Assessing Lettuce Exposure to a Multi-Pharmaceutical Mixture in Soil: Insights from LC-ESI-TQ Analysis and the Impact of Biochar on Pharmaceutical Bioavailability

Agricultural practices introduce pharmaceutical (PhAC) residues into the terrestrial environment, potentially endangering agricultural crops and human health. This study aimed to evaluate various aspects related to the presence of pharmaceuticals in the lettuce-soil system, including bioconcentration factors (BCFs), translocation factors (TFs), ecotoxicological effects, the influence of biochar on the PhAC bioavailability, persistence in soil, and associated environmental and health risks. Lettuce (Lactuca sativa L.) was exposed to a mixture of 25 PhACs in two scenarios: initially contaminated soil (ranging from 0 to 10,000 ng·g–1) and soil irrigated with contaminated water (ranging from 0 to 1000 μg·L–1) over a 28-day period. The findings revealed a diverse range of BCFs (0.068–3.7) and TFs (0.032–0.58), indicating the uptake and translocation potential of pharmaceuticals by lettuce. Significant ecotoxicological effects on L. sativa, including weight change and increased mortality, were observed (p < 0.05). Interestingly, biochar did not significantly affect PhAC uptake by L. sativa (p > 0.05), while it significantly influenced the soil degradation kinetics of 12 PhACs (p < 0.05). Additionally, the estimated daily intake of PhACs through the consumption of L. sativa suggested negligible health risks, although concerns arose regarding the potential health risks if other vegetable sources were similarly contaminated with trace residues. Furthermore, this study evaluated the environmental risk associated with the emergence of antimicrobial resistance (AMR) in soil, as medium to high. In conclusion, these findings highlight the multifaceted challenges posed by pharmaceutical contamination in agricultural environments and emphasize the importance of proactive measures to mitigate the associated risks to both environmental and human health.


QuEChERS Extraction of Lettuce Samples
The lettuce leaves and roots were extracted separately using already validated and published method in previous study. 4Briefly, 0.1 g of lyophilized and homogenized lettuce leaves were accurately weighed and placed into a 50 mL PE centrifugation tube.In the extraction step, ceramic homogenizers were initially introduced, followed by pipetting of 5 mL of the extraction medium (MeOH:McIlvaine buffer, pH 2.6:ACN in a ratio of 8:20:72).Following this, the sample was vortexed for 1 min.Subsequently, separation salts (2 g anhydrous Na 2 SO 4 and 0.5 g NaCl) were added, and the mixture was further vortexed for 1.5 min before centrifugation at 3,500 rpm for 10 min at 20 °C.In the purification step, following centrifugation, 2 mL of the organic phase was carefully pipetted into a 15 mL PE centrifugation tube preloaded with dSPE sorbents (12.5 mg DSC-18, 12.5 mg PSA, and 225 mg of anhydrous Na 2 SO 4 ).Subsequently, the sample was vortexed for 1 min, followed by another centrifugation at 3,500 rpm for 10 min at 20 °C.Finally, the sample was filtered through 0.22 μm nylon syringe filters (diameter 13 mm) into a 2-mL glass vial, ready for LC-MS/MS analysis.

Extraction of pharmaceuticals from soil
The soil samples were extracted using already validated and published method in previous study. 4 subjected to evaporation under a nitrogen stream in a thermostatic metal block heated to 40°C until each vial's weight loss reached 6 g.Subsequently, the contents of both vials were combined and transferred into a 600-mL beaker, and the soil extract was diluted by adding 480-mL Milli-Q water to decrease the percentage of the organic phase (no additional pH adjustment was made).In the solid phase extraction step, the pre-concentrated soil extracts were purified using Chromservis HLB cartridges (200 mg; 6 mL; Particle diameter 25-35 μm; Chromservis Czech Republic) using a Baker vacuum system (J.T. Baker, Deventer, The Netherlands).To outline the procedure briefly, the SPE column was conditioned with 6 mL of MeOH, followed by 6 mL of Milli-Q water, with a flow rate of approximately 1 mL•min -1 .Subsequently, the diluted soil extract was loaded onto the column at a flow rate of 5 mL•min -1 .The washing step was performed with 15 mL of Milli-Q water at a flow rate of 1 mL•min -1 , followed by 2 min of vacuum drying of the sorbents.Finally, the elution of PhACs was achieved by passing 9 mL of 0.1% formic acid (FA) in MeOH, and the eluate was collected into 20 mL glass vials.This was followed by evaporation under the nitrogen stream in the thermostatic metal block heated to 40°C to dryness.
Subsequently, 5 μL of an internal standards mixture (concentration of mixture 10 μg•mL -1 ) was introduced, followed by the addition of 995 μL of 0.1% FA in H 2 O:ACN (95:5, v/v).The sample was filtered through 0.22 μm nylon syringe filters (diameter 13 mm) into a 2-mL glass vial.The prepared sample was then subjected to LC-MS/MS analysis.

Appendix 2. LC-MS/MS method
Both lettuce and soil sample extracts were analyzed using a previously validated and published method for over 40 pharmaceuticals in previous study. 4However, in this study, only 20 pharmaceuticals were analyzed, which allowed for lower limits of detection (LoDs) and quantification (LoQs) to be achieved, while maintaining similar recovery rates, as the extraction method remained the same.
Instrumental analysis for the quantification of PhACs in lettuce and soil extracts was performed using ultraperformance liquid chromatography (UHPLC Agilent 1290 Infinity LC) coupled with a triple quadrupole mass spectrometer (Bruker EVOQ LC-TQ) with electrospray ionization (ESI).The gas sources of nitrogen and air were provided by an external gas generator (Peak Scientific -Genius 3045).
Chromatographic separation was accomplished using a Luna Omega Polar C18 Phenomenex column (100 x 2.1 mm, 1.6 µm).The column temperature was optimized at 35°C, and the flow rate was set to 0.5 mL•min - 1 .The mobile phases consisted of A) 0.1% FA in H 2 O and B) ACN, following a gradient program for the A eluent (%): t(0 min) = 90, t(0.5 min) = 90, t(13.0 min) = 35, t(14.0 min) = 10, and t(15.5 min) = 90.The LC method was set to a stop time of 16 min, with a 2-min re-equilibration time.The injection volume for all analyses was 7 µL.
To prevent carry-over, an external needle wash was performed using a wash solvent composed of FA:H 2 O:ACN at a ratio of 1:9:90 for 30 s.
The MS conditions were set as follows for electrospray ionization in positive mode: spray voltage: 4,500 V; cone temperature: 350°C; cone gas flow: 15 arbitrary units (a.u.); heated probe temperature: 500°C; probe gas flow: 25 a.u.; nebulizer gas flow: 45 a.u.; and exhaust gas: ON.For both quantitative and qualitative analysis of PhACs, the multiple reaction monitoring (MRM) mode was employed, using the specific MRM transitions outlined in Table S4.Argon served as the collision gas at a pressure of 1.5 mTorr.Consequently, we searched scientific literature for acceptable daily intake (ADI) values [9][10][11][12][13][14] to establish benchmarks for calculating risk quotients (RQs) (Table S6).Using these values, we calculated the RQ for each pharmaceutical separately with Eq. 3. Next, we computed the Hazard Index (HI) as the sum of all RQs for each soil treatment using Eq. 4 (Table S6), as described in previous studies. 6,15A human health risk is considered negligible when the RQ or HI is less than 0.01, considerable when either RQ or HI exceeds 0.01, and distinct when either value is greater than 0.05. 15
where HI [-] stands for hazard index and RQ [-] denotes risk quotient.

S12
Table S6.Estimation of Health Risk due to Exposure to Pharmaceutical Residues in Lettuce -Acceptable daily intake (ADI) for selected pharmaceuticals, their estimated daily intake (EDI) at different soil treatments (soil spike or soil irrigation), with calculated risk quotients (RQ) for each compound and total sum of RQs as hazard risk (HI) at different levels of soil contamination [9][10][11][12][13][14]16 , N.D. -not determined.

Firstly, 1
.0 g of soil was precisely weighed and placed into a 50 mL polyethylene (PE) centrifugation tube.The extraction procedure (Steps 1-2) involved pipetting 5 mL of methanol (MeOH) and 5 mL of phosphate buffer (pH 3) into the sample, followed by vortexing for 30 s. Subsequently, PhACs were extracted using an ultrasound bath for 10 min at 12°C.After sonication, the mixture was centrifuged at 4,800 rpm for 8 min at 20 °C.The resulting supernatant was transferred into a 30-mL dark glass vial.The extraction process was then repeated using the same extraction medium and extraction conditions.The extracts obtained from extraction rounds (1-2) were combined and placed in the same vial.In the subsequent extraction steps (3-4), 0.6 g of EDTA was added to the soil along with 7.5 mL of acetonitrile (ACN), 7.5 mL of McIlvaine buffer (pH 8), 4.8 mL of Mg(NO 3 ) 2 •6H 2 O aqueous solution (concentration 0.5 g•mL -1 ), and 0.2 mL of 2.5% NH 3 aqueous solution in the centrifugation tube.The mixture was vortexed for 30 s, followed by PhAC extraction using an ultrasound bath for 10 min at 35°C.After sonication, the solution was centrifuged at 4,800 rpm for 8 min at 20 °C.Subsequently, the supernatant was transferred to a dark 30 mL glass vial.The extraction process was repeated with half the volume of the extraction medium (EM) without the addition of EDTA (3.75 mL of McIlvaine buffer, 3.75 mL of ACN, 2.4 mL of Mg(NO 3 ) 2 solution, and 0.1 mL of 2.5% NH 3 ) under the same extraction conditions.The extracts obtained from extraction rounds (3-4) were combined and placed in the same vial.During the pre-concentration step, both vials were

Table S2 .
Sampling location and physico-chemical properties of the soil

Table S3 .
3hysico-chemical properties of the biochar, data were obtained from study3

Table S4 .
MRM transitions of selected pharmaceuticals for LC-MS/MS analysis

Table S5 .
Comparison of Time-Weighted Average (TWA) Soil Concentrations with Different Routes of Pharmaceutical Contamination