Multi-residue analysis of 36 priority and emerging pollutants in marine echinoderms (Holothuria tubulosa) and marine sediments by solid-liquid extraction followed by dispersive solid phase extraction and liquid chromatography–tandem mass spectrometry analysis

1 Multi-residue Analysis of 36 Priority and Emerging Pollutants in Marine Echinoderms (Holothuria tubulosa) and Marine Sediments by Solid-Liquid Extraction Followed by Dispersive Solid Phase Extraction and Liquid Chromatography–Tandem Mass Spectrometry Analysis J. Martín(1), A. Zafra-Gómez(2)*, F. Hidalgo(3), A.J. Ibáñez-Yuste(4), E. Alonso(1), J.L. Vilchez(2) (1) Department of Analytical Chemistry, Superior Polytechnic School, University of Seville, C/ Virgen de África 7, E-41011 Seville, Spain (2) Research Group of Analytical Chemistry and Life Sciences, Department of Analytical Chemistry, University of Granada, Campus of Fuentenueva, E-18071 Granada, Spain (3) Department of Zoology, University of Granada, Campus of Fuentenueva, E-18071 Granada, Spain (4) Agency of "Gestión Agraria y Pesquera" of Andalusia. Administrative Central Services. Av. of Greece s/n. 41012, Seville, Spain ABSTRACT Marine echinoderms are filter-feeding invertebrates widely distributed along the coasts, and which are therefore extensively exposed to anthropogenic xenobiotics. They can serve as good sentinels for monitoring a large variety of contaminants in marine ecosystems. In this context, a multi-residue analytical method has been validated and applied to Holothuria tubulosa specimens and marine sediments for the determination of 36 organic compounds, which belong to some of the most problematic groups of emerging and priority pollutants (perfluoroalkyl compounds, estrogens, parabens, benzophenones, plasticizers, surfactants, brominated flame retardants and alkylphenols). Lyophilization of samples prior to solvent extraction and clean-up of extracts with C18, followed by liquid chromatography– tandem mass spectrometry analysis, is proposed. A Box–Behnken design was used for optimization of the most influential variables affecting the extraction and clean-up steps. For validation, matrix-matched calibration and recovery assay were applied. Linearity (% r2) higher than 99 %, recoveries between 80 and 114 % (except in LAS and NP1EO), RSD (precision) lower than 15 % and limits of quantification between 0.03 and 12.5 ng g-1 dry weight (d.w.) were achieved. The method was applied to nine samples of Holothuria collected along the coast of Granada (Spain), and to marine sediments around the animals. The results demonstrated high bioaccumulation of certain pollutants. A total of 25 out of the 36 studied compounds were quantified, being surfactants, alkylphenols, perfluoroalkyl compounds, triclocarban and parabens the most frequently detected. Nonylphenol was found in the highest concentration (340 and 323 ng g-1 d.w. in sediment and Holothuria samples, respectively). Keywords: Anthropogenic pollution; Priority and emerging pollutants; Holothuria tubulosa; Sediment; Sample preparation; LC–MS/MS _________________ * Corresponding author: E–mail address: [email protected] (A. Zafra-Gómez) 2 1. Introduction A large variety of anthropogenic pollutants tend to accumulate in marine ecosystems. In the last decades, many previously unrecognized pollutants, usually called emerging contaminants, have been identified [1-2]. Many of these substances are especially relevant, in particular those with the capacity to disrupt the hormonal (endocrine) system of living organisms [4-9]. Although the consequences of this pollution are becoming increasingly apparent, their actual impact on ecosystems is poorly known, particularly concerning the risk of bioaccumulation in different trophic levels [1, 10-13]. In this respect, some marine organisms are used as target samples because they are extensively exposed to xenobiotics [1,14-17]. Recently, Huerta et al. [14] found diclofenac, ibuprofen, estrone (E1), bisphenol A (BPA), and nonylphenol (NP) in concentrations up to 183 ng g-1 dry weight (d.w.) in macroinvertebrates from the Segre River in the Pyrenees (Iberian Peninsula). Ruhí et al. [1] detected pharmaceutical compounds and endocrine disruptors in water, biofilm, and three aquatic macroinvertebrate of different taxa. The flame retardant tributoxyethyl phosphate was found in all taxa and showed magnification across trophic levels. Gago-Ferrero et al. [18, 19] found UV filters (octocrylene) in marine mammals in concentrations from 89 to 782 ng g-1 lipid, in mussels and fish in concentrations up to 7112 and 3100 ng g-1 lipid, respectively, along the Brazilian coastal area. Sea cucumbers, echinoderms from the class Holothuroidea are common filter-feeding organisms widely distributed along the coasts. According to some authors, holothurians can serve as good sentinels for monitoring organic micropollutants because they may take up these pollutants through the gills (dissolved contaminants in water) and/or digestive tract [20- 23]. The impact of environmental contaminants on holothurians is important to assess, since these organisms are functionally important in transferring environmental contaminants to higher trophic levels as they are a major source of food for fish, waterfowl, and humans. Sea cucumbers have been well recognized as a tonic and traditional remedy in Asian countries. In recent years, the increasing demand has prompted sea cucumber aquaculture in some countries [23]. Holothuria organisms have been used extensively as sentinel to assess ecotoxicologycal risk of contaminated sediments and water by heavy metals [20, 24-27] and organic compounds such as biocides [28], polychlorinated biphenyls (PCBs) [29-31], polybrominated diphenylethers [29] and some endocrine disruptors (alkylphenols) [32]. Sugni et al. [21] carried out a complete and excellent review on the presence of endocrine disruptors in marine echinoderms. The work focused on PCBs, alkylphenols, hormones, and pesticides, among others. Despite this, an important lack of information about emerging pollutants has 3 been identified in the literature. There has been growing interest regarding the effects of endocrine disruptors in vertebrate organisms, but the effects in invertebrate organisms have been little studied [33] and few papers have been published concerning the analytical methods developed for the determination of contaminants in echinoderms. To date, published methods are limited to chemical families of pollutants and most of them were performed on aquatic vertebrates, mainly fish [34, 35], and macro-invertebrates, mainly mollusks [36, 37]. Extraction and purification are key steps for reducing matrix effects and optimizing extraction. Ultrasound assisted extraction (UAE) and pressurized liquid extraction (PLE) have been the most common techniques used to extract organic micropollutants from marine organisms [14, 19, 22, 38, 39]. Although these techniques are now automatized, they are time-consuming and require large volumes of solvents. Currently, sample preparation is moving towards a more environmentally friendly, low cost, miniaturized, automated and simpler approach [40]. In recent publications, modified dispersive extraction QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedures have been developed [37]. The study by Berlioz- Babier et al. [33] is one of the few that reports on endocrine disruptors in benthic invertebrates. The study is based on a modified QuEChERS procedure that involves salting out liquid–liquid extraction followed by nano-liquid chromatography–nano electospray ionization coupled with tandem mass spectrometry (nanoLC-MS/MS). For purification of extracts, the most often applied technique is SPE (solid-phase extraction), but MSPD (matrix solid-phase dispersion) has been successfully applied to determine PCBs, pesticides and personal care products [36] or perfluoroalkyl compounds [41] in mollusks. The aim of this study was to develop an innovative, rapid, robust and sensitive analytical method for trace analysis of priority and emerging pollutants in Holothuria tubulosa specimens and marine sediments. A total of 36 target compounds were chosen because of their environmental occurrence and persistence, ecotoxicological risks, inclusion in different prioritization lists, and physicochemical properties. 2. Experimental 2.1. Chemicals and reagents All reagents were analytical grade unless otherwise specified. The estrogens estrone (E1), 17β-estradiol (E2), estriol (E3), and 17α-ethinylestradiol (EE2) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Linear alkylbenzene sulfonate (LAS) mixture 4 containing C10 (12.3 %), C11 (32.1 %), C12 (30.8 %) and C13 (23.4 %) and alcohol sulfates (AS) were supplied by Cepsa Química S.A. (Madrid, Spain). 4-nonylphenol (4-NP) technical grade mixture of isomers, nonylphenol monoethoxylate (NP1EO) and nonylphenol diethoxylate (NP2EO) Igepal® CO-210 technical mixture were obtained from Sigma Aldrich (Milwaukee, WI, USA). The industrial micropollutants perfluorobutanoic acid (PFBuA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), phenylparaben (PhP), benzophenones 1, 2, 3, 6, 8 and 4-hydroxy (BP1, BP2, BP3, BP6, BP8, 4-OHBP), triclosan (TCS), triclocarban (TCB), bisphenol A (BPA) and hexabromocyclododecane (HBCDD) were purchased from Sigma-Aldrich (Steinheim, Germany). Bis(2-ethylhexyl)phthalate (DEHP) was obtained from Riedel-de Haën (Seelze, Germany). The internal standards (IS) ethylparaben-d5 (EtP-d5), benzophenone-d10 (BP-d10), perfluorooctanoic acid-13C4 (PFOA-13C4) and bisphenol A-d16 (BPA-d16), were supplied by Cambridge Isotope Laboratories (MA, USA). Individual stock standard solutions of each compound were prepared in methanol and stored at 4ºC. Mixtures of target compounds at different concentration levels were freshly prepared immediately before use by diluting the stock standard solutions with methanol. Water, Methanol (MeOH), acetonitrile (AcN), ethyl acetate (AcOEt), and acetone (all chromatographic analysis grade) were purchased from Romil Ltd. (Barcelona, Spain). Alumina, fluorisil, primary-secondary amine bonded silica (PSA), silica and octadecyl functionalized silica (C18) were provided by Sigma-Aldrich (Steinheim, Germany). Acetic acid and ammonium acetate were obtained from Panreac (Barcelona, Spain). 2.2. Instrumentation and software Chromatographic analyses were performed on an Agilent 1200 series HPLC (Agilent, USA) equipped with a vacuum degasser, a binary pump, an autosampler, and a thermostated column compartment. Two types of chromatographic columns were tested, an HALO C-18 Rapid Resolution (50 × 4.6 mm i.d., 2.7 µm particle size) from Teknokroma (Barcelona, Spain) and a Zorbax Eclipse XDB–C18 Rapid Resolution HT (50 × 4.6 mm i.d.; 1.8 µm) from Agilent (Barcelona, Spain) . The HPLC system was coupled to a 6410 triple quadrupole (QqQ) mass spectrometer (MS) equipped with an electrospray ionization source (ESI) (Agilent). Ionization of analytes was carried out using the following settings: MS capillary voltage 3000 V, drying-gas flow rate 9 L min-1, drying-gas temperature 350 ºC and nebulizer pressure 0.28 MPa. A vortex-mixer (IKA, Staufen, Germany), an ultrasound-HD bath (Selecta, 5 Barcelona, Spain), a Spectrafuge™ 24D centrifuge from Labnet International, Inc. (Edison, NJ, USA) and a sample concentrator (Stuart, Staffordshire, UK) were also used. Statgraphics Plus software version 5.1 (Statpoint Technologies Inc., Warrenton, VA, USA) was used for statistical treatment of data. 2.3. Sample collection Specimens of Holothuria tubulosa (n=9) and samples of associated marine sediments were hand collected by scuba divers at random locations in March 2016 (mode of underwater diving in which the scuba diver uses a self-container underwater breathing apparatus (scuba) to breathe underwater, completely independent of surface supply), along a transect (50 m long and 7-13 m deep ) in the infralittoral zone of Marina del Este beach (Almuñecar, Southern Spain; coordinates: 36.720528, -3.728383). Specimens were similar in size (average 100 g). Holothuria specimens and sediment samples were frozen at -20 ºC until dissection and analyses. Prior to the dissection with scalpel, the holothurians were washed with distilled water to remove foreign matter and dirt. For the analyses, the digestive and respiratory tracts and gonads of the holothurians were used but the body wall was discarded. These samples were kept in sterile polythene bags and stored in the laboratory deep freezer (-20 ºC) to prevent deterioration until further analysis. Holothurian viscera and sediments around the sampling area were then freeze-dried, homogenized and grounded into powder. Each sample was transferred to a clean and opaque glass bottle and stored in the dark until extraction and analysis. 2.4. Basic procedure 2.4.1. Preparation of matrix-matched calibration curves Matrix-matched calibration standards were prepared at eight different analyte concentration levels. Taking into account the substance specific responses and the concentration levels present in natural samples, different concentration ranges were used (from 0.03 to 25 ng g-1 d.w. for most of analytes, except for alkylphenols ethoxylates, HBCDD and DEHP where a range from 1.25 to 250 ng g-1 d.w. and for estrogens and NP where a range from 12.5 to 500 ng g-1 d.w. were used). The mixtures were vortexed for 2 min and then left to stand for 24 h at 4 ºC in the dark before analysis. This allows the analytes to 6 come into full contact with the sample. Ideally, a matrix-matched standard should not contain any of the selected compounds, but in practice this is not always possible. Working with matrix-matched standard in environmental samples could lead to representativeness problems, therefore a pool of different holothurians were used. The areas of the compounds which are present in the blank extract have to be subtracted to the area obtained from the matrixmatched standards to construct the calibration curves and to calculate the limits of quantification (LOQs). These compounds (mainly LAS and NP) were found at low concentration levels in blank matrices. Therefore, the uncertainty associated with blank subtraction, assuming a uniform S/N ratio, is probably lower than the uncertainty associated with the entire method including composition of matrix extracts. 2.4.2. Sample treatment Aliquots of the samples (0.5 g) were weighed into 12 mL glass vials, containing 100 µL of a methanol solution (250 ng mL-1) of the four surrogate standards selected: EtP-d5, BP-d10, PFOA13C4 and BPA-d16 (final concentration 50 ng g-1 d.w. in sample). The samples were vortexed for homogenization twice in 7 mL of acetonitrile (2 min) and centrifuged for 10 min at 4050 × g. For clean up, disperse solid phase extraction (d-SPE) was applied. The supernatants obtained from the two extractions of each sample were combined in a 50 mL polypropylene conical tube containing 800 mg of C18 sorbent. The mixture was hand-shaken for 2 min and centrifuged for 5 min at 4050 × g. The solvent was then evaporated to dryness at room temperature under a nitrogen stream and the extract was reconstituted in 0.25 mL of methanol:water (50:50, v/v) and filtered through a 0.22 µm nylon filter. A 20 µL aliquot of the extract was injected into the HPLC instrument. 2.4.3. High performance liquid chromatography–tandem mass spectrometry operating conditions Chromatographic separation of compounds was performed using a HALO C-18 Rapid Resolution (50 × 4.6 mm i.d., 2.7 µm particle size) column. The compounds were separated using a gradient mobile phase consisting of an aqueous buffer solution of acetic acid/ammonium acetate (pH 4.4) (solvent A) and methanol buffered with the same composition (solvent B). The gradient program was as follows: 0-14 min, linear gradient from 7 28 to 70 % of solvent B, from 70 % to 80 % of solvent B in 5 min, and then increased to 100 % in 6 min and held for 2 min. Moreover, two MRM transitions were selected for each analyte for quantification and confirmation of compounds. The selected transitions and the intensity ratio (Quantification/Confirmation ions) for each analyte are shown in Table 1. The mass spectrometer was operated in both negative and positive ESI modes. Optimized fragmentor and collision energy for each MRM transition are also shown in Table 1. Flow rate was fixed at 0.6 mL min-1. The injection volume was 20 µL. The column temperature was maintained at 30 ºC. Table 1 2.5. Box-Behnken experimental design The volume of extraction solvent, the amount of C18 and the number of extraction steps were optimized by using a Box–Behnken experimental design. The objective of a design of experiment approach is to provide enough tests to fit the second-order equations correlating the response function with independent parameters. To this purpose a Box-Behnken response surface design followed by mapping the mathematical models corresponding to each response were used. By combining a fractional factorial with incomplete block designs, each factor requires only three levels instead of the five required for central composite designs, which results in fewer design points. In addition, Box-Behnken designs do not have axial points, thus all design points fall within the safe operating zone. Although this avoids factor-level combinations that are prohibitively expensive or technically impossible to perform, Box- Behnken designs are not suitable when one wishes to know the responses at the extremes, i.e., where all the factors are at their highest or lowest levels. Moreover, they are less economical than Doehlert designs, especially when the number of factors increases. Nevertheless, Box- Behnken designs are considered as efficient options in response surface methodology and ideal alternatives to central composite design [42]. The importance of each term in the mathematical models was assessed using ANOVA. The magnitudes of the coefficients in the regression equations were utilized as the basis for judging statistical significance and illustrating the relative effects of linear, quadratic and cross product interactions between the parameters. Pareto charts were also obtained to analyze statistically significant effects of the variables were screened using a Student's t-test for 8 ANOVA. Variables having a confidence greater than 95.0 % were considered to have a significant effect on the extraction efficiency. 2.6. Validation requirements The method was validated according to the International Conference on Harmonization (ICH) guidelines for analytical method validation (ICH Quality Guidelines) [43]. A matrixmatched calibration curve was recorded for each of the target compound at eight concentration level. Linearity of the calibration graphs was tested using the determination coefficients (r2). Before the assessment of matrix effect, two calibration curves were obtained for each compound: one in solvent (initial mobile phase) and the other one in the matrix. A Student’s t test was applied to compare the slopes of calibration curves. The t calculated was compared with the two-tailed tabulated value, ttab for the appropriate number of degrees of freedom at P % confidence. Typical values are k = 2 for 95 % confidence [44], so: If t < k, the ratio of the slopes is not significantly different from 1; and, if t > k, the ratio of the slopes is significantly different from 1 and the matrix-matched calibration must be used. Limit of detection (LOD) and limit of quantification (LOQ) are fundamental aspects that need to be examined in the validation of the analytical method in order to determine whether the analyte is present in the sample. The LOD is the minimum amount of analyte detectable in the sample while the LOQ is the minimum amount that can be quantified. In this work, these parameters were calculated from the signal-to-noise ratio (LODs = 3, LOQ = 10) obtained from injecting several blank samples fortified with decreasing amounts of the compounds of interest. A recovery assay was carried out to validate the accuracy of the method in terms of trueness and precision. Fortified blank samples for each compound at three concentrations levels were analyzed. Precision, expressed as relative standard deviation (%, RSD) was determined from triplicate spiked samples during the same day and in six different days, and the trueness was evaluated using the recovery data. The selectivity of the method was determined by comparing the chromatograms of blank with the corresponding spiked sample. Finally, a quality assurance and quality control was also developed. Procedural blanks were injected to monitor for background contamination. These blanks were processed in the same way as the samples and injected into the HPLC-MS/MS system. Target compounds were not determined above LOQs. On the other hand, in order to evaluate possible 9 contaminations and the variability of the instrumental analysis, standards (spiked samples at 0, 50 and 100 ng g-1 d.w.) and a standard in the initial mobile phase (100 ng mL-1) were injected by triplicate every 20 samples. 3. Results and discussion 3.1. Liquid chromatographic-mass spectrometric analysis To obtain the highest sensitivity and selectivity for the chromatographic separation in a shorter run time, the main variables affecting chromatographic separation and signal intensity were studied. A standard mixture of the selected analytes (1 µg mL-1 for each compound) was used. First, Zorbax Eclipse XDB–C18 Rapid Resolution HT (50 × 4.6 mm i.d.; 1.8 µm) and a HALO C-18 Rapid Resolution (50 × 4.6 mm i.d., 2.7 µm particle size) column were tested in order to obtain a high separation performance. The HALO C-18 Rapid Resolution column provided better peak shape in the shortest time with similar resolution for all the studied analytes. Consequently, this column was the one we selected for further experiments. The effect of the mobile phase on chromatographic separation was also studied. MeOH and AcN were evaluated as organic modifiers (solvent B). As solvent A, deionized water with different additives was studied. The use of formic acid, acetic acid, ammonium formate, ammonium acetate and mixtures of these acids and the corresponding salts were assayed as additives in the mobile phase. Since most of the analytes showed higher responses with better peak shapes, a gradient elution with an aqueous buffer solution containing acetic acid/ammonium acetate (pH 4.4) (A) and buffered methanol (B) was selected as mobile phase. 3.2. Optimization of the extraction procedure The extraction step (solvent, time, number of extraction steps and technique [vortex vs UAE] and the clean-up step [type and amount of sorbent]) were optimized using aliquots of 0.5 g of sample spiked with the selected compounds at a concentration level of 125 ng g-1 d.w. Each experiment was carried out in triplicate. 3.2.1. Selection of the extraction solvent Several solvents covering a wide range of polarities were tested: MeOH, AcN, AcOEt, 16 Acknowledgements This study was supported by the Spanish Ministry of Education, Culture and Sports (Project CTQ2011-24210/PPQ) and the Spanish Ministry of Economy and Competitiveness (Project No. CGL2013-44402-R). References [1] A. Ruhí, V. Acuña, D. Barceló, B. 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González, M.A. Herrador, A practical guide to analytical method validation, including measurement uncertainty and accuracy profiles, Trends Anal. Chem. 26 (2007) 227–238. [45] B. Oliver-Rodríguez, A. Zafra-Gómez, F.J. Camino-Sánchez, J.E. Conde-González, J.P. Pérez-Trujillo, J.L. Vílchez, Multi-residue method for the analysis of commonly used commercial surfactants, homologues and ethoxymers, in marine sediments by liquid chromatography-electrospray mass spectrometry, Microchem. J. 110 (2013) 158–168. [46] M.M. González, J. Martín, D. Camacho-Muñoz, J.L. Santos, I. Aparicio, E. Alonso, Degradation and environmental risk of surfactants after the application of compost sludge to the soil, Waste Manag. 32 (2012) 1324–1331. [47] I. Aparicio, J.L. Santos, E. Alonso, Limitation of the concentration of organic pollutants in sewage sludge for agricultural purposes: A case study in South Spain, Waste Manag. 29 (2009) 1747–1753. 21 Figure captions Fig. 1. Optimization of solvent (A) and clean-up sorbent (B) on the extraction procedure (n = 3). Fig. 2. Response surface plots, corresponding to the desirability function, grouped by chemical classes of pollutants, when optimizing the following pair of factors from the extraction procedure: (A) AcN volume vs amount of C18; (B) AcN volume vs extraction steps. Holothuria samples were spiked with 125 ng g-1 of each pollutant. Fig. 3. Global response surface plots, corresponding to the desirability function, when optimizing the following pair of factors from the extraction procedure: (A) AcN volume vs amount of C18; (B) AcN volume vs extraction steps; (C) extraction steps vs amount of C18. Results were evaluated using a 95 % confidence interval. Fig. 4. HPLC–MS/MS chromatograms of a contaminated Holothuria sample (number 2). Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.81 C18 amount 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.63 Extraction steps 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.63 Extraction steps 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.81 C18 amount 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Desirability Desirability DesirabilityDesirability Estimated Surface Response Extraction steps= 3 Estimated Surface Response C18 amount= 0.55 g Estimated Surface Response Extraction steps= 3 Estimated Surface Response C18 amount= 0.55 g Desirability Desirability Desirability Desirability Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.63 Extraction steps 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.81 C18 amount 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Desirability Desirability Estimated Surface Response Extraction steps= 3 Estimated Surface Response C18 amount= 0.55 g Desirability Desirability A B Perfluoroalkyl compounds Preservatives LAS PFBuA MeP PFPeA EtP PFHxA BPA PFHpA PFOA PFOS BP3 AS C12 LAS C10 LAS C11 TCB AS C14 LAS C12 LAS C13 NP AS C16 NP1EO NP2EO AS C18 DEHP Table S1. Box–Behnken design matrix for optimization of extraction procedure. Experiment AcN Volume (mL) Extraction steps C18 amount (g) 1 7 3 0.55 2 5 1 1 3 3 3 0.55 4 7 1 0.55 5 5 3 1 6 7 2 0.1 7 3 2 1 8 5 1 0.1 9 3 2 0.1 10 7 2 1 11 5 3 0.1 12 3 1 0.55 13 5 2 0.55 14 5 2 0.55 15 5 2 0.55 Figure S1. Optimization of extraction procedure. Standardized effect main Pareto charts and response surfaces for target compounds according to Box–Behnken experimental design. Holothuria samples were spiked with 125 ng g-1 d.w. of each pollutant. PFBuA Diagrama de Pareto Estandarizada para PFBuA 0 2 4 6 8 10 Efecto estandarizado CC AC BC AA AB C:C18 amount BB A:AcN Volume B:Extraction steps + - Standardized effect Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 45 55 65 75 85 PFBuA Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 45 55 65 75 85 PFBuA Estimated Surface Response Extraction steps=3 Recovery (%) Estimated Surface Response C18 amount=0.55 g Recovery (%) PFPeA Estimated Surface Response C18 amount=0.55 g Recovery (%) Diagrama de Pareto Estandarizada para PFPeA 0 2 4 6 8 10 Efecto estandarizado CC AA BC AC AB C:C18 amount BB A:AcN Volume B:Extraction steps + - Standardized effect Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 52 62 72 82 92 PFPeA Estimated Surface Response Extraction steps=3 Recovery (%) Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 52 62 72 82 92 PFPeA Estimated Surface Response C18 amount=0.55 g Recovery (%) PFHxA Diagrama de Pareto Estandarizada para PFHxA 0 2 4 6 8 10 Efecto estandarizado AA CC AC BC AB BB C:C18 amount A:AcN Volume B:Extraction steps + - Standardized effect Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 51 61 71 81 91 PFHxA Estimated Surface Response C18 amount=0.55 g Recovery (%) Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 51 61 71 81 91 PFHxA Recovery (%) Estimated Surface Response Extraction steps=3 Diagrama de Pareto Estandarizada para PFHpA 0 2 4 6 8 Efecto estandarizado AC CC AB BB AA BC C:C18 amount B:Extraction steps A:AcN Volume + - Standardized effect Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 35 38 41 44 47 50 53 PFHpA Estimated Surface Response Extraction steps=3 Recovery (%) Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 35 39 43 47 51 55 PFHpA Estimated Surface Response C18 amount=0.55 g Recovery (%) PFHpA PFOA Diagrama de Pareto Estandarizada para PFOA 0 1 2 3 4 5 6 Efecto estandarizado BC CC AC BB AA AB C:C18 amount B:Extraction steps A:AcN Volume + - Standardized effect Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 24 28 32 36 40 44 PFOA Estimated Surface Response C18 amount=0.55 g Recovery (%) Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 24 27 30 33 36 39 42 PFOA Estimated Surface Response Extraction steps=3 Recovery (%) PFOS Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 58 68 78 88 98 PFOS Estimated Surface Response C18 amount=0.55 g Recovery (%) Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 58 68 78 88 98 PFOS Estimated Surface Response Extraction steps=3 Recovery (%) Diagrama de Pareto Estandarizada para PFOS 0 3 6 9 12 15 Efecto estandarizado CC AA BC AC AB BB C:C18 amount A:AcN Volume B:Extraction steps + - Standardized effect Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.81 C18 amount 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Estimated Surface Response Extraction steps=3 Desirability Desirability Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.63 Extraction steps 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Estimated Surface Response C18 amount=0.55 g Desirability Desirability Perfluoroalkyl compounds: Desirability function MeP Diagrama de Pareto Estandarizada para MeP 0 2 4 6 8 10 12 Efecto estandarizado BC AA AC CC AB BB A:AcN Volume B:Extraction steps C:C18 amount + - Standardized effect Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 52 62 72 82 92 102 MeP Estimated Surface Response C18 amount=0.55 g Recovery (%) Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 52 62 72 82 92 102 MeP Estimated Surface Response Extraction steps=3 Recovery (%) EtP Diagrama de Pareto Estandarizada para EtP 0 2 4 6 8 10 12 Efecto estandarizado AA CC BC AC AB A:AcN Volume BB B:Extraction steps C:C18 amount + - Standardized effect Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 51 61 71 81 91 EtP Estimated Surface Response Extraction steps=3 Recovery (%) Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 51 56 61 66 71 76 81 EtP Recovery (%) Estimated Surface Response C18 amount=0.55 g BP6 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 39 49 59 69 79 BP-6 Estimated Surface Response Extraction steps=3 Recovery (%) Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 39 49 59 69 79 BP-6 Recovery (%) Estimated Surface Response C18 amount=0.55 g Diagrama de Pareto Estandarizada para BP-6 0 2 4 6 8 Efecto estandarizado CC BC AB AC BB AA A:AcN Volume B:Extraction steps C:C18 amount + - Standardized effect BP8 Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 46 56 66 76 86 96 BP-8 Estimated Surface Response C18 amount=0.55 g Recovery (%) Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 46 56 66 76 86 96 BP-8 Estimated Surface Response Extraction steps=3 Recovery (%) Diagrama de Pareto Estandarizada para BP-8 0 2 4 6 8 10 12 Efecto estandarizado BC AB CC AC AA BB A:AcN Volume C:C18 amount B:Extraction steps + - Standardized effect 4OHBP Diagrama de Pareto Estandarizada para 4OH-BP 0 2 4 6 8 Efecto estandarizado CC AC AA A:AcN Volume B:Extraction steps BC AB BB C:C18 amount + - Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 41 51 61 71 81 91 4OH-BP Estimated Surface Response Extraction steps=3 Recovery (%) Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 41 51 61 71 81 91 4OH-BP Estimated Surface Response C18 amount=0.55 g Recovery (%) Standardized effect Benzophenones: Desirability function Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.63 Extraction steps 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Desirability Estimated Surface Response C18 amount=0.55 Desirability Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.81 C18 amount 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Desirability Estimated Surface Response Extraction steps=3 Desirability E3 Diagrama de Pareto Estandarizada para E3 0 1 2 3 4 5 Efecto estandarizado AC BC AB CC BB C:C18 amount AA A:AcN Volume B:Extraction steps + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 24 34 44 54 64 74 E3 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 24 34 44 54 64 74 E3 Estimated Surface Response C18 amount=0.55 g Estimated Surface Response Extraction steps=3 Recovery (%) Recovery (%) Standardized effect E2 Diagrama de Pareto Estandarizada para E2 0 1 2 3 4 5 Efecto estandarizado AB A:AcN Volume BC AC AA BB CC C:C18 amount B:Extraction steps + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 19 22 25 28 31 34 37 E2 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 19 22 25 28 31 34 37 E2 Estimated Surface Response C18 amount=0.55 g Estimated Surface Response Extraction steps=3 Recovery (%) Recovery (%) Standardized effect E1 Diagrama de Pareto Estandarizada para E1 0 1 2 3 4 5 6 Efecto estandarizado CC AB AC BC AA BB A:AcN Volume C:C18 amount B:Extraction steps + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 28 31 34 37 40 43 46 E1 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 28 31 34 37 40 43 46 E1 Estimated Surface Response C18 amount=0.55 g Estimated Surface Response Extraction steps=3 Recovery (%) Recovery (%) Standardized effect EE2 Diagrama de Pareto Estandarizada para EE2 0 2 4 6 8 Efecto estandarizado AA C:C18 amount BC AC BB AB B:Extraction steps CC A:AcN Volume + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 8 11 14 17 20 23 EE2 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 8 11 14 17 20 23 EE2 Estimated Surface Response C18 amount=0.55 g Estimated Surface Response Extraction steps=3 Recovery (%) Recovery (%) Standardized effect Estrogens: Desirability function Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.63 Extraction steps 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.81 C18 amount 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55 Desirability Desirability Desirability Desirability TCB Diagrama de Pareto Estandarizada para TCB 00.5 11.5 22.5 3 Efecto estandarizado AB AA BC BB C:C18 amount CC B:Extraction steps AC A:AcN Volume + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 37 42 47 52 57 62 67 TCB Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 36 41 46 51 56 61 66 TCB Recovery (%) Recovery (%) Estimated Surface Response C18 amount=0.55 g Estimated Surface Response Extraction steps=3 Standardized effect LAS: Desirability function Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.63 Extraction steps 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.81 C18 amount 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55g Desirability Desirability Desirability Desirability AS C12 Diagrama de Pareto Estandarizada para AS C12 0 2 4 6 8 10 Efecto estandarizado BC BB AC CC AB C:C18 amount AA A:AcN Volume B:Extraction steps + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 90 110 130 150 170 190 210 AS C12 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 90 110 130 150 170 190 210 AS C12 Standardized effect Recovery (%) Recovery (%) Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55g AS C14 Diagrama de Pareto Estandarizada para AS C14 0 2 4 6 8 Efecto estandarizado AC CC AA BC AB C:C18 amount BB A:AcN Volume B:Extraction steps + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 46 56 66 76 86 96 AS C14 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 46 56 66 76 86 96 AS C14 Standardized effect Recovery (%) Recovery (%) Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55g AS C16 Diagrama de Pareto Estandarizada para AS C16 0 2 4 6 8 10 12 Efecto estandarizado C:C18 amount AC AA CC BB AB BC B:Extraction steps A:AcN Volume + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 21 26 31 36 41 46 AS C16 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 20 25 30 35 40 45 50 AS C16 Standardized effect Recovery (%) Recovery (%) Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55g AS C18 Diagrama de Pareto Estandarizada para AS C18 0 2 4 6 8 Efecto estandarizado AA BB CC AB AC C:C18 amount BC B:Extraction steps A:AcN Volume + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 8 12 16 20 24 28 AS C18 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 0 10 20 30 40 AS C18 Standardized effect Recovery (%) Recovery (%) Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55g AS: Desirability function Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.63 Extraction steps 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.81 C18 amount 0 0.2 0.4 0.6 0.8 1 Deseabilidad Deseabilidad 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55g Desirability Desirability Desirability Desirability HBCDD Diagrama de Pareto Estandarizada para HBCDD 0 1 2 3 4 Efecto estandarizado CC AB BB C:C18 amount BC AA B:Extraction steps AC A:AcN Volume + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 0 10 20 30 40 HBCDD Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 0 10 20 30 40 HBCDD Recovery (%) Recovery (%) Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55g Standardized effect NP Diagrama de Pareto Estandarizada para NP 00.5 11.5 22.5 3 Efecto estandarizado AC AB CC BB AA A:AcN Volume C:C18 amount BC B:Extraction steps + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 0 0.4 0.8 1.2 1.6 2 (X 1000) NP Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 0 0.4 0.8 1.2 1.6 2 (X 1000) NP Standardized effect Recovery (%) Recovery (%) Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55g NP1EO Diagrama de Pareto Estandarizada para NP1EO 0 1 2 3 4 5 Efecto estandarizado AB CC AA BB AC BC C:C18 amount A:AcN Volume B:Extraction steps + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 52 62 72 82 92 102 112 NP1EO Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 52 72 92 112 132 NP1EO Standardized effect Recovery (%) Recovery (%) Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55g NP2EO Diagrama de Pareto Estandarizada para NP2EO 00.5 11.5 22.5 3 Efecto estandarizado BB AB BC AA CC AC C:C18 amount B:Extraction steps A:AcN Volume + - Superficie de Respuesta Estimada C18 amount=0.55 34567 AcN Volume 11.4 1.8 2.2 2.6 3 Extraction steps 33 38 43 48 53 58 63 NP2EO Superficie de Respuesta Estimada Extraction steps=3.0 34567 AcN Volume 00.2 0.4 0.6 0.8 1 C18 amount 33 38 43 48 53 58 63 NP2EO Standardized effect Recovery (%) Recovery (%) Estimated Surface Response Extraction steps=3 Estimated Surface Response C18 amount=0.55g