Environ. Sci. Technol, 1994, 28, 1320-1324

Environmental Monitoring of Polychlorinated Biphenyls Using Pine Needles as Passive Samplers

Flenrlk Kylln

Department of Environmental Chemistry, Stockholm University, 106 91 Stockholm, Sweden

Eva Grlmvallt

Department of Analytical Chemistry, Stockholm University, 106 9 1 Stockholm, Sweden

Conny Ostman' Analytical Chemistry Division, National Institute of Occupational Health, 171 84 Solna, Sweden

Pine needles were used as passive samplers for monitoring polychlorinated biphenyls (PCBs) in the environment. A method for the determination of PCB in pine needle wax was developed. By applying an HPLC-based cleanup procedure to wax extracts of pine needles, a high selectivity toward PCB was obtained. High precision and accuracy was achieved, as well as high relative (91-108 f 4-8 % ) and absolute overall recoveries (81 i 14%). Pine needle wax from the central and northern parts of Europe were examined. High concentrations of PCBs with a profile shifted toward low molecular species were found in Western Germany (C 9 CBs = 47 ng/g of wax) when compared to the other investigated geographical sites (c 9 CBs = 4-7 ng/g of wax).

Introduction A large body of indirect evidence for the long-range

atmospheric transport of persistent organic environmental pollutants exists by the presence of residues of these compounds in biota in remote areas. Vegetation in remote areas has been used as a means to characterize atmospheric concentrations of lipophilic air pollutants (1-9). Data on organochlorine residues in various plants have also been used for the modeling of global distribution of these compounds ( I 1.

Accumulation of lipophilic organic trace substances in plants is attributed to uptake from the atmosphere (1). Rot uptake followed by translocation within the plant is not significant for substances with log octanol-water partitioning coefficients larger than 3 (10). The cuticles of the green parts of higher plants are covered by a wax layer functioning as a protection against desiccation. This epicuticular wax, which consists mainly of long-chain esters, polyesters, and paraffins (111, has been shown to accumulate lipophilic compounds (2,3). Air contaminants in the vapor phase are adsorbed to and accumulate in the epicuticular wax of conifer needles, thus acting as diffusive samplers (4) . The waxy surface of the pine needles also traps particulates and, thus, pollutants associated to the particles to a certain extent.

Conifer needles have been used for monitoring both local and regional distribution of lipophilic air pollutants. In this way, the presence of polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) has been investigated in the vicinity of wood-preserving sites in the United States

* Corresponding author. Present address: Analytical Chemistry Division, National In-

stitute of Occupational Health, 171 84 Solna, Sweden.

1920 Envlron. Scl. Technol., Vol. 28, No. 7, 1994

(9). Scots pine (Pinus sylvestris), a species with wide- spread distribution on the Northern Hemisphere, has been found to be a suitable monitor of atmospheric pollution (7,8). Usually three or more year-classes of needles can be identified on a single specimen. In a project aimed at mapping the distribution of organochlorine compounds in Europe, data regarding the concentrations of a number of organohalogens in pine needles have been obtained (8). Pine needles were collected in a transect from the French- Spanish border in southern Europe, up to the northern border of Sweden in northern Europe. From this data, it was possible to locate a large source of DDT in the southern parts of the former East Germany (8). Some information on PCB was also included. However, the chromatograms obtained for the determination of organochlorines were so complex that the quantification of compounds occurring at low levels, such as individual chlorinated biphenyls (CBs), were unsatisfactory. Presently, there are not any statistical materials regarding atmospheric concentrations of known contaminants such as PCB and organochlorine pesticides (12), and their trends over time are therefore largely unknown.

When determining halogenated pollutants in environ- mental samples, gas chromatography-mass spectrometry (GC-MS) and gas chromatography-electron capture de- tection (GC-ECD) are the most commonly applied analytical techniques. The electron capture detector offers a high selectivity and low detection limits regarding halogenated pollutants. However, its use is often com- plicated by the large differences in both concentration and detector response for the compounds of interest. Further, a large number of interfering compounds exhib- iting positive or negative ECD responses are present in many samples. This adds substantially to the complexity of the chromatograms. Negative peaks have been a major problem in connection with the analysis of pine needle extracts with respect to PCB (13). Cleanup by solvent extraction, sulfuric acid extraction, and open column chromatography was not sufficient for obtaining a quality GC-ECD chromatogram of the PCB in pine needle epicuticular wax. Thus, a cleanup procedure with a high selectivity toward the compounds of interest had to be developed.

By operating a high-performance liquid chromatography (HPLC) aminopropyl column in the straight-phase mode, hydrocarbons can be separated according to the size of the conjugated a-electron system. Paraffinic and olefinic compounds elute prior to aromatic species. The latter are then separated according to the number of fused aromatic rings (14). These properties have been used for the

0013-938X/94/0928-1320$04.50/0 0 1994 Amerlcan Chemlcal Soclety

separation of PCDD and PCDF from aliphatic and aromatic hydrocarbons (15, 16) and for cleanup of Poly- chlorinated biphenyls in marine samples (1 7).

This paper presents an analytical procedure for the selective analysis of PCB in the complex pine needle epicuticular wax matrix. A study on the recovery of PCB congeners has been performed. The method includes normal-phase HPLC in order to isolate a PCB fraction and capillary GC with ECD detection for the separation and quantification of individual PCB congeners. A rather simple method was obtained, making it possible to map the distribution of PCB using the pine needle as a passive sampler. The levels of selected CBs that are often used as indicator compounds are determined in pine needles collected in central and northern Europe.

Experimental Section Chemicals. Individual PCB congeners identified in

samples or used for recovery experiments were CB-28 (2,4,4’-trichlorobiphenyl), CB-44 (2,2’,3,5’-tetrachlorobi- phenyl), CB-52 (2,2’,5,5’-tetrachlorobiphenyl), CB-99 (2,2’,4,4’,5-pentachlorobiphenyl), CB-101 (2,2’,4,5,5’-pen- tachlorobiphenyl), CB-110 (2,3,3/,4’,6-pentachlorobiphe- nyl), CB-118 (2,3’,4,4’,5-pentachlorobiphenyl), CB-138 (2,2’,3,4,4’,5’-hexachlorobiphenyl), CB-153 (2,2’,4,4’,5,5’- hexachlorobiphenyl), CB-156 (2,3,3/,4,4’,5-hexachlorobi- phenyl), CB-170 (2,2’,3,3’,4,4’,5-heptachlorobiphenyl), and CB-180 (2,2’,3,4,4’,5,5’-heptachlorobiphenyl) (18). Indi- vidual PCB congeners of 99 % purity obtained from Ultra Scientific (North Kingstown, RI) were used as quan- tification standards. The internal standard CB-189 (2,3,3’,4,4’,5,5/-heptachlorobiphenyl) (19) and l*C-labeled CB-101 (2.45 Ci/mol, 1.55 pCi added to the sample) used for recovery experiments were synthesized at the Depart- ment of Environmental Chemistry, Stockholm University. Pesticide standards consisted of EPA reference materials. The technical PCB mixture Aroclor 1254 (Monsanto, St. Louis, MO) was used for testing the analytical procedure. All solvents were pesticide-grade (Fisons, Loughborough, England) except hexane (HPLC grade, Rathburn Inc., Walkerburn, Scotland), which was distilled in an all-glass apparatus prior to use. The silica gel (Kieselgel60,0.063- 0.200 mm, Merck, Darmstadt, Germany) was Soxhlet extracted with dichloromethane during 24 h, dried, and activated overnight at 180 “C. Analytical-grade concen- trated sulfuric acid was also obtained from Merck. n-Dodecane (Janssen, Geel, Belgium) was cleaned on activated neutral aluminium oxide (Merck, Darmstadt, Germany) prior to use as a keeper in the solvent evapora- tion steps. The laboratory glassware were washed in acid and basic detergents, rinsed with distilled water and ethanol, and then heated to 300 “C overnight prior to use.

Sampling. In a program to monitor organic air pollutants by the analysis of pine needles, samples of Scots pine (Pinus sylvestris) have been collected in the western and northern parts of Europe (4, 7, 8). Samples were collected from pines situated more than 20 km away from the nearest municipal or industrial center and more than 2 km away from any major road. Samples have been collected from pines a t the edge of forest stands facing the southwest or from solitary trees on the southwest-facing side with at least 500 m of open land in front of the sampling site. Sampling was primarily done at a height of 2-3 m on trees 25-30 years of age. Collection of samples was done in the spring during bud shooting. In this way, the youngest needles had been exposed to ambient air for

approximately 1 year before sampling. All the needles collected from Scots pine were of year-class one, collected in the spring/early summer of 1989. In May 1990, needles that ranged from year-class one to year-class eight divided in separate samples were collected from a mountain pine (Pinus mugo) near Zakopane (Tatra Mountains, Poland).

Extraction. A total of 20 g of fresh pine needles was immersed in 50 mL of dichloromethane containing 10 ng of the internal standard, CB-189. The sample was shaken for 3 min and then left standing for 48 h. After decanting the solvent, the needles were shaken once more with a fresh portion of dichloromethane. The combined solutions were filtered through a dichloromethane-washed filter paper (Whatman No. 1). After evaporation of the solvent, a hard off-white waxy residue remained. The dry weight was determined by constant weight, after extraction of the wax layer by letting the remaining needles dry in open air. The weight obtained plus the wax weight constituted the dry weight.

Preseparation. In order to remove the wax matrix, the extract was dissolved in 1 mL of dichloromethane and loaded onto a column containing 10 g of dry-packed activated silica gel. The organochlorines were eluted with 10 mL of dichloromethane. After evaporation of the solvent by gentle blow-down under nitrogen, the extract was dissolved in 500pL of benzene:hexane (1:l) and further fractionated using a column containing 1 g of a dry-packed mixture of silica ge1:concentrated sulfuric acid (2:l w/w). By elution with 5 mL of benzene:hexane (l:l), the organochlorine fraction was obtained. After adding dode- cane as a keeper, the eluate was concentrated to 100-150 pL prior to injection on the HPLC.

Fractionation was performed using an HPLC system consisting of a pump (Model 590, Waters Associates, Milford, MA), an electrically actuated switching valve (ELV 7000, W- Krannich, Gottingen, Germany), a UV detector (SPD-BAS, Shimadzu, Japan) operated at 254 nm, an aminopropyl silica column (p-Bondapak, 300 mm X 3.9 mm, 5 pm, Waters Associates, Milford, MA) and an injector (Model 7125, Rheodyne, Cotati, CA) equipped with a 200-pL loop. Injection volumes did not exceed 150 pL. Hexane was used as the mobile phase at a flow rate of 1.0 mL/min.

A PCB fraction was obtained by heart-cutting the HPLC eluent between the start of the elution of the CB-153 and the end of the elution of the last eluting PCB congener, CB-77 (20). A small number of highly chlorinated PCB congeners, such as CB-204, were shown to elute prior to this PCB fraction (20). However, these CBs were regarded to be insignificant for this study since these components are minor constituents of the technical PCB mixtures and are also regarded to have low biological activity. After the elution of CB-77, the flow through the column was reversed and the remaining compounds were eluted in a single back- flush peak. Dodecane was added to the collected PCB fraction prior to evaporation and subsequent redissolvation in 0.5-1 mL of hexane.

In order to investigate the efficiency of the method for eliminating possible interferences by other common chlorinated environmental pollutants, the fractionation procedure was characterized using a number of such compound groups.

Gas Chromatography. The gas chromatograph (Vari- an 3400 or 3700, Varian, Walnut Creek, CA) was equipped with a split/splitless injector, an electron capture detector,

Envlron. Sci. Technol., Vol. 28, No. 7, 1994 1321

and a Varian 8100 autosampler. Hydrogen at a column head pressure of 12 psi was used as a carrier gas with nitrogen as a makeup gas a t a flow rate of 40 mL/min. The column was a DB-5 (30 m X 0.25 mm, J&W Scientific, Folsom, CAI. Temperature settings for the injection port and the detector were 250 and 360 "C, respectively. An initial column temperature of 80 "C was held for 2 min, followed by a linear temperature increase of 10 "C/min up to a final temperature of 280 "C, which was held for 10 min. All injections were done in splitless mode. Identi- fication of PCB congeners was done by comparison with reference compounds using relative retention and mass spectra (21). All chromatograms were registered, stored, and processed by an ELDS 900 chromatography data system (Chromatography Data Systems Inc., Svartsjo, Sweden).

Gas chromatography-mass spectrometry in the negative ion chemical ionization selective-ion monitoring mode was performed on a Finnigan 4500 mass spectrometer equipped with a Finnigan 9610 gas chromatograph. Chromato- graphic conditions were as given above, except that helium was used as the carrier gas. Methane at a pressure of 0.45 Torr was used as the reactant gas. The ion source was heldat 125 "C, and the electron energy was 125 eV. Tetra- to octachlorobiphenyls were monitored by using three major peaks in each molecular ion cluster.

Gas chromatography-atomic emission detection (GC- AED) (Hewlett Packard 5890, Avondale, PA) monitoring the sulfur and carbon signals was used for analysis of HPLC fractions with respect to elementalsulfur. The GC column was a HP-1(5 m X 0.53 mm, Hewlett Packard, Avondale, PA). The temperature of the detector was 320 "C. The column temperature program started at 65 "C for 1 min, increasing 10 "C/min to 260 "C, which was kept for 10 min. Helium was used both as the carrier gas, a t a linear velocity of 30 cm/s, and as the makeup gas, a t a flow rate of 30 mL/min. The injection was made using an on column injector.

Radioactivity Measurement. Measurement of ra- dioactivity in the recovery experiments was done on a Packard TriCarb 460C liquid scintillation counter (Pack- ard Instruments, Downers Grove, IL). Results and Discussion

By a HPLC cleanup method utilizing an aminopropyl silica bonded-phase column, it is possible to remove many environmental pollutants and anthrophogenic compounds that may interfere with gas chromatographic analysis of PCB. An HPLC chromatogram of the fractionation of a pine needle wax sample is shown in Figure 1. In the collected PCB fraction, a number of PCB congeners were easily detected using GC-ECD analysis. Two GC-ECD chromatograms obtained from a sample prior to and after HPLC fractionation are shown in Figure 2. Analysis of the HPLC prefraction and the back-flush fraction showed that most of the interfering material giving rise to negative response in the ECD were found in the prefraction eluting prior to the PCB.

Elemental sulfur, s8, which has been shown to be present in higher plants (221, is a possible interference for lower chlorinated CB congeners. To test for S8, fractions from the HPLC were analyzed by GC-AED. This showed s8 to elute in the prefraction.

GC-MS analysis was used to confirm the identity of the peaks in the PCB fraction as being PCB compounds. It was found that the large peak eluting at 18.5 min was

Figure 1. Fractionatlon of a plne needle wax extract. After collection of a heart-cut fraction Containing the PCB, the flow is reversed, and the remaining material is eluted as one peak.

I b r I

L

17 M 10 M 91 M _I"

Figure 2. GC-ECD chromatogram of the same pine needle wax sample prior to (a) and after (b) HPLC fractionation.

Table 1. Characterization of Fractions from Aminopropyl Silica Column with Respect to Some Chlorinated Compounds.

prefraction PCB fraction back-flush fraction

hexachlorobenzene PCB CP PCN 33%b PCN67%b toxaphene aldrin toxaphene 10% p,p'-DDD & p,p'-DDT mirex p,p'-DDE a- & y-chlordane

a- and y-chlordene heptachlorepoxide heptachlor a-,b-, y-, & &HCH

oxychlordane dieldrin & endrin p,p'-methoxychlor transnonachlor

0 PCB, polychlorinated biphenyls; CP, polychlorinated paraffins; PCN, polychlorinated naphthalenes; HCH, hexachlorocyclohexane. b Account to UV response. c Account to ECD response.

p,p'-DDE, which is not possible to remove using this method. A few minor peaks in the PCB region of the chromatogram were shown not to be PCB.

The aminopropyl silica column was also characterized regarding the retention of a number of chlorinated environmental pollutants in order to check which chlorine- containing compounds might appear in a PCB fraction. As shown in Table 1, most of the organochlorine com- pounds elute in the back-flush fraction. This fraction can thus be collected and used for further analysis of a number of chlorinated pesticides and other organohalogens.

Recovery Tests. Absolute recovery for the overall workup procedure was obtained by adding 14C-labeled

1322 Envlron. Scl. Technol., Vol. 28, No. 7, 1994

Table 2. Recovery of 1qC-Labeled CB-101 after Different Cleanup Steps (n = 5)

treatment recovery ( % ) RSD

starting material 100 silica gel column 98 2 sulfuric acid column 82 13 HPLC cleanur, 81 14

Table 3. Recovery Relative to Internal Standard of Some Selected PCB Congeners Subject to Entire Cleanup Procedure (n = 5)

PCB recovery PCB recovery congener (%) RSD congener (%) RSD CB-52 99 4.6 CB-138 102 6.3 CB-44 107 5.1 CB-153 100 4.2 CB-99 104 5.0 CB-170 108 7.8 CB-110 91 4.9 CB-180 95 7.9

CB-101 to a pine needle wax extract. Five replicate analyses were made involving the complete method. Radioactivity was measured by scintillation counting after eachstep in the cleanup procedure (Table 2). The overall recovery was found to be 81 f 14% (n = 5). About 80% of the loss of material was accounted for by the silica gel/ sulfuric acid column separation. This step also accounted for the major variance in absolute recovery. The HPLC fractionation did not introduce any significant additional losses to the workup procedure. It should be pointed out that in order to avoid losses by sample handling in the HPLC fractionation step, it is necessary to dissolve the sample in a t least 100 pL of solvent, which requires an injection loop of not less than 150 pL in order to inject the entire sample.

The recovery of PCB relative to the internal standard (CB-189) for the entire analytical procedure was tested by using a standard addition procedure. Aroclor 1254, corresponding to approximately four times the levels of the analyzed PCB congeners in a selected wax sample, was added together with the internal standard. The concentration of total PCB as well as eight selected CBs, listed in Table 3, were quantified in both the spiked and unspiked sample after the cleanup procedure. Recovery of the Aroclor 1254 technical mixture relative to the internal standard was found to be 102 f 2%. For the selected CBs, the recovery relative to the internal standard varied between 91 and 108% with relative standard deviations between 4 and 8%, Table 3.

The HPLC fractionation procedure was also evaluated regarding the recoveries of the organochlorine pesticides listed in Table 4. Yields in the range of 95-105% were demonstrated for all compounds except for 0- and 6-HCH. These two compounds are sensitive to degradation, especially in a basic environment. Since the aminopropyl column is regarded as a basic ion-exchange column, the reduced yields of these two compounds could be due to dehydrochlorination catalyzed by the stationary phase. However, the recoveries of other compounds, such as a-HCH, r-HCH, and DDT, that also can undergo dehy- drochlorination in a basic environment were good.

Analysis of PCB in Pine Needle Epicuticular Wax, A pilot study was performed in order to estimate the concentrations of CBs found in pine needle epicuticular wax (Table 5). Six samples of 1-year-old pine needles from central Germany and southern and central Sweden

Table 4. Recovery of Some Organochlorine Pesticides for HPLC Fractionation (n = 5)

compound

hexachlorobenzene mirex a-HCH &HCH T-HCH (3-HCH aldrin dieldrin endrin heptachlorepoxide oxychlordane a-chlordane @chlordane y-chlordene 6-chlordene heptachlor transnonachlor p,p’-DDT p,p’-DDD p,p’-DDE p,p’-methoxychlor

recovery ( % )

105 96 98 71 98 70 97

102 98

101 99 98 99

101 100 100 98 97 99

100 100

RSD 5 3 5

13 5

12 3 3 5 6 3 4 4 3 4 3 3 5 2 2 3

Table 5. Levels of Some Selected PCB Congeners in Pine Needle Epicuticular Waxs

Sweden central

Germany south central Giessen Bebra Smygehuk Lyngsjo FunLdalen Vemdalen

CB 28 25.56 0.21 0.13 0.18 0.10 0.10 CB 52 5.73 0.42 0.70 0.59 0.33 0.23 CB 101 5.38 2.17 2.07 1.31 0.41 0.44 CB 118 2.43 0.60 1.00 2.95 1.43 1.32 CB 138 3.75 1.65 0.82 0.65 0.51 0.50 CB 153 2.27 1.51 1.60 0.72 0.67 0.69 CB 156 0.27 0.07 0.14 0.12 0.16 0.10 CB 170 0.85 0.11 0.17 0.09 0.10 0.11 CB 180 0.43 0.17 0.38 0.14 0.32 0.27 sum 46.66 6.92 7.02 6.74 4.03 3.77

Concentration in ng of congener/g of dry pine needles.

DDE 153

Flgure 3. GC-ECD chromatogram of the PCB fraction obtained from a pine needle sample collected in Smygehuk in southern Sweden. The CB peaks are identified by IUPAC numbers. IS, internal standard. DDE, l,l-dichloro-2,2-bis(4-chlorophenyl)ethene.

were analyzed with respect to PCB using the described method. Five of the samples exhibited similar PCB profiles. A GC-ECD chromatogram of a sample with a typical PCB profile, collected in Smygehuk situated in the south of Sweden, is shown in Figure 3. The sixth sample was collected outside Giessen in Germany, in the vicinity of the Ruhr industrial area. It exhibited a

Envlron. Scl. Technol., Vol. 28, No. 7, I994 1323

180 (IS)

17.00 19.00 21.00 mln

Figure 4. GC-ECD chromatogram of the PCB fraction obtained from a pine needle sample collected in Giessen in Germany, in the vicinity of the Ruhr area. The CB peaks are identified by IUPAC numbers. IS, internal standard. DDE, l,ldichloro-2,2-bis(4-chlorophenyl)ethene.

nu9

1 2 3 4 5 6 7 8

Flgure 5. Plot showing the accumulation of CB-138 in pine needles of different age. completely different PCB profile (Figure 4). When comparing the sampling sites using the sum of the concentrations of the nine selected PCB congeners, the concentrations varied from 47 ng/g in central Europe to 4 ng/g in central Scandinavia. I t should be pointed out that some of the determined compounds might coelute with other usually less abundant PCB congeners due to insufficient separation on single capillary columns. On slightly polar columns like 5 % phenyldimethylsilicone, the CBs 28/31,138/163,101/90/84, and 105/132 are known to coelute (23).

Accumulation of PCB in Epicuticular Wax. To investigate the PCB accumulation in the epicuticular wax of pine needles, eight year-classes of needles from a single tree were analyzed. The samples were collected from a mountain pine growing in the Tatra Mountains, in the vicinity of Zakopane, Poland. In Figure 5, the concentra- tion of one of the dominating CB congeners, CB-138, in the epicuticular wax layer is plotted versus pine needle age. It is our experience from previous investigations of other chlorinated pollutants that the concentrations increase for each year-class, except for the last year-class available (4 ) . In this class, the concentrations decrease in comparison to the previous year. Presumably, this phenomenon is connected to the inception of senescence of the pine needles. A decrease in the accumulated concentration of PCB in the fifth year-class needles was observed. The needles of this year class were muchshorter and stouter than the other year-classes, and it is conceivable that adverse growth conditions have affected the surface properties of those needles. With the exception of the fifth and the last year-classes, the accumulated PCB

Year-class

concentration was found to increase with age. The results from this preliminary study have to be

further supported by an extended investigation. Work is in progress in order to determine the concentrations of various lipophilic air pollutants, including PCB, in indi- vidual samples in a program aimed at mapping the distribution of organochlorines in Europe.

Acknowledgments The authors wish to thank Professor Emeritus Soren

Jensen for being instrumental in developing the pine needle project and for supplying the needle samples. Janis Athanasiadis, Lotta Hovander, and Karel Janak are acknowledged for technical assistance. Useful comments on the manuscript from Ake Bergman are highly acknowl- edged. This project was financially supported by the Swedish National Environmental Protection Board, Grants 5316191-5 and 532630-9.

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Received for review November 3, 1993. Revised manuscript received March 21, 1994. Accepted March 28, 1994.'

Abstract published in Advance ACS Abstracts, May 1, 1994.

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