Environ. Sci. Technol. 1998, 32, 2113-2118
Figure 1. Pyrolysis/ mass spectrometry has indicated a
Chloroaniline/Lignin Conjugates as
Model System for Nonextractable
Pesticide Residues in Crop Plants
preferential binding of 3-chloroaniline and 3,4-dichloroa-
niline (DCA) to the R-carbon of lignin side chains (4). This
position of chloroaniline binding has also been demonstrated
1
13
by H and C NMR spectroscopy of in vitro lignins (dehy-
drogenation polymers, DHP), which were synthesized by
2 2
peroxidase/ H O -mediated polymerization of coniferyl al-
cohol in the presence of 4-chloroaniline or DCA (5).
B . M A R K U S L A N G E , † , ‡
N O R B E R T H E R T K O R N , A N D
H E I N R I C H S A N D E R M A N N , J R . * , †
§
The ecotoxicological significance of nonextractable pes-
ticide residues depends on the bioavailability to an animal
that ingests it and the potential release of low molecular
weight xenobiotic fragments. Bound pesticide residues in
plants generally have but low bioavailability in animals (6,
GSF Forschungszentrum f u¨ r Umwelt und Gesundheit GmbH,
Institut f u¨ r Biochemische Pflanzenpathologie, D-85764
Oberschleissheim, Germany, and GSF Forschungszentrum f u¨ r
Umwelt und Gesundheit GmbH, Institut f u¨ r O¨ kologische
Chemie, D-85764 Oberschleissheim, Germany
7
). Feeding of a nonextractable wheat metabolite fraction
1
4
of [ C] DCA to rats and lambs led to the excretion of 11-20%
of the bound xenobiotic in the form of soluble metabolites.
Under comparable conditions, lignin DHPs containing 14 or
44 mol % chloroanilines released about 65% of the bound
xenobiotic (8, 9), indicating that structural differences existed
between the wheat and the enzymatic chloroaniline/ lignin
conjugates. So far, no experimental evidence exists to explain
the difference in bioavailability. It has also remained open
whether the case of high bioavailability (8, 9) is relevant for
pesticidal crop plant residue levels of chlorinated anilines.
In vitro lignins formed by the peroxidase/H2O2-mediated
polymerization of coniferyl alcohol in the presence of 3,4-
15
1
13
dichloroaniline or [ N]aniline were studied by H, C,
15
and N NMR spectroscopy. The anilines were >95% bound
to the benzylic R-position of lignin side chains. Mild acid
hydrolysis under simulated stomach conditions (0.1 M HCl,
37 °C) was studied as a first estimate of animal bioavail-
Here, we present an improved DHP model system that
ability. The two extremes of facile or slow acid hydrolysis
that are known for chloroaniline/lignin complexes could be
reproduced by using low or high incorporation ratios of
aniline to coniferyl alcohol (10 or 40 mol %, respectively). The
case of facile acid hydrolysis and high animal bioavailability
may be due to the high mole ratios used and may not be
relevant for pesticidal crop plant residue levels of 3,4-
dichloroaniline. The latter are typically in the parts per
15
utilizes derivatives of [ N]- and unlabeled aniline derivatives
1
13
15
and their characterization by H, C, and N NMR spec-
troscopy. Mild acid hydrolysis under simulated stomach
conditions (0.1 M HCl, 37 °C) is used to mimick the digestion
in the animal stomach. Variation of the aniline incorporation
rate allowed us to produce and characterize chloroaniline/
lignin conjugates with high or low acid sensitivity.
15
Experimental Section
million range. On the basis of N NMR spectral fine structure,
we propose that the acid-labile linkage may be due to
anchimeric assistance in conformers formed at the high
aniline molar ratio. The optimized methods presented
here allow the use of in vitro lignin copolymers as a reference
system for the structural features and the bioavailability
of nonextractable pesticide residues in crop plants.
Chem icals. Coniferyl alcohol was purchased from Fluka
(Neu-Ulm, Germany), 3,4-dichloroaniline was from Riedel
1
5
de Haen (Seelze, Germ any), [ N]aniline was from
Chemotrade (Leipzig, Germany), and horseradish peroxidase
1
4
was from Boehringer (Mannheim, Germany). [Ring U- C]-
DCA (Sigma, St. Louis, MO) was purified by HPLC to 99%
(
10). All reagents for NMR spectroscopy were obtained from
Merck (Darmstadt, Germany).
Introduction
Synthesis of Model Com pounds. erythro-1-(4-Hydroxy-
A wide range of commercially important herbicides contains
chlorinated anilines as structural components, e.g., acyla-
nilide, N-phenylurea, and carbamyl derivatives. The free
chloroanilines, which are formed as primary metabolites,
are conjugated in plants, yielding soluble as well as insoluble
3
-m ethoxyphen yl)-2-(2-m ethoxyphen oxy)propan e-1,3-
diol (2a) was synthesized according to Nakatsubo et al. (11).
-(4-Hydroxy-3-methoxyphenyl)-3-(3,4-dichlorophenylami-
3
15
no)-2-(2-methoxyphenoxy)propan-1-ol (2b) and [ N]-3-(4-
hydroxy-3-m ethoxyphenyl)-3-phenylam ino-2-(2-m ethox-
yphenoxy)-propan-1-ol (2c) were synthesized as follows. In
a 100-mL two neck round-bottom flask, 0.1 mmol of 2a in
(
nonextractable) conjugates (1, 2). In intact plants and plant
cell cultures, the operationally defined lignin fraction has
been identified as a major covalent binding site for nonex-
tractable chloroaniline residues (3-5).
Lignins are formed by an oxidative polymerization of
monolignol precursors such as coniferyl alcohol (1 in Figure
4
0 mL of dichloromethane was placed under a nitrogen
atmosphere. At room temperature, 1 mmol of trimethylsilyl
bromide was added within 1 min to the stirred solution with
a syringe through a septum, and the reaction was allowed
to proceed for 4 min to yield the R-brominated product. The
reaction mixture was washed twice with 20 mL of saturated
NaCl. The organic phase containing the quinone methide
was dried over 3 g of Na
removed by a stream of dry nitrogen at 0 °C. Chloroform (20
mL) was added to the residue, and Na SO was removed by
filtration. To the organic solution, 0.2 mmol of 3,4-dichlo-
roaniline or [ N]aniline was added during a 20-min period
under a nitrogen atmosphere for the synthesis of 2b and 2c,
respectively. The mixture was incubated for 2 h at 22 °C. An
aliquot was examined for product purity by analytical HPLC
1
). The proposed reaction mechanism for lignin biosynthesis
involves the formation of a mesomeric monolignol radical
that gives rise to achiral and polydisperse polymers with a
multitude of binding types. The main interunit linkage
present in lignins is the â-aryl ether structure shown in 2 of
2 4
SO for 30 min, and the solvent was
2
4
*
Corresponding author e-mail: sandermann@gsf.de; telephone:
+ †
49 89 3187 2285; fax: +49 89 3187 3383.
15
Institut f u¨ r Biochemische Pflanzenpathologie.
‡
Present address: Institute of Biological Chemistry, Washington
State University, Pullman, WA 99164-6340.
§
Institut f u¨ r O¨ kologische Chemie.
S0013-936X(98)00158-8 CCC: $15.00
Published on Web 05/30/1998
1998 Am erican Chem ical Society
VOL. 32, NO. 14, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
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