Enantiomerization and enantioselective bioaccumulation of metalaxyl in tenebrio molitor larvae

Article abstract of DOI:10.1002/chir.22269

The enantiomerization and enantioselective bioaccumulation of metalaxyl by a single dose of exposure to Tenebrio molitor larvae under laboratory condition were studied by high-performance liquid chromatography tandem mass spectroscopy (HPLC-MS/MS) based on a ChiralcelOD-3R [cellulosetris-tris-(3, 5-dichlorophenyl-carbamate)] column. Exposure of enantiopure R-metalaxyl and S-metalaxyl in Tenebrio molitor larvae exhibited significant enantiomerization, with formation of the R enantiomers from the S enantiomers, and vice versa, which might be attributed to the chiral pesticide catalyzed by a certain enzyme in Tenebrio molitor larvae. Enantiomerization was not observed in wheat bran during the period of 21 d. In addition, bioaccumulation of rac-metalaxyl in Tenebrio molitor larvae was enantioselective with a preferential accumulation of S-metalaxyl. These results showed that enantioselectivity was caused not only by actual degradation and metabolism but also by enantiomerization, which was an important process in the environmental fate and behavior of metalaxyl enantiomers.

Full text of DOI:10.1002/chir.22269

CHIRALITY 26:88–94 (2014)
Enantiomerization and Enantioselective Bioaccumulation of Metalaxyl
in Tenebrio molitor Larvae
YONGXIN GAO, HUILI WANG, FANG QIN, PENG XU, XIAOTIAN LV, JIANZHONG LI, AND BAOYUAN GUO*
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences Haidian District, Beijing, China
ABSTRACT
The enantiomerization and enantioselective bioaccumulation of metalaxyl by a
single dose of exposure to Tenebrio molitor larvae under laboratory condition were studied by
high-performance liquid chromatography tandem mass spectroscopy (HPLC-MS/MS) based
on a ChiralcelOD-3R [cellulosetris-tris-(3, 5-dichlorophenyl-carbamate)] column. Exposure of
enantiopure R-metalaxyl and S-metalaxyl in Tenebrio molitor larvae exhibited significant
enantiomerization, with formation of the R enantiomers from the S enantiomers, and vice versa,
which might be attributed to the chiral pesticide catalyzed by a certain enzyme in Tenebrio
molitor larvae. Enantiomerization was not observed in wheat bran during the period of 21 d. In
addition, bioaccumulation of rac-metalaxyl in Tenebrio molitor larvae was enantioselective with
a preferential accumulation of S-metalaxyl. These results showed that enantioselectivity was
caused not only by actual degradation and metabolism but also by enantiomerization, which
was an important process in the environmental fate and behavior of metalaxyl enantiomers.
Chirality 26:88–94, 2014. © 2013 Wiley Periodicals, Inc.
KEY WORDS: enantiomerization; enantioselectivity; bioaccumulation; metalaxyl; Tenebrio molitor
larvae
INTRODUCTION
used in the control of plant diseases caused by pathogens of
5,16
the Oomycora division in several crops.1
It was initially
The most notorious environmental pollutants are pesti-
1
–3
marketed in the form of a racemate containing two enantio-
mers, R and S, although its fungicidal activity mostly origi-
nates from the R-enantiomer. At present, R-metalaxyl has
cides, which are ~40% chiral molecules. A chiral pesticide
with one chiral center exists as two mirror image isomers
called enantiomers possessing the same physical and chemi-
cal properties. The enantiomers of chiral pesticides may react
at different rates with achiral molecules in the presence of
chiral catalysts. Since constituents of living organisms are
usually chiral, there are greater chances of the chiral pollut-
ants to react at different rates, which may lead to variations
in bioactivity, toxicity, biological uptake, metabolic pathways,
17,18
been used in place of rac-metalaxyl in many countries.
To date, a number of investigations have reported metalaxyl
1
9–22
enantioselective behavior in a variety of ecosystems
animals,
and
23,24
and the results indicated that both environmen-
tal media and conditions could greatly influence the direction
and rate of enantioselective degradation and transformation of
the metalaxyl enantiomers. However, to our knowledge, ex-
4
,5
degradation, and so on. Recently, a growing concern about
the enantioselective behavior of chiral pesticides in the envi-
ronment and the side effects of chiral pesticides on nontarget
organisms have promoted the use of enantiomerically enriched or
pure compounds as a means to diminish the amount of chemicals
2
0
cept for the study of enantiomerization of metalaxyl in soil
25
and sunflower plants, data concerning the enantiomerization
and enantioselective bioaccumulation of metalaxyl in other
nontarget organisms and especially in insects are limited,
although this pesticide is widely applied. (Figure 2)
6
,7
released into the environment. The enantio-specific profiles
of chiral pesticides have become significant issues in the field
of environmental chemistry and toxicology research.
As is known, insects are the most diverse groups of animals
distributed in nature, which are largely responsible for the
breakdown of organic materials such as plants, animals, and
human remains and the elimination of animal wastes. They
are essential food sources for many birds, fishes, reptiles,
Moreover, previous studies has suggested that some chiral
pesticides were configurationally instable under certain con-
ditions and might be easy to occur in enantiomerization,
which was observed for mecoprop, dichlorprop, triadimefon,
phenthoate, malathion, fenpropathrin, fenvalerate, cypermethrin,
and amphibians and they also constitute a significant part of
the human diet in some parts of the world.2
6,27
With a large
and so on.8
–13
number of pesticides widely abused in China, insects can be
infected through touching the agricultural products contami-
nated by the various insecticides, herbicides, or fungicides
and transferred to the higher trophic level of predatory in-
sects. Tenebrio molitor larvae are a kind of lepidoptera insects
in stored grain products and are often used as a model
Enantiomerization of chiral pesticides could give
rise to different influences on the environment. On the one hand,
an active enantiomer could be converted to an inactive one with
more toxicity and persistency; on the other hand, an inactive
enantiomer might partly be converted into the active one. This
could make the environmental behaviors and ecological effects
of chiral pesticides complicated and unexpected. A detailed
knowledge of the environmental behavior of biologically active
chiral compounds must, therefore, contain extensive stereo-
*
Correspondence to: Baoyuan Guo, Research Center for Eco-Environmental
Sciences, Chinese Academy of Sciences, Shuangqing RD 18, Haidian District,
Beijing 100085, China. E-mail: guoby@rcees.ac.cn
Received for publication 25 July 2013; Accepted 13 October 2013
DOI: 10.1002/chir.22269
1
4
chemical information on enantiomeric stability.
Metalaxyl [(R,S)-methyl-N-(2-methoxyacetyl)-N-(2,6-xylyl)-
DL-alaninate] (Fig. 1) is a chiral acylanilide fungicide widely
Published online 3 December 2013 in Wiley Online Library
(wileyonlinelibrary.com).
©
2013 Wiley Periodicals, Inc.

BIOACCUMULATION OF METALAXYL IN T. MOLITOR LARVAE
89
600 pump, a 20 μL and a 2 μL flow cell. Enantiomers were separated on
a ChiralcelOD-3R [cellulosetris-tris-(3, 5-dichlorophenyl-carbamate)] col-
umn. The mobile phase was a mixture of 75% acetonitrile and 25% water
with a flow rate of 0.4 mL/min. Chromatographic separation was
conducted at 20°C and the injection volume was 1 μL.
TSQ QUANTUM ACCESS MAX was used for LC-MS/MS analysis
(Thermo Electron). Quantification was achieved in positive-ion mode
(ESI+) with selected reaction monitoring (SRM). The signals were
received and processed with Thermo Xcalibur 2.2 SP1.48 software. The
optimized major working parameters were as follows: Spray Voltage
Fig. 1. Structures of furalaxyl enantiomers.
3200 V, Vaporizer Temperature 250°C, Sheath Gas Pressure 30 psi, Aux
Gas Pressure 10 arbitrary units, Capillary Temperature 350°C, Capillary
Offset 35 V, and Q2 Gas Pressure 1.5 psi. For metalaxyl, transition m/z
280 > 220 was used for confirmation, m/z 280 > 160 was used for quanti-
fication, and collision energies were 7 eV and 18 eV, respectively.
species in the fields of environmental toxicology, genetics,
28,29
and so on.
So far, no research has yet been reported for
the enantiomerization and enantioselective bioaccumulation
of the metalaxyl enantiomers in Tenebrio molitor larvae.
In this article, an effective method for the stereoselective
determination of metalaxyl residues in Tenebrio molitor larvae
and wheat bran was developed by high-performance liquid
chromatography tandem mass spectroscopy (HPLC-MS/
MS). Enantiopure metalaxyl enantiomers were employed to
elucidate potential enantiomerization of metalaxyl in Tenebrio
molitor larvae. In addition, enantioselective bioaccumulation
of metalaxyl in Tenebrio molitor larvae was investigated. The
purpose of the present study is to emphasize the importance
of enantiomerization in the environmental fate and behavior
of chiral pesticides and to accurately evaluate the potential
effects of the metalaxyl enantiomers on organisms.
Synthesis of the Two Enantiomers of Metalaxyl
To better explore the enantiomerization of chiral pesticides in organ-
isms, obtaining sufficient enantiopure enantiomers standards is neces-
sary. In the study, the two metalaxyl enantiomers were prepared via
chiral synthesis methods.
3
0,31
General Procedure
Synthesis of N-xylyl-D-methyl alaninate. Pyridine (1.42 mL, 17.6 mmol,
.1 equiv) in 30 mL of CH Cl was cooled to 0°C, trifluoromethanesulfonic
1
2
2
anhydride (2.8 mL, 16.2 mmol, 1.05 equiv) was added, and the resulting
mixture was stirred for 10 min. Methyl (S)-(ꢀ)-lactate (1.53 mL, 16.0 mmol,
1
3
equiv) was added dropwise. The reaction mixture was stirred at 0°C for
0 min and then warmed to room temperature. The mixture was filtered
and the filter cake was washed with ether. The filtrate was evaporated in
vacuo (<25°C), and the residue was triturated with ether and filtered.
After evaporating the solvent, the residue was dissolved in 20 mL of
dichloromethane and 2, 6-dimethyl aniline (1.97 mL, 16.0 mmol, 1 equiv)
was added. The reaction solution was stirred at room temperature over-
night. Removal of the solvent in vacuo followed by flash chromatography
gave a light yellow oil (2.76 g).
MATERIAL AND METHODS
Chemicals and Reagents
The fungicide of rac-metalaxyl (>98.0% purity) was provided by the
China Ministry of Agriculture Institute for Control of Agrochemicals.
Two metalaxyl enantiomers were prepared via chiral synthesis methods,
and the optical purities of the R-enantiomer and S-enantiomer were both
about 97.0%. Water was purified by a Milli-Q system. Methanol (HPLC
grade), n-Hexane (HPLC grade), and acetonitrile (HPLC grade) were
obtained from Dikma (Lake Forest, CA). Pyridine, trifluoromethanesulfonic
anhydride, methyl (S)-(ꢀ)-lactate, methyl (R)-(+)-lactate, 2, 6-dimethyl aniline,
Synthesis of R-metalaxyl. Sodium bicarbonate (0.5 g, 6 mmol, 1.1 equiv)
were added to a solution of N-xylyl-D-methyl alaninate (1.12 g, 5.5 mmol,
1
1
equiv) in 30 mL of toluene, cooled to a temperature ranging from 5°C-
0°C, and subsequently 2-furoyl chloride (0.65 g, 6 mmol, 1.1 equiv) was
2
(
-methoxyacetyl chloride, sodium bicarbonate, toluene, dichloromethane
CH Cl ), ether, ethyl acetate, acetone, acetonitrile (analytical grade),
2
2
slowly added dropwise. After 4 h at room temperature, the above solution
was washed with saturated sodium chloride and the organic phase was
evaporated in vacuo. The obtained crude product was crystallized with
hexane to give 1.2 g of white crystalline solid alaninate with an enantiomeric
R/S ratio = 97 (yield 68%).
2 4
and anhydrous sodium sulfate (Na SO ) were purchased from Beijing
Chemical Reagent (Beijing, China).
HPLC-MS/MS Conditions
HPLC was performed using Thermo ACCELA series (Thermo Elec-
tron, Hopkinson, MA) equipped with ACCELA Autosampler, ACCELA
Synthesis of S-metalaxyl with methyl (R)-(+)-lactate as a starting mate-
rial followed the same experimental procedure.
Fig. 2. Enantiomerization of enantiopure R-metalaxyl (a) and enantiopure S-metalaxyl (b) in Tenebrio molitor larvae.
Chirality DOI 10.1002/chir

9
0
GAO ET AL.
evaporated to dryness by a vacuum rotary evaporator. The alumina-N-
Tenebrio molitor Larvae
solid-phase extraction (SPE) (1.0 g) on a cartridge (6 mL) was used to
clean up other interfering substances. The cartridge was preconditioned
by rinsing with 5 mL of ethyl acetate followed by 5 mL of n-hexane and
equilibrated with 10 mL of 1:2 ethyl acetate:n-hexane. The sample of
dry extract was dissolved in 2 mL of 20% ethyl acetate in n-hexane, then
the solution was passed through the SPE cartridge. The cartridge was
eluted with an additional 8 mL of 1:2 ethyl acetate:n-hexane. The eluates
were combined with the loading eluates. The combined 10 mL of eluate
was collected in a pear-shaped flask and evaporated to dryness by a
vacuum rotary evaporator at 35°C and the dry extract was diluted to
1.0 mL with acetonitrile (HPLC grade) and passed through a filter
membrane (pore size, 0.22 μm).
Tenebrio molitor larvae were purchased from Beijing jie matt biological
technology (Beijing, China) and were reared in ventilated plastic terrariums
25× 15 × 20 cm). A layer of wheat bran covered the terrarium floor. The
population was kept at 25°C and the light cycle adopted was 16 h light
and 8 h dark.
(
Wheat Bran Formulation and Tenebrio molitor Larvae
Exposure
To ensure that the rac-metalaxyl was dispersed uniformly in the 100 g
dry weight of wheat bran, we did the procedure in steps (dilution spike).
First, rac-metalaxyl was dissolved in acetonitrile to make a stock solution
at a concentration of 1000 mg/L. Then 1 mL of acetonitrile solution (1000
mg/L) was dissolved into 50 mL of acetone; meanwhile, 100 g of wheat
bran was soaked in the 50 mL of acetone solution of rac-metalaxyl,
followed by drying in a fuming cupboard overnight. The wheat bran
contaminated with enantiopure R-metalaxyl and S-metalaxyl followed
the same experimental procedure.
Method and Validation
A stock solution (100 mg/L) of rac-metalaxyl and two enantiomers
were prepared in acetonitrile,. A series of standard working solutions at
10, 50, 100, 500, and 1000 μg/L concentrations were prepared from the
stock solution by serial dilution in acetonitrile. According to the proce-
dure described above, matrix-matched standard solutions were obtained
at the same concentrations by adding blank Tenebrio molitor larvae and
wheat bran sample extracts to each serially diluted standard solution. Cal-
ibration curves were generated by plotting the peak area of quantification
ion transition against the concentrations of the enantiomers with regres-
sion analysis.
Blank samples (Tenebrio molitor larvae and wheat bran) analysis was
performed to check interference from the matrix. The slope ratios of
the linear calibration functions were calculated to differentiate between
the extraction efficiency and the matrix-induced signal suppression/
enhancement (SSE). The SSE caused by matrix effects was determined.
Recovery of metalaxyl enantiomers was measured in a blank sample
that was fortified at different levels (0.1, 0.5, and 1mg/L based on five rep-
licates). The samples were left for 1 h to ensure that the spiked samples
were evenly distributed. The fortified samples were analyzed and the
recoveries were calculated by comparing the measured concentration to
the fortified concentrations. The precision in the condition for repeatability,
expressed as the relative standard deviation (RSD), was determined at the
entire calibration range.
In this experiment, Tenebrio molitor was used in the larval stage (0.1 g
mean body weight). Molting or pupating larvae were excluded from the
experiment. Before the worms were introduced, they were fed with
uncontaminated wheat bran for 1 week to acclimate. A piece of paper rest-
ing on top of the bran provided cover for the worms. 30 g wet weight of
Tenebrio molitor larvae were exposed to the chemical in each ventilated
plastic terrarium containing 7 g wet weight of contaminated wheat bran.
Food was renewed once a week. All of the plastic terrariums were placed
in the dark in an environmental chamber.
For the uptake experiment, 1 g of Tenebrio molitor larvae and 0.5 g of
wheat bran were collected after an exposure period (1, 3, 9, 24, 72, 168,
3
36, and 504 h). Wheat bran on the surface of worms was filtered by a
sieve, and the worms were frozen at ꢀ20°C in 50 mL of a polypropylene
centrifuge tube. Wheat bran samples from each plastic terrarium were
also stored at ꢀ20°C. All experiments were performed at 25 ± 0.5°C and
were run in triplicate at each sample point.
Analysis of Metalaxyl Residue
For the sample pretreatment of wheat bran, 15 mL of acetonitrile was
added to a 50 mL polypropylene centrifuge tube containing 0.5 g wet
weight of incubated wheat bran sample. Next, the tube was stirred for 3
min on a vortex mixer, exposed to ultrasonic vibration for 10 min, and
then centrifuged at 3000 rpm for 5 min. The extract was transferred to a
new tube. The remaining part was extracted again following the same ex-
traction step, and the extract was combined. The combined extract was
filtered by a funnel with 5 g of anhydrous sodium sulfate to a pear-shaped
flask and evaporated to dryness by a vacuum rotary evaporator at 35°C.
The alumina-N-solid-phase extraction (SPE) (1.0 g) on a cartridge (6 mL)
was used to clean up other interfering substances. The cartridge was
preconditioned by rinsing with 5 mL of ethyl acetate followed by 5 mL of
n-hexane and equilibrated with 5 mL of 1:2 ethyl acetate:n-hexane. The sam-
ple of dry extract was dissolved in 2 mL of 20% ethyl acetate in n-hexane,
then the solution was passed through the SPE cartridge. The cartridge
was eluted with additional 8 mL of 1:2 ethyl acetate: n-hexane. The eluates
were combined with the loading eluates. The combined 10 mL of eluate
was collected in a pear-shaped flask and evaporated to dryness by a vacuum
rotary evaporator at 35°C and the dry extract was diluted to 1.0 mL with
acetonitrile (HPLC grade) and passed through a filter membrane (pore size,
The stability of the metalaxyl enantiomers were evaluated in the stock
solution and matrix extracts. The stock solutions of the enantiomers,
which were prepared two months previously, were tested by comparison
of injections of a newly prepared working solution. The stability of the
spiked Tenebrio molitor larvae and wheat bran samples for the enantio-
mers were determined, and all the samples used in the stability test were
stored at ꢀ20°C.
RESULTS AND DISCUSSION
Calibration and Method Validation
The chiral LC-MS/MS method described above was vali-
dated for the analysis of metalaxyl in Tenebrio molitor larvae
and wheat bran. The typical chromatogram of R-metalaxyl
and S-metalaxyl was baseline separated with no endogenous
interference peaks (Fig. 3). Good linearity was obtained over
the concentration range 0.01–1 mg/kg, and the correlation
2
coefficients (r ) of R-metalaxyl and S-metalaxyl were 0.9961
0.22 μm).
and 0.9968, and the equations of the linear regression are
6
6
For analysis of the Tenebrio molitor larvae, the samples were thawed for
y = 9.0 × 10 x + 268332 and y = 9.0 × 10 x + 255584, respectively.
If the concentrations of the analytical extract exceed the
linear range of the analysis, the analytical extract should be
diluted or concentrated. Tenebrio molitor larvae samples were
fortified with 0.1, 0.5, and 1 mg/kg of the metalaxyl enantio-
mers. The matrix effect was calculated by comparing the
slope of matrix-matched standard curve with the slope of
the standard calibration curve, and the slope ratios were in
the range of 0.85–1.14, so there was no significant matrix
effect for metalaxyl determination with HPLC-MS/MS based
about 15 min at room temperature. 15 mL of acetonitrile was added to
each tube containing 1 g wet weight of Tenebrio molitor larvae sample.
Next, the tube was stirred for 3 min on a vortex mixer, exposed to ultra-
sonic vibration for 10 min, and then centrifuged at 3000 rpm for 5 min.
The sample was reextracted in the same way and the supernatants were
combined. The combined extract was filtered by a funnel with about 5 g
of anhydrous sodium sulfate to a pear-shaped flask and evaporated to
dryness at 35°C. For cleanup (fat destruction), 3 × 10 mL of n-hexane
was added for liquid–liquid partition to extract most of the lipid. The
upper layer of n-hexane was discarded, and the layer of acetonitrile was
Chirality DOI 10.1002/chir

BIOACCUMULATION OF METALAXYL IN T. MOLITOR LARVAE
91
Fig. 3. Representative HPLC-MS chromatogram of the expections of rac-metalaxyl enantiomers in Tenebrio molitor larvae after 21 days of exposure. Flow
rate = 0.4 mL/min, ultrapure water: acetonitrile = 25: 75 (V/V).
on the proposed method. The average recoveries for both
enantiomers ranged between 86% and 94% in Tenebrio molitor
larvae and between 90% and 95% in wheat bran, and the RSDs
ranged from 2.8%–7.9% in Tenebrio molitor larvae, and 2.5% to
With respect to enantiopure S-metalaxyl, the enantiomerization
from S to R in Tenebrio molitor larvae was also observed and the
change trend of the concentration was similar to that of the
enantiopure R-metalaxyl. According to the above results, appar-
ently the metalaxyl enantiomers underwent enantiomerization
at a fast rate in Tenebrio molitor larvae. It was not surprising
that the biotransformation process of chiral pesticides could
lead to an inversion of configuration, but so a fast conversion
rate in Tenebrio molitor larvae exceeded our expectations.
We considered that the rapid enantiomerization of metalaxyl
enantiomers exhibited a strong excitability for Tenebrio
molitor larvae. At the later phase of exposure, the extent of
enantiomerization got smaller, due to continuous uptake of
the major enantiomer and degradation of the minor enantio-
mer. Although the rate of enantiomerization of enantiomer
R-metalaxyl and S-metalaxyl could not be directly calculated,
it seemed that the rate of enantiomerization (R to S) was
different from that of enantiomerization (S to R) based on
the degree of conversion. It has been reported that in
chemically mediated reactions, both enantiomers of a chiral
compound interconvert, and the rates of enantiomerization
5
.6% in wheat bran. Based on the peak-to-peak signal-to-noise
ratio of 3, the limit of detection of the method was 0.02 mg/kg
both in Tenebrio molitor larvae and wheat bran.
Enantiomerization in Tenebrio molitor Larvae
To accurately investigate enantiomerization of metalaxyl
enantiomers in Tenebrio molitor larvae, enantiomerization
was evaluated first in wheat bran. Figure 4 shows that no
enantiomerization occurred for the metalaxyl enantiomers in
wheat bran during 21 d, demonstrating that metalaxyl enantio-
mers were configurationally stable in wheat bran. Then the
wheat bran contaminated with enantiopure R-metalaxyl and
S-metalaxyl was fed to Tenebrio molitor larvae,. The results after
exposure for 21 d are shown in Figure 4a,b. With respect to
enantiopure R-metalaxyl, the concentration of R-metalaxyl rap-
idly increased to 0.23 mg/kg in 9 h, and then quickly decreased
and reached a steady-state level in 3–21 d. Besides, obvious
enantiomerization from R to S was observed at each point of
exposure time. Concentration of S-metalaxyl reached up to
8
(R to S) and (S to R) are equal, and in enzyme-catalyzed
reactions, these rates generally differ, and the formation of
one enantiomer over the other is favored.32 Therefore, to
some extent, the results of the experiment illustrated
enantiomerization could be attributed to enzyme-catalyzed
reactions with enantiomers of metalalxyl in Tenebrio molitor
larvae.
0
.06 mg/kg in 24 hours, and then the degree of conversion
gradually became weak and reached a steady-state level.
Enzyme-mediated enantiomerization had been extensively
studied for pharmaceuticals,3
3,34
amino acids, and sugars
35
with respect to its mechanism. A plausible explanation of
the relationship between enantiomerization and chemical
structure was given. The inversion of stereochemistry in a
chiral compound was generally catalyzed by racemases or
epimerases through cleavage of the methane C-H bond
adjacent to a carbonyl group followed by its reformation or
36
successive oxidation-reduction at a chiral center. As seen
in Figure 1, the chiral carbon in metalaxyl holds an electron-
withdrawing carbonyl group and a conjugated acetanilide
functional group. In the light of the principle of structural
chemistry, the proton on the chiral center is relatively acidic
and is thus easily lost to create an intermediate carbanion.
Under protic environment, reprotonation regenerates the
parent compound from either the top or the bottom face of
the anion, thus interconverts enantiomers.
Fig. 4. Representative HPLC-MS chromatogram of the expections of (a)
enantiopure R-metalaxyl in wheat bran and (b) enantiopure S-metalaxyl in
wheat bran after 21 d of exposure. Flow rate = 0.4 mL/min, ultrapure water:
acetonitrile = 25:75 (V/V).
Chirality DOI 10.1002/chir

9
2
GAO ET AL.
We believed that there should be more chiral pesticides
undergoing such enantiomerization that may occur in a chiral
environment and this deserves further investigation. This was
particularly important with more and more enantiopure prod-
ucts being introduced to the market.
Enantioselective Bioaccumulation in Tenebrio molitor
Larvae
Enantioselective bioaccumulation in Tenebrio molitor larvae
was investigated with rac-metalaxyl. The concentrations of
the two metalaxyl enantiomers in Tenebrio molitor larvae
were determined. The result of bioaccumulation of Tenebrio
molitor larvae is shown in Figure 5. The rapid uptake of the
two metalaxyl enantiomers was observed in Tenebrio molitor
larvae at the initial phase. Concentrations of the two enantio-
mers both reached the highest level in 9 h. But the rapid
decrease of concentrations was observed between 9 to 24 h
of exposure. Finally, the concentrations tended to stability
after 3 d. In Figure 5, a significant difference was observed
between the concentrations of two enantiomers in Tenebrio
molitor larvae tissue at all sample points, with the concentra-
tion of S-metalaxyl higher than that of R-metalaxyl. Another
article has reported that the concentration of S-metalaxyl
was higher than that of R-metalaxyl from soil to earth-
worms, the result of which is in accordance with that of
the experiments.
The enantiomer fraction (EF) was employed to measure
the enantioselectivity of the bioaccumulation of enantiomers
of benalaxyl in Tenebrio molitor larvae. The EF values defined
range from 0 to 1, with EF = 0.5 representing the racemic
mixture. EF was expressed as follows:
Fig. 6. Enantiomeric fraction (EF) of rac-metalaxyl residue in Tenebrio
molitor larvae and wheat bran.
disappeared faster than the R-enantiomer, so the bioaccumulation
of metalaxyl in Tenebrio molitor larvae for this treatment was
expected to be an enantioselective process. The result was in ac-
cordance with what the earlier paper reported on enantioselective
24
bioaccumulation of metalaxyl in earthworm.
24
In this work, the bioaccumulation factor (BAF) is used to
express the bioaccumulation of the metalaxyl enantiomers.
BAF is a function of the relative sorptive capacities of organ-
ism versus the surrounding food, and is defined as:
BAF ¼ C =C
T
WB
where CT is the concentration of compound in Tenebrio
molitor larvae tissue and CWB is the concentration of com-
pound in wheat bran. Since the experiment was confined to
one species, the BAF was not normalized to the lipid content
of the organism.37 Steady state was defined as the time only
when concentrations did not continue to obviously increase
or decrease over three consecutive sampling intervals.38
The calculated BAF was 0.02 for R-metalaxyl and 0.015 for
S-metalaxyl in the steady state. A paired t test for the steady
state between R-metalaxyl and S-metalaxyl yielded a P value
of 0.013, showing that there was a significant difference in
BAF between the two enantiomers of metalaxyl. The BAF value
of the S-enantiomer was higher than that of the R-enantiomer,
which revealed that the S-enantiomer was preferentially accumu-
lated over the R-enantiomer in Tenebrio molitor larvae. It could
be assumed that the bioaccumulation was enantioselective.
From the results of the experiments, the metalaxyl enantio-
mers exhibited a low capacity of bioaccumulation in Tenebrio
molitor larvae and the characteristics observed were that the
metalaxyl enantiomers were taken up rapidly and the concen-
trations of metalaxyl reached the highest level in a short time
and then decreased quickly until equilibration. The low ca-
pacity of bioaccumulation of metalaxyl could be attributed to
a slow uptake rate by Tenebrio molitor larvae, the low lipophi-
licity of metalaxyl, and a high degradation rate.
EF ¼ ½Sꢁ=½Rꢁ þ ½Sꢁ
The EF values calculated in Tenebrio molitor larvae tissue
are shown in Figure 6. It was observed that the EF values
deviated from 0.5 in the bioaccumulation experiment during
1 d of exposure, reached the highest level in 9 h, and finally
maintained at ~0.58. A paired t test for the same timepoints
between R-metalaxyl and S-metalaxyl yielded a P value of
2
0
.002. The paired t test exhibited a significant difference
between the concentrations of two enantiomers in Tenebrio
molitor larvae during the course of the experiment. The change
of EF indicated an initial predominance of S-enantiomer in
comparison to R-enantiomer, but with time the S-enantiomer
As obvious enantiomerization of R-metalaxyl and S-metalaxyl
was observed in Tenebrio molitor larvae before, enantiomerization
should be considered one of the possible factors which brought
about enantioselective bioaccumulation of metalaxyl in Tenebrio
molitor larvae. This showed that the enantioselective behavior
of chiral pesticides in organisms was the result of a combination
of enantioselective degradation and enantiomerization. The
use of each enantiomer as a starting material can clearly
Fig. 5. Concentrations of rac-metalaxyl enantiomers in Tenebrio molitor
larvae.
Chirality DOI 10.1002/chir

BIOACCUMULATION OF METALAXYL IN T. MOLITOR LARVAE
93
1
1
1
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demonstrate the involvement of an enantiomerization process
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of chiral pesticides. Hence, the behavior of each enantiomer
in ecotoxicology on the most sensitive species should be ideally
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CONCLUSION
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In our experiment, the enantiomerization and enantioselective
bioaccumulation of the metalaxyl enantiomers in Tenebrio
molitor larvae from wheat bran were investigated by HPLC-
MS/MS based on a ChiralcelOD-3R column. The results indi-
cated that exposure of enantiopure R-benalaxyl and S-benalaxyl
in Tenebrio molitor larvae revealed significant enantiomerization
with formation of the R enantiomers from the S enantiomers,
and vice versa; enantiomerization was not observed in wheat
bran during the period of 21 d. In addition, there was an obvious
enantioselective bioaccumulation in the larvae with a preferen-
tial accumulation of S-metalaxyl at 10 mg/kg exposure.
Much more attention should be paid to enantiomerization for
a better understanding of the chiral profiles of relevant chiral
pesticides in the environment.
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ACKNOWLEDGMENTS
1. Jarman JL, Jones WJ, Howell LA, Garrison AW. Application of capillary
electrophoresis to study the enantioselective transformation of five
This work has been funded by the Innovative Program of the
Chinese Academy of Sciences (KZCX2-YW-JS403) and National
High Technology Research and Development Program (863) of
China (No. 2012AA06A302).
chiral pesticides in aerobic soil slurries.
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SUPPORTING INFORMATION
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Additional supporting information may be found in the on-
line version of this article at the publisher’s web-site.
4. Xu P, Diao J, Liu D, Zhou Z. Enantioselective bioaccumulation and toxic
effects of metalaxyl in earthworm Eisenia foetida. Chemosphere
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