METABOLISM OF TERBACIL IN ORANGE

SEEDLINGS
LOWELLS. JORDAN. AHMED A. ZURQIYAH,WlLHELMUS A, CLERX and J AMESG. LEASCH1
Department of Plant Sciences, University of California
Riverside, California 92502

Orange seedlings were cultured in aqueous solutions of 3-tert-butyl-5-chloro-6-methyluracil

(terbacil and terbacil-2A4C). Radioactivity was distributed throughout the plant with the largest
amount in the roots and the smallest amount in the leaves. Terbacil was metabolized to form
3-tert-butyl-5-chloro-6-hydroxymethyl uracil, which was conjugated to form a/3-glucoside as
the conjugate was hydrolyzed by fl-glucosidase. Identification of the metabolite was made by in-
frared and mass spectrometry after isolation and purification by column and thin layer
chromatography. An additional unidentified water-soluble material was accumulated in the
plant. 5-chloro-uracil was not detected as a metabolite of terbacil.

Terbacil (3-tert-butyl-5-chloro-6-methyluracil) is used for selective weed control of an-

nual and some perennial weeds in citrus, apples, peaches, and sugarcane. It is most readily
absorbed through the root system and transported upwards into the leaves where it acts as
an inhibitor of photosynthesis (Barrentine and Warren 1970, Gardiner et al. 1969, Herholdt
1968). Citrus is very tolerant to terbacil. We have applied up to 32 kg/ha without injury to
trees under field conditions while weeds are killed at rates as low as two kg/ha. Residues of
terbacil were not found in leaves or fruit of trees treated with 32 kg/ha (Jordan et al. 1969).

Few studies were made on the metabolism of terbacil in plants. Gardiner et aL (1969)
found that when terbacil-2-14C is injected into sugarcane, 90% of the radioactivity could be
recovered from the foliar portions of the plant. They determined that 5-chlorouracil was
not a metabolite of terbacil in sugarcane. Barrentine and Warren (1970) reported that ter-
bacil, applied either to the foliage or the root, was partially metabolized in peppermint and
ivy leaf morning glory. No water soluble metabolites were detected in peppermint.
Herholdt (1968) isolated terbacil metabolites from orange seedlings and beans, but did not
identify them.

Herholdt (1968) studied the physiological action, movement, and metabolism of terbacil
in Citrus sinensis L. He observed that the Hill reaction was equally inhibited in isolated
chloroplasts from susceptible Phaseolus vulgaris L. and tolerant citrus. Therefore, the
mechanism of citrus tolerance does not reside in the oxygen evolution system of photo-
system II. As a result, Herholdt proposed that citrus tolerance to terbacil result from less
transport to the leaves and greater metabofism of the herbicide by citrus than by sensitive
species.

IPresent address: USDA, ARS, Market Quality Research Division, Stored-Product Insects Research and Develop-
ment Laboratory, P.O. Box 125, Savannah, GA 31403.

Archives of Environmental Contamination 268

and Toxicology Vol. 3,268-277 (1975)
9 1975 by Springer-Verlag New York Inc.
 Metabolism of Terbacil in Orange Seedlings 269

We performed research to obtain further information relevant to terbacil metabolism in

orange seedlings to determine if metabolism is sufficient to determine tolerance of citrus
to terbacil and the nature of residues from terbacil in citrus. Special effort was made to
determine the possibility of metabolism of terbacil to 5-chorouracil which is closely related
to the mutagen 5-bromouracil.

Procedure

Treatment. Orange seedlings (var. Koethen Sweet orange), approximately 12 months

old, were removed from the steam-sterilized soil in which they were grown. The roots were
thoroughly washed with water to remove all soil. For each treatment, two uniform groups
of three plants were placed in separate 250 ml Erlenmeyer flasks containing 200 ml of
water. The flasks were wrapped with aluminum foil and the water was continuously aer-
ated. The flasks were then placed in a growth chamber with 12 hr of light and 12 hr of
darkness. Light intensity ranged from 25800-26900 lux. Relative humidity was maintained
between 50 and 60%, and temperatures were set at 20~ and 180C for the light and the
dark periods respectively. The plants were allowed to become adapted to the new environ-
ment for three days before treatment.

Terbacil-2-14C, terbacil and potential metabolites were obtained from E. I. duPont de

Nemours, Wilmington, Delaware. The specific activity of terbacil-2-14C was 1.10 mc/m
mole and its purity was better than 99% as determined by two-dimensional TLC. Stock
solutions of terbacil-2-14C and nonlabeled terbacil were prepared in concentrations of one
mg/ml in 100% ethanol. Radioactive stock solutions contained 5.11/~c terbacil-I4C per ml.
Equal amounts (0.4 ml) of the two stock solutions were combined in 200 ml of water to
make a four mg/L solution. The roots of intact orange seedlings were placed in the solu-
tions for 2, 4, 9, and 12 weeks. The water level was maintained at 200 ml throughout each
experiment. Plants from each treatment period were autoradiographed by the method de-
scribed by Crafts and Yamaguchi (1964).

Extraction of terbacil and metabolites. Plants were removed from the treatment solu-
tions and the roots of each plant were washed by swirling with 200 ml of distilled water for
one min, followed by rinsing with water from a wash bottle. The plants were then divided
into leaves, upper stem (above water level), lower stem, and roots. Roots were excised at
the root-stem axis. Stems were excised at the point reached by the water level during treat-
ment (about three cm above the root-stem axis). The lower portion of the stem is called
"lower stem", and the remainder of the stem is called "upper stem" in this paper. Each
plant part was chopped into small pieces, weighed, and placed into an Erlenmeyer flask
with 20-30 ml of 80% aqueous ethanol and soaked for 24 hr in a refrigerator at 5~ to allow
the tissue to soften. The tissues were homogenized three times with 30 ml of 80% ethanol
in a high speed De Virtis blender. The efficiency of the extraction was 88 to 96%. The total
radioactivity in each extract was determined by liquid scintillation counting using Bray's
cocktail (Bray 1960). The 14C in the solid residues, after extraction, was determined by ox-
ygen flask combustion (Davidson and Oliverio 1967).
270 Lowell S. Jordan et al.

Terbacil is soluble in chloroform and slightly soluble in water (710 rag/L). Ten ml ali-
quots of each extract were used for determination of partition coefficients of the radioac-
tive components between chloroform and water. The aliquots were evaporated in a
nitrogen atmosphere to near-dryness and the residue redissolved in a two-phase system of
I0 ml chloroform and 10 ml water. After shaking and centrifuging, the i'adioactivity of
each phase was determined (aliquot 1 ml). Toluene-14C was used as an internal standard to
determine counting efficiency.

Preparation of extracts for column chromatography. Ethanol extracts from leaves were
evaporated on a rotary evaporator to near-dryness. The residue was redissolved by re-
peated additions of acetone and water. The solution was made up to 50% aqueous acetone
(12-15 ml). A precipitate of chlorophyll and other plant materials was formed while stand-
ing overnight in a refrigerator. This precipitate was filtered off and discarded since it con-
tained no radioactivity. The filtrate was then evaporated to near-dryness and the residue
redissolved in 3 to 5 ml of 50% aqueous acetone. This procedure was also employed for the
preparation of extracts from upper stems. Ethanol extracts from roots and lower stems, not
containing high amounts of chlorophyll, were evaporated to near-dryness and the residue
redissolved to 3 to 5 ml of 66% aqueous acetone.

Column chromatography. Polyethylene powder (FN-510 from U.S. Industrial Chemi-

cals, particle size 30: melt index 5) was cleaned by washing three times with acetone and
three times with hexane. The powder was then vacuum dried and packed directly in a col-
umn of 2.5 cm i d to a height of 25-30 cm. The concentrated 50% aqueous acetone extracts
from leaves and upper stems were applied to the column and developed under slightly
reduced pressure with 50% acetone until the chlorophyll band was 3 to 4 cm from the bot-
tom of the column. The eluate at this time contained xanthophylls, terbacil, and terbacil
metabolites. Subsequent sequential applications of 80% acetone and hexane eluted
chlorophyll and carotenes; however, these fractions did not contain any radioactivity. All
of the radioactivity from terbacil and its metabolites was in the first nine ml eluate as deter-
mined by liquid scintillation counting. Extracts in 66% acetone from roots and lower stems
were chromatographed as above but the first elution was made with 66% acetone.

The eluate containing radioactivity was evaporated to near dryness and partitioned be-
tween chloroform and water. Aliquots were taken for liquid scintillation counting to deter-
mine total radioactivity in each phase. The chloroform and water phases were concen-
trated by evaporation in a nitrogen atmosphere for further analysis by thin-layer
chromatography.

TLC analysis of terbacil and metabolites. The thin-layer plates used were commercially
prepared 250 tz silica gel plates with fluorescent indicator and calcium sulfate binder.
Kieselguhr plates were prepared in our laboratory. The plates were prewashed in the
developing solvents used, and stored in a desiccator. The plates were activated for one hr at
l l 0 ~ before use. Aliquots of the chloroform, and water phases were spotted and
developed for 15 cm in chloroform, ethyl acetate, and methanol (10:10:1 v/v/v). The plates
were scanned on a Berthold radioscanner to locate radioactive spots. Radioactive compo-
nents of alcohol extracts were cochromatographed with terbacil and standard potential
 Metabolism of Terbacil in Orange Seedlings 271

metabolites. The Rf values of terbacil, potential metabolites and t h r e e 14C-labeled com-

pounds from orange seedlings treated with terbacil-2-t4C are shown in Table I.

Enzyme treatments, a-Glucosidase and /3-glucosidase were purchased from Sigma

Chemical Co. The water-soluble metabolites were treated with the enzymes c~-glucosidase
and/3-glucosidase in sodium acetate buffer (pH 4.8) and incubated at 35~ for 24 hr. The
solution was then partitioned between chloroform and water. Aliquots of both phases were
spotted on SGF plates together with standards and developed in benzene:ethyl acetate
(50:50 v/v).

The total chloroform soluble fraction was applied as a streak on a SGF plate and
developed in benzene:ethyl acetate (50:50 v/v). A yellow pigment cochromatographed
with the major metabolite. The radioactive area was scrapped and extracted several times
with acetone. The concentrated acetone extract was streaked on a kieselguhr plate and
developed for 15 cm in water. The plate was then turned 180~ and redeveloped in
benzene:ethyl acetate (80:20 v/v). The interference of the yellow pigment was eliminated
by this method.

After extraction with acetone and concentration, enough pure material was collected
for incorporation into a KBr pellet. The pellet was run on a Beckman infrared
spectrophotometer (IR- 12) to identify the major metabolite. A sample of the same material
was dissolved in acetone and analyzed in a Finnigan 1015 S/L mass spectrometer.

Attempted conjugation of major metabolite in vitro. Finely divided roots of fresh

orange seedlings were placed in 80% ethanol. The major identified radioactive metabolite
obtained from our experiment was added in this mixutre and the culture was left soaking

Table I. R f valuesa o f terbacil, potential metabolites and three 14C-labeled compounds from
orange seedlings treated with terbacil-14C by placing roots in water solutions

Chloroform: Ethyl acetate:

Standard ethyl acetate: butanol: Benzene:
or extracted water water ethyl acetate
compounds (I0:I0:I v/v) (20:8:1 v/v) (50:50 v/v)
terbacil 0.42 0.82 0.40
5-chlorouracil 0.I I 0.65 0.09
3-tert-butyl-5-chloro-
6-h ydroxymeth yluracil 0.34 0.81 0.21
3-tert-butyl-6-methyluracil 0.39 0.66 0.25
Compound A (from chloroform) 0.43 0.82 0.40
Compound Bb and Cc(from
chloroform) 17.34 0.81 0.21
Compound D c in H20 0 streak 0

a250tx silica gel F TLC plates

bbefore hydrolysis with/3-glucosidase
eafter hydrolysis with/3-glucosidase
272 Lowell S. Jordan et al.

in the refrigerator for 24 hr. Subsequent extraction and analysis by TLC were made to
determine if conjugation occurred during processing.

Results
Distribution of 14C-labeled materials. The uptake and distribution of terbacil-14C or its
metabolites in orange seedlings over a 12-week period is shown in Table II. The plants ab-
sorbed the applied radioactivity for nine weeks. Based on concentration per unit weight
(/zg/g), the 14C- components accumulated most rapidly in the roots, less rapidly in the
stems, and least rapidly in the leaves. The largest amount of radioactive material was
located in the roots. As time progressed, the proportion of radioactivity in the leaves in-
creased. Autoradiographs revealed that the 14C was located mostly in the veinal areas of
the leaves.

The partition of the extracted radioactive material between chloroform and water is
shown in Figure 1. Moving from the roots to the leaves, the 14C-containing compounds
became more water soluble. After nine weeks, the 14C-containing compounds in all plant
parts were almost entirely water-soluble.

After chloroform-water partition, the water-soluble fraction was treated with /3-
glucosidase. The partition of the components in the original water-soluble fraction from
the plants changed after enzyme treatment (Figure 2). From 55 to 62% of the water soluble
components from the two-week treatment were converted into chloroform soluble
materials. The amount of material which changed from water-soluble to chloroform-solu-
ble decreased as the time of treatment increased. After 12 weeks, from 70 to 90% of the
water-soluble component Containing 14C from terbacil was not hydrolized by the /3-
glucosidase.

Characterization of 14C=labeled material in plants. Separation and final purification of

the chloroform-soluble and water-soluble 14C-labeled material was carried out by TLC in
th ree solvent systems (Table i). The chloroform-soluble material consisted of a major com-
pound (A) and traces of a minor compound (B). The water-soluble material did not
chromatograph.

The water-soluble material was treated with/3-glucosidase and the reaction mixture ex-
tracted with chloroform. The 14C-labeled material from both phases was spotted on silica
gel plates and developed with benzene:ethyl acetate (50:50 v/v). The chloroform phase
contained only one radioactive compound (C). The 14C-material in the water phase after
enzyme treatment did not chromatograph (D).

In Table I the Rf-values are presented for terbacil, 5-chlorouracil, 3-tert-butyl-5-

chloro-6 hydroxymethyluracil and 3-tert-butyl-6-methyluracil in comparison with the Rf
values of compounds A, B, C and D. The Rf value and infrared spectra of compound A
were identical to that of terbacil. Compounds B and C chromatographed the same as 3-tert-
butyl-5-chloro-6-hydroxymethyluracil. Insufficient quantities of B were recovered for
further analysis but because of the same Rf values with three solvent systems on TLC, at
 T a b l e 1I. Distribution of terbacil and its metabolites in orange seedlings maintained 2, 4, 9, and 12 weeks in culture solutions treated with 2 mg/L
terbacil-2-14C. Data and presented as ixg labeled compounds extracted (80% ethanol) and residual (determined by oxygen flask combustion) in
leaves, stems, roots, and whole plants
O

Time after treatment (weeks)

o
Ave. 2 4 9 12
Wt. a
Plant part (g) Ext. b Res. c Tot. d Ext. Res. Tot. Ext. Res. Tot. Ext. Res. Tot.

Leaves 2.8 0.9 0.1 1.0 2.8 0.8 3.6 18.5 1.5 20.0 20.2 1.1 21.3 ~"
Upper stem 0.9 1.2 0.1 1.3 5.1 0.3 5.4 12.4 0.5 12.9 12.6 0.4 13.0 9
Lower stem 0.7 1.8 0.1 1.9 5.3 0.3 5.6 6.8 0.4 7.2 8.3 0.2 8.5
Roots 1.6 12.0 0.4 12.4 48.3 2.6 50.9 55.3 5.1 60.4 53.4 5.6 59.0
Whole plant 6.0 15.9 0.7 16.6 61.5 4.0 65.5 93.0 7.5 100.5 94.5 7.3 101.8
t~

aThe average weight of plants and plant parts is shown to facilitate conversion of concentration to/zg labeled material per gram of plant tissue.
bExt. - extracted; ores. - residual; dTot. - total. r~
 274 Lowell S. Jordan et al.

o~o H20 i~i CHCl 3

Roots Lower Upper Leaves

stem stem

100 O-O 100

O
j 0#o....~ ~ 0 - 0
o-v--~ o

80

o
60

a.
4G 40

20 20

I I
o,
I '~
~ r162

24 912 2 4 9 12 2 4 9 12 2 4 9 12
Time (wk)

Figure 1. Chloroform-water partition of ]4C extracted with 80% aqueous ethanol from parts of orange
seedlings treated for 2, 4, 9 and 12 weeks with terbacil-2-14C in water culture solutions. Data is pre-
sented as % z4C in each phase.

the present time we assume it to be the same as compound C. The infrared and mass
spectra for compound C, from the chloroform-soluble fraction after/3-glucosidase treat-
ment, were identical to the specta for 3-tert-butyt-5-chloro-6-hydroxymethyluracil as
published by Rhodes et al. (1969). The mass spectrum indicated a molecular ion of 232
mass units and a fragmentation pattern identical to 3-tert-butyl-5-chloro-6-hydrox-
ymethyluracil. The 5-chlorouracil was not detected in extracts from citrus with any of the
systems tried.

Attempts to further characterize the water-soluble compound (D) remaining after/3-

glucosidase treatment were not successful. Treatments with o~-glucosidase, ficin, and
clarase did not release chloroform-soluble materials.

The incubation of 3-tert-butyl-5-chloro-6-hydroxymethyluracilwith root tissue did not

result in the formation of conjugated water-soluble compounds such as extracted from
seedlings. The 3-tert-butyl-5-chloro-6-hydroxymethyturacil was recovered unchanged
from the incubation solution after 24 hr.
 Metabolism of Terbacil in Orange Seedlings 275

O~OH20 I~ 9 CHCI3

roots lower upper leaves

stem stem

100 100

/o
8O
/ /
0 o J ~
80

1--
60 oj ~ 60

40 40

\ \
20 20
\
1 9 I I I I I ! t | I
2 4 9 12 2 4 912 24 9 12 24 912
Time (wk)

Figure 2. Chloroform-water partition of 14C-compounds of the water soluble fraction (from 80%
aqueous ethanol extracts of terbacil-2-14C treated orange seedlings) which had been treated with /3-
glucosidase. Data is presented as % I4C in each phase. Terbacil treatments were per 2, 4, 9 and 12
weeks in water culture solutions.

The chloroform-soluble 14C labeled compound from the first solvent partition was in-
tact terbacil (compound A). The amount of 14C recovered as intact terbacil in the roots
decreased from 64% at two weeks to 3% at 12 weeks (Figure 1). The amount of terbacil
recovered from the stems also decreased with time. In the leaves, the amount of terbacil
stabilized at about 10% of the extracted 14C-labeled material.

The water-soluble fraction did not contain more than traces of terbacil or its free
metabolite. The water-soluble fraction from the first partition was treated with /3-
glucosidase. Partition between chloroform and water resulted in a new chloroform-soluble
14C-labeled compound which was determined to be 3-tert-butyl-5-chloro-6-hydrox-
ymethyluracil (compound C). The amount of the hydroxymetabolite released was 40 to
60% in all plant tissue at two weeks (Figure 2). After 12 weeks the a m o u n t of hydroxylated
material released by the enzyme decreased to about 25% for stem, and 10% for leaf ex-
tracts.
276 Lowell S. Jordan et al.

The water-soluble 14C-labeled material, remaining after partition of the e n z y m e treated

fraction, was not hydrolized by treatments with the enzymes previously m e n t i o n e d . At the
end of the treatment period, the unknown water-soluble 14C-component c o n t a i n e d about
80% for roots, 75% for stems, and 90% for leaves of the 14C-label originally as t4C-terbacil.

Discussion
The orange seedlings (var. Koethen Sweet) were treated with aqueous solutions of ter-
bacil. In previous research we noted no visual differences among orange plants grown in
either water or half-strength Hoagland's solution over a period of four m o n t h s . In the pre-
sent research, there were no symptoms of nutrient deficiency or terbacil toxicity.

Terbacil was taken up by orange roots from water solutions and the 14C label dis-
tributed throughout the plant. The first step in its metabolism was the hydroxylation o f the
6-methyl substituent. The 3-tert.butyl-5-chloro-6-hydroxymethyluracil did n o t remain in
a free state very long because only trace amounts could be found in the plant parts. It was
rapidly conjugated with a carbohydrate moiety, most likely glucose, through a b e t a linkage.

The absence of injury symptoms on oranges under field conditions at e l e v a t e d treat-

m e n t rates and in this research indicates the tolerance of citrus to terbacil. Tolerance does
not reside in the chloroplasts because Herholdt observed that the Hill reaction was in-
hibited in citrus chloroplasts (Herholdt 1968). Resistance probably results from two
sources, restricted m o v e m e n t to the site of action (chloroplasts) and metabolism to a non-
toxic compound.

Over 50% of the 14C label was restricted to the roots and translocation to the leaves was
slow. Once in the leaves, the 14C was concentrated around the veins and not in the
mesophyll where the bulk of the chloroplasts are located. Over 90% of the 14C f o u n d in the
veins of the leaves was in the form of water-soluble metabolites and not free terbacil.
Metabolism occurred in all parts of the plant and free terbacil was converted first to the hy-
droxy analogue and then to a complex conjugate. Lowen (1972) states that hydroxylation of
the 6-methyl substituent decreases phytotoxic activity of terbacil and that conversion to
water-soluble metabolites is a detoxication process. Plants (such as orange trees) able to
make the conversion are likely to be more tolerant to terbacil than those which do not
readily make the conversion.

The rapid conjugation of terbacil in orange seedlings probably accounts for the lack of
ability to detect terbacil in leaves and fruit of trees treated even at elevated rates under
field conditions (Jordan et al. 1969). The residue method was designed to isolate and detect
terbacil but would not detect hydroxylated or conjugated terbacil. A valid assessment of
total residues from terbacil in citrus would include methods for determination o f the 6-hy-
droxymethyl metabolite and the nature and the quantities of the unidentified water-solu-
ble compounds disclosed in this research. F r o m the standpoint of residues, t h e most im-
portant conclusion from this research is that 5-chorouracil was not a metabolite resulting
from terbacil applications to citrus.
 Metabolism of Terbacil in Orange Seedlings 277

Acknowledgements

The terbacil, terbacil-2-14C, and standards of potential metabolites were furnished by E.

I. du Pont de Nemours & Co. We thank J. A. Gardiner and Warren K. Lowen for their con-
tributions.

References

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morning glory. Weed Sci. 18, 373 (1970).
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scintillation counter. Anal. Biochem. 1, 279 (1960).
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Man. 35, 143 (1964).
Davidson, Jack D., and V. T. Oliverio: Tritium and C 14 by oxygen flask combustion.
Atomlight 60, 1 (1967).
Gardiner, J. A., Ro C. Rhodes, J. B. Adams Jr., and E. J. Soboczenski: Synthesis and studies
with 2-C14-1abeled bromacil and terbacil. J. Agr. Food Chem. 17, 980 (1969).
Herholdt, J. A.: The mode of action and metabolism of terbacil (3-tert-butyl-5-chloro-6-
methyl-uracil) in bean (Phaseolus vulgaris L.) and citrus (Citrus sinensis L. Osbeck and
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tors in citrus. Proc. Ist Intern. Citrus Symp. 2, 1063 (1969).
Lowen, W. K.: Private communication. E. I. du Pont de Nemours & Co., Inc., Wilmington,
Del. 19898 (1972).
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Manuscrip t received May 17, 1974; accepted July 23, 1974.