- Email: [email protected]
Role of loosely bound humic substances and humin in the bioavailability of phenanthrene aged in soil
Environmental Pollution 118 (2002) 427–433 www.elsevier.com/locate/envpol
Role of loosely bound humic substances and humin in the bioavailability of phenanthrene aged in soil K. Nam*, J.Y. Kim School of Civil, Urban and Geosystem Engineering, Seoul National University, Seoul 151-742, Republic of Korea Received 1 May 2001; accepted 4 September 2001
‘‘Capsule’’: Major sequestration sites for phenanthrene may reside in the humin-mineral fraction of soil, while humic and fulvic acids may act as a barrier, limiting the compound’s bioavailabilty. Abstract A study was conducted to determine a possible role of loosely bound humic substances (i.e., humic and fulvic acids) in bioavailability of aged phenanthrene with time. In this study, long-term residence of phenanthrene in soil is defined as aging or sequestration, and the effect was determined by the declined bioavailability to bacteria of the polycyclic aromatic hydrocarbon with increased residence time. After 1, 7, and 100 days of aging of phenanthrene in Lima loam, about 90–93% of initial phenanthrene was recovered from the humin-mineral fraction of Lima loam whereas less than 12% was found in humic and fulvic acids of the same soil. Mineralization rates of phenanthrene aged in the humin-mineral fraction significantly decreased with time by the test bacterium P5-2. In terms of extents of mineralization, the difference with time was not appreciable, but still significant at P< 0.05. Additional decreases in the rates and extents of mineralization were observed with the whole soil (i.e. Lima loam) to which phenanthrene had been aged. Data suggest that major sequestration sites for phenanthrene may reside in the humin-mineral fraction, and probably humic and fulvic acids may act as a physico-chemical barrier to bacterial degradation so that the compound’s bioavailability may be limited. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Phenanthrene; Humin; Humic acid; Fulvic acid; Bioavailability; Aging
1. Introduction It is now widely accepted that aging or sequestration phenomenon of some hydrophobic contaminants exists in soil, sediment, and aquifer (Alexander, 1995; Luthy et al., 1997). Also, it is well known that behavior of aged compounds is different from that of freshly added chemicals in the environment. It is typical that aged compounds show resistance to solvent extraction (Kelsey et al., 1997) and desorption (Kan et al., 1994). Such resistance results in reduced availability to bacteria (Nam et al., 1998) and higher organisms as well including plants (Bowmer, 1991), earthworms (Kelsey and Alexander, 1997), and guinea pigs (Umbreit et al., 1986). Despite the current consensus on the aging of hydrophobic contaminants in the natural environment, it is * Corresponding author. Tel.: +82-2-888-6966; fax: +82-2-8870349. E-mail address: [email protected] (K. Nam).
still not clear which mechanisms are involved in the aging phenomenon. Among the possible mechanisms are the association of organic compounds with natural organic matter (Carroll et al., 1994) and the penetration of contaminants into small pores in soil (Wu and Gschwend, 1986). An additional model has been proposed originating from observations of different competitive effects in the sorption of organic contaminants (Xing et al., 1996). According to this model, natural organic matter has two different sorptive domains that interact with organic contaminants; partitioning domain and hydrophobic hole domain. The hydrophobic hole domain exhibits competitive sorption behavior and may be responsible for the desorptionand extraction-resistant fractions of aged contaminants. This concept is consistent with the findings that small pores with hydrophobic surfaces are responsible for resistant desorption (Werth and Reinhard, 1997) and declined bioavailability of contaminants to bacteria (Nam and Alexander, 1998).
0269-7491/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(01)00296-2
428
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433
Weber and Huang (1996) proposed that the hydrophobic hole domain is located between loose, amorphous humic materials and mineral surfaces and is composed of highly compact humic materials, which is typical to humin. Earlier studies have demonstrated that humin has macromolecular aliphatic chains as major constituents (Almendros and Gonzalez-Vila, 1987; Almendros and Sanz, 1991) and has significant amounts of small pores on its surface (Malekani et al., 1997). In these regards, it seems reasonable to hypothesize that the hydrophobic hole domain, which is proposed to be responsible for persistence of organic compounds, may exist in humin fraction of soil organic matter. The present study was thus conducted to determine a potential role of humin and alkalineextractable humic substances in the sequestration and bioavailability of phenanthrene in soil.
the method described by Swift (Swift, 1996) with some modifications. After 1, 7, and 100 days of aging in Lima loam, the phenanthrene-aged soil was transferred to a 250-ml Teflon centrifuge bottle and 100 ml of 0.1 N NaOH solution was added to the bottle. The suspension was shaken on a horizontal shaker (200 rpm) for 24 h under nitrogen at room temperature. The dark brown colored supernatant containing humic and fulvic acids was separated from the residual soil solid by centrifugation (12, 860 g for 20 min). The precipitated solid was considered as a humin-mineral fraction. The solid was recovered and washed with distilled water until its pH reached about 7. The supernatant was acidified with concentrated hydrochloric acid (pH < 1) to precipitate humic acid fraction. The resulting solution contained fulvic acid. Phenanthrene was extracted from the three fractions, and concentrations of the compound were determined by high-pressure liquid chromatography. More detailed procedures are described in Section 2.6.
2. Materials and methods 2.4. Biodegradation of aged phenanthrene 2.1. Soil samples Lima loam was collected from the Aurora Experimental Farm of Cornell University (Ithaca, NY). The soil is composed of 32.0% sand, 45.5% silt, and 22.5% clay (pH 6.89) and has 2.99% of organic carbon. The soil was passed through a 2-mm sieve, air-dried, and sterilized by gamma irradiation (2.5 Mrad) from a 60Co source (Ward Laboratory, Cornell University, Ithaca, NY). The samples were stored in 50-ml screw cap tubes at room temperature until use. 2.2. Aging of phenanthrene Unlabeled and [9-14C] phenanthrene (Sigma Chemical Co., St. Louis, MO) were added to soil samples for aging. Ten gram portions of soil samples were aseptically added to 50-ml test tubes, and 1.0 mg of labeled phenanthrene (ca. 1.0105 dpm; 8.3 mCi/mmol, > 98% purity) and 99.0 mg of unlabeled phenanthrene in dichloromethane were added to the soils. The total volume of dichloromethane added was less than 100 ml. The soil samples were then placed in a hood mixed for 1 h with a vortex mixer at 15-min intervals to evaporate dichloromethane and to disperse phenanthrene-spiked soil particles. Sterile distilled water was then added to bring the moisture level to the 80% of the water-holding capacity of the soil. The tubes were closed with Teflonlined screw caps and stored at room temperature in the dark for aging. 2.3. Fractionation of humic substances Humic substances were fractionated into fulvic acid, humic acid, and humin-mineral fraction according to
The humin-mineral fraction isolated as described above was sterilized by Gamma irradiation and used for aging and biodegradation experiments. One hundred micrograms of phenanthrene were aged in 10 g of humin-mineral fraction (1.52% organic carbon). After aging for 0, 30, and 100 days, biodegradation experiments were performed with the phenanthrene-aged humin fractions. Phenanthrene-aged samples were transferred to 50-ml flasks to which 10-ml portions of inorganic salts solution (0.10 g CaCl2 2H2O, 0.01 g FeCl3, 0.10 g MgSO4 7H2O, 0.10 g NH4NO3, 0.20 g KH2PO4, and 0.80 g K2HPO4 l 1 sterile distilled water; pH 7.0) were added. After shaking the soil slurries for 1 h at room temperature on a rotary shaker (100 rpm), a soil isolate P5-2 was inoculated into each flask at a level of 107 cells per gram of soil. The bacterium (Gram negative and small rod) has been isolated from soil because of its ability to use phenanthrene as a sole carbon and energy source (Tang et al., 1998). To determine the amounts of 14CO2 evolved during the biodegradation, each flask was sealed with a Teflon-wrapped silicon stopper through which was placed an 18-gauge needle and a 16-gauge steel cannula. From the cannula was suspended a small vial containing 1.5 ml of 0.5 N NaOH to trap 14CO2 released from mineralization. The NaOH solution was periodically removed and replaced with fresh solution, and the amount of evolved 14 CO2 was determined by a liquid scintillation counter (Model LS 7500; Beckman Instruments, Irvine, CA). The flasks were incubated at 30 C for 30 days on a rotary shaker (120 rpm) for biodegradation. The maximum rate of mineralization was determined by conducting a linear regression analysis on the points that formed the steepest section of the mineralization
429
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433
curve. At least three data points were used for each regression. The regression coefficient (r) was greater than 0.95. Mineralization extent represented an accumulative value of the amounts of 14CO2 evolved during the 30-day biodegradation experiment. 2.5. Determination of porosity and surface area Porosity and surface area were calculated from the data obtained by using a mercury-intrusion porosimeter (Pore Sizer 9300, Micromeritics Inc., Norcross, GA). A sample of 1.5 g of soil was placed in a 3-ml penetrometer and the soil was dried for 16 h at 105 C. The penetrometer containing the soil was filled with mercury (> 99.99% purity; Aldrich Chemical, Milwaukee, WI) in a preparation chamber and transferred to a high pressure chamber. The change in the volume of mercury was measured by increasing the chamber pressure from 0.1 to 213.7 MPa. Detailed procedures are previously described elsewhere (Nam et al., 1998). By this procedure, pores having diameters ranging from 0.007 to 10 mm were determined. 2.6. Extraction and chemical analysis After aging of phenanthrene in Lima loam for 1, 7, and 100 days, the soil was fractionated into fulvic acid, humic acid, and humin-mineral fraction as described above, and phenanthrene was then extracted from each fraction. The acidified solution containing fulvic acid was mixed with 20 ml of hexane in a 250-ml Teflon centrifuge bottle, and the mixture was shaken on a horizontal shaker (200 rpm) at room temperature. After shaking for 24 h, 10 ml of hexane layer was recovered and concentrated to less than 1 ml by using a rotary evaporator (Bu¨chi Rotavapor; Buchler Instruments Fort Lee, NJ). Phenanthrene from humic acid fraction was recovered using the mixture of hexane and n-butanol. Twenty milliliters of hexane and 5 ml of n-butanol were mixed with the phenanthrene-humic acid fraction, and the suspension was shaken for 16 h at room temperature on a horizontal shaker (200 rpm). After shaking, the solvent mixture was recovered from the humic acid fraction by centrifugation (18 600 g for 20 min) and the solvent was concentrated to less than 1 ml by evaporation as described above. For extraction of phenanthrene from humin-mineral fraction, 20-ml of n-butanol was added to a 50-ml Teflon centrifuge tube containing the solid, and the suspension was mixed with a vortex mixer for 1 min. The solvent–soil mixture was then shaken vigorously on a horizontal shaker (200 rpm) for 16 h at room temperature and centrifuged at 18,600 g for 20 min. The supernatant was saved for further analysis. The soil sample extracted with n-butanol was transferred to a cellulose extraction thimble (2580 mm, Whatman Fisher Scientific, Springfield, NJ) and
placed in a Soxhlet extractor (Ace Glass Vineland, NJ). The extractor was fitted with a 250-ml round bottom flask containing 90 ml of hexane amended with 2 ml of n-butanol, and the extraction was performed for 16 h. The n-butanol and Soxhlet extracts were combined and concentrated to less than 1 ml by evaporation as described above. Solvent extracts from each component of humic substances were passed through 0.20-mm Teflon filters (Millex-FG13; Millipore Co., Bedford, MA) to remove particulates and analyzed by high-pressure liquid chromatography (Hewlett-Packard series 1050; HewlettPackard Co., Avondale, PA). A Spherisorb ODS-2 octadecyl-bonded silica column (5 mm, 2504 mm; Hewlett-Packard) was used. Acetonitrile-water (86:14) was used as a mobile phase at a flow rate of 0.8 ml/min. Phenanthrene was detected by its UV absorbance at 254 nm.
3. Results 3.1. Distribution of phenanthrene in humic substances after aging An experiment was conducted to determine the distribution of phenanthrene in humic substances after aging. After 1, 7, and 100 days of aging of phenanthrene in Lima loam, the soil was treated with alkali to separate humic and fulvic acids from humin-mineral fraction. Phenanthrene was extracted from each component of humic substances, and the amounts were determined chromatographically. As shown in Table 1, more than 90% of initial amount was found in humin-mineral fraction regardless of aging period. Although more phenanthrene was present in humic acid than in fulvic acid, the amounts of phenanthrene present in two fractions were not appreciable. About 11% of initial phenanthrene was recovered from the humic and fulvic acids in 1-day aged soil samples, and 5.87 and 5.70% were recovered from the same fractions which were separated Table 1 Distribution of freshly added or aged phenanthrene in isolated humic substances of Lima loama Aging days
1 7 100
Phenanthrene extracted (%) Fulvic acid
Humic acid
Humin-mineral fraction
Total
3.2Aa 2.4Aa 201Aa
8.3Ba 3.4Ab 3.6Ab
90.1Ca 93.2Ba 90.9Ba
101.6a 99.0a 96.6a
a Values are the means of triplicate determinations and values in columns followed by the same lowercase letters and values in rows followed by the same uppercase letters are not significantly different (P<0.05).
430
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433
from 7- and 100-day aged samples, respectively. After 100 days of aging, a total of 96.6% of initial phenanthrene was recovered by combined solvent extraction, which was not significantly different from the total recovery from 1-day aged samples, indicating that abiotic transformation, volatilization, and formation of nonextractable residues had not occurred significantly during the aging period.
When the same polycyclic aromatic hydrocarbon was aged in the whole soil (i.e. Lima loam), which maintained undisturbed physicochemical structure of organic matter, additional decreases in bioavailability with increased residence time: both maximum rates and extents were presented in Table 2.
3.2. Biodegradation of phenanthrene aged in huminmineral fraction
Porosity and surface area were determined with Lima loam and humin-mineral fraction of the loam by using mercury porosimetry. The results indicate that removal of humic and fulvic acids did not cause significant changes in total porosity (i.e. 0.284 m3 g 1 for Liam loam and 0.272 m3 g 1 for the humin-mineral fraction) and surface area (Table 3). Pore volume resulting from pores having 7- to 100-nm diameter did not change even after humic and fulvic acids had been removed. However, changes in pore volumes were obvious in pores with larger diameters. In humin-mineral fraction, pore volume originating from pores having a diameter of 0.1–1 mm increased by 43%, and pore volume from pores with a diameter of 1–10 mm decreased by 28% compared to the intact Lima loam.
Since most freshly added and aged phenanthrene was found in humin-mineral fraction from the distribution experiments, the ability of humin-mineral fraction to decrease biodegradability of organic compounds was determined. Phenanthrene was aged in the fraction, and biodegradation was performed after 0, 30, and 100 days of aging (Fig. 1). Phenanthrene aged for 0, 30, and 100 days was mineralized with the extents of 66.8, 63.1, and 57.3%, respectively, by the test bacterium P5-2. After 2 days of biodegradation experiments, extents of mineralization were different up to 14.8% between freshly added and 100-day aged samples. However, the difference gradually decreased with prolonged biodegradation and, at the end of 30-day biodegradation, it was about 9.4%, which was still significantly different (P < 0.05). Appreciable decreases in the maximum rates of mineralization were observed with phenanthrene aged in humin-mineral complexes. The maximum rate of 100-day aged samples was 19.8% day 1, which was about 1.2- and 1.7-fold less than the rates of 30-day aged and freshly added samples, respectively (Table 2).
3.3. Porosity and surface area
4. Discussion The data show that most phenanthrene was found mainly in humin-mineral complex rather than in humic and fulvic acids whether the polycyclic aromatic hydrocarbon was freshly added or aged. Operationally, humin is defined as the fraction of mineral-bound humic materials that can not be extracted with either alkali or acid. Since humin often comprises more than 50% of organic carbon in soil (Rice and MacCarthy, 1990) it may play an important role in the association of organic contaminants in soil. It has been reported that pesticides such as atrazine adsorb tightly to humin and such interactions often result in bound residues (Capriel et al., 1985). The formation of nonextractable bound residues Table 2 Extents and maximum rates of mineralization of phenanthrene aged in humin-mineral fraction and whole soil (i.e. Lima loam)a Aging (days)
0 30 100 Fig. 1. Biodegradation of phenanthrene aged in humin-mineral fraction separated from Lima loam or in the original Lima loam by bacterium P5-2. Values are the means of triplicate determinations and statistical analyses are shown in Table 2.
a
Extent (%)
Maximum rate (%)
Huminmineral
Whole soil
Huminmineral
Whole soil
66.7Aa 63.1Aa 57.3Ab
65.4Aa 60.9Ab 47.8Bc
32.9Ca 23.3Cb 19.8Cc
32.C8a 21.4cb 12.9Dc
Values are the means of triplicate determinations and values in columns followed by the same lowercase letters and values in rows followed by the same uppercase letters are not significantly different (P<0.05).
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433 Table 3 Porosity and surface area determined by mercury porosimetrya Soils
Lima loam Humin-mineral fraction
Pore volume (m g 1)b
Surface area (m g 1)
0.007–0.1 mm
0.1–1 mm
1–10 mm
0.030Aa 0.030Aa
0.083Ba 0.119Bb
0.171Ca 0.123Bb
6.042a 6.018a
a Values are the means of triplicate determinations and values in columns followed by the same lowercase letters and values in rows followed by the same uppercase letters are not significantly different (P<0.05). b Pore sizes in diameter.
also has been observed with polycyclic aromatic hydrocarbons. Anthracene and hexadecane were shown to produce nonextractable fractions in soil (Ka¨stner et al., 1995). These bound residues often require structural changes of the parent compounds and are essentially not active biologically, and are mainly associated with humin fraction (Capriel et al., 1985; Heim et al., 1995). Recently, however, bound pesticide residues were shown to be noncovalently bound to soil by cation exchange and hydrophobic interactions (Lerch et al., 1997). Considering the facts that aging of organic compounds does not involve any types of structural alteration, and aged compounds can be recovered by exhaustive solvent extraction (Alexander, 1995), nonextractable residues covalently bound to soil may not truly represent aged or sequestered compounds. In the present study, aged phenanthrene associated with humin was fully extractable by the combination of solvent and Soxhlet extraction methods. Thus, the compound found in the huminmineral fraction in this study was literally sequestered in the soil matrix. This is consistent with the finding that pyrene, when aged in sediments, was found primarily in humin, and interactions between pyrene and humin are noncovalent in nature (Guthrie et al., 1999). Organic carbon content in soil humin has been estimated in the range of 10–45% (Rice and McCarthy, 1989; Malekani et al., 1997) and this organic fraction was attributed to the retention of organic contaminants in humin (Kohl and Rice, 1998). Nieman et al. (1999) also reported that about 64% of the solvent-nonextractable 14C was associated with the lipid and humic acid bound to soil mineral fraction. Recently, Guthrie and Pfaender (1998) showed that about 29 to 73% of recovered pyrene was found in humin fraction of a soil and up to 26% in humic/fulvic acid extracts, regardless of the pyrenedegrading activity of the soil. It is well known that aging causes a decreased bioavailability of a compound of interest in soil. In this study, phenanthrene aged in both humin-mineral fraction and a whole soil showed decreased rates and extents of mineralization with time although the extents
431
decreased were much greater in the whole soil than in humin-mineral fraction. A recent study showed that removal of humic and fulvic acids caused a decrease in the rate of desorption of phenanthrene (White et al., 1999). Alkaline extraction of soil organic matter produces a highly crystallized, denser, higher-molecular-weight humic material (i.e. humin), and sorption of hydrophobic compounds to such a so called humin fraction is greater and less linear than the unaltered soil organic matter (Pignatello and Xing, 1996). Our data showed that a significant amount of pores with a diameter of 0.1–1 mm are present on the surfaces of humin-mineral fraction. Pore volume originating from the 0.1–1-mm sized pores increased by 43% after removing humic and fulvic acids from the original soil. They were newly exposed by the fractionation procedure. A previous study (Malekani et al., 1997) demonstrated that such high porosity on humin-mineral complex mainly came from humin, not from mineral surfaces. The pore sizes of 0.1–1 mm seem to be larger compared to generally believed size ranges that might contribute to the formation of sequestered or resistant compounds. However, it is plausible that such seemingly larger pores can serve as sequestration sites for organic pollutants considering that humin is thought to be located in between loosely structured humic substances such as humic and fulvic acids and mineral surfaces. Loosely bound humic substances may impede desorption by increasing additional interactions or tortuosity and provide barriers to biological attack. Extracting humic substances from soil has clearly increased the accessibility of 0.1–1-mm-sized pores (Table 3). These were presumably inaccessible to due to the presence of the humic substances that essentially blocked entry to the pores. Phenanthrene spiked into the isolated humin-mineral fraction will be able to diffuse more readily into these sites compared to whole soil, thereby reducing bioaccessibility to degrading microbes. Considering the facts that pores less than 1 mm in diameter are dominant on humin-mineral surfaces and such pores are usually interconnected to much smaller pores, phenanthrene, once enters into such pores during aging, is not likely to be readily desorbed and thus is less available to enzymatic attack and direct bacterial degradation. A recent study (Cornelissen et al., 1998) with model sorbents demonstrate that organic compounds diffused through pores in soil organic matter or pores coated with organic materials are resistant to desorption. The importance of hydrophobic small pores in the reduced bioavailability of organic compounds has also been demonstrated with model solids (Nam and Alexander, 1998). It is worthwhile considering that phenanthrene might have been re-distributed during the base extraction. When humic and fulvic acids are extracted with using NaOH (i.e. pH > 12), functional groups such as
432
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433
carboxylic groups will predominantly be in a dissociated form. This may produce uncoiled macromolecules of humic and fulvic acids in solution and such shift is likely to influence the retention of nonpolar hydrophobic contaminants associated with these phases. If this is the case in our study, phenanthrene initially sorbed to fulvic and humic acids might have been re-distributed during the extraction procedure, and thereby the chemical might have re-located to humin-mineral fraction. Nonetheless, the present findings are significant in that they show relative distribution of aged phenanthrene among humic substances and demonstrate a potential role of humin-mineral complex and base-extractable humic substances in the sequestration and bioavailability of hydrophobic organic compounds in soil.
Acknowledgements The authors wish to thank Dr. Martin Alexander for financial support and guidance for this work and Dr. Joseph J. Pignatello for invaluable discussion during the preparation of this manuscript. We also thank the financial support by the Research Division of Seoul National University/Hanyang University for Social Infrastructure and Construction Technology.
References Alexander, M., 1995. How toxic are toxic chemicals? Environmental Science and Technology 29, 2713–2717. Almendros, G., Gonzalez-Vila, F.J., 1987. Degradative studies on a soil humin fraction-Sequential degradation of inherited humin. Soil Biology and Biochemistry 19, 513–520. Almendros, G., Sanz, J., 1991. Structural study on the soil humin fraction-Boron trifluoride-methanol transesterification of soil humin preparations. Soil Biology and Biochemistry 23, 1147–1154. Bowmer, K.H., 1991. Atrazine persistence and toxicity in two irrigated soils of Australia. Australian Journal of Soil Research 29, 339–350. Capriel, P., Haisch, A., Khan, S.U., 1985. Distribution and nature of bound (nonextractable) residues of atrazine in a mineral soil nine years after the herbicide application. J. Agricultural Food and Chemistry 33, 567–569. Carroll, K.M., Harkness, M.R., Bracco, A.A., Balcarcel, R.R., 1994. Application of a permeant/polymer diffusional model to the desorption of polychlorinated biphenyls from Hudson river sediments. Environmental Science and Technology 28, 253–258. Cornelissen, G., Van Noort, P.C., Grovers, H.A.J., 1998. Mechanism of slow desorption of organic compounds from sediments: A study using model sorbents. Environmental Science and Technology 32, 3124–3131. Guthrie, E.A., Pfaender, F.K., 1998. Reduced pyrene bioavailability in microbially active soils. Environmental Science and Technology 32, 501–508. Guthrie, E.A., Bortiatynski, J.M., Van Heemst, J.D.H., Richman, J.E., Hardy, K.S., Kovach, E.M., Hatcher, P.G., 1999. Determination of [13C]pyrene sequestration in sediment microcosms using flash pyrolysis-GC-MS and 13C NMR. Environmental Science and Technology 33, 119–125.
Heim, K., Schuphan, I., Schmidt, B., 1995. Behavior of [14C]-4nitrophenol and [14C]-3,4-dichloroaniline in laboratory sedimentwater system: Part 2. Desorption experiments and identification of sorptive fraction. Environmental Toxicology and Chemistry 14, 755–761. Ka¨stner, M., Lotter, S, Heerenklage, J., Breuer-Jammali, M, Stegmann, R, Mahro, B., 1995. Fate of 14C-labeled anthracene and hexadecane in compost-manured soil. Applied Microbiology and Biotechnology 43, 1128–1135. Kan, A.T., Fu, G., Tomson, M.B., 1994. Adsorption/desorption hysteresis in organic pollutants and soil/sediment interaction. Environmental Science and Technology 28, 859–867. Kelsey, J.W., Alexander, M., 1997. Declining bioavailability and inappropriate estimation of risk of persistent compounds. Environmental Toxicology and Chemistry 16, 582–585. Kelsey, J.W., Kottler, B.D., Alexander, M., 1997. Selective chemical extractants to predict bioavailability of soil-aged organic chemicals. Environmental Science and Technology 31, 214–217. Kohl, S.D., Rice, J.A., 1998. The binding of contaminants to humin: A mass balance. Chemosphere 36, 251–261. Lerch, R.N., Thurman, E.M., Kruger, E.L., 1997. Mixed-mode sorption of hydroxylated atrazine degradation products to soil: A mechanism for bound residue. Environmental Science and Technology 31, 1539–1546. Luthy, R.G., Aiken, G.R., Brusseau, M.L., Cunningham, S.D., Gschwend, P.M., Pignatello, J.J., Reinhard, M., Traina, S.J., Weber Jr., W.J., Westll, J.C., 1997. Sequestration of hydrophobic organic contaminants by geosorbents. Environmental Science and Technology 31, 3341–3347. Malekani, K., Rice, J.A., Lin, J.-S., 1997. The effect of sequential removal of organic matter on the surface morphology of humin. Soil Science 162, 333–342. Nam, K., Alexander, M., 1998. Role of nanoporosity and hydrophobicity in sequestration and bioavailability. Environmental Science and Technology 32, 71–74. Nam, K., Chung, N., Alexander, M., 1998. Relationship between organic matter content of soil and the sequestration of phenanthrene. Environmental Science and Technology 32, 3785–3788. Nieman, J.K.C., Sims, R.C., Sims, J.L., Sorensen, D.L., Mclean, J.E., Rice, J.A., 1999. [14C]Pyrene bound residue evaluation using MIBK fractionation method for creosote-contaminated soil. Environmental Science and Technology 33, 776–781. Pignatello, J.J., Xing, B., 1996. Mechanisms of slow sorption of organic chemicals to natural particles. Environmental Science and Technology 30, 1–11. Rice, J.A., MacCarthy, P., 1990. A model of humin. Environmental Science and Technology 24, 1875–1877. Swift, R.S., 1996. Organic matter characterization. In: Sparks, D.L. (Ed.), Methods of Soil Analysis, Part 3—Chemical Methods. Soil Science Society of America, Madison WI, pp. 1011–1069. Tang, W.-C., White, J.C., Alexander, M., 1998. Utilization of sorbed compounds by microorganisms specifically isolated for that purpose. Applied Microbiology and Biotechnology 49, 117–121. Umbreit, T.H., Hesse, E.J., Gallo, M.A., 1986. Bioavailability of dioxin in soil from a 2,4,5-T manufacturing site. Science 232, 497–499. Weber Jr., W.J., Huang, W., 1996. A distributed reactivity model for sorption by soils and sediments. 4. Intraparticle heterogeneity and phase-distribution relationships under nonequilibrium conditions. Environmental Science and Technology 30, 881–888. Werth, C.J., Reinhard, M., 1997. Effect of temperature on trichloroethylene desorption from silica gel and natural sediments. 2. Kinetics. Environmental Science and Technology 31, 697–703. White, J.C., Hunter, M., Nam, K., Pignatello, J.J., Alexander, M., 1999. Correlation between biological and physical availabilities of phenanthrene in soils and soil humin in aging experiments. Environmental Toxicology and Chemistry 18, 1720–1727.
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433 Wu, S., Gschwend, P.M., 1986. Sorption kinetics of hydrophobic organic compounds to natural sediments and soils. Environmental Science and Technology 20, 717–725.
433
Xing, B., Pignatello, J.J., Gigliotti, B., 1996. Competitive sorption between atrazine and other organic compounds in soils. Environmental Science and Technology 30, 2432–2440.
Role of loosely bound humic substances and humin in the bioavailability of phenanthrene aged in soil K. Nam*, J.Y. Kim School of Civil, Urban and Geosystem Engineering, Seoul National University, Seoul 151-742, Republic of Korea Received 1 May 2001; accepted 4 September 2001
‘‘Capsule’’: Major sequestration sites for phenanthrene may reside in the humin-mineral fraction of soil, while humic and fulvic acids may act as a barrier, limiting the compound’s bioavailabilty. Abstract A study was conducted to determine a possible role of loosely bound humic substances (i.e., humic and fulvic acids) in bioavailability of aged phenanthrene with time. In this study, long-term residence of phenanthrene in soil is defined as aging or sequestration, and the effect was determined by the declined bioavailability to bacteria of the polycyclic aromatic hydrocarbon with increased residence time. After 1, 7, and 100 days of aging of phenanthrene in Lima loam, about 90–93% of initial phenanthrene was recovered from the humin-mineral fraction of Lima loam whereas less than 12% was found in humic and fulvic acids of the same soil. Mineralization rates of phenanthrene aged in the humin-mineral fraction significantly decreased with time by the test bacterium P5-2. In terms of extents of mineralization, the difference with time was not appreciable, but still significant at P< 0.05. Additional decreases in the rates and extents of mineralization were observed with the whole soil (i.e. Lima loam) to which phenanthrene had been aged. Data suggest that major sequestration sites for phenanthrene may reside in the humin-mineral fraction, and probably humic and fulvic acids may act as a physico-chemical barrier to bacterial degradation so that the compound’s bioavailability may be limited. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Phenanthrene; Humin; Humic acid; Fulvic acid; Bioavailability; Aging
1. Introduction It is now widely accepted that aging or sequestration phenomenon of some hydrophobic contaminants exists in soil, sediment, and aquifer (Alexander, 1995; Luthy et al., 1997). Also, it is well known that behavior of aged compounds is different from that of freshly added chemicals in the environment. It is typical that aged compounds show resistance to solvent extraction (Kelsey et al., 1997) and desorption (Kan et al., 1994). Such resistance results in reduced availability to bacteria (Nam et al., 1998) and higher organisms as well including plants (Bowmer, 1991), earthworms (Kelsey and Alexander, 1997), and guinea pigs (Umbreit et al., 1986). Despite the current consensus on the aging of hydrophobic contaminants in the natural environment, it is * Corresponding author. Tel.: +82-2-888-6966; fax: +82-2-8870349. E-mail address: [email protected] (K. Nam).
still not clear which mechanisms are involved in the aging phenomenon. Among the possible mechanisms are the association of organic compounds with natural organic matter (Carroll et al., 1994) and the penetration of contaminants into small pores in soil (Wu and Gschwend, 1986). An additional model has been proposed originating from observations of different competitive effects in the sorption of organic contaminants (Xing et al., 1996). According to this model, natural organic matter has two different sorptive domains that interact with organic contaminants; partitioning domain and hydrophobic hole domain. The hydrophobic hole domain exhibits competitive sorption behavior and may be responsible for the desorptionand extraction-resistant fractions of aged contaminants. This concept is consistent with the findings that small pores with hydrophobic surfaces are responsible for resistant desorption (Werth and Reinhard, 1997) and declined bioavailability of contaminants to bacteria (Nam and Alexander, 1998).
0269-7491/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(01)00296-2
428
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433
Weber and Huang (1996) proposed that the hydrophobic hole domain is located between loose, amorphous humic materials and mineral surfaces and is composed of highly compact humic materials, which is typical to humin. Earlier studies have demonstrated that humin has macromolecular aliphatic chains as major constituents (Almendros and Gonzalez-Vila, 1987; Almendros and Sanz, 1991) and has significant amounts of small pores on its surface (Malekani et al., 1997). In these regards, it seems reasonable to hypothesize that the hydrophobic hole domain, which is proposed to be responsible for persistence of organic compounds, may exist in humin fraction of soil organic matter. The present study was thus conducted to determine a potential role of humin and alkalineextractable humic substances in the sequestration and bioavailability of phenanthrene in soil.
the method described by Swift (Swift, 1996) with some modifications. After 1, 7, and 100 days of aging in Lima loam, the phenanthrene-aged soil was transferred to a 250-ml Teflon centrifuge bottle and 100 ml of 0.1 N NaOH solution was added to the bottle. The suspension was shaken on a horizontal shaker (200 rpm) for 24 h under nitrogen at room temperature. The dark brown colored supernatant containing humic and fulvic acids was separated from the residual soil solid by centrifugation (12, 860 g for 20 min). The precipitated solid was considered as a humin-mineral fraction. The solid was recovered and washed with distilled water until its pH reached about 7. The supernatant was acidified with concentrated hydrochloric acid (pH < 1) to precipitate humic acid fraction. The resulting solution contained fulvic acid. Phenanthrene was extracted from the three fractions, and concentrations of the compound were determined by high-pressure liquid chromatography. More detailed procedures are described in Section 2.6.
2. Materials and methods 2.4. Biodegradation of aged phenanthrene 2.1. Soil samples Lima loam was collected from the Aurora Experimental Farm of Cornell University (Ithaca, NY). The soil is composed of 32.0% sand, 45.5% silt, and 22.5% clay (pH 6.89) and has 2.99% of organic carbon. The soil was passed through a 2-mm sieve, air-dried, and sterilized by gamma irradiation (2.5 Mrad) from a 60Co source (Ward Laboratory, Cornell University, Ithaca, NY). The samples were stored in 50-ml screw cap tubes at room temperature until use. 2.2. Aging of phenanthrene Unlabeled and [9-14C] phenanthrene (Sigma Chemical Co., St. Louis, MO) were added to soil samples for aging. Ten gram portions of soil samples were aseptically added to 50-ml test tubes, and 1.0 mg of labeled phenanthrene (ca. 1.0105 dpm; 8.3 mCi/mmol, > 98% purity) and 99.0 mg of unlabeled phenanthrene in dichloromethane were added to the soils. The total volume of dichloromethane added was less than 100 ml. The soil samples were then placed in a hood mixed for 1 h with a vortex mixer at 15-min intervals to evaporate dichloromethane and to disperse phenanthrene-spiked soil particles. Sterile distilled water was then added to bring the moisture level to the 80% of the water-holding capacity of the soil. The tubes were closed with Teflonlined screw caps and stored at room temperature in the dark for aging. 2.3. Fractionation of humic substances Humic substances were fractionated into fulvic acid, humic acid, and humin-mineral fraction according to
The humin-mineral fraction isolated as described above was sterilized by Gamma irradiation and used for aging and biodegradation experiments. One hundred micrograms of phenanthrene were aged in 10 g of humin-mineral fraction (1.52% organic carbon). After aging for 0, 30, and 100 days, biodegradation experiments were performed with the phenanthrene-aged humin fractions. Phenanthrene-aged samples were transferred to 50-ml flasks to which 10-ml portions of inorganic salts solution (0.10 g CaCl2 2H2O, 0.01 g FeCl3, 0.10 g MgSO4 7H2O, 0.10 g NH4NO3, 0.20 g KH2PO4, and 0.80 g K2HPO4 l 1 sterile distilled water; pH 7.0) were added. After shaking the soil slurries for 1 h at room temperature on a rotary shaker (100 rpm), a soil isolate P5-2 was inoculated into each flask at a level of 107 cells per gram of soil. The bacterium (Gram negative and small rod) has been isolated from soil because of its ability to use phenanthrene as a sole carbon and energy source (Tang et al., 1998). To determine the amounts of 14CO2 evolved during the biodegradation, each flask was sealed with a Teflon-wrapped silicon stopper through which was placed an 18-gauge needle and a 16-gauge steel cannula. From the cannula was suspended a small vial containing 1.5 ml of 0.5 N NaOH to trap 14CO2 released from mineralization. The NaOH solution was periodically removed and replaced with fresh solution, and the amount of evolved 14 CO2 was determined by a liquid scintillation counter (Model LS 7500; Beckman Instruments, Irvine, CA). The flasks were incubated at 30 C for 30 days on a rotary shaker (120 rpm) for biodegradation. The maximum rate of mineralization was determined by conducting a linear regression analysis on the points that formed the steepest section of the mineralization
429
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433
curve. At least three data points were used for each regression. The regression coefficient (r) was greater than 0.95. Mineralization extent represented an accumulative value of the amounts of 14CO2 evolved during the 30-day biodegradation experiment. 2.5. Determination of porosity and surface area Porosity and surface area were calculated from the data obtained by using a mercury-intrusion porosimeter (Pore Sizer 9300, Micromeritics Inc., Norcross, GA). A sample of 1.5 g of soil was placed in a 3-ml penetrometer and the soil was dried for 16 h at 105 C. The penetrometer containing the soil was filled with mercury (> 99.99% purity; Aldrich Chemical, Milwaukee, WI) in a preparation chamber and transferred to a high pressure chamber. The change in the volume of mercury was measured by increasing the chamber pressure from 0.1 to 213.7 MPa. Detailed procedures are previously described elsewhere (Nam et al., 1998). By this procedure, pores having diameters ranging from 0.007 to 10 mm were determined. 2.6. Extraction and chemical analysis After aging of phenanthrene in Lima loam for 1, 7, and 100 days, the soil was fractionated into fulvic acid, humic acid, and humin-mineral fraction as described above, and phenanthrene was then extracted from each fraction. The acidified solution containing fulvic acid was mixed with 20 ml of hexane in a 250-ml Teflon centrifuge bottle, and the mixture was shaken on a horizontal shaker (200 rpm) at room temperature. After shaking for 24 h, 10 ml of hexane layer was recovered and concentrated to less than 1 ml by using a rotary evaporator (Bu¨chi Rotavapor; Buchler Instruments Fort Lee, NJ). Phenanthrene from humic acid fraction was recovered using the mixture of hexane and n-butanol. Twenty milliliters of hexane and 5 ml of n-butanol were mixed with the phenanthrene-humic acid fraction, and the suspension was shaken for 16 h at room temperature on a horizontal shaker (200 rpm). After shaking, the solvent mixture was recovered from the humic acid fraction by centrifugation (18 600 g for 20 min) and the solvent was concentrated to less than 1 ml by evaporation as described above. For extraction of phenanthrene from humin-mineral fraction, 20-ml of n-butanol was added to a 50-ml Teflon centrifuge tube containing the solid, and the suspension was mixed with a vortex mixer for 1 min. The solvent–soil mixture was then shaken vigorously on a horizontal shaker (200 rpm) for 16 h at room temperature and centrifuged at 18,600 g for 20 min. The supernatant was saved for further analysis. The soil sample extracted with n-butanol was transferred to a cellulose extraction thimble (2580 mm, Whatman Fisher Scientific, Springfield, NJ) and
placed in a Soxhlet extractor (Ace Glass Vineland, NJ). The extractor was fitted with a 250-ml round bottom flask containing 90 ml of hexane amended with 2 ml of n-butanol, and the extraction was performed for 16 h. The n-butanol and Soxhlet extracts were combined and concentrated to less than 1 ml by evaporation as described above. Solvent extracts from each component of humic substances were passed through 0.20-mm Teflon filters (Millex-FG13; Millipore Co., Bedford, MA) to remove particulates and analyzed by high-pressure liquid chromatography (Hewlett-Packard series 1050; HewlettPackard Co., Avondale, PA). A Spherisorb ODS-2 octadecyl-bonded silica column (5 mm, 2504 mm; Hewlett-Packard) was used. Acetonitrile-water (86:14) was used as a mobile phase at a flow rate of 0.8 ml/min. Phenanthrene was detected by its UV absorbance at 254 nm.
3. Results 3.1. Distribution of phenanthrene in humic substances after aging An experiment was conducted to determine the distribution of phenanthrene in humic substances after aging. After 1, 7, and 100 days of aging of phenanthrene in Lima loam, the soil was treated with alkali to separate humic and fulvic acids from humin-mineral fraction. Phenanthrene was extracted from each component of humic substances, and the amounts were determined chromatographically. As shown in Table 1, more than 90% of initial amount was found in humin-mineral fraction regardless of aging period. Although more phenanthrene was present in humic acid than in fulvic acid, the amounts of phenanthrene present in two fractions were not appreciable. About 11% of initial phenanthrene was recovered from the humic and fulvic acids in 1-day aged soil samples, and 5.87 and 5.70% were recovered from the same fractions which were separated Table 1 Distribution of freshly added or aged phenanthrene in isolated humic substances of Lima loama Aging days
1 7 100
Phenanthrene extracted (%) Fulvic acid
Humic acid
Humin-mineral fraction
Total
3.2Aa 2.4Aa 201Aa
8.3Ba 3.4Ab 3.6Ab
90.1Ca 93.2Ba 90.9Ba
101.6a 99.0a 96.6a
a Values are the means of triplicate determinations and values in columns followed by the same lowercase letters and values in rows followed by the same uppercase letters are not significantly different (P<0.05).
430
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433
from 7- and 100-day aged samples, respectively. After 100 days of aging, a total of 96.6% of initial phenanthrene was recovered by combined solvent extraction, which was not significantly different from the total recovery from 1-day aged samples, indicating that abiotic transformation, volatilization, and formation of nonextractable residues had not occurred significantly during the aging period.
When the same polycyclic aromatic hydrocarbon was aged in the whole soil (i.e. Lima loam), which maintained undisturbed physicochemical structure of organic matter, additional decreases in bioavailability with increased residence time: both maximum rates and extents were presented in Table 2.
3.2. Biodegradation of phenanthrene aged in huminmineral fraction
Porosity and surface area were determined with Lima loam and humin-mineral fraction of the loam by using mercury porosimetry. The results indicate that removal of humic and fulvic acids did not cause significant changes in total porosity (i.e. 0.284 m3 g 1 for Liam loam and 0.272 m3 g 1 for the humin-mineral fraction) and surface area (Table 3). Pore volume resulting from pores having 7- to 100-nm diameter did not change even after humic and fulvic acids had been removed. However, changes in pore volumes were obvious in pores with larger diameters. In humin-mineral fraction, pore volume originating from pores having a diameter of 0.1–1 mm increased by 43%, and pore volume from pores with a diameter of 1–10 mm decreased by 28% compared to the intact Lima loam.
Since most freshly added and aged phenanthrene was found in humin-mineral fraction from the distribution experiments, the ability of humin-mineral fraction to decrease biodegradability of organic compounds was determined. Phenanthrene was aged in the fraction, and biodegradation was performed after 0, 30, and 100 days of aging (Fig. 1). Phenanthrene aged for 0, 30, and 100 days was mineralized with the extents of 66.8, 63.1, and 57.3%, respectively, by the test bacterium P5-2. After 2 days of biodegradation experiments, extents of mineralization were different up to 14.8% between freshly added and 100-day aged samples. However, the difference gradually decreased with prolonged biodegradation and, at the end of 30-day biodegradation, it was about 9.4%, which was still significantly different (P < 0.05). Appreciable decreases in the maximum rates of mineralization were observed with phenanthrene aged in humin-mineral complexes. The maximum rate of 100-day aged samples was 19.8% day 1, which was about 1.2- and 1.7-fold less than the rates of 30-day aged and freshly added samples, respectively (Table 2).
3.3. Porosity and surface area
4. Discussion The data show that most phenanthrene was found mainly in humin-mineral complex rather than in humic and fulvic acids whether the polycyclic aromatic hydrocarbon was freshly added or aged. Operationally, humin is defined as the fraction of mineral-bound humic materials that can not be extracted with either alkali or acid. Since humin often comprises more than 50% of organic carbon in soil (Rice and MacCarthy, 1990) it may play an important role in the association of organic contaminants in soil. It has been reported that pesticides such as atrazine adsorb tightly to humin and such interactions often result in bound residues (Capriel et al., 1985). The formation of nonextractable bound residues Table 2 Extents and maximum rates of mineralization of phenanthrene aged in humin-mineral fraction and whole soil (i.e. Lima loam)a Aging (days)
0 30 100 Fig. 1. Biodegradation of phenanthrene aged in humin-mineral fraction separated from Lima loam or in the original Lima loam by bacterium P5-2. Values are the means of triplicate determinations and statistical analyses are shown in Table 2.
a
Extent (%)
Maximum rate (%)
Huminmineral
Whole soil
Huminmineral
Whole soil
66.7Aa 63.1Aa 57.3Ab
65.4Aa 60.9Ab 47.8Bc
32.9Ca 23.3Cb 19.8Cc
32.C8a 21.4cb 12.9Dc
Values are the means of triplicate determinations and values in columns followed by the same lowercase letters and values in rows followed by the same uppercase letters are not significantly different (P<0.05).
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433 Table 3 Porosity and surface area determined by mercury porosimetrya Soils
Lima loam Humin-mineral fraction
Pore volume (m g 1)b
Surface area (m g 1)
0.007–0.1 mm
0.1–1 mm
1–10 mm
0.030Aa 0.030Aa
0.083Ba 0.119Bb
0.171Ca 0.123Bb
6.042a 6.018a
a Values are the means of triplicate determinations and values in columns followed by the same lowercase letters and values in rows followed by the same uppercase letters are not significantly different (P<0.05). b Pore sizes in diameter.
also has been observed with polycyclic aromatic hydrocarbons. Anthracene and hexadecane were shown to produce nonextractable fractions in soil (Ka¨stner et al., 1995). These bound residues often require structural changes of the parent compounds and are essentially not active biologically, and are mainly associated with humin fraction (Capriel et al., 1985; Heim et al., 1995). Recently, however, bound pesticide residues were shown to be noncovalently bound to soil by cation exchange and hydrophobic interactions (Lerch et al., 1997). Considering the facts that aging of organic compounds does not involve any types of structural alteration, and aged compounds can be recovered by exhaustive solvent extraction (Alexander, 1995), nonextractable residues covalently bound to soil may not truly represent aged or sequestered compounds. In the present study, aged phenanthrene associated with humin was fully extractable by the combination of solvent and Soxhlet extraction methods. Thus, the compound found in the huminmineral fraction in this study was literally sequestered in the soil matrix. This is consistent with the finding that pyrene, when aged in sediments, was found primarily in humin, and interactions between pyrene and humin are noncovalent in nature (Guthrie et al., 1999). Organic carbon content in soil humin has been estimated in the range of 10–45% (Rice and McCarthy, 1989; Malekani et al., 1997) and this organic fraction was attributed to the retention of organic contaminants in humin (Kohl and Rice, 1998). Nieman et al. (1999) also reported that about 64% of the solvent-nonextractable 14C was associated with the lipid and humic acid bound to soil mineral fraction. Recently, Guthrie and Pfaender (1998) showed that about 29 to 73% of recovered pyrene was found in humin fraction of a soil and up to 26% in humic/fulvic acid extracts, regardless of the pyrenedegrading activity of the soil. It is well known that aging causes a decreased bioavailability of a compound of interest in soil. In this study, phenanthrene aged in both humin-mineral fraction and a whole soil showed decreased rates and extents of mineralization with time although the extents
431
decreased were much greater in the whole soil than in humin-mineral fraction. A recent study showed that removal of humic and fulvic acids caused a decrease in the rate of desorption of phenanthrene (White et al., 1999). Alkaline extraction of soil organic matter produces a highly crystallized, denser, higher-molecular-weight humic material (i.e. humin), and sorption of hydrophobic compounds to such a so called humin fraction is greater and less linear than the unaltered soil organic matter (Pignatello and Xing, 1996). Our data showed that a significant amount of pores with a diameter of 0.1–1 mm are present on the surfaces of humin-mineral fraction. Pore volume originating from the 0.1–1-mm sized pores increased by 43% after removing humic and fulvic acids from the original soil. They were newly exposed by the fractionation procedure. A previous study (Malekani et al., 1997) demonstrated that such high porosity on humin-mineral complex mainly came from humin, not from mineral surfaces. The pore sizes of 0.1–1 mm seem to be larger compared to generally believed size ranges that might contribute to the formation of sequestered or resistant compounds. However, it is plausible that such seemingly larger pores can serve as sequestration sites for organic pollutants considering that humin is thought to be located in between loosely structured humic substances such as humic and fulvic acids and mineral surfaces. Loosely bound humic substances may impede desorption by increasing additional interactions or tortuosity and provide barriers to biological attack. Extracting humic substances from soil has clearly increased the accessibility of 0.1–1-mm-sized pores (Table 3). These were presumably inaccessible to due to the presence of the humic substances that essentially blocked entry to the pores. Phenanthrene spiked into the isolated humin-mineral fraction will be able to diffuse more readily into these sites compared to whole soil, thereby reducing bioaccessibility to degrading microbes. Considering the facts that pores less than 1 mm in diameter are dominant on humin-mineral surfaces and such pores are usually interconnected to much smaller pores, phenanthrene, once enters into such pores during aging, is not likely to be readily desorbed and thus is less available to enzymatic attack and direct bacterial degradation. A recent study (Cornelissen et al., 1998) with model sorbents demonstrate that organic compounds diffused through pores in soil organic matter or pores coated with organic materials are resistant to desorption. The importance of hydrophobic small pores in the reduced bioavailability of organic compounds has also been demonstrated with model solids (Nam and Alexander, 1998). It is worthwhile considering that phenanthrene might have been re-distributed during the base extraction. When humic and fulvic acids are extracted with using NaOH (i.e. pH > 12), functional groups such as
432
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433
carboxylic groups will predominantly be in a dissociated form. This may produce uncoiled macromolecules of humic and fulvic acids in solution and such shift is likely to influence the retention of nonpolar hydrophobic contaminants associated with these phases. If this is the case in our study, phenanthrene initially sorbed to fulvic and humic acids might have been re-distributed during the extraction procedure, and thereby the chemical might have re-located to humin-mineral fraction. Nonetheless, the present findings are significant in that they show relative distribution of aged phenanthrene among humic substances and demonstrate a potential role of humin-mineral complex and base-extractable humic substances in the sequestration and bioavailability of hydrophobic organic compounds in soil.
Acknowledgements The authors wish to thank Dr. Martin Alexander for financial support and guidance for this work and Dr. Joseph J. Pignatello for invaluable discussion during the preparation of this manuscript. We also thank the financial support by the Research Division of Seoul National University/Hanyang University for Social Infrastructure and Construction Technology.
References Alexander, M., 1995. How toxic are toxic chemicals? Environmental Science and Technology 29, 2713–2717. Almendros, G., Gonzalez-Vila, F.J., 1987. Degradative studies on a soil humin fraction-Sequential degradation of inherited humin. Soil Biology and Biochemistry 19, 513–520. Almendros, G., Sanz, J., 1991. Structural study on the soil humin fraction-Boron trifluoride-methanol transesterification of soil humin preparations. Soil Biology and Biochemistry 23, 1147–1154. Bowmer, K.H., 1991. Atrazine persistence and toxicity in two irrigated soils of Australia. Australian Journal of Soil Research 29, 339–350. Capriel, P., Haisch, A., Khan, S.U., 1985. Distribution and nature of bound (nonextractable) residues of atrazine in a mineral soil nine years after the herbicide application. J. Agricultural Food and Chemistry 33, 567–569. Carroll, K.M., Harkness, M.R., Bracco, A.A., Balcarcel, R.R., 1994. Application of a permeant/polymer diffusional model to the desorption of polychlorinated biphenyls from Hudson river sediments. Environmental Science and Technology 28, 253–258. Cornelissen, G., Van Noort, P.C., Grovers, H.A.J., 1998. Mechanism of slow desorption of organic compounds from sediments: A study using model sorbents. Environmental Science and Technology 32, 3124–3131. Guthrie, E.A., Pfaender, F.K., 1998. Reduced pyrene bioavailability in microbially active soils. Environmental Science and Technology 32, 501–508. Guthrie, E.A., Bortiatynski, J.M., Van Heemst, J.D.H., Richman, J.E., Hardy, K.S., Kovach, E.M., Hatcher, P.G., 1999. Determination of [13C]pyrene sequestration in sediment microcosms using flash pyrolysis-GC-MS and 13C NMR. Environmental Science and Technology 33, 119–125.
Heim, K., Schuphan, I., Schmidt, B., 1995. Behavior of [14C]-4nitrophenol and [14C]-3,4-dichloroaniline in laboratory sedimentwater system: Part 2. Desorption experiments and identification of sorptive fraction. Environmental Toxicology and Chemistry 14, 755–761. Ka¨stner, M., Lotter, S, Heerenklage, J., Breuer-Jammali, M, Stegmann, R, Mahro, B., 1995. Fate of 14C-labeled anthracene and hexadecane in compost-manured soil. Applied Microbiology and Biotechnology 43, 1128–1135. Kan, A.T., Fu, G., Tomson, M.B., 1994. Adsorption/desorption hysteresis in organic pollutants and soil/sediment interaction. Environmental Science and Technology 28, 859–867. Kelsey, J.W., Alexander, M., 1997. Declining bioavailability and inappropriate estimation of risk of persistent compounds. Environmental Toxicology and Chemistry 16, 582–585. Kelsey, J.W., Kottler, B.D., Alexander, M., 1997. Selective chemical extractants to predict bioavailability of soil-aged organic chemicals. Environmental Science and Technology 31, 214–217. Kohl, S.D., Rice, J.A., 1998. The binding of contaminants to humin: A mass balance. Chemosphere 36, 251–261. Lerch, R.N., Thurman, E.M., Kruger, E.L., 1997. Mixed-mode sorption of hydroxylated atrazine degradation products to soil: A mechanism for bound residue. Environmental Science and Technology 31, 1539–1546. Luthy, R.G., Aiken, G.R., Brusseau, M.L., Cunningham, S.D., Gschwend, P.M., Pignatello, J.J., Reinhard, M., Traina, S.J., Weber Jr., W.J., Westll, J.C., 1997. Sequestration of hydrophobic organic contaminants by geosorbents. Environmental Science and Technology 31, 3341–3347. Malekani, K., Rice, J.A., Lin, J.-S., 1997. The effect of sequential removal of organic matter on the surface morphology of humin. Soil Science 162, 333–342. Nam, K., Alexander, M., 1998. Role of nanoporosity and hydrophobicity in sequestration and bioavailability. Environmental Science and Technology 32, 71–74. Nam, K., Chung, N., Alexander, M., 1998. Relationship between organic matter content of soil and the sequestration of phenanthrene. Environmental Science and Technology 32, 3785–3788. Nieman, J.K.C., Sims, R.C., Sims, J.L., Sorensen, D.L., Mclean, J.E., Rice, J.A., 1999. [14C]Pyrene bound residue evaluation using MIBK fractionation method for creosote-contaminated soil. Environmental Science and Technology 33, 776–781. Pignatello, J.J., Xing, B., 1996. Mechanisms of slow sorption of organic chemicals to natural particles. Environmental Science and Technology 30, 1–11. Rice, J.A., MacCarthy, P., 1990. A model of humin. Environmental Science and Technology 24, 1875–1877. Swift, R.S., 1996. Organic matter characterization. In: Sparks, D.L. (Ed.), Methods of Soil Analysis, Part 3—Chemical Methods. Soil Science Society of America, Madison WI, pp. 1011–1069. Tang, W.-C., White, J.C., Alexander, M., 1998. Utilization of sorbed compounds by microorganisms specifically isolated for that purpose. Applied Microbiology and Biotechnology 49, 117–121. Umbreit, T.H., Hesse, E.J., Gallo, M.A., 1986. Bioavailability of dioxin in soil from a 2,4,5-T manufacturing site. Science 232, 497–499. Weber Jr., W.J., Huang, W., 1996. A distributed reactivity model for sorption by soils and sediments. 4. Intraparticle heterogeneity and phase-distribution relationships under nonequilibrium conditions. Environmental Science and Technology 30, 881–888. Werth, C.J., Reinhard, M., 1997. Effect of temperature on trichloroethylene desorption from silica gel and natural sediments. 2. Kinetics. Environmental Science and Technology 31, 697–703. White, J.C., Hunter, M., Nam, K., Pignatello, J.J., Alexander, M., 1999. Correlation between biological and physical availabilities of phenanthrene in soils and soil humin in aging experiments. Environmental Toxicology and Chemistry 18, 1720–1727.
K. Nam, J.Y. Kim / Environmental Pollution 118 (2002) 427–433 Wu, S., Gschwend, P.M., 1986. Sorption kinetics of hydrophobic organic compounds to natural sediments and soils. Environmental Science and Technology 20, 717–725.
433
Xing, B., Pignatello, J.J., Gigliotti, B., 1996. Competitive sorption between atrazine and other organic compounds in soils. Environmental Science and Technology 30, 2432–2440.