Environ. Sci. Technol. 1997, 31, 2464-2468
Chlorinated Structures in High Molecular Weight Organic Matter Isolated from Fresh and Decaying Plant Material and Soil CARINA FLODIN,† EMMA JOHANSSON,† H A N S B O R EÄ N , † A N D E R S G R I M V A L L , * , † OLOF DAHLMAN,‡ AND ROLAND MO ¨ RCK‡ Department of Water and Environmental Studies, Linko¨ping University, SE-581 83 Linko¨ping, Sweden, and Swedish Pulp and Paper Research Institute, P.O. Box 5604, SE-114 86 Stockholm, Sweden
During the past few years, surveys of AOX (adsorbable organic halogens) in water and TOX (total amount of organic halogens) in soil have demonstrated that natural halogenation of organic macromolecules is responsible for the widespread occurrence of organohalogens in seemingly unpolluted environments. This study revealed the presence of several chlorinated aromatic structures in organic matter derived from different types of decaying plant material and soil. In samples derived from fresh plant matter, however, there was normally no evidence of such structures. Two types of samples were analyzed: (i) lignin materials isolated by acidic solvolysis of fresh and decaying spruce wood, birch leaves, peat moss (Sphagnum), and meadow grass and (ii) high molecular weight organic matter leached with base from spruce forest soil and meadow grass soil. An oxidative degradation technique was used to render the studied structures amenable to gas chromatography with atomic emission detection (GC-AED) and mass spectrometric detection (GC-MS). The identified degradation products were methyl esters of 3-chloro- and 3,5-dichloro4-ethoxybenzoic acid, 5-chloro-4-ethoxy-3-methoxybenzoic acid, dichloro- and trichlorobenzoic acids, and 3,5-dichloro4-methoxybenzoic acid.
Introduction Measurements of the group parameters AOX (adsorbable organic halogens) and TOX (total amount of organic halogens) in samples of water and soil, respectively, have revealed the presence of large amounts of organohalogens in seemingly unpolluted environments (1-3). Furthermore, simple mass balance calculations and analysis of samples of organic matter of pre-industrial origin have demonstrated that this widespread occurrence of organohalogens in water and soil is caused primarily by natural halogenation of organic matter (4). More specific studies have revealed the following: humic substances are naturally halogenated (5, 6), chlorine and bromine can be bound to aromatic rings in humic substances (7, 8), and a chloroperoxidase-like catalyst is present in soil (9). In addition, it was recently shown that the amount of organically bound halogens in spruce litter can increase during certain stages of the degradation (10, 11), i.e., degradation * Corresponding author telephone: +46 13 671836; fax: +46 18 673502; e-mail:
[email protected]. † Linko ¨ ping University. ‡ Swedish Pulp and Paper Research Institute.
2464
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 9, 1997
and halogenation of organic matter in soil seem to be concurrent processes. We have already shown that certain halogenated aromatic structures in aquatic humus are similar or identical to those of chlorolignins found in bleached kraft mill effluents (7, 8, 12). Moreover, we have published preliminary data indicating the presence of such structures in soil organic matter (13). In the present paper, we report the results of a study that was undertaken to more thoroughly investigate (i) whether the structures previously detected in the aquatic environment can be traced back to organic matter present in the terrestrial environment and (ii) whether chlorine and other halogens are bound to lignin materials isolated from plant materials. The samples that were analyzed were selected to represent a broad spectrum of organic matter found in the terrestrial environment. In light of the above-mentioned results regarding simultaneous degradation and halogenation of organic matter (10, 11), we sampled both fresh and decaying wood, leaves and moss, and also soil. Furthermore, we analyzed rather nonspecific mixtures of organic matter obtained by base-leaching as well as more specific mixtures of organic substances obtained by using methods designed for isolation of lignin.
Experimental Section Samples and Sample Preparation. Samples of different types of plant material and soil were collected in southeastern Sweden at sites that were unaffected by local sources of pollution. The main characteristics of the different samples are summarized in Table 1. One pooled sample was analyzed for each combination of site and sample type. Intact wood was sampled from an air-dried log of Norway spruce (Picea abies). Decaying wood was collected from the interior of a spruce stump. Spruce forest soil was taken in the A horizon of a podzolic soil under a spruce tree. Yellow birch leaves were picked directly from a tree (Betula sp.), and brown birch leaves were collected from a rock where no other plant materials were present. The brown leaves were somewhat decomposed but still had a clear leaf structure. Fresh Sphagnum moss and Sphagnum peat were collected at the same site in a peat bog, the latter at a depth of approximately 10 cm. The fresh sample was free from visible traces of material from other plant species, whereas the peat sample contained some fragments of other types of plants, such as cranberry. Grass, mostly timothy (Phleum sp.) and fescue (Festuca sp.), was harvested from a meadow where no chlorinated pesticides had been applied. At the same site, soil was collected by digging up a piece of turf and shaking the soil off the roots of the grass. Prior to the isolation of organic matter, the samples of fresh spruce wood and fresh meadow grass were air dried. All other samples were dried at 105 °C for 12 h. After drying, the samples were ground through a 0.5 mm mesh. Model Compounds and Reagents. Methyl esters of a selection of chlorinated benzoic acids were either purchased or synthesized. The compounds 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5-dichlorobenzoic acid and 2,4,6-trichlorobenzoic acid (Lancaster Synthesis Ltd., United Kingdom) and 2,3,5trichlorobenzoic acid (Aldrich, Germany) were purchased as acids and methylated in diethyl ether solution by addition of diazomethane. We synthesized 2,3,4- and 2,3,6-trichlorobenzoic acid methyl ester from 2,3,4-trichloroacetophenon and 2,3,6-trichlorobenzaldehyde (Chemicon, Sweden) by employing the haloform reaction and dichromate oxidation, respectively. The 3,5-dichloro-4-methoxybenzoic acid methyl ester was purchased from Dr. J. Hyo¨tyla¨inen, Department of
S0013-936X(96)00374-4 CCC: $14.00
1997 American Chemical Society
TABLE 1. Sampled Environmental Compartments or Plant Species, Sample Types, and Isolation Methods Used To Obtain High Molecular Weight Organic Matter environmental compartment or plant species Norway spruce birch sphagnum meadow grass
type of sample
isolation method
fresh wood decaying stump forest soil yellow leaves brown leaves, decomposing fresh moss peat fresh grass soil
acidic solvolysis acidic solvolysis base leaching acidic solvolysis acidic solvolysis acidic solvolysis acidic solvolysis acidic solvolysis base leaching
Chemistry, University of Jyva¨skyla¨, Finland, and 5-chloro4-ethoxy-3-methoxybenzoic acid methyl ester was synthesized from 5-chlorovanillin (12). Mixtures of the methyl esters of 3-chloro- and 3,5-dichloro-4-ethoxybenzoic acid were obtained by chlorination of 4-ethoxybenzoic acid, and 2-chloro- and 2,6-dichloro-3,5-dimethoxy-4-ethoxybenzoic acid were synthesized from 3,5-dimethoxy-4-hydroxybenzaldehyde. The diethyl ether used for extraction was distilled prior to use. TMS reagent for silylation of organic acids was prepared by adding 2.4 mL of bis(trimethylsilyl)trifluoroacetamide and 0.24 mL of chlorotrimethylsilane to 2.4 mL of pyridine. Total Amount of Halogens and Total Amount of Organically Bound Halogens. TX (total amount of halogens) and TOX were determined by using a Euroglas AOX-analyzer (Model 84/85). With this instrument, samples are incinerated in an atmosphere of oxygen, and the amount of hydrogen halides formed is measured by microcoulometric titration with silver ions. The results are calculated as if all of the halides formed are chloride. It should also be noted that fluoride escapes detection and that bromide and iodide are only partly determined by this method. Moreover, not all inorganic halogens are volatile, and it is possible that part of the chloride is not determined by the TX analysis. The TX analysis measures the total amount of organic and inorganic halides present in the sample. In our experiments, aliquots (10-30 mg) of dried and ground plant material or soil were introduced directly into the AOX-analyzer for TX determinations. The TOX analysis is designed to measure the amount of organic halides present in the sample. When performing this analysis, we washed aliquots (30-50 mg) of dried and ground soil or plant material with an acidic nitrate solution before introducing them into the AOX-analyzer. A detailed description of this TOX analysis has been reported by Asplund et al. (14). Isolation of Solvolysis Products by Acidic Solvolysis. Samples of dried and ground plant materials (4-17 g) were subjected to continuous extraction with ethanol:toluene (1:1 v/v) for about 48 h to remove extractives. The extracted material was then refluxed in 0.2 M sulfuric acid in dioxane: water (9:1) for 6 h to dissolve high molecular weight organic matter, i.e., to obtain solvolysis products. Thereafter, particulates were removed by filtration, the filtrate was concentrated, and the solvolysis products were precipitated with Na2SO4. Details regarding the isolation method have been reported by Pepper et al. (15). The cited author used hydrochloric acid in the acidolysis, whereas we used sulfuric acid to eliminate the possibility that chloroorganics would be produced during the isolation procedure (16). Isolation of High Molecular Weight Organic Matter (HMW OM) from Soil. Samples of dried and ground soil (5-10 g) were first leached with 100 mL of 0.1 M sodium hydroxide for 1 h under nitrogen. After centrifugation, the
FIGURE 1. Reaction sequence for ethylation of free phenolic groups, oxidative degradation of organic macromolecules, and methylation of formed carboxylic acid groups.
FIGURE 2. Nonhalogenated degradation products investigated in this study. The compounds are the methyl esters of 4-ethoxybenzoic acid (A), 4-ethoxy-3-methoxybenzoic acid (B), 3,4-diethoxybenzoic acid (C), 3,5-dimethoxy-4-ethoxybenzoic acid (D), benzoic acid (E), and 4-methoxybenzoic acid (F). supernatant (which contained dissolved organic matter) was acidified to pH 3, and low molecular weight neutral and acidic organic compounds were removed by extracting the aqueous phase four times with equal volumes of diethyl ether. To ensure that all extractable chloroorganics had been removed by the four extractions, the last ether extract was dried and evaporated to a volume of 100 µL and, after addition of 50 µL of TMS reagent, the response to the injected ether extract was recorded in the chlorine channel of a GC-AED system (see below). Lastly, the extracted leachate was neutralized with sodium hydroxide, evaporated to a final volume of 4-5 mL, and transferred to the reaction vessel for oxidative degradation. Permanganate Oxidation and Derivatization of Formed Degradation Products. To render aromatic structures in the analyzed macromolecules amenable to gas chromatographic analysis, we used an oxidative degradation method based on the sequence of reactions shown in Figure 1. This method was originally developed for studies of lignin structures, and it has been described in detail by Erickson et al. (17), Gellerstedt and Gustafsson (18), and Gellerstedt (19). The final product is a dichloromethane extract that contains methyl esters of low molecular weight carboxylic acids formed during the degradation procedure. For each sample examined in our study, about 100 mg of solvolysis products or baseleached organic matter was degraded. In the original version of the oxidative degradation method, potassium periodate was used to reoxidize the permanganate consumed. We preferred to add small amounts of permanganate during the entire degradation, because periodate has been shown to cause formation of iodinated organic compounds that may interfere with determinations of the total amount of organic halogens (8). Analytical Procedures and Quantification of Degradation Products. The chemical characterization of degradation products was focused on gas chromatographic analysis of the structures shown in Figure 2 and the chlorinated and brominated analogues of those structures. Nonchlorinated structures were analyzed and quantified by gas chromatography-mass spectrometry in the full scan mode (GC-MSTIC). Chlorinated and brominated structures were analyzed and quantified by gas chromatography with microwaveinduced plasma atomic emission detection (GC-AED). The identification of detected compounds was based on the following: GC-AED analysis to ascertain whether or not a detected compound contained chlorine or bromine; GCMS analysis in the selected ion monitoring mode to obtain structural information; and the use of model compounds to determine retention times. Ethyl cinnamate (100 ng/µL) was used as internal standard in the GC-MS analysis, and 1-chlorodecane (20 ng/µL) and
VOL. 31, NO. 9, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
2465
TABLE 2. Original Samples Analyzed for Dry Weight, Total Amount of Halogens (mg of TX/g dw), and Total Amount of Organic Halogens (mg of TOX/g dw)a sample
dry weight (%)
TX (mg/g dw)
TOX (mg/g dw)
extractives (%)
solvolysis productsb or OMc (%)
spruce, fresh wood spruce, stump spruce, forest soil birch, yellow leaves birch, brown leaves sphagnum, fresh moss sphagnum, peat meadow grass, fresh meadow grass, soil
nmd 74 78 37 36 6 3 nm 76
95 530 150 600 230 700 1030 3200 60
10 150 90 10 120 60 230 10 40
nm 8 -e 30 6 5 1 20 -
2.4b 3.8b 3.8c 4.6b 2.0b 0.5b 0.8b 6.8b 2.0c
a The values in the last two columns represent the amount of extractives removed from dried plant material during extraction with ethanol:toluene and the amount of solvolysis products or organic matter (OM) obtained from pre-extracted plant material or dried soil. b % solvolysis products obtained from pre-extracted fresh and decaying plant material. c % OM base-leached from dried soil. d nm ) not measured. e - ) not measurable.
1-bromononane (20 ng/µL) were used as internal standards in the GC-AED analysis. Experiments performed on model compounds of lignin indicate that the average yield of nonchlorinated benzenecarboxylic acids can vary strongly and that the average yield is about 60% if one carbon substituent is attached to the aromatic ring (17, 20). For simplicity, we assumed 100% recovery of both nonchlorinated and chlorinated benzenecarboxylic acids in the oxidative reaction sequence. Gas Chromatographic Parameters. The GC-MS analyses were performed on a Shimadzu QP-2000 mass spectrometer equipped with a fused silica column (HP Ultra-1, 50 m × 0.32 mm, 0.17 µm phase thickness). Helium at a velocity of 35 cm/s was used as carrier gas. Temperature program: start at 40 °C, hold for 5 min, increase at 5 °C/min to 250 °C, and then hold for 20 min. Extracts of 1.6 µL were introduced by split injection with a split ratio of 1:40. The GC-AED analyses were performed on an HP 5890 gas chromatograph equipped with an HP 5921A microwaveinduced plasma atomic emission detector. All GC parameters were the same as in the GC-MS analyses. Furthermore, split injection was used for all dichloromethane extracts of degradation products, whereas splitless injection (1.1 µL) was used for the ether extracts obtained during removal of low molecular weight organic compounds from the original samples of organic matter. The responses to carbon, chlorine, and bromine in the AED system were measured at 496, 479, and 478 nm, respectively.
Results Group Parameters. TX and TOX values for the dried and ground samples of plant materials and soil are presented in Table 2. The TOX values for samples of fresh plants were found to be markedly lower than the values for soil and decaying plant materials. It is also noteworthy that there is no obvious correlation between TX and TOX values. The amount of extractives removed by extraction with ethanol: toluene varied strongly among samples, whereas the yield of solvolysis products from pre-extracted samples was invariably rather low (see Table 2). Nonchlorinated Aromatic Structures. By using GC-MS in the full scan mode, we were able to identify several benzoic acid derivatives in all of the dichloromethane extracts of organic matter that were subjected to oxidative degradation. We paid special attention to degradation products that had been observed in our previous studies (7, 8, 13) or that appeared in both chlorinated and nonchlorinated forms in the present investigation (see below). The nonchlorinated members of this group of compounds are illustrated in Figure 2, and the amounts detected of each of these compounds are shown in Figure 3. Chlorinated Aromatic Structures. By using GC-AED and GC-MS, we were able to identify chlorinated aromatic
2466
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 9, 1997
degradation products in most of the analyzed samples of organic matter subjected to oxidative degradation; these compounds are listed in Table 3. Closer examination of data showed that chlorinated benzoic acid derivatives were present in all samples derived from soil and decaying plant material collected on the ground, whereas such reaction products were not detected in samples derived from fresh spruce wood, yellow birch leaves, and meadow grass. The methyl esters of 3-chloro- and 3,5-dichloro-4-ethoxybenzoic acid (ACl1 and ACl2, respectively) were the major chlorinated compounds in degraded samples of solvolysis products from decaying plant materials and base-leached organic matter from spruce forest soil. Trichlorobenzoic acid methyl ester (ECl3) was the dominating product obtained from meadow grass soil, and this compound was also found in high molecular weight organic matter from spruce forest soil, brown birch leaves, and Sphagnum peat. In addition, methyl esters of dichlorobenzoic acids (ECl2) and 3,5dichloro-4-methoxy benzoic acid (FCl2) were present in several of the samples of decaying plants and soils, and 5-chloro-4-ethoxy-3-methoxy benzoic acid methyl ester (BCl1) was found after degradation of decaying spruce wood and Sphagnum peat. The GC-AED chromatogram in Figure 4 shows the response in the chlorine channel to a sample of degraded solvolysis products of brown birch leaves. As can be seen, most of the large peaks in the chlorine chromatogram represent the compounds identified in the present study, although one large peak was caused by a compound that has not yet been identified. In contrast to the sample derived from brown leaves, the degraded sample of yellow leaves did not elicit a response in the chlorine channel of the AED system. Furthermore, it is interesting that no brominated compounds were detected in any of the extracts. The degree of chlorination of the analyzed compounds, i.e., the mole ratio of the chlorinated forms of the compound under consideration to the sum of the nonchlorinated and chlorinated forms, was found to vary from 0.02 to 0.9% for most of the investigated compounds and samples. Substantially higher degrees of chlorination were observed for the following methyl esters: 3,5-dichloro-4-ethoxybenzoic acid (3.7%), 3,5-dichlorobenzoic acid (2.5%), and 3,5-dichloro4-methoxybenzoic acid (20%) in spruce forest soil; 3,5dichloro-4-methoxy benzoic acid (20%) in the spruce stump; and trichlorobenzoic acid (2.8%) in grass soil.
Discussion This study of chlorinated aromatic structures in plant material and soil organic matter clearly showed that, for a majority of the analyzed samples, chlorinated monoaromatic reaction products were present after oxidative degradation of macromolecules. In a previously published study involving degradation of humic substances in the presence of chloride
FIGURE 3. Nonhalogenated degradation products obtained upon oxidative degradation of high molecular weight organic matter isolated from various environmental compartments and plant species. Yields (µg/g of organic matter) of compounds A-F as presented in Figure 2.
TABLE 3. Chlorinated Methyl Estersa Found after Oxidative Degradation of Solvolysis Products from Fresh and Decaying Plants and HMW OM from Soilb spruce, fresh wood spruce, stump spruce, forest soil birch, yellow leaves birch, brown leaves sphagnum, fresh moss sphagnum, peat meadow grass, fresh meadow grass, soil
ACl1
ACl2
BCl1
ECl2a
ECl2b
ECl2c
ECl3
FCl2
-c 2 3 5 4 11 1
1 19 13 5 2
1 2 -
3 -
