Chromatographic methods for the determination of a broad spectrum of UV filters in swimming pool water

This paper describes an analytical approach based on solid-phase extraction (SPE) followed by analysis using liquid and gas chromatography coupled to mass spectrometry detectors for a determination of 18 organic UV filters from water samples. Extraction method parameters were optimized: 250 ml of water sample loaded on Chromabond C18 cartridges after adjustment to pH 4 and then eluted with acetonitrile. The mobile phase and the parameters of the mass spectrometer, as well as those of the ionization source, were tested to enhance detection sensitivity. During method validation, the extracted target compounds showed good recoveries (> 68%) with acceptable values in terms of repeatability (RSDr) and reproducibility (RSDR), where relative standard deviations values were lower than 20%. The validated method was applied to 10 water samples collected from different swimming pools located in Lebanon from which eight UV filters among the eighteen targets compounds were detected at concentrations ranged between 1 and 2526 µg L−1. The most detected compounds were padimate-O (OD-PABA) and octocrylene (OCR). This study represents the first available data on the occurrence of UV filter residues in Lebanese swimming pool opening hence future perspectives and insights to evaluate their degradation by-products and their toxicity on human health and marine ecosystem.


ILQ
Limit of quantification R 2 Coefficients of determination

Introduction
For the past decade, having a tanned complexion has been a popular esthetic concern. However, this attraction causes massive exposure to ultraviolet UV radiation which can affect negatively the skin and the immune system of the humans. In fact, every year, around one million persons are diagnosed with skin cancers worldwide (Suozzi et al. 2020). For this reason, the prevention of prolonged exposure to solar UV radiations might occur by applying sun protection products which comprise UV filters whose main role is to block or absorb UV light which are called UV filters. These products are utilized as sunscreens and in other cosmetic products they aim at protecting the skin from the noxious effects of sun radiation. In addition, they have been used to produce some plastics, industrial products, vehicle maintenance products, and pesticides (Eriksson et al. 2008).
The determination of UV filters in water system at trace levels requires the use of efficient extraction method that concentrates and enriches the extracts before their analysis by chromatographic techniques. Many analytical methods are previously published concerning the assessment of particular classes of UV filter compounds in different aqueous matrices (Liu et al. 2010;Khalikova et al. 2018;Chisvert et al. 2018). Different sample preparations such as liquid-liquid extraction (LLE) (Tarazona et al. 2013), solidphase extraction (SPE) (Díaz-Cruz et al. 2008;He et al. 2017;Nurerk et al. 2020), and solid-phase micro-extraction (SPME) (Negreira et al. 2009;Zhang and Lee 2012) were reported. LLE was the most frequently utilized method for aqueous samples. However, this method is a time-consuming and laborious technique that requires large volumes of organic solvents. Many studies have substituted LLE method with SPE, since it aims at diminishing significantly the utilization of organic solvents (Capriotti et al. 2014;da Silva et al. 2015;Ekowati et al. 2016;He et al. 2017). Different SPE cartridges, containing sorbent materials, have been used to extract UV filters from water such as hydrophilic-lipophilic balanced HLB (Ekowati et al. 2016;He et al. 2017), Strata X (Cuderman and Heath 2007;da Silva et al. 2015), and C18 for non-polar and polar compounds He et al. 2017). SPME was introduced as miniaturization technique of the conventional SPE which is based on equilibrium processes. Although it aims at reducing solvent consumption and manipulation time, SPME represents some drawbacks represented by the expensive cost of the fiber constituting the sorbent, and the prolonged time needed to achieve the equilibrium attributed to the small surface area between the donor and the acceptance phases (Jiménez-Díaz et al. 2014;Chisvert et al. 2018). More recently, stir bar sportive and nonporous membrane-assisted extractions, as micro extraction techniques, have been also used (Jiménez-Díaz et al. 2014). On the other hand, the analytical methods utilized to assess the UV filters in environmental matrices are mostly limited to chromatographic techniques, including gas chromatography (GC) (Cuderman and Heath 2007;da Silva et al. 2015;Nurerk et al. 2020), and LC-MS/MS (Meinerling and Daniels 2006;Zhang and Lee 2011;He et al. 2017;Kędziora-Koch and Wasiak 2018).
To date, no multiresidue analytical technique that covers mostly the detection of all UV filters in a single method has been developed yet. In fact, most of published studies are limited to 4 (Giokas et al. 2004;da Silva Meirelles et al. 2006), 5 (He et al. 2017, and 6 (Chisvert et al. 2001;Cuderman and Heath 2007;Vela-Soria et al. 2014;Locatelli et al. 2019). Only few researchers have extended their analysis to 14 (Ekowati et al. 2016) and 16 (Capriotti et al. 2014) UV filters.
Different legislations were implemented to define the UV filter compounds allowed to be present in sunscreen cosmetics whose aim is protecting the skin from solar harmful UV light and avoiding or minimizing the damage on human health caused by this radiation. The European Union, the USA, and Japan have nowadays approved different compounds at different levels for authorized substances (Khalikova et al. 2018).
Lebanon imports all its needed sunscreen products to different countries all over the world, i.e., the USA and Europe. This might increase in turn the probability to find a lot of sunscreen products containing different UV filters in the Lebanese market, allowing hence the possibility to detect a wide variety of UV filter substances within the Lebanese environmental samples. To date, no conducted studies are performed to assess the presence of UV filters in Lebanese water system. In this context, target compounds chosen in this study aim at covering the mostly active ingredients present in the market for further assessment.
To this end, the present study aims at developing a highly sensitive analytical method for the assessment of 18 UV filters in swimming pool water samples using the SPE technique and LC-MS/MS and GC/MS for the first time in Lebanon. The parameters affecting the extraction performance will be optimized, and the method will be validated before being applied to different swimming pool waters in Lebanon proving its suitability and performance.
Deionized water with a resistivity of 18.2 MΩ·cm −1 was obtained from Barnstead-Easy Pure II from Thermo Fisher Scientific (Hudson, USA). The cartridges for sample extractions were obtained from Waters Technologies (Millipore, USA) for Oasis HLB (200 mg, 6 mL) and from Machery-Nagel (Germany) for Chroma bond C18 (200 mg, 3 mL).
Stock standard solutions were prepared in MeOH except EMT, ET, and DBT in DMF and stored in the dark at − 15 • C . Working standard solutions of 1 mg L −1 were daily prepared with appropriate dilution in ACN and stored in the dark at − 4 • C.

Liquid chromatography/tandem mass spectrometry
An Agilent 1200 HPLC System (Agilent Technologies, USA) equipped with a reverse phase Zorbax Eclipse C8, with 3.5-µm particles (2.1 mm × 100 mm), was used for the separation of the selected analytes from aqueous samples. The HPLC system was interfaced to an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technologies) through an electrospray ionization source that was run in positive mode (ESI +). The parameters including the drying gas temperature (250, 300, and 350 °C) and the nebulizing gas pressure (5, 10, 15, 20, and 30 psi) were optimized. The cone voltage and collision energy (CIV) for each UV filter product were optimized to give the highest abundance. One milligram per liter of standard solution was injected directly into the mass spectrometer to optimize the CIV and the fragmentor for each analyte. Afterwards, a series of preliminary experiments were carried out to optimize the mobile phase, consisting of water and MeOH or water and ACN without or with formic acid at different concentrations (0.01%, 0.02%, 0.03%, 0.05%, and 0.1%) or ammonium acetate (5 mM).
The capillary voltage, temperature, nebulizer pressure, and gas flow rate that constitute the MS/MS parameters were set after optimization at 4000 V, 350 °C, 20 psi, and 9 L min −1 , respectively. The optimized mobile phase of the system, composed of pure water and ACN, was set at a flow rate of 0.3 mL/min and regulated according to a gradient elution consisted as follows: 0-0.5 min; 95:5 water/ACN, 0.5-10 min; 100% ACN and 15-20 min; 95:5 water/ACN. The necessary time for the analysis, the column temperature, and the injection volume of the extract were set at 20.5 min, 35 °C, and 5 μL, respectively.

Gas chromatography/mass spectrometry
A GC Agilent Technologies 6890 N series (Agilent, Waldbronn, Germany) coupled to an Agilent single quadrupole mass spectrometer 5973 N series (Santa Clara, CA, USA) was used for the analysis. The column used was an HP-5MS (Hewlett Packard, Palo Alto, CA, USA) capillary column (30 m × 0.25 mm i.d., 0.25-μm film thickness). The oven temperature program was monitored as follows: a starting temperature of 100 °C for 1 min, followed by a gradient increase of 25 °C/min to reach 290 °C. The total run time and the temperature of the injection port were 23.6 min and 250 °C. The volume of the samples to be injected into GC-MS following the spitless mode was 1 μL using the Helium as carrier gas flowed at 0.4 mL min −1 . The electron impact (EI) ionization mode with 70 eV was applied within the mass spectrometry detector (MSD) with a temperature of ion source set at 230 °C. Selective ion monitoring (SIM) mode was used to perform the analysis on MSD where one target and two or three qualifier ions were selected for each compound.
Sample pre-treatment and SPE procedure SPE extraction was performed with a Visiprep SPE manifold (Supelco, Bellefonte, PA, USA). For SPE optimization, different parameters in terms of cartridge type, sample pH, elution solvent, and breakthrough volume were studied. In detail, two different adsorbent materials Oasis HLB (200 mg) and Chromabond C18 (200 mg) were tested. The effect of Na 2 EDTA addition on extraction efficiency was also evaluated. Moreover, the extraction efficiency of target compounds was studied after adjusting the pH of water samples using formic acid at four pH: 2, 4, 7, and 9. Then, three solvents were tested (MeOH, ACN, and ACN/MeOH 60/40) in order to choose the performant one to be used for the elution step. Finally, different sample volumes (100 mL, 250 mL, 500 mL, and l000 mL) were investigated to assess the breakthrough volume. All these investigations were conducted using tap blank water samples previously scanned and resulted negative for the target compounds included in this study. Samples were doped at the same concentration (60 ng mL −1 ) for all analytes and then processed for extraction.
The optimized experimental protocol consisted of conditioning the cartridge of Chromabond C18 (200 mg) at atmospheric pressure using ACN (5 mL) followed by water adjusted at pH 4 with formic acid (5 mL). Afterwards, each water sample (250 mL) adjusted to pH 4 and supplemented with 10.375 ml of EDTA (250 mM) was doped with 15 µL from 1 mg L −1 of the surrogate standard (BP-d10 and BA-d5) to better control the quality of the extraction process and the daily performance of the method. The cartridge was then dried under vacuum for 1 h. The compounds of interest were eluted, under atmospheric pressure, using ACN (5 mL). Finally, nitrogen flow was used at 45 °C to let the eluent evaporate that was reconstituted, in a 300-μL insert, with ACN spiked with 15 μL of atrazine D5 (500 µg L −1 ), used as internal standard, for both LC-MS/MS and GC-MS analysis.

Validation of the method
Tap water samples, resulted free of UV filters, were used as blank samples to assess the applicability of the optimized SPE-LC-MS/MS and SPE-GC/MS methods, before being applied to real water samples to determine their contamination levels with UV filters. Table 2 shows the investigated parameters in terms of linearity, precision, instrumental limit of detection (ILD) and quantification (ILQ), and method detection limit (MDL).
The ILD and ILQ were determined by injecting UV filter standards prepared in solvent at different concentrations. The lowest concentrations of each UV filter that shows a signalto-noise ratio (S/N) for every target ion (m/z) equal to 3 are defined as ILD while ILQ is the lowest concentration of a compound that can be quantified in a sample with acceptable precision equivalenting to a S/N of 10.
Blank water samples were fortified at 6 concentration levels (5, 10, 20, 40, 80, and 100 ng mL −1 ) from which the linearity (Table 2) was assessed using the matrix-matched calibration curve. Four water samples were spiked with each analyte (30 ng) and analyzed in parallel to determine the repeatability of the method. The repeatability, known as intra-day trueness, was calculated within the same experiment at the same day, using the repeatability relative standard deviation (RSD r ), by comparing the mean measured concentration with the fortified concentration of the samples. The assessment of inter-day trueness, also named reproducibility, determined by measuring the relative standard deviation (RSD R ), was performed by analyzing the spiked blank tap water samples at different days. Fifteen microliters of Atrazine D5 (30 ng) were added to each sample, after each extraction, as internal standard. The MDL of each compound, defined as signal-to-noise ratio set at 3, was obtained after fortifying, extracting, and analyzing blank tap water samples at low concentration to assess method performance.
To ensure the reliability of the results, procedures of quality assurance/quality control were applied. To this end, the real water samples, collected from swimming pool, were spiked with deuterated standards, as surrogate standard, before extraction while no spiked blank water samples were considered blank samples to exclude any cross-contamination during the process. To monitor the instrumental and the potential cross-contamination during LC-MS/MS detection, instrumental blank and a calibration solution were analyzed before and after each sample sequence. Twenty blank water samples were analyzed to check method selectivity and to verify the presence of potential interfering substances around the retention times of the compounds of interest.

Sample collection and pre-treatment
Ten water samples were collected in amber glass bottles, previously washed with acetone, MeOH, and Milli-Q water, from swimming pools situated in Beqaa (Lebanon) in 2019 during swimming period. Samples of 1 L each were placed in the glass bottle to which 100 mg of Na 2 S 2 O 3 was immediately added for neutralization of the chlorine and other chlorinating agents present in the samples, and for avoiding any reaction that might take place with the target compounds. Once in the laboratory, the water samples were directly filtered through cellulose filters (pore size 0.45 μm) and conserved at 4 • C until analysis within the 48 h.
After sample processing and analysis, the quantification of UV filter compounds in real samples was achieved by comparing the analyte/internal standard peak area ratio from matrix-matched calibration curve to the analyte/ internal standard peak area ratio in the analyzed samples. The matrix-matched calibration curve was prepared in blank tap water sample fortified at six concentration levels as described before.

Statistical analysis
All experiments concerning SPE and LC-MS/MS and GC-MS were conducted at least in triplicate. The obtained results were expressed as mean ± RSD and analyzed using GraphPad 9 (GraphPad Software, San Diego, CA, USA).

LC-MS/MS analysis
Among the 18 compounds, seven (PABA, BP-4, BDM, PDSA, EMT, DBT, and ET) were analyzed by LC-ESI-MS/MS since the analytes showed efficient ionization in ESI + (Supplementary Material S1). Many publications used LC-MS/MS as technique of choice for the detection of UV filters (Capriotti et al. 2014;Ekowati et al. 2016;He et al. 2017). For method development, all parameters were optimized by directly injecting each standard at a level of 1 mg L −1 , prepared in ACN, to identify the ionization mode for detection of all the analytes. Afterwards, the precursor and the product ions for each compound were identified by optimizing the collision energy and the cone voltage (Table 1). Consequently, the conditions of LC were also optimized. An Agilent Zorbax Eclipse XDB-C8 column was utilized with particle size of 3.5 µm and a length of 100 mm. After mobile phase optimization, gradient elution with pure water and ACN has been found to be the most efficient mobile phase for compounds analyzed in positive mode. This result can be related to the lower viscosity of the mixture of water/ ACN compared to water/MeOH, which reduce the pressure problems mostly frequent in LC. Additionally, the intensities were better without additives (formic acid and ammonium acetate) for all compounds. In contrast, He et al. found that high extraction recoveries of 4 UV filters (4-MBC, BP-3, EHMC, and OCR) were obtained when NH 4 OH in MeOH was used as mobile phase (He et al. 2017). Although He et al. considered the obtained results unexpected since abundant [M + H] + ions were generated during ionization in positive mode due to the phenomena named "wrong-way-wrong" ionization (He et al. 2017), the limited number of tested UV filters compared to the 18 compounds selected in this study may greatly influence the method performance. The parameters of the drying gas temperature and the nebulizing gas pressure were adjusted, and the optimized intensities were established at 350 °C and 20 psi respectively. Multiple reaction monitoring was utilized, and Table 1 shows the specific MS/MS parameters for the targeted compounds and their retention times.

GC-MS analysis
The remaining 11 compounds (BP-3, BP-8, IMC, CIN, OD-PABA, OCR, ES, ET, HMS, EHMC, and DHB) were analyzed using GC-MS (Supplementary Material S2). Data acquisition was done using the SIM and the full-scan modes (Supplementary Data). Full scan mode was selected with a wide range of m/z. Two monitored ions were chosen for each target compound, the first for confirmation while the second for quantitation, based on the scanning chromatograms of UV filter standards (Table 1).

Optimization of the extraction procedure
The SPE was chosen for extraction among the other extraction techniques. This technique is considered the key step in sample pre-concentration due to its best extraction recovery and low solvent consumption rendering it the most utilized to extract pharmaceutical compounds from aquatic samples (da Silva Meirelles et al. 2006;Cuderman and Heath 2007;da Silva et al. 2015;Ekowati et al. 2016). The filtration step did not influence the SPE extraction. Subsequently, the extraction process was assessed in terms of nature of sorbent, pH of sample, elution solvent, and breakthrough volume.

Choice of cartridge type
The extraction efficacy of the SPE technique is highly affected by the type of the adsorbing material. Oasis HLB cartridge constitutes two combined monomers, lipophilic divinylbenzene, and hydrophilic N-vinylpyrrolidone, permitting the retention of polar and non-polar compounds (Khalikova et al. 2018). Moreover, this cartridge type has shown also a good performance in extracting acidic, neutral, and basic compounds (Khalikova et al. 2018). Chromabond C18 is made by silica material, permitting the retention of polar and non-polar compounds (Khalikova et al. 2018). In this study, the assessment of the two sorbent types on the extraction efficiency of UV filters was performed under the following conditions: sample volume (200 mL), addition of EDTA (5 mL at 250 mM), pH (4), and elution solvent (ACN). The obtained results exhibited good extraction recoveries for all compounds with acceptable range (60-120%) using Chromabond C18 compared to Oasis HLB (Fig. 1a).
Moreover, once the cartridge type C18 was selected, the effect of EDTA salt addition, which acts as metal chelates, in the water sample on improving the extraction recoveries of UV filters was also studied. Figure 1b shows that the extraction recoveries of most of the UV filter compounds were enhanced when EDTA salt was added to the extracted samples except for PABA and BP-4. This result can be explained by the disruption of bonds between the studied target compounds and the metals presented in the water and by the decrease of the pH of the water samples allowing better retention of the compounds of interest on the cartridges.

Effect of pH
The adjustment of the pH of the water samples before being loaded into the cartridges consists of another critical point of SPE procedure to be optimized. The blank tap water samples were adjusted with formic acid to different pH and extracted through C18 cartridges. In Fig. 2a, it can be noticed that extraction efficiency is highly affected by the adjustment of the pH of the sample. The lowest recoveries can be detected when extraction was performed at pH 7 followed by pH 9. The obtained results showed that the adjusted water samples with pH 4 revealed the best average extraction recoveries (> 77%) for all the target compounds compared to the other tested pH (Fig. 2bb). These latter can be explained by the fact that when increasing the pH to 7 and 9, the target compounds such as BP-3 and BP-8 acquired an ionization charge, which impair their interaction with the solid phase and lead to a drastic decrease in extraction recoveries. Instead, at acidic pH values (2 and 4), the ionic charge of the target compounds reduced, favoring the retention of UV filters on the SPE C18 cartridge allowing hence the obtention  Fig. 2 a Extraction efficiency at different pH (2, 4, 7, and 9) for each target UV filter. b Overall extraction efficiency of all UV filters at each pH (2, 4, 7, and 9) of better extraction recoveries. These results are in accordance with previously published works (Giokas et al. 2004).

Optimization of the elution solvent
Furthermore, to eluate the target compounds from the C18 cartridges, it is necessary to use a solvent whose interaction with compounds is higher than the adsorbents allowing then their desorption. It can be noticed from Fig. 3 that the average yields of the extracted compounds obtained in the case of MeOH and ACN/MeOH (60/40) are around 51 and 41%, respectively, while those obtained when pure ACN is used increase up to 76% (Fig. 3). In the procedure of extraction, the pure ACN was adopted due to the fact of ensuring the highest recoveries of all compounds together with its short evaporation time in the final extraction step compared to the other eluents.

Optimization of breakthrough volume
Finally, a last experiment was carried out to assess the breakthrough volumes. It corresponds to the maximum volume, which can be percolated on the SPE cartridge without modifying the extraction yields. As shown in Fig. 4, the recoveries tended to decrease with a sample volume higher than 250 mL. For this reason, 250 mL has been selected as a sample volume to perform the extractions, thus a maximum enrichment. Similar results were obtained in a previously conducted study, which proved that breakthrough happened when increasing the sample loading volume (> 500 mL) (Giokas et al. 2004).

Evaluation of the method performance
The developed method for the extraction of 18 UV filters in water samples was assessed for its analytical performance by evaluating the quality parameters: the linearity, precision, ILD, and ILQ (Table 2). According to the optimum conditions, the method displayed good linearity with coefficients of determination (R 2 ) higher than 0.9 for all the UV filters studied in the 0.13-400 μg L −1 range, and repeatability, considered percentage relative standard deviations (RSD r < 20%). For GC and LC performance, the calculated ILD was between 0.03 and 20 ng L −1 while calculated ILQ was between 0.08 and 60 ng L −1 .   The MDL was assessed by spiking water samples with low concentrations of UV filters. Calculated MDL was between 2.5 and 50 ng L −1 .

Application to water samples from swimming pool
To assure the suitability of the validated method, 10 recreational water samples, collected from swimming pools located in the region of Bekaa in Lebanon, were worked on and analyzed (Al Rihab, Bekaa Joy, Sunny Land, Al Tilal, Park Hotel, Kadery, Water Park, Serenity, Shams Palace, and Mountajaa Al Sharek). Eight out of the 18 target UV filters were detected in the analyzed samples (BP-3, BP-8, OCR, OD-PABA, EHMC, DBT, EMT, and IMC) and the found concentrations were reported in the Supplementary data Table S1. As shown in Fig. 5, OD-PABA is found in 80% of swimming pools at concentrations ranged between 1.05 and 518 µg L −1 . Other UV filters, i.e., BP-3, BP-8, IMC, and EMT, were detected at concentrations between 20 and 476 µg L −1 . The last category including OCR, EHMC, and DBT were detected at high concentration between 11 and 2526 µg L −1 . OCR has been reported to be found in 70% of the studied swimming pools water. The difference among the concentrations of UV filters detected in the swimming pool water samples varies according to the persistence of each UV filter in the light and its release in water.
In this study, the concentrations of detected UV filters, BP-3, BP-8, EHMC, and OD-PABA were found at higher concentration compared to those found in swimming pool in Spain (Ekowati et al. 2016), water treatment plants in São Paulo State in Brazil (da Silva et al. 2015), and in river water in Slovenia (Cuderman and Heath 2007). PB-8 represents a human metabolite derived from the PB-3 suggesting that swimmers continue urinating in the pools. The presence of UV filters at high concentrations in the pool swimming water constitutes an alert to establish new measures allowing the control of quality of water and implying the necessity in performing further investigations.
Despite the limited number of real swimming pool samples analyzed in this study, the obtained results represent the first available data in Lebanon concerning the contamination of swimming pool with UV filters. The developed method will be extended to be applied on more environmental samples to cover not only Lebanese swimming pools, but also surface (river, lake, wastewater treatment plants) and ground (well, fountains) water after assessing their matrix effect.

Conclusion
A sensitive analytical method based on SPE followed by LC/MS/MS and GC/MS has been developed for the determination of eighteen UV filters in water matrix. The parameters affecting pre-concentration step, the mobile phase, the parameters of the MS for LC and GC, and those of the ionization source have been all assessed and optimized. Moreover, the parameters of the SPE have also been evaluated. The optimized method demonstrated to be sensitive enough in detecting trace levels of UV filters allowing its application for environmental assessment studies. The best recoveries for all compounds were found when Chromabond C18 cartridges were used for extraction with ACN as elution solvent. The method was satisfactorily validated in terms of linearity, precision, repeatability, and reproducibility. Its detection limits are between 2.5 and 50 ng L −1 with recoveries higher than 69% and RSD for repeatability and reproducibility values below 20%. The validated method was applied to 10 samples collected from different swimming pools located in Bekaa region in Lebanon. Nine out of the 18 target UV filters were found in the analyzed samples. OD-PABA was detected in 80% of swimming pools at low concentrations ranged between 1 and 518 µg L −1 . OCR, EHMC, and DBT were detected in high concentration between 11 and 2526 µg L −1 . Our results reported here the first data for the occurrence of UV filters in swimming pools in Lebanon. Future studies are required to assess the presence of different UV filter compounds in swimming pools located in different regions in Lebanon. This study will be the building block for both long-and short-time applications. Shortly, it will contribute to the design of future monitoring programs of water quality assessment for water systems such as drinking water and wastewater. On the other hand, measuring the concentrations of these chemicals in water will help to guide future studies to deal with their removal using water treatment processes and to evaluate their eco-and cytotoxicity.