Diphenyl ethers

Masako Ueji

National Institute for Agro-Environmental Sciences, Tsukuba, Japan

1

Introduction

Diphenyl ethers are both systemic and contact herbicides and are used for the selec-
tive control of annual broad-leaved weeds and grasses in a variety of crops (such as
soybeans, maize, rice, wheat, barley, peanuts, cotton, onions and ornamental trees)
under different application scenarios. This class of herbicides contains a diphenyl
ether moiety as the core substructure. Acifluorfen, bifnox, chlomethoxyfen, chlorni-
trofen, fluoroglycofen-ethyl and fomesafen, etc., are representative compounds of the
diphenyl ether herbicides (Figure 1).

The mode of action of diphenyl ether herbicides is the inhibition of protopor-

phyrinogen oxidase (PPO) and this inhibitory action is light-activated. The herbicides
absorbed by plants inhibit PPO in the system of porphyrin–chlorophyll synthesis, and
the chlorophyll precursor protoporphyrinogen IX is accumulated in the plants. The
excess protoporphyrinogen IX in the thylakoid membrane leads to oxidation of proto-
porphyrin IX, which is the strong photosensitizer for producing singlet oxygen. The
reactive singlet oxygen disrupts the plasma membrane and the breakdown of mem-
brane unsaturated fatty acids, resulting in the loss of chlorophyll and carotenoids and
in leaky membranes.

1

The diphenyl ether herbicides are nonvolatile compounds, generally very lipophilic

and insoluble in water. Solubility in water and octanol–water partition coefficients
(log K

ow

) of the various diphenyl ether herbicides range from 120 mg L

−1

(aciflu-

orfen) to 0.16 mg L

−1

(oxyfluorfen) and from 2.9 (fomesafen) to 5.4 (acifluorfen),

respectively. Diphenyl ether herbicides are stable in an acidic or alkaline condition,
but some compounds are gradually degraded under the sunlight.

2

Because of the limited root uptake and slow rate of systemic translocation, the

diphenyl ether herbicide residues detected in the aerial plant portion are low.

In Japan, bifenox is the only registered diphenyl ether herbicide. The tolerance

and/or maximum residue limits (MRLs) are established at 0.1 mg kg

−1

for cereals such

as rice grain, barley and wheat, and 0.05 mg kg

−1

for potatoes (Ministry of Health,

Labour and Welfare, Japan). The California Department of Food and Agriculture
(CDFA) established the minimum detectable quantity of diphenyl ether herbicides at
0.1 mg kg

−1

for bifenox, nitrofen and oxyfluorfen.

3

Handbook of Residue Analytical Methods for Agrochemicals.

C

2003 John Wiley & Sons Ltd.

452

Compound class

F

O

OR

CI

F

F

O

NO

2

R=Na, Acifluorfen-sodium;
R=CH

3

,Acifluorfen-methyl;

R=C

2

H

5

, Acifluorfen-ethyl

F

O

N

H

CI

F

F

O

NO

2

S

O

O

Fomesafen

F

O

O

CI

F

F

O

NO

2

O

O

Fluoroglycofen-ethyl

F

O

O

CI

F

F

O

NO

2

O

O

Lactofen

F

O

CI

F

F

O

NO

2

Oxyfluorfen

CI

O

CI

O

O

NO

2

Bifenox

CI

O

CI

NO

2

Nitrofen

CI

O

CI

NO

2

CI

Chlornitrofen

CI

O

CI

NO

2

O

Chlomethoxyfen

O

NO

2

NH

2

CI

Aclonifen

Figure 1

Structures of diphenyl ether herbicides

A residue analytical method for diphenyl ethers in soil and water samples and in crop

samples has been developed. The basic principle of the residue method consists of the
following steps: extraction from the samples with acetone or other organic solvents,
purification using liquid–liquid partition and column chromatography including solid-
phase extraction (SPE), and quantitative analysis by gas chromatography/electron
capture detection (GC/ECD), gas chromatography/nitrogen–phosphorus detection
(GC/NPD) or high-performance liquid chromatography (HPLC).

Diphenyl ethers

453

2

Analytical methodology for plant materials

2.1

Nature of the residues

Diphenyl ethers in the soil are absorbed by roots with limited translocation generally
to the foliage. Low levels of herbicide residues can be expected when the compound
is used in accordance with good agricultural practice. The parent diphenyl ether
compound is defined as the residue of analytical and regulatory concern.

2.2

Analytical method

A 20-g homogenized cereal or vegetable sample is extracted with an organic sol-
vent such as acetone. After filtration, the solvent extract is concentrated by rotary
evaporation to about 20 mL, below 40

◦

C. The residue is transferred with 5% sodium

chloride solution and partitioned twice with n-hexane. The n-hexane extracts are dried
by anhydrous sodium sulfate, which is subjected to a cleanup procedure by Florisil
or silica gel column chromatography. The eluate is concentrated to dryness and the
residue is dissolved in an appropriate amount of acetone for GC/ECD (Ministry of
the Environment, Japan).

2.2.1

Extraction

(1) Vegetables

20-g of chopped vegetables and 100 mL of acetone are placed in a blender cup and
shaken vigorously on a mechanical shaker for 30 min. The homogenate is filtered
under vacuum through a funnel fitted with a filter paper, and the residue is rehomog-
enized with 50 mL of acetone and then filtered again. The filtrates are combined and
concentrated to about 20 mL using a vacuum rotary evaporator below 40

◦

C.

(2) Brown rice, wheat and soybean

Cereal samples are milled with an ultracentrifuge mill and sieved through a 42-mesh
screen, then 20 g of the homogenized sample are transferred into a 300-mL Erlenmeyer
flask and soaked in 40 mL of distilled water for 60 min. After 100 mL of acetone have
been added to this flask, the same procedure as described in the case of vegetables is
carried out.

Acifluorfen (an acidic diphenyl ether herbicide) is often difficult to extract from the

complex soybean matrix. Therefore, Nemoto and Lehotay

4

developed the method of

pressurized liquid extraction [(PLE), Hydromatrix (diatomaceous earth material) as a
sample dispersant ingredient is used] followed by capillary electrophoresis (CE). PLE
was performed with an ASE 200 instrument (Dionex, Sunnyvale, CA, USA), with the
operating conditions of 2000 psi, 100

◦

C, a 5-min extraction time, 100% solvent flush

of the vessel (one cycle), and a 1-min purge with nitrogen. Approximately 24 mL of
acetonitrile–0.05 N HCl (7 : 3, v/v; pH 2) were added to 3 g of soybean sample which
was ground using a centrifugal mill to pass through a 60-mesh screen and 1.5 g of
Hydromatrix. The mixed solution was extracted by PLE.

454

Compound class

2.2.2

Cleanup

(1) Liquid–liquid partition

(a) NaCl solution–n-hexane

A 100–200-mL volume of 5% NaCl aqueous solution and 100 mL of n-hexane are
added to the extracts prepared in Section 2.2.1, and the mixed solution is shaken
vigorously for 5 min. The n-hexane layer is separated and a further 50 mL of n-
hexane are added to the aqueous layer (lower layer) and shaken again. The n-hexane
layers are collected, dehydrated with ca 20 g of anhydrous Na

2

SO

4

, and concentrated

using a vacuum rotary evaporator below 40

◦

C, and the residue is dried under a gentle

stream of pure nitrogen and dissolved in ca 20 mL of n-hexane.

(b) Acetonitrile–n-hexane

The acetonitrile–n-hexane partitioning is an additional procedure in the residue anal-
ysis of plant samples having high oil content (e.g., rice grain, bean, and corn). A
30-mL volume of acetonitrile is added to the above-mentioned n-hexane layer of
plant extract and the mixed solution is shaken vigorously. The acetonitrile layer is
separated, a further 30 mL of acetonitrile are added to the n-hexane layer, and the
mixed solution is shaken vigorously. The combined acetonitrile layers are carefully
concentrated to dryness.

(c) Microporus diatomaceous column (MDC)

The MDC is an effective procedure for the cleanup of the sample. The sample residue
obtained in Section 2.2.1 is transferred to the MDC (Chem Elut, etc.) and the column
is left at room temperature for 10 min. Bifenox is eluted with 80 mL of n-hexane, and
the eluate from the MDC is concentrated to near dryness below 40

◦

C.

5

(2) Column chromatography

(a) SPE silica gel cartridge cleanup

An SPE silica gel cartridge is prewashed with 10 mL of n-hexane to remove any
contaminants from the cartridge. The sample residue in the flask is loaded on the car-
tridge with 6 mL of acetone–n-hexane (1 : 49, v/v) and the eluate is discarded. Bifenox
is eluted with 8 mL of acetone–n-hexane (1 : 9, v/v) and the eluate is concentrated
to near dryness below 40

◦

C, then dissolved with acetone to an appropriate volume

for analysis by gas chromatography (GC).

6

Several types of SPE cartridge such as

C

18

(octadecylsilane), alumina-N (alumina-neutral), silica, NH

2

(aminopropyl) and

SAX (anion exchange) were evaluated for the cleanup of acifluorfen from soybean
extracts. SPE using SAX seems not to be appropriate among the five cartridges to
obtain a high recovery. For example, SPE using the C

18

cartridge recovered

>90%

of acifluorfen when 10 mL of 0.05 N HCl, 10 mL of water, and 7 mL of acetone were
loaded sequentially. The procedure using the C

18

cartridge was able to remove salts

from the soybean extracts effectively.

4

Diphenyl ethers

455

(b) Silica gel column chromatography

In the case of silica gel column chromatography, 10 g of Kieselgel 60 suspended in ad-
equate amounts of diethyl ether–n-hexane (1 : 19, v/v) are placed in a chromatographic
tube plugged with cotton wool at the bottom. On the top of the silica gel column,
10 g of anhydrous Na

2

SO

4

are placed and the solvent is drained. A 5-mL volume of

the n-hexane solution prepared by partitioning between NaCl solution and n-hexane
[(1) (c)] is concentrated to dryness and the residue is transferred completely to a
column with two portions of 5 mL of diethyl ether–n-hexane (1 : 19, v/v). Bifenox is
eluted with 100 mL of diethyl ether–n-hexane (3 : 17, v/v), the eluate is concentrated
using a rotary evaporator, and acetone is added to the concentrated residue for GC
analysis.

(c) Florisil column chromatography

In place of silica gel, Florisil is also used as the adsorbent in column chromatography.
Purification of chlornitrofen using a Florisil column is as follows: after installing
a column packed with 10 g of Florisil suspended in n-hexane, the sample solution
is added continuously to the column and the initial eluate is discarded. A 100-mL
volume of diethyl ether–n-hexane (1 : 19, v/v) is charged to the Florisil column and
the eluate is discarded. Chlornitrofen is eluted with 30 mL of this mixture and the
eluate is concentrated to dryness before the addition of acetone for GC analysis.

6

Examination of the elution solvent from the Florisil column to purify bifenox

and chlomethoxyfen was carried out. Bifenox is eluted with 100 mL of acetone–
n-hexane (1 : 9, v/v) after discarding 100 mL of diethyl ether–n-hexane (3 : 7, v/v)
eluate, and chlometoxyfen is also eluted with 100 mL of acetone–n-hexane (5 : 95,
v/v) after discarding 100 mL of n-hexane eluate.

7

(3) Extraction and cleanup of diphenyl ether herbicide metabolites in plants

The purification of chlornitrofen and the reduced metabolite, 2,4,6-trichlorophenyl
4-aminophenyl ether (CNP-NH

2

) in brown rice and vegetables was investigated.

8

A 20-g amount of the milled brown rice or minced vegetable is transferred into

a 300-mL Erlenmeyer flask. After 100 mL of 0.2 M KOH–acetone (1 : 9, v/v) have
been added to the flask, the mixture is shaken vigorously on a mechanical shaker for
30 min. The homogenate is filtered under vacuum through a funnel fitted with a filter
paper, and the residue is rehomogenized with 70 mL of the same solution and filtered
again. The filtrates are combined and concentrated to about 40 mL using a vacuum
rotary evaporator below 40

◦

C.

The extracts are transferred to a flask which contains 100 mL of 2% Na

2

SO

4

in 0.1 M

KOH aqueous solution and 100 mL of n-hexane, and the flask is shaken vigorously
for 5 min. The n-hexane layer is separated, a further 50 mL of hexane are added to
the aqueous layer and the mixed solution is shaken. The combined n-hexane layers
are transferred into a separatory funnel containing 100 mL of 0.2 M HCl and shaken
vigorously on a mechanical shaker for 5 min. The two layers are separated for the
determination of chlornitrofen in the n-hexane layer and CNP-NH

2

in the aqueous

layer.

The n-hexane layer is dried with ca 50 g of anhydrous Na

2

SO

4

, filtered through

a funnel fitted with a filter paper, concentrated to about 1 mL under vacuum below

456

Compound class

40

◦

C and dried under a gentle stream of pure nitrogen. The residue is dissolved

in 5 mL of n-hexane and loaded on the column suspended with 10 g of Florisil in
an adequate volume of n-hexane with ca 10 g of anhydrous Na

2

SO

4

on the top of

the Florisil. An additional 5 mL of n-hexane are transferred to the column, which is
drained. Chlornitrofen is eluted with 70 mL of diethyl ether–n-hexane (3 : 17, v/v).
The eluate is concentrated to about 1 mL under vacuum below 40

◦

C, dried under a

gentle stream of pure nitrogen and dissolved in an appropriate amount of n-hexane
for GC/ECD.

The aqueous layer (CNP-NH

2

layer) collected by liquid–liquid partitioning is trans-

ferred into a separatory funnel containing 20 mL of 4 M KOH aqueous solution and
100 mL of n-hexane, and shaken vigorously for 5 min. The n-hexane layer is collected.
A further 100 mL of n-hexane are added to the aqueous layer and shaken again. The
n-hexane layers are collected, dried with ca 50 g of anhydrous Na

2

SO

4

, concentrated

to dryness carefully, and n-hexane solution of the residue is prepared for GC analysis.

2.2.3

Determination

(1) Gas chromatography

To determine the diphenyl ether herbicides in the samples, GC/ECD or GC/NPD is
used in general. GC/ECD is preferred to GC/NPD owing to its higher sensitivity. An
aliquot of GC-ready sample solution is injected into the gas chromatograph under the
conditions specified below. In addition, multi- and confirmatory analysis of residues is
carried out using gas chromatography/mass spectrometry (GC/MS) in the selected-ion
monitoring (SIM) mode.

(a) GC/ECD

Bifenox: column, SPB-5 (15 m

× 0.53-mm i.d., 1.0-µm film thickness); column, inlet

and detector temperature, 250, 250 and 280

◦

C, respectively; gas flow rates, He carrier

gas 20 mL min

−1

, N

2

makeup gas 40 mL min

−1

; injection volume, 2-µL. The retention

time for bifenox is about 3 min.

6

Chlornitrofen and CNP-NH

2

: column, DB-1 (10 m

× 0.53-mm i.d., 1.0-µm film

thickness); column, inlet and detector temperature, 200, 250 and 280

◦

C, respectively;

N

2

gas flow pressure, 1.6 kg m

−2

; injection volume, 2 µL. The retention times for

chlornitrofen and CNP-NH

2

is about 5 and 3.5 min, respectively.

8

Simultaneous determination of three diphenyl ethers: column, 5% DC-200 (0.5–

1 m

× 2–3-mm i.d.); temperature, column 210–230

◦

C, inlet and detector 260

◦

C; gas

flow rates, N

2

carrier gas 30–50 mL min

−1

; injection volume, 2 µL. The retention

times are approximately 4.5 min for bifenox, 2.5 min for chlornitrofen and 3.4 min
for chlomethoxfen.

7

(b) GC/NPD

Multiresidue analysis of 72 pesticides including three diphenyl ethers was carried out
by GC/NPD under the following conditions: column, 5% DB-5 (30 m

× 0.53-mm i.d.,

0.53-µm film thickness); temperature, column 100

◦

C (1 min) increased at 5

◦

C min

−1

to 280

◦

C (10 min), inlet and detector 280

◦

C; gas flow rates, He 11.2 mL min

−1

,

H

2

3.5 mL min

−1

, air 110 mL min

−1

; injection volume, 2 µL. The retention times

Diphenyl ethers

457

are approximately 35.5 min for bifenox, 34.6 min for chlornitrofen and 32.7 min for
chlomethoxyfen.

Oxyfluorfen: column, fused-silica capillary column coated with cross-linked methyl

silicone (25 m

× 0.3-mm i.d., 0.52-µm film thickness); temperature, column 200

◦

C

(1 min), 10

◦

C min

−1

to 250

◦

C (5 min), inlet and detector 250 and 300

◦

C, respec-

tively; gas flow rates, N

2

carrier gas 30 mL min

−1

, N

2

makeup gas 30 mL min

−1

, H

2

3.5 mL min

−1

, air 110 mL min

−1

; injection volume, 2 µL.

9

(c) GC/MS

Chlornitrofen and nitrofen: conditions for GC/MS: column, cross-linked methyl sili-
cone capillary (12 m

× 0.22-mm i.d., 0.33-µm film thickness); column temperature,

60

◦

C (1 min), 18

◦

C min

−1

to 265

◦

C; inlet, transfer line and ion source temperature,

260, 200 and 200

◦

C, respectively; He gas column head pressure, 7.5 psi; injection

method, splitless mode; solvent delay, 3 min; electron ionization voltage, 70 eV; scan
rate, 0.62 s per scan cycle; scanned mass range, m

/z 100–400. The retention times

for chlornitrofen and nitrofen were 11.8 and 11.3 min, respectively. The main ions of
the mass spectrum of chlornitrofen were at m

/z 317, 319 and 236. Nitrofen presented

a fragmentation pattern with the main ions at m

/z 283, 202 and 285.

10

(2) HPLC

Okumura et al.

3

reported State regulatory programs for pesticide residues in food

crops analyzed by the CDFA. In the multiresidue analysis of several organochlorine
pesticides including diphenyl ether herbicides, bifenox, nitrofen and oxyfluorfen,
HPLC has also been used.

Bifenox, nitrofen and oxyfluorfen: HPLC conditions with post-column fluorescence

reactor system: column, C-18 reversed-phase (25 cm

× 4.6-mm i.d.); temperature,

40

◦

C; flow rate, 1 mL min

−1

; flow composition, acetonitrile–water (1 : 4, v/v) (2 min),

with increase in acetonitrile at 5% min

−1

to 90% acetonitrile to acetonitrile–water

(9 : 1, v/v) (2 min).

(3) CE

The determination of acifluorfen in soybean was performed using CE,

4

under the

following conditions: capillary column (total length 83 cm, 65 cm to the detector,
with a 3-mm pathlength, 75-µm i.d.); absorbance detector, 240 nm; capillary oven
temperature, 20

◦

C; running buffer, 50 mM ammonium acetate buffer (pH, 4.75);

applied voltage, 17 kV; injection, 0.4 min at 4 mbar; migration time, 20 min.

2.2.4

Evaluation

Quantitative analysis is performed by the calibration technique. A new calibration
curve with a standard solution of each diphenyl ether herbicides is constructed, plot-
ting the peak area against the amount of standard solution injected. Each diphenyl
ether herbicide in the sample is measured by using the peak area for each standard.
Before each set of measurements, the GC and HPLC system is checked by injecting
more than one standard solution containing ca 0.01–2 mg L

−1

of each compound.

458

Compound class

2.2.5

Recoveries, limit of detection and limit of quantitation

The MDCs are estimated from an S/N of the diphenyl ether peaks of at least of 3
in the recovery test. With fortification levels between 0.2 and 0.5 mg kg

−1

, recov-

eries of bifenox from brown rice matrices ranged from 85 to 102% with the limit
of detection (LOD) and limit of quantitation (LOQ) being 0.010 mg kg

−1

accord-

ing to the analytical method of the Notification of the Ministry of the Environment,
Japan. By the residue analysis method described in Section 2.2.2(3), recoveries of
chlornitrofen and CNP-NH

2

from brown rice and vegetables with fortification lev-

els of 0.04–0.10 mg kg

−1

ranged from 82 to 98%. The LOD for each sample was

0.005 mg kg

−1

for chlornitrofen and CNP-NH

2

.

8

The recoveries of nitrofen and oxyflourfen from green bean, bell pepper, lettuce

and carrot fortified with 0.25 mg kg

−1

were obtained using GC/MS. The average

recoveries ranged from 106 to 127% and the LOD was 0.05 mg kg

−1

.

10

In the HPLC method for the regulatory system of the CDFA, the MDCs are

0.1 mg kg

−1

for each of bifenox, nitrofen and oxyfluorfen.

3

In the determination

of acifluorfen residues in soybeans using PLE and CE, the recovery of acifluorfen
fortified with 0.1 mg kg

−1

was between 70 and 72%.

4

2.2.6

Calculation of residues

The residual amount (R, mg kg

−1

) of diphenyl ether herbicides in the sample is

calculated by the following equation:

R

= (W

i

/V

i

)

× (V

f

/G)

where

G

= sample weight (g)

V

i

= injection volume into gas chromatograph (µL)

V

f

= final sample volume (mL)

W

i

= amount of diphenyl ether herbicide for V

i

read from the calibration curve (ng)

3

Analytical methodology for soil

3.1

Nature of the residues

The degradation of diphenyl ether herbicides in treated soil is rapid and mainly
facilitated by soil microorganisms. Diphenyl ethers herbicides degrade much faster
under flooded paddy field conditions than under upland conditions, and the value
of the half-life (DT

50

) in anaerobic soil conditions is about 4 days for bifenox

and nitrofen and 6–7 days for chlornitrofen.

11

Water et al.

12

investigated the

persistence of fluorodifen under aerobic soil conditions, and estimated that the

DT

50

was between 1.5 and 3.5 months for sandy loam and clay soils, respec-

tively. It was reported that under Brazilian Savanna conditions, the dissipation
time of fomesafen (DT

50

= 38 days) was longer than that of acifluorfen (DT

50

=

28 days).

13

Diphenyl ethers

459

In flooded soils, the main metabolites of diphenyl ether herbicides are p-amino

derivatives, adhering tightly to soil particles. The soil-bound residue in soil could be
extracted with an organic solvent at 80–100

◦

C under alkaline conditions in

>4 h. It

was reported that at the earlier stages of degradation of chlornitrofen, nitrofen and
chlomethoxyfen, the reduction rate of these herbicides increased with increase in
the ferrous ion concentration of the system, and decreased with the redox potential
of the soil.

14

From May to July, chlornitrofen was applied at the rate between 2.7

and 3.6 kg ha

−1

to paddy fields in Hokkaido, Japan, and in the following spring

the residues of chlornitrofen and CNP-NH

2

were found at levels of 0.18–1.33 and

1.16–3.36 kg ha

−1

, respectively.

15

In nonflooded soils, however, the reduction of the

metabolites of diphenyl ether herbicides is difficult to achieve.

The principal degradation products of bifenox are the free acid, 5-(2,4-

dichlorophenoxy)-2-nitrobenzoic acid, and the amino derivatives, methyl 5-(2,4-
dichlorophenoxy)anthranilate and its free acid, in flooded soil. A free acid is observed
in nonflooded soil.

16

When [

14

C]chlomethoxfen was used to treat rice field soil,

chlomethoxfen was extensively transformed into unextractable products with or-
ganic solvents; however, the amine, the N -demethylated compound and the formyl-
amino and acetylamino compounds were isolated and identified as the metabolites of
chlomethoxfen.

17

3.2

Analytical method

Air-dried soil samples were screened through a 2-mm sieve, and the water content in
the soil was calculated after holding at 105

◦

C for 5 h.

Diphenyl ether herbicides are generally extracted from 10 to 50 g of air-dried

soil with an organic solvent such as acetone, methanol and benzene by sonication,
mechanical shaking or Soxhlet extraction. If necessary, the extracts are then cleaned
by column chromatography or SPE. The extract is evaporated completely to dryness
and the residue is dissolved in an appropriate volume of the solvent for GC analysis.
The reduced amine metabolites are extracted under alkaline conditions.

3.2.1

Extraction and cleanup

A 100-mL volume of benzene is added to the 20 g of air-dried soil and the mixture
is shaken vigorously for 2 h. After extraction twice with 100 mL of benzene, the
combined extract is filtered through filter paper and the filter cake is washed with an
additional 20 mL of benzene. The benzene extracts are dried over anhydrous Na

2

SO

4

and concentrated to dryness using a vacuum rotary evaporator. The residue is dis-
solved in an appropriate volume followed by GC/ECD analysis. For the monitoring
of pesticide residues in soil, methanol for bifenox and oxyfluorfen and acetonitrile
for nitrofen were recommended as the solvents for efficient extraction.

18

Florisil column chromatography is effective in eliminating interfering substances in

soil. The organic solvent extracts from soil samples are charged to a column plugged
with Florisil which has been activated at 130

◦

C overnight before use. The effluents

from the column with a mixed solvent such as n-hexane–acetone are concentrated to

460

Compound class

dryness and the residue is dissolved in an appropriate amount of n-hexane for GC
analysis.

The extraction efficiency of the SPE procedure for oxyfluorfen in soil was compared

with that of Soxhlet extraction.

19

Ten grams of soil including the natural water contents

were added to 5 mL of water and the mixture was shaken vigorously for 1 h. After
extraction with 15 mL of methanol by sonication at 60

◦

C for 15 min, the mixture was

subsequently shaken for a further 15 min by a mechanical shaker at room temperature.
The extracts were transferred into an extraction reservoir containing 1 L of water and
acidified to pH

< 3 with 6 N HCl and then passed through an SPE (C

18

) disk at a flow

rate of about 50 mL min

−1

. The absorbed oxyfluorfen was eluted with 2

× 5 mL of

ethyl acetate and the eluate was concentrated and analyzed by gas chromatography/ion
trap mass spectrometry (GC/ITDMS). In the case of Soxhlet extraction, 10 g of soil
were extracted with 200 mL of n-hexane–acetone (1 : 1, v/v) for 24 h. The extract
was dried through an anhydrous Na

2

SO

4

column and concentrated for GC/ITDMS

analysis. With a fortification level of 0.2 mg kg

−1

, the mean recovery of oxyfluorfen

was 80% for SPE and 97% for Soxhlet extraction.

3.2.2

Determination and evaluation

The residue levels of 46 pesticides, including oxyfluorfen in soil, were determined us-
ing GC/ITDMS as described in Section 3.2.1. The conditions for GC/ITDMS were as
follows: column, fused-silica capillary (30 m

× 0.25-mm-i.d.) with a 0.25-µm bonded

phase of DB-5; column temperature, 50

◦

C (1 min), 30

◦

C min

−1

to 130

◦

C, 5

◦

C min

−1

to 270

◦

C; inlet and transfer temperature, 270 and 220

◦

C, respectively; He gas with

column head pressure, 12 psi; injection method, splitless mode. The retention time
and quantitation ion of oxyfluorfen were 23.9 min and m

/z 252, respectively.

19

3.2.3

Recoveries, limit of detection and limit of quantitation

In analyses at fortification levels of 1 and 10 mg kg

−1

of chlornitrofen, nitrofen and

chlomethoxyfen in soil, the recoveries varied from 96 to 103% for GC/ECD (2 m

×

3-mm i.d. spiral glass column packed with 1.5% silicone GE SE-30; temperature
of column, injector and detector, 220, 230 and 220

◦

C, respectively); the LOD was

0.1 mg kg

−1

.

17

In the method reported by Bao et al.

19

using a combination of disk

SPE with GC/ITDMS, the recovery of oxyfluorfen at fortification levels ranging from
0.01 to 0.4 mg kg

−1

was between 100 and 102%; the LOD was 0.004 mg kg

−1

.

3.2.4

Calculation of residues

Calculation of residues in soil was carried out as described in Section 2.2.6.

3.3

Analytical method for the metabolites of diphenyl ether
herbicides in soil

Under flooded soil conditions, the diphenyl ether herbicides are substantially trans-
formed into the amino derivatives, which are incorporated tightly into the soil particles.
An analytical method for these amino derivatives in soil has been developed.

Diphenyl ethers

461

For the simultaneous residue determination of chlornitrofen and CNP-NH

2

, 10 g

of soil are placed in a flask containing 5 g of Na

2

SO

4

· 9H

2

O and 20 mL of 10 M

NaOH aqueous solution and the mixture is refluxed overnight at 80

◦

C. After cooling,

30 mL of water, 5 g of diatomaceous earth, 2 g of copper and 100 mL of acetone are
added and the mixture is shaken for 30 min. A further 50 mL of n-hexane are added
to the aqueous layer and the mixed solution is shaken again. The combined n-hexane
layers are filtered through a funnel fitted with a filter paper. The n-hexane extract is
concentrated to ca 50 mL under reduced pressure. A 200-mL volume of 5% NaCl
aqueous solution and 50 mL of n-hexane are added to the concentrates and shaken
vigorously for 5 min. A further 50 mL of n-hexane are added and shaken again. The n-
hexane layer is transferred to a separatory funnel. The cleanup procedure and residue
determination are carried out as described in Section 2.2.3.

Niki and Kuwatsuka

17

reported a method involving trifluoroacetylation of the

amino derivatives of chlornitrofen, nitrofen and chlomethoxyfen. A 1-mL volume
of 10 M NaOH solution was added to 50 g of soil and the mixture was extracted with
100 mL of benzene. After separation and drying over anhydrous Na

2

SO

4

, the benzene

solution was trifluoroacetylated by adding successively 1 mL of 0.1% trifluoroacetic
anhydride in benzene and 1 mL of 0.1% triethylamine in benzene. The mixture was
shaken for 30 s and diluted to 10 mL with benzene. To remove the excess of tri-
fluoroacetic anhydride, about 2 mL of water were added to the mixture and shaken
for 30 s. The benzene layer was dried over anhydrous Na

2

SO

4

and injected for gas

chromatography/flame ionization detection (GC/FID).

The GC/FID conditions were as follows: column, 1.5% OV-17 (2 m

× 3-mm i.d.)

glass column; N

2

carrier gas flow rate, 45 mL min

−1

; temperature of injection port,

column and detector, 240, 235 and 235

◦

C, respectively. The recoveries of these amino

derivatives with fortification level ranging from 0.5 to 10.0 mg kg

−1

were 62–101%

for chlornitrofen, 62–101% for nitrofen and 58–101% for chlomethoxyfen, and satis-
factory recoveries from soil were obtained at high concentrations, but the recoveries
at lower concentration averaged about 66% for the least recovered compound. Inter-
ference from other substances in the soil extracts derived from the acetylation reaction
was negligible.

4

Analytical methodology for water

4.1

Nature of the residues

Environmental pollution caused by pesticides has become a serious problem. Espe-
cially during and/or after pesticide application to crops, the pesticides are released
into sensitive environmental areas, and also into ground and surface water, and could
be harmful or dangerous to humans and other species. Therefore, very low concen-
trations of diphenyl ether herbicides in environmental waters must be monitored.

The concentration of chlornitrofen in river water released from flooded paddy

fields 30–60 days after application was detected in the range 0.039–1.25 µg L

−1

.

20

Further, it was reported that the DT

50

of diphenyl ether herbicides in groundwater,

river water and seawater were 17–84, 14–140 and 10–88 days for chlornitrofen, and
18–131, 4–206 and 6–23 days for bifenox, respectively. Diphenyl ether herbicides in

462

Compound class

water are hydrolytically stable in the dark, but in light are rapidly degraded, and the

DT

50

of acifluofen and bifenox are ca 2 h and 24 min, respectively, at 250–400 nm.

2

In anaerobic conditions, chlornitrofen was decreased to below 5% of the dose by
microorganisms by 7 days after application, and the metabolites identified were CNP-
NH

2

and 4-aminophenyl 2,6-dichlorophenyl ether.

21

4.2

Analytical method

Water samples of 500–1000 mL are extracted and purified simultaneously through an
SPE cartridge such as Carbograph-1, C

18

and RP-18, usually followed either by HPLC

with ultraviolet (UV) or photoconductivity detection or by GC/ECD. The acidic-type
diphenyl ether herbicides are derivatized with diazomethane and various kinds of
chloroformates and determined by GC and HPLC.

4.2.1

Extraction and cleanup

In recent years, the extraction of diphenyl ether herbicides from water samples
such as river water, groundwater and drinking water by SPE has increased in popu-
larity.

Water samples (1000 mL of groundwater and drinking water, 500 mL of river water)

are stirred artificially and drawn through a Carbograph-1 cartridge (LARA, Rome,
Italy), which is fitted into a side-arm filter flask, at flow rates of 30–50 mL min

−1

.

The cartridge is then washed with 7 mL of water. Most of the water remaining in
the cartridge is expelled under vacuum for ca 5 min and the residual water content
is further decreased by slowly passing 1 mL of methanol through the cartridge. The
neutral diphenyl ether herbicides (aclonifen, bifenox, fluoroglycofen, lactofen and
oxyfluorfen) are then eluted with 8 mL of dichloromethane–methanol (4 : 1, v/v).
Thereafter, to elute the acidic diphenyl ether herbicides (acifluorfen, bifenox acid and
fomesafen), the cartridge is turned upside-down, and the herbicides are reverse-eluted
by passing 10 mL of dichloromethane–methanol (4 : 1, v/v) acidified with formic
acid through the cartridge at a flow rate of ca 5 mL min

−1

. A 40-µL volume of 30%

aqueous ammonia solution–methanol (1 : 1, v/v) is added to the eluate, and the latter
is then concentrated to about 200 µL at 40

◦

C, under a gentle stream of nitrogen.

The walls of the vials are washed sequentially with 100 µL of acetonitrile–water
(1 : 1, v/v) and 100 µL of formic acid–acidified methanol (25 mM). The combined
concentrate and washings containing neutral diphenyl ether herbicides are further
concentrated to ca 200 µL and the final volume is carefully measured. The fraction
containing the acidic diphenyl ether herbicides and metabolites is concentrated to
dryness, and the residue is reconstituted with 250 µL of acetonitrile–methanol (1 : 1,
v/v)–water (3 : 2, v/v) acidified with 100 mM formic acid. Volumes of 100 µL of both
the solutions containing neutral and acidic diphenyl ether herbicides and the acidic
compounds are injected on to the LC column.

22

Chlornitrofen in river water (1000 mL) was determined using two Sep-Pak C

18

cartridges connected together (Waters), which were rinsed with 5 mL of methanol,
and cleaned and conditioned with 10 mL of water. Chlornitrofen was eluted from the
cartridge with 10 mL of methanol after being rinsed with 3 mL of water–methanol

Diphenyl ethers

463

(7 : 3, v/v). The eluate was evaporated to dryness and the residue was dissolved in
acetone, and injected into the GC/MS system.

23

Acifluorfen, an acidic diphenyl ether herbicide, was extracted from 100 mL of the

water samples after adjusting the pH to 1.0 with sulfuric acid and was eluted through
47-mm C

18

and polystyrene–divinylbenzene (PS-DVB) resin extraction disks (Ana-

lytical International, PA, USA) with 20 mL of methanol–methyl tert-butyl ether (1 : 9,
v/v). After drying the extract by passing it through a large Pasteur pipet containing 4 g
of acidified anhydrous sodium sulfate, acifluorfen was esterified with diazomethane
for analysis by GC.

24

Water samples were treated with diazomethane gas by a mi-

cromolar generation procedure. The methyl ester of acifluorfen was determined by
GC/ECD. Butz and Stan

25

reported a simple method for the determination of aciflu-

orfen residues in water. The water sample (100 mL) acidified to pH 1.5 was drawn
through an RP-18 cartridge (Baker, Germany) at a flow rate of ca 8 mL min

−1

and

acifluorfen was eluted with 2 mL of methanol after drying the cartridge for 2–3 h
under a gentle stream of nitrogen. Thereafter, the extract was treated with methyl,
ethyl or butyl chloroformate to give the corresponding methyl, ethyl or butyl esters
of acifluorfen and the esters were determined by GC/MS.

4.2.2

Determination and evaluation, recoveries, limit of detection, limit of
quantitation and calculation of residues

The procedure for the determination, evaluation and calculation of residues of diethyl
ether herbicides in water is carried out fundamentally by a similar procedure to the
plant material method described in Sections 2.2.3 and 2.2.4.

Lagana et al.

22

developed a new analytical method combining off-line SPE with liq-

uid chromatography/ultraviolet/diode-array detection (LC/UV/DAD). The conditions
for LC/UV/DAD were as follows: column, C

18

packed Alltima (25 cm

× 4.6-mm i.d.);

precolumn, Supelguard, C

18

(2 cm

× 4.6-mm i.d.); absorbance, 290 nm by diode-array

detection; mobile phase: for neutral diphenyl ether herbicides, acetonitrile–water (lin-
ear increase in the proportion of acetonitrile from 62 to 75% in 30 min, and to 100%
in the following 5 min); for acidic diphenyl ether herbicides, phase (A) acetonitrile–
methanol (1 : 1, v/v) acidified with 20 mM formic acid and phase (B) water acidified
with 50 mM formic acid (linear increased in the proportion of phase A from 55 to
70% in 30 min, followed by 5 min at 100%); flow rate, 1 mL min

−1

for both groups.

The retention times for the five diphenyl ether herbicides, aclonifen, bifebox, fluoro-
glycofen, lactofen and oxyfluorfen were 12–24 min and for the three acidic diphenyl
ether herbicides acifluorfen, bifenox acid and fomesafen they were 22–27 min. Re-
coveries and LODs from different natural drinking water and groundwater samples
fortified at 50 µg L

−1

and of river water fortified at 200 µg L

−1

with aclonifen were

96–97% and 6.0–19.8 ng L

−1

and 94–96% and 6–22 ng L

−1

for bifenox, 90–91% and

7–10 ng L

−1

for fluorglycofen, 91–96% and 6–32 ng L

−1

for oxyfluorfen, 85–93%

and 7–32 ng L

−1

for lactofen, 96–100% and 5–26 ng L

−1

for acifluorfen, 93–102%

and 5–30 ng L

−1

for bifenox acid and 94–97% and 4–18 ng L

−1

for fomesafen. In

groundwater and river water samples, higher detection limits were observed than in
drinking water owing to the interference of matrix compounds.

Chlornitrofen in water samples was determined by GC/MS using an SPB-1 fused-

silica capillary (15 m

× 0.53-mm i.d., 0.5-µm film thickness), with the ionization

464

Compound class

voltage and the ion-source temperature set at 70 eV and 150

◦

C, respectively.

23

He-

lium was used as the carrier gas at a flow rate of 30 mL min

−1

. The temperatures of

the column, separator and injection port were maintained at 170, 250 and 250

◦

C,

respectively. The molecular ions of m

/z 317, 236 and 173 of chlornitrofen were mon-

itored. The recovery from the water samples at a fortification level of 2.5 µg L

−1

was

79% and the LOD was 50 ng L

−1

.

In the above-mentioned method by Hodgeson et al.,

24

acifluorfen was determined

by GC/ECD after the extraction with a 47-mm PS-DVB disk and derivatization with
diazomethane. The conditions for GC/ECD were as follows: column, DB-5 fused sil-
ica (30 m

× 0.32-mm i.d., 0.25-µm film thickness); He carrier gas velocity, 25 cm s

−1

(210

◦

C), detector makeup gas, methane–argon (5 : 95), 30 mL min

−1

; column tem-

perature, 50

◦

C (5 min), 10

◦

C min

−1

to 210

◦

C (5 min) and to 230

◦

C (10 min); injec-

tion port and detector temperature, 220 and 300

◦

C, respectively; injection method,

splitless mode. The recovery of acifluorfen from purified water, dechlorinated tap
water and high humic content surface water fortified at 0.5–2.0 µg L

−1

was 59–150%

and the LOD was 25 ng L

−1

. Acifluorfen after derivatization with various chlorofor-

mates was also determined by GC/MS using an SE-54 column (25 m

× 0.20-mm i.d.,

0.32-µm film thickness), and the average recovery of acifluorfen fortified between
0.05 and 0.2 µg L

−1

was 78%.

25

4.2.3

Enzyme-linked immunosorbent assay (ELISA) determination of
acifluorfen and chlornitrofen

Miyake et al.

26

reported an ELISA method for the determination of pesticide residues

in the aquatic environment. The polyclonal antibody and three monoclonal anti-
bodies of acifluorfen were prepared by immunization of rabbits and mice with
acifluorfen–bovine serum albumin conjugates. The polyclonal antibody reacted with
acifluorfen at concentrations of 1.5–800 mg L

−1

, while the monoclonal antibodies re-

acted with acifluorfen at concentrations of 1.5–144 mg L

−1

. Among three monoclonal

antibodies, AF 75-144 reacted with chlornitrofen, which did not react with the other
two antibodies. It seems that the ELISA method is effective for the determination of
herbicide residues in the aquatic environment.

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