bioreclamation of chlorophenol-contaminated soil by composting

Appl Microbiol Biotechnol (1986) 25:68--75 Applied Microbiology

Biotechnology © Springer-Verlag 1986

Bioreclamation of chlorophenol-contaminated soil by composting

R. Valo and M. Salkinoja-Salonen

University of Helsinki, Department of General Microbiology, Mannerheimintie 172, SF-00280 Helsinki 28, Finland

Summary. Microbiological decontamination of technical chlorophenol-containing soil by corn- posting was studied. In two 50 m 3 windrows the concentration of chlorophenols went down from 212 mg kg -1 to 30 mg kg -~ in 4 summer months and after the second summer of composting it was only 15 mg kg -~. All chlorophenol congeners present in the technical chlorophenol were degraded, but the main dimeric impurities, polychlorinated phenoxyphenols were recalcitrant. The contaminated soil was found to contain chlorophenol-degrading microbes, 5 x 10 6 cfu g-~ of dry windrow soil. Laboratory experiments with samples from the windrow compost showed that chlorophenols were truly degraded and that chlorophenol loss by evaporation was less than 1.5% under the circumstances studied. Laboratory experiments also showed that degradation of chlorophenols (120 mgkg -1) was accelerated when sterilized contaminated soil was inoculated with Rhodococcus chlorophenolicus (mineralizer of several chlorophenols) or naturally occurring microbes of the field composts. Biomethylation of chlorophenols in the composts was insignificant compared to biodegradation.

Introduction

Technical chlorophenols are efficient fungicides still widely used at sawmills, although to a de- creasing extent, because several countries have banned or restricted their use. The formulation (Ky-5) used in Finland until 1984 consisted mainly of 2,3,4,6-tetrachlorophenol, 2,4,6-trichlorophenol and pentachlorophenol.

Offprint requests to: R. Valo

Technical chlorophenol contains usually polychlorinated phenoxyphenols (PCPPs) polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) as impurities (Ar- senault 1976; Humppi et al. 1984), of which some are more toxic than the chlorophenols themselves. These impurities have accumulated in soil near wood-preserving facilities using technical chlorophenol (Kitunen et al. 1985). Biological or other degradation of these impurities has not been observed in the environment.

We studied soil around dipping basins of sawmills (Valo et al. 1984) and found up to 500 mg k g - l d wt of the chlorophenols, contamination extending to several meters' depth until ground water table. It seemed that chlorophenols were stable under the environmental conditions concerned (inorganic soil), maybe because conditions for microbiological attack were unfavoura- ble. Even after discontinuation of the use of chlorophenols, the soil remained contaminated thus forming a future threat to the ground water because of leaching (Valo et al. 1984; Valo et al. 1985b; Kitunen et al. 1985). Ground water pollu- tion by wood preservatives, pentachlorophenol and components of creosote, near an abandoned wood-creosoting plant was also described by Lee et al. (1984).

Successful small scale experiments on microbiological clean up of chlorophenol contaminated soil were reported by Edgehill (1982) and Craw- ford and Mohn (1985) in laboratory scale or small windrow composts. Successful large scale work on microbiological decontamination of chlorophenol contaminated soil in field conditions has apparently not been reported so far, except for our preliminary report (Valo and Salkino~a-Sa- lonen 1984). In this paper we described the details and final results of composting of 100 m 3 of

R. Valo and M. Salkinoja-Salonen: Bioreclamation of chlorophenol-contaminated soil 69

heavily chlorophenol contaminated soil collected around the dipping basin of a sawmill.

Materials and methods

Field experiments

In 1981, 70m 3 of heavily chlorophenol-contaminated soil (400--500 mg of chlorophenols kg -~ of fresh soil) was removed from a wood preservation facility of a sawmill and has been stored since then in windrows at an abandoned landfill site at Heinola, south Finland. The sawmill had diptreated timber of decades using a chlorophenol-based fungicide, com- posed of 60% of chlorophenols (by wt) as sodium salts (80% of 2,3,4,6-tetrachlorophenol, 10% of 2,4,6-trichlorophenol, 8% of pentachlorophenol and minor amounts of other isomers of trichlorophenol and dichlorophenol). This fungicide contained as impurities polychlorinated phenoxyphenols (1--5% of wt), of which nine different congeners were identified (Humppi et al. 1984; Kitunen et al. 1985).

The soil consisted of gravel and sand with little organic matter (<2% by wt). During the 3-year storage period, the quantity of chlorophenols in soil did not decrease.

In the experiment described here, the contaminated soil was transferred to a new site, which was protected against leaching of chlorophenols by plastic lining. Furthermore, 35 m 3 of softwood bark and 3 m 3 of ash from a fiberboard factory boiler were mixed with the contaminated soil (70 m 3) of pro- vide for organic matter and mineral nutrients and to buffer the pH near to neutral.

In June 1984, this mixture was piled into two windrows, 50 m 3 of each, and into two 1.5 m 3 pilot composts. A mixed culture of laboratory-grown chlorophenol degrading bacteria (Valo et al. 1985a) was added to one 1.5 m 3 pilot compost (I × 105 g-1 of compost). The pilot size composts were sampled in 1984 at monthly intervals between June and October, and the 50 m 3 windrow was analysed in June and in October after four months of composting. In 1985, all field composts were analysed in October. The initial concentration of chlorophenols in the windrows and pilot composts was 200--300 mg/kg d wt. The 50-m 3 windrows were sampled three times only, because representative sampling from the large windrow was only possible with a heavy-duty excavator. Fertilizer (1 kg m-3 of compost) containing 16% of N (5.4% as nitrates, 10.6 as ammonium), 7% of P and 3.3% of K dissolved in water was added. The composts were irrigated weekly from June to October 1984.

Laboratory experiments. Figure 1 shows the apparatus used for laboratory experiments. It allows calculation of the mass ba- lance of chlorophenol degradation using ~4C-labelled compounds. Each 3-1jar held 1 1 of soil. Sterile inflow air was pre- moistened by passing through water. Any phenolic compounds in the exhaust air were trapped onto XAD-4 resin (Rohm & Haas, 0.2--0.4 ram). Before use the resin was was- hed in soxhlett apparatus for 24 h with methanol and for 24 h with acetonitrile. The purified resin was kept in methanol and dried in nitrogen stream before use. The carbon dioxide resul- ting from mineralization of 14C-labelled pentachlorophenol was trapped in 1 M NaOH. The composts were held at room temperature in the dark and stirred weekly by rotating the jars. At the end of the incubation period the content of the jars was extracted and analysed for chlorinated phenols and phenoxyphenols and their methylated analogs.

C

Fig. 1. Set up of laboratory scale composting. A, aeration pump; B, 0.45 I.tm filter; C, moistening of the inflow air; D, compost jar (volume 3 litres, contains 1 litre of material); E, adsorption resin (0.8 cm x 5 cm) for volatile organics (XAD- 4); F, 50 ml of 1 M NaOH to trap the ~4CO2 released from labeled PCP

Soil taken from one 50-m 3 windrow and from both pilot composts was used for laboratory experiments. It contained 20--30 mg kg - ~ dry wt. of chlorophenols at the time of sampling (October 1984). A composite sample of these three composts was gamma sterilized (3.5 Mrad) and used as a control for non-biological degradation and for the bacterization experiments. To each jar were added 120 mg kg -1 dry wt. of technical chlorophenol (Ky-5, sterilized by filtration) and a mineral salts solution (Salkinoja-Salonen et al. 1983) containing 0.1% of yeast extract so that the water holding capacity was 70%. The jars were supplemented with 5 x 105 cmp of uni- formly 14C-labelled PCP (specific activity 25.6 mCi/g). The rate of aeration was 100 ml rain -1 and the pH was about 7 in each jar.

Rhodoeoceus chlorophenolieus PCP-1 DSM 43826 (J. Apa- jalahti et al. 1986), capable of mineralizing several choloro- phenols, was grown in the medium described above and added to two jars containing sterilized compost, 6 x 10 H cells per jar (1 × 10 9 cells g-~ dry wt. of the mass).

Calculation of chlorophenol-degradin9 mierobes. Chlorophenol mineralizing bacteria in the composts were measured using MPN method (Manual of methods for general bacteriology, 1981) in shake flasks (Valo et al. 1985a) in October 1984. Also two reference samples were assayed: agricultural soil (clay) and chlorophenol-contaminated soil from another sawmill (sawmill A, Valo et al. 1984). 14C-PCP (7000 cpm) was added to each flask at the rate of 1 mg 1- ~. Degradation was interpre- ted as positive if at least 25% of the added 14C-PCP was recovered as 14COz within 3 weeks.

Analysis of the chlorinated compounds. Chlorinated phenols (CP) and polychlorinated phenoxyphenols (PCPP) were extracted from soil samples and acetylated as described elsewhere (Kitunen et al. 1985). Chloroanisoles (CA) and methylated polychlorinated phenoxyphenols (polychlorinated phenoxyanisoles, PCPA) were extracted as follows: 1 1 of soil was acidi- fied to pH 2 with concentrated H2SO4, 1 mg of 2,4,6-tribromo- anisole and 200 I-tg of 2-(pentachlorophenoxy)anisole, were added as internal references followed by extraction for 1 h with 800 ml of acetone in a Bransonic 32 bath sonicator and for 1 h in a mechanical shaker. One volume of the acetone phase was mixed with two volumes of 1 M NaOH and CAs and PCPAs were extracted into hexane.

The XAD-4 resins (0.75 g each) used for entrapment of organics in laboratory experiments were eluted with 5 ml of acetone. CPs and PCPPs were acetylated as above. For CA and PCPA analysis the eluate was extracted with hexane.

The compounds in hexane were quantitated by GLC as described earlier (Valo et al. 1984) except for the gas chroma- tograph used which was a Hewlett Packard-5890 A, equipped

70 R. Valo and M. Salkinoja-Salonen: Bioreclamation of chlorophenol-contaminated soil

with two parallel, fused silica capillary columns (SE-54 and OV-1701, 25 m each) and two 63Ni EC detectors, both connec- ted to a Shimadzu CR3-A integrator. Identification was based on retention times at the two columns with a different solid phase using authentic reference compounds of CPs, CAs, PCPPs and PCPAs.

Other procedures. Total combined nitrogen was assayed by the Kjelldahl method (Standard Methods, 1985) including a pre- treatment with salicylic acid and sodium thiosulphate to re- duce nitrate to amino compounds.

The content of organic matter was calculated as ignition loss at 750°C of the dry samples; 40% of organic matter was assumed to be organic carbon.

Ca, K and Mg were analysed from ammonium acetate extracts (1 N, pH 4.65) and As, Co and Mo from 2 M H/SO4 extracts using AAS-method; Hg, Cd, Pb, Ni, Cu, Cr, AI, Zn, Fe from ignited samples (750°C) using DC plasma emission (Spectra Span VI, Beckman); B, F, P and conductivity accor- ding to the routine methods used for agricultural soil analysis by Viljavuuspalvelu Oy (Helsinki, Finland).

Results

Field-scale composts

Figure 2 shows the rate of disappearance of chlorophenols during outdoor composting in the summer of 1984 and the residual level of chlorophenols on 10. 10. 1985 after the period of 500 days of composting. In both the 50-m 3 windrow and the pilot composts the level of chlorophenols de- creased to about one tenth of the initial level during the first summer. During the first 2 months, disappearence of chlorophenols was fast, but later in summer and in the autumn it slowed down. A1-

300

200

100

SUM OF CHLOROPHENOLS rag. kg4 dw

n 50 m3 WINDROW

O 1.5 m3 PILOT COMPOST

O 1.5 m3 PILOT COMPOST, INOCULATED

o O o n

o ~'Z e. 50 I00 560

DAYS Fig. 2. Disappearance of chlorophenols in field composting from June 1st 1984 (day 1) to October 1985 (day 500)

though the content of chlorophenols in the different composts was somewhat different at the be- ginning, at the end 20--30 mg kg- 1 was found in all composts. During the second summer, little further decrease in chlorophenol content was observed. The final level of chlorophenols in all composts was about 15 mg kg -1 dry wt. in Octo- ber 1985.

Temperature of the composts was about 5-- 15°C above ambient temperature; the highest temperatures were measured close to the surface, 32°C in midsummer and 15°C in October. Also in the center of the windrows temperature was more than 5°C above that of ambient air. In the winter of 1984--1985, the surface of all windrows froze. No measures were taken to maintain biological activity during the winter.

Table 1 shows the composition of the three composts in October 1984. The necessary inorganic nutrients seem to have been present at a sa- tisfactory level for microbial activity while the amounts of inhibiting agents (especially heavy metals As, Hg, Pb, Cd) were low. The amount of fluoride (110--320 mg kg -1) was high indicating earlier use of fluoride-containing diptreatment fungicide.

Table 2 shows the densities of chlorophenol- mineralizing bacteria in the composts and reference soils, both contaminated and uncontaminated. The results show that while total bacterial colony counts in the different soils was quite similar, varying from 1 × 106 to 9 x 107 cfu g-~, the count of PCP-degrading bacteria varied widely, from 1 x 102 to 5 x 106 g - 1. The density of PCP- degrading bacteria in contaminated soils was 102 to 104 fold higher than in uncontaminated soil.

Laboratory experiments

In field scale experiments it is impossible to esti- mate what portion of the chlorophenol was truly degraded and how much was biotransformed (for instance methylated) or evaporated. Laboratory experiments were designed to assay for these. Fig- ures 3 and 4 and Tables 3 and 4 summarize the results. Figure 3 shows time course of mineralization of PCP in technical chlorophenol in 40 days of incubation. Figure 4 shows the sum of chlorophenols, chlorinated phenoxyphenols and their analogous methylated compounds found in each jar at the end of the 68 days incubation. Tables 3 and 4 show the amounts of each chlorinated con- gener analysed from the jars after 68 days of laboratory incubation.


Table 1, Analysis of the field composts in October 1984. Fig- ures are calculated to dry weight if not otherwise expressed

Parameter 50-m 3 1.5-m 3 pilot 1.5-m 3 pilot windrow compost compost

inoculated

Organic C (%) 7.6 10 6 Total N (%) 0.19 0.30 0.25 Water soluble N (%) 0.08 0.17 0.10 Water soluble B (mg/kg) 3 11 11 pH 6 6 6 Conductivity (10 × mS/cm) 14 39 22 Extractable in 1 N ammonium acetate (pH 4.65) solution (mg/kg) P 160 530 250 K 390 1500 860 Ca 3900 9800 2700 Mg 330 490 340 Extractable in 2 M H2SO4 (mg/kg) F 110 320 170 As 8 16 9 Co 8 7 9 Ash (% of dry weight) 69 43 58 Extractable in 6 M HNO3 (mg/kg) Na 840 640 740 Fe 23000 9800 19000 A1 18000 7800 12000 Zn 130 77 77 Cr 85 52 45 Cu 56 55 49 Ni 37 <20 <20 Pb 47 27 36 Hg < 8 < 8 < 8 Mo 1 1 1 Cd <19 <19 <19

As is seen in Fig. 3, soil samples of the windrow and pilot composts mineralized PCP also in laboratory. A lag period of 15 to 20 days pre- ceeded the onset of mineralization of PCP in the

pilot compost samples, while samples of the 50 m 3 windrow started PCP mineralization with- out lag; 30--40% of the added PCP was mineralized into CO2 in 40 days b y the field compost samples.

No mineralization of PCP was observed in the sterilized soil. Adding 109 chlorophenol-mineralizing Rhodococci per g to the sterilized soil at day 0 or day 23 slowly initiated degradation leading to mineralization of 0.018 m g d a y -1 and 0.023 mg day -1 of 1 4 C - P C P respectively, equivalent to 0.03 mg d a y - 1 kg - ~ dry wt. The soils taken from 50-m 3 windrow and 1.5-m 3 composts (unsterilized) mineralized PCP even better, 0.07--0.09 m g d a y -a , equivalent to 0.1 m g d a y -1 kg -~ dry wt.

Table 3 and Fig. 4 show that the other chlorophenols (2,3,4,6-tetrachlorophenol and different isomers of trichl0rophenol and dichlorophenol) also were efficiently removed in jars 3, 4 and 5, which contained unsterilized soil from 50-m 3 windrow and 1.5-m 3 field composts. Mineralization to CO2 could not be tested because the respective 14C-compounds are not available. A part of chlorophenols seems to have become immobilized in the soil because only 80--90 mg kg-~ of chlorophenols were recovered after sterile incubation of 68 days of the input of 120 mg kg -1 on day 0. No significant increase in the amounts of chloroanisoles was observed during the incubation (Table 3, Fig. 4) (Sum 5 to 6 mg kg -1 in sterilized sample, 1 to 5 mg kg -a in unsterilized sample). Me- thylation of chlorophenols was thus not a major route of chlorophenol removal.

Figure 4 shows that the amount of PCPPs remained the same as in the sterilized uninoculated soil in all composts, indicating resistance of PCPPs to microbial attack. The contents of PCPP was high (30 mg kg -1 dry wt.) in the composts, equal or higher than that of chlorophenols at the end of composting after chlorophenols were de-

Table 2. Numbers of PCP-degrading and colony forming bacteria in various composts and soils a

Compost Compost /Soi l Total colony PCP-mineralizing Organic matter number count g - I dry wt. microbes g - ~ dry wt. % of dry wt.

3 50-m 3 Windrow 7.2 × 107 5 ::< 106 19 4 1.5-m 3 Inoculated pilot compost 8.7 × 10 7 4 × 105 15 - Sawmill A soil, gravel

(Valo et al., 1984) 1.2 × 10 6 4.5 × 10 4 2 - Agricultural soil, clay 1.2 × 10 7 1.5 × 10 2 9

Composts 3 and 4 were sampled in October 1984, sawmill A soil (containing 150 mg kg - I of chlorophenols) was sampled in May 1984 and the agricultural soil having no previous contact with chlorophenolic compounds was sampled in September 1985. PCP-mineralizing microbes were counted by MPN-method and total colony counts on Standard Plate Count Agar (Standard Methods 1985)


O STERILIZED COtdPO~[ SOL A STERILIZED ~ T SOILj ~ L I L A T E D AT 0 D • STERILIZED COIMPOST SOL, INOCULATED AT 2~ D

" 1 /+ O SOIL FROM 1.5 m3 PLOT COMPOST / O

30,, 2 > .......O.----O

/

I o.O, / / I ~ t ' L _ ~ - -o -I_, m~ ~ .o 9,

10 20 30 /+0 DAYS

Fig. 3. Mineralization of pentachlorophenol in technical chlorophenol formulation during laboratory composting. Each jar (Fig. 1) contained 1 litre of the field compost from October 1984; 120mg of technical chlorophenol/kg d wt (as Ky-5) were added to every jar together with ' 14C-PCP (500000 cpm) and evolution of ]4CO2 was followed for 40 days

graded. Most of the 30 mg kg -1 of PCPPs was present in the compost samples before the laboratory experiment was started, because the Ky-5 added (120 mg kg-~) contained only 2 mg of PCPPs kg- 1 as impurity.

mg. kg-ldw 80 -

60

40.

1 2 3 4 . 5 POL YCHLORINATEO

PHENOLS

i STERILIZED COMPOST SOIL

2 STERILIZED COMPOST SOIL, INOCULATED AT 0 D

) SOIL FROM 50 m} WINDROW

tL SOIL FROM 1.5 m3 INOCULATED PILOT COMPOST

5 SOIL FROM 1.5 m] PILOT COMPOST

] METHYLATED COMPOUND

1 2 3 / . , 5 POLYCHLORINATEO

PHENOXYPHENOLS

Fig. 4. Sum of chlorinated phenols, chlorinated phenoxyphenols and their methylated analogs in various composts after 68 days of laboratory incubation

Table 4 shows the contents of main PCPP- congeners and their methylated analogs in compost soil. In all cases the amount of PCPAs remained at the level of 1 to 5 mg kg-1. The results show that PCPPs were not biomethylated into PCPAs to any significant degree during the incubation of 68 days.

Table 3. Chlorophenol and chloroanisole composition of composts after 68 days of laboratory incubation S

Compost 2,6 2,4 2,4,6- 2,4,5- 2,3,4- 2,3,4,6- Compost DCP DCP TCP TCP TCP TeCP number per cent of total

PCP Total

mg kg - 1 dry wt.

Chlorinated phenols 1 Sterilized compost

soil 0.2 2.2 5.8 0.7 2 Sterilized compost soil, inoculated at 0 D

0.1 5.4 1.6 3 Soil from 50-m 3 windrow

5.5 7.8 3.2 4 Soil from inoculated 1.5-m 3 compost

0.5 4.1 4.6 0.5 5 Soil from uninoculated 1.5-m 3 compost

7.3 4.2

Chlorinated anisoles 1 Sterilized compost

soil 21.9 32.8 2 Sterilized compost soil, inoculated at 0 D

22 16 3 Soil from 50-m 3 windrow

8.3 25 4 Soil from inoculated 1.5-m 3 compost

11.8 29.4 5 Soil from uninoculated 1.5-m 3 compost

3.8 23.1

0.2 81.7 9.2 83.4

0.2 82.2 10.5 83.1

0.5 80.2 2.8 21.7

0.5 74.3 15.6 21.8

71.9 10.7 9.6

12.5 31.3 1.6 6.4

18 40 4 5.0

< 1 58.3 8.3 1.2

13.7 33.3 11.8 5.1

19.2 38.5 15.4 2.6

At day 0, 120 mg of technical chlorophenol mixture (Ky-5) was added to each of 5 jars. DCP, dichlorophenol; TCP, trichlorophenol; TeCP, tetrachlorophenol; PCP, pentachlorophenol


Table 4. Polychlorinated phenoxyphenols and polychlorinated phenoxyanisoles in laboratory composts after 68 days of composting. Structures of compounds 1--VIII are shown below.

Compost I II III IV V VI VII VIII Total Number per cent of total mg k g - 1 dry wt.

Polychlorinated phenoxyphenols 1 Sterilized compost soil 9.2 8.8 7.1 13.6 10.5 2 Sterilized compost soil, inoculated at 0 D

7.4 7.8 7.8 14.2 8.9 3 Soil from 50-m 3 windrow 9.3 9.0 9.3 8.7 7.2 4 Soil from inoculated 1.5-m 3 compost

9.0 8.1 7.1 13.7 7.6 5 Soil from uninoculated 1.5-m 3 compost

2.4 2.7 44.2 15.2 10.1

Polychlorinated phenoxyanisoles 1 Sterilized compost soil 16.7 24.1 7.4 35.2 11.1 2 Sterilized compost soil, inoculated at 0 D

9.1 18.2 9.1 18.2 27.3 3 Soil from 50-m 3 windrow 14.3 14.3 14.3 14.3 14.3 4 Soil from inoculated 1.5-m 3 compost

11.8 17.6 5.9 17.6 29.4 5 Soil from uninoculated 1.5-m 3 compost

9.1 18.2 9.1 36.4 27.3

6.1 23.5 21.1 29.4

7.4 25.9 20.6 28.2 8.7 18.7 29.0 32.1

7.6 24.6 22.3 21.1

3.9 15.5 6.0 33.5

5.6 5.4

18.2 1.1 14.3 14.3 0.7

17.6 1.7

1.1

Structures of PCPPs analyzed in the composts. PCPAs differ from PCPPs by replacement of a hy- droxyl by a methoxyl

CL CL

CL OH CL

I

CL CL

CL - ~ } - 0 - ~ CL

CL OH CL

IV \

CL CL CL

CL OH CL

Vll

CL CL CL CL CL

CL CL CL OH CL

II I l l

CL CL CL CL CL CL

CL CL CL CL

V Vl

CL CL CL CL

CL CL

VIII

Table 5. Volatilization of chlorophenols (CP) and chloroanisoles (CA) in 68 days of laboratory scale incubation a

Compost Exhaust chlorophenols Exhaust chloroanisoles Number l.tg % of input btg % of input

1 Sterilized compost soil 168 0.2 58 10 2 Sterilized compost soil,

inoculated at 0 D 111 0.1 112 2.1 3 Soil from 50-m 3 windrow 50 0.2 18 1.5 4 Soil from inoculated

1.5-m 3 compost 112 0.5 83 1.6 5 Soil from uninoculated

1.5-m 3 compost 98 1.3 31 1.2

CAs and CPs were t rapped in XAD-4 resins (Fig. 1) and the results given as micrograms and as percent of the amounts extractable from each jar after 68 days of incubation. The XAD-4 resin was found to trap the volatile phenolic compounds completely, as nothing was found in the NaOH-flasks (Fig. 1). Sum of all CPs and CAs are included. Input CP was 120 mg kg -1


Table 5 shows that loss of chlorophenols by volatilization was insignificant compared with biodegradation. In all but one case, less than 1% of the input chlorophenol (120 mg kg-1) escaped in 68 days by volatilizing. The chloroanisoles were somewhat more volatile, as 1--2% of those present in the composted mass was later recovered in the adsorption resin. No dimeric compounds were found to evaporate.

Discussion

Chlorophenol degrading microbes are frequently found in soil (review by Kaufman 1978; Wata- nabe 1977). We found that at a CP-contaminated site (150 mg CP kg -1 of soil) the number of CP- degrading microbes was 4.5 x 104 g-1, which was up to 4% of the total colony count, whereas in agricultural soil with no known previous exposure to CPs their share was only 0.001% (Table 2). Sim- ilarly high numbers (105 g-1 of soil) of CP-degraders were reported by Watanabe in agricultural soil after three, annually-repeated exposures to CP (Watanabe 1977). Despite the fact that CP- degrading microbes thus occurred in CP contaminated soil, these organisms did not degrade the CPs in situ or, if they did, the rate of degradation was inadequate to cope with soil contamination.

In our previous study (Valo et al. 1985a) we showed that CP-degraders of a mixed culture iso- lated from the environment required oxygen, suf- ficient nutrients (especially nitrogen), suitable pH, adequate temperature and moisture for activity. Since some PCPP components are bactericidic, e.g. 5-chloro-2-(2,4-dichlorophenoxy)phenol, trade name Irgasan DP 300, a commercial disin- fectant, PCPP compounds may inhibit biodegradation of chlorophenols. Arsene-, chromium- and copper-compounds contained in some wood preservatives may also be fatal for the activity of CP- degrading microbes.

In this paper we have shown that conditions for activity of chlorophenol-degrading microbes could be created in the field and that these can lead to 80% removal of CPs within 4 months (Ta- bles 1, 2, 3). The first two months of composting were most effective, with a half-life for chlorophenols of 25--50 days (Fig. 2).

Addition of chlorophenol-degrading bacteria to sterilized soil speeded up degradation in the laboratory. In the field test we added 1 x 105 CP- degrading bacteria g-1. This was too low to in- duce any observable effect Since the total number of chlorophenol degraders in the soil to be com-

posted was 4 x 105. This result was, however, not available at the moment when the windrows had to be constructed.

The importance of organic matter for degradation of chlorophenols was demonstrated by Ku- watsuka and Igarashi (1975) who observed no degradation in soil with 0.04% of organic matter, while in soil containing 7--8% of organic matter, over 90% of input PCP (100 ppm) was degraded under otherwise identical conditions. Bark chips may promote CP-degradation by several different mechanisms: bark was shown to protect CP degraders against toxicity of CP (Apajalahti and Salkinoja-Salonen 1984); it provides for organic matter (temperature maintenance of compost); it may serve as a source of CP degrading microorganisms (Salkinoja-Salonen et al. 1983) and pro- motes aeration in the compost.

Temperature maintenance is important in a cold climate like that of Finland, where the temperature of the surface soil reaches about + 16°C in July, falling to below zero in the winter. When the contaminated soil was built into compost heaps, its temperature rose 10-- 15 ° C above that of the surrounding soil. Crawford and Mohn (1985) found no mineralization of PCP in soil inoculated with Flavobacterium (PCP degrader) at +12°C or +40°C. Our previous experience shows that CP-degradation ceases when the temperature is below 8°C (Valo et al. 1985a).

Edgehill and Finn (1983) added Arthrobacter (106 g- l ) to chlorophenol-containing soil (34 mg PCP d m - 3 ) ; this resulted in 85% disappearance of PCP in 12 days at 8--16°C comparred with 30% disappearance in uninoculated soil. Craw- ford and Mohn (1985) decontaminated PCP-containing soil (10--300 ppm of PCP) by adding F/a- vobacterium (103--106 g - l ) , but 500 ppm of PCP was toxic for the bacteria with no degradation. In both studies the residual concentration of CP in the treated soil was 10--30 mg kg-1, and a similar figure was obtained in our study. The reason why the microbial decontamination process halted at this level is not known.

Methylation of 2,3,4,6-tetrachlorophenol by several fungi has been reported (Gee and Peel 1974) and methylation of PCP to pentachloroanisole (PCA) in soil was shown by Murthy et al. (1979) together with demethylation of PCA to PCP. We found about 5 mg kg -1 of chloroanisoles in the CP-contaminated compost soil. Dur- ing the study period, however, a total of 200-- 300 mg of CPs kg- 1 was removed, so that biomethylation appears not to have been the major route of CP removal. Chlorinated anisoles may be


harmful, because of their tendency to bioaccumu- lation (Lech et al. 1978).

PCPP compounds were not observed to decrease at composting. These compounds (including other dimeric impurities of technical chlorophenol) are sparsely soluble in water and there- fore tend to stick onto soil rather than leach (Ki- tunen et al. 1985; Valo et al. 1985 b) to ground water. As the CP concentration went down at the end of our experiments, the composts contained as much PCPP compounds as chlorophenols.

We found a significant amount of PCPAs in compost soil (PCPP:PCPA about 6, Table 4). These compounds were not found in the commercial wood preservative and may thus have origi- nated from PCPPs by slow rate (bio)methylation. Humppi (1985) found PCPAs (0.4 ppm) in Ky-5 contaminated soil, where the ratio PCPP:PCPA was about 60. A biological methylation product of PCPP (Irgasan) was found in fish and shellfish collected from Tokyo Bay (Miyazaki et al. 1984).

Acknowledgements. The authors are grateful to Dr. Tarmo Humppi (University of Jyv~iskyl~i) for synthesis of PCPP and PCPA model compounds; Prof. Dr. M. Fischer (Institut for Wasser-, Boden- und Lufthygiene des Bundesgesundheits- amtes, Berlin, FRG) for his gift of 14C-labelled PCP; Sinikka Marmo for metal analysis; Veikko Kitunen for help in GLC- analysis; Ekokem OY of use of their equipment; Enso-Gutzeit OY and Martti Tolonen for help in field scale composting. This work was financially supported by the Finnish Work and Environmental Fund and Maj and Tor Nessling Fund.

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Received February 5, 1986/Revised May 28, 1986

bioreclamation of chlorophenol-contaminated soil by composting

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