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Introduction

Natural and anthropogenic sources are the two major areas from where arsenic (As) may contaminate the normal environment¹. Contamination of arsenic can be toxic and fatal if it will enter the food web via water and soil routes. Previous researchers also observed the contamination of groundwater and foods via arsenic in different parts of the world^2,3. Contamination of soil by arsenic may result from agricultural activities, various anthropogenic activities, etc. According to Huang et al., (2006) inorganic-As is the most predominant toxic form presents in soil and water.⁴然而,一些之前s studies reported about As- methylation in the soil-paddy system, where they found that the inorganic form of arsenic may be converted from organic arsenic by microorganisms⁵. The uptake of arsenic by a plant species depends on many factors like soil properties, the concentration of arsenic present in the soil, and most importantly depends on the plant species itself⁶. Physical characteristics of plant species is an important aspect which is evident from few previous studies that found the concentration of arsenic was estimated high in root parts followed by leaves, stems, fruits, and seeds^7、8. However, Farid et al., (2003) have found aerial parts of leafy vegetables can accumulate high concentration of arsenic than any other plant body parts⁹. So, it is clear that the bioavailability of arsenic and ability to translocate these two factors were primary which determine the arsenic distribution in the plant body¹⁰. Studies on arsenic availability in the plant body are important as they can bioaccumulate in human tissue through the food chain¹¹. The effects of chronic arsenic exposure on humans can cause serious health effects like keratosis, hepatomegaly, pigmentation, diabetes mellitus, cardiovascular and cerebrovascular problems, peripheral neuropathy, kidney, bladder, and skin, etc.^12,13.

Keeping the facts in mind, this study urge to explore the sensitivity and tolerance against the arsenic toxicity in a common plant species, which is widely used as a food ingredient in most of the part of India, and it also will assess the health risk among the population consuming this edible plant species.

Materials and Methods

Experimental Methodology

Seeds of fenugreek (Trigonella foenum-graecum)were procured from the grocery stores. Generally, fenugreek leaves were used as vegetables, and seeds were used as common Indian spices. The entire experiment was performed in the laboratory condition. Three different concentrations of sodium arsenite (NaAsO₂) and sodium arsenate (Na₂HAsO₄, 7H₂O) (dissolved in distilled water only) solutions were used for this study. The sterilization process of the experimental seeds was carried out with 1% sodium hypochlorite (NaOCl) solution for a minute and then washed with double distilled water (DDW)¹⁴. The process of seeds germination and growth was made in the soil collected from the institute campus using Petri dishes. Petri dishes were covered with lids and were incubated at room temperature (30°C). Stock solutions of the As⁺³和As⁺⁵were prepared by the required amount of arsenic salts in DDW to make 100 ppm solution for each As⁺³和As⁺⁵. Required dilutions were made to get the working solutions i.e. 1 mg/L, 2 mg/L, and 3 mg/L. The experimental study was performed using respective arsenic solutions. The Petri dishes were periodically nourished and moisturized during the entire experimental period with 10 ml of the solution once a day. Control sets of experiments were set up by using only DDW. Three replicates were taken for each treatment and control. Germination and growth of the seeds were observed for 10 days. Estimation of chlorophyll, Carotenoid contents, dry and wet weight was recorded after 10 days of the experiment (Fig. 1).

Figure 1: Growth of Control and Arsenic-Treated Fenugreek Plants in Laboratory Condition. Click here to view Figure

Determination of Fresh and Dry Weights

A total of 10 fully grown seedlings with almost uniform size were taken from each dish (both from control and treatments) and were thoroughly washed with DDW to remove the excess soil. Tissue papers were used to soak the excess water. Fresh weights were taken after the seedling was room dried and dry weights were taken after the seedlings were dried at 70°C for 48 hr in an oven¹⁴.

Estimation of Chlorophyll and Carotenoid

After 10 days of the experiment, fenugreek leaves were taken for the estimation of chlorophyll and carotenoid. Standard methodologies were followed for the estimation of chlorophyll and carotenoid¹⁵.

Total Arsenic Estimation in Plants

For estimation of total arsenic, plants were weighed to measure their mass. Two grams of dry plant sample was digested in the tri-acid mixture (HCl-HNO₃-HClO₄) until the mixture became a muddy-like texture. The digestion mixture was diluted with DDW to make up the volume to 50 ml and then it was filtered with filter papers. Finally, the filtrate was used for the estimation of arsenic in Atomic Absorption Spectroscopy (Bruker, ARTAX).

Bioaccumulation Factor Calculation

Bioaccumulation factors (BAF) were calculated by taking the ratio of concentrations found in plants to the soil arsenic concentration.



Where C_As-plantis the concentration of arsenic in the plant body and C_As-Soilis the concentration of arsenic in the soil.

Chronic Intake Dose (CDI) Estimation

CDI is calculated taking the arsenic concentration found in the fenugreek plant and following the formula:



Where, C_As= As-concentration (mg/kg), IR_veg= Intake rate of contaminated plant (mg/day), CF = Conversion factor, BW = Body weight

Risk Assessment

The mean value of total arsenic concentrations was used in the estimated daily dose (EDD) calculation (Eq. 1)^16,17.



Where, C_As= As-concentration (mg/kg), IR_veg= Intake rate of contaminated palnt (mg/day), CF = Conversion factor, EF = Exposure factor, ED = Exposure duration (years), LE = Lifetime expectancy, BW = Body weight

Determination of Hazard Quotient and Hazard Index

A Hazard Quotient (HQ) is the proportion between potential exposures to the safe level of exposure. Hazard Quotient less than 1predicted to be the safest level. The reference dose (RfD) is the limit above which any chemicals may pose a toxic effect on the health of the human. HQ was estimated by following Eq. 3.The hazard index (HI) was calculated as the sum of average HQ values¹⁸.





Incremental Life Time Cancer Risk Assessment

For assessment of the incremental lifetime cancer risk (ILTCR), the established formula (Eq. 2) was used¹⁸. According to USEPA (2010), ILTCR value > 10⁻⁴indicates high-potential cancer risk, and < 10⁻⁴indicates probable health risk.



Where, Where, C_As= As-concentration (mg/kg), IR_veg= Intake rate of contaminated plant (mg/day), CF = Conversion factor, EF = Exposure factor, ED = Exposure duration (years), LE = Lifetime expectancy, BW = Body weight, CFS = cancer slope factor (per mg/kg/day)

Results and Discussion

Table 1 represents the fresh and dry weight of the control and treatment plant samples. After the experimental period was over, it was observed that control plants showed higher weight than that of treatments. Among all the treatments, 3 mg/L treatments in both As⁺³和As⁺⁵显示最低的湿重(4.899±0.700克;和4.558 ± 0.088 g) as well as dry weight (0.612 ± 0.030 g; and 0.497 ± 0.039 g), respectively. Chlorophyll and carotenoid contents were estimated in both the control and treatment groups. It was observed from table 2 that chlorophyll and carotenoid contents were decreased irrespective of treatments than that of the control group. However, results found that with the increasing concentrations of arsenic the chlorophyll and carotenoid contents were decreased in both the treatment groups. Results revealed that in 3 mg/L treatment of As⁺³, the chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid were found 0.413 ± 0.093 mg/g, 0.163 ± 0.028 mg/g, 0.609 ± 0.124 mg/g, and 0.109 ± 0.030 mg/g, respectively. This decrease in concentration indicates that the chlorophyll synthesis system and chlorophyllase activities might be affected by the exposure to a high level of toxic metal concentrations⁵. The same trends have been observed in 3 mg/L of As⁺⁵treatment (0.532 ± 0.064, 0.2 ± 0.038, 0.768 ± 0.109, and 0.145 ± 0.035 in case of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid, respectively. Although there was no previous report on the effect of arsenic concentration on the chlorophyll content in the fenugreek plant, however, Miteva and Merakchiyska (2002) reported that arsenic may put an effect on the photosynthetic process in bean plants¹⁹. The uptake potential of arsenic by a crop depends on the concentration of arsenic in water, soil and it also depends on soil and crop’s physiological properties³. The concentration of soil arsenic and in the plant shoot portion in each treatment were represented in Table 3. Probably these higher arsenic contents might have resulted from the direct absorption of arsenic from the soil environment. Similar results have been observed by previous researchers, who reported that the concentrations of arsenic in the plant's body might increase more or less linearly with the total concentration of arsenic available in the soil^2,1,20. Moreover, it was also reported by previous researchers that the high concentration of arsenic in the soil might result in a high concentration of arsenic in the plant body⁴.

Table 1:Fresh and Dry Weight of Fenugreek Plants.

Table 2:Chlorophyll and Carotenoid Content of the Control, As⁺³, and As⁺⁵Treated Fenugreek Plants.

Table 3:Concentration of Arsenic in Soils and in Fenugreek Plants and Respective Bioaccumulation Factors.

BAF: Bioaccumulation Factor

On the other hand, soil arsenic concentration was also estimated to find out the ability of arsenic accumulation by fenugreek plants. However, bioaccumulation factor (BAF) analysis results showed that an independent relationship with soil arsenic concentration (Table 3). The highest bioaccumulation factor was found in 2 mg/L As⁺³treatments (1.028) and 3 mg/L As⁺⁵treatments (1.573). The study results showed that the increased concentration of soil Arsenic may not certainly represent higher arsenic accumulation by the fenugreek plants. This result may differ in different plants due to the difference in physiological characteristics. Moreover, our present study results were very similar to the study reported by Mandal and Suzuki (2002)²¹. On the other hand, Das et al., (2004) was also observed that the concentration of arsenic in potato, vegetable leaves, rice, spinach, the gourd was in the range between 0.02 to 3.99 mg kg⁻¹while estimated from an arsenic-contaminated field²². Pendergrass and Butcher (2006) were also have found the bioaccumulation of arsenic in the range of 1.5 to 178 mg kg⁻¹in plants²³. Correlation study between soil and plant arsenic concentration with pigment contents were represented in Table 4. The study result showed that soil and plant arsenic concentration had a negative correlation with all estimated pigment contents i.e., chlorophyll a: -0.699; chlorophyll b: -0.474; total chlorophyll: -0.638; carotenoid: -0.783 and chlorophyll a: -0.445; chlorophyll b: -0.081; total chlorophyll: -0.277; carotenoid: -0.124. It was clear from the study results that pigments contents were affected by the arsenic concentrations which invariably means that fenugreek is not an arsenic resistant species but it can be used as an indicator plant variety in arsenic contaminated areas. It is important to measure the level of toxicity by quantifying the arsenic exposure pathways to determine the human health risk. To estimate the potential human health risk associated with fenugreek consumption, chronic daily intake (CDI) was estimated. Table 5 summarized the CDI and HQ for adults and children upon consumption. The results revealed that the CDI values for adults and children were relatively high in both arsenic (As⁺³和As⁺⁵) concentrations. However, all CDI values are < 1. HQ is used often to evaluate a possible non-cancerous health risk from toxic pollutants. It was observed that most of the HQ values for children were high than the adults irrespective of treatments. It might be due to higher intake rates and lower body weights for the children. Some previous community-level studies reported that children and teenagers might pose a high risk due to the consumption of arsenic-contaminated vegetables²⁴. EDD and ILTCR were estimated to determine the cancer risks associated with arsenic-contaminated fenugreek shoot ingestion by the common people (Table 6). The EDD values were found greater than the acceptable limits for daily exposure of total arsenic. The study results revealed that the potential cancer risk was found from the estimation of ILTCR for both adults and children as the values found to be > 10⁻⁴.

Table 4:Correlation Between Plant Chlorophyll and Carotenoid Contents, Plant and Soil Arsenic Concentration.

** Significant at the 0.01 level (2-tailed)

* Significant at the 0.05 level (2-tailed)

Table 5:CDI和总部值计算的浓度n Estimated from Fenugreek in Different Treatments.

Table 6:CalculatedEDD and ILTCR Values from the Concentration Estimated from Fenugreek in Different Treatments.

Conclusion

Findings from this research will add some new information on arsenic toxicity assessment in an edible vegetable plant. The present study showed that the pigment content decreased in fenugreek with the increasing concentration of both As⁺³和As⁺⁵treatments. This study also concluded that a high concentration of soil arsenic content does not necessarily mean a higher accumulation rate in plant body parts and it may depend on the plant physiological properties. Results also indicated that fenugreek grown in arsenic-contaminated areas may pose a potential health risk upon consumption as a vegetable by adults and children. This experimental study results concluded that fenugreek may be used as an indicator plant species in arsenic-contaminated areas which will be helpful for easy health risk assessment.

Acknowledgment

The authors want to thank the Department of Environmental Health Engineering, Faculty of Public Health, Sri Ramachandra Institute of Higher Education and Research (DU), Chennai for permitting to conduct the study.

Funding Source

作者真诚地感谢“总理的夏er Fellowships for UG of Sri Ramachandra Institute of Higher Education and Research (DU) – 2019” for funding the research study.

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