In the current study, the effect of using TW on the metal contents of two alfalfa cultivars was examined. Concentrations of macronutrients (N, P, K, Ca, Mg, and S) and micronutrients (Na, Cl, Cu, Zn, Fe, Mn, Pb, Ni, and Co) were also estimated. The obtained results showed that all of the studied independent variables (water type, cultivar, and cutting time) had a significant (
P ≤ 0.05) effect on macronutrient and micronutrient contents in alfalfa plants. Furthermore, the interactions between each pair of independent variables, and the overall interactions between the studied parameters, produced significant (
P ≤ 0.05) differences in nutrient contents in the plants. Although K and Ca contents were higher in WW as compared to TW (
Table 1), irrigation with TW produced plants with slightly higher K and Ca contents, while no differences in S content were observed between plants irrigated with WW and plants irrigated with TW. This agrees with the results obtained by Chávez, et al. [
11], who found that irrigation with untreated wastewater led to higher S levels in alfalfa tissues than that with partially TW. Enhancement of nutrient uptake was reported as one of beneficial gains of irrigation with TW [
29,
30]. Nevertheless, plants irrigated with TW had lower N, P, and Mg contents than those irrigated with WW, regardless of cutting time and cultivar (
Table 3). Furthermore, irrigation with TW led to slight decreases in the contents of all studied micronutrients apart from Cl, which increased by an average of 3.2% compared to plants irrigated with WW. Surprisingly, the contents of Mn and Cd in plants irrigated with TW were lower than in those irrigated with WW; however, TW was characterized by higher Mn and Cd contents as compared to WW. Hussain et al. [
31] showed that levels of different heavy metals (i.e., Cd, Co, Cu, Mn, Zn, Pb, Ni and Cr) in radish, spinach and carrot plants irrigated with TW were below toxic limits, but plants irrigated with 100% TW had lower contents of heavy metals as compared to others irrigated with 25%, 50% and 75% TW. In general, metals uptake could be affected by several factors including plant cultivar (genotype), external concentrations, and soil pH. Although soils irrigated with TW showed a roughly 50% decrease in Pb content (
Table 2), no differences in Pb content were observed between treatments (
Table 4). Such a result could be partially explained by the ability of Pb to move in the soil solution and leach to the underground water [
32], in addition to its high dependability on the solubility and pH of soil [
33]. Chávez, et al. [
11] studied alfalfa tissues and found that Cu and Zn were the most sensitive micronutrients to irrigation with wastewater; however, in the present study, significant decreases in N, P, and K contents were observed after irrigation with TW. Variations were also detected in the macronutrient and micronutrient contents of the different cultivars, regardless of cutting time and the type of water used for irrigation. Plants of the Alhassawy cultivar contained higher N, P, and K contents but lower Ca, Mg, and S contents than plants of the CUV101 cultivar (
Table 3). In general, cultivar variation affects several alfalfa yield traits, including morphology, forage quality, overall yield [
34], and elemental composition of the plant shoots [
35]. It is perhaps unsurprising, then, that plants of the Alhassawy cultivar had higher contents of all micronutrients apart from Cu, Zn, and Co than plants of the CUV101 cultivar (
Table 4). James et al. [
36] examined five different cultivars of alfalfa and found significant variations in P, Ca, Na, Mn, Sr, and Zn contents. The plant material obtained in the first cutting period (2013/2014) had higher contents of the majority of macronutrients and micronutrients than plant materials obtained during the second cutting period (2014/2015). This could be correlated with the reduction observed in contents of macronutrients in the soils irrigated with TW or WW at the end of the experiment. It was previously reported that long-term application of TW led to significant reductions in plants’ contents of heavy metals [
37]. Another study that sampled alfalfa cultivated lands that had been irrigated with TW since 80 years found that levels of Fe, Cu, Zn and Mn were higher if compared with their levels in soils irrigated with potable water [
38]. The exceptions to this were S, Na, Ni, and Co, which were present in higher contents in plants obtained during the second cutting period than the first cutting period (
Table 3 and
Table 4). Other studies have also shown that cutting time significantly affects the yield, nutritive value, and elemental composition of alfalfa plants [
39].
The interaction between each pair of independent variables significantly (
P ≤ 0.05) affected the nutrient content of alfalfa plants.
Table 5 and
Table 6 show the interactive effects between each pair of independent variables on alfalfa macronutrient and micronutrient contents, respectively. Alhassawy cultivar plants irrigated with WW showed the highest contents of all macronutrients (apart from S, as no significant differences in S content were observed in any of the studied plants), and most micronutrients (except Na and Ni) compared to Alhassawy plants irrigated with TW, CUV101 plants irrigated with TW, and CUV101 plants irrigated with WW. CUV101 plants irrigated with WW contained the highest Na and Ni contents out of any of the treatments. The interaction between cutting time and water type used for irrigation did not produce significant variations in micronutrient contents among the different treatments; however, plants obtained in the second cutting period had lower nutrient contents than those obtained in the first cutting period. Plants from the first cutting period irrigated with WW contained the highest micronutrient contents apart from Na and Ni, which were highest in plants from the second cutting period irrigated with WW. There was no significant variation in Pb content among any of the treatments. CUV101 plants obtained in the first cutting period had the highest contents of all macronutrients except S and Ca, which were highest in CUV101 plants of the second cutting period and in Alhassawy plants obtained during the first cutting period, respectively. The contents of several micronutrients (Cl, Fe, Mn, and Co) were highest in Alhassawy plants from the first cutting period. However, the contents of other micronutrients (Cu, Zn) were higher in CUV101 plants of the first cutting period. No significant changes in Pb content were observed among the treatments, and the highest Co content was found in CUV101 plants obtained during the second period. The results obtained in this study indicate that the effect of irrigation water quality significantly depends on other factors, mainly cultivar and cutting time. For example, irrigation with saline water has been shown to affect total forage production and iron relations (including iron uptake and translocation inside the plant tissues) in alfalfa plants, and this effect varies from one cultivar to another [
40]. Nevertheless, usage of untreated wastewater increases yield as well as N, P, K, Ca, Mg, Na, Fe, Mn, Zn, Cu, Pb, Ni, and Cd contents in cauliflower (
Brassica oleracea L. var. botrytis) and red cabbage (
Brassica oleracea L. var. rubra), without contaminating the soil or plant tissues with heavy metals [
41].
The results obtained in the current study demonstrate that the overall interaction between the studied variables, namely, water type used for irrigation, cultivar, and cutting time, significantly (
P ≤ 0.05) affected alfalfa macronutrient and micronutrient contents. CUV101 plants obtained in the first cutting period that were irrigated with WW showed the highest N, P, and Mg contents. There were no differences in P content for first cutting period CUV101 plants irrigated with TW (
Table 7). Furthermore, first cutting period CUV101 plants irrigated with TW contained the highest K content among all the studied treatments. Moreover, Alhassawy plants irrigated with TW obtained in the first and the second cutting periods showed the highest Ca and S contents, respectively. N, K, Mg, and Na contents in alfalfa plants irrigated with TW were higher than in plants irrigated with tap water [
42]. Regarding the present study, the effects of the three independent variables on alfalfa micronutrient contents are shown in
Table 8. Alhassawy plants obtained in the first cutting period that were irrigated with WW had the highest Fe, Mn, and Pb contents. In contrast, Al-Karaki [
42] found that Pb and Ni contents were higher in plants irrigated with TW than in plants irrigated with tap water. Irrigation with TW did not increase Cl content in the tissues of Alhassawy plants obtained in the first cutting period. The highest Cu and Zn contents were found in tissues of CUV101 plants irrigated with WW and harvested in the first cutting period. Higher Zn content in alfalfa tissues is considered an undesirable trait because it can lead to problems in crop yield. This is mainly due to the antagonistic effect of Zn against N in different parts of the plant [
43], which might lead to N deficiency and, consequently, affect yield. In contrast, our results showed a significantly strong positive association between Zn content and N content (
Table 9). The highest Ni and Co contents were found in Alhassawy plants irrigated with WW that were harvested in the second cutting period. The highest Na content was observed in Alhassawy plants irrigated with WW that were harvested in the second cutting period. The crucial nutrients for the health of alfalfa plants are N, P, K, Ca, Mg, and Na [
44]. Our results demonstrate that irrigation with TW does not lead to dramatic decreases in the contents of these nutrients within plants. Nevertheless, irrigation with TW does lead to significant increases in K and Ca contents, without any adverse effects on P content. These results agree with those found in a study by Chávez et al. [
11], who studied alfalfa irrigated with untreated or partially TW. Kiziloglu et al. [
41] concluded that untreated wastewater can be used confidently, on a short-term basis, in agricultural land, while primary TW is suitable for long-term use in sustainable agriculture. Irrigation with TW has been shown to greatly enhance the physiological status, enzymatic activity, and nutritional composition of alfalfa plants, compared to irrigation with WW [
45]. Changes in the contents of macro and micronutrients in plants irrigated with TW could be attributed to several factors including the accumulation of heavy metals inside the plant tissues, which hamper the uptake of other essential ions. In the current study, a negative correlation between Ni and Mg contents was observed, evidence for the effect of heavy metals leading to decreased uptake of essential nutrients. Another factor is the presence of contaminants (e.g., pharmaceutical) that might stimulate or hinder the uptake of specific nutrients [
46]. Such contaminants might bind to a plant cell’s membrane fraction and thus facilitate or obscure the flux of several ions, e.g., Ca
2+ [
47,
48]. Moreover, some nutrients play critical roles in a plant’s tolerance to heavy metals, which might explain the vast increase in their levels in plant tissues. In the current study, increased levels of certain heavy metals (i.e., Fe, CU, Zn and Mn) were associated with an increase in magnesium levels. Mg was shown to have a potential role in heavy metal stress tolerance in mangrove [
49] and soybean [
50] plants.