Environ. Sci. Technol. 2009, 43, 3569–3573
Temperature Sensitivity Indicates That Chlorination of Organic Matter in Forest Soil Is Primarily Biotic D A V I D B A S T V I K E N , * ,†,‡ TERESIA SVENSSON,‡ ´ N,‡ SUSANNE KARLSSON,‡ PER SANDE AND GUNILLA O ¨ BERG§ Department of Geology and Geochemistry, Stockholm University, SE-106 91 Stockholm, Sweden, Department of Water and Environmental Studies, Linko¨ping University, SE-581 83 Linko¨ping, Sweden, and Institute for Resources, Environment and Sustainability, University of British Columbia, 428-2202 Main Mall, Vancouver, B.C., Canada, V6T 1Z4
Received December 17, 2008. Revised manuscript received March 5, 2009. Accepted March 11, 2009.
Old assumptions that chloride is inert and that most chlorinated organic matter in soils is anthropogenic have been challenged by findings of naturally formed organochlorines. Such natural chlorination has been recognized for several decades, but there are still very few measurements of chlorination rates or estimates of the quantitative importance of terrestrial chlorine transformations. While much is known about the formation of specific compounds, bulk chlorination remains poorly understood in terms of mechanisms and effects of environmental factors. We quantified bulk chlorination rates in coniferous forest soil using 36Cl-chloride in tracer experiments at different temperatures and with and without molecular oxygen (O2). Chlorination was enhanced by the presence of O2 and had a temperature optimum at 20 °C. Minimum rates were found at high temperatures (50 °C) or under anoxic conditions. The results indicate (1) that most of the chlorination between 4 and 40 °C was biotic and driven by O2 dependent enzymes, and (2) that there is also slower background chlorination occurring under anoxic conditions at 20 °C and under oxic conditions at 50 °C. Hence, while oxic and biotic chlorination clearly dominated, chlorination by other processes including possible abiotic reactions was also detected.
Introduction Natural chlorination of organic matter in soils has been discussed for several decades (e.g., 1, 2). It has been a controversial topic partly due to previous assumptions that (a) chloride is inert in soil environments, and (b) that all organochlorines found in nature were toxic and of anthropogenic origin. Now that natural formation of chlorinated organic matter in soils is recognized (3-5), questions about rates and mechanisms of chlorination as well its implications are in focus. The mechanisms behind the formation of most chlorinated soil organic matter are unclear and while there is * Corresponding author e-mail:
[email protected]; phone: +46 8 6747324. † Stockholm University. ‡ Linko¨ping University. § University of British Columbia. 10.1021/es8035779 CCC: $40.75
Published on Web 04/01/2009
2009 American Chemical Society
evidence of both biotic and abiotic processes (6-10), their relative importance is unknown. The underlying processes and their rates are of interest for several reasons. First, based on the assumption that Clin (chloride) is inert in soil, Clin is commonly used to trace water movement (e.g., 11-13). Recent estimates suggest that transformation of Clin to organically bound chlorine (Clorg) may cause considerable retention of Clin in soil (4, 5), which in turn may bias hydrological modeling. Second, the major part of Clorg represents chlorinated organic matter with unknown functions, but compounds of ecotoxicological concern such as chloroacetic acid, chloromethane, and chlorinated aromatic compounds are also formed (14, 15). Given the extensive presence and formation of Clorg in soils (Clorg g Clin in many forest soils; e.g., (4)), and that some of these compounds may be toxic, a deeper understanding of underlying Clorg formation mechanisms is important. A fundamental step in exploring this is to study how formation rates depend on environmental variables and to what extent Clorg formation is biotic or abiotic. The aim of the present study was to investigate the incorporation of 36Cl into soil organic matter at different temperatures and in the presence and absence of molecular oxygen (O2). The results were used to discuss to what extent the chlorination was biotic or abiotic.
Materials and Methods Soil Sampling. Soil was collected at the Stubbetorp catchment (58°44′ N, 16°21′ E) in SE Sweden in July 2006. The catchment is covered with coniferous forest dominated by Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) and the soil is of the spodosol type. Detailed catchment budget estimates of Clorg and Clin in the soil and the stream draining the area have been conducted (5, 16, 17) and the storage of Clorg in the catchment was three times larger than the storage of Clin (18 g Clorg m-2 and 6 g Clin m-2). At the site of soil collection, the uppermost centimeters of the soil were dominated by litter. Below the litter layer there was a dark brown to black organic rich layer down to approximately 15 cm depth (referred to as the A horizon). Below the A horizon, there was a leached gray layer up to 5 cm thick (E horizon), and farther below the soil was rust colored (B horizon). The catchment area is 0.87 km2 with a broken topography and a granite-dominated bedrock that is poor in chloride (18). The long-term annual mean precipitation in the region is approximately 600 mm and the annual mean temperature is about 6 °C. Soil from the A horizon, where chloride retention were previously found to be higher than in the other layers (4), was collected with a spade and transported in plastic bags (polyethylene). In the laboratory, the soil was sieved through 2 mm mesh, transferred to a wide mouth plastic bottle (2 L, polyethylene), and stored moist in the dark at 2 °C until the start of the experiments (storage time 10 days). Experimental Setup. The fate of Clin was studied in a 198 day long radiotracer experiment using soil incubated with 36 Cl. The experiment was conducted at five different temperatures (4, 10, 20, 37, and 50 °C). Two treatment series, one oxic and one anoxic, were incubated at 20 °C and were sampled on six occasions (day 0 representing initial samples for all treatments, and days 23, 55, 138, and 198). The other series incubated at the other temperatures (all oxic) were sampled on three occasions (days 23, 55, and 198). Three replicates were collected each time for each treatment series. The experimental design followed the design of one of our previously conducted experiments (4); 2.0 g of fresh soil and 36Cl-chloride (36Clin) were added to 50 mL centrifuge tubes (Sarstedt, Germany). The 36Clin (Amersham Biotech; VOL. 43, NO. 10, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Percentage of added 36Clin recovered as 36Clorg over time in the different treatments (see text for details). Error bars denote ( 1 SD (n ) 3). For some treatments the position along the x-axis has been modified ( 1-2 days in the figure to make it easier to distinguish individual treatments. 0.52 MBq mg Cl1-) was diluted in Milli-Q water so that 36Clin corresponding to 318 000 disintegrations per minute (DPM; 1 Bq ) 60 DPM) was added to each centrifuge tube. Given the specific activity of the source, 318 000 DPM of 36Clin represented a mass of 9.6 µg Cl. The total added radioactivity was the same for all replicates. The volume of the 36Clin solution added to each tube (1.2 mL) was adjusted to make the soil semifluid. Thereafter homogeneous distribution of the isotope could be achieved by stirring the wet soil with a syringe needle. After stirring, two slots in the soil crossing each other were made with the needle allowing penetration of air to the bottom of the test tube, increasing the soil area in contact with the head space gas in the tubes. The tubes were dried under a fan at room temperature (20 °C) to the original weight. The whole procedure, from addition of 36Clin solution to drying, took 18 h. The tubes were divided into six series and placed in climate chambers in the dark with different temperatures (4, 10, 20, 37, and 50 °C). In the oxic treatments, oxic conditions were maintained in the tubes by timer-regulated aeration using aquaria pumps (4 × 30 min each day). The air pumped through the tubes was first pumped through a similar tube filled with water to increase the water content of the air and prevent drying of the soil in the tubes. The water content was also checked every week and evaporated water was replaced with water additions to the initial fresh weight. A second set of tubes kept at 20 °C was incubated under anoxic conditions in a glovebox with a through flow of N2. This set of tubes was kept in the glovebox under the same conditions as the oxic tubes (i.e., timed gas flushing using aquaria pumps and dark conditions). Soil Extractions. Twenty mL of Milli-Q water was added to each tube at sampling and the tubes were placed on an end-over-end shaker for 30 min, centrifuged at 6000g for 12 min, and the supernatant was transferred by pipet to new centrifuge tubes (water extract no. 1). An additional 20 mL of water was added to the residual soil and the shaking and centrifugation were repeated to yield water extract no. 2. The extraction procedure was repeated twice more (yielding extracts 3 and 4). Through this procedure, each original tube yielded four water extracts and residual soil. The extracts, as well as the residual soil, were frozen until further analyses. Based on findings of significant microbial uptake of 36Clin in a previous experiment (4), the residual soil was then thawed, dried in 60 °C, milled, rewetted with H2O, sonicated, and then extracted four more times (two times with water and two times with 0.01 M KCl to enhance exchange with possible adsorbed 36Clin) according to above yielding extracts 5-8. Previous tests confirmed that this procedure also leached the 36Clin possibly taken up by microbial cells and that the remaining 36Cl in the residual soil was organically bound (4). 3570
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FIGURE 2. Percentage of added 36Clin recovered as 36Clorg by day 55 of the experiment at different temperatures. Black diamonds show oxic treatments while open squares show anoxic treatments. Individual replicates are shown (n ) 3). See text for details. Extracts 5-8 were frozen, and the residual soil was dried at 60 °C until further analysis. Inorganic and Organic 36Cl in Extracts. The extracts contained both 36Clin and 36Cl bound to the extracted organic matter (36Clorgex). These two Cl pools were separated according to the AOX analysis procedure (19). In brief, a volume of 1 mL of acidified nitrate solution (0.22 M KNO3, 0.02 M HNO3) and approximately 4 drops of concentrated HNO3 (yielding a pH 98% of the 36Cl present in the sample prior to combustion. Organic 36Cl in the Residual Soil (36Clorgsoil). After all 8 extractions, the organically bound 36Cl in the residual soil was determined by combustion of 0.2 g of soil and the formed H36Cl was trapped in NaOH as described above for 36Clorgex. Previous tests confirmed that the Cl in the residual soil was organically bound to both humic and fulvic acid parts of the organic matter (4). Analytical Procedures. Soil contents of water (after drying at 105 °C for 31 h), soil organic matter, total organic chlorine, and extractable Clin were determined before the start of the experiment. Soil organic matter was measured by loss on ignition at 550 °C for 8 h. Soil without added 36Cl was extracted according to the same procedure as described above for the 36 Cl amended samples and subportions of nonfrozen extracts from soil were analyzed for total extractable organic carbon content using a Shimadzu 5000 TOC analyzer. Remaining extracts were frozen and after thawing were analyzed for chloride concentrations by ion chromatography with chemical suppression (MIC-2, Metrohm) according to the standard procedure for determination of Clin of water with low contamination (Standardization 1995). A calibration standard was analyzed approximately every 10th sample. The detection limit was 0.1 mg L-1. The original soil was dried and milled, and then analyzed for total organic halogen (TOX) content according to ref 20 using an ECS3000 analyzer (Euroglas).
TABLE 1. Specific and Estimated Actual Chlorination Rates in Different Treatments of the Experiments Day 0-55 a
Day 55-198 c
treatment
specific chlorination rateb (d-1)
chlorination rate (µg g-1 dry weight d-1)
specific chlorination rateb (d-1)
chlorination ratec (µg g-1 dry weight d-1)
4 °C 10 °C 20 °C 37 °C 50 °C anoxic 20 °C
0.0017 0.0027 0.0037 0.0020 0.0007 0.0006
0.035 0.057 0.078 0.042 0.015 0.012
0.0009 0.0004 0.0010 0.0007 0.0003 0.0002
0.018 0.009 0.020 0.014 0.006 0.004
a
c
If O2 regime is not specified, treatments were oxic (see text). b Fraction of present Clin turned into Clorg per day. Calculated from specific rates and the measured extractable Clin concentration of 21 µg g-1 dry weight.
Soil samples were analyzed for 36Cl by LSC (Beckman LX 6300), correcting for quench using standard curves prepared from solutions with the same chemical composition as the samples. Scintillation cocktail (Ultima Gold XR, Chemical Instruments AB) was added to all 36Cl sample extracts as well as to extracts without added 36Cl as blank controls. Blank controls were always run together with actual samples and all 36Cl measurements were corrected for background radiation.
Results and Discussion Soil Characteristics. The soil-water content was 33 ( 0.4% (average ( 1 SD; n ) 6) and the dry weight organic matter content was 27 ( 0.3%. The initial total organic chlorine content in the soil was 133 ( 27 µg g-1 dry weight and the extractable inorganic chlorine was 21 ( 2.5 µg g-1 dry weight. Clorg Formation Rates. 36Cl was incorporated in all samples and the amounts incorporated generally increased over time (Figure 1). The change over time was clearly temperature dependent and peaked at 20 °C (Figure 1). The lowest rates were found at 50 °C and in the anoxic treatment. Substantial chlorination occurred in all oxic treatments above 4 °C between day 0 and 23 while the 4 °C and anoxic 20 °C treatments showed lower initial rates of chlorination. After day 23, chlorination rates were highest in the oxic 20 °C treatment, followed by the 10 °C, the 37 °C, and the 4 °C treatment. A low but seemingly steady increase of 36Clorg occurred also in the anoxic 20 °C treatment. The amount of 36 Clorg in the 50 °C treatment first increased to 12% of the added 36Clin between day 0 and day 23, but by day 55 the amount had decreased to 6% of the addition. Thereafter the chlorination rate in the 50 °C treatment occurred at a similar level as in the anoxic treatment (Figure 1; Table 1). 36Clexorg always constituted a minor fraction ranging from 2% of 36Clorg in the treatments with lowest chlorination to 10% of 36Clorg in the treatments with highest chlorination rates. The formation of 36Clexorg over time was proportional to overall 36 Clorg formation. Biotic Chlorination under Aerobic Conditions. Our experiment clearly shows that temperature influences chlorination rates under aerobic conditions (Figure 2). The fact that the temperature dependency shows a bell shaped curve with a clear optimum suggests a strong biological component. It is well-known that enzymes increase their catalytic ability up to a certain temperature optimum. Thereafter the catalytic ability decreases because the protein is disrupted. In contrast, the rate of a purely physicochemical reaction is more likely to continuously increase (or possibly level off and become constant) with increasing temperature in the studied range. The enzymes thought to be most important for chlorination of soil organic matter are oxygenases (such as chloroperoxidases; e.g., (22)) meaning that presence of O2 is required for proper functioning. Our results show that oxic conditions favor chlorination. The observations of the present
study are in line with previous observations of soil organic matter chlorination under conditions likely to be oxic (4, 23-25). The chlorination rates declined over time, which is reasonable for a biotically driven process since biotic chlorination depends on availability of degradable organic matter (OM) to be used as energy source and the supply of such OM should decline over time in closed vessels. The rate of chlorination in the 50 °C treatment differed from that of the other aerobic treatments in that it was comparably high initially, thereafter declining to the lowest rates. It is reasonable to assume that a transformation rate that is catalyzed by enzymes depends on temperature in at least two different ways: (a) direct effects on the enzyme activity and (b) the rate at which new enzymes are produced by soil microorganisms. The microorganisms may be more sensitive to elevated temperatures than the enzymes. If so, initial activity of the enzymes already present could be high due to the high temperature, while at the same time the higher temperatures inhibit the microorganisms that are producing the enzymes. This could explain the high initial rates of chlorination at 50 °C followed by very low rates after the initially present enzymes had been depleted during which time no new enzymes were produced. Chlorination at High Temperatures and under Anoxic Conditions? The pattern of the 50 °C treatment suggests that the slow chlorination observed after day 23 in the 50 °C treatment was due to nonenzymatic processes. Few organisms are active at 50 °C and a previous study of soil from the sampled site showed no microbial activity at 50 °C (4), indicating that the chlorination at this temperature was primarily abiotic. However, it cannot be excluded that the soil contained enzymes which were active also at this temperature. Low rates of chlorination were also observed under anoxic conditions when oxygenase enzymes are inactive. Hence, processes being independent of O2 were partly responsible for the chlorination. The temperature effect on this anaerobic formation of organic chlorine was not studied and the present study cannot be used to conclude to what extent this process was biotic or abiotic. The anaerobic chlorination rates were similar to the rates observed at 50 °C (Table 1). Hence, while our data does not provide information about specific mechanisms, it is possible that abiotic and anaerobic processes with limited sensitivity to temperature within the studied range could explain chlorination at both 50 °C and anoxic conditions. It has previously been proposed that abiotic formation of volatile organohalogens from halide ions and organic matter is driven by the reduction of Fe3+ (9). Further evidence that iron is involved in abiotic chlorination processes is provided in a study of abiotic production of chloroacetic acids from soil humic matter after addition of Fe (10). The suggested mechanism involves redox reactions with Fe and is independent of O2 as long as Fe3+ is available. Such mechanisms may consequently be responsible for the VOL. 43, NO. 10, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Comparison between biotic (i.e., enzymatic and O2 dependent) and abiotic specific chlorination rates. The average rate at 50 °C was considered representative of the maximum possible abiotic chlorination (the dashed line; see text for details). This rate was subtracted from the other rate estimates resulting in an optimum curve for biotic chlorination (the gray line, the equation, and the black diamonds), which dominated overall chlorination between 4 and 40 °C in the studied forest soil. chlorination observed at higher temperatures and/or anoxic conditions in the present study. It should be noted though that chlorination driven by Fe redox processes may in the long run not be strictly abiotic or O2 independent since Fe cycling often depends on microbial activity and O2 is needed to regenerate Fe3+ from Fe2+. Specific and Actual Chlorination. When calculating process rates from incubation experiments, it is often argued that data from the early phase should be used to avoid incubation effects. For example, depletion of degradable OM will yield increasingly biased results over time. On the other hand, as argued above, temperature effects on the microbiota may be concealed by the presence of extracellular enzymes and the soil processes are likely disturbed by the experimental setup itself. Hence results obtained any time during an incubation experiment must be interpreted with caution. We therefore provide two sets of chlorination rates dividing the data set into an early phase (before day 55) and a later phase (after day 55). Specific chlorination rates (proportion of Clin turned into Clorg per time unit) and actual chlorination rates (µg Cl g-1 dry weight d-1) are shown for the different treatments in Table 1. For calculating the actual chlorination rates we assumed that all extractable Clin originally present in the soil was transformed at similar rates as the added 36 Clin. It should also be noted that the addition of 36Clin corresponding to 33% of the initial Clin levels means addition of a potentially rate limiting compound and this should be considered if the rates given in Table 1 are used to discuss possible in situ rates. Relative Contribution of Biotic and Abiotic Chlorination. Given the discussion above, the net chlorination rate amounts to the sum of oxygen dependent biotic, and the background (i.e., the chlorination taking place under anaerobic conditions and at high temperatures). The maximum possible abiotic chlorination rate in our experiment is obtained by assuming (a) that all of the background chlorination is abiotic, and (b) that the abiotic chlorination rate is independent of temperature and equal to the rate observed at 50 °C (the dashed line in Figure 3). The biotic chlorination rate was then estimated by subtracting the abiotic rate from the total rate. We also assumed zero biotic chlorination at 0 °C to enable extrapolation from 4 to 0 °C. The estimates suggest that biotic chlorination dominates in the temperature interval 4 to 40 °C (Figure 3). Hence, when assuming that all background chlorination is abiotic, the biotic chlorination dominates at temperatures being common in soils. If the assumption is 3572
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invalid meaning that the chlorination observed at 50 °C or under anoxic conditions is partly biotic, the biotic part of the chlorination dominates even more. 36 Cl Recovery and Formation of Volatile Clorg. At the start of the experiment (after one day of incubation), and in treatments with low chlorination rates close to 100% of the 36 Cl could be accounted for by summing all measured Cl pools. However, in the 20 °C treatment the amount that could not be accounted for increased over time and corresponded to approximately 10% of the added 36Cl at the end of the incubation period. One potential methodological reason for not recovering 100% of the added isotopes could be that the 36 Cl content of some pools such as Clin and Clorgsoil was derived from subsamples taken from a greater total sample. If the subsample were not representative of the total sample, this would result in errors when calculating the total 36Cl budget. However, we made great efforts to thoroughly mix samples before sampling and errors due to subsampling should be random resulting in deviation from 100% recovery in both directions (i.e., both more and less than 100% recovery could be expected). In addition, there should be no reason to expect decreasing recovery over time based on subsampling errors. In this case, when we consistently recovered less than 100% and when the recovery decreased with time, it seems more reasonable to suspect that we did not capture one or several Cl pools incorporating 36Cl. The only pool of Cl we know of that was not measured was volatile organochlorines (VOCl). Several VOCl compounds, including various chloromethanes and chloroethanes, are known to be produced in forest soils (26-28). Our presumed rate for the total VOCl production in the 20 °C treatment corresponds to 11 ng g-1 dry weight d-1 (0.2 g Cl m-2 yr-1 or 5 mmol Cl m-2 yr-1 and a specific rate of 5.1 · 10-4 d-1). These rate estimates appear reasonable given previous studies (29-32). Implications. This study provides further evidence that natural chlorination of organic matter represents a quantitatively important part of chlorine cycling in soil, and that most of this chlorination is driven by microbiological processes at temperatures between 4 and 40 °C. Our results show that the presence of O2 is crucial for the dominating processes, indicating that haloperoxidase enzymes play an important role. The results also indicate that there is also a slower background chlorination being independent of O2 and occurring at temperatures as high as 50 °C. This suggests that haloperoxidase independent and/or abiotic chlorination may contribute to the chlorination of bulk soil organic matter. Altogether, the results have implications for the use of Clin as a tracer of soil-water, represent a step toward increased understanding of the rates of organochlorine formation in the environment, and raise questions about chlorination mechanisms and the ecological reasons for the extensive biological chlorination.
Acknowledgments We thank Monica Petersson, Lena Lundman, and Hildred Crill for valuable assistance and are grateful for financial support from the Swedish Research Council (VR) (2004-4361 and 2006-5387) and the Natural Sciences and Engineering Research Council of Canada (NSERC RGPIN 341994-07).
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