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Environ. Sci. Technol. 1999, 33, 3781-3787

Relationships between Structure and Binding Affinity of Humic Substances for Polycyclic Aromatic Hydrocarbons: Relevance of Molecular Descriptors
IRIN A V . P ERMINO VA,* N A TA LIY A YU. GRECHISH CHEVA, AND V A LE R Y S. PET RO SYAN Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia

To address this problem, we have derived structure-PAH binding affinity relationships for a larger set of humic materials of different origin (26 samples, compared to the 5-17 sources used in previous studies; 10, 12) encompassing three different PAHssanthracene, fluoranthene, and pyrene. We have broadened the range of molecular descriptors to include more specific structural descriptors derived from 13 C NMR data. The latter provide the most reliable information on the structure of such a complex matter as HS (14). We have examined a relevance of the molecular descriptors for prediction of Koc by deriving the correlation relationships for the different subsets of the target humic materials. Our objectives were to (1) measure Koc of three different PAHs to a variety of humic sources, (2) derive the correlation relationships between the PAH binding affinities and molecular descriptors of HS, and (3) compare significance of the relationships obtained for different subsets of the target humic materials.

Partition coefficients for the binding affinities of pyrene, fluoranthene, and anthracene to 26 different humic materials were determined by fluorescence quenching. Sources included isolated humic acids, fulvic acids, and combined humic and fulvic fractions from soil, peat, and freshwater as well as Aldrich humic acid. Each of the humic materials was characterized by elemental composition, ultraviolet absorbance at 280 nm, molecular weight, and for 19 samples, composition of main structural fragments determined by 13C solution-state NMR. The magnitude of the K values oc correlated strongly with the independent descriptors of aromaticity of humic materials, including atomic H/C ratio, absorptivity at 280 nm, and three interdependent 13C NMR descriptors (CAr-H,R, CAr, CAr/CAlk). Statistical comparison of humic sources grouped by the origin revealed that binding affinities were best predicted by the 13C NMR descriptors, with a slight prevalence of CAr/CAlk ratio, while molecular weight was the poorest predictor. The latter produced either direct or inverse significant correlation with the Koc values depending upon the origin and/ or fractional composition of the grouped humic materials.

Materials and Methods
The PAHs used were anthracene (Aldrich, 98+% pure), fluoranthene, and pyrene (Aldrich, 97% pure). Humic materials (humic acids (HA), fulvic acids (FA), and a combination of HA and FA (HA + FA)) used were isolated from different natural sources (freshwater, soil, and peat, Table 1). Aquatic HA + FA were isolated from the River Moscow (FMX) and North Dvina (WM3X) and from swamp water (SWA) using Amberlite XAD-2 resin as described elsewhere (15). Peat HA + FA were isolated from seven peat samples of different geobotanical composition. The peat types were Sphagnum-Fuscum (T1), Sphagnum (T4, T5), sedge (T6), woody (T7), and woody-herbaceous (HTL, TTL). Isolation procedure was as described elsewhere (16) and included a preliminary treatment of a peat sample with an ethanolbenzene (1:1) mixture followed up by an alkaline (0.1 M NaOH) extraction. One sample (HTW) was a concentrated water extract of woody-herbaceous peat. Soil HA were extracted from eight soils. These included sod-podsolic soils nearby Moscow (HBW, HBP, HBG) and Novgorod (HBWN), two grey wooded soils nearby Tula (HGW, HGP), and typical and meadow chernozemic soils nearby Voronezh (HST and HS, respectively). The HA extraction was carried out according to Orlov and Grishina (17). This included pretreatment of a soil sample with 0.1 M H2SO4, follow up alkaline extraction (0.1 M NaOH), and acidification of the extract to pH 1-2. The precipitated HA were desalted by dialysis. Soil FA were extracted from five out of the eight described above soils: three sod-podzolic soils nearby Moscow (FBW, FBP, FBG), grey wooded soil (FGW), and typical chernozem (FST). To isolate FA, the supernatant after precipitation of HA was passed through Amberlite XAD-2 resin. Further treatment was as described elsewhere (15). Soil HA + FA (SEL) was isolated by alkaline extraction (0.1 M NaOH) from typical Chernozem nearby Stavropol. The extract was desalted by cation-exchanging. Commercial Aldrich humic acid (AHA) was used as purchased from the supplier. Concentrated stock solutions of humic materials (100500 mg of organic carbon (OC)/L) were prepared by evaporation of the corresponding cation-exchanged isolates or by a dissolution of a weight of a dried material. Content of organic carbon in the stock solutions was measured using a Shimadzu 5000 TOC analyzer (18).
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Introduction
Polycyclic aromatic hydrocarbons (PAHs) belong to one of the most hazardous classes of hydrophobic organic contaminants. The fate of PAHs released into the environment is affected by humic substances (HS), which comprise from 50 to 80% of natural organic matter in water and soil ecosystems (1, 2). The binding to HS can result in increased contaminant mobility (3, 4) and a decrease in bioavailability and toxicity of PAHs (5-7). In turn, the binding affinity of HS for PAHs (Koc) has been related to the composition and structure of HS (8-12). The simplest constitutional descriptors (13) such as the content of aromatic carbon, H/C and O/C atomic ratios, molar absorptivity, and molecular weight have been used for deriving the corresponding correlation relationships (8-12). However, the predictive power of these molecular descriptors varied greatly among the different studies. This variability could result from differences in the amount, type (isolated humic fractions or unfractionated dissolved organic matter of natural water), and source of target materials used for deriving quantitative relationships.
* Corresponding author phone/fax: 7 (095) 9395546; e-mail: iperm@org.chem.msu.ru.
10.1021/es990056x CCC: $18.00 Published on Web 09/21/1999 © 1999 American Chemical Society

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TABLE 1. Partition Coefficients of the Three PAHs for Humic Substances Used in This Study, KOc в 10-5, L/kg C
sample FMX WM3X SWA T1 T4 T5 T6 T7 HTL TTL HTW source of HS River Moscow River North Dvina swamp water Sphagnum-Fuscum peat Sphagnum peat Sphagnum peat sedge peat woody peat woody-herbaceous peat woody-herbaceous peat water extract of peat sod-podzolic soil, forest sod-podzolic soil, forest sod-podzolic soil, plough sod-podzolic soil, garden gray wooded soil, forest gray wooded soil, plough meadow chernozem typical chernozem sod-podzolic soil, forest sod-podzolic soil, plough sod-podzolic soil, garden grey wooded soil, forest typical chernozem typical chernozem Aldrich humic acid pyrene Aquatic Humic Substances (HA + FA) 0.7 ( 0.1a 0.4 ( 0.1 1.2 ( 0.2 Peat Humic Substances (HA + FA) 1.2 ( 0.2 1.4 0.8 0.7 1.7 1.4 ( ( ( ( ( 0.1 0.2 0.1 0.1 0.2 fluoranthene 0.5 ( 0.1 0.2 ( 0.1 0.9 ( 0.2 0.9 ( 0.2 0.9 0.6 0.7 1.1 0.9 ( ( ( ( ( 0.1 0.2 0.1 0.2 0.2 anthracene <0.1 <0.1 <0.1
b

0.12 ( 0.05 0.22 ( 0.05 0.16 ( 0.03 0.25 ( 0.07 0.6 ( 0.2 0.5 ( 0.1 <0.1 <0.1

1.0 ( 0.2 <0.1 Soil Humic Acids 1.0 ( 0.1 1.3 ( 0.1 1.2 ( 0.2 0.7 ( 0.1 1.4 ( 0.5 1.8 ( 0.2 2.2 ( 0.2 2.4 ( 0.3 Soil Fulvic Acids 0.13 ( 0.08 <0.1 <0.1 0.5 ( 0.1 1.1 ( 0.2 Soil Humic Substances (HA + FA) 1.0 ( 0.1 Commercial Preparation 2.3 ( 0.3

0.8 ( 0.1 <0.1

HBW HBWN HBP HBG HGW HGP HS HST FBW FBP FBG FGW FST SEL AHA
a b

0.8 ( 0.1 0.8 ( 0.1 0.8 ( 0.1 0.5 ( 0.1 0.9 ( 0.2 1.2 ( 0.1 1.3 ( 0.2 1.6 ( 0.3 <0.1 <0.1 <0.1 0.3 ( 0.1 0.7 ( 0.1 0.6 ( 0.1 1.8 ( 0.2

<0.1 0.5 ( 0.1 0.5 ( 0.1 <0.1 0.5 ( 0.2 0.7 ( 0.1 1.0 ( 0.1 1.0 ( 0.4 <0.1 <0.1 <0.1 <0.1 <0.1 0.5 ( 0.1 1.0 ( 0.2

( value corresponds to a confidence interval of the slope of the Stern-Volmer plot for the corresponding HS sample at n ) 7 and P ) 95%. Value of <0.1 в 105 corresponds to the minimum detectable Koc value estimated in this study and is given for HS samples which did not cause fluorescence quenching of the PAHs.

Elemental analyses (C, H, N) were performed on a Carlo Erba Strumentazione elemental analyzer. S, H2O, and ash contents were determined manually. Oxygen contents were calculated as a difference. The H/C and O/C atomic ratios were derived from the contents of the elements calculated on ash- and water-free basis. Size exclusion chromatography (SEC) analysis was performed at the facilities of the Division of Water Chemistry, Engler-Bunte Institute, University of Karlsruhe, Germany, according to (19). Toyopearl HW-50S resin (Japan) was used as a column packing. Polydextranes were used for calibration. HS solution was equilibrated with the SEC mobile phase (0.028 M phosphate buffer) prior to the analysis. A value of peak molecular weight (M) of HS was used for correlations with Koc coefficients. UV-absorbance data were recorded on a Varian DMS100-S spectrophotometer. Absorbance of an HS solution was measured at 280 nm in a 1-cm quartz cuvette. All the
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measurements were made in 0.028 M phosphate buffer. Absorbance values were normalized to a concentration of HS in milligrams of C/L to produce an ABS280 value used for correlations with Koc coefficients.
13 C solution-state NMR spectra of 19 HS samples were measured on solutions of humic materials in 0.1 M NaOD/ D2O at an approximate concentration of 30 g/L. Measurements were made on a Varian VXR-400 spectrometer operating at 100 MHz 13C observation frequency using inverse gate decoupling. Each spectrum is a result of 12 000-14 000 scans. Sodium trimethylsilylpropanesulfonate was used as an internal standard. All the spectra were recorded at 4-s delay time. These conditions were shown to provide quantitative determination of carbon distribution among the main structural fragments of HS (20). The corresponding relationships between delay time (0.5, 1, 2, 3, 4, and 8 s) and integral intensities of the signals of carbon with different chemical surroundings for five humic materials are described in (20,

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21); the manuscript is now in preparation for publication. To quantify the observed spectra, the assignments were made after Kovalevskii (20) and were as follows (in ppm): 5-50, aliphatic H and C-substituted C atoms (CAlk); 50-108, aliphatic O-substituted C atoms (CAlk-O); 108-145, aromatic H and C-substituted atoms (CAr-H,C); 145-165, aromatic O-substituted C-atoms (CAr-O); 165-187, C atoms of carboxylic and esteric groups (CCOO); and 187-220, C atoms of quinonic and ketonic groups (CCdO). To derive the molecular descriptors, the percentage of carbon in the given structural fragments was used. Fluorescence measurements were performed on a laser fluorimeter consisting of a nitrogen laser LGE-21 (excitation wavelength 337.1 nm) and an optical multiplier analyzer OMA-1 (PARC, U.S.A.). Fluorescence measurements on PAHs were made as described by Yashchenko et al. (22). The emission wavelengths (nm) used were 400, 476, and 393, with slit widths of 100, 25, and 25 µm for anthracene, fluoranthene, and pyrene, respectively. Koc coefficients were determined by fluorescence quenching (23). Aqueous PAH solutions below the solubility limit were prepared by spiking 1 L of double-distilled water with 20-60 µL of concentrated solutions of PAH in acetonitrile (Fischer, spectroanalyzed). The resulting solutions were equilibrated overnight and filtered through the precombusted glass fiber filters (GF/F, Whatman). Final concentrations were 1 в 10-7, 5 в 10-7, and 0.6 в 10-7 M for anthracene, fluoranthene, and pyrene, respectively. The stock solutions were stored in the dark at 4 °C. For measuring fluorescence quenching, a series of seven solutions of PAHs in the presence of HS was prepared in 25 mL volumetric flasks, and the necessary amount of concentrated (1 в 10-3 kg C/L) solutions of HS was added to make up a concentration of HS in the range of (0.2-6) в 10-6 kg C/L. The solutions were thoroughly mixed and fluorescence measured after 15 min. Increase of the contact time up to 3 h did not influence a magnitude of the fluorescence quenching, indicating that 15 min was sufficient for reaching an equilibrium between PAH and HS. This is in agreement with the results reported in ref 24 for quenching of benz[a]pyrene. The measured fluorescence included a background component from the HS. To correct for this, the fluorescence of solutions containing HS only was measured under the same conditions as for the HS and PAH together. Absorbance measurements at the excitation and emission wavelengths required to correct for inner filter effects (23) were taken for each series. The fluorescence measurements were corrected for the background fluorescence of HS and inner filter effect. The corrected fluorescence intensity of PAHs in the presence of HS (F) and absence of HS (F0) was used in the Stern-Volmer equation (23), which described the decrease in fluorescence in the presence of quencher (HS). Values of F0/F formed linear plots against concentration of HS, with Koc values calculated from the slopes.

FIGURE 1. Stern-Volmer plots for quenching pyrene, fluoranthene, and anthracene fluorescence with Aldrich HA. determined by fluorescence quenching have ranged from 0.23 в 105 to 5.5 в 105 (8, 23, 25-27) and for anthracene from 0.15 в 105 to 0.64 в 105 (23, 25). A reported fluoranthene Koc value of 0.9 в 105 for a soil HA (28) agreed well with the range of (0.7-1.6) в 105 for soil HA in this study. At the same time, some of the soil FA and water extract of peat did not cause any fluorescence quenching of any of the PAHs. For anthracene, quenching was not detected for some soil HA as well. The lower range of the potential binding affinity of these HS was estimated by calculating the minimum detectable Koc value. The latter was considered to be equal to a value of a factor of 3 higher than a standard deviation of the lowest Koc coefficient determined from three parallel series of measurements (29). The weakest binding was observed for anthracene with T1. The resulting mean Koc value and standard deviation were 0.12 в 105 and 0.034 в 105, respectively. This indicates a minimum detectable Koc value of 0.1 в 105 which is given in Table 1 as an estimate for the humic materials that did not cause any detectable fluorescence quenching of PAHs. In general, pyrene Koc values are a factor of 1.5-2 higher than for fluoranthene and from 2- to 10-fold higher than for anthracene. This can be related to the lower hydrophobicity of anthracene (log Kow ) 4.45) in comparison with pyrene and fluoranthene (log Kow ) 4.88 and 5.16, respectively, 30), as has been reported previously (23, 31, 32). Based on the measured Koc values, the target humic materials can be arranged in the following descending order: AHA and chernozemic HA . sod-podzolic and gray wooded soil HA = peat HA+FA > aquatic HA+FA = chernozemic FA . sod-podzolic and gray wooded soil FA. The above trend with the source of HS is in agreement with that reported in the literature. For example, very high binding affinity of AHA for the three PAHs (Table 1) is consistent with the strong binding capacity of this material observed in previous studies (9, 12). Only the chernozemic HA had the equally high binding affinity for PAHs. In general, Koc values of soil HA were a factor of 2-3 higher than those of soil FA. These results corroborate the findings of the other authors (8, 11, 23). Hence, the binding affinity for PAHs of target humic materials depends greatly both on the properties of PAHs and on the source of HS. Establishing structure-PAH binding affinity relationships for HS was our next goal. Relationships between Structure and Binding Affinity of HS for PAHs. A wide range of data were used for characterization of the composition and structure of the target humic materials (Tables 2 and 3). In deriving molecular descriptors, the 13C NMR data were given primary importance as the most meaningful descriptors of the structure of HS. Correlation with Koc values were evaluated for both individual descriptors reflecting the carbon content in each of the main structural fragments as well as combined descriptors reflecting the sums, ratios, and prodVOL. 33, NO. 21, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Results and Discussion
Partition Coefficients. Stern-Volmer plots for fluorescence quenching of pyrene, fluoranthene, and anthracene with AHA are given in Figure 1. Pyrene had the highest affinity of AHA and anthracene the lowest. In addition, anthracene has the larger uncertainty in the estimated Koc value, probably due to the smaller slopes and lower reliability in determination of the extent of binding. Similar results were obtained on all the other target humic materials (Table 1). The Koc values of the humic materials able to exhibit a detectable fluorescence quenching lay in the range of (0.132.4) в 105, (0.27-1.8) в 105, and (0.12-1.0) в 105 L/kg C for pyrene, fluoranthene, and anthracene, respectively. The observed Koc values agree well with the literature. Reported Koc values for pyrene binding to dissolved humic materials

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TABLE 2. Molecular Descriptors (Other Than 13C NMR) of Humic Substances Used in This Study
sample source of HS H/C
a

O/C

a

ABS

b 280

M

c

Aquatic Humic Substances (HA FMX River Moscow 1.14 WM3X River North Dvina 1.41 SWA swamp water 0.86 T1 T4 T5 T6 T7 HTL TTL HTW HBW HBWN HBP HBG HGW HGP HS HST FBW FBP FBG FGW FST SEL AHA
a

+ FA) 0.54 0.030 1.00 0.035 0.93 0.025 FA) 0.48 0.55 0.54 0.53 0.52 0.49 0.48 0.62 0.57 0.45 0.39 0.53 0.78 0.62 0.39 0.48 0.91 0.74 0.75 0.63 0.64 0.066 0.064 0.064 0.031 0.072 0.046 0.060 0.020 0.082 0.092 0.071 0.110 0.075 0.080 0.068 0.114 0.035 0.036 0.034 0.044 0.054 0.026 0.045

6.1 6.6 9.8 18.5 18.5 16.4 18.2 18.2 17.3 19.8 6.3 12.2 14.3 16.1 17.3 16.4 14.5 12.0 12.6 7.9 7.9 10.6 11.0 9.6 13.5 13.0

Peat Humic Substances Sphagnum-Fuscum peat Sphagnum peat Sphagnum peat sedge peat woody peat woody-herbaceous peat woody-herbaceous peat water extract of peat

(HA + 1.01 1.07 0.98 0.93 0.93 0.89 0.89 1.21

Soil Humic Acids sod-podzolic soil, forest 0.93 sod-podzolic soil, forest 0.95 sod-podzolic soil, plough 0.86 sod-podzolic soil, garden 1.01 gray wooded soil, forest 0.97 gray wooded soil, plough 0.88 meadow chernozem 0.62 typical chernozem 0.51 Soil Fulvic Acids sod-podzolic soil, forest 0.90 sod-podzolic soil, plough 1.06 sod-podzolic soil, garden 0.92 gray wooded soil, forest 0.98 typical chernozem 0.81

Soil Humic Substances (HA + FA) typical chernozem 1.15 0.57 Commercial Preparation Aldrich humic acid 0.74 0.28

H/C and O/C ratios are calculated on ash- and water-free basis. b Absorptivitiy values are listed in L/(mg C в cm). c Molecular weight values are determined by SEC with calibration by polydextranes (19) and listed in kDalton.

ucts of the individual descriptors. These more complex expressions may reveal interaction features within the HS structure such as the prevalence of aromatic core over aliphatic periphery or lipophilic-lipophobic balance of the molecule (indicated by the CAr/CAlk ratio, where CAr ) CAr-O + CAr-H,R, CAlk ) CAlk-O + CAlk-C,H), the degree of oxidation of aromatic core (estimated as CAr-O/CAr ratio), substitution of aromatic rings with electron-withdrawing carboxyl and donor methyl groups (CAr-H,R в CCOO and CAr-H,R в CAlk, respectively), and others. The full correlation matrix included seven individual and eight combined 13C NMR descriptors (Table 4). Among the individual descriptors, the strongest correlation with Koc values was observed for CAr-H,C. The use of the combined descriptor CAr/CAlk resulted in the best fits to the experimental Koc values shown in Figure 2. The corresponding regression equations are given below:

Koc в 10 Koc в 10 Koc в 10

-5

) (1.3 ( 0.3) в CAr/C ) (0.8 ( 0.4) в CAr/C ) (0.6 ( 0.2) в CAr/C

Alk

- (0.3 ( 0.3) (pyrene) - (0.1 ( 0.5) (fluoranthene) - (0.4 ( 0.3) (anthracene)

-5

Alk

-5

Alk

The values of given confidence intervals of the slopes (n ) 19, P ) 95%) demonstrate a statistical relevance to the
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observed trend between Koc value and CAr/CAlk ratio. The same is true for other parameters of aromaticity of HSsCAr-H,R and CAr. This can indicate a key role of aromatic core in binding of HS to PAHs. The strongest correlation with CAr/ CAlk ratio suggests the highest binding affinity in the least sterically hindered aromatic structures that permits coplanar orientation of the PAH molecules. This could have been interpreted as indicative of the donor-acceptor character of interaction between HS and PAHs. However, the CAr/CAlk ratio could also reflect the lipophilic-lipophobic balance of the humic molecules, determined by a prevalence of hydrophobic aromatic core over hydrophilic (mostly polysaccharidic) aliphatic periphery. This suggests that the molecules with least sterically hindered aromatic core may, at the same time, be the most hydrophobic. Hence, hydrophobic interactions with PAHs may be reinforced by interactions with aromatic moieties in HS molecules to result in higher binding affinity. These results are consistent with previous studies (8, 12) correlating Koc values with the percentage of aromatic carbon in the humic material. Correlations between Koc and two indirect estimates of the aromaticity of HSsatomic H/C ratio and molar absorptivity ABS280 (Table 4) are much poorer than those for 13C NMR descriptors. However, they remain significant at P ) 95%. Our studies show that a usage of H/C ratios calculated without a correction for water content in the sample caused an additional decrease in r 2 of about 0.1 (from 0.5 to 0.4). In general, the significance of the correlations with absorptivity was comparable with those obtained with H/C ratio. These results contradict previous findings on the greater predictive power of absorptivity compared to the H/C ratio (8, 12). The correlation between Koc values and O/C ratio which indicates oxidation of a humic carbon backbone was much poorer than for H/C. This can reflect the much greater impact of the degree of unsaturation of humic backbone compared to the oxidation degree on its affinity for binding PAHs. Relationships between the PAH binding coefficients and molecular weight of the target humic materials are given in Figure 3. According to the values of the corresponding r 2 (Table 4), the linear relationship between the molecular weight of HS (M) and its ability to bind any of the three PAHs is significant at P < 99%. Each of the obtained relationships has a not distinctive maximum in the range of M from 10 to 15 kDalton. HS molecules less than 10 kDalton bind very weakly to PAHs (Koc ) (0.1-0.6) в 105, with the singular exception of FST with M ) 9.6 and pyrene Koc ) 1.1 в 105). In contrast, molecules larger than 10 kDalton are capable of much stronger PAH binding. For the samples with M > 15, a slight decrease in Koc is observed at increasing molecular weight. It should be stressed that the absolute values of molecular weight of HS can differ from the apparent values reported here (19). However, the relative molecular weights of the different HS should be adequately represented by the M values, and thus the correlation coefficients should be unaffected. The results do not corroborate the findings of Chin et al. (12) on an existence of the strong positive relationship between molecular weight and binding affinity of HS for PAHs. A lack of the strong linear relationship between Koc values and molecular weight of HS can be explained by heterogeneity and irregular structure intrinsic to humic macromolecules (33). As a result, and in contrast to regular polymers, humic macromolecules of different molecular weight can have various quantitative ratios of main structural fragments. This effect is especially apparent for humic materials formed under different environmental conditions. To illustrate this, we have compared the data on contents of structural fragments and molecular weight of a set of seven soil HA used in this study (except for HBW). For these humic materials, there was a

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TABLE 3. Percentage of Carbona in the Main Structural Fragments of Humic Substances Used in This Study (13C NMR Descriptors)
sample FMX T1 T4 T5 T6 T7 HTL TTL HTW HBW HBWN HBP HBG HGW HGP HS HST SEL AHA
a

source of HS River Moscow Sphagnum-Fuscum peat Sphagnum peat Sphagnum peat sedge peat woody peat woody-herbaceous peat woody-herbaceous peat water extract of peat sod-podzolic soil, forest sod-podzolic soil, forest sod-podzolic soil, plough sod-podzolic soil, garden gray wooded soil, forest gray wooded soil, plough meadow chernozem typical chernozem typical chernozem Aldrich humic acid

C

CdO

C

COO

C

Ar-O

C

Ar-H,C

C

Alk-O

C

Alk-H,C

Aquatic Humic Substances (HA + FA) 2 19 11 Peat Humic Substances (HA + FA) 1 15 10 1 16 12 1 16 10 2 14 11 2 16 15 2 15 14 3 15 13 0 11 3 1 2 2 1 1 3 2 3 Soil Humic Acids 17 15 16 15 19 14 14 15 12 13 12 13 13 12 9 11

25 29 31 27 38 34 31 32 13 32 30 32 33 34 34 48 43 32 43

19 25 24 22 22 18 20 20 58 20 20 21 21 16 18 14 15 22 7

24 20 16 24 14 14 17 17 15 18 20 17 17 17 19 14 13 14 21

Soil Humic Substances (HA + FA) 1 18 13 Commercial Preparation 1 15 13

Percentage of carbon equals to an integral intensity of the following regions of the 13C NMR spectrum of HS recorded at delay time of 4 s (assignments are after Kovalevskii (20), in ppm): 5-50 (CAlk), 50-108 (CAlk-O), 108-145 (CAr-H,C), 145-165 (CAr-O), 165-187 (CCOO), 187-220 (CCdO).

TABLE 4. Squares of Correlation Coefficients (r 2) between the Koc Values of the Three PAHs and Molecular Descriptors of HS Used in This Study
molecular descriptors Individual CCdO CCOO CAr-O CAr-H,C CAlk-O CAlk-H,C
13C

pyrene

fluoranthene
a

anthracene 0.14 0.0025 0.048 0.56 0.29 0.091 0.40 0.49 0.66 0.14 0.49 0.49 0.22 0.39 0.25 0.41 0.11

NMR Descriptors 0.25 0.23 0.0009 0.0049 0.16 0.18 0.77 0.69 0.52 0.56 0.063 0.0064

Combined 13C NMR Descriptorsa CAlk ) CAlk-O + CAlk-H,C 0.66 0.59 CAr ) CAr-O + CAr-H,C 0.74 0.67 CAr/CAlk 0.83 0.74 CAr-O/CAr 0.091 0.041 CAr-H,C в CCOO 0.71 0.64 (CAr + CCOO + CCdO)/CAlk 0.55 0.64 CAr-H,C в CAlk-H,C 0.40 0.50 H/C O/C ABS M Other Than 13C NMR Descriptors 0.51 0.48 0.31 0.34 0.49 0.45 0.25 0.25
b

FIGURE 2. Correlation between the Koc values of PAHs used in this study and aromaticity of humic substance expressed as CAr/CAlk.

b

280

a Correlation for 19 humic materials. materials.

Correlation for 26 humic

significant inverse relationship (P ) 95%) between molecular weight and CAr (r 2 ) 0.53), while there was direct correlation with CAlk-O (r 2 ) 0.53). Hence, the larger molecules were enriched in carbohydrate structures, whereas the smaller onesswith aromatic moieties. The observed relationship does not agree with the findings of Chin et al. (34) that there is a strong positive relationship between molecular weight and aromaticity of HS. The discrepancy may reflect differences in the type of target humic materialssaquatic HSswhich were used by authors (34). In case of soil HA, the trend observed in this study is consistent with the theory of genesis

FIGURE 3. Correlation between the Koc values of PAHs used in this study and molecular weight of humic substance ((9) pyrene, (2) fluoranthene, and (O) anthracene)). of soil HA (2, 35). Among three soil types under study, chernozems have the highest level of microbial activity. This results in a high degree of a degradation of carbohydrate periphery of a humic macromolecule while simultaneously
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FIGURE 4. Correlation coefficients of the pyrene Koc values with the molecular descriptors of humic substances for the whole set of target humic materials and for the subsets grouped by the origin and/or fractional composition. promoting a well developed aromatic core (2). The opposite situation is observed in sod-podzolic and gray wooded soils, where the carbohydrate complex of humic precursors is much better preserved in humic macromolecules. As a result, the large, but carbohydrate-enriched molecules of sod-podzolic and gray wooded soil HA would be predicted to have lower binding affinity for PAHs than the chernozemic ones possessing less hindered and more condensed aromatic structures. Indeed, the corresponding pyrene Koc values correlated inversely with M and directly with CAr/CAlk (r 2 ) 0.83 and 0.69, respectively). Very similar results were obtained for fluoranthene and anthracene. However, if the binding strength of different molecular weight humic materials formed under the same conditions are compared (for example, FA and HA from the same soil), Koc values of the higher molecular weight HA are much higher than that of FA. Comparison of the Predictive Power of the Descriptors. The last observation prompted us to subdivide the initial data set of 26 HS samples into the subsets according to the source and/or fractional composition of HS. By doing so, we hoped to reveal the general structure-PAH binding affinity relationships intrinsic to humic materials from different sources as well as specific relationships intrinsic only to HS of certain genesis. This could also allow us to evaluate the predictive power and relevance of different molecular descriptors in relation to binding affinity of HS for PAHs. Twenty-six humic materials were subdivided into four not overlapping subsets of 8 soil HA, 8 peat (HA + FA), 5 soil FA, and 5 humic materials of various origin (FMX2, WM3X, SWA, SEL, AHA). A set of 19 humic materials which were characterized with 13C NMR spectroscopy was also included as a subset of 26 samples. To evaluate the correlations between Koc values and the molecular descriptors within the given above subsets of HS, r value was preferred to r 2 as indicating not only significance but a direction of the correlation as well. The correlation coefficients between pyrene Koc and CAr, CAr-H,R, CAr/CAlk, H/C, ABS280, and M obtained for the listed above subsets of HS are given in Figure 4. Very similar results were obtained for fluoranthene and anthracene. The highest correlation and lowest variability of r-values among the different subsets of HS were provided by all three of the 13C NMR descriptors of aromaticity. Correlation with H/C ratio was, in general, lower but remained rather stable for all six subsets of HS (for pyrene Koc values, r varied from -0.67 to -0.98). Correlation with absorptivity was characterized with a much wider range of variation of the r-value among the different subsets of humic materials (from
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0.46 for the subset of eight soil HA to 0.98 for the subset of five various HS). The most sensitive descriptor indicating the impact of the composition of HS set was molecular weight. Correlations between Koc and M were significant for subsets of both eight soil HA and eight peat (HA + FA), but correlations were in opposite directions. As a result, there was very poor correlation for the whole set of the humic materials used. This is another confirmation of the discussed above complex character of relationship between molecular weight and properties of HS. Hence, extreme caution must be exercised in choosing a descriptor such as molecular weight for prediction of binding affinity of HS for PAHs. This concern is particularly relevant to prediction of the properties of HS of different origin. For these purposes, the best descriptors are aromaticity indexes derived from 13C NMR data. If 13C NMR data are not available, an atomic H/C ratio is to be used. Environmental Implications. The results reported in this paper are of major importance for developing a QSAR (quantitative structure-activity relationship) approach for predicting HS properties from their structure. In the case of binding affinity for PAHs, it was shown that among the constitutional descriptors evaluated here, the most powerful are those derived from 13C NMR data. The quantitative relationships (e.g. Koc versus CAr/CAlk) allow prediction of the influence of structural variations of the humic substance on its ability to bind PAHs. In addition, they provide deeper insight on the nature of interactions underlying binding of HS to PAHs. We believe that significant progress in structural investigation of HS by NMR spectroscopy will make it possible to define characteristic sets of structural descriptors intrinsic to HS of different origin and create the database on structure of HS. The results of our work in this direction are very promising (20) and are now in preparation for publication. This can provide an opportunity to predict the binding affinity for PAHs based on known origin of the humic material without requiring preliminary investigation of its structure with a use of 13C NMR spectroscopy. Another notable environmental benefit arising from this study is the observed strong correlation among Koc values of the three different PAHs. There was a strong correlation between the Koc values of pyrene and fluoranthene and between pyrene and anthracene (r 2 of 0.996 and 0.885, respectively) observed. This relationship may justify the use of one PAH (e.g. pyrene as strongly binding to HS and best studied among the other PAHs) as a probe to estimate the binding affinity of HS for other PAHs. The estimated Koc values can be used to predict the reduction in bioavailability (5, 6) and toxicity (7) of PAHs in the presence of HS reported by many authors. Our immediate goal is an extrapolation of the approach described above to the prediction of the detoxifying properties of HS to PAHs.

Acknowledgments
Our thanks to Prof. Fadeev and Dr. Filippova from the Department of Physics, Lomonosov Moscow State University for providing access to laser fluorimeter and many useful discussions. We wish to thank Drs. Kovalevskii and Permin from our lab for invaluable help both in recording and interpreting the 13C NMR spectra. We extend our deepest appreciation to Dr. J. McCarthy (Oak Ridge National Laboratory, U.S.A.) for useful comments and editorial work on the manuscript, which substantially improved its quality. Two anonymous referees are sincerely appreciated for constructive critique and useful suggestions on the data analysis and data presentation. This research was financially supported by the grant of the Russian Fund for Basic Research No. 98-03-33186a and of INTAS 97-1129.

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Received for review January 20, 1999. Revised manuscript received May 28, 1999. Accepted July 7, 1999. ES990056X

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