Environ. Scl. Technol. 1991, 25, 1728-1731
Effect of Surfactants at Low Concentrations on the Desorption and Biodegradation of Sorbed Aromatic Compounds in Soil Borls N. Aronsteln, Yolanda M. Calvillo, and Martln Alexander* Laboratory of Soil Microbiology, Department of Soil, Crop. and Atmospheric Sciences, Cornell University, Ithaca, New York 14853
A study was conducted to determine the effect of low concentrations of surfactants on the biodegradation of sorbed aromatic compounds in soil. The nonionic alcohol ethoxylate surfactants Alfonic 810-60 and Novel I1 1412-56 increased the extent of desorption of phenanthrene from a mineral soil. Alfonic 810-60 enhanced desorption of biphenyl from this soil at one concentration tested, but Novel I1 1412-56 did not. Less than 0.01%0of the added phenanthrene and biphenyl was present in solution after their introduction into an organic soil, and the surfactants did not promote desorption. The two surfactants a t 10 pg/g of soil markedly increased the extent of biodegradation of phenanthrene in both the mineral and the organic soil; the stimulation was greater in the organic soil. Biphenyl mineralization in the mineral soil was not affected by either surfactant, but biodegradation in the organic soil was enhanced by Alfonic 810-60 at 100 pg/g. We suggest that surfactants a t low concentrations may promote the mineralization of sorbed aromatic compounds in polluted soils, even when surfactant-induced desorption is not appreciable. Introduction
Many anthropogenic aromatic compounds represent important environmental pollutants, and many enter and persist in sediments and soils ( I , 2). In these environments, such compounds are frequently extensively sorbed ( 3 , 4 ) , so that little is present in the water phase. The native organic matter of sediments and soils is the active fraction involved in this sorption ( 4 , 5 ) , and the clay and larger particles are not as important in sorption of the hydrophobic aromatics (6, 7). Because of the persistence and sorption of many aromatics and the fact that the nonsorbed compounds are often readily biodegradable, it is plausible that sorption results in a protection of such chemicals from microbial attack. Attention has been given to the possible use of surfactants for the removal of pollutants that are sorbed to contaminated aquifers (8). Similarly, the effect of several nonionic and anionic surfactants on the solubilization of anthracene, phenanthrene, and pyrene sorbed to soil has been tested with soil-water suspensions, the octyl- and nonylphenylethoxylates being effective for this purpose (9). If a surfactant solubilizes the sorbed compound, the molecule may become readily available for microbial utilization (IO),and the consequent biodegradation might obviate the necessity of removing and then destroying the desorbed chemical. A stimulation of microbial degradation of lubricating oil was noted when a dispersant was introduced into a slurry of contaminated soil (12). In view of the possible usefulness of surfactants for promoting the microbial destruction of organic pollutants sorbed to soil, a study was conducted of the effects of several surfactants on both desorption and biodegradation. Low concentrations of the potential desorbing agents were chosen because of the possibility that the sorbed aromatics might become available to soil microorganisms a t levels below the critical micelle concentrations of the surfactants. Phenanthrene and biphenyl were selected as test compounds. 1728 Environ. Sci. Technol., Voi. 25, No. 10, 1991
Materials and Methods
Samples of Lima silt loam (7.6% organic matter) from Aurora, NY, and Edwards muck (32.9% organic matter) from a reclaimed marsh in Newfield, NY, were dried in air and passed through a 2.0-mm sieve. Soil was sterilized for some studies by autoclaving for 2 h. No growth was evident when samples of the autoclaved soil were added to Trypticase-soy broth or Trypticase-soy agar and incubated at 30 "C for 48 h. Before use, water was passed through a series of deionizing columns (Milli-Q;Millipore Corp., Bedford, MA). All glassware was soaked overnight in concentrated sulfuric acid containing Nochromix oxidant (Godax Laboratories, New York, NY). Reagent-grade phenanthrene, [9J4C]phenanthrene (13.1 mCi/mmol, purity >98%), and [U-14C]biphenyl (14.5 mCi/mmol, purity >97 %) were purchased from Sigma Chemical Co. (St. Louis, MO), and reagent-grade biphenyl and Triton X-100 were purchased from Aldrich Chemical Co. (Milwaukee, WI). The critical micelle concentration of Triton X-100 is reported to be approximately 195 mg/L (12). Anionic surfactants Neodol 25-7 [CnH2n+l(OCHzCH2),0S03Na;n = 12-15, average x = 71, Neodol n = 12-15, average 25-38 [C,H2,+1(0CH2CH2)xOS03Na; x = 31, and Enordet LXS-814 (C,H2,+lCEHES03Na;n = 8-15, CEH, is xylyl) were obtained from Shell Chemical Co. (Geismar, LO), and nonionic surfactants Alfonic 810-60 [C,Hz,+,(OCH2CH2),0H; n = 8-14, average x = 4.51 and Novel I1 1412-56 [C,H2,+1(0CH2CH2)xOH;n = 12-14, average x = 5.61 were obtained from Vista Chemical Co. (Austin, TX). The critical micelle concentrations, which were measured by determining the surface tension of aqueous solutions of the surfactants, were 275 f 25 and 50 f 5 pg/mL for the last two surfactants. The values for hydrophile-lipophile balance, average moles of ethylene oxide per mole of ethylene, and average weight percent of ethylene oxide are 11.7,4.51, and 58.6% for Alfonic 810-60 and 10.9, 5.57, and 54.3% for Novel I1 1412-56. The biodegradation of phenanthrene and biphenyl was determined by adding 50 pg of unlabeled compound and approximately 100 000 dpm of labeled compound to 50 g of soil contained in 250-mL biometer flasks (Bellco Glass Inc., Vineland, NJ). The test compounds were added in a CH,Cl, solution to the flasks. The CH2C1, was allowed to evaporate before addition of the soil to prevent the possible toxicity of CH2C12to the soil microflora, and 0.5 mL of an aqueous solution containing the surfactant then was added directly to the soil to final concentrations of 10 and 100 pg/g of air-dried soil. Deionized water was added to bring the soils to 70% of field capacity, which was 34% for Lima silt loam and 74% for Edwards muck. The soils were then mixed with a glass rod. The flasks were immediately sealed with silicone stoppers covered with Teflon tape. The 14C02evolved was trapped in 2.2 mL of 0.5 M NaOH contained in the sidearm of the biometer flask, and the NaOH was replaced with fresh alkali. The NaOH removed was mixed with 3.5 mL of Liquiscint scintillation fluid (National Diagnostics, Sommerville, NJ) in 7-mL scintillation vials, and the radioactivity was determined
0013-936X/91/0925-1728$02.50/0
0 1991 American Chemical Society
with a liquid scintillation counter (Model LS 7500; Beckman Instruments Inc., Irvine, CA). To determine the effect of the surfactants on desorption rates, 25 g of sieved air-dried soil was placed in 250-mL Erlenmeyer flasks and autoclaved. Labeled and unlabeled phenanthrene or biphenyl and 100 mL of sterile deionized water was added aseptically to the flasks. The surfactants had been sterilized by passage through 0.2-pm pore-size membrane filters (Nalge Go., Rochester, NY). Each flask contained 25 pg of the test compound and 100000 dpm of labeled chemical added in the same manner as in studies of biodegradation. The flasks were incubated at 30 "C for 24 h on a rotary shaker operating at 45 rpm. Preliminary experiments showed that the solution reached equilibrium within 24 h. At equilibrium, the amount of chemical in the aqueous phase of the soil-water mixtures was 4 9 0 of the amount added. The soil particles were allowed to settle, and an amount of particulate matter corresponding to 1.0 g of air-dried soil was removed with a spatula and added together with 100 mL of a sterile solution containing 0, 0.01, 0.1, 1.0, 10, or 100 pg of surfactant per milliliter to sterile 125-mL Erlenmeyer flasks. The flasks were incubated at 30 "C on a rotary shaker operating at 45 rpm. At 0.5 h and regular intervals thereafter, 5 mL of liquid was removed aseptically and replaced with a sterile solution of the surfactant. The liquid was passed through sterile nylon syringe filters (0.22-pm pore size; MSI, Westboro, MA). Filtration rather than centrifugation was used to avoid the possibility of rapid readsorption of the test chemicals by the soil (13). The filtrate (4 mL) was mixed with 5 mL of scintillation fluid in 20-mL scintillation vials, and the radioactivity was determined. Duplicate flasks were used in studies of mineralization and desorption, and only the mean values are presented. The surfactant concentrations are expressed per milliter of solution in studies of desorption and per gram of airdried soil in tests of biodegradation. The data were analyzed statistically a t the 95% confidence level. Results Tests were conducted of the effect of several surfactants on the release of phenanthrene from sterile Lima silt loam suspended in water. Neodol25-7, Neodol25-39, Enordet LXS-814, and Triton X-100 did not enhance the extent of desorption of phenanthrene at surfactant concentrations of 0.01, 0.10, 1.0, 10, and 100 pg/mL. On the other hand, the extent of desorption of phenanthrene from Lima silt loam was affected by the nonionic surfactants, Alfonic 810-60 and Novel I1 1412-56 (Figure 1). Alfonic 810-60 a t 100 pg/mL appreciably increased the extent of desorption of phenanthrene as compared to treatments with no surfactant, and almost 20% of the hydrocarbon that had been held by the soil was desorbed by this surfactant. Novel I1 1412-56 a t concentrations of 10 and 100 pg/mL also promoted the desorption of phenanthrene. The intermediate concentrations of surfactants did not have appreciable effects, and the lowest level (0.01 pg/mL) reduced the extent of desorption. Similar tests were conducted with biphenyl as the test compound. Alfonic 810-60 surfactant at 100 pg/mL caused a statistically significant increase and Novel I1 1412-56 a t 10 Mg/mL caused a statistically significant decrease in the extent of desorption from Lima soil (Figure 2). The two surfactants did not have a significant effect on the extent of release at the other concentrations. At all time intervals up to and including 10 h, the rate of desorption was stimulated by Alfonic 810-60 at 10 and 100 but not 1.0 pg/mL. At no concentration did Novel I1 1412-56 significantly
15 10
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Flgure 1. Effect of concentration of Alfonic 810-60 (A) and Novel I1 1412-56 (B) on the desorption of phenanthrene from Llma silt loam. The values on the plots are micrograms of surfactants per milliliter.
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Flgure 2. Influence of concentration of Alfonic 810-60 (A) and Novel I I 14 12-56 (B) on the desorption of biphenyl from Lima silt loam. The values shown on the plots are micrograms of surfactant per milliliter.
enhance the rate of desorption, and an inhibition a t the 10 pg/mL level was evident beginning a t 5 h. Little phenanthrene and biphenyl were desorbed from Edwards muck in 120 h,
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Figure 4. Mineralization of phenanthrene in Lima soil and Edwards muck in the presence of 0, 10, or 100 pg of Novel I1 1412-56 per gram of airdried soll.
effect on biodegradation was still tested to determine whether the biodegradation of sorbed chemicals could be enhanced without their desorption. With no surfactant present, mineralization of phenanthrene was slow in the mineral soil and scarcely detectable in the organic soil (Figure 3). However, the addition of Alfonic 810-60 at 10 pg/g of air-dried soil had a pronounced stimulatory effect on the biodegradation of phenanthrene in both soils. The most marked enhancement at this surfactant concentration was observed in the organic soil, in which case nearly 50% of the added phenanthrene was mineralized in 495 h in the presence of Alfonic 810-60 compared to only 4.8% in the absence of the surfactant. However, the same surfactant at 100 pg/g did not cause a statistically significant stimulation in the organic soil. Similarly, Novel I1 1412-56 at 10 pg/g of soil stimulated phenanthrene mineralization in both soils (Figure 4). The influence was again especially marked in the organic soil. On the other hand, 100 pg of the surfactant per gram of soil caused a statistically significant inhibition of phenanthrene biodegradation in the mineral soil, although this 1730 Environ. Sci. Technol., Vol. 25, No. 10, 1991
200
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Figure 6. Mineralization of biphenyl in Lima soil and Edwards muck in the presence of 0, 10, or 100 pg of Novel I1 1412-56 per gram of air-dried soil.
concentration induced a slight but statistically significant increase in the rate in the organic soil. A study was also conducted of the influence of the surfactants on the biodegradation of biphenyl. Alfonic 810-60 did not have a statistically significant effect at 10 or 100 pg/g of the mineral soil (Figure 5 ) . Linear regression analysis showed that the surfactant at 100 pg/g of soil significantly enhanced the rate of the biodegradation in Edwards muck but that the lower surfactant concentration did not significantly affect the rate. Novel I1 1412-56 at 10 and 100 pg/g of soil did not have a statistically significant effect on biphenyl mineralization in Lima silt loam (Figure 6). In the organic soil, on the other hand, the surfactant at both concentrations depressed the rate; linear regression analysis revealed that these suppressions were statistically significant.
Discussion Only a small percentage of a hydrophobic organic compound sorbed to soil is partitioned to the aqueous phase
(14), and the surfactants tested to increase the mobility of such chemicals usually have been used at levels much above their critical micelle concentrations (9,15,16). The surfactants a t such concentrations are not only expensive but they may inhibit the microorganisms that have the capacity to metabolize the polluting compounds. Hence, this study was designed to determine the effect of relatively low concentrations of surfactants. The data show an enhancement in the biodegradation of phenanthrene resulting from the addition of two nonionic surfactants a t low levels. It is not certain that the stimulation occurred below their critical micelle concentrations because the aromatic compounds and probably the surfactants were sorbed and not in the aqueous phase. The results also show that the extent of desorption following addition of the surfactants to sterile soil did not predict their influence on the extent of phenanthrene biodegradation. It is possible, nevertheless, that the surfactant increased the hStanMeous rate, rather than the extent, of desorption and that the microorganisms thus had more substrate continuously made available to them as they transformed the chemical; i.e., the equilibrium concentration of the chemical in the aqueous phase in the presence of the surfactant is not important but rather the rate of removal of the substrate from the soil solids determines the rate of microbial destruction of the aromatic hydrocarbon. Because some bacteria are able to use sorbed aromatic hydrocarbons that do not appear in detectable amounts in the aqueous phase (R. Araujo and M. Alexander, unpublished data), it is also possible that the surfactants alter the strength of sorption or complex the substrate in some way that the compound becomes more available a t microorganisms without appearing in solution. Several factors influence the effectiveness of a surfactant in promoting biodegradation. The identity of the sorbed chemical is of importance, as indicated by the effects of Alfonic 810-60 on the biodegradation of phenanthrene and biphenyl. Characteristics of the soil are also important, as shown by the markedly different effects on biodegradation in the mineral and the organic soils. The role of organic matter in binding hydrophobic compounds added to soil is well-known (5, 61, but the reason surfactants markedly enhanced microbial activity in the organic soil is unknown. The concentration of the surfactant also determines whether biodegradation will be promoted. The toxicity of the surfactants to microorganisms may explain the reduced effectiveness of the higher surfactant concentration. Thus, Irkhin et al. (17) showed that Escherichia coli is sensitive to several detergents but not to a nonionic detergent. Considerable information exists on the effects of surfactants on microbial communities (18). Moreover, linear alcohol ethoxylates are readily degradable in sbils (19) and subsoils (20). Further research is required to determine the sorbed chemicals, surfactants, and soil properties that will result in the greatest enhancement of biodegradation. Furthermore, the mechanism by which surfactants promote microbial activity without a parallel increase in detectable
desorption should be defined. Nevertheless, the data presented show that the use of surfactants at low concentrations represents a means of stimulating the microbial decomposition of environmental pollutants. Acknowledgments We gratefully acknowledge the assistance of John D. Morton. Registry No. Biphenyl, 92-52-4; phenanthrene, 85-01-8.
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Received for review January 4,1991. Revised manuscript received May 16,1991. Accepted May 30,1991. Support for this research was provided by Grant R-816109-01 from the U.S. Environmental Protection Agency. Atzy opinion, findings, and conclusions or recommendations in this publication are those of the authors and do not necessarily reflect the views of the awarding agency.
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