Study area

The study was conducted in the "Sierra Norte" of Oaxaca, Mexico, located in the sub-province of the Southern Sierra Madre of Mexico, and in the hydrological region RH28, "Papaloapan", the second largest watershed in the country¹⁴. The research was carried out in the towns of Ixtlán de Juárez and Capulálpam de Méndez. The municipality of Ixtlán de Juárez (17° 20' N, 96° 29' W), is located at an altitude of 2,030 m asl; the climate is C (w) temperate sub-humid, and the area has a rugged orography; the average rainfall is 900 to 1,100 mm per year; the average annual temperature is 20 ^oC¹⁵, and the soil type is humic Acrisol (HA). The municipality of Capulálpam de Méndez (17° 18' N, 96° 27' W) is located at an altitude of 2,040 m asl; the climate is classified as C (w2) (w) b (i ') g temperate sub-humid; the average precipitation is 1,115 mm per year and occurs between June and October; the average annual temperature is 15.2 ^oC¹⁶; the soil type is cambisol.

Experimental design and sowing

A complete randomized design (CRD) was used in each municipality (Ixtlán de Juárez and Capulálpam de Méndez). The treatments consisted of a 2 x 2 factorial, i.e. the two forage crops (Chenopodium quinoa Willd and Zea Mays L) and two types of irrigation: fresh water (DW) and treated wastewater (TWW), with four replicates per treatment. The study was established in March 2017. The cultivated plot in Ixtlán was established in an area of 300 m² (20 x 15 m) divided into four sections of 60 m² each (15 x 4 m), with a slope of 3 %. The cultivated area in Capulálpam was 400 m² (20 x 20 m) divided into four subplots of 80 m², with a slope of 1 %. The cultivated area of both Ixtlán and Calpulálpam was subdivided into two parts: in the first subplot, sown with quinoa, was irrigated with freshwater and treated wastewater; in the second section, corn was grown and also irrigated with treated wastewater and freshwater (FW). The subplots were divided by five unsown furrows for the application of the types of irrigation. The soil was prepared with a tractor, the distance between furrows of both crops was 80 cm, drawn parallel to the slope. The quinoa variety Ontifor was sown by hand continuously in the bottom of the furrow, at a depth of less than 3.0 cm approximately, with an approximate density of 450,000 plants ha^-1(8,17. Corn (creole Zea mays) was planted by hand continuously at the bottom of the furrow for forage, at a depth of approximately 4.0 cm, with a density of 60,000 plants ha^-1.

Obtainment of treated wastewater (TWW)

The wastewater treatment plants (WWTPs) receive all the domestic water from the same localities; Ixtlán receives a flow rate of 3.3 L s^-1 and Capulálpam receives a flow rate of 1.0 L s^-1. The WWTPs have a pretreatment system based on grids of different diameters, which are located at the inlet of the reception channel to retain solid wastes, such as soda caps, hair, wood, PET, etc. The water passes through the sand trap (3.0 m long channel), where the first process of sedimentation of solids takes place. The wastewater enters the biodigesters by gravity; each drop has a free fall of 3.10 m (this is where most of the sludge settles) and, by laminar flow, the water reaches the tubular sedimentation area (inner tank) and gradually rises until it overflows into the biodigester area (outer tank). The biodigester area is composed of polyethylene fabrics to facilitate the accommodation of anaerobic bacteria, which is why they are called hosts. Bacteria generally form granules at the apexes of the hosts, and they take care of the biodigestion of the wastewater, transforming it into treated (biodigested) wastewater. The process takes 14 h.

Obtainment of fresh water (FW)

In Capulálpam, fresh or clean water was extracted directly from a well and piped to the plot. In Ixtlán de Juárez, fresh water was obtained from the potable water system. Neither synthetic fertilizers nor pest or disease control substances were applied. Crops were established during the dry season following the World Health Organization's Guidelines and Recommendations for the Safe Use of Wastewater in Agriculture⁶. Irrigations with fresh water and with treated wastewater were carried out at the same time, for five days, using 2" diameter hoses to flood the furrows.

Soil analysis

Soil samples were collected from each study site for analysis¹⁸. Four soil samples were obtained at the beginning stage, four at the intermediate stage, and four at the end of the cultivation process, adding up to a total of 12 samples in the cultivated area of Ixtlán. Likewise, a total of 12 soil samples were extracted from the cultivated area of Capulálpam. The initial samples were taken before planting and irrigation, and the intermediate and final samples, 45 and 90 d later, respectively. Two kg of each sample were collected at a depth of 30 cm from each site. The pH and electrical conductivity (EC) were determined with a potentiometer (Conductronic PC45), organic matter (Walkley and Black method), texture (Bouyoucos method), nitrogen (by micro Kjeldahl), and phosphorus (Bray and Kurtz 1 method), the latter using a UV-Vis spectrophotometer (GBC CITRA10).

Measurement of plant growth variables

For each treatment, ten Quinoa plants and six corn plants of an easy-to-handle size were randomly selected from each crop, discarding the plants on the edges. Their height was measured with a flexometer from ground level to the last main leaf; their stem diameter was measured with a vernier at 3.0 cm from the ground, and the number of leaves on each plant was counted. Variables were read at 60 and 90 d in quinoa and corn, respectively. The plants were harvested after 90 d to obtain forage because more than 50 % of the plants no longer showed increased growth in height and stem diameter. To account for biomass, four plants in each treatment were destructively sampled and leaves, stems and roots were separated 90 d after planting. The fresh material was placed in forced air circulation ovens at 65 ^oC until a constant weight was reached. The fresh and dry weight of each plant was obtained. The nitrogen content was measured with an organic elemental analyzer (PerkinElmer, series II, model 2400), and the phosphorus content, by the vanadomolybdic method in a UV-Vis spectrophotometer (GBC, model CITRA10)¹⁹.

Statistical analysis

Analyses of variance and Tukey's mean tests (P≤0.05) were performed on the data obtained in the field for the variables studied in the quinoa and corn crops. The effect of the factors and treatments, i.e., the type of irrigation on both crops established at each site, was evaluated.

Physical and chemical properties of soil

The pH of the soils before crop establishment was "moderately acidic", i.e. pH 5.1 - 6.5¹⁹; at 45 and 90 d after planting. In some cases, the pH increased to a "neutral" level (pH 6.6 - 7.3); such pH increase could be due to nitrates being transformed to atmospheric nitrogen (N2)²⁰. This condition is contrary to that of other studies where soils irrigated with secondary treatment water show decreases in pH of 8.2 to 7.6²¹ and 8.0 to 7.7²². The electrical conductivity (EC) in the soils before planting was 0.31 to 0.44 dS m^-1, which, according to the norm NOM-021-2000, is considered as "negligible salinity effects" (< 1.0 dS m-¹) in both the Ixtlán and Capulálpam soils^23,24. 45 and 90 d after planting, the EC increased mainly when irrigated with TWW, still within the "negligible effects of salinity"; only two soil samples increased to 1.05 and 1.27 dS m^-1, the latter value being categorized as "very slightly saline" (1.1 - 2.0 dS m-¹). The cause of this could be the fact that soils retain cations, expressed as cation exchange capacity (CEC), due to the increase in clay and organic matter²⁵. For the same reason, an increase of a few tenths was observed in soils irrigated with TWW. This confirmed the findings of other researchers in the sense that soil EC increased when TWW irrigation was applied: 0.34 - 0.42 dS m^-1(21) and 2.73 - 4.70 dS m^-1(22.

A similar trend was obtained with soil organic matter. In the initial sampling, the values indicated a "medium" (1.6 - 3.5 %) to "high" (3.6 - 6.0 %) content; after sowing and irrigation (45 d) the percentages increased in both crops and remained in the same "medium" and "high" ranges. In this regard, some researchers indicate that wastewater contributes organic matter to the soil, helping to maintain soil fertility^24,26,27. However, in the final stage (90 d), the organic matter content decreased. In general, TWW improves soil fertility by providing nutrients and other benefits to crops, thereby reducing the use of chemical fertilizers²⁸. The soils have average fertility, average erosion capacity, and average organic matter mineralization capacity, all easy to manage for the farmer. The region where these localities are located has a rugged orography.

Plant growth

Quinoa, a domesticated Andean crop, belongs to the Amaranthaceae family and is also considered a forage crop²⁹. Corn belongs to the Poaceae family, a crop with different uses such as fodder for livestock³⁰. The type of growth and vegetative development differs between the two forage species. An analysis of variance showed significant differences (P≤0.01) in the factor "type of water" for the variables height, stem diameter, and number of leaves in quinoa plants, at 60 and 90 d after planting; the same occurred in corn plants except for the variable number of leaves.

Plant height, stem diameter, and number of quinoa leaves were significantly higher (P≤0.05) when irrigation was with treated wastewater at 60 and 90 days after planting (Table 2). The results for corn were similar (Table 3), except for the number of leaves, which showed no significant difference, which means that the number of leaves was similar when the plants were irrigated with treated wastewater and with freshwater. Plant height was significantly greater (P≤0.05) in plants that received treated wastewater. The results show that treated wastewater should no longer be seen as a waste product, but as a water resource that can be treated and reused productively¹².

Biomass

An analysis of variance showed significant differences (P≤0.05) in root, leaf, and stem fresh and dry weight of the studied quinoa and corn plants when irrigated with fresh water versus treated wastewater. The fresh and dry weights of the quinoa and forage corn crops were significantly higher (P≤0.05) in plants irrigated with treated wastewater (Tables 4 and 5). The fresh and dry weight of the organs of both crops was root < leaves < stems. In quinoa plants irrigated with treated wastewater, the total fresh weight of the leaves + stems were 3.4 times higher, and the dry weight was 3.2 higher. Likewise, the fresh weight of corn was 2.3 higher, and the dry weight 2.6 times higher, when irrigated with treated wastewater. This may be due to the higher nutrient content provided by the TWW, as other research has shown^26,31,32. In a similar study to the present one, the N content in the leaves of corn irrigated with TWW was shown to increase from 1 to 3 % with respect to the crops irrigated with aquifer water³³, and the corn plot irrigated with TWW had a yield of 2.58 t ha^-1, while the yield of the control plot was 1.61 t ha^-1(34.

The water content was significantly higher in quinoa and forage corn plants that were irrigated with TWW than in plants that were irrigated with fresh water (Table 6); the leaves showed lower percentage values of water and a significantly higher water content. In corn plants, the water content ranged between 80 and 90 %; in quinoa, it was 70 to 85 %, depending on the organ concerned. These results express the influence of TWW on the water content.

Plant nitrogen and phosphorus content

In general, the nitrogen and phosphorus content were significantly higher in the organs of quinoa and feed corn plants irrigated with TWW, with the exception of some organs in both crops (Table 7). Research on citrus fruit trees³⁵ and vegetables³⁶ reports a higher N concentration in leaves and a higher crop growth, due to the higher amount of nutrients and organic matter in wastewater¹². The concentration of N and P in the organs of the plants irrigated with TWW does not represent a danger for the plants themselves, and, therefore, neither for the human or animal that consumes it¹². In Almeria, Spain, wastewater is reused because of its moderate salt content and its high content of nutrients for plants, particularly of N, P, and K³⁷. These results indicate that the plants were able to grow more easily when they obtained more N and P. The highest N and P content in quinoa plants was found in the leaves, followed by the stem and nutrient the root, respectively. In forage corn plants, P content was second to that of quinoa, and the N content was higher in the stem, followed by the leaves and the root (Table 7). Khaskhoussy et al²², found higher N content in corn irrigated with TWW: leaves 1.2 % and root 0.6 %, as compared to corn irrigated with FW: leaves 1.0 % and root 0.45 %, Munir et al³⁸ showed that the N content of TWW-irrigated corn plants was significantly higher than that of TWW-irrigated corn plants -of 1.08 %-, compared to plants irrigated with FW (0.66 %). A similar tendency was reflected in the P content of TWW-irrigated plants: 0.19 %, and FW-irrigated plants: 18 %. Therefore, the use of treated wastewater could be considered as an alternative that will help reduce the use of chemical fertilizers on forage crops in the region and replicate them in another site.