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

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

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

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

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

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

Fenitrothion insecticide 2004.0 14 (30℃) N

Pretilachlor herbicide 334.4 50 (20℃)

Tricyclazole fungicide 296.6 1,600 (25℃)

-Pyroquilon fungicide 275.7 4,000 (20℃)

-Isoxathion insecticide 215.6 1.9 (25℃)

-Iminoctadine-triaetate fungicide 212.4 -

-Buprofezin insecticide 171.2 0.9 (25℃)

-Pyridiphenthion insecticide 74.1 100 (20℃)

-N ; Mutagenicity of the sample before the chlorination is reported to be negative in CCRIS.

- ; No imformation is availale.

a ; TA1535 strain

Commercial name Purpose Usage

[t, kL]

Dataum in CCRIS

Aqueous solubility [mg/L]

Abbreviation

FNT BTZ PTL DZN TCZ PQL IXT ICT BPF BST PPT PAP FMZ MIP IBP NPP PPZ IPP

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

Fig 1 - Chemical structures of agricultural chemical

O

H N N

SO 2 CH(CH 3 ) 2 O

H N N

SO 2 CH(CH 3 ) 2

CH 2 CH 2 O(CH 2 ) 2 CH 3

COCH 2 Cl

CH 2 CH 3 N

CH 2 CH 3

CH 2 CH 2 O(CH 2 ) 2 CH 3

COCH 2 Cl

CH 2 CH 3 N

CH 2 CH 3 OP(OCH 3 ) 2

H 3 C

O 2 N

S

DZN

CH 3 N

S N N

CH 3 N

S N N

TCZ

PQL

IXT

NH 2 CN(CH 2 ) 8 NH 2 (CH 2 ) 8 NHCNH 2

+ ・3CH 3 COO－

NH 2 CN(CH 2 ) 8 NH 2 (CH 2 ) 8 NHCNH 2

NH + 2

NH + 2 NH NH + + 2 2

+ ・3CH 3 COO－

NC(CH 3 ) 2

S

N

O CH(CH 3 ) 2

N

NC(CH 3 ) 2

S

N

O CH(CH 3 ) 2

N

ICT

BPF

SO 2 S

SO 2 S

CH 2

CH 2

CH N(CH 3 ) 2

SO 2 S

SO 2 S

CH 2

CH 2

CH N(CH 3 ) 2

N

N

CH 3

(CH 3 ) 2 CH

OP(OCH 2 CH 3 ) 2 O

N

N

CH 3

(CH 3 ) 2 CH

OP(OCH 2 CH 3 ) 2 O

OP(OCH 2 CH 3 ) 2

N O

S OP(OCH 2 CH 3 ) 2

N O

S

O O P(OCH 2 CH 3 ) 2

S

O O P(OCH 2 CH 3 ) 2

S

CH 2 SP[OCH(CH 3 ) 2 ] 2

O

CH 2 SP[OCH(CH 3 ) 2 ] 2 O

CHSP(OCH 3 ) 2

S

CO 2 CH 2 CH 3

CHSP(OCH 3 ) 2

S

CO 2 CH 2 CH 3

CH 3

N N H

CH 3

N N

CH 3

CH 3

CH 3

N N H

CH 3

N N

CH 3

CH 3

CONH

Cl

Cl

CH 3

CH 3

CH CONH

Cl

Cl

Cl

Cl

CH 3

CH 3

CH

O

CH 3 CHCON(CH 2 CH 3 ) 2 O

CH 3 CHCON(CH 2 CH 3 ) 2

(CH 3 ) 2 CHOCO

OPOCH 2 CH 3 S

NHCH(CH 3 ) 2

(CH 3 ) 2 CHOCO

OPOCH 2 CH 3 S

NHCH(CH 3 ) 2

CH 3 NHCO 2

CH(CH 3 ) 2

CH 3 NHCO 2

CH(CH 3 ) 2

Fig 2 - Preparation procedure of composite sample

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

Add chlorine of the water sample volume × (3 ~ 4) TOC using ca 5,000 mg-Cl/L NaClO while agitating the sample

is necessary

Leave standing for 24 ± 2 hours

in 10 ~ 30 degree C

Adjust pH of the water sample to 7.0 ± 0.2

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

Filtrate the test solutions with paper filter

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)

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]

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)

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

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

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

0 50 100

150

200

250

300

350

Dose [L/plate]

0 50 100 150 200 250 300 350

Dose [L/plate]

-40 -20 0 20 40 60 80 100

Day [d]

BPF

0

10

20

30

40

50

60

70

80

90

100

Day [d]

PAP

-10 0 10 20 30 40 50 60 70 80 90 100

Day [d]

NPP

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

Table 2 - Summary of the test results

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

Mutagenicity MFP

Iminoctadine-triacetate ICT N.D N.D 75

N D ; Not detected.

Biodegradation [%]

Commercial name Specific activity of

AC- decomposition [net rev./mg]

Abbreviation