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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
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 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: takanashi@be.kagoshima-u.ac.jp Received February 6, 2008, Accepted February 25, 2008 - 19 - 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 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 - 20 - 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 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 - 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 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 - 21 - 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 mL of ethanol and 400 µL of the solution was then added to 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 (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 - 22 - 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 - Chemical structures of agricultural chemical - 23 - 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 - 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 ± hours in 10 ~ 30 degree C Fig - 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 - 24 - 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 mL eluent after water is displaced by DMSO Sterilize by filtration Evaluate by the Ames Salmonella mutagenicity assay (preincubation method) Fig - 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 However, Fig 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) 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] - 25 - 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 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 300 Run 300 Response [rev./plate] Response [rev./plate] 350 250 200 150 100 50 250 200 150 100 50 0 0.05 Dose [L/plate] 0.1 0.05 Dose [L/plate] 0.1 PAP 14 21 Day [d] 28 100 90 80 70 60 50 40 30 20 10 -10 100 80 Biodegradation [%] 100 90 80 70 60 50 40 30 20 10 Biodegradation [%] Biodegradation [%] Fig - Dose-response lines of blank test: ○ mutagenicity; ● MFP NPP 60 40 BPF 20 -20 -40 14 21 Day [d] 28 14 21 Day [d] Fig - Examples of biodegradation test results on aniline and ACs: ○ aniline; ● ACs - 26 - 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 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 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 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 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 - 27 - Journal of Water and Environment Technology, Vol 6, No.1, 2008 Table - 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 53 19 -33 52 56 100 75 41 -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 - 28 - 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 0.5 Dose[mg/plate] 300 200 800 600 400 NPP 1200 1000 800 600 400 0.5 Dose[mg/plate] 0.5 Dose[mg/plate] Response [rev./plate] PTL 400 300 200 100 0.5 Dose [mg/plate] 500 BPF 400 300 200 600 400 300 200 0.5 Dose [mg/plate] 0.5 Dose [mg/plate] 1 FNT 400 300 200 0.5 Dose [mg/plate] 0.5 Dose [mg/plate] 600 500 IBP 400 300 200 500 IPP 400 300 200 100 0 500 100 100 0.5 Dose [mg/plate] 0 Response [rev./plate] Response [rev./plate] DZN 200 100 600 500 300 600 100 MIP 400 600 500 500 Response [rev./plate] 600 0 0.5 Dose [mg/plate] 100 0 400 200 200 600 0.5 Response [rev./plate] Response[rev./plate] 1000 800 600 1400 BST 1200 1000 0.25 Dose [mg/plate] 1600 1400 BTZ 1200 200 1600 Response[rev./plate] 400 0 Response [rev./plate] 1400 PPT 100 200 Response [rev./plate] 500 Response [rev./plate] Response [rev./plate] Response[rev./plate] FMZ 1200 1600 600 1400 0.5 Dose [mg/plate] 0 Fig - Dose-response lines of MFP-positive sample: ○ mutagenicity; ● MFP - 29 - 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 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 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 - 30 - 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 0.5 Dose [mg/plate] 500 450 400 350 300 250 200 150 100 50 DZN 0.5 Dose [mg/plate] Fig - Changes of MFP by biodegradation: ○ mutagenicity of composite sample; ● MFP of composite sample; ▲ MFP of AC Rb [-] 0 20 40 60 DOC removal [%] Fig - 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 - 31 - 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 REFERENCES Arai T., Kamata M., Shimazaki D., Asami M and Aizawa T (2005) Degradation of Pesticides by Chlorination According to Their Basic Structures J Jap Soc Water Environ., 28(7), 437-444 (in Japanese) Chemicals Inspection & testing institute, Japan (1992) Date of existing chemicals based on the cscl Japan, Japan chemical industry ecology-toxicology & information center, Tokyo, Japan (in Japanese) Guidelines for drinking-water quality, 2nd edition, Volume (1999) Japan Water Works Association, Tokyo, JAPAN (in Japanese) Kamoshita M., Kosaka K., Asami M and Aizawa T (2007) Reactivities of Organophosphorus Pesticides in Relation to Their Chemical Classifications and Their Transformation to Oxons by Chlorination J Jap Soc Water Environ., 30(3), 145-154 (in Japanese) Komatsu T., Manabe Y., Himeno S., Harada A., Murakami M and Furumai H (2007) Distribution of Mutagen Formation Potential in Nationwide River Waters in Japan and Relationship with General Water Quality or River Basin Characteristics J Jap Soc Wat Environ, 30(8), 433-440 (in Japanese) Matsushita T., Matsui Y., Sakuma S and Inoue T (2005) Fate of mutagenicity produced during anaerobic biodegradation of the herbicide chlornitrofen (CNP) J Environ Sci Health Part B-Pestic Contam Agric Wastes, 40(6), 851-861 Matsushita T., Matsui Y., Taniwaki S and Inoue T (2002) Changes in mutagenicity during biodegradation of fenitrothion Chemosphere, 47(1), 9-14 Ministry of Agriculture, Forestry and Fisheries of Japan, Consumption and Safety Bureau, Agricultural Protection Safety Control Division, Plant Protection Division (2002) Annual Inventory of Registered Pesticides and Their Use 2002, Japan Plant Protect Association, Tokyo, Japan (in Japanese) - 32 - Journal of Water and Environment Technology, Vol 6, No.1, 2008 Ministry of Health, Labour and Welfare of Japan (2003) Summary and Individual Agricultural Chemicals Date Base for Revision of Water Quality Standard (in Japanese) Ministry of Labour Japan, Mutagenicity assay for the occupational safety and health act (1991) Test guideline and GLP, Occupational Health Service Center, Tokyo, Japan (in Japanese) Nishimura T, Tahara M., Kubota R., Shimizu K., Magara Y and Tokunaga H (2007) Behavior of Organophosphorus Pesticides after Chlorination Treatment and Effect of Its Products on Cholinesterase Activity Pro 2nd International Water Association-Asia Pacific Regional Group Conference & Exihibition, 009 NLM － National Library of Medicine (2006) Chemical Carcinogenesis Research Information System Onodera S., Maeda S and Saitoh T (1995) Mutagenicity and Mass Spectrometric Characterization of Oxygen Analogs (P=O) derivatized from Organothiophosphorus Pesticides J Environ Chem., 5(3), 617-624 (in Japanese) Peschka M., Pstrovic M., Knepper T P and Barcelo D (2007) Determination of two phototransformation products of bentazone using quadrupole time-of-flight mass spectrometry Anal Bioanal Chem., 388(5/6) 1227-1234 Setsuda S., Itoh S and Naito S (1992) Decomposition of Thiram by Chlorination Bull Kanagawa P H Lab., 22, 42-44 (in Japanese) Takahashi Y., Onodera S., Morita M and Terao Y (2003) Detected Pesticides and their Concentrations in Raw Water and Tap Water Samples J Environ Chem., 13(1), 89-101 (in Japanese) Takanashi H., Kishida M., Hidaka Y., Kofune H., Ohyama K., Nakajima T and Ohki A (2007) Mutagenicity of chlorinated agricultural chemicals Pro 2nd International Water Association-Asia Pacific Regional Group Conference & Exihibition, P023 Takanashi H., Urano K (1998) Statistical procedures for estimating the detection limit and determination limit of the Ames Salmonella mutagenicity assay Sci Total Environ., 221(1), 31-42 Takanashi H., Urano K., Hirata M., Hano T and Ohgaki S (2001) Method for measuring mutagen formation potential (MFP) on chlorination as a new water quality index Water Res., 35(7), 1627-1634 Urano K., Igarashi I and Takanashi H (1997) Guidance Manual of the Ames Mutagnenicity Assay for Water Samples (I) Easy and High Performance Method for Concentration of Mutagens J Water and Waste, 39(2), 163-168 (in Japanese) Urano K., Okabe F., Takanashi H and Fujie K (1995) Ames Mutagenicity of Tap Water III Analysis of Mutagenicity Level of Tap Water in Japan J Jap Soc Water Environ., 18(12), 1001-1011 (in Japanese) - 33 - ... 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. .. 150 100 50 DZN 0.5 Dose [mg/plate] Fig - Changes of MFP by biodegradation: ○ mutagenicity of composite sample; ● MFP of composite sample; ▲ MFP of AC Rb [-] 0 20 40 60 DOC removal [%] Fig - Relationship... 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 mL of ethanol and 400 µL of