Identification of degradation products of the avicide 3-chloro-p- toluidine hydrochloride in louisiana rice fields

Article abstract of DOI:10.1021/es9600789

An investigation of the migration of 3-chloro-p-toluidine hydrochloride (CPTH) on treated rice baits to soils in Louisiana rice fields was completed. The persistence of free CPTH in these soils was evaluated with field and laboratory experiments. These so

Full text of DOI:10.1021/es9600789

Environ. Sci. Technol. 1997, 31, 346-350
In coastal parishes of Louisiana, depredating blackbirds
Identification of Degradation
Products of the Avicide
generally roost more than 3 mi from the nearest possible bait
site. Glahn and Wilson (3) showed that the flight lines of
blackbirds emanating from coastal roosts could be decoyed
to simulated rice fields. Studies by West (4) and Knittle et al.
3
-Chloro-p-toluidine Hydrochloride in
(
5) showed that, with proper timing and judicious baiting
Louisiana Rice Fields
techniques, large roosts of starlings can be selectively reduced
by baiting with 3-chloro-p-toluidine hydrochloride (CPTH)
baits. Additionally, both laboratory and field studies showed
no secondary hazards to non-target species (6, 7).
,
†
T H O M A S M . P R I M U S , *
†
J E A N N E N . T A W A R A ,
J O H N J . J O H N S T O N ,
†
In conjunction with a study to evaluate the efficacy of
CPTH-baited decoy rice fields to reduce localized blackbird
populations, we investigated the decrease in CPTH bait
concentrations versus time, the migration of CPTH from baits
to soil, the decrease in free CPTH soil concentrations versus
time, and the formation of CPTH degradation products in
soil. We ascertained the decrease in CPTH concentrations
in baits and the propensity of CPTH to leach from rice baits
to soil by quantifying the CPTH content of the rice bait as
well as soil in rice-baited plots at pre-baiting and 1, 3, 6 and
†
J O H N L . C U M M I N G S ,
S T E P H A N I E A . V O L Z ,
†
†
M A R G A R E T J . G O O D A L L ,
†
D A N I E L B . H U R L B U T ,
†
D O R E E N L . G R I F F I N , A N D
‡
S H E R R I T U R N I P S E E D
USDA APHIS Denver Wildlife Research Center, Building 16,
Denver Federal Center, Denver, Colorado 80225, and
FDA Animal Drugs Research Center, P.O. Box 25087,
Denver Federal Center, Denver, Colorado 80225
1
3 days post-baiting. We ascertained the persistence of free
CPTH in Louisiana rice fields by applying CPTH directly to
soil plots and determining the acetonitrile-extractable CPTH
content of soil at the same sampling intervals. Finally, we
screened the soil and bait extracts by high-performance liquid
chromatography (HPLC) for the presence of CPTH degrada-
tion products. Greater quantities of the degradation products
were produced by fortifying soils with CPTH at exaggerated
levels and incubating under simulated field conditions in an
environmental chamber. Extraction of the incubates and co-
chromatography with synthesized standards permitted ten-
tative identification of CPTH degradation products. Iden-
tification was finalized by gas chrom atography/ m ass
spectrometry (GC/ MS) and/ or liquid chromatography/ mass
spectrometry (LC/ MS).
An investigation of the migration of 3-chloro-p-toluidine
hydrochloride (CPTH) on treated rice baits to soils in Louisiana
rice fields was completed. The persistence of free CPTH
in these soils was evaluated with field and laboratory
experiments. These soils were also screened for the presence
of CPTH degradation products. Soils treated with CPTH-
fortified rice bait and aqueous solutions of CPTH were
exposed under actual field conditions for a 13-day period.
Control soil samples were also treated with CPTH and
incubated under simulated field conditions in an environmental
chamber. Soil samples were then screened for CPTH
residues and degradation products by HPLC. Possible
degradation products were identified with the assistance of
LC/MS and GC/MS. The concentration of 2% CPTH on
treated rice baits placed in Louisiana rice fields decreased
by approximately 55% over 3 days. Several CPTH
degradation products were detected in rice field soils, treated
with an aqueous solution of CPTH, at concentrations too
low for spectral identification. Multiple CPTH degradation
products were detected in a laboratory soil metabolism
study including the previously unreported compounds, cis-
and trans-azo-3-chloro-p-toluidine (azo-CPT). Neither azo
compound was detected in soils collected from fields that
were treated with CPTH rice baits.
Experimental Section
Chem icals. All solvents and chemicals were reagent grade
unless otherwise specified: acetone, HPLC grade, Fisher
Scientific; acetonitrile, optima grade, Fisher Scientific; ben-
zene, ACS grade, Fisher Scientific; diethyl ether, J. T. Baker;
ethanol, denatured, Fisher Scientific; ethyl acetate, HPLC
grade, Fisher Scientific; hexane, optima grade, Fisher Sci-
entific; CPTH (99.1%), Purina Mills, Inc.; acetyl chloride,
Aldrich; 2-chloro-4-nitrotoluene, Chem Service; lithium alu-
minum hydride, Aldrich; sodium sulfate, ACS grade, 10-60
mesh, Fisher Scientific; deuterated chloroform and deuterated
methanol, Aldrich; TLC plates, aluminum backed silica gel,
Whatman, 250 µm, 20 × 20 cm, fluorescent at 254 nm; silica
gel, Whatman, 70-230 mesh.
Baits. A 2% CPTH-treated rice bait was formulated at the
U.S. Department of Agriculture, Animal Damage Control,
Pocatello Supply Depot (Pocatello, ID). CPTH concentration
was determined by HPLC. The baits were then shipped by
truck to the study site in Crowley, LA, and stored in a climate-
controlled building.
Introduction
Red wing blackbirds (Agelaius phoeniceus) frequently con-
gregate in large spring roosts and cause damage estimated
in excess of 3.7 million dollars annually to sprouting rice (1).
Damage is particularly severe in Louisiana and tends to be
localized and proportional to the size of nearby roost sites
Quantification of CPTH. All HPLC analyses were con-
ducted on a Hewlett Packard 1090M HPLC system equipped
with a diode array detector and a 250 × 4.6 mm Deltabond
(
2). As attempts to alleviate damage via habitat manipulation,
mechanical and pyrotechnic devices and shooting have been
less than successful; recent research efforts have concentrated
on the development of an environmentally safe avicide for
reducing blackbird populations in localized areas suffering
from blackbird-induced rice damage.
(
Keystone Scientific) 5 µm C-8 column. Injection volumes of
1
0 µL were used with a water:acetonitrile (70:30) mobile phase
at 1 mL/ min. CPTH was quantified by comparing absorbance
at 241 nm with that of a standard curve generated by analyses
of seven calibration standard solutions ranging from 0.02 to
2
.0 µg of CPTH/ mL of mobile phase. HPLC run times of 14
*
Corresponding author telephone: (303)236-7867; fax: (303)236-
min were used to quantify the degradation of CPTH. HPLC
run times of 35 min were used to analyze soil extracts for the
formation of CPTH degradation products.
7
863.
†
‡
USDA APHIS Denver Wildlife Research Center.
FDA Animal Drugs Research Center.
S0013-936X(96)00078-8 This article not subject to U.S. copyright.
Published 1997 by the Am erican Chem ical Society.
3
4 6
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 2, 1997

The CPTH content of bait samples was quantified following
the extraction of approximately 2.0 g of bait with 25 mL of
mobile phase in a 50-mL centrifuge tube. The tubes were
capped, aligned horizontally, and mechanically shaken at
high speed for 10 min, followed by centrifugation at 2000g
for 2 min. Extractions were repeated two more times with
Soil Testing Laboratory for characterization. Conductivity,
pH, percent organics, cation exchange capacity, texture
analysis, and moisture content were determined for each
sample.
Soil Laboratory Incubation. Untreated soils from three
sites were combined and mixed, and 10-g aliquots were placed
in 60-mL polypropylene centrifuge tubes. The soil was then
fortified with CPTH at 300 ppm. Control soils were not
fortified with CPTH. The uncovered tubes were incubated
in a Revco Model PG-8-1045-A environmental chamber at 25
°C and exposed to a 12:12 h light:dark cycle with an average
relative humidity of 65%. The mouths of the uncapped tubes
were positioned approximately 0.3 m from a bank of
fluorescent and incandescent light bulbs. Approximately 1
mL of deionized water was added to each tube on alternate
days. Soil samples were removed on days 0, 1, 3, 6 and 13,
extracted, and analyzed by HPLC using the same procedure
as was used for the field soil samples.
Mass Spectrom etry. HPLC/ MS was conducted on a
Hewlett Packard (HP) 1090 liquid chromatograph/ 5989 mass
spectrometer equipped with a HP 5990A particle beam
interface and operated in the electron impact (EI) ionization
mode. Mass spectrometry conditions were as follows: helium
nebulizer pressure, 38 psi; ionization potential, 70 eV;
desolvation chamber temperature, 40 °C; source temperature,
250 °C. The mass spectrometer was manually tuned using
perfluorotributylamine and monitoring ions m/z 69, 219, and
502 in EI mode. Analyses were conducted in the full scan
mode (100-300 m/z) and selected ion monitoring mode for
the detection of azo-CPT (m/z 125, 153, 278, 280), acetyl-CPT
(m/z 106, 141, 183, 185), and 3-hydroxy-p-toluidine (m/z 77,
94, 122, 123). Chromatographic separation was conducted
with a 250 cm × 4.6 mm Octyl/ H HPLC column (Keystone
Scientific). A mobile phase of acetonitrile:water (92:8) was
used at a flow rate of 0.5 mL/ min.
1
0 mL of mobile phase; supernatants were combined and
diluted to a final volume of 50.0 mL with mobile phase.
Aliquots were filtered through 0.45 µm nylon filters prior to
analysis by HPLC.
For analyses of soil samples, approximately 10 g of soil
was added to 50-mL centrifuge tubes containing 10.0 mL of
acetonitrile. The tubes were capped, aligned horizontally,
and mechanically shaken at high speed for 15 min. The soil-
containing tubes were then centrifuged at 1000g for 2 min.
Aliquots of the supernatant were removed, filtered through
0
.45 µm filters, transferred to two 2-mL amber autosampler
vials, and capped. CPTH content of the extracts was
determined by HPLC. Recoveries were determined by the
analysis of quality control (QC) samples containing 0.2, 0.5,
and 2.0 µg of CPTH/ g of soil. Soil moisture content was
determined by calculating mass difference following the
drying of 10.00-g aliquots of soil at 110 °C for at least 12 h.
Site Preparation. The rice fields at three different study
sites were prepared identically. Fields were divided into 0.3
m × 0.3 m treatment areas. Each treatment area was at least
1
m from any other treatment area. Two application methods,
aqueous and seed treatment, were evaluated at each site. For
each sampling day, triplicate treatment areas and a control
plot were required for each treatment method. Each study
site required 24 treatment and eight control areas.
Application Methods. All treatment areas were fortified
on the same day by one of two methods. For the rice
application method, 30 grains of 2% CPTH-treated rice bait
2
were placed randomly over each 0.09 m treatment area. The
rice bait on each treatment area contained approximately
GC/ MS was conducted on a HP 5890 gas chromatograph
and 5970 mass selective detector. The GC was equipped with
a 0.25 mm × 15 m DB-5 column (J&W Scientific) and operated
with helium carrier gas at a flow rate of approximately 45
mL/ min at an initial temperature of 50 °C, ramped at 10 °C/
min to 160 °C, held at 160 °C for 0.1 min, ramped at 30 °C/
min to a final temperature of 290 °C, and held at the final
temperature for 2.5 min. Mass spectrometry conditions were
as follows: ionization energy, 70 eV, mass range, m/z ) 70-
400; ion source temperature, 280 °C.
1
3.2 mg of CPTH. This application rate is equal to 10× the
maximum permitted application rate. No bait was applied
to control areas. To ensure that the initial quantity of CPTH
in contact with the soil was known, an aqueous treatment
method was also employed in which 1.00 mL of a 13.2 mg of
CPTH/ mL of water solution was evenly applied with a syringe
over each aqueous treatment area. Control areas received
no CPTH solution. The field application experiments were
completed in the spring of 1994 from March 28 to April 10.
The ambient temperatures ranged from 58 to 82 °F with
predominately clear skies except for the occurrence of rain
on day 12.
Nuclear Magnetic Resonance Spectroscopy. NMR spec-
tra of the synthesized standards of acetyl-CPT and cis- and
trans-azo-CPT were collected on a Bruker-ACE spectrometer,
1
13
Sam ple Collection. Soil samples were collected one day
prior to treatment, several hours after treatment, and 1, 3, 6,
and 13 days post-treatment. On each sampling day, a control
and three treated samples were collected for each application
method; eight samples were collected per sampling day at
each site. Each sample was obtained by collecting the top
300 MHz ( H) and 75 MHz ( C).
Synthesis of Standards. Postulated degradation products
of CPTH were synthesized for use in identification of actual
CPTH degradation products. Acetyl-CPT (N-acetyl-3-chloro-
p-toluidine hydrochloride) (Figure 1) was prepared from the
free base of CPTH. The free base was prepared by differential
pH extraction on 489.2 mg of CPTH. A total of 130 mg of the
free base CPT (0.92 mmol) was taken up in anhydrous diethyl
ether, and 1 mL of acetyl chloride (14.2 mmol) was added
dropwise. The reaction mixture was stirred at room tem-
perature for 30 min. The reaction was quenched by the
addition of distilled water, and the ether was evaporated under
vacuo. The white crystals that formed in the aqueous layer
were filtered (86.8 mg, 0.47 mmol, 51% yield). Analysis by
GC/ MS and HPLC indicated a purity of >99%. Mass spectral
3
cm deep layer of soil in the desired treatment or control
area in a plastic bucket. The soil-containing buckets were
covered and mechanically shaken to provide a homogeneous
sample of which a 500-g subsample was removed and placed
in a 500-mL polyethylene jar. The samples were placed
immediately in a freezer, stored for no longer than 4 days,
and shipped frozen overnight to our laboratory in Denver.
Samples were stored at -26 °C until analyzed.
Storage Stability. Soil samples were stored in our
laboratory for 19-33 days prior to analysis. To estimate the
loss of CPTH during this storage period, control soils from
each study site were combined, homogenized, and fortified
at 0, 0.20, 0.50, and 2.00 µg of CPTH/ g and stored at identical
conditions to the actual field samples. CPTH content of the
soils was determined after 0, 8, 15, 26, and 33 days of storage.
Soil Characterization. Representative control soil samples
from each study site were sent to the Colorado State University
1
data agreed with published data (8). Analysis by H NMR
3
(CDCl , 7.24 ppm) yielded the following data: H-2, 7.55 ppm
(s, 1 p); H-5 and H-6, overlapping, 7.12 and 7.23 ppm (d, 8.4
Hz, d, overlapping solvent peak, 2 p); H-7, 2.29 ppm (s, 3 p);
acetyl methyl, 2.14 ppm (s, 3 p).
Azo-CPT (3,3′-chloro-4,4′-methylazobenzene) (Figure 1)
was synthesized by following the procedure for the reduction
of halogenonitroarenes with lithium aluminum hydride by
VOL. 31, NO. 2, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3 4 7

FIGURE 1. CPTH and degredation products.
FIGURE 2. CPTH storage stability curve: ppm CPTH vs days of storage.
Calculated with soil dry weight and recovery corrected. (9) 0.20,
Corbett and Holt (9). A total of 301.2 mg of 2-chloro-4-
nitrotoluene (1.76 mmol) was taken up in 30 mL of anhydrous
diethyl ether and stirred, with the slow addition of 0.2 g of
(2) 0.52, and (b) 2.0 ppm.
LiAlH
4
. The yellow solution was stirred under nitrogen for
3
0 min. When distilled water was added to quench the
TABLE 1. Soil Characterization
reaction, a white precipitate formed and separated from the
clear orange-colored organic layer. The organic layer was
decanted and evaporated under vacuo leaving an orange solid.
Analysis of the orange solid by GC/ MS revealed that the
product was a mixture of two major compounds: the azo-
CPT compound and what appeared to be the azoxy analogue
of the azo-CPT compound.
sand silt clay
water
site pH conda OMb CECc
(
g/kg) (g/kg) (g/kg) texture estimate (g/kg)
1
2
3
6.9 0.7 34 188 150 550 300 silty clay loam 173
6.4 0.8 25 142 120 660 220 silt loam
6.2 0.6 27 151 170 610 220 silt loam
122
124
a
Conductivity (m m hos/cm ). b Organic m atter (g/kg). c Cation ex-
change capacity (m m ol /kg).
c
The azo-CPT compound was separated from the azoxy
compound by silica gel flash chromatography with hexane
as the eluant. Twenty-two fractions of 8-10 mL each were
collected. The azo-CPT compound eluted in fractions 1-11.
With exposure to light during the separation procedure, the
trans-azo-CPT isomerized to the corresponding cis isomer.
The cis- and trans-azo-CPT compounds were separated using
All residue data were corrected for QC recoveries generated
by analyzing CPTH-fortified soil on the same day of analysis.
The mean QC recovery for the three fortification levels over
the duration of the study was 43.2 ( 14.1%. In method
development experiments, acetonitrile extractions of CPTH-
fortified soils gave superior recoveries to other extraction
solvents, which included methanol, ethyl acetate, and ac-
etonitrile:water ratios of 50:50 to 100:0. Extractions of CPTH-
fortified soil samples with acetonitrile:water (80:20) at pH 3,
silica gel TLC plates, eluted with 9:1 hexane/ acetone. The R
f
values for trans- and cis-azo-CPT were 0.81 and 0.58,
respectively. Each band was scraped from the TLC plate,
and the compounds were recovered from the silica gel by
extraction with ethanol.
6
, and 9 also yielded inferior recoveries to acetonitrile.
The storage stability data for the 0.20, 0.52, and 2.0 ppm
When analyzed by HPLC, the retention times of the cis-
and trans-azo-CPT compounds were 11.1 and 30.1 min,
respectively. By GC/ MS, the cis- and trans-azo-CPT com-
CPTH-fortified soils were very similar. When compared by
analysis of variance (11), the mean storage losses in soil from
each study site at the three spiking levels were not significantly
different (R ) 0.05). Also, the storage-associated losses at
each fortification level in soil from different study sites were
not significantly different. The mean percent loss ( standard
deviation for the three soil types during 33 days of storage
were 32 ( 4%, 37 ( 4%, and 33 ( 7% for sites 1, 2, and 3,
respectively. The data for each site were combined to generate
the storage stability curves (ppm of CPTH vs days of storage)
presented in Figure 2. All residue data were also corrected
for estimated storage stability losses.
+
pounds were indistinguishable. m/z: M 278 (5), M + 2 280
(
3), 153(16), 155 (5), 125 (100), 127 (33), 89 (62).
NMR analysis (CDCl
3
, 7.24 ppm). trans-Azo-CPT: H-2,
7
7
.88 ppm (d, 2.1 Hz, 1 p); H-5, 7.36 ppm (d, 8.1 Hz, 1 p); H-6,
.71 ppm (dd, 8.1, 2.1 Hz, 1 p); H-7, 2.43 ppm (s, 3 p); cis-
Azo-CPT: H-2, 6.99 ppm (d, 2.4 Hz, 1 p); H-5, 7.07 ppm (d,
.1 Hz, 1 p); H-6, 6.52 ppm (dd, 2.4, 8.1 Hz, 1 p); H-7, 2.31
8
ppm (s, 3 p).
The synthesis of 3-hydroxy-p-toluidine (Figure 1) was
performed by the iron-activated reduction of 5-nitro-o-cresol
and confirmed by NMR as described by Tawara et al. (10).
The soil moisture content between sites generally varied
by less than 2.5%. On the day of treatment, the mean water
content for the soil sampled from the three study sites was
Results and Discussion
1
6.8 ( 1.0%. The mean water content decreased to 8.6 (
The soil characterization data are presented in Table 1. The
soil from site 1 was classified as a silty clay loam while the
soil from sites 2 and 3 were characterized as silt loams.
1.8% on sampling 6 days post-treatment. Due to rain on day
12, the mean water content increased to 28.7 ( 2.4% in the
soil sampled on day 13 post-treatment. Due to this variation
3
4 8
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 2, 1997

experiment apparently contributed to a more rapid decrease
of CPTH rice bait residues as the mean half-life for CPTH on
the bait was 1.1 ( 0.02 days between the days 6 and 13
sampling periods.
CPTH was the only compound detected in the soil extracts
from the 10× rice seed bait-treated areas from the field
experiment. However, the HPLC chromatograms of extracts
of treated soil collected 1-13 days after treatment with the
aqueous solution of CPTH indicated the formation of several
new compounds. The retention time of CPTH was ap-
proximately 4.3 min. Additional chromatographic peaks with
retention times of approximately 11.1 and 29.7 min, which
were not evident in the day 0 samples, were first detected in
the day 1 samples. The relative area of these peaks increased
in magnitude through the last sampling date (day 13) when
the peak areas of the degradates were approximately 2-10
times that of the parent compound. However, the quantities
of degradation products in the soil extracts were insufficient
to obtain mass spectral confirmations.
To obtain sufficient quantities of the degradation products
for identification by mass spectrometry, control soil samples
from the study site were fortified with CPTH and incubated
as outlined in the Experimental Section. Comparison of the
chromatograms from the analysis of a 13-day fortified soil vs
a 13-day control soil again indicated the presence of two
CPTH degradates with retention times of 11.1 and 29.7 min.
The peak areas of both of these compounds increased over
time as the area of the CPTH peak decreased. Also, the area
of the 29.7-min peak was approximately five times that of the
FIGURE 3. CPTH soil degradation curve: ppm CPTH vs days post-
treatment. Calculated with soild dry weight and recovery corrected.
(b) Rice, (s) mean rice, (9) aqueous, (- -) mean aqueous.
1
1.1-min peak.
in soil moisture, residue data were calculated on a dry weight
basis.
As CPTH had a reversed-phase HPLC retention time of 4.3
min, these degradation products were less polar than CPTH.
Co-injection of the cis-azo-CPT, trans-azo-CPT, acetyl-CPT,
and 3-hydroxy-p-toluidine standards indicated that the peaks
with retention times of 11.1 and 29.7 min were due to the
presence of cis- and trans-azo-CPT in the fortified soil extracts.
The presence of both azo isomers in the soil extract was
confirmed by HPLC/ MS. Mass spectra of the compounds
eluting at these retention times were identical and matched
the spectra of the synthesized azo-CPT standard: m/z (relative
intensity) 125 (100), 153 (21), 278 (23), 280 (15). The 0.65
ratio of the m + 2 (m/z ) 280) to the molecular ion m/z ) 278
is consistent with a dichloro compound. The base ion (m/z
) 125) corresponds to a fragment formed by the cleavage of
the C-N bond and subsequent loss of the azo-chlorotoluene
Concentration (ppm) of free CPTH vs days post-treatment
is presented in Figure 3. As the trends in all three sites were
similar, the data were plotted as mean residues for all three
plots vs days post-treatment. No free CPTH residues were
detected in the pre-treatment soil samples. Comparison of
the soil residues in the aqueous vs rice bait-treated areas
indicated that approximately 40% of the CPTH on the rice
seed had migrated to the soil by day 1. Analyses of the CPTH-
treated rice bait from the three test sites indicated that the
mean half-life of CPTH on the rice seed for the first 6 days
of the field test was 2.5 ( 0.1 days. These data indicated that
the concentration of CPTH on rice seed decreased by
approximately 55% by day 3. The rain on day 12 of the
FIGURE 4. Mass spectra of azo-CPT.
VOL. 31, NO. 2, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3 4 9

moiety (m/z ) 153). Loss of N
2
via further fragmentation of
with rice bait could be due to cleavage of the acetyl-CPT to
CPTH by microbial acylamidases. Such enzymes have been
implicated in the degradation of the herbicide propanil (3,4-
dichloropropionanilide) to 3,4-dichloroaniline (21). Likewise,
the substitution of hydroxy moieties for halogens on halo-
genated benzenes has been documented for numerous
compounds under a variety of environmental conditions (22,
23). No soil metabolites coeluted with the 3-hydroxy-p-
toluidine standard. The rapid degradation of CPTH under
actual field conditions indicates that the propensity for
accumulation of the toxicant and the formation of degradation
products are minimal.
this azo-chlorotoluene moiety gives a m/z ) 125 ion,
additionally contributing to the base peak (Figure 4). None
of the other standards co-eluted with peaks indicative of
degradation products either by HPLC/ UV or HPLC/ MS. The
greater peak area of the later eluting trans isomer in the
laboratory incubated soil extracts suggests that the less
sterically hindered trans-azo-CPT is favored over the cis
isomer.
The presence of azo-CPT was also confirmed by GC/ MS.
However, it was not possible to resolve both isomers by GC/
MS, presumably due to conversion of the cis isomer to the
more stable trans isomer at injection port temperatures. The
formation of azo metabolites in soils has been reported for
a variety of chlorinated anilines. Bartha et al. (12) detected
the formation of dichloroazobenzenes from all three isomers
of chloroaniline and tetrachloroazobenzene (TCAB) from a
variety of dichloroanilines. Kearney and Plimmer (13)
detected both cis- and trans-tetrachloroazobenzene in soils
fortified with 3,4-dichloroaniline. Azo soil metabolites have
also been detected for a variety of pesticides that produced
chloro- and dichloroanilines as intermediate metabolites
including phenylcarbamates, phenylureas, and acylanilides
Acknowledgments
We would like to thank Allen Wilson of the United States
Department of Agriculture in Crowley, LA, and the Vermillion
Rice Growers Association for arranging study sites and
providing control soil samples. Jim Davis and Patty Pochop
of the Denver Wildlife Research Center, Bird Research Section,
were very helpful in the collection of field samples. We are
grateful for the statistical assistance provided by Heather
Krupa. Mention of commercial products is for identification
only and does not constitute endorsement by the United
States Department of Agriculture.
(
14).
It has been reported that the degradation of chloroanilines
Author-Supplied Registry Num bers: CPTH, 7745-89-3;
to azo compounds in soil is mediated by microbial peroxidase
activity (12). As peroxidase activity has been reported in a
variety of soils (15), the formation of azo compounds from
aniline-containing pesticides and environmental contami-
nants may be widespread. However, it appears that the
toxicological significance of these azo compounds is minimal.
While most azo compounds are innocuous, a few have been
found to be associated with liver tumors in animals (16).
Carcinogenicity studies with TCAB, which is structurally
similar to azo-CPT, suggest that TCAB does not appear to
have the required molecular geometry for carcinogenicity
3
-hydroxy-p-toluidine, 2835-95-2; acetyl-CPT, 7149-79-3; azo-
CPT, not available.
Literature Cited
(
(
(3) Glahn, J. F.; Wilson, E. A. Proc. East. Wildl. Damage Control
Conf. 1992, 5, 117-123.
(
(
1) Besser, J. F. Denver Wildlife Research Center Report 340, 1985.
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D. J. Proc. Vertebr. Pest. Conf. 1980, 9, 31-37.
(
6) DeCino, T. J.; Cummingham, D. J.; Schafer, E. W. J. Wildl. Manage.
(
17). Rats fed TCAB at 4 mg/ week for 3 weeks and 10 mg/
1
966, 30, 249-253.
week for 37 weeks and then finally sacrificed at 60 weeks
produced no tumors (18).
(7) Besser, J. F.; Royall, W. C.; DeGrazio, J. W. J. Wildl. Manage.
1967, 31, 48-51.
(
8) Wallnofer, P. R.; Tillmanns, G.; Engelhardt, G. Pest. Biochem.
Physiol. 1977, 7, 481-485.
9) Corbett, J. F.; Holt, P. F. J. Chem. Soc. 1963, 2385-2387.
To determine the amount of TCAB absorbed and trans-
located in plants, Still (19) conducted feeding experiments
(
1
4
with rice plants in saturated solutions of [ C]TCAB. Of the
(
10) Tawara, J. N.; et al. J. Agric. Food Chem. In press.
1
4
C-TCAB available to the rice plants, 5.6% was absorbed;
(11) Anderson, R. L. Practical Statistics for Analytical Chemists; Van
Nostrand Reinhold Company: New York, 1987; pp 122-156.
however, less than 0.2% was found in foliar tissues. Although
TCAB was absorbed by rice plants, the results indicated that
translocation was minimal. Due to the similarities in
structures between TCAB and azo-CPT, these data suggest
that if it were available to rice plants, translocation of azo-
CPT would be insignificant.
Although azo-CPT was present in the aqueous CPTH-
treated soils, no azo-CPT was detected in soils treated with
CPTH rice bait at 10× the maximum application rate. The
low localized concentration of free CPTH in the rice bait-
treated soils may reduce the possibility of polymerization of
CPTH to azo-CPT. Therefore under actual field conditions,
it appears unlikely that azo-CPT residues would accumulate
in soils.
Acetylation of anilines during incubation in soil has been
reported for a variety of substrates including 3,4-dichloro-
aniline (13, 20). Acetyl-CPT was detected on the treated rice
baits following exposure to field conditions. The concentra-
tion of acetyl-CPT increased on rice sampled from days 1-6
and then decreased from days 6 through 13. The ratio of
acetyl-CPT to CPTH increased throughout the study. How-
ever, we failed to detect acetyl-CPT in any of the CPTH fortified
soil incubates. The failure to detect acetyl-CPT in soils treated
(
(
(
12) Bartha, R.; Linke, H. A. B.; Pramer, D. Science 1968, 161, 582-
83.
13) Kearney, P. C.; Plimmer, J. R. J Agric. Food Chem. 1972, 20, 584-
85.
14) Lee, J. K.; Kim, K. C. J. Kor. Chem. Soc. 1978, 21, 197-203.
5
5
(15) Bartha, R.; Pramer, D. Soil Biol. Biochem. 1969, 1, 139-143.
(16) Weisburger, J. H.; Weisburger, E. K. Chem. Eng. News 1966, 44,
1
24-142.
(
(
17) Arcos, J. C.; Arcos, M. In Fortschritte der Arzneimittelforschung;
Junker, E., Ed.; Berghauser Verlag: Stuttgart, Germany, 1962; pp
4
07-581.
18) Bartha, R.; Pramer, D. Adv. Appl. Microbiol. 1970, 13, 317-341.
(19) Still, G. G. Weed Res. 1969, 9, 211-217.
(20) Lee, J. K.; Fornier, J. C. J. Kor. Agric. Chem. Soc. 1978, 21, 71-80.
(
(
(
21) Bartha, R.; Pramer, D. Science 1967, 156, 1617-1618.
22) Plimmer, J. R. Residue Rev. 1970, 33, 47-74.
23) Miller, G. C.; Miille, M. J.; Crosby, D. G.; Sontum, S.; Zepp, R. G.
Tetrahedron 1979, 35, 1797-1800.
Received for review January 24, 1996. Revised manuscript
received July 5, 1996. Accepted September 20, 1996.
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ES9600789
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Abstract published in Advance ACS Abstracts, December 1, 1996.
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 2, 1997