Effect of llama (Lama glama) manure and Trichoderma strain T1R3 on arsenic uptake by Swiss chard and broad bean crops. A strategy to minimize health risks


 The town of Pastos Chicos (Jujuy-Argentina), presents arsenic (As) concentrations in soil (49 mgAs kg−1) and water (1.44 mgAs L−1) significantly above the maximum allowable limits set by National Laws No 24,585 and N° 24,051. This study aimed to evaluate the effect of llama manure (Lama glama) and Trichoderma strain T1R3 on As uptake and toxicity in Swiss chard (Beta vulgaris var. cicla) and broad bean (Vicia faba L.) crops, while assessing potential human health risks. Results indicated that Trichoderma strain T1R3 inoculation stimulated broad bean plant growth by reducing As stress. Swiss chard crops treated with 5% manure and a manure/Trichoderma T1R3 combination reduced As absorption from 32.46 to 64.02% in roots, and from 35.2 to 44.5% in leaves. Broad bean crops inoculated with Trichoderma T1R3 showed a significant mitigation of toxicant accumulation in the leaves (67.42%). Also, the manure/Trichoderma T1R3 combination reduced As accumulation (57.46%) in broad bean roots. The efficacy of llama manure and of the llama manure/Trichoderma T1R3 combination in reducing health hazards that derive from As intake by consuming chard leaves was also reflected in Hazard Quotient < 1 values. Although Cancer Risk values decreased considerably, these showed there was a considerable carcinogenic risk for humans consuming chard leaves. These observations reveal that adding llama manure and Trichoderma T1R3 might mitigate As uptake by crops, thus reducing human health risks. This study advanced our understanding the complex llama manure/Trichoderma strain interactions in As-contaminated soils, which are imperative for developing the effective mitigations strategies.


Introduction
Arsenic (As) is a hazardous element, which represents a signi cant risk to human health when introduced into the food chain, skin exposure, and inhalation (Farooq et al. 2016). Prolonged exposure to As by consuming water or food that contains certain As levels causes serious toxic effects, including skin lesions (pigmentation and keratosis) and lung and bladder cancer (Mandal et al. 2019). Therefore, immobilizing As in contaminated soils may constitute an effective strategy to reduce its accumulation in plants and food (Arco-Lázaro et al. 2016). Actions to mitigate As toxicity and mobility in plants involve the use of organic amendments, and the inoculation of soil with bene cial microorganisms, such as bacteria and ectomycorrhizal fungi (Luo et al. 2016;Nawab et al. 2018). In particular, the genus Trichoderma tolerates heavy metals, and using it to immobilize and remove As constitutes an eco-friendly Page 4/23 proteins, vitamins, amino acids, and minerals, among others. These crops are produced in areas with high As contents in water and soil, exceeding the allowable limits established by the National Law N° 24,051 (0.10 mgAs L −1 in irrigation water), and by National Law N o 24,585 (20 mgAs kg −1 in agricultural soil) (Yañez et al. 2018 and. Vegetables are generally highly sensitive to metal stress, and become a source of As poisoning for humans who grow and consume them. This has caused interest amongst scientists, who are exploring some sustainable and eco-friendly option for remediation and restoration of As-contaminated soils (Mehmood et al. 2017). Therefore, adding organic amendments and bene cial fungi to soil could reduce As accumulation in crops grown in areas polluted with this toxicant, diminishing its translocation to edible plant parts, and thus making agriculture safer and more sustainable.
This study aimed to evaluate the effect of llama manure (Lama glama) and Trichoderma strain T1R3 on As uptake and toxicity in Swiss chard (Beta vulgaris var. cicla) and broad bean (Vicia faba L.) crops, as well as assessing potential risks to health of humans consuming these vegetables.
Electrical conductivity was measured using the saturation extract method. Other determinations were total nitrogen, according to Bremner and Mulvaney (1982); bioavailable phosphorus content, by means of Bray and Kurtz's method (1945); and sodium and potassium complexes by ame photometry (Eaton et al. 2005). The soil and llama manure were processed for total As quanti cation using acid digestion according to USEPA method 3050 B (1996). Pulverized samples (0.5 g) were transferred to Te on beakers, where 10 mL of 50% HNO 3 was added. The solutions were heated on a hot plate at 95°C±5 with a watch glass for 2 h, until they evaporated (without boiling) to about 5 mL. Subsequently, 3 mL of hydrogen peroxide (30% H 2 O 2 ) was added, and the solutions were again heated until the sample suffered no further changes in its appearance. Finally, 10 mL of concentrated HCl was added to the solutions, followed by hot plate heating (95°C±5) for 15 min. After digestion, total As concentration was determined with a hydride generation-atomic absorption spectrometer (HG-AAS), methodology described below.
Soil, water, and llama manure were collected in a single sampling to avoid variations in physical-chemical properties and As content.

Microorganism and culture conditions
The fungus used was As tolerant Trichoderma strain T1R3 previously selected by Yañez et al. (2017). To obtain an appropriate inoculum (spore suspensions), strain T1R3 was pre-cultivated on potato dextrose agar (PDA) medium, and incubated for 7 days at 28 ° C. Subsequently, 10 mL of sterile 0.85% NaCl was added to release the chlamydospores from the surface of the medium. The ltered suspension was vigorously stirred and subsequently centrifuged at 9000xg, for 10 min. The supernatant was discarded, and the precipitated chlamydospores were re-suspended in sterile 0.85% NaCl (10 7 log units/mL CFU).

Aqueous extract of llama manure
An aqueous manure extract was prepared by nely disintegrating the manure in a mortar, and sieving it with a 2 mm pore size mesh to remove stones, plastic, paper, among other elements. Afterwards, a suspension was made in 5% distilled water, and stirred for 12 h at 150 rpm. This aqueous suspension was then ltered to remove the non-solubilized material, and the resulting extract was kept cold until its use.

Arsenic uptake mitigation experiments
The broad bean seeds used in the trial belonged to the Agua Dulce variety and were provided by the "Pro Huerta" program of Ministerio de Desarrollo Social de la Nación Argentina, whereas the Swiss chard (Beta vulgaris var. cicla) seeds were supplied by the company Emeral Seeds-USA.
The chard seeds were germinated in multipot trays and transferred to black polyethylene bags, each containing 1 kg of soil. Each broad bean seed was kept in a bag too, and sown directly into 3 kg of soil.
The chard and broad bean tests lasted 60 and 180 days, respectively. The crops were grown in soil typical of Pastos Chicos and irrigation water came from the local river (Yañez et al. 2018). Constant volumes of irrigation waters were added to each pot of the experiment, in order to maintain the soil moisture at 70% of the eld capacity, avoiding any phenomenon of leaching. Different pot experiments were carried out in a greenhouse at ambient temperature, with a natural light and darkness regime. The seeds were surface sterilized and dipped in a Trichoderma T1R3 chlamydospores suspension inoculum, at a CFU of 10 7 log units/mL. One month after cultivating chards, and two months after cultivating broad beans, these were again inoculated with 1 mL of the spore suspension, and with 10 mL of the aqueous llama manure extract. In this study, As uptake mitigation experiments had a completely randomized design, with a total of 60 pots, including the amended and control soils, was prepared in triplicate.
The treatments were as follows: 1) Chard seedlings and broad bean seeds in soil with As, irrigated with As-contaminated water (control).
2) Chard seedlings and broad bean seeds in soil with As, irrigated with As-contaminated water and applied with 5% (w/w) llama manure.
3) Chard seedlings and broad bean seeds in soil with As, irrigated with As-contaminated water and applied with a Trichoderma T1R3 spore suspension. 4) Chard seedlings and broad bean seeds in soil with As, irrigated with As-contaminated water and application of 5% llama manure (w/w) and a Trichoderma T1R3 spore suspension.
Salinity reduces plant growth, development, yield and seed quality when the concentration the salts reaches 4 dS m −1 (Acosta-Motos et al. 2017). Thus, the 5% llama manure was chosen according to electrical conductivity of 3.42 dS m −1 in the manure-soil combination.
For dry weight and As content analysis, roots and aerial parts different plant parts were oven dried at 70°C for 72 h. Samples had been dried following the As quanti cation methodology (as described below), but the dry weight values were turned into wet weight, so as to reveal the potential health risks brought about by consuming produce from these crops. The As concentration in wet weight was determined considering a humidity of 90.6%, according to the following equation: wet weight concentration = dry weight concentration × (1 -% humidity) (Costa et al. 2003).
Arsenic mobility in the chard and broad bean crops was evaluated by estimating translocation factors (TF) according to following equation:

AsT root
Where, AsT aerial and AsT root represent the total As concentration in the aerial and root plant (Sharma et al. 2020).

Non-carcinogenic health risks
The health risk represented by the intake of As through consumption of Swiss chard was assessed in terms of the hazard quotient (HQ), which was calculated with the following Eq. (the corresponding parameters are explained in Table 1):

Cancer risk assessment
The Cancer Risk (CR) which derives from consuming Swiss chard was determined using the following equation, as described by Shahid et al. (2017) (its parameters are explained in Table 1).
A CR lower than 10 −6 is considered to be negligible, one between 10 −6 and 10 −4 is generally considered acceptable, whereas a CR above 10 −4 is deemed unacceptable, with a high potential for causing cancer (Muñoz et al. 2017).

Total arsenic determination
For the analysis of total As, 1 g dried sample of plant was digested in a mixture of the mineralizing agent, 20% w/v magnesium nitrate [Mg(NO 3 ) 2 ] and 2% w/v magnesium oxide (MgO). Then, 5 mL of 50% v/v nitric acid (HNO 3 ) was added to promote organic matter oxidation, and the sample was heated on a hot plate at 90°C. Finally, the preparation was mu ed at 550°C for 24 h until white ash was formed. This was resuspended in 10% v/v hydrochloric acid (HCl) to measure total As. The suspension was subjected to a prereduction step with a solution of potassium iodide/ascorbic acid. Arsine (AsH 3 ) was then formed by reaction with a solution of sodium borohydride (BH 4 Na) in an alkaline medium, together with a solution of HCl as the source of hydrogen ions using a HG-AAS (PerkinElmer AAnalyst 100, interfaced with the FIAS 400 hydride generator). The detection limit of the method was 0.1 µgAs L −1 and quanti cation limit was 0.3 µgAs L −1 , with a linear response of up to 5 µgAs L −1 (r = 0.9996). Relative error amounted to 10%, and equipment sensitivity was checked with external certi ed standards (0.1 µgAs L −1 , Certipur-Merck).

Statistical analysis
The results are expressed as mean values with standard deviations (SDs) as a measure of dispersion (means ± SD). The differences between individual means were compared by one-way analysis of variance (ANOVA). When signi cant differences were found, Duncan's post-test was used to separate the effects among treatments. Tests were considered signi cantly different at p < 0.05. These statistical analyses were performed using professional versions of Infostat software.

Soil, llama manure and irrigation water characterization
The physicochemical properties of the soil, llama manure, and irrigation water used in this study are presented in Table 2. The soil from Pastos Chicos presented a sandy-loan texture. As reported by Mehmood et al. (2017), a higher sand amount in the soil contributes to low As retention, which represents a greater amount of As available for the crops. Considering its electrical conductivity value (1.98 dS m −1 ), the soil had the characteristics of an alkaline and slightly saline type, according to Richards' criteria (1982). The soil presented a moderately alkaline pH (8.3), 5.12% organic matter content, and a high proportion of phosphorus (72.8 mg kg −1 ). It also had a total As concentration of 49 mg kg −1 , exceeding the maximum As concentration level of 20 mg kg −1 recommended by National Law Nº 24,585 for agricultural soils.
The llama manure presented a slightly alkaline pH (7.6), and a content of organic matter (23%) which represents a good quality organic fertilizer (Chan et al. 2016). According to Richards (1982), the electrical conductivity (12.76 dS m −1 ) of the saturation extract indicated a manure with high salinity. In addition, the manure revealed high contents of nitrogen (1.6%), phosphorus (418 mg kg −1 ), and potassium (23.20 cmol c kg −1 ), important nutrients for plant growth and development (Shrivastav et al. 2020). The chemical analysis of the llama manure showed a total As concentration of 13.3 mg kg −1 , this re ects that water and vegetation are a source of As transfer in the food chain.
The water had a moderately alkaline pH (8.25), and an electrical conductivity of 2.58 dS/m, which indicates very high salinity according to Richards (1954). A sodium absorption ratio (SAR) of 9.92 was found, which represents a high risk of soil salinization (Yañez et al., 2018). In addition, water analysis showed a total As content of 1.44 mg L −1 , which was 14-fold higher than the maximum limit allowed for irrigation water (National Law Nº 24,051).
3.2 Growth of Swiss chard and broad bean crops exposed to arsenic, llama manure and Trichoderma T1R3 Dry biomass is a critical parameter for assessing As effects on crop growth . The results showed that in the chard crops treated with llama manure alone, the total dry biomass of plants was signi cantly lower than the control (Fig. 1). In the T1R3 strain treatment and in those with the llama manure/Trichoderma T1R3 combination no produced signi cantly differences among them and the control (Fig. 1). The As content in llama manure (13 mg kg −1 ), the soil (49 mg kg −1 ), and irrigation water (1.44 mg L −1 ) negatively in uenced plant growth and led to a lower biomass production. It is well known that As has negative effects on plant metabolic functions, reducing growth, and changing nutrient balance and assimilation (Mirza et al. 2016). Besides, it can interfere with photosynthetic activity, metabolic processes, and water absorption (Gusman et al. 2013). Additionally, it causes oxidative stress and lipid peroxidation due to the overproduction of reactive oxygen species (ROS), such as hydrogen peroxide, superoxide, and hydroxyl radicals (Tripathi et al. 2017). For example, Tripathi et al. (2013) showed that As stress negatively affected chickpea (Cicer arietinum L.) germination (25.9%), and stem length (15%) and diameter (30%) in comparison to the control treatment. Also, they registered a signi cant decrease in root growth, which also affected lateral roots (58%), and in root dry weight (66%) and length (49%). In addition, the high calcium content in the Pastos Chicos soil may reduce phosphate availability to plants due to the possible formation of Ca-phosphate precipitates (Mehmood et al. 2017). According to the soil texture (sandy loam), phosphorus contents added through the llama manure amendments could have displaced As ions adsorb onto sand particles, thus increasing As uptake and reducing plant growth (Anawar et al. 2018). Similarly, Klaber and Barker (2014) demonstrated that growth of rice cutgrass (Leersia oryzoides Sw.) and tall fescue (Festuca arundinacea Schreb.) was not enhanced by phosphorus fertilization.
The broad bean crops inoculated with Trichoderma T1R3 strain showed signi cant differences respect to the control (Fig. 1). The fungus application considerably stimulated broad bean plant growth, and possibly increased phosphorus and nitrogen uptake and the production of auxin and siderophores, ameliorating the adverse effects of As toxicity (Zhang et al. 2018;Khoshmanzar et al. 2019). These results are in accordance with those reported by Caporale et al. (2014), who published the bene cial effects of Trichoderma harzianum strain T22 and Trichoderma atroviride strain P1 on lettuce growth. These authors also reported a reduction of As toxicity when the plants were irrigated with Ascontaminated water (5 and 10 mg L −1 ). In this sense, Anawar et al. (2018) revealed that irrigating with Asrich water may change the As-phosphorus balance in the soil solution, causing the mobilization and availability of phosphorus for the plant nutrition. Furthermore, Tripathi et al. (2017) suggested that As is methylated in soils that have been inoculated with Trichoderma, and this could alleviate As stress in chickpea. Besides, as mentioned before, broad bean and chard crops growth might be determined by the synergistic relationships among the Trichoderma T1R3 strain, changes in As availability, and the physiological responses of the crops.

In uence of llama manure and Trichoderma strain T1R3 on arsenic absorption by Swiss chard and broad bean crops
In this study, the results showed that the application of llama manure and llama manure/Trichoderma T1R3 combination, signi cantly reduced As concentration in chard roots and leaves. The Trichoderma strain application, signi cantly decreases the As concentration in chard root, compared to the controls (Table 3).
In the broad bean crops with llama manure amendments and the addition of llama manure/Trichoderma T1R3 combination, signi cantly reduced the As content in bean roots. Also, in bean leaves, the addition of llama manure and Trichoderma T1R3 inoculation signi cantly lowered As concentration, as compared with the control (Table 4). In contrast, Trichoderma strain led to the highest As accumulation in broad bean roots, with concentrations signi cantly higher in comparison to the other treatments (Table 4).
The lower As absorption in chard and broad bean (roots and leaves), can be attributed to the combined effect of microbial activity, As adsorption to soil particles and organic material, such as manure.
Adsorption is the rst process that takes place when As are in contact with a soil, affecting processes such as leaching, bioavailability or toxicity (Morillo and Villaverde, 2017). This strategy leads to a reduced availability of As, which in turns improves plant growth (Mehmood et al. 2017;Nawab et al. 2018). In this sense, Mehmood et al. (2017) reported that in the (sandy loam) soil in Narwala, contaminated with As (0, 40, 80, 120 mg kg −1 ), the addition of compost (2.5%) decreases As concentration in maize shoot, signi cantly improving shoot dry biomass. In addition, Nawab et al. (2018) demonstrated that applying 5% farmyard manure to the soil showed the highest reduction in As bioaccumulation in pea (21 to 37%) and chili (18 to 36%), respect to the control treatments. Also, Trichoderma induced As methylation in soil could be the reason for less As uptake in Trichoderma inoculated plants. This loss of As could be due to volatilization of methylated As, such as in the form of trimethylarsine (TMA) or trimethylarsine oxide (TMAO) (Wang et al. 2015;Tripathi et al. 2017). Lower root uptake of methylated As species has been reported by several studies (Mishra et al. 2016 and. Furthermore, organic matter decomposition results in a release of simple aliphatic acids, sugar acids, amino acids, phenols, phosphates and carbonate minerals, which act as adsorption sites for As ions. This reduces their mobility in the soil solution, mitigating As toxicity hazards and making the toxicant unavailable for plants (Mehmood et al. 2017;Nawab et al. 2018;Mandal et al. 2019). The presence of phosphorus in the soil (72.8 mg kg −1 ) and in the llama manure (418 mg kg −1 ) may have positive effects on plant growth and reduce As uptake. This is possibly due to the fact that in plant cells, phosphorus can compete with As (As(V)) in different important biochemical processes, where As substitutes for phosphorus .
The pH of the soil plays an important role in the As bioavailability, and its subsequent bioaccumulation in plants (Nawab at al. 2018). In the present study, the alkaline pH of the soil and water (Table 2) could have contributed to As coprecipitation with sulfate or calcium, reducing its availability (Natasha et al. 2020). According to Chaoua et al. (2019), in alkaline pH soils, the concentrations of metal ions drop due to an increased surface oxide charge, or on account of either processes of precipitation of metal hydroxides, or of formation of insoluble organic complexes.
In addition, calcium is generally known to reduce As accumulation in plants by forming stable Caarsenate precipitates (Hassan et al. 2014). In this study, high calcium concentrations in the soil (8.8 meq L −1 ) and in the irrigation water (4 meq L −1 ) could form stable precipitates, such as calcium arsenate, and reduce As uptake by crops. This was observed by Liu et al. (2014), reported that applying CaO 2 to the soil signi cantly reduced As accumulation in celery shoots. Similarly, Shahid et al. (2017) reported that the application of Ca (1, 5 and 10 mM) signi cantly reduced As transfer to spinach aerial parts.
In this study, the high levels of As (590.83 mg kg −1 ) accumulated in broad bean roots (Trichoderma strain treatment) could be attributed to the fact that Trichoderma T1R3 released organic acids, such as gluconic acid, fumaric acid, and citric acid, which decreased soil pH and caused the dissolution of phosphate and As, among other compounds, thus resulting in a greater bioavailability of the toxicant and of nutrients in the rhizosphere (Stewart et al. 2014;Anawar et al. 2018). In the two crops studied in this work, the roots were the organs with the highest accumulated As, probably attributed to the toxicant was compartmentalized in root vacuoles, gets complexed with sulfhydryl (-SH) groups of peptides, such as γglutamylcysteine, glutathione and phytochelatins (Mishra et al. 2016 and. These phenomena were observed in crops such as rice, tomato, beans, chard and lettuce Pigna et al. 2013;Yañez et al. 2018 and. The results presented in this work are consistent with previous studies by Kumwimba et al. (2013), these authors reported values of 534.06 mg kg −1 As in roots of hydroponic lettuce crops, showed that the average As concentration in roots was 19-26 times higher than in shoots. Also, studies conducted by Babu et al. (2014), who inoculated a metal-polluted mining ground with Trichoderma virens chlamydospores, and grew corn plants to evaluate the mobility of toxicants, found that the fungus signi cantly increased As accumulation in corn roots (31%), compared with plants grown in soils without inoculation.
In our study, broad bean crops did not develop pods with seeds, so As could not be quanti ed. The salinity of llama manure (12.76 dS m −1 ), the soil (1.98 dS m −1 ), and the irrigation water (2.58 dS m −1 ) could have negatively affected crop development. Saline medium has several adverse effects on plant growth, as a result of a low osmotic potential of the soil solution (osmotic stress), speci c ion effects, nutritional imbalance, or a combination of these factors Parvez et al. 2020). Moreover, chlorosis symptoms were observed in the leaves, which were later affected by foliar necrosis.

Arsenic translocation factors in Swiss chard and broad bean crops applied with llama manure and Trichoderma T1R3
The ability of plants to mobilize As from roots to leaves was calculated as a translocation factor (TF). Due to high accumulation of As in plant roots, As translocation from root to leaves was low (TF < 1) in all As treatments (Table 5). According to Sharma et al. (2020), a TF < 1 indicates poor As translocation from roots to the aerial parts. It could be observed that FT= 0.01 value for broad bean crop treated with Trichoderma strain showed a considerably lower translocation of the toxicant from the roots to the leaves, respect to the control treatment. In this sense, Khan et al. (2009) reported that a TF value ≤ 0.1 would indicate that the plant reduces the amount of accumulated toxicant by expelling from the plant tissue, as a detoxi cation mechanism. The same phenomenon was reported by Caporale et al. (2014), who demonstrated that lettuce plants inoculated with two Trichoderma strains, and irrigated with Ascontaminated water (5 or 10 mg L −1 ), showed a signi cantly lower concentration of As in leaves, respect to the non-treated control. Also, Tripathi et al. (2017) published that As concentration decreased in chickpea plants (root, stem, seeds) inoculated with Trichoderma sp. Thus, a low As concentration in the leaves could be due to limited translocation at a systemic level (Smith et al. 2009). In addition, the higher retention of As in roots could be caused by a process saturation, where plants exceed their capacity of translocating the toxicant to aerial parts (Gusman et al. 2013. The TF of 0.33 determined for the broad bean crop treated with the llama manure/Trichoderma T1R3 combination showed a higher As translocation from root to leaves, respect to the control (TF of 0.15).
Considering that the As is translocated from the root to the leaves through phosphate channels (Niazi et al. 2016), this may be attributable to better solubilization of phosphorus caused by the T1R3 strain and to the bioavailable phosphorous present in llama manure. Yao et al. (2009), also observed that the application of 4% chicken manure and 4% pig manure to water spinach enhanced As translocation. Also, Niazi et al. (2017) obtained higher TF values in B. napus, which suggests that this plant species is e cient in transferring As from roots to shoots in presence of phosphate.
In our study, broad bean plants showed a greater capacity of translocate As than chard plants, except in the Trichoderma T1R3 treatment. In the llama manure treatments and in those with the llama manure/Trichoderma T1R3 combination, the bean crops had As TF values which were two-fold and threefold higher than those of chard crops, respectively.
3.5 Potential health risks associated with the consumption of Swiss chard leaves from crops exposed to arsenic, and supplemented with llama manure and Trichoderma T1R3 The food chain is an important pathway for human exposure to As. The risk to human health by the intake of As through consuming chard crops was assessed using the hazard quotient (HQ). As shown in Table 6, the applications with llama manure and the llama manure/Trichoderma T1R3 combination to chard crops grown in As-contaminated soil and irrigated with As-contaminated water brought HQ indices below 1, compared to the control treatment. According to USEPA (2000) guidelines, potential adverse impact on human health would occur when HQ ≥ 1, whereas HQ < 1 values mean that the exposed population is unlikely to experience adverse health effects. Recently, Mandal et al. (2019) showed that adding farmyard manure to a soil contaminated by As (10, 20, 30, and 40 mgAs kg −1 ) reduces hazard quotient values for intake of As through the consumption of wheat grown in contaminated soil.
The carcinogenic risk (CR) posed by consuming As-contaminated chard leaves is shown in Table 6.
Applications of llama manure and the llama manure/Trichoderma T1R3 combination to chard crops signi cantly decreased the CR, compared to the control. However, CR values obtained for As were not within the acceptable range, and exceeded the threshold value (1×10 −4 ), which suggests that consuming leafy vegetables involves a considerably high risk of developing cancer (Muñoz et al. 2017). This CR values were consistent with those reported by Ma et al. (2017), who evaluated the consumption of leafy vegetables from thirteen different crops, reporting CR values between 1.28×10 −4 and 4.57×10 −4 . Also, risk assessment studies conducted by Nawab et al. (2018) showed that applying 1, 2 and 5% farm manure and peat to agricultural soils contaminated with Ni, Cr, As, Zn, Cd and Pb decreased their daily intake of these metals, as well as the cancer risks associated with rice consumption. The major source of human exposure to As is through consumption of As-accumulating crops and vegetables. However, the inhalation of soil particles, drinking water and dermal contact are important pathways for human exposure to As (Li et al. 2017).

Conclusion
In the present study evaluated the effect of llama manure amendment and Trichoderma strain T1R3 inoculation on As uptake and toxicity in chard an broad bean crops, together with human health risks associated with the consumption from these crops. In both crops, the combined addition of llama manure and Trichoderma T1R3 was the treatment that most e ciently reduced As accumulation in chard leaves and in the roots of both crops, showing a great potential as an As complexing agent, and a capacity to reduce plant As uptake and its availability in the soil. The TFs of broad beans were also higher than those of chard; however, speci cally in broad bean, treatments with Trichoderma T1R3 strain led to a considerably lower translocation of the toxicant from the roots to the leaves. In addition, the llama manure/Trichoderma T1R3 combination was more effective in reducing Hazard quotients (HQ) and Carcinogenic risk (CR) represented by consuming chard leaves. However, CR values obtained for As were higher than those acceptable, which means that there is a considerable carcinogenic risk in consuming leafy vegetables.
This work indicates that it is possible combine bio-fertilization and mitigation of As toxicity in important food crops by using selected Trichoderma strains. Further research is needed in relation to the role llama manure and Trichoderma T1R3 play in As immobilization/mobilization and its uptake by different plant species that are grown in a range of As-contaminated soils. Therefore, if becomes of primary importance to perform detailed studies and development of strategies that minimize the water-soil-plant transfer of arsenic or restrict As contamination of edible plant parts, preventing root-to-shoot translocation. These strategies would be su cient to become another route for increasing food safety.

Declarations
Declarations Con ict of interest On behalf of all authors, the corresponding author states that there is no con ct of interest.

Authors Contributions
LMY: designed the study, and analyzed the data, discussion of the results, and writing of the manuscript.
JAA: designed the study, conducted the experiments and data compilation, discussion of the results.
NMEAC: analyzed the data, acquisition and administration of funds GBM: methodology planning, acquisition and administration of funds.
All authors reviewed and approved the nal manuscript.  Total As (mg kg -1 ) 49 Total As (mg L -1 ) 1.44 EC: electrical conductivity; P: phosphorus; Ca: calcium; Mg: magnesium; C: carbon; N: nitrogen; Na: sodium; K: potassium; SAR: sodium absorption ratio Data are expressed as mean values ± SD (n = 3). The different letters within a column indicate a signi cant difference at p ≤ 0.05 according to Duncan's multiple range tests. The % reduction was determined considering the As concentration of the control treatment as 100% As content. Data are expressed as mean values ± SD (n = 3). The different letters within a column indicate a signi cant difference at p ≤ 0.05 according to Duncan's multiple range tests. The % reduction was determined considering the As concentration of the control treatment as 100% As content.