Leaching properties of some degradation products of alachlor and metolachlor

Article abstract of DOI:10.1016/S0045-6535(99)00513-5

Once in soil, pesticides undergo degradation processes that give rise to a complex pattern of metabolites. Those presenting a significant percentage of formation, genotoxic and leaching properties may pose a threat to human health associated with the consumption of drinking water. The aim of this study is to assess the hazard potential of some metabolites that may occur in ground water. 2,6-diethylaniline, 2-chloro-2',6'-diethylacetanilide, 2- hydroxy-2',6'-diethylacetanilide, metabolites of alachlor and 2-ethyl-6- methylaniline, metabolite of metolachlor, were chosen for their genotoxic properties. Under laboratory conditions, these metabolites showed DT₅₀ = 1- 5 days and K(oc) = 45-357. Their leaching potential, calculated according to Gustafson, is very low and, therefore, they should not be regarded as contaminants of ground waters. Aged residue leaching studies as well as preliminary studies on well waters seem to confirm these findings. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0045-6535(99)00513-5

Chemosphere 41 (2000) 1503±1508
Leaching properties of some degradation products of alachlor
and metolachlor
L. Fava, P. Bottoni, A. Crobe, E. Funari *
Laboratorio di Igiene Ambientale, Istituto Superiore di Sanit aꢀ , Viale Regina Elena 299, 00161 Rome, Italy
Received 26 March 1999; accepted 4 October 1999
Abstract
Once in soil, pesticides undergo degradation processes that give rise to a complex pattern of metabolites. Those
presenting a signi®cant percentage of formation, genotoxic and leaching properties may pose a threat to human health
associated with the consumption of drinking water. The aim of this study is to assess the hazard potential of some
0
0
0
0
metabolites that may occur in ground water. 2,6-diethylaniline, 2-chloro-2 ,6 -diethylacetanilide, 2-hydroxy-2 ,6 -die-
thylacetanilide, metabolites of alachlor and 2-ethyl-6-methylaniline, metabolite of metolachlor, were chosen for their
genotoxic properties. Under laboratory conditions, these metabolites showed DT50 ˆ 1±5 days and Koc ˆ 45±357. Their
leaching potential, calculated according to Gustafson, is very low and, therefore, they should not be regarded as
contaminants of ground waters. Aged residue leaching studies as well as preliminary studies on well waters seem to
con®rm these ®ndings. Ó 2000 Elsevier Science Ltd. All rights reserved.
1
. Introduction
In spite of this, some legislative measures have been
established in order to control the possible impact of
pesticide metabolites on human health and the
environment. In Europe, in order to achieve authorisa-
tion to place new pesticides on the market, environmental
studies on metabolites that form at signi®cant percent-
ages are mandatory (Council Directive 91/414/EEC).
The aim of our activity is to identify metabolites that
pose a potential hazard to human health. Through an
extensive survey of the scienti®c literature, we take note
of those that show biological properties of concern (i.e.
genotoxic activity). Then, we look at their chemiody-
namic characteristics, the extension, rate and mode of
application of their parent compounds, and their oc-
currence in soil, leachates and waterbodies. On the basis
of this information, we select those of particular concern
and de®ne their leaching potential mostly under labo-
ratory conditions (Donati et al., 1994; Bottoni et al.,
After agricultural application, pesticides undergo
biotic and abiotic processes in soil that give rise to a
complex pattern of degradation products, mostly me-
tabolites. Many of them have been determined in
groundwaters (Lagas et al., 1989; Pereira et al., 1990), at
concentrations that range from some units to tens of lg/l
(
Funari et al., 1995). The possible human health impli-
cations associated with the use of these waters for
drinking are generally dicult to assess, as too few data
are available on the biological properties of pesticide
metabolites.
The World Health Organization has de®ned drinking
water quality guidelines for many pesticides, but not for
their metabolites (World Health Organization, 1993),
and these products are not usually monitored in waters
used for drinking.
1
996).
This paper presents the results of laboratory studies
*
on four metabolites of acetanilide herbicides. These
studies included (i) the determination of mobility (ex-
pressed as Koc), (ii) persistence in soil (expressed as
Corresponding author. Tel.: +39-6-4990-2307; fax: +39-6-
938-7083.
E-mail address: funari@iss.it (E. Funari).
4
0
045-6535/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 5 1 3 - 5

1
504
L. Fava et al. / Chemosphere 41 (2000) 1503±1508
0
0
DT50) in order to de®ne their leaching potential ac-
cording to Gustafson (1989), and (iii) aged residue
leaching experiments. Koc and DT50 were determined for
the metabolites, their parent compounds and atrazine,
used as reference compound.
diethylacetanilide and 2-hydroxy-2 -6 -diethylacetanilide
were prepared in our Institute as described below.
0
0
2-Chloro-2 ,6 -diethylacetanilide: 0.1 moles of 2,6-
diethylaniline were dissolved in a solution containing
3
50 ml of acetic acid and 50 ml of a saturated CH COONa
Moreover, preliminary studies have been conducted
in order to analyse the metabolites in a limited number
of well waters in an agricultural area where their parent
compounds are extensively used.
liquid, 0.12 moles of chloroacetyl chloride were dripped
in an ice bath under stirring. The mixture was stirred for
30 min. The precipitated solid was collected by ®ltration,
washed with a solution of NaHCO
dried in a hot air furnace and crystallized.
0
3
and water, then
The metabolites selected for these studies were 2,6-
0
0
0
diethylaniline (DEAN), 2-hydroxy-2 ,6 -diethylacetani-
0
2-Hydroxy-2 -6 -diethylacetanilide: 1 g of 2-chloro-
0
0
0
lide (2OH), and 2-chloro-2 ,6 -diethylacetanilide (2CL),
which form in soil from alachlor (Tiedje and Hagedorn,
2 ,6 -diethylacetanilide was dissolved in a solution con-
3
taining 10 ml of water with 1 g NaCO and 10 ml of
1
975), and 2-ethyl-6-methylaniline (EMA), which forms
ethanol. The solution was bubbled for 6 h. The reaction
was monitored by thin-layer chromatography (ethyl
acetate/hexane 1:2) and stopped when the Rf was lower
than that of the starting material. Then the bulk of
ethanol was removed by rotary evaporation under vac-
uum and extracted with ethyl acetate. The precipitated
solid was collected by ®ltration, washed with a solution
in soil from metolachlor (Guzzella et al., 1996). DEAN
and EMA are intermediates in the metabolism of ala-
chlor and metolachlor in the rat liver where they are
converted to nitroso metabolites, which are highly mu-
tagenic in the Ames test (Kimmel et al., 1986). 2CL and
2
OH are positive in the same test after metabolic acti-
vation with microsomes (Tessier and Clark, 1995).
As for their environmental fate and occurrence, only
few data are available that show the presence of low
levels of DEAN, 2OH, and 2CL in groundwater (Pereira
et al., 1990; Potter and Carpenter, 1995; Kolpin et al.,
3
of NaHCO and water, then dried in a hot air furnace
and crystallised.
The purity degree of these metabolites was P 98%.
Their identity was con®rmed by Nuclear Magnetic
Resonance Spectroscopy (NMR).
1
996). Their chemiodynamic characteristics are poorly
known (Lyman, 1982; Liu et al., 1987; Heyer and Stan,
995).
The parent compounds of these metabolites are
Distilled water for high performance liquid chroma-
tography (HPLC) was further puri®ed through a Nor-
ganic cartridge (Millipore, Bedford, MA). All the
organic solvents used were of HPLC grade (acetone ±
Baker, Holland; methanol ± Sigma Aldrich, Germany;
acetonitrile ± Merck, Germany).
1
widely applied herbicides. Alachlor has been classi®ed
by the WHO as a human carcinogen, metolachlor is
carcinogenic in one mammal species (World Health
Organization, 1993).
2.3. Determination of Koc
The Koc of the examined compounds were deter-
mined by HPLC, according to published procedures
2
. Materials and methods
(
Donati et al., 1994; Kordel et al., 1995; Hong et al.,
2
.1. Soils
1997), using nine reference compounds: atrazine, diclo-
fop-methyl, fenamiphos, isoproturon, linuron, methio-
carb, monuron, quintozene and tri¯uralin (from Dr
Ehrenstorfer, GmbH, purities P 97%). These com-
pounds were chosen because their Koc cover a sucient
range of values and are available in the literature
(Kordel et al., 1995).
DT50s were determined using fresh soil samples col-
lected from the surface layer (0±10 cm depth) of an ag-
ricultural ®eld in the province of Modena (Italy). Their
characteristics were: 29% clay, 49% silt, 22% sand, 1%
organic carbon, pH 7.6 and 22% moisture (w/w).
For aged residue leaching experiments, a fresh soil
sample coming from the province of Perugia (Italy) was
used. Its characteristics were: 12% clay, 17% silt, 71%
sand, 1% organic carbon, pH 8 and 12% moisture (w/w).
2.4. Determination of soil half-life (DT50
)
Degradation tests were conducted according to SE-
TAC procedures (Lynch, 1995), in two replicates. The
examined substances were applied at rates of 2.5 mg/kg
and incubated at 21°C, keeping the soil moisture con-
stant for all the experiments. All the residues were ex-
tracted from soil with methanol at a ratio of 2 g/8 ml,
but for alachlor, for which 5 g/20 ml was used. The re-
2.2. Products and reagents
Atrazine and metolachlor were purchased from Ciba-
Geigy (99.2% and 99.5% of purity, respectively), ala-
chlor from Monsanto (99.5%), 2,6-diethylaniline and
0
2-ethyl-6-methylaniline from Fluka (Fluka Chemie AG,
Switzerland) (98 and 97%, respectively). 2-chloro-2 ,6 -
sulting suspensions were vigorously shaken for 10 and
0
0
0
then centrifuged for 10 at 2420g. Supernatants were

L. Fava et al. / Chemosphere 41 (2000) 1503±1508
1505
reduced to 1 ml under nitrogen ¯ow, and then analysed
by HPLC.
at 80%, then linearly decreases to 20% from 25 to
30 min, and remains stable till 35 min.
The mobile phase ¯ow was of 1 ml/min; the column
was kept at 30°C and the detector set at the wavelength
of 210 nm.
2
.5. Determination of the leaching potential
The leaching potentials of the examined compounds
were calculated as groundwater ubiquity score, using the
Each analytical data represents the mean value of
three HPLC determinations.
following equation: GUS ˆ logDT  ꢀ4 � logK † of
5
0
oc
DT50 and Koc (Gustafson, 1989).
3
. Results
2.6. Aged residue leaching
3
.1. Koc
These experiments were carried out according to
SETAC procedures (Lynch, 1995) with some modi®ca-
tions. Glass columns 45 cm high and with an inner di-
ameter of 4 cm were ®lled with untreated soil up to a
height of 30 cm.
0
Fig. 1 shows the relationship between the Koc and k
values determined for the nine reference compounds.
The correlation coecient of the linear relationship be-
tween these values is not particularly high but it can be
considered sucient for the purpose of this study.
Alachlor and metolachlor were separately incubated
in soil samples for a period of time corresponding to
their half-lives, under the above conditions.
K
oc values for the metabolites and the parent com-
pounds, obtained by applying the regression equation of
Fig. 1, are shown in Table 1. Koc values for atrazine,
alachlor and metolachlor are within those published in
the literature (Bottoni and Funari, 1992; Tomlin, 1995).
Only 2OH has a mobility higher than that of atr-
azine. The other metabolites as well as their parent
compounds show a limited mobility.
Then, aliquots of incubated soils were transferred to
the top of the columns in order to ®ll an additional
2
height of 5 cm; 2000 ml of 0.01 M CaCl solution were
added on the top of each column for 4 days, by means of
a peristaltic pump. This volume is equivalent to 300 mm
rainfall per day and simulates a worst-case scenario.
The experiments were conducted in two replicates in
the dark. Leachates were collected, ®ltered through
cellulose acetate (0.45 lm), and concentrated using
disposable solid phase extraction (SPE) cartridges
3
.2. DT50
The degradation rates of the examined substances
(
LiChrolut EN 200 mg, Merck).
follow a ®rst order kinetics (Figs. 2 and 3). All the ex-
amined substances have DT50 values lower than that of
atrazine.
2
.7. HPLC apparatus
The metabolites show very low DT50 values that
never exceed three days (Table 1). These values are much
A liquid chromatograph Model 9010 (Varian, Wal-
nut Creek, CA) equipped with a Rheodyne injector,
having a 50 ll loop, a UV detector Model 9050 (Varian)
and an autosampler Model 9100 (Varian) were used.
The chromatographic raw data were elaborated with the
software Star Integrator 4.1 (Varian).
0
For Koc experiment, the capacity factors (k ) of all the
substances were derived from retention times measured
with a 25 cm ´ 4.6 mm i.d. column ®lled with 5 lm of
LC-CN (cyano) packing (Supelco Inc., Bellofonte, PA).
The mobile phase was methanol±water (55:45 v/v), ac-
cording to published procedures (Kordel et al., 1995).
For DT50 determination and aged residue leaching
experiments, a 25 cm ´ 4.6 mm i.d. column ®lled with
5
lm, average particle size, of Supelcosil LC-18 packing
(
Supelco) was used.
Metabolites were separated from their parent com-
pounds by applying a gradient program at the following
conditions. The mobile phase was a mixture of aceto-
nitrile (A) and water at the following percentages: from
Fig. 1. Regression line derived from the correlation between
0
0 to 5 min A keeps at 20%; from 5 to 20 min A linearly
increases from 20% to 80%; from 20 to 25 min A remains
Koc and capacity factor (k ) of the reference pesticides selected
for this study.

1
506
L. Fava et al. / Chemosphere 41 (2000) 1503±1508
Table 1
DT50, Koc values and GUS index for the examined parent compounds, their metabolites, and atrazine
a
Substances
Atrazine
DT50 (days)
59.2
K
oc
GUS
66
3.87
Alachlor
5.2
2.4
0.8
1.3
312
148
45
1.07
0.7
0 0
0
2
2
2
-Cl-2 ,6 -diethylacetanilide (2CL)
0
-OH-2 ,6 -diethylacetanilide (2OH)
,6-Diethylaniline (DEAN)
)0.18
0.19
357
Metolachlor
-Ethyl-6-methylaniline (EMA)
17.4
1.7
244
197
2.00
0.38
2
a
GUS index ˆ ꢀlogDT †  ꢀ4 � logKoc†; GUS > 2:8 leacher; 1:8 < GUS < 2:8 transient GUS < 1:8 nonleacher. These values have
50
been calculated using mean DT50 values.
Fig. 2. Decrease in soil concentrations of alachlor, its metabolites and atrazine.
Fig. 3. Decrease in soil concentrations of metolachlor, its metabolite and atrazine.
lower than those for alachlor and metolachlor. From
tions. For DEAN and EMA, a signi®cant percentage of
the dissipation is due to non biotic factors, e.g. chemical
degradation or bound residues formation. DT50 for
alachlor and metolachlor are similar to those found in
the literature (Bottoni and Funari, 1992).
further experiments on thermally sterilized soil (Hsu and
Bartha, 1974), 2OH and 2CL do not show important
variations of concentration, meaning that the observed
degradation in nonsterilized soil is due to biotic reac-

L. Fava et al. / Chemosphere 41 (2000) 1503±1508
4. Discussion and conclusion
1507
3
.3. Leaching potential (GUS)
The results show that only atrazine can be classi®ed
The experimental approach used in this study in or-
der to de®ne the leaching potential of the examined
compounds seems correct as the results obtained for
alachlor, metolachlor and atrazine are in the range of
those published in the scienti®c literature.
as a leacher, as it is shown in Table 1 and Fig. 4. Met-
olachlor has a GUS value which is characteristic of
transition compounds. Alachlor and all the metabolites
show leaching potentials belonging to non-leachers.
The examined metabolites have very short half lives
and consequently low leaching potentials; aged residue
leaching studies show that they occur in the leachates at
low concentrations and percentages, and are unlikely to
contaminate ground water at concentrations signi®cant
to human health.
3
.4. Aged residue leaching columns
Table 2 reports the residue concentrations and rela-
tive percentages of the examined compounds in the
leachates at the end of the soil column experiment. The
metabolites occur in the leachates at very low percent-
ages with respect to their parent compounds. Only 2CL
reached a percentage close to 10% of the applied dose.
Taking into account that alachlor and metolachlor have
been rarely found in ground water, and only at low
concentrations, their metabolites represent a minor risk
of groundwater contamination.
In conclusion, they may not be regarded as con-
taminants of groundwater. In spite of their biological
properties, these metabolites do not represent a threat to
human health when ground water resources are used for
drinking.
These ®ndings are con®rmed by a preliminary study
on a limited number of well waters from agricultural
areas where alachlor and metolachlor have been widely
used in the last years (data not shown).
Acknowledgements
We thank Dr. F. Gatta, from the Laboratory of
Pharmaceutical Chemistry of our Institute, for having
kindly synthetized and provided us with two acetan-
ilide metabolites. We thank also Dr. L. Barbieri,
Regional Agency for Prevention and Environment
(
ARPA) of the Emilia Romagna Region (Italy) and
Prof. M. Businelli, Institute of Agricultural Chemis-
try, University of Perugia (Italy), for having charac-
terized and provided us with soil samples. Finally, we
are grateful to Ms. M. Brocco, who revised this
manuscript.
Fig. 4. GUS index of metabolites, their parent compounds and
atrazine.
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