Biodehalogenation. Reductive Dehalogenation of the 'Biocides Ethylene Dibromide, 1,2-Dibromo-3-chloropropane, and 2,3-Dibromobutane m Soil C. E. Castro and N. 0. Belser Department of Nematology, University of California, Riverside, Calif. 92502
• Soil-water cultures reductively dehalogenate the vicinal dibromides :ethylene dibromide. 1,2-dibromo-3-chloropropane, and the 2.3-dibromobutanes. The products of the conversion of these widely employ'.!d so1l biocides, ethylene, n-propanol, and butcnes, respectively, and the scope, nature, and speed of the processes have been outlined.
T
he nature of the interaction of polyhalo-organic biocides with the soil matrix in which they are placed must be considered an important feature in the understanding of man's alteration of thi~ :'spect of his environment. Unlike cis- and trans-1,3-dichloropropane (Castro and Belser, 1966) the broad range biocides ethylene dibromide and 1,2-dibromo-3-chloropropane, widely employed as nematocidal soil fumigants, are not readily hydrolyzed in aqueous milieu. Consequently the persistence of these substances in soil would be expected to be limited by their volatility rather than by any chemkal or biological conversion in the soil matrix. Indeed, the relatively long term effectiveness of 1,2dibromo-3-chloropropane as a nematocide has been noted (Baines, 1962; Ichikawa, Gilpatrick, eta/., 1955). However, neutron activation analyses (Castro and Schmitt, 1962) of a variety of extracted plant materials (Thomason, Baines, et a/., 1963) confirm earlier findings that no organic bromide is present in plants grown in soil which has been treated with either of these substancLs. There is, however, an increase in the bromide ion content of such plants, and residue tolerances have been set on this basis. Although preliminary greenhouse experiments from these laboratories indicate orange seedlings can take up these substances intact, an assessment of the dominance of the dehalogenation of these entities in soil or in plants remains to be made. The present work demonstrates that a significant biological dehalogenation of these toxicants as well as other vicinal dibromides does occur in soil. E:,.perimentul
Soil Screen. A variety of approaches was initially employed. The method deS\:ribed provided the most rapid and reliable procedure. Typically 25 ml. of soil, 50 mi. of water containing 0.5% glycerol, 0.005 mg. of biotin, 0.5 mg. of thiamine, and substrate at ,..,.,1 X 10-sM were incubated at room temperature in 4-ounce prescription bottles. Bromide ion content was assayed at weekly intervals. One milliliter of inoculum
from most active soils (Table I, group C) was transferred into each of four bottles containing fresh autoclaved soil and the above ingredients. The amount of inoculum could be reduced to 0.1 ml. after a year of bimonthly transfers. Some 100 soils from orchards and fields throughout southern California were tested for their ability to dehalogenate 1,2dibromo-3-chloropropene. Materials. All substances were reagent grade. Alkyl halide substrates were carefully distilled, had the physical constants reported in the literature, and they were gas chromatographically pure. Ethylene dibromide 1,2-C 14 (New England Nuclear Corp.), was diluted with cold ethylene dibromide. The material employed had a specific activity of 4.31 X w-a me. per mmole .. Radiolabelled 1,2-dibromo-3-chloropropane 1,2-C 14 (specific activity 3.04 X 10-a me. per mmole)and l,2-dibromo-3chloropropane-3-C14 were synthesized from ethylene dibromide according to the sequence outlined (Equations 1 to 4). This route was chosen because the intermediate 2,3-dibt:omopropanol was desired for related studies of enzymatic dehalogenation (Castro and Bartnicki, 1968). VINYL BROMIDE 1,2-C 14 was prepared by the rapid addition of ethylene dibromide-1,2-C 14 into a warm (80°), stirred solution of alcoholic KOH. The product was distilled through an ice water condenser and calcium chloride tube into a dry cold trap as rapidly as it was formed (Kogermann, 1930). ALLYL ALCOHOL-2,3-C 14 OR 1-C 14• Into a 1-liter three-neck flask equipped with a stirrer, an addition funnel fitted with a nitrogen inlet, and a C02 condenser connected to a mercury trap, was placed 14.5 grams (0.60 mole) ofmagnesiumturnings under nitrogen. A solution of labelled or non:labelled. vinyl bromide, 66 grams (0.618 mole), of trap contents from the preceding experiment, which had been dissolved in 200 mi. of tetrahydrofuran and dried over calcium chloride overnight, was employed. Initially the vinyl bromide solution was added slowly in 20-ml. increments at room temperature until the Grignard reaction commenced. A tO-minute induction period was usual. When the Grignard reaction began, 175 ml. of tetrahydrofuran was added to the mixture, the flask. was immersed in an ice bath, and the rest of the vinyl bromide solution was added slowly. When the addition was complete,the flask contents were allowed to warm to room temperature with stirring and finally were refluxed for 1/2 hour. Upon cooling, under a slow stream of nitrogen, 40 grams of parafortnaldehyde (labelled or nonlabelled) was decomposed at gJOo and passed through a calcium chloride tube and bubbled into the stirred Grignard solution. The contents began -reflilxing
after 1;" hour. At the end of 1 hour the solution cooled and the color changed to a light gray. The flask was swept with nitro• gen and allowed to remain stoppered overnight. Saturated ammonium chloride, 100 mi., was added to the mixture with stirring. The whole was filtered and the white precipitate was dissolved in 350 mi. of ammonium chloride-HCI and extracted thril·e with ether. The ether extracts and tetrahydrofuran filtrate were combined, washed once with saturated ammonium sulfate. and dried over potassium carbonate. The solution was concentrated by distillation through a Vigreaux column and fractionated through a ::;Jinning band apparatus. A 20-ml. fraction boiling at 94-95° was collected and contained 2":~ tetrahydrofuran by gas chromatography. A minimum yield for this process was 49~{. 2,3-Dibromopropanol2.3-C 14 or 1-C 04 was prepared from the corresponding allyl alcohols and cupric bromide in methanol (Castro, Gaughan. e1 a/., 1965). Average yields for purified material boiling at 71 o (0 I mm.) were 70~~. 1,2-DIBROMo-3-CHLOROPROPANE-1.2-C 14 AND 3-C 14 . Typically, 13 grams of the corresponding dibromopropano!, 20 grams of thionyl chloride, and 2 drops of pyridine were \Varmed at llO'' for 8 hours. The cooled solution was poured into 100 mi. of ice water and 100 mi. of carbon tetrachloride and stirred for I hour. The separated carbon tetrachloride solution was washed onct: each with water, saturated sodium bicarbonate. and water, and was dried over sodium sulfate. The solution was concentrated and the residue distilled through a small Vigreux column to yield II grams of product boiling at 76-78 o (15 mm.). Highest purity material was obtained byrefractionation of this distillate through the spinning band column. Anulytical ProcedureJ
Bromide Ion. Bromide was determined by direct potentiometry in the manner previously described (Castro and Bartnicki, 1965). Ethylene. Portions of the gas phase from serum capped bottles were analyzed by gas chromatography on a 30-foot dimethylsulfolane (OMS) column at room temperature. A peak co-emergent with ethylene was obtained. A charge like that noted in Table III was placed in a calibrated 200-ml. three-neck flask fitted with a serum capped stopcock and connected to an open ended manometer. Pressure change was monitored manometrically, and the gas containing co~ and ethylene was analyzed gas chromatographically. An infrared spectrum of the gases was identical with that of a mixture of CO: and ethylene. Furthermore, utilizing ethylene . dibromide-1,2-C 14 , the ethylene peak was trapped from the gas chromatograph by passing the effluent into a saturated aqueouo; solution of KBra. The excess bromine was destroyed with sodium thiosulfate and the solution was ether extracted. The ether extracts were radioactive~ Cis- and Trans-Butene-2. In· similar fashion the products resulting from the consumption. of meso and d/-2,3-dibromobutane were co-emergent With the respective butenes upon gas
chromatography on a 30-foot OMS column. The~e olefins could not be detected in the absence of substrate. n-Propanol. With I ,2-dibromo-3-chloropropane 1,2-C 14 or 3-C 14 direct gas chromatography of the aqueous. phase of 'a culture bottle (Table IV) afforded a multiplicity of peaks. Only one major peak was radioactive. This was determined by employing a splitter on un A 600 C Aerograph Hi-Fy with a flame ionization detector and passing the effluent corresponding to each peak into counting solution. This peak was coemergent with n-propanol on a 20-foot diethylene glycol succinate (DEGS) column and a 6-foot Porapak P column a' all temperatures. It was the only entity including all vadants of three carb0ns and one or two oxygens to do this. At the low concentrations of the unknown, it .:ould not l'le extracted with eth.:r. The product was concentrated by collecting the first few drops from the distillation of the aqueo(ts solution. These small amounts were combined and gas chromatographed. The desired peak was collected in a small amount of water. The subs!:mce was collected and purified from many runs in this fashion. 1t had no visible or ultraviolet spectrum. Additional support for this structural assignment was obtained by a series of qualitative tests performed with this solution. Model compounds at the approximate concentrations of the unknown ( ...... }0- 3M) were made so as to give the same peak height as the n-propanol peak upon gas chromatography (included in parentheses). In all cases the peak corresponding to the model compound upon gas chromatography of the product solution was either completely invisible or greatly diminished by the reagent employed. These results attest to the general validity of the qualitative tests. The unknown peak (n-propanol) was not diminished by any of the follo·,ving: warming at 90° for 8 hours with IN NaO:H (ethyl acetate-acetic acid); room temperature treatment with saturated Bre in water for 3 hours (allyl alcohol); room temperature potassium permanganate for 8 hours (allyl alcohol); reaction with dimedone and base (CH 3CH2CHO but only diminished slightly); treatment with strong CrS04 under N~ fCHaCH2CHO). However, warming the solution with strong potassium permanganate and acidifying with H 2S04 resultect in a peak on a 1-foot Porapak P column that co-emerged with propionic acid. This peak was eliminated with base. The ilpropanol was quantitated in the reaction mixture by direct gas chromatography employing dioxane as the calibrated marker. Ethylene Dibromide and l,l-Dibromo-3-chloropropane. The amount of unreacted DBCP was determined by gas chromatographic analysis of a hexane extract on the 6-foot Porapak P column. Bromobenzene was employed as the calibrated marker. Unreacted EDB was monitored by radiocounting the hexane extract. Radiocounting. All counting was performed with a Packard · automatic liquid sl:inlillalion ~pectrometer. The n~propanol peak was ;trapped from the gas chromatograph by bubbling the effiuent through a dioxane scintiJiator solution. This solution and the counting techniques were previously described (Castro and Bartnicki, 1965). ·. ·
Rt>sulrs
Soil Screen. BeL·uuse of its wide use. high toxinly, and chemicul inertne~s. 1,2-dihromo-3-chloropropane (DBCP) was employed as the scanning substrate for the soil screen. Approximately 75°~ of the samples examined showed some capacity to convert DBCP to Br··. These varying capacities are illustrated in Table I along with the influence of pH on the conversion. These results reflect the frequency of the conversion and the range of responses noted. One of the most effective soils was obtained from an old lemon grove and all subsequent work was done with it.
Table l. \'ariability of Soil to Decomposition of DBCP·• Fraction No. of Molt:s Br- Produced of Samples Soil 6 Weeks pH Group Screened A
5 () 6 0 7 0
7.5 !\3 50%
B
7 05
7 .I 7 R5 8.3
25%
c
7 5
77 8.2
8 25
6 3 X \0 ' 3 24 X 10 6 7 95 X \0 " 2 X 10 -• 5 X 10 " 8.9XI0 6 2 5 X to·> 7.95 X 10 6 2 X tO-; 6.3X10' 3.2 X 10 6. 5 x to-"
I . 25 X tO I 0 X 10 7 I 6 3 6.3 1
X 10 X 10 X llP X 10 :.
5 o x w--; 6 3 X 10 ·•
5.oxiO-" 4
o x to-- 5
I. 25 X
to-~
9 2 X J0- 5 2 x to·-~ . I X I0- 4
''Initial charge· :!5 mi. of soil, 50 mi. of H,O, 0.5 ",; glycerol, and :!.5 X JO-• moles of DBCP. A bl:tnk composed nf stcrik soil--water + DBCP atrordcd only 5 X JO-·' nwlcs of Br- in 12 weeks.
Conversion Conditions. The most rapid dehalogenation of DBCP (20% in 1 week) occurred with whole soil suspension-> at pH 8. During a three-year period, beginning with the original cultures, bimonthly transf~rs of I mi. and eventually 0.1 mi. were made into sterilized soil (25 ml.}-water (50 ml.) suspensions containing o.so;~ glycerol. 0.005 mg. of biotin , 0.5 mg. of thiamine, and substrate at I0- 3M. Near the end' of this time the reactivity approached that of the original whole soil. Intensive efforts over a four-year period to isolate a pure cuhure or reproducibly effect the dehalogenation in the absence of soil failed. Soil which had been Soxhlet extracted for 48 hours with either water, acetone, or benzene retained its capacity to support dehalogenation when it was inoculated from an active culture. Under no conditions would charcoal, sand, fireb~ick. or diatomaceous earth support dehalogenation. Neither would any of the above concentrated extracts effect reaction when added to a nonactive soil suspension. Moreover, although many bacteria could be obtained from the active cultures no single or mixed colony was effective when incubated with sterile soil-water or any other media. A neutralized sulfuric acid extract of the soil would support bromide release through four transfers two out of three times. The biological nature of the conversion was deduced from a series of experiments which are summarized inTable II. Thus, halide release occurs only under conditions that will support growth and only. when the substrate is present during the growth phase. It is inhibited by sterilization of the soil with ethylene oxide or by autoclaving, by omission of a carbon source (glycerine), or by treatment of active cultures with sodium azide or heat. Hence we conclude the transformation may be a detoxification process or the result of a secondary interaction of biologically engendered substances with the substrate.
Conditions for Br- Releasl' from 1,2-Dibromo-3-chloropropane by Soil-Water Suspensions Components of Reaction Mixture Br- Release DBCP Soil-water' ·----------- cilycerol !0.5~~)--After 3 Weeks Inoculum Table II.
Whole soil Whole soil \Vhole soil Sterilized soil Sterilized soil Sterilized soil Sterilized soil
+ + A· A., A,. A'
+ +
Sterilized soil A' Sterilized soil s.r Sterilized soil Bi
+ +
+
+
+ +
+
+
• 25 mi. of the indicated soil + 50 rnl. of H,O. b This result is unchanged if glycerol added after I week. 'Soil suspension sterilized by autoclaving at 121 o C. for 15 minutes. d Boiled inoculum before adding to the mixture. • This result is unchanged if DBCP is added after I week. f Ethylene oxide sterilized soil. ·
+
A similar p\,2-C14, 1,2-dibromo-3-chloropropane-l,2-C 11 wert: employed to characterize the products and ensure that the observed ''metabolites" did emanate from the starting substrates. Quantitation of the reactions was usually accomplished with cold substrates. Both 1,2-dibrorilo-3-chloropropane-1 ,2-C 14 and 1,2-dibromo-3-chloropropane-3-C 14 were prepared from ethylene dibromide by the following sequence of reactions. BrCH~
O~J~=CHBr
CH"Br
c\!fo.
the mixture except that when less than 5 ml. of soil is employed; no appreciable quantity ofBr~- can be detected after 3 weeks. ' Dibromobutanes. Both meso- and d/-2,3-dibromohutane were examined to assess the stereochemical consequence of tht! reductive dehalogenation. These halides were not studied quantitatively because over an 8-week period significant hydrolysis occurred in the blanks. Nonetheless, inoculated soil water suspensions containing the dibromobutanes re.stilted in butene production. No butenes nor an)' gaseous products could be Jetected in the blanks.
]~011
-~ CH~- ~CHBr
:\l~,t
CHa
(1)
---+ CH~, ccCHMgBr ~~)··· ,)
Cll1
,;,,jl~llcfl
-~
":
'(
CH"~=CHo
CH~==CH---CH~OH
'-. /Br
Br /
liCIIU
·CH~OH
(2)
·CH~OH
(3)
(6)
''vf'"'-cH~
CHa 1111.'SO
:\I,.o II
+ CuBr.. ----+ M
;o~·;
BrCH"CH
I
Br
CH:1
/~/Br
-~
.
Br
(7)
CHa·
""-/
CHa
d.! Br
Br Ethylene Dibromide. In about two months. ethylene dibromide is converted almost completely and quantitatively to ethylene (Equation 5). (5)
The material balance for three separate runs is presented in Table III. These experiments and all other conversions noted herein were performed with sterilized soil that was inoculated. The variation in conversion no doubt reflects the heterogeneity of the inoculum. Separate experiments show that there is no correlation of conversion with the amount of soil present in
Table III.
Elimination of bromine from each of the isomers (E4uations 6 and 7) is stereospecifically trans. 1,2-Dibromo-3-chloropropane. In addition to its relative inertness, studies with DBCP were complicated by its volatility, lower solubility, and the ease with which it was taken up in bottle cap liners and rubber serum caps. Moreover, it is difficult to extract quantitatively this substance frorri a soil-water suspension. fhus, the amount of this halide present at the end of a run is more an attribute of its volatility than its biodegradability. Nevertheless, the substance. is cleanly converted by soil water cultures (Table II) to npropanol (Equation 8).
Br
Material Balance for Ethylene Dibromide (Equation 5)·• Experiment -~---;c--
I
Substance EDB BrCHz=CH2 % conversion Br-;EDB consumed CH~H2/EDB consumed Br-/2 CHpoCH2
.... o··
-- -------sWeeks
5. 75 Y. I0- moles 0 4
0
• Initial charge: 50 mi. of soil, 100 mi. of HoD, 5.75 X
------~--------
782
Time
- - -3
0.17 X 101.10 X I0- 4 5.5 X 10- 5 97 1.97 1.0 1.0
w-•M EDB.
-. .
~T~
~--
weeks ~
--~--------
4 -~-
5
Tweeks_______nveeks ·
4
6.9 X IQ- 5
3.2 X I0- 5
24 X I0- 4 I .4 X 10- 4
1.08
0.86
2.6
x w-•
1.5XI0
0,87
4
Table IV.
Material Balance for 1,2-Dibromo-3-chloropropane (Equation 8)m .. ___ ---~xp~rime~ 2 time ___
Substance
• Initial charge:
~5
I . 78 X 10
4
moles
()
12 X 0. 39 X 0 22 X to-• 0 !N
.13 X IO-• 0.79 X to-• 0.56 X to·-• 0.71
0.38 X to-• o.66 x w--· 0.30 X 10 1.1
12 63
11
63
22 63
19 21
10
3 .\1
Discussion The reductive dt:halogenations (Equations 5, 6, and 7) typ1fied by Equation 9 would suggest a most plausible sequence for the reduction of DBCP (Equation 8) as shown in Equations 10, 11, and 12. The allyl chloride (Equation 10) emanating -· -C· - ----·C·-·- - -C-'""C/
/
Br
(9)
-,,
Br
CH~=CH2CH20H CH~-=CH2---CH20H
+ Cl-
_., CHaCH2CH20H
4
Day~
w-• w·-•
•
DBCP, 0.5 ~~glycerol, 0.005 mg. of b'otin, and 0.5 mg. of thiamine.
The variability of the rates of this surprising process and the stoichiometry of it are illustrated in Table IV. Assuming a 10~~ error in the gas chromatographic estimation of n-propanol at these levels (see experimental section) and a 5~{ error in Branalyses, the stoichiometry of Equation 8 is valid throughout the conversion period. The loss of DBCP through volatilization is particularly evident after. three weeks. Furthermore, the presence or absence of n-propanol in the media follows the identical pattern for Br- noted in Table II. In contrast to ethylene dibromide, 1.2-dibromo-3-chloropropane was never wholly con:;umed. Indeed, the maximum conversion (Br-;2 DBCP) we have generally observed is 63':'~ in the course of 4 weeks.
""'
·- ---3 weeks
13 X 10 ·• 0.44 X to-• 0.28 X IQ-4 0.79
l)
X
..
--~--
12 X IO-• 0. 33 X to-• 0 16 X to-• 1 ()
63
ml. of soil. 50 ml. of HzO. 3.56
---···---
4 Days
3
()
6 bays
---------···-5
-··
Days
0
DBCP Br··· n-PrOH Bn'2 n-PrOH ~~ conversion (Br-;2 DBCPo)IOO % DBCP remaining
4
3
(11) (12)
from a process like that shown in Equation 9 would certainly hydrolyze even nonenzymatically under these conditions. A soil matrix will not impede this process for the allylic halide isomers cis and trans-1,3-dichloropropene are hydrolyzed under identical conditions (Castro and Belser, 1966). Moreover, a small gas chromatographic peak which does correspond to allyl alcohol can be seen in some experiments, and allyl alcohol is converted to n-propanol (Equation 12) by these cultures. We have not, however, rigorously established its intermediacy.
Taken together the transfonmttions (Equations 5, 6, 7, and 8) are chemically difficult to accomplish under these conditions. Nonetheless, chemical analogies do exist in the reduction of organic molecules by low valent metal specie. Thus, aqueous solutions of CrH salts will convert vicinal dihalides to the corresponding olefins (Equation 9) at room temperature (Castro and Kray, 1964). Olefins can also be reduced to alkanes by these salts under similar conditions (Castro, Stephens, et a/., 1966), a process analogous to that shown in Equation 12. Indeed a model which more doselyintegrates with the biosphere is the reduction of alkyl halides by low valent iron porphyrins (Castro, 1964). The rapid conversion of DDT to DDD by Fe·r2 porphyrins in very dilute solutions is a process closely akin to that illustrated in Equation 9. The conversion of ethylene dibromide to ethylene by soil organisms (Equati~n 5) is particularly interesting because-ethylene is well known to be a plant hormone (Lyons, McGlasson, et a/., 1962). These findings could partially account for the beneficial effects of ethylene dibromide treatment. Literature Cited Baines, R. C., University of California, Riverside, unpublished data, 1962. Castro,_ C. E., J. Am. Chem. Soc. 86, 2310 (1964). Castro, C. E., Bartnicki, E. W., Biochim. Biophys. Acta 100, 384 (1965), Castro, C. E., Bartnicki, E. W., Biochem. 7, 3213 (1968). Castro, C. E., Belser, N. 0., J~ Agr. FoodChem. 14, 69 (1966). Castro, C. E., Gaughan, E. J., Owsley, D. C., J. Org. Chem. 30, 587 (1965). . . Castro, C. E., Kray, W. C., Jr., J. Am. Chem. Soc. 86, 4603 (1964). Castro, C. E., Schmitt, R. A., J. Agr. Food Chem. 10, 236 (1962). Castro, C. E., Stephens, R. D., Moje, S., J. Am. Chern. Soc. 88, 4964 (1966). . Ichikawa, S. T., Gilpatrick, J.D., McBeth, C. W., Phytop'ath. 45, 576 (1955). . ' Kogermann, P. N., J. Am. Chern. Soc. 52, 5062 (1930); Lyons, J. M., McGlasson, W. B., Pratt, H. K., PlantPkysiol. 37, 31 (1962). . .. ··-···--· Thomason, I. J., Baines, R. C., Castro, C. E., University of , California, Riverside, unpublished results, 1963. ·· Received for ·review January 19,1968. Accepted August ,21, 1968. The .authors are grateful to the Nation(lf Jn~Ututes of Health (ES-00169)for generous support. · · _
