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Environ. Sci. Technol. 2001, 35, 3841-3848

Quantification and Prediction of the Detoxifying Properties of Humic Substances Related to Their Chemical Binding to Polycyclic Aromatic Hydrocarbons
IRIN A V . P ERMINO VA,* , N A TA LY A YU. GRECHISH CHEVA, DMITRII V . KO VAL EVSKII, A LE X E Y V . KUD RYAVT SEV, V A LE R Y S. PET RO SYAN , AN D DMITRII N . MAT O RIN Departments of Chemistry and Biology, Lomonosov Moscow State University, Moscow 119899, Russia

Effects of 27 different humic materials on the toxicity of polycyclic aromatic hydrocarbons (PAH) were studied for crustacean Daphnia magna. Sources included isolated humic acids, fulvic acids, and their combination from soil, peat, and freshwater. The PAH used were pyrene, fluoranthene, and anthracene. The observed reduction in toxicity of PAH in the presence of humic substances (HS) was shown to be a result of the detoxification effect caused by the chemical binding of PAH to HS and of the direct effect of HS on D. magna. An approach was developed to quantify the detoxifying impact of humic materials related to their chemical binding to PAH with a use of the "constant of detoxification" or "toxicological partition coefficient" KocD. The latter was proposed to determine by fitting the experimental relationships of the detoxification effect versus concentration of HS. The obtained KocD values were well tracked by the corresponding partition coefficients determined by the fluorescence quenching technique (Kocfq): KocD ) b в Kocfq, b (mean ( CI, n ) 26, P ) 95%) ) 2.6 ( 0.3, 4.6 ( 0.6, and 6.0 ( 1.4 for pyrene, fluoranthene, and anthracene, respectively. The predictive relationships between the structure and detoxifying properties of humic materials in relation to PAH were developed. It was shown that the magnitude of the KocD values correlated closely with the aromaticity of humic materials characterized with the 13C NMR descriptors (CAr, CAr/CAlk) and atomic H/C ratio. The obtained relationships showed the highest detoxifying potential of the humic materials enriched with aromatics and allowed a conclusion on the chemical binding as the governing mechanism of the mitigating action of HS on the toxicity of PAH.

are mitigated via their binding to humic substances (HS). The latter causes a reduction in the concentration of freely dissolved PAH in the water that makes the contaminant less bioavailable for the aquatic biota. This has been shown by numerous studies on the bioaccumulation (2-7) as well as on toxicokinetics (2, 8-10) of PAH in the presence of HS. According to the reported data (3, 11, 12), the bioconcentration factor (BCF) in the presence of HS is directly proportional to the fraction of PAH freely dissolved in the water. The authors based the conclusion on the good match of the partition coefficients of PAH (Koc) determined from the bioaccumulation data and measured by equilibrium dialysis. The uptake/depuration studies (8, 9) have also demonstrated a good correlation between the partition coefficients determined from chemical and biological data sets. The similar studies on the toxicity of PAH are missing. In addition to our previous results which displayed the detoxifying effects of HS on phenanthrene and fluoranthene (13), the only estimates we are aware of concern a reduction in phototoxicity of anthracene (14) in the presence of HS. In contrast to bioaccumulation, determination of the partition coefficient from a reduction in toxicity of PAH can be hampered by the physiological activity of HS. The latter was numerously reported for the higher plants (15, 16). The corresponding studies on the aquatic biota are much more scarce (17-20) and report on the varying nature (stimulating as well as inhibiting) of the direct effects of HS upon different aquatic organisms. This prompts a development of the quantification approach to evaluate the mitigating impact of HS on the toxicity of PAH related to the chemical binding that would account for the direct effects of HS on the test organism. Using this approach, a predictive estimate of the detoxifying properties of HS in relation to PAH can be obtained independently on the specific response of the test organism to the presence of HS. This could facilitate a directed use of the humic materials for the purposes of remediation of the PAH contaminated media. Our objectives were to (1) develop an approach to quantify the detoxifying properties of HS related to their chemical binding to PAH; (2) evaluate the detoxifying properties of 27 humic materials from freshwater, soil, and peat in relation to three different PAH (anthracene, fluoranthene and pyrene); and (3) establish quantitative relationships between the structure of humics and their detoxifying properties in relation to PAH.

Experimental Section
The PAH used were anthracene (Aldrich, 98+% pure), fluoranthene (Aldrich, 97% pure), and pyrene (Aldrich, 97% pure). Preparation of Water Solutions of PAH. The batch technique described elsewhere (21) was used for preparation of water solutions of the selected PAH. PAH were dissolved in acetone and placed into a 1 L flask. The added amount of each PAH was below its water solubility. The acetone was then evaporated. One liter of water prepared for culturing the Daphnia magna (see below) was added into the flask and shaken overnight. The obtained PAH solutions were filtered through the precombusted glass fiber filters (GF/F, Whatman). The concentration of the PAH in the filtrates was determined with a use of laser fluorimetry. It was 1.7 в 10-7, 7 в 10-7, and 5 в 10-7M for anthracene, fluoranthene, and pyrene, respectively. The prepared solutions were stored in the dark. Humic materials (27 samples) used included humic acids (HA), fulvic acids (FA), and their nonfractionated mixtures
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Introduction
Polycyclic aromatic hydrocarbons (PAH) expose toxic, cancerogenic, and mutagenic effects on the living organisms (1). The adverse affects of PAH in the aquatic environments
* Corresponding author phone/fax: 7(095)939 5546; e-mail: iperm@cityline.ru. Department of Chemistry. Department of Biology.
10.1021/es001699b CCC: $20.00 Published on Web 08/30/2001 © 2001 American Chemical Society

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TABLE 1. Molecular Parameters of Humic Substances Used in This Study
sample FMX FMC WM3X SWA T1 T4 T5 T6 T7 HTL TTL HTW HBW HBP HBG HGW HGP HS HST FBW FBP FBG FGW FST SEL AHA AGK source of HS River Moscow 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, plough sod-podzolic soil, garden grey wooded soil, forest grey 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 coal humic acid H/C
a

O/C

a

ABS

b 280

M

c

C

COO

d

C

Ar

d

C

Alk

d

Aquatic Humic Substances 1.14 0.54 1.21 0.62 1.41 1 0.86 0.93 Peat Humic Substances 1.01 0.48 1.07 0.55 0.98 0.54 0.93 0.53 0.93 0.52 0.89 0.49 0.89 0.48 1.21 0.62 0.93 0.86 1.01 0.97 0.88 0.62 0.51 0.9 1.06 0.92 0.98 0.81

(HA+FA) 0.03 0.015 0.035 0.025

6.1 6.4 6.6 9.8 18.5 18.5 16.4 18.2 18.2 17.3 19.8 6.3 12.2 16.1 17.3 16.4 14.5 12 12.6 7.9 7.9 10.6 11 9.6 13.5 13 12.5

21 nd nd nd 16 17 17 16 18 17 18 11 18 18 16 20 17 16 18 nd nd nd nd nd 19 16 14

36 nd nd nd 39 43 37 49 49 45 45 16 44 44 46 47 46 57 54 nd nd nd nd nd 45 56 58

43 nd nd nd 45 40 46 36 32 37 37 73 38 38 38 33 37 28 28 nd nd nd nd nd 36 28 28

(HA+FA) 0.066 0.064 0.064 0.031 0.072 0.046 0.06 0.02

Soil Humic Acids (HA) 0.57 0.082 0.39 0.071 0.53 0.11 0.78 0.075 0.62 0.08 0.39 0.068 0.48 0.114 Soil Fulvic Acids (FA) 0.91 0.035 0.74 0.036 0.75 0.034 0.63 0.044 0.64 0.054

Soil Humic Substances (HA+FA) 1.15 0.57 0.026 Commercial Preparations 0.74 0.28 0.045 0.79 0.32 0.050

a 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 Peak molecular weight values are determined by SEC (calibration by polydextranes) and listed in kDaltons. d Content of carbon in the structural fragments is determined by 13C NMR spectroscopy as the integral intensity (%) of the following spectral regions, ppm: 220-185 (CCOO), 185-108 (CAr), and 108-5 (CAlk).

(HA+FA). They were isolated from different natural sources (freshwater, soil, and peat). Aquatic humic substances (HA+FA) were isolated from the River Moscow (FMX), North Dvina (WM3X), and from the swamp water (SWA) using Amberlite XAD-2 resin as described elsewhere (22). Peat humic substances (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). The isolation procedure was as described elsewhere (23) and included a preliminary treatment of a peat sample with an ethanol-benzene (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 HTL. Soil humic acids (HA) were extracted from seven soils. These included sod-podsolic soils nearby Moscow (HBW, HBP, HBG), two gray wooded soils nearby Tula (HGW, HGP), and typical and meadow chernozemic soils (mollisols) nearby Voronezh (HST and HS, respectively). The HS extraction was carried out according to ref 24. 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 fulvic acids (FA) were extracted from five of the described above soils: sod-podzolic (FBW, FBP, FBG), gray wooded (FGW), and typical chernozem (mollisol) (FST). To isolate FA, after precipitation of HA, the supernatant was passed through Amberlite XAD-2 resin. Further treatment was as described for aquatic HS.
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Nonfractionated soil HA+FA was isolated by alkaline extraction from typical chernozem (mollisol) nearby Stavropol (SEL). Commercial Aldrich humic acid (AHA) and activated coal humic acid (AGK) (the latter is produced by the Biotechnology Ltd. Moscow, Russia) were used as obtained from the supplier. Concentrated stock solutions of HS (1-5) в 10-4 kg C/L were prepared by evaporation of the corresponding cationexchanged 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 as described elsewhere (25). Chemical characteristics of the target humic materials and the corresponding determination techniques are described in details in our previous publication (26). The most important ones (contents of elements, molecular weight, molar absorptivity, content of aromatic carbon) are summarized in Table 1. Elemental analyses (C, H, N) were performed on a Carlo Erba Strumentazione elemental analyzer. S, H2O, and ash contents were determined manually. Size exclusion chromatography analyses were performed according to ref 27 with a use of Toyopearl HW-50S resin (Japan) as a column packing, 0.028 M phosphate buffer as a mobile phase, and polydextrans as the calibrants. Peak molecular weight (M) was used for calculations. UVabsorbance was measured on solutions of HS in 0.028 M buffer at 280 nm in a 1-cm quartz cuvette and normalized to a concentration of HS in mg C/L to produce ABS280 values. 13C NMR spectra were measured on solutions of humic

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TABLE 2. Partition Coefficients of the Three PAH for Humic Substances Used in This Study Determined by Fluorescence Quenching Technique, Kocfq в 10-5, L/kg C (26)
sample FMX WM3X SWA T1 T4 T5 T6 T7 HTL TTL HTW HBW HBP HBG HGW HGP HS HST FBW FBP FBG FGW FST SEL AHA
a

source of HS River Moscow River North Dvina swamp water

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 ( 0.1 0.8 ( 0.2 0.7 ( 0.1 1.7 ( 0.1 1.4 ( 0.2 1.0 ( 0.2 <0.1 Soil Humic Acids 1.0 ( 1.2 ( 0.7 ( 1.4 ( 1.8 ( 2.2 ( 2.4 ( (HA) 0.1 0.2 0.1 0.5 0.2 0.2 0.3

fluoranthene 0.5 ( 0.1 0.2 ( 0.1 0.9 ( 0.2 0.9 0.9 0.6 0.7 1.1 0.9 0.8 <0.1 0.8 0.8 0.5 0.9 1.2 1.3 1.6 ( ( ( ( ( ( ( 0.2 0.1 0.2 0.1 0.2 0.2 0.1

anthracene <0.1 <0.1 <0.1
b

sphagnum-fuscum peat sphagnum peat sphagnum peat sedge peat woody peat woody-herbaceous peat woody-herbaceous peat water extract of peat

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

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 gray wooded soil, forest typical chernozem typical chernozem Aldrich humic acid

( ( ( ( ( ( (

0.1 0.1 0.1 0.2 0.1 0.2 0.3

( 0.1 ( ( ( ( 0.2 0.1 0.1 0.4

Soil Fulvic Acids (FA) 0.13 ( 0.08 <0.1 <0.1 0.5 ( 0.1 1.1 ( 0.2

<0.1 <0.1 <0.1 0.3 ( 0.1 0.7 ( 0.1 0.6 ( 0.1 1.8 ( 0.2

Soil Humic Substances (HA+FA) 1.0 ( 0.1 Commercial Preparations 2.3 ( 0.3

( 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%. b Value of <0.1 в 105 corresponds to the minimum detectable K oc value estimated in this study and is given for HS samples which did not cause fluorescence quenching of the PAH.

materials in 0.1 M NaOD/D2O on a Varian VXR-400 spectrometer operating at 100 MHz 13C observation frequency. All the spectra were recorded at 4-s delay time using inverse gate decoupling. These conditions were shown to provide quantitative determination of carbon distribution among the main structural fragments of HS (28). The assignments of the total carbonylic, aromatic, and aliphatic carbon were as follows (in ppm): 5-98saliphatic nonsubstituted and O-substituted C atoms (CAlk), 108-165s aromatic nonsubstituted and O-substituted C-atoms (CAr), and 165-220sC atoms of carboxylic, esteric, and ketonic groups (CCdO). Chemical binding of PAH to dissolved humic materials was characterized by partition coefficients determined by a fluorescence quenching technique as described elsewhere (29). The details of the experimental design are given in ref 26. Shortly, the PAH solutions below the water solubility limit (0.6 в 10-7, 1 в 10-7, and 5 в 10-7 M for pyrene, fluoranthene, and anthracene, respectively) were prepared by solubilization technique. The concentration of HS was in the range of (0.2-6) в 10-6 kg C/L. The slopes of the obtained Stern-Volmer plots yielded the Kocfq valuesspartition coefficients, characterizing binding affinity of HS for PAH. The obtained Kocfq values for the target humic materials are summarized in Table 2. Acute toxicity tests were performed according to the procedure described in refs 30-32. Crustacean Daphnia magna was used as a test organism, its feeding activity (averaged filtration rate per dapnid, mL/h)sas a target response. D. magna were obtained from a stock maintained at the Division of Hydrobiology of Lomonosov

Moscow State University. Daphnids were cultured at 20 °C with a light:dark rhythm of 16 h:8 h in the tap water which was previously filtered through activated charcoal and stored for 2 weeks under room conditions. Suspension of green microalgae Chlorella vulgaris was used to feed D. magna daily. For the toxicity tests, 5-6 day old animals were used. Five to six daphnids were transferred into a 50 mL glass beaker containing 25 mL of the test solution. Three replicates were made for each assay. Water pH, checked at the beginning and the end of the test, was 7.5 ( 0.2 in all assays and the temperature 20 ( 2 °C. Daphnids in test solutions were kept for 24 h without feeding. The previous investigations (30) showed that 1 day starving increases the filtration rate of daphnids and improves its reproducibility (sr ) 15-20%, n ) 3). The animals were fed with a culture of algae C. vulgaris that was added into the test solution at the concentration of about 105 cells/L. The beakers were gently shaken in the middle and at the end of the feeding period to prevent the settling of algae. Detoxification tests included measurements of the feeding activity of daphnids in the presence of the working concentration of PAH and varying concentration of HS. The working solutions of PAH were prepared at the maximum achievable concentrations of, nominally, 5 в 10-7,7 в 10-7, and 1.7 в 10-7 M for pyrene, fluoranthene, and anthracene, respectively. Concentration of HS varied in the range from 1.5 till 25 mg C/L. Each experimental series included the following treatments: control-water, PAH solution at the working concentration, HS solutions at five different concentrations, solutions of PAH+HS at the working concentraVOL. 35, NO. 19, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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tion of PAH, and five above concentrations of HS. Three replicates were made for each assay. Data Treatment. The feeding activity (R) was determined by a decrease in the concentration of algae grazed out by daphnids. It was calculated via a ratio of the fluorescence intensity (F) of algal suspension at the start (F0) and end point (Ft) of the feeding period. The latter was set at 1 h for all the tests. The F values were corrected for the background fluorescence of the components of the test solutions (Fbg). It was measured on each test solution before adding the algae. The ratio of the corrected F0 and Ft values was further corrected on the change in the cell concentration due to the growth of algae during the feeding period. For this purpose the control was prepared at the same initial concentration of algae which was kept over the whole feeding period without daphnids. The fluorescence intensities of this solution measured at the start and end point of the feeding period were used for calculation of the correction coefficient b. Given the exponential character of cell concentration change due both to grazing out by daphnids and growth of algae (3032), the feeding activity was calculated as follows

Quantification of the Detoxifying Properties of Humic Materials. Toxicity of the PAH (TPAH) was estimated as a relative decrease in the feeding activity of daphnids in the presence of PAH

T

PAH

)

R0 - RPAH R0

(2)

where R0 and RPAH are the feeding activity of D. magna in control-water and in the presence of PAH, respectively. In the concentration range of (1-5) в 10-7, (0.5-7) в 10-7, and (0.2-1.7) в 10-7 M used in the toxicity tests with pyrene, fluoranthene, and anthracene, respectively, the corresponding dose-response relationship could be fit satisfactorily by the following linear model

TPAH ) k в C

PAH

(3)

R)

F0 - Fbg V в ln b в nt Ft - Fbg

(

)

(1)

where V is the volume of the test solution, mL; t is the duration of the feeding period, h; n is the number of species of D. magna; Fbg is the fluorescence intensity of the test solution without algae; F0 and Ft are the fluorescence intensity of the test solution with the algae grazed out by the daphnids at the starting and end point of the feeding period, respectively; b ) F t - Fbg/F - Fbg is the correction coefficient on the 0 change in fluorescence intensity during the feeding period due to the growth of algae, where F and F is the 0 t fluorescence intensity of the algal suspension in the controlwater without daphnids at the starting and end point of the feeding period, respectively. The fluorescence intensity of algal chlorophyll was measured at 680 nm using the excitation wavelength of 450 nm. Given that the maximum of fluorescence spectra of the PAH used and HS lay in the UV and short visible region (350-450 nm), this resulted in the low magnitude of Fbg fluorescence at all the concentrations of the PAH and HS tested. As a rule, it did not exceed a small percentage of F0 and Ft values. Of particular importance is that the inner filter effect (of HS at high concentrations, in particular), which can weaken the fluorescence intensity of algae, did not interfere with the measurements on feeding activity of daphnids. The latter is calculated as a ratio of the fluorescence intensities (F0 and Ft); both of those were measured at the same concentration of the light absorbing compound in the solution. Hence, the attenuation factor is the same for both fluorescent measurements, and their ratio reflects a ratio of the fluorophor concentration in the corresponding solutions. To check if the presence of HS or PAH in the test-solution influences the photosynthesis or growth of algae, the fluorescence intensity on the corresponding solutions and control-water with the added algae, but without daphnids (F), was measured at t ) 0 and 1 h. The ratios (F - Fbg/F i 0 - Fbg)HS and (F - Fbg/F - Fbg)PAH were compared to these t 0 of the control-water (correction coefficient b). There was no statistically significant differences (P ) 95%) found between the ratios calculated for the solutions of the model PAH, HS at different concentrations and control-water. The obtained values varied in the range of 1.05-1.1 (SD ) 0.1 at n ) 3). A lack of the direct impact of HS of different origin on the photosynthetic activity of C. vulgaris was also shown in our previous studies (33). This allowed to determine the correction factor b on the control-solution once for each batch of algae.
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where k is the slope of the dose-response relationship in the tested range of CPAH. The slope k (mean ( SD, n ) 5) was (0.94 ( 0.05) в 108, (0.64 ( 0.03) в 108, and (3.3 ( 0.2) в 108 M-1 for pyrene, fluoranthene, and anthracene, respectively. The corresponding r2 were 0.96, 0.97, and 0.97. Due to physiological activity of HS, the reduction in toxicity of PAH in the presence of HS reflects a combined action of two effectssthe sequestration of toxicity caused by a reduction in the freely dissolved PAH due to their binding to HS and the direct effect of HS on the test organism. To estimate a sequestration of toxicity, or detoxification effect, the toxicity in the presence of HS (TPAH+HS) was related to the response of D. magna in the presence of HS alone instead of the HSand PAH-free control. The corresponding expression is

TPAH

+HS

)

RHS - RPAH RHS

+HS

(4)

where RHS is the feeding activity of D. magna in the test solution containing HS alone and RPAH+HS is the feeding activity of D. magna in the test solution containing HS and PAH. Then, the sequestration of the initial toxicity, or detoxification effect, D can be calculated as follows:

D)

TPAH - TPAH TPAH

+HS

(5)

Taking into consideration the linearity of the doseresponse relationships obtained for the model PAH in the range of concentrations studied (eq 3), the above expression for D can be rewritten as

D)1-R

(6)

where R ) [PAH]/CPAH is the portion of the freely dissolved PAH in the presence of HS and [PAH] is the equilibrium concentration of the freely dissolved PAH. Consequently, the dependence of D on total concentration of HS (CHS) in the test system ("detoxification curve") can be described via partition coefficient or detoxification constant KocD similarly to chemical binding:

D)

KD в C oc 1+ KD oc

HS HS

вC

(7)

In practice, the KocD values were calculated by fitting the experimental D versus CHS relationships with a use of the nonlinear regression procedure.

Results and Discussion
Detoxification Partition Coefficients. The typical dependencies of the toxicity of PAH upon the concentration of HS

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FIGURE 1. model PAH HS present ( SD (n )

The typical relationships of the toxicity of the three determined according to eq 4 versus concentration of in the test system (on the example of T6). Bars represent 3): в pyrene, 0 fluoranthene, and 2 anthracene.

FIGURE 3. The typical relationships of the detoxification coefficient D versus concentration of HS in the test system (on the example of T6) for the three model PAH: в pyrene, 0 fluoranthene, and 2 anthracene. Bars represent ( SD (n ) 3). who reported the beneficial effect of Aldrich HA on D. magna up to 10 mg C/L, whereas 22 mg C/L dropped the beneficial effect up to 0. The obtained data allowed for one to account for the direct effect of HS on the test organism while estimating the true sequestration of toxicity of PAH as a result of their chemical binding to HS. For this purpose the detoxification effect D was calculated according to eq 5. The D values plotted against the concentrations of HS yielded the detoxification curves of the PAH. The typical examples for pyrene, fluoranthene, and anthracene are given in Figure 3. By the solid lines the best fits are shown corresponding to the KocD values calculated by eq 7. The determined KocD values and r2 of the corresponding fits are summarized in Table 3. The comparable magnitudes of KocD values were obtained for pyrene and fluoranthene; the lower ones were observed 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) (34). The similar trends have been previously reported for the bioconcentration of the PAH in the presence of HS as well (2, 7, 9, 12). Based on the determined KocD values, the target humic materials can be arranged in the following descending order: coal HA, 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 the bioaccumulation of PAH (8, 35, 36) as well as with the trend in Kocfq values obtained by the fluorescence quenching technique (Table 2) for the same humic materials. However, in general, the magnitude of KocD was a factor of 2-6-fold higher than that of Kocfq for the same humic material. The scatter plots of KocD versus Kocfq are given in Figure 4. The use of t-statistics for a comparison of the two means under study is feasible because of a significant difference in the absolute values of their standard deviations (the average ratio accounted for 20) (37). The plots yielded rather strong positive correlation (r2 ) 0.74, 0.72, and 0.58 for pyrene, fluoranthene, and anthracene, respectively). The intercepts of the regression lines were equal to zero and the slopesssignificantly different from one (mean ( CI, P ) 95%, n ) 26):

FIGURE 2. The typical relationships of the feeding activity of D. magna versus concentration of HS of different origin. Bars represent ( SD (n ) 3); (a) peat HA+FA: [ T7, 9 TTL, 2 T6, O T5, в T1, ] T4, + HTL; (b) soil HA: [ HS, 9 HBP, 2 HST, O HGP, 4 HBG, ] HBW, + HGW; (c) soil FA: [ FGW, 9 FST, 2 FBW, O FBP, 0 FBG; (d) aquatic HA+FA, [ WM3X, 9 SWA, O FMX, 2 FMC; and (e) various origin HS: [ AGK, 9 AHA, O HTW, 2 SEL. present in the system are given in Figure 1. The toxicity decreases with an increase in a concentration of HS. This effect has been observed for all the humic materials used, except for soil FA that did not cause any influence on the toxicity of PAH. The relationships of the feeding activity of D. magna versus concentration of HS are given in Figure 2a-e for all the target humic materials grouped by the origin. In general, HS in the range of concentration from 1.5 until 6 mg C/L caused a predominantly stimulating effect on D. magna. The biggest stimulating effect (up to 140% of control) was observed for peat HA+FA (Figure 2a). Soil HA and FA (Figures 2b,c) caused a much less stimulating effect (up to 120% of control). Aquatic HA+FA showed quite diverse effects (Figure 2d). While XADisolates (FMX and WM3X) caused the stimulation effect of 120%, DEAE-cellulose isolate (FMC) did not cause any effect. No stimulating effect was displayed by commercial coal HA (AGK), while Aldrich HA stimulated D. magna up to concentration of 6 mg C/L. The further increase in concentration of Aldrich HA lead to a decrease in the feeding activity of D. magna until control (Figure 2e). The obtained results corroborate well the findings of Petersson and Persson (18)

pyrene: K

D oc

) (2.6 ( 0.3) в K

fq oc fq oc fq oc

(8) (9) (10)

fluoranthene: KocD ) (4.6 ( 0.6) в K anthracene: KocD ) (6.0 ( 1.4) в K

Thus, Kocfq accounts for about 74, 72, and 58% of the variability in the KocD of the dissolved humic materials to pyrene, fluoranthene, and anthracene, respectively. However, the slopes of the regression lines indicate a constant bias
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TABLE 3. Detoxification Constants of Humic Substances Used in This Study for the Three PAH Determined from Toxicological Data, KocD в 10-5 L/kg C
sample FMX FMC WM3X SWA T1 T4 T5 T6 T7 HTL TTL HTW HBW HBP HBG HGW HGP HS HST FBW FBP FBG FGW FST SEL AHA AGK
a

source of HS River Moscow 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, 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 gray wooded soil, forest typical chernozem typical chernozem Aldrich humic acid coal humic acid

pyrene Aquatic Humic 2.8 ( 1.1 ( 1.9 ( 1.9 ( Peat Humic 3.9 3.7 3.7 3.7 4.3 3.6 4.2 0.8 Substances (HA+FA) 1.1a (0.91b) 0.3 (0.91) 0.5 (0.93) 0.7 (0.98)

fluoranthene 2.3 0.4 2.5 2( 3.4 4.7 3.4 3.0 5.9 3.9 4.0 0.3 4.5 3.8 2.1 3.8 4.7 8.6 6.0 ( 0.5 (0.96) ( 0.1 (0.96) ( 0.5 (0.88) 0.6 (0.91) ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( 1.2 (0.89) 1 (0.88) 2.5 (0.98) 0.8 (0.91) 0.3 (0.96) 0.8 (0.92) 1 (0.96) 0.1 (0.97) 1.3 1.5 0.2 2.2 0.8 4.8 3.5 (0.95) (0.86) (0.93) (0.88) (0.90) (0.95) (0.91)

anthracene <0.3 <0.3 <0.3 <0.3 nd 1.2 1.0 1.3 nd 1.8 0.3 <0.3 5.9 3.9 1.9 3.9 3.8 5.6 5.6 <0.3 <0.3 <0.3 <0.3 <0.3 2.0 ( 0.5 (0.98) 5.1 ( 1.3 (0.91) nd
c

Substances (HA+FA) ( 1.1 (0.86) ( 0.6 (0.81) ( 0.8 (0.91) ( 0.6 (0.96) ( 2.6 (0.96) ( 1.7 (0.98) ( 2.7 (0.97) ( 0.2 (0.95)

( 0.5 (0.88) ( 0.2 (0.91) ( 0.6 (0.86) ( 0.5 (0.87) ( 0.1 (0.96)

Soil Humic Acids (HA) 3.9 ( 1.4 (0.95) 3.3 ( 0.8 (0.92) 2.1 ( 0.5 (0.90) 3.6 ( 1.3 (0.91) 4.1 ( 0.8 (0.96) 6.0 ( 3.1 (0.96) 5.1 ( 1.6 (0.86) Soil Fulvic Acids (FA) <0.3 <0.3 <0.3 <0.3 3.0 ( 0.8 (0.89) Soil Humic Substances (HA+FA) 3.6 ( 0.6 (0.91) Commercial Preparations 5.8 ( 3.4 (0.84) 5.5 ( 0.6 (0.97)

( ( ( ( ( ( (

2.4 1.3 0.6 1.1 1.2 2.4 2.5

(0.91) (0.95) (0.86) (0.88) (0.93) (0.88) (0.88)

<0.3 <0.3 <0.3 <0.3 2.2 ( 0.6 (0.94) 3.4 ( 0.4 (0.93) 5.8 ( 2.8 (0.94) 6.7 ( 0.8 (0.87)

( SD.

b


r2 ) 1 i)1 n

n

(Di - Di/)

2


i)1

(Di - D) h

2

where r2 is the portion of explained variance, Di is the experimental value, Di/ is the calculated value, D is the mean experimental value, and n is a number of experimental points. c <0.3 в 105 corresponds to the minimum detectable KocD value estimated as a 3-fold standard deviation of the lowest determined KocD value (37). The latter was (0.3 ( 0.1) в 105 L/kg C ((SD, n ) 3) for fluoranthene and HTW. This yields a value of KocD(min) of 0.3 в 105 L/kg C.

FIGURE 4. The partition coefficients determined from the toxicity data (KocD) are directly proportional to those determined by fluorescence quenching technique (Kocfq). The constant bias between the values apparently results from the different conditions (aqueous chemistry) of their determination: в pyrene, 0 fluoranthene, and 2 anthracene. between the partition coefficients estimated by the two techniques used.
3846
9

The observed bias between the Kocfq and KocD values, both of those are the conditional constants, can be prescribed to a systematic error resulting from the differences in conditions of their determination. The most probable reason is that the fluorescence quenching determinations were conducted on the distilled water, whereas the toxicity testsson the filtered tap water. To test this hypothesis, few determinations of the Kocfq were conducted on the tap water on the example of anthracene and T6. The obtained data yielded a mean value of Kocfq of 0.8 в 105 L/kg C. It is a factor of approximately 3-4-fold higher than 0.25 в 105 L/kg C determined in the distilled water and is much closer to the mean value of KocD of 1.3 в 105 L/kg C. The distilled water differs from the tap water in pH (5.5 and 7, respectively) and in the ionic strength (I ) 0 and 4 mM, respectively). Our previous investigations on the influence of pH and ionic strength on the Kocfq values (38) conducted for the same model PAH showed that the variations in pH from 5 till 8 did not effect substantially the Kocfq value. The remarkable increase in Kocfq (of a factor of 2-3-fold) was observed only at pH 2. At the same time, a reduction in Kocfq value of a factor of 2-3-fold was detected along with an

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 19, 2001


FIGURE 5. Correlation between the KocD values and aromaticity of humic materials expressed as CAr/CAlk: в pyrene, 0 fluoranthene, and 2 anthracene. increase in ionic strength up to 10-2 M. This suggests that neither differences in pH nor ionic strength itself could cause the observed bias between the Kocfq and KocD. However, the ionic strength in the tap water is provided mostly by the divalent metal ionssCa2+ and Mg2+, while in our experiments (38) NaCl was used for this purpose. According to the findings of Schlautman and Morgan (39), the presence of 1 mM Ca2+ at 0.1 M total ionic strength and pH 7 caused a substantial increase (30%) in the Kocfq values of pyrene and anthracene. This can explain the higher values of Kocfq and KocD obtained on the tap water in comparison with the Kocfq determined on the distilled water. Hence, the detoxification effect described by the KocD partition coefficient is provided mostly by the chemical binding of PAH to HS. To facilitate the predictive estimates of the detoxifying properties of HS in relation to PAH, the relationships between the structure and detoxification partition coefficient were established. Relationships between Structure and Detoxifying Properties of HS to PAH. The quantitative relationships between the structure of HS and detoxification partition coefficients KocD were derived using the molecular descriptors of the target humic materials given in Table 1. The ratio of aromatic to aliphatic carbon content reflecting the prevalence of aromatic core over aliphatic periphery or lipophilic-lipophobic balance of the molecule (indicated by the CAr/CAlk ratio) was also used for correlation with KocD values. Its use resulted in the best fits to the experimental KocD values shown in Figure 5. The corresponding regression equations are given below:

thene, and anthracene, respectively. Much poorer correlation was observed for another indirect estimate of the aromaticitysmolar absorptivity (ABS280). No correlation was found with the peak molecular weight of humic materials (r2 ) 0.011, 0.012, and 0.001). Environmental Implications. The obtained structureproperty relationships point out that the aromatics enriched humic materials are the most efficient detoxifying agents in relation to PAH. The most abundant source of such material is brown coal which contains up to 70-80% of humic acids. The humates isolated from brown coal can be widely used for the remediation of the PAH contaminated media. Given that the governing mechanism of interaction between HS and PAH is hydrophobic binding, the similar action of HS on the other hydrophobic organic compounds (petroleum or polychlorinated hydrocarbons) can be expected. The advantage of humates application as detoxifying agents for the remediation purposes is a lack of danger of the secondary pollution of the contaminated sites. Another advantage is a combinatory action of humates, that is, they not only expose a detoxifying impact on the hydrophobic contaminants but also can impose a direct beneficial effect on the biotic community at the contaminated site. The quantification approach to detoxifying properties of humic material and the obtained structure-property relationships facilitate an effective use of humates as detoxifying agents toward PAH as well as to the other contaminants.

Acknowledgments
We extend our deepest appreciation to Dr. A. V. Garmash (Department of Chemistry, Lomonosov MSU, Russia) for very useful discussions on the statistics used in the manuscript. The constructuve criticism of the anonymous reviewers is greatly acknowledged. This research was financially supported by the grant of INTAS 97-1129.

Literature Cited
(1) Neff, J. M. Polycyclic Aromatic Hydrocarbons in the Aquatic Environment; Applied Science Publishers Ltd: London, U.K., 1979. (2) McCarthy, J. F.; Jimenez, B. D. Environ. Toxicol. Chem. 1985, 4, 511-521. (3) Kukkonen, J.; McCarthy, J. F.; Oikari, A. Arch. Environ. Contam. Toxicol., 1990, 19, 551-557. (4) Kukkonen, J.; Oikari, A. Sci. Total Environ. 1987, 62, 399-402. (5) Oikari, A.; Kukkonen, J. Bull. Environ. Contam. Toxicol. 1990, 45, 54-61. (6) Leversee, G. J.; Landrum, J. P.; Fannin, T. Can. J. Fish. Aquat. Sci. 1983, 40, 63-69. (7) Haitzer, M.; Hoess, S.; Transpurger, W.; Steinberg, C. Environ. Toxicol. Chem. 1999, in press. (8) Landrum, P. F.; Reinhold, M. D.; Nihart, S. R.; Eadie, B. J. Environ. Toxicol. Chem. 1985, 6, 11-20. (9) Landrum, P. F.; Sheila, R. N.; Eadie, B. J.; Herche, L. R. Environ. Toxicol. Chem. 1987, 6, 11-20. (10) Black, M. C.; McCarthy, J. F. Environ. Toxicol. Chem. 1988, 7, 593-600. (11) McCarthy, J. F.; Jimenez, B. D.; Barbee, T. Aquat. Toxicol. 1985, 7, 15-24. (12) Kukkonen, J.; Oikari, A. Water Res. 1991, 25, 455-463. (13) Perminova, I. V.; Kovalevsky, D. V.; Yashchenko, N. Yu.; Danchenko, N. N.; Kudryavtsev, A. V.; et al. In Humic Substances and Organic Matter in Soil and Water Environments: Characterization, Transformations and Interactions; Clapp C. E., Hayes M. H. B., Eds.; IHSS, Inc.: St. Paul, MN, 1996; pp 398406. (14) Oris, J. T.; Hall, A. T.; Tylka, J. D. Environ. Toxicol. Chem. 1990, 9, 575-583. (15) Batalkin, G. A.; Galushko, A. M.; Makhno, L. Yu.; Khristeva, L. A. In Proceedings of the IVth International Symposium on "Peat: Properties and Perspectives of Application"; Minsk, USSR, 1982; pp 115-117 (in Russian). (16) Visser, S. A. In Humic Substances. Effects on Soil and Plants; Visser S. A., Ed., REDA: Rome, 1986; pp 89-135.
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pyrene K

D oc

в 10

-5

) (2.2 ( 0.3) в CAr/C
-5

Alk

+

(1.0 ( 0.4) r2 ) 0.76 (11) fluoranthene KocD в 10 ) (3.2 ( 0.4) в CAr/C
Alk

-

(0.01 ( 0.6) r2 ) 0.75 (12) anthracene KocD в 10
-5

) (3.5 ( 0.6) в CAr/C

Alk

-

(2.0 ( 0.8) r2 ) 0.7 (13)
The values of given confidence intervals of the slopes (n ) 19 for pyrene and fluoranthene and n ) 17 for anthracene, P ) 95%) demonstrate a statistical relevance to the observed trend between KocD value and CAr/CAlk ratio. The same is true for the other parameters of aromaticity of HS s CAr. This can indicate a key role of interactions between aromatic core of HS and PAH for detoxification effect under study. These results are consistent with our previous findings (26) and reported in the literature (8, 12) correlating the binding affinity of PAH for HS (Koc values) with the percentage of aromatic carbon in the humic material. Correlations between KocD and atomic H/C ratio (Table 1) is poorer than those for 13C NMR descriptors. However, they remain significant at P ) 95%. The corresponding r2 values account for 0.56, 0.49, and 0.67 for pyrene, fluoran-

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(17) Hargeby, A.; Petersen, R. C. Fresh. Biol. 1988, 19, 235-247. (18) Petersen, R. C.; Persson, U. Sci. Total Environ. 1987, 62, 387398. (19) Petersen, R. C. In Humic Substances in the Aquatic and Terrestrial Environment; Allard, B., Ed.; Springer-Verlag: Berlin, Heidelberg, 1991; pp 369-389. (20) Tulonen, T.; Salonen, K.; Arvola, L. Hydrobiol. 1992, 229, 239252. (21) Hashimoto, Y.; Tokura, K.; Ozaki, K.; Strachan, W. M. J. Chemosphere 1982, 11, 991-1001. (22) Mantoura, R. F. C.; Riley, J. P. Anal. Chim. Acta 1975, 76, 97106. (23) Lowe, L. E. Sci. Total Environ. 1992, 113, 133-145. (24) Orlov, D. S.; Grishina, L. A. Handbook of humus chemistry; Moscow State University Publisher: Moscow, 1981 (in Russian). (25) Sugimura, Y.; Suzuki, Y. Mar. Chem. 1988, 24, 105-131. (26) Perminova, I. V.; Grechishcheva, N. Yu.; Petrosyan, V. S. Environ. Sci. Technol. 1999, 33, 3781-3787. (27) Perminova, I. V.; Frimmel, F. H.; Kovalevskii, D. V.; Abbt-Braun, G.; Kudryavtsev, A. V.; Hesse, S. Water Res. 1998, 32, 872-881. (28) Kovalevskii, D. V.; Permin, A. B.; Perminova, I. V.; Petrosyan, V. S. Moscow State University Bulletin (Vestnik MGU) Series 2 (Chemistry) 2000, 41, 39-42. (29) Gauthier, T. D.; Shane, E. C.; Guerin, W. F. Environ. Sci. Technol. 1986, 20, 1162-1166. (30) Matorin, D. N.; Vavilin, D. V.; Venediktov, P. S. Biol. Sci. (USSR) 1990, 1, 146-151 (in Russian). (31) Matorin, D. N.; Vavilin, D. V.; Popov, I. V.; Venediktov, P. S. In Methods of Biotesting of Aquatic Media Quality; Filenko O. F.,

(32) (33)

(34)

(35) (36)

(37) (38)

(39)

Ed.; Moscow State University Publisher: Moscow, 1989; pp 1021 (in Russian). Polynov, V. A. Ph.D. Thesis. Lomonosov Moscow State University, 1992; Moscow (in Russian). Perminova, I. V.; Kulikova, N. A.; Lebedeva, G. F. In Proceedings of the 10th Meeting of IHSS, 24-28 July, Toulouse, France; 2000; pp 112-115. Hansch, C.; Leo A.; Hoekman, D. Exploring QSAR Hydrophobic, Electronic, and Steric Constants; American Chemical Society: Washington, DC, 1995. Kukkonen, J.; Oikari, A.; Johnsen, S.; Gjessing, E. T. Sci. Total Environ. 1989, 79, 197-207. McCarthy, J. F. In Aquatic Humic Substances: Influence on Fate and Treatment of Pollutants; Suffet I. H., MacCarthy P., Eds.; American Chemical Society: Washington, DC, 1989; pp 263277. Doerffel, K. Statistik in der analytischen Chemie; Deutscher Verlag fuer Grundstoffindustrie GmbH: Leipzig, Germany, 1990. Yashchenko, N. Yu.; Perminova, I. V.; Petrosyan, V. S.; Filippova, E. M.; Fadeev, V. V. Moscow State University Bulletin (Vestnik MGU), Series 2 (Chemistry) 1999, 40, 188-193. Schlautman, M. A.; Morgan, J. J. Environ. Sci. Technol. 1993, 27, 961-969.

Received for review September 25, 2000. Revised manuscript received April 25, 2001. Accepted May 14, 2001. ES001699B

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