Evaluation of a new, effective method to extract polycyclic aromatic hydrocarbons from soil samples

Chemosphere, Vol. 28, No. 4, pp. 683-692, 1994

Pergamon

Copyright © 1994 Elsevier Science Lid Printed in Great Britain. All rights reserved 0045-6535/94 $6.00+0.00

0045-6535(94)E0002-B

EVALUATION OF A NEW, EFFECTIVE METHOD TO EXTRACT POLYCYCLIC AROMATIC HYDROCARBONS FROM SOIL SAMPLES A. Eschenbach 1, M. Kistner 1, R. Bierl 2, G. Schaefer 1 and B. Mahro I* 1 Technische Universit~tHamburg-Harburg, Biotechnologie2, Denickestr.15, 21073 Hamburg, Germany 2 Universi~itTrier, FBIV Geowissenschafien,Abt. Hydrologie, 54286 Trier, Germany * corresponding author (Receivedin Germany29 September1993; accepted4 November1993) Abstract: A new and efficient protocol for the extraction of polycyclic aromatic hydrocarbons (PAH) from soil samples is described. The method is based on two successive steps, including an organic solvent extraction and a methanolic hydrolysis (saponification) of the soil sample. By use of the new technique the microbial degradation of PAH in soil batch cultures was monitored. The application of the method helps to recover significant higher amounts of PAH from soil than it was possible with an organic solvent extraction only. An explanation for the observed effect could be that PAH molecules which got trapped in the humic polymer are released due to the cleavage of ester bonds which form parts of humic macromolecules. Introduction Many industrialized countries have large areas of soil contaminated by unreflected use or disposal of manmade chemicals. Soil remediation became therefore a task of high priority. One group of environmental pollutants which requires particular consideration in that respect is the group of polycyclic aromatic hydrocarbons (PAH). Several of these compounds are considered as genotoxic (Yang & Siiverman 1988) and were therefore included in the EPA li~t of environmental priority pollutants. One promising way to eliminate the genotoxic potential of PAH is to induce microbial activities in soil which may lead to the complete degradation or transformation of these compounds (Sims and Overcash 1983; Mueller et al. 1989; Mahro & Kastner 1993). It has been shown repeatedly that these processes may contribute to a significant extent to the PAH depletion in soil (Hosler et al. 1988; Van Afferden et al. 1991; Weissenfels et al. 1990). The success of such a microbial clean up can be measured by HPLC or GC analysis of organic soil sample extracts which are usually obtained by Soxhlet or ultrasonic treatment (Blankenhorn 1990). If the concentration of the PAH compounds decreases with time, the remediation is generally considered successful. However, one problem that is frequently overlooked in practice is that parts of the PAH contamination might be bound to the soil matrix so strongly that they are not extractable any longer by conventional organic solvent extraction. Experiments which were carried out with radiolabelled PAH confirm that a large fraction of the xenobiotic substances can stick to the soil as non-extractable, bound residues (Park et al. 1988; Mahro & Kastner 1993; Qiu & McFarland 1991). One way to improve the reliability of the PAH determination would be, therefore, to design more rigid protocols for the extraction of PAH from soil. Grimmer et al. (1975) have shown that the extraction of PAH from food with high protein or lipid content could be optimized significantly by application of a methanolic saponification procedure. Based on this observation we designed a new extraction protocol for the PAH analysis in soil samples which includes a combination of both, a solvent extraction and a methanolic hydrolysis. This paper evaluates the use of the new technique by monitoring the microbial degradation of PAH in soil batch cultures. 683

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Material and methods

Soil characteristics The experiments were carried out with two different soils. Soil A, material from the Ap-horizont of a gleyic Luvisol, was collected near Trier. The second soil (soil B) is material from a AhAp-horizont of a Luvisol near Hamburg. Some physical and chemical characteristics of the soils are given in Tab. 1. Betbre use, the soils were homogenized by sieving at 2ram mesh size. Tab. 1: Characterization of the soils Soil A

Soil B

texture

loamy silt

loamy sand

clay

16.4%

6.3%

organic carbon

0.85%

1.10%

pH

6.5

4.5

water holding capacity

36.95%

31.06%

Chemicals Solvents and other chemicals were obtained from Merck (Darmstadt, FRG) at the highest purity grade available The SPE-columns which were used in the course of the experiments (type Cyano(CN)-column (1000mg) and Octadecyl(C18)-column (1000mg)) were purchased from J.T. Baker (Grol3-Gerau, FRG) Set up of soil batch cultures The extraction method was evaluated with two different types of soil batch cultures. The first series of experiments were carried out with soil A (Tab. 1) being contaminated at a final PAH concentration of 5 and 100mg of fluoranthene or with 1 and 10mg of benzo(a)pyrene per kg of dry soil, respectively. The depletion kinetics were investigated for each compound and concentration with five parallel batch cultures. For each parallel experiment 40g of dry soil were contaminated with 03ml PAil-acetone solution. The soil was incubated for two months in closed 500ml glass vessels at 20°C. The moisture content of the soil was adjusted at 55-60% of the maximum water holding capacity. The second series of soil batch experiments were carried out with a mixture of different PAH compounds (naphthalene, fluoranthene, phenanthrene, anthracene and pyrene) and with soil type B (Tab. 1). The single PAH were dissolved in acetone, mixed and dripped into 100 g of soil. The final concentrations of the individual PAH werel00mg/kg of soil for naphthalene and 25mg/kg of soil (dry weight) for each of the other PAH. Three parallel soil batch cultures were incubated for 25 days in 11 glass incubation vessels at 20°C The moisture content was adjusted to 60% of the water holding capacity.

Extraction and analysis of PAH from soil samples This section will describe the basic extraction protocol as it was applied in the experiments with soil A The handling of this extraction protocol has been simplified significantly in the course of our experiments. The major modifications of the optimized protocol will be described in the results section. Soil extraction with organic solvent: Before extraction the soil samples were freeze-dried and homogenized by crushing. The first step of the extraction was carried out with acetone. Preliminary examinations of different organic solvents had shown that the extraction efficiency and reproducibility was better with acetone than with

685

toluene or dichlormethane. Between 0.5 and I g of the homogenized soil was filled in a glass centrifugation-tube (23ml volume) and mixed with 20ml acetone. The samples were shaken once by hand and then extracted by ultrasonic treatment (ultrasonic bath: Sonorex RK 106S, Bandolin; Berlin FRG) for 30rain. Soil and solvent mixture was separated by centrifugation (3000 rpm for 15rain; GPKR Centrifuge, Beckmann Instruments; Mtinchen FRG). The organic supernatant was removed with a pipette. The extraction procedure was repeated two more times, but the third extraction was carried out with 10 ml acetone only. The three organic extracts were combined and evaporated to nearly dryness by rotary evaporation (RF 121 Rotavapor with vaccum/destillation controller, B~ichi; Goppingen FRG). The dried soil extracts were redissolved in lml hexane. Soil extraction with alkaline methanol (saponification): After the organic solvent extraction the soil sample pellet was transferred into a 100ml-retort and mixed with 80ml methanolic potassium leach (2N KOH : methanol, 1:4 (v:v)). The mixture was heated for 5 hours at a temperature slightly above the boiling point of the methanolic potassium lye. The solvent was recirculated by using a condenser. The resulting soil-liquid mixture was subsequently extracted twice by liquid/liquid partitioning with 30ml hexane in a glass separation funnel (150ml volume). These hexane extracts were concentrated by rotary evaporation down to a volume of 1ml. Purification of extracts: The extracts from both steps, the organic solvent-extraction and the saponification were purified with a prechromatography system consisting of two coupled separation columns (first the CNcolumn and second the C 18-column). The hexane extracts were loaded on the top of the CN-column and the columns were rinsed with 2ml of hexane by vaccum suction subsequently the residual PAH-fraction was eluated from the columns with 2ml toluene. The toluene eluat was carefully evaporated under a N2-gas-stream to nearly dryness and was redissolved with 1ml methanol. HPLC-analysis: The purified extracts were analyzed by high percussion liquid chromatography (HPLC). The separation-system consisted of a Gynkotek gradient former (type 250B), a high pressure pump (Gynkotek 300B) and a RP 18 column. The signal detection was obtained by use of a Gynkotek UV/VIS spectrophotometer SP-4 at 254 nm (Gynkotek;Braunschweig FRG). An isocratic elution was carried out using a mixture of methanol and water (90:10 (v:v), 1ml/min). The second part of the experiments were carried out with a HPLC-system from Pharmacia LKB (Freiburg, FRG) (Pump 2248, Autosampler 2157, column oven 2155, UV-Detector VWM 2141). At this system a linear elution gradient of methanol/water (70:30(v:v) and methanol (70-100% from 8rain to 15rain, fow: lml/min) was applied.

Results

Determination of PAH-recovery in soil batch experiments The major idea of our new extraction method was to include an additional saponification step into the extraction protocol to improve the extraction efficiency for PAH from soil samples. For method evaluation we determined the PAll-recovery rates. The experiment was carried out with freshly contaminated soil. The soil (type A) was contaminated with fluoranthene or benzo(a)pyrene, each at a low and high concentration (Tab. 2). Five replicates were investigated for each compound and each concentration. The subsequent extraction was repeated for each parallel experiment three times. The results of the experiment are given in Tab. 2.

686

Tab. 2:

Recovery rates in % and coefficients of variation for two different PAIl after extraction from soil (five replicates for each single experiment and extraction was repeated three times)

fluoranthene

benzo(a)pyrene

5 mg/kg

100 mg/kg

1 mg/kg

10 mg/kg

98.60 +1.91

91.83 :~2.75

78.50 ±3.55

80.37 ±5.89

The data show that the recovery rates varied between 78.5% and 98.6% if the saponification step was included in the protocol. Usually the additional saponification step extracted 20-30% more than the solvent extraction itself These data indicate also that the overall reliability of the chosen ultrasonic extraction was very high as long as the ultrasonic conditions were kept constant (i.e. same amount of water, same field intensity etc.). In another experiment soil of type A contaminated with 100mg fluoranthene per kg dry soil was extracted by a three step extraction procedure. Following the aceton extraction, the solvent extraction was repeated using toluene and subsequently the me~hanolic hydrolysis was applied. Fig. I shows that the aceton extraction could extract 72% of total extractable PAIl, the toluene extraction yields smal amounts of fluoranthene (5%), while the additional hydrolysis extracted further 24% of the total extractable PAH content. This suggested that the saponification allows the extraction of compounds which are not accessible any more to organic solvents.

acetone

toluene

saponification

Fig. 1: Extractable PAH amounts using a three step extraction procedure (1. step: aceton extraction, 2. step toluene extraction, 3. step: saponification) soil A contaminated with 100 mg/kg

The second important question was to check whether the relative amounts of PAH one can extract with the different extraction procedures would change with time or due to microbial activities in the non sterile soil samples. Therefore we incubated the contaminated soil samples (soil A) for two months at 20°C under non sterile conditions to induce microbial degradation. The results of this experiment are summarized in Fig. 2.

687

fluoranthene

benzo(a)pyrene

% 120

%

r

/

j

Coefficient...of

100

80

60

40

20

0

TO T56 TO T56 5 mg/kg 100 mg/kg • solvent extraction [ ] hydrolysis

v

TO T56 TO T56 1 mg/kg 10 mg/kg • solvent extraction [ ] hydrolysis

Fig. 2: Content of fluoranthene and benzo(a)pyrene extractable by the two step extraction procedure after a two months incubation. Data are given as % in relation to the initial concentrations (mean of five replicate soil cultures, three extractions each, coefficients of variation of the sum of solvent extraction and hydrolysis)

The PAH-content being extractable by acetone decreased significantly during the two month incubation period (e.g. up to 40 % for fluoranthene). This result could be observed with both concentrations of fluoranthene and benzo(a)pyrene (Fig. 2) and suggested that the PAH might have undergone (microbial) degradation. The results were completely different if the saponification step was implemented in the extraction protocol. Based on this extraction protocol, the absolute depletion of PAH in soil was much less significant. This time the detectable residual amount of fluoranthene, for example, decreased by only 20% rather than by 40% as with acetone extraction. The absolute amount ofbenzo(a)pyrene in the soil batch experiment with 10mg benzo(a)pyrene/kg of soil could also be recovered completely by methanolic saponification though the acetone extraction after 56 days had indicated a degradation by 30%. Another important observation was the fact that the absolute amount of PAH being extractable by saponification increased with time. An amount of 34% of flouranthene content was extractable by saponification at the beginning of the incubation (TO). This amount had increased to 56% of the total extractable fluoranthene content at the end of the incubation (T56). Similar results were obtained with benzo(a)pyrene The saponifiable benzo(a)pyrene content increased from 29% to 51% of the respective extractable PAIl amount in the experiment with l mg benzo(a)pyrene, anf from 18% to 38% during incubation with 10mg benzo(a)pyrene per kg soil (Fig 2).

688

The data given supported our assumption that the detectable amounts of PAH in soil may differ significantly, depending on the applied extraction method and on the time exposed to microbiall activity. It is obvious that a relevant part of the PAH could only be recovered by the additional saponification-extraction.

Optimization of the basic extractionprotocol A drawback of the developed two-step extraction method was the time consuming different steps of purification, solvent exchange etc. This could become a severe handicap if many samples must be handled in the course of routine analysis programs. Therefore we tried to simplify the proposed protocol at several steps. A summarized scheme of the simplified method is given in Fig. 3.

Fig. 3: Process scheme of the simplified extraction method

[

3g soil

] 3ml ethylacetate

I ultrasonic extraction 30rain

$

I

centrifugation 3000rpm, 15rain soil pellet II

hydrolysis 95-99°C, lh

) HPLC 2800~tl methanol + 200kt I KOH ]

$ centrifugation 3000rpm, 15rain

I

]

> HPLC

The first simplification included the omission of the drying of the soil samples. If ethyhl acetate was used, the extractable amounts of PAH were very similar, no matter whether the soil samples had been dried by sodium sulfate or whether they were used with its natural moisture content (data not shown). The second simplification step was to scale down the volumes of the extraction solvents and to carry out the complete extraction procedure in heat and pressure resistant centrifugation tubes with buthyl rubber septa (Hungate glass tubes; 15 ml volume, Bellco International; Feltham UK). The organic solvent being used during the first extraction step was removed from the tube after centrifugation and quantified volumetrically with a microliter syringe. This extract could be used for HPLC

analysis without further purification. The residual soil pellet was taken for further extractions. The saponification leach (2.8ml methanol + 0.2ml 2N KOH) was added directly to the soil pellet in the Hungate tube and mixed thoroughly. The samples were incubated in a water bath for one hour at 95-99°C, were let cool down and centrifuged at 3000 rpm for 15 min. The supernatant, representing the alkaline soil extract, could be analyzed and quantified directly by the HPLC system. The quantification of the total PAH concentration were done according to equation (1).

689

(Equation 1) C= Cs.Vs+ Ch'(Vh+R)-Cs.R M M C - PAH-content of the soil sample in mg/kg soil dry weight Cs - PAH concentration measured by solvent extraction (me/l) Vs -volume of organic solvent (ml) Ch -PAH concentration measurde by methanolic hydrolyse (mg/i) Vh -Volume of saponification leach (ml) R - Volume of the residual solvent from solvent extraction (ml) M - amount of soil (g dry weight) The equation pays regard to the fact that parts of the PAH in the saponified extract are actually dissolved in the residual trace amounts of the organic solvent being carded over from the first extraction step. An accurate volumetric measurement of the organic solvent taken out after step 1 of the extraction is therefore essential (see term R in equation 1). Another change of the original protocol concerned the solvent being used in step 1. The originally used solvent acetone was replaced now by ethyl acetate. The higher polarity of ethyl acetate should help to extract putative metabolites of the microbial PAH-degradation more efficiently (Pothuluri et al. 1992). It was found in comparative experiments that the extraction of the original PAH compounds was not effected by the solvent change (data not shown). naphthalene mg/kg 120

mg/k!

30

,-=/kO 30 ]

25t

100

25

2O

80

20

15

60

15

10

40

lo

5

20

s

~0

o.

v

0

'

'

'

'

5

:

I

i

i

I

I

I

E

10

I

I

P

I

f

15

~

20

I

I

T~-

25

N

A

PH

F

PAH

days -e-N -*-A -+-PH + F

I

-v-p

P 25

d

• solvent extraction [ ] hydrolysis

A B Fig. 4: PAH disappearance and recovery in soil samples in dependance on the applied extraction protocol. A.: Kinetics of PAH depletion based on a one step extraction with ethyl acetate B: Residual PAl{ concentrations after 25 days of incubation analyzed by solvent extraction or the simplified saponification extraction respectively (N: naphthalene, A: anthracene, PH: phenanthrene, F: fluoranthene, P: pyrene)

690

The simplified extraction protocol was further tested in series of soil batch experiments. Nonsterile air dried soil (soil type B) was artificially contaminated with either naphthalene, anthracene, phenanthrene, fluoranthene or pyrene and incubated for 25 days at 20°C after adjusting the moisture content at 60% of the maximum water holding capacity. The resulting PAH-depletion kinetics are given in Fig. 4. The left part of Fig. 4 documents the disappearence of PAH over the 25 days period as it was measured after organic solvent extraction. At the end of the incubation period, four of five PAH-compounds were detectable only with less than 50%, naphthalene with less than 5% of its original concentration. If the soil was extracted once more by methanolic alkaline hydrolysis we obtained completely different results. Except for naphthalene, which could be recovered only with about 25% probably due to volatile losses, all other PAH were recovered with 80-90% of its original concentration (Fig.4B). The conclusions which would have been drawn from the data obtained by the one step organic extraction would have been therefore completely misleading. These results confirmed our previous assumption that the one step solvent extraction may lead to a significant underestimation of the actual PAH-concentrations in soil.

Discussion It could be shown that the application of a two-step saponification extraction method yields significantly higher PAH-recoveries from soil samples than it was possible with organic solvent extractions only. The given results support experiments carried out in several laboratories with radiolabelled 14C-PAH which had shown that the depletion of PAH over time does not necessarily reflect real degradation of these substances (Hosler et al. 1988; Park et al. 1988; Qui & McFarland 1991). It could be demonstrated here that a PAH depletion might be caused to some extent by a strong but reversible association of the organic xenobiotics to the soil, especially to the organic soil matrix. The mechanisms of the binding or sorption processes of PAH in soil are still poorly understood. It is assumed that both, organic xenobiotics in general and PAH can be sorbed or bound to humic compounds by various mechanisms like van der Waals forces, hydrogen bonds, formation of charge transfer complexes, ionic bonds or covalent binding (Haider et al. 1992; Ziechmann 1980; Botlag et a1.1992; Scheunert et al. 1992; Mahro & Kastner 1993). The xenobiotics may become trapped in molecular sieve or cavity like structures of the organic macromolecules (Chiou 1989; Haider et al. 1992; Scheunert et al. 1992; Bollag & Loll 1983). The major part of these adsorbed or bound molecules cannot be extracted by a simple solvent extraction. One possible explanation for the observed effect of saponification on the PAH extraction could therefore be, that the different types of ester bonds which help to form the macromolecular humic entity get cleaved by the applied alkaline hydrolysis (Parsons 1989). The alkaline hydrolysis might therefore help to release the trapped PAH molecules or other xenobiotics by dissolving the macromolecular structur of the humic acids polymer. As our results with artificially contaminated soils show, the reversible sorption or binding of PAH to the soil matrix is also a time dependent process. The relative portion of PAH which became non-extractable by conventional solvent extraction increased with time. The phenomenon of an increasing sorptive PAH depletion in soil can therefore easily be misinterpreted as (microbial) PAH degradation and should be checked carefully. The described protocol is a powerful tool to extract these stronger bound compounds. However, the sorption or binding which is reversible by alkaline hydrolysis contributes probably only to some extent to the whole binding potential of the soil. Experiments which we carried out with 14C-labelled PAH have shown that the whole amount of irreversibly bound residues may be even larger than the amount of radioactivity

691

which is recoverable by either of both types of soil extraction (Kastner et al. 1993). The binding of PAH remaining reversible by saponification seems therefore to represent only an early stage of a long time process, leading to irreversibly bound components at the end. This hypothesis fits well with our observations, that the amount of PAH which can be recovered by saponification from soil being exposed to PAH for several years ranged only between 10 and 20% (unpulished data) while soil being contaminated with PAH recently led in some cases to a PAH recovery of more than 50%. This indicates that the soils with long term contaminations had reached a kind of sorption equilibrium. This paper presented two different protocols for an alkaline saponification of soil samples. The simplified extraction protocol (Fig. 3) combined the optimized efficiency of the saponifying soil extraction with some other important advantages. Due to the simplified handling of the samples the time for performing extractions is reduced significantly. The scale down of the extraction volumes allows also to reduce the necessary amount of organic solvents and to carry out all extraction steps within the same closable vessels. This minimized the risk of getting into contact with (geno-)toxic compounds substantially. It remains to be shown whether the alkaline hydrolytic extraction can be effectively applied also to other types ofxenobiotics or with other types of soil.

References: Blankenhorn I. (1990): Ein Methodenvergleich zur Analytik der PAK in Feststoffproben. In: Arendt, F., Hinsenveld, M., van den Brink, W.J. (eds.): Altlastensanierung 90. Kluwer Acad. Publ., Dodrecht. 915919 Bollag J.-M., Myers C.J., Minard, R.D. (1992): Biological and chemical interactions of pesticides with soil organic matter.- in: Sci. Total Environ., 123/124, 205-217 Bollag J.-M., Loll, M.J. (1983): Incorporation ofxenobiotics into soil humus.- Experientia, 39, 1221-1231 Chiou C.T. (1989): Theoretical Considerations of the partition uptake of nonionic organic compounds by soil organic matter.-Reactions and movement of organic chemicals in soil (SSSA Spec. Publ. 22, Madison, USA, 1989)Chap. 1, 1-29 Grimmer, G, Hildebrandt, A., B6hnke, H. (1975): Profilanalyse der polycyclischen aromatischen Kohlenwasserstoffe in proteinreichen Nahrungsmitteln, Olen und Fetten (gaschromatographische Bestimmungsmethode).- in: Deutsche Lebensmittel-Rundschau, 3, 93-100; Halder, K., Spiteller, M., Reichert, K., Fild, M (1992): Derivatization of humic compounds: An analytical approach for bound organic residues.- Intern. J. Environ. Anal. Chem, 46, 201-211 Hosler, K R , Bulman, TL., Fowlie, P.J.A. (1988): Der Verbleib von Naphthalin, Anthrazen und Benzpyren im Boden bei einem fiat die Behandlung von Raffinerieabfallen genutzten Gelande.- in: Wolf, K., van den Brink, W.J., Colon, F.J. (Hrsg): Altlastensanierung "88, zweiter internationaler TNO/BMFT-Kongress ~iber Ahlastensanierung-111-113, Bonn; K~stner, M., Lotter, S., Heerenklage, J., Breuer-Jammali, M., Stegmann, R.,Mahro, B. (1993): Fate of 14C labelled carbon from anthracene and hexadecane in non-saturated soil cultures.- Environ. Sci. Technol. (submitted)

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Mahro, B. & K~istner, M. (1993): Der mikrobielle Abbau polyzyklischer aromatischer Kohlenwasserstoffe (PAK) in B6den und Sedimenten: Mineralisierung, Metabolitenbildung und Entstehung gebundener Rtickstande.- Bioengineering, Vol. 9, 1, 50-58. MOiler, J.G., Chapman, P.J., Pritchard, P.H. (1989): Creosote-contaminated sites.-Environ. Sci. Technol., Vol 23, 1197-1201 Parsons J.W. (1989): Hydrolytic degradation ofhumic substances.- MH.B. Hayes, MacCarthy, P., Malcom,RL., Swift, R.S.: Humic Substances II, Chp. 4, 99-120 Park, K.S., Sims, R.C., Doucette, W.J., Matthews, J.E. (1988): Biological transformation and detoxification of 7,12-dimethylbenz(a)anthracen in soil systems.- J. Water Pollut. Control Fed. 60, 1822-1825 Pothuluri J.V., Heflich, R. H., Cemiglia, C. E. (1992): Fungal metabolism and detoxification of fluoranthene.Appl. Environ. Microbiol., 58, 3, S. 937-941. Qui, X., McFarland, M.J. (1991): Bound residue formation in PAH contaminated soil composting using Phanaerochaete chrysosporium.- Hazardous waste hazardous materials 8 (2), 115-126 Scheunert I., Mansour, M., Andreux, F. (1992): Binding of organic pollutants to soil organic matter.- Intern J. Environ. Anal. Chem., 46, 189-199 Sims, R.C. & Overcash, MR. (1983): Fate of polynuclear aromatic compounds in soil-plant system.- Residue Rev., 88, 1-68. Van Afferden, M., Weissenfels, W.D., Beyer, M., Klewer, H.J., Klein, J., Lanhoff,J. (1991): Einflul3 der "Bioverftigbarkeit" auf den mikrobiellen PAK-Abbau in BOden.- DECHEMA-Fachgespr/ich Umweltschutz, "Mikrobiologische Reinigung von BOden", 27-28.2. 1991 Frankfurt. Weissenfels,W.D., Klewer, H-J., Langhoff, J. (1992): Adsorption of polycyclic aromatic hydrocarbons (PAHs) by soil particles: influence on biodegradability and biotoxicity.- Appl. Microbiol. Biotechnol, 36, 689-696 Yang, SK., Siiverman, B.D. (eds.) (1988): Polycyclic aromatic hydrocarbon carcinogenisis, structure-activity relationships. CRC Press, Boca Raton. Ziechmann, W. (1980): Huminstoffe. Probleme, Methoden, Ergebnisse. Verlag Chemie, Weinheim