Elimination of racemic and enantioenriched metalaxyl based fungicides under tropical conditions in the field

Article abstract of DOI:10.1016/j.chemosphere.2007.02.055

The elimination has been studied of racemic and enantioenriched metalaxyl applied as an emulsifiable concentrate and wettable powder combined with copper in a Cameroonian field site. The kinetics of the degradation/dissipation of metalaxyl and its acid metabolite were investigated using reversed phase HPLC-MS/MS, while the enantiomeric ratios were measured by HPLC-MS/MS using a Chiralcel OD-H HPLC column. Some soil enzymes activities were determined concurrently for 120 d. The elimination of racemic metalaxyl was shown to be enantioselective, with the R-enantiomer being degraded more slowly than the S-enantiomer. Dissipation followed approximate square root first-order kinetics (R > 0.98) without lag phases. The enantiomers of metalaxyl have different elimination rates, with half-lives ranging from only 0.8 to 1.5 days. After application to soil, the elimination of metalaxyl in the copper containing formulation was slower. The activities of acid phosphatase, alkaline phosphatase, and alkaline glucosidase were monitored throughout the experiments. No significant influence of metalaxyl and copper could be observed on these parameters. The significantly shorter half-life values of all forms of metalaxyl under field conditions, compared to the previously reported laboratory derived ones, may have implications for the plant disease control with these fungicides in tropical rainforest areas.

Full text of DOI:10.1016/j.chemosphere.2007.02.055

Chemosphere 69 (2007) 655–663
www.elsevier.com/locate/chemosphere
Elimination of racemic and enantioenriched metalaxyl based
fungicides under tropical conditions in the field
1
1
Adolphe Monkiedje , Sebastian Zuehlke, Saurelle Jacqueline Ngouopiho Maniepi ,
Michael Spiteller *
Institute of Environmental Research (INFU), University of Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany
Received 8 September 2006; received in revised form 23 February 2007; accepted 24 February 2007
Available online 9 April 2007
Abstract
The elimination has been studied of racemic and enantioenriched metalaxyl applied as an emulsifiable concentrate and wettable pow-
der combined with copper in a Cameroonian field site. The kinetics of the degradation/dissipation of metalaxyl and its acid metabolite
were investigated using reversed phase HPLC-MS/MS, while the enantiomeric ratios were measured by HPLC-MS/MS using a Chiralcel
OD-H HPLC column. Some soil enzymes activities were determined concurrently for 120 d. The elimination of racemic metalaxyl was
shown to be enantioselective, with the R-enantiomer being degraded more slowly than the S-enantiomer. Dissipation followed approxi-
mate square root first-order kinetics (R > 0.98) without lag phases. The enantiomers of metalaxyl have different elimination rates, with
half-lives ranging from only 0.8 to 1.5 days. After application to soil, the elimination of metalaxyl in the copper containing formulation
was slower. The activities of acid phosphatase, alkaline phosphatase, and alkaline glucosidase were monitored throughout the experi-
ments. No significant influence of metalaxyl and copper could be observed on these parameters. The significantly shorter half-life values
of all forms of metalaxyl under field conditions, compared to the previously reported laboratory derived ones, may have implications for
the plant disease control with these fungicides in tropical rainforest areas.
ꢀ
2007 Elsevier Ltd. All rights reserved.
Keywords: Enantioselective pesticide analysis; LC-MS/MS; Degradation behavior
1
. Introduction
contains a stereogenic centre in the carboxy alkyl moiety
Fig. 1) so it consists of a pair of enantiomers, S(+) and
(
Metalaxyl [methyl N-(2,6-dimethylphenyl)-N-(methoxy-
R(ꢀ) (Ari e¨ ns et al., 1988). It is a stable compound resistant
to a broad range of pH, temperature, and light (Singh
et al., 1982). On account of these properties and its
broad-spectrum activity, this compound has been regis-
tered for use on a wide range of crops and in many coun-
tries and in both temperate and tropical regions.
acetyl)-DL-alaninate], an important acylanilide fungicide
with residual and systemic activity, is used to protect crops
against diseases caused by pathogens of the orders Peron-
osporales (Peronospora, Pseudoperonospora, Plamopara,
and Bremia) and Pythiales (Pythium and Phytophthora)
(
Kerkenaar and Sijpestijin, 1981). It is apoplastically trans-
Metalaxyl was initially marketed as a racemic product,
even though its fungicidal activity is almost entirely due
to the R-enantiomer. The product enriched with the R-
enantiomer (trade names: Metalaxyl-M, Mefenoxam; M =
minus; laevorotatory; Nuninger et al., 1996), typically
comprises 97.5% of the R-enantiomer and 2.5% of S-enan-
tiomer and has the same biological activity as racemic met-
alaxyl at ꢁ50% of the application rate. Wettable powder
combined with copper formulated rac-metalaxyl, which
ported and selectively interferes with the synthesis of ribo-
somal RNA (Hassal, 1990). Metalaxyl is chiral because it
*
Corresponding author. Tel.: +49 231 755 4080; fax: +49 231 755 4085.
E-mail address: spiteller@infu.uni-dortmund.de (M. Spiteller).
Laboratory of General Biology, Department of Animal Biology and
1
Physiology, Faculty of Science, University of Yaounde I, P.O. Box 812,
Cameroon.
0
045-6535/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2007.02.055

656
A. Monkiedje et al. / Chemosphere 69 (2007) 655–663
mic metalaxyl by enantioenriched metalaxyl means that
O
O
knowledge of and data on the persistence of the latter and
its effect on soil biology under field conditions have to be
reassessed too. This is essential for use, management and
registration of enantioenriched metalaxyl and it’s copper
based formulation, especially in the tropical regions of
Africa. Knowledge concerning the elimination mechanism
is a prerequisite for registration in new field applications.
The elimination of pesticides in soil does not always fol-
low first-order kinetics, but shows a pattern where the pes-
ticide concentration declines more rapidly in an initial
phase. The decline of pesticide residues in soil was investi-
gated in 420 experiments in order to validate possible non-
linear behavior. Specifically the square root 1st order
model showed a better fitting than the linear model in
35% of the investigations (Timme et al., 1986). Several
studies have substantiated the relevance of the square root
first order kinetic (Korpraditskul et al., 1993; Di et al.,
1998; Banerjee et al., 2006).
The purpose of the present work was to investigate the
behavior of racemic and enantioenriched metalaxyl in
two different formulations under field conditions in tropical
rainforest soil, and to verify and monitor any changes in
the enantiomeric ratio (ER) of the racemic pesticide formu-
lations. Indeed, a stereo specific biodegradation may lead
to changes in the enantiomeric ratios of parent compounds
and metabolites. Further, in light of the fact that enzymes
play a role in the biological degradation of metalaxyl, we
monitored the effect of these compounds on the activities
of selected soil enzymes with a view to determining the rela-
tionship between the elimination rate of various forms of
metalaxyl and the activities of these enzymes.
H
H C
3
O
O
O
O
H C
H
3
N
N
O
O
S-Metalaxyl
R-Metalaxyl
Fig. 1. Structures of S- and R-metalaxyl.
has been massively used for the protection of cash crops
cocoa) and subsistence crops in Cameroon (Monkiedje
(
et al., 2000; Duguma et al., 2001; Fontem, 2001), is cur-
rently being replaced in the market by the enantioenriched
substitute metalaxyl-M. Recent projections of higher cocoa
production rates by 2010 in the main farming regions in
Africa (FAO, 2003), mean that the consumption rate of
this new product is likely to increase in the future in
Cameroon.
Metalaxyl is readily biodegraded in soil, plants, and in
animals by cleavage of the ester and by a concurrent series
of oxidative biotransformations (N-dealkylation, alkyl and
aryl hydroxylation Roberts and Hutson, 1999). The major
metabolite of metalaxyl in soil is metalaxyl carboxylic acid
{
MX-acid; 2-[(2,6-dimethylphenyl) methoxyacetylamino]
propionic acid}, with further metabolites also being formed
from this primary hydrolysis product. MX-acid is also
chiral (Buser et al., 2002).
Many reports have documented the microbially medi-
ated degradation of metalaxyl in soils (Sukul and Spiteller,
2
001), and the more rapid degradation of the R-metalaxyl
2
. Experimental section
in slightly acidic to alkaline soils in temperate climate
under controlled laboratory conditions (Buser and M u¨ ller,
1
2.1. Experimental site
995; M u¨ ller and Buser, 1995; Buser et al., 2002; Buerge
et al., 2003). Further studies such as the correlation
between the biodegradation rate of the compounds and
enzyme activities and the influence of the type of formula-
tions on degradation under tropical field conditions remain
to be made. However, under specific field conditions, these
compounds are applied as formulated products consisting
of complex mixtures of a number of potentially hazardous
and potentially interacting agents for which there is, as a
whole, a lack of toxicity data relating to soil biology.
Recent work has dealt with the degradation behavior of
metalaxyl in soils performed under controlled laboratory
conditions. S-Metalaxyl was degraded more rapidly than
R-metalaxyl in tropical soils (Sukul and Spiteller, 2001)
and in aerobic soils with pH < 4 as well as in most anaerobic
soils (Buerge et al., 2003), when these soils were spiked with
racemic metalaxyl. These laboratory findings, if validated
by field data, may have far reaching consequences for agri-
cultural practice. The application rate as well as the applica-
tion frequency of metalaxyl in the main regions of use, may
have to be reassessed. Moreover, the replacement of race-
The study was conducted in Yaounde (Cameroon) on an
agricultural plot which had not received any pesticide
application for at least ten years. Selected physicochemical
properties of the soil are reported in Table 1. This site has
Table 1
Selected physicochemical properties of the soils investigated
Texture analysis (USDA)
Value
Clay (<2 lm) (%)
Silt (2–50 lm) (%)
Sand (50–2000 lm) (%)
pH (water, ratio 1:2.5)
2
pH (0.01 M CaCl , ratio 1:2.5)
51.2
16.1
32.7
4.75
3.7
Corg. (% dry soil)
C/N
5.01
17.5
21.3
0.04
52.6
1.27
0.40
0.12
ꢀ1
P (mg P O Kg dry weight)
2 5
ꢀ1
Cation exchange capacity (meq g dry weight)
Maximum water holding capacity (MWHC) (%)
Bulk density (g ml
Field capacity (cm water cm soil)
Available water (cm water cm soil)
ꢀ
3
1
)
ꢀ3
ꢀ3
3

A. Monkiedje et al. / Chemosphere 69 (2007) 655–663
657
been under continuous cultivation since 1990. The crops
grown, irrespective of seasons, have included maize, pea-
nut, sweet potato, and cassava. This site, which is located
in the equatorial rainforest region, receives an annual rain-
fall of 1400–1600 mm, while the annual temperature varies
between 19 and 28 ꢁC. The study site was divided into four
blocks. Each block included replicates of all fungicide
treatments and a control plot. Plots within the blocks were
organized according to the ‘‘Latin Square’’ model of ran-
domized blocks (Rohrmoser, 1986). This arrangement of
the plots represents complete randomization, as both treat-
ments and controls were distributed at random in blocks
and in columns. In order to control the undesired influ-
ences such as soil differences amongst blocks and border
effects, each treatment occurred once in each soil block.
Each plot measured 1 m · 1.5 m. The experiments began
at the onset of the rainy season and ended at the beginning
of the following dry season.
mulations were pipetted or weighed and mixed thoroughly
and separately in 1500 ml of distilled water. The rates of
ꢀ
1
application of the racemic metalaxyl were 1085 g a.i. ha
ꢀ
1
of the emulsifiable formulation and 1096 g a.i. ha of the
wettable powder formulation. Application rates of the
enantioenriched metalaxyl were 585 g a.i./ha of the emulsi-
ꢀ
1
fiable formulation and 550 g a.i. ha of the wettable pow-
der formulation. These solutions were uniformly applied
once by hand spray (7-l compression sprayer, Intereko 7)
to the prepared surface of the experimental plots. No
attempt was made to incorporate the chemicals into the soil
by mechanical mixing. The plots were managed with mini-
mum tillage. The plots were not cultivated and weed control
was performed by manual weeding.
2.4. Soil sampling and preparation
Six soil cores were taken at random from each plot at
each incubation period (0, 1, 3, 15, 30, 60, 90 and 120 d)
from 0 to 5 cm depth using a plastic coring tube (5 cm
diameter). The samples were taken at different positions
within the plot to get better statistical distribution. No
sampling was done at the border of the experimental plots
(20 cm minimum distance). The sampling areas were
marked with wooden sticks and refilled with untreated soil
in order to avoid preferential flow. The samples were
bulked and homogenized in disposable plastic buckets.
Whenever necessary, the soil was gently air-dried for a
few days to reduce the moisture content of the soil and
make it suitable for sieving to a maximum particle size of
<2 mm. Enzyme activities were determined immediately
after sampling of field moist and sieved sub-samples. The
soils for fungicide residue analysis were kept field moist
and stored frozen (ꢀ20 ꢁC) until analysis. A determination
of gravimetric soil moisture content was performed concur-
rently with each parameter analysis.
2
.2. Chemicals
The standards racemic metalaxyl [(R,S)-metalaxyl]
(
chemical purity >99.3%) and enatioenriched metalaxyl
[
R(ꢀ) enantiomer (chemical purity, 96.5%)], were pur-
chased from Dr. Ehrenstorfer GmbH (Germany). The
commercial products Fonganil Neu (formulated racemate
metalaxyl) and Fonganil Gold (formulated R-enantiomer
enriched metalaxyl) were obtained as emulsifiable concen-
trate formulations containing 24% and 45% of racemic
and enantiomer enriched metalaxyl, respectively, from Syn-
genta Agro, Frankfurt, Germany. The compounds that
make up the remaining 76% and 55% of the respective
emulsifiable concentrate formulations include a petro-
leum-derived solvent, a glycol-derived solvent, naphtha-
lene, trimethylbenzene, alkyl-aryl-poly-oxy-ethylenate, and
emulsifiers whose exact structures are kept confidential by
the company. Ridomil Plus 72 (formulated racemic metal-
axyl combined with copper) and Ridomil Gold Plus/Cop-
per (formulated enantiomer enriched metalaxyl combined
with copper) were obtained from a local vendor as wettable
powder formulations containing 12% and 6% of racemic
and enantioenriched metalaxyl and 60% copper oxide,
respectively,. Metazachlor, used as internal standard, was
purchased as analytical grade from Riedel-de Haen (Ger-
many). C18 SPE cartridges (500 mg, 6 ml) were from
Baker, Deventer, Netherlands. Formic acid,, hydrochloric
acid, and the HPLC grade solvents (ethyl acetate,
n-hexane, 2-propanol, acetonitrile, and methanol) were
purchased from Merck, Darmstadt, Germany.
2.5. Preparation of the metalaxyl acid metabolite
N-(2,6-Dimethylphenyl)-N-(methoxyacetyl)alanine (meta-
laxyl acid) was synthesized according to a previously
described procedure (Sukul and Spiteller, 2001). Racemic
metalaxyl (100 mg, purity >99%) was added to 2 N KOH
(5 ml) and the mixture was refluxed (110–115 ꢁC) for 1 h
before cooling to ambient temperature. 2 N HCL (7 ml)
was added to the reaction mixture, which was then stored
in a freezer overnight. The resulting white crystals were col-
lected and washed with ice-cold water. When poor crystal-
lization was observed, the whole solution was extracted
three times with ethyl acetate (50 ml aliquotes). The com-
bined extracts were gently evaporated to dryness and the
resulting white crystals were collected. The identity of the
metalaxyl acid metabolite obtained was confirmed from
NMR data. The NMR spectra were recorded using a Var-
2
.3. Application of the fungicides on the field
The dose calculations were based on the assumption of a
ꢀ
3
penetration depth of 0.05 m and a soil density of 1.5 g cm
.
Calculated amounts, corresponding to recommended field
ian 200 MHz spectrometer equipped with a 5 mm probe.
ꢀ
1
1
application rates of roughly 1 kg ha of racemic metalaxyl
H NMR (CD OD, 200 MHz): d = 7.13 (m, 3H, H-3,H-
3
ꢀ
1
0
0
0
and 0.5 kg ha of enantioenriched metalaxyl in both for-
4,H-5), 4.37 [q, J = 7.5 Hz, 1H, H-2 ], 3.45 [m, 2H, H-2 ],

658
A. Monkiedje et al. / Chemosphere 69 (2007) 655–663
0
0
3
(
.16 [s, 3H, H-3 ], 2.35 (s, 3H, H-8), 2.06 (s, 3H, H-7), 0.91
100
9
.07
0
NL: 2.18E5
66.1-> 60.1 [22V]
d, J = 7.5, Hz, 3H, H-3 ).
2
5
0
2
.6. Extraction and clean-up of treated samples
Metalaxyl-acid 1
1
1
1
1
1
00
9.07
NL: 2.02E5
66.1->192.2 [17V]
The procedure described previously was employed
Monkiedje et al., 2003). Here, extraction of the acidified
2
5
0
(
Metalaxyl-acid 2
soil (100 g) with 200 ml methanol was carried out in Erlen-
meyer flasks containing the incubated soil and the internal
standard (IS) Metazachlor (5 lg abs.) on an overhead sha-
ker. After allowing the sediment to settle, the clear super-
natant was decanted through a glass fibre filter. After a
second extraction step, the pooled methanolic extracts were
evaporated to approx. 10 ml on a rotary evaporator and
cleaned up using C18 SPE cartridges. The final methanolic
eluates were concentrated to about 5 ml and aliquots were
subjected to HPLC-MS analysis.
00
10.29
10.29
9.98
NL: 4.00E5
78.1->105.0 [38V]
2
5
0
IS Metazachlor 1
IS Metazachlor 2
Metalaxyl 1
00
NL: 1.48E6
278.1->133.1 [20V]
5
0
00
NL: 7.53E6
2
80.1->220.2 [13V]
5
0
2
.7. Quantitation via reversed phase HPLC-MS/MS
00
9.98
The quantitation of metalaxyl and the acid metabolite
NL: 2.78E6
2
80.1->160.0 (21V]
was performed by reversed phase HPLC-MS/MS using a
Survior HPLC system (Thermo Finnigan, San Jose, CA,
50
Metalaxyl 2
˚
USA). A Luna C18 100 A column from Phenomenex (Tor-
6
8
10
12
rance, CA, US; 150 · 2 mm, 3 lm particle size, free exposed
silanols virtually eliminated by complete bonding and end-
capping) was used for non-chiral separations. The mobile
phase (flow rate = 0.25 ml min ) consisted of water and
acetonitrile, both HPLC grade (Baker, Deventer, Nether-
lands) with 0.1% formic acid. The gradient was pro-
grammed as follows: 0–2 min: 10% acetonitrile; 2–3.5 min:
Time (min)
Fig. 2. SRM chromatograms of metalaxyl, the acid metabolite and the
internal standard (IS) Metazachlor of a soil extract after 15 days of
incubation; quantifier and qualifier traces are shown.
ꢀ
1
2.8. Enantioselective analysis by normal phase
HPLC-MS/MS
1
0 ! 30%; 3.5–8 min: 30 ! 100%; 8–15 min: 100%, 15–
1
6 min: 100 ! 10%, and equilibration for another 8 min.
All separations were performed at 22 ꢁC. The calibration
Aliquots of the extracts prepared for RP-HPLC (0.5 ml)
were conditioned for chiral HPLC by solvent exchange to
n-hexane/2-propanol by means of careful evaporation to
dryness at 60 ꢁC under vacuum using an Eppendorf
Concentrator 5301 (Eppendorf, Hamburg, Germany).
The residues were then re-dissolved in 0.1 ml of n-hex-
ane:2-propanol (80:20). A 5 ll aliquot of this solution
was then analyzed by chiral normal phase HPLC-MS/MS
using a Gynkotek/Dionex HPLC system consisting of a
degassing unit (ERC-3822), a Gina 50 automatic sampler,
and a Dionex P 580 pump. Separations were performed
at room temperature which was maintained at 22 ꢁC.
The enantiomers of racemic metalaxyl, and those of the
corresponding racemic MX-acid, were separated on a Chi-
ralcel OD-H HPLC (5 lm, 25 cm · 4.6 mm i.d.; Daicel
Chemical Industries, Tokyo, Japan) column. The mobile
phase consisted of n-hexane:2-propanol (50:50) with 0.3%
formic acid (isocratic elution). The flow rate was set at
was performed as a multilevel internal standard calibration
(
IS metazachlor). The ratio of peak areas (analyte/IS) was
ꢀ
1
ꢀ1
linear within the range 0.04–100 lg ml (0.002–5 lg g
soil). The procedure gave recoveries of 114.0% with 9.3%
RSD for metalaxyl and 104.0% with 6.2% RSD for its acid
metabolite (recovery rates obtained from spiked soil;
ꢀ
1
ꢁ
1 lg g
dry soil). The limit of determination was
ꢀ
1
0
.002 lg g soil for both metalaxyl and its acid metabolite.
Blanks were found to be below the limit of detection. The
data reported are uncorrected for recoveries.
The identity of the compounds was confirmed by com-
paring retention times and multiple reaction monitoring
in tandem mass spectrometry (TSQ quantum ultra AM
equipped with an APCI source, Thermo Finnigan, San
Jose, Ca, USA). For (+)APCI, the ionization conditions
were optimized for every compound and two selected prod-
uct ions were monitored for each precursor compound.
Nitrogen was used as both drying and nebuliser gas. The
drying gas temperature was set at 200 ꢁC and a pressure
of 124 kPa. The collision gas (argon) pressure was set to
ꢀ1
0.3 ml min . The calibration was performed by using high
purity metalaxyl and metalaxyl-M standards prepared in
HPLC grade n-hexane:2-propanol (80:20).
0
.2 Pa. For the selected reaction monitoring (SRM) scans,
Mass spectra were obtained using a Varian 1200L LC-MS
(Varian, Inc. Palo Alto, CA, USA) equipped with an ESI
source operating in positive mode. For ESI, the operating
a dwell time of 0.15 s was applied. MS/MS parameters for
every SRM chromatogram are reproduced in Fig. 2.

A. Monkiedje et al. / Chemosphere 69 (2007) 655–663
659
conditions were as follows: needle voltage at 4 kV, shield at
00 V and tube lens at 4 V. The drying and nebuliser gas
was nitrogen, which was set at 200 ꢁC and a pressure of
8 psi. The acid metabolite was detected as both the sodium
describe the non-linear decline of the pesticide residues in
soil, a number of non-linear equations, like the square root
1st order model have been developed (Timme et al.,
1986; Matsumura, 1989; Korpraditskul et al., 1993; Di
et al., 1998). In this study the 1st order model has been
compared with the square root 1st order model.
Racemic and enantiomer enriched metalaxyl in both for-
mulation types were degraded in soils to levels of <2% of
their initial concentration within 30 d of application. The
concentrations of four replicate applications of R- and S-
metalaxyl and R- and S-metalaxyl acid remaining in the
soils are summarized in Tables 2 and 3. Because of the very
low concentrations of the fungicide after 60 days and more,
the uncertainty in measurement is high. Thus, regression
analysis was only performed from time 0 up to 30 days.
In contrast to further investigations of laboratory experi-
ments (Buser et al., 2002; Monkiedje et al., 2003; Buerge
et al., 2003) approximate first order kinetics were not
observed after field application. The biodegradation of the
parent compounds (both formulations) in soil appears to
6
1
+
adduct [M + Na] and, after fragmentation, as the product
ion (288 ! 113). Intensive fragments of the [metal-
+
axyl + Na] were not observed; so the sodium adduct only
was monitored for this compound. The selectivity of the
mass spectrometric detection was high, no additional peaks
with the same precursor/product ion could be observed in
the range of the expected retention time in all soil extracts.
The enantiomer ratios in the samples were determined
from their peak area ratios. Racemic metalaxyl acid yields
an exact peak area ratio of 1:1 in both standard solutions
and spiked to soil extracts. But the ratio differed for metal-
axyl on account of peak broadening at the higher retention
time of the R-metalaxyl on normal phase separation. To
ensure a matrix-independent result, the effect of the soil-
matrix was checked by injection of racemic metalaxyl
standards and soil extracts spiked with racemic metalaxyl.
Samples from all four experiments (both formulations, both
metalaxyl and enantioenriched metalaxyl) were checked by
this procedure and confirmed the correction factor for the
ER of metalaxyl which ranged from 1.2 to 1.3 (average
follow square root first order kinetics over the first 30 days.
pffiffi
Fig. 3 shows the superior linear fit for ln(Ao/A ) versus
t
t
(Timme et al., 1986) than for ln(Ao/A ) versus t of R- and
t
S-metalaxyl after application of the racemic metalaxyl to
soil in emulsifiable concentrate. The comparison of 1st
order model and Square root 1st order model is shown in
Fig. 3 using the application of emulsifiable concentrates of
racemic metalaxyl as an example. The following Eq. (1)
(Timme et al., 1986) allows the calculation of the residue
concentration over the time of the field experiment, here
1
.25). This factor of 1.25 was applied to all samples to yield
corrected enantiomeric ratios (racemates corrected to the
ratio of 1:1). All concentrations of the chiral separation
were tested for concentration dependencies concerning the
peak area ratios. Ratios (R:S) were determined for both
the racemic mixtures (metalaxyl and acid metabolite) for
the entire range of concentrations analyzed. Ratios were
also constant for different injection volumes of soil extracts.
ꢀ
1
[A] is the concentration (lg kg soil) of A at time t (days)
t
and [A] is the initial concentration of A at time 0.
0
½
Aꢂ
0
2
.9. Determination of enzyme activities
½Aꢂ ¼
p
ffi
ð1Þ
t
t
k
Acid and alkaline phosphatase activities were deter-
Rate constants for each experiment were derived from ‘‘ln
pffiffi
mined according to methods described in the literature
Tabatabai and Bremmer, 1969; Eivazi and Tabatabai,
982) with slight modifications. Toluene was not included
(
A /A ) versus t’’ plots by linear regression analysis (fit-
t
0
(
1
ting without any weighting). The half-life (t1/2, days) was
calculated from
in the procedure as it has been shown to increase the
observed activities of both acid and alkaline phosphatases
and can be used as source of carbon by most soil microor-
ganisms. b-Glucosidase activity was measured using a pub-
lished method (Eivazi and Tabatabai, 1988). The results of
the enzyme activity studies are reported on an oven dry
weight basis, determined by drying the soils at 105 ꢁC for
ꢀ
ꢁ
2
ln 2
t1=2
¼
ð2Þ
ln k
The enantiomeric ratio (ER) is defined by the relation (3)
where R and S are the concentration of R- and S-enantio-
mers, respectively. The ER values define a range from 0
(
R = 0, S = 100%) to >1 (R > S).
2
3
3
4 h.
½
Rꢂ
ER ¼
ð3Þ
. Results and discussion
½
Sꢂ
.1. Elimination kinetics
Thus, elimination rate constants were determined using
regression plots of ln (Ao/A ) versus square root of the
t
pffiffi
Many published degradation studies assume simple first-
time ð tÞ (Fig. 3). Correlation coefficients are in the range
from 0.984 to 0.999 for R- and S-metalaxyl in racemic for-
mulations, and for R-metalaxyl in the enantiomer enriched
formulations. Elimination kinetics of S-metalaxyl in the
enantioenriched formulations was not determined, because
order degradation kinetics. Often, however, the elimination
of pesticides in soil does not follow first-order kinetics, but
shows a pattern where the pesticide declines at an initial
phase rapidly and in a second phase less rapidly. To

660
A. Monkiedje et al. / Chemosphere 69 (2007) 655–663
Table 2
Concentrations of R- and S-metalaxyl, together with the R- and S-enantiomer of its metabolite, in experimental plots treated with emulsifiable concentrate
after the various soil sampling intervals; values represent means ± standard deviation of 4 independent samples; concentration (lg/100 g of dry soil)
Day
Total metalaxyl
R-metalaxyl
S-metalaxyl
Total MX acid
R-MX acid
S-MX acid
Emulsifiable concentrate of racemic metalaxyl
0
1
3
5
0
0
0
249 ± 38
133 ± 16
77 ± 20
17 ± 4.5
5.3 ± 5.1
2.5 ± 2.1
0.7 ± 0.2
0.6 ± 0.3
120 ± 10
64 ± 3.2
36 ± 5
10.3 ± 1.1
3.1 ± 0.7
1.1 ± 0.05
0.3 ± 0.05
0.4 ± 0.05
129 ± 11
68 ± 5
40 ± 4
6.4 ± 1.0
2.1 ± 0.2
1.3 ± 0.05
0.3 ± 0.05
0.15 ± 0.05
12 ± 2.8
6.6 ± 1.4
5.3 ± 1.6
2.0 ± 0.8
1.6 ± 1.1
1.9 ± 1.3
1.5 ± 1.0
0.3 ± 0.25
7.1 ± 0.3
3.5 ± 0.7
3.1 ± 0.4
1.3 ± 0.1
1.2 ± 0.4
1.1 ± 0.05
0.8 ± 0.05
0.19 ± 0.05
4.9 ± 0.4
3.1 ± 0.7
2.2 ± 0.05
0.8 ± 0.05
0.8 ± 0.05
0.7 ± 0.05
0.7 ± 0.05
0.13 ± 0.05
1
3
6
9
1
20
Emulsifiable concentrate of enantioenriched metalaxyl
0
1
3
5
0
0
0
147 ± 5
86 ± 12
49 ± 10
10 ± 3.0
6.2 ± 2.4
2.5 ± 0.9
1.7 ± 0.5
0.9 ± 0.3
133 ± 6
73 ± 7
43 ± 6
13.2 ± 2.0
12.7 ± 3.1
5.9 ± 2.3
1.6 ± 0.05
0.8 ± 0.05
0.7 ± 0.05
0.3 ± 0.05
0.1 ± 0.05
4.6 ± 0.5
3.0 ± 0.3
2.9 ± 0.2
2.2 ± 0.6
1.9 ± 0.8
1.8 ± 0.7
1.4 ± 0.6
0.7 ± 0.2
4.2 ± 0.5
2.7 ± 0.1
2.6 ± 0.05
2.0 ± 0.6
1.9 ± 0.8
1.8 ± 0.6
1.4 ± 0.7
0.7 ± 0.2
0.46 ± 0.05
0.36 ± 0.05
0.32 ± 0.05
0.18 ± 0.05
<0.02 ± 0.05
<0.02 ± 0.05
<0.02 ± 0.05
<0.02 ± 0.05
1
3
6
9
8.5 ± 2
5.4 ± 1.5
1.8 ± 0.5
1.3 ± 0.4
0.8 ± 0.05
1
20
Table 3
Concentrations of R-and S-metalaxyl, together with the R- and S-enantiomer of its metabolite, in experimental plots treated with copper containing
wettable powder, material after the various soil sampling intervals; values represent means ± standard deviation of 4 independent samples; concentration
(
lg/100 g of dry soil)
Day Total metalaxyl
Copper containing wettable powder of racemic metalaxyl
R-metalaxyl
S-metalaxyl
Total MX acid
R-MX acid
S-MX acid
0
1
3
1
3
6
9
1
249 ± 31
131 ± 10
77 ± 12
20 ± 7.9
9.5 ± 3.6
3.4 ± 1.7
2.6 ± 1.2
1.1 ± 0.6
130 ± 23
74 ± 6.2
42 ± 7.3
12.7 ± 2.4
6.2 ± 1.1
2.2 ± 0.6
1.0 ± 0.7
0.7 ± 0.7
119 ± 32
57 ± 6.3
34 ± 19
7.2 ± 0.9
3.3 ± 0.3
1.1 ± 0.05
1.5 ± 0.05
0.4 ± 0.05
14 ± 3.3
12 ± 4.3
5.5 ± 4.0
5.2 ± 1.7
2.9 ± 0.6
2.8 ± 1.5
2.8 ± 1.0
0.8 ± 0.6
5.7 ± 0.3
5.7 ± 0.4
2.4 ± 0.2
2.4 ± 0.4
1.4 ± 0.05
1.1 ± 0.05
1.2 ± 0.05
0.4 ± 0.05
8.2 ± 0.2
6.2 ± 0.2
3.2 ± 0.2
2.9 ± 0.4
1.5 ± 0.05
1.7 ± 0.1
1.6 ± 0.05
0.4 ± 0.05
5
0
0
0
20
Copper containing wettable powder of enantioenriched metalaxyl
0
1
3
1
3
6
9
1
105 ± 4.5
64 ± 9.0
43 ± 9.3
12 ± 7.8
6.2 ± 2.5
1.7 ± 0.6
1.7 ± 0.6
0.6 ± 0.2
99 ± 1.9
59 ± 3.4
39 ± 3.5
9.3 ± 1.4
4.6 ± 0.6
1.3 ± 0.2
1.2 ± 0.05
0.3 ± 0.05
5.9 ± 0.3
5.5 ± 0.6
5.0 ± 0.1
3.2 ± 0.1
2.0 ± 0.7
1.8 ± 0.85
1.0 ± 0.21
0.5 ± 0.05
0.2 ± 0.05
4.9 ± 0.5
4.5 ± 0.1
2.6 ± 0.05
1.7 ± 0.2
1.6 ± 0.05
0.9 ± 0.05
0.4 ± 0.05
0.2 ± 0.05
0.6 ± 0.05
0.5 ± 0.05
0.5 ± 0.05
0.3 ± 0.05
0.2 ± 0.05
0.1 ± 0.50
0.1 ± 0.05
<0.02 ± 0.50
5.0 ± 0.05
4.1 ± 0.05
2.8 ± 0.05
1.5 ± 0.05
0.4 ± 0.05
0.5 ± 0.05
0.3 ± 0.05
5
0
0
0
20
of the very low concentrations of S-metalaxyl in the soil
samples (even immediately after application).
Considerably higher reaction constants and shorter
half-lives (0.8–1.5 days) were observed in the field studies
ronmental factors, such as variations in temperature,
moisture (Matsamura, 1989) and light, which have a
strong influence on microorganisms in their degradation
preferences for metalaxyl enantiomers in experimental
plots, may account for the observed differences in elimina-
tion rates, especially since these products were applied to
the surface of the experimental plots without manual
incorporation into deeper soil layers. One reason for the
shorter half-lives in the field compared to the laboratory
experiments may be the wash out of the fungicides by
rain. Leaching into deeper soil must also be considered,
especially as sampling was only performed until a depth
of 5 cm.
(
Table 4) compared with those measured in the earlier lab-
oratory experiments (Monkiedje et al., 2003: 17–39 days;
Buerge et al., 2003: 3–60 days). This may be the result
of the more complex processes of elimination in the field
(
degradation, hydrodynamic flow, runoff), compared to
degradation alone in laboratory experiments. An inconsis-
tent relationship between elimination rates of pesticides,
including metalaxyl, measured in the laboratory or in
the field has already been reported (Di et al., 1998). Envi-

A. Monkiedje et al. / Chemosphere 69 (2007) 655–663
661
first-order: R-metalaxyl
metalaxyl in particular is a strong indication that the for-
mation of the acid metabolite is at least partly biologically
mediated. Given the nature of the soil studied (clay and
acidic, Table 1), reactions occurring in the soil pores (e.g.
acid-catalysis) or on the surfaces of clay minerals (hetero-
geneous surface catalysis) may also contribute to the
hydrolytic degradation of these compounds as observed
for other pesticides (Korpraditskul et al., 1993). In this soil,
and with both formulations tested, the production of the
acid metabolite was less pronounced, thus confirming our
previous laboratory findings (Monkiedje et al., 2003). A
maximum acid metabolite concentration of ꢁ6% the initial
concentration of metalaxyl was observed at the start of the
experiment (0 d). The concentration of the metabolite
decreased with incubation time (Tables 2 and 3). Possibly,
the metalaxyl was transformed to metabolites other than
the acid metabolite or was mineralized in the soils. Never-
theless, no other metabolites could be found using HPLC-
MS in full scan mode as well as in precursor ion scan mode
first-order: S-metalaxyl
5
4
3
2
1
0
5
4
3
2
1
0
y = 0.113x + 0.4884
y = 0.1315x + 0.5041
2
2
R = 0.9428
R = 0.9419
0
20
t
40
0
20
t
40
square root first-order:
R-metalaxyl
square root first-order:
S-metalaxyl
5
5
4
3
2
1
0
4
3
2
1
0
(LC-MS/MS) of typical metalaxyl fragments. These field
observations of metalaxyl elimination contrast with those
made from our previous laboratory incubation study using
racemic and enantioenriched metalaxyl with German and
Cameroonian soils (Monkiedje et al., 2003), where a grad-
ual formation of MX-acid (0% ! <7%) occurred in the
course of 14 d incubation in a similar Cameroonian soil.
Under field conditions with continuous rainfall it can be
assumed that the highly polar metabolite will be leached
into deeper soil.
y = 0.6689x - 0.0205
y = 0.7784x - 0.0878
2
2
R = 0.9989
R = 0.9976
0
2
4
6
0
2
4
6
Square root of t
Square root of t
pffiffi
3
.3. Soil hydrolase activities
Fig. 3. Linear fit of ln(Ao/A
t
) versus t as well as ln(Ao/A
t
) versus t
(days) of R- and S-metalaxyl after 0–30 days of application of racemic
metalaxyl in emulsifiable concentrate.
Many studies have reported microbial metabolism of
pesticides in soil as a result of the broad-spectrum enzymes
(hydrolases, oxidases, etc.) (Matsamura, 1989) generally
present. In order to evaluate the relationship between the
elimination of the various forms of metalaxyl and the activ-
ities of soil enzymes, we monitored the effect of these com-
pounds on the activities of selected soil hydrolase enzymes.
The results for soil enzyme activities (acid phosphatase,
alkaline phosphatase, and b-glucosidase) in untreated and
treated experimental plots differed only within the range
of the uncertainty of measurement and sampling. No sig-
nificant changes were observed for the parameters moni-
tored; which were chosen to reflect the general microbial
activity of the soil samples.
3
.2. Formation of acid metabolite
In all formulations, the acid metabolite was detected
almost immediately after application of the parent com-
pound (Tables 2 and 3). It is not possible to speculate on
the kinetics of elimination of the metabolite on account
of the continued degradation of metalaxyl and simulta-
neous elimination of the acid metabolite. Biological pro-
cesses, especially the initial attack of compounds can be
very rapid. Thus, the degradation of metalaxyl may be bio-
logically mediated. The non-racemic composition of metal-
axyl acid immediately after application of racemic
Table 4
pffiffi
2
Degradation rate constants (k) and half-lives (t1/2) as derived from the regression line from the ln(C
values for the degradation of total metalaxyl and its enantiomers in soil with two different formulations
0
/C
t
) versus t plot plus correlation coefficient (R )
Formulation
R-metalaxyl
ln(k)
S-metalaxyl
2
2
R
t
1/2 (d)
ln(k)
R
t1/2 (d)
Emulsifiable concentrate of racemic metalaxyl
0.669
0.614
0.576
0.564
0.999
0.984
0.994
0.995
1.07
1.27
1.45
1.51
0.778
–
0.680
–
0.998
0.79
–
1.04
–
Emulsifiable concentrate of enantioenriched metalaxyl
Copper containing wettable powder of racemic metalaxyl
Copper containing wettable powder of enantioenriched metalaxyl
0.992

662
A. Monkiedje et al. / Chemosphere 69 (2007) 655–663
3
.4. Enantioselective investigation
The ratios of R:S enantiomers in field plots were inves-
3
day 0
day 1
day 3
day 15
day 30
tigated in order to compare the fate of the two enantiomers
of metalaxyl and of their respective acid metabolites. The
ERs of the racemic metalaxyl standard and of the racemic
acid metabolite were both 1.0.
The enantio enriched products investigated were highly
enriched in R-metalaxyl (R:S; 96:4 and 94:6) with an enan-
tiomeric ratio of 15.7 and 24.0. The ER of these products is
not discussed because of the differing initial concentrations
of R-metalaxyl and S-metalaxyl in the pesticide formula-
tions used. Nevertheless, the elimination of the S- and
R-metalaxyl is also presented in the Tables 2 and 3. The
degradation rate constants and half-lives of the applied
metalaxyl are reported in Table 4.
2
1
0
In the experimental plots, S-metalaxyl was degraded
much more quickly (t1/2 0.8 and 1.0 days) than R-metalaxyl
(
t1/2 1.1 and 1.5 days) when racemic metalaxyl was applied
in both formulations (Table 4). This is in agreement with
previous reports on the behavior of metalaxyl in aerobic
soils with pH < 4 and in most anaerobic soils (Buerge
et al., 2003). After application to soil the S-metalaxyl
degraded much faster than R-metalaxyl, independent of
the type of formulation applied. The elimination of both,
the R- and S-enantiomer in emulsifiable concentrate was
faster than in the copper containing (Table 4). This
suggests that copper exerts some toxic effect on soil
micro-organisms. The toxic effects of copper on soil
micro-organism activity are well known (Varlet and Berry,
Metalaxyl
Metalaxyl
acid metabolite acid metabolite
emulsifiable wettable powder emulsifiable wettable powder
concentrate concentrate
Fig. 4. Enantiomeric ratios (ER) of metalaxyl and metalaxyl acid after
application of different formulations containing racemic metalaxyl to soil;
day 0–30; values represent means ± standard deviation of 4 independent
samples.
could be different microbial populations in the soil studied.
These produce different enzymes, which preferentially
degrade the different enantiomers. Our findings show that
the degradation of racemic metalaxyl in the soil studied is
clearly enantioselective, with S-metalaxyl being eliminated
more rapidly than R-metalaxyl (residues enriched in
R-metalaxyl). The physicochemical behavior, such as
sorption, mobility and leaching, ought to be identical for
R- and S-metalaxyl, as well as for R- and S-metalaxyl acid.
Metalaxyl seems to be configurationally stable in soil under
field conditions, since there is no evidence from the ER val-
ues to suggest interconversion of R- to S-enantiomer,
which had already been reported for earlier reported labo-
ratory studies (Buser et al., 2002). The influence of the pos-
sible leaching of metalaxyl and metalaxyl acid ought to be
similar for both the enantiomers (R- or S-) on account of
the similarity in their physicochemical properties.
The field study presented here yielded in significantly
shorter half-lives for metalaxyl than those previously
reported from laboratory incubation studies (Buser et al.,
2002; Monkiedje et al., 2003; Buerge et al., 2003). The
application rates and frequencies of application employed
by farmers are much higher than those recommended by
the makers of metalaxyl-based products (Varlet and Berry,
1997; Fontem, 2001). The rapid field dissipation of these
products in the field reported here may justify this heavy
application in part. As a consequence, cases of metalaxyl
resistance of fungal pathogens have been reported (Fontem
et al., 2005).
1
997). This is in agreement with a previous report (Klodka
and Nowak, 2004) showing adverse effects on soil enzyme
activities after application of pesticides in formulations.
The elimination behavior of the R-and S-enantiomers, as
well as those of R- and S-metalaxyl acid, was assessed by
plotting the ER at each sampling time from 0 up to 30 days
after application of racemic mixtures (Fig. 4). After applica-
tion to field plots, the ER of racemic pesticide formulations
changed, showing that the two enantiomers are present in
different proportions. This indicates that there is enantiose-
lective degradation of metalaxyl in these soils in the same
sense as in our previous study (Monkiedje et al., 2003).
On account of the more rapid degradation of S-metal-
axyl, the ER of Metalaxyl is >1 for both formulations and
increases with time. A significant increase in the ER is first
apparent at day 15 for the emulsifiable concentrate and at
day 1 for the copper containing metalaxyl. On the other
hand, differences were observed for the formation of the acid
metabolite. ER > 1 was measured with emulsifiable concen-
trate but ER < 1 was observed with the copper containing
powder. The S-metalaxyl may generally be degraded more
quickly in the emulsifiable concentrate. Thus, more of the
S-metalaxyl-acid is formed and if the degradation of both
the enantiomers of the acid metabolites is in the same range,
more S-metalaxyl-acid is the result (Fig. 4).
One reason for the different ranking order of degrada-
tion rate constants of the two enantiomers of metalaxyl

A. Monkiedje et al. / Chemosphere 69 (2007) 655–663
663
4
. Conclusion
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eight pesticides in surface and subsurface soils under laboratory and
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The elimination has been studied of racemic and enan-
Duguma, B., Gockowski, J., Bakala, J., 2001. Smallholder Cacao
tioenriched metalaxyl applied as emulsifiable concentrate
or as a wettable powder combined with copper to a Came-
roonian field site. Analysis of the degradation/dissipation
of metalaxyl and its acid metabolite were investigated using
reverse phase HPLC-MS/MS, while the enantiomeric
ratios were measured by normal phase HPLC-MS/MS.
The dissipation of racemic metalaxyl is enantioselective,
with the R-enantiomer being degraded more slowly than
the S-enantiomer. Elimination followed square root first-
order kinetics with different with half-lives of the enatio-
mers ranging from only 0.8 to 1.5 days. Metalaxyl was
eliminated more slowly after application of the copper con-
taining formulation to soil. The activities of acid phospha-
tase, alkaline phosphatase, and alkaline glucosidase
revealed no significant influence of metalaxyl and copper.
The significantly shorter half-lives of all forms of metalaxyl
under field conditions, compared to those previously
reported for laboratory investigations, may have implica-
tions for the plant disease control with these fungicides in
tropical rainforest areas.
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We thank the Alexander von Humboldt Foundation for
a renewed period of research spent by the first author in
Germany, under the Georg Forster Research Fellowship.
We also thank Syngenta Agro (Frankfurt, Germany), for
providing the emulsifiable concentrate of enantiopure R-
metalaxyl and racemic metalaxyl employed, and Dr. Her-
bert Resseler from Syngenta Agro (Frankfurt, Germany)
for his valuable comments on the field experimental design.
We thank Dr. P. Sukul and Dr. M. Lamsh o¨ ft of the Uni-
versity of Dortmund for preparing the metalaxyl acid
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