2.1.

Arsenic in the environment

2.2. 2.3.

Arsenic and plants Arsenic toxicity

15 23

2.4.

Phytoremediation

27

rsenic, a silver-gray brittle crystalline solid represented by symbol As, has an atomic weight of 74.9216, atomic number of 33, specific gravity of 5.73, melting point of 817C (28 atm) and sublimes at 613C. It is 20th abundant element in the earths crust (NAS, 1977; ATSDR, 0031998). A notorious poisonous metalloid it exists in different allotropic forms that are yellow, black and grey (Adriano, 1969; Budavari et al., 1989). In the natural environment, arsenic is rarely encountered as a free element (Fig. 2.I). It usually occurs as a component of sulfiolic ores as metal arsenide (NAS, 1977). Arsenic exists in variable oxidation states in the environment i.e., -3, 0, +3 and +5 (Adriano, 1986; Welch et al., 1988). Under aerobic conditions arsenic exists as As (+5) whereas reducing environment is congenial for existence of elemental Arsenic (0), Arsenite (+3) and Arsine (-3) (Dukes et al., 2005). Arsenicals, both trivalent and pentavalent, are soluble over a wide pH range (Bell, 1998) and are found routinely in surface as well as ground water (Feng et al., 2001).

2.1. ARSENIC IN THE ENVIRONMENT

2.1.1. Geogenic Sources of Arsenic Arsenic is ubiquitous in nature and small amount of this element can be found in every environmental compartment. Most arsenic in the environment exists in rock or soil (ATSDR, 1998). Arsenic is a major constituent of more than 245 minerals. Arsenicsulphides (FeAsS), the most common mineral readily oxidize when exposed to air, yielding inorganic arsenic salts which are highly water-soluble (Woolson, 1973). A list of some of the most common As minerals is given in Table 2.1. The average arsenic content of igneous rocks is 2 to 3 mg/kg (up to 100 mg/kg) and varies from small

amounts in limestone and sandstone up to 15,000 mg/kg in some manganese ores (YanChu, 1994).

Figure 2.I. Arsenic compounds commonly detected in the environment (Abbreviations are in parenthesis) (Goessler and Kuehnelt, 2002).

Table: 2.1. Major arsenic minerals occurring in nature (Smedley and Kinniburgh, 2002). Mineral Native arsenic Niciolite Realgar Composition As NiAs AsS Occurrence Hydrothermal veins Vein deposits and norites Vein deposits, often associated with orpiment, clays and limestones, also deposits from hot springs. Hydrothermal veins, hot springs, volcanic sublimation products. High-temperature deposits, metamorphic rocks. The most abundant As mineral, dominantly in mineral veins. Hydrothermal veins. Hydrothermal veins. Secondary mineral formed by oxidation of arsenopyrite, native arsenic and other As minerals. Secondary mineral formed by oxidation of realgar, arsenopyrite and other As minerals. Secondary mineral
2

Orpiment

As S

2 3

Cobaltite Arsenopyrite

CoAsS FeAsS

Tennantite Enargite Arsenolite

(Cu, Fe) As S
12

4 13

Cu AsS
3

As O
2

Claudetite

As O
2

Scorodite Annabergite Hoernesite Haematilite Conichalcite Pharmacosiderite

FeAsO .2H O
4 2

(Ni,Co) (AsO ) .8H O

3 4 2

Secondary mineral Secondary mineral, smelter wastes.

8

Mg (AsO ) .8H O
3 4 2 2

(Mn,Mg) Al(AsO )(OH)

4 4

CaCu(AsO )(OH)
4

Secondary mineral
2

Fe (AsO ) (OH) .5H O

3 4 2 3

Oxidation product of arsenopyrite and other As minerals.

Arsenic concentrations in soil depend in part on the parent materials from which the soils were derived, although they may be enriched by other sources. Natural concentrations of arsenic in the earths crust vary, but average concentrations are generally reported to range from 1.5 to 5 mg/kg (ATSDR, 1998; Cullen and Reimer, 1989; NAS, 1977). The level of arsenic in soil derived from basalts tends to be higher than in soils of gigantic origin and concentrations of 20 to 30 mg/kg may be found in the soils derived from sedimentary rocks (Yan-Chu, 1994). The uncontaminated soils contain between 0.1 and 40 mg As/kg (average 5 to 6 mg/ kg) (Colburn et al., 1975; O'Neill, 1995). Soils overlying sulphide ore deposits may have concentrations up to 8'000 mg As/kg (Chilvers and Peterson, 1987). Tamaki and Frankenburgur (1992) have suggested that natural arsenic emission exceeds industrial emissions while other studies maintain that industrial emissions of arsenic are significantly greater than natural emissions (Loebenstein, 1994; Pacyna et al., 1995). 2.1.2. Anthropogenic Sources Arsenic, naturally present in most lead, copper, and gold ores, and is released during the smelting through gaseous and solid waste emission (Woolson, 1973) and is accumulated in the soils around the emission source (Peterson et al., 1981). Another important source of As emission into the atmosphere is coal-burning during electrical power production and heating. Arsenic exists largely as arsenopyrite in coal and is emitted as arsenic trioxide from power plants. Arsenic concentrations in coal from the USA, Australia and the UK range from around 0.5 to 93 mg As/kg and fly ash particles can contain up to 1'700 mg As/ kg (Peterson et al., 1981). Brown coal (from the Czechoslovakia) was found to contain up to 1'500 mg As kg"1 (Piver, 1983). Coal from Coalfield, Northeastern India has been reported to contain 3.27 mg/kg of As with a maximum value of 40 mg/kg from (Khandelar et al., 1999; Warwick et al., 2001). Arsenic and its compounds find a variety of industrial applications. Arsenic metal is used in the lead-acid storage batteries and formation of some copper alloys (Reese, 1998). Smelting activities generate the single largest source of anthropogenic arsenic input into the atmosphere (Chilvers and Peterson, 1987).

Arsenic is added into the environment by burning of fuels and wastes, mining and smelting, pulp and paper production, glass manufacturing, cement manufacturing, paint industry, detergent production (USEPA, 1998 b; Ning, 2002; Goh and Lim, 2005; Pierzynski et al., 2005; Pichler et al., 2001; Moldovan et al., 2003; CCME, 2001; IPCS, 2001; ATSDR, 2007; Kabata-Pendias and Mukherjee, 2007). Agricultural uses of arsenic and arsenic compounds include pesticides, herbicides, insecticides, defoliants and soil sterilants (Azuce and Nriagu, 1994; EPA, 2000). Pesticides are the major sources of As in agricultural soils (Merry et al., 1986; Peterson et al., 1981; Woolson et al., 1971a; Jiang and Singh, 1994). Dichlorodiphenyltrichloroethane (DDT), lead arsenate (PbAs04), calcium arsenate (CaAs04), magnesium arsenate (MgAs04), zinc arsenate (ZnAs04), and Paris green [Cu(CH3COO)2*3Cu(As02)2] are used extensively as pesticides in agriculture (Anastasia and Kender, 1973; Merry et al., 1983). Excessive use of arsenic compounds as insecticides and defoliant has been known to increase its concentration in the respective soils (Steevens et al., 1972; Hess and Blanchar, 1977; Maclean and Langille, 1981; Davenport and Peryea, 1991; EPA, 2000). The leaching of arsenic from agricultural topsoil to subsoil has been reported by NAS (1977) and Peryea (1991). Due to the essential role of As in animal nutrition, organic arsenicals play an important role as food additives to promote the growth of farm animals (Christen, 2001). Organic arsenicals are frequently used as desiccants and defoliants in the cotton industry and for weed control (Woolson, 1975). Despite immense controversy arsenic is still used as an ingredient of wood preservatives, for debarking trees, in cattle and sheep dips and in aquatic weed control (Azcue and Nriagu, 1994). 2.1.3. Arsenic Pollution More than thirty countries all over the world (Table 2.2, Fig. 2.II) have been reported to be affected due to presence of excessive As in their environment (Matschullat, 2000; Saad et al., 2001; Gurzao, 2001; Berg et al., 2001; Rahman et al., 2001; Smedley, 2001, 2002; Tun, 2003; Pfeifer and Zobrist, 2002; Chakraborti et al., 2002, 2003; Sengupta et al., 2003; Mosaferi et al., 2003, Shreshtha et al., 2003; Saltori, 2004; Islam et al., 2004; Nickson et al., 2005; Stagner et al., 2005; Olias et al., 2006).

The arsenic contaminant has reached mammoth levels in many countries namely, Afghanistan, Bangladesh, Cambodia, China, India, Lao PDR, Mongolia, Myanmar, Nepal, Pakistan, Thailand and Vietnam (ENVIS, 2009). The limit for As in drinking water, set by the WHO, was 50 g As/L (WHO, 1993) which was later revised to 10 g As/ L (WHO, 2005). In India, the presence of elevated levels of As in groundwater has been confirmed in states of West Bengal, Bihar, Uttar Pradesh, Jharkhand, the seven north eastern states, Andhra Pradesh, parts of Chattisgarh, Haryana, Himachal Pradesh and Punjab (Garai et al., 1984; Varsanyi, 1989; Rahman et al., 2001; Mukherjee et al., 2003; Chowdhury et al., 2001; Datta and Kaul, 1976; Chakraborti et al., 1998, 1999, 2003; Pandey et al., 1999; Guha et al., 1992; Kishan, 2001; Dhar et al., 2001; Gurundha et al., 2001; Govil et al., 2001; Chandra et al., 2003; Mosaferi et al., 2003). Chatterjee and Banerjee (1999) reported the presence of As ranging from 17.500.52 mg/kg to 9740226 mg/kg in soil in vicinity of lead industry in Kolkata. Arsenic contamination was reported from Chandigarh and some villages of Punjab, Haryana and Himachal Pradesh (Datta and Kaul, 1976). Hundal et al., (2007, 2008) analysed water samples from different regions of Punjab and reported the presence of As ranging from 0.3 to 2.89 ppb in the water samples from various canals and the ground water of the arid south-western region of Punjab i.e., Sangrur, Mansa, Faridkot, Muktsar, Bathinda and Ferozepur was reported to contain 11 to 688 g/l As in water. Presence of As in most regions is geogenic in origin but in case of Talwandi Sabo block of Punjab, which is an important cotton producing region, the presence of As in soil and water is attributed to natural sources as well as contamination from agricultural inputs (ENVIS, 2009).

Fig 2.II: Arsenic-affected areas in the world (Source: British Geological Survey, 2001. http://www.bgs.ac.uk/.)

Table: 2.2. Arsenic affected areas round the world (Mukherjee et al. 2006).
Country Afghanistan Argentina Sources Natural Natural References Saltori, 2004 Arguello et al. 1938; Astolfi et al. 1981; De Sastre et al. 1992;Hopenhayn et al. 1996, 1998; Nicolle et al. 1989; Smedley et al. 1998, 2002; Vahter et al. 1995 Australia Bangladesh Anthropogenic Natural Hindwood et al. 1998; Smith et al. 2003; Tallis , 1989 Arnold et al. 1990; Chakraborti et al. 2004; Dhar et al. 1997; Ioanid et al. 1961;Sengupta et al. 2003 Brazil Anthropogenic Matschullat et al. 2000

Bulgaria

Anthropogenic

Nilsson et al. 1993

Canada

Natural and Anthropogenic

Boyle et al. 1998; Granthum et al. 1977; Meranger et al. 1999;Temple et al. 1977

Cambodia Chile

Natural Natural and Anthropogenic

Stagner et al. 2005 Borgono et al. 1971; Caceres et al. 1992; Ferreccio et al. 1996; Smith et al. 1998, 2000; Zaldivar , 1974; Zaldivar et al. 1978

PR China

Natural and Anthropogenic

Cao, 1996; Gao, 1997; Hotta, 1989;Lianfang et al. 1994; Sun et al. 2001; Xiau, 1997; Zheng and Long, 1994 Bencko and Symon, 1977

Czech Republic

Anthropogenic

Egypt Finland Germany

Natural Natural Anthropogenic

Saad and Hassanien, 2001 Kurttio et al. 1998 Butzengeiger, 1940; Luchtrath, 1972; Roth, 1957;1983; Wolf, 1974

Ghana

Natural

Bowell et al. 1992

Greece Hungary

Natural Natural

Yiannis, 2004 Egyedi and Pataky, 1978; Nagy et al. 1983; Pataky and Lusztig, 1958; Varsanyi, 1989

India

Natural and Anthropogenic

Akai et al. 2004; Chakraborti et al. 1998, 1999, 2001, 2002, 2003; Chandra et al. 2003; Chatterjee et al. 1993, 1999; Chowdhury et al. 1999, 2001; Das et al. 1996; Datta and Kaul , 1976; Datta, 1976; Dhar et al. 1998;Garai et al. 1984; Govil et al. 2001; Guha et al. 1992; Gurunadha et al. 2001; Harvey et al. 2002; Islam et al. 2004; Kishan, 2001; Nickson et al. 1998, 2000;Mukherjee et al. 2003; Pandey et al. 1999; Rahman et al. 2001;

Iran Japan

Natural Natural and Anthropogenic

Mosaferi et al. 2003 Horiguchi et al. 1977; Kondo et al. 1999; Tsuda et al. 1989, 1990;

Mexico

Natural and Anthropogenic

Armienta et al. 1997;Cebrian et al. 1983, 1994; Diaz et al. 1993; Del razo et al. 1990; Gomez et al. 1997; Tun et al. 2002 Shrestha et al. 2003; Tandukar et al. 2001 Grimmet and NcIntosh, 1939; Ritchie, 1961

Myanmar Nepal New Zealand Pakistan

Natural Natural Natural

Natural

Nickson et al. 2005

Romania Sri Lanka Spain Sweden Switzerland Taiwan China

Natural Natural Natural Anthropogenic Natural Natural

Gurzau and Gurzau, 2001 Senanayake et al. 1972 Olias et al. 2006 Aschengrau et al. 1989; Nordstrom et al. 1979 Pfeifer and Zobrist, 2002 Bates et al. 1992;Guo et al. 1997; Lu et al. 1957; Lu, 1990 Thornton and Farago, 1997; Tsai et al. 1998;Tseng et al. 1968; Wu et al. 1989;Yeh, 1963;

Thailand USA

Anthropogenic Natural and Anthropogenic

Choprapawon et al. 1977; Pavittranon et al. 2003; Oshikawa et al. 2001 Bates et al. 1995; Birmingham et al. 1965; Davis et al. 1994; Feinglass, 1973; Goldsmith et al. 1972; Harrington et al. 1978; Hawkins and Swain, 1907; Hoover et al. 1963; Karagas et al. 2002; Korte and Fernando, 1991; Lee and Fraumeni, 1969; Lewis et al. 1999; Matisoff et al. 1982; Milhan and Strong, 1974; Morton et al. 1976; Page, 1981; Peters et al. 1999; Pinto et al. 1953; Southwick et al. 1983; Viz et al. 1984; Wilson et al. 1978; Welch et al. 1982, 1999

United Kingdom

Anthropogenic

Farrago et al. 1997; Hill and Faning, 1948; Lloyd, 1978; Smith and Lloyd, 1986

2.2.

ARSENIC AND PLANTS

2.2.1. Arsenic Uptake The uptake of arsenic by wild as well as cultured plants has been reported by many workers (Woolson, 1973; Helegesen and Larsen, 1998; Asher and Reay, 1979; Sanders and Osman, 1985; Meharg and Macnair, 1991b; Onken and Hossner, 1995; Truong and Baker, 1998, Truong, 2000). Uptake of arsenic (between 0.035 to 18.53 mg/kg) by tolerant plants was reported by Casado et al., (2007) from three polluted mining soils (Cabacomine, Spain). Cistus monospeliensis (18.53 mg/kg) has the maximum arsenic content followed by Teesdalia nudicaulis (13.69 mg/kg As), Leontodon crispus (9.93 mg/kg As), Rumex crispus (4.66 mg/kg As), Hippocrepis comosa (4.47 mg/kg As), Lotus corniculatus (3.98 mg/kg As) and minimum arsenic content was reported in Erica arborea (0.035 mg/kg As). Uptake of arsenate (10 mg/L) by Hylocomium splendens (moss) was reported by Wells and Richardson (1985). Asher and Reay (1979) in his studies reported uptake of arsenate (1mg/L) in Hordeum vulgare (barley). Sanders and Osman (1985) reported uptake of arsenic by Spartina alterniflora. ptake of arsenic in plants of Vaccinium angustifolia (blue berry) accumulated 0.78-15 mg/kg in leaves, 0.27-13.3 mg/kg in stems and 2.4164.2 mg/kg in roots (Antasia and Kender ,1973). The uptake of arsenic has also been reported in Pteris vittata (Ma et al., 2001), Vaccinium angustifolium (Anastasia and Kender, 1973), Urtica dioica and Phragmites australis (Otte et al., 1990), Oryza sativa (Onken and Hossner, 1995), Tumbleweed (Jorge et al., 2006), Spirodela polyrhiza (Rahman et al., 2007), Isatis capadocica and Hesperin persica (Karimi et al., 2010). A pot experiment was conducted by Srisatit et al. (2003) on two ecotypes of vetiveria grasses, the arsenic accumulation in the leaves of V. zizanioides and V. nemoralis was 0.463 and 0.494 mg As/kg DW, respectively. The maximum arsenic accumulation in the roots (9.79 and 11.77 mg As/kg DW). Hyperaccumulation of arsenic: The term hyperaccumulators was first used for plants accumulating Ni and was later generalized to plants accumulating a metal more than

100-fold relative to its concentration in the soil (Reeves and Baker, 2000). First arsenic hyperaccumulate discovered was Pteris vittata L. (Ma et al., 2001); followed Pityrogramma calomelanos L. (Francesconi et al., 2002) and many other species of the Pteris genus such as P. cretica L., P. longifolia L., P. umbrosa L. and P. argyraea L. (Zhao et al., 2002) and P. quadriaurita L., P. ryiunkensis L. and P. biaurita (Srivastava et al., 2005). Some other plants growing on mine wastes from various sites in the United Kingdom (Porter and Peterson, 1977) and on smelter wastes in northeast Portugal (De Koe, 1994) have also been reported as arsenic hyperaccumulators (>1,000 mg/kg As). Ma et al. (2002) found that Pteris vittata (brake fern) accumulated 7,234 mg As/kg in the fronds. Francesconi et al. (2002) identified another arsenic hyperaccumulator, Pityrogramma calomelanos which accumulated up to 8,350 mg As/ kg (dry weight) in the fronds while the roots contained only 88-310 mg As/ kg. Zhao et al. (2002) reported high (27,000 mg As/kg dry wt.) accumulation of arsenic in Pteris vittata fronds in an 18-day hydroponic experiment. 2.2.2. Mechanism of Arsenic Uptake Arsenate, chemically very similar to phosphate, is thought to enter the root cell by the same uptake mechanism as phosphate in a variety of organisms (Asher and Reay, 1979; Meharg and Macnair, 1994; Schachtman et al., 1998). This mechanism seems to have, however, a lower affinity for As than for P (Asher and Reay, 1979). Phosphate is taken up as H2PO4 at low soil pH and at high pH as HPO4. Similarly arsenate is taken up at low pH as H2ASO4 and at high pH as HASO4 (Schachtman et al., 1998). The root system is the primary site of damages when As reaches phytotoxic levels. Compared to P, translocation of As to shoots is generally low. Typical As concentrations in aerial parts are < 2 mg As/ kg (O'Neill, 1995), and crop damage is usually expected before As reaches concentrations which are considered critical for human health (Peterson et al., 1981). The degree of As uptake varies widely from species to species (Woolson, 1975). Roots accumulate higher concentrations than stems, leaves or seeds. Uptake increases

with increasing arsenic concentration in the soil. While As concentrations generally remain below 1 mg /kg fresh weight in food crops, grasses were found to contain up to 3'460 mg As/kg (dry weight) grown on spoil with As concentrations up to 26'430 mg As/ kg. Grasses on urban soils containing 20 mg As/ kg were found to accumulate up to 3 mg As per kg dry weight (Porter and Peterson, 1975). 2.2.3. Factors Affecting Arsenic Uptake Elemental uptake and accumulation by plants is influenced by many factors including element species, growth medium and concentration of the element, pH, presence or absence of other elements and organisms as well as abiotic changes. Arsenic uptake and accumulation from soils by plants are influenced by such factors as plant species (Matschullat, 2000), soil arsenic concentration (Jiang and Singh, 1994), soil properties (Jiang and Singh, 1994; Matschullat, 2000), the presence of other ions (Khattak et al., 1991), exposure time and the age of the plants. Soil pH: pH of soil is the master variable controlling soil chemical processes and reactions (Mc Bride, 1994). Several scientists have also reported that arsenic distribution in soils is associated with pH (Akins and Lewis, 1976; Adriano, 2001; Bech et al., 1997). Akins and Lewis (1976) examined the effects of pH from (4 to 8) on arsenic sorption by soils using sequential fractionation procedure and found that at low pH (pH=4) Fe-As is the most abundant form followed by Al-As whereas at high pH (pH 6 to 8) Ca-As is the predominant form. Fayiga et al. (2007) reported a positive correlation between soil pH and Ca-As fraction in the soils after 8 weeks of plant growth. His data shows that ca-As fraction increased with increasing soil pH which is consistent. As concentrations: Many researchers have attempted to relate As concentrations in soil to plant growth. Total soil arsenic concentration is not a good predictor of As phytotoxicity when soils with widely differing properties are compared (Jacobs et al., 1970). According to Woolson et al. (1971a) the correlation between plant growth and water-soluble As was better than between plant growth and total As concentration in the

soil. Sadiq (1986) reported that growth of maize was significantly correlated with the water-extractable As but not with the total As concentrations in calcareous soils. Arsenic is generally considered phytotoxic and is expected to negatively affect plant growth (Kabata-Pendias and Pendias, 1991). As a result of many negative effects on plants, arsenic caused a reduced growth of plants. In Indian mustard, arsenic extraction by plants increased significantly with increasing arsenic concentrations in soil (Chaturvedi, 2006). The accumulation of inorganic arsenic species (arsenate and arsenite) in Spirodela polyrhiza from solutions treated with EDTA was higher than without EDTA (Rahman et al., 2007). Soil Microorganisms: Mycorrhiza has been reported in plants growing on heavy metalcontaminated soils (Shetty et al., 1994; Chaudry et al., 1998, 1999). These symbiotic associations have the potential to enhance root absorption area and stimulate the acquisition of plant nutrients including metal ions (Khan et al., 2000). Arbuscular mycorrhizal fungi are known to colonize ferns (Sharma, 1998) suggesting a possible role of mycorrhizal associations in the recently reported arsenic hyperaccumulation (Ma et al., 2001). Effect of Phosphate: Phosphate (PO4) is an analogue of As (V), making it an important factor in the behaviour of As in aerobic soils (Lambkin and Alloway, 2003; Mahimairaja et al., 2005; Williams et al., 2003). As (III) is not an analogue of PO4, making the presence of PO4 probably less relevant to As behaviour under flooded soil conditions (Takahashi et al., 2004). Physiological and electrophoresis experiments have shown that As (V) competes weekly with Pi for uptake (Asher and Reay, 1979; Esteban et al., 2003; Hurd-Karrer, 1939; Lee et al., 2003; Pickering et al., 2000; Ulrich-Eberius et al., 1989). Work with Arabidopsis mutants has confirmed that, Pi transporters also mediate As (V) uptake (Shin et al., 2004). Both synergistic and antagonistic effects of P on As uptake by plants have been reported (Woolson et al., 1973). Several nutrient culture studies have demonstrated that the amount of P relative to As (P/As ratio) influences plant growth (Hurd-Karrer, 1936).

In soil culture, several investigators noted reduction in phytotoxicity as P levels increased on a wide variety of crops (Benson, 1953; Woolson and Kearney, 1973). Woolson et al. (1973) reported that additions of 100 mg As/kg soil significantly increased corn growth at low P but decreased yield at high P levels in soil. Whether P additions to soils enhance or decrease As phytotoxicity will depend on soil as well as plant conditions (Otte et al., 1990; Clarkson and Lttge, 1991). 2.2.4. Phytotoxicity of Arsenic Toxicity of As in plants is well documented. Injury symptoms from higher quantities of arsenic in soil were reported by Thompson and Batjer (1950) who recognized this metalloid as the reason for necrotic spots, discoloration of leaves and ultimately defoliation in young peach trees planted in soils contaminated with lead arsenate. Kabata-Pendias (2001) has also reported that wilting, violet colour (anthocyanin), discoloration of roots and cell plasmolysis as the toxicity symptoms for arsenic in plants. Symptoms of arsenic toxicity have been studied in much details and it has been established that it manifests initially as chlorosis, retarded growth, gradual browning and ultimately death of the plant occurs (Rumberg et al., 1960; Lasko, 1973; Kempen, 1968). Visual symptoms of phytotoxicity include leaf wilting, followed by retardation of root and shoot growth (Liebig, 1965). This is often accompanied by root discoloration and necrosis of leaf tips and margins, indicating inhibition of root water uptake and ultimately resulting in death from wilting (Woolson et al., 1971a). Toxicity of arsenic in growth medium on the chlorophyll content of leaves has been reported in rice (Rahman et al., 2001), maize (Stoeva et al., 2003), bean (Stoeva et al., 2005) and tea (Shi et al., 2008). Phytotoxicity of arsenic depends on the form and availability of arsenic in the soil. Organic arsenic compounds are less toxic than inorganic compounds and the toxicity decreases in the following order: arsenite > arsenate > organic As compounds (Adriano, 1986). The phytotoxicity of As in soils depends primarily on soil texture and secondly on the pH. There is an increase in As phytotoxicity as soils become more acid,

particularly when the pH drops below 5 and As sorbents such as Fe- and Al- oxides are dissolved (O'Neill, 1995). Sheppard (1992) concluded that phytotoxicity of inorganic As is influenced by soil type. It was reported that inorganic As was five times more toxic to plants in sand (mean = 40 mg As/ kg) than in clay (mean = 200 mg As/ kg) soils. Arsenic phytotoxicity is expected to be greater in sandy soils than in other soil types, because sandy soils generally contain low amounts of Fe and Al oxides and clay minerals (Peterson et al., 1981). Arsenic is known to disturb uptake and transport of mineral nutrients in plants (Paivoke and Simola, 2001). Disturbance of plant mineral nutrition is the main cause for yield decrease, the most frequent sign of As toxicity. According to Liebig (1965) the phytotoxicity of arsenic is attributed to its ability to substitute for phosphate in enzyme catalyzed reactions particularly to uncouple phosphorylation reactions and thus to interfere with the energy status of the plant. 2.2.5. Arsenic Resistance in Plants Although the physiological basis of metalloid resistances has been investigated in some detail, it is only for arsenate resistance that a full mechanism understanding is available (Meharg and Macnair, 1992). Intracellular compartmentalization has a major role to play in adaptation for some elements (Clemens, 2001). Arsenate is an analogue of the macronutrient phosphate, thus plants growing on arsenate contaminated soils will assimilate high levels of arsenate unless they have altered phosphate transport mechanisms (Meharg and Macnair, 1992; Sharples et al., 2000a). Arsenates primary modes of toxic action is competing with phosphate (such as in ATP formation), cytoplasmic concentrations of arsenate (and arsenite which is readily formed from arsenate in cytoplasm) must be kept low to maintain cellular function (Meharg, 1994). Arsenate resistance has been identified in a number of species on arsenic-contaminated soils including Andropogon scoparius (Rocovich and West, 1975), Agrostis castellana, A. delicatula (De koe and Jacques, 1993), A. capillaries,

Deschampsia cespitosa (Meharg and Macnair, 1994), H. lanatus (Macnair and Cumbes, 1987), S. vulgaris (Sharples et al., 2000 b). In populations of H. lanatus from uncontaminated soils across the UK approximately 45% of seeds gave rise to arsenic-resistant plants (Meharg et al., 1993), whilst individuals of S. vulgaris (Paliouris and Hutchinson, 1991) and P. lanceolate (Pollard, 1980) from uncontaminated soils also exhibited resistance to arsenic. Bacteria and yeast reduce arsenate to arsenite and then efflux the arsenite from their cells through arsenite transporters (Rosen, 1999). Saccharomyces cerevisae, as well as organisms exhibiting arsenite extrusion can complex As(III) with glutathionine to give As (GS)3 which is then actively transported into the vacuole through a As(GH)3 transporter (Rosen, 1999). Arsenate-resistant plants differ from non-resistant phenotypes in their reproductive behaviour, with resistant phenotypes flowering earlier and investing more in their reproductive biomass (Fitter et al., 1998; Wright et al., 2000). Arsenate-resistant plants have higher shoot P status, despite exhibiting suppressed phosphate uptake, and it has been suggested that suppressed uptake is a feedback mechanism in response to high shoot P (Meharg et al., 1994b; Wright et al., 2000). Plants growing on arsenic-contaminated soils tend to be mycorrhizal (Meharg and Cairney, 1999; Gonzalez-Chaney, 2000). Gonzalez-Chaney (2000) reported that infection of both tolerant and non-tolerant plants by fungi from arsenic contaminated sites further enhanced resistance to arsenate. Generally plants use intracellular and extracellular mechanisms to enhance nutrient uptake. The external mechanisms include the exudation of substances that can bind the metal ions and change the rhizosphere pH. Consequently, this action affects metal speciation or the production of ion exchange sites on the cell walls, both acting to facilitate the uptake (Rathinasbapathi et al., 2006). Root exudates depend on the inherent plant biology and the external environment, the quantity and chemical make up can be different from plant to plant. Tu et al. (2004) compared the nature of root exudates in Pteris vittata fern and non

accumulating specie Nephrolepis exaltata (Boston fern) on the mobilization of aluminium arsenate, ion arsenate and chromate copper arsenate. It was found that predominant components of the root exudates were two organic acids, phytic acid and oxalic acid for both ferns. Both ferns had comparable phytic acid exudation in the absence of arsenic. On the other hand, Pteris fern exuded about 3-5 times more oxalic acid than the Boston fern under the experimental conditions used in the study (Tu et al., 2004). Root exudates have been observed to dissolve significant amounts of arsenic from minerals. Root exudates from Pteris fern mobilized 3-4 times more arsenic from aluminium arsenate, 4-6 times more arsenic from iron arsenate and 6-18 times more arsenic from chromated copper arsenate than Boston fern exudates (Tu et al., 2004). 2.2.6. Arsenic detoxification in plants Hyperaccumulating plants possess efficient mechanism for detoxification of these accumulated metals. According to Salt et al., 1998 these mechanisms include chelation, compartmentalization, biotransformations and cellular repair. Carbohydrates (such as melate, oxalate, citrate etc.) are commonly the major charge balancing anion present in the cell vacuoles of photosynthetic tissues. Several of these carbohydrates have been associated with high metal concentrations in plants (Ma et al., 1997; Gabbrielli et al., 1997; Homer et al., 1995). The tripeptide glutathione (GSH) is synthesized by gamma-glutamylcysteine synthetase (-ECS) and glutathione synthetase (GS). Increasing GSH synthesis is considered a mean to increase cellular defence against oxidation stess. Since GSH is precursor of phytochelatin (PC) overexpression of -ECS or GS leads to high PC accumulation under metal exposure (Mendoza-Cozatt et al., 2005; Verbruggen et al., 2009). Glutathionine conjugates with Arsenic (As III- GS3) are transported into vacuoles of plants by ABC transports (Mendoza-Cozatt et al., 2005; Bleeker et al., 2005). Phytochelatin synthesis has been induced on exposure to arsenate in number of plant species (Snellar et al., 1999; Schmoger et al., 2000; Zhao et al., 2003). Intact

phytochelatins-arsenic complexes have been isolated from plant tissues (Schmoger et al., 2000) suggesting that phytochelatins are also involved in arsenic detoxification in plants. Phytochelatin (PC) synthesis was induced on exposure to arsenate in P. vittata (Zhao et al., 2003). Plant possess arsenate reduction to reducing As(V) to As(III) showing homology with yeast arsenate reductase acr2 which seem to be essential for As(V) tolerance (Bleeker et al., 2006; Ellis et al., 2006). Studies on the acr2 mutant showed that phenotypes are strongly dependent on plant P-status (Verbrugger et al., 2009). According to Dhankher et al. (2002) over expression of - ECS and Ars C (arsenate reductase) substantially increased As (V) tolerance in A. thaliana. Efflux of As roots represents another potential stratergy for metal detoxification. The process involves uptake of As (V) an intracellular reduction to As (III) and followed by efflux of As (III) (Xu et al., 2007). Methylation of As in plants is another potential detoxification mechanism because methylated arsenic are less toxic than inorganic As (Norton et al., 2008). This is less abundant in plants than in animals.

2.3.

ARSENIC TOXICITY
The toxic effects of arsenic in humans, plants and animals have been well

characterized. 2.3.1. Toxicological Effects in Animals Acute toxic effects seen in animals after oral exposure are similar to effects seen in humans (USEPA, 1984). The signs of acute arsenic poisoning in humans include intense abdominal pains, staggering, weakness, trembling, salivation, gastrointestinal effects such as vomiting and diarrhoea, fast feeble pulse, prostration, hypothermia, collapse and death (Marcus and Rispin, 1988). Arsenite has been shown to induce oxidative DNA damage in human vascular smooth muscle cells in vitro (Lynn et al., 2000). Oral LD50 values for various arsenic compounds range from 15 to 293 mg/kg in

rats and from 10 to 150 mg/kg in other animals (USEPA, 1984). ATSDR (2000) lists oral LD50 for inorganic As ranging from 15 to 175 mg/kg for rats and 26 to 39 mg/kg for mice. Most deaths occurred within one day of exposure. Studies have shown that MMAIII is more toxic than arsenite in cultured human cells in vitro (Petrick et al., 2000; Styblo et al., 2000). The ones most affected by arsenic are those involved in absorption, accumulation, and/or excretion, i.e., the gastrointestinal tract, circulatory system, skin, liver, and kidney. However, other organs or systems that are particularly sensitive to the effects of arsenic, such as the nervous system, and those that are affected secondarily, such as the heart, are also affected (Squibb and Fowler, 1983). Histopathological changes have been observed in liver tissue as a result of arsenic exposure (Mohelka et al., 1980; Rozenshtein, 1970). Arsenic impaired mitochondrial respiration in rats injected with arsenite (Ishinishi et al., 1980; Ghatghazi et al., 1980). 2.3.2. Human Exposure Food chain represents an important pathway for arsenic exposure to the consumers. Food constituents the largest source of As intake (Chen and Lin, 1994). Daily intake of total As from the consumption of food and beverages lies between 20 and 30 g/day (Abernathy, 2001). A study conducted in Mexico revealed that 30% of inorganic As came from intake of this element in food (Del Razo et al., 2002). The plants raised over soils contaminated with As are known to accumulate this element thus exposing the consumer to its ill effects. In Bangladesh and West Bengal (India) rice has become an important dietary source of As for the people (Schoof et al., 1999; Bae et al., 2002; Meharg and Rahman, 2003; Das et al., 2004; Williams et al., 2005; Ackerman et al., 2005; Sengupta et al., 2006; Rahman et al., 2006). Various traditional medicinal concoctions have also been identified as a source of dietary arsenic (Mitchell et al., 1990; Kew et al., 1993; Shen et al., 1999; Ernst & Coon, 2001; Chakraborti et al., 2003 a).

People who work in arsenic processing industries such as non-ferrous metal smelting, pesticide manufacture and application of pesticides or wood preservatives can be exposed to higher levels of arsenic by the inhalation of arsenic containing particles (WHO, 2001). 2.3.3. Toxicological Effects on Humans Arsenic toxicity (Arsenicosis) is manifested through symptoms ranging from non-specific abdominal pain, diarrhoea, nausea to multiorgan disorders and more serious consequences as malignancy (ATSDR, 1999; NRC, 1999; EPA, 2000; WHO, 2005; ENVIS, 2009). Arsenic is specifically associated with skin cancer, hyperkeratosis, melanosis, blackfoot disease, damage to blood vessels and sensation of pins & needles in hands and feet reported to be diabetogenic (Navas-Acien et al., 2006; Coronado-Gonzaliza et al., 2007). Cases among pregnant women are available where arsenite (As2O3) readily passes through the placenta. Several studies have reported association between prolonged low-dose As exposure, reports of arsenic foetotoxicity and adverse pregnancy outcomes in terms of live birth, still births, spontaneous abortion, fetel loss, premature delivery and preterm birth are also available (Lugo et al., 1969; Kagey et al., 1977; Bollinger et al., 1992; Ahmad et al., 2001; Chakraborti et al., 2003 b; Von Ehrenstein et al., 2006). 2.3.4. Genetic Toxicity The earlier genetic toxicity data on arsenic are summarized in the Genetic Activity Profiles (GAP) database for short-term tests based on data of the U.S. EPA and the IARC monographs. For trivalent arsenic (AsIII), the GERMCELL and IARC

databases list 11 positive findings in 25 non-human animal, plant, or microbial test systems. These include chromosomal aberrations in vitro and in vivo (Armstrong et al., 1984), micronuclei induction in mice in vivo (Abernathy et al., 1993), SCEs in mammalian cells (ACGIH, 1986), and cell transformation in vitro (Armstrong et al., 1984). For As(V), the IARC database lists 6/13 positive findings: chromosome aberrations in vitro (Armstrong et al., 1984); SCEs in vitro (ACGIH, 1984); and cell

transformation in vitro (Abernathy et al., 1993). In general, the lowest effective doses (LEDs) for As(III) in vitro were in the 1-10 M range, whereas for As(V) the LEDs were usually 10-50 M. Jacobson-Kram and Montalbano (1985) and Basu et al., (2001) have published comprehensive reviews of arsenic genetic toxicity. Studies assessing the ability of arsenic to induce gene mutations have largely produced negative results. The results of earlier studies suggested positive mutagenic activity (Nishioka, 1975; Oberly et al., 1982). Yamanaka et al., (1989) reported the mutagenicity of dimethylarsinic acid (DMA) in E. coli B tester strains. The study by Nakamuro and Sayato (1981) evaluated the genotoxic activity of six arsenic compounds and found the following order of potency (from highest to lowest) was observed: As2O3 > AsC13, NaAsO2 > Na2HAsO4 > H3AsO4,As2O5. Crossen (1983) indicated that arsenic is only clastogenic when present during the cell phase of DNA replication (i.e. the S-phase). Wang and Huang (1994) observed that active oxygen species are involved in the induction of micronuclei by arsenite in XRS-5 cells, an X-ray sensitive Chinese hamster ovary cell line. Zhao et al., (1997) demonstrated an association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression in rat liver epithelial cell line. Exposure to arsenic trioxide increased the frequency of chromosomal aberrations in the peripheral lymphocytes of smelter workers (Bechman et al., 1977; Nordenson et al., 1978). There is no conclusive evidence that arsenic causes point mutations in any cellular system and arsenic is mutagenic (Belton et al., 1985; Dekundt et al., 1986). However, Li and Rossman (1974) have shown that arsenite causes inhibition of DNA repair after the incision step in Chinese hamster V79 cells. Arsenic has been known to cause chromosomal damage but most investigators have been unable to induce direct gene mutation (Goyer, 1991; Squibb and Fowler, 1983). Arsenic promotes genetic damage in large part by inhibiting DNA repair (Bencko et al., 1988; Leonard and Lauwerys, 1980; Nordenson et al., 1978; Rossman et al., 1977). The comparisons of chromosome aberration frequencies induced by trivalent

and pentavalent arsenic have indicated that the trivalent forms are far more potent and genotoxic than the pentavalent forms (Barrett et al., 1989; Nakamuro and Sayato, 1981; Nordenson et al., 1981). Epidemiological studies have demonstrated an evident causal relationship between environmental, occupational and medical exposure of man to inorganic arsenic and cancer of the skin and lungs (Fowler and Weissburg, 1974; IARC, 1980; Leonard and Lauwerys, 1980; Lerman et al., 1980; NAS, 1977; Neubauner, 1947; Pershagen, 1981). Most animal experiments, however, were not able to demonstrate concinogenicity, except for very few observations of increased incidence of leukaemia and lung cancer (Fishbein, 1987; Sunderman, 1986). Epidemiological studies in Argentina, Chile, Canada, and Taiwan suggest correlations between drinking water arsenic and Blackfoot disease, Bowens disease and skin cancer (Fishbein, 1987).

2.4.

PHYTOREMEDIATION
Current methods for remediation of arsenic-contaminated soils include soil

removal and washing, physical stabilization, and/or the use of chemical amendments, all of which are expensive and disruptive, with an average cost of $ 404,700 per ha (Raskin et al., 1997). USEPA (2002) recommended excavation, capping, solidification and stabilization, vitrification, soil washing/acid extraction, soil flushing, phytoremediation, etc. as current remediation technologies for arsenic-contaminated soil. Phytoremediation includes any remediation method that uses plants to either remove pollutants or render them harmless in soil and water systems, and can be applied for both organic and inorganic pollutants present in soil, water, and air (Salt et al., 1998).This practice is gaining popularity because of its overall cost-effectiveness (Watanabe, 1997; Salt et al., 1998; Kabata-Pendias and Pendias, 2001). The term phytoremediation includes several strategies: a) Phytoextraction - the use of pollutant- accumulating plants capable to extract and translocate pollutants to the harvestable parts. It uses tolerant plants that concentrate soil

contaminant sin their above ground biomass, so that the contaminant-enriched biomass can be properly disposed (Kramer, 2005). b) Phytostabilization the use of pollutant-tolerant plants for mechanical stabilization of polluted land in order to prevent bulk erosion, reduce air-borne transport, and leaching of pollutants. It is used to provide a cover of vegetation for a moderately to heavily contaminated site, thus preventing wind and water erosion (Kramer, 2005). Plants suitable for phytostabilization have extensive root system, provide good soil cover, possess tolerance to the contaminant metals and ideally immobilize the contaminant in the rhizosphere. Arsenic-tolerant plants that can be used for phytostabilisation purposes have been known for a long time (Rocovich and West, 1975; Porter and Peterson, 1977; Benson et al., 1981). c) Phytoimmobilisation- the use of plants to decrease the mobility and bioavailability of pollutants by altering soil factors that lower pollutant mobility (formation of precipitates, insoluble compounds and sorption on roots). Based on the chemical similarities of arsenic and phosphorus, there may be precipitate formation of arsenic/lead compounds as shown for phosphorus-lead precipitates in the rhizosphere of A. capillaries (Cotter-Howells et al., 1999). Other plant-mediated processes of arsenic immobilisation at the soil-root interface e.g. accumulation of arsenic on iron plaque in the oxidized rhizosphere of salt marsh plants (Doyle and Otte, 1997). d) Phytovolatilization - the use of plants to volatilize pollutants and has been demonstrated for Hg and Se. Volatilization of arsenic is also known to occur in natural environments (Frankenberger and Arshad, 2002), but rhizosphere studies have not been reported. Available information on arsenic volatilization suggests that the volatile compounds account only for small proportions of total arsenic (Turpeinen et al., 1999). Phytoextraction of Arsenic Arsenic is a nonessential and highly phytotoxic element for plants. Under normal conditions, arsenic concentrations in terrestrial plants are usually less than 10 mg/ kg (Matschullat, 2000). He observed that plants contain arsenic in the following order: cabbage (0.020 0.050 mg/ kg) < carrots (0.040 0.080 mg/ kg) < grass (0.020

0.160 mg/ kg) < potatoes(0.020- 0.200 mg/ kg) < lettuce (0.020 0.250 mg/ kg) < mosses and lichens (0.26 mg/ kg) < ferns (1.3 mg/ kg). An average toxicity threshold of 40 mg kg-1was established for crop plants (Sheppard et al., 1992). The transfer of arsenic from soil to plant is low for most plant species. This is probably due to the restricted uptake of arsenic by plant roots, the limited translocation of arsenic from root to shoot, arsenic phytotoxicity and the low bioavailability of arsenic in soil (Wang et al., 2002). Unfortunately, most metal-hyperaccumulator plants are slow growing and have low biomass, while the plants which quickly produce high biomass are usually sensitive to high metal concentrations. According to Ma et al. (2001) phytoextraction can be accomplished by using either tolerant, high-biomass plant species or hyperaccumulator plant species. Both bioaccumulation factor (BF) and translocation factor (TF) have to be considered while evaluating whether a particular plant is a metal hyperaccumulator (Ma et al., 2001). Therefore, an arsenic hyperaccumulator plant should have BF > 1 and TF > 1 as well as total accumulation> 1,000 mg/ kg arsenic in plant biomass (Tu and Ma, 2002). While some plants can survive in environments containing extremely high concentrations of metals, they may not show high ability of accumulating metals. For example, Agrostis tenuis concentrated 3,470 mg/kg of arsenic when grew in a soil containing as high as 26,500 mg/ kg of arsenic. Even though the arsenic concentration in the plant was very high, it cannot be characterized as a suitable arsenic hyperaccumulator because the bioconcentration factor (BF) and translocation factor (TF) were lower than 1 (Gonzaga et al., 2006). Saraswat and Rai (2009) conducted pot experiment to compare the phytoextraction potential of six plant species for metal removal. The results revealed that metal accumulation was plant specific. A. thaliana accumulated maximum Zn and Cd where as B. juncea and C. dactylon accumulated Ni and Cr in their shoots, respectively. Karimi et al. (2010) studied the arsenic hyperaccumulation potential of the native plant species collected from Zareshuras area (N-W Iran) which has a long history

of As accumulation from mining. Among 89 plant species the highest foliar As concentration were found in Isatis capadocica (upto 300 mg/kg) and Hesperis persica (upto 1500 mg/kg). These are the first terrestril angiosperms shown to possess As hyperaccumulation traits. Successful application of phytoextraction to arsenic contaminated soils depends on many factors. Plant species used to extract arsenic should be responsive to agricultural practices designed to enhance arsenic accumulation and to allow repeated planting and harvesting of arsenic-rich biomass (Tu and Ma, 2002). The continuous phytoextraction depends on the arsenic bioavailability in the soil and the natural ability of a plant to accumulate, translocate and tolerate high concentrations of arsenic over the whole growth cycle (Garbisu and Alkorta, 2001). Following harvest of arsenic-enriched plants, the weight and volume of contaminated material can be further reduced, transported and disposed off site as hazardous material (Salt et al., 1998; Ma et al., 2001). Identification of candidates for removal of arsenic by phytoremediation is still at its preliminary stage. No such work is available from the polluted areas of Punjab. Keeping in view the presence of arsenic in the soils of the study area (Talwandi Sabo Block of Bathinda, Punjab) the present problem was taken up to identify the species suitable for remediation of the soil.