Single and joint acute toxicity of isocarbophos enantiomers to Daphnia magna

Article abstract of DOI:10.1021/jf073535l

Although enantioselectivity in the toxicity of chiral pesticides has received considerable attention over recent years, how coexisting enantiomers interact with each other during their toxic action remains unknown. In this study, we attempted to resolve the enantiomers of a chiral organophosphate insecticide, isocarbophos, and investigated the acute toxicity of individual enantiomers and various enantiomer mixtures. Baseline enantiomeric separation of isocarbophos was achieved on a Chiralcel OD column with the mobile phase of n-hexane/isopropanol (90/10, v/v) at a flow rate of 0.8 mL/min. The resolved enantiomers were differentiated by their responses on a circular dichroism detector. The median lethal concentrations (LC₅₀) of racemate, (+)-enantiomer, and (-)-enantiomer of isocarbophos toward Daphnia magna were 13.9, 7.08, and 353 μg/L, respectively, after 48 h of static exposure, displaying a 50-fold difference between the enantiomers. Toxic unit (TU) analysis was employed to evaluate the joint toxicity of isocarbophos enantiomer mixtures. The calculated TU_mix for the acute toxicity (48 h test) of various binary mixtures ranged from 0.83 to 1.04, suggesting a mode of additive effect. Further evaluation of available literature data for chiral organophosphorus insecticides showed that the joint toxicity of enantiomers may be additive, synergistic, and antagonistic. Therefore, when significant enantioselectivity exists for a chiral pesticide, it is important to also evaluate the interaction of enantiomers in the joint toxicity effect when enantiomers are present in a mixture.

Full text of DOI:10.1021/jf073535l

J. Agric. Food Chem. 2008, 56, 4273–4277 4273
Single and Joint Acute Toxicity of Isocarbophos
Enantiomers to Daphnia magna
†
,†
†
‡
KUNDE LIN, WEIPING LIU,* LING LI, AND JAY GAN
Research Center of Green Chirality, Zhejiang University of Technology, Hangzhou 310032, China,
and Department of Environmental Sciences, University of California, Riverside, California 92521
Although enantioselectivity in the toxicity of chiral pesticides has received considerable attention over
recent years, how coexisting enantiomers interact with each other during their toxic action remains
unknown. In this study, we attempted to resolve the enantiomers of a chiral organophosphate
insecticide, isocarbophos, and investigated the acute toxicity of individual enantiomers and various
enantiomer mixtures. Baseline enantiomeric separation of isocarbophos was achieved on a Chiralcel
OD column with the mobile phase of n-hexane/isopropanol (90/10, v/v) at a flow rate of 0.8 mL/min.
The resolved enantiomers were differentiated by their responses on a circular dichroism detector.
The median lethal concentrations (LC50) of racemate, (+)-enantiomer, and (-)-enantiomer of
isocarbophos toward Daphnia magna were 13.9, 7.08, and 353 µg/L, respectively, after 48 h of static
exposure, displaying a 50-fold difference between the enantiomers. Toxic unit (TU) analysis was
employed to evaluate the joint toxicity of isocarbophos enantiomer mixtures. The calculated TUmix
for the acute toxicity (48 h test) of various binary mixtures ranged from 0.83 to 1.04, suggesting a
mode of additive effect. Further evaluation of available literature data for chiral organophosphorus
insecticides showed that the joint toxicity of enantiomers may be additive, synergistic, and antagonistic.
Therefore, when significant enantioselectivity exists for a chiral pesticide, it is important to also evaluate
the interaction of enantiomers in the joint toxicity effect when enantiomers are present in a mixture.
KEYWORDS: Chiral; pesticide isocarbophos; enantiomeric separation; joint toxicity; enantioselective
toxicity
INTRODUCTION
specific acute toxicity toward Daphnia magna. However,
enantioselectivity in the ecotoxicity of isocarbophos has not been
studied.
Organophosphorus pesticides (OPs) are one of the most
important classes of currently used pesticides. OPs are also a
pesticide class containing many chiral compounds. A survey
showed that 30 compounds of 70 OP standards available from
a popular commercial source for pesticide standards are chiral
compounds (1). In OPs, both the phosphorus and the carbon
atoms may be the chiral center. Isocarbophos [(RS)-(O-2-
isopropoxycarbonylphenyl O-methyl phosphoramidothioate]
with an asymmetric center at the phosphorus atom contains one
pair of enantiomers (Figure 1). Isocarbophos was introduced
into agricultural use in 1981 in China. Isocarbophos is a potent
acetylcholinesterase inhibitor and is used to control chewing
and sucking insects and spider mites on a variety of crops such
cotton, tobacco, and rice. Because of its high toxicity and high
residue, isocarbophos was banned from use on vegetables in
China. Other chiral OPs such as methamidophos (2), chlora-
midophos (3), fosthiazate (4), trichloronate (5), leptophos (6),
and fenamiphos (7) have been found to display enantiomer-
Studies increasingly show that the toxicity of chiral pesticides
is enantiomer-specific, suggesting that the environmental risk
of chiral pesticides should be evaluated using individual
enantiomers (2–10). However, most chiral OPs are currently
applied and released into the environment in their racemic forms,
that is, an equimolar mixture of enantiomers. Because micro-
organisms and the associated enzymes constitute a chiral
environment, biodegradation of chiral pesticides is often
enantioselective (11, 12). As a result, the enantiomeric ratio
(
ER), defined as the ratio of the concentration of the (+)-
enantiomer and the (-)-enantiomer, will often deviate from the
racemic composition (ER ) 1.0) (13, 14). When enantioselective
*
To whom correspondence should be addressed. Tel: 0571-
8
8320666. Fax: 0571-88320884. E-mail: wliu@zjut.edu.cn.
†
Zhejiang University of Technology.
Figure 1. Enantiomers of isocarbophos (the asterisk indicates the chiral
center).
‡
University of California.
1
0.1021/jf073535l CCC: $40.75  2008 American Chemical Society
Published on Web 05/20/2008

4274 J. Agric. Food Chem., Vol. 56, No. 11, 2008
Lin et al.
degradation occurs, it is inaccurate to assess the toxicity of
enantiomeric mixtures with enantiomeric excesses using the data
derived from the racemate of chiral pesticides.
organisms were originally obtained from the Chinese Academy of
Protection and Medical Science (Beijing, China). The medium was
renewed three times a week, and daphnids were fed daily with the alga
Scenedesmas obliquus, which were cultured in the laboratory using a
nutrient medium. The test animals used in this experiment were
juveniles aged between 6 and 24 h. Prior to the test, a sensitive test for
daphnids to potassium dichromate was performed as a positive control
and the LC50 (24 h) value was in the range of 0.6-1.7 mg/L. The overall
acute toxicity test was conducted according to the standard protocol
(23). Briefly, five neonates were transferred into glass beakers filled
with 20 mL of blank or test solutions of known enantiomer concentra-
tions. The test solutions with the highest concentrations were prepared
by adding a known amount of enantiomer to the dilution water.
Subsequent dilution was made from the highest concentration to derive
the lower concentration solutions. The nominal concentrations were
Studies considering the enantioselectivity of chiral xenobiotics
in toxicity effects so far have neglected the evaluation of
interaction of enantiomers. The toxicity of compound mixtures
is usually determined by the mode of concentration addition or
response addition. Response addition is based on the prerequisite
that toxicants have different physiological systems in the
organism and is commonly used in assessing the joint effect of
pharmaceuticals (15, 16). Concentration addition is employed
to predict the toxicity of organic compounds that usually pose
a nonspecific mode of action, for example, narcosis (17–19).
In the mode of concentration addition, toxicity of mixtures can
be expressed as toxic units of the mixtures (TUmix), which is a
ratio of the measured concentration of a chemical in a mixture
to the corresponding effect concentration of the single compound
in the same medium (20, 21). Assuming concentration addition,
the TUs for individual components in a mixture can be added
to estimate the total toxicity (21).
1.0, 5.0, 10.0, 20.0, and 40.0 µg/L for (+)-isocarbophos, 50, 125, 250,
500, and 1000 µg/L for (-)-isocarbophos, and 1.0, 5.0, 10.0, 20.0, and
40.0 µg/L for the racemate. For enantiomer mixtures, the nominal
concentrations were chosen according to the ER. For example, the
nominal concentrations for the mixture with an ER of 1:5 were 1.0/
5.0, 2.5/12.5, 5.0/25.0, 10.0/50.0, and 20.0/100.0 as µg/L (+)-
isocarbophos to (-)-isocarbophos. Four replicates were prepared for
each treatment. The test animals were not further fed and were incubated
at 22 ( 1 °C for 48 h. Mortality of daphnids was observed after
incubation for 24 and 48 h. The LC50 values were determined from the
survival data with ToxCalc (Version 5.0) (Tidepool Scientific Software,
McKinleyville, CA).
Joint Toxicity Assay. The extent of toxic interaction was evaluated
by converting the LC50 estimates associated with the mixture to TUs
based on the LC50 estimates associated with the individual enantiomers
when tested alone. The TUmix could be determined by the following
equation:
The main objectives of this study were to develop an
analytical method for the separation of isocarbophos enantiomers
and to evaluate the acute aquatic toxicity of the resolved
enantiomers and the mode of toxicity interaction for the
coexisting enantiomers.
MATERIALS AND METHODS
Chemicals. An analytical standard of racemic isocarbophos (99.6%)
was obtained from Kefa New Technology Development (Shenyang,
China). Other chemicals and solvents were of analytical or high-
performance liquid chromatography (HPLC) grade. For the enantiomeric
separation, isocarbophos was dissolved in the mobile phase at 1000
mg/L.
LC5
0 (+)-isocarbophos (mix)
LC50 (-)-isocarbophos (mix)
LC50 (-)-isocarbophos (alone)
TUmix
)
+
LC50 (+)-isocarbophos (alone)
(1)
Chromatographic Conditions and Resolution of Enantiomers.
Enantiomer separation and preparation were carried out on a Jasco LC-
Accordingly, 1 TU is considered equivalent to the LC50 of an individual
enantiomer when tested alone. Assuming a strictly additive effect, the
LC50 concentration of a mixture should equal unity when expressed as
TUs (24). For example, if the (+)-isocarbophos concentration associated
with the LC50 of a mixture is 12.2 µg/L, 0.87 TU of (+)-isocarbophos
would be present at this concentration, assuming the LC50 for (+)-
isocarbophos alone is 14.1 µg/L. A TUmix value equal to 1.0 ( 0.2
indicates concentration addition. A TUmix less than 0.8 indicates a
synergistic effect (more than additive effect), while a TUmix greater
than 1.2 indicates an antagonistic effect (less than additive effect) (25).
2
2
000 series HPLC system (Jasco, Tokyo, Japan) equipped with a PU-
089 quaternary gradient pump, a mobile phase vacuum degasser, an
AS-1559 autosampler with a 100 µL loop, a CO-2060 column
temperature control compartment, a variable-wavelength CD-2095
circular dichroism (CD) detector, and an LC-Net II/ADC data collector.
Separation was achieved at 25 °C on a Chiralcel OD column (250 mm
×
4.6 mm) with the enantioselective phase [cellulose tris(3,5-dimeth-
ylphenyl carbamate)] coated onto a 5 µm silica-gel substrate. The
injection volume was 20 µL. The flow rate of the mobile phase was
0
.8 mL/min. The detection wavelength of CD was set at 230 nm. The
RESULTS AND DISCUSSION
light source for the chiral detector was a 150 W Hg-Xe lamp, and the
tapered cell path was 25 mm with a volume of 44 µL. The rotation
sign (“+” or “-”) was indicated by a positive or negative peak on the
chromatogram. Identification of the resolved enantiomers was performed
by measuring their CD spectra and by qualitative analysis with an
Agilent 6890 gas chromatograph coupled with an Agilent 5975 mass
spectrum detector (gas chromatography-mass spectrometry, GC-MS)
Enantiomer Separation and Preparation. Successful sepa-
ration of isocarbophos enantiomers was obtained on the Chiral-
cel OD column with a mobile phase of n-hexane/isopropanol
(
90/10, v/v) at the flow rate of 0.8 mL/min. A typical
chromatogram is shown in Figure 2. The separated enantiomers
were easily differentiated from the response in the CD chro-
matograms. According to their CD responses at 230 nm, the
first and second eluted enantiomers were designated as (+)-
isocarbophos and (-)-isocarbophos, respectively. To confirm
that the resolved two peaks corresponded to a pair of enanti-
omers, their CD spectra were scanned by the online CD detector.
The CD spectra of the two peaks (Figure 3) were mirror images
of each other when taking the axis of CD ) 0 as a mirror,
indicating that the resolved chemicals were a pair of enanti-
omers. The resolved enantiomers were separately collected, and
an aliquot of the solutions was analyzed with GC-MS. Their
mass spectra were found to be consistent with that of the racemic
isocarbophos (data not shown).
(
Agilent, Wilmington, DE).
The preparation of individual enantiomers was achieved by manually
collecting the eluent corresponding to the resolved peaks at the HPLC
outlet of the CD detector while observing the UV absorbance. The
collected individual enantiomer solutions were evaporated to dryness,
redissolved in acetone, and used as the stock solutions for bioassays
(
the final amount of acetone in assay solution was <0.04%). The
concentrations of enantiomers in the stock solutions were determined
by analyzing an aliquot on an Agilent 6890 GC coupled with a nitrogen
phosphorus detector (Agilent, Wilmington, DE) assuming the same
response factor for both enantiomers as for the racemate.
Bioassays. The acute toxicity was measured for individual enanti-
omers and racemate and enantiomer mixtures using D. magna as the
test organism. The test organisms were obtained from a continuous
culture maintained at 22 ( 1 °C in M4 culture medium (22) with a
photoperiod of 12 h/day and a density of <50 animals per liter. Stock
The developed method was employed to prepare the pure
enantiomers of isocarbophos for the subsequent bioassays. A

Toxicity of Isocarbophos Enantiomers
J. Agric. Food Chem., Vol. 56, No. 11, 2008 4275
Table 2. Acute Median Lethal Concentrations (LC50, µg/L) of Isocarbophos
Enantiomers in Their Binary Mixtures for Daphnia magna and the
Calculated TUs
24 h test
48 h test
a
ER
(+)
(-)
TU
(+)
(-)
TU
1
1
1
1
1
1
1
2
5
:200
:100
:50
:25
:5
:2
:1
:1
:1
2.22
3.49
6.31
8.30
10.5
11.8
11.8
12.2
12.2
444
349
316
158
50.3
23.6
11.8
6.10
2.40
0.78
0.74
0.89
0.81
0.81
0.87
0.85
0.87
0.87
1.29
1.91
3.31
3.92
6.53
7.08
6.95
7.07
7.07
258
191
166
98.0
32.6
14.2
6.95
3.54
1.41
0.91
0.81
0.93
0.83
1.03
1.04
1.00
1.01
1.00
a
The concentration ratio of (+)-isocarbophos and (-)-isocarbophos.
dophos (3) and fosthiazate (4), which have two chiral centers,
were 13.0- (pk2/pk4) and 3.1-fold (pk1/pk3), respectively.
However, the 50-fold difference between isocarbophos enanti-
omers in the acute toxicity toward D. magna is the largest
between enantiomers for chiral OPs that have been considered
thus far.
Figure 2. Typical HPLC chromatogram for the enantiomeric separation
of isocarbophos on the Chiralcel OD column. Other chromatographic
conditions: mobile phase, n-hexane/isopropanol (90/10, v/v) at the flow
rate of 0.8 mL/min (25 °C).
The significant enantioselectivity strongly suggests that the
aquatic toxicity of isocarbophos enantiomers should be consid-
ered individually. Currently, the environmental risk of most
chiral pesticides is evaluated based on their racemates. Thus,
overestimation or underestimation may result because of the
enantioselective toxicity to nontarget organisms. After a race-
mate of a chiral pesticide is applied, its ER may change due to
enantioselective biodegradation in the environment, leading to
an enantiomeric excess (11, 26). Taking the toxicity of isocar-
bophos to D. magna as an example, the predicted acute toxicity
(
24 h) based on the racemate might be overestimated by 30
times if only the (-)-enantiomer were present in the residue.
However, it might cause a 1.7-fold underestimation if only the
(+)-enantiomer were present in the residue. It is unknown if
enantioselective attenuation occurs to isocarbophos; however,
studies on other chiral pesticides suggest a frequent occurrence
of enantioselective degradation in soil (9). For example,
deforestation in Brazil caused soils to shift from preferentially
removing the less toxic (-)-ruelene to exclusively transforming
the (+)-enantiomer (9). Soil warming in Norway, on the other
hand, caused soils to shift from preferentially removing the (+)-
ruelene to preferentially removing the less toxic enantiomer
Figure 3. CD spectra of resolved isocarbophos enantiomers. Chromato-
graphic conditions were the same as given for Figure 2.
Table 1. Acute Median Lethal Concentrations (LC50, µg/L) of Racemate
and Enantiomers of Isocarbophos for Daphnia magna
LC50 and 95% confident interval
compound
24 h
48 h
(()-isocarbophos
(+)-isocarbophos
(-)-isocarbophos
23.5 (18.9-28.1)
14.1 (13.2-15.0)
709 (648-776)
13.9 (11.1-17.4)
7.08 (6.45-7.71)
353 (302-413)
(
9).
Joint Acute Toxicity of Isocarbophos Enantiomer Mix-
resolution factor (Rs) of 1.5 can theoretically provide 99.7%
separation. With Rs ) 9.9, the pair of isocarbophos enantiomers
was completely resolved on the Chiralcel OD column (with Rs
tures. To understand the interaction of enantiomers in the joint
acute toxicity of isocarbophos enantiomer, the LC50 values at
2
4 and 48 h for binary enantiomer mixtures with ERs ranging
)
9.9) under the experimental conditions and warranted the
sufficient purity of the enantiomers prepared for bioassays.
from 1:200 to 5:1 were determined. On the basis of the LC50
values from the tests on the individual enantiomers and binary
mixtures, the results for the mixtures were converted to TUs of
the individual enantiomer and summed (Table 2). In 24 h test,
the values of calculated TUmix ranged between 0.74 and 0.89,
with an average of 0.83 (n ) 9). Similarly, the values of
calculated TUmix in the 48 h test ranged between 0.83 and 1.04,
with an average of 0.95 (n ) 9). Collectively, these values were
very close to the hypothetical value of 1.0, suggesting that
isocarbophos enantiomers exhibited additive toxicity to D.
magna when both were present in the solution. The additive
effect between isocarbophos enantiomers may be reasonably
explained by the fact that both enantiomers have a similar mode
of toxic action by binding with acetylcholinesterase. This
observation implies that the toxicity to D. magna for isocarbo-
Acute Toxicity of Enantiomers and Racemate. The LC50
values obtained from the tests are summarized in Table 1. The
order of toxicity potency at both 24 and 48 h was (+)-
isocarbophos > racemate > (-)-isocarbophos. The (+)-isocar-
bophos enantiomer exhibited acute toxicity to D. magna was
about 50 times of that for (-)-isocarbophos after either 24 or
4
8 h of exposure (Table 1). It is common for the acute aquatic
toxicity of a chiral OP to be associated primarily with only one
of the enantiomers. For example, the toxicity differences against
D. magna for methamidophos (2), fenamiphos (7), leptophos
(
6), fonofos (8), profenofos (8), and trichloronate (5), were 7.0-,
.6-, 20.0-, 15.0-, 6.6-, and 8.2-fold, respectively. In addition,
the enantioselectivity among the stereoisomers of chlorami-
2

4276 J. Agric. Food Chem., Vol. 56, No. 11, 2008
Lin et al.
Table 3. Calculated TUs for Chiral Organophosphorus Insecticides with One Chiral Center Using Reported Data
LC50(µg/L)
a
compound
exposure time (h)
24
racemate
(+)-enantiomer
(-)-enantiomer
TU
ref
2
2
methamidophos
1.09 × 10
70.7
33.8
1.60
3.90 × 10
2.77 × 10
0.97
0.57
0.75
0.55
1.35
1.13
1.68
2
2
7
6
8
8
5
2
48
34.0
2.38 × 10
fenamiphos
leptophos
fonofos
profenofos
trichloronate
24
24
96
96
96
1.90
4.10 × 10
6.10
0.80
0.23
0.35
0.17
-
2
-2
0.58
0.69
3.45
2.32
1.39
0.51
a
TU was calculated as TU ) [LC50 (+)-enantiomer in racemate/LC50 (+)-enantiomer alone] + [LC50 (-)-enantiomer in racemate/LC50 (-)-enantiomer alone].
Table 4. Calculated TUs for Chiral Organophosphorus Insecticides with Two Chiral Centers Using Reported Data
LC50 (mg/L)
a
a
a
a
b
compound
exposure time (h)
48
racemate
pk1
pk2
pk3
pk4
TU
ref
c
chloramidophos
1.31
1.12
1.10
0.66
0.33
0.26
0.34
0.16
0.50
0.22
0.16
0.16
0.04
0.02
0.65
0.32
0.29
0.16
0.13
0.05
0.87
0.68
0.51
0.40
0.40
0.26
0.57
0.49
0.34
0.28
12.9
18.2
1.77
1.85
1.20
1.21
3
3
2
2
2
2
96
d
fosthiazate
24
48
72
96
a
b
pk1 to pk4 are the first, second, third, and fourth eluted stereoisomers on a chiral column. More detailed information is available in the original references. TU was
4
c
d
calculated as TU ) ∑i)1 [LC50 pk(i) in racemate/LC50 pk(i) alone]. The molar ratio for the pk1:pk2:pk3:pk4 in racemic chloramidophos was 1:2.7:1:2.7. The molar ratio for the
pk1:pk2:pk3:pk4 in racemic fosthiazate was 1:1:1:1.
phos enantiomer mixtures with different ERs can be predicted
by the calculation of TUmix.
may facilitate or hinder the other enantiomer’s binding to the
active sites in an organism simply because the enantiomers have
the same mode of toxic action.
The joint toxicity of enantiomers of a chiral compound is
usually thought to be additive effect, as observed for isocarbo-
phos in the present study. Using the TU analysis approach, we
further calculated the values of TUmix for some other chiral OPs
with one or two asymmetric centers for which toxicity values
were reported (Tables 3 and 4). Judging from the values of
TUmix, the joint acute toxicity to D. magna for methamidophos
In conclusion, racemic isocarbophos was enantiomerically
separated on a Chiralcel OD column, which allowed the
preparation of small quantities of pure enantiomers. About a
5
0-fold difference was observed between the two enantiomers
of isocarbophos in eliciting acute toxicity to D. magna, with
the (+)-isocarbophos being more active than the (-)-enantiomer.
Analysis of the joint toxicity of binary mixtures of isocarbophos
enantiomers toward D. magna clearly showed an additive effect
from the individual enantiomers. Applying the same approach
to literature available toxicity data showed that while an additive
effect was found also for methamidophos and profenofos,
antagonistic and synergistic effects were observed for chlora-
midophos and leptophos, respectively. These results together
imply that the joint acute toxicity for enantiomer mixtures of
chiral xenobiotics may show different modes of interactions
when present in a mixture. Given that most chiral pesticides
are released into the environment as racemates and that
enantiomeric excesses may occur from enantioselective degra-
dation, it is important to understand not only the toxicity
differences between enantiomers but also the mode of toxicity
interaction between enantiomers. More comprehensive studies
are needed to further understand the mechanisms involved in
the different interactions of enantiomers in causing toxicity to
nontarget organisms.
(24 h test), fenamiphos, profenofos, and fosthiazate (72 and 96 h
tests) can also be explained by the mode of additive effect.
However, a synergistic effect between the enantiomers is evident
for methamidophos (48 h test) and leptophos, with TUmix values
being 0.57 and 0.55, respectively. On the other hand, an
antagonistic effect between isomers was found for trichloronate,
chloramidophos, fonofos, and fosthiazate (24 and 48 h test).
The values of TUmix for racemic mixtures of trichloronate,
fonofos, and fosthiazate (24 and 48 h test) showed small
deviations from 1.0, displaying a limited antagonistic effect.
However, a significant antagonistic effect was observed in
racemic mixtures of chloramidophos, with TUmix being 12.9 and
1
8.2 in 48 and 96 h tests, respectively.
No studies so far have attempted to understand such
antagonistic or synergistic interactions between enantiomers. It
may be attributable to in vivo enantiomerization or other in vivo
enantioselective biological reactions. Enantiomers of a chiral
compound may undergo chiral inversion or enantiomerization
in organisms (27, 28), implying that the ER of a mixture binding
to active sites may be different from that of the exposure
mixture. In this case, TUmix does not accurately reflect the joint
toxicity of enantiomer mixtures. For example, enantiomerization
from the less toxic enantiomer to the more toxic enantiomer
would lead to an underestimated TUmix, while the opposite chiral
inversion would lead to an overestimated TUmix. Enantioselec-
tivity in the biological activity may be another important factor.
Before the enantiomers bind to the active sites in D. magna,
they may undergo many enantioselective biological processes
such as uptake, bioconcentration, transformation, and meta-
bolism (11, 26). Furthermore, the presence of one enantiomer
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Received for review December 4, 2007. Revised manuscript received
March 21, 2008. Accepted March 23, 2008. This work was financially
supported by the National Basic Research Program of China
(
(
(
(2003CB114400), the Program for Changjiang Scholars and Innovative
Research Team in University (No. IRT 0653), the National Natural
Science Foundation of China (30500304) and the Opening Foundation
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