.42, 0.6 and 0.8 cm), leaf length (5.3, 5.7, 6.6 and 7.3cm), Leaf breadth (1.9, 2.3, 2.6 and 2.9cm), Leaf area (6.2, 6.9, 7.2 and 9.6cm2) have significantly different at all four sites 200, 400, 600 and 5000m significantly. These result resemble with the findings of Pyatt and Haywood, (1989); Leghari et al., (2019b) and Mughal et al., (2018). Whereas, the dust load on plant leaf surface was (1.16, 0.84, 0.62 and 0.13µg/cm2) which is inversely proportional to 28 Bazai et al. Int. J. Biosci. 2020 the distance from the crusher. Similar findings have been reported by Prajapati and Tripathi, (2008); Leghari et al., (2019a); Shafiqet al., (2009) and Preeti, (2000). Various plants have shown decrease in leaf length and breadth in highly polluted areas as reported by Leghari and Zaidi, (2013). Other researchers Bahttiand Iqbal, (1988); Gupta and Ghous, (1988); Iqbal and Shafiq, (1999); Shafiqand Iqbal, (2003). Dineva, (2004) have also reported that the dust deposition causes decrease in leaf length, breadth and petiole length. Table 2. Summary of pair sample t-test at 95% Confidence level for variation in leaf morphological characteristics and dust load on leaf area. Parameters Distance from stone crusher 200m 400m 600m 5000 m (Control) Petiole Length (cm) Mean T value Sig (2 tailed) 0.3±0.01 19.1 0.003 0.42±0.02 12.22 0.006 0.6±0.02 4.6 0.034 0.8±0.2 Leaf Length (cm) Mean T value Sig (2 tailed) 5.3±0.28 22.2 0.002 5.7±0.34 18.23 0.006 6.6±0.46 8.21 0.009 7.3±0.01 Leaf Breadth (cm) Mean T value Sig (2 tailed) 1.9±0.04 18.2 0.003 2.3±0.05 13.2 0.005 2.6±0.11 7.44 0.013 2.9±0.2 Leaf Area cm2 Mean t value Sig (2 tailed) 6.2±0.18 22.1 0.002 6.9±0.19 16.4 0.005 7.2±0.33 8.2 0.021 9.6±1.5 Leaf Dust Content µg/cm2 Mean t value Sig (2 tailed) 1.16±0.21 18.1 0.003 0.84±0.23 8.1 0.019 0.62±0.22 3.1 0.166 0.13±0.02 Fruit yield and quality The facts reported in Table 3 revealed that number of fruits/tree (29.64, 56.52, 87.2 and 149) and Fruit Size (5.14, 5.64, 6.73 and 9.32cm) was varied significantly at all four locations 200, 400, 600 and 5000m respectively. Similarly, Mughal et al., (2018) observed that the number of fruits/ plant and fruit size is increased as the dust deposition is decreased. The excessive influence of CO2 content and leaf injuries cause reduction in the yield and quality of fruits. A significant decrease was observed in fruit diameter of Malus pumila. Analogous findings in the biomass, number of fruit/tree, number of branches/tree, fresh weight of fruit and number of fruit/ branches in Prunus armeniaca L. due to stone crushing dust. Leghari et al., (2018). Furthermore, Adbel-Rehman and Ibrahim, (2012) reported decrease in number of fruits in Ficuscarica due to cement dust load. Table 3. Summary of pair sample T-test at 95% Confidence level for the variation in yield and fruit quality. Parameters Distance from stone crusher 200m 400m 600m 5000m Control) No of Fruits/ Tree Mean t value Sig (2 tailed) 29.64±3.23 9.33 0.004 56.52±11.64 7.2 0.016 87.2±14.3 3.35 0.034 149±2.32 Fruit Size Mean t value Sig (2 tailed) 5.14±0.25 20.4 0.002 5.64±0.41 15.22 0.011 6.73±0.51 4.3 0.041 9.32±1.3 29 Bazai et al. Int. J. Biosci. 2020 Leaf physiological characteristics The data given in Table 4 regarding leaf relative water content (75.23, 62.77, 56.54 and 41.83%) and fruit moisture content (58.21,63.36 and 77.43%) were significantly different at all four investigated sites (200, 400, 600 and 5000 m) respectively. Moreover, it was found that as the distance from the stone crushing increases the LRWC and FMC% also increases. Whereas the number of closed stomata/ cm2 (70.2, 50.88, 37.64 and 2.54) at location A, B, C and D in the leaves lower epidermis was decreased with respect to the distance. Table 4. Summary of pair sample T-test at 95% confidence level for the physiological characteristics. Parameters Distance from stone crusher 200m 400m 600m 5000m Control) Leaf relative water Content % Mean t value Sig (2 tailed) 75.23±14.54 22.4 0.002 62.99±15.12 10.3 0.018 56.54±14.03 6.8 0.033 41.83±1.34 Fruit moisture content % Mean t value Sig (2 tailed) 58.21±3.2 39.33 0.001 63.36±4.7 18.4 0.002 72.82±2.9 9.21 0.006 77.43±1.32 No; of closed stomata/cm2 Mean t value Sig (2 tailed) 70.2±12.79 24.2 0.001 50.88±13.60 12.11 0.009 37.64±13.09 2.66 0.018 2.54±0.92 Table 5. Summary of pair sample T-test at 95% confidence level for photosynthetic, transpiration rate and water use efficiency characteristics. Parameters Distance from stone crusher 200m 400m 600m 5000m Control Photosynthetic Rate µmole CO2 Mean t value Sig (2 tailed) 7.27±0.41 25.22 0.002 7.92±0.58 18.32 0.004 8.72±0.36 8.33 0.008 9.32±0.32 Transpiration Ratemmole H2O Mean t value Sig (2 tailed) 0.78±0.08 27.22 0.004 0.82±0.08 14.76 0.013 0.92±0.08 6.67 0.062 1.02±0.03 Water Use Efficiency µmole CO2/mmole H2O Mean t value Sig (2 tailed) 9.36±0.47 5.37 0.033 9.68±0.41 5.2 0.035 9.47±0.69 5.11 0.031 9.15±0.32 These results of this experimental research agrees with the reports of Leghari et al., (2011). They resulted that the relative moisture content increased at polluted site in Fraxinus xanthoxyliods and Vitus vinifera. Plants have shown variation in the RWC of from season to season and specie to specie Ogunkunle et al., (2015) and Saura-Mas and Lloret, (2007). Similarly, Paulsamy et al., (2000) reported that the increase in RWC may help the plant to maintain their physiological balance under dust pollution stress. The alteration number of opened and closed stomata cause damage in the leaf and this alteration in stomatal density may reduce the physiological processes in the plant as described by Leghari et al., (2015) during study plants near house hold waste burning sources in six plants. Larcher, (1995) reported that the oxides of sulphur, Nitrogen and toxic particulate matters pierce in the photosynthetic tissues through stomatal pores and clog them which further effects the physiological processes. 30 Bazai et al. Int. J. Biosci. 2020 Table 6. Summary of pair sample T-test at 95% confidence level for the plant leaf photosynthetic pigment Composition. Parameters Distance from stone crusher 200m 400m 600m 5000m (Control) Chlorophyll a (mg/g.f.wt) Mean t value Sig (2 tailed) 0.66±0.06 8.31 0.016 0.69±0.07 5.5 0.021 0.80±0.05 1.6 0.137 1.01±0.2 Chlorophyll b (mg/g.f.wt) Mean t value Sig (2 tailed) 0.33±0.03 9.2 0.02 0.37±0.03 6.1 0.028 0.42±0.01 3.2 0.000 0.49±0.01 Total Chlorophyll (mg/g.f.wt) Mean t value Sig (2 tailed) 0.99±0.06 8.2 0.014 1.06±0.10 5.1 0.103 1.22±0.05 1.2 0.211 1.5±0.3 Carotenoids (mg/g.f.wt) Mean t value Sig (2 tailed) 0.29±0.3 11.5 0.026 0.36±0.02 7.86 0.050 0.41±0.03 3.99 0.14 0.49±0.02 Gas exchange characteristics The findings in Table 5 illustrated that the photosynthetic rate (7.27, 7.92 8.92 and 9.32 µmole CO2) and transpiration rate (0.78, 0.82, 0.92 and 1.02mmole H2O) are significantly different in site 200, 400, 600 and 5000 m respectively. It is found that the photosynthetic rate and transpiration rate has increased as the distance has increased from the crushing plant. However, Water use Efficiency (9.36, 9.68, 9.47 and 9.15) at site 200, 400, 600 and 5000 m have not shown any significant alteration in plants at all sites. These findings are in the lines of the findings many researchers including Bhatti and Iqbal, (1988); Gupta and Ghous, (1988); Iqbal and Shafiq, (2000); Dineva, (2004); Mughal et al., (2018) have reported that dust particles penetrate in the leaf photosynthetic tissues and result decrease in the gaseous exchange and morphological characteristics. Fig. 1. Abbaseen stone crushing plant at Kuchlak Quetta. 31 Bazai et al. Int. J. Biosci. 2020 Photosynthetic pigment composition The consequences in Table 6 regarding Chlorophyll a at Site 200, 400, 600 and 5000 m were (0.66, 0.69, 0.80 and 1.01 mg/g.f.wt) chlorophyll b (0.33,0.37, 0.42 and 0.49 mg/g.f.wt) and Total Chlorophyll 0.99, 1.06, 1.22 and 1.5 mg/g.f.wt), carotenoid content (0.29, 0.36, 0.41 and 0.49 mg/g.f.wt) at location 200 m, 400m, 600m and 5000m respectively. It was observed during the study that the chlorophyll content has increased with respect to the decrease in dust load on the leaf surface. Similarly many researchers as Chauhan, (2010) found 43.36% reduction in chlorophyll a concentration in Ficus religiose. Leghari et al., (2011; 2014; 2015) found reduction in chlorophyll a, b in many plants. Missanjo et al., (2015); Saini et al., (2011) stated that it is due to decline in to phaeophtin by loss Mg ion. Moreover, decrease in carotenoid is due to pollutants from releasing sources. Sharma and Tripathi, (2009) reported that carotenoid is a photo protective agent in chloroplast. Fig. 2. Stone crushing dust on leaves and fruits Punica granitum L. This chemical protects the chloroplast machinery against photo oxidative destruction. Many plants have shown decrease in Chlorophyll a, b and carotenoids as Ficus religiose Mir et al., (2008), E. angustifolia L. Vitis vinifera L. 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