Study area

The research was conducted in the municipality of Tezontepec de Aldama, Hidalgo, Mexico, in the Mezquital Valley region (20° 11’ 33” N and 99° 16’ 21” W, at 2007 m a. s. l.). This area is surrounded by hills and mountains composed of volcanic and calcareous sedimentary rocks (^{Lesser et al., 2018}). The climate is semi-arid, with temperatures ranging from 9 to 39.5 °C. The rainy season is from May to October, while the dry season extends from November to April, with an annual rainfall of 400 to 700 mm (^{National Institute of Statistics and Geography [INEGI], 2017}).

The study area was delimited using the ArcGis 10.6 cartographic analysis program (^{Esri, 2018}) and two field tours, where the surface irrigated with wastewater with the least obstruction from population and infrastructure was selected. The study area covered 37.3 ha (20° 13’ 20.05” N and 99° 15’ 19.9” W), located within the wastewater irrigation system of DR-100 Alfajayucan.

Soil and plant material sampling

Within the 37.3 hectares, 70 mesquite trees were recorded, from which 24 trees (30 %) were randomly selected to ensure population representativeness. Soil, root, leaf, and fruit samples were collected from each tree. Sampling was conducted in the summer of 2019. The samples were placed in brown paper bags and transported to the laboratory for analysis. A total of 96 samples were obtained: 24 soil samples and 72 plant material samples (roots, leaves, and fruit), corresponding to the 24 trees evaluated.

Soil sample analysis

Soil sample analysis was performed according to NOM-021-RECNAT-2000 (^{Secretaría de Medio Ambiente y Recursos Naturales [SEMARNAT], 2002}). The analyzed variables were: pH (method AS-02), electrical conductivity (EC) in 1:2 aqueous medium, texture (Bouyoucos method AS-09), cation exchange capacity (CEC) and exchangeable bases (Ca, Mg, Na and K) (method AS-13), organic matter (OM; Walkley and Black method AS-07), inorganic nitrogen (Ni; method AS-08) and extractable concentration of Pb (method AS-14, with DTPA 0.005 M). The determination of Pb was performed by atomic absorption spectrophotometry (SavantAA, GBC, Australia) from a calibration curve with a detection limit of 0-20 mg∙kg^-1 (^{Amezcua-Ávila et al., 2020}).

Analysis of plant samples

Plant samples of roots, leaves, and fruits were washed with distilled water. Roots and leaves were oven-dried at 60 °C, while fruits were left to dry at room temperature. Once dry, the materials were ground. 0.5 g of each sample was weighed and placed in 30 mL Kjeldahl flasks. Five milliliters of the diacid mixture (80 % HCl and 20 % HClO₄) were added to each flask, the mixture was shaken and heated on a hot plate at 300 °C until gas emission was observed, at which point 1 mL of 30 % hydrogen peroxide was added. This procedure was repeated until the mixture became cloudy or until a total of 5 mL of hydrogen peroxide had been added. The samples were then cooled for 24 h, diluted with distilled water, filtered, and brought to a final volume of 50 mL. The quantification of Pb was determined by atomic absorption spectrophotometry (^{Amezcua-Ávila et al., 2020}).

Pb bioconcentration and translocation factors

The bioconcentration factor (BCF) was calculated as the ratio between the Pb concentration in plant tissues (roots, leaves, or fruits) and the Pb concentration in the soil. The translocation factor (TF), on the other hand, was determined as the ratio between the Pb concentration in the aerial parts (leaves and fruits) and the Pb concentration in the roots (^{Ali et al., 2013}).

Statistical analysis

Statistical analysis was performed using the R software (^{R Development Core Team, 2019}). Measures of central tendency and dispersion (minimum, maximum, mean, median, range, and coefficient of variation) were calculated for all variables. Data normality was assessed using normal probability plots and the Shapiro-Wilk test. Additionally, Spearman's rank correlation coefficient was estimated between Pb concentration in the soil and in different plant organs (root, leaf, and fruit).

Since the data did not follow normal distribution, the Kruskal-Wallis test was applied to identify significant differences in Pb concentrations among plant organs. Subsequently, paired comparisons (root-leaf, leaf-fruit, and root-fruit) were performed using the Mann-Whitney test, with significance correction.

Characterization and concentration of Pb in soils

The measures of central tendency and dispersion of the physical and chemical variables of the evaluated soils are shown in Table 1. The mean pH was 7.6, corresponding to a neutral to slightly alkaline value (7.15 - 8.48) according to NOM-021-RECNAT-2000 (^{SEMARNAT, 2002}). In the Mezquital Valley, ^{Siebe et al. (2016)} reported a neutral pH (7.3) in soils with 102 years of irrigation with wastewater, which indicates that this type of irrigation does not significantly modify this variable. The pH observed in this study, after 47 years of irrigation with wastewater, reinforces the stability of the pH under these conditions. This stability is attributed to the alkaline nature of the soil, resulting from the presence of calcareous materials characteristic of the arid regions of Mexico (^{Ortiz-Solorio, 2019}).

The mean EC was 0.42 dS∙m^-1, classifying the soil as negligible to slightly saline (0.22-1.42 dS∙m^-1), according to NOM-021-RECNAT-2000 (^{SEMARNAT, 2002}) (Table 1). The results indicated that irrigation with wastewater did not generate salinity in the soils where mesquite trees grow. ^{Soto-Gonzáles et al. (2021)} indicate that this species tolerates up to 11.10 dS∙m^-1, demonstrating its capacity to adapt to saline environments. ^{Muñoz-Iniestra et al. (2017}), on the other hand, recorded an EC of 1.57 dS∙m^-1 in soils not irrigated with wastewater where mesquite grows, a value higher than that obtained in the present study. These results confirm the high resistance of mesquite to salinity and the electrical stability of the soil.

The soil texture showed an average of 18.66 % clay, 15.5 % silt, and 65.8 % sand (Table 1), which corresponds to the sandy loam class according to the soil texture triangle (^{Ortiz-Solorio, 2019}). Similar results were reported by ^{Carrillo-González et al. (2018)} and ^{Siebe et al. (2016)}, who documented clay concentrations of 18.3 and 23 %, respectively. A high sand content compared to clay limits the ability of plants to adsorb heavy metals from the soil (^{Keçeci et al., 2020}).

The organic matter (OM) content was 1.28 %, classified as low to medium (0.67-2.51 %) according to NOM-021-RECNAT-2000 (^{SEMARNAT, 2002}) (Table 1). This value, after 47 years of irrigation with wastewater, reveals persistently low OM concentrations, in contrast to previous studies conducted in the region. ^{Carrillo-González et al. (2018)} and ^{Siebe et al. (2016)} reported minimum values of 3.6 and 2.2 % OM, respectively, in soils irrigated with wastewater for more than 10 years. Similarly, ^{Sánchez-Hernández et al. (2016)} recorded low OM concentrations (0.27-1.23 %) in soils of DR-028, Tulancingo, Hidalgo, irrigated with wastewater. The differences detected can be attributed to the organic load of the irrigation water, the time and frequency of application, as well as the type of crop (corn, beans, and alfalfa).

The CEC showed a mean of 14.17 cmol∙kg^-1, considered low to medium (9.13-22.77 cmol∙kg^-1) according to NOM-021-RECNAT-2000 (^{SEMARNAT, 2002}) (Table 1). ^{Carrillo-González et al. (2018)} obtained similar values (9.44-22.88 cmol∙kg^-1), while ^{Siebe et al. (2016)} reported values between 16 and 30 cmol∙kg^-1 in soils irrigated with wastewater for more than 100 years in the Mezquital Valley. These results indicate a limited capacity of the soil to retain and exchange cations, which suggests that if Pb is added through wastewater, it could be leached.

The mean K content was 0.08 cmol∙kg^-1, Ca content was 9.54 cmol∙kg^-1, and Mg content was 2.51 cmol∙kg^-1, classified as very low for K and medium to high for Ca and Mg (Table 1). The low K concentration is associated with the sandy crumb texture, which favors cation leaching due to its low CEC and high permeability. In contrast, the high Ca and Mg contents are explained by the calcareous origin of the soil (^{Carrillo-González et al., 2018}).

Inorganic nitrogen showed an average of 33.97 mg∙kg^-1, categorized as medium to very high (21.72-63.62 mg∙kg^-1) according to NOM-021-RECNAT-2000 (^{SEMARNAT, 2002}) (Table 1). These concentrations can be attributed to the nitrification of ammoniacal nitrogen incorporated through irrigation with wastewater (^{Siebe et al., 2016}). The availability of inorganic N contributes to soil fertility, as it is one of the main sources of N available to plants.

Extractable Pb showed a mean of 1.99 mg∙kg^-1, with a range of 0.28 to 12.4 mg∙kg^-1. These values are below the maximum permissible limits for uncontaminated soils established by ^{MAPA (1990)}, NOM-021-RECNAT-2000 (^{SEMARNAT, 2002}), and ^{USEPA (1993)}. ^{Carrillo-González et al. (2018)} obtained similar values in soils irrigated for 35 and 102 years (1.16 and 2.91 mg∙kg^-1, respectively) with wastewater in the Mezquital Valley. Low Pb availability is associated with a neutral to slightly alkaline pH, sandy texture, low organic matter content, low cation exchange capacity (CEC), and high calcium concentration, as these conditions reduce its availability in the soil (^{Kabata-Pendias, 2011}). Likewise, the Pb content in the water used for irrigation, the frequency and duration of irrigation with wastewater determine the concentration of extractable Pb in the soil.

Bioconcentration of Pb in mesquite trees

Lead levels averaged 79.61 mg∙kg^-1 in the root, 21.4 mg∙kg^-1 in the leaf, and 64.13 mg∙kg^-1 in the fruit. In the absence of a Mexican standard for mesquite plant material, the European Union regulation was considered, which establishes a maximum limit of 0.2 mg∙kg^-1 for legume fruits intended for human consumption (^{Reyes et al., 2016}). The values obtained exceed this limit by 320 times, representing a potential health risk for the local population that consumes these fruits. Therefore, it is recommended to avoid their consumption until specific toxicological studies are conducted. In the Mezquital Valley, high concentrations of Pb have also been reported in crops such as corn (^{Ponce-Lira et al., 2022}), wheat, oats, beans, tomatoes, chili peppers, alfalfa, and sunflowers (^{Ponce-Lira et al., 2020}). Consumption of contaminated crops can cause toxic effects, especially in children and vulnerable populations (^{Angon et al., 2024}).

Spearman's rank correlation coefficients between Pb in soil and root (ρ = -0.06; p = 0.77), soil and leaf (ρ = 0.001; p = 0.99), and soil and fruit (ρ = 0.30; p = 0.14) showed no significant associations (p > 0.05), suggesting that Pb accumulation in mesquite is not linearly dependent on its concentration in the soil. However, the high concentrations in root, leaf, and fruit confirm its accumulation capacity.

The coefficient of variation was higher in leaves (243 %) than in fruit (106 %) and roots (92 %), which could be attributed to differences in tree biomass, wastewater quality, and irrigation time and frequency. ^{Ponce-Lira et al. (2020)} indicate that irrigation depth and continuous irrigation can influence metal accumulation.

The Kruskal-Wallis test showed significant differences (p < 0.05) in Pb concentration among plant organs, while the Mann-Whitney test revealed significant differences (p < 0.05) between leaf and root and leaf and fruit, but not between root and fruit (p > 0.05), reflecting the mesquite's capacity to absorb and distribute Pb in its different organs. ^{Buendía-González et al. (2012)} found a greater accumulation of Pb in the roots of woody seedlings, and ^{Muro-González et al. (2020)} reported that mesquite bioconcentrates Pb in roots and leaves. Unlike these studies, the present investigation also detected a high concentration of Pb in the fruit, possibly due to the translocation of nutrients and metals through the xylem during fruit development. According to ^{Kumar and Prasad (2018)}, Pb becomes immobilized in cell walls and vacuoles, which generates a “final accumulation” effect in the fruit.

The mean BCF was 102.86 (> 1), classifying mesquite as an accumulator species (^{González-Chávez et al., 2017}). ^{Buendía-González et al. (2010)} reported an BCF of 43 in mesquite seedlings under in vitro conditions. The high Pb accumulation capacity in mesquite trees may be due to a combination of physiological factors (such as the species' tolerance mechanisms, cell wall modifications to immobilize metals, the activity of specific membrane transporters [^{Skuza et al., 2022}], biomass accumulation [^{Xu et al., 2023}], and tree age) and environmental factors (such as exposure time [^{Trejo-Calzada et al., 2015}], soil physical and chemical properties, wastewater quality, and interaction with rhizospheric microorganisms).

The average TF was 9.52 (> 1), classifying mesquite as a hyperaccumulator species (^{Covarrubias & Peña-Cabriales, 2017}). ^{Muro-González et al. (2020)} obtained an TF of 0.75 in one-year-old mesquite plants grown in a greenhouse on mining tailings, indicating limited translocation in that environment. In contrast, the results of the present study, obtained in soils irrigated with wastewater, confirm the hyperaccumulator capacity of mesquite under these conditions.

The variation in heavy metal concentration among plant organs depends on the efficiency of transport processes and internal redistribution (^{Kabata-Pendias, 2011}). Therefore, Pb translocation from the root to the shoot may be regulated by physiological mechanisms (metal mobility processes, as well as absorption and transport pathways), the plant species, and environmental conditions (^{Shi et al., 2022}; ^{Vrsaljko, 2023}). This explains why in different environments, such as mine tailings and soils irrigated with wastewater, mesquite shows a different capacity to mobilize Pb to the leaves and fruit.

To our knowledge, this is the first study to report BCF and TF values for mesquite under natural irrigation conditions using wastewater. The results highlight the importance of investigating this species in its natural habitat under prolonged contamination. This is because the environmental conditions in the Mezquital Valley may have induced specific physiological mechanisms that favor Pb accumulation and translocation in this species.

Future research should address the physiological and molecular mechanisms that regulate Pb absorption, accumulation, and translocation in mesquite, as well as symbiotic interactions with rhizospheric microorganisms, which could play a key role in heavy metal tolerance and accumulation. This is to establish the risks and impact of mesquite in contaminated environments.