compost mixture

Chemosphere 40 (2000) 359±367

E€ects of heterocyclic PAHs (N, S, O) on the biodegradation of typical tar oil PAHs in a soil / compost mixture Susanne Meyer, Hans Steinhart* Institut fur Biochemie und Lebensmittelchemie, Universit at Hamburg, Grindelalle 117, D-20146 Hamburg, Germany Received 27 January 1999; accepted 8 June 1999

Abstract The interaction phenomena during the biodegradation of typical coal tar polycyclic aromatic hydrocarbons and their heterocyclic analogues (N, S, O) were investigated in an arti®cially contaminated AhA1-horizon / compost mixture. All compounds were partly or completely biodegraded. Degradation of two- to ®ve-ring PAHs was inhibited by the presence of hetero-PAHs, whereas degradation of just some hetero-PAHs was inhibited by the presence of PAHs. Among the hetero-PAHs the sulphur-containing compounds were less susceptible to degradation than the corresponding oxygen- or nitrogen-containing analogues. The basic azaarene acridine showed an extreme persistence and strong sorption to the soil matrix proved by an increase of recovery after saponi®cation of the soil matrix. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Biodegradation; Heterocyclic PAHs; PAHs

1. Introduction Soil contamination based on coal tar or tar oil represents a frequent and widespread environmental problem at former coal gasi®cation or tar oil distillation plants as well as at wood-preserving facilities. Biological treatment of such contaminated soils using hydrocarbon degrading microorganisms is a frequently applied remediation technique because of its environmental compatibility and its cost-e€ectiveness. The biodegradation of polycyclic aromatic hydrocarbons (PAHs), which represent with contents of up to 85% the predominant chemical class of coal tar and tar oil (Collin and Zander, 1985), has been extensively investigated in soil (Weissenfels et al., 1992; Erickson et al., 1993; Wilson and

* Corresponding author. Tel.: +49-40-4123-4356/7; fax: +4940-4123-4342. E-mail address: [email protected] (H. Steinhart).

Jones, 1993; K astner and Mahro, 1996; Wischmann et al., 1996). Few studies have investigated interaction phenomena ± synergistic as well as antagonistic ± between di€erent PAHs in culture media (Bouchez et al., 1995; Tiehm and Fritzsche, 1995). Only little attention has been paid to the biodegradability of heterocyclic PAHs containing nitrogen, sulphur, or oxygen (PANHs, PASHs, PAOHs), which comprise a smaller but nevertheless important part of coal tar with contents ranging from 5% to 13% (Collin and Zander, 1985; Wright et al., 1985; Mueller et al., 1991; Wischmann and Steinhart, 1997). Di€erent studies in culture media have shown that PANHs, PASHs, and PAOHs can have a signi®cant inhibiting e€ect on the biodegradation of PAHs and monoaromatic hydrocarbons (Arcangeli and Arvin, 1995; Dyreborg et al., 1996a; Lantz et al., 1997). However, it remains uncertain whether corresponding e€ects between PAHs and hetero-PAHs occur during biodegradation in complex soil mixtures as well. Besides the described interaction phenomena, several hetero-PAHs have been reported to show toxic, mutagenic, and

0045-6535/00/$ - see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 2 3 7 - 4

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carcinogenic activities, even when present in low concentrations. Especially the nitrogen-containing PAHs from the basic fraction of coal tars and tar oils contribute considerably to the mutagenicity of tar oils (Later et al., 1983). Due to the higher polarity of heteroPAHs, the possibility of their incorporation into the humic polymer should be taken into account. Such incorporated contaminants ± well known from PAH biodegradation and unavailable by conventional solvent extraction ± can be released by methanolic hydrolysis (saponi®cation) of the humic matrix (Wischmann and Steinhart, 1997; Eschenbach et al., 1994). However, the fate of tar oil hetero-PAHs in soil is of great environmental concern. The aim of this study was to investigate the e€ects of hetero-PAHs on the biodegradation of typical tar oil PAHs in a soil material from an AhA1-horizon supplemented with compost. The organic matrix of compost is well known to enhance degradation of PAHs (K astner and Mahro, 1996). The soil / compost mixture was arti®cially contaminated with nine typical tar oil PAHs (soil ``PAH'') or with these PAH compounds and 10 typical tar oil hetero-PAHs (soil ``PAH + NSO''). For the degradation experiments, the autochthonous micro¯ora of the soil / compost mixture was used under aerobic conditions. In a second laboratory approach, the biodegradability of hetero-PAHs was investigated (soil ``NSO''). The residues of the PAHs and hetero-PAHs ± solvent extractable as well as saponi®able ± during an investigation period of 203 days were determined. 2. Experimental 2.1. Reagents PAHs, hetero-PAHs, and internal standards (purity >95%) were obtained from Aldrich (Steinheim, Germany), Fluka (Buchs, Switzerland), Merck (Darmstadt, Germany) and Promochem (Wesel, Germany). The solvents used were of HPLC grade (n-hexane, n-heptane; Biomol, Hamburg, Germany) or of analytical grade (hydrochloric acid (30%); Merck, Darmstadt, Germany). Dichloromethane, acetonitrile and methanol (Merck, Darmstadt, Germany) were of synthetic grade and distilled before use. Sodium sulphate, potassium dihydrogenphosphate and potassium hydroxide were of analytical grade (Merck, Darmstadt, Germany). The SPE materials ± 8 ml borosilicate glass SPE columns, PTFE frits, stainless steel brass taps with PTFE cones and silica gel (average particle size 40 lm, particle size distribution 30±60 lm) ± were purchased from Baker (Grob-Gerau, Germany). The strong basic anion exchange material Chromabondâ SB was supplied by Macherey and Nagel (D uren, Germany).

2.2. Soil / compost mixture An AhA1 soil (1.1% organic carbon, 6.5% kaolin, density 2.66 g/cm3 , maximum water capacity 35.9% (w:w)) was sieved at 2 mm mesh size and mixed with biocompost (sieved at 4 mm mesh size, degree of maturity at V, maximum water capacity 213% (w:w)) resulting in a ratio of 9:1, related to dry weight. Soil humidity was adjusted to 55% of the maximum water capacity. 2.3. Spiking procedure and degradation experiments 150.0 g dried soil material from an AhA1-horizon was ®lled into a 1±1 glass jar and spiked with 10 ml PAH standard solution and / or 5 ml PASH / PAOH standard solution and 10 ml PANH standard solution in dichloromethane, respectively. The solvent was evaporated under a gentle stream of nitrogen. Subsequently 357.9 g dried AhA1-horizon, 78.1 g wet compost and 114.0 g bidistilled water were added and mixed thoroughly. The resulting concentrations are listed in Table 1. The jars were covered with polyethylene foil and stored at room temperature in the dark. Under these aerobic conditions, degradation was carried out by the autochthonous micro¯ora. Before sampling after 1, 6, 13, 20, 30, 48, 64, 83, 111 and 203 days, the jars were weighed and evaporated water was compensated. All systems were prepared in triplicate. Poisoned controls were prepared to estimate the in¯uence of abiotic processes on the decrease of the organic contaminants such as evaporation. Therefore soil / compost mixtures were autoclaved (120°C, 1,8 bar, 15 min), spiked with contaminants as described above, and humidity was adjusted with a solution of mercury(II)chloride in water, resulting in a ®nal concentration of 1 g HgCl2 / kg wet soil. 2.4. Extraction of contaminants 20 g contaminated soil was mixed with 1 ml 1 M hydrochloric acid. Subsequently the soil was dried with sodium sulphate and soxhlet extracted for 7.5 h with a mixture of dichloromethane (210 ml) and n-heptane (10 ml). The resulting extract was concentrated to about 5 ml by rotary evaporation (40°C, 600 mbar). The SPE columns were prepared by ®lling 0.7 g Chromabondâ SB and 2.0 g of silica gel (inactivated with 10% water (w:w)) between three PTFE frits. After equilibration with n-hexane, the Soxhlet extracts were transferred to the columns. The fractions were eluted as follows: PAHs, PASHs and PAOHs were eluted with 3 ml nhexane, 12 ml n-hexane / dichloromethane (85:15; v:v), and 2 ml dichloromethane (fraction 1). Subsequently PANHs and neutral metabolites were eluted with 1 ml dichloromethane, 6 ml methanol and 3 ml 0.05 N

S. Meyer, H. Steinhart / Chemosphere 40 (2000) 359±367

361

Table 1 Initial concentrations of PAHs and hetero-PAHs in di€erent soil systems Compound (number of rings)

Soil ``PAH'' (mg/kg)

Soil ``NSO'' (mg/kg)

Soil ``PAH + NSO'' (mg/kg)

PAHs Naphthalene (2) Acenaphthene (3) Fluorene (3) Phenanthrene (3) Anthracene (3) Fluoranthene (4) Pyrene (4) Benz[a]anthracene (4) Benzo[a]pyrene (5)

400 202 204 300 196 251 175 58 75

± ± ± ± ± ± ± ± ±

400 202 204 300 196 251 175 58 75

Hetero-PAHs Indole (2) Quinoline (2) Benzothiophene (2) Benzofuran (2) Carbazole (3) Acridine (3) Dibenzothiophene (3) Dibenzofuran (3) 1-Cyanonaphthalene (2) 9-Cyanoanthracene (3)

± ± ± ± ± ± ± ± ± ±

76 77 75 176 174 77 75 176 53 51

76 77 75 176 174 77 75 176 53 51

hydrochloric acid in methanol (fraction 2). This analytical procedure was described in more detail previously (Meyer et al., 1999). The fractions were diluted, internal standards were added (fraction 1: 9-chloroanthracene and indeno[1,2,3-cd]¯uoranthene; fraction 2: 4-methylquinoline). The fractions were analysed by GC-FID. At day 111 and 203 soxhlet extraction was followed by saponi®cation of the soil matrix. This procedure avoids underestimation of contaminants, which are incorporated into the soil humic matrix and are less available to conventional solvent extraction (Wischmann and Steinhart, 1997; Eschenbach et al., 1994). The soxhlet extracted soil residues were ®lled with 50 ml 0.5 N potassium hydroxide in methanol / water (4:1; v:v) into a centrifugation tube, which was tightly closed and sonicated for 60 min at 60°C (Elma Digital Ultrasonic Cleaner T480 H-2, Singen, Germany). After centrifugation an aliquot was neutralised. HPLC was used for determination of PAHs and hetero-PAHs in the saponi®ed extracts with their high contents of humic substances as this enables analysis without an additional clean-up step. 2.5. Gas chromatography ± ¯ame ionisation detection (GC-FID) Quantitative determination of fraction 1 (PAHs, PAOHs, PASHs) and fraction 2 (PANHs) was performed with a Carlo Erba HRGC 5160 gas chromatograph equipped with a ¯ame ionisation detector and the

Chromstar evaluation unit (SCPA, Stuhr±Brinkum, Germany). A 30 m ´ 0.32 mm i.d. column coated with a 0.25 lm ®lm of DB-5 (J&W Scienti®c, Folsom, USA) was used. For fraction 1, the initial oven temperature was 60°C (3 min isothermal) and was then programmed at 7°C/min to 200°C and at 13°C/min to 300°C (6 min isothermal). For fraction 2 the initial oven temperature was 70°C and was then programmed at 6°C/min to 220°C and at 25°C/min to 300°C (4 min isothermal). Split injection (split ratio 1:4) was performed at an injector temperature of 300°C, and a detector temperature of 320°C was employed. 2.6. High performance liquid chromatography ± diodearray detection (HPLC-DAD) The HPLC system consisted of an L-6200 low pressure gradient system, a T-6300 column thermostat and an AS-4000 autosampler (all Merck / Hitachi, Darmstadt, Germany). For detection, a Millipore / Waters (Eschborn, Germany) 994 diode-array detector and the evaluation software DART (Ifas, Hamburg, Germany) were connected. The system was equipped with a 150  5 lm) mm ´ 4 mm i.d. Nucleosilâ C18-PAH (100 A, â  column and an 8 mm ´ 4 mm i.d. Nucleosil C18 (120 A, 5 lm) guard column (Macherey and Nagel, D uren). The separation of PAHs and hetero-PAHs was performed at 30°C with a ¯ow rate of 1 ml/min and a ternary gradient consisting of acetonitrile (A), phosphate-bu€er (0.68 g potassium dihydrogenphosphate and 40 ml 0.1 N

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Table 2 HPLC-gradient for separation of saponi®able contaminants

3. Results and discussion

Time (min)

A (%)

P (%)

M (%)

3.1. Degradation of PAHs a€ected by hetero-PAHs

0 1 14 28 38 45

12 12 20 49 70 70

55 55 35 22 0 0

33 33 45 29 30 30

During the investigation period of 203 days, all nine PAHs were partly or completely biodegraded in the only PAH-contaminated (``PAH'') as well as in the PAH- and hetero-PAH contaminated soil systems (``PAH + NSO''). The study shows that hetero-PAHs can have a signi®cant inhibiting e€ect on the biodegradation of typical tar oil PAHs. The inhibition was identi®ed by change of at least one of the following three degradation characteristics in the presence of hetero-PAHs: (1) increase in the lag-phases, (2) decrease in the degradation rates and/or (3) increase in the residual concentrations at the end of the investigation period. Table 3 shows exemplarily the residual concentrations of all nine spiked PAHs after 111 days in both soil systems. Only naphthalene was degraded completely in both soil systems within this time. However, in contrast to the ``PAH + NSO'' soil systems, where naphthalene was eliminated within 111 days, degradation in the ``PAH'' soil systems was completed after 48 days (Fig. 1(a)). Likewise lag-phases of three- and four-ring PAHs increased and degradation rates slowed down in the presence of hetero-PAHs. In the ``PAH'' soil systems after 111 days, residues between 0% (acenaphthene and ¯uorene) and 57% (benz[a]anthracene) of the initial concentrations were detected. In contrast in the ``PAH + NSO'' soil systems phenanthrene, anthracene and the four-ring PAHs showed almost no or only little biodegradation (residual concentrations between 89% and 94%, Table 3). Only the ®ve-ring PAH benzo[a]pyrene was not in¯uenced by the presence of the heteroPAHs until the 111th day as nearly the same residual

potassium hydroxide per litre bidistilled water, adjusted to pH 7.5) (P), and methanol (M) (Table 2) and the detector operating simultaneously at 225, 235, 251 and 286 nm. 2.7. Estimating colony-forming units (CFU) A suspension of 1 g soil / compost mixture in 10 ml physiological sodium chloride solution was prepared and decimal dilutions were incubated on inhibitor-free plate-count agar for 48 h at 30°C. 2.8. Quality assurance The analytical method was validated in a ®ve level matrix-calibration as described previously (Meyer et al., 1999). Average percent recoveries of the aromatic compounds used in this study varied between 78% (acenaphthene) and 102% (benz[a]anthracene) except for the low molecular weight and relatively volatile 2-ring compounds benzofuran, benzothiophene, indole and naphthalene (30±64%).

Table 3 Concentration of PAHs in ``PAH''-contaminated and ``PAH + NSO''-contaminated soil samples after 111 days Compound

Naphthalene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benz[a]anthracene Benzo[a]pyrene P 9 PAHs a

Day 1 ca spiked (mg/kg) 400 202 204 300 196 251 175 58 75 1861

Day 111 Soil ``PAH'' ca residualb (perc.c ) (mg/kg (%))

Soil ``PAH + NSO'' ca residualb (perc.c ) (mg/kg (%))

N.d.d (0) N.d.d (0)e N.d.d (0)e 5 ‹ 3.9 (2)e 23 ‹ 26.7 (12)e 48 ‹ 53.4 (19)e 39 ‹ 43.8 (22)e 33 ‹ 18.0 (57) 69 ‹ 4.2 (92)

N.d.d (0) 51 ‹ 1.6 (25)e 145 ‹ 34.2 (71)e 279 ‹ 7.9 (93)e 184 ‹ 6.1 (94)e 234 ‹ 9.8 (94)e 158 ‹ 3.6 (91)e 52 ‹ 2.0 (89) 69 ‹ 3.2 (91)

216 ‹ 147.6 (12)e

1172 ‹ 37.8(63)e

Concentration. Mean ‹ standard deviation of three replications (three di€erent jars). c Percent of initial spiked concentration. d Not detectable. e Concentrations in ``PAH'' and ``PAH + NSO'' are signi®cantly di€erent (t-test, a ˆ 0.05). b

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363

Fig. 1. Degradation of PAHs in the ``PAH'' and the ``PAH + NSO'' soil systems: (a) naphthalene, (b) ¯uorene, (c) ¯uoranthene, (d) benzo[a]pyrene (di€erences between black data points are signi®cant; t-test, a ˆ 0.05).

concentrations were observed in both soil systems. A statistical treatment of the data at day 111 proved that with the exception of naphthalene, benz[a]anthracene and benzo[a]pyrene the residual concentrations of PAHs were signi®cantly higher in the ``PAH + NSO'' soil systems (t-test, a ˆ 0:05). Exemplarily the elimination of some selected PAHs in the two di€erent soil systems are presented in Fig. 1. Naphthalene (Fig. 1(a)) was eliminated rapidly in both soil systems without a lag period. A slight in¯uence of the hetero-PAHs on the naphthalene elimination rate could be observed as residual naphthalene concentrations in the ``PAH + NSO'' soil systems at day 20 and between day 48 and 64 were con®rmed as signi®cantly higher by statistical analysis (t-test, a ˆ 0.05; s. black data points in Fig. 1). Concentrations of naphthalene also decreased in the poisoned controls though to a smaller extent. Residual concentrations of 320 mg/kg (20 days), 168 mg/kg (48 days), and 81 mg/kg (111 days) were determined, respectively. The estimation of <10 CFU/g soil con®rmed sterile conditions. As a representative of the degradation of three-ring PAHs, ¯uorene is shown in Fig. 1(b). Its composition was signi®cantly a€ected by the presence of hetero-PAHs. In the ``PAH'' soil systems, ¯uorene showed a lag period of about 13 days, followed by a rapid depletion and a complete elimination after 111 days. In contrast, the lag-period was prolonged until day 30 in the ``PAH + NSO'' soil systems followed by a decreased degradation rate. As demonstrated with ¯uoranthene (Fig. 1(c)), and benzo[a]pyrene (Fig. 1(d)) degradation of four- and ®ve-ring

PAHs was negatively a€ected by the hetero-PAHs as well, though to a lower extent than for three-ring PAHs. However, although degradation of PAHs was inhibited by the hetero-PAHs, the order of biodegradability 2-ring PAH > 3-ring PAH > 4-ring PAH > 5-ring PAH was maintained (Table 4). In contrast to the two- and threering PAHs none of the four- and ®ve-ring PAHs could be eliminated completely within the investigation period of 203 days, neither in the ``PAH'' nor in the ``PAH + NSO'' soil systems. Saponi®cation of the soil matrix at days 111 and 203 resulted in no substantial release of PAHs (saponi®ed part <4% of the initial concentration). The interaction phenomena described above are consistent with the results of Dyreborg et al. (1996b), who reported that heteroaromatic compounds such as thiophene, benzothiophene, benzofuran and 1-methylpyrrole can have an inhibiting e€ect on the degradation of benzene and naphthalene in culture media with a creosote adapted mixed bacteria culture. In a similarly designed experiment pyrrole, 1-methylpyrrole, thiophene, benzofuran and indole inhibited toluene degradation while no e€ects of benzothiophene and quinoline on toluene degradation were observed (Dyreborg et al., 1996a). In the same way, creosote compounds such as quinoline, methylated quinolines, acridine, carbazole, dibenzofuran and dibenzothiophene inhibited the activity of a ¯uoranthene-degrading bacterium in culture media (Lantz et al., 1997). To the best of our knowledge there is no report about the in¯uence of hetero-PAHs on biodegradation of PAHs in soil.

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Table 4 Order of susceptibility of PAHs or hetero-PAHs to microbial degradation in di€erent soil systems Soil system PAHs ``PAH'' Hetero-PAHs ``NSO'' PAHs and hetero-PAHs ``PAH + NSO''

Order of susceptibility to microbial degradation (highest degradable to least degradable) Naphthalene (2) > acenaphthene (3)  ¯uorene (3)  phenanthrene (3) > anthracene (3) > ¯uoranthene (4)  pyrene (4) > benz[a]anthracene (4) > benzo[a]pyrene (5) Indole (2)  benzofuran (2)  quinoline (2) > benzothiophene (2) > dibenzofuran (3)  carbazole (3) > dibenzothiophene (3)  1-cyanonaphthalene (2) > 9-cyanoanthracene (3) > acridine (3) Indole (2)  benzofuran (2) > naphthalene (2)  benzothiophene (2) > quinoline (2) > acenaphthene (3) > dibenzofuran (3)  carbazole (3) > ¯uorene (3)  dibenzothiophene (3)  1-cyanonaphthalene (2) > phenanthrene (3)  anthracene (3)  ¯uoranthene (4)  pyrene (4) > 9-cyanoanthracene (3)  acridine (3)  benz[a]anthracene (4) > benzo[a]pyrene (5)

3.2. Degradation of hetero-PAHs The biodegradation of hetero-PAHs was investigated only in the hetero-PAHs containing (``NSO'') and in the ``PAH + NSO'' soil systems. In contrast to biodegradation of PAHs susceptibility to microbial degradation was a€ected not only by the number of condensed rings but also by the heteroatom itself. Hetero-PAHs with the same number of condensed rings showed considerably di€erent degradation rates. While the analogous PANHs and PAOHs showed similar biodegradability, the replacement of nitrogen or oxygen by sulphur resulted in a decrease in the biodegradation rate (Fig. 2 and Table 4). Thus benzothiophene was eliminated more slowly than indole and benzofuran and dibenzothiophene was eliminated more slowly than carbazole and dibenzofuran, respectively. This relatively high resistance of sulphur-containing heteroaromatics to microbial degradation ± about twice as high as for the hydrocarbon analogues ± is a well-known phenomenon in different environmental matrices (Kuhn and Su¯ita, 1989; Fedorak, 1990). Therefore, dibenzothiophene and its alkylated derivatives have already been suggested as markers for oil pollution (Friocourt et al., 1982).

Fig. 2. Degradation of analogous nitrogen-, sulphur-, and oxygen-containing hetero-PAHs with two and three condensed rings in the ``NSO'' soil systems.

While the degradation of the two-ring hetero-PAHs indole, benzofuran and benzothiophene was almost una€ected by the presence of PAHs, degradation rates of the remaining two-ring hetero-PAHs quinoline and 1cyanonaphthalene were substantially slower due to the presence of PAHs. As an example, the decay of quinoline is shown in Fig. 3(a). The lag-phase of six days in the ``NSO'' soil systems was prolonged by about two weeks, followed in each case by a rapid depletion of quinoline. In contrast to the two-ring hetero-PAHs, an in¯uence of PAHs on all three-ring hetero-PAHs was observed, though to quite di€erent extents. Strong antagonistic inhibition phenomena of PAHs were observed for carbazole and dibenzofuran degradation. As an example in Fig. 3(b) the degradation of carbazole is shown. Dibenzothiophene degradation was inhibited less (Fig. 3(c)). While after 111 days a residual concentration of 81% of the initial concentration was still present in the ``PAH + NSO'' soil systems, it was completely degraded by the end of the investigation period. These results concerning the in¯uence of PAHs on the degradation of hetero-PAHs match in some respects those of Millette et al. (1995), who observed that p-cresol and phenanthrene inhibited the aerobic degradation of carbazole in groundwater. From the factorial experiments it was concluded that biodegradation of the more hydrophobic compound was a€ected by the presence of other creosote compounds to a larger extent (Millette et al., 1995). This might be the reason why the strongest polar and therefore best bioavailable two-ring hetero-PAHs indole, benzothiophene and benzofuran are not a€ected by the presence of the more hydrophobic PAHs. In contrast, the in¯uence of PAHs on the degradation of the as well polar two-ring compound quinoline can be explained by its basicity (pKa 4.92), which causes sorption of quinoline to the soil matrix via a cationexchange reaction and a reduction of its bioavailability (Smith et al., 1992).

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365

Fig. 3. Degradation of hetero-PAHs in the ``NSO'' and the ``PAH + NSO'' soil systems: (a) quinoline, (b) carbazole, (c) dibenzothiophene, (d) acridine (di€erences between black data points are signi®cant; t-test, a ˆ 0.05).

The extreme persistence of acridine ± whose structure di€ers from that of carbazole by a pyridine-ring instead of a pyrrole-ring ± in both soil systems might be due to its basicity, as well. Decay of solvent extractable amounts of acridine is shown in Fig. 3(d). While at day 111 similar amounts of about 90% of the initially spiked concentrations were observed, a further decrease to 75% (``NSO'') and 60% (``PAH + NSO'') was observed by the end of the investigation period. In addition, considerable amounts of acridine were released from the soil matrix by its saponi®cation, which was carried out at days 111 and 203 (Fig. 4). Thus the decrease in the solvent extractable part of acridine were accompanied by an in-

Fig. 4. Extractable and saponi®able amounts of acridine in the ``NSO'' and the ``PAH + NSO'' soil systems at day 111 and day 203.

crease in the incorporated part, resulting in total acridine contents of 102% (``NSO'') and 80% (``PAH + NSO'') of the initially spiked concentration. Recoveries of all other hetero-PAHs were only slightly in¯uenced by the saponi®cation (saponi®ed part <4% of the initial concentration) or not at all, respectively. Information about the biodegradability of acridine is rare in the literature even in culture media. In a laboratory microcosm, acridine was degraded under di€erent anaerobic conditions (Knezovich et al., 1990), but in an aquifer contaminated by wood treatment chemicals basic three-ring azaarenes such as acridine were not transformed (Pereira et al., 1987). Due to the pKa values of acridine (5.68) and quinoline (4.92) and the pH value of the soil solution of the Ah-horizon / compost mixture used (pH 5.5), acridine sorption via a cation exchange reaction can be supposed to exceed quinoline sorption in the present study (Traina and Onken, 1991). This sorption might lead to a decrease in bioavailability and therefore exhibits a protective function against biodegradation such as is already known from the sorption of quinoline to clay surfaces (Smith et al., 1992). Degradation of cyano-PAHs was observed in both soil systems but they displayed a higher persistence than the corresponding unsubstituted PAHs or hetero-PAHs with the same number of condensed rings (except acridine) (Table 4). The e€ect of a cyano-substitution might be comparable to a methylation of the aromatic ring system, as the latter is known to cause a decreased susceptibility to microbial degradation as well (Elmendorf et al., 1994).

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3.3. Decay of total contents of contaminants Total solvent extractable contents of PAHs and hetero-PAHs are shown in Fig. 5. After 13 days total PAHs decreased more rapidly in the ``PAH'' soil system to residual concentrations of 216 mg/kg after 111 days and 57 mg/kg after 203 days. In the ``PAH + NSO'' soil systems residual concentrations of 1172 mg/kg (111 days) and 151 mg/kg (203 days) were observed, respectively. Similar results were achieved from summarising the hetero-PAHs, which were as well degraded faster in the ``NSO'' soil systems. Fig. 6 displays the total degraded contaminants of each soil system. Until day 30 only slight di€erences existed between the total degraded amounts of contaminants of the three soil systems. The following decrease in the elimination rate in the ``NSO'' soil systems might be due to approximation to the maximal degradable amount of contaminants. The decrease in the elimination rate in the ``PAH + NSO'' soil systems (signi®cant at days 64 and 111; t-test, a ˆ 0.05), might result from several reasons such as interaction or rather inhibition phenomena. However, from the present experimental design it cannot be excluded that the delay of degradation is caused by the higher absolute amount of contaminants in the ``PAH + NSO'' soil systems. Nevertheless, as the substances in the ``PAH + NSO'' soil systems are degraded in order of decreasing polarity (except for basic PANHs quinoline and acridine) (Table 4), it is supposed, that losses of PAH degradation capacity are caused by the presence of hetero-PAHs. This assumption matches the results of Millette et al. (1995), who pointed out, that the order of biodegradability of single substances in complex mixtures is determined by their polarity and bioavailability. However, further studies are required to get more detailed information about dependence of concentration or in¯uence of di€erent kinds of hetero-PAHs. As the presence of concomitant organic substances can limit the eciency of bioremediation processes, predictions about duration and success of remediation processes must take the presence of such compounds into account. Therefore, the determination of only the

Fig. 5. Degradation of total solvent extractable contents of PAHs and hetero-PAHs (di€erences between black data points are signi®cant; t-test, a ˆ 0.05).

Fig. 6. Total amount of degraded contaminants in the three soil systems (di€erences between black data points are signi®cant; t-test, a ˆ 0.05; broken lines: initially spiked concentrations).

16 PAHs in the EPA list of environmental priority pollutants is insucient. However, a determination of at least major coal tar compounds is essential to increase the success of biological treatment of contaminated soil.

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