Mutagen Formation Potential of Composite Samples Prepared by Biodegradation of Agricultural Chemicals

Journal of Water and Environment Technology, Vol. 6, No.1, 2008

Mutagen Formation Potential of Composite Samples
Prepared by Biodegradation of Agricultural Chemicals
M. Kishida*, H. Takanashi*, H. Kofune*, T. Nakajima* and A. Ohki*
*Department of Bioengineering Kagoshima University, Kagoshima 890-0065 Japan
ABSTRACT
Ten mg-AC/L aqueous solutions of 18 kinds of agricultural chemicals (ACs), such as
organophosphorus, organochlorine and amidic chemicals, were prepared and underwent the
biodegradation test. The samples attained through the test were considered to contain various
decomposition products, hereafter referred to as composite samples. Mutagenicity and mutagen
formation potential (MFP) were measured for the composite samples, and the test results
revealed that the ACs tested in the present study do not produce mutagens as a result of
undergoing the biodegradation test. However, 12 out of 18 samples manifested statistically
significant MFP. All of the 12 ACs, except for iminoctadine-triacetate, were aromatic compounds.
Specific activities for thiram and DDVP, which were reported to be mutagenic, were measured,
showing 320 net rev./mg and 190 net rev./mg respectively. Compared with these values, MFP of
the composite samples attained from ferimzone, pyridiphenthion, bentazone, bensultap, and
napropamide were greater. Accordingly, it was suggested that some ACs, though they were
non-mutagenic compounds, could form strong mutagens when they were biodegraded in a water
environment, and the decomposition products subsequently intruded into the raw water for water
supply, and the water was then chlorinated at a purification plant.
Keywords: agricultural chemicals, biodegradation, mutagen formation potential, mutagenicity.

INTRODUCTION
Tap water in Japan is among the safest in the world. However, the toxicity of
disinfection by-products (DBPs) produced by chlorination during the water purification
process has been noted. Among these DBPs are substances that manifest mutagenicity
such as chloral hydrate (Japan Water Works Association, 1999). The authors, therefore,
had surveyed the mutagenicity of Japanese tap water from 2001 through 2002 by means
of the Ames assay, which is one of the most popular methods for measuring

mutagenicity (Urano et al., 1995). The results revealed the existence of mutagens in
many water samples. Mutagens are considered to form when non-toxic organic matters
like humic substances, which are common in water environments, react with chlorine at
a purification plant. Therefore, in many cases, the mutagen formation potential (MFP)
of surface waters show some correlation with the organic matter concentration of raw
waters (Komatsu et al., 2007). It is commonly known that the mutagenicity of tap water
does not vary excessively because the organic matter concentration of raw water does
not vary excessively. For example, the survey made by the authors from 1992 through
1993 showed that the mutagenicity observed ranged from less than the detection limit
up to 9,200 net revertant/L (Urano et al., 1995). However, in another survey conducted
by the authors from 2002 through 2005, high mutagenicity of 16,000 net revertant/L
(hereafter referred to as an outlier) was detected. This finding was 5.2 times as high as
the mean mutagenicity of the above tap water samples. These results suggested that the
mutagenicity of tap water was not only affected by the substances that were consistently
Address correspondence to Hirokazu Takanashi, Department of Bioengineering, Kagoshima
University, Email:
Received February 6, 2008, Accepted February 25, 2008.
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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

contained in raw water for water supplies, but also by substances inconsistently
contained in raw water.
The authors thus focused on agricultural chemicals (ACs) and their biodegradation
products (AC-decompositions) because these were the substances inconsistently
contained in raw waters. ACs are typical chemicals that are actively sprayed on
agricultural fields and subsequently permeate the water environment. Actually, ACs and
AC-decompositions were detected in many studies (Takahashi et al., 2003, Barcelo et
al., 2007). We were intrigued as to whether mutagens are formed when ACs and

AC-decompositions go through chlorination treatments at water purification plants.
The production, sale, and usage of ACs in Japan are regulated by Japan’s Agricultural
Chemicals Regulation Law. Before ACs are permitted to be registered, they must
undergo various toxicity tests, including the Ames assay. According to the law, the
major derivatives of ACs, such as hydrolysates or metabolic products produced by
vegetation, are also required to undergo the tests. However, substances produced
through the chlorination process are exempt from the law.
Although many reports have been done about the degradability or mutagenicity of ACs
(Arai et al., 2005; Kamoshita et al., 2007), there are only a few studies on mutagenicity
change when ACs are decomposed in a water environment. Some of the studies about
mutagenicity changes during the decomposition process are reported. In a study by
Onodera et al. (1995), by analyzing the decomposition products of fenitrothion,
fenitrothion-oxon was detected, showing that it was non-mutagenic. In a study by
Setsuda et al. (1992), the mutagenicity lowered when thiram was chlorinated, and in
that conducted by Matsushita et al., under the anaerobic condition, fenitrothion
(Matsushita et al., 2002) and CNP (Matsushita et al., 2005) aminated and increased
mutagenicity. However, the kinds of ACs studied in these reports were limited. There
were even fewer studies on the formation of mutagens during the chlorination process
of AC-decompositions.
Therefore, in this study, composite samples of AC-decompositions attained through the
biodegradation test were prepared to measure their MFP. A composite sample was
considered to contain various decomposition products from an AC. MFP was defined as
mutagenicity that was measured when the composite samples went through the
chlorination process with similar pH, contact time, and chlorine dosage as those found
during the chlorination process at actual water purification plants. For these tests, 18
composite samples were prepared from 18 different kinds of ACs. Thus, the significance
of MFP measurement under the law for the composite samples was discussed.
MATERIALS AND METHODS
Agricultural Chemicals
Table 1 shows the commercial names of the 18 kinds of ACs tested in this study, their

purposes, usage amounts from Dec. 2001 through Sep. 2002, aqueous solubility, and the
data provided by CCRIS (Chemical Carcinogenesis Research Information System)
(National Library of Medicine, 2006), a database consisting of peer- reviewed test
results for carcinogenicity and mutagenicity. Ten of these ACs were selected because

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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

they were the chemicals recommended for spraying on the paddy fields at the times of
sampling around a certain river examined during the 2002-2005 survey. During the
survey, the authors had found that the tap water, which originated from the river and
was subsequently processed through a water purification plant, had manifested
excessively high mutagenicity (identified as an outlier). The other 8 ACs were selected
out of 101 ACs designated as Items Used to Set Targets for Water Quality Management
in Japan’s Waterworks Law, for the reason that they have aqueous solubility of more
than 10 mg/L. The AC usage amounts were determined by subtracting the exported
amounts from the total amount of production and imports shown in the ACs directory
(Japan Plant Protect Association, 2002). A negative figure was attained for IBP usage
presumably because its storage amounts before and after the survey varied considerably.
According to the Abstract of Water Quality Standard Revision and Individual ACs Data
provided by the Ministry of Health, Labour and Welfare (The Japanese Ministry of
Health, Labour and Welfare, 2003), the presumed shipment of IBP in 2002 was 165.1 t,
showing its considerable usage. Aqueous solubility was thus attained from the
individual AC data above.
Table 1 - Agricultural chemicals used in the study
Commercial name

Abbreviation

Purpose

Fenitrothion
Bentazone
Pretilachlor
Diazinon
Tricyclazole
Pyroquilon
Isoxathion
Iminoctadine-triaetate
Buprofezin
Bensultap
Pyridiphenthion
PAP
Ferimzone
MIPC
IBP
Napropamide
Propyzamide
Isophenphos

FNT
BTZ
PTL
DZN
TCZ
PQL
IXT
ICT

BPF
BST
PPT
PAP
FMZ
MIP
IBP
NPP
PPZ
IPP

insecticide
herbicide
herbicide
insecticide
fungicide
fungicide
insecticide
fungicide
insecticide
insecticide
insecticide
insecticide
fungicide
insecticide
fungicide
herbicide
herbicide
insecticide

Usage
[t, kL]

Aqueous
solubility
[mg/L]

2004.0
14 (30℃)
435.8
570 (20℃)
334.4
50 (20℃)
306.0
60 (20℃)
296.6 1,600 (25℃)
275.7 4,000 (20℃)
215.6
1.9 (25℃)
212.4
171.2
0.9 (25℃)
79.6
74.1
100 (20℃)
65.7
10 (25℃)
64.6
26.6
265 (20℃)

-492.0
430 (20℃)
73 (25℃)
15 (25℃)
18 (20℃)

Dataum in
CCRIS
N
N
N
a
N
-

N ; Mutagenicity of the sample before the chlorination is reported to be negative in CCRIS.
- ; No imformation is availale.
a
; TA1535 strain

Fig. 1 shows the chemical structures of 18 ACs. As is shown, various types of ACs, such
as organophosphorus, organochlorine, and amide, were examined in this study. All the
ACs tested were purchased in the purity grade of Standards for Pesticide Residue
Analysis from Wako Pure Chemical Industries, Ltd.

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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

Preparation of Composite Samples
To prepare 10 mg-AC/L of aqueous solution, 100 mg of AC was dissolved into 2 mL of
ethanol and 400 µL of the solution was then added to 2 L of distilled water. The
composite sample was prepared by subjecting the AC aqueous solution to the
biodegradation test. The biodegradation test was conducted based on the Biodegradation
Test of Chemical Substance by Microorganisms etc, stipulated in the Order Prescribing
the Items of Test Relating to the New Chemical Substance (hereafter referred to as the
guideline), and its procedure is shown in Fig. 2 (Chemicals inspection & testing institute
Japan, 1992). The sludge used for the degradation test was collected from the aeration
tank of a sewage treatment plant in Kagoshima Prefecture, and used after more than 28
days of accumulation with the basal culture medium shown in the guideline. After the
activated sludge was added to the AC aqueous solution, the time courses of dissolved
organic carbon (DOC) and pH were observed on the 0th, 7th, 14th, and 28th day. The
validity of the test was confirmed by the fact that the degradation degrees of aniline
were respectively more than 40% and 60% on the 7th and 14th day. The sample solution
was stirred under a light-shielding condition and kept at 25 degree C. On the 28th day of
the test process, the sample solution was filtrated with a No.5C paper filter and
subsequently served as the composite sample.
Chlorination Procedure for Measuring Mutagen Formation Potential
As shown in Fig. 3, the composite samples were chlorinated in order to measure MFP
(Takanashi et al., 2001). The pH of the test solutions was adjusted to 7.0 ± 0.2. Then 3-4
mg-Cl/mg-C of chlorine was added to each solution with a sodium hypochlorite
aqueous solution. The chlorination process was completed after the solution had sat for
24 hours under a light-shielding condition at room temperature. The existence of more
than 0.1 mg/L of residual chlorine was confirmed by the DPD colorimetric method after
the chlorination process.
Concentration of Mutagens in Water Samples
As shown in Fig. 4, the mutagens produced by chlorination were concentrated 1,000
times from the composite samples by an adsorption-desorption method in order to be
served for the Ames assay (Urano et al., 1997). The pH of the solution was adjusted to

2.0 ± 0.2 using 2.5 M sulfuric acid and the mutagens were adsorbed by passing it
through a Sep-Pak Plus CSP-800 cartridge which was purchased from Nippon Waters,
Ltd. The dimethyl sulfoxide (DMSO) was applied to the cartridge in order to desorb the
adsorbed mutagens.
In general, by implementing the above method, it has been proven that more than 90%
of the mutagens in tap water can be recovered on the basis of mutagenic activity (Urano
et al., 1997). However, whether the mutagens were recovered with high percentage is
unknown in this study because the AC concentration was quite high compared to that in
tap waters. Accordingly there is a possibility that the actual MFP is higher than the MFP
measured in this study. However, the method does not negate the importance of the
discussion regarding MFP measurement of composite samples under Japan’s
Agricultural Regulation Law, because the purpose of this study was to examine whether
AC-decompositions with high MFP exist.

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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

H

H3 C

N

S
O2 N

N

OP(OCH3)2

COCH2Cl

CH3

OP(OCH2 CH3 )2

N

N

N

N

DZN

PQL

TCZ

N

+

S

+

NH2

NH2

・3CH3COO－

+

OP(OCH2CH3)2

NH2 CN(CH2 )8 NH2 (CH2 )8NHCNH2

IXT

ICT

S

SO2 S

N

NC(CH3 )2

CH2
CH

N
O

O

N

CH3

O

CH2 CH2 O(CH2 )2CH3
CH2 CH3

PTL
S

O

(CH3 )2 CH

N

CH(CH3)2

O

BTZ

FNT

N

CH2 CH3
SO2

SO2 S

CH(CH3 )2

BPF

N(CH3)2

N
O

CH2

O

BST
S

N

H
N

PPT
CH(CH3)2

N

N

CO2CH2CH3
CH3

PAP

CH3NHCO2
CH3

FMZ

MIP
CH3 CHCON(CH2 CH3 )2

O

O

CH2 SP[OCH(CH3 )2 ]2

IBP

NPP

Cl
CH3
CONH

C

(CH3)2 CHOCO
C

P(OCH2 CH3 )2

CH3

CH3

CHSP(OCH3)2

S

N

CH

S
OPOCH2 CH3
NHCH(CH3 )2

CH3
Cl

IPP

PPZ

Fig. 1 - Chemical structures of agricultural chemical.

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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

Prepare aqueous solution
of agricultural chemical at 10 mg/L
Add activated sludge at 30 mg-SS/L
Stir the solutions for 28 days under the light
shading condition at 25 degree C
Measure dissolved organic carbon and pH
of the test solutions on 0th, 7th, 14th, 28th day
Filtrate the test solutions with paper filter

Fig. 2 - Preparation procedure of composite sample.

Adjust pH of the water sample to 7.0 ± 0.2
with ca. 0.25 M-H2SO4 or 0.5 M-NaOH
Add chlorine of the water sample volume ×
(3 ~ 4) TOC using ca. 5,000 mg-Cl/L
NaClO while agitating the sample
If [NH4+-N] is above 0.2 mg/L,
further addition of Cl to nine-fold [NH4+-N]
is necessary
Leave standing for 24 ± 2 hours
in 10 ~ 30 degree C

Fig. 3 - Chlorination procedure for measuring MFP.

Ames Assay
The Ames mutagenicity assay (preincubation method) was conducted according to the
guidebook (Mutagenicity Assay for the Occupational Safety and Health Act. Test
Guideline and GLP) published by Japan’s Ministry of Labour and Welfare (Japan’s
Ministry of Health, Labour and Welfare, 1991). The assay was performed with the
method using Salmonella typhimurium TA100 strains, without exogenous activation
(S9), with 3-6 dose steps, and with duplicate plates for each step. Quadruplet plates
were used for the negative control tests. A positive control substance of
4-nitroquinoline-1-oxide, 4NQO, was used to confirm the strains’ specific activities. At
9,000-11,000 net rev./mg-4NQO, the strains’ specific activities were quite consistent
throughout all the runs. The negative test results were also quite consistent, showing
95-181 rev./plate. From these results, all the MR values attained in the different runs of
the Ames assay could be compared with each other.

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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

Adjust pH of the sample to 2.0 ± 0.2 with
ca. 2.5 M-H2SO4 without chlorine reduction
Pass the water sample through one of the
CSP-800 cartridge in an upward flow
at ca. 50 mL/min
Turn the cartridge upside down and apply
DMSO in an upward flow at 0.2 mL/min
and collect 2 mL eluent after water is
displaced by DMSO
Sterilize by filtration
Evaluate by the Ames Salmonella

mutagenicity assay (preincubation method)

Fig. 4 - Concentration procedure for Ames assay.
RESULTS AND DISCUSSION
Mutagen Formation Potential of Organic Compounds from Activated Sludge
There was a possibility that the test results were misleadingly heightened because of the
elution of mutagens or mutagen precursors from the activated sludge, which was used
during the biodegradation test. Therefore, a control test, or a test without the addition of
ACs, was conducted in order to study the influence of organic matter originating from
the activated sludge. The results showed that the specific activity of mutagens eluted
from the sludge was less than the detection limit as shown in Fig. 5. However, Fig. 5
also shows that the MFP measurement results manifested some weak mutagenicity, with
MR values of 2.0 and 2.1. This indicated that in the measurement of
AC-decompositions’ MFP, the MFP of the composite samples should be attained by
subtracting 1.0 in MR value from the actual measured value.
Accordingly, the value calculated by subtracting 1.0 from the MR value observed for
the AC-decomposition sample was defined as MRb as shown in equation (1). The test
result that exceeded 1.4 in MRb value was considered positive (Takanashi and Urano,
1998), and its specific activity (MFP) was calculated by equation (2).
MRb = Nｄ / Nｓ - 1

(1)

MFP = a - Nｓ / d

(2)

where Nd the mean number of revertant colonies at the maximum dosage of the samples
[rev./plate], and Ns the mean number of revertant colonies in the negative control tests, a
the slope of dose-response line at the MFP measurement of composite samples [net

rev./mg-AC], and d the maximum dosage of AC [mg-AC/plate].

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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

Mutagenicity and MFP of Composite Samples
In addition to the AC biodegradation tests, decomposition tests for aniline were
conducted according to the guideline, in order to confirm that the activity of the
activated sludge met the guideline. Fig. 6 also shows the time course of the
biodegradation degrees of aniline. Aniline showed more than 40% biodegradation on
the 7th day and more than 60% on the 14th day for every test, thus meeting requisite in
the guideline.
For the purpose of the biodegradation tests on 18 kinds of ACs, the ACs could be
separated into three groups; the ACs that showed significant biodegradation degrees
such as PAP; the ACs that did not show significant biodegradation percentages such as
NPP; and the ACs that showed DOC increases due to contact with bacteriolysis, such as
BPF. Three out of the 18 ACs indicated DOC increases; two of them were insecticides
and the rest was a fungicide. As for the tested herbicide, a DOC increase was not
observed. When their mutagenicity or MFP results were positive, the test results
assessment for the samples with DOC increases needed to be made carefully, because it
was not possible to know whether the mutagen originated from the ACs or from the
activated sludge.
350

Run 1

300

Run 2

300
Response [rev./plate]

Response [rev./plate]

350

250
200
150
100
50

250
200
150
100
50

0

0
0

0.05
Dose [L/plate]

0

0.1

0.05
Dose [L/plate]

0.1

PAP

0

7

14
21
Day [d]

28

100
90
80
70
60
50
40
30
20
10

0
-10

100
80
Biodegradation [%]

100
90
80
70
60
50
40
30
20
10
0

Biodegradation [%]

Biodegradation [%]

Fig. 5 - Dose-response lines of blank test: ○ mutagenicity; ● MFP.

NPP

60
40

BPF

20
0
-20
-40

0

7

14
21
Day [d]

28

0

7

14
21
Day [d]

Fig. 6 - Examples of biodegradation test results on aniline and ACs: ○ aniline; ● ACs.

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28

Journal of Water and Environment Technology, Vol. 6, No.1, 2008

Mutagenicity of the composite samples was also measured. As shown in Table 2, no
significant mutagenicity was observed for the tested composite samples in this study.
This means that the ACs used for the tests in this study do not form mutagens.
Nishimura et al. reported that the ChE activity inhibition for the oxone-form of an
organophosphorus AC is higher than the AC itself (Nishimura et al., 2007). It is also
well known that some substances easily form oxone in their aqueous solutions. It is not
appropriate to compare the results of this study with those found by Nishimura et al.,
because the mechanism of toxicity formation in organophosphorus is mostly based on
oxone formation. However, the study results by Nishimura et al. show that an endpoint
where the toxicity strength changes exists because the chemical structures of the ACs
change during the biodegradation test. Therefore, it is suggested that the toxicity
formation mechanism needs to be studied for mutagenicity.
The MFP of the composite samples was also studied. Out of 18 tested samples, 12
samples manifested statistically significant MFP. Fig. 7 shows the dose-response lines
for the 12 samples with statistically significant MFP. As is shown, they all had good
linearity. Statistically significant MFP was detected from the composite sample made
out of buprofezin (BPF) and a DOC increase was observed when BPF underwent the
biodegradation test. This did not reveal whether the mutagen precursor in the BPF
composite sample originated from BPF or from the activated sludge.
Table 2 shows the summary of mutagenicity and MFP measured in this study, as well as
the biodegradation degrees on the 28th day of the test process. When focusing on the
chemical structures of the ACs shown in Fig. 1, all the ACs tested were noted to be
aromatic compounds except iminoctadine-triacetate (ICT). When ACs biodegrade, the
bonds between the benzene rings and the substituents will break prior to the breaking of
the benzene rings themselves, thereby forming decomposition products possessing
benzene rings. AC decompositions that possess benzene rings active to electrophilic

addition are likely to cause chloride substitution reactions, presumably forming
mutagens as a result (Takanashi et al., 2007).
The ACs commercially available at present contain substances that display mutagenicity.
For example, the mutagenicity of thiram and DDVP is reported to be mutagenic in the
CCRIS data base. The mutagenicity for the 10 mg/L aqueous solution of thiram and
DDPV was measured because CCRIS does not give the specific activities for these
substances. The mutagenicity attained were 320 net rev./mg and 190 net rev./mg
respectively for thiram and DDPV. Compared with these values, the MFP of the
composite samples of FMZ, PPT, BTZ, BST, and NPP were high, as shown in Table 2.
The above findings suggested that even though the original ACs are non-mutagenic,
some ACs manifest high mutagenicity during the chlorination process at water
purification plants, when decomposition products in water environments intrude into
raw water. Therefore, the chemical structures of the formed mutagen precursors and the
existence of these substances in raw water needs to be identified. In the case that such
substances are identified, it is considered significant to add MFP measurement for the
composite samples to the Agricultural Chemicals Regulation Law.

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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

Table 2 - Summary of the test results.
Commercial name

Ferimzone
Pyridiphenthion
Bentazone
Bensultap
Napropamide

MIPC
Pretilachlor
Buprofezin
Fenitrothion
Diazinon
IBP
Isophenphos
PAP
Iminoctadine-triacetate
Pyroquilon
Propyzamide
Tricyclazole
Isoxathion
N. D. ; Not detected.

Abbreviation

FMZ
PPT
BTZ
BST
NPP
MIP
PTL
BPF
FNT
DZN
IBP
IPP
PAP

ICT
PQL
PPZ
TCZ
IXT

Specific activity of
AC- decomposition
[net rev./mg]
Mutagenicity
MFP
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.

1,200
1,200
1,100
760
510
260
240
220
200
170
160
130
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.

Biodegradation
[%]

27
5
5
53
2
9
19
-33

52
7
56
9
100
75
41
4
-2
-44

In this study, mutagenicity and MFP were assessed by means of the net number of
revertant colonies per unit of mass of the added ACs [net rev./mg-AC]. Composite
samples are presumed to contain various kinds of AC-decompositions produced through
biodegradation test as well as undecomposed ACs. However, it is difficult to both
identify every substance and measure the mutagenicity and MFP for each substance.
When the measurements of mutagenicity and MFP for many ACs are required, it is
effective to make a collective assessment for the composite, which is produced after the
biodegradation test. In this manner, the mutagenicity and MFP of the composite
samples were measured for this study. When the results of collective assessment
indicate the existence of ACs with low mutagenicity but high MFP, it is considered
significant to add the MFP measurement of composite samples to the present
examination required by the law. It is therefore valid, based upon the results attained by
the above manner, to discuss the significance of adding MFP measurement of composite
samples as an addition to the Agricultural Chemicals Regulation Law.
AC concentration in this study was 10 mg/L, which was over 10,000 times greater than
actual AC concentration in raw water. This is why the chlorination process in this study
differed from that conducted in actual water purification plants. However, the ratio of
the chlorine dosage to ACs concentration [mg-Cl/mg-C] is considered to be similar to
the one at real purification plants because chlorine dosage was decided on residual free

chlorine concentration. Though the reaction rate could be faster than under actual
conditions because of the higher concentration of substrates, the concentration ratio and
the temperatures were almost identical, thus presumably maintaining the same
equilibrium. The purpose of this study is to reveal the existence of composite samples

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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

that manifest high MFP and accordingly discuss the importance of MFP measurement of
composite samples in Japan’s Agricultural Chemicals Law. It is not to discuss the effect
of AC decompositions on the MFP of actual raw waters or to discuss the measuring
method of the MFP of composite samples. That is why, for the purpose of attaining
accurate test results, it was effective and correct to conduct the experiments with high
concentration aqueous solutions of ACs.
1600

1000
800
600
400

0

0.5
Dose[mg/plate]

300
200

800
600
400

NPP

1200
1000
800
600
400

0

0.5
Dose[mg/plate]

0.5
Dose[mg/plate]

Response [rev./plate]

PTL

400
300
200
100
0

0.5
Dose [mg/plate]

500

BPF

400
300
200

0

1

600

400
300
200

0.5
Dose [mg/plate]

0.5
Dose [mg/plate]

1

1

FNT

400
300
200

0

0.5
Dose [mg/plate]

1

0.5
Dose [mg/plate]

1

600

500

IBP

400
300
200

500

IPP

400
300
200
100

0
0

500

1

100

100

0.5
Dose [mg/plate]

0
0

Response [rev./plate]

Response [rev./plate]

DZN

200

100

600

500

300

600

100

0

MIP

400

0

600

500

500

1

Response [rev./plate]

600

1

0

0

1

0.5
Dose [mg/plate]

100

0

0

400

0

200

200

600

0.5

Response [rev./plate]

Response[rev./plate]

1000

800

600

1400

BST

1200

1000

0
0.25
Dose [mg/plate]

1600

1400

BTZ

1200

200
0

1

1600
Response[rev./plate]

400

0

0

Response [rev./plate]

1400

PPT

100

200

Response [rev./plate]

500

Response [rev./plate]

Response [rev./plate]

Response[rev./plate]

FMZ

1200

0

1600

600

1400

0

0.5
Dose [mg/plate]

1

0

0

Fig. 7 - Dose-response lines of MFP-positive sample: ○ mutagenicity; ● MFP.

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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

Change of MFP through Biodegradation Test
The biodegradation treatment requires a duration of 28 days, which is time-consuming,
laborious, and costly. Therefore, in order to expedite the procedure, the possibility of
utilizing a simplified measuring method for the MFP of ACs was studied. This
simplified method consisted of two steps: measuring the MFP of all the ACs without
undergoing biodegradation tests, and then giving biodegradation tests only to the ACs
that manifested high MFP after the first measurement.
The experiment results of MFP measurement of AC aqueous solutions without
biodegradation tests and the test results of this study were compared. Fig. 8 shows an
example of the comparison (Takanashi et al., 2007). Nine AC samples manifested
higher MFP for their aqueous solutions than the MFP for their composite samples as
FMZ. In contrast, three AC samples manifested lower MFP for their aqueous solutions
than for their composite samples as DZN. The majority of ACs tends to manifest lower
MFP after the biodegradation test, but some ACs have been observed as manifesting
higher MFP after the biodegradation test. This means that it is not possible to adopt the
simplified measuring method for the MFP of ACs. Instead, it is necessary to subject
every targeted AC to the biodegradation test and measure MFP for each composite
sample attained.
The study result that some ACs manifest higher MFP after the biodegradation test also
implied that some ACs form strong mutagens from their decomposition products. In
order to confirm the implication, the correlation between MFP change Rb [-] and DOC

removal before and after the biodegradation test was studied for the 9 ACs with
composite samples manifesting statistically significant MFP before and after the
biodegradation test, and showing no DOC increase after the biodegradation test. Rb [-]
was attained by equation (4).
Rb = Md / Ma

(4)

where Md the MFP [net rev./mg-AC] of the composite sample and Ma the MFP [net
rev./mg-AC] of the ACs.
The study results, as shown in Fig. 9, indicated that some ACs displayed a large Rb
value despite the fact that the DOC removal was high and significant mineralization was
observed. This meant that the MFP increase caused by the chlorination of
biodegradation products was larger than the MFP decrease caused by the biodegradation
of the ACs. For example, diazinon etc. manifested high Rb despite their high DOC
removals. Therefore, their decompositions are thought to be precursors of strong
mutagens.

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1800
1600
1400

Response [rev./plate]

Response [rev./plate]

Journal of Water and Environment Technology, Vol. 6, No.1, 2008

FMZ

1200
1000
800
600
400
200
0
0

0.5
Dose [mg/plate]

1

500
450
400
350
300
250
200
150
100
50
0

DZN

0

0.5
Dose [mg/plate]

1

Fig. 8 - Changes of MFP by biodegradation: ○ mutagenicity of composite sample; ●
MFP of composite sample; ▲ MFP of AC.
7
6

Rb [-]

5
4
3
2
1
0
0

20

40

60

DOC removal [%]

Fig. 9 - Relationship between DOC removal and MFP change.
CONCLUSIONS
Composite samples of 18 ACs were prepared for mutagenicity and MFP measurement.
The results did not show that the ACs tested in this study formed mutagens as a result of
undergoing biodegradation tests. Meanwhile, statistically significant MFP were detected
for 12 out of the 18 composite samples used for the test. However, as in the case of the
mutagen detected in the composite sample made out of buprofezin, it was impossible to
identify whether it originated from buprofezin itself or from the activated sludge. The
ACs from which statistically significant MFP was detected were all aromatic
compounds, except for iminoctadine-triacetate. Specific activities for thiram and DDVP,
which were reported to be mutagenic, were calculated, showing the respective values of
320 net rev./mg and 190 net rev./mg. Compared with these values, the MFP for the
composite samples of FMZ, PPT, BTZ, BST, and NPP was higher.
Some ACs manifested higher MFP after the biodegradation test despite the fact that
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Journal of Water and Environment Technology, Vol. 6, No.1, 2008

their DOC removals were quite large and mineralization was observed. These results
indicated that the MFP increase caused by the chlorination of biodegradation products
was larger than the MFP decrease caused by the decomposition of ACs, and accordingly
revealed that strong mutagen precursors are formed through the biodegradation test.
As shown above, it has been implied that some agricultural chemicals manifest
statistically significant mutagenicity when their decompositions, produced in a water
environment, intrude into raw waters and go through the chlorination process at water
purification plants. Therefore, the chemical structures of the formed mutagen precursors
need to be identified, and the presence of these substances in raw waters needs to be
confirmed. If the presence is confirmed, it is considered necessary to conduct MFP

measurement for composite samples under Japan’s Agricultural Chemicals Regulation
Law.
The possibility of a simplified measuring method, by eliminating the biodegradation test,
was also studied. However, the study results clarified that even though a majority of
ACs decrease their MFP values after the biodegradation test, some ACs increase their
MFP values, indicating that the simplified test method is not available. Therefore, every
AC concerned needs to be subjected to the biodegradation test and the MFP must be
measured for each composite sample attained.
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