Impacts of Long-Term Treated Wastewater Irrigation and Rainfall on Soil Chemical and Microbial Indicators in Semi-Arid Calcareous Soils

Abstract

Frequent and severe droughts intensify water scarcity in arid and semi-arid regions, creating an urgent need for alternative water resources in agriculture. Treated wastewater (TWW) has emerged as a sustainable option; however, its long-term use may alter soil properties and pose risks if not carefully managed. This study tested the hypothesis that long-term TWW irrigation increases soil salinity, alters fertility, and affects microbial quality, with rainfall partially mitigating these effects. Soil samples (n = 96 at each time point) were collected from two calcareous soils in Jordan, silt loam (Mafraq) and silty clay loam (Ramtha), under four treatments (control and 2, 5, and 10 years of TWW irrigation) at three depths (0–30, 30–60, and 60–90 cm). Sampling was conducted at two intervals, before and after rainfall, to capture the seasonal variation. Soil indicators included the pH, electrical conductivity (EC), sodium (Na⁺), chloride (Cl⁻), calcium (Ca²⁺), magnesium (Mg²⁺), exchangeable sodium percentage (ESP), sodium adsorption ratio (SAR), organic matter (OM), total nitrogen (TN), and microbial parameters (total coliforms (TC), fecal coliforms (FC), and Escherichia coli). Data were analyzed using a linear mixed-effects model with repeated measures, and significant differences were determined using Tukey’s Honest Significant Difference (HSD) test at p < 0.05. The results showed that rainfall reduced Na⁺ by 70%, Cl⁻ by 86%, EC by 73%, the ESP by 28%, and the SAR by 30%. Furthermore, the TC and FC concentrations were diminished by almost 96%. Moderate TWW irrigation (5 years) provided the most balanced outcomes across both sites. This study provides one of the few long-term field-based assessments of TWW irrigation in semi-arid calcareous soils of Jordan, underscoring its value in mitigating water scarcity while emphasizing the need for monitoring to ensure soil sustainability.

1. Introduction

Currently, the world is facing many challenges in achieving sustainable agricultural production and maintaining water and food security. Climate change, the rapidly increasing population, urbanization, economic growth, frequent droughts, imbalances in the rainfall distribution, and the degradation of water quality are all leading to increases in the water demand and causing severe limitations of fresh water resources for agricultural use in many countries around the world, especially in arid and semi-arid regions [1,2,3]. The Middle East and North Africa (MENA) region is among the most water-stressed globally, characterized by severely limited freshwater resources and a heightened susceptibility to droughts [4,5]. Jordan epitomizes this crisis: over 90% of the nation receives less than 200 mm of annual rainfall, and the per capita water availability is among the lowest worldwide [6,7,8]. Jordan acquires roughly 78 cubic meters (m³) of renewable water resources per capita annually, representing only about 1% of the global per capita average water availability [9]. The previously mentioned limits, along with demographic pressures and political instability, place Jordan among the countries that are most affected by water scarcity [10,11]. Agriculture is the main sector affected by water scarcity, utilizing around 70% of freshwater resources in Jordan and the wider MENA region, highlighting the sector’s susceptibility to shortages [6,7,12]. Consequently, agriculture in Jordan relies on irrigation to attain sufficient yields [13]. Frequent and severe droughts raise the challenge of meeting the agricultural water demand and increase the need to reuse TWW as a non-conventional water resource for non-potable purposes.

Irrigation with TWW represents a key factor for sustainable agriculture and prospective production in arid and semi-arid regions. In addition, it is a reliable and sustainable source of water and nutrients for crops to achieve sustainability and food security [7,14,15,16]. The current technology has the ability to treat wastewater to any level required. The reuse of TWW typically involves varying levels of plant nutrients, influenced by the quality of the municipal water source, the nature of the additional wastes, the extent of the treatment, and the type of technology used [13,17]. Nutrients in TWW will reduce the use of costly chemical fertilizers and enhance soil fertility and crop productivity [18,19]. Although the reuse of TWW could help meet the demand for water in the agriculture sector, it would carry both environmental and agronomic hazards that must be given more consideration [20]. Therefore, long-term irrigation with TWW has both positive and negative impacts [21]. Long-term irrigation with TWW may have effects either on crops or on physical, chemical, and biological properties of soils [22]. The characteristics of soil, including the salinity, pH, nutrient concentrations, micro- and macronutrients, soil microbial populations, and organic matter, will be altered by TWW irrigation [18,21,23]. Higher salinity and sodicity in TWW could cause a serious soil concern, and they would reduce agricultural productivity and threaten food security [14].

Globally there are several field experiments and studies regarding the reuse of TWW for irrigation. Nonetheless, limited studies have examined the varying impacts of TWW reuse on diverse soil textures, including silt loam and silty clay loam, in semi-arid areas such as Jordan. This disparity is especially notable considering the region’s growing dependence on alternate water sources due to escalating water scarcity. This research aimed to assess the effects of prolonged irrigation with TWW on two soil types under varying precipitation levels in northern Jordan (Mafraq and Ramtha). The results of our investigation offer essential insights into the long-term chemical, physical, and biological alterations in soils exposed to TWW irrigation.

These findings can guide sustainable agriculture practices by providing evidence that can guide policymakers in developing safe reuse regulations and farmers aiming to sustain soil productivity under water-limited conditions.

2. Materials and Methods

Study area and experimental conditions:

The experiment was conducted on land in the cities of Ramtha and Mafraq in northern Jordan, Figure 1.

The city of Ramtha:

The city of Ramtha is located 88 km north of Amman city, where it rises 590 m above the sea level [24,25,26]. The mean annual rainfall ranges from 450 mm in the northwest to less than 150 mm in the southeast [27,28]. The mean annual minimum and maximum temperatures are 10.7 and 23.7 °C, respectively [29,30]. Al-Ramtha WWTP is about 4 km northwest of the Ramtha city center (32°35′ north latitude and 35°59′ east longitude), with an elevation of 490 m above sea level [31]. Farmers utilize land next to the WWTP on the western side to raise fodder crops, including barley, corn, and alfalfa. The geomorphological parent materials consist of undulating plains and deep colluvial and aeolian mantles weathered into cracking clays. The slope ranges from 0.4% to 4.8%, with the altitude ranging from 420 to 610 m.

The City of Mafraq:

The city of Mafraq is located in northern Jordan, within the western part of Mafraq governorate. It is located about 65 km from Amman [32]. The annual rainfall is less than 150 mm/year. This region is characterized by dryness as a result of the mean annual minimum and maximum temperatures for Mafraq being 9.3 °C and 24.0 °C, respectively [33]. The Mafraq WWTP is located approximately 6 km north of Mafraq City (32°24′ north latitude and 36°13′ east longitude). Farmers nowadays lease around 24 hectares of the land for the cultivation of wheat, corn, sorghum, alfalfa, and olives. The geomorphological parent material consists of a gently undulating basalt plain, characterized by relatively deep to deep colluvial and aeolian deposits, which are somewhat calcareous. The mean annual rainfall is 140 to 190 mm. The altitude is between 620 and 780 m, and the slope is 0.4%.

Sampling sites:

Soil samples were obtained from fields surrounding the two wastewater treatment plants (WWTPs) cultivated with alfalfa and irrigated with TWW throughout three distinct durations (2 years, 5 years, and 10 years). Each treatment was represented by a standard 100 m² plot, from which four composite samples were randomly collected with an auger at three depths (0–30, 30–60, and 60–90 cm). Each composite consisted of three subsamples taken at random locations within the plot, which were homogenized to obtain a representative sample per depth. Adjacent rainfed fields that had never been irrigated with wastewater or groundwater served as controls [34,35,36]. All plots exhibited a uniform agricultural history, being continuously planted with alfalfa under conventional tillage.

Sampling was carried out twice, before the rainy season (17–20 October 2022) and after its end (27–30 April 2023), to capture the maximum seasonal variance between pre-irrigation and post-rainfall circumstances. The average rainfall received during this period was approximately 278 mm in Ramtha and 146 mm in Mafraq, based on meteorological data. In total, 96 samples were collected at each time point across both locations. Additional undisturbed cores were collected individually using stainless steel cylinders for bulk density analysis at three depths: 0–30 cm, 30–60 cm, and 60–90 cm. Microbiological samples were freshly collected under aseptic circumstances; each plot was treated as an independent sampling unit to minimize cross-contamination risks. Soil cores were collected using disinfected augers, with tools sterilized using 70% ethanol between samples. Sterile gloves were replaced at each plot, and all samples were transferred into sterile polyethylene bags, stored in an insulated icebox for transport to the laboratory within two hours. Samples were processed within 24 h after collection and maintained at 4 °C until analysis to mitigate alterations in microbial activity [35], while other samples were air-dried, crushed, and sieved through a 2 mm screen for physical and chemical examination preparation. The saturation paste extract was prepared following the guidelines of the United States Salinity Laboratory Staff (USSLS) [37] and thereafter employed for the measurement of cations and anions.

Climatic and Environmental Covariates:

This study was conducted at two semi-arid locations (Mafraq and Ramtha) that differ in long-term temperature, evapotranspiration (ET), and rainfall regimes. No statistical adjustments were made for temperature or ET; however, within each location, both control (rainfed) and TWW-irrigated fields were exposed to the same local weather conditions during common sampling windows, minimizing potential confounding. Rainfall was explicitly addressed as a seasonal factor in the sampling design (pre- and post-rainfall), while broader climatic differences between Mafraq and Ramtha were considered as a location effect in the analysis.

Soil analysis:

Soil texture was assessed using the hydrometer method [38]. The soil’s bulk density (BD) was determined using the core technique [39]. Soil pH was measured using a digital pH meter (Edge pH, Hanna Instruments, Padova, Italy), with a resolution of 0.01 pH units and an accuracy of ±0.01. Electrical conductivity (ECe) was determined using conductivity meter (HD 3406.2, Delta OHM, Caselle di Selvazzano, Italy), with a resolution of 0.01 dS m⁻¹ and an accuracy of ±0.5%. Both measurements were performed on saturated paste extracts, following the methodologies described in [40,41]. Sodium (Na⁺) was quantified using a flame photometer (PFP7, Jenway Ltd., Staffordshire, UK), with a detection limit of 0.02 mg L⁻¹ following the standard methodology [42]. Soluble chloride ions (Cl⁻⁾ were determined by silver nitrate titration using potassium chromate as an indicator, according to the standard method [37], with a practical detection limit of approximately 0.5 meq L⁻¹. Organic matter was measured by wet combustion method as described in [43]. Total nitrogen (TN) was measured by Kjeldahl method with an automatic distillation unit (UDK 149, VELP Scientifica, Usmate Velate, Italy) according to [44,45], with a detection limit of 0.1 mg N (0.01% N in soil). Calcium (Ca²⁺) and magnesium (Mg²⁺) were determined by EDTA titration method as outlined by [46], with practical detection limits of approximately 1 mg L⁻¹ for Ca²⁺ and Mg²⁺. Total coliforms, fecal coliforms, and Escherichia coli (E. coli) were assessed using the multiple tube fermentation (MTF) technique as outlined by [47], and the results were presented in Most Probable Number (MPN/g) according to [48]. The soil sodium adsorption ratio (SAR) was calculated according to [49], using the following formula:

$$\mathrm{S}\mathrm{A}\mathrm{R}={\displaystyle \frac{{[\mathrm{N}\mathrm{a}}^{+}]}{\sqrt{\frac{\left(\right[{\mathrm{C}\mathrm{a}}^{2+}]+{[\mathrm{M}\mathrm{g}}^{2+}])}{2}}}}$$

(1)

where [Na⁺], [Ca²⁺], and [Mg²⁺] are the concentrations of sodium, calcium, and magnesium ions expressed in meq L⁻¹. Exchangeable sodium percentage (ESP) was calculated according to [25] using the following equation:

$$\mathrm{E}\mathrm{S}\mathrm{P}={\displaystyle \frac{100 (-0.0126 + 0.01475 \times SAR) }{1 + (-0.0126 + 0.01475 \times SAR)}}$$

(2)

All measurements were conducted in triplicate, incorporating equipment calibration, blanks, and standard solutions for analytical quality control; mean results are presented, with a relative standard deviation consistently below 5%. For microbial analysis, 10 g of fresh soil was aseptically weighed and transferred into 90 mL of sterile buffered peptone water (1% w/v) in a sterile flask, then homogenized by shaking for 30 min to detach microorganisms from soil particles. The suspension was serially diluted (10⁻¹ to 10⁻⁵), and aliquots from each dilution were inoculated into selective broth media using the (MTF) technique. Presumptive coliforms were detected in Lauryl Tryptose Broth (LTB) with Durham fermentation tubes (Thermo Fisher Scientific, Waltham, MA, USA) for gas formation, confirmed in Brilliant Green Bile Broth (BGBB) for total coliforms and in EC broth at 44.5 °C for fecal coliforms. E. coli was further confirmed by fluorescence in EC-MUG medium. Microbiological analyses incorporated sterile blanks as negative controls and standard reference strains (E. coli ATCC 25922, E. aerogenes) as positive controls to guarantee analytical quality assurance [50,51].

Statistical analysis

Statistical analyses were conducted using a linear mixed-effects model (LMM) (Mixed-model ANOVA with repeated measures) implemented in JMP software, Version 16 (SAS Institute Inc., Cary, NC, USA, 2021). The model was fitted using Restricted Maximum Likelihood (REML) with a variance components covariance structure, and degrees of freedom were calculated using the containment method. Fixed effects included location (Mafraq, Ramtha), irrigation treatment (control and 2, 5, and 10 years), soil depth (0–30, 30–60, and 90 cm), and season (before vs. after rainfall), together with their interactions. Replicate plots were treated as random effects nested within location × treatment, while season was modeled as a repeated measure on the same plots. A total of 192 observations (4 replicates × 2 seasons × 2 locations × 4 treatments × 3 depths) were analyzed as illustrated in

Table 1. Factors and levels included in the experimental design for statistical analysis.

Class	Levels	Values
Replicates	4	1, 2, 3, 4
Time	2	Before rainfall, after rainfall
Location	2	Mafraq, Ramtha
Treatment	4	10 years, 5 years, 2 years, control
Depth	3	0–30 cm, 30–60 cm, 60–90 cm

. Type III tests of fixed effects were used to evaluate significance, and when differences were detected, pairwise comparisons were carried out using the Tukey–Kramer Honest Significant Difference (HSD) test at a significance threshold of p < 0.05.

To ensure validity of the analyses, model assumptions were verified by assessing the normality of residuals (Shapiro–Wilk test and Q–Q plots) and the homogeneity of variances (Levene’s test) prior to conducting HSD comparisons. Residual diagnostics and boxplots were used to identify potential outliers, but no extreme values required removal. Missing values were minimal and were excluded listwise, with the REML procedure providing valid estimates under these conditions. When the assumptions of ANOVA were not met, data were analyzed using the Kruskal–Wallis test, followed by Bonferroni-adjusted Mann–Whitney U tests for pairwise comparisons (p < 0.05).

In addition to the mixed-model ANOVA, regression analyses were conducted to examine irrigation duration as a quantitative predictor of soil response. This complementary approach facilitated both statistical testing of treatment effects and visualization of long-term response trends.

3. Results and Discussion

3.1. Treated Wastewater Quality

The physical, chemical, and biological properties of the TWW effluents from the Ramtha and Mafraq WWTPs are illustrated in

Table 2. Average chemical and biological characteristics of TWW effluents from the Ramtha and Mafraq WWTPs during 2022–2023, compared with Jordanian Standards (JS 893:2006) [52] and Food and Agriculture Organization (FAO).

Parameter	Unit	Ramtha WWTP Effluent	Mafraq WWTP Effluent	Jordanian Standards (JS 893:2006)	FAO
pH	-	7.95	7.91	6–9	6.5–8.4
EC	dS m−1	2.29	1.95	-	<3 (no restriction), 3–9 (slight to moderate restriction), >9 (severe restriction)
Ca+2	mg L−1	28.50	80	230	-
Mg+2	mg L−1	69.90	37	100	-
Na+	mg L−1	314.94	245	230	≤200 (slight), 200–400 (moderate), >400 (severe)
TN	mg L−1	98	53	100	-
Cl−	mg L−1	490.30	320	400	≤140 (slight), 140–350 (moderate), >350 (severe)
COD	mg L−1	93	118	500	-
BOD5	mg L−1	21.2	20	300	-
TSS	mg L−1	39	30	300	-
TDS	mg L−1	1553	1257	1500	2000
E. coli	MPN/100 mL	8.8 × 106	2.95 × 105	-	-

Source: Yarmouk Water Company (YWC) and the National Agricultural Research Center (NARC) for TWW data. Maximum allowable limits for fodder crop irrigation according to the Jordanian Standard JS 893:2006 (Water Authority of Jordan, 2006) and FAO guidelines [53].

. The majority of parameters, such as the pH, Ca²⁺, Mg²⁺, TN, chemical oxygen demand (COD), biochemical oxygen demand (BOD₅), and total suspended solids (TSS), adhered to the Jordanian Standard for utilized domestic wastewater reuse in forage crop irrigation (JS 893:2006).

Several exceedances were documented: Na⁺ concentrations were 314.9 mg L⁻¹ in Ramtha and 245 mg L⁻¹ in Mafraq, both surpassing the permissible limit of 230 mg L⁻¹; Cl⁻ levels were elevated in Ramtha at 490.3 mg L⁻¹, exceeding the standard of 400 mg L⁻¹; and total dissolved solids (TDS) surpassed the limit of 1500 mg L⁻¹ in Ramtha, measuring 1553 mg L⁻¹. Prior research has documented similar challenges regarding Na⁺ and Cl⁻ concentrations in the Ramtha and Mafraq WWTPs [25,26,54]. The majority of the salt and chloride present in treated wastewater originates from domestic activities, particularly detergents and water softeners. The unusual chloride levels detected at the Ramtha plant indicate that part of the fluctuations could originate from alterations in the facility’s chlorine application during the last phase of disinfection [26]. Based on the FAO classification [53], Na⁺ levels in Ramtha (314.9 mg L⁻¹) and Mafraq (245 mg L⁻¹) fall within the moderate to severe restriction range, while Cl⁻ concentrations place Ramtha in the severe restriction category (>350 mg L⁻¹) and Mafraq in the moderate range (140–350 mg L⁻¹). The increased values represent significant problems for the safe reuse of TWW in agricultural irrigation and underscore possible concerns of soil sodicity and salinity accumulation with prolonged TWW irrigation.

3.2. Alterations in Soil’s Physical Properties Resulting from Long-Term TWW and Rainfall

3.2.1. Soil Texture

The soil texture analysis was conducted using the USDA soil texture triangle [55], which revealed considerable variances between the two research locations.

In Mafraq, as illustrated in

Table 3. Soil texture classification before and after rainfall across treatments, depths, and locations.

Location	Treatment	Depths (cm)	Before Rain	After Rain
Mafraq	10 years	0–30, 30–60, and 60–90	Silt Loam	Silt Loam
	5 years	0–30, 30–60, and 60–90	Silt Loam	Silt Loam
	2 years	0–30, 30–60, and 60–90	Silt Loam	Silt Loam
	Control	0–30, 30–60, and 60–90	Silt Loam	Silt Loam
Ramtha	10 years	0–30, 30–60, and 60–90	Silty Clay Loam	Silty Clay Loam
	5 years	0–30, 30–60, and 60–90	Silty Clay Loam	Silty Clay Loam
	2 years	0–30, 30–60, and 60–90	Silty Clay Loam	Silty Clay Loam
	Control	0–30, 30–60, and 60–90	Silty Clay Loam	Silty Clay Loam

, the soils across all treatments and depths were consistently classified as silt loam, with the silt content ranging from 52.0% to 62.6% and the clay content ranging from 16.6% to 25.7%. The relatively high proportion of silt and the low clay content in Mafraq indicates a moderately fine texture, which has moderately structured soil and moderate permeability, with good water retention and aeration characteristics [56]. In contrast, soils in Ramtha were predominantly classified as silty clay loam, with higher clay contents, averaging above 30% across most depths and treatments, and correspondingly lower sand fractions. Ramtha’ soil has a denser structure, lower permeability, and a greater tendency for water retention and potential salinity accumulation, particularly under long-term TWW irrigation [57]. The differences in soil texture are primarily attributed to the parent material and depositional environment of each region [58].

These findings in Table 3 confirm the stability of the soil texture across treatments, depths, and locations, indicating that neither long-term TWW irrigation nor rainfall events resulted in significant changes in the particle size distribution [59].

3.2.2. Bulk Density (BD)

As shown in

Table 4. Effect of location on bulk density (Tukey–Kramer HSD, p < 0.05).

Location	Bulk Density Estimate (g/cm3) (Mean ± SE)	Letter Group
Mafraq	1.2167 ± 0.01426	a
Ramtha	1.1446 ± 0.01426	b

Note: Values are means ± SE (n = 48 samples per location). Means followed by different letters are significantly different at p < 0.05 according to Tukey–Kramer HSD test at p < 0.05.

, there is a considerable significant difference in the bulk density (BD) between Mafraq (1.217 ± 0.014 g/cm³, group a) and Ramtha (1.144 ± 0.014 g/cm³, group b) after using the Tukey–Kramer HSD test at p < 0.05. This aligns with their distinct soil textures, silt loam in Mafraq and silty clay loam in Ramtha, which fundamentally differ in their compaction and porosity.

The substantial effect occurred post-rainfall as illustrated in

Table 5. Effect of time on BD and salinity-related soil parameters (Na⁺, Cl⁻, EC, ESP, and SAR).

Time	BD (g/cm3) (Mean ± SE)	Na+ (ppm) (Mean ± SE)	Cl− (ppm) (Mean ± SE)	EC (dS/m) (Mean ± SE)	ESP (%) (Mean ± SE)	SAR (Mean ± SE)
Before rain	1.1565 ± 0.01426 b	1279.21 ± 113.05 a	4107.65 ± 433.11 a	6.5200 ± 0.6600 a	2.5167 ± 0.05438 a	12.1740 ± 0.2841 a
After rain	1.2048 ± 0.01426 a	373.96 ± 113.05 b	563.19 ± 433.11 b	1.7408 ± 0.6600 b	1.8051 ± 0.05438 b	8.5608 ± 0.2841 b

Note: Values are means ± SE (n = 96 observations per time). Means followed by different letters are significantly different at p < 0.05 according to Tukey–Kramer HSD test at p < 0.05.

, where the BD significantly increased from 1.1565 ± 0.01426 before the rainfall to 1.2048 ± 0.01426 after rainfall. This finding aligns with a previous study [60,61] that indicated that successive rainfall events lead to an increased BD due to the surface compaction and soil crusting caused by the raindrop impact on exposed soil surfaces. Conversely, several studies have indicated a decline in bulk density during wet times [56]. On the other hand, all salinity parameters (Na⁺, Cl⁻, EC, ESP, and SAR) were leched and significantly reduced after rainfall, Table 5. The rainfall significantly reduced the Na⁺ concentration from 1279.21 ± 113.05 ppm before rain to 373.96 ± 113.05 ppm after rain (p < 0.05). Moreover, there was a significant reduction in Cl⁻ levels from 4107.65 ± 433.11 ppm (group a) before rain to 563.19 ± 433.11 ppm (group b) after rain, indicating a strong leaching effect.

As shown in Figure 2, BD values across all treatments ranged between approximately 1.05 and 1.3 g/cm³, reflecting moderate compaction levels characteristic of silt loam and silty clay loam soils. There was considerable variation between sites, treatments, and rainfall occurrences. In Mafraq, the BD was generally higher compared to Ramtha, consistent with the coarser silt loam texture.

Bulk density values showed significant variation between locations, irrigation durations, and rainfall conditions, as indicated by the HSD test, p < 0.05. Prior to rainfall, the highest BD in Mafraq was observed under the 2-year TWW treatment (1.253 ± 0.04 g/cm³), followed closely by the 10-year treatment (1.2453 ± 0.04 g/cm³). Interestingly, the control showed the lowest BD (1.069 ± 0.04 g/cm³). After rainfall, the BD was increased for all treatments, peaking with the 10-year treatment (1.2988 ± 0.04 g/cm³; group a), suggesting moisture-induced compaction or swelling effects. In contrast, before rainfall in Ramtha, the 5-year treatment exhibited the highest BD (1.2446 ± 0.04 g/cm³), which significantly differed from the 2-year treatment (0.9914 ± 0.04 g/cm³), indicating that BD accumulation in this silty clay loam soil may occur more gradually. The control and 10-year treatments showed intermediate bulk density values and shared letter groups (ab). After rainfall the BD ranged from 1.1279 to 1.1825 g/cm³, and no treatment showed a statistically distinct mean. All treatments were grouped under (ab). Overall, the results support the hypothesis that long-term TWW irrigation increases the bulk density, particularly in Mafraq [62].

3.3. Salinity Dynamics Under Long-Term TWW Irrigation and Rainfall Event

This section presents the salinity-related parameters (Na⁺, Cl⁻, EC, ESP, and SAR) analyzed before and after rainfall across different TWW irrigation durations, soil depths, and locations. Each parameter is discussed independently due to distinct trends and statistically significant interactions.

3.3.1. Sodium

As illustrated in

Table 6. Mean values ± standard error (SE) of salinity parameters (Na⁺, Cl⁻, EC, and ESP) under different TWW irrigation durations at two locations: Mafraq and Ramtha.

Location	Treatment	Na+ (ppm) (Mean ± SE)	Cl− (ppm) (Mean ± SE)	EC (dS/m) (Mean ± SE)	ESP (%) (Mean ± SE)	SAR (Mean ± SE)
Mafraq	10 years	403.29 ± 230.96 b	283.26 ± 697.10 b	1.8018 ± 1.5362 b	1.8716 ± 0.1088 b	8.8758 ± 0.5682 b
	5 years	328.70 ± 230.96 b	276.60 ± 697.10 b	1.3490 ± 1.5362 b	1.5553 ± 0.1088 b	7.3504 ± 0.5682 b
	2 years	421.23 ± 230.96 b	312.10 ± 697.10 b	1.9308 ± 1.5362 b	1.7492 ± 0.1088 b	8.2825 ± 0.5682 b
	Control	3877.43 ± 230.96 a	8469.71 ± 697.10 a	22.2967 ± 1.5362 a	4.3380 ± 0.1088 a	21.4212 ± 0.5682 a
Ramtha	10 years	537.14 ± 230.96 a	414.91 ± 697.10 a	1.8425 ± 1.5362 a	2.7111 ± 0.1088 a	12.9926 ± 0.5682 a
	5 years	472.63 ± 230.96 a	392.72 ± 697.10 a	1.6716 ± 1.5362 a	2.3331 ± 0.1088 a	11.1246 ± 0.5682 a
	2 years	403.32 ± 230.96 a	337.99 ± 697.10 a	1.5715 ± 1.5362 a	1.7243 ± 0.1088 b	8.1647 ± 0.5682 b
	Control	168.93 ± 230.96 b	80.6146 ± 697.10 a	0.5794 ± 1.5362 a	1.0047 ± 0.1088 c	4.7275 ± 0.5682 c

Note: Values are means ± SE (n = 48 observations per location). Means followed by different letters within the same column are significantly different at p < 0.05 according to Tukey–Kramer HSD.

, the Na⁺ concentration in the control was significantly higher in Mafraq than Ramtha (HSD, p < 0.05). All TWW treatments showed significantly lower sodium concentrations compared to the control, which displayed the highest Na⁺ level (3877.43 ± 230.96 parts per million (ppm), group a). Conversely, Ramtha exhibited higher Na⁺ concentrations across all TWW treatments (ranging from 403.32 to 537.14 ± 230.96 ppm, group a), while the control displayed a significantly lower sodium concentration (168.93 ± 230.96 ppm, group b).

The polynomial regression confirmed that salinity parameters in Mafraq followed a quadratic trend, decreasing from the control to 5 years and then increasing at 10 years, whereas in Ramtha the variables exhibited a positive linear trend with the irrigation duration (Figure S1a–e).

These patterns can be attributed to soil texture, which influences both sodium retention and leaching capacity, as reported in previous studies. The soils of Ramtha possess smaller particles and reduced permeability, which restrict sodium leaching and improve cation retention, resulting in increased Na⁺ buildup over time and potentially leading to waterlogging and salinity accumulation, especially in semi-arid environments [63]. The reduced Na⁺ concentrations in the Mafraq TWW treatments indicate that the TWW had diluted sodium concentrations over time and enhanced leaching in this lighter textured soil.

As shown in Figure 3, salinity indicators (Na⁺, Cl⁻, EC, ESP, and SAR) exhibited clear regression trends with the irrigation duration in both Mafraq and Ramtha soils. In Mafraq, polynomial regressions showed strong fits (R² = 0.82–0.83), with the Na⁺, Cl⁻, EC, ESP, and SAR generally decreasing up to 5 years of irrigation, followed by a slight increase at 10 years.

In Ramtha, regression models (R² = 0.91–0.99) indicated positive linear-to-quadratic trends, with salinity indicators increasing steadily with the irrigation duration. Rainfall sampling locations consistently exhibited reduced values compared to pre-rainfall samples, underscoring the leaching effect.

In Mafraq, Figure 3a, Na⁺ concentrations followed a quadratic trend, decreasing from the control to the 5-year TWW treatment, before rising again at 10 years. This pattern suggests that in the early years, salts were more effectively leached in the coarse-textured silt loam, limiting sodium buildup. However, under prolonged irrigation, cumulative Na⁺ loading and reduced leaching efficiency led to renewed sodium accumulation. The post-rainfall decline in Na⁺ further confirms the role of rainfall in temporarily flushing sodium from the root zone. This aligns with previous research indicating that rainfall can mobilize and wash away soluble salts from the upper soil layers [63].

In contrast in Ramtha, Figure 3b, Na⁺ increased steadily with the irrigation duration, as indicated by the strong positive regression fit. The finer texture and higher clay content of Ramtha soils enhance sodium retention and limit leaching, leading to a progressive rise in Na⁺ with long-term TWW irrigation [64]. Even after rainfall, reductions were modest, underscoring the limited mobility of sodium in the silty clay loam [65].

3.3.2. Chloride

As shown in Table 6, the Cl⁻ concentration in Mafraq was significantly higher in the control treatment (8469.71 ± 697.10 ppm, group a) compared to all treated plots (ranging from 276.60 to 312.10 ppm, group b). This finding demonstrates that long-term TWW irrigation may have facilitated Cl⁻ leaching or redistribution within the soil profile. The findings in Ramtha indicate no statistically significant variations in the chloride Cl⁻ content across the irrigation treatments, ranging from the control to 10 years (80.61 to 414.91 ppm), which are classified within the same letter group. Although, there was an increase in values, chloride levels accumulated after the TWW irrigation.

As illustrated in Figure 3c, Cl⁻ followed a similar quadratic trend to sodium, declining from the control to 5 years of irrigation and then rising again at 10 years. This indicates initial leaching effectiveness in the silt loam profile but also shows that under extended TWW irrigation chloride accumulates, which is consistent with its high solubility and presence in wastewater effluents. Rainfall reduced Cl⁻ concentrations significantly, highlighting its high mobility compared with sodium. This reduction was statistically significant at the 0–30 cm soil depth, as confirmed by the HSD test (p < 0.05).

In Ramtha, Figure 3d, Cl⁻ exhibited a strong linear increase with the irrigation duration. The restricted leaching capacity of silty clay loam soils contributes to chloride buildup, even though chloride is normally considered highly mobile. The accumulation trend in Ramtha suggests that limited infiltration and slower percolation counteract chloride’s leaching potential, leading to long-term buildup under continuous TWW use. These findings align with prior research indicating that Cl⁻ dynamics are affected by both water inputs and the soil’s physical qualities that regulate the infiltration and drainage behavior [66,67,68,69].

3.3.3. Electrical Conductivity

In this study, the data indicate that the EC significantly varied between Mafraq and Ramtha. The EC was significantly higher in Mafraq (6.84 ± 0.77 deciSiemens per meter (dS/m), group a) compared to Ramtha (1.42 ± 0.77 dS/m, group b). As presented in Table 6, the Mafraq control had an extremely high EC (22.2967 ± 1.5362 dS/m), significantly higher than all other treatments. In contrast, Ramtha treatments showed smaller variation, and all values belonged to the same letter group.

The primary and most significant effect on EC in this research was time (F = 40.62, p < 0.0001), demonstrating that EC levels are markedly sensitive to rainfall events. According to Table 5, a significant reduction in EC was observed following rainfall (p < 0.05). The overall EC decreased from 6.52 ± 0.66 dS/m prior to rainfall to 1.74 ± 0.66 dS/m after rainfall.

As illustrated in Figure 3e, EC values exhibited a quadratic trend, decreasing from the control, which had a high EC prior to rainfall, to the 5-year treatment and then increasing at 10 years. This pattern reflects the combined behavior of soluble salts (Na⁺, Cl⁻, and others), where the early leaching reduced salinity levels, but prolonged irrigation led to renewed salt accumulation [70]. Rainfall substantially lowered EC across all treatments, consistent with its flushing effect on soluble salts. Salt accumulation was stratified and concentrated in the upper layers, particularly at a depth of 0–30 cm in Mafraq prior to rainfall, based on the HSD test (p < 0.05).

Conversely in Ramtha, the EC increased consistently with the irrigation duration, as shown by the strong regression fits in Figure 3f. The steady rise in EC highlights the cumulative effect of long-term TWW irrigation in a soil with limited leaching potential. Despite some reductions after rainfall, the overall upward trajectory indicates salt accumulation that could pose risks to the soil structure and crop productivity if the irrigation continues without management interventions.

3.3.4. Exchangeable Sodium Percentage

There was a significant difference between the two research sites; Mafraq had significantly higher ESP values (2.3785 ± 0.05%) compared to Ramtha (1.9433 ± 0.05%).

As shown in Table 6, the ESP values in Mafraq were significantly decreased in all TWW treatments (2, 5, and 10 years) compared to the control, which exhibited the highest ESP (4.3380%, group a). The decrease in the ESP under the long-term TWW irrigation indicates that sodium was efficiently leached from the silt loam soil. Conversely, in Ramtha, ESP values markedly increased during extended periods of TWW irrigation. The control had the lowest ESP at 1.0047%, whereas the 10-year treatment demonstrated the highest value at 2.7111%. The gradual elevation of the ESP over the 2-, 5-, and 10-year treatments aligns with prior research indicating that clay-rich soils impede water movement and facilitate sodium retention within the soil matrix, making them more vulnerable to sodium accumulation under prolonged irrigation [71].

Time had a highly significant effect on the ESP (F = 85.61, p < 0.0001). As illustrated in Table 5, the ESP decreased from 2.52 ± 0.05% prior to rainfall to 1.81 ± 0.05% following rainfall (p < 0.05). As observed in Figure 3g, the ESP displayed a quadratic trend, with values decreasing up to 5 years of TWW irrigation and rising again at 10 years. This indicates that, in the short term, Na⁺ was either leached or equilibrated by Ca²⁺ and Mg²⁺ in the soil exchange complex. Prolonged irrigation with TWW resulted in an ongoing sodium influx, displacing Ca²⁺ and Mg²⁺ and elevating sodicity levels. Precipitation episodes diminished ESP values, underscoring the impact of leaching in temporarily mitigating exchangeable sodium accumulation.

In Ramtha, the ESP increased progressively with the irrigation duration, following a strong positive regression fit, Figure 3h. The fine texture and elevated cation exchange capacity of Ramtha soils promote sodium retention, whereas the restricted leaching under TWW irrigation exacerbates the sodicity development in the absence of effective management. Although precipitation diminished the ESP a little, the overall ascending trend underscores the enduring risk of soil structural deterioration and permeability reductions.

The depth significantly affected ESP values (p < 0.05), with a more pronounced reduction observed in Mafraq, particularly in the upper 0–30 cm layer of the control treatment. In contrast, Ramtha soils irrigated with TWW for 10 years exhibited the highest ESP values prior to rainfall, especially in the deepest layer (60–90 cm).

3.3.5. Sodium Adsorption Ratio

According to Table 6, in Mafraq the control treatment exhibited the highest SAR value (21.42 ± 0.57, group a), surpassing all TWW-treated plots. In contrast, the SAR values in the 10-, 5-, and 2-year TWW treatments ranged from 7.35 to 8.87, which are all significantly lower (group b). In comparison, in Ramtha the control had the lowest value (4.73 ± 0.57, group c), while the SAR increased with the irrigation duration, reaching 12.99 ± 0.57 in the 10-year treatment.

As shown in Table 5, a significant reduction in the SAR was observed following rainfall events (p < 0.05). Before rainfall, the SAR averaged 12.17 ± 0.28, while after rainfall it dropped to 8.56 ± 0.28, indicating notable sodium leaching from the soil profile. The observed trend aligns with the findings of previous studies, which reported reduced SAR and ESP values during wet periods due to enhanced leaching processes from the soil surface [72].

In Mafraq, as illustrated in Figure 3i, the SAR prior to rainfall reached almost 30 in the control group, markedly exceeding all TWW treatments. This indicates a heightened sodicity risk in non-irrigated soil. The SAR exhibited a quadratic trend, decreasing until the 5-year irrigation treatment and subsequently rising again at 10 years. This reflects the balance between Na⁺ inputs and the mitigating influence of Ca²⁺ and Mg²⁺ in the soil solution during the initial years. With extended irrigation, Na⁺’s dominance over divalent cations became more pronounced, elevating the SAR. Post-rainfall, the SAR in the control significantly dropped to 10, reflecting the strong leaching of soluble sodium relative to Ca²⁺ and Mg²⁺. All TWW treatments after rainfall exhibited SAR values between seven and eight, suggesting that TWW irrigation maintained relatively stable SAR levels with less fluctuation following rainfall.

In contrast, Ramtha showed more moderate SAR values overall. Before rainfall, the SAR increased linearly with the irrigation duration, consistent with the steady rise in the Na⁺ and ESP in these soils. After rainfall, the SAR decreased across all treatments, with the control reaching 5 and the long-term TWW treatments ranging from 7 to 10. The lower SAR values in Ramtha reflect the influence of the finer soil texture, which retains divalent cations and reduces sodium mobility compared to the Mafraq soil.

To improve interpretability, the measured salinity and sodicity parameters were compared with international guidelines, including FAO irrigation water quality standards as well as the Jordanian Standard JS 893/2006. The results indicate that while pre-rainfall values in Mafraq often exceeded the recommended thresholds (e.g., EC > 20 dS m⁻¹, SAR > 20, Na⁺ = 3877 ppm, Cl⁻ = 8469 ppm), rainfall events substantially reduced these parameters to levels closer to international safety limits. Post-rainfall microbial loads were well below WHO thresholds, confirming that rainfall plays a key role in restoring soil safety.

As shown in Figure S2, in Mafraq (silt loam) the SAR showed moderate correlations with clay (R² = 0.64 before and 0.31 after rainfall), where values rose at low clay contents but declined beyond 20–25%, reflecting both the sodium retention by the CEC and leaching after rainfall. Strong positive relationships with silt (R² > 0.83) indicated limited leaching in finer fractions, while negative associations with sand (R² = 0.87–0.99) confirmed enhanced Na⁺ removal in coarser textures. At Ramtha (silty clay loam), the SAR was higher overall due to the finer texture. Clay showed a rise then a decline after rainfall (R² = 0.59), silt maintained strong positive effects (R² = 0.78–0.97), and sand reduced the SAR (R² = 0.41–0.81), highlighting the restricted leaching capacity of heavy soils compared with Mafraq.

3.4. Soil Fertility Responses to TWW Irrigation and Rainfall in Semi-Arid Conditions

3.4.1. Soil pH

The treatment factors had a significant effect on pH levels (F = 32.71, p < 0.0001). Soils irrigated for 2, 5, or 10 years demonstrated consistently elevated pH values (between 7.85 and 7.98), all categorized under group (a), while the control displayed a significantly lower pH (7.50) and was assigned to a distinct group (b). The results indicate that extended irrigation with TWW significantly increases the soil pH relative to the control group, as reported in previous studies [73]. This indicates that the utilization of TWW leads to soil alkalization over time, presumably due to the accumulation of basic cations, including Ca²⁺, Mg²⁺, and Na⁺, often present in treated effluents [74].

The primary effect on the pH was time, which was extremely significant (F = 1092.03, p < 0.0001). Rainfall significantly affected the soil pH, with a rise from 7.35 before rainfall to 8.30 after, as demonstrated in

Table 7. Effect of Time on soil fertility indicators (pH, OM, TN, Ca²⁺, and Mg²⁺).

Time	Soil pH (Mean ± SE)	OM (%) (Mean ± SE)	TN (%) (Mean ± SE)	Ca2+ (ppm) (Mean ± SE)	Mg2+ (ppm) (Mean ± SE)
Before rain	7.3486 ± 0.02410 b	1.6093 ± 0.03006 a	0.5781 ± 0.02197 a	320.64 ± 33.3128 a	190.19 ± 22.9975 a
After rain	8.2993 ± 0.02410 a	1.3693 ± 0.03006 b	0.2660 ± 0.02197 b	101.04 ± 33.3128 b	39.001 ± 22.9975 b

Note: Values are means ± SE (n = 96 observations per time). Means followed by different letters are significantly different at p < 0.05 according to Tukey–Kramer HSD test at p < 0.05.

. This change in pH was consistent at both sites, indicating an extensive buffering impact of rainwater, either attributable to the leaching of acidic constituents or the dilution of salts particularly in calcareous soils [75,76].

Ramtha before rain displayed a marginally lower pH of 7.25 in contrast to Mafraq’s pH of 7.45. Following precipitation, both sites exhibited a significant increase in pH, attaining alkaline levels (>8.1), with Ramtha being more alkaline (8.40) than Mafraq (8.20). This finding aligns with prior studies indicating that rainfall events lead to soil alkalization, especially in areas receiving long-term TWW irrigation, and that the soil texture may influence the magnitude of the pH change [59,77].

As shown in Figure 4a, the pH in Mafraq exhibited a slight quadratic trend, with values increasing from the control to 5 years of irrigation and then stabilizing by 10 years. Before rainfall, the pH curve was lower, whereas after rainfall, the values shifted upward to values over 8.0 across all treatments. This suggests that the rainfall enhanced alkalinity, likely due to the leaching of soluble salts and a relative increase in the carbonate/bicarbonate buffering capacity. The rise in pH is consistent with calcareous soils, where the dissolution of CaCO₃ and the removal of acidic ions (H⁺, Al³⁺) during rainfall events reduce soil acidity.

In Ramtha, Figure 4b, the pH followed a positive linear trend, remaining more stable compared to Mafraq. Before rainfall, values were lower, while after rainfall, a consistent upward shift occurred. The clay-rich soil has a higher cation exchange capacity (CEC), which buffers changes in the pH more strongly. The increase after rainfall reflects the reduced ionic strength of the soil solution due to salt leaching, leading to a relatively more alkaline reaction.

The regression analysis in Figure 4 showed stronger fits in Mafraq (R² = 0.73–0.99) than in Ramtha (R² = 0.27–0.96), indicating the greater sensitivity of silt loam soils to the irrigation duration and rainfall. Ramtha soils exhibited weaker and more variable regressions due to clay buffering.

3.4.2. Organic Matter

The soil OM accumulation varied significantly across locations and treatments. The location significantly influenced the soil OM level (p < 0.05), with Mafraq (1.6158%) exhibiting marginally greater OM than Ramtha (1.3629%). Moreover, the highest OM content was recorded in soils irrigated with TWW for 5 years (1.8809 ± 0.05%, group a), which was significantly greater than other treatments. The 2-year treatment followed with intermediate OM levels (1.5148 ± 0.05%, group b), while the 10-year treatment and control exhibited the lowest and statistically similar OM contents (1.2561 and 1.3055%, respectively, in group c), which aligned with [59].

It is important to note that the OM enrichment in both sites is not solely attributable to TWW inputs but also to other sources, including the root biomass from continuous alfalfa cultivation, microbial turnover, and natural soil organic inputs. These combined processes indicate that TWW contributes to soil fertility, but the magnitude and stability of OM retention are strongly regulated by the soil texture and management conditions.

The primary effect on OM was observed with the depth (F = 172.56, p < 0.0001). The OM was highest in the 0–30 cm layer for both locations prior to rainfall (1.9449 ± 0.04, group a) and significantly decreased with depth, reaching its minimum at 90 cm (1.1119 ± 0.04, group c). Following precipitation, OM diminished in most treatments at shallow depths in Mafraq. In Ramtha, the greatest concentration of OM was detected in the 0–30 cm layer after five years of TWW application subsequent to rainfall.

Time had a secondary significant effect on OM, indicating that precipitation considerably influences its quantity. Following rainfall and according to Table 7, there was a significant reduction in the OM content, from 1.6093 ± 0.03 before the rainfall to 1.3693 ± 0.03 after the rainfall.

As shown in Figure 4c, in Mafraq, the OM content exhibited a quadratic decline with irrigation duration. Prior to rainfall, OM levels were elevated, particularly under the 5-year TWW treatment. However, OM content was significantly higher before rainfall than after, indicating that rainfall accelerated the decomposition and loss of organic residues. The sharp reduction in OM after rainfall can be attributed to the soil texture: in silt loam, the OM is less stabilized by clay particles, making it more susceptible to leaching and microbial breakdown when the soil moisture increases.

In contrast, Ramtha, Figure 4d, displayed a parabolic increase in OM with the irrigation duration, peaking around the 5-year treatment, before declining slightly at 10 years. After rainfall, the OM increased more sharply than before rainfall, suggesting that clay particles protected OM through aggregation and reduced decomposition rates. Rainfall here likely enhanced the incorporation of residues while preventing rapid loss, reflecting the stabilizing role of silty clay loam.

3.4.3. Total Nitrogen

The main effect on the TN was time (F = 152.65, p < 0.0001). As shown in Table 7, the mean TN levels declined significantly from 0.58 ± 0.02% before rainfall (group a) to 0.27 ± 0.02% after rainfall (group b). In Mafraq, the TN significantly declined from 0.5897 ± 0.03% before rainfall to 0.2952 ± 0.03% after rainfall (p < 0.05), with means grouped into distinct letter categories (a and b, respectively). In Ramtha, TN values decreased from 0.5664 ± 0.03107% before rain to 0.2368 ± 0.03107% after rain (p < 0.05).

The TN was also significantly affected by depth (p < 0.05), whereas the location, treatment, and their interactions had no significant effect. The TN varied significantly by depth before rainfall, with the highest values observed at 30–60 cm (0.6991%), followed by 60–90 cm and 0–30 cm depths (0.5241% and 0.5110%, respectively). After rainfall, TN values decreased across all depths (range: 0.2310–0.2879%) and no longer showed significant depth-based differences, indicating leaching and redistribution effects.

Therefore, while TWW irrigation can improve nitrogen availability over time, periodic rainfall can alter this balance, potentially affecting both soil fertility and nitrogen loss pathways. The significant reduction in the TN observed after rainfall in our study may be attributed to several interrelated mechanisms, including plant uptake, increased leaching under elevated soil moisture, and microbial shifts. These processes have been similarly reported in other arid and semi-arid ecosystems, where rainfall events drive nitrogen losses and reallocation [78,79].

3.4.4. Calcium and Magnesium

Time had the most significant influence on both Ca²⁺ and Mg²⁺, as evidenced by the greatest F-values (F = 24.94 for Ca²⁺ and F = 38.09 for Mg²⁺; p < 0.0001 for both). Table 7 illustrates that both Ca²⁺ and magnesium Mg²⁺ contents decreased markedly following rainfall episodes. Ca²⁺ concentrations were reduced from 320.64 ± 33.31 ppm prior to rainfall to 101.04 ± 33.31 ppm, and Mg²⁺ levels diminished from 190.19 ± 22.99 ppm to 39.00 ± 22.99 ppm.

According to Figure 4e, Ca²⁺ concentrations declined steeply with the irrigation duration. Before rainfall, values were substantially higher, following an exponential decrease from the control to 10 years. After rainfall, Ca²⁺ consistently declined, particularly in the control group, highlighting the leaching of exchangeable Ca²⁺ under rainfall events. This decrease is typical for silt loam soils with a moderate retention capacity, where Ca²⁺ is more prone to being displaced by the Na⁺ from TWW.

In Ramtha, Figure 4f, Ca²⁺ followed a parabolic increase, peaking around 5 years, with higher concentrations before rainfall than after. After rainfall, values declined across treatments, indicating partial leaching, but the clay fraction slowed the loss. This pattern reflects a stronger Ca²⁺ retention in clayey soils due to the higher CEC, with rainfall reducing soluble fractions but not completely depleting exchangeable pools.

As illustrated in Figure 4g, Mg²⁺ displayed a sharp decline with irrigation duration, particularly before rainfall. After rainfall, Mg²⁺ concentrations were drastically reduced across all treatments, confirming their high mobility and susceptibility to leaching. In calcareous soils, Mg²⁺ is less strongly adsorbed than Ca²⁺, which explains its more pronounced reduction following rainfall. The silt loam texture further promotes percolation losses.

In contrast to Mafraq, the Ramtha site, Figure 4h, showed that Mg²⁺ levels were relatively stable, showing slight fluctuations across irrigation durations. Before rainfall, concentrations were higher, whereas after rainfall, values dropped but less dramatically than in Mafraq. The clay matrix likely provided a stronger adsorption of Mg²⁺, buffering against rainfall-induced losses. This suggests that Ramtha soils have a greater resilience to cation depletion, which is critical for maintaining fertility under long-term TWW irrigation.

These findings support the hypothesis that clay-rich soils have a higher CEC, leading to a greater retention of divalent cations like Ca²⁺ and Mg²⁺ due to their stronger electrostatic binding to negatively charged clay particles [80]. The depth significantly influenced Ca²⁺ and Mg²⁺ concentrations (p < 0.05), with a more substantial decrease noted in Mafraq, especially within the upper 0–30 cm layer of the control treatment.

3.5. Soil Microbial Dynamics Under Long-Term TWW Irrigation and Rainfall Events

3.5.1. Total Coliforms

The main effect on the TC was time (F = 134.20, p < 0.0001). According to

Table 8. Effect of time on TC and FC concentrations in soil.

Time	TC (MPN g−1) (Mean ± SE)	FC (MPN g−1) (Mean ± SE)
Before rain	691.35 ± 46.7414 a	135.81 ± 25.4885 a
After rain	25.9156 ± 46.7414 b	0.3062 ± 25.4885 b

Note: Values are means ± SE (n = 96 observations per time). Different letters within each column indicate significant differences at p < 0.05 according to the Tukey–Kramer HSD test. The sharp decline in TC and FC following rainfall reflects leaching and dilution processes that override the enrichment effect of long-term TWW irrigation.

, the mean TC concentration declined significantly from 691.35 ± 46.74 MPN g⁻¹ prior to rainfall (group a) to 25.92 ± 46.74 MPN g⁻¹ after rainfall (group b).

The TC were significantly affected by the time and location (p < 0.05). Prior to precipitation, Mafraq exhibited a TC value of 579.66 MPN g⁻¹, which is significantly higher than the post-precipitation level of 38.48 MPN g⁻¹. A similar trend was observed in Ramtha: TC levels diminished from 803.04 MPN g⁻¹ before rainfall to 13.35 MPN g⁻¹ after rainfall.

The TC concentrations were significantly affected by the interaction between the time, location, and TWW irrigation treatment (p < 0.05). Before rainfall, TC levels were substantially higher in TWW treatments than in controls across both sites. In Mafraq, the 2-year and 5-year treatments recorded the highest values (1294.17 and 790.54 MPN g⁻¹, respectively), while in Ramtha, the greatest TC levels were observed under the 10-, 2-, and 5-year treatments (1260.00, 951.67, and 864.48 MPN g⁻¹, respectively). Control plots in both locations showed significantly lower TC levels. Following rainfall, TC levels declined sharply across all treatments and locations and converged into a single statistical group.

This pronounced homogenizing effect illustrates the pre-eminent influence of rainfall-induced leaching and dilution, which supersedes variations caused by prolonged irrigation. The contrast between Mafraq’s silt loam soil and Ramtha’s silty clay loam offers a mechanistic explanation: the larger pore spaces in the silt loam facilitate the rapid percolation and expulsion of microbes, whereas the finer texture of the silty clay loam retains greater moisture, restricting microbial transport and resulting in a comparatively enhanced persistence of coliforms post-rainfall. Thus, rainfall not only reduces overall microbial loads but also mediates site-specific differences in microbial dynamics through soil physical properties [81,82].

3.5.2. Fecal Coliforms

Time significantly affected FC concentrations (p < 0.05), with notably higher values observed before rainfall (135.81 ± 25.4885) compared to those obtained after rainfall (0.3062 ± 25.4885), as shown in Table 8.

The concentrations of FC were significantly affected by the interaction of the time, location, and treatment (p < 0.05). In Ramtha, the 10-year treatment before rainfall recorded the highest FC concentration (851.32 MPN g⁻¹, group a), significantly higher than the 2- and 5-year treatments (77.08 and 58.10 MPN g⁻¹, group b). After rainfall, FC levels dropped sharply across all treatments and became statistically similar (group b), indicating effective microbial leaching due to precipitation. In Mafraq, no statistically significant differences were detected in all FC values.

These findings underscore that prolonged TWW irrigation enhances soil OM and nutrient levels suitable to coliform survival; nevertheless, precipitation swiftly alters these advantageous environments by mechanical flushing, dilution, and the alleviation of osmotic stress. The greater persistence of FC in Ramtha compared with Mafraq highlights the influence of the soil texture and bulk density; the silty clay loam in Ramtha limits infiltration and oxygen diffusion, creating microhabitats that enable facultative anaerobes such as FC to endure longer following precipitation events.

The sharp decline in TC and FC observed after rainfall may be partly explained by a high-intensity storm event that occurred one week prior to sampling, which generated widespread runoff across northern Jordan. Such runoff likely enhanced the scouring and transport of microbes from the soil surface, while infiltration promoted leaching into deeper layers. In addition, post-storm sunny and dry conditions would have further reduced microbial survival through UV exposure and desiccation. These combined processes provide a mechanistic explanation for the pronounced reduction in microbial indicators observed in our study.

3.5.3. Escherichia coli

The statistical analysis revealed that none of the examined main effects (location, treatment, depth, or time) nor their interactions had a statistically significant effect on E. coli concentrations in the soil (p > 0.05).

This lack of response suggests that, the survival of E. coli in semi-arid soils is constrained more by intrinsic microbial traits than by external irrigation or rainfall inputs. E. coli is known to be sensitive to pH shifts, osmotic stress, desiccation, and predation by native soil microorganisms. Consequently, while TC and FC concentrations respond strongly to nutrient enrichment and leaching processes, E. coli fails to persist under the fluctuating moisture and aeration regimes of semi-arid soils, especially in the well-aerated silt loam of Mafraq.

4. Conclusions

This study demonstrated that long-term TWW irrigation and rainfall have contrasting effects on soil quality in semi-arid calcareous soils. Rainfall played a dual role: alleviating salinity by leaching the Na⁺, Cl⁻, EC, ESP, and SAR, while simultaneously accelerating nutrient and microbial losses.

• In Mafraq (silt loam): Salinity indicators declined strongly under TWW irrigation, with rainfall enhancing leaching. Regression fits were consistently high (R² = 0.82–0.99), showing predictable leaching responses.
• In Ramtha (silty clay loam): Salinity indicators increased gradually with irrigation, reflecting slower leaching and higher retention due to clay. Regression fits were extremely strong (R² = 0.91–0.99), confirming stable accumulation trends.
• In Mafraq, fertility indicators showed strong regression responses (R² = 0.73–0.99) and sharper nutrient losses after rainfall, especially for Ca²⁺ and Mg²⁺. While in Ramtha, regression relationships were weaker (R² = 0.27–0.96), reflecting soil buffering, higher OM stability, and the slower leaching of base cations.
• Rainfall reduced Na⁺ by 70%, Cl⁻ by 86%, EC by 73%, the ESP by 28%, and the SAR by 30, confirming a strong leaching effect of salts.
• Moreover, in Mafraq the pH increased with the irrigation duration, rising from 7.35 to 8.20 after rainfall, with strong regression fits (R² = 0.83–0.96), while in Ramtha, the pH also increased, reaching 8.40, but regression relationships were weaker (R² = 0.39–0.62), reflecting greater buffering in clay soils.
• The total nitrogen declined sharply by 54%, from 0.58% to 0.27%, confirming the high mobility of nitrogen and its susceptibility to rainfall-induced leaching in semi-arid soils.
• In Mafraq before rainfall, the control had a significantly lower BD (1.05 g/cm³) than irrigated plots (1.20–1.25 g/cm³), indicating that long-term TWW irrigation increased compaction relative to rainfed conditions. After rainfall, the BD increased slightly in Mafraq (1.25–1.30 g/cm³), suggesting surface sealing and compaction from the raindrop impact.
• Rainfall reduced microbial loads sharply, with TCs and FCs decreasing by more than 96%. The sharp post-rainfall decline in TCs and FCs observed in this study underscores the critical role of extreme storm events, such as the high-intensity rainfall that occurred prior to sampling, in accelerating microbial scouring and leaching processes in semi-arid soils irrigated with TWW.
• The five-year treatment consistently demonstrated the most equitable enhancements, characterized by elevated OM, moderate EC levels, and appropriate ESPs.
• The findings indicate that TWW may serve as a sustainable irrigation supply in semi-arid areas when there is adequate monitoring and a consideration of the soil type and meteorological circumstances.

Implications for field management and future research

• The adoption of moderate TWW irrigation periods (5 years) appears most sustainable, balancing soil fertility, salinity, and microbial safety.
• The regular monitoring of soil salinity and fertility parameters is essential to prevent long-term degradation.
• Rainfall patterns must be integrated into irrigation scheduling and risk assessments, as they strongly influence both leaching and nutrient depletion.

Study limitations

This study was conducted under alfalfa cultivation in semi-arid calcareous soils. Results may vary under different cropping systems, in soils with contrasting mineralogy (e.g., non-calcareous or sandy soils), or under extreme climatic conditions such as prolonged droughts or high-intensity rainfall events. Further research is needed to assess long-term impacts on crop productivity, soil microbial ecology, and nutrient cycling under diverse field conditions.