Enantioselective acute toxicity effects and bioaccumulation of furalaxyl in the earthworm (Eisenia foetida)

Article abstract of DOI:10.1002/chir.22323

The enantioselectivities of individual enantiomers of furalaxyl in acute toxicity and bioaccumulation in the earthworm (Eisenia foetida) were studied. The acute toxicity was tested by filter paper contact test. After 48 h of exposure, the calculated LC₅₀ values of the R-form, rac-form, and S-form were 2.27, 2.08, and 1.22 μg cm^-2, respectively. After 72 h of exposure, the calculated LC₅₀ values were 1.90, 1.54, and 1.00 μg cm^-2, respectively. Therefore, the acute toxicity of furalaxyl enantiomers was enantioselective. During the bioaccumulation experiment, the enantiomer fraction of furalaxyl in earthworm tissue was observed to deviate from 0.50 and maintained a range of 0.55-0.60; in other words, the bioaccumulation of furalaxyl was enantioselective in earthworm tissue with a preferential accumulation of S-furalaxyl. The uptake kinetic of furalaxyl enantiomers fitted the first-order kinetics well and the calculated kinetic parameters were consistent with the low accumulation efficiency. Chirality 26:307-312, 2014. 2014 Wiley Periodicals, Inc.

Full text of DOI:10.1002/chir.22323

CHIRALITY 26:307–312 (2014)
Enantioselective Acute Toxicity Effects and Bioaccumulation of
Furalaxyl in the Earthworm (Eisenia foetida)
FANG QIN, YONGXIN GAO, BAOYUAN GUO, PENG XU, JIANZHONG LI AND HUILI WANG
*
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China
ABSTRACT
The enantioselectivities of individual enantiomers of furalaxyl in acute toxicity
and bioaccumulation in the earthworm (Eisenia foetida) were studied. The acute toxicity was
tested by filter paper contact test. After 48 h of exposure, the calculated LC50 values of the R-form,
rac-form, and S-form were 2.27, 2.08, and 1.22 μg cm-2, respectively. After 72 h of exposure, the calcu-
lated LC50 values were 1.90, 1.54, and 1.00μg cm-2, respectively. Therefore, the acute toxicity of
furalaxyl enantiomers was enantioselective. During the bioaccumulation experiment, the enantiomer
fraction of furalaxyl in earthworm tissue was observed to deviate from 0.50 and maintained a range of
0.55–0.60; in other words, the bioaccumulation of furalaxyl was enantioselective in earthworm tissue
with a preferential accumulation of S-furalaxyl. The uptake kinetic of furalaxyl enantiomers fitted the
first-order kinetics well and the calculated kinetic parameters were consistent with the low accumu-
lation efficiency. Chirality 26:307–312, 2014. © 2014 Wiley Periodicals, Inc.
KEY WORDS: furalaxyl; enantioselectivity; earthworm; acute toxicity; bioaccumulation
INTRODUCTION
In this study, we selected the commonly used test species,
Eisenia foetida, for acute toxicity and bioavailability tests un-
der laboratory conditions. The objectives of this study were
to (1) evaluate the acute toxicities of rac-furalaxyl and its en-
antiomers to earthworm using a filter paper contact test, (2)
investigate the enantioselective bioaccumulation of furalaxyl
in earthworm from soil.
Furalaxyl (R, S)-methyl-N-(2, 6-dimethylphenyl)-N-(2-
furanylcarbonyl)-DL -alaninate, belonging to the acylanilide
fungicides, was first introduced in 1977 and is still widely
used in the control of plant diseases caused by infection with
Albugo candida, Fusarium solani, Peronospora parasitica,
Phytophthora sp., and Pythium sp.1,2 It can be absorbed by
the roots, stalk, and leaves and can move to all parts of the
plant with protective, curative, and eradicant action for vegeta-
bles and fruits.3 Furalaxyl is a chiral fungicide and consists of
a pair of enantiomers with R- and S-configuration (Fig. 1). Pre-
vious studies have shown that the enantiomers of a chiral
pesticide have identical physical and chemical properties
but they may perform different in the processes of absorption,
accumulation, and degradation when confronted with a chiral
environment.4,5 This makes it necessary to study the biologi-
cal activity, toxicity, and environmental behavior of the two
enantiomers of furalaxyl individually. Sulimma et al. reported
that the R-furalaxyl decreased more rapidly than the S-isomer
by Brevibacillus brevis.6 Thus, application of commercially
available racemic mixtures poses an inevitable biological
burden for treated plants and soils.6 To further research the
effects of chiral pesticides on environmental safety and public
health, we checked the uptake and translocation of furalaxyl
enantiomers by soil organisms in biological systems. This
helps us to address the suitability of replacing pesticide race-
mic mixtures with single biologically active enantiomers for a
more sustainable pesticide use.7–10
Earthworms are widely distributed in topsoil and frequently
dominant in soil communities. They are appropriate model
organisms for bioavailability as they live in close contact with
the soil, have a thin and permeable cuticle, and also consume
large amounts of soil. Earthworms can thus be considered
model organisms for assessing the potential biological effect
of the chemicals released into the environment.11–13 Several
articles have been reported that earthworms were utilized in
both toxicity and bioaccumulation studies,14–16 but until now
there is no report about the enantioselective transfer or
bioaccumulation of furalaxyl from soil to earthworm and the
toxicity of furalaxyl on earthworm.
MATERIALS AND METHODS
Chemicals and Reagents
Racemic furalaxyl and its two enantiomers were synthesized by our lab-
oratory; the purity of rac-enantiomer was 98.0% and both the enantiomeric
purities of R-enantiomer and S-enantiomer were 97.0%. Acetonitrile (high-
performance liquid chromatography [HPLC] grade) was obtained from
Fisher Scientific (Fair Lawn, NJ). Silica-based sorbents including C18
(40 μm particle size) and primary secondary amine (PSA, Si-(CH2)3-NH-
(CH2)2-NH2) (40 μm particle size) were obtained from Agela Technolo-
gies (Tianjin, China). Water was purified by a Milli-Q (Bedford, MA) sys-
tem. All other chemicals and solvents were of analytical reagent grade
and commercially available.
Synthesis of Racemic and Two Enantiomers of Furalaxyl
The two furalaxyl enantiomers were prepared via chiral synthesis
methods,17–19 and the synthetic procedure was the same as synthesis of
benalaxyl enantiomers we have discussed in detail.17
The preparation of R-furalaxyl with methyl S-(-)-lactate (0.77 mL,
8.0 mmol, 1.0 equiv) as a starting material followed the experimental pro-
cedure we reported17 to obtain the light yellow oil N-xylyl-D-methyl
alaninate (1.38 g). Then sodium bicarbonate (0.26 g, 3.1 mmol, 1.1 equiv)
was added to a solution of N-xylyl-D-methyl alaninate (0.58 g, 2.8 mmol,
1.0 equiv) in 15 mL of toluene, cooled to a temperature ranging from
5–10°C, and subsequently 2-furoyl chloride (0.40 g, 3.1 mmol, 1.1 equiv)
Contract grant sponsor: National High Technology Research and Develop-
ment Program (863) of China; Contract grant number: 2012AA06A302.
Contract grant sponsor: National Natural Science Foundation of China; Con-
tract grant numbers: 21277163.41301569.
*Correspondence to: Huili Wang, Research Center for Eco-Environmental Sci-
ences, Chinese Academy of Sciences, Shuangqing Road 18, Haidian District,
Beijing 100085, China. E-mail: huiliwang@rcees.ac.cn
Received for publication 18 November 2013; Accepted 27 February 2014
DOI: 10.1002/chir.22323
Published online 28 April 2014 in Wiley Online Library
(wileyonlinelibrary.com).
© 2014 Wiley Periodicals, Inc.

308
QIN ET AL.
vehicle control replicates. Earthworms were exposed to each jar
containing 340 g contaminated soil after evacuating their gut contents
on moist filter paper for 3 h at 20°C. The jars containing contaminated soil
and worms were weighed and the loss of water by evaporation was com-
pensated by addition of water every 2 d. All of the vessels were placed in
an incubator and maintained at 20 2°C with a photoperiod of 16 h light
and 8 h dark. Worms were collected after exposure periods (0.25, 0.5, 1,
2, 3, 5, 7, 10, 14, 21, and 31 d), rinsed with distilled water, and allowed
to depurate most of their gut contents. Water on the surface of the worms
was dried by absorbent paper cautiously, and then the worms were
weighed and frozen at -20°C. Soil samples (6.8 g wet weight) from each
jar were also stored at -20°C.
Fig. 1. Structures (absolute configurations) of R- and S-furalaxyl.
were slowly added dropwise. After 4 h at room temperature, the above
solution was washed with saturated sodium chloride solution and the
organic phase was evaporated in vacuo. The obtained crude product
was crystallized with hexane to give 0.57 g of white crystalline solid
R-furalaxyl with an enantiomeric R/S ratio = 97 (yield 67%).
The preparation of S-enantiomer with methyl R-(+)-lactate as a starting
material followed the same experimental procedure, S/R ratio = 97 (yield
67%). The preparation of rac-enantiomer with methyl ( )-lactate as a
starting material followed the same experimental procedure, R/S ratio = 1
(yield 67%).
Sample Extraction and Purification Procedure
All the samples were thawed for about 15 min at room temperature. For
soil samples (6.8 g per sample), they were weighed into a 30-mL polypro-
pylene centrifuge tube, followed by addition of 10 mL acetonitrile
containing 1% of acetic acid. After the mixture was vortexed for 1 min
and exposed to ultrasonic vibration for 20 min, anhydrous MgSO4
(2.0 g) and CH3COONa (1.0 g) were added. Then the tube was shaken
vigorously for 1 min using a vortex mixer, exposed to ultrasonic vibration
for 20 min, and centrifuged at 5000 rpm for 3 min. 1.0 mL aliquot of the
Earthworms
acetonitrile extracts was transferred into
a 2-mL centrifuge tube
Mature earthworms (Eisenia foetida), purchased from a northern sub-
containing PSA (50 mg), C18 (50 mg), and anhydrous MgSO4 (100 mg)
for cleanup. After shaking and centrifugation, the resulting solution was
filtered through a 0.22-μm filter. Then 0.1 mL solution was taken and
diluted to 10 mL with acetonitrile for HPLC-dual mass spectrometry
(MS/MS) analysis.
urbs farm, Beijing, were maintained in
a wooden breeding box
(50 × 50 × 20 cm3) containing a mixture of soil and cattle manure. Active,
sexually mature worms with body masses between 200 and 300 mg were
used in the experiment.
For analysis of the earthworms, the samples (2.0 g per sample) were
blended with 8 mL of acetonitrile containing 1% of acetic acid. The mix-
ture was homogenized with an Ultra-Turrax T18 homogenizer for 30 s,
vortex-mixed for 1 min, exposed to ultrasonic vibration for 20 min, and
then anhydrous MgSO4 (1.6 g) and CH3COONa (0.8 g) were added. Then
the purification procedure was the same as the above soil sample. Finally,
0.05 mL solution was taken and diluted to 1 mL with acetonitrile for
HPLC-MS/MS analysis.
Toxicity Test
According to the OECD guideline 207, a paper contact toxicity assay
was used to test the acute toxicity of rac-furalaxyl and its two enantiomers
to earthworms.20 A range of known concentrations of test substances
were prepared with acetone as the solvent. After the depuration period
of 24 h on wet filter paper under dark conditions to evacuate the earth-
worm gut content, earthworms were rinsed in distilled water and cau-
tiously dried by absorbent paper. One milliliter of solution was pipetted
and added to the filter paper (5.5 × 11.5 cm2) placed in flat-bottomed glass
vial. The concentrations of S-furalaxyl on filter papers were 0.63, 0.95,
1.26, 1.58, 1.90, 2.21, and 2.53 μg cm-2. The concentrations of rac-furalaxyl
and R-furalaxyl on filter papers were 1.26, 1.58, 1.90, 2.21, 2.53, 2.85, and
3.48 μg cm-2. After drying of the solvent under a stream of compressed air,
deionized water (1.0 mL) was added to each vial. Controls were also run
in parallel with the carrier solvent alone. Ten replicates for each treatment
and each vial containing one worm were done. Each vial was sealed with
plastic film with several ventilation holes. After that, all the vials were
placed in a room at 20 2°C, and mortality of earthworms was assessed
after incubation for 48 and 72 h, respectively.
Chemical Analysis and Method Validation
HPLC-MS/MS analyses were performed on a TSQ QUANTUM ACCESS
MAX triple quadrupole MS and an Accela 600 pump/autosampler HPLC
(Thermo Electron, Hopkinson, MA). The system was controlled and data
were collected and analyzed by the Thermo Fisher LC Quan software
package (v. 2.7). Enantiomers were separated on a Chiralpak IC column
(4.6 × 250mm i.d., Daicel, Japan) with 5μm particle size. The mobile phase
was a mixture of 80% acetonitrile and 20% water with a flow rate of 0.35mL
min-1. Chromatographic separation was conducted at 20°C and the injection
volume was 10μL.
The electrospray ionization (ESI)-MS interface was operated in the pos-
itive ion mode with selected reaction monitoring (SRM). The ESI source
conditions were as follows: spray voltage, 3200 V; sheath gas pressure,
40 Arb; auxiliary gas pressure, 5 Arb; collision gas (Ar): 1.5 mTorr; vapor-
izer temperature, 250°C; capillary temperature, 350°C. For furalaxyl, m/z
302 > 242 was used for quantification, m/z 302 > 95 wase used for confir-
mation, and collision energies were 30 eV and 16 eV, respectively. Race-
mic furalaxyl was ideally baseline separated and no enantiomerization
was observed for furalaxyl under this analytical condition (Fig. 2). The
first eluted enantiomer was S-form (12.83 min) and the second one was
R-form (14.11 min). Good linear calibration curves were obtained over
the concentration range of 1–500 ng mL-1 for S-furalaxyl and R-furalaxyl.
The calibration curves of matrix-matched standard solutions (1–500 ng
mL-1) by adding blank earthworm and soil sample extracts were also
obtained. Comparing the slope of solvent standard curve with matrix-
matched standard curve, the slope ratios ranged from 0.86–0.98, so there
was no significant matrix-induced ion signal suppression / enhancement
(SSE).
Soil Collection and Earthworm Exposures
The site to collect soil was a farm of Changping District, Beijing, China.
After the superficial vegetation was removed, the top soil (0–10 cm) was
collected. The soils were sieved (2 mm) and air-dried at room tempera-
ture and kept in a dark, dry place. Physicochemical properties of the soil
were as follows: sand loam, organic matter, 3.22 0.12 %; clay, 4.23 0.11
%; silt, 38.04 1.14 %; sand, 55.12 1.26 %; and pH (water, ratio 1:2.5),
7.1 0.2.
To ensure that 250 g of medium was spiked homogeneously with rac-
furalaxyl, S-enantiomer, R-enantiomer, the procedure was as follows.
First, the chemicals were dissolved respectively in acetone (10 mL), and
then the acetone solution was slowly added to dry soil (50 g). The spiked
soil was left in a fume cupboard overnight to remove acetone. Next, the
contaminated dry soil (50 g) was mixed thoroughly with 200 g of
uncontaminated medium. The final concentrations of rac-furalaxyl in soil
were at 20 and 50 mg kg-1 soil. S-form and R-form were at 10 mg kg-1 soil.
Then water (90 g) was added to each jar to restore the 36% water content.
Earthworms used in the bioaccumulation test weighed between 200
and 300 mg and were acclimated to the test soils for 1 week prior to the
start of the test. We used three replicates per concentration and 10
Recovery of furalaxyl enantiomers was determined in blank soil and
earthworm samples by being fortified at different concentration levels
(0.01, 0.1, 1.0, and 10 mg kg-1 based on five replicates). The samples were
left for 1 h to ensure that the spiked pesticides were evenly distributed
Chirality DOI 10.1002/chir

TOXICITY AND BIOACCUMULATION OF FURALAXYL
309
and k2 (d-1) denoted the rate constant characterizing the sum of elimina-
tion, growth dilution, and metabolic transformation of the two enantio-
mers in the earthworm, K was the constant ratio of k1 and k2.
The bioaccumulation factors (BAF) was used to express the relative
bioaccumulation capacities of pesticides in the organisms versus the
surrounding environmental and was calculated as:
À
Á
Cworm
Csoil
BAF kg dry kgꢀ1wet weight
¼
(4)
When the uptake and elimination reached steady state during the
accumulation phase (i.e., t = ),13 Eq. (2) may be reduced to:
Cworm ¼ KCsoil
(5)
(6)
Cworm
K ¼
Csoil
when steady state was reached, according to Eqs. (3), (4), and (6):
k1
BAF ¼ K ¼
k2
(7)
The enantiomer fraction (EF) was used as measure the enantioselective
behaviors of furalaxyl during the experiment. The EF values range from
0 to 1, with EF = 0.5 representing the racemic mixture. It was defined as:
ðSÞ
EF ¼
(8)
ððSÞ þ ðRÞÞ
where (S) was the concentration of S-furalaxyl and (R) was that of
R-furalaxyl. A paired t-test was carried out to compare the means of the
EF values in earthworm and soil samples with EF = 0.5.
RESULTS AND DISCUSSION
Acute Toxicity Assay
The acute toxicity for furalaxyl individual enantiomers and
racemate was measured by a filter paper contact test and
used earthworms (Eisenia foetida) as the test organism.
Earthworms take up organic compounds through their skin
as well as from feeding on soil particles in gut. Hydrophobic
chemicals with a log Kow above 5 mainly were absorbed from
the gut contents, and chemicals with a log Kow below 5 mainly
were absorbed through the skin in the earthworms.11,24 As
the log Kow for furalaxyl is 2.70, skin absorption is the main
route for furalaxyl in earthworm tissue. So the filter paper
contact test is sufficient for assay of the acute toxicity of
furalaxyl enantiomers to earthworm.
In the filter paper contact test, the LC50 values calculated
are shown in Table 1. From these results, the LC50 values
generally decreased over exposure time, and the calculated
LC50 values at both 48 and 72 h exposure of R-furalaxyl, rac-
furalaxyl, and S-furalaxyl were 2.27, 2.08, and 1.22 μg cm-2
(48 h); 1.90, 1.54 and 1.00 μg cm-2 (72 h), respectively. These
results indicated that the acute toxicity of furalaxyl enantio-
mers was enantioselective to earthworm, and R-furalaxyl
was the least toxic compared with racemate and S-form. The
order of toxicity potency at both 48 and 72 h was S-form
racemate > R-form. However, for benalaxyl and metalaxyl,
which have a similar structure with furalaxyl, the fungicidally
active R-benalaxyl was more toxic to earthworm than
S-benalaxyl, and rac-metalaxyl exhibited more than two times
the toxicity than R-metalaxyl.14,24 After 48 h of exposure,
the calculated LC50 values of R-benalaxyl, rac-benalaxy and
S-benalaxyl were 4.99, 5.08, and 6.66 μg cm-2, and the LC50
values of R-metalaxyl and rac-metalaxyl were 52 and 22 μg cm-2,
respectively.14,24 Thus, the order of acute toxicity potency of
Chirality DOI 10.1002/chir
Fig. 2. Simultaneous stereoselective separation of furalaxyl. (a) rac-furalaxyl
(b) S-furalaxyl (c) R-furalaxy.
and then extracted and determined as previously described. The recover-
ies were calculated by comparing the measured concentrations to the for-
tified concentrations and the relative standard deviation (RSD) revealed
the method precision. The average recoveries for furalaxyl enantiomers
ranged between 88 and 97% in soil, between 80 and 95% in earthworm tis-
sue with RSD below 16%. There was no significant matrix effect for
furalaxyl enantiomers determination with the HPLC-MS/MS method. In
this study, the limit of detection (LOD) and limit of quantitation (LOQ)
were estimated to be 0.07 μg kgꢀ1 and 0.22 μg kgꢀ1 for each enantiomer
in earthworm tissue and soil sample. Compared with the newly prepared
solutions, the stability test of furalaxyl enantiomers was also evaluated in
the stock solutions and matrices, which were prepared 2 months previ-
ously and stored at -20°C. Both enantiomers were stable under the stock
solutions and the spiked earthworm and soil samples.
Equilibrium Partitioning Model and Data Analysis
Equilibrium partitioning theory model was introduced to describe the
uptake process of furalaxyl enantiomers.21–23 The uptake kinetic parameters
and the uptake curve were performed by using the following model equation:
dCWorm=dt ¼ k1CSoil ꢀ k2CWorm
(1)
(2)
(3)
k1CSoil
k2
CWorm ¼
½1 ꢀ expðꢀk2tÞꢁ ¼ KCSoil½1 ꢀ expðꢀk2tÞꢁ
k1
K ¼
k2
Where t was the exposure time, CWorm (mg kg-1) was the concentration of
furalaxyl enantiomers in earthworm tissues, CSoil (mg kg-1) was the con-
centration in soil, k1 (d-1) denoted the uptake rate constant from soil,

310
QIN ET AL.
TABLE 1. Calculated LC50 values for enanatiomers of furalaxyl
Exposure time (48 h)
Exposure time (72 h)
Chemicals
LC50 (μg cm-2)
95% Confidence intervals
R2
LC50 (μg cm-2)
95% Cofidence intervals
R2
R-furalaxyl
rac-furalaxyl
S-furalaxyl
2.27
2.08
1.22
1.87-2.74
1.72-2.40
0.55-1.76
0.965
0.959
0.952
1.90
1.54
1.00
1.54-2.19
1.06-1.82
0.69-1.23
0.933
0.960
0.954
R
2 represents the correlation coefficient.
acylanilide fungicides to worms was S-furalaxyl > rac-furalaxyl
R-furalaxyl > R-benalaxyl > rac-benalaxyl > S-benalaxyl > rac-
metalaxyl > R-metalaxyl. The change of acyl side chains in
acylanilide fungicides may result in the chemicals’ toxicology
difference, and furanylcarbonyl toxicity potency may be
higher than phenylacetyl and methoxyacetyl.
The EF values in earthworm tissue were observed to deviate
from 0.50 and maintained a range of 0.55–0.60, as shown in
Figure 4, and these deviations were significant (P < 0.001).
The results implied that the bioaccumulation of furalaxyl in
earthworm tissue was enantioselective, with preferential
accumulation of S-enantiomer. The possible metabolite of
furalaxyl acid was not found in any dose of earthworm tissue.
According to Figure 3a,b, a higher rate of application of
furalaxyl resulted in a higher concentration in earthworm tis-
sue, and the concentration of both enantiomers reached the
highest levels on the 2–3rd day. After a short-time decrease,
the concentrations increased gradually and obtained the max-
imum values again on the 10th day. Thereafter, the concentra-
tions in earthworm tissue declined and reached steady state
as the duration of exposure increased. The peak-shaped accu-
mulation curves were observed in the bioaccumulation of
furalaxyl by earthworm (Fig. 3). According to Xu et al.,24
peak-shaped accumulation curves happened while organic
compound bioavailability decreased. Although the exact
causes are not known yet, sorption and desorption from soil
organic matter, or pesticides biodegradation and excretion
may all play important roles in the peak-shaped accumulation
patterns in our study.25–27
Bioaccumulation and Elimination in Earthworm
The concentrations of the two furalaxyl enantiomers in
earthworm tissue and soil were determined. The concentra-
tions in soil samples appeared to decrease 20–30% after
31 days exposure at different concentrations (Fig. 3). The
chemicals’ degradation and biotransformation may be the
main reasons for the loss of soil concentrations. Residue of
the main metabolite, furalaxyl acid, was lower in soil. The con-
centrations of furalaxyl enantiomers were almost the same in
spiked soil (20 and 50 mg kg-1) and the EF values were 0.5 at
the beginning and remained so with almost no change during
the course of the experiment (P > 0.050).
The concentrations of furalaxyl individual enantiomers
were observed in earthworm tissue at the same sample point
during the bioaccumulation period. The concentrations of
S-form were higher than that of R-form in spiked soils (Fig. 3).
Fig. 3. Average concentrations of S-furalaxyl and R-furalaxyl in soil and earthworm.
Chirality DOI 10.1002/chir

TOXICITY AND BIOACCUMULATION OF FURALAXYL
311
concentration group were no different from that at a high
concentration level. The BAF values of S-form were larger
than that of R-form (P < 0.001), indicating that the S-form
was preferentially accumulated over the R-form in earthworm
tissue. It can be concluded that the accumulated concentra-
tion of furalaxyl in earthworm tissue via single skin exposure
and gut absorption was enantioselective. The BAF values
of furalaxyl were smaller than benalaxyl and larger than
metalaxyl. The results should relate to the chemicals hydro-
phobicity and lipophilicity, since it was already confirmed that
the chemicals hydrophobicity and lipophilicity can affect the
chemicals transformation and bioaccumulation in earthworm
tissue.12,28 Although both enantiomers showed low accumula-
tion efficiency in earthworms, furalaxyl was dissipated with
DT50 31-65 d in soil29 and was relatively persistent in the envi-
ronment and earthworms. Therefore, the result of furalaxyl
bioaccumulation in earthworms could help provide data for
evaluating the environmental risk. Earthworms are widely
distributed in topsoil and consumed by predator organisms
and so furalaxyl enantiomers may have trophic transfer
and/or bioaccumulation and/or harmful effects on organisms
through food chains.
In this work, we also investigated whether furalaxyl enantio-
mers in earthworm tissue were caused by enantiomerization
or not by exposing earthworm to the soil contaminated with
enantiopure R-furalaxyl and S-furalaxyl, respectively. The
results (Fig. 3c,d) showed that furalaxyl was configurationally
stable in earthworm tissue and soil during a period of 31 d,
showing no interconversion of R- to S-enantiomers, and vice
versa. We have reported that17 benalaxyl was enantiomerization
and enantioselective bioaccumulated in Tenebrio molitor
larvae from wheat bran and the rate of enantiomerization of
R-benalaxyl was higher than that of S-benalaxyl. So furalaxyl
enantiomers may have enantiomerization in other organisms
or higher animals, and it is important to utilize enantiopure en-
antiomers of chiral pesticides for more precise risk assess-
ment in biological systems.
Fig. 4. Enantiomeric fraction (EF) of furalaxyl residues in soil and earthworm.
In the bioaccumulation phase, the concentrations in earth-
worm tissue showed peak-shaped curves around 2 to 10 d
for furalaxyl enantiomers (Fig. 3). At the initial stage, the con-
centrations of furalaxyl enantiomers in soil were high, and the
earthworms responded to these in availability with a high up-
take rate. After the uptake of organic pollutants by earthworm
increased rapidly in the body at first, it increased slowly or
even decreased (Fig. 3), then increased rapidly to reach the
highest level. This process was governed by an increase of
sorption and slow desorption furalaxyl from soil organic mat-
ter, biotransformation, or a decreasing concentration in the
soil, or elimination or metabolic transformation of compounds
from earthworm induced by enzymes. At the last
bioaccumulation stage, the concentrations of furalaxyl enan-
tiomers in earthworm reached steady state, and the uptake-
elimination rate approached a steady state smoothly at a body
residue consistent with the expected basis of equilibrium.
This conclusion was consistent with previous studies.24,27
The appearance of peak-shaped accumulation curves in our
experiment was affected by the change of bioavailability in
earthworms and the chemicals’ transportation and fate in
the soil.
Kinetic Analysis
We tested if the temporal course of furalaxyl uptake into
earthworms from the spiked soil can be described by the
equilibrium partitioning model. By fitting the average concen-
trations in earthworm and time into Eq. (2), a nonlinear
regression fitting technique provided by SPSS 16.0 (Chicago,
IL), and the regression results are shown in Table 2.
The calculated BAFs according to Eq. (4) of two enantio-
mers are shown in Figure 5. The values of BAFs at the low
The trends of S-furalaxyl and R-furalaxyl fitted the first-or-
der kinetics under different exposure concentration groups
(r = 0.797–0.877, 0.819–0.858, respectively). The results indi-
cated that furalaxyl enantiomers in earthworm tissue were
proportional to the concentration in soil, and the accumula-
tion from soil was an equilibrium partition process.30 The rate
constants were calculated applying the estimated values
obtained from the regression results. The approximations of
the rate constants for S-furalaxyl were 0.605–0.934 (k1),
1.227–2.079 (k2), and 0.449–0.493 (K), and the rate constants
for R-furalaxyl were 0.500–0.893 (k1), 1.347–2.372 (k2) ,and
0.342–0.377 (K). The uptake rate (k1) of furalaxyl was much
lower than the rate of elimination and metabolism (k2) in
earthworm tissue and this estimate was in accord with the
low accumulation efficiency (BAF) we have discussed. The
K values (Table 2) of furalaxyl enantiomers according to
Eq. (2) were consistent with the BAF for earthworms (Fig. 5)
when earthworm bioaccumulation reached steady state. A
Chirality DOI 10.1002/chir
Fig. 5. Calculated bioaccumulation factors (BAF) for the two enantiomers
of furalaxyl by sampling day at different spiked concentration.

312
QIN ET AL.
TABLE 2. Calculated kinetic parameter of furalaxyl enantiomers
Exposure group
Enantiomers
K
k1
k2
r
50 mg kg-1 rac-furalaxyl
S-furalaxyl
R-furalaxyl
S-furalaxyl
R-furalaxyl
S-furalaxyl
R-furalaxyl
0.449 0.026
0.377 0.019
0.493 0.034
0.342 0.017
0.466 0.026
0.371 0.025
0.934 0.286
0.893 0.238
0.605 0.192
0.623 0.159
0.635 0.171
0.500 0.162
2.079 0.676
2.372 0.668
1.227 0.419
1.820 0.494
1.363 0.394
1.347 0.470
0.797
0.819
0.846
0.858
0.877
0.844
20 mg kg-1 rac-furalaxyl
10 mg kg-1 S-furalaxyl
10 mg kg-1 R-furalaxyl
11. Jager T, Fleuren RHLJ, Hogendoorn EA, De Korte G. Elucidating the
routes of exposure for organic chemicals in the earthworm, Eisenia andrei
(Oligochaeta). Environ Sci Technol 2003;37:3399–3404.
12. Jager T, Van Der Wal L, Fleuren RHLJ, Barendregt A, Hermens JLM.
Bioaccumulation of organic chemicals in contaminated soils: evaluation
of bioassays with earthworms. Environ Sci Technol 2005;39:293–298.
paired t-test showed that there was a significant difference in
the values between the two furalaxyl enantiomers
(P = 0.045). The results also indicated that the accumulation,
degradation, and elimination of furalaxyl in earthworm tissue
were enantioselective.
K
13. Wang H, Chen J, Guo B, Li J. Enantioselective bioaccumulation and
metabolization of diniconazole in earthworms (Eisenia fetida) in an artifi-
cial soil. Ecotoxicol Environ Saf 2014;99:98–104.
CONCLUSION
In this study, the acute toxicity and bioaccumulation of
furalaxyl enantiomers to earthworm were assayed. The re-
sults of acute toxicity showed that S-furalaxyl was the more
toxic compared with the racemate and R-furalaxyl to the non-
target organism, earthworm. The concentrations of two
furalaxyl enantiomers in soil and earthworm were also deter-
mined and there was a significant trend of enantioselective
bioaccumulation in the earthworm with a preferential accu-
mulation of S-furalaxyl at four levels of exposure. Thus, we
advise that R-furalaxyl with fungicidal activity higher than
the S-enantiomer replace rac-furalaxyl for a more sustainable
pesticide use for environmental safety and public health.
14. Xu P, Diao J, Liu D, Zhou Z. Enantioselective bioaccumulation and toxic
effects of metalaxyl in earthworm Eisenia foetida. Chemosphere
2011;83:1074–1079.
15. Yu D, Li J, Zhang Y, Wang H, Guo B, Zheng L. Enantioselective
bioaccumulation of tebuconazole in earthworm Eisenia fetida. J Environ
Sci 2012;24:2198–2204.
16. Diao J, Xu P, Liu D, Lu Y, Zhou Z. Enantiomer-specific toxicity and
bioaccumulation of alpha-cypermethrin to earthworm Eisenia fetida. J Hazard
Mater 2011;192:1072–1078.
17. Gao Y, Chen J, Wang H, Liu C, Lv X, Li J, Guo B. Enantiomerization and
enantioselective bioaccumulation of benalaxyl in Tenebrio molitor larvae
from wheat bran. J Agric Food Chem 2013;61:9045–9051.
18. Effenberger F, Burkard U, Willfahrt J. Enantioselektive synthese
N-substituierter α-Aminocarbonsäuren aus α-Hydroxycarbonsäuren. Liebigs
Ann Chem 1986;2:314–333
LITERATURE CITED
19. Palla O, Mirenna L, Colombo L, Zini G, Filippini L, Zanardi G. Fungicidal
compositions based on (N-phenylacetyl-N-2, 6-xylyl) methyl alaninate. U.
S. 6228885 B1; 2001 (English).
20. Organization for Economic Cooperation and Development. Guidelines for
testing of chemicals, earthworm, acute toxicity tests. Washington, DC:
OECD; 1984.
1. Davidse LC, Gerritsma OCM, Ideler J, Pie K, Velthuis GCM. Antifungal
modes of action of metalaxyl, cyprofuram, benalaxyl and oxadixyl in
phenylamide-sensitive and phenylamide-resistant strains of Phytophthora
megasperma f. sp. medicaginis and Phytophthora infestans. Crop Prot
1988;7:347–355.
21. Connell DW, Markwell RD. Bioaccumulation in the soil to earthworm sys-
2. Zadra C, Marucchini C, Zazzerini A. Behavior of metalaxyl and its pure
R-enantiomer in sunflower plants (Helianthus annus). J Agric Food Chem
2002;50:5373–5377.
3. Zhang H, Wang X, Jin L, Qian M, Wang X, Xu H, Qi P, Wang Q, Wang M.
Enantioselective determination of acylamino acid fungicides in vegetables
and fruits by chiral liquid chromatography coupled with tandem mass
spectrometry. J Sep Sci 2012;35(15):1869–1876
tem. Chemosphere 1990;20:91–100.
22. Krauss M, Wilcke W, Zech W. Availability of polycyclic aromatic hydro-
carbons (PAHs) and polychlorinated biphenyls (PCBs) to earthworms in
urban soils. Environ Sci Technol 2000;34:4335–4340.
23. Armitage JM, Gobas FA. A terrestrial food-chain bioaccumulation model
for POPs. Environ Sci Technol 2007;41:4019–4025.
4. Williams A. Opportunities for chiral agrochemicals. Pestic Sci
1996;46:3–9.
5. Hegeman WJM, Laane RWPM. Enantiomeric enrichment of chiral pesti-
24. Xu P, Liu D, Diao J, Lu D, Zhou Z. Enantioselective acute toxicity and
bioaccumulation of benalaxyl in earthworm (Eisenia fedtia). J Agric Food
Chem 2009;57:8545–8549
cides in the environment. Rev Environ Contam Toxicol 2002;173:85–116.
25. Reinecke AJ, Nash RG. Toxicity of 2,3,7,8-TCDD and short term
bioaccumulation by earthworms (Oligochaeta). Soil Biol Biochem 1984;16:45–49.
6. Sulimma L, Bullach A, Kusari S, Lamshöft M, Zühlke S, Spiteller M.
Enantioselective degradation of the chiral fungicides metalaxyl and
furalaxyl by Brevibacillus brevis. Chirality 2013;25:336–340.
7. Garrison AW. Probing the enantioselectivity of chiral pesticides. Environ
Sci Technol 2006;40:16–23.
26. Ma WC, Immerzeel J, Bodt J. Earthworm and food interactions on
bioaccumulation and disappearance in soil of polycyclic aromatic hydro-
carbons: studies on phenanthrene and fluoranthene. Ecotoxicol Environ
Saf 1995;32:226–232.
27. Jager T, Antón Sánchez FA, Muijs B, Van der Velde EG, Posthuma L.
Toxicokinetics of polycyclic aromatic hydrocarbons in Eisenia andrei
(Oligochaeta) using spiked soil. Environ Toxicol Chem 2000;19:953–961.
28. Jager T. Mechanistic approach for estimating bioconcentration of organic
chemicals in earthworms (Oligochaeta). Environ Toxicol Chem
1998;17:2080–2090.
8. Magrans JO, Alonso-Prados JL, García-Baudín JM. Importance of consid-
ering pesticides stereoisomerism-proposal of a scheme to apply Directive
91/414/CEE framework to pesticide active substances manufactured as
isomeric mixtures. Chemosphere 2002;49:461–469.
9. Ye J, Zhao M, Liu J, Liu W. Enantioselectivity in environmental
risk assessment of modern chiral pesticides. Environ Pollut
2010;158:2371–2383.
29. The e-Pesticide Manual, version 3.0. British Crop Protection Council; 2003.
10. Celis R, Gámiz B, Adelino MA, Hermosín MC, Cornejo J. Environmental
behavior of the enantiomers of the chiral fungicide metalaxyl in
Mediterranean agricultural soils. Sci Total Environ 2013;444:288–297.
30. Liang X, Zhu S, Chen P, Zhu L. Bioaccumulation and bioavailability of
polybrominated diphenyl ethers (PBDEs) in soil. Environ Pollut
2010;158:2387–2392.
Chirality DOI 10.1002/chir