Bull Environ Contam Toxicol DOI 10.1007/s00128-015-1545-1

Application of Focused Ultrasound-Assisted Extraction to the Determination of Persistent Organic Pollutants (POPs) in Soil Samples R. Flores-Ramı´rez1 • S. E. Medellı´n-Garibay3 • C. G. Castillo2 C. A. Ilizaliturri-Herna´ndez1 • B. A. Zuki-Orozco1 • L. Batres-Esquivel1 • F. Dı´az-Barriga1

•

Received: 2 December 2014 / Accepted: 7 April 2015 Ó Springer Science+Business Media New York 2015

Abstract A simple and rapid focused ultrasound extraction (FU) based method is presented for the determination of persistent organic pollutants (POPs) in soil using a gas chromatography coupled to a mass detector with electron impact ionization. The main experimental parameters affecting the FU step have been optimized by applying a PERMANOVA and PCO analysis allowing us to obtain a maximum amount of information with a minimum number of assays. The limits of detection for POPs fell within the 0.9–6.8 ng/g d.w. interval; a linear method was used with correlation coefficients (r) higher than 0.99. Recovery percentages at low concentrations (25 ng/g d.w.) were 75.8 %–110 %, and at high concentrations (75 ng/g d.w.) 82.3 %–109 %; the evaluated precision as RSD% of repeatability and reproducibility were within a range of 0.5 %–11 % and 0.3 %–18 %, respectively. Keywords Persistent organic pollutants  Focused ultrasound extraction  Soil  GC–MS–EI Persistent organic pollutants (POPs) are organic chemicals, which are generated naturally or due to human activities. They have specific physical and chemical characteristics & F. Dı´az-Barriga [email protected] 1

Centro Nacional de Bioana´lisis (CENBIOS)-Centro de Investigacio´n Aplicada en Ambiente y Salud, CIACYT, Universidad Auto´noma de San Luis Potosı´, Av. Sierra Leona 550, 78210 San Luis Potosı´, S.L.P., Mexico

2

Departamento de Bioquı´mica, Facultad de Medicina, Universidad Auto´noma de San Luis Potosı´, Av. Venustiano Carranza 2405, 78210 San Luis Potosı´, S.L.P., Mexico

3

Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramo´n y Cajal, 28040 Madrid, Spain

that allow them to remain chemically intact in the environment for long periods of time; they can be dispersed through different environmental matrices (soil, water, sediment, and air), stored in fatty tissues and biomagnified in the food chain; furthermore, they are toxic to humans and wildlife (Eskenazi et al. 2009; Wikstrom et al. 2004; Mullerova and Kopecky 2007). Current methodologies used for quantifying POPs in environmental matrices result from a great deal of research in the field of analytical chemistry. The classical method for extraction of POPs from environmental samples is Soxhlet extraction which requires large amounts of solvent and about 20 h for the extraction to be completed. Ultrasound (Martens et al. 2002; Nikonova and Gorshkov 2011) and microwave assisted extractions (MAE) (Itoh et al. 2008; Xiong et al. 2000) have gained wide acceptance due to their high temperature requirement, high extraction rate, automation and the possibility of simultaneously extracting different types of samples. Focused ultrasound solid liquid extraction (FU) is a relatively new technique based on the application of high power focused ultrasonic waves using a micro-tip immersed directly in the extraction mixture. When ultrasound waves cross the liquid solvent, numerous tiny gas bubbles (cavitation bubbles) are produced that implode and locally produce very high temperatures, pressures and velocities of solvent micro-jets. It has been successfully applied for determining POPs (PAHs, PBB, PBDEs) in biota (Trier et al. 2011), indoor dust (Begley et al. 2005), soil (SanzLandaluze et al. 2010; Yu and Hu 2007) and recycled paper material (Pe´rez-Palacios et al. 2012). However, in the literature review, there were no references on the technique potential for simultaneous extraction from soil of POP pesticides and industrial products, and from compounds listed as future POPs such as atrazine.

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The aim of this study was to develop and validate an analytical method to determine POPs in soils samples using focused ultrasound probe coupled with gas chromatography–mass spectrometry–electron impact (GC–MS–EI).

Materials and Methods For PCBs analysis was employed a mixture of standard references (28, 52, 99, 101, 118, 138, 153 and 180) and individual (105, 128, 170, 183, 187, and 156) PCBs, with 95 % purity at a concentration of 100 lg/mL in hexane, and use surrogate standard of PCB 141 (C13-isotopically labeled) at a concentration of 40 lg/mL in nonane. For POP pesticides (OCPs), a mixture (-a,-b,-c,-R-HCH, 4,40 DDD; DDE; DDT, aldrin; heptachlor; heptachlor epoxide; -a,-b and sulfate endosulfan) and individual standards (HCB, atrazine) were used, with 99 % purity at a concentration of 100 lg/mL in hexane–toluene and 1000 lg/ mL for HCB. With a surrogate standard of a-HCH (C13), DDE (C13) and PCB 141 (C13) isotopically labeled at a concentration of 100 lg/mL in nonane; all standards were purchased from Chemservice. Based on reference standards, independent solutions of 1000 ng/mL were prepared for PCBs, OCPs and three surrogate standards in hexane. Solutions remained stored at -20°C. For extraction, was used an Ultrasonic Processor GEX130 (115 V 50/60 Hz, Ultrasonic Processor) equipped with a 3 mm titanium tip, mechanical agitators (Thermolyne) and a Zymark evaporator. Samples and calibration curves were analyzed in a gas chromatography (GC) 6890 (Agilent) equipped with a split/ splitless injector coupled with a mass spectrometry detector (MS) 5975 (Agilent) with electron impact ionization (EI). The injection port was operated in splitless mode with a 0.75 mm liner without glass wool. Injection port temperature fixed at 230°C; helium used as carrier gas at a pressure of 36 psi with a constant flow of 1 mL/min. The chromatographic separation was done through a HP 5 ms (60 m 9 0.25 mm 9 0.25 lm) column (Agilent). The setting of the oven was as follows: 90°C (2 min), 180°C (30°C/min), 200°C (1°C/min), 265°C (2°C/min), 310°C (30°C/min) with a run time of 59 min. Blank samples, non-spiked soil samples, were analyzed to check for any contamination that could have happened throughout the analytical procedure. After verifying the blank matrix, one gram of soil was fortified with PCBs and OCPs with independent concentrations from the quantification limit up to 400 ng/g dry weight (ng/g d.w.) and 20 ng/g d.w. of surrogate standards; it was stored at 4°C for 2 days to stabilize the compounds. Extractions to test solvents were done using fortified soil at 100 ng/g d.w. (Fig. 1).

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A multivariate analysis (PERMANOVA) was performed to infer the difference between treatment patterns; the experiment was based on the three factors ‘‘Time’’ (0.5, 1 min, fixed factor), ‘‘Temperature’’ (4, 20, 37°C, fixed factor) and; Amplitude (20 %, 30 %, 60 %, fixed factor) with three replicates for each treatment at a concentration of 100 ng/g d.w. The analysis were based on Euclidean distance matrices calculated from normalized data of POPs responses (30 analyzed compounds); test probability values (p) of the model were calculated by 9999 random permutations (Monte Carlo analysis). Significant values (p \ 0.05) were researched with pairwise comparisons (post hoc), which also use 9999 random permutations to obtain p values. An ordination diagram (principal co-ordinate analysis-PCO) was constructed in order to visualize patterns in the treatments and evaluate their consistency with results provided by PERMANOVA. All multivariate analysis were performed in PRIMERv6 Software with the add-on package PERMANOVA (Anderson et al. 2008; Clarke and Gorley 2006). Under optimal conditions 1 g of sample were placed together with 10 mL of an hexane: dichloromethane (hexane:DCM) (75:25) mixture in a 25 mL vessel and surrogate standards [a-HCH (C13), DDE (C13) and PCB 141 (C13)] were added (20 ng/g d.w.). The FU step was performed for 1 min in duplicate, and at 60 % of irradiation power. Extractions were carried out at 20°C. Between samples, cleanup tip was performed using the same method of sonication. The cleanup tip consisted a wash using 2 mL of hexane and drying with a clean duster, and as internal quality control, we verified contamination between samples. After the extraction step, the supernatant was centrifuged for 5 min at 3500 rpm and FU extract was evaporated to *1 mL under a nitrogen stream using a Turbovap LV Evaporator depending on the clean-up selected. The extract obtained from soil underwent a cleaning procedure using columns packed with florisil. The soil extract went through the conditioned column and was eluted with 10 mL of hexane: dichloromethane (75:25); and 10 mL hexane:acetone (85:15) were applied. Finally, it was evaporated to 100 lL with N2 for its analysis in the GC–MS–EI. The internal quality control and validation of the method were performed based on the Guide for the Validation of Analytical Methods for the Determination of Organic Compounds at Trace Levels (AOAC/FAO/IAEA/IUPAC 2000), by evaluating the following parameters: limit of detection (LOD) and limit of quantification (LOQ), linearity (r), sensitivity, percentage of recovery and precision (repeatability and reproducibility). LOD and LOQ were calculated using results obtained from the triple calibration curve of each compound

Bull Environ Contam Toxicol

Fig. 1 Extraction efficiencies for pollutants groups depending on the extraction solvent. Each figure represents the optimization of distinctive compounds in each group of analytes. R-HCH: isomers a-, b-, c- and R-HCH; HCB; atrazine; R-heptachlor: heptachlor and heptachlor epoxide; aldrin; R-endosulfan: endosulfan sulfate, -a and -b; R-DDX: DDE, DDT and DDD; R-PCBs: 28, 52, 99,101, 105, 118,

128, 138, 153, 156, 170, 180, 183 and 187. Mean ± SD. Extraction solvent: acetone; hexane; hex:Et Ac (50:50): hexane:ethyl acetate (50:50); hex:DCM (50:50): hexane:dichloromethane (50:50); hex:DCM (75:25): hexane:dichloromethane (75:25). ANOVA (*) p \ 0.05 compare versus hex:DCM (75:25)

determined in a concentration range of 2.5 to 15 ng/g d.w. The linearity expressed by the correlation coefficient (r) and the sensitivity determined by the slope of the working range curve (LOQ-400 ng/mL) resulted from the average of eleven curves registered for five days. The percentage of recovery of the method for each analyte was derived from evaluating 10 different fortified points on the calibration curve of a low (25 ng/g d.w.) and high (75 ng/g d.w.) concentration (Table 1). The method’s precision was measured based on repeatability and reproducibility, evaluating different concentrations in triplicate on the same day, and in duplicate on five different days. Accuracy for DDTs (DDT, DDE, DDD) in soil was evaluated, using analytical reference material CRM 105-100 BNA/Pesticide soil (Resource Technology Corp).

to improve the sensitivity of the method by increasing extraction efficiency (best extraction in matrix), hexane:DCM (75:25) was chosen as the extraction solvent for further experiments. The chemometric strategy (factorial design and PERMANOVA), developed in a previous work (FloresRamirez et al. 2014), was employed to optimize extraction conditions using a focused ultrasonic probe (FU). In this study, blank and spiked (100 ng/g d.w. of POPs) soil samples were used. Hexane:DCM (75:25) was found to have a better capability of penetrating into soil, which is considered a high complexity matrix, and thus enabling effective isolation of non-polar analytes. Figure 2 represents the main coordinate analysis graph related to the Euclidean distance matrix of the normalized responses for the 29 compounds that were analyzed. Each point was obtained from a triplicate of each compound. An increase in the response occurs when there is a shift towards the right of the PCO diagram and the difference among treatments is visualized in the separation of both the aggregate and individual variables. Based on the PERMANOVA analysis, all experimental factors and their interactions significantly influence the response of the compounds tested (Table 2). The amount of variation was explained in the following order of importance: temperature [ time [ amplitude [ all interactions. The diagram shows the importance of the PCO temperature factor (PCO1 axis, 88.4 % of

Result and Discussion Five extraction solvents were tested according to the literature: Acetone, Hexane, Hexane:Ethyl Acetate (hexane: Et Ac) (50:50), hexane:DCM (50:50) and hexane:DCM (75:25). The experiments were performed in triplicate (each analyte had a concentration level of 100 ng/g d.w.). Figure 1, shows that the signals (normalized to the area signal) were significantly higher when using hexane:DCM (75:25) and hexane for all compounds. Therefore, in order

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Bull Environ Contam Toxicol Table 1 Parameters of analytical method validation FU–GC–MS in soil fortified

Compound

Ion

rt

LOD

LOQ

r

m

Low

High

*a-HCH C13

187

14.9

–

–

–

–

–

–

*DDE C13

258

34.5

–

–

–

–

–

–

*PCB 141 C13

372

40.9

–

–

–

–

–

–

a-HCH

183, 181

14.9

2.8

6.8

0.99

0.43 ± 0.29

88.0

95.0

HCB

284, 286

15.4

1

3.7

0.99

0.54 ± 0.15

95.0

98.0

Atrazine

200, 215

15.8

3.1

6.2

0.99

0.40 ± 0.01

80.4

85.2

b-HCH

183, 181

16.3

2.5

5.1

0.99

0.42 ± 0.01

85.3

89.6

c-HCH

183, 181

16.7

2.4

5.1

0.99

1.86 ± 0.55

92.5

96.3

d-HCH

183, 181

18.2

2.4

5.7

0.99

1.00 ± 0.25

91.6

96.8

PCB 28

256, 258

20.5

0.9

2.8

0.99

1.70 ± 0.69

110

100.6

Heptachlor

272, 256

21.8

2.5

5.2

0.99

0.18 ± 0.20

91

98.5

PCB 52

292, 290

23.3

1.4

3.4

0.99

1.38 ± 0.22

102

109

Aldrin

263, 261

24.7

2.4

5.2

0.99

0.49 ± 0.16

87.2

88.6

Heptaclor epoxide

353, 355

28.5

3.1

6.2

0.99

0.43 ± 0.18

96.8

102.5

PCB 101 a-Endosulfan

326, 324 241, 239

31.6 31.9

2.5 2.9

5.1 6.2

0.98 0.99

1.79 ± 0.58 0.18 ± 0.09

99.4 78.3

105.8 82.3

PCB 99

326, 324

32.1

2.5

5.3

0.99

1.96 ± 0.05

105.4

102.3

DDE

246, 248

34.5

2.2

4.6

0.99

1.77 ± 0.26

99.8

101.5

b-Endosulfan

241, 239

37.4

2

4.1

0.99

1.63 ± 0.28

75.8

82.4

PCB 118

326, 324

37.6

2.5

5.1

0.99

1.34 ± 0.11

98.6

103.4

DDD

235, 237

38.5

2.3

5

0.99

2.81 ± 0.15

100.6

99.5

PCB 153

360, 362

39.8

2.1

5.2

0.99

1.07 ± 0.17

98.6

103.5

PCB 105

326, 324

40.1

2.2

5.1

0.99

1.14 ± 0.18

99.3

106.9

Endosulfan sulfate

272, 282

41.5

3.2

6.4

0.99

0.84 ± 0.11

82.3

86.9

DDT

235, 237

42

3.2

6.8

0.99

0.58 ± 0.35

98.1

100.5

PCB 138

360, 362

42.4

2.5

5.1

0.98

1.25 ± 0.02

99.3

106.8

PCB 187

394, 396

44

2.4

5.2

0.99

0.89 ± 0.16

98.2

103.7

PCB 183

394, 396

44.4

2.5

5

0.99

0.97 ± 0.26

97.6

99.7

PCB 128

360, 362

44.9

2.4

5.1

0.99

0.90 ± 0.27

98.5

106.5

PCB 156

360, 362

47.1

2.4

5.1

0.99

0.65 ± 0.37

99.8

108

PCB 180 PCB 170

394, 396 394, 396

48.6 51.2

2.2 2.3

4.8 4.7

0.98 0.99

0.66 ± 0.08 0.51 ± 0.23

100.8 102.9

105.7 106.1

rt: retention time (min); LOD: limit of detection (ng/g d.w.); LOQ: limit of quantification (ng/g d.w.); r: coefficient of correlation; m: sensibility expressed as a linear slope; recovery percentage at low concentration: 25 ng/g d.w. and high concentration: 75 ng/g d.w. (*) surrogate standard

variation) and the time (PCO2 axis, 8.0 % of variation) in generating the distribution pattern in the treatments. An increase in the response of the tested compounds was detected by treatment towards the right PCO diagram (Fig. 2). The comparisons between abundances of the pairs of factors were: time 1 [ 0.5 min (t = 6.81, p = 0.0001), temperature 20 [ 4°C (t = 10.16, p = 0.0001), [37°C (t = 2.53, p = 0.0004) and amplitude 60 [ 30 % (t = 3.00, p = 0.001), [20 % (t = 1.94, p = 0.003). According to the PCO analysis and peer comparison, the best treatment was identified at 1 min, 20°C and 60 % amplitude. Additionally, responses to 4 and 20°C were significantly influenced by the amplitude in the time going

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from 0.5 to 1 min (pair wise PERMANOVA Table 3). Transient bubble collapsing is considered to be the main source of the mechanical effects of ultrasonic energy. Each collapsing bubble can be considered as a microreactor in which temperatures of several degrees and pressures higher than on thousand atmospheres are created instantaneously. Solvent temperature plays two roles in ultrasonication (Mason 1992). On the one hand, the use of high temperatures helps to disrupt strong solute-matrix interactions, which involve Van der Waals forces, hydrogen bonding and dipole attractions between the solute molecules and active sites on the matrix. Moreover, faster diffusion rates occur at higher temperatures. However, as the bulk solution

Bull Environ Contam Toxicol

Fig. 2 Ordination of centroids derived from principal coordinate analysis (PCO) of the Euclidean distance matrix based on normalized abundance data for the 29 analyzed compounds. Treatments are

Table 2 Three-factor PERMANOVA main test of abundances from POPs on environmental samples in response to the time, temperature and amplitude factors

represented by time (0.5 and 1 min): temperature (4, 20 and 37°C): amplitude (20 %, 30 % and 60 %)

Factor

d.f

Time

1

Temperature

2

Amplitude Time 9 temperature

SS

Pseudo-F

p (MC)

217.9

46.4

0.0001

676.9

72.0

0.0001

2

112.8

12.0

0.0001

2

50.3

5.3

0.0001 0.0055

Time 9 amplitude

2

23.0

2.4

Temperature 9 amplitude

4

53.7

2.8

0.0001

Time 9 temperature 9 amplitude

4

47.9

2.5

0.0002

Residual

34

159.7

Total

51

1411.8

Conditions: (1) time (0.5, 1 min), (2) temperature (4, 20, 37°C) and amplitude (20 %, 30 %, 60 %) Bold values indicate significant differences at p \ 0.05 df Degrees of freedom, SS sum of squares, p (MC) probability of Monte Carlo Test

Table 3 PERMANOVA pairwise interaction comparisons of response abundances of POPs on environmental samples for the factor amplitude within time–temperature interactions

Time (min)

Comparisons

Temperature (°C) 4

0.5

1.0

20

37

t (perm)

p (MC)

t (perm)

p (MC)

t (perm)

p (MC)

Amp 20 vs. 30

2.54

0.0147

9.86

0.0001

1.06

0.3421

Amp 20 vs. 60

1.87

0.0516

3.51

0.0068

1.21

0.2851

Amp 30 vs. 60

3.37

0.0041

2.15

0.0523

1.48

0.1251

Amp 20 vs. 30

1.54

0.1674

1.38

0.1997

0.65

0.6209

Amp 20 vs. 60

4.17

0.0033

3.07

0.0142

10.49

0.0036

Amp 30 vs. 60

1.52

0.1551

2.48

0.0331

1.39

0.2147

Bold values indicate significant differences at p \ 0.05 p (MC) probability of Monte Carlo Test

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Bull Environ Contam Toxicol

temperature rises, the performance of treatment deteriorates. This is because lower cavitation efficiency is obtained as the density of the liquid decreases with the increasing temperature caused by ultrasonication (Vale et al. 2007). Furthermore, cavitation is better attained at lower temperatures when the ultrasonic power of the generator is constant. This is because as the temperature of the solvent rises so to does its vapor pressure and so more solvent vapor fills the cavitation bubbles which then tend to collapse less violently, that is, the sonication effects are less intense than expected (Capelo et al. 2005). FU’s results are comparable or even better than those obtained using other techniques for extracting POPs in soil. For instance, compared to methods such as Soxhlet, we reduced the volume of solvent five times and the extraction 960 times (Gai et al. 2014). Additionally, FU achieved better results in terms of the sample size (reduced by 10 times), the time (by 6.6 times) and the solvent (by 2 times) with regard to the ultrasonic bath (Tor et al. 2006). On the other hand, unlike the ultrasonic bath, FU is immersed directly into the sample container and can deliver a much higher ultrasonication intensity than the one provided by the ultrasonic bath (100 times higher) allowing rapid extractions of organic compounds to take place from liquid and solid matrixes (Capelo and Mota 2005). However, the choice between baths and FU depends on the requirements for the particular analysis of contaminants. If the aim is the total solid–liquid extraction, the use of a powerful probe could be better because the necessary time for extraction is lower (20 samples in 25 min), but, when a great number of samples need to be analyzed the bath is the better option (depends on the size of the ultrasonic bath, can reach 60 samples in 30 min). On the other hand, FU also showed comparable results regarding the pressurized liquid

extraction (PLE), especially in the results of the efficiency of extraction, note that PLE further decreases the volume of the solvent, however it also requires more time and energy consumption (uses heat and pressure for extraction), it should be noted that the equipment are more expensive (Albero et al. 2012). A selected ion monitoring mode was used for quantification purposes. Selectivity of the developed method was assessed by the analysis of six blank samples of each of the soils extracted using the two proposed methods. No interfering peaks at the retention time of target compounds were detected. The linearity range for the soil samples was calculated using matrix-matched calibration solutions using six increasing concentrations. Linear regression, using peak area ratios; was used to evaluate results. Good correlation (r [ 0.99) was obtained for almost compounds except for PCB 101, 138 and 180 (Table 1). LOD and LOQ were determined by a linear curve at low concentrations, adding a confidence limit of 95 % (Hubaux and Vos 1970; Miller and Miller 2000). Values were obtained through the slope method, which consists on measuring variation in the lower areas of the curve (where there is more uncertainty) to verify that response values obtained are different from a blank response. This is calculated from the intercept (YB) value plus the deviation that estimates random errors in the direction of the intersection (Sy/x), YB ? 3 Sy/x for LOD and YB ? 10 Sy/x for LOQ. The 95 % confidence limit of the slope variation in the interpolation area is added to the obtained value, thus the final value contemplates the largest uncertainty in the lowest areas of the curve and ensures analyte presence and quantification with acceptable precision. The compounds’ LOD interval was 0.9–6.8 ng/g d.w.; these results are similar to those reported by Rhind for PCBs (Rhind et al.

Fig. 3 Chromatogram FU–GC–MS (SIM mode) fortified soil to 10 ng/g dry weight. Compounds: (1) a-HCH, (1*) a-HCH (C 13), (2) HCB, (3) atrazine, (4) b-HCH, (5) c-HCH, (6) d-HCH, (7) PCB 28, (8) heptachlor, (9) PCB 52, (10) aldrin, (11) heptachlor epoxide, (12) PCB 101, (13) a-endosulfan; (14) PCB 99, (15) DDE, (15*) DDE (C

13), (16) PCB 118, (17) b-endosulfan; (18) DDD; (19) PCB 153, (20) PCB 105, (21*) PCB 141 (C 13), (22) endosulfan sulfate, (23) DDT, (24) PCB 138, (25) PCB 187, (26) PCB 183, (27) PCB 128, (28) PCB 156, (29) PCB 180 and (30) PCB 170

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Bull Environ Contam Toxicol Table 4 Repeatability and reproducibility of the analytical method FU–GC–MS in soil fortified

Compounds

Repeatability (%RSD)

Reproducibility (%RSD)

5

10

25

50

100

5

10

25

50

100

R-HCHs

3–10

1–7

1–4

6–9

1–4

3–14

3–9

1–15

1–5

2–3

HCB

2

0.8

1

9

4

5

6

15

7

1

Atrazine

–

6.2

4.8

8

4

–

4

1

4

3

R-Heptachlors

1.6–2.4

1–1.6

1.6–5.3

7–8

2

4–16

3–7

8–12

5–6

2–16

Aldrin

4.11

2.1

4

8

0.5

10

8

8

2

2

R-Endosulfan

0.5–11

2

6–7

2–4

1–2

6

11

13

11.7

4

R-DDXs

1–5

1–6

6–7

1–4

1.9–2

10–14

12

9

2–15

0.3–1

R-PCBs

3–6

0.5–1

0.7–3

5–7

0.1–4

6–12

3–11

2–17

3–18

0.3–16

%RDS Acceptablea

17.7

16

13.9

12.5

10.1

26.6

21

18.5

16.7

15

a

Horwitz 1982. Concentration: ng/g d.w.; isomers a-, b-, c-, d-HCH; HCB: hexachlorobenzene; R-heptachlor: heptachlor, heptachlor epoxide; R-endosulfan: -a, -b, endosulfan sulfate; DDTs: DDD, DDE, DDT; PCBs: 28, 52, 99, 101, 105,118, 128, 138, 153, 156, 170, 183, 180 and 187

2013) (10 ng/g) using GC–MS–EI. In another study, LOD for OCPs and PCBs, intervals were 0.04–0.71 pg/g, the differences among these values are due to the type of detector used (HRGC–HRMS) (Gai et al. 2014). Nevertheless, LOD are consistent with the reference values for the compounds analyzed in soil. A typical chromatogram corresponding to the analysis of the POPs in a soil sample under these experimental conditions is shown in Fig. 3. For DDTs in soil, analytical reference material CRM 105-100 BNA/Pesticide soil (Resource Technology Corp) was used. Our extraction efficiency was 90 %–110 % for all tested analytes. For the other compounds, in the absence of a certified reference material (CRM) for POPs in soil (concentrations below the detection limits of the developed method), we follow a recovery approach in fortified samples for method validation. In terms of obtained recovery from fortified samples, FU provided acceptable results for a matrix studied at two spiking levels. Recovery ranges of 75.8 %–110 % were obtained at the lowest concentration level (25 ng/g d.w.) and 82.3 %–109 % at the highest (75 ng/g d.w.) (Table 1). Precision was measured as repeatability and reproducibility, and each point in the calibration curve was evaluated as relative standard deviation (RSD%) of the area ratios; all compounds are below the acceptable RSD% (Horwitz 1982), which shows that the method was reliable throughout different working days (Table 4). Finally, a rapid and simple ultrasonic-based methodology to determine POPs in soil samples has been proposed. The main experimental parameters affecting the FU step have been optimized by applying a PERMANOVA and PCO analysis allowing us to obtain a maximum amount of information with a minimum number of assays. On the other hand, in accordance to environmental values of POPs (Dominguez-Cortinas et al. 2013; Martinez-Salinas et al. 2011; Pe´rez-Maldonado et al. 2010; Trejo-

Acevedo et al. 2009), the detection and quantification limits found in this work are acceptable for monitoring. Acknowledgments The authors acknowledge grants and fellowships (Posdoctoral fellowship of Flores-Ramı´rez, R) from National Council for Science and Technology sectorial funds CONACYTSALUD (#182121).

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