Mesocosm Evaluation of the Safety in the Use of Reclaimed Water Regarding Emerging Pollutants in Murcia, Spain

doi:10.21203/rs.3.rs-1642968/v1

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Research Article

Mesocosm Evaluation of the Safety in the Use of Reclaimed Water Regarding Emerging Pollutants in Murcia, Spain

https://doi.org/10.21203/rs.3.rs-1642968/v1

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The increasing shortage of conventional water for crop irrigation in arid and semiarid regions is encouraging the use of non-conventional resources. Nevertheless, concern about the presence and possible negative effects of Contaminants of Emerging Concern (CECs) in reclaimed waters can put in danger the possible use of this water source. To clarify the guarantees that reclaimed water offers, a mesocosm study was carried out. Pots with a lettuce were watered with three different types of water (distilled, reclaimed and spiked with CECs). The results showed a low concentration of these compounds in leachates and in plant tissues when reclaimed water was used. However, their concentrations were high when pots were watered with spiked water, mainly for carbamazepine (CBZ), reaching a maximum of 2982 ng L^− 1 in leachates and 45.2 ng g^− 1 in plant roots. In lettuces watered with spiked water, CBZ was found in edible part at very low concentrations that did not imply any human risk. Finally, an acute toxicity test was performed with the leachates, which resulted only slightly toxic for spiked water. This work confirms that, with the current technical improvements of active sludge water treatment, reclaimed water can be used for irrigation without risk for CECs contamination.

Reclaimed water

Irrigation

WWTPs

CECs

PPCPs

Mesocosm

Ecotoxicological assay

Due to the problems derived from water scarcity and the competition for a resource as necessary as drinking water, in arid and semiarid regions there has been a growing interest, for decades now, in search for new and alternative water sources to reduce the use of clean/potable water for diverse applications (Gil-Meseguer et al. 2019). Already in 1958, the UN Economic and Social Council (ECOSOC 1958) boosted water reuse by instructing not to consume high-quality water for uses that could tolerate lower qualities, such as for industrial activities or for soil irrigation. In the European Union (EU), the current tendency towards a circular economy has led to the promotion of further efforts to make a greater consumption of alternative water sources. However, at present, only 4% of all the water consumed in the world is reused and 80% of wastewaters return to the ecosystems, even without being treated (WWAP 2017). This data contrasts with the high-water demand in regions with a serious water deficit, where the number of cities that are reclaiming water for direct or indirect potable water use is steadily increasing (Scruggs et al. 2020). In European regions, only 2.4% of treated wastewater is reused, while an increase is expected in the near future, mainly in the southern regions (Ungureanu et al. 2020). Currently, Spain reuses 11.2% of its waters, but there is a great disparity in this reuse between north and south parts of the country. For example, in the Region of Murcia (SE Spain), 95.6% of the treated wastewater is used as for irrigation in the agriculture (INE 2018).

In the Region of Murcia, the annual average precipitation is approximately 300 mm per year, which is clearly insufficient for the agricultural production in the area, as there is a water deficit. The use in the local agriculture of water from different sources, including desalinated and reclaimed waters, is a key factor for its sustainability (Aldaya et al. 2019). This region reused 117 hm³ year^− 1 of treated water during 2020 (data from ESAMUR) being the leader in terms of the use of reclaimed water, and in agricultural production, exporting 2.5 million tons of fruit and vegetables per year (Martin-Gorriz et al. 2020). The use of reclaimed water has become part of the solution to prevent water scarcity and the effects produced by extreme climate events like droughts (WWAP 2017; WWDR 2020).

However, the possible presence of the so called Contaminants of Emerging Concern (CECs) in reclaimed waters has generated certain alarm, as this may affect human and environmental health (Al-Baldawi et al. 2021, Golovko et al. 2021; Corrêa et al. 2021; Wang et al. 2021). Therefore, these compounds are candidates for being included in future regulations regarding reclaimed water reuse, as a function of their ecotoxicity, their occurrence in the different environmental compartments, etc. Examples within the list of CECs are surfactants, flame retardants, pharmaceutical and personal care products (PPCPs), gasoline additives and their degradation products, biocides, polar pesticides and their degradation products and various compounds tested and/or suspected of endocrine disruption. Several studies have determined their presence and ecological risk assessment in waters (Martínez-Alcalá et al. 2021; Corrêa et al. 2021; Molnar et al. 2021; Wang et al. 2021), in soil (Urra et al. 2019; Jauregi et al. 2021) and in plants (Keerthanan et al. 2021), receiving reclaimed water irrigation and/or sewage sludge on agricultural soils as fertilizer (Bourdat-Deschamps et al. 2017). Nevertheless, the concern about the possible soil contamination due to the use of reclaimed water (Chiaia-Hernández et al. 2020) and on the potential PPCPs transport to groundwater settings via runoff and leaching (Renau-Pruñonosa et al. 2020) persists.

The knowledge about the concentrations of CECs that can be found in wastewater treatment plants (WWTP) effluents, and the repercussions that could derive from the use of these effluents in crop irrigation, becomes prevalent to be able to refute any restriction that may arise on its use. The objective of this work was to assess, with an advisory, predictive and/or confirmatory nature, the possible consequences or ecotoxicological effects of the application of reclaimed water on the soil-organism-plant system. For that reason, water with CECs was used to irrigate plant crops in a mesocosm experiment and their leaching risk, accumulation in the leaves and roots of the test plants (lettuce) and remaining in the soil was determined. Also, an ecotoxicological evaluation of the regenerated and spiked waters was carried out and compared with clean control water.

Mesocosm experiments were designed following the example of Carbonell et al. (2009). They consist of plastic pots of a height of 10 cm and an internal diameter of about 12 cm; a hole for drainage was made at the lower part of each pot where a nylon mesh was placed to avoid soil loss. Each pot had a system for the collection of leachates (a large beaker (600 mL) covered with tin foil to prevent light exposure). The pots were filled with unpolluted agricultural soil and lettuce seedlings were planted in the soil (one plant per pot; Fig. 1).

In this type of mesocosm systems, the soil-air interface, the water transport, and the kinetics of absorption/degradation are reproduced better than in standard soil bioassays. The system allows a realistic incorporation of CECs such as PPCPs, which resembles the agricultural practices expected from the use of reclaimed water.

A total of 24 pots were settled in a growth chamber using a completely randomized design with 6 replicate pots per treatment. The treatments consisted in irrigation with distilled water (CW), reclaimed water (RW), spiked water with 5000 ng L^− 1 of PPCPs (SW) and spiked water with 5000 ng L^− 1 of PPCPs but without plant (SW-P).

2.1. Chemicals, soil, plants, and waters

A total of 5 pharmaceutical compounds (1 psychiatric drug and 4 Nonsteroidal Anti-Inflammatory Drugs (NSAIDs)) and a cosmetic preservative were included in the spiked water (Table S.I.1). The selection was based primarily on previous studies of the research group and their occurrence in treated wastewater (Martínez-Alcalá et al. 2017). The pharmaceutical compounds selected for the experiment were the anticonvulsant and mood stabilizing drug carbamazepine, four non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac, ibuprofen, ketoprofen and naproxen and the cosmetic preservative triclosan (CBZ, DCF, IBU, KTP, NPX and TCS, respectively).

The spiked concentration of each compound was 5000 ng L^− 1 to facilitate measurements although it can be considered a severe contamination in treated water. Lower concentrations of PPCPs such as carbamazepine, diclofenac and naproxen have been previously detected (around 500 ng L^− 1) in treated wastewater (Kim et al. 2007). However, Shenker et al. (2011) reported that in Israel the level of carbamazepine in treated wastewater used for irrigation could be as high as 3000 ng L^− 1. The sources of the chemicals and the deuterated standards are described in the Supplementary Information (S.I.1). Individual stock solutions were prepared in methanol at 500 mg L^− 1 and stored at -20 ºC in the dark. An intermediate combined solution was prepared in methanol with all the chemicals at a concentration of 2500 µg L^− 1. The spiked water (SW) consisted on distilled water with5000 (ng L^− 1) of each selected PPCPs (CBZ, DCF, IBU, KTP, NPX and TCS).

The reclaimed water (RW) was collected from the Murcia-East WWTP (UTMX (ETRS89): 670034, UTMY (ETRS89): 4207420), where an active sludge with modified A2O and disinfection treatment plant is running to 553451 population equivalents (data from 2019). The modified A2O process consisted of one anaerobic stage, two anoxic stages and two oxic stages. The reclaimed water contained 25.6, 27.3 and 134 (ng L^− 1) of CBZ, DCF and KTP, respectively.

An uncontaminated calcaric fluvisol (IUSS 2015) was taken from the facilities of the Catholic University of Murcia (UTM coordinates: X: 659338; Y: 4206255, SE Spain) for this study. The soil (with clay loam texture) was collected from the surface (top 20 cm), air-dried and passed through a 2 mm sieve before its use in the experiment and the corresponding analyses. The main physicochemical characteristics of the soil were: silt loam texture (29.0% sand, 55.8% silt, 15.2% clay); pH 7.63; Eh 226 mV; 3.1% organic matter content; 0.22% total nitrogen; and 33.0% CaCO₃ concentration. Pots were filled with 500 g (dry weight) of soil and introduced in a growth chamber under controlled conditions (22/18 ± 2 ºC, environmental humidity of 60%, photoperiod of 16/8 h light/dark and illumination between 250 and 300 µmol m^− 2s^− 1). Deionized water was added to the pots to adjust the overall moisture content to 35% of the water holding capacity (WHC) of the soil. The pots were irrigated with distilled water every 2 days before planting.

After 14 days of stabilization, the pots were wetted to 80% of their WHC with distilled water and then a mini romaine lettuce (Lactuca sativa var. Jabera) seedling of 1 month old, acquired from Deitana nursery (Murcia, Spain), was placed in each pot. From that moment, the pots were irrigated everyday with 100 mL of the corresponding treatment water. The experiment was carried out for 21 days under the same conditions in the growth chamber. Then, the plants were harvested and the different soil and leachate samples collected.

2.2. Sampling procedure and PPCPs analysis.

After plant harvesting (21 days), soil samples were taken from the pots, mixed to homogeneity, freeze dried (Freeze Dryer Christ alpha 1–2/LD plus), ground to a fine powder with a mortar, and then stored at -20°C until PPCPs extraction. Lettuce plants were split into roots and aerial part and rinsed with distilled water; half of the aerial part was weighted (fresh weight and dry weight after 24 h at 60 ºC in an aerated oven) and the other half was transferred to liquid nitrogen, freeze dried and ground to a fine powder, and then stored at -20°C until PPCPs extraction. The leachate samples (one sample per pot) were divided in two subsamples, one to analyze the PPCPs and another to perform an ecotoxicity bioassay with the aquatic crustacean Thamnocephalus platyurus.

PPCPs in soil were extracted following the method of Martín et al. (2010) with slight modifications. Briefly, aliquots (2 g) of lyophilized soil samples were accurately weighed directly in centrifuge tubes (12 mL). Afterwards, the samples were successively extracted with 5 and 2 mL of methanol and 2 mL of acetone. In each extraction step, the sample was vigorously shaken during 30 s, sonicated for 15 min and centrifuged at 4000 rpm (15.1 g) during 20 min. The supernatants from each extraction step were combined and evaporated to 0.2mL under a N₂ stream (TurboVap LV concentrator). The extract was diluted to 250 mL with distilled water acidified to pH 2 with sulfuric acid and subjected to a cleanup procedure, where the aqueous mixture was loaded onto a hydrophilic-lipophilic balance cartridge (HLB, 60 mg, Dublin, Ireland), which was preconditioned with 3 mL of acetone, 3 mL of methanol and 3 mL of deionized water acidified to pH 2 with sulfuric acid. Samples were percolated through the cartridges using a vacuum manifold system (Waters) connected to a vacuum pump. The loaded cartridges were rinsed with 6 mL of water/methanol (95:5 v/v) and 3 mL of n-hexane. The elution was performed with three aliquots of 1 mL of acetone. The combined aliquots were evaporated to dryness by a gentle nitrogen stream and the evaporated extract was dissolved to a final volume of 1 mL with methanol.

Freeze dried and ground plant tissue samples were extracted and analyzed for PPCPs (Wu et al. 2012). Briefly, a 0.2 g (dry weight, DW) aliquot of plant sample was placed in a 50 mL glass centrifuge tube, spiked with deuterated PPCPs as recovery surrogates and then extracted with 20 mL of methyl tert-butyl ether (MTBE) in an ultrasonic water bath (50/60 Hz, Fisher) for 20 min, followed by centrifugation at 3000 rpm (15.1 g) for 20 min. The supernatant was decanted into a 40 mL glass vial and the residue was extracted once more using 20 mL of acetonitrile. The combined extracts were dried under N₂ stream at 30°C and re-dissolved in 1 mL methanol, followed by the addition of 20 mL distilled water. The aqueous mixture was then loaded onto a HLB cartridge (60 mg, Dublin, Ireland), which was preconditioned with 7 mL of methanol and 7 mL of distilled water. After the cartridge was dried with N₂, the analytes were eluted using 7 mL of methanol. The extract was further condensed under a gentle N₂ stream and reconstituted to 1 mL with methanol.

In the leachates, PPCPs extraction was performed according to method described by Martínez-Alcalá et al. (2017). A volume of 500 mL of leachate samples was preserved by adjusting to pH 2 with concentrated sulfuric acid. Then the leachates were loaded onto HLB cartridges (60 mg, Dublin, Ireland), which were preconditioned with 5 mL of MTBE, 5 mL of methanol and 5 mL of distilled water. After that, the cartridges were rinsed with reagent water and eluted with 5 mL of 10/90 (v/v) methanol/MTBE followed by 5 mL of methanol. The resulting extract was evaporated to dryness under vacuum at 40–50 ºC using a TurboVap LV concentrator. Finally, the extracts were brought to a volume of 1 mL using methanol.

All the samples were filtered through polytetrafluoroethylene (PTFE) filters (13 mm, 0.2 µm, Millipore, Carrigtwohill, Cork, Ireland) before instrumental analysis. The final samples were analyzed using an ACQUITY UPLC Waters I-Class system (Waters Corporation, Milford, MA, USA) coupled to a Bruker Daltonics QToF-MS mass spectrometer (maXis impact Series, Bruker Daltonics, Bremen, Germany). Details of the instrumental analysis and quality control are provided in the supplementary information section (S.I.2).

2.3. Estimation of human exposure and acute toxicity tests

The human exposure (HE) to PPCPs through the dietary intake was evaluated according to the corresponding concentrations in the plants produced in the different water treatments. The daily human exposure for the PPCPs accumulated in the aerial part of lettuce was calculated using the formula by Beltrán et al. (2020) (Eq. 1):

HE = C·10^− 6 × I

Where HE is the human exposure (mg day^− 1), C is the concentration of the corresponding PPCP (ng g^− 1, Fresh Weight (FW)) in the edible part of the plant (data obtained in the present mesocosm experiment), while I is the romaine lettuce daily intake recommended values (edible portion in grams (uncooked weight) per day) from the U.S. EPA (2018).

The acute toxicity test with the crustacean Thamnocephalus platyurus (Thamnotoxkit F, MicroBioTest Inc., Gent, Belgium) was performed according to the ISO norm 14380 (2011), using larvae hatched from cysts 20–22 h before the assay in diluted (1:8) standard freshwater at 25°C under continuous illumination (3000–4000 lx). So, for the analysis of the leachate samples, a 12 mL aliquot from each treatment was frozen and stored until its use. The tests were performed in 24-well plates, placing 10 crustaceans per well containing 1.0 mL of the corresponding test solution (leachates), in three replicates. Serial dilutions from 0 to 100% of the solutions were tested and the results are presented as a percentage (%) of survival.

3.1. PPCPs concentrations in leachates and soils

Once PPCPs are introduced into soil, they can suffer both biotic (biodegradation and/or phytodegradation) and abiotic (soil adsorption, hydrolysis, photolysis, volatilization, etc.) transformations (Qiao-ling et al. 2020). Although these degradation processes can chief the PPCPs reduction or complete removal, it is also possible that transformation products, which can result more toxic and/or persistent than the parent contaminants, can be formed in the soil (Andreu and Picó 2004; Zheng et al. 2019). After degradation, the remaining (non-degraded) compounds can migrate to the groundwater system (especially hydrophobic PPCPs) and cause contamination. PPCPs retention and/or mobility in the soil is mainly affected by the properties of the soil components, of the particular PPCP and of the surrounding environment (Xu et al. 2021). Therefore, particular soil properties and composition play a very important role with respect to PPCPs migration and transformation (Qiao-ling et al. 2020). In the present experiment, the PPCPs contained in the different irrigation waters used were completely degraded in or leached from the soil, sincethe concentrations of the different PPCPs in the soil extracts were all below the corresponding limits of detection (DL) of the equipment used for the analyses (Table S.I.3). Consequently, under the experimental conditions, irrigation both with the reclaimed water from the East Murcia WWTP and with the spiked water, did not produce any accumulation of the target compounds in the soil. PPCPs may have been either leached, photo-degraded under the light and temperature conditions of the growth chamber, and/or directly degraded by the action of soil microorganisms (Al-Baldawi et al. 2021).

Ibuprofen (IBU) was not detected in any of the leachate samples studied (the limits of detection and quantification in our experimental conditions can be found in Table S.I.2) and naproxen (NPX) was only detected in one replicate sample of the leachates from SW-P treatment (Table 1). These particular compounds seem to be well degraded in the soil. In agreement with this, Edwards et al. (2019) indicated that rapid rates of degradation and relatively slow rates of infiltration may explain why several analytes were not detected in groundwater. Also, the fact that both are relatively hydrophobic (log K_ow of 3.97 and 3.18, respectively) could favor their adsorption to soil limiting their percolation to groundwater. Other authors (Stuart et al. 2012; Fram and Belitz 2011) have suggested that compounds with a log K_ow value of around 3–4 adsorb strongly onto soil particles, which may reduce their potential leaching and contamination of the groundwater. However, the fact that these compounds were not either detected in the soils suggests the complete degradation of these PPCPs, as these compounds are relatively easy to decompose in the soil (Martínez-Alcalá et al., 2017).

Table 1

Concentrations (ng L^− 1) of detected compounds in the leachates from the pots irrigated with distilled (CW), reclaimed (RW), and spiked water (with plant (SW) or without plant (SW-P)): carbamazepine (CBZ), diclofenac (DCF), ketoprofen (KTP), triclosan (TCS).
	CBZ	DCF	KTP	NPX	TCS
CW
RW	104*
SW	1749 ± 1590	1567 ± 1677	532 ± 222
SW-P	1943 ± 978	575 ± 345	140 ± 92	225*	11.5 ± 0.7
*One replicated sample, other replicates were below detection limits.

Contrastingly, the anticonvulsant and mood stabilizing drug carbamazepine (CBZ, log K_ow of 2.45) was found in leachate samples from pots watered both with reclaimed (only in one replicate sample, with a concentration of 104 ng L^− 1) and spiked water (1749 and 1943 ng L^− 1 with and without plant, respectively; Table 1). Several studies have shown that CBZ is highly stable in water (Martínez-Alcalá et al. 2017, 2021). Renau-Pruñonosa et al. (2020) conducted an experiment where CBZ was found in aquifers in all the samples analyzed, although at extremely low concentrations (< 0.2–1.9 ng L^− 1). These authors calculated as 98.5% the percentage of degradation during the transfer of treated water from the WWTP to groundwater.

In this experiment, DCF was only detected in leachates from pots irrigated with spiked water (Table 1). Average concentrations of 1567 ng L^− 1 were detected in the presence of plant and 575 ng L^− 1 without plant, with a very high variability among replicate samples. These results agree with those reported by Renau-Pruñonosa et al. (2020), where DCF was found in the WWTP effluent, but only in some samples of the aquifers analyzed, at maximum concentrations of 4.6 ng L^− 1, observing an almost complete degradation of this compound during the transfer of water from the WWTP to the groundwater level.

Regarding the NSAID ketoprofen (KTP) concentration in the leachates, all the samples showed concentrations below the DL (40 ng L^− 1), except those collected from pots watered with spiked water (Table 1). Ketoprofen has been previously found to be almost completely removed in WWTPs (Martínez-Alcalá et al. 2017). In the case of the spiked water, the concentration found in the leachates amounted for 2.8–10.6% of the original concentration, which indicates the high tendency of this compound to be degraded in the soil.

Finally, the triclosan (TCS) compound was only detected in SW-P leachates at very low concentrations (Table 1). TCS is highly hydrophobic, with a log K_ow of 4.90 (Table S.I.1), so it is expected to be retained in the soil, but it also seems to be easily degraded, especially when compared to other PPCPs such as CBZ. The half-life time of TCS biodegradation was calculated as 18 days under aerobic conditions and 70 days under anaerobic conditions (Ying et al., 2007), which indicate the easy biodegradation of TCS under the aerobic conditions prevailing in the soil. Also, in the case of TCS, the presence of plants provoked further degradation or retention of this compound since its concentration in the leachates from pots with lettuce were found to be below the limit of detection (Table S.I.4). Plants may excrete some substances capable of degrading or immobilizing triclosan, or this compound may have also been adsorbed on or absorbed by plant roots, preventing the leaching of this PPCP (Zhao et al. 2016).

Regarding the acute toxicity test, the larvae of Thamnocephalus platyurus used barely showed any negative effect when exposed to the leachates coming from the different treatments. In both distilled (CW) and reclaimed (RW) water, no mortality was observed whatsoever, while with the spiked water (SW) leachates, some larvae died when exposed to the non-diluted (100%) samples. The calculated mortality of T. platyurus in spiked water was 10%, while reclaimed water did not show toxicity towards this organism.

3.2. PPCPs concentration in lettuces and human exposure risk assessment

Plants can remove soil/water PPCPs through mechanisms such as root absorption and the action of root exudates, but also the compounds absorbed by the roots can be translocated to the aerial part of the plants or the fruits. Nevertheless, these processes are complicated since they are governed by the PPCPs physico-chemical properties as well as by soil and plant particular characteristics (Beltrán et al. 2020; Abril et al. 2021). In this experiment, the plants did not show any symptom of impaired growth in any of the treatments, and no significant differences were found for plant yield (fresh and dry weights) among the different treatments (Table 2).

Table 2

Plant fresh weight (FW) and dry weights (DW) (g), and concentrations (ng g^− 1 DW) of detected compounds: CBZ and DCF in the roots and aerial part (AP) of the lettuces irrigated with distilled (CW), reclaimed (RW) and spiked water (SW).
	FW	DW	FW/DW	CBZ_Root	CBZ_AP	DCF_Root
CW	31.1 ± 3.8	3.5 ± 0.2	8.9 ± 0.9
RW	28.9 ± 2.2	3.1 ± 0.5	9.3 ± 1.3	37.1*
SW	34.1 ± 4.1	3.4 ± 0.6	10.2 ± 0.9	40.0 ± 20.5	41.6 ± 4.1	13.9 ± 7.8
*One replicated sample, other replicates were below detection limits.

The concentrations of some of the studied PPCPs (IBU, KTP, NPX and TCS) were below the corresponding DL (Table S.I.4) in the different plant tissues (aerial part or roots) coming from the different treatments. The low concentrations obtained for these compounds suggested a scarce tendency of plant absorption by plant roots and translocation to the aerial parts of the plants. However, CBZ (with relatively low hydrophobicity; Table S.I.1) was accumulated in both plant tissues (Table 2). The CBZ concentration in the roots of plants irrigated with reclaimed water (detected only in one replicate sample) was very close to that in the roots of plants irrigated with spiked waters, despite the huge difference in CBZ concentration in both irrigation waters.

Certain translocation of CBZ from the roots to the aerial part could occur when the plants were watered with spiked water, since the average concentration found in the aerial part (41.6 ng g^− 1, DW) was similar to that determined in the roots of the plants from this water treatment (SW). In anexperiment with atenolol, carbamazepine and triclosan, Beltrán et al. (2020) found that, CBZ hadthe highest accumulation in lettuce, radish, and maize roots and leaves of the contaminants studied (atenolol, CBZ and TCS). They found that factors such as non-ionic property and relatively low hydrophobicity of CBZ could support this tendency, due to the absence of interaction with the organic colloids present in soil. This could also favor its potential for internal plant movement from the xylem to the phloem in the direction of the transpiration stream, and accumulation in leaves. In fact the same authors found translocation factors (root to leaves) for CBZ greater than 3 in maize and of 5 in radish irrigated with highly contaminated water. In a hydroponic experiment with lettuces, González-García et al. (2018) observed an accumulation of CBZ and DCF in plant leaves of up to 1456 and 82 ng g^− 1, respectively. Those concentrations are much higher than the ones observed in the present experiment, likely as a consequence of the very high concentrations used in their spiked water (both compounds were added to the growing solution at a concentration of 210000 ng L^− 1). This indicates the capacity of this plant to accumulate these compounds in its tissues. Nevertheless, these water concentrations are difficult to find in WWTPs effluents. In the spiked water, the concentration of those compounds was 5000 ng L^− 1, which can be already considered high, since CBZ and DCF normal concentration observed in WWTPs of the same area are between 10.1 and 98 ng L^− 1 for CBZ and between 6.64 and 307 ng L^− 1 for DCF (Martínez-Alcalá et al. 2017, 2021).

Regarding DCF, it was only detected in the roots of plants irrigated with spiked water (Table 2), with an average concentration of 14 ng g^− 1. In a hydroponic experiment with lettuce (González-Garcia et al. 2018), a root concentration of DCF of 100 ng g^− 1 was found when the growing media was spiked with 30000 ng L^− 1 of DCF, a concentration 89% higher than that determined in the leaves. Diclofenac properties, like their high log K_ow (> 4, Table S.I.1), facilitate their absorption by roots, which generally show higher concentrations than leaves.

In light of these results, it can be said that this variety of lettuce, irrigated with reclaimed water under these experimental conditions, was not able to accumulate the compounds studied in the aerial part, and only some CBZ was retained in plant roots. However, when lettuces were watered with spiked water, CBZ and DCF were accumulated in the roots, and CBZ could even be translocated to the aerial and edible part of the plants. The plants did not absorb the rest of the contaminants studied (IBU, KTP, NPX and TCS), or maybe they were absorbed and transformed into other compounds that the analytical procedure used were not able to identify. In an experiment carried out by Abril et al. (2021) with radish, neither triclosan translocation or bioconcentration were measured, this fact being considered a consequenceof their metabolization after plant uptake. In addition, other authors (Kinney and Heuvel 2020) have attributed the low concentrations of PPCPs detected in plant tissues to their biodegradation by rhizosphere microorganisms.

Taking into account the concentrations obtained in the edible part of lettuce, the potential human exposure was evaluated. No PPCPs accumulation was observed in the aerial part of the lettuces watered with the reclaimed water, so they are safe for their consumption. Whereas, in lettuce plants watered with spiked water, a CBZ maximum concentration of 45.2 ng g^− 1 (DW) was obtained; if the average weight obtained for the edible part of lettuce is considered (Table 2), this corresponds to 4.51 ng g^− 1 (W/W), The daily intake of romaine lettuce (U.S. EPA 2018) is estimated to be 7.0 g day^− 1. The human exposure (HE) estimated value per person for this PPCP was 31.6·10^− 6 mg day^− 1, lower than that found by Beltrán et al. (2020) in lettuces watered with spiked water (which ranged from 484·10^− 6 to 6784·10^− 6 mg day^− 1). If the CBZ maximum permissible concentration for daily human intake (1200 mg person^− 1 day^− 1; Vademecum (2019)) is considered, the value obtained in the present experiment is several orders of magnitude below it, indicating no risk of human consumption of these lettuces, even when watered with spiked water.

The irrigation with reclaimed water from the studied WWTP did not contribute to concentrate the studied PPCPs in leachates or the edible part of lettuces under our experimental conditions, while spiked water provoked the presence of CBZ, DCF, KTP and TCS in the leachates, and also some CBZ was detected in the edible part of lettuce. Nevertheless, this concentration may not imply human risk of consumption.

In the soils, PPCPs were not detected, not even when they were irrigated with the spiked water. These compounds were degraded or transformed in the soil, likely by soil microorganisms and/or by light induced degradation in the growth chamber. The acute toxicity test run indicated only some toxicity for spiked waters.

Therefore, it can be said that the use of reclaimed water from the studied WWTP is safe for watering crop species such as lettuce, since they do not imply any risk in terms of the transference of the compounds analyzed in this experiment to their edible part or to other water bodies or their accumulation in the soil.

Data availability

All data are available for the submitted journal.

Acknowledgements

The authors are grateful to Dr. Juan J. Alarcón from CEBAS-CSIC for allowing the use of the CEBAS-CSIC facilities for the experiment, and to Dr. José Enrique Yuste for the PPCPs analysis.

Funding

This work was supported by the Regional Ministry of Water, Agriculture, Livestock, Fisheries and by the ESAMUR, Entity of the Autonomous Community of the Murcia Region (Project No. CV.00A.2018 “Evaluación de efectos de contaminantes emergentes de aguas regeneradas en microcosmos”).

Author information

Affiliations:

Department of Civil Engineering, Catholic University of Murcia (UCAM), Av. de los Jerónimos, 135, 30107, Guadalupe, Murcia, Spain.

Isabel Martínez-Alcaláand Francisco Pellicer-Martínez.

Centro de Edafología y Biología Aplicada del Segura, CEBAS-CSIC, Campus Universitario de Espinardo, 30100 Murcia, Spain.

María Pilar Bernaland Rafael Clemente.

Regional Entity for Sanitation and Wastewater Treatment in the Region of Murcia (ESAMUR), C. Santiago Navarro, 4, 30100, Espinardo, Murcia, Spain.

Agustín Lahora.

Contributions

Conceptualization (Martínez-Alcalá, Lahora); data curation (Martínez-Alcalá, Pellicer-Martínez, Bernal); formal analysis (Martínez-Alcalá, Pellicer-Martínez, Clemente); funding acquisition (Martínez-Alcalá); investigation (Martínez-Alcalá, Lahora); methodology (Martínez-Alcalá, Bernal); project administration (Martínez-Alcalá); resources (Martínez-Alcalá, Bernal); supervision (Martínez-Alcalá); validation (Lahora, Clemente, Bernal); visualization (Bernal); writing, original draft (Martínez-Alcalá,); writing, reviewing and editing (Martínez-Alcalá, Pellicer-Martínez, Lahora, Clemente, Bernal).

Corresponding author

Correspondence to Martínez-Alcalá, Isabel.

Ethical approval

This study has been approved by all the shared departments and the authors.

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Mesocosm Evaluation of the Safety in the Use of Reclaimed Water Regarding Emerging Pollutants in Murcia, Spain

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Version 1

Abstract

Figures

1. Introduction

2. Material And Methods

2.1. Chemicals, soil, plants, and waters

2.2. Sampling procedure and PPCPs analysis.

2.3. Estimation of human exposure and acute toxicity tests

3. Results And Discussion

3.1. PPCPs concentrations in leachates and soils

3.2. PPCPs concentration in lettuces and human exposure risk assessment

4. Concluding Remarks

Declarations

References

Supplementary Files

Status:

Version 1