Biodegradation of 3,4,4′-trichlorocarbanilide, TCC®, in sewage and activated sludge

Water Research Vol. 9, pp. 649 to 654. Pergamon Press 1975. Printed in Great Britain.

BIODEGRADATION OF 3,4,4'-TRICHLOROCARBANILIDE, TCC ®, IN SEWAGE A N D ACTIVATED SLUDGE W. E. GLEDHILL Monsanto Co. St. Louis, Missouri, U.S.A. (Received 30 August 1974) Abstract--As part of the studies to elucidate the environmental consequences from bacteriostat usage the extent of biodegradation of 3,4,4'-trichlorocarbanilide, TCC ®, in sewage systems was examined. TCC sampies uniformly labeled in either the p-chloroaniline ring (xac PCA TCC) or the dichloroaniline ring (~4CDCA-TCC) were monitored in activated sludge systems by measurements of ~4CO2 evolution. As was expected, the p-chloroaniline (PCA) ring of TCC was more rapidly degraded than the dichloroaniline (DCA) ring. In a continuous flow activated sludge system (10 h retention time, 200 #g 1- ~ TCC) acclimation to primary biodegradation was readily gained. ~4CO2 evolution from 14C-PCA TCC was consistent with complete metabolism of the PCA ring while that from ~4C-DCA-TCC indicated about 50% biodegradation of the DCA ring. Analysis of effluents from continuous flow activated sludge units established that TCC undergoes primary biodegradation to its chloroaniline components which are in turn biodegraded.

INTRODUCTION

T C C ®, 3,4,4'-trichlorocarbanilide (Compound 1), is employed exclusively as a bacteriostat in bar soaps. The compound shows bacteriostatic activity towards Gram-positive bacteria, such as staphylococci, in the 100-200ppb range, while similar activity towards Gram-negative species and fungi generally requires 1000 ppm. The major route for T C C disposal is, therefore, through domestic sewage into natural waters either with or without intermediate waste treatment. Because of the rising numbers of various minor chemicals in waste water it is becoming increasingly more important to establish their environmental impact. In this vein an investigation of the extent of T C C biodegradation in sewage and activated sludge was undertaken. ct

Compound l. Studies of the biodegradation of chemicals with structures similar to T C C have been restricted primarily to the plant herbicide area. Numerous chlorophenyl substituted urea and carbamate herbicides have been found to be readily decomposed in soil through their chloroaniline intermediates. The chloroanilines in turn either undergo complete biodegradation (Geissbuhler, 1969; Herrett, 1969; Kauffman and Blake, 1973; Kearney, et al., 1967; Kearney and Kaufman, 1971) or detoxification via condensation reactions (Alexander and Lustigman, 1966; Bordeleau and Bartha, 1972a; Burge, 1972; 1973; Tweedy, et al., 1970). If biodegradTCC ® is a registered trademark of Monsanto Company. 649

able, T C C would be expected to be metabolized in a manner similar to these plant growth regulators. However, the mechanism by which T C C is metabolized is currently unknown and is also a subject of this investigation. EXPERIMENTAL

Test materials Two 14C-labeled T C C products, TCC uniformly labeled in the p-chloroaniline ring (14C-PCA-TCC), specific activity 13.8 m Ci m-mole- 1, and TCC uniformly labeled in the 3.4-dichloroaniline ring (14C DCA TCC), specific activity 4.5 m Ci m-mole-I, were synthesized by Mallinckrodt Chemical Company (St. Louis, Missouri). Uniformly labeled glucose (U-14C-Glucose), specific activity 257M Ci m-mole (International Chemical and Nuclear Corp., City of Industry, California) was also employed for comparative purposes. Test units and procedures (a) Shake flask system. Figure 1 depicts the shake flask apparatus designed to study ultimate biodegradation (~4CO2 evolution) of TCC in sewage and activated sludge. Aqueous solutions, 100 ml, were incubated in 300 ml Bellco baffled Erlenmeyer flasks which were sealed with a number 7 rubber stopper containing a 15 × 12.5 cm screw cap test tube and an air inlet tube. The rest tube contained a 1 cm hole just below the rubber stopper and 3.0 ml of 0.5 N KOH. After the desired concentration of TCC was added the flasks Aeration tube Rubber stopper 1 ' 5 x 12.5 cm tube 1.0 cm hole 3 ' 0 rn!, - - 0 " S N

KOH

IOOmL activated sludge

Fig. 1. Shake flask system employed for activated sludge die-away tests.

650

W.

S

~

[!. GLIi|)H1LI

Effluent

('ompound 2.

Ct-~N 64C02 t r a p s -

=N~Ct

15 ml

('ompotmd 3.

M e t h y l cellosolve : 7 Monoet hanolamine

I

feed

Settling area 7 5 m (

A

Influent

30mr

h -t

B

Activated sludge-$OOmt-MLSS

C

CO~ f r e e

4 0 0 0 pprn

air inlet

Fig. 2. Continuous flow activated sludge units for monitoring ~'~CO, evolution from 14Cqabeled TCC.

were sparged with a 70--30 0 2 N2 mixture, sealed, and incubated on a rotary shaker at room temperature (18 20°C). Periodically. the KOH from the center tube was removed, fresh base was added, and the flasks were resparged with 70°~i O2. Scintillation counting was performed by adding 1.5 ml of the KOH from the center tube to 15 ml of Instagel (Packard Instrument Co., Downers Grove, Ill.) in a counting vial. The effect of chemiluminescence was eliminated by permitting samples to stand for at least 48 h at room temperature before counting. Counting was conducted on a Nuclear Chicago Isocap 300 counter with external standardization. Corrections for background and chemical quenching were made. (b) Continuous flow activated sludge ( CFAS) system. Ultimate biodegradation of TCC in CFAS involved a modification of the biodegradation apparatus of Swisher, et al. (1964) shown in Fig. 2. Influent feed consisted of raw sewage which was obtained weekly from the Sugar Creek wastewater treatment plant, filtered through Whatman Number l filter paper and refrigerated in an ice chest. The influent feed rate. 30 ml h-x (10 h retention time in the activated sludge), and effluent removal were controlled by use of a Manostat cassette pump (Manostat, New York, N.Y.). MLSS were maintained at 4000 mg 1 ~. The units were aerated with CO,-free air (0.05 SCFH) and exit gas was passed through a series of 3 tubes containing 3 mm glass beads to disperse air bubbles and 15 ml of a methylcellosolve: monoethanolamine (7:1) absorbent to trap ~4CO2. Daily the first >*CO2 trapping tube was removed, the two end tubes moved forward, and a fresh tube replaced on the end. The volume of absorbent in the tube removed was ac[iusted back to its original weight by addition of fresh absorbent. Radioactivity was measured by transferring 1.0 ml of absorbent to 15 ml of Instagel followed by scintillation counting. Radioactivity of feed, settling chamber and effluent was measured daily with 1.0 ml samples in lnstagel. Periodically, radioactivity of the washed activated sludge was assessed by the combustion method of Peterson. ct al. (1969). Three such CFAS units were employed for this study. One received 200#g 1-~ unlabeled TCC in the influent raw sewage, another 200 #g 1- ~ 14C -PCA TCC (0.631 l* Ci 1 J). and the third 200#gi ~ ~'*C DCA TCC (0.2071~ Cil tl. The 200/~g I ~ level of TCC was chosen since, based upon the quantity manufactured, this would be the maximum amount anticipated in sewage. (c) Enzymology, thin-layer chromatography and autoradio~traphy. Enzymatic preparation of aniline condensation products, azobenzenes, was carried out according to the procedure of Bordeleau and Bartha (1972b) using horseradish peroxidase (type II, Sigma Chem. Co.. St. Louis, Mo.). Enzymatic reaction with p-chloroaniline (PCA) yielded primarily two products. 4-chloro-4'-(4-chloroanilino)-azobenzene (Compound 2) and 4,4'-dichloro-azobenzene (Compound 3). which were identified using the above publication. The pro-

ducts were readily separated by thin-layer chromatography using Eastman 6060 silica gel sheets containing a fluoroscent indicator and either hexane: benzene: acetone (7: 3: 1) or n-butyl ether:hexane:acetic acid (80: 16:4). In addition the latter solvent system revealed the presence of 2 minor unknown reaction products from the PCA system. Only one product, presumably 3,3',4,4'-tetrachloroazobenzene, was detected from the dichloroaniline (DCA) system. All azobenzene reaction products traveled ahead of the chloroanilines in both solvent systems. Determination of intermediate degradation products from TCC metabolism was accomplished by daily extracting the effluent from the CFAS units with 100ml ethyl acetate per liter of effluent. Extraction flasks were shaken for 1 h. The ethyl acetate layer was separated and concentrated at room temperature by blowing nitrogen over the surface. Composite 2-week effluent extracts were concentrated to 2(X)itl and 10 ld were spotted on the thin layer chromatograms. Chromatograms were prepared and developed in duplicate, one set being visualizcd by autoradiography, carried out by exposing Kodac no-screen X-ray film (NS-2T) to TLC plates for 3-4 weeks, and the other quantitated after eluting chromatogram sections into Instagel. RESt LTS

AND i)ISCUSSIONS

TCC hiodeqradation in the actit~ated slud#e die-away ~sysfem

Figure 3 depicts the ratc of ~4CO2 evolution from 200 ~g I ~ ~4C P C A T C G in activated sludge and raw sewage using the shake flask apparatus. Activated sludge for this study was o b t a i n e d from a l a b o r a t o r y s e m i c o n t i n u o u s activated sludge unit that h a d been receiving only natural sewage as the daily feed. This activated sludge was adjustecl to a M L S S conc e n t r a t i o n of 1000 mg I ~ by a d d i t i o n o f a dilute minimal salts solution. B O D m e d i u m (American Public Health Assoc., et al., 1965). F o r c o m p a r a t i v e p u r p o s e s

g

JOG

--

90

--

m 6C

0

4c 3c

o zc I-

I0 2

4

6

a

10

Time, weeks

12

- e - 1 4 C- PCA -TCC - 2 0 0 ppb- activated sludge - A - 14C - P C A - T C C - 2 0 0 ppb- raw sewage

- -Fig.

U-14C - glucose - 5 0 0 ppb - activated sludge

3. Biodegradation of " ; ( PCA TCC U t4C glucose.

and

Biodegradation of TCC in sewage

O~ RtL)

40

30

o

zo io

o

oc 9o D 80

l

1

6

e

Time,

_,

,

~...-a----" •

O~ 60 ~U 50 _

• /,-

~o~

--A-- 200 ppb TCC

/

-4-- 2000 ppb TCC

/

bm,-~,k--;'-2 4

/

°°'°f/

/

//

60 50

=

,oo

'-I

90

651

I ,o

l ,2

#

l ,4

O

2

4

weeks

6 Time,

8 PO weeks

12

14

Fig. 5. Effect of TCC concentration on biodegradation of 14C~PCA-TCC in activated sludge.

--a- Lob I activated sludge

-•-- Lab 2 a c t i v a t e d sludge

mittent low levels or no TCC at all. This may arise because of the low water solubility of TCC. It may adsorb onto the primary sludge and undergo initial metabolism in the primary clarifiers with subsequent transport to the anaerobic digestors. The effect of TCC concentration on the rate of 14CPCA-TCC biodegradation in laboratory activated sludge is shown in Fig. 5. The results reported are those at a MLSS concentration of 1000 mg 1- *; however, activated sludge levels from 500-2500mg1-1 behaved similarly. Inhibition in the rate of biodegradation was noted at both the higher and lower TCC concentrations. Although the reasons for these results have not been established experimentally it is believed that inhibition at higher TCC levels may be due to its bacteriostatic effects, while that at lower TCC levels may be caused by TCC binding to the walls of the vessel or to that portion of the activated sludge not involved with TCC metabolism making it less available to those organisms concerned with its biodegradation. With regard to the latter the affinity of TCC for fresh activated sludge was assessed by adding 20 or 200 #g 1- 1 lac_PCA_TC C to various activated sludge concentrations in shake flasks. The flasks were shaken on a rotary shaker for 2 h at room temperature after which the suspensions were centrifuged and the residual dissolved TCC in the supernatant assayed. TCC at TCC/activated sludge ratios up to 0.4 #g TCC mgactivated sludge were efficiently bound ( > 90 per cent) to the activated sludge (Table l). A log-log plot of the /~g TCC adsorbed per mg activated sludge against the /tg TCC dissolved per liter, of solution obeys the Freundlich equation for adsorption by a bacterial system (Swisher, 1970) and yields a straight line, Binding

- - e - Fresh activated sludge - - Q - - Fresh activated sludge r a w sewage feed

Fig. 4. Biodegradation of 14C PCA TCC in different activated sludges. the biodegradation of 500/~g 1- 1 U ~4C-glucose in activated sludge is also shown. In activated sludge ~4CO2 production from TCC proceeded without an apparent lag with the rate during the first 2 weeks being about 70 per cent of that of glucose. In raw sewage a two-week lag occurred before significant TCC biodegradation was attained. Final CO2 evolution values of 80-90 per cent indicate substantially complete biodegradation of at least the p-chloroaniline (PCA) ring of TCC in the sewage and activated sludge systems examined. During this investigation differences were noted in the ability of various activated sludges (MLSS = 1000mgl -~) to metabolize TCC (200#g1-1) as is shown in Fig. 4. Activated sludges obtained from two laboratory semicontinuous units possessed the ability to rapidly metabolize the PCA ring of TCC without an apparent lag. On the other hand, the activated sludge obtained from a local treatment plant displayed an eight to 10-week lag before acclimation was gained. However, with suspension of the field activated sludge in raw sewage, instead of minimal salts medium, only a 2-week acclimation period was necessary. Differences between the laboratory and field activated sludges were unexpected since both sludge types were receiving essentially the same raw sewage feed. The apparent non-acclimation of the field activated sludge may be an indication that the field aeration tanks see only inter-

Table 1. Binding of TCC to activated sludge TCC TCC

Added

~g/1 Z00

Activated Sludge

mg/1

TCC/Act.

Sludge

Ftg/mg

0

Dissolved

Adsorbed

~tg/1

%

~g/mg

go

Z00

100

0

0

200

I00

Z

78

39

I.Z2

61

200

500

0.4

18

9

0.36

91

Z00

i000

0.Z

I0

5

0.19

95

200

2000

0. I

8

4

0.096

96

20

I000

0.0Z

1

5

0.019

95

652

W.E. GLEDHnl. (a)

I00'_

(b)

14C-PCA-TCC

~ 50

P4C-DCA-TCC

90

'ii 80

..-..-4

,or:

lot,, 0

-~ 40

112 2 4 6 Time,

8 I0 12 weeks --o--

0

°~" 2 4 6 Time,

8

I0 12 weeks

No supplement

..--~-- Glucose, molate, peptone , ethanol - 2 , I 0 , tO0 ppm weeRly ~ll~ p - chloroaniline, 2 p p m weekly

Fig. 6. Effect of co-substrate addition on biodegradation of ~4C-PCA..TCC and ~'*CDCA-TCC in activated sludge.

reduces the available soluble TCC and may explain the slower rate of TCC metabolism at the 20 #g 1- * TCC level in Fig. 5. Since in natural systems, such as activated sludge, readily degradable carbon sources would be present, the effect of supplemental carbon sources (co-oxidation) on the rate of biodegradation of TCC was examined. Figure 6 shows the comparative biodegradation rates of ]4C-PCA-TCC and 14C-DCA TCC in the shake flask system at a MLSS level of 625 mg 1- ~, and the effects of weekly supplemental feeding of readily degradable carbon sources to the flasks. Figure 6(a) indicates PCA ring biodegradation to be comparatively rapid, reaching 74 per cent within the 12-week period. Weekly addition of a mixture of glucose, sodium malate, ethanol and peptone, yielding a final concentration of 2, l0 or 100 mg 1- ~ each, gave ~4CO2 curves almost identical to this (not shown). The DCA ring of TCC degraded at a considerably slower rate than did the PCA ring. In Fig. 6(b), DCA ring biodegradation reached only 7 per cent of theoretical in 12 weeks with no co-substrate addition. Addition of the previously mentioned co-substrates resulted m a threefold increase in the rate. Weekly supplemental addition of 2 ppm PCA resulted in 44% >*CO2 from the DCA ring of TCC in this particular activated sludge. The results, therefore, indicate co-oxidation to significantly improve the rate of biodegradation of the DCA ring of TCC and to have no effect on the rate of biodegradation of the PCA ring.

TCC hiodegradation in continuous flow activated sludge units Because the previous experiments were conducted in the closed flask activated sludge die-away systems, they do not simulate natural conditions probably after the first few days due primarily to nutrient exhaustion. The shake flask data might indicate poor TCC biodegradation under actual sewage treatment conditions were retention times in activated sludge plants are generally less than 10h and activated sludge levels generally exceed 1500ppm. However, based on the

not-too-different rates of glucose and 14C PCA-TCC biodegradation in closed shake flask systems (Fig. 3), such extrapolations may not be valid. In order to examine TCC biodegradation under conditions more closely approximating the "'real world" situation ultimate biodegradation of TCC was investigated during a 3-month study in continuous llow activated sludge (CFAS) units (Fig. 71. Acclimation to biodegradation of the PCA ring of TCC was rapid, requiring 8 days to achieve a steady state 56'I,; '4CO2 evolution with the 10h retention time. CO_, evolution values of this magnitude are what would be expected if fairly complete biodegradation of a compound to CO2 and microbial cells were occurring. The DCA ring of TCC was slower to acclimate and ~4CO2 evolution averaged only 15 per cent for the first 40 days. At 41 days (arrow) the feed lines to the units receiving DCA and PCA-labeled TCC were switched so that the unit which had been receiving '4C PCA-TCC was now receiving ~4C-DCA TCC, and vice versa. During the last 50 days, 14CO2 evolution values from 14C" PCA TCC averaged 54 per cent while the corresponding values from ~4C DCA TCC averaged 26 per cent. These amounts are consistent with nearly complete metabolism of the PCA ring and perhaps 50 per cent of the DCA ring during the 10h exposure time. Table 2 depicts a material balance for the location of radioactivity during a 5-week period from day 50 to 85. Approximately 3 per cent of the ~4C-PCA TCC left the unit in the effluent while 30 per cent of the 14C DCA TCC label was found in the effluent. Similar percentages of radioactivity were associated with the activated sludges in each unit. The activated sludge-associated radioactivity presumably represents anabolic cellular products from TCC biodegradation; however, the possibility of adsorbed intact TCC or primary catabolites of TCC is also a possibility. In excess of 90 per cent of the radioactivity fed to the units during this time period was accounted lbr.

Metabolic products,tin'meal during TCC biodeqradatiot~ Identification of the metabolic products leaving the CFAS units was assessed by ethyl acetate extraction of the effluent followed by thin-layer chromatographic and autoradiographic analysis during a 2-week period of operation. Table 3, a representation of the thin layer autoradiographic plates (hexane:benzene:acetone, ioo r

[

90 "5 80 ~U 0

"~ ~4C-PCA - TCC ~- ~4C-DCA- TCC

l

60 50 40

"/I

ro

~'-I

20

30

I

]

40 50 60 Time, days

I

70

I

80

i°

9

Fig. 7. Biodegradation of ~4(, PCA TCC and ~4C DCA TCC in continuous flow activated sludge units.

Biodegradation of TCC in sewage

653

Table 2. Fate of radioactivity in CFAS system Total DPM

Total

Influent

3 . 3 7 x 10 ~

15.18

Effluent

1 . 0 8 x 10 s

0.49

3.2

14C0 2

1 . 8 9 x 10 v

8.51

56.1

1 . 1 5 x 10 v

5.18

34.1

Location of Radioactivity

Substrate

14C - P A - T C C

Activated

Sludge z

% of Influent

% Recovered

14C - D C A - T C C

93.4

Influent

I. IZ x 10~

5.05

Effluent

3.40 x 106

1.53

30.3

'4CO z

2.90 x I06

1.31

25.9

Activated Sludge

3,95 x 106

1.78

35.2

% Recovered

I Measured

during

a 5 week period

91.4

f r o m d a y 50 to 8 5 .

z Activated sludge was washed once with deionized and analyzed via the combustion procedure.

7: 3:1), indicates the effluent associated label from the CFAS sludge unit receiving 14C-PCA-TCC consisted primarily (88 per cent) of radioactivity at the origin, presumably metabolic products, and TCC. Less then 3 per cent of the extract radioactivity was PCA. In contrast, radioactivity at the origin and at the TCC area from the CFAS unit receiving ~4C-DCA-TCC represented only 54 per cent of the extract. The labeled constituent chloroaniline, DCA (16.7 per cent), and chloroaniline condensation products (7.9 per cent) were present in greater concentration in the effluent of the unit receiving ~4C-DCA-TCC. In addition, several

water

other unidentified spots were present in significant quantities in the a4C-DCA-TCC effluent. Product recovery from the extraction and chromatographic procedures was 41.3 per cent for the 14C-DCA-TCC effluent and 55.6 per cent for the 14C-PCA-TCC effluent. Label unaccounted for may have been lost due to volatility during concentration or chromatography of the extract, incomplete extraction, or insolubility in ethyl acetate. The chromatograms support the premise that TCC undergoes primary biodegradation to its component chloroanilines with the PCA being substantially

Table 3. Thin-layer chromatographic and autoradiographic analysis of effluents from CFAS units Unit Receiving 14C-DCA-TCC F~I OI Autoradio o Spot Fraction graphic Spots Identity Fraction DPM z

Unit Receiving 14C-PCA-TCC go of T o t a l Activit~r

5326

0.760

Aniline C ondens ation Products

O. 531

0. 394 0.277

<

>

<

>

<

>

44407

7.88

15223

2.70 16.71

3.05

?

20727

3.68

?

7873 33095

1.40 5.87

< 0.515

PCA

0,390

?

~

20910

3.71

89619

15.90

0.112

O. 014

2.15095

38.16

0.020

Total DPM

563683

0.104

TCC

% of Total Influent % Recovery 1, Z. 3.

from Effluent~

>

Fraction

<

17188

(

Spot Identity

0.94

994220

DCA

K10l Autoradio g r a p h i c SDota

563683 4.5x-'~ 12.5 30.3

TCC

Fraction DPM z

%of Total Actlvit~

357

0.15

3440

1.44

>

>

2356

0.99

6486

Z. 72 1.81

>

4315

< ' ~

352Z

1.47

~

1612 Z736

0.67 1.15

f

3206

1.34

15O616

63.07

6O166

25.19

238811 =

12.5

=

41.3

~wo - w e e k c o m p o s i t e e t h y l a c e t a t e e x t r a c t s f r o m day 57 to 71. A m o u n t of l a b e l c o r r e c t e d f o r the e n t i r e e x t r a c t . B a s e d on p e r c e n t of l a b e l in e f f l u e n t f r o m T a b l e 2.

238811 1.78

1.78

55.6

654

W.E. GLEDHILL

degraded. The DCA is also degraded but at a slower rate. Consequently, its presence together with its condensation products is observed in the effluent. The environmental consequence of dichloroaniline condensation product formation from T C C a n d herbicide metabolism has not been elucidated but has been assigned the teleological role as an aniline detoxification mechanism in nature (Ross a n d Tweedy, 1973). CONCLUSION In summary, acclimation of activated sludge to primary biodegradation of T C C was shown to occur rapidly in systems where T C C was added as a minor c o m p o n e n t of sewage, a situation simulating that which would occur in nature. The chloroaniline products of primary biodegradation in turn underwent ultimate biodegradation. W i t h a 10 h retention time in CFAS units a n d 200/~gl-~ T C C the CO2 released from the PCA moiety of T C C was consistent with nearly complete biodegradation while that from the DCA ring represented a b o u t 50 per cent degradation. Longer activated sludge retention times a n d / o r lower T C C concentrations in sewage would be expected to yield more complete DCA metabolism. It would also be expected that biodegradation of material leaving the waste treatment plant would continue in river water. Thus, from a biodegradation standpoint, the relatively small a m o u n t of T C C added to the environment should present insignificant consequences. Acknowledoement The excellent technical assistance of P. D. McDonald is gratefully acknowledged. Thanks also extended to Dr. R. D. Swisher for valuable discussions both during the investigation and in preparation of the manuscript. REFERENCES

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Bartha R. and Pramer D. (1967) Pesticide transformation to aniline and azo compounds in soil. Science. 156, 1617 1618. Bordeleau L. M. and Batha R. (1972a) Biochemical translormations of herbicide-derived anilines in culture medium and soil. Can. J. Microbiol. 18, 1857- 1864. Bordeleau L. M. and Bartha R. (1972b) Biochemical transformations of herbicide-derived anilines: requirements of molecular configuration. Can. J. Mk'rohiol. 18, 1873 1882. Burge W, D. {1972) Microbial populations hydrolyzing propanil and accumulation of 3,4-dichloroaniline and 3,Y.4.4'-tetrachloroazobenzene in Soils. Soil Biol. Biochem. 4~ 379 386. Burge W. D. [ 1973) Transk)rmation of propanit derived 3,4dichloroaniline in soil to 3,Y,4,4'-tetrachloroazobenzene as related to soil peroxidase activity. Proe. Soil Set. Soc. Amer. 37, 392 395. Chisaka H. and Kearney P. C. (1970) Metabolism of propanit in soils. J. Atlric. Food Chem. 18~ 854 858. Geissbuhler H. (1969)The substituted ureas In Degradation of Herbicides. (Edited by P. C. Kearney and D. D. Kaufman). Chapter 3, pp. 79 11 {. Marcel Dekker, New York. Herrett R. A. (1969) Methyl- and phenylcarbamates. In Degradation o/Herbicides. IEdited by P. C. Kearney and D. O. Kaufman). Chapter 4. pp. {13 145. Marcel Dekker, New York. Kaufman D. D. (1967) Degradation of carbamate herbicides in soil. J. A,qrie. Food Chem. 15, 582 591. Kaufman D. D. and Blake J. (1973) Microbial degradation of several acetamide, acylanilide, carhamate, toluidine, and urea pesticides. Soil Biol. Biochem. 5, 29"7 308. Kaufman D. D., Plimmer J. R. and Klingebiel U. I. (1973) Microbial oxidation of 4-chloroaniline. J. Aclric. Food Chem. 21, 127 132. Kearney P. C. and Kaufinan D. D. (1971) Microbial degradation of some chlorinated pesticides. Proc. Conference, De:lradation o/' Synthetic Or qanic Molecules in the Biosphere, pp. 166-189, San Francisco, Calil\, June 12 13. Kearney P. C., Kaufman D. D. and Alexander M. 119671 Biochemistry of herbicide decomposition in soils. In Soil Biochemistry. (Edited by A. D. McLoren and G. H. Peterson). pp. 318 342. Marcel Dekker, New York. Peterson J. I., Wagner F., Siegel S. and Nixon W. (1969) A system for convenient combustion preparation of tritiated biological samples for scintillation analysis. Anal. Biochem. 31, 189-203. Ross J. A. and Tweedy B. G. (1973) Malonic acid conjugation by soil microorganisms of a pesticide-derived aniline moiety. Bldl, Envion. Contain. Toxicol. 10, 234 236. Swisher R. D., O'Rourke J. T. and Tomlinson H. D. {1964) Fish bioassays of linear alkyiatc sulfonates (LAS) and intermediate biodegradation products. J. Amer. Oil Chert1. Soc. 41,746 752. Swisher R. D. (1970) Sut[/iwtant Biodecjradatiml, p. I II) 121. Marcel Dekker, New York. Tweedy B. G., Loeppky C. and Ross J. A. (1970) Metabromuron: acelylation of the aniline moiety as a detoxification mechanism. Science. 168, 482 483.