Journal of Geochemical Exploration 144 (2014) 267–272

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Journal of Geochemical Exploration

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New method for benzo[a]pyrene analysis in plant material usingsubcritical water extraction

Svetlana N. Sushkova a,⁎, Galina K. Vasilyeva b, Tatjana M. Minkina a, Saglara S. Mandzhieva a, Irina G. Tjurina a,Sergei I. Kolesnikov a, Ridvan Kizilkaya c, Tayfun Askin d

a Southern Federal University, Rostov-on-Don, Russiab Institute of Physicochemical and Biological Problems in Soil Science of Russian Academy of Sciences, Pushchino, Moscow Region, Russiac Ondokuz Mayis University, Samsun, Turkeyd Ordu University, Ordu, Turkey

⁎ Corresponding author: Russian Federation, Rostov-room 106.

E-mail address: svetlana.sushkova.sfedu@gmail.com (

http://dx.doi.org/10.1016/j.gexplo.2014.02.0180375-6742/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 October 2013Accepted 17 February 2014Available online 26 February 2014

Keywords:Benzо[a]pyrenePlantsSubcritical water extractionHexane extraction

A newmethod for benzo[a]pyrene (BaP) analyses in plantmaterialwas developed using subcriticalwater extrac-tion followed by HPLC analyses of the extracts. BaP extraction efficiency was determined by spiking grass vege-tation collected from a preserve in Rostov Oblast (Russia). BaP recovery was optimal with a 30-min extraction bywater in a special steel cartridge at 250 °C and 100 atm. More than 98% of the BaP was recovered from the plantmaterial using subcritical water extraction, compared to 72% recovery by saponification of the sample with con-ventional hexane extraction. Other advantages of subcritical water extraction are the use of water as an environ-mentally friendly solvent instead of large volumes of organic solvents as well as a shorter analysis time.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are significant contami-nants in the environment. PAHs comprise the largest group of chemicalcompounds known to be cancer-causing agents and some PAHs arealsomutagenic. Environmental contaminationwith PAHs is usually char-acterized by the presence of BaP (I) as amarker compound (Wenzl et al.,2006). The International Agency for Research on Cancer (IARC) lists BaPas a Group I carcinogen. The BaP content of all environmental matricesand food is under obligatory regulations world-wide (Department forEnvironment, Food and Rural Affairs and the Environment Agency,2002; GOST, 1986, 2004; Jian, 2004; Wenzl et al., 2006).

on-Don, Stachki street, 194/1,

S.N. Sushkova).

Recent reviews (Camel, 2001; Ong et al., 2006; Rivas, 2006; Smith,2002; Wenzl et al., 2006) and research papers (Ayala and Luque deCastro, 2001; Camel, 2001; Dyke, 1999; Hawthorne et al., 2000a,b; Liet al., 2010; Oleszczuk and Baran, 2007; Ratola et al., 2006, 2012; Zitkaet al., 2012) describe and evaluate modern methods for extracting BaPand other PAHs in environmental matrices and food. Common method-ologies for PAH quantification in environmental samples include solventextraction and analysis by high-performance liquid chromatography(HPLC) with fluorescence detection or by GC–MS. Solvent-based extrac-tion methods of soil, sediments or sludge samples are generally carriedout using Soxhlet apparatus (Hawthorne et al., 2000a), ultrasonication(Domeno et al., 2006),microwave-assisted extraction, pressurized liquidextraction or accelerated solvent extraction (Smith, 2002). Solventsinclude n-hexane, acetone, dichloromethane, toluene and others. Theextract volume is reduced under nitrogen or by rotary evaporation to afinal volume less than 1 ml. The procedure may also include extractcleanup by solid phase extraction (typically using Florisil, aluminumoxide, or silica gel) before HPLC or GC–MS analysis.

There are standardized United States Environmental ProtectionAgency (US EPA) methods for extracting organic pollutants, includingPAHs, from environmental solids (soil, sediment and sludge). For exam-ple, in method 3540C, PAHs are extracted for more than 8 hwith a mix-ture of acetone and n-hexane in a Soxhlet extractor (US EPA, 1996b). Inmethod 3550C, solid samples are extracted with organic solvents com-bined with ultrasonic treatment (US EPA, 2007b). USEPA PAH extrac-tion methods 3545A and 3561 are based on extraction with organicsolvents (US EPA, 2007a) or organic solvents (dichloromethane in

268 S.N. Sushkova et al. / Journal of Geochemical Exploration 144 (2014) 267–272

particular) in combination with supercritical carbon dioxide (31.2 °С,72.8 atm) (US EPA, 1996a). One of the widespread methods for the de-termination of PAHs in environmental samples is Soxhlet extraction,using 150 ml per sample by CH2Cl2–acetone extraction for a periodof 18 h (Hawthorne et al., 2000a).

Procedures for environmental monitoring of BaP-contaminatedsoils in the Russian Federation include four certified methods basedon extraction with n-hexane (Methodical Instructions…, 2003;Procedure of measurements, 2008b) or benzenewith ultrasonic treat-ment (Volkotrub and Baushev, 1994). The saponification method wasdeveloped for determination of PAH and other petroleumhydrocarbonsin sediments with a high content of organic substances. It is based onsample treatment with a boiling solution of alkali in alcohol, with sub-sequent extraction of pollutants using n-hexane (Directive Document,2002). Currently there is no certified method for PAH determinationin vegetation. However, there are certified methods for BaP in foodbased on extraction with n-hexane (Procedure of Measurements,2008a) with subsequent extract cleanup on a silica gel column.

All of the above-listed methods are characterized by long and multi-stage procedures for sample preparation using a large volume of toxic or-ganic solvents, typically 50–450ml per sample (Basova and Ivanov, 2011).Some prospective methods were recently developed for PAH extractionfrom various solid matrices using water or carbon dioxide under sub- orsupercritical conditions. Sub- and supercritical water extractions have be-come popular green extractionmethods for various classes of compoundsin numerous environmental, food and plantmatrices (Ayala and Luque deCastro, 2001; Hawthorne et al., 2000a). Sub- and supercritical waterextractions are also used to extract organic contaminants for food safetyanalyses and from soils/sediments for environmental monitoringpurposes. Themain parameters influencing extraction efficiency are tem-perature, extraction time, and the addition of modifiers and/or additives.Fluid extraction of PAHs from food, soil, water and solid deposits by sub-and supercritical CO2 and water are discussed in reviews (Anitescu andTavlarides, 2006; Barco-Bonilla et al., 2009; Camel, 2001; Hsueh et al.,2013; Machmudah et al., 2008; Ratola et al., 2006; Smith, 2002; Sunarsoand Ismadji, 2009; Teo et al., 2010; Turrio-Baldassarri et al., 2003).

Subcritical water extraction is one of the most recent techniques de-veloped for extracting organic compounds, including pollutants from en-vironmentalmatrices and food (Anitescu and Tavlarides, 2006; Carr et al.,2011; Carro et al., 2013; Drugov and Rodin, 2002; Hawthorne et al.,2000a; Miller et al., 1998; Ong et al., 2006; Ramos et al., 2002; Rivas,2006; Turrio-Baldassarri et al., 2003; Wenzl et al., 2006). This method isbased on the use of superheated water (100 to 374 °C and b22.4 ×106 Pa pressure) as a solvent in place of organic solvents. Subcriticalwater has unique characteristics; high temperature and pressure greatlyreduce its dielectric constant, surface tension, and viscosity, therebyweakening the hydrogen bonding network of water molecules (Carret al., 2011; Galkin and Lunin, 2005). Increasing temperature from25 °C to 350 °C at a pressure of 10.1× 106 Pa decreases the dielectric con-stant (ε) of water from 73 to 2. Therefore, the solubility of nonpolar com-pounds increases as temperature increases in this range. For example, thedielectric constant of superheatedwater is 27 at 250 °C and 10.1 × 106 Papressure, which is between that of ethanol (ε= 24 at 25 °C) and aceto-nitrile (ε=36.2 at 25 °C), one of the best solvents for BaP. Because super-heatedwater acts as an organic solvent, subcriticalwater extraction couldbe categorized as a solvent extraction process (Islam et al., 2012). More-over, superheatedwater is readily available, non-toxic, reusable and verylow in cost as well as environmentally benign. Thus subcritical water ex-traction has been suggested as an alternative to organic solvents or toxicaqueous liquid media (Carro et al., 2013).

Presently there are only a fewpublications on subcriticalwater extrac-tion of PAHs, and in particular BaP, from solid environmental matrices.Subcritical water extraction of (PAH)-contaminated soil (Hawthorneet al., 2000a) was investigated in conditions using 30 ml water and10 ml of toluene (for the 300 °C extractions) or 60 ml water and 20 mlof toluene (for the 250 °C extractions). In the present study, we have

developed a method for coupling static subcritical water extraction withcollection on styrene-divinylbenzene (SDB) SPE of disks for quantitativeextractions of PAHs from soils, sediments, and air particulate matter(Hawthorne et al., 2000b). Two papers describe the use of subcriticalwater extraction of fluoranthene, phenanthrene and pyrene from soiland sediments (Islam et al., 2012; Latawiec and Reid, 2010), but to ourknowledge there is no information on the use of these methods for anal-ysis of plant samples and all of them describemethods used in subcriticalwater extraction with organic solvents added.

The main aim of this study was to develop a method for subcriticalwater extraction of BaP from vegetation. We determined the optimumconditions for subcritical water extraction of BaP and compared the ef-fectiveness of the technique with that of a standardized method usingorganic solvent extraction.

Because there are no standardizedmethods for BaP determination invegetation, subcritical water extraction was evaluated by comparisonwith the standardmethod for BaP determination in sediments using sa-ponification followed by re-extraction into n-hexane (Directive docu-ment 52.10.556-95, 2002). The saponification method was used forcomparison with the method being developed because the previous re-searches showed high efficiency on soils, plants and sediments at small-er labor input in comparisonwith the Soxhlet. At the same time all thesemethods demand a large amount of organic solvents. In this regard wecarried out the comparative analysis of a saponification method with anew environmentally friendly method of subcritical water extraction.The main objective of this work was to use modern effective extractionmethods with a high extent of BaP extraction with theminimum quan-tity of organic solvents and minimal time of extraction.

2. Materials and methods

Subcritical water extraction of BaPwas developed using natural veg-etation with background contamination growing in the Persianovskayasteppe (natural preserve) of the Rostov region, located far from possiblesources of pollution. For analysis every average plant sample represent-ed a typical harvest of herbs growing on somemonitoring areas and in-cluded aboveground plant parts of couch-grass (Elytrigia repens L.),Austrian wormwood (Artemisia austriaca Jacq.), common yarrow(Achillea millefolium L.), costmary (Tanacetum vulgare L.), common rag-weed (Ambrosia artemisiifolia L.), and common chickory (Cichoriumintybus L.). The plant samples were taken in triplicate field frequencyand ninefold analytical frequency.

Solvents and reagents were HPLC grade and included ethanol (96%,analytical grade), n-hexane (99%, analytical grade), potassium hydrate(98%, analytical grade), acetonitrile (99.9%, analytical grade), NaOH(97%, analytical grade), and anhydrous Na2SO4 (purchased fromAquatest, Rostov on Don, Russia). A BaP standard in acetonitrile (GSO7515-98; Aquatest) was used to prepare standards for HPLC analyses.

2.1. Subcritical water extraction

Subcritical water extraction of BaP from plant samples was conduct-ed in a specially developed extraction cartridge made of stainless steeland equipped with screw-on caps at both ends. It was also equippedwith amanometer that included a valve for pressure release tomaintainan internal pressure of 10.1 × 106 Pa. The extraction cartridge contain-ing a sample andwater was placed into an oven connected to a temper-ature regulator. This device is schematically represented on Fig. 1.

The process of BaP analyses in plants based on subcritical water ex-traction is schematically shown on Fig. 2. It consisted of the followingstep-by-step operations. An air-dried sample of the natural vegetationwas ground in a porcelain mortar and passed through a 1 mm sieve.One gram of sample was placed into the extraction cartridge and 8 mlof double-distilled water was added. The extraction cartridge wassealed from both sides with the screw caps. The cartridge was placedinto an oven held at 230, 240, 250, 260 or 270 °С for 20, 30 or 40 min.

Cartridge

Pressure sensor

Pressure release valve

Reheat steam

attemperator

Thermostat

Fig. 1. Scheme of the device used for subcritical water extraction of BaP from solid matrix samples.

269S.N. Sushkova et al. / Journal of Geochemical Exploration 144 (2014) 267–272

Subsequent extractions were conducted under optimum conditions(30 min at 250 °С and 10.1 × 106 Pa).

After cooling, the content of the cartridgewasfiltered (Whatman no.1) into a conical glass flask and washed with 2 ml of double-distilledwater. This operation was repeated two or three times, until the filtratewas clear. The aqueous extract was re-extracted three times with 5 mlof n-hexane by shaking for 15 min in a separatory funnel. The hexaneextracts were combined and filtered through anhydrous Na2SO4 andevaporated to dryness in a pear-shaped flask on a vacuum evaporatorin a 40 °С water bath. The residue was dissolved in 1 ml of acetonitrileby shaking for 30 min. The BaP concentration in the acetonitrile extractwas determined by HPLC.

The efficiency of BaP extraction from plants was determined using amatrix spike (Procedure of Measurements, 2008b). The air-dried vege-tation sample (1 g) was placed into a round-bottom flask and BaP stan-dard solution in acetonitrile was added to give BaP concentrations of 2,4, 6, 8, 16 or 32 ng/g. After evaporating the solvent for 30 min under ahood under ambient conditions, the BaP-spiked plant samples were in-cubated for 24 h at 7 °C. The sampleswere then analyzed by the subcrit-ical extraction method described above.

2.2. Method of saponification

The method of saponification involves treating solid material with aboiling mixture of alkali and alcohol, and subsequent extraction of pol-lutants with n-hexane (Directive document 52.10.556-95, 2002). Ascheme for the procedure is shown in Fig. 3.

A 1-g aliquot of air-dried vegetation sample was placed in a conicalflask and 30ml of 2% solution of NaOH in ethanol was added. Themixturewas boiled on a water bath for 3 h. After cooling, the liquid layer wasdecanted into another flask and 5 ml of double-distilled water and 15 ml

Dry, grind and s

Subcritical water

Re-extract with n

Dry with anhydr

Average plant sample

Air-dry sample (1 g) + 8 ml double-distilledplaced into an extraction cartridge

Aqueous filtrate

Hexane extract

HPLC analysis

Fig. 2. Scheme of BaP analysis in plant material using subcri

of n-hexane were added. The flask was placed on a rotary shaker for10 min, then transferred to a separator funnel to collect the hexanelayer. The hexane extraction was repeated twice. The combined hexaneextract was transferred to a conical flask and washed with water until aneutral reaction (using indicator paper), transferred to an amberflask con-taining 5 g anhydrous Na2SO4 and allowed to stand overnight at 5 °C. Thedehydrated extractwas transferred to a round-bottomflask and evaporat-ed to dryness by rotary evaporation in a 40 °C water bath. The extractresidue was dissolved in 1 ml of acetonitrile and analyzed by HPLC.

2.3. Analyses of BaP in extracts and calculation of BaP concentration inplants

BaP in the extracts was quantified by HPLC (Model 2000, ThermoSeparation Products, Waltham, MA, USA) with simultaneous ultraviolet(UV-1000) and fluorescence (FL-3000) detection following ISO 13877requirements (ISO, 2005). Excitation wavelength of the FD is 263 nm,and emissionwavelength of the FD is 408 nm. TheBaPpeak on chromato-grams of plant sample extracts was identified by comparing retentiontime to that of the analytical standard sample using the two detectors.The limit of BaP detection and quantification was determined using stan-dard solutions and calibration curves. A calibration standardwas insertedafter every six samples to correct for drift in retention time within a run.

BaP concentrations in plant samples (A, ng/g) were calculated asfollows:

A ¼ kSI � Cst � V= Sst �mð Þ ð1Þ

where Sst and SI = respective areas of BaP peaks in chromatograms ofstandard and sample solutions; Cst = BaP concentration in standard so-lution (ng/ml); k = coefficient of BaP recovery from a sample; V =

ieve (1 mm)

extraction (250 , 100 atm, 30 min), filter

-hexane using a 3 × 5 ml separatory funnel

ous Na2SO4

water

tical water extraction and HPLC analyses of the extract.

Dry, grind and sieve through a 1-mm sieve

Boil 3 h in a conical flask equipped with a backflow condenser h

Re-extract with n-hexane (3 × 15 ml) with shaking for 1 h 15 min

Wash with distilled water at 7 and filter through anhydrous Na2SO4 for 1 h

Plant sample

Air-dry sample (1 g) + 30 ml 2% NaOH in ethanol

Alkaline/alcohol extract

Hexane extract

HPLC analysis

Fig. 3. Scheme for determination of BaP in plant material by saponification followed by hexane extraction and HPLC analysis.

270 S.N. Sushkova et al. / Journal of Geochemical Exploration 144 (2014) 267–272

volume of acetonitrile extract used for HPLC (ml); andm=mass of thesample (g). Data handling and statistical analyseswere conducted usingMicrosoft EXEL.

3. Results and discussion

Fig. 4 shows HPLC chromatograms of plant extracts after subcriticalwater extraction (A) and for hexane extraction following saponification(B). The chromatograms contain similar peaks, with BaP retention timecoinciding with that of the BaP standard. The same number of back-ground peaks in chromatogram (B) compared to chromatogram (A) in-dicates that subcritical water extraction is also effective as well as the

Fig. 4. Chromatograms of extracts from a representative plant sample p

saponification/hexane method. Fewer co-extractives using subcriticalwater extraction allow satisfactory results without additional purifica-tion of the extract.

Concentrations of BaP in plants from the sampling site determinedby subcritical water extraction under varying temperatures and timesare given in Table 1. Results show that extraction conditions significant-ly affected BaP recovery, with amounts of BaP in the representativeplant sample ranging from 2.8 to 7.2 ng/g. Optimum conditions formaximum BaP recovery from the plant sample were 30 min of subcrit-ical water extraction at 250 °C and 10.1 × 106 Pa pressure. BaP extrac-tion efficiency decreased by 5 or 20% when temperature varied by 10or 20 °C and was greatly reduced when both temperature and

repared by subcritical water extraction (A) and saponification (B).

Table 1BaP concentration (ng/g) in a representative plant sample after subcritical waterextraction at varying temperatures and extraction times (n = 27).

Extraction temperature, °С Extraction time, min LSD0,95

20 30 40

230 4.9a 6.6 3.5 0.7240 5.7 6.8 3.2 0.5250 6.4 7.2 4.7 0.4260 5.6 6.4 3.6 0.6270 4.7 5.8 2.8 0.4LSD0,95 0.6 0.3 0.5

a The value means average from 27 replications.

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extraction time were changed. Islam et al. (2012) reported the sameoptimum extraction time and temperature for subcritical water ex-traction of PAHs from spiked sea sand. Up to 95% of fluoranthene,phenanthrene and pyrene were recovered after a 30-min extractionat 250 °С and 10.1 × 106 Pa. PAH underestimation by subcriticalwater extraction at higher temperatures can be attributed to thermaldegradation (Andersson et al., 2003; Yang and Hildebrand, 2006).Latawiec and Reid (2010) found a similar relationship between tem-perature and extraction efficiency for fluoranthene and phenan-threne in spiked soils and sediments.

The efficiencies of BaP extraction from spiked natural vegetation bythe two describedmethods are shown in Table 2. For all BaP concentra-tions in the plant samples (10–70 ng/g), subcritical water extraction re-covered substantially more BaP than the saponification method. Morethan 98% of the BaP was recovered from the plants using subcriticalwater extraction compared to 72% recovered by conventional hexaneextraction combined with preliminary saponification of the sample.The other advantages of subcritical water extraction are a shorter anal-ysis time and the use ofwater as an environmentally friendly solvent in-stead of large volumes of organic solvents.

4. Conclusions

Most conventional methods of analysis for organic pollutants in en-vironmental matrices rely on the use of organic solvents for Soxhlet ex-traction or ultrasonication. However, there is increasing interest anddemand for more rapid, environmentally-friendly, and cost-effectivemethods of extracting pollutants. Subcritical water extraction is agood alternative.

A new method for BaP analyses in plants was developed based onsubcritical water extraction followed by HPLC analyses of the extractwith no additional cleanup steps. The efficiency of BaP extractionfrom plant material under optimal conditions (30 min at 250 °С and10.1 × 106 Pa) was 98% compared to 72% using saponification and hex-ane extraction. Aside from its effectiveness, the use of subcritical waterallows a large reduction in volume of organic solvents and extractiontime from that of conventional methods. Thus a very simple, inexpen-sive, and field-rugged headspace method was developed for quantita-tively extracting and collecting organic pollutants in a form thatsimplifies their transport to a standard laboratory for analysis.

Table 2BaP concentrations in natural vegetation and extraction efficiencies comparing subcritical wate

Initial BaP in sample (ng/g) BaP content by method of extractio

Background Spike Subcritical water

7.2 ± 0.5 2.0 9.0 (1.8)a

8.0 14.9 (2.3)16.0 22.8 (3.3)32.0 38.4 (4.7)64.0 69.7 (5.3)

a The value means average from 9 replications and standard deviation is given in brackets.

Acknowledgements

The authors are grateful to academic staff of Laboratory of Sub- andSupercritical Fluid Technologies of Southern Federal University, espe-cially to Dr. Nikolay I. Borisenko, Anna V. Lekar, for granting the uniquelaboratory equipment, valuable scientific materials, consultations ona research subject. We appreciate Prof. Pat Shea for the help in themanuscript preparation. This research was supported by projects ofthe Ministry of Education and Science of Russia, no. 5.885.2014/К, theRussian Foundation for Basic Research, no. 14-05-00586_a, and theLeading Scientific School, no. 2449.2014.4 andno. 274.2014.3. Analyticalwork was carried out on the equipment of Centers for collective useof Southern Federal University "High Technology", "Biotechnology,Biomedical and Environmental Monitoring".

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