Dynamics of Pharmaceuticals in the Soil–Plant System: A Case Study on Mycorrhizal Artichoke

Abstract

Contaminants of emerging concern, such as pharmaceuticals (PhACs), are continuously introduced into agro-ecosystems through irrigation with treated wastewater (TWW). While this practice is increasingly common in drought regions, only limited information is available on the fate of PhACs within the soil–plant system. For this purpose, a two-year study was conducted by irrigating artichokes, non-inoculated and inoculated with different arbuscular mycorrhizal fungi, with water containing PhACs at different concentrations. The experiment, conducted in both open field and pot conditions, aimed to evaluate their potential accumulation in the soil and plant tissues. Results showed that PhACs concentrations varied according to the physicochemical properties of the compounds and the duration of irrigation. The study revealed minimal accumulation of contaminants in the soil and non-edible plant parts. This was observed only at the end of the second growing cycle, when the plants were irrigated with TWW containing trace PhAC levels. In contrast, during both pot cultivation cycles, PhACs accumulated in the soil were translocated into plant organs when irrigated with water enriched to 200 μg L⁻¹ with eight PhACs. At the end of the trial, climbazole had the highest concentration in soil, while carbamazepine and fluconazole showed greater accumulation across all plant organs compared to other PhACs. In both trials, plants inoculated with Septoglomus viscosum absorbed less PhACs compared to those inoculated with Rhizophagus irregularis + Funneliformis mosseae. These results suggest that, while the long-term use of TWW containing PhACs may improve artichoke yield, it could present different degrees of risk to both environmental and human health, depending on the concentration levels of contaminants.

1. Introduction

Water supply sources are not always able to meet the demands of various sectors due to climate change and rapid population growth, particularly in drought regions and countries using non-innovative water management practices [1]. Non-conventional water resources, such as urban and industrial treated wastewater, can address such water shortages [2]. However, the use of non-conventional water resources, especially in agriculture, requires careful and strategic planning to minimize potential risks to public health and the environment. Recently, the European Commission introduced the Water Reuse regulation, which defines the minimum quality requirements for wastewater (WW) used for crop irrigation [3]. This regulation primarily focuses on physicochemical parameters and microbiological risks, but there is no mention of risks posed by contaminants of emerging concern (CECs), such as Pharmaceutical Active Compounds (PhACs), which may be present in reclaimed water. The EU market has registered around 3000 commonly used pharmaceuticals, with global usage still on the rise [4]. As a result, most WW discharges continue to be contaminated with PhACs and personal care products [5]. Although environmental risks associated with water reuse in agriculture are a top priority, there is still limited understanding of the environmental fate of wastewater-borne biological and chemical contaminants and their potential ecotoxicological impacts on soil-dwelling organisms and eco-physiological functions. Akhter et al. [6] and Wei et al. [7] found that the uptake and translocation of PhACs from soil to plant roots and aerial parts can be influenced by factors such as the physicochemical properties of PhACs, environmental factors affecting transpiration, plant physiology, and soil or matrix properties. Thus, the evaluation of the fate of PhACs within the plant–soil system is crucial, especially in drought-prone areas like the Mediterranean basin, where plants can increasingly be irrigated with reclaimed wastewater.

A crop that could benefit from this kind of irrigation is globe artichoke (Cynara cardunculus var. scolymus (L.) Fiori), an important crop in the Mediterranean Basin [8], grown for its edible part, the “head”. The annual global production of artichoke heads is approximately 1700 kt, with significant contributions from southern Europe (e.g., Italy, Spain, France, Greece), the Middle East (e.g., Turkey, Syria, Israel), North Africa (e.g., Egypt, Morocco, Algeria, Tunisia), and recently, China as well [9]. Italy is the largest producer globally, yielding around 477 kt per year, with artichoke cultivation mainly occurring in southern Italy (i.e., Sicily, Sardinia, Apulia).

Several microorganisms, or their byproducts, can be used for the bioremediation of PhACs and pesticides using in situ or ex situ approaches [10]. Fungi are promising because they can absorb several organic contaminants and/or produce enzymes that degrade them, although they grow slowly and are sensitive to environmental conditions [11]. The symbiosis between arbuscular mycorrhizal fungi (AMF) and roots can help keep fungal metabolism at its best, as well as enhance root access to water and essential inorganic nutrients (e.g., P, N, K, Ca, S, Zn, Cu) leading to increased plant biomass [12], crop yield [13], and consequently, crop quality [14,15]. In addition to modifying soil structure [16], the extraradical mycelium can also influence the uptake of contaminants, including heavy metals [17] and CECs, thereby reducing their potential negative impact on ecosystems [18]. Thus, the application of AMF can be a promising phytoremediation strategy for restoring contaminated soils and rehabilitating degraded ecosystems [19]. AMF can also shield plants from the harmful effects of CECs by modulating the transport and distribution of these contaminants within plant tissues. Previous research has reported that AMF affect the dynamics of organic contaminants in the soil–plant system [20,21], helping to reduce CEC concentrations in plant shoots by promoting CEC accumulation in the roots [22,23]. Furthermore, mycorrhizal fungi may contribute to the biodegradation and metabolization of CECs. Recent studies by Yu et al. [24] and Małachowska-Jutszet et al. [25] indicate that AMF can enhance the removal of polycyclic aromatic hydrocarbons and petroleum compounds from soils. Despite incomplete knowledge of the mechanisms of action of AMF [26], it is likely that these fungi play a key role in CEC-contaminated environments. The main objective of this study was to address these knowledge gaps by investigating the behavior of selected organic contaminants within the water–soil–plant system for the specific conditions of artichoke grown in pots and open fields over multiple crop cycles.

2. Materials and Methods

2.1. Site Description and Climate Conditions

The experiment was conducted concurrently in both open field and pot setups in Noci, Puglia, southern Italy (40°79′18″ N, 17°08′13″ E; altitude: 420 m a.s.l.) over two crop cycles (2020–2021 and 2021–2022). The area was characterized by a typical Mediterranean climate, with air temperatures dipping below 0 °C in winter and exceeding 40 °C during the summer. The historical average annual rainfall is approximately 590 mm, with precipitation unevenly distributed throughout the year and primarily concentrated between October and April (Figure 1).

2.2. Experimental Design and Crop Management

Globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori, cultivar Brindisino) plantlets, inoculated and not inoculated (control) with AMF in the greenhouse, were transplanted into the open fields and pots in October 2020. For the open field test, each plot (5 m × 5 m) was characterized by loamy-clayey-loamy soil and contained 20 plants (about 8000 plants ha⁻¹). Since mycorrhizal hyphae generally spread at a rate of 1.5–2 m per year in soil [27,28], plots were placed 10 m apart to inhibit mycorrhizal colonization of the other artichoke plants. The experiment followed a split-plot design with two irrigation treatments (GW: locally sourced groundwater and TWW: treated wastewater), as main factors, and three AMF inoculation treatments, as sub-factors. Inoculations of AMF were performed in accordance with the protocol described by De Mastro et al. [29], and included (1) a crude inoculum of Septoglomus viscosum (syn. Glomus viscosum) (MSE), (2) an inoculum of Rhizophagus irregularis (syn. Glomus intraradices) and Funneliformis mosseae (syn. Glomus mosseae) (MSY), and (3) a non-inoculated control (CON).

The pot trial was conducted using GW, TWW, and GW spiked with eight commonly detected PhACs in wastewater (SGW) at a concentration of 200 μg L⁻¹. The PhACs were three antibiotics (CLR: clarithromycin, SMX: sulfamethoxazole, and TMP: trimethoprim), one antiepileptic (CBZ: carbamazepine), two anti-inflammatories (KET: ketoprofen and NPX: naproxen), and two antifungals (FCZ: fluconazole and CLZ: climbazole) (

Table 1. Physicochemical properties (Mw: molecular weight; K_OW: octanol/water coefficient; pKa: acid ionization constant; K_d: adsorption coefficient) of the selected PhACs.

PhACs	Mw (g mol−1)	Chemical Structure	Chemical Class	Water Solubility (mg L−1)	KOW	pKa	Literature Kd (Reference)
Carbamazepine (CBZ)	236.27		anti- depressants	18 at 25 °C	2.45	13.9	12–20 [30] 0.53–16.7 [31]; 0.43 [32]; 0.08 [33]
Clarithromycin (CLR)	748		antibiotic	1.693 at 25 °C	3.16	8.99	262–400 [30]
Climbazole (CLZ)	292.76		antifungal	58 at 25 °C	3.76	6.49	123–200 [30]
Fluconazole (FCZ)	306.27		antifungal	4.363 at 25 °C	0.25	2.27	16.04 [34]
Ketoprofene (KET)	254.28		anti- inflammatory	51 at 22 °C	3.12	4.45	0.09–9.59 [31]; 1.26–8.24 [35]
Naproxen (NPX)	230.26		anti- inflammatory	15.9 at 25 °C	3.18	4.15	0.23–17.5 [31]; 2.39–4.4 [36]
Sulfamethoxazole (SMX)	253.28		antibiotic	610 at 37 °C	0.89	1.6	0.6–4.9 [30]
Trimethoprim (TMP)	290.32		antibiotic	400 at 25 °C	0.91	7.12	1.16 [32]; 7.42 [37]; 7.06–9.2 [38]

). Details of the preparation of the pot trial are reported elsewhere [29].

Each combination of irrigation and AMF inoculation was replicated in four blocks. For both experiments, a drip irrigation system with a single plastic pipe and two 2 L h⁻¹ drippers was used for all treatments. Soil moisture was monitored with smart sensors (X-Farm Technologies srl, Valmacca, Italia) placed in the top and middle soil layers for each treatment and replication, triggering irrigation when moisture fell to 25% of the available water capacity. During the growing season, in addition to weed and pest management, fertilizers were applied to artichoke plants using the rates reported by De Mastro et al. [29].

2.3. Water, Soil and Plant Sampling and Analysis

During each growing season, irrigation water, soil and plants were sampled multiple times to monitor variations during crop cycles. GW and TWW samples were collected monthly in triplicate using sterile 1000 mL glass bottles, transported to the laboratory and analyzed within 24 h to determine conventional physical and chemical properties according to APHA [39]. PhACs concentration in GW and TWW samples was analyzed with the analytical technique of high-resolution mass spectrometry and ultra-pressure liquid chromatography using an online solid phase extraction method with previously optimized analytical settings [40], and the operating conditions reported by De Mastro et al. [29].

Soil samples were collected in triplicate at a depth of 0–0.3 m in the areas wetted by the drippers before the beginning of the test and at the end of the first and second crop cycles for both experimental trials. The main soil chemical and physical parameters were determined according to the analytical methods of Swift et al. [41].

Roots, leaves, stems, and heads of artichokes were collected in triplicate at the same time as the soil samples and immediately transported to the laboratory for analysis.

Modified QuEChERS methods [29,42] were used for PhACs extraction from soil and different parts of the artichoke plant. The same analytical technique used for water samples was applied to determine the concentrations of PhACs in all plant organs.

The number of heads per plant, head diameter, and the weight of fresh heads were measured only for plants grown in the open field, and were calculated as cumulative values of all main and second-order flower head harvests after complete removal of the floral stem.

Root colonization by AMF (mycorrhizal frequency in %) was assessed at the end of the first and second crop cycles of the open field test and at the end of the second crop cycle of the potted trial. Three root samples per plant were collected, washed with tap water to remove soil residue, and prepared for microscopic observation according to the method of Phyllips and Hyman [43]. The percentages of colonization were measured under an optical microscope (Leica DMLB100, Buccinasco, Italy) following the method outlined by Trouvelot et al. [44].

2.4. Pharmaceutical Active Compounds (PhACs) Uptake and Translocation Factors

The propensity of each PhAC to translocate from the soil to the artichoke plant was assessed by the uptake factor (UF), calculated using Equation (1) [45]:

UF = C_plant/C_soil

(1)

where C_plant is the concentration of each contaminant in the plant and C_soil is its corresponding concentration in the soil.

Each PhAC can accumulate itself in the root apparatus or can translocate to other organs of the plant. This behavior was assessed by the translocation factor (TF), calculated according to Equation (2) [46]:

TF = C_ao/C_root

(2)

where C_ao is the concentration of each PhAC in the aerial organs of the plant, such as leaves and heads, and C_root is its corresponding concentration in the roots.

2.5. Statistical Data Analysis

All experimental data were tested against the normal distribution of variables (Shapiro–Wilk test) and the homogeneity of variance (Bartlett test) using R studio software, version 4.1.3. The variables were then subjected to the suitable ANOVA and post-hoc test. The difference was significant when the p value ≤ 0.05.

3. Results and Discussion

3.1. Assessment of Mycorrhizal Frequency

The values of the mycorrhizal root colonization, measured at the end of the first and second crop cycles of the open field test, are reported in Figure 2a. The results, expressed as mycorrhizal frequency, show that the highest values of mycorrhizal colonization were observed at the end of the first growing season, while a reduction of about 10% of infection for all the treatments and AMF tested occurred in the second cycle. The highest colonizations were obtained in the presence of TWW, achieving values of more than 60% of frequency in the first year, and between 55% and 58% in the second year. Between MSY and MSE, the colonization of the former AMF was slightly lower than the latter (on average 59.2% vs. 63.33% in the first year, and 48.0% vs. 53.0% in the second year, respectively).

In contrast, the application of TWW in the pot trial resulted in a significant reduction in MSE with respect to MSY, and a similar trend was observed in the pots irrigated with SGW, where the frequency of MSE was numerically lower than MSY (Figure 2b). The declining trend of MSE colonization can be attributed to the presence of two fungicides, i.e., FCZ and CLZ, in the cocktail of PhACs added to GW, that may have exerted a slow but more selective inhibitory effect on Septoglomus viscosum than the other two fungi present in MSY.

3.2. Characteristics of Soil and Water Used for Irrigation

3.2.1. Soil

The main chemical and physical properties of the soils and their changes over time are given in

Table 2. Main properties of irrigated soils (mean ± STD of three samples for GW and TWW soils). TWW: tertiary treated wastewater irrigated soil; GW: groundwater irrigated soil; EC: electrical conductivity; CaCO₃: calcium carbonate; OC: organic carbon; OM: organic matter; N_tot: total nitrogen; P_ava: available phosphorus.

Parameters	TWW						GW
	1st Year			2nd Year			1st Year			2nd Year
	CON	MSY	MSE	CON	MSY	MSE	CON	MSY	MSE	CON	MSY	MSE
pH (H2O)	7.6 ± 0.2	7.4 ± 0.1	7.6 ± 0.2	7.9 ± 0.0	7.4 ± 0.3	7.5 ± 0.1	7.9 ± 0.2	7.4 ± 0.2	7.6 ± 0.0	7.5 ± 0.1	7.6 ± 0.0	7.4 ± 0.3
pH (KCl)	5.7 ± 0.1	6.5 ± 0.1	6.5 ± 0.2	7.1 ± 0.1	6.7 ± 0.2	6.9 ± 0.3	7.0 ± 0.0	6.6 ± 0.3	6.7 ± 0.1	6.4 ± 0.0	6.7 ± 0.1	6.4 ± 0.0
EC (μS cm−1)	1769.0 ± 32	1844.0 ± 45	1708.0 ± 25.9	1863.9 ± 56.9	1863.4 ± 42.8	1695.4 ± 72.5	629.5 ± 20.9	789.6 ± 21.8	678.0 ± 15.2	601.9 ± 12.9	741.9 ± 41.1	628.1 ± 28.0
CaCO3tot (%)	3.0 ± 0.4	2.2 ± 0.30	2.4 ± 0.10	3.1 ± 0.1	2.1 ± 0.2	2.3 ± 0.1	3.0 ± 0.30	2.4 ± 0.40	2.3 ± 0.50	2.9 ± 0.2	2.0 ± 0.3	2.2 ± 0.2
OC (g kg−1)	7.8 ± 1.1	7.7 ± 1.1	6.7 ± 0.8	8.2 ± 1.2	8.0 ± 0.9	7.7 ± 1.1	7.5 ± 1.0	7.2 ± 0.2	5.7 ± 1.5	7.4 ± 0.6	7.3 ± 0.5	6.0 ± 1.1
OM (%)	13.5 ± 1.3	13.3 ± 1.3	11.6 ± 1.1	14.1 ± 1.1	13.8 ± 0.9	13.3 ± 1.2	13.0 ± 1.3	12.4 ± 1.2	9.8 ± 1.9	12.8 ± 0.8	12.6 ± 0.8	10.3 ± 1.6
Ntot (g kg−1)	1.3 ± 0.1	0.9 ± 0.4	1.1 ± 0.1	1.7 ± 0.2	1.3 ± 0.3	1.4 ± 0.2	0.8 ± 0.4	0.6 ± 0.1	0.7 ± 0.2	0.9 ± 0.2	0.5 ± 0.2	0.7 ± 0.1
Pava (mg kg−1)	73.7 ± 4.4	95.5 ± 3.7	68.9 ± 5.9	82.4 ± 2.3	124.6 ± 2.1	76.2 ± 1.6	49.5 ± 4.7	86.8 ± 4.6	48.2 ± 6.4	53.8 ± 1.3	79.6 ± 2.2	49.8 ± 5.2

.

The water used for irrigation affected all soil parameters evaluated except for pH and total CaCO₃. Similar results were reported by Rusan et al. [47] after the long-term WW irrigation of soil. TWW increased electrical conductivity (EC), total nitrogen (TN), and available phosphorus (Pava) in the soil at the end of the first crop cycle, while an increase in soil organic matter (SOM) content was observed at the end of the experimental trial. These results can be ascribed to the chemical and physical properties of WW. The significant EC increase can be attributed to the higher salt and total dissolved solids (TDSs) concentrations in TWW, which is in agreement with what has been reported in the literature [48]. Additionally, the use of TWW improved TN and Pava, which is notably higher especially in the second year. Similar trends have been observed by Li et al. [49] and Castro et al. [50]. According to Alrajhi et al. [51], using TWW for irrigation, total N percentage increased by 4% in soil due to the presence of urea and other nitrogen compounds commonly contained in urban wastewater [52]. In our study, the SOM content increased with TWW irrigation only at the end of the second cycle. This is likely due to an initial boost in microbial input from TWW, which enhanced soil microbiological activity and contributed to SOM losses [53]. Relative to mycorrhizal treatment, as found by Brunetti et al. [17], no significant differences were observed for some soil physicochemical parameters, such as pH, Ntot, OC, and OM. However, EC and Pava content were notably higher in MSY soil. In fact, AMF play a crucial role in improving P availability in soil since they accelerate the transformation of P into bioavailable forms through various chemical reactions and biological interactions [54]. Recent studies revealed that AMF lack the capacity to release phosphatases into the soil [55]. However, they recruit phosphate-solubilizing bacteria which produce phosphatases that mineralize organic P [56,57]. Additionally, AMF contribute to phosphorus solubilization by converting inorganic phosphates into soluble forms through processes such as acidification, chelation, exchange reactions, and the production of organic acids, and other metabolites [58,59].

Finally, no difference was observed between soil parameters irrigated with SGW and GW.

3.2.2. Water

Characteristics of the analyzed TWW and GW during the two growing seasons of the artichoke are given in

Table 3. Physicochemical and microbiological analysis of TWW and GW used for the irrigation of the artichoke crop during the two years. TWW: treated wastewater; GW: locally sourced groundwater.

Parameters	TWW		GW		Recommended Value
	1st Year	2nd Year	1st Year	2nd Year	Italy [60]	EU [3]	FAO [61]
Temperature (°C)	19.88 ± 5.75	20.1 ± 5.8	22.30 ± 0.14	21.80 ± 0.10	-	-	-
pH	7.22 ± 0.27	7.30 ± 0.20	7.50 ± 0.06	7.48 ± 0.02	6.0–9.5		6.50–8.50
EC (dS m−1)	1.08 ± 0.10	0.90 ± 0.10	0.35 ± 0.00	0.32 ± 0.10	3.0		(0.7–3) SM
TSSs (mg L−1)	2.36 ± 1.63	2.0 ± 0.50	0.83 ± 0.8	0.75 ± 0.10	10	35	<50.00
Turbidity (NTU)	1.22 ± 0.72	1.11 ± 0.10	1.39 ± 015	1.32 ± 0.08	-	-
N-NH4+ (mg L−1)	2.24 ± 4.86	2.10 ± 0.20	0.04 ± 0.02	0.03 ± 0.02
N-NO2− (mg L−1)	0.79 ± 1.77	0.80 ± 0.0	ND	ND
N-NO3− (mg L−1)	5.89 ± 2.44	6.10 ± 1.20	0.93 ± 0.31	0.88 ± 0.18
Ntot (mg L−1)	12.65 ± 7.16	13.10 ± 1.90	2.89 ± 0.25	2.77 ± 0.20	15	-
Ptot (mg L−1)	4.75 ± 1.88	5.10 ± 1.90	0.03 ± 0	0.03 ± 0	2	-
COD (mg O2 L−1)	22.57 ± 7.25	20.10 ± 3.3	0.70 ± 0.20	0.60 ± 0.20	100
BOD5 (mgO2 L−1)	9.80 ± 1.04	10.80 ± 1.0	ND	ND	20	25	<30
Free Chlorine (mg L−1)	0.13 ± 0.24	0.10 ± 0.0	ND	ND
Tot. Chlorine (mg L−1)	1.07 ± 2.50	1.10 ± 0.0	0.1 ± 0.0	ND
TC (MPN 100 mL−1)	1556.19 ± 752.32	1456.20 ± 485.2	ND	ND
E. coli (MPN 100 mL−1)	596.69 ± 230.58	606.10 ± 189.47	ND	ND	100	1000	<200

EC: electrical conductivity; TSSs: total suspended solids; N-NH₄: nitrogen in ammonium form; N-NO₂: nitrogen in nitrite form; N-NO₃: nitrogen in nitrate form; N_tot: total nitrogen; P_tot: total phosphorus; COD: chemical oxygen demand; BOD₅: biochemical oxygen demand for 5 days; TC: total coliform; MPN: most probable number; E.coli: Escherichia coli; ND: not detected; SM: slight to moderate restrictions; water quality values are mean ± STD (n = 3).

.

Regarding the pH main values, which greatly influence the dissociation of studied PhACs, the analyzed TWW samples were moderately alkaline (7.22–7.30) and within the National (6.0–9.5) and FAO guidelines (6.5–8.5) for irrigation use. The EC mean values ranged from 0.9 to 1.08 (dS m⁻¹), indicating that the analyzed TWW samples were within the National maximum allowable EC value (3 dS m⁻¹) and fall within the FAO specified range (0.7–3.0 dS m⁻¹) for slight to moderate restrictions on irrigation water. These results are partially in line with Muscarella et al. [62] and Urbano et al. [63] who found slightly higher pH values in TWW used to irrigate tomato and lettuce plants, while EC were very similar to the values observed in our experimental test. The mean TSS concentration ranged from 0.75 to 2.36 mg L⁻¹, which is well within the limits set by Italian (10 mg L⁻¹) and EU (35 mg L⁻¹) guidelines. The mean biochemical oxygen demand (BOD₅) and chemical oxygen demand (COD) levels suggest that the TWW has higher organic content compared to GW. This may affect the bioavailability of weakly acidic PhACs, as polar interactions between acidic PhACs and dissolved organic matter in the WW can lead to the formation of water-soluble complexes. These complexes are less available for uptake and/or co-sorption onto solid phases in the soil, which in turn reduces their concentration in the soil solution [64,65]. The mean values of BOD₅ and COD measured (9.80–10.80 and 22.57–20.10 mg L⁻¹, respectively) were below the limits established by the Italian guidelines (20 and 100 mg L⁻¹, respectively) and in agreement with Cirelli et al. [66], who found similar values in TWW used to irrigate eggplant and tomato plants. Finally, bacteriological results showed higher loads of TC (1556.19–1456.20 MPN 100 mL⁻¹) and E. coli (596.69–606.10 MPN 100 mL⁻¹) in the examined TWW collected samples (

Table 4. Concentration values of PhACs detected in GW and TWW. GW: locally sourced groundwater; TWW: treated wastewater.

Pharmaceuticals	Formula	GW	TWW	TWW
		I and II Years	I Year	II Year
		µg/L
Clarithromycin	C38H69NO13	<LOQ	0.8 ± 0.001	<LOQ
Sulfamethoxazole	C10H11N3O3S	<LOQ	<LOQ	0.28 ± 0.001
Trimethoprim	C14H18N4O3	<LOQ	<LOQ	<LOQ
Ketoprofen	C16H14O3	<LOQ	0.2 ± 0.01	<LOQ
Carbamazepine	C15H12N2O	<LOQ	0.54 ± 0.04	0.51 ± 0.01
Fluconazole	C13H12F2N6O	<LOQ	0.07 ± 0.002	0.25 ± 0.001
Climbazole	C15H17ClN2O2	<LOQ	0.03 ± 0.001	0.1 ± 0.001
Naproxen	C14H14O3	<LOQ	0.21 ± 0.02	<LOQ

). TC is a heterogeneous group of different bacterial species having some common characteristics, whose E. coli species is a member. The mean E. coli levels exceeded the Italian regulatory limit but remained within the EU limit of 1000 MPN/100 mL.

3.3. Pharmaceutical Active Compounds (PhACs) in Irrigation Water

Table 4 reports the PhACs concentration in GW and TWW.

The GW did not have any PhACs, while several PhACs were detected in TWW in two years of investigation. The presence of these compounds in TWW used for irrigation raises concerns about their potential accumulation in crops and subsequent transfer into the food chain. In literature, PhACs concentrations in TWW range from low ng L⁻¹ to low μg L⁻¹ [67,68,69]. In our trial, the concentrations of PhACs were about a few µg L⁻¹. The highest concentration was observed for CBZ (0.54 µg L⁻¹ in the first year and 0.51 µg L⁻¹ in the second year) followed, in the second year, by SMX and FCZ. This concentration range aligns with findings from other studies that reported PhACs in WW [69], and in reclaimed wastewater used for irrigation [68], suggesting that the detected PhACs are relatively persistent. Our results are in agreement even with the findings of Ternes et al. [70], who identified 52 CECs in secondary reclaimed wastewater used for irrigation in Braunschweig, Germany. Detected compounds included trimethoprim, sulfamethoxazole, clarithromycin, erythromycin, carbamazepine, metoprolol, clofibric acid, diclofenac, and caffeine.

Some PhACs, however, were not detected in our second-year experimental samples. In general, the water solubility of a compound, its pKa (acid dissociation constant) and K_ow (octanol/water coefficient) are key factors influencing its behavior in the environment.

In particular, the pKa values of PhACs affect their interaction with other molecules. The charge state of pollutants, determined by pKa under different pH conditions, significantly influences their absorption, distribution, metabolism, excretion, and toxicity [71]. When the pKa of a compound is higher than the water pH value, as in the case of CLR, the molecule exists predominantly in its non-ionized form. This form is generally less soluble in water due to reduced interactions with water molecules. Among the PhACs investigated, CBZ also has a pKa higher than the pH of the WW. Its presence, however, can be attributed to the high dosages administered to patients (800–1200 mg day⁻¹) [72]. Furthermore, the absence of CLZ, TMP, NPX, and KET in the TWW used for irrigation of artichoke may be due to their Kow, since molecules with a logKow > 3, such as the previous PhACs, are classified as hydrophobic and not retained by soils [73].

3.4. Dynamics of the Pharmaceutical Active Compounds (PhACs) in the Soil–Plant System

3.4.1. Pharmaceutical Active Compounds (PhACs) in Soil

Soil is considered as one of the final destinations of chemical wastes [74]. The uptake, degradation, and accumulation of PhACs within the soil–plant system are influenced by their physical and chemical properties [75]. However, research on the behavior and dynamics of PhACs in soil and plants irrigated with reclaimed water remains limited. In this study, soils irrigated with GW showed no detectable PhACs, either in open field or in pot experiment. In contrast, when TWW was used to irrigate open field and pots trials, only trace amounts of CBZ were found at the end of the first cycle. At the end of the second experimental year, trace residues of three of the eight compounds searched were detected in soil samples from both open field and pot trials (Figure 3a,b).

The concentration of the three PhACs detected in soils irrigated with TWW was in the range of a few ng/g and was consistent with that reported in numerous other studies that have detected PhACs in soil environments. For example, Ben Mordechay et al. [67] found that CEC concentrations in soils irrigated with reclaimed WW ranged from low ng g⁻¹ to tens of ng g⁻¹. In particular, the range of the mean concentrations of CBZ, CLR, and CLZ was very similar to each other. This result, especially for the antibiotic and antifungal, was closely correlated with their relatively high k_d values (262–400 and 123–200 L kg⁻¹, respectively), which explained their retention in the soil. Regarding the CBZ concentration, it was very similar between the two experimental trials, confirming that this pharmaceutical is one of the most frequently and consistently detected in WW due to a limited removal efficiency (<10%) during conventional WW treatment processes [76,77]. Similarly, Kinney et al. [78] and Gielen et al. [33] identified CBZ as one of the most commonly detected PhACs in their study sites. The low concentration or absence of the other PhACs in soil samples can be attributed to the low detection frequencies of pharmaceuticals in water samples used for irrigation (Table 2). In addition, degradation processes in the soil and on its surface, due to sunlight, could justify the low concentrations observed.

Most of the PhACs were detected at the end of the second year in soil samples irrigated with SGW in the pot trial (Figure 4).

The concentration of these PhACs was much higher than those reported by De Mastro et al. [29] at the end of the first production cycle. These results confirm that prolonged irrigation with reclaimed water inevitably leads to the accumulation of pollutants in soil. In this regard, Christou et al. [65] reported that after three years of irrigation with reclaimed water, the concentration of PhACs in the topsoil layer was significantly influenced by both the duration of irrigation and the origin of the WW used. KET, NPX, and SMX exhibited lower concentrations in soil compared to the other contaminants. De Mastro et al. [29] and Mininni et al. [34] also reported low concentrations or absence of KET, NPX, and SMX in their experimental soils. These three compounds, which share similar molecular weight and negative charge at pH > 7, are characterized by weak interactions and limited sorption in soil, as indicated by their low soil–water sorption coefficients (Table 1) [64,79,80]. Furthermore, these compounds are characterized by high water solubility and were reported to be readily degradable in agricultural soils [81]. Other studies reported a similar behavior for SMX probably due to its low octanol/water partition coefficient (Kow) and low adsorption capacity, which may contribute to its high mobility and removal, assuming rapid degradation of sulfonamide antibiotics in the soil [82]. The other five PhACs were found at concentrations above 100 ng g⁻¹ in both control and mycorrhizal soils. CLZ and CLR were the most abundant in MSY soil (765 and 532 ng g⁻¹, respectively), followed by TMP, CBZ, and FCZ. This result was well correlated with the relatively low kd values of the latter PhACs (Table 1), which explain their poor retention in soil [30]. Similar results were found by Gallego et al. [5] in a greenhouse experiment involving two successive lettuce cultivation campaigns, using the same soil and irrigated with deionized and spiked water containing a mixture of 14 compounds.

In addition, all contaminants were more prevalent in MSY soil than in control and MSE soils, either in open field or in pot trial. This suggests that MSY and MSE, present at a comparable mycorrhizal frequency (Figure 1), differently influenced the fate of PhACs. MSE may have promoted a greater degradation of the contaminants compared to MSY due to the high antioxidase activities of Septoglomus viscosum and its higher adaptability to changing environments [83]. The MSY treatment may have favored the adsorption of PhACs on soil colloids and/or their uptake in plants, possibly due to a synergistic effect of the two fungi, present in its composition, on the aggregates stability [84].

It is known that the benefits of the plant–mycorrhiza association can differ depending on both the plant and the AMF species involved [85]. Regarding the artichoke, Avio et al. [86] reported that different mycorrhizae modulated the plant’s secondary metabolism in distinct ways, thereby influencing the microbial community in the soil responsible for degrading organic contaminants. Several studies have reported that mycorrhizal fungi can differently stimulate the release of root exudates and enzymes and/or improve soil structure, which in turn influences the degradation of contaminants [87]. In addition, the concentrations observed in MSE and CON soils were similar. In the control soil, the absence of competition with the mycorrhizal fungus may have favored a greater microbial presence, responsible for a rapid PhACs degradation. Lindahl et al. [88] demonstrated that mycorrhizal fungi can also suppress microbial activity to better compete for soil nutrients, confirming our thesis.

3.4.2. Pharmaceutical Active Compounds (PhACs) in Plant Organs

Only a few studies have investigated the uptake and accumulation of organic contaminants by plants irrigated with TWW over several cycles, both in pots and open fields, under realistic agricultural conditions. In our study, under TWW irrigation, no PhACs were found in all artichoke organs after the first year of irrigation, either in open fields or in pots, probably due to the low concentration of contaminants detected in TWW. Instead, at the end of the second experimental year, traces of CBZ were found in artichoke roots and leaves in both experimental trials (Figure 5 and Figure 6), but not in the edible parts, the heads.

Ben Mordechay et al. [67] found traces of PhACs in crops irrigated with recovered wastewater, with concentrations ranging from low ng g⁻¹ to low µg g⁻¹, depending on the plant and its organs (e.g., leaves, roots, fruits, or tubers). In roots and tubers, they confirmed concentrations <10 ng g⁻¹. However, the results of our study revealed that two years of irrigation with TWW can lead to the uptake and bioaccumulation of some pharmaceuticals, such as CBZ, in all organs of artichoke except for the heads. Increased PhACs concentrations due to prolonged WW use were also confirmed by Christou et al. [65]. They observed higher concentrations of SMX and TMP (5.26 and 3.40 μg kg⁻¹, respectively) in tomato berries harvested during the third year of the study.

The use of SGW determined a greater translocation of PhACs across all organs of the artichoke plants, even heads, in the pot test (Figure 7).

CBZ and FCZ showed higher concentrations in roots than other compounds, likely due to their chemical properties. In fact, these molecules have an intermediate lipophilicity (0 < Kow < 3) and a molecular weight < 500, that allow them to be easily absorbed by plant roots [89]. Higher concentrations of CBZ and FCZ were also measured in the roots in experiments conducted on tomato [90] and olive [34]. The increased root uptake capacity of CBZ and FCZ was further confirmed by their UF indexes (

Table 5. Uptake factors (UFs) for each PhAC calculated in the organs of the artichoke plant irrigated with SGW. CON: not mycorrhizal artichoke; MSY: artichoke inoculated with Rhizophagus irregularis and Funneliformis mosseae; MSE: artichoke inoculated with Septoglomus viscosum.

PhACs	Inoculation	Plant Organs
		Roots	Leaves and Stems	Head
CBZ	CON	0.69 ± 0.10	3.80 ± 0.34	0.33 ± 0.04
	MSY	0.68 ± 0.05	3.37 ± 0.48	0.27 ± 0.02
	MSE	0.99 ± 0.15	4.51 ± 0.50	0.44 ± 0.03
CLR	CON	0.03 ± 0.01	<LOQ	<LOQ
	MSY	0.05 ± 0.00	<LOQ	<LOQ
	MSE	0.10 ± 0.01	<LOQ	<LOQ
CLZ	CON	0.02 ± 0.00	<LOQ	<LOQ
	MSY	0.07 ± 0.00	<LOQ	<LOQ
	MSE	0.10 ± 0.02	<LOQ	<LOQ
FCZ	CON	0.46 ± 0.06	3.55 ± 0.49	0.39 ± 0.08
	MSY	0.56 ± 0.05	4.03 ± 0.59	0.49 ± 0.10
	MSE	0.59 ± 0.10	4.16 ± 0.43	0.49 ± 0.02
TMP	CON	0.05 ± 0.00	<LOQ	<LOQ
	MSY	0.13 ± 0.01	<LOQ	<LOQ
	MSE	0.26 ± 0.06	<LOQ	<LOQ

). In fact, the CBZ and FCZ UF values were much higher than those of the other PhACs, ranging from 0.66 to 0.99 and 0.46 to 0.59, respectively.

These two PhACs were also the only ones found at the end of the first growing season, as shown by De Mastro et al. [29], but at much lower concentrations. Furthermore, in contrast to the first year, the highest concentration was observed in the roots inoculated with MSY, rather than in the control, resembling the trend found in soils (Figure 4). CLR, on the other hand, was the pharmaceutical with the lowest concentration in the roots likely due to its high volume and molecular weight, factors that influence the translocation of the compound. Smaller molecules have been shown to more easily cross the Casparian strip in the root [91,92]. In contrast, anionic compounds such as KET, NPX, and SMX were not detected in the roots. This result can be attributed firstly to their low concentration in soil and secondly to the low permeability of cell membranes to ionic organic compounds in general [93].

Climbazole and FCZ were the only PhACs detected in artichoke leaves, stems, and heads when irrigated with SGW (Figure 7b,c) and their accumulation was directly related to the irrigation duration. The concentration of CBZ and FCZ at the end of the second year was more than double that observed after one year of SGW irrigation [29]. For example, in the leaves of plants inoculated with MSY, the concentration of CBZ increased from 400 ng g⁻¹ in 2021 to 934.9 ng g⁻¹ in 2022, while FCZ levels rose from 208.3 ng g⁻¹ to 644.8 ng g⁻¹ during the same period. The concentration of these two PhACs was significantly higher in the leaves and stems with respect to that in the roots and heads, as indicated by their UF values (Table 5). These results reflect those of Carter et al. [45], who also found higher UF values for CMZ in ryegrass, radish leaf, and radish bulb. In general, small-sized and neutrally charged compounds like CBZ and FCZ can be absorbed by plants and, moving through the symplastic pathway, can easily cross the Casparian strip, enter the xylem, and reach the aerial parts of the plant [94,95]. Several studies have also reported higher concentrations of CBZ in leaves compared to roots [96,97], suggesting a passive uptake of this compound not restricted by root membranes.

Climbazole and FCZ were also detected in the edible parts of the plant but only at the end of the second experimental year and at much lower concentrations than those found in the leaves (Figure 7c). This trend is also confirmed by their TF (Figure 8).

Specifically, both PhACs showed TF > 1 in leaves, indicating a stronger tendency for these compounds to accumulate in the leaves. Moreover, FCZ showed higher TF values (7.45, mean value) than CBZ (5.10, mean value) in the aerial parts of the plant. This difference can be attributed to their different logKow values (0.25 for FCZ and 2.45 for CBZ). More hydrophilic compounds, as FCZ, are translocated to a greater extent than the more hydrophobic compounds [98].

Regarding mycorrhiza, similar concentrations for both PhACs (about 75 ng g⁻¹) were detected in the artichoke head of MSY plants, while significantly lower levels were observed in the other two treatments.

3.5. Artichoke Agronomic Parameters

Table 6. Effects of water irrigation treatments, growing seasons, and mycorrhiza on artichoke agronomic parameters. GW: locally sourced groundwater; TWW: treated wastewater; SGW: spiked groundwater; CON: not mycorrhizal artichoke; MSY: artichoke inoculated with Rhizophagus irregularis and Funneliformis mosseae; MSE: artichoke inoculated with Septoglomus viscosum.

Experimental Factor	Number of Heads (n. ha−1)		Heads Mean Weight (g)		Diameter Heads (cm)
	Main	Secondary	Main	Secondary	Main	Secondary
Growing seasons	**	*	n.s.	n.s.	n.s.	n.s.
First	11,155.56 b	37,111.11 b	132.92 a	73.48 a	73.08 a	53.31 a
Second	12,177.78 a	38,066.67 a	135.62 a	73.74 a	73.34 a	53.08 a
Irrigation treatment	***	***	n.s.	n.s.	n.s.	n.s.
GW	10,466.67 b	36,800.00 b	133.53 a	72.07 a	73.91 a	53.06 a
TWW	12,866.67 a	38,377.78 a	135.01 a	75.16 a	72.51 a	53.33 a
Inoculum	***	***	n.s.	n.s.	n.s.	n.s.
CON	11,400 b	37,000.00 b	129.93 a	73.97 a	71.49 a	52.72 a
MSY	10,600 b	36,566.67 b	141.76 a	73.87 a	73.79 a	53. 66 a
MSE	13,000 a	39,200.00 a	131.12 a	72.99 a	74.35 a	53.20 a

The values in each column followed by a different letter are significantly different according to Tukey’s test. n.s.: not significant; *, **, and *** are significant at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively.

shows the effects of growing seasons, irrigation treatments, and mycorrhiza on the number of primary and secondary heads per hectare, weight, and diameter of heads.

The yield of primary and secondary artichoke heads, expressed in terms of head count per hectare, varied significantly across the two growing seasons, irrigation treatments, and mycorrhizal fungi.

Over the two experimental years, the number of primary heads per hectare was higher in 2022, reaching approximately 12,177 heads compared to 11,155 heads in 2021. A similar trend was observed for secondary heads, which constituted approximately 76% of the total production by the conclusion of the first production season and 78% by the end of the second. This variation in artichoke yield between growing seasons likely reflects the influence of climatic conditions during the trial (Figure 1). Higher average rainfall in the second growing season appears to have positively impacted yield. Previous studies have confirmed that climate variability can significantly affect crop yields [99,100]. According to Rezaei et al. [101], under severe climate change scenarios, crop yield losses could range from 7% to 23%.

Total marketable heads yield per hectare increased significantly under TWW. Specifically, plants irrigated with TWW produced a higher number of primary and secondary heads compared to those irrigated with GW (12,866 vs. 10,466 primary heads and 38,377 vs. 36,800 secondary heads, respectively). This difference was due to the higher concentration of plant nutrients of TWW (Table 4). The positive effect of TWW was much more evident for the second growing season for both types of heads (Figure 9a,b), confirming the positive correlation between TWW irrigation frequency and soil nutrient concentration.

Our results agree with those reported in other similar studies, which have highlighted the positive effects of TWW irrigation on crop yields. The application of TWW improved carrot plant growth and increased soil nutrient levels in the experimental trial conducted by Ofori et al. [102]. Similarly, Du et al. [103] reported that irrigating cucumbers with pig WW significantly improved yields and increased the content of vitamin C, soluble sugars, and proteins. An optimal balance of the supply of these nutrients, combined with the selection of suitable artichoke genotypes, can represent an effective approach to boost yield performance [104]. In particular, a higher nitrogen supply can influence protein synthesis in plants, by promoting the increase in growth characters [105].

Regarding mycorrhizal treatment, plants inoculated with MSE produced a significantly higher number of primary and secondary heads compared to those treated with MSY and the control. This result confirms a possible greater interaction between the AMF Septoglomus viscosum (syn. Glomus viscosum) and the artichoke plant compared to other mycorrhizal fungal species present in the same soil with similar mycorrhizal frequencies (Figure 2). Improved plant–AMF affinity could lead to higher productivity through more efficient resource use, supporting key physiological processes in plants and promoting root system growth. This hypothesis is confirmed by Ancona et al. [106], who found that the pure mycorrhizal inoculum of S. viscosum, three months after transplanting, had a positive impact on leaf and root growth of the artichoke cultivar “Troianella”. A potential functional specificity between MSE and artichoke plant was also demonstrated by Campanelli et al. [107]. In their study, the mycorrhizal fungus Glomus viscosum was found to better support the growth of artichoke plantlets. Enhanced morphological responses were observed due to the greater increase in the surface area of the host root systems.

However, no significant differences were observed for other morphological parameters, such as the weight and diameter of the artichoke heads (Figure 9c–f). These results are partly in agreement with those of Gatta et al. [108], who found no variation in head weight and diameter between the two growing seasons, although these parameters were significantly influenced by irrigation treatments.

4. Conclusions

The present study sheds light on the fate of PhACs in real agricultural systems subjected to irrigation with TWW, as well as in pot trials where it was possible to use artificially enriched waters with high concentrations of PhACs. The results showed that the concentration of the studied PhACs in soils and various organs of the artichoke plant was influenced by the duration of irrigation, the type of water applied, and the physicochemical properties of the PhACs. Notably, short-term irrigation with TWW containing low concentrations of contaminants can facilitate the accumulation into the soil of PhACs traces characterized by high Kd values such as CLR and CLZ and the translocation of very persistent PhACs as CBZ into the non-edible organs of the plants. On the other hand, the use of TWW with higher PhACs concentrations poses a significant risk to human health. In addition, the presence and concentration of PhACs in soils irrigated with SGW increased significantly compared to data collected at the end of the first crop cycle; some PhACs translocated up to the artichoke head after only two crop cycles. Therefore, our study confirms the hypothesis of a positive correlation between the risk of PhACs accumulation in soil and plants and the duration of irrigation with TWW. In addition, mycorrhizal activity also influenced PhACs uptake. The species of AMF applied played an important role in reducing contaminant concentrations, as well as microbial activity in soil controls. Finding the best affinity between the AMF species and crop could be a great opportunity not only to increase productivity, but also to ensure greater food security. To reduce human exposure to PhACs and other CECs from WW, irrigation with TWW could be used for non-edible crops or for tree crops. Another strategy to minimize risk would be to alternate TWW and GW for the irrigation of perennial crops in order to reduce soil contamination or apply TWW only during the early part of the crop cycle for annual crops. Furthermore, the potential risk of using these unconventional waters could be reduced by irrigating plants grown in soils rich in organic matter or by amending the soil with organic soil conditioners. Further research is needed today to fully understand the environmental risks of TWW irrigation, particularly regarding the long-term impact of PhACs on soil quality and plant uptake.