Arsenic accumulation in lentil (Lens culinaris) genotypes and risk associated with the consumption of grains

Arsenic (As) is a toxic metalloid. As phyto-toxicity is manifested by its accumulation in different tissue types and subsequent growth inhibition in plants. Despite the vital role of leguminous crops in providing proteins to human diets, a little is known about the As accumulation in lentil. In this study, the rate of As uptake and transport from soil to root, shoot and grain of lentil as well as associated risks with the consumption of As contaminated food were examined. Biomass accumulation of lentil genotypes pardina, red chief and precoz drastically decreased when treated with As at 6 mg kg−1 concentration in comparison to 0 and 3 mg kg−1 As. Quantification of As concentrations following different treatment periods showed that As accumulation in roots and shoots of 0, 3 and 6 mg kg−1 As-treated lentil genotypes was statistically different. Arsenic content in grains of red chief genotype was found significantly lower than pardina and precoz. Moreover, As transport significantly increased in roots and shoots compared to the grains. Due to the high concentrations of As in biomass of lentil genotypes, animal as well as human health risk might be associated with the consumption of the As contaminated legume crops.


Results
Dry weight of root, shoot and pod. Dry weights of pardina roots were found 0.333, 0.389 and 0.264 g in 0, 3 and 6 mg kg −1 As treated pots, respectively at week 6. Similarly, dry weights of red chief roots were found 0.349, 0.497 and 0.301 g at 0, 3 and 6 mg kg −1 As treated pots, respectively. Chronologically, average dry weights of root, shoot and pod were found lower in 6 mg kg −1 As treated lentil cultivars at week 6, 10 and 13. On the other hand, average dry weight of red chief root, shoot and pod at 0, 3 and 6 mg kg −1 As treated condition were found significantly higher than other lentil genotypes at week 6, 10 and 13 (Figs 1a-3b). Treatment and lentil varieties both showed significant differences on the dry weight of root at week 6. Similarly, treatments and varieties both were found significantly different on the effect of dry weight of lentil shoot at week 6, 10 and 13. In week 10, treatment and varietal effects on the dry weight of pod were found statistically different. On the other hand, only treatment effect was found significantly different on the pod dry weight at week 13 (Table 1).
Arsenic accumulation in root. Treatment, interaction result of variety and treatment on As uptake in root of lentil genotypes were found significantly different at week 6 and 10. On the other hand, only treatment effect on As accumulation in root was found significantly different at week 13 (Table 2). Arsenic accumulation in root was found significantly higher at A 6 (6 mg kg −1 ) treated pardina, red chief and precoz lentil genotypes than control (0 mg kg −1 ) and A 3 (3 mg kg −1 ) during week 6, 10 and 13. Based on the treatment period, accumulation of As in root of control, A 3 and A 6 treated lentil genotypes were found statistically different. For instance, As uptake in root of pardina, red chief and precoz lentil genotypes at week 6 was found significantly higher than week 10 and 13 (Table 3). However, As uptake was found at similar rate during week 6 and 10 in root of these lentil genotypes. In week 13, As uptake in red chief genotype was lower than precoz and pardina (Table 3). Arsenic uptake in roots in A 6 treated lentil genotypes was more than other As treatments.
Arsenic accumulation in shoot. Treatment effect on As accumulation in shoot of lentil genotypes was found statistically significant during week 6, 10 and 13 according to significance test. On the other hand, varieties were found statistically different for As uptake in shoot at week 6 rather than week 10 and 13 (Table 2). Arsenic accumulation in the shoot was found higher at A 6 treated pardina, precoz and red chief lentil genotypes during www.nature.com/scientificreports www.nature.com/scientificreports/ week 6, 10 and 13 (Table 3). On the other hand, accumulation of As at week 6 and 13 in the shoot of the pardina, and red chief genotype was found significantly different between A 6 and control, and A 6 and A 3 treated lentil genotypes. According to week, accumulation of As in shoot of control, A 3 and A 6 treated lentil genotype was found statistically different. Red chief was found significantly different at week 13 for uptake of As in its shoot from week 6 and 10. Arsenic accumulation in shoots of pardina, red chief and precoz genotypes were found statistically week 13. Arsenic treatments indicate concentration of As in peat moss; A, control, 0 mg As kg −1 peat moss; A 3 , 3.0 mg As kg −1 peat moss; A 6 , 6.0 mg As kg −1 peat moss. Means denoted by different letters under the same As level indicate significant difference at 0.1% level of significance.  Table 1. Significance test on the dry weight of lentil root, shoot and pod at different As treated lentil plant at week, 6, 10 and 13. ***Indicates significant difference at p < 0.001 level of significance **Indicates significant difference at p < 0.01 level of significance, *Indicates significant difference at p < 0.05 level of significance, (@) Indicates significant difference at p < 0.1 level of significance. www.nature.com/scientificreports www.nature.com/scientificreports/ insignificant at week 6 and 10 ( Table 3). Arsenic uptake in shoot with A 6 treated lentil genotypes was more than other As treatments (Table 3).
Arsenic accumulation in grain. Treatment and varietal effects on As accumulation in grains of lentil genotypes were found statistically significant during week 10 and 13 ( Table 2). Arsenic accumulation in grains was found statistically insignificant between control, A 3 and A 6 treated pardina and precoz lentil genotype during week 10 and 13. At week 10 and 13, As accumulation in grains of red chief genotype was found statistically different between the control and A 6 treatment. According to week, accumulation of As in grain between control, and A 6 treated lentil genotypes was found statistically dissimilar. This uptake in the grains was found statistically insignificant between A 3 and A 6 treatment. Arsenic uptake in grains of red chief was found significantly lower than pardina and precoz at week 13. Arsenic uptake in grains showed insignificant difference between pardina and precoz (Table 3). Arsenic accumulation in grains was found lower than root and shoot during week 10 and 13. Arsenic in grains increased by 20% and 40% in red chief and precoz with 6 mg As kg −1 peat moss as compared to 3 mg As kg −1 peat moss after 13 week of growth. Arsenic in grain was found 17% higher by the treatment of 6 mg As kg −1 peat moss in comparison to 3 mg As kg −1 peat moss after week 13. Red chief genotype was found low As accumulator in contrast to pardina and precoz (Table 3).

Discussion
Arsenic (As) is a lethal metalloid. Its accumulation in plant tissues and associated health risk with the consumption of As contaminated grains are matters of huge public concern. Among food crops, rice as well as lentil is also grown in As contaminated areas in Bangladesh and other contaminated areas in the world. Arsenic is translocated from soil to root shoot and grains of lentil and other food crops 33 . As a result, As reduces the biomass accumulation of lentil genotypes. Lentil seedlings showed normal growth in As free pots. The seedling growth was negatively affected by increasing the rate of As concentration in pardina, red chief and precoz genotypes. Similarly 34 , conducted research on As toxicity in food crops. They found a high concentration of As decreased the plant growth and development by inducing phyto-toxicity. Due to the As toxicity, germination, plant height, number of roots and shoot growth were negatively affected which might eventually lead to the death of food crops [35][36][37][38][39] . The transportation and accumulation of As in plants followed the order, roots > shoots > grains 40,41 . Arsenic contamination in growing media (0.2 mg kg −1 ) causes negative effects on food crops 42,43 . Similarly, 0.6 mg kg −1 As in soil affected soybean growth 44 . Likewise, biomass of 3 mg kg −1 and 6 mg kg −1 As-treated pardina, red chief and precoz lentil genotypes significantly decreased compared to the control plants grown in As free medium (Figs 1a-3b).
Arsenic is one of the most toxic elements for the reduction of biomass production in food crops. In this experiment, dry weight of lentil genotypes was found lower in As treated lentil crops than As free crops (Figs 1a-3b). It is in agreement with the previous studies as As contamination could reduce dry weight of root and shoot in maize and sunflower plants 45 . Similarly, tomato (Lycopersicum esculentum) plants grown under different levels of As show As toxicity. Arsenic exposure resulted in a drastic decrease in plant growth parameters (e.g., maximum decrease of 76.8% in leaf fresh weight) and fruit yield in tomato crops (maximum reduction of 79.6%) 46 . Upon translocation of As can rigorously constrain plant growth by slowing or arresting expansion and biomass production as well as compromising plant reproductive capacity through losses in fertility, yield, and fruit production 20 .
Consequently, As accumulation significantly increased in the root of lentil genotypes from soil solutions. Pardina, red chief and precoz, varieties were shown to have significant uptake of As in their roots ( Table 3). The results are in agreement with a study on chickpea (Cicer arietinum L.), a major supplementary food in many areas throughout the world 47 . On the other hand, in mangrove plants such as, (Aegiceras corniculatum L.), seedlings grown in As contaminated soils, showed increased As concentrations in roots stems and leaves with increasing treatment concentrations of As, but the As accumulation rates in the roots were found 74.54-89.26% of the total As accumulation in the plants 48 . A similar range of As in the roots (14.5-27.4 mg kg −1 ) was found in maize (Zea mays) 49 . Arsenic treatments indicate concentration of As in peat moss; A, control, 0 mg As kg −1 peat moss; A 3 , 3.0 mg As kg −1 peat moss; A 6 , 6.0 mg As kg −1 peat moss. Means denoted by different letters under the same As level indicate significant difference at 0.1% level of significance.
www.nature.com/scientificreports www.nature.com/scientificreports/ Notably, arsenic can be translocated from the roots to the shoots of plants. In the present study, As concentration significantly increased in the shoot of lentil genotypes (Tables 2 and 3). Similarly, As is transported significantly to the shoot, which impairs the growth, and biomass accumulation in rice plants 26,50 . In general, As accumulation in the plant increases with the increasing As concentrations in soils. Nonetheless, terrestrial plants such as legume crops show a higher concentration of As in shoot to root compared with emergent plants 51 .
Arsenic can be accumulated in grains of lentil crops, but this is not a level that is significant for all genotypes (Table 3). In this context, precoz genotype uptakes a significant level of As compared to pardina and red chief cultivars ( Table 3). As well as lentil genotypes, As can be accumulated in the edible grains of Phaseolus vulgaris. It is also found that increasing the concentration of As in plants decreases the plant growth as well in the lentil plants 52,53 . Research was also conducted on As uptake in several pulse crops such as pea which showed the highest As uptake (1.30 mg kg −1 ) 54 . Arsenic in selected paddy soils of China causes toxicity to bean, lentil, oats and other food crops 55,56 .
Extreme uptake of this metalloid may cause physiological changes in food crops, producing a wide range of detrimental effects, such as suppression of photosynthesis and pigment synthesis, oxidative stress, and other metabolic disturbances 57 . This metal (As) induces oxidative damage in lentil and other food crops due to its excessive uptake in their biomass. Oxidative stress is associated with the increasing levels of reactive oxygen species (ROS) and osmolytes in As stress condition 58 . Although the accumulation of As in roots, shoots were found higher than grains in lentil (Table 3), this element can be transferred to human and animal bodies through the food chains 59 . In human beings, inorganic As species arsenate (AsV) and arsenite (AsIII) are strongly cytotoxic and may lead to As-induced skin hyperkeratosis and cancer 60,61 . In Southern Asia, groundwater contaminated with As is used for the irrigation in food crops 8,62 . Lentil grown on As contaminated soil contains considerable amounts of As in shoots tissues and grains 63 . Persistence of this As within soil and its toxicity to plants, animals and human are  Table 3. Arsenic accumulation (mg kg −1 ) in root, shoot and grain in Pardina, Red Chief and Precoz lentil varieties after 6, 10 and 13 weeks of growth. Arsenic treatments indicate concentration of As in peat moss; control, 0 added arsenic; A 3 , 3.0 mg As kg −1 peat moss; A 6 , 6.0 mg As kg −1 peat moss. Means within each variety and time followed by a different letter indicate significant difference at p ≤ 0.001.
www.nature.com/scientificreports www.nature.com/scientificreports/ of grave concern. Long-term exposure to low concentrations of As can lead to skin, bladder, lung, and prostate cancer. Non-cancer effects of ingesting As at low levels include cardiovascular diseases, diabetes, and anemia 64,65 .
It is well understood that As is a threat for the development of lentil crops in As contaminated regions in the world. For this reason, good agricultural practices are significantly important to get an optimum yield in this food crop 66 . The optimum water and N doses both are important components as recognized good agricultural practices for attaining higher yield, which may enhance grain yield under abiotic stress 66 . As well, optimum temperature and radiation both are important indicator for the increasing of biomass 67 . Good agricultural practices such as optimum temperature, water use efficiency, radiation, and nutrient availability are all affected due to As stress in soils. On the other hand, drought, salinity, lead (Pb), Cd, Cu, Cr and As stress in food crops disrupt the photosynthesis and its associated metabolic activities 57 . This type of stress severely decreases the photosynthesis, water use efficiency, stomatal conductance, chlorophyll contents, and antioxidant defense mechanism in crops 68,69 . Likewise, oxidative damage and osmotic stress increase in food crops due to As toxicity 70,71 . In this situation, mycorrhyzal association might mitigate different abiotic stresses and minimize metal toxicity as well as associated health hazards [72][73][74][75] . Mycorrhizal fungi colonized with the root cortex and extended the network of its hyphae into the surrounding environment. These external hyphae can contribute to improving plant nutrients for increasing the biomass growth as well as can alleviate heavy metal toxicity by modulating the metal acquisition in plants from contaminated soils 76 . In addition, agronomic and civil engineering methods such as, judicious use of water, management of soil and plant-nutrients might be recommended in As prone crop growing areas to mitigate the building up of As in human food chain and thus minimizing the negative impact on the environment 77 . Chitosan (CH) and biochar (BC) can be used to reduce mobility and bioavailability of heavy metals and to facilitate plant growth by improving the antioxidant system 78,79 . It is to be noted that biomass of lentil should be toxin free to the end users. If biomass becomes affected in the As contaminated region, the associated risk with the consumption of As contaminated food would definitely increase. In this circumstance, reduction of As transportation from soil to food crops towards the human food chain is significantly important for a sustainable global environment.

Conclusion
Lentil is an important leguminous crop that provides protein to human diets. Arsenic accumulated in tissues of lentil genotypes and its reallocated to grains enhance health risk with the consumption of contaminated tissue. In this study, we found that concentration of As transport significantly increased from soils to root and shoot tissues and grains in lentil genotypes. Due to such high As transport, biomass of the crops was negatively affected in their entire life cycle. As a result, root and shoot mass of lentil genotypes was found significantly affected. Pardina, red chief and precoz lentil genotypes responded remarkably in terms of As uptake from soils to their root, shoot and grains. Arsenic in grains was increased 17% by the treatment of 6 mg As kg −1 peat moss in comparison to 3 mg As kg −1 peat moss after 13 week of growth. Thus, the toxic metalloid (As) might transfer to the human body through the consumption of grains, thereby increasing health risks. Therefore, development of As mitigation technologies that could improve plant growth by restricting As transport to plant tissue is urgently needed to expand lentil production in the As contaminated regions throughout the world as well as the reduction of health risk with the consumption of this food crop.

Methods
Peat Moss and Pot. Peat moss was collected from the local market in the USA. This growing media was brought to the greenhouse in the Department of Crop and Soil Sciences at Washington State University (WSU) for the growing of different lentil genotypes. The sizes of pots were 275-300 ml volume. All pots were made of toxin free plastic.
Lentil genotypes and nutrient added from fertilizers. Precoz, red chief, and pardina lentil genotypes were collected from ICARDA (International Center for Agricultural Research in the Dry Areas) for this pot experiment. Slow released mixed fertilizer (Osmocote plus) was purchased from the local market in the USA. The ratios of N P K 15: 9: 12 were found in this slow released fertilizer. Out of the 15% total Nitrogen (N), 8.4 and 6.6% were added to its fertilizer from the source of ammoniacal and nitrate nitrogen, respectively. On the other hand, available phosphate (P 2 O 5 ), soluble potash (K 2 O), total magnesium (Mg), sulfur (S), boron (B), cupper (Cu), total iron (Fe), manganese (Mn), molybdenum (Mo), and zinc (Zn) at 9, 12, 1.3, 5.9, 0.02, 0.05, 0.46, 0.06, 0.02 and 0.05% were added from this slow released fertilizer in this pot experiment, respectively.
Sowing of lentil seeds. Precoz, red chief, and pardina seeds were sown in the pots on 13 th March, 2014. Seven to 8 seeds from each variety were spread at 2-3 cm depth in peat moss in each pot. After the emergence of the lentil seeds, only 5 seedlings were kept in each pot for further sampling during different stages of lentil plant. Recommended doses (2 g pot −1 ) of slow released fertilizers were applied during seedling and flowering stages of lentil plants.

Preparation and application of arsenic diluted solution in lentil plants. Sodium arsenate dibasic
heptahydrate (Na 2 HAsO 4 .7H 2 O) was used as the source of As. Deionized water was used for the preparation of As diluted solutions. Arsenic solutions were applied from seedling to mature stages at intervals of about every 7 days. About 0.012494 g sodium arsenate dibasic heptahydrate was added to 1liter water for the preparation of 3 mg L −1 concentrated As solution. On the other hand, 0.02498 g of sodium arsenate dibasic heptahydrate was taken for the preparation of 6 mg L −1 concentrate As dilute solution. These diluted solutions were kept in bottles with proper labeling and preserved in a refrigerator for further application in the pot experiment.
Treatments and replications in pot experiment. Arsenic free peat moss was used as the growing media in this pot experiment. Three treatments were followed such as, control (A) = 0 mg As kg −1 peat moss, A 3 = 3 mg As kg −1 peat moss and A 6 = 6 mg As kg −1 peat moss. Three varieties of lentils, precoz, red chief and pardina were selected for this pot experiment. Three replications with three lentil varieties were followed at week 6, 10 and 13 sampling point and the total number of pots was 81 for this experiment.
www.nature.com/scientificreports www.nature.com/scientificreports/ Collection of plant sample at week 6, 10 and 13. Precoz, red chief and pardina seedlings were collected from each of the treatment at week 6, 10 and 13. Three pots including seedlings of each genotypes were removed from each treated tray randomly at week 6. After collection of seedlings, roots were washed with distilled water. Then water was removed from the washed roots using tissue paper. Roots were separated from the shoot of each collected lentil seedlings using scissors. All root and shoot samples were kept separately inside an envelope with proper labeling on each sample. After that, all root and shoot samples were kept in a drying oven for 72 hours at 55-65 °C. Similarly, root, shoot and pod samples of lentil genotypes from each of the treated pots were collected at week 10 and 13 also.

Dry weight, grinding and sieving of plant samples. Dried samples were brought into the Laboratory of
Crop and Soil Sciences at WSU. Dry weight of root, shoot and pod were taken separately. Meanwhile, grains were separated from pod of lentil manually by hand with gloves. Hand gloves were changed during the separation of grain for each sample. Then the samples were ground separately by a coffee grinder using liquid nitrogen. The grinder was cleaned between the samples with ethyl alcohol (C 2 H 5 OH) and tissue papers. These ground root, shoot and grain samples were sieved with 250 µ mesh. Then all samples were kept in envelopes with proper labeling.
Digestion of samples. Lentil roots, shoots and grains (seeds) were digested separately following heating block digestion procedure 80 . Of the plant samples, 0.1 g ground root, shoot and grains samples were put into clean digestion vessels and 5 ml concentrate HNO 3 was added to it. The mixture was allowed to stand overnight under a fume hood. On the following day, this vessel was digested in a digestion block for 1 hour at 120 °C. This content cooled and 3 ml HClO 4 was added to it. Again, samples were put into the heating block for 3-4 hours at 140 °C. Generally, heating stopped whenever white dense fume of HClO 4 was emitted into air. Then cooled samples were diluted to 25 ml with de-ionized water and filtered using Whatman 42 filter paper. Finally, samples were stored in polyethylene bottles. Prior to samples digestion, all glassware was washed with 2% HNO 3 followed by rinsing with de-ionized water and drying.
Analysis of total arsenic. Digested samples were analyzed for the determination of total arsenic in the lentil root, shoot, and grains. The total arsenic in root and shoot tissue and reallocation to grains of the lentil plants was analyzed by flow injection hydride generation atomic absorption spectrophotometry (FI-HG-AAS, Perkin Elmer A Analyst 400-USA) using external calibration 81 . The optimum HCl concentration was 10% v/v and 0.4% NaBH 4 which produced the maximum sensitivity. For each sample of the digested lentil's root, shoot, and grains, three replicates were taken and the mean values obtained based on the calculation of those three replicates. Standard Reference Materials (SRM) from the National Institute of Standards and Technology (NIST), USA was analyzed using the same procedures from the start of the experiment, during and at the end of the measurements to ensure continued consistency and accuracy. Method detection limit (MDL) for As was 0.02 µg/l or ppb (parts per billion).

Statistical analysis.
The experiment was carried out following Completely Randomized Design (CRD).
Level of significance was analyzed between average dry weight of root, shoot and pod at different As levels in lentil genotypes using software R. Accordingly, the significance test for the comparison of As uptake in root, shoot and grains of lentil plants was performed using software R as well.

Data Availability
Data supporting the findings of the current study are available from the corresponding author on reasonable request. All data analyzed during this study are included in this published article.