Microbial degradation by the autochthonous soil population of alpha and beta HCH under anaerobic field conditions in temperate regions

Chemosphere, Vol.17, No.2, Printed in G r e a t B r i t a i n

MICROBIAL

DEGRADATION

pp

481-487,

1988

BY THE AUTOCHTHONOUS

ANAEROBIC

0045-6535/88 $3.00 + .00 Pergamon Journals Ltd.

SOIL POPULATION

FIELD CONDITIONS

OF ALPHA AND BETA HCH UNDER

IN TEMPERATE

REGIONS*

P. Doelman, L. Haanstra & A. Vos

Research Institute

for Nature Management,

Department

of Environmental

Pollution Research

ABSTRACT

Microbial soil sanitation technology was applied to calcareous, alkaline, sandy loam soil, polluted with a mixture of hexachlorocyclohexane isomers (alpha 400; beta 230; gamma 15 and delta 17 mg.kg- ). Under anaerobic conditions microbial degradation in the Dutch climate (soil temperature 5-17°C) failed, and even the low concentration of the easily degradable gamma HCH failed to be affected. The well homogenized soil contained a normal number of bacteria and in the laboratory (at 9 and 20°C) proved to possess a sanitation potential for alpha HCH, the main pollutant. The open literature mainly refers to anaerobic degradation of low concentrations of gamma and alpha HCH, though.sometimes also to that of beta HCH. For degrading higher concentrations (>400 mg.kg -i ) in lower temperature regions aerobic soil conditions are preferable. It was concluded that the failure of microbial sanitation in the field was due to combined environmental conditions of anaerobic soil plus low soil temperature. INTRODUCTION

Of the four hexachlorocyclohexane degradation research. equivalent peratures

of the gamma isomer,

Its degradation

isomers

(alpha,

beta, gamma and delta)

which is the pesticide

at concentrations

applicable

Lindane,

the microbial

has been subject to most

in agriculture

(I-6 kg.ha -I being

to 1-15 mg.kg -1 of soil) was studied mainly under anaerobic conditions between 20 and 37°C (I). Aerobic or semi-aerobic

investigated

(2). Anaerobic

at 30°C was recorded anaerobic degradation

degradation

conditions

of 15 mg.kg -I of alpha, beta, gamma and delta HCH

by McRay et al. (1967). Siddaramappa

and Sethunathan

(1975) measured

of alpha and beta HCH in five soil types at 28°C. Addition of orga-

nic matter both accelerated

(4; 5) and retarded

(4; 6) degradation

of the four isomer

types.

*Research was made possible Planning

at tem-

had scarcely been

and Environmental

through a grant from the Ministry Management.

481

of Housing,

physical

482

In alkaline soils with a very low organic matter content gamma HCH at a concentration of I00 mg.kg -1 was degraded to half value at 25°C under flooded and non-flooded conditions after 35-46 days (7). In heavily polluted HCH soil (5300 mg of alpha HCH per kg-l),

at 25°C, d e g r a d a t i o n of

alpha HCH by 55 and 35% was measured under aerobic (moist) and anaerobic (flooded) conditions respectively during 20 weeks

(8).

The above data on microbial d e g r a d a t i o n of HCH isomers,

the presence of tens of

thousands of cubic metres of mainly alpha and beta H C H - p o l l u t e d soil, and the expectation that microbial s a n i t a t i o n is e c o n o m i c a l l y preferable to both soil combustion and soil extraction,

have led to field experiments.

the soil appeared to be very heterogeneous. extra homogenization,

In the field plots the HCH distribution

through

Therefore the aim was to study the effects of

aeration and the addition of organic matter, on the microbial degra-

dation of HCH isomers under Dutch climate conditions.

Since large quantities

of soil can

better and e a s i e r be h o m o g e n i z e d as a w a t e r - s a t u r a t e d slurry, and since anaerobic conditions are p r e p o n d e r a n t l y flooded (anaerobic)

referred

to in the literature,

the experiments were started with

soil.

MATERIALS and METHODS H C H - p o l l u t e d soil was obtained excavated,

from a polluted soil storage yard. After having been

the soil had been stored, wrapped in plastic foil and provided w i t h a soil

cover for six years. The soil was a sandy loam with a pH-H20 of 7.6, an organic matter content of < I%, a Ca content of 30% (caused by dumping lime together with the HCH),

a

water content at field capacity of 14-18%, and alpha, beta, gamma and delta HCH contents of approx.

400, 250,

15 and 17 m g . k g -I respectively. The soil was collected,

and enriched with n i t r o g e n (0.1%) and phosphorus thirty kg of soil (Schnellkutter,

sieved (8 mm)

(0.05%). In a food h o m o g e n i z e r containing

TNO-Apeldoorn),

three units of soil and one unit of

water were mixed for i0 min (at i000 rpm). In some tests extra homogenization was obtained by passing the material through a 4-mm sieve. In some tests extra organic matter was given by adding 0.2% of powdered wheat

straw (see Table i; the codes H, A and 0 are used consis-

tently in the tables). 90 kg of w a t e r - s a t u r a t e d ners

(I00 x 35 x 35 cm) to prevent

soil (slurry) was put in plastic contai-

leakage of intermediates to groundwater. The samples

were buried in soil to a depth of 90 cm. During the first two months aeration of the slurry by air pressure through single hole tubes failed to succeed. Starting from the third month the slurry was aerated through thirty-hole schedule,

tubes. Table I gives the treatment

all tests being carried out in duplicate.

Table I. Treatment schedule of the field experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Container

Extra h o m o g e n i z a t i o n

(H)

Aeration

E x t r a organic matter

(A)

(0)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I III V VII IX

& & & & &

II IV VI VIII X

+ + +

+ +

+ + +

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

483

The field experiment was started in the beginning of April, and the ten slurry containers were sampled six times using five replicates. The temperature was recorded at regular intervals. Soil samples for HCH determination were taken 5, 11, 19, 25 and 30 weeks after the start of the experiment. To determine the contents of the four HCH isomers the soil was air-dried and ground,

25 g of soil was shaken with 50 ml of toluene for 2 h. This

fluid phase was diluted with hexane and used for HCH determinations gas-chromatographic

conditions:

in GC-ECD. Relevant

glass column 3 m, diameter 4 mm with 2% OV-17½% QF-I on

GC-Q~ temperature of oven 200°C, carrier gas N 2 (45 ml.min), make-up N 2 (15 ml.min-l). The numbers of bacteria in the containers were determined by the miniaturized Most Probable Number method (9) using the nutrient medium of Bunt und Rovira (1955). From the untreated soil used in the field experiment subsamples were taken for laboratory testing (25 g of soil in glass bottles) at a constant temperature of 9 and 20°C to verify the degradation ability of the soil.

RESULTS

and D I S C U S S I O N

The average slurry temperature (°C) at a depth of 15 cm is given in Figure I.

20i 10 ~ .........

~ .............

~. . . . . . . . . . . . . . . . . . . . . . . . . . . .

......V

.

.

.

.

.

.

.

0 IT~,T,rFT~'--r~,,T,JJr~-T~=~I~r'I,ITTr~Tll 1957

1958

Tr A V - - T T I ' ~ T ' ' ~ r l 1959

1985

Year Fig. I. Monthly average soil temperature in Tubbergen (1985) compared with other years in the same area (I0)

The soil temperature curve for 1985 was normal for the Dutch climate,

compared with the

data from Witkamp & Van der Drift (1961). Only during approximately 90 days the temperature tended to be slightly higher than 15°C, while during another 120 days it ranged between 5 and 15°C. After ii weeks the temperature reached levels above lO°C and around the 19th week the temperature reached 15°C. Table 2 gives the alpha HCH level and standard error during thirty weeks. The alpha HCH content at the beginning of the experiment varied between 363+23 and 457~57, and after 30 weeks this was between 372+27 and 442+17 mg.kg-l° First, the relatively small standard errors

indicate well homogenized

degradation occurred.

and therefore comparable soil systems, and secondly no

In similar soil,

alpha HCH concentration,

from the same problem area with a 13 times higher

both anaerobic and aerobic degradations

occurred at 25°C (8). In

the present study the soil temperature is lower (maximum 17°C, mostly between 5 and 15°C) and the scale is much bigger (90 kg in the field versus 25 g in the laboratory).

484

Table 2. Average alpha HCH level (n = 5) (mean ~ standard error) for 30 weeks .

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Treatment .

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Time in weeks

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5

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11

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19 .

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25 .

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30 .

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384+21

374+12

363+9

312+8

378+8

413+8

-

395+14

381+24

370+5

410+110

396+9

442+17

H

389+17

340+17

370+15

330+4

374+4

392+14

-

H

387+13

318+12

359+32

326+25

357+4

390+12

A+O

394+72

341+11

370+20

361+8

390+32

372+27

A+O

412+34

382+57

382+32

386+8

377+34

401+24

H+O

363+23

335+16

388+8

374+17

382+15

426+22

H+O

370+15

351+11

374+14

346+10

375+9

373+12

A+H+O

410+19

381+29

395+13

372+8

389+22

394+12

A+H+O

457+57

387+14

378+9

369+12

373+12

408+8

In laboratory studies in soil (2) and in aerated thin slurry (13) complete degradation wa~ proven by measuring their complete dechlorination.

In this field experiment, however,

the

slurry (aerated flooded soil) never reached an aerobic stage for a long time. Technical problems prohibited the continuous aeration of slurry. Table 3 shows that within 24 hours after stopping the aeration the aerobic system (from oxygen saturation level of 10-12 mg.l -I) changed to anaerobic.

Table 3. Course of oxygen content (mg.l -I) in slurry after stopping aeration .

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Treatment .

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A+O

11.2

II.i

8.4

0.i

A+O

8.5

3.2

1.2

0. I 0.1

9.2

7.8

3.4

A+H+O

10.0

9.6

6.9

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24

A+H+O

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Time in hours

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0. I .

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Since the aeration factor did not meet the expectations,

the whole experiment was further

carried out anaerobically. Figure 2 shows the average concentration curves for the ten containers of all four HCH isomers in the course of time. The HCH concentrations slightly fluctuate around their original values. The curves for alpha and beta are relatively high (400 and 230 mg.kg-l),

those for gamma and delta being relatively low (15 and 17 mg.kg -I)

Besides a low temperature and lack of oxygen a third limiting factor could have been an insufficient microbial biomass in the slurry. Even the easily degradable gamma isomer was not affected, which was unexpected and shows that there is a big gap in knowledge of the degradability of compounds at high and low temperatures.

For the same soil,

though as a

485

1 2003 ........ + . . . . . . .

#. . . . . . . . . .

+ . . . . . . . + . . . . . "+ /3-HC H

E co

I00-

rco -7

2sl

"r 2o

"~- H C H

!

1

5

IoI

~

]

i

'

......

1

~5 HCH

3~0

315

, ...... . ...... + ........ ¢"

0

5

li0

1'5 210 Time{ weeks}

25

Figure 2. Alpha, beta, gamma and delta HCH concentrations

thin slurry,

in flooded soils

Bachmann et al. (1987) showed a tendency towards generally,

of the slurry under anaerobic The numbers of micro-organismes

conditions,

which indicated

(Table 4) in the containers,

with the average numbers of micro-organisms

per unit weight

however,

of aerobic and facultative

(per g) in slurry with various

clearly correspond

anaerobic micro-organlsms

treatments

(6 = 106 )

......................................................................... H H A+O A+O H+O H+O A+H+O A+H+O ......................................................................... aerobic

5.7

6.2

7.0

7.7

6.5

7.5

7.9

7.0

7.3

toxicity

activity.

of normal unpolluted

(106-10 8 ) (12).

Table 4. Numbers

higher

a general microbial

7.4

facultatlve anaerobic 6.3 6.5 7.7 7.0 7.0 7.4 7.7 7.0 8.0 7.9 ........................................................................

soils

486

The total number of micro-organisms

is no ~ a r a n t e e

that a specific HCH degradation will

have been achieved at the end of the field experiment. Therefore, subsamples of 25 g were taken to the laboratory to which 25 ml of H20 was added,

incubated at 9 and 20°C.

Laboratory test were carried out under four main conditions,

i.e. flooded,

permanently

aerated, anaerobic (under N 2 pressure) and alternately in cycles of two days of aeration and [2 days without aeration. After 24 weeks the alpha HCH concentrations were determined six times using. The results are given in Table 5.

Table 5. Alpha HCH concentration after 24 weeks at 9 ° and 20°C .................................................................... Flooded Aerated N2 An/ae (6/I) ............................................................... 20 ° 9° 20 ° 9° 20 ° 9° 20 ° 9°C .................................................................... to

364

t24

3 7 6 + _ 1 7 360~28

364

364

364

364

364

364

364~I0

37~6

174~4

360~i0

352~16

90~19

276~72

......................................................................

The data of Table 5 prove that the soil is able to degrade alpha HCH, so the microbial population is present, but the environmental field conditions their activity. Under anaerobic conditions

of this experiment

inhibit

(flooded or N2-pressure ) degradation did not

occur at 9° or 20°C, whereas under aerated conditions at 20°C and even at 9°C alpha HCH was degraded,

although to different

extents:

37 and 174 mg.kg -1 respectively.

In the

intermittently aerated soil slurry degradation also occurred (to 90 mg.kg -I) and even slightly at 9°C (to 276 mg.kg-l). So these HCH-polluted

soils, after having been stored for years without extra nutrients

and water, had still maintained their purifying power for alpha HCH, but the environmental conditions in the first field trial were not appropriate to achieve a successful sanitation.

CONCLUSION In temporate areas such as the Netherlands

the low temperature

degradation of alpha HCH. HCH-polluted soils,

inhibits the anaerobic

stored under unfavourable conditions,

maintain an HCH-degrading population which revives as soon as the proper environmental conditions,

such as aeration and a higher temperature,

are obtained.

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1983-1987.

P. Duraisamy, M. Balasubramanian

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(Received

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W. Roelofsen,

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