Influence of sediment grain size on the efficiency of focused microwave extraction of polycyclic aromatic hydrocarbons M. Letellier and H. Budzinski* LPTC, UPRESA 5472 CNRS, 351 cours de la libération, 33405 Talence, France Received 25th September 1998, Accepted 6th November 1998 The efficiency of focused microwave (FMW)-assisted extraction of polycyclic aromatic hydrocarbons (PAHs) at atmospheric pressure was investigated for sediments with different grain size distributions. The PAH contents and distribution profiles obtained by FMW extraction for a dry matrix and a remoistened dry matrix were compared with those obtained by Soxhlet extraction for a bulk matrix and six fractions.The effect of moisture depended on the composition of the matrix and the grain size: an improvement in PAH recovery with the addition of water was noted for coarse fractions, but not for fine fractions. Application to other matrices of different grain sizes and contamination levels showed that FMW-assisted extraction is a good alternative to Soxhlet extraction.FMW extraction efficiency was tested on a naturally moist sediment. PAH concentrations were compared with those obtained by extraction of dry and remoistened dry matrices by FMW extraction and with those obtained by extraction of a dry matrix by Soxhlet extraction. PAH recoveries, compared with those obtained by Soxhlet extraction, were satisfactory. Therefore, it is possible to avoid the drying step with the FMW method.The FMW technique might be suitable for field studies, for example, on a boat during an oceanographic cruise. The developed procedure cosists of an extraction step of 10 min with a few millilitres of solvent, reconcentration steps and micro-column purification. The treatment of the sample can be performed immediately after sampling. The method affords good recovery. The reproducibilities are comparable to, or better than, those obtained by conventional extraction. Introduction Contaminants of anthropogenic or natural origin such as polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment.1–3 Such persistent compounds damage the entire ecosystem and especially the aquatic environment. Sediment, because of its accumulation capacity, is a huge sink for contaminants arising from atmospheric contaminated particles, gas transfer and effluents. Because of the bioavailability of contaminants, present in the sedimentary matter, there is a toxicological risk to fauna and flora.In this respect, monitoring of contamination levels and research into the association of contaminants with sediment have been conducted. In environmental studies, the improvement in analytical methods has allowed more accurate quantification of individual compounds of different toxicity levels, which allows an estimation of the exposure of organisms to contaminants. However, the limiting factor in the analysis of numerous samples is the treatment of the sample.The conventional procedure consists in drying the sample followed by Soxhlet extraction, which involves the percolation of the sample for 8–72 h by a solvent. This method is time and solvent consuming and is not easy to automate. The development of new extraction methods, based on microwave irradiation, showed that such methods might be a good alternative to Soxhlet extraction.4–12 A focused microwave (FMW)-assisted extraction method at atmospheric pressure gives satisfactory results with a reduction of time (10 min), a reduction of solvent (30 mL) and with safety.11 The procedure of extraction of PAHs has been optimised on a certified matrix (NIST SRM 1941a).12 This preliminary study12 has shown the influence of moisture on the extraction efficiency.Indeed, the specific interaction of microwaves with polar compounds allows local heating of the moist matrix and an overall improvement in extraction recovery.10,12 The optimum amount of moisture in sediment has been determined (30%) and allows a significant improvement in recovery.12 However, the effect of moisture varies depending on the matrix. The preliminary study12 could not show a relationship between the nature of the matrix and water content.One interesting characteristic of a matrix appears to be the grain size. The efficiency of microwave procedures according to grain size was studied in this paper. Firstly, it is important to study the selectivity of the FMW extraction towards compounds of different origin and associated with different particles.Some studies showed a difference in the distribution profile with the grain size of the sediment for different classes of contaminants, such as PAHs and PCBs.13,14 Secondly, it is interesting to study the influence of moisture content on the microwave extraction efficiency. In this study, efficiencies obtained by Soxhlet extraction and FMW extraction were compared for a bulk muddy sediment and for six sub-fractions of this matrix. The effect of moisture for each sub-fraction and for different compounds was studied. Two other sediments with different levels of contamination and different grain sizes were also studied.Finally, the extraction of a naturally moist sediment was compared with the extraction of the freeze-dried matrix to determine whether a drying step is necessary for the FMW method. Experimental Standards, solvents and reagents The PAHs studied ranged from three-ring aromatics (phenanthrene) to six-ring aromatics (benzo[ghi]perylene).The Standard Reference Material, SRM 2260, Aromatic Hydrocarbons in Toluene (Nominal Concentration 60 mg mL21), a standard solution of 24 aromatic hydrocarbons (23 are certified), was provided by the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) and used for calibration. Analyst, 1999, 124, 5–14 5The compounds used as internal standards were perdeuterated PAHs.Phenanthrene-d10, benzo[a]pyrene-d12 and benzo[ ghi]perylene-d12 were purchased from Cambridge Isotope Laboratories (CIL, Andover, MD, USA), and fluoranthene-d10, pyrene-d10 and chrysene-d12 from MSD Isotopes (Division of Merck Frost Canada, Montreal, Canada). Pestinorm dichloromethane was purchased from Prolabo (Fontenay-sous-Bois, France). HPLC-grade isooctane and extra-pure pentane (Scharlau) were purchased from ICS (St Médard en Jalles, France).Pentane was distilled. Copper (40 mesh, 99.5% purity) (Aldrich, Saint Quentin Fallavier, France) was activated with hydrochloric acid (7 M), then washed with water, acetone and dichloromethane. Alumina (150 basic, Type T, 0.063-0.2 mm) and silica (silica gel, 0.063–0.2 mm) (Merck, Darmstadt, Germany) were washed with dichloromethane, deactivated at 150 °C overnight and then stored at 150 °C. Sediment sampling For the study of grain size fraction and of the influence of the matrix state (dry or moist), a sediment was sampled in the superficial layer (0–5 cm) in the harbour of Arcachon (south west Atlantic coast of France).It was homogenised and frozen (220 °C) until treated. For grain size study (sand or silt), two sediments (‘OPCB 5b’ and ‘OPCB 9b’) were sampled in the Gironde estuary (south west of France) by Flusha or Shipeck grab. They were chosen for their different grain sizes and contamination levels.Sediment fractionation For the study of grain size fraction, bulk sediment was wetsieved: sediment samples were gently shaken by hand in sieves with water (2000, 500, 300, 125, 63, 40, 15 mm). Six grain-size sub-fractions were defined as follows: 500–300 mm (coarse sands), 300–125 mm (medium sands), 125–63 mm (fine sands), 63–40 mm (silts), 40–15 mm (medium silts), 15–0 mm (fine silts and clays). Wet-sieving and manual stirring were employed to avoid possible carry-over of finer particles, which might reach the coarser fractions if dry-sieving was performed.Each subfraction was centrifuged to remove water and freeze-dried. The weight of each sub-fraction of sediment was carefully recorded to estimate the relative weight contribution. During the sieving, attempts were made to minimise sediment weight loss. For the study of the influence of the matrix state (dry or moist), bulk sediment was wet-sieved in sieves of 300 mm to remove large debris in order to improve homogeneity.The 0–300 mm sub-fraction was centrifuged to remove as much water as possible and to keep about 30% of water as in the natural bulk sediment. Half of the material was stored at 220 °C until treatment. The other half was freeze-dried. For the study of grain size (sand or silt), bulk sediments were freeze-dried and then sieved at 500 mm. Soxhlet extraction procedure Soxhlet extractions with dichloromethane (2 3 250 mL) were performed using 0.3–10 g of sediment (Table 1) spiked with perdeuterated internal standards for quantification. The extraction lasted 48 h.Blank experiments were performed. The extract was reduced to a small volume (a few millilitres) using a rotary evaporator. The organic extract was desulfurized on activated copper and then purified as described below (Fig. 1). Microwave extraction procedure Apparatus. FMW-assisted extractions in open cells were performed at a frequency of 2450 MHz using a SOXWAVE 100 Table 1 Extracted amount of sediment by Soxhlet and FMW-assisted methods and weight and number percentage, organic carbon and mineral carbon content (weight-%) for each sub-fraction of Arcachon sediment.Sediment Extracted amount/g Weight distribution (%) Number distribution (%) Organic carbon content (weight-%) Mineral carbon content (weight-%) Arcachon sediment: < 500 mm 1 Arcachon sediment: 500–300 mm 1 25 24 0.27 0.01 Arcachon sediment: 300–125 mm 1 45 32 0.30 0.00 Arcachon sediment: 125–63 mm 1 14 11 0.97 0.10 Arcachon sediment: 63–40 mm 0.5 5 5 2.45 0.25 Arcachon sediment: 40–15 mm 0.5 9 17 7.60 0.35 Arcachon sediment: 15–mm 0.3 2 11 7.90 0.25 Silty sediment: OPCB 5b 10 Sandy sediment: OPCB 9b 10 Naturally moist Arcachon sediment 2 Fig. 1 Schematic diagram of the sample preparation procedure. 6 Analyst, 1999, 124, 5–14apparatus (Prolabo) with a programmable heating power (from 30 to 300 W). The use of focused microwaves allowed homogeneous and reproducible treatment of samples.The SOXWAVE 100 operates at atmospheric pressure. Procedure. The procedure has been optimised on a certified matrix, NIST SRM 1941a.12 The FMW extractions were performed using conditions as close as possible to those of Soxhlet extractions using 0.3–10 g of sediment (Table 1). A solution containing the perdeuterated PAHs used for quantification was added to the matrix prior to the extraction. A level of 30% of moisture (g of water per g of sediment) was added to the freeze-dried sediment in the vessel, after which 30 mL of dichloromethane were added.The power (30 W) and time (10 min) were programmed. Blank experiments were performed. The extract was filtered and reduced to a small volume (a few millilitres) using a rotary evaporator. The organic extract was desulfurized on activated copper and then purified (Fig. 1). Purification The extract was purified on a micro-column containing alumina by eluting with dichloromethane. The purified extract was then fractionated on a micro-column containing silica in order to collect separately saturated and aromatic compounds eluted with, respectively, pentane and pentane–dichloromethane (65 + 35, v/v).15 The purified aromatic fraction was finally reconcentrated to a few microlitres in isooctane and analysed by gas chromatography-mass spectrometry (GC-MS). Gas chromatography-mass spectrometry conditions The analyses were performed on an HP 5980 Series II gas chromatograph (Hewlett-Packard, Palo Alto, CA, USA) equipped with a splitless injector (purge delay: 1 min; purge flow: 60 mL min21). The injector was maintained at 270 °C.The temperature program was: 50 °C (2 min) to 290 °C (20 min) at 5 °C min21. The carrier gas was helium at a constant flow rate of 1 mL min21. The capillary column used was a PTE-5 (Supelco, Bellefonte, PA, USA), 60 m 3 0.25 mm id (0.25 mm film thickness). The gas chromatograph was coupled to an HP 5972 Mass Selective Detector (MSD) (electron impact: 70 eV, voltage: 2000 V) operated in the single ion monitoring (SIM) mode using the molecular ion of each compound at 1.23 scans s21.The interface temperature was maintained at 290 °C. The PAHs were quantified relative to perdeuterated PAHs. The response factors of the different compounds were measured by injecting a solution of SRM 2260, containing 23 PAHs at certified concentrations and spiked with perdeuterated compounds used as internal standards for the extraction of the samples.Some co-elutions were noted between structural isomers: (i) chrysene co-eluted with triphenylene; (ii) benzo[b]fluoranthene co-eluted with benzo[j]fluoranthene and benzo[k]fluoranthene; (iii) dibenz[a,h]anthracene co-eluted with dibenz[a,c]anthracene. The concentrations given for the compounds suffering from these co-elutions take this factor into account: Chry* = Chry + Trip, BF = BbF + BjF + BkF, DaA = DahA + DacA.Carbon analyses Each fraction was analysed with a LECO CS 125 carbon analyser (LECO, MI, USA), according to Cauwet,16 to evaluate the total carbon and organic carbon content. Results and discussion Extraction efficiency of PAHs from a muddy sediment and from sub-fractions Characteristics of sediment. Arcachon sediment is a silt. The grain size distribution was determined by sieving (% in Table 2 PAH concentrations (ng g21) of the different fractions of Arcachon sediment obtained by Soxhlet extraction.Conc. = concentrationa < 500 mm 500–300 mm 300–125 mm 125–63 mm 63–45 mm 45–15 mm 15–0 mm Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) P 182 ± 52 28 279 ± 257 92 101± 21 20 224 ± 11 5 246 ± 9 4 283 ± 16 6 234 ± 45 19 Fluo 423 ± 100 24 637 ± 477 75 244 ± 72 30 566 ± 45 8 627 ± 45 7 500 ± 45 9 280 ± 90 32 Pyr 641 ± 88 14 550 ± 398 72 243 ± 64 27 595 ± 77 13 944 ± 125 13 959 ± 126 13 598 ± 220 37 BaA 179 ± 16 9 296 ± 223 75 128 ± 32 25 309 ± 12 4 322 ± 29 9 239 ± 48 20 136 ± 58 43 Chry* 443 ± 16 4 353 ± 241 68 179 ± 40 22 466 ± 16 3 656 ± 40 6 792 ± 16 2 555 ± 77 14 BF 569 ± 129 23 678 ± 477 70 307 ± 71 23 946 ± 39 4 1305 ± 10 1 1641 ± 123 7 1226 ± 137 11 BeP 254 ± 16 6 261 ± 183 70 120 ± 29 24 377 ± 19 5 520 ± 10 2 680 ± 30 4 530 ± 70 13 BaP 230 ± 24 10 337 ± 250 74 160 ± 44 27 399 ± 30 8 457 ± 87 19 339 ± 98 29 220 ± 96 44 Per 86 ± 4 4 107 ± 80 75 51 ± 13 26 125 ± 7 6 152 ± 18 12 151 ± 17 12 157 ± 33 21 IP 324 ± 25 8 306 ± 235 77 141 ± 36 25 401 ± 6 2 599 ± 53 9 850 ± 111 13 736 ± 54 7 BP 225 ± 5 2 253 ± 185 73 120 ± 33 28 347 ± 8 2 470 ± 19 4 602 ± 3 1 521 ± 70 13 DaA 64 ± 2 4 57 ± 39 69 27 ± 5 18 88 ± 2 2 129 ± 12 9 159 ± 17 10 135 ± 35 26 SPAHs 3619 ± 413 11 4115 ± 297372 1822 ± 449 25 4843 ± 214 4 6427 ± 414 6 7196 ± 254 4 5329 ± 939 18 a PAH identification: P = phenanthrene; Fluo = fluoranthene; Pyr = pyrene; BaA = benzo[a]anthracene; Chry* = chrysene (Chry) + triphenylene (Trip); BF = benzo[b]fluoranthene (BpF) + benzo[j]fluoranthene (BjF) + benzo[k]fluoranthene (BkF); BeP = benzo[e]pyrene; BaP = benzo[a]pyrene; Per = perylene; IP = Indeno[1,2,3-cd]pyrene; BP = benzo[ghi]perylene; DaA = dibenz[a,h]anthracene (DahA) + dibenz[a,c]anthracene (DacA).Fig. 2 Correlation between organic carbon content and the concentration of the sum of PAHs in sub-fractions of Arcachon sediment.Analyst, 1999, 124, 5–14 7weight) and by laser diffraction (% in number) (Table 1). In both cases, the major fraction is medium sands (300–125 mm). If the weight of bulk matrix which has been sieved, corrected for the percentage of moisture, is compared with the sum of the weight of each freeze-dried sub-fraction, the difference is 7.5%. This error is due to the loss of the finer particles during the removal of water by centrifugation.Organic and mineral carbon contents of each fraction were determined (Table 1). Fractions of the finest particles (40–15 and 15–0 mm) have a significant organic carbon content. PAH content in bulk sediment. The study of the bulk sediment (sieved at 2 mm to remove parts of shell or stone) was not possible because of problems of homogeneity. RSDs of PAH concentrations were above 50%. The sample amount of a few grams was not representative owing to the presence of vegetal fragments.Hence, the studied sediment was not strictly bulk but was sieved at 500 mm for this study. PAH concentrations obtained by Soxhlet extraction are given in Table 2. The RSD is under 28% for individual compounds and 11% for the sum of PAHs. The level of contamination is a few mg g21 for the sum of the PAHs studied. The profile of contamination shows that the contamination is both pyrolytic and petrogenic. There is a wide range of the parent PAHs in similar abundance which is characteristic of pyrolytic input and the presence of alkylated compounds shows an additional input of petrogenic contaminants. 17 Indeed, this area is fairly urbanised and the sample location is a yachting harbour. Grain-size distribution of PAHs. Concentrations of PAHs for each sub-fraction obtained by Soxhlet extraction are given in Table 2. If the contamination of the bulk sediment (0–500 mm) is calculated according to the weight percentage and the concentration of each sub-fraction, the result (3602 ng g21) is in agreement with the experimental value (3619 ± 413 ng g21) for the sum of PAHs.This shows that the fractionation is accurate and that the extractions performed on bulk sediment or subfractions are correct. The RSDs of concentrations obtained by Soxhlet extraction for the different sub-fractions are very different and depend on Fig. 3 Relative distribution of PAHs in sub-fractions of Arcachon sediment. PAH identification as in Table 2.Table 3 PAH concentrations (ng g21) of the < 500 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture. Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 145 ± 39 27 202 ± 44 22 80 ± 21 111 ± 24 Fluo 385 ± 106 27 610 ± 364 60 91 ± 25 144 ± 86 Pyr 581 ± 134 23 778 ± 287 37 91 ± 21 121 ± 45 BaA 187 ± 45 24 333 ± 201 60 104 ± 25 186 ± 112 Chry* 334 ± 60 18 532 ± 200 38 75 ± 14 120 ± 45 BF 600 ± 129 21 960 ± 413 43 105 ± 23 169 ± 73 BeP 241 ± 50 21 382 ± 157 41 95 ± 20 150 ± 62 BaP 233 ± 60 26 404 ± 232 57 101 ± 26 176 ± 101 Per 75 ± 18 24 145 ± 86 59 87 ± 21 169 ± 100 IP 293 ± 73 25 413 ± 126 30 90 ± 23 127 ± 39 BP 219 ± 51 23 339 ± 140 41 97 ± 23 151 ± 62 DaA 58 ± 21 36 83 ± 41 50 91 ± 33 130 ± 64 · PAHs 3351 ± 778 23 5180 ± 2275 44 93 ± 21 143 ± 63 a PAH identification as in Table 2.Table 4 PAH concentrations (ng g21) of the 500–300 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture.Conc. = Concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 85 ± 18 22 557 ± 476 86 30 ± 6 200 ± 171 Fluo 278 ± 69 25 1170 ± 878 75 44 ± 11 184 ± 138 Pyr 251 ± 52 21 977 ± 727 74 46 ± 9 178 ± 132 BaA 137 ± 31 23 572 ± 381 67 46 ± 10 193 ± 129 Chry* 174 ± 45 26 592 ± 367 62 49 ± 13 168 ± 104 BF 322 ± 87 27 1106 ± 705 64 47 ± 13 163 ± 104 BeP 118 ± 30 25 426 ± 276 65 45 ± 11 163 ± 106 BaP 165 ± 45 27 658 ± 454 69 49 ± 13 195 ± 135 Per 51 ± 12 24 222 ± 174 79 48 ± 11 207 ± 163 IP 137 ± 42 30 506 ± 350 69 45 ± 14 165 ± 114 BP 120 ± 35 29 460 ± 325 71 47 ± 14 182 ± 128 DaA 30 ± 9 31 93 ± 55 59 53 ± 16 163 ± 96 · PAHs 1866 ± 467 25 7340 ± 5166 70 45 ± 11 178 ± 126 a PAH identification as in Table 2. 8 Analyst, 1999, 124, 5–14the composition of the fraction.The RSD for the 500–300 mm fraction for the sum of PAHs is 72%. This might be explained, as stated previously, by the non-representative nature of the small amount (a few grams) of sediment owing to the presence of vegetal fragments. The RSD for the 300–125 mm fraction is lower but remains significant (25%). The concentrations obtained for the extracts of the 125–63, 63–40 and 15–40 mm fractions are very reproducible ( < 6%). The RSD obtained for the 0–15 mm fraction is more significant (18%) and might be explained by the very small sample amount (0.3 g) which is limited by the total amount of this fraction (3.2 g).Each sub-fraction has a different contamination level. Concentrations in the 500–300 mm fraction are significant and can be explained by the presence of vegetal fragments which Table 5 PAH concentrations (ng g21) of the 300–125 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture.Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 152 ± 3 2 135 ± 24 18 150 ± 3 134 ± 24 Fluo 324 ± 10 3 351 ± 33 10 133 ± 4 144 ± 14 Pyr 282 ± 1 0 321 ± 26 8 116 ± 0 132 ± 11 BaA 151 ± 16 10 177 ± 30 17 118 ± 13 138 ± 23 Chry* 214 ± 10 5 235 ± 36 15 120 ± 6 131 ± 20 BF 355 ± 59 17 451 ± 92 20 116 ± 19 147 ± 30 BeP 134 ± 20 15 174 ± 36 21 112 ± 17 145 ± 30 BaP 173 ± 26 15 229 ± 51 22 108 ± 16 143 ± 32 Per 55 ± 6 12 73 ± 21 28 108 ± 12 143 ± 41 IP 145 ± 14 9 208 ± 55 27 103 ± 10 148 ± 39 BP 127 ± 17 13 169 ± 43 26 106 ± 14 141 ± 36 DaA 32 ± 7 20 35 ± 1 3 119 ± 26 130 ± 4 · PAHs 2145 ± 180 8 2559 ± 387 15 118 ± 10 140 ± 21 a PAH identification as in Table 2.Table 6 PAH concentrations (ng g21) of the 125–63 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture. Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 204 ± 13 7 233 ± 70 31 91 ± 6 104 ± 31 Fluo 516 ± 24 5 581 ± 115 20 91 ± 4 103 ± 20 Pyr 567 ± 44 8 651 ± 86 13 95 ± 7 109 ± 14 BaA 283 ± 13 5 302 ± 51 17 92 ± 4 98 ± 17 Chry* 449 ± 28 6 475 ± 58 12 96 ± 6 102 ± 12 BF 845 ± 45 5 940 ± 159 17 89 ± 5 99 ± 17 BeP 335 ± 26 8 373 ± 61 16 89 ± 7 99 ± 16 BaP 368 ± 15 4 428 ± 71 17 92 ± 4 107 ± 18 Per 126 ± 6 5 142 ± 31 22 101 ± 5 114 ± 25 IP 375 ± 32 8 431 ± 62 14 94 ± 8 107 ± 15 BP 324 ± 21 6 370 ± 60 16 93 ± 6 107 ± 17 DaA 75 ± 11 15 85 ± 2 2 85 ± 13 97 ± 2 · PAHs 4467 ± 234 5 5002 ± 774 15 92 ± 5 103 ± 16 a PAH identification as in Table 2.Table 7 PAH concentrations (ng g21) of the 63–40 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture. Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 186 ± 24 13 220 ± 12 5 76 ± 10 89 ± 5 Fluo 556 ± 41 7 649 ± 33 5 89 ± 7 104 ± 5 Pyr 823 ± 22 3 927 ± 106 11 87 ± 2 98 ± 11 BaA 288 ± 23 8 371 ± 32 9 89 ± 7 115 ± 10 Chry* 623 ± 35 6 718 ± 42 6 95 ± 5 109 ± 6 BF 1166 ± 99 8 1346 ± 108 8 89 ± 8 103 ± 8 BeP 481 ± 57 12 563 ± 30 5 93 ± 11 108 ± 6 BaP 391 ± 8 2 494 ± 54 11 86 ± 2 108 ± 12 Per 153 ± 13 8 169 ± 15 9 101 ± 9 111 ± 10 IP 520 ± 33 6 671 ± 92 14 87 ± 6 112 ± 15 BP 439 ± 44 10 526 ± 29 6 93 ± 9 112 ± 6 DaA 97 ± 14 15 127 ± 15 12 75 ± 11 98 ± 12 · PAHs 5724 ± 359 6 6780 ± 218 3 89 ± 6 105 ± 3 a PAH identification as in Table 2.Analyst, 1999, 124, 5–14 9greatly adsorb lipophilic PAHs. Raoux and Garrigues13 showed that concentrations in vegetal debris are 10–25 times higher than those in the sediment. Fractions less than 63 mm are also highly contaminated. The PAH level can be compared with the organic carbon content (Fig. 2). The two are not strictly correlated but a tendency can be observed.The relationship between organic carbon content and the contamination level based on hydrophobicity is generally accepted.18 Karickhoff et al.19 and Rao et al.20 showed the association of compounds with the finest and richest organic matter particles. However, such relationships are not always strictly observed. Raoux and Garrigues13 observed an enrichment of fine particles for sediments with low contamination but an enrichment in coarse fractions for highly contaminated sediments. The association of PAHs in sediment is not simply a result of their hydrophobicity but also the result of the characteristics of the different sedimentation processes which affect each specific location.The adsorption of contaminants on to particles rich in organic carbon content may be followed by internal diffusion as is the case for most lipophilic compounds. Concerning individual compounds, the fingerprints are different. Fig. 3 represents the weight percentage of each PAH in each fraction. Concentrations of phenanthrene, fluoranthene, benzo[a]anthracene and benzo[a]pyrene are more significant in coarse fractions. Concentrations of benzo[b+j+k]fluoranthene, benzo[e]pyrene, indeno[1,2,3-cd]pyrene, benzo[ghi]perylene and dibenz[ah+ac]anthracene are more significant in fine fractions. This might be explained by the atmospheric source of pyrolytic high molecular weight PAHs associated with fine particles.21,22 Such preferential relative enrichment of high molecular weight compounds has been observed in the smallest particles ( < 10 mm) and might also be explained by preferential adsorption of the more hydrophobic higher molecular weight compounds.23 Table 8 PAH concentrations (ng g21) of the 40–15 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture.Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 257 ± 4 2 272 ± 42 16 91 ± 1 96 ± 15 Fluo 474 ± 33 7 441 ± 15 3 95 ± 7 88 ± 3 Pyr 976 ± 42 4 941 ± 67 7 102 ± 4 98 ± 7 BaA 258 ± 26 10 251 ± 33 13 108 ± 11 105 ± 14 Chry* 743 ± 32 4 767 ± 82 11 94 ± 4 97 ± 10 BF 1562 ± 68 4 1490 ± 83 6 95 ± 4 91 ± 5 BeP 675 ± 29 4 629 ± 41 7 99 ± 4 93 ± 6 BaP 370 ± 60 16 289 ± 97 33 109 ± 18 85 ± 29 Per 154 ± 7 5 138 ± 27 20 102 ± 5 91 ± 18 IP 760 ± 30 4 684 ± 35 5 89 ± 4 80 ± 4 BP 628 ± 22 4 597 ± 40 7 104 ± 4 99 ± 7 DaA 156 ± 6 4 180 ± 12 7 98 ± 4 113 ± 8 · PAHs 6929 ± 162 2 6680 ± 320 5 96 ± 2 93 ± 4 a PAH identification as in Table 2.Table 9 PAH concentrations (ng g21) of the 15–0 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture. Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 122 ± 26 21 160 ± 16 10 52 ± 11 68 ± 7 Fluo 249 ± 39 15 240 ± 26 11 89 ± 14 86 ± 9 Pyr 569 ± 95 17 530 ± 63 12 95 ± 16 89 ± 11 BaA 160 ± 7 4 132 ± 11 8 118 ± 5 97 ± 8 Chry* 487 ± 4 1 483 ± 13 1 88 ± 1 87 ± 2 BF 1098 ± 47 4 1197 ± 98 8 90 ± 4 98 ± 8 BeP 454 ± 17 4 518 ± 40 8 86 ± 3 98 ± 8 BaP 258 ± 37 14 188 ± 46 24 117 ± 17 85 ± 21 Per 121 ± 12 10 136 ± 3 2 77 ± 8 87 ± 2 IP 521 ± 44 8 606 ± 38 6 71 ± 6 82 ± 5 BP 482 ± 24 5 518 ± 52 10 93 ± 5 99 ± 10 DaA 95 ± 6 6 151 ± 32 21 70 ± 4 112 ± 24 · PAHs 4615 ± 333 7 4697 ± 125 3 87 ± 6 88 ± 2 a PAH identification as in Table 2.Fig. 4 Comparison of recoveries of the sum of PAHs for FMW extraction without and with 30% of moisture compared with Soxhlet extraction for each sub-fraction of Arcachon sediment. 10 Analyst, 1999, 124, 5–14Hence, there is a difference in the association of compounds. An extraction method must be efficient for all compounds whatever their origin and the grain size of the particles. Comparison of Soxhlet extraction and FMW-assisted extraction.Concentrations of PAHs for each sub-fraction obtained by FMW-assisted extraction for a dry matrix or a remoistened dry matrix (30% of moisture) are given in Tables 3–9. The extraction recovery for the 0–500 mm fraction obtained by FMW is good compared with Soxhlet extraction being 93% for the sum of PAHs. With 30% of moisture, the recovery is higher, but the RSD is very high (44%). Hence, in this case, the results are not significant.For the 500–300 mm fraction, the results are not interpretable because of the heterogeneity of this fraction which is rich in vegetal matter, which was also the case with Soxhlet extraction. For the fractions from 300–125 to 15–0 mm, RSDs for FMW extraction with or without water are equal to, or lower than, those obtained by Soxhlet extraction (except for FMW extraction with 30% of moisture for the 125–63 mm fraction). Data for the sum of PAHs are summarized in Fig. 4, which shows the recovery for FMW compared with Soxhlet extraction.FMW extraction affords better recoveries than Soxhlet extraction for the 300–125 mm fraction (recoveries of 118 and 140%, respectively, for FMW extraction and FMW extraction with 30% of moisture). For the 125–63 and 63–40 mm fractions, recoveries obtained by FMW extraction are acceptable (92 and 89%, respectively), but can be improved by adding moisture to the matrix, giving recoveries of 103 and 105%, respectively.For the 40–15 and 0–15 mm fractions, moisture content does not seem to affect extraction recoveries. Maximum recoveries are 96% for the 40–15 fraction and 88% for the 15–0 mm fraction. All the recoveries are above 80%, which is acceptable when considering the benefits in terms of time and solvent. Fine fractions seem to be more difficult to extract by FMW extraction whereas coarse fractions are better extracted by FMW than Soxhlet extraction. FMW recoveries of the sum of PAHs vary between 140 and 88%.However, this selectivity linked to grain size is not very important, especially if one considers the difference in the RSDs obtained for each fraction. The effect of moisture is not the same for each fraction. For fine and high organic carbon content particles, containing in particular clays (40–15; 15–0 mm), the effect of water on the recovery is not significant. Improvements of up to 22% are observed for coarse and low organic carbon content particles (300–125; 125–63; 63–40 mm).This dismisses the hypothesis that an improvement in the extraction efficiency is related to a specific interaction of clays with water under microwave irradiation. The dependence of the extraction efficiency on the analyte was studied. Fig. 5 shows the recoveries of a tricyclic (phenanthrene), a tetracyclic (chrysene), a pentacyclic (benzo[ a]pyrene) and a hexacyclic compound (benzo[ghi]perylene) for the different fractions. The compounds can be associated with different particles in different ways according to their origin.Paschke et al.24 showed the influence of the nature of a sample on the extraction efficiency by supercritical fluid extraction for nitro-PAHs from diesel exhaust particles and diesel soot. Diesel exhaust particles, where PAHs are formed at the same time as the growth of the particles and are adsorbed in the internal structure, are more difficult to extract than diesel soot, where PAHs are adsorbed on the surface.The pyrolytic compounds (associated with finer particles) may thus be more difficult to extract than petrogenic compounds, which are adsorbed on the surface. For phenanthrene, recoveries vary from 52 to 152% depending on the fraction, whereas for the other compounds, recoveries only vary between 86 and 120%. This difference can be explained by the different origin of these compounds (pyrolytic, petrogenic and diagenetic). Benzo[ghi]perylene, the heaviest compound, is well extracted whatever the fraction.This contradicts the perceived idea that high molecular weight compounds are more difficult to extract than low molecular weight compounds. The recoveries of phenanthrene are influenced by moisture whatever the fraction but the reproducibility is lower. The extraction of chrysene in the 300–125, 125–63 and 63–40 mm fractions is improved with moisture, but the improvement in recovery for the 40–15 and 0–15 mm fractions is not significant.For benzo[ghi]perylene, the same tendency is apparent, except for the 0–15 mm fraction for which there is a slight improve- Fig. 5 Comparison of recoveries for FMW extraction without and with 30% of moisture compared with Soxhlet extraction for each sub-fraction of Arcachon sediment for (a) phenanthrene, (b) chrysene, (c) benzo[a]pyrene and (d) benzo[ghi]perylene. Analyst, 1999, 124, 5–14 11ment. For benzo[a]pyrene, the extraction of the 40–15 and 0–15 mm fractions is better without moisture, but the RSD is significant.Other pentacyclic compounds (benzo[e]pyrene, perylene) do not show the same behaviour. Application to other sediments The efficiency and selectivity of FMW extraction were tested for other matrices with different contamination levels and different grain sizes. The method can be validated if the treatment of the sample does not influence the contamination level determination and does not change the biomarker parameters useful for the determination of contaminant origin.Table 10 shows the results for a muddy sediment, ‘OPCB 5b’ for which 94% of particles are under 63 mm. Three Soxhlet and three FMW extractions of the dry matrix and the remoistened dry matrix (30% of moisture) were performed. The RSDs are comparable. Recoveries of the sum of PAHs obtained for FMW extraction are 84% without water and 101% with 30% of water. Individual recoveries for FMW extraction without water are comparable and close to 90–100%, except for phenanthrene, anthracene and perylene which are, respectively, 64, 53 and 73%.With 30% of moisture, recoveries are better and vary between 87 and 120%. In this case, there is no extraction selectivity. This is confirmed by the biomarker parameters (concentration ratios of isomers) calculated for each extraction technique, which are comparable and generate the same interpretation of contamination origin. Table 10 shows the results for a sandy sediment, ‘OPCB 9b’ in which 15% of particles are under 63 mm.Whatever the extraction technique, the reproducibility is very poor, owing to the heterogeneity of the matrix, the relatively small sampling amount and the presence of vegetal debris. The ratio of phenanthrene to anthracene is influenced the most by the Table 10 Comparison of PAH concentrations (ng g21) of ‘OPCB 5b’ (silty sediment) and ‘OPCB 9b’ (sandy sediment) obtained by FMW extraction without and with 30% of moisture and by Soxhlet extraction (SOX).Conc. = concentrationa OPCB 5b FMW FMW (30% of moisture) SOX FMW (30%) FMW: of moisture: Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) SOX (%) SOX (%) P 68 ± 2 3 127 ± 7 6 106 ± 14 14 64 ± 2 120 ± 7 A 11 ± 1 5 23 ± 4 16 21 ± 6 28 53 ± 3 112 ± 18 Fluo 102 ± 3 3 96 ± 7 7 102 ± 3 2 100 ± 3 95 ± 7 pyr 82 ± 5 6 83 ± 5 6 84 ± 2 2 98 ± 6 99 ± 6 BaA 58 ± 5 8 55 ± 5 10 60 ± 1 2 97 ± 8 92 ± 9 Chry 68 ± 6 9 66 ± 6 10 71 ± 3 4 97 ± 9 93 ± 9 BF 117 ± 6 5 107 ± 8 7 116 ± 9 8 101 ± 5 92 ± 7 BeP 49 ± 3 5 49 ± 3 5 53 ± 1 2 92 ± 5 93 ± 5 BaP 61 ± 3 5 57 ± 4 7 65 ± 4 6 95 ± 5 87 ± 6 Per 105 ± 1 1 153 ± 8 5 143 ± 2 1 73 ± 1 107 ± 6 IP 42 ± 2 5 56 ± 3 4 58 ± 4 7 72 ± 5 97 ± 6 BP 49 ± 2 4 50 ± 3 6 55 ± 2 4 90 ± 4 91 ± 6 DaA 12 ± 1 6 12 ± 1 8 14 ± 2 12 86 ± 5 88 ± 7 SPAHs 824 ± 37 4 933 ± 53 6 927 ± 51 6 89 ± 4 101 ± 6 ratio ratio ratio P :A 6.2 ± 0.3 5 5.6 ± 0.6 11 5.2 ± 0.7 13 Fluo : Pyr 1.2 ± 0.0 0 1.2 ± 0.0 0 1.2 ± 0.0 0 Chry : BaP 1.2 ± 0.0 0 1.2 ± 0.0 0 1.2 ± 0.0 0 BeP : BaP 0.8 ± 0.0 0 0.9 ± 0.0 0 0.8 ± 0.1 13 OPCB 9b FMW FMW (30% of moisture) SOX FMW (30%) FMW: of moisture: Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) SOX (%) SOX (%) P 28 ± 11 39 36 ± 6 17 48 ± 17 35 58 ± 23 75 ± 12 A 4 ± 2 50 6 ± 3 50 10 ± 6 60 40 ± 22 60 ± 29 Fluo 53 ± 36 68 24 ± 8 33 66 ± 50 76 80 ± 54 36 ± 12 pyr 41 ± 30 73 18 ± 7 39 46 ± 37 80 89 ± 66 39 ± 15 BaA 26 ± 25 96 16 ± 16 100 34 ± 29 85 76 ± 75 47 ± 49 Chry 29 ± 24 83 19 ± 18 95 35 ± 23 66 83 ± 69 54 ± 52 BF 48 ± 45 94 26 ± 24 92 44 ± 25 57 109 ± 101 59 ± 54 BeP 19 ± 17 89 11 ± 9 82 18 ± 10 56 106 ± 90 61 ± 48 BaP 28 ± 28 100 15 ± 14 93 24 ± 17 71 117 ± 116 63 ± 57 Per 17 ± 10 59 10 ± 5 50 20 ± 6 30 85 ± 52 50 ± 23 IP 16 ± 15 94 12 ± 8 67 15 ± 7 47 107 ± 98 80 ± 55 BP 18 ± 16 89 9 ± 6 67 14 ± 7 50 129 ± 108 64 ± 42 DaA 5 ± 4 80 2 ± 2 100 4 ± 2 50 125 ± 102 50 ± 52 SPAHs 332 ± 260 78 204 ± 126 62 378 ± 235 62 88 ± 69 54 ± 33 ratio ratio ratio P :A 9.4 ± 4.1 44 6.7 ± 1.9 28 4.9 ± 1.2 24 Fluo : Pyr 1.4 ± 0.3 21 1.4 ± 0.1 7 1.5 ± 0.1 7 Chry : BaP 1.2 ± 0.2 17 1.3 ± 0.1 8 1.2 ± 0.3 25 BeP : BaP 0.8 ± 0.2 25 0.8 ± 0.1 13 0.8 ± 0.2 25 a PAH identification as in Table 2. 12 Analyst, 1999, 124, 5–14vegetal debris, but the difference is not significant. The biomarker parameters generate the same interpretation. Therefore, it is impossible to test the precision of the method with this matrix. However, the order of magnitude, between 100 and 600 ng g21, is in agreement with that obtained by Soxhlet extraction.Extraction of a naturally moist matrix The extraction of a naturally moist matrix (30% of moisture) by FMW extraction was compared with the extraction of a freezedried matrix by the same technique and by the conventional method of Soxhlet extraction and with the extraction of a freezedried matrix to which 30% of water had been added before the extraction (remoistened dry matrix).Drying of the matrix is generally used to improve the extraction efficiency of Soxhlet extraction. Moreover, a dry matrix is easier to store than a frozen matrix and avoids alteration of the matrix by bacterial degradation, for example. However, this step is very long and can lead to a loss of volatile compounds. Table 11 shows the concentrations obtained for a dry matrix, a remoistened dry matrix and a naturally moist matrix by FMW extraction and for a dry matrix by Soxhlet extraction (n = 3).Compared with Soxhlet extraction, the recovery obtained for the dry matrix by FMW extraction is 91% for the sum of PAHs. The recovery is improved by the addition of 30% of moisture before extraction. Concentrations are comparable to those obtained by Soxhlet extraction with a recovery of 104% for the sum of PAHs. The extraction of the naturally moist matrix is less efficient than for the remoistened dry matrix with a recovery of 84% for the sum of PAHs.This might be because some of the water is trapped in the pores and prevents access of the solvent (dichloromethane). The use of other solvent mixtures, richer in ethanol to dissolve the water better or having a higher boiling-point, such as heptane–ethanol (80 + 20, v/v), does not improve the recovery. The concentration obtained with heptane–ethanol (80 + 20, v/v) for the sum of PAHs is 3.7 ± 0.6 mg g21 compared with 3.6 ± 0.3 mg g21 obtained with dichloromethane.The use of a drying agent, such as anhydrous sodium sulfate, was also tested but the concentration obtained (3.8 ± 0.4 mg g21) was not significantly better. However, the extraction efficiency is acceptable for environmental matrices. The RSDs are comparable to those obtained for a dry matrix (under 20%, except for anthracene) and lower than those obtained by Soxhlet extraction. Table 11 also shows the biomarker parameters calculated in each case from the concentrations of isomers. The FMW treatment of the sample has no influence on the parameters, whatever the state of the matrix.There is no selectivity for the compounds studied. The ratios of the concentrations of phenanthrene to anthracene are all less than 10. The ratios of the concentrations of benzo[e]pyrene to benzo[a]pyrene are all less than 2. This suggests a pyrolytic contamination. The ratios of the concentrations of fluoranthene to pyrene are all less than 1, whereas the ratios of the concentrations of chrysene to benzo[a]anthracene are all greater than 1.This suggests a petrogenic contamination. In conclusion, the analysis of a naturally moist matrix can be performed rapidly by FMW extraction without a drying step with good precision and reproducibility in terms of individual concentrations and for biomarker studies. The FMW apparatus is not bulky and might be used for field work during an oceanographic cruise on a boat for example. The only requirement is refrigeration with water circulation.No gas supply is necessary. It should be possible to perform homogenisation of the matrix (removal of stones and shells), extraction, reconcentration under microwave irradiation, and purification on micro-columns as the sampling is performed. Results could then be obtained rapidly after sampling. Conclusions FMW extraction at atmospheric pressure is a good alternative to Soxhlet extraction for the analysis of PAHs in sediments with various grain size distributions.There is no extraction selectivity concerning contaminants or particles. Good recoveries and good reproducibility are obtained. The procedure allows a Table 11 Comparison of PAH concentrations (ng g21) obtained by FMW extraction without and with 30% of moisture for the freeze-dried Arcachon sediment, by FMW extraction for the naturally moist sediment and by Soxhlet extraction (SOX) for the freeze-dried sediment. Conc. = concentrationa FMW (dry matrix) FMW (remoistened dry matrix) FMW (naturally moist matrix) SOX (dry matrix) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) FMW (dry matrix): SOX (%) FMW (remoistened dry matrrix): SOX (%) FMW (naturally moist matrix): SOX (%) P 457 ± 78 17 515 ± 8 2 327 ± 65 20 357 ± 231 65 128 ± 22 144 ± 2 92 ± 18 A 84 ± 11 13 79 ± 9 11 66 ± 18 28 72 ± 18 25 116 ± 15 109 ± 13 91 ± 25 Fluo 357 ± 68 19 447 ± 23 5 354 ± 57 16 388 ± 59 15 92 ± 18 115 ± 6 91 ± 15 pyr 587 ± 46 8 666 ± 15 2 579 ± 33 6 646 ± 68 11 91 ± 7 1`03 ± 2 90 ± 5 BaA 213 ± 38 18 229 ± 16 7 212 ± 28 13 243 ± 46 19 88 ± 16 95 ± 7 88 ± 12 Chry 393 ± 51 13 425 ± 6 1 340 ± 31 9 442 ± 58 13 89 ± 12 96 ± 1 77 ± 7 BF 626 ± 76 12 718 ± 3 0 556 ± 40 7 722 ± 118 16 87 ± 10 99 ± 0 77 ± 6 BeP 260 ± 29 11 290 ± 2 1 224 ± 13 6 293 ± 25 9 89 ± 10 99 ± 1 76 ± 5 BaP 238 ± 33 14 282 ± 6 2 238 ± 20 8 290 ± 55 19 82 ± 11 97 ± 2 82 ± 7 Per 77 ± 10 13 79 ± 4 5 64 ± 5 7 99 ± 11 11 78 ± 10 80 ± 4 64 ± 5 IP 297 ± 28 9 416 ± 38 9 337 ± 5 1 311 ± 17 5 95 ± 9 134 ± 12 108 ± 1 BP 237 ± 34 14 282 ± 8 3 203 ± 14 7 264 ± 40 15 90 ± 13 107 ± 3 77 ± 5 DaA 56 ± 7 13 76 ± 13 17 65 ± 4 6 77 ± 14 18 73 ± 9 99 ± 17 84 ± 5 SPAHs 3880 ± 484 12 4421 ± 81 2 3565 ± 269 8 4243 ± 625 15 91 ± 11 104 ± 2 84 ± 6 ratio ratio ratio P :A 5.4 ± 0.4 8 6.6 ± 0.8 13 5.1 ± 0.4 8 4.3 ± 5.4 125 Fluo : Pyr 0.6 ± 0.1 11 0.7 ± 0.0 7 0.6 ± 0.1 11 0.4 ± 0.1 14 Chry : BaP 1.9 ± 0.1 5 1.9 ± 0.1 6 1.6 ± 0.1 6 1.2 ± 0.2 20 BeP : BaP 1.1 ± 0.0 4 1.0 ± 0.0 2 0.9 ± 0.0 5 0.6 ± 0.1 13 a PAH identification as in Table 2.Analyst, 1999, 124, 5–14 13reduction of time and solvent amount. 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