Gas chromatographic–mass spectrometric method for polycyclic aromatic hydrocarbon analysis in plant biota

Journal of Chromatography A, 1108 (2006) 240–247

Gas chromatographic–mass spectrometric method for polycyclic aromatic hydrocarbon analysis in plant biota A. Meudec a,∗ , J. Dussauze b , M. Jourdin b , E. Deslandes a , N. Poupart a a

Laboratoire d’Ecophysiologie et de Biotechnologie des Halophytes et des Algues Marines (EA 3877 LEBHAM), Institut Universitaire Europ´een de la Mer, Universit´e de Bretagne Occidentale (UBO), Technopˆole Brest Iroise, Place Nicolas Copernic, 29280 Plouzan´e, France b Laboratoire d’Analyse de Brest Oc´ ean-Pˆole Analytique des Eaux, 120 rue Alexis de Rochon, B.P. 52, 29280 Plouzan´e, France Received 6 October 2005; received in revised form 2 January 2006; accepted 4 January 2006 Available online 26 January 2006

Abstract Using gas chromatography–mass spectrometry, a new method was developed for the identification and the quantification of polycyclic aromatic hydrocarbons (PAHs) in plants. This method was particularly optimised for PAH analyses in marine plants such as the halophytic species, Salicornia fragilis Ball et Tutin. The saponification of samples and their clean up by Florisil solid-phase extraction succeeded in eliminating pigments and natural compounds, which may interfere with GC–MS analysis. Moreover, a good recovery of the PAHs studied was obtained with percentages ranging from 88 to 116%. Application to the determination of PAH in a wide range of coastal halophytic plants is presented and validated the efficiency, the accuracy and the reproducibility of this method. © 2006 Elsevier B.V. All rights reserved. Keywords: Polycyclic aromatic hydrocarbons (PAH); Saponification; Florisil solid-phase extraction; GC–MS; Coastal plants; Oil spill

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. The United States Environmental Protection Agency (EPA) has recommended them as priority pollutants. Their occurrence is due to both natural and anthropogenic sources. Among these sources, pyrogenic (mainly through incomplete combustion of organic materials, such as coal, oil, vegetation or fossil fuels) and petrogenic inputs are the two main sources of PAHs [1,2]. PAHs constitute a variable group of compounds composed of carbon and hydrogen atoms, characterized by two to seven fused aromatic (benzene) rings. This family includes over 100 substances and their physical and chemical characteristics vary with their molecular weight. PAH resistance to oxidation, reduction, and vaporization, increases with molecular weight, whereas their aqueous solubility decreases. PAHs are characterised by “low to very low” water solubility and “low to moderate” volatility [3].

∗

Corresponding author. Tel.: +33 2 98 49 86 68; fax: +33 2 98 49 87 72. E-mail addresses: [email protected] (A. Meudec), [email protected] (N. Poupart). 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.01.010

As consequences, persistence, behaviour and distribution of the PAHs in the environment differ due to these physico-chemical properties. Plant exposure to PAHs may occur through numerous ways. In the neighbourhood of urban and industrial areas, crops and natural vegetation can be polluted by atmospheric deposition on leaves or by root contact with soil particle [4]. In various food categories including vegetables, such as lettuce, potato, carrot, cabbage, endive, spinach or celery, PAHs were detected [5,6]. Indeed, plants grown under PAH-polluted substrates or atmosphere, were shown to be able to take up these compounds, through roots or cuticle, and bioaccumulate them in their tissues [5,6]. In marine environment, one of the main sources of PAHs is accidental oil spill, often leading to pollution of coastal vegetation, in particular in salt marshes. For instance, littoral halophytic plants growing along the Atlantic French coast have been largely affected after Erika oil spill [7]. Petroleum coated to plant shoots and mixed with plant substratum, so symptoms of chemical toxicity were observed on coastal plants exposed to fuel oil [7]. Many methods exist for the extraction and the subsequent quantification of PAHs in soil, sewage sludges, water or air, including solid-phase extraction (SPE), sonication, sequential

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supercritical fluid extraction (SSFE) and focused microwave assisted extraction (FMW) [8–12]. However, these methods cannot be directly applied to PAH analysis in plant. The main problem for analysis of organic pollutants in plants comes from the complexity of the matrix. Plants have a particular tissue structure, which depend on the species and the age, and are highly rich in pigments, essential oil, fatty acids or alcohols. In addition, they contain natural compounds strictly similar in structure to PAHs, well known as hormone (auxins like naphthalene acetic acid) in plants. As these compounds are co-extracted with the PAHs, a clean-up procedure of the extracts may be useful before the quantification. Moreover, halophytic plants growing in salt environment contain high amounts of sodium chloride that could interfere with the extraction procedure. Therefore, there is a need to develop standardised methods for determining PAHs in plants and especially in marine plants. The aim of this study was to provide a new method for routine analysis of PAHs in plants. The development of the GC–MS method was performed using Salicornia fragilis. This plant was chosen as a model for further studies about bioaccumulation of PAHs and physiological impact of fuel oil. Salicornia fragilis (Chenopodiaceae), or glasswort, is an annual, vascular, flowering, apparently leafless halophytic plant that carries articulated, succulent stems [13]. This species, distributed in salt-marshes along the Atlantic coast of Europe, is an obligatory halophyte, containing concentrations of NaCl at concentration similar to the seawater one. Glassworts are edible wild plants, collected by local population for food use as a vegetable. In addition, their low localisation on intertidal area potentially exposed them to accidental marine pollution, such as oil spill. This paper describes a simple and rapid method for PAH extraction and clean-up adapted to plant biota. Extraction was performed using a saponification of the sample. Clean up was based on solid-phase extraction with Florisil column. Method was improved by the study of PAH recoveries in spiked Salicornia samples compared to standard control solution. The developed method was then applied for the quantification of PAHs in different plant samples collected onto areas polluted by the Erika’s oil spill. 2. Experimental 2.1. Chemicals As PAH analyses were carried out in a laboratory accredited by the French agency COmit´e FRanc¸ais d’ACcr´editation (COFRAC), all chemicals were of analytical grade. 2.1.1. PAH standards A polycyclic aromatic hydrocarbons mix containing the 16 priority PAHs (5.00 ± 0.03 mg l−1 stocked in acetone) selected by EPA [1], was purchased from Promochem (Molsheim, France). These 16 PAHs are described in Table 1. Deuterated pyrene {[2 H10 ] pyrene (pyrene-d10 ), 5.00 ± 0.03 mg l−1 in acetone) and benzo[a]pyrene ([2 H12 ] benzo[a]pyrene (benzo[a]pyrene-d10 ), 5.00 ± 0.03 mg l−1 in acetone) with a purity of at least 99.5% (CIL Cluzeau Info Labo, Puteaux,

241

Table 1 List of PAHs studied, code numbers, molecular weights and retention times PAH

Code numbera

m/z fragments

Retention time (min)

Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene-d10 Pyrene Benzanthracene Chrysene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene-d12 Benzo[a]pyrene Indenopyrene Dibenz[a,h]anthracene Benzo[ghi]perylene

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

128 152 154 166 178 178 202 212 202 228 228 252 252 264 252 276 278 276

16.55 19.85 20.19 21.25 23.23 23.33 25.66 25.80 26.13 28.96 29.07 32.71 32.84 33.50 33.64 40.91 41.12 42.96

a

64 153 153 165 176 176 201 106 201 114 114 126 126 132 126 138 139 138

Numbers specified in chromatograms.

France) were used as internal standards for the determination of PAHs into unknown polluted plants samples. 2.1.2. Organic solvents and reagents Ethanol, hexane and dichloromethane of special grade for pesticide residue analysis were purchased from Carlo Erba (Val de Reuil, France). Potassium hydroxide (KOH) and sodium chloride (NaCl) from Prolabo (Paris, France) were of analytical grade. Florisil adsorbent, a registered trade name of US Silica, was purchased from SDS (Peypin, France). 2.1.3. Plant samples Plants of Salicornia fragilis, established as uncontaminated by PAHs, were collected in a natural salt marsh at the “Aber du Conquet” (Finist`ere, France). Fresh aerial green parts of plants were cut off and stored in aluminium foil at −20 ◦ C until analysis. A 5 g sample was used for the PAH extraction and quantification. As PAH concentrations were expressed in ␮g kg−1 dry weight (DW), the dry weight was determined by drying an aliquot of plant shoots at 70 ◦ C until a constant weight was obtained. To our knowledge, no standard reference material (SRM) was available for PAH quantification in halophytic plants. 2.2. Instrumentation A warring blender laboratory shaker with 100 ml bucket (VWR International, France) was used for the plant samples blend. A Turbovap II evaporator (Zymark, Germany) was used for solvent evaporation. Quantification of PAH compounds was performed by highresolution gas chromatography using a HP-5890 gas chromatograph (Hewlett-Packard, USA) equipped with a HP-DB 5 MS column (60 m × 0.25 mm, 0.25 ␮m film thickness) and coupled to a mass spectrometer detector (HP 5972). The initial oven

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temperature was 50 ◦ C. Upon analysis, it was held for 5 min, and then grew steadily to 300 ◦ C at 12 ◦ C min−1 with a final holding of 20 min. The injector, transfer lines (from column to ion-source) and ion-source temperatures were set at 260, 305 and 186 ◦ C, respectively. Helium was used as carrier gas with constant flow (0.9 ml min−1 ). Injection of 1 ␮l of the extract was performed with a HP autosampler Model G 1512A in splitless mode. Data acquisition and integration were carried out with the HPCHEM chromatography software.

ular masses and specific ion m/z ratio of the PAHs (Table 1). For each extraction series, procedural blank (deuterated PAHs) and control sample (PAH standard solution without mattrix) were run. In a way to assign exact retention times to individual PAHs and to calculate relative response factors, PAHs in the control were analysed before samples. Typical chromatograms of total PAHs in standard solution and in spiked-plant sample were shown in Fig. 2. 2.5. Recovery study

2.3. Extraction and clean up of the plant samples Extraction was performed by saponification of 5 g spiked Salicornia. Frozen plant tissues were cut off into small pieces in a 100 ml warring blender with 40 ml of absolute ethanol. The samples were crushed and homogenised for 2 × 1 min and transferred into a 100 ml bottle. After addition of 2 g of KOH, samples were incubated either at 60 ◦ C or at 80 ◦ C (see details in Section 2.5). PAHs were recovered by addition of 30 ml of hexane. A single phase was obtained, and then distilled water (30 ml) was added to fraction the extract into two phases, i.e. saponifiable (water/ethanol) and insaponifiable (hexane) phases. The hexane upper phase, including the emulsion, was transferred into a new bottle (60 ml). Because of its ionic strength, 1 g of NaCl was added in order to break the emulsion phase and to improve the recovery of a reduced amount of hydrophobic compounds. A second wash with distilled water (30 ml) was performed to eliminate traces of ethanol from the hexane phase. Then, 20 ml of the hexane extract was deposited onto a manually prepared Florisil column (6 cm × 6 mm). Previously, Florisil has been baked out overnight at 450 ◦ C to remove organic contaminants and then, deactivated by 5% of distilled water. The column was conditioned with 10 ml of hexane. After depositing the extract, elution was carried out with 20 ml of hexane, followed by 10 ml of hexane:dichloromethane and 10 ml of dichloromethane:ethanol in different proportions. The eluate was finally vacuum-concentrated and resuspended in 500 ␮l of dichloromethane. A 150 ␮l aliquot was used for the determination of PAH concentrations with GC–MS.

Recoveries of tested PAHs were determined in spiked samples of Salicornia fragilis. A 5 g amount of plant tissues were spiked with 1 ml of the standard solution four-fold concentrated (20 ␮g ml−1 ). Samples were immediately processed as described above in Section 2.3. After the saponification steps, recoveries of PAHs were determined as referred to an external standard consisting of a spiked plant sample for which saponification was stopped immediately by addition of hexane (no incubation time). Total recovery rates for each PAH after Florisil purification was calculated referred as PAHs of the standard solution. The concentrations of individual PAHs in each elution fraction were normalized to their respective total concentration in the sample studied. 2.6. Limits of detection The limits of detection were estimated from the signal (S) of PAH peaks determined from a spiked sample and the integration of the noise (N) situated at the retention time of the corresponding PAH on a chromatogram of a non-spiked Salicornia extract. The limits of detection were calculated according to the equation: 3NC LD = S where C is the concentration of the PAH concerned in the spiked sample. 3. Results and discussion

2.4. Gas chromatography calibration On one hand, PAHs were identified from a standard solution of 16 PAHs by GC–MS detecting in scanning mode. By this program, a screening of the molecular weights from 45 to 550 of all compounds present in the standard solution was established. PAHs exhibited relatively simple mass spectra, with specific m/z ratios, as the molecular ion was often the most intense ion (Fig. 1). The mass chromatograms of the individual PAHs were used to establish retention times and appropriate retention time windows. They were finally integrated and the peak height/mass/time data were used for subsequent calibration and quantification. On the other hand, quantification of PAHs in polluted samples was carried out using the selected ion monitoring mode (SIM). The mass spectrometer was calibrated to detect nominal molec-

3.1. Development of extraction and clean up by saponification The assay of PAHs in the living matter can generate many problems in relationship with the effects of matrix and with the low contents of the PAHs compared to those of the co-extracted natural products. The advantages of the saponification method are that it allows to extract the total PAHs with the membrane lipids and to carry out a first step of purification. Indeed, the saponifiable compounds will be transferred to the aqueous phase, whereas the insaponifiable compounds, including PAHs, will be recovered in the organic phase (hexane). This separation will draw aside a very large proportion of the awkward products for the GC analysis. In particular, chlorophylls are present in large quantities in the shoot parts of

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Fig. 1. Examples of specific ion m/z ratios mass spectra in spiked-plant sample: (a) pyrene with 202 and (b) benzo[a]pyrene with 252 as dominant ion.

plants. Because of their non-polar nature and high molecular weight, chlorophylls may interfere with PAH analyses and may be extracted in large amounts into the organic solvent [14]. Through saponification treatment, chlorophylls acquire a hydrophilic character (due to their two ester functions) that makes them go into the water fraction after the first addition of distilled water. In the same way, as shown in Table 2, less than 1% ethyl ester fatty acid was recovered after 2 h at 60 ◦ C or one hour at 80 ◦ C, whereas they were largely present in plant samples. This indicates that the fatty acids and their derivatives (ethyl ester compounds, triglycerides, etc.) were rapidly degraded during the saponification process.

However, separation by saponification is not complete. Compounds such as fatty alcohols, tocopherols, sterols and their derivatives, were rapidly liberated upon incubation. Accordingly, after 2 h at 60 ◦ C, increases of 34 and 81% were observed for sterols and fatty alcohols amounts, respectively. After 30 min at 80 ◦ C, content of these compounds increased by 35 and 117% (Table 2). The extracts need to undergo a second stage of clean up by a purification of the insaponifiable fraction on Florisil. Compared to a magnesium silicate (SiO2 : 84%, MgO: 15.5% and Na2 SO4 : 0.5%), Florisil is an extremely white, hard powdered gel (200 ␮m mesh). Because of the polar properties of the matrix, it is a highly selective adsor-

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Fig. 2. GC–MS chromatograms of the total PAHs in the control sample (PAH standard without matrix) (a) and in a spiked-plant sample (b). The number of each peak indicates an individual PAH noted in Table 1.

bent and may be used to separate compounds from interfering molecules before the chromatographic step, as solid-phase extraction. Florisil has already been used for the cleanup of pesticide residues and other chlorinated hydrocarbons, for the separation of nitrogen compounds from hydrocarbons, for the separation of aromatic compounds from aliphatic–aromatic mixtures, and for similar applications using fats, oils, and waxes [15]. During the extraction of PAHs from plant tissues, Florisil allowed interactions responsible for a good selectivity towards PAHs. It also retained products more polar than the aromatic heavy ones such as benzo[ghi]perylene. The used of fractionated clean-up permits the separation of different chemical family of compounds including degraded PAHs as the hydroxyPAH.

3.2. Optimization of saponification conditions During the saponification process, high temperatures (80–100 ◦ C) are generally used in order to accelerate the chemical transformation of the fatty acid to glycerol and soap. Therefore, we have tested the recoveries of PAHs after different times of incubation at two temperatures (60 and 80 ◦ C). Results are shown in Table 2. For the control sample, recoveries of PAHs under 80 ◦ C were high and ranged from 79 to 104%. In the presence of the plant matrix, extraction of PAHs was more difficult and the percentage of recovery of each PAH was reduced. In order to improve the PAH recuperation, time of incubation was increased but no improvement was observed (Table 2). In the same way, when extracts were

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245

Table 2 Mean recovery rate (±5%) of PAHs from control sample and spiked Salicornia samples with 16 PAH mixture (5 mg l−1 ) after 0, 2, 4, 8 and 16 h at 60 ◦ C or 0, 0.5, 1, 2 and 3 h at 80 ◦ C (n = 3) Samples Plant sample 0a

60a

0b

2b

Control sample 80a 4b

8b

16b

0.5b

1b

2b

3b

0a

80a

0b

0.5b

1b

2b

3b

Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzanthracene Chrysene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene Indenopyrene Dibenz[a,h]anthracene Benzo[ghi]perylene

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

79 70 74 68 66 68 63 77 65 66 67 66 66 70 69 63

72 61 72 60 65 64 61 76 68 63 68 69 64 67 70 63

68 61 67 58 64 61 62 76 67 63 69 69 65 69 69 66

65 68 56 68 62 61 63 72 68 67 63 65 62 62 64 63

70 65 70 64 65 62 60 74 66 63 69 67 64 65 66 64

76 66 70 67 67 76 60 79 68 64 73 67 64 69 74 65

76 66 70 62 68 67 66 79 69 66 69 68 66 62 70 67

69 60 67 58 64 61 59 74 66 64 68 67 65 65 73 66

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

80 85 92 87 93 92 99 90 95 93 93 89 90 90 87 81

80 91 93 83 96 96 100 93 99 100 95 92 95 89 86 83

88 93 97 86 100 98 102 97 102 104 101 98 97 93 89 84

79 87 92 83 91 93 98 92 99 99 94 95 92 87 88 80

Psi–psi carotene Phytol Sterols Fatty alcohol Ethyl ester fatty acid

100 100 100 100 100

77 151 134 181 1

79 122 160 211 1

70 133 159 222 1

68 81 164 228 0

76 158 135 217 2

80 177 155 314 1

81 115 170 324 0

78 106 177 327 0

– – – – –

– – – – –

– – – – –

– – – – –

– – – – –

a b

Temperature (◦ C). Time (h).

incubated at 60 ◦ C, no differences were observed in the percentages of recovery. The monitoring of natural compounds contained in plants showed that saponification tended to liberate alcohol-type compounds such as phytol, sterol or fatty alcohol. After the first hours of incubation, phytol, a residue of the saponification of chlorophylls, showed high concentrations (percentage >100%). Then it decreased with the progress of incubation. In the same way, sterols or fatty alcohols present high percentages of recovery after the saponification process. They increased with time of incubation. The release of these compounds proves the lyses of cellular membrane and supports the intracellular access to the quantification of PAHs. Globally, less compounds originating from plants were recovered upon incubation at 60 ◦ C than at 80 ◦ C. However, saponification at 60 ◦ C is softer than that at 80 ◦ C and allows preserving some compounds such as degraded PAHs more sensible to temperature than PAHs. Accordingly, we have chosen 16 h at 60 ◦ C and 3 h at 80 ◦ C as incubation times for the saponification of Salicornia samples. 3.3. Optimization of SPE clean-up conditions Compositions of the eluting solvent were tested in order to establish the most powerful eluent for the recovery of PAHs during the clean up step on Florisil (Table 3). Therefore, mix of 95:5, 90:10, 80:20, 70:30, 55:45 and 30:70 (v/v) of hexane:dichloromethane, and 75:25 and 0:100 of

dichloromethane:ethanol were used to augment the solvent polarity and the affinity of the compounds for it. The results showed that the recovery of high molecular weight PAHs increased with the solvent polarity. Low (two and three benzene rings: from naphthalene to acenaphthene) and medium (four rings: from fluorene to chrysene) molecular weight PAHs were primarily eluted with 100% of hexane, whereas high molecular weight compounds (more than five rings: from benzo[b]fluoranthene to benzo[ghi]perylene) were eluted adding dichloromethane in hexane. As a result, the recuperation of 100% of each PAH was complete by elution with 70/30 of hexane/dichloromethane. However, fatty alcohols, tocopherols, sterols and their derivatives are co-extracted with PAHs. Most of them are strongly adsorbed onto Florisil. Their elution began with 70/30 of hexane/dichloromethane. Therefore, as elution with 80/20 hexane/dichloromethane did not removed them and allowed a good recovery of PAHs, this solvent combination was retained as the more suitable for the purification procedure of Salicornia extracts. In those conditions, the extract was clear enough to avoid contamination of the ion source, while many compounds were still present in the extract injected for GC–MS analysis. 3.4. Detection limits Detection limits of the improved method are reported in Table 4. The values ranged between 3.5 and 18.0 ␮g of PAH per

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246

Table 3 Recovery rates (±standard deviation (%)) of PAH spiked Salicornia samples with 16 PAH mixture (5 mg l−1 ) after purification on florisil column (n = 3) PAHs

Eluting solvent ratios

Total purification recovery

Hexane:DCM

DCM:ethanol

100:0

95:5

90:10

80:20

70:30

Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzanthracene Chrysene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene Indenopyrene Dibenz[a,h]anthracene Benzo[ghi] perylene

98 ± 2 96 ± 3 96 ± 4 94 ± 2 93 ± 3 93 ± 3 87 ± 4 89 ± 4 70 ± 2 61 ± 2 4±1 3±1 3±1 – – –

2±1 4±1 4±1 6±1 7±1 7±1 13 ± 1 11 ± 1 29 ± 2 38 ± 2 87 ± 4 88 ± 4 88 ± 3 31 ± 2 11 ± 1 39 ± 1

– – – – – – – – 1±1 1±1 9±1 8±1 9±1 64 ± 3 78 ± 2 59 ± 2

– – – – – – – – – – – – 1±1 5±1 11 ± 2 2±1

– – – – – – – – – – – – – – – –

Psi-psi carotene Phytol Sterols Fatty alcohol Ethyl ester fatty acid

64 ± 4 – – – –

14 ± 3 – – – –

12 ± 3 – – – –

4±2 – – – –

3±1 – – 3±1 1±1

55:45 – – – – – – – – – – – – – – – – 2±1 3±1 84 ± 5 40 ± 3 3±1

30:70 – – – – – – – – – – – – – – – – – 5±1 14 ± 2 38 ± 3 9±2

75:25

100:0

– – – – – –– – – – – – – – – – – – 69 ± 5 2±1 16 ± 3 24 ± 4

– – – – – – – – – – – – – – – – – 23 ± 3 – 3±1 63 ± 5

112 ± 9 96 ± 3 95 ± 6 93 ± 1 90 ± 1 88 ± 8 112 ± 10 116 ± 6 107 ± 1 106 ± 8 93 ± 6 107 ± 6 98 ± 6 112 ± 7 108 ± 5 104 ± 1 – – – – –

The concentrations of individual PAHs in each elution fraction were normalized to their respective total concentration in the sample studied. The total purification recovery rates were calculated referred as PAHs of the standard solution.

kg of dry Salicornia, which are compatible with the PAH levels found in plant tissues following fuel exposure [7]. If needed, such levels of sensitivity may be further improved by reducing the volume of the final hexane extract (from 500 to 250 ␮l or less). Besides, traces of PAHs may be quantified more precisely by increasing the quantity of polluted sample to be analysed (from 5 to 10 g of fresh weight).

Table 4 Detection limits for the 16 PAH mixture in control sample (␮g l−1 ) and spiked Salicornia samples (␮g kg−1 DW) PAH

Limit of detection in standard sample

Limit of detection in Salicornia sample

Limit of detection admitted

Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzanthracene Chrysene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene Indenopyrene Dibenz[a,h]anthracene Benzo[ghi]perylene

0.66 0.99 1.02 0.74 0.47 0.60 0.51 0.44 0.24 0.27 0.57 0.60 0.78 1.74 1.28 1.16

3.5 8.0 8.0 8.6 4.6 4.6 5.0 5.0 8.0 8.0 17.0 17.0 17.0 18.0 18.0 18.0

<5.0 <10.0 <10.0 <10.0 <5.0 <5.0 <5.0 <5.0 <10.0 <10.0 <20.0 <20.0 <20.0 <20.0 <20.0 <20.0

3.5. Application of the method To demonstrate the efficiency and accuracy of the developed method, it was applied to the quantification of PAHs in different plant samples collected onto areas polluted by the Erika’s oil spill. Approximately 300 samples of Armeria maritima, Atriplex hastata, Arthrocnemum perennis, Crithmum maritimum, Glaux maritima, Inula crithmoides, Juncus maritimus, Sedum acre, Beta maritima, Spergularia rupicola, Sarcocornia perennis, Sueda maritima, and Salicornia sp. were analysed [7]. Analyses were performed on a wide range of plant tissues: leaves, roots, fresh or lignified stems Moreover, the method was used in parallel studies in order to quantify PAHs in Salicornia fragilis plants grown on artificially fuel oil-polluted sediments. A set of 20–25 plant samples were extracted at the same time. Quantification of PAHs was performed by GC–MS in selected ion monitoring (SIM) mode. After the identification of 16 PAH compounds and determination of their retention times from control sample (standard PAHs with out matrix), quantification of PAHs was performed using the HPCHEM chromatography software through response factors calculated as referred to pyrene-d10 ) and benzo[a]pyrene-d12 as internal standards. Deuterated pyrene (d10 -pyrene) was used to determine the quantity (␮g kg−1 DW) of low- or medium-molecular-weight PAHs: naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene. The following eight PAHs (from benzanthracene to benzo[ghi]perylene) were quantified as referred to benzo[a]pyrene-d12 . Since the deuterated PAH solution is added as an internal standard at the beginning

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of the extraction process, correction for incomplete recoveries was made. Corrections is also evaluated and rectified compared to control sample (PAH standard without matrix) analysed at the same time. If needed, quantification may be improved by the use of more than two deuterated PAHs (i.e. one for each class of PAHs, related to the number of rings) as internal standards. Whatever the plant species, our method was successful allowing the quantification of PAHs in tissues without any additional improvement. Indeed, whereas some species such as C. maritimum contained high contents of aromatic compounds such as essential oil potentially co-extracted with PAHs, these natural molecules were well-separated from PAHs on GC–MS spectra, which allowed a valid identification and quantification of PAH compounds. This method also permits to measure low as well as high PAH amounts from a small quantity of plant material. Indeed, total PAH content measured in samples exposed to Erika’s fuel oil ranged from 50 to 7000 ␮g kg−1 dried plant. Moreover, for each plant sample, extraction procedure and quantification of PAHs were performed in two and three replicates, respectively, and revealed a high reproducibility of the method. 4. Conclusion Numerous studies report the quantification of PAHs by GC–MS in tissues of various organisms. However, to our knowledge, no study describes precisely a technical and complete method to quantify PAHs into tissues of marine plants. By using saponification and Florisil-SPE clean up of the extract, we have reduced significantly the amount of a number of compounds including plant pigments, sterols, fatty acid and alcohol, which may interfere with GC–MS analysis. Best recoveries of plant PAHs (ranging from 88 to 116%) were obtained with: (1) saponification for 16 h at 60 ◦ C or 3 h at 80 ◦ C; (2) clean up on Florisil column; (3) hexane:dichloromethane 80:20 (v/v) as elution solvent. Limits of detection were lower than 20 ␮g kg−1 of plant dry weight. This procedure was being successfully applied to the monitoring of PAHs in coastal plants exposed to fuel oil. The results demonstrated the reproducibility of this method and its possible application for PAH analyses in a wide range of halophytic plants [7,8]. Some authors used Soxhlet or Dionex methods for PAHs extraction from vegetables samples [5,6]. Saponification extraction of PAHs from halophyte samples was chosen since this method is less time- and solvent-consuming than soxhlet, and it needs no specific device unlike dionex method. Moreover, it allowed the elimination of saponified compounds in the aqueous phase. Then, SPE on Florisil was performed to clean up extracts from plant compounds potentially interfering through GC–MS analysis. This method is easily automatisable, uses less solvent and is less expensive than method of purification such as gel permeation chromatography [16]. After an ultrasonic extraction and the saponification of the samples, Dugay et al. also carried out a SPE using Oasis HLB extraction cartridges [17]. How-

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ever, these columns need a long time to load the sample onto the cartridge [9]. Therefore, SPE on Florisil columns is less time consuming than extraction cartridges. This new method has been initially developed for assays of PAHs in marine plants, in a frame of an environmental project and is currently improved in investigations concerning the identification and the quantification of degraded PAHs. However, as it is easy, rapid and considers the complexity and the impact of the plant matrix, it is also of interest for scientists working in several important fields of research: environmental, ecotoxicological, human health (etc.). It could be applied to the detection of PAHs in other plant biota, enlarged to crop plants, wild plants or algae. When grazing animals or humans consume such plant materials, its application may be extended to human health-related studies. Acknowledgement The work was partially supported by the “Minist`ere de l’Ecologie et du D´eveloppement Durable” and the “Institut National de l’Environnement Industriel et des Risques” (INERIS). Contribution N◦ 983 of the IUEM, European Institute for Marine Studies (Brest, France). References [1] J.W. Hodgeson, Polynuclear aromatic hydrocarbons: EPA Method 550.1, US Environmental Protection Agency, Cincinnati, OH (1990) 143. [2] X.-C. Wang, Y.-X. Zhang, R.F. Chen, Mar. Pollut. Bull. 42 (2001) 1139. [3] L.E. Sverdrup, P.H. Krogh, T. Nielsen, C. Kjaer, J. Stenersen, Chemosphere 53 (2003) 993. [4] S.L. Simonich, R.A. Hites, Environ. Sci. Technol. 29 (1995) 2905. [5] A.M. Kipopoulou, E. Manoli, C. Samara, Environ. Pollut. 106 (1999) 369. [6] J. Fismes, C. Perrin-Ganier, P. Empereur-Bissonnet, J.L. Morel, J. Environ. Qual. 31 (2002) 1649. [7] N. Poupart, A. Meudec, Final report for the program “Suivi Erika”, IUEM-UBO, Brest (2005) 91. [8] N. Fladung, J. Chromatogr. A 692 (1995) 21. [9] P.A.d.P. Pereira, J.B.d. Andrade, A.H. Miguel, Anal. Sci. 17 (2001) 1229. [10] O.H.J. Szolar, H. Rost, D. Hirmann, M. Hasinger, R. Braun, A.P. Loibner, J. Environ. Qual. 33 (2004) 80. [11] M. Letellier, H. Budzinski, P. Garrigues, ISPAC Newslett. 9 (1998) 4. [12] G. Northcott, K.C. Jones, J. Environ. Qual. 32 (2003) 571. [13] C. Lahond`ere, Les salicornes s.l. (Salicornia L., Sarcocornia A.J. Scott et Arthrocnemum Moq.) sur les cˆotes franc¸aises, Bulletin de la Soci´et´e Botanique du Centre-Ouest, 2004, p. 24. [14] EPA, Polynuclear aromatic hydrocarbons. Method 8100, in Test methods for evaluating solid waste, SW-846, US Environmental Protection Agency, Office of Solid Waste and Emergency Response, Washington, DC, 1986, p. 10. [15] Florisil cleanup: Method 3620, in Test methods for evaluating solid waste, SW-846, US Environmental Protection Agency, Washington, DC, 1996, p. 25. [16] M. Tomoniova, J. Hajslova, J. Pavelka Jr., V. Kocourek, K. Holadova, I. Klimova, J. Chromatogr. A 827 (1998) 21. [17] A. Dugay, C. Herrenknecht, M. Czok, F. Guyon, N. Pages, J. Chromatogr. A 958 (2002) 1.