Environ. Sci. Technol. 1903, 27, 1880-1884
Biotransformations and Toxicity Changes of Chlorolignins in Soil Robert Brerny,'vt Thomas W. Joyce,* Bernard0 Gonraler,§ and Mllan Sllmakt
Department of Wood, Pulp, and Paper Technology, Faculty of Chemical Technology, Slovak Technical University, 812 37 Bratislava, Slovakia, Department of Wood and Paper Science, North Carolina State University, Box 8005, Raleigh, North Carolina 27695, and Department de Biologia Celular y Molecular, Laboratorio de Microbiologia, Facultad de Ciencias Biologicas, Casilla 114-D, Santiago, Chile The fate of high molecular weight and medium molecular weight chlorolignins (MW > 10K and MW > 1K but < 10K,respectively) in aerobic soil under in vitro conditions was studied. After several days of incubation, less than 5 7% of the chlorolignin absorbance could be recovered by water extraction. This indicates a rapid immobilization into the soil. About 117% of the organic chlorine content of chlorolignins was mineralized to chlorides a t both 1000 and 2000 ppm concentrations after 90 days of incubation in soil. GC-ECD and GC-MS analysis of organic solvent extracts of soil incubated with chlorolignins (1000 ppm) showed no detectable accumulation of low molecular weight chlorinated compounds. There was no significant difference in viable bacterial counts between control soils and those incubated with chlorolignins. Aqueous extracts of soils incubated with chlorolignins showed no toxicity above the detection limit of the Microtox system. Organic solvent extractions recovered toxicity more completely. Since all these toxicities are very low (ECK,> 100 g of soil/L), it was concluded that chlorolignins do not release significant toxicity during incubation in the soil.
Introduction Chlorolignins are formed during the bleaching of wood pulp with chlorine and chlorine derivatives. They occur in the effluents from the bleaching plants. Although the biological treatment of the effluents is very efficient in removing toxicity, chlorolignins are degraded to only a small extent (1, 2), and most of them pass through a biological wastewater treatment system essentially unchanged and enter the environment. The chlorolignins in wastewater streams, together with chlorolignin in sludges from the water treatment, thus represent another form of organic chlorine in the environment in addition to chlorinated pesticides, wood preservatives, and other anthropogenic sources. Because of the lack of appropriate analytical methods, there are no estimates of their quantities, movement, and transformations in the environment. There are concerns about chloroligninsreleasing toxic or bioaccumulating fragments or being transformed into biologically recalcitrant compounds. Several studies have reported spontaneous release of chlorinated phenolics from chlorolignins in aqueous medium under sterile conditions as well as by microbial activity (3-5). Using 14C-labeledchlorolignin, Eriksson and Kolar (6) measured a 4 5% mineralization rate when chlorolignin was exposed to mixed bacterial cultures from secondary waste treatment plant sludges. Incubation of the white-rot fungus Sporotrichum pulverulentum resulted in 40% mineralization of the chlorolignin in 6 weeks. While + Slovak Technical University.
North Carolina State University. Facultad de Ciencias Biologicas. 1880
Environ. Sci. Technol., Vol. 27, No. 9, 1993
studying pure cultures of several Pseudomonas and Enterobacter species, bacteria which can be found in lake and soil environments, the same investigators found no evidence of the ability to depolymerize chlorolignin. Bacteria were able to remove chlorinated catechols and guaiacols from aqueous solution, but a fraction was transformed to more persistent veratrole-type compounds. No studies on the pathways of decomposition of chlorolignins have been performed, mainly because of their structural complexity. Some studies have shown essentially no toxicity of chlorolignins (7,8). We have previously found about 7 % mineralization of organic chlorine in high molecular weight chlorolignin in soil during 30 days (9). There were no indications of accumulating metabolites. The objective of the present study was to compare the behavior of two chloroligninsof different molecular weights in soil under in vitro conditions in order to evaluate the potential hazards after the disposal of paper mill bleach plant sludges or concentrates. The immobilization of chlorolignin into the soil matrix, the rate of mineralization of organic chlorine, the potential release of low molecular weight chlorolignin fragments, the toxicity changes, and the effect on soil microflora were examined.
Experimental Section High Molecular Weight Chlorolignin (HM-CL, MW > 10K). The chlorolignin was isolated from the extraction stage effluent of a bleach plant (CEDED sequence) for softwood kraft pulp. Further hydrolytic reactions in the effluent were substantially reduced by adjusting the pH to 6 with concentrated sulfuric acid 1day after collection. A total of 16.2L of the effluent was ultrafiltered (Millipore) with a nominal 10K molecular weight cutoff membrane. The retentate (1.3 L) was washed and ultrafiltered four times with an equal volume of distilled water until the filtrate was almost colorless. The retentate was then concentrated on a vacuum evaporator, dried in air, pulverized, and finally dried under vacuum (2 kPa) a t 45 "C for 2 h. A total of 11.0 g of high molecular weight chlorolignin was recovered. Elemental analysis revealed this preparation to contain 43.19% C, 5.26% H, 0.61% S, and 4.90% C1. Residual chlorides, as measured by specific ion electrode, were 60 pmollg. Medium Molecular Weight Chlorolignin (MM-CL, MW > 1K but < 10K).The permeate from the preparation of the HM-CL was further ultrafiltered (Millipore) with a 1K nominal molecular weight cutoff membrane. The retentate (about 1L) was washed four times with an equal amount of distilled water and worked up similar to the HM-CL. A total of 3.4 g of medium molecular weight chlorolignin was recovered. Elemental analysis revealed this preparation to contain 35.62 % C, 3.79% H, 7.65 % C1, and 1.76% S. Residual chlorides, as measured by specific ion electrode, were 840 vmollg. Oxidative Hydrolysis of HM-CL. High molecular weight chlorolignin (HM-CL) (800 mg) was dissolved in 0013-936X/93/0927-1880$04.0010
0 1993 Amerlcan Chemical Society
water (20 mL), and aqueous 2 N NaOH was added (20 mL). While heating at 80 "C, sodium peroxosulfate (600 mg) was added*over 30 min. The mixture was kept a t 80 "C for 2 h. Sodium sulfite (200 mg) was then added, and after cooling, the mixture was neutralized by the addition of 13 g of air-dried cation exchanger in the H-form with about 100 mL of water. After the removal of cation exchanger, the pH was adjusted to 7 with 2 N NaOH, and the final solution was concentrated to about 80 mL in vacuum. The stock solution was prepared by filling the volume to 100 mL with deionized water (1mL of solution contained oxidized chlorolignin corresponding to 8 mg of the starting HM-CL). Soil. The soil taken from a potential landspreading site in Maine was stored without drying in plastic bags at 4 "C. The soil had not been previously exposed to industrial wastes or wastewater treatment sludges. The soil has been classified as a fine, sandy loam of the spodosol type formed from marine sediments. Its pH (with CaClz 1:l) was 6.25, organic carbon content 4.25%, and bulk density 0.95 g/cm3. Before spiking, the soil was air-dried and sifted (1-mm i.d.) to remove large pieces of organic material and stones. Incubation in Soil. Soil samples (20 g) were mixed with 4 mL of aqueous chlorolignin stock solution (5 or 10 mg/mL) and blended with 2 mL of distilled water. The samples were kept in 100-mLbeakers (soil layer thickness: 10-12 mm) at 25 "C and sprayed with distilled water every other day to keep the soil moisture a t the field capacity. For analysis of aqueous extracts, duplicates and control soil samples without chlorolignin were set up, and another two duplicates were set up for organic solvent extraction to examine the release of low molecular weight chloroaromatic compounds. Incubation with enhanced aeration, thorough mixing every 7 days, was performed in separate beakers. Analysis of Aqueous Soil Extracts. After the incubation time had elapsed, the soil samples were dried a t ambient temperature for 24 h. Deionized water (25 mL) and 5 M sodium nitrate (0.5 mL) as an ionic strength adjuster were then added. The slurry was stirred 1 h in a closed vessel, sonicated for 3 min (300 W), and centrifuged. The supernatant was used for the measurement of chloride ions concentration and UV spectra. Analysis of Organic Solvent Extracts of Soil. The analysis of soil for the presence of low molecular weight compounds was performed on extracts of soil obtained by an acetylating extraction technique (IO). After the incubation time elapsed, the soil samples were mixed with anhydrous sodium sulfate (25 g) and then with a mixture containing ethyl acetate (60 mL), acetic anhydride (10 mL), and pyridine (1mL). The mixture was homogenized with a spatula and subjected to sonication (300 W) for 3 min. The extract was separated by filtration through a sintered glass funnel, and the extraction procedure was repeated three more times. The combined extracts were concentrated on a rotary evaporator; small amounts of methanol (10 mL) and toluene (2 X 10 mL) were added to decompose excess acetic anhydride and to assist in the evaporation of the solvents. The volume was reduced to about 6 mL and spiked with the quantitative internal standard, 4,5-dichloroveratrole (DCV). About 0.7 mL was reacetylated by the addition of acetic anhydride (0.1 mL) and pyridine (0.02 mL) and analyzed by gas chromatography on a Hewlett-Packard 8990A system equipped with
an automatic sampler and an electron-capture detector. An open tubular column (30 m X 0.31 mm i.d., DB-5 phase of film thickness 0.25 pm; J&W Scientific) with helium as a carrier gas was used. Temperature program: 1 min a t 45 "C, from 45 to 120 "C at 15 "C/min, from 120 to 230 "C a t 2 "C/min. Purification and Enrichment of Soil Extract. A soil extract prepared by the acetylating procedure (6 mL) and containing the internal standard (0.01 mg of DCV) was concentrated on a vacuum evaporator almost to dryness. It was reacetylated by the addition of acetic anhydride (0.5 mL) and pyridine (0.1 mL) to the residue. After 1h, methanol was added to destroy the acetylating agent, and the acetylated sample was concentrated with the aid of toluene (several additions of 0.3 mL). The dry residue was dissolved in toluene (0.4 mL) and put on a column of silica gel (Silica TSC Woelm, activity 111, 1cm i.d. X 1cm). With the aid of a slight vacuum, the column was eluted with 3 mL of toluene. The eluate was concentrated to a small volume (50 pL) and injected onto the chromatographic column. Chloride Ion Determination. The chloride concentration in aqueous soil extracts was measured by a selective chloride ion electrode (Orion 96-17B) with an Orion 520A pX-meter. Three independent readings for each measurement were averaged. The system was calibrated with aqueous solutions of potassium chloride after the adjustment of ionic strength. UV spectra of aqueous soil extracts were measured on a Perkin-Elmer UV-VIS spectrophotometer after a 3:lO dilution. GC-MS analysis with electron impact spectra (70 eV) was performed on a Hewlett-Packard 5985b system; chromatographic conditions were the same as in GC-ECD analyses. Automated library searching (NBS) was used for the tentative identification of the chromatographic peaks. Microbial Counts. About 2 g of soil was weighed and resuspended in 10mL of sterile distilled water. The slurry was agitated for 1h, and then proper dilutions (from 0 to 1:lOOO) were prepared in sterile conditions. Aliquots (0.1 mL) of each dilution were plated and incubated at 25 "C for 2-5 days. All dilutions were duplicated. Total microbial counts were determined on plates containing 40 g/L of Luria broth, a tryptone-yeast extract solidified with agar. Chloramphenicol-resistant microorganisms were estimated in Luria broth with 5 mg/L of chloramphenicol plates. Molds and yeasts were estimated in LB plates containing 7 mL/L of a mixture of antibiotics (penicillin 5000 units/mL, streptomycin 5 mg/mL, and neomycin 10 mg/mL; Sigma Chemicals). Microbial counts were expressed as colony-forming units per gram of soil. A control with nonincubated soil was performed with the air-dried and sifted soils kept for 1 month a t room temperature. Microtox Toxicity Measurements. Aqueous soil extracts for toxicity measurements were prepared as described above, except that no ion adjustor was added. Water/organic solvent extraction was the combination of water extraction with the organic solvent extraction described by ref 11. The soil was first extracted with deionized water (25 mL) as described above. The water extract was collected after centrifuging (10 min a t 15 000 rpm). Acetonitrile (8 mL) and 1.14 M aqueous L-ascorbic acid (2 mL) were next mixed into the soil sample, and the mixture was shaken for 1h. Then 20 mL of water and 1 Environ. Sci. Technol., Vol. 27,
No. 9, 1993 1881
Table I. Chlorolignin Absorbance i n Aqueous Soil Extracts incubation time (days)
Azso
corrected Azso
% of starting
Control Soil (without Chlorolignin) 0
0.25 5 15 30 60 90
0.010 0.028 0.003 0.053 0.008 0.083 0.041
0.207 0.061 0.012 0.065 0.016 0.075 0.049
0.196 0.033 0.009 0.008 0.008 -0.008 0.008
76.9 12.8 3.5 3.1 3.1 -3.1 3.1
High Molecular Weight Chlorolignin (2 mg/g) in Soil; Theoretical A = 0.51 60 90
0.1 0.054
0.017 0.013
3.4 2.6
High Molecular Weight Chlorolignin (2 mg/g) in Aerated Soil; Theoretical A = 0.51 60 90
0.088 0.049
0.005 0.008
1.0 1.5
drop of concentrated sulfuric acid were added. The soil suspension was extracted with a mixture of hexane/t-butyl methyl ether (2:1, 10 mL) for 15 min. The liquid was collected after centrifuging, and the organic layer was separated by a separatory funnel. The extraction was repeated once again, and the combined extracts were mixed with 1drop of 1M sodium acetate and concentrated under vacuum almost to dryness. The residue was transferred with two portions of 50% aqueous DMSO (0.6 mL) to a 50-mL volumetric flask, the aqueous extract (about 20 mL) was added, and the volume was increased to 50 mL with deionized water. The toxicity of aqueous and water/ organic solvent soil extracts were measured with aMicrotox Model 500 toxicity test system (MicrobicsCorp., Carlsbad, CA). Determinations were performed using the 100% protocol for low toxicity samples,followingthe instructions given by the supplier in the manual. Results were expressed as ECb0 in gram of soil per liter using the Microtox software. Results and Discussion
UV Spectrophotometry of Chlorolignin in Soil. UV spectrophotometry was used to monitor the presence of chlorolignin in aqueous soil extracts during incubation. Extracts of control soil without chlorolignin (spectrum 3) show some absorption in the 280-nm region where chlorolignin has a typical absorption maximum. Table I shows corrected A280, calculated as the differences in A280, of extracts of soils incubated with and without chlorolignin. Fluctuations in absorbance of control soils indicate ongoing changes in the organic matter of soil. However, these changes do not overshadow the rapid decrease in chlorolignin UV absorption in soil extracts, indicating a rapid immobilization of chlorolignin into the soil matrix. Extraction of soil immediately after applying chlorolignin recovered 77 % of the lignin absorbance, but after 6 h only 1882
time (days)
chloride (ppm)
0 5 15 30 60 90
5.23 f 0.1 5.51 f 0.1 4.88 f 0.11 6.10 f 0.12 6.20 f 0.05 7.17 f 0.20
total chloride (pg)
chlorolianin-derived cgloride (pg) ( % of TCP)
Control Soil (without Chlorolignin)
High Molecular Weight Chlorolignin (1mg/g) in Soil; Theoretical A = 0.255 0 0.25 5 15 30 60 90
Table 11. Mineralization of Chlorolignin Organic Chlorine i n Soil (20 g Air-Dried)
Environ. Sci. Technol., Voi. 27, No. 9, 1993
0 5 15 30 60 90 0
15 30 60 90
133.4 f 2.6 140.5 f 2.6 124.4 f 2.8 155.6 f 3.1 158.1 f 1.3 182.8 f 5.1
High Molecular Weight Chlorolignin (1mg/g) in Soil 6.27 f 0.08 166.2 f 2.1 33 f 4 3.3 7.53 f 0.24 7.62 f 0.12 9.88 f 0.19 11.03 f 0.14 12.66 f 0.15
192.0 f 6.1 194.3 f 3.1 251.9 f 4.8 281.3 f 3.6 322.8 f 3.8
52 f 7 70 f 4 96 f 6 123 f 4 140 f 6
5.2 7.1 9.8 12.5 14.2
Control Soil (without Chlorolignin) 97.1 f 2.3
3.81 f 0.10 4.41 f 0.05 4.96 f 0.05 6.2 f 0.13 6.4 f 0.07
112.5 f 1.3 126.5 f 1.3 158.1 f 3.3 163.2 f 1.8
High Molecular Weight Chlorolignin (2 mg/g) in Soil 0 30 60 90
7.14 f 0.21 12.3 f 0.39 15.89 f 0.15 17.02 f 0.23
182.1 f 5.4 313.7 f 9.9 405.5 f 3.8 434 f 5.9
85 f 5 187 f 10 247 f 5 271 f 6
4.3 9.6 12.6 13.8
High Molecular Weight Chlorolignin (2 mg/g) in Well-Aerated Soil 30 60 90
12.46 f 0.25 17.21 f 0.56 17.85 f 0.12
317.7 f 6.4 438.9 f 14.3 455.2 f 3.1
191 f 6 281 f 14 292 f 4
9.8 14.3 14.9
Oxidized High Molecular Weight Chlorolignin (2 mg/g) in Soil 0 34.3f 0.2 874.7 f 4.3 778 f 4 39.6 30 60 90
36.01 f 0.6 45.86 f 0.88 44.4 f 0.9
918.3 f 15.3 1169.4 f 22.4 1132.2 f 23
792 f 15 1011 f 22 969 f 23
40.4 51.6 49.4
Medium Molecular Weight Chlorolignin (2 mg/g) in Soil 0 30 60 90 a
51.5 f 0.91 69.86 f 2.0 66.32 f 0.99 65.6 f 0.93
1313.3 f 23 1781.4 f 51 1691.2 f 25.2 1672.8 f 23.7
1216 f 23 1654 f 51 1533 f 25 1510 f 24
39.7 54.1 50.1 49.3
Total chlorine (from elemental analysis).
13% of the lignin absorbance could be recovered. As seen in Table I, chlorolignin or its transformation products could not be recovered from soil by water extraction over the entire incubation period. These data suggest that chlorolignin mobility in soil under natural conditions will be very limited. This influences chlorolignin availability for biodegradation, and the leaching of chlorolignins from soil into groundwaters is improbable. The rapid immobilization further suggests that a simple adsorption is the mechanism responsible for the loss of absorbance recovery rather than chemical reactions catalyzed by enzymes or soil constituents, such as transition metals. Mineralization of Organic Chlorine. The measurement of chloride ion concentrations in soils incubated with and without chlorolignin showed a release of chlorides from chlorolignin during incubation. As seen in Table 11, the mineralization of organic chlorine is most rapid at the beginning of the incubation. After 90 days of incubation, about 11% of the organic chlorine was mineralized in high molecular weight (MW >10K) chlorolignin. Almost the
r
2 0 0 ~y
I
; C L in soil: difference 90 day - 0 day
10
20 Tlme
30
1
2
8
I
52
1
7
4 5
I
40
50
(rntn.)
Oxidized HM-CL in soil: difference 90 day - 0 day
I
1%
I
ll
! 30 Tlme
40
50
60
'
(rnrn.)
I I
30 Time
40
50
60
(min.1
same rates of mineralization were found for 1000 and 2000 ppm chlorolignin concentrations in soil. Improved aeration of the soil layer had no positive effect on rate of mineralization. Assuming that a degradative pretreatment would result in a material more amenable to decomposition in soil, we attempted to accelerate mineralization by oxidative hydrolysis of the chlorolignin before incubation. Oxidative alkaline pretreatment of high molecular weight chlorolignin caused about 36 % conversion of organic chlorine to chlorides, but it did not accelerate biological mineralization of organic chlorine in soil, and the rate of organic chlorine release was found to be essentially the same as for the unmodified material (about 10% after 90 days). Medium molecular weight chlorolignin, containing more residual chloride (almost 40% of total chlorine), was mineralized to almost the same extent (about 10%1. Although the rates of mineralization of organic chlorine to chloride in both chlorolignins might seem slow, it is comparable with natural carbon mineralization of nonchlorinated lignins. Measuring the evolution of carbon dioxide, 1-10% mineralization was found for lignins in lignocellulose materials incubated in various soils for 30 days (12). GC Analysis of Soil Extracts. As we reported earlier, no generation and accumulation of low molecular weight chlorinated products was detected by GC-ECD chromatography in soil incubated with high molecular weight chlorolignin for 30 days (9). The detection limits ranged from 0.7 (for monochloro) to 0.002% (for tetrachloro compounds) based on chlorolignin applied to soil at 1000 ppm concentration. In this study, we came to the same conclusion with the same chlorolignin incubated at two concentrations and under enhanced aeration conditions for 90 days, as well as for medium molecular weight chlorolignin. This is illustrated by the difference chromatograms in Figure 1. The differences were obtained by subtraction of chromatograms of soil extracts corresponding to final incubation times and the beginning of incubation. To lower the detection threshold, the acetylated soil extract, obtained after 60 days incubation of high molecular weight chlorolignin in soil, was purified and concentrated by short column chromatography. From the increased
I
8
12
16
20
24
28
32
36
44
40
48
Figure 2. GC-ECD and GC-MS chromatograms (A and B, respectlvely) of extract of soil incubated with high molecular weight chlorolignin (2000 ppm) for 60 days. Chlorinated compounds, designated by numbers, were identified by MS spectra with the aid of NBS library searching ( % matching): 1,2,2dichloroethenyl-4-chlorobenzene(not found): 2, DCV (internal standard): 3, Dursban (76); 4, Dieldrin (90): 5, l,ldichloroethenylldene-bis(4-chlorobenzene)(99); 6, 1,1'-(2,2dichloroethylidene)-bis(4-~hlorobenzene)(87); 7, o,p'-DDT (90); 8, DDT (94).
Table 111. Microbial Counts (X 104) per Gram of Soil Incubated with Chlorolignin (Calculated from Three Replicates) chloramphenicolresistent bacteria yeast/molds
incubation conditions
total bacteria
control soil, 0 day control soil; 30 days aerobic anaerobic soil with HM-CL, 1000 ppm chlorolignin aerobic anaerobic
8.71 f 0.97
3.29 f 0.45
2.08 f 1.25
2.29 f 0.68 3.47 f 0.86
0.80 f 0.08 1.87 f 0.23
1.68 f 0.72 1.46 f 0.25
3.19 f 0.45 4.69 .+ 0.70
0.48 f 0.01 0.71 f 0.06
1.64 f 0.41 1.79 f 0.35
response of the internal quantitative standard, it was estimated that the detection limit was improved by purification about seven times. The GC-ECD and GCMS patterns of the purified and enriched soil extract are shown in Figure 2. Mass spectra of the compounds present were compared with those in the NBS mass spectra library. Most of the peaks represented nonchlorinated compounds with long hydrocarbon chains. In addition to dichioroveratrole as the internal standard, seven chlorinated compounds were found. They are designated by numbers in Figure 2. None of them could be related to chlorolignin, and obviously they are present as residual chlorinated pesticides (DDTs, Dieldrin, Dursban) or metabolites of DDT, such as dichloroethenylidene- and dichloroethylidene-bis(4-chlorobenzene)and dichloroethylene-4-chlorobenzene. Microbial Counts and Toxicity of Soil. To illustrate the effect of chlorolignin on soil microflora, viable counts of total bacteria, Gram-positive bacteria, and yeasts and molds were made (Table 111). As is obvious, laboratory incubation decreased the number of bacteria in the control soil by a factor of 4. This can be ascribed to the incubation conditions which were different from those in nature. The presence of chlorolignin slightly increased the total bacterial population in soil, but decreased the contribution of Gram-negative bacteria to the total. There was no significant difference in the number of yeasts and molds in the soil after 30 days of incubation with chlorolignin. Environ. Sci. Technol., Vol. 27,
No. 9, 1993 1883
Table IV. Toxicities of Chlorolignins and Organic Solvent Extracts of Soil Incubated with Chlorolignins* incubation time (days)
5-min ECw (g of soil/L)
15-min ECm (g of soil/L)
relative soil toxicityb
Aqueous Extracts control soil 30 high molecular weight chlorolignin in soil 30
not toxic
not toxic
not toxic
not toxic
Water/Organic Solvent Extracts control soil 0
329 f 29
299 f 28
1
High Molecular Weight Chlorolignin (2000 ppm) in Soil 137 f 5 147 f 5 2.2 5 193 f 27 201 f 13 1.6 15 230 f 91 250 87 1.3 30 172 f 33 187 f 1 1.7
and medium molecular weight chlorolignins at 2000 ppm concentration. Expressed as relative soil toxicity, defined as the ratio of EC50 values for control and noncontrol soils, HM-CL showed a decrease in relative soil toxicity during the first 15 days of incubation (from 2.2 to 1.3). Over the next 15days, the relative toxicity slightly increased to 1.7. The final toxicity is still low and lower than the starting one. For medium molecular weight chlorolignin, relative soil toxicities lower than control soil (lo0 g of soil/L) were found for organic solvent extracts of soils incubated with high
1884
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Literature Cited (1) Pellinen, J.; Salkinoja-Salonen, M. S. In Organic micropollutants in the aquatic environment;Bjorseth, A., Angeletti, G., Eds.; D. Reidel Publishing Co.: Dodrecht, The Netherlands, 1986; p 354. (2) Eriksson, K.-E.; Kolar, M.-C.; Ljungquist, P. 0.;Kringstad, K. P. Environ. Sci. Technol. 1986, 19 (12), 1219. (3) O’Connor, B. I.; Voss, R. H. Environ. Sci. Technol. 1992, 26 (3), 556-560. (4) Neilson, A. H.; Allard, A.-S.; Hynning, P.-A,; Remberger, M.; Landner, L. Appl. Environ. Microbiol. 1983,45 (3), 774783. (5) Allard, A.-S.; Remberger, M.; Viktor, T.; Neilson, A. H. Water Sci. Technol. 1988, 20 (2), 131-141. (6) Eriksson, L.-E.; Kolar, M.-C. Environ. Sci. Technol. 1985, 19 ( l l ) , 1086-1089. (7) Salkinoja-Salonen, M. S.;Saxelin, M.-L.; Pere, J.; Jaakhola, T.; Saarikoski, J.; Hakulinen, R.; Koistinen, 0.In Advances in the Identification a n d Analysis of Organic Pollutants in Water; Keith, L. H., Ed.; Ann Arbor Science: Ann Arbor, MI, 1981; Vol. 2, p 1131. (8) Sagfors, P.-E.; Starck, B. Water Sci. Technol. 1988,20 (2), 49. (9) Joyce, T. W.; Brezny, R.; Gonzales, B.; Palo, N. Pap. Celul. 1992,47 (6), 124. (10) Brezny, R.; Joyce, T. W. Chemosphere 1992,24 (B), 1031. (11) Remberger, M.; Allard, A.-S.; Neilson, A. H. Appl. Environ. Microbiol. 1986, 51 (3), 552. (12) Crawford, R. L. In Lignin Biodegradation and Transformation; Crawford, R. L., Ed.; John Wiley & Sons: New York, 1981; p 61. Received for review December 14, 1992. Revised manuscript received March 23, 1993. Accepted April 23, 1993.
