## 4 Discussion  
To date, little is known regarding the phytotoxicity of GLY-contaminated soils on non-target plants, including cover crop species, such as _M. sativa_. Although these plants are not intentionally treated with GLY, they can still be affected by its application through leaching, runoffs or even wind in the case of spraying. Moreover, GLY strongly adsorbs to solid particles [13; 14] and accumulates in soils [15], resulting in a serious problem of diffuse contamination. Indeed, several studies were  
Figure 5: Effect of increased concentrations of GLY, on the AOX (antioxidant) system of M. sativa shoots after 21 days of exposure. **(a)** Total antioxidant capacity (TAC); **(b)** Total phenol content (TPC); **(c)** Proline (Pro). Error bars correspond to the standard deviation. Statistically significant differences compared to the CTL, considering \(p\leq 0.05\), are marked with a * above bars. conducted in order to determine GLY levels in soils around the world and despite many of them reporting levels lower than 3 mg kg\({}^{-1}\) for agricultural soils or soil located nearby agricultural areas in South America [13; 40; 41; 42] and Europe [7; 43; 44; 45; 46], other studies have reported values of 5.0 mg kg\({}^{-1}\) in soybean-cultivated areas in Argentina [47], reaching concentrations as high as 40.6 mg kg\({}^{-1}\) in olive groves from Greece [45] or even 608 mg kg\({}^{-1}\) in a crop fields from Mexico [48]. Therefore, the main goal of the present study was to assess the effects of soil contamination by a GBH on the growth responses and redox homeostasis of alfalfa plants, at environmentally relevant concentrations of the a.i. In fact, despite recent studies having been conducted to evaluate the effects of GLY application in non-target plants, most of these works applied GLY as foliar spray [49; 50; 51; 52; 53; 54; 55] or as a supplement to the nutrient solution [12; 56; 57; 58; 59; 60] rather than simulating soil contamination scenarios. The present study showed that, after 21 days of exposure, Roundup UltraMax(r) severely repressed the growth of _M. sativa_ in a dose-dependent manner, inhibiting both organ elongation and biomass production. Actually, given the already accentuated reduction in shoot fresh weight upon exposure to the lowest concentration tested (8 mg kg\({}^{-1}\) of a.i. ), it can be suggested that even lower levels would be capable of impairing plant growth. When GLY is absorbed by the plant, it is translocated through vascular tissues, namely by phloem, reaching active metabolite sites, such as root and shoot meristems, following the same pathway as photoassimilates [18; 61], which could explain the repression of shoot growth. The fact that GLY is an herbicide that inhibits an enzyme from the shikimate pathway, 5-enolpyruvateylshitz-3-phosphate synthase (EPSPS; EC 2.5.1.19), can also explain the results obtained. EPSPS plays a role in the synthesis of the aromatic amino acids tryptophan, phenylalanine, and tyrosine that are crucial for the growth and survival of plants and which function as the precursors of many secondary metabolites, such as pigments, auxins and lignin [62]. As a result of the shikimic acid pathway being blocked, there will be an accumulation of shikimate in plant tissues, which will lead to a deficit in important end products, such as lignin, alkaloids and flavonoids, and a reduction in CO\({}_{2}\) fixation and biomass production in a dose-dependent manner [63]. The decrease in root and shoot length and biomass can also be due to the impact that GLY has on i) indole-3-acetic-acid (IAA) metabolism which is the main endogenous auxin in the plant, as well as on ii) the interference with plant-water relationships [30; 58; 64]. Another hypothesis that can explain these results is the fact that GLY can impair the absorption of several macro and micronutrients, such as Ca, Mg, N, P, Fe and Zn, among others, as reviewed by [PERSON] et al. [18].