Global water scarcity is intensifying due to climate change, increasing demand, and inadequate management, with the Mediterranean Basin emerging as a major hotspot (Eekhout et al., 2024; Tappi et al., 2023a, 2023). As agriculture accounts for nearly 70% of global freshwater use and 1.8 billion people are expected to face limited water access by 2025, innovative and integrated water management strategies are urgently needed to safeguard food security (Hashem and Qi, 2021; Lahlou et al., 2021; Hamdan et al., 2022; Carucci et al., 2023a).

In this context, the reuse of treated wastewater for irrigation is emerging as an increasingly explored and adopted agronomic strategy, especially in areas facing water scarcity, expanding urban populations, and increasing demand for irrigation water (Winpenny et al., 2010).

Supported by EU Directive 2020/741 (Official Journal of the European Union, 2020), TWW reuse promotes sustainable, climate-resilient farming. Globally, over 20 million hectares are already irrigated with TWW or untreated wastewater, a practice set to grow (Jiménez, 2008; Seidi et al., 2020). Beyond conserving freshwater resources, TWW reuse can enhance soil fertility and crop yields, reduce dependence on chemical fertilizers, and contribute to lower greenhouse gas emissions (Abou Jaoude et al., 2025; Gatta et al., 2015). Despite the steady increase in TWW production and its recognized benefits, its reuse in agriculture remains limited at the global scale. A key barrier to wider TWW use is the presence of unregulated contaminants like pharmaceutical residues and metabolites, which conventional treatment often fails to remove. Antibiotics, antifungals, and antidepressants are especially concerning due to their persistence. Repeated TWW irrigation can lead to their accumulation in soils and potential uptake by edible plants (Mordechay et al., 2022; Ponce-Robles et al., 2022).

Therefore, the greatest risk associated with pharmaceutical active compounds (PhACs) contamination may occur in regions where vegetables are intensively cultivated and irrigated with reclaimed wastewater.

This concern is supported by findings from Tadić et al. (2021), who detected ten antibiotics and their metabolites in lettuce samples collected from fields irrigated with TWW. Similarly, Mordechay et al. (2021) reported the highest number and concentrations of PhACs in leafy vegetables. Specifically, analysis of samples collected from commercial fields irrigated with reclaimed wastewater in Israel revealed that antiepileptics-namely carbamazepine, lamotrigine, and gabapentin-were the most dominant therapeutic group detected across the reclaimed wastewater-soil-plant continuum. These results highlight the importance of understanding the mechanisms underlying the uptake and translocation of pharmaceuticals in plants, usually governed by the physicochemical properties of the compounds (Carter et al., 2014), as well as environmental factors such as soil temperature, pH, and plant-specific parameters like transpiration rates (García et al., 2019).

PhACs uptake depends on factors like biodegradability, which influences soil concentration (Dalkmann et al., 2014; Grossberger et al., 2014), and chemical properties such as lipophilicity and charge (Miller et al., 2016; Li et al., 2019). Moderately lipophilic compounds (octanol/water distribution coefficients, log D 1.5–3) are more likely to move from roots to bulbs, leading to higher concentrations in leaves and fruits (Riemenschneider et al., 2016; Madikizela et al., 2018). Therefore, assessing the fate of PhACs within the plant-soil system is essential, particularly in arid and semi-arid regions of the Mediterranean, where the use of recycled wastewater for irrigation is expected to become increasingly common. This practice may offer a sustainable alternative water source, particularly for high water-demand crops such as fennel. Research on bulbous crops is limited, and to date, fennel has not been studied. The assessment of the uptake and translocation of pharmaceutical active compounds (PhACs) in this crop could be particularly interesting, as it exhibits traits of both root and leafy vegetables. Experimental evidence indicates that pharmaceutical accumulation in crops generally follows the order: fruiting vegetables < cereals and forages < root vegetables < leafy greens (Christou et al., 2019), placing fennel in an intermediate position in terms of potential PhAC accumulation. Fennel (Foeniculum vulgare Mill.), a member of the Apiaceae family, is one of the oldest cultivated herbs in the Mediterranean basin. It is a valuable edible, medicinal, and cosmetic plant predominantly grown in arid and semi-arid environments, where water availability is a key factor limiting growth and yield (Bahmani et al., 2015).

Although previous studies have demonstrated that fennel possesses a notable biosorption capacity for certain contaminants, including heavy metals such as cadmium (Cd), nickel (Ni), and chromium (Cr) (Endalamaw and Chandravanshi, 2015; Rao et al., 2010), to the best of our knowledge, this is the first study into the uptake of PhACs by fennel plants irrigated with contaminated water.

In this context, several studies have evaluated the uptake and accumulation of pharmaceutical contaminants in agricultural crops irrigated with treated wastewater, consistently highlighting strong crop- and compound-specific variability (Wu et al., 2015; Christou et al., 2019; Sleight et al., 2023). These studies consistently report a lack of data for aromatic and medicinal crops, including fennel, and very limited information for compounds such as climbazole and flecainide. Consequently, substantial knowledge gaps persist regarding the behavior, uptake, and translocation of these pharmaceuticals in soil–plant systems involving Mediterranean horticultural species such as fennel.

Gaining insight into the accumulation of PhACs in soil and plant tissues is critical not only for environmental risk assessment but also for evaluating their potential impact on irrigated horticultural crops and informing remediation strategies.

Accordingly, the main objective of this study was to address these knowledge gaps by: (i) investigating the fate and behavior of three widely used PhACs as carbamazepine, climbazole, and flecainide and their metabolites within the soil–plant system under fennel cultivation, and (ii) assessing the plant’s capacity to uptake and translocate PhACs.

The recent EU Regulation 2020/741 on treated wastewater reuse for irrigation is based on the estimation and management of environmental impacts with particular reference to the agricultural systems. Therefore, PhACs in treated wastewater and their persistence, bioaccumulation in the soil-plant system, and potential toxicity highlight the need for effective monitoring and management strategies. To the best of our knowledge, no literature is available about the effects of PhACs derived to treated wastewater irrigation on fennel crop. Thus, our study aimed to evaluate the fate of selected pharmaceuticals at different concentrations applied to the fennel crop through irrigation, also considering important factor as BCF and TF. Several studies have reported that a BCF ≤ 1 suggests that, although the plant may uptake pollutants (Ackah et al., 2014; Gatta et al., 2018), including emerging contaminants (Sunyer-Caldú et al., 2022), there is no significant accumulation in plant tissues. Conversely, a BCF>1 indicates that the plant accumulates PhACs in the plant parts. Therefore, low BCF values suggest limited or no uptake of PhACs, implying that the plant tends to exclude these compounds from its tissues.

Under our experimental conditions, all PhACs (carbamazepine, climbazole, and flecainide) were detected in soil following irrigation with spiked fresh water, depending on the different concentrations utilized.

Carbamazepine accumulated in the soil only at High₂₀₀ and High₆₀₀ treatments. In contrast, climbazole and flecainide were detectable in the soil even at lower concentrations (Low_0.5 and Low_2.0). Although a complete quantitative mass balance was beyond the scope of this study, a qualitative evaluation suggests that the majority of the applied pharmaceuticals were retained in the soil, particularly for climbazole (about 94.1% and 72.6% for High₂₀₀ and High₆₀₀ treatments, respectively) and flecainide (about 64.8% and 83.4% for High₂₀₀ and High₆₀₀ treatments, respectively), while a smaller fraction was taken up by plant roots (<0.03%) and only negligible amounts were translocated to edible tissues. The mass balance for climbazole confirms its high sorption affinity for the soil matrix and its low mobility (Denora et al., 2023).

Additional fractions may have undergone degradation, irreversible sorption to soil organic matter, or remained unaccounted for due to transformation processes, as previously reported for pharmaceutical contaminants in soil–plant systems (Dalkmann et al., 2014; Meffe et al., 2021; Mordechay et al., 2022).

The soil behavior of flecainide and climbazole may be attributed to similar octanol/water partition coefficient (i.e., log K_OW) values to those of carbamazepine (3.76 and 3.78 versus 2.45). This means that they could interact strongly with the organic component of the soil (Richter et al., 2013), increasing their adsorption in the soil and explaining the high concentration found also in the soil.

These results are confirmed by the multivariate HCPC procedure since climbazole was highly represented in cluster 2 characterized mainly by soil samples. This is a significant result since this compound showed the lowest BCF factor in the roots, highlighting the lowest uptake from the soil to the root. The high flecainide soil content is in accordance with Meffe et al. (2021), who reported a high affinity of flecainide accumulating in the soil. However, our results showed a higher uptake of flecainide from soil to root with respect to climbazole, even if the bioaccumulation coefficient (BCF) was<1 (0.48 and 0.45 for High₆₀₀ and High₂₀₀ irrigation treatment, respectively), thus showing no significant accumulation in this plant part. Flecainide is most represented in cluster 2 characterized mainly by root samples, but the compost seems to remain localized in the root since there was no translocation from the root to the edible bulb (TF_(bulb/root)=0). These results are relevant since soil-root uptake and plant translocation of pollutants is one of the fundamental processes through which humans are exposed to PhACs through the food chain (Prosser and Sibley, 2014; Fernandes et al., 2024; Petruzzelli et al., 2025).

In our experimental trial, no compounds attributable to the transformation products of flecainide and climbazole were detected in the soil, likely due to different metabolic processes compared to those occurring in humans. The degradation pathways of climbazole and flecainide have primarily been documented in human plasma and urine (Schönrath et al., 2024; Benijts et al., 2003). Climbazole is metabolized through the reduction of its ketone group to a secondary alcohol function, resulting in OH-climbazole (Schmidtkunz et al., 2021), while flecainide undergoes biotransformation via O-dealkylation, producing meta-O-dealkylated flecainide and the corresponding meta-O-dealkylated lactam derivative (Benijts et al., 2003). Furthermore, the lack of metabolites attributable to these two molecules may also be attributable to the duration of the experiment.

The lower concentration of carbamazepine in soil, as compared to climbazole and flecainide, may be attributed to its neutral form and relatively low octanol-water partition coefficient (log K_ow=2.45), which indicates higher hydrophilicity. A low log K_ow value reflects a greater affinity for the aqueous phase, suggesting that carbamazepine is more likely to remain in the soil solution and therefore more inclined to leaching into groundwater (Wojsławski et al., 2019). Due to its higher pKa (13.9) respect to flecainide (9.3) and climbazole (6.49), carbamazepine remains mostly in its neutral (non-ionized) form under typical soil pH conditions, resulting in weaker interactions with soil constituents. For this reason, PCA results showed a high correlation of carbamazepine with PC₂ representing ‘low soil-absorbed form’ factor.

Regarding carbamazepine metabolites, the detection of acridine in soil, even at low concentrations and as the sole metabolite, raises concerns about its potential long-term entry into the food chain and associated toxic effects. Acridine is known to inhibit DNA repair and impair cellular growth. Notably, previous research has shown that acridine is significantly more toxic than carbamazepine across multiple trophic levels (De Mastro et al., 2024).

Among the three compounds, carbamazepine appears to be more readily taken up by roots; indeed, also this compound was highly represented in cluster 3 characterized mainly from root samples. Its concentration in root tissues was approximately 4.8-fold and 2.6-fold higher than respect to soil for the High₂₀₀ and High₆₀₀ irrigation treatments, respectively. BCF was therefore found to be higher for High₂₀₀ than for High₆₀₀. This behavior has also been observed by some authors (Sunyer-Caldú et al., 2022; Fernandes et al., 2022), highlighting how obtaining higher BCF values in experimental treatments with lower contaminant exposure was possible. On the other hand, if a compound’s plant uptake is considered a rate-limited process, BCF values will become progressively smaller as greater contaminant inputs are applied (Fernandes et al., 2024).

Carbamazepine, climbazole, flecainide and related metabolites were not quantified (below the limit of quantification) in the different plant parts (roots, leaves, and bulb) under low-concentration irrigation treatments (Supplementary Table S2). Therefore, these compounds’ translocation factor (TF) values were not determined under such treatments (Supplementary Table S3). In contrast, under high-concentration irrigation treatments (High₂₀₀ and High₆₀₀), only carbamazepine and climbazole exhibited translocation and accumulation in the aboveground plant parts (leaves and bulb), although the TF values remained well below unity (<1). Since the TF is defined as the ratio of the PhAC concentration in the aerial parts to that in the roots, these results indicate that carbamazepine and climbazole had very low concentrations in the edible bulb compared to the roots, reflecting limited root-to-bulb translocation. These findings are consistent with previous studies (Prosser et al., 2014; Gatta et al., 2025), which reported TF values for aboveground crop parts mostly below 1. For instance, TF values for green pepper, carrot, cucumber, tomato, radish, and lettuce were ≤0.33. These results suggest a physiological barrier that limits PhAC accumulation in edible fennel tissues, potentially mitigating human health risks.

The limited translocation to edible parts (bulbs) of the evaluated compounds (carbamazepine, climbazole, flecainide and their derivatives) represents a crucial factor to mitigate the potential health risks related to these PhACs.

This study provides the first analysis of the behavior of selected pharmaceutical compounds in fennel crop irrigated with water containing PhACs. The results highlight that: i) all three PhACs accumulated in soil at high irrigation concentrations, with climbazole and flecainide showing greater persistence even at low doses; ii) carbamazepine was more readily absorbed by roots, exhibiting BCF values >1, yet its movement to aerial plant parts remained limited; iii) climbazole and flecainide showed low bioaccumulation (BCF<1) and negligible translocation to edible tissues (TF≈0), remaining largely confined to soil or roots; iv) the low translocation rates of all tested PhACs suggest the possible presence of “physiological barriers” that limit their movement to edible fennel parts, potentially lowering the risk of human exposure via consumption; v) regarding PhACs metabolites, no compounds attributable to the transformation products of flecainide and climbazole were detected; however, the detection of acridine in the soil, even at low concentrations and as the sole metabolite of carbamazepine, raises concerns regarding its potential long-term entry into the food chain, suggesting the need for frequent evaluation of that compound in the soil-plant system.

The limited translocation of the investigated PhACs to the edible bulb suggests that fennel may represent a low-risk crop for treated wastewater reuse. These findings support the implementation of crop-specific risk assessments within current wastewater reuse regulations (e.g., EU Regulation 2020/741), emphasizing that not all crops pose the same level of risk for human exposure. This aspect is particularly important given that, in many European countries, integrated systems for managing non-conventional water sources, including treated wastewater irrigation, are currently under evaluation.

Future research should extend this evaluation across multiple crop cycles, various environmental conditions, and additional pharmaceutical compounds to better understand the fate of PhACs, and their metabolites, in soil-plant system.