3.1 Physical and chemical characteristics of Soil
Results of the texture shows that soil varied from sandy loam to loamy sand (Table 1). Higher EC value was measured for Mansehra (7.53 mS/cm) and lower in Upper Dir (0.77 mS/cm). Highest soil OM was observed in DI Khan (4.7%) and lower in Lower Dir (2.0%). Maximum pH value was observed for Banu (8.1) and minimum for DI Khan (7.4). Low pH of the DI soil could be attributed to higher OM. Higher values of P were found in DI Khan (0.47 g/kg) and lower in Charsadda (0.15 g/kg) and that of Fe were in Upper Dir (3.37 g/kg) and lower in Charsadda (2.06 g/kg).
3.2 Arsenic in soil
Figure 1 represent the values of Asi and Aso found in the soils of Khyber Pakhtunkhwa. Overall results shows that the percentage of Asi is more as compared to Aso while in some areas percentage of the Aso is higher than the Asi. The highest value of Asi was recorded for 79.6% for Buner, while the lowest was 34.6% for Swat district. Also, for Aso, the higher percentage 72.3%, was found in Malakand and the lowest was 20.2% for Buner. This is due to the fact that As3+ can be converted into organic forms of As by the process of methylation encouraged by microbial activities in soil paddies (Islam et al. 2004).
In soil the mean Asi ranged from 2.27-4.54 mg/kg, while it was higher in Upper Dir and lower in Banu district. As far as Aso is concerned, it was higher in Swat district (mean=5.73 mg/kg) and lower in Buner (mean=1.11 mg/kg). Similarly, mean AsT was higher in Chitral district (mean=13.72 mg/kg) and lowest in Banu (mean=4.31 mg/kg). The higher values of As in Chitral and swat was due to the mining activities in those areas. All these concentrations were below normal level of As in soil (30 mg/kg) (SEPA 1995). Roychowdhury et al. (2005) reported the highest concentration of As in West Bengal soil (19.4 mg/kg). It is due to the fact that paddy soils are naturally abundant in As (Kato et al. 2019) that further is worsen by contaminated water irrigation (Gan et al. 2019). Flooded paddies soils had the characteristics that resulted in the mobilization of As, thus enhancing its bioavailability and uptake of rice plant (Yamaguchi et al. 2011). Jehan et al. 2019, reported As concentration values in Chitral soil was 10.24 mg/kg, which is much lower as compared to the present study result (13.72 mg/kg). Marin et al. 1992 analyze that bioavailability of As to rice plant shows the following order As3+ > MMA >As5+ > DMA. All the species of As are taken up through rice roots, however organic form of As is taken up slower than the inorganic form (Abedin et al. 2002).
3.3 Arsenic in rice
The rice samples collected from 16 different districts of Khyber Pakhtunkhwa show variation in As species concentration and are presented in Fig 2 (S10) of SI. The Asi concentration in the rice ranged from 0.05-0.24 mg/kg with high level in DI Khan and low level in Shangla district (Fig 2). Similarly, the Aso ranged from 0.002-0.02 mg/kg with maximum concentration in Charsadda and minimun in Mardan. Also, the AsT was ranged 0.06-0.28 mg/kg, which was higher in DI Khan and lower in Shangla.
Flooded rice paddies has the characteristics of reducing environment, and this anoxic condition enhances the availability and mobility of As3+ as compared to that of As5+, because of the process of dissolution of Fe (Niazi et al. 2011; Shakoor et al. 2018). Earlier studies showed that Asi and DMA were the most common species of As detected in rice (Meharg et al., 2009; Zhu et al. 2008), while MMA occurred rarely with low content (Zhao et al. 2013b). Wu et al. 2019) also found similar result that the concentration of different species of As (As3+, As5+, and DMA) dominates in rice grains, but, no MMA was found in the samples of rice. For AsT the highest concentration was in DI Khan rice 0.28 mg/kg and lower in Shangla rice 0.062 mg/kg, which is much less than the study conducted by Jehan et al. 2019 (3.33 mg/kg). The studies had explained that in Pakistan the rice are cultivated in paddy soils that could possible host higher As concentration (Kato et al. 2019). Also, this contamination increases because of irrigation of soil with As-contaminated water irrigation (Gan et al. 2019). Another reason may be the capacity of rice to accumulate As double than other grains i.e. wheat and barley (Jehan et al. 2019; Mitani et al. 2009; Ma et al. 2008). Selenite and selenate are found to facilitate the arsenate adsorption by paddy soil also they inhibited the uptake of arsenate by rice roots. Further selenate has stronger inhibition on iAs transfer factors than selenite (Pokhrel et al. 2020).
Numerous researches on speciation of As in South and South East Asia found Asi which is highly toxic constitute about 42%-91% of AsT in rice while in America rice, the major specie of As is DMA (Schoof et al. 1999; Meharg et al. 2009). Other researches also showed that Asi (75%-90%) is high in different products of rice such as, baby rice, rice crackers, breakfast cereals, rice milk, and other products of rice (Sun et al. 2009).
3.4 Relation of Arsenic and soil physicochemical characteristics
Clayey and silty soils have finer texture, more surface area and higher As scavenging capability as compared to sandy soils, which is because of the Fe oxides presence. Hence, clayey soils plants show less toxic effects of As; As phytotoxicity is five times high in loamy and sandy soils (Quazi et al. 2011).
The highest OM value is present in soils containing dolomite, while those with greater pyrite and phyllosilicates levels showed very less OM content. The results shown that samples with higher OM content, displayed lower As content. Soil OM is responsible for the mobility of As also its complexes (soluble or insoluble) and chemical nature (William et al. 2011). Pikaray et al. (2005) testified that solubility of As decreased in soils with high value of OM, which affect its accessibility to plant, as OM has a high affinity for sorption of As because of the formation of different OM complex. However, on the other side, there is a positive correlation between As accumulation and soil OM in rice grain (Jia et al. 2013). OM increase in the soil can increase the As mobility with the help of increasing microbial activities and by decreasing redox potential of the soil (Turpeinen et al. 1999), which is a favorable condition for the reduction of Fe-oxyhydroxides which is linked to OM (Reza et al. 2010).
The highest adsorption was nearby pH 7 (Miyatake and Hayashi 2011). Tabassum et al. 2018 and Bibi et al. (2015b) presented strong positive correlation of As contents with pH, in Rawalpindi and Hasilpur areas. The leaching of As also its speciation depends upon the soil pH, therefore the availability of As as well as its solubility depends on soil pH (Chatterjee et al. 2013; Quazi et al. 2011). Some researches (Signes-Pastor et al. 2007) showed that the uptake and accumulation of As by rice plant is affected by both low and high pH. The reason may be that at low pH (pH < 5), Fe-oxyhydroxide compounds which are As-binding species, becoming highly soluble and increase the uptake of As by plants. Bhattacharya et al. 2010 also supported the negative relationship between soil pH and As concentration in rice. On the other hand, many authors supported the idea of a positive relationship between soil pH and As accumulation (Ahmed et al. 2011; Campbel et al. 1985). increasing soil pH (such as pH 8.5) rises the negative surface charges (hydroxyl ions), which facilitates As desorption from Fe-oxides that leads to mobilization of As in the root, which, in turn, increases accumulation of As in the plant (Ahmed et al. 2011). As the pH decreases As3+ solubility increases with the soil pH range (pH 3-9), whereas in the case of As5+ this pattern is reversed. In flooded paddy soils As3+ predominates. In this regard, for the current study, a decrease in soil pH can enhance the mobility of soil As, which gives the reason why we observed almost equal concentrations of As in rice grain irrespective of the levels of soil As (Sahoo and Kim 2013).
By the application of external P, the uptake of As is reduced and the toxicity symptoms of As5+ alleviates. Different researches (Pigna et al. 2010; Rahman and Hasegawa 2011) have described that with the increasing P value in nutrient solution, decreases As5+ uptake by the rice plant. In plant physiology and biochemistry, P plays a significant role (Shin et al. 2004). In As contaminated soil, there are some properties such as As concentration in soil solution, application of phosphatic fertilizers and other soil physicochemical properties control As uptake by rice plant (Geng et al. 2005; Farooq et al. 2016; Liu et al. 2004) described that if there is sufficient P in soil it will result in less Fe-plaques formation on the rice roots, while the lack of P in the soil solution enhances the plaque formation on the rice roots. Less amount of P in plant tissues can be useful in increasing oxygen transportation in rice roots (Kirk and Van Du 1997) which may encourage Fe plaque formation on the rice roots. Several studies supported the idea of application of phosphate to soil, lowers the As content in Fe-plaques leading to increase in solubility of As and bioavailability to the soil rhizosphere (Azam et al. 2016; Smith et al. 2002).
The Matsumoto et al. (2015) investigated that if the rice field is amended with Fe-oxide and metallic Fe, it will reduce As content in rice grain by 47% and 51%, respectively. This may be due to the fact that As binds with Fe-(hydr)/oxides in soil (Inskeep et al. 2002) which has a great effect on the mobility of As in soil solution (Marin et al. 1993). The strong affinity of Fe (an essential mineral of plants) towards As helps to decrease the As adsorption in rice (Yamane 1989; Nath et al. 2014). Some of the effects by the application of external Fe and its compounds in soil include: (i) Fe (Fe-oxide) deposits around rice roots reduces the uptake of As in rice roots, (ii) the co-precipitation of Fe and As enhances and (iii) it reduces the desorption of soluble As because of the adsorption of As5+ on the surface of Fe. Anaerobic condition for rice cultivation encourage the Fe-plaque formation around the roots of the rice plant (Armstrong 1964). Ferrihydrate (63%), siderite (5%) and goethite (32%) constitute Fe-plague (Hansel et al. 2002). The concentration of free Fe around the rice roots also rises its concentration in the rhizosphere that further lowers uptake of As in rice plants (Geng et al. 2005; Syu et al. 2014). Fe-plaques has high affinity towards As5+, therefore it plays an important part in decreasing the As uptake in rice. For the sequestration of As, Fe-plague is probably the best solution which ultimatle reduces the transport of As from roots to shoot in rice plant (Liu et al. 2004). Leonardite had a higher efficiency to adsorb As than biochar’s because of the iron contents in leonardite which strongly bind to As anions (Dolphen and Thiravetyan 2019).
3.5 Comparison of As level in rice of mining and non-mining areas
One of the most important human activities that contaminate our environment with As is mining (Williams et al. 2009). Due to the presence of As in many minerals and other human activities such as mining, As-contaminated waste water irrigation, fertilizers and pesticides application containing As, As became one of the most common contaminants affecting human health via food chains (Zhu et al. 2014). As is present naturally in many ores such as Cu, Pb, Au and Zn ores. Therefore, in mine impacted areas, As is found in soils at higher levels, posing a risk to not only human health but also ecosystem health (Li et al. 2014). Current study reported high Asi, Aso and AsT in mining areas. In non-mining areas, the Asi concentration is higher in Peshawar (0.14 mg/kg) and while lower in Mardan (0.06 mg/ kg). Aso was higher in Charsadda (0.02 mg/kg) while lower in Mardan (0.002 mg/kg). While the concentration of AsT is higher in Peshawar (0.018 mg/kg) and lower in Mardan (0.07 mg/kg). By comparing these results with the mining areas result it was concluded that for Asi the highest value was detected in rice grown in DI Khan (0.24 mg/kg) and lowest in Shangla (0.05 mg/kg). For Aso the maximum concentration was detected in the rice of Lower Dir 0.03 mg/kg while the lowest concentration was found in Shangla (0.002 mg/kg). For AsT highest value is (0.28 mg/kg) in rice of DI Khan while lowest value is (0.06 mg/kg) in Shangla rice as shown in Fig 3. The overall results depicted that the highest values for the species of As as well as the AsT was in the rice of mining areas such as DI Khan. But there are some non-mining areas such as (Peshawar AsT= 0.18 mg/kg) which shows a little higher concentration than the mining areas (Buner AsT =0.09 mg/kg, Shangla AsT = 0.06 mg/kg and Upper Dir AsT = 0.08 mg/kg). The reason behind this may be due to higher level of pollution in Peshawar district. This gap need to be filled through further research that why non mining areas show higher concentration of As than the mining areas.
Both the natural and anthropogenic activities are the sources of As (Duan et al. 2013). Among the natural sources, volcanism and bed rocks weathering are important while among anthropogenic sources, metal mining and smelting, the use of As-pesticides, herbicides, feed additives, wood preservatives and irrigation with As-contaminated water are important sources that results in elevated levels of As in soil (Zhao et al. 2009). One of the main pathways of human exposure to As is the transport of As in soil-plant systems (Dave 2013). Most of the samples in mining sites shows a higher trend of As and its different species as compared to non-mining sites. The concentration of As in soil of mining area was higher (0.624 mg/kg) than the non-mining area (0.096 mg/kg) in Hunan Province, China (Zhu et al. 2008). As is a natural constituent some ores such as ores of Cu, Pb, Zn, and Au. This is the reason why As is mostly found at elevated levels, in mining areas soils posing a risk to health of both ecosystem and human (Li et al. 2014). None of the district of the study area exceeds the acceptable range (0.5 mg/kg) of AsT by (WHO 2011). Kwon et al., 2017 also stated that the values of As was high (0.247 mg/kg) in the rice of mining areas of Korea as compared to other lands.
3.6 As concentration in different rice types of Khyber Pakhtunkhwa
Arsenic concentration in different types of rice are presented in Fig 4, which shows that Asi is higher in basmati rice type (0.21 mg/kg) while low in khetai rice (0.03 mg/kg). Aso higher concentration was found in garma seela (0.3 mg/kg) while lower in china haripaka, china naray and khetai (0.00 mg/kg). Also the AsT concentration was high in basmati rice type (0.25 mg/kg) as compare to others, while lowest in Khetai rice (0.04 mg/kg). An overall assessment of As concentration was as follow; Asi basmati > garma seela > china polawal/ seela > china > aripaka (garma) > begamay/ naray > china naray/ china haripaka/ china begamay > aripaka > aripaka (yakha) khetai. Aso, garma seela > seela/ aripaka (garma) / china polawala > china / begamay/ basmati/ china begamay/ aripaka/ naray/ aripaka (yakha)/ china naray/ khetai/ china haripaka. AsT, Basmati > garma seela > seela > china polawala > china > aripaka (garma) > begamay > naray > china begamay > china haripaka > china naray > aripaka > aripaka (yakha) > khetai. Previous findings revealed that the average As values in American rice type (white Basmati) from Texas was (0.26 ± 0.08 mg/kg) (Zavala and Duxbury 2008).
3.7 Chronic Health Risk Assessment
3.7.1 Estimation of EDI
The local people were assumed to consume the local rice. In Pakistan, per capita consumption of rice is very low only 20.8 kg due to high cost of rice (Bashir et al. 2010). The mean estimated daily intake (EDI) and its standard deviation of Asi through rice consumption ranged from lowest 0.000273 mg/kg/day in Shangla to highest 0.00131mg/kg/day in DI Khan (Fig. 5). While, the estimated daily intake of Aso through rice consumption were ranged from 1.09x10-5 mg/kg/day in Mardan to 2.03x10-4 mg/kg/day in Lower Dir. Also, estimated daily intake for AsT showed that it was lower in Shangla (0.0003 mg/kg/day) higher in DI Khan (0.0015 mg/kg/day).
Previous findings indicate that EDI values for AsT were equivalent to those values calculated in Bangladesh rice (5.00×10-2–5.00×10-1 mg/kg/day), Vietnam (1.1 × 10-3 –4.3 × 10-3 mg/kg/day) and Turkey (2.3×10-5–5.21×10-3 mg/kg/day) (Caylak 2012; Karim 2000; Nguyen et al. 2009).
3.7.2 Cancer Risk Assessment
The cancer risk was calculated by the formula of ILTCR, which is attained by multiplying EDI of Asi with the CSF which is 1.5 for AsT. The ILTCR risk calculated was high for DI Khan district (1.9E-3 mg/kg/day). The lowest calculated cancer risk was for district Shangla (4.1E-4 mg/kg/day) as shown in Fig 6. Earlier risk assessments for As exposure via Indian rice consumption described risk results almost similar to this study using Indian intake values i.e. 7 adults in population of 10,000 (Meharg et al. 2009; Mondal and Polya 2008). In Bangladesh, studies (Meharg et al. 2009) also report same levels of cancer risk (with 19, 22 men and women 423 in10, 000 population) in adults.