DISSIPATION AND DEGRADATION OF ALAR ®

IN SOILS UNDER GREENHOUSE CONDITIONS

LELAND E. DANNALS, R. JAMES PUHL, and NORBERT KUCHARCZYK
Agricultural Chemicals Research,
Uniroyal Chemical, Division o f Uniroyal, Inc.,
Elm Street,
Naugatuck, Connecticut 06770

The rate of dissipation of ALAR -14C (succinic acid 2,2-dimethylhydrazide) was

studied on four soils under greenhouse conditions. The half-life of ALAR was 3 to
4 days on all soils.
Data derived from experiments with sterilized sandy loam indicate that microbial
degradation is the major route of dissipation of ALAR from soil. Trapping experi-
ments showed that the major degradation product from ALAR (N-methyl-14C)is
1 4 C O 2 , which is liberated in amounts of up to 84% in 14 days. Of the 14 C remain-
ing on soil, most is associated with soil organic matter.

ALAR (succinic acid 2,2-dimethylhydrazide) is a plant growth regulator currently

being used on a variety of food crops and fruit trees. Since the chemical is applied as a
foliar spray, direct soil contamination results. Also, since ALAR is water soluble, it can be
washed off the plants onto the soil.

Earlier experiments in these laboratories, using growth inhibition of peanuts as an

indicator, revealed that the biological activity of ALAR in soil is undetectable after 14
days, indicating that ALAR is undergoing a rather rapid change. Here we report studies
on the rate of dissipation of ALAR from four soils under greenhouse conditions using
14C-radioassay. Also presented is evidence that indicates that ALAR-14C is degraded
primarily by microbial pathways, the major degradation product being 14CO2.

Experimental
Synthesis of 14C-ALAR. Succinic acid, 2,2-dimethylhydrazide-2,2-14C (ALAR, N-
methyl tagged).

Ninety-one mg (two mCi, sp. activity 1.32 mCi/mM) of 2,2-dimethylhydrazine-2,2-14C

(from Mallinckrodt Labeled Compounds) was added to one ml of anhydrous reagent grade
acetonitrile at 30°C. A solution of 155 mg of freshly recrystallized succinic anhydride
(m.p. 121.5-122°C) in one ml of acetonitrile was added and the resulting mixture allowed
to stay for 12 hours at room temperature. The crystalline product was filtered, washed
with 0.5 ml of acetonitrite and dried in vacuum. (All operations were conducted under
nitrogen.) The yield was 86% (208 mg; t.72 mCi, sp. activity 1.30 mCi/mM), and the
m.p. 163-164°C. The radio-purity by thin-layer chromatography on cellulose in 1-butanol-

Archives of Environmental Contamination 213

and Toxicology, Vol. 2, No. 3, 19"74, © 1974
214 Leland E. Dannals et al.

acetic acid-water (4:1:5) as described below, was 98%; the 2,2-dimethylhydrazine con-
tent was less than 0.1%.

Succinic acid-l,4-14C-2,2-dimethylhydrazide (ALAR, carbonyl tagged) was prepared

similarly from succinic anhydride-14C and 2,2-dimethylhydrazine in approximately the
same yield and purity.

Radioactivity determinations. Determination of 14C of soil extracts, solutions, and

thin-layer chromatogram sections was conducted in a PPO-toluene-based cocktail, con-
taining 18 g of Permablend II (98% PPO, 2% POPOP) in 2000 ml of toluene and 1000 ml
of 2-ethoxyethanol with a Beckmann LS-250 liquid scintillation spectrometer, with 81
to 85 percent 14C counting efficiency. In soils 14C was determined by dry combustion
on a modified Peterson apparatus (Peterson 1969) using a cocktail consisting of five g
of Permablend II in 430 ml of xylene and I70 rnl of methanol and containing 140 ml of
/3-phenethylamine for 14CO2 trapping and counting; the counting efficiency was 83 to
89 percent. The 14C in soil extracts containing NaOH was counted in the latter cocktail.

Dissipation of ALAR-14C from soil. Descriptions and analyses of four soils used in our
experiments are given in Table I. The soil samples (70 to 90 g) were placed in plastic pots
in the greenhouse and an aqueous ALAR-14C (sp. activity 0.2 mCi/mM)solution was
applied evenly to the surface with a syringe at a rate equivalent to 0.85 lb active ingredient/
acre. The soils were kept at 20 to 30°C and watered twice a week. At desired time intervals

Table I. Chemical and Physical Characteristics of Soils Used

Percent of
Organic b CECc
Soil type Sand a Silt a Claya matter pH me/lOOg

Connecticut
sandy loam 52.6 33.0 t4.4 5.8 5.0 9.0

Mississippi
silt loam 11.6 68.0 20.4 4.2 6.4 13.8

Illinois
clay loam 39.6 32.0 28.4 12.8 7.0 25.7

North Carolina
sandy loam 59.6 32.0 8.4 2.2 5.8 5.3

aAir dry basis, Bouyoucos hydrometer method

b Loss-on-ignition, water-free basis
cCation Exchange Capacity, air-dry basis, copper acetate method
 ALAR in Soils 215

duplicate samples were removed for analysis. The soil was stirred for two hours with a
7:3 mixture of 1-propanol-water, containing one volume percent of aqueous ammonia,
then filtered and dried at room temperature. Aliquots of the filtrate were analysed by
liquid scintillation counting. Residual unextracted activity in soil was determined by
liquid scintillation following dry combustion. The extraction of freshly treated soils with
1-propanol-water-ammonia gave quantitative recoveries on the Connecticut and North
Carolina sandy loams, 87% on the lllinois clay loam and 67% on the Mississippi silt loam.

Degradation of ALAR-14C (N-methyl tagged) on sterilized and non-sterile sandy

loam (14C02 trapping experiments). The 14C02 evolution experiments were conducted
in two interconnected flasks each having one separate septum-containing inlet. One of the
flasks contained soil; the other contained 50 ml of a 10 percent aqueous NaOH solution.
With the non-sterile soil the ALAR-14C solution (rate equivalent to four lb active in-
gredient/acre based on the surface area or 16 ppm in soil-l.6 nag or 13 vCi on I00 g of
soil) was applied to the surface with a syringe through the septum. The NaOH solution
was agitated with a magnetic stirrer and 0.1 ml samples of solution were taken at appro-
priate intervals for liquid scintillation counting. After the completion of the experiment,
the soil was extracted with 1-propanol-water-ammonia and then analyzed by dry com-
bustion as described above.

In the sterilized soil experiment the apparatus was assembled with soil in the flask but
without the NaOH solution and with septums removed. For sterilization it was placed in
an oven with hypodermic syringes, septums, etc., and heated to 121-127°C for 30 min.
After heat treatment the NaOH solution was added and septums inserted as rapidly as
possible. The ALAR-14C was dissolved in sterile water and injected onto the soil surface
through the septum. The NaOH solution was then sampled with a hypodermic syringe
and sterilized needle through the septum after various periods of exposure.

After completion of the sterilized soil experiment a portion of the 14CO2-containing

NaOH solution was diluted with distilled water, a saturated solution of BaC12 was added
and the resulting mixture was stirred for 15 min in a counting vial. After 24 hr, the super-
natant was decanted, the precipitate was washed with water and suspended with the help
of Cab-O-Sil in 15 ml of counting solution and counted to determine the amount of
Bal 4CO3 collected.

The 1-propanol-water-ammonia extract of the soil was analyzed by thin-layer chroma-

tography on Baker-flex cellulose sheets in 1-butanol-acetic acid-water (4: I:5). After de-
velopment to ten cm the strip was cut into twenty 0.5 cm sections which were placed in
the toluene-cellosolve-PPO counting solution for determination of radioactivity in each
section by liquid scintillation counting. In this system ALAR had a n R f = 0.82 and was
detected with a one percent acidic aqueous spray solution of K M n O 4 .

A part of the extract was analyzed by a colorimetric method involving hydrolysis of

ALAR and distillation of the released 1,t-dimethylhydrazine (Lane 1967). Also, the
14C in the distillate was determined by liquid scintillation counting.
216 Leland E. Dannals et al.

Extraction and fractionation of soil bound 14C residue with NaOH according to
Stevenson (1965). Soil (2000 rag) from the CO2 trapping experiment (12 percent bound
t 4C) was extracted twice with 0.5N NaOH followed by a water wash. The NaOH extracts
and the water wash were combined and the resulting solution was acidified with concen-
trated HC1 to pH 1 causing precipitation of humic acids. The mixture was centrifuged and
the liquid (fulvic acids) was removed by pipet. The solid humic acids were reprecipitated
twice by dissolving them in 0.5N NaOH followed by acidification with concentrated HC1
to pH 1 and centrifugation. After each centrifugation, the supernatant was combined
with the first fulvic acid fraction. The humic acid fraction was then washed twice with
water, dried and weighed.

The combined fulvic acids were further fractionated by stirring with activated charcoal
(prewashed with 0.IN HC1) and centrifuged. The supernatant was removed by pipet and
the charcoal washed once with 0.1N HC1, twice with 90 percent acetone, followed by two
washes with distilled water and two washes with 0.5N NaOH.

Results a n d discussion
Dissipation rate studies of ALAR-14C (N-methyl label) on four soils in the greenhouse
indicated a half-life of 3 to 4 days for all soils (Table II). After four weeks the total 14C
residue on three of the soils was less than 15 percent of the amount applied, while the
residue on the Illinois clay loam was 27 percent. The extractable t 14C, which would in-
clude unchanged ALAR, decreased to 50 percent within three days and to less than
10 percent in seven days (Figure l, Table II). This rate of disappearance of ALAR can be
considered rapid, particularly when compared to the persistence of other pesticide
chemicals.

Dissipation studies were run on sandy loam with two labeled forms of ALAR(N-methyl-
14C and carbonyl-14C). 14C-Disappearance curves (Figure 1) were similar for the two
labels. The fact that volatile 14 C is formed from both tagged molecules indicates that the
ALAR molecule is extensively degraded. Since the rate of disappearance of the 14C from
soil is similar in both cases (half-life of 3 to 4 days) it is evident that complete degradation
of the ALAR molecule occurs rapidly.

The identity of the volatile degradation products from the N-methyl-labeled ALAR
was investigated by trapping experiments. Potential gaseous 14C-containing products
include 1,1-dimethylhydrazine, monomethylhydrazine, dimethylamine, methane, formal-
dehyde, carbon dioxide and formic acid. In preliminary experiments no appreciable
amount of 14 C was found in a sulfuric acid trap which would trap basic compounds or in
a silicone oil trap, which would trap neutral compounds. After 14 days, however, 75 to
84% of the initially applied 14C was trapped in a sodium hydroxide trap (Fig. 2). The
trapped 14C was identified as 14CO 2 by precipitation of 97 percent of the activity as

1The terms "extractable" and "unextractable" as used in the text refer to extraction with the solvent
system 1-propanol-water (7:3), containing one volume percent aqueous ammonia.
 T a b l e II. Residues o f 14C in Soils After ALAR-14 C Treatment

A v e r a g e % o f a p p l i e d 14 C
Carbonyl tag N-Methyl tag
Connecticut Connecticut N. C a r o l i n a Illinois Mississippi
Time sandy sandy sandy clay si l t
Fraction (days) loam loam loam loam loam

Extractable
4 - 8.6 12,9 22.9 6.1
7 7.0 4.2 5.6 8.8 5.5
14 5.3 3.2 5,1 3.9 3,1
28 - - 4.4 2.0 2.5 ;>
38 2 .8 2.4 - - - t"

120 - 3,6 - - -

Unextractable
G
4 ,- 21.7 8,2 27.1 t5.3
7 16.8 14.3 10,0 26.0 12,5
14 7.0 9.5 8.4 25.9 15,9
28 - - 9.3 24.8 12,3
38 5.8 9.1 ,---
120 - 5.8 . . . . .

Total

4 - 30,3 21,1 50,0 21.4

7 23,8 18.5 15,6 34.8 t8,0
14 12,3 12,7 13,5 29,8 t9,0
28 - - 13,7 26,8 14,8
38 8.6 11.5 - - -
120 - 9.4 . . . . .
tO
"-3
218 Leland E. Dannals e t al.

Bal 4CO3. Of the non-volatile t4C in this experiment 17 percent remained on the soil
after 14 days, of which 4 to 5 percent was unchanged ALAR (determined by thin-layer
chromatography) and 12 percent was unextractable.

A comparison of the above 14CO 2 trapping experiment was made with a similar
experiment on sterilized sandy loam (Table III). On sterilized soil a negligible amount of
14CO 2 (0.2 percent) was produced in ten days. Of the applied 14C, about 93 percent
remained on the soil, as compared to only 17 percent for non-sterile soil. Unchanged
ALAR represented 78 percent of the initially applied 14C, compared to only 4 to 5 per-
cent on the non-sterile soil. These data imply that chemical hydrolysis or volatility of
ALAR is not contributing significantly to 14 C disappearance and that microorganisms
are largely responsible for the degradation of ALAR on soil.

In the above experiment, the residual 14 C activity left on non-sterile sandy loam after
extraction with propanol-water-ammonia was examined further, After 38 days only
14 percent of the unextractable 14C residue could be released with water. Extraction
with ten percent aqueous HC1 removed approximately the same amount of 14C as water.
However, 83 percent of this residue was released from soil by 0.5N NaOH, indicating that
it is associated with the soil organic matter. Therefore, a method of chemical fractionation
of organic matter (Stevenson 1965) was used for analysis of the sodium hydroxide-
soluble residue. Fractionation into insoluble humic acids and 5 soluble fulvic acid fractions
(Table IV) shows that the 14C is distributed among all the fractions. The ratio (0.31) of
extracted soil material in the humic acid fraction (27 mg) to that in the whole fulvic

I I I I I

A B
8O

60

'S
40

2oi

I
v I ,~___ I
8 16 24 32 8 16 24 32
Days Days

Fig. 1. Disappearance curves of t 4 C activity from ALAR -l 4C treated Connecticut sandy

loam. A: N-Methyl-I4C labeled. Curve 1: Activity extractable with propanol-water-
ammonia. Curve lI: Total 14C activity on soil (extractable + bound). B: Carbonyl-14C
labeled. Curve I: Activity extractable with propanol-water-ammonia.
 ALAR in Soils 219

Table III. A L A R - 1 4 C (N-Methyl Tag) Degradation on

Sterilized vs. Non-Sterile Sandy Loam

Average % of applied 14C radioactivity

Extractable from
soil with In extracted
Exposure propanol-water- soil (by dry In NaOH
(days) NH 3 combustion) trapping solution Total

Sterilized 10 85.1 a 7.8 0.2 93.1

Non-sterile 14 5.0 b 11.8 75.4 92.2

a92% ALAR by thin-layer chromatography

bEighty percent of this amount was determined to be ALAR by the colorimetric test
(Lane 1967).

acid fraction (88 mg) is the same order of magnitude as the ratio of 14C in these fractions
(0.51), suggesting that no preferential accumulation of t 4 C takes place.

In summary, in the CO2 trapping experiment, more than 90 percent of the ALAR
applied to non-sterile sandy loam can be accounted for after 14 days: 75 to 84 percent as
liberated 14CO2, 12 percent as fixed or utilized carbon in organic matter and 4 to 5 per-
cent as unchanged ALAR.

Oxidative N-demethylation leading to CO2 evolution has been reported for amide
(diphenamid), urea and carbamate herbicides on soil (Kearney and Kaufman 1969). Our
data suggest that this reaction is the major pathway in the degradation of the 1,1-
dimethylhydrazine moiety of ALAR by soil microorganisms and could be of importance
for other alkyl hydrazides as well. Formation of CO2 from 1,1-dimethylhydrazine itself
has been reported by Dost et al. (t966) to occur in rats after intraperitoneal application
of subacute doses.

It is apparent that ALAR is a good substrate for soil microorganisms as evidenced by

the high percent conversion of both the dimethylhydrazine and succinic acid portion of
the molecule to CO2, and by the rapid decrease in total t 4C activity in soils (Figure 2). It
is of interest to note that the rate of evolution of 14CO2 from ALAR on soil is very
similar to that observed for ~4C-labeled amino acids (Schmidt et al. 1960). Since ALAR
can be degraded as rapidly as amino acids, which are integrally involved in natural meta-
bolic pathways, it seems reasonable to expect that ALAR would not be a serious soil
contaminant.

Martin and Ervin (1970) have reported that aerobic bacteria utilize about ten percent
of the carbon in substrates for synthesis of cell constituents; fungi from 10 to 40 percent.
220 Leland E. D a n n a l s et al.

T a b l e IV. Fractionation of Alkali Extractable Soil Organic Matter Components

Containing ALAR-i 4C (Methyl Tag) Derived Residues

T y p e s of
chemicals % of
Method of in t h e applied 14 C
N a m e of f r a c t i o n aquisition fraction a radioactivity

Alkaline s o l u t i o n f r o m soil NaOH extraction m

10.0
H u m i c Acid c Insoluble 3.4
at p H = 1
Fulvic A c i d - w h o l e d Soluble 6.6
at pH = 1
Fulvic Acid A Not adsorbed Small sugars 2.7
by charcoal & a m i n o acids
Fulvic A c i d - B Eluted from Phenolic 1.1
charcoal by glycosides or
90% a c e t o n e tannins
Fulvic Acid - C Eluted from Polysaccharides, 0.4
charcoal by a m i n o sugars
water
Fulvic Acid - D Eluted from P e n t o s e sugars 0.6
charcoal by N orP
0.5N N a O H compounds
Fulvic Acid - R Remaining on 1.8b
charcoal

a A c c o r d i n g to S t e v e n s o n ( 1 9 6 5 )
bCalculated by difference
c23% of e x t r a c t e d soil m a t e r i a l
d 7 7 % of e x t r a c t e d soil m a t e r i a l

a. 20

i I I J .....

3 6 9 12
Days

Fig. 2. A m o u n t o f ] 4CO2 f o r m e d from A L A R - 1 4 C ( N - M e t h y l l a b e l e d ) o n C o n n e c t i c u t

s a n d y loam. A L A R - 1 4 C applied as free acid (I) a n d as its p o t a s s i u m salt (II).
 ALAR in Soils 221

On soil, this incorporation into cell components would result in subsequent binding into
soil organic matter. This was demonstrated in studies with 14C.acetat e (Stevenson and
Ivarson 1964) and 14C.glucose (Mayaudon and Simonart 1958) on soil, where the respec-
tive carbon atoms were partially converted to 14C02, while the remainder were bound,
as cell constituents, to soil humus fractions. We, therefore, have reason to expect that a
portion of the carbon in the ALAR molecule is incorporated this way into the soil
organic matter. Distribution of this 14C in all soil organic matter fractions is consistent
with incorporation as harmless products.

References
Dost, F. N., D. J. Reed, and C. H. Wang: The metabolic fate of monomethylhydrazine
and unsymmetrical dimethylhydrazine. Biochem. Pharmacol. 15:1325 (1966).
Kearney, P. C., and D. D. Kaufman: Degradation of Herbicides. New York: M. Dekker,
Inc., pp. 89 and 130 (1969).
Lane, J. R.: Analytical Methods for Pesticides, Plant Growth Regulators and Food
Additives, edited by G. Zweig. Acad. Press, pp. 502-505 (1967).
Martin, J. P., and J. O. Ervin: Decomposition and transformation of herbicides in soils.
Proc., 22nd Ann. Calif. Weed Conf. 22, 83 (1970).
Mayaudon, J., and P. Simonart: Study of the decomposition of the organic matter in the
soft by means of radioactive carbon. II. Decomposition of radioactive glucose in the
soil. A. Distribution of the radioactivity in the humic fractions of soil. P1. Soil 9,
376 (1958).
Peterson, J. I.: Carbon dioxide collection accessory of the rapid combustion apparatus
for preparation of biological samples for liquid scintillation analysis. Anal. Biochem.
31,204 (1969).
Schmidt, E. L., H. D. Putnam, and E. A. Paul: Behavior of free amino acids in soil. Proc.
Soil Sci. Soc. Am. 24, 107 (1960).
Stevenson, F. J.: Methods of Soil Analysis, edited by B. A. Black, Am. Soc. Agron.,
Vol. 2, pp. 1409-21 (1965).
Stevenson, I. L., and K. C. Ivarson: The decomposition of radioactive acetate in soils.
Can. J. Microbiol. 10, 139 (1964).

Manuscript received September 12, 1973; accepted November 4, 1973