Heavy metal pollution from vehicular emissions and its phytomonitoring along two roads i.e. Pindi Bhattian to Kala Shah Kaku and Lahore to Gujranwala By NAILA HADAYAT M. Sc. M. Phil. (UAF) A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN BOTANY DEPARTMENT OF BOTANY, FACULTY OF SCIENCES UNIVERSITY OF AGRICULTURE, FAISALABAD, PAKISTAN 2018 Abstract The increase in heavy metal pollution along the roadside environment has become a major global concern recently. Therefore, current project was planned to monitor the level of heavy metals along the roads. For this study, five wild plant species (Calotropis procera, Cenchrus ciliaris, Cynodon dactylon, Nerium oleander and Parthenium hysterophorus) generally growing near the two roads [a segment of Motorway (M-2) from Pindi Bhattian to Kala Shah Kaku and a segment of Grand Trunk road (G.T. road) from Lahore to Gujranwala] in Punjab, Pakistan, were collected. The plant leaves and soil samples were collected from five sites along each road. The control samples of leaves and soil were collected at a distance of 50 meter away from roadside. The collected samples were analyzed during the four seasons of the year (2015-2016). Metals such as lead (Pb), cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) were examined in all the plant leaves and soil samples using Atomic Absorption Spectrophotometer (AAS). Some plant physiological parameters such as gas exchange characteristics [photosynthetic rate (A), stomatal conductance (gs), transpiration rate (E), sub- stomatal CO2 concentration (Ci) and water use efficiency (WUE)] were evaluated. Plant biochemical attributes such as the contents of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids), total free amino acids, total soluble proteins and total antioxidant activity were also determined. Significantly higher contents of the studied metals were detected in soil and plants along the roadside as compared to their control samples and they indicated clear spatio-temporal variations. The highest contamination of metals in both plants and soil samples was noted during summer, whereas, minimum was observed during winter. The contents of metals were found in the order: Cd < Pb < Ni < Cu < Zn. The higher contents of all the metals were noted along G.T. road as compared to M-2. However, Kala Shah Kaku site along M-2 and Muridke site along G.T. road appeared as the more polluted sites. The correlation between metal contents in soil/plants and traffic density was significantly positive for almost all the sites during all the seasons. Significant positive correlation between metal contents in soil and metal content in plants was also observed. The high contents of metals were also obtained in petrol, diesel, soot and used motor oil samples. Physiological parameters such as A, E and gs were significantly lower while Ci and WUE were higher in all the plant species along the roadsides. The contents of photosynthetic pigments and total soluble proteins were significantly lower whereas total antioxidant activity and total free amino acids were significantly higher in roadside plant species under metal stress. Among plant species, C. procera accumulated the maximum contents of Cd, Ni and Pb whereas, N. oleander had potential to accumulate high concentrations of Cu and Zn, hence, these plants can be suggested as the best choice as phytomonitors and/or phytoremediators of the metal pollution. CHAPTER 1 INTRODUCTION Environmental pollution due to rapid urbanization and industrialization has become a major concern for living organisms almost all over the world. Anthropogenic activities emit a number of pollutants in huge quantity into the environment (Kho et al., 2007; Sarala and Vidya, 2012). Among these pollutants, metals particularly pose very deleterious effects on biota (Islam et al., 2015; Maanan et al., 2015; Pons-Branchu et al., 2015). Although metals are natural constituents of soil (Hutton and Symon, 1986), anthropogenic activities such as industrial discharges, metalliferous mining and smelting, agricultural materials (pesticides and fertilizers) and waste disposal activities, as well as roadside traffic also emit large quantities (Pam et al., 2013; Qin et al., 2014; Cao et al., 2014; Zhao et al., 2014; Ngole-Jeme, 2016). Traffic emissions being the largest source of metal pollution along the roadside has become a burning topic for study worldwide (UNEP/GPA, 2004; Modrzewska and Wyszkowski, 2014). It has been observed that several factors affect the emission of metals from vehicular sources. However, the quantity of metals emitted depends upon age of vehicle and quality of its maintenance, type of fuel (e.g. natural gas, diesel and gasoline), type of tire (e.g. friction or studded tires), quality of road infrastructure, road condition, implementations of assessment, maintenance and other emission control programs (Nirjar et al., 2002; EEA, 2011) as well as traffic density on road (Aslam et al., 2013; Rolli and Gadi, 2015). Therefore, a progressive degradation in roadside environment due to rapid urbanization, increase in number of motor vehicles, poor maintenance of vehicles, badly maintained roads and ineffective environmental regulations has become a global phenomenon (Joshi and Chauhan, 2008). The widespread use of automobiles for transportation, in the absence of enforcement of standards for pollution emission is resulting in release of huge amounts of metals besides other pollutants (Ibrahim, 2009; Irvine et al., 2009; Morton-Bermea et al., 2009) like oxides of nitrogen (NOX), carbon (CO, CO2) and sulphur (SOX) as well as hydrocarbons (Laschober et al., 2004). The unlimited population growth in Pakistan is accompanying rapid increase in number of vehicles and unchecked vehicular emissions. So the roadside environment in the entire country especially in the urban areas is plagued with metal pollution (Farrukh et al., 2005). As a consequence, Pakistan is included in the most polluted countries of the world NFEH report (2005). 1 Metals being non-biodegradable persist in the environment for long period of time (Babin-Fenske and Anand, 2011) and can pose many health hazards to both plants and animals (Wuana and Okieimen, 2011; Mathur and Kumar, 2013; Waoo et al., 2014; Mtunzi et al., 2015). Among metals, cadmium (Cd), nickel (Ni), copper (Cu), lead (Pb) and zinc (Zn) emitted by vehicular sources have been extensively examined along roadside environment. These metals have been found in petroleum products, tanks of fuel, engines and several other components of vehicles for example tires, catalytic converters, brake pads in addition to surface materials of roads (Zehetner, 2009; Popoola et al., 2012). Vehicular sector releases these metals during different operations such as from wearing and tearing of tires, mechanical abrasion of brake linings (Zhang et al., 2009; Ugwu et al., 2011; Raj and Ram, 2013), catalytic convertors (Zereini et al., 2007), abrasion of pavement, burning of fossil fuel in the internal combustion engines, leakage of oils, corrosion of batteries as well as metallic vehicle parts (Dolan et al., 2006; Aslam et al., 2013; Ghimire, 2015). Among the metals released by motor vehicles, Pb proves the major toxic element for biota along the roadside environment. It is mostly emitted from the exhaust pipes of the vehicles resulting after the combustion of fuel where it is deliberately added as an anti- knocking agent (Sheng and Peart, 2006; Suzuki et al., 2009; Atayese et al., 2009). Although, the use of leaded gasoline has been reduced in various countries of the world, however it is still being widely used in most developing countries like Pakistan (Parekh et al., 2002). Lead is used in some other products such as wheel weights (Root, 2000) and in yellow road paint (Adachi and Tainosho, 2004). Plants growing along road ways have relatively increased Pb content (Bu-Olayan and Thomas, 2002). Cadmium (Cd) is mostly emitted from consumption of lubricating oil, wearing of tires and the burning of fossil fuels (Suzuki et al., 2009; Chen et al., 2010). It has been listed as number 7 among the top 20 toxins (Yang et al., 2008) because it is highly mobile in soil and vegetation and is taken up readily by the root system (Ciecko et al., 2001; Renella et al., 2004; Popova, 2013), as a result disturbs the uptake of other elements (Ciecko et al., 2004, 2005). Nickel (Ni) in vehicular emission mainly comes from fluid leakage and wearing of engine. It is one of the most important metal pollutants because its concentration is rapidly increasing in the environment which adversely affects plant growth and development (Faryal et al., 2007; Atiq-ur-Rehman and Iqbal, 2008). 2 Zinc (Zn) is extensively emitted from tire abrasion, lubricating oil (Adachi and Tainosho, 2004) and galvanized parts of vehicles such as fuel tanks (Falahi-Ardakani, 1984). Abrasion of brake linings is a main source of Cu as well as Zn and Ni (Lough et al., 2005; Hjortenkrans et al., 2007). The wear and tear of various vehicular components is also a cause of Cu and Fe emission (Polkowska et al., 2001; Preciado and Li, 2006). Metals emitted from vehicles not only become deposited on the soil and vegetation along the roadside (Werkenthin et al., 2014) but may persist in the air for some time and can penetrate plants directly by dust or rain (Jozic et al., 2009). Plants growing near the roadsides show high concentrations of heavy metals as they are irreversibly incorporated into the cuticle of the plants (Corsmeier et al., 2005). The large amount of metals accumulated in the soil may also be transported to aerial parts of plants via roots (Gall and Rajakaruna, 2013; Nadgorska- Socha et al., 2013; Neilson and Rajakaruna, 2014; Bourioug et al., 2015). Although some metals (Zn, Ni, Fe, Cu) are essential micronutrients required by plants at low concentration for normal metabolic functions, growth and development (Dixon et al., 2004; Marschner, 2012) but at excess they become toxic (Seregin and Kozhevenikova, 2006; Chen et al., 2009; Chaffai and Koyama, 2011), cause metabolic abnormalities and growth reduction in plant species (Rengel, 2004; Sinha et al., 2005; Bragato et al., 2009).