Effect of arsenate on germination and early develop ment parameters of three forage leguminous plants

The Chacopampean plain is one of the most arsenic affected areas in Argentina, especially in groundwater, used both for animal drinking and forage irrigation. The main objective of this study was to determine the effect of arsenic (As) present in irrigation water on the germination parameters of red clover (Trifolium pratense L.), white clover (Trifolium repens L.), and alfalfa (Medicago sativa L.) seeds exposed to different concentrations of As(V). The germination and viability parameters of seeds from the three species were not affected by elevated concentrations of As. However, As significantly reduced the radicle and hypocotyl lengths of the three forage specimens. The inhibition level of the plants increased with the increase in the concentration of As. Regarding to hypocotyl length/radicle length ratio, the results evidenced that both the hypocotyl and radicle of clover species are affected in the same degree, while alfalfa radicles seemed more affected than hypocotyls at higher concentrations of As. Our results showed that irrigation with solutions containing As affect seedling growth parameters differently in the three species, and the effect of As is mostly evidenced when in high concentrations.

. Most of the organic species are less toxic than the inorganic species (BUNDSCHUH et al. 2008, CHEN et al. 2016, LI et al. 2016.
High As levels in groundwater have been registered in several countries worldwide. In Latin America, the most affected countries are Argentina, Bolivia, Peru, Chile, and Mexico (BECH et al. 1997, BUNDSCHUH et al. 2012, ALARCÓN-HERRERA et al. 2013, ALONSO et al. 2014. In Argentina, the Chacopampean plain is one of the most important areas for agriculture. In this zone, groundwater As originates from aquifer rocks (NICOLLI et al. 1989, SMEDLEY & KINNIBURGH 2002. Plants may be exposed to As through both irrigation water and soil. Arsenic is involved in several chemical processes within plants, including in reduced, detoxified, and sequestered forms. It mimics phosphorous, binds sulfhydryl groups, and causes oxidative stress (ISLAM et al. 2015, HUANG et al. 2012. As (III) and As (V) flow in plants through different pathways. As (III) enters plant cells via aquaporins (MA et al. 2008, ZHAO et al. 2009). The aquaporin transporter AtNiP1-2 is highly expressed especially in seeds (LI et al. 2016). As (V) is taken up via phosphate transporters, using a co-transport mechanism with two protons (ESTEBAN et al. 2003, ZHAO et al. 2009). Inside the cell, arsenate is reduced to arsenite by arsenate reductase, which consumes reduced glutathione (ZHAO et al. 2010, ZHU et al. 2014. As (III) tends to be complexed due to its high affinity for protein SH groups. It binds to phytochelatins, glutathione oligomers produced by the enzyme phytochelatin synthase and is stored into vacuoles in root cells (BATISTA et al. 2014). Also, part of this arsenite is translocated to other plant tissues (ZHAO et al. 2009, PÉREZ-CARRERA & FERNÁNDEZ-CIRELLI 2014. Despite this arsenic resistance, uptake, translocation to different parts of the plant, and bioaccumulation rate, it has not yet been well studied (LI et al. 2016). Several studies show that these phenomena depend on several factors, such as soil characteristics, microbiological activity, other ions present in the water and soil, plant species, genetic background, and others (HARTLEY & LEPP 2008, SMITH et al. 2010, RAHMAN & HASEGAWA 2011, RAI et al. 2011, FU et al. 2011, MARQUEZ-GARCIA et al. 2012, AMARAL et al. 2013, IRIEL et al. 2015. Arsenic levels in soil or in irrigation water can affect forage growth and its nutritional characteristics and can represent a risk to foraging cattle (PÉREZ-CARRERA & FERNÁNDEZ-CIRELLI 2014).
Among the deleterious effects of As on plants, we highlight a decrease in phosphorus uptake since both elements compete for phosphate transport systems. For instance, arsenate acts like P (FARNESE et al. 2014). LIU et al. (2008) showed that As also interferes with the metabolism of other elements, such as calcium (Ca), magnesium (Mg), potassium (K), manganese (Mg), and zinc (Zn). In several plant species, many effects occurred on growth, biomass production, number of leaves, root length, plant height, diverse morphological changes, disorganization of photosynthesis, generation of reactive oxygen species, and declination on germination rate (CARBONELL-BARRACHINA et al. 1998, MITEVA 2002, SRIVASTAVA et al. 2005, AHMED et al. 2006, SHAIBUR et al. 2008, SMITH et al. 2010, PÉREZ-CARRERA & FERNÁNDEZ-CIRELLI 2014. Despite this, MA (2001) reported that in low concentrations, As may stimulate the growth of many plants. However, the mechanism is still unclear.
It has been proposed that the effect of As in legumes can depend on the interaction between nodule bacteria and plant (CHEN et al. 2007, XU et al. 2008. MACUR et al. (2001) has reported that rhizobial bacteria can grow in soils with high As levels. However, As also causes the reduction in nodule numbers due to a declination in plant infection (PAJUELO et al. 2008). This process depends on the specie of nodule bacteria (CHRISTOPHERSEN et al. 2012). Due to these characteristics, legumes with As resistant nodules, have been proposed for remediation strategies on As contaminated areas (CARRASCO et al. 2005, KHAN et al. 2009). The effect of As on plants could depend of mycorrhizal fungus, because As speciation in plants change with the presence of fungus, increasing the percentage of As (III) in plant tissues (LOMAX et al. 2012, ZHANG et al. 2015. However, the effect of arsenic in forages hasn't been well studied. In the Chacopampean plain in Argentina, the clover (Trifolium pratense L.), white clover (Trifolium repens L.), and alfalfa (Medicago sativa L.) are the three leguminous species most commonly used as forage for cattle due to their high protein content. Therefore, the aim of this study was to determine the effect of As present in irrigation water on the germination parameters of three forage leguminous plant seeds exposed to different concentrations of As (V).
The seeds used in this work (Trifolium repens L., Trifolium pratense L. and Medicago sativa L.) were supplied by a national provider of commercial seeds. Their viability was confirmed through a germination test before beginning the experiments. Seeds showed a 90% of germination by the fifth day. Ideal stocking conditions (temperature and relative humidity) were guaranteed in all experiments. Sterile plastic Petri dishes and 80 g/m 2 filter paper, 250µm thick, with pores of 14µm and a permeability of 14 l/s/m 2 (measured by method DIN 53887) were used. The arsenic exposure experiment took place under controlled conditions, in darkness, and at a constant temperature of 22 ºC ± 2 ºC. A modified version of an international protocol was used -EPA 600/3 (GREENE JC et al. 1988). Sodium arsenate heptahydrate (NaHAsO 4 .7H 2 O) from Biopack (CAS#10048-95-0) was used to prepare the As solutions at the concentrations of 50, 100, 500, 1000, and 5000 µg/L. Potassium dichromate 2% m/v was used as positive control. The selected concentrations of As used in this experiment resemble natural concentrations of As in irrigation water in Argentina (NICOLLI et al. 1989, FARIAS 2003, PÉREZ-CARRERA & FERNÁNDEZ-CIRELLI 2013. Groups of 25 seeds were placed in the Petri dishes and soaked with two ml of each solution. A positive control of potassium dichromate 0.2% and a negative control of distilled water were used. All treatments were conducted using three replicates. The Petri dishes were kept in darkness and under controlled humidity and temperature conditions for five days. All the germinated seeds were counted and the radicle and hypocotyl lengths were measured. All seedlings with a radicle longer than 2 mm were considered germinated. The seedlings were photographed with a high-resolution camera and their radicle and hypocotyl length were measured by digital software. The experiments were conducted using the completely randomized design. All the results were analyzed by a blind system. Analysis of variance (ANOVA) was used to determine the statistical significance of the differences between the means of the treatments in all experiments. For the germination analysis, arc sin transformation was used before ANOVA (ZAR et al. 1996).
The No Observable Adverse Effect Concentration (NOAEC) values for seed germination and hypocotyl and root elongation were determined using Dunnett's procedure for multiple comparisons. ANOVAs and other statistical parameters were calculated using InfoStat Student Version. The EC 50 for seedling growth was determined by expressing each treatment as a proportion of the control seedling growth (HAMILTON et al. 1977, USEPA 1999. Basal germination rates were 69, 80, and 88% for alfalfa, white clover, and red clover, respectively. The effect of arsenic on seed germination was analyzed for the different As solutions. For the three species, there was no significant variation on germination percentage between treatments (p>0.05). Despite this, there was a decrease in the tendency for seed germination at high concentrations of As (Figure 1). Figure 1. Normalized percentage of germination for the three legume species: white clover (white), red clover (grey), and alfalfa (dark). There were no significant differences (p<0.05) between treatments for each species. Infostat.
Rev. Ciênc. Agrovet., Lages, SC, Brasil (ISSN 2238-1171) 239 Toxicity to forage can be evidenced by the decrease on hypocotyl and radicle lengths. A significant difference among the treatments was observed, showing a decreasing tendency in both hypocotyl and radicle lengths when As levels increased. The effect was higher in both alfalfa and red clover when compared to white clover. The analysis of the hypocotyl elongation changes in alfalfa showed a significant difference when compared to the control (p<0.05) at 500 µg/L or higher concentrations of As. In contrast, red clover appears to be more sensitive, with significant differences (p<0.05) in hypocotyl elongation appearing at concentrations equal or above 100 µg/L. In the case of white clover, a significant reduction (p<0.05) was observed from 1000 µg/L (Figure 2). Regarding the effect on radical elongation, alfalfa showed a significant difference (p<0.05) from the control at 1000 µg/L. Red clover also appears to be more sensitive, showing significant differences (p<0.05) in radicle elongation at concentrations equal or above 100 µg/L. In the case of white clover, a significant reduction in radicle length (p<0.05) was observed with As concentration higher than 1000 µg/L (Figure 3). The hypocotyl length/radicle length ratio (HL/RL) was measured for the three studied forage species as another toxicity indicator. There were no significant differences between treatments on both clover species. Alfalfa showed a tendency to increase the HL/RL ratio with the increase in As concentration. This legume showed a significant difference (p<0.05) to the control at 1000 µg/L (Figure 4). Asterisks denote significant differences (p<0.05) between the treatments and the control.
No observable adverse effect concentration (NOAEC) and 50% and 10% effect concentration (EC 50 and EC 10 ) were calculated for the three species. EC 50 , EC 10 , and R 2 , for root and shoot growth and the percentage of germination were calculated by expressing each treatment as the inhibition percentage in relation to the control treatment. The toxic effects of As on germination and growth parameters are summarized in Table1.   Table 1. NOAEC, EC 50 , and EC 10 values for germination and growth parameters of seeds exposed to As (V) for 5 days, and R 2 value of the regression line. Arsenic accumulation and toxic effect were reported for different plants species, such as sorghum, fern, grass, and rice (SRIVASTAVA et al. 2005, SHAIBUR et al. 2008, SHRI et al. 2009, SMITH et al. 2010. Studies conducted with alfalfa in our laboratory (PÉREZ-CARRERA & FERNÁNDEZ-CIRELLI 2014) showed that the increase in the soil concentration of As also increased As concentration in leaves, stems, and roots, demonstrating the As transference in alfalfa. The accumulation of As in the roots was higher than in other parts of the plant (stem and leaves). Similar results were observed by MASCHER et al. (2002) in red clover plants, who found that As levels in the shoot tissues increased when arsenate concentration in the soil increases. They also found that there is a significant reduction of shoot growth at high arsenate levels, concluding that As toxicity was superior in shoots than in roots. These results are comparable to those obtained in the present study. Regarding the increase in plant growth at low concentrations of arsenate, similar results were reported by MASCHER et al. (2002) andIRIEL et al. (2015).
The reduction on the percentage of germination was documented for several plant species (TALUKDAR 2011, UPADHYAYA et al. 2014. VAZQUEZ ALDANA et al. (2013) reported an adverse effect on the germination of Festuca rubra seeds exposed to As that was significant only when its concentration was >12 mg/L, a value more elevated than the As levels used in this experience. Nevertheless, our results suggest that seeds can germinate in a wide range of As concentrations present in irrigation water. Furthermore, As has shown deleterious effects on other germination parameters. LUAN et al. (2008) showed that As has an inhibitory effect on soybean growth parameters. The same effect was reported by YOON et al. (2015) for other plant species. Toxic effects of As on germination and seedling growth of several plant species used as animal feed are presented in Table 2. Table 2. EC 50 value for growth parameters of seeds exposed to As(V) for 5 days (units: soil mg/kg and water µg/L). Other authors also report similar results in other forage species. A study regarding the effects on seed germination and seedling growth of Medicago sativa L., Vicia villosa, and Cajanus cajan L. reported that As levels over 5 mg kg show deleterious effects on germination and seedling growth for all tree species (MA et al. 2009). MARQUEZ et al. (2007 reported that crimson clover (Trifolium incarnatum L.) exposed to As had an inhibitory effect on the germination and growth.

Specie
Both genetic and environmental factors affect seed germination and early seedling growth. Different plant species have developed a variety of mechanisms to adapt to adverse conditions, becoming more or less susceptible to certain substances or environmental conditions (IBRAHIM 2016). Many researchers have evaluated the seed germination process regarding several stress factors (LARA-NUÑEZ et al. 2015, IBRAHIM 2016, CHAMORRO et al. 2016. The response of legumes to As-induced toxicity was investigated in some forage species (MASCHER et al. 2002, DONG et al. 2008, LA FUENTE et al. 2010, PEREZ-CARRERA & FERNÁNDEZ-CIRELLI 2014. However, the studies concerning the impact of As on germination and early Rev. Ciênc. Agrovet., Lages, SC, Brasil (ISSN 2238-1171) 242 plants development are scarce (LI et al. 2007). Regarding the obtained results, even if the effect on seedling growth was significant, a direct effect on the percentage of germination was not observed. This fact could suggest that there is a different response of the plant embryo to As exposure before and after seed germination. This was studied by LI et al. (2005) who reported that the isolated embryos of the plants studied were much more sensitive than intact seeds to the exposure to heavy metals, and in fact, the seeds could germinate in the presence of high concentrations of metals, although growth was inhibited once germination began and the seed coat was broken. As presented by other authors (LI et al. 2005, SEREGIN & KOZHEVNIKOVA 2005, our study supports the idea that the effect of As on forage legumes can depend on the seed coat structure of each plant species. In the present study, we analyzed the impact of exposure to As on the early development of forage seeds to improve feed production and provide information for management practices to protect livestock from As toxicity. This investigation showed that seed germination and viability were not affected by high concentrations of As (V). However, As significantly reduced the radicle and hypocotyl lengths of the three forage specimens studied, and their inhibition level increased with the increase of As concentration in irrigation water. Regarding to hypocotyl length/radicle length ratio, the results demonstrated that, in clover species, both hypocotyl and radicle are affected in the same degree, while alfalfa radicles seemed more affected than hypocotyls at As concentrations higher than 1000 µg/L. The NOAEC, EC 50 , and EC 10 values are dissimilar between species. This emphasizes the need to evaluate the effect of arsenic on each forage species.
In this context, the present study provides information on the impact of As on forage germination and development. Our results showed that As affect seedling growth parameters in the three species studied, and also suggest that the effect of As on forage legume seeds can depend on the structure and integrity of the seed coat. It was also observed that the impact of As on these plants is mostly evidenced at high As concentrations. Further studies are in progress to understand As uptake, translocation, and effects on forage plants, as well as their impact on livestock health and production and the mechanisms through which As affect germination and seedling growth to clarify the impact of As on agriculture ecosystems.