Genetic Toxicology ELSEVIER
Mutation Research 343 (1995) 109-119
Stability of 1-nitropyrene and 1,6-dinitropyrene in environmental water samples and soil suspensions Isao Tahara, Keiko Kataoka, Takemi Kinouchi, Yoshinari Ohnishi
*
Department of Bacteriology, School of Medicine, The University of Tokushima, Tokushima 770, Japan Received 10 May 1994; revised received 8 October 1994; accepted 7 January 1995
Abstract
This study examined the stability of mutagenic 1-nitropyrene (1-NP) and 1,6-dinitropyrene (1,6-diNP) in environmental water samples and various soil suspensions containing 0.1% peptone and in water samples containing no peptone. The water samples or the soil suspensions were mixed with NPs and incubated at 30°C. The stability of NPs was expressed as mutagenic activity remaining in the test solutions. The mutagenicity decreased rapidly when 1-NP or 1,6-diNP was incubated in unautoclaved test solutions containing 0.1% peptone but not when incubated in autoclaved test solutions. The mutagenicity in the soil suspensions, especially in the sludge, decreased faster than in the water samples. This was due to the large number of colony-forming units (CFU) in the soil suspensions. In the water samples containing 0.1% peptone, the mutagenicity of NPs in the polluted Tamiya River water decreased faster than in the unpolluted Yoshino River water. The rate of decrease was dependent on the number of CFU in the water samples. A large number of CFU decreased the mutagenicity more rapidly than did a small number of CFU in samples. The disappearance of mutagenicity was dependent on the initial concentrations of NPs. The periods required for a 50% decrease in the mutagenicity of 1-NP at the low concentration ( 0 . 2 / z g / m l ) was shorter than that at the high concentration (3 /~g/ml). 1-Aminopyrene was detected in the 1-NP test solution after incubation when it was analyzed by high-pressure liquid chromatography. In the water samples containing no peptone, the mutagenicity of 1-NP (0.2 /zg/ml) decreased gradually during 30 days of incubation. After incubation for 1540 days, the remaining mutagenicity of 1-NP in the water samples was almost the same as that in autoclaved water samples. On the other hand, the mutagenicity of 1,6-diNP (10 n g / m l ) decreased and the remaining mutagenicity, except in the Yoshino River water, was less than 20% after 30 days of incubation and was completely lost during the 1540-day incubation. However, the mutagenicity of 1,6-diNP in autoclaved water samples was very stable and almost all mutagenicity, except in sea water, remained after 1540 days of incubation at 30°C. These results suggest that the microflora in the environment plays an important role in the primary degradation and decontamination of relatively low concentrations of NPs. Keywords: Biodegradation; 1-Nitropyrene; 1,6-Dinitropyrene; Mutagenicity; Soil; Aquatic environment
* Corresponding author. Tel.: 81-886-33-7069; Fax: 81-88633-0771; e-mail:
[email protected]. 0165-1218/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-1218(95)00013-5
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1. Introduction
Nitropolycyclic aromatic hydrocarbons (nitroPAHs) are a widely distributed class of environmental contaminants, and most of them are produced as a result of various incomplete combustion processes (Tokiwa and Ohnishi, 1986; Tokiwa et al., 1993). Nitropyrenes (NPs) are some of the most common nitro-PAHs and are found in the organic extracts of diesel engine exhaust (Manabe et al., 1985; Tahara et al., 1994), kerosene heater exhaust (Kinouchi et al., 1988), wastewater of oil-water separating tanks of gasoline stations (Manabe et al. 1984), used crank-case oil (Manabe et al., 1984) and yakitori (Kinouchi et al., 1986). Their widespread occurrence is not surprising, because NPs are readily formed not only by incomplete combustion but also by exposure of pyrenes to nitrogen dioxide in vitro and in uivo (Tokiwa et al., 1981; Tokiwa and Ohnishi, 1986; Ohnishi et al., 1990). NPs are direct-acting, frame-shift type mutagens in bacteria and mammalian cells and carcinogens in experimental animals (Tokiwa and Ohnishi, 1986). NPs are metabolized via oxidative and reductive pathways to form intermediates which bind to DNA and are genotoxic (Tokiwa and Ohnishi, 1986; Ohnishi et al., 1991). These studies are important in predicting risk for directly exposed persons. However, nitro-PAHs are not necessarily stable in the environment because they undergo photochemical and chemical reactions or microbial metabolism in the environment. Therefore, biological instability, which causes a decrease of mutagenicity of NPs in the environment, is an important factor for assessment of risk of NPs to humans. 1-Nitropyrene is photodecomposed in the atmospheric environment and changes to less or non-mutagenic compounds, such as hydroxypyrene, pyrene quinone and dihydroxypyrene (Benson et al., 1985; Stark et al., 1985). 1-NP is also biotransformed to non-mutagenic compounds, glycoside conjugates of 6-hydroxy-l-NP and 8-hydroxy-l-NP by Cunninghamella elegans, a soil microorganism (Cerniglia et al., 1985). However, there is little information on the stability and biotransformation of NPs in the aquatic and soil environment.
This study was an attempt to find out how microbial communities in the aquatic environment and in the soil are responsible for the degradation of 1-NP and 1,6-diNP under controlled laboratory conditions.
2. Materials and methods 2.1. Chemicals
[3H]I-NP (CAS No. 5522-43-0, specific activity 100 mCi/mmol) and [3H]I,6-diNP (CAS No. 42397-64-8, specific activity 183.7 mCi/mmol) were produced by 3H-exchange and obtained from Amersham Radiochemical Centre, Buckinghamshire, UK. 1-NP and 1-aminopyrene (1-AP; CAS No. 1606-67-3) were purchased from Aldrich Chemical, Milwaukee, WI, USA. [3H]I-NP, [3H]I,6-diNP, 1-NP and 1,6-diNP were purified as previously described (Kinouchi et al., 1982, 1983). The purity of 1-NP and 1,6-diNP was 99.8% and 97.6% (contaminated with 1.5% 1-NP), respectively. For test chemicals, 1-NP or 1,6-diNP was mixed with [3H]I-NP or [3H]I,6-diNP, respectively. Other standard compounds were synthesized as described previously (Manabe et al., 1985). Peptone (Poylpeptone) and GAM medium were obtained from Nihon Pharmaceutical, Tokyo and Nissui Pharmaceutical, Tokyo, Japan, respectively. All other chemicals were reagent grade or higher and were purchased from Wako Pure Chemical Industries, Osaka, Japan. 2.2. E n v i r o n m e n t a l water and soil samples
Two river water samples (the Yoshino River and the Tamiya River in Tokushima City), one river sludge sample (the Tamiya River), one sea water sample (Tsuda Harbor in Tokushima City, the Kii Channel) and one pond water sample (Jizoin Temple, Tokushima City) were collected and used in this study. The soil samples were collected from the playing field of the School of Medicine, The University of Tokushima (red soil), in the mountain woods of Shiroyama (black soil), in Kuramoto Park (sandy soil). The samples (100-150 g) of the various soils and the river sludge were mixed with five volumes (500-750
L Tahara et aL/Mutation Research 343 (1995) 109-119
ml) of sterilized water and then the suspension was filtered through filter paper (Toyo filter paper No. 2). The filtrates were used in these experiments.
2.3. Measurement of degradation The procedure of the degradation test was a slight modification of the method of Kondo et al. (1988). The procedure was as followes (Fig. 1): aliquots of the water samples or the filtrates of soil suspensions (2.5 ml) were mixed with equal volumes of 0.2% autoclaved peptone (Polypeptone, Nihon Pharmaceutical). The sea water sample alone was further mixed with 3% sodium chloride. The mixtures (5 ml) or 5-ml quantities of the water samples containing no peptone were used as test solutions. To elucidate the role of
Samples, 2.5 ml (Water and filtrate of soil suspension)
\
0.2% Peptone solution, 2.5 ml (pH7.0) /
J
Incubated at 30°(; Extracted with benzene-ethanol (4:1), 5 ml (3 times)
I
I Water layer
Benzene-ethanol layer
Evaporated Residue Dissolved in DMSO
/
Mutagenicity test
microorganisms in biodegradation of NPs we used the untreated and autoclaved (no microorganisms) samples and also added peptone to increase growth of the microorganisms and obtain the results rapidly. The test solutions were mixed with 50/zl of [3H]I-NP or [3H]I,6-diNP dissolved in dimethylsulfoxide (DMSO). The final concentrations of [3H]I-NP in the test solutions were 3 /~g/ml (specific activity, 255.6 ~Ci/mmol), 1 /~g/ml (specific activity, 766.8 /~Ci/mmol) and 0.2 /~g/ml (specific activity, 511.2 /~Ci/mmol). The final concentration of [3H]I,6-diNP was 10 ng/ml (specific activity, 29.7 mCi/mmol). The test solutions were incubated at 30°C at selected times and then extracted with an equal volume of a mixture of benzene/ethanol (4 : 1, V / V ) three times. The benzene/ethanol layers were combined and evaporated. The residue was dissolved in DMSO for chemical analysis by high-pressure liquid chromatography (HPLC) and the Salmonella mutagenicity preincubation assay (Maron and Ames, 1983; Yahagi et al., 1975).
2.4. Mutagenicity assay
Mixture (Sml) t-NP (final concentration : ~ug/ml, f ljJg/ml, 0.21ug/ml)or 1,6-diNP (lOng/ml) in DMSO 50 ~JI
I
111
\
Mutagenicity was measured with Salmonella typhimurium strain TA98, kindly supplied from Dr. B. N. Ames, in the absence of $9 mix. The mutagenicity test was performed as preincubation test (Maron and Ames, 1983; Yahagi et al., 1975) with a preincubation time of 20 min at 37°C. 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2, CAS No. 3688-53-7, 0.1 /~g/plate) was used as positive control. We used two plates for each sample from each reaction mixture and the values were shown as mean values. The number of spontaneous revertants/plate and 0.1 /~g AF-2-induced revertants/plate were 44.4 + 9.3 and 436.6 _+ 60.6, respectively.
HPLC analysis
Fig. 1. Procedure of the degradation test of 1-NP and 1,6-diNP by the water samples and the filtrate of soil-suspensions containing 0.1% peptone. Various samples were mixed with 1-NP (final concentration: 3 p,g/ml, 1/xg/ml and 0.2/~g/ml) or 1,6-diNP (10 ng/ml) and incubated at 30°C. At the selected time the mixtures were extracted with organic solvents. Mutagenicity of the extracts was assayed for Salmonella typhimurium strain TA98 in the absence of $9 mix. Reaction products were also measured by HPLC.
2.5. High-pressure liquid chromatography HPLC was conducted with a Shimadzu LC-5A equipped with a Shimadzu SPD-2A variablewavelength UV detector. The column temperature was maintained at 50°C. At times, a Hewlett-Packard HP 1040A high-speed spectrophotometric detector was used to obtain on-
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L Tahara et al. / Mutation Research 343 (1995) 109-119
line spectra of metabolites. Metabolites in the DMSO solution were separated with a reverse phase column, Chemcosorb 7-ODS-H (4.6 mm i.d. × 250 mm, Chemco Scientific, Osaka) and the metabolites were eluted at a flow rate of 1.0 m l / m i n with 70% methanol.
solution ( K H 2 P O 4 , 4 g; N a 2 H P O 4 , 6 g; Tween 80, 1 g; L-cysteine, 1 g; distilled water, 1000 ml) (Kinouchi and Ohnishi, 1986). After appropriate dilution, samples were plated on GAM agar plates (containing 3% NaC1 for the sea water) and incubated aerobically or anaerobically at 37°C for 48 h. We used two agar plates for each dilution point of each samples and values were shown as mean values. Cultures for anaerobic bacteria were incubated under an atmosphere of 80% N 2, 10% H2, and 10% CO2 in a Forma Scientific anaerobic system model 1024. GAM medium (peptone, 10 g; soya peptone, 3 g; protease peptone, 10 g; digested serum, 13.5 g; yeast extract, 5 g; beef extract, 2.2 g; liver extract, 1.2 g; dextrose, 3 g; sodium chloride, 3 g; monopotassium phosphate, 2.5 g; soluble starch, 5 g; L-cysteine-HC1, 0.3 g; sodium thioglycolate, 0.3 g; distilled water, 1,000 ml, pH 7.1) is used for anaerobic bacteria. As this medium contained the digested serum which has heroine, it is successfully used for cultivation of anaerobic bacteria and other microorganisms.
2.6. Characterization of the water samples Properties of the water samples and soil suspensions, namely pH, dissolved oxygen (DO), suspended solid (SS), chemical oxygen demand (COD) and biological oxygen demand (BOD) were determined by standard methods for hygienic chemists (Pharmaceutical Society of Japan, 1990) (Table 1). The pH of the Yoshino River water and the Tamiya River water was neutral and that of the sea water and the pond water was alkaline. The DO levels of all water samples were higher than 5 rag/1 and that in the pond water was the highest. The COD and BOD were low in the unpolluted Yoshino River water and the sea water and comparatively high in the polluted Tamiya River water and the pond water.
3. Results
2. 7. Determination of colony-forming units (CFU) of the water samples and the filtrates of various soil suspensions
The properties of the water samples and the soil suspensions are shown in Table 1. The number of CFU in the polluted Tamiya River water was higher than that in the unpolluted Yoshino
The water samples and the filtrates of various soil suspensions were diluted with an anaerobic Table 1 Properties of water samples and soil suspensions Sample
Water sample Sea water Yoshino River water Tamiya River water Pond water Filtrate of soil suspension Red soil Sandy soil Black soil Sludge of Tamiya River
pH
DO (mg/l)
SS (mg/l)
COD (mg/1)
BOD (mg/l)
Number of microorganisms recovered Aerobically
Anaerobically
CFU/ml
C F U / g of soil
CFU/ml
C F U / g of soil
1.6 x 3.7 x 3.1 x 9.2 x
N.D. N.D. N.D. N.D.
8.02 6.85 7.11 9.11
5.8 7.4 5.3 7.7
< 0.1 < 0.1 < 0.1 0.3
2.7 1.5 4.3 8,3
1.7 0.6 9.8 6.7
3.4 x 102 2.2 x 103 4.1 X 104 3.1 x 103
N.D. N.D. N.D. N.D.
6.95 6.50 6.27 6.61
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.
3.8 X 106 1.4 x 105 1.7 × 106 4.0 x 106
1.9 x 7.0 × 8.5 x 2.0 x
107 105 106 107
102 102 103 102
2.6 X 106 5.9 × 104 1.3 × 106 4.2 x 106
DO, Dissolved oxygen; SS, suspended solid; COD, chemical oxygen demand; BOD, biochemical oxygen demand. N.D., not done.
1.3 x 107 3.0 X 10 s 6.5 X 106 2.1 x 107
I. Tahara et aL /Mutation Research 343 (1995) 109-119
River water. The number of CFU in the sea water was low. The CFU in all the water samples were more numerous after aerobic incubation than that after anaerobic incubation. The mutagenicity of 1-NP and 1,6-diNP decreased in all eight unautoclaved samples containing 0.1% peptone (Figs. 2 and 3). Decrease in mutagenicity in the soil suspensions was equal to or faster than that in the water samples. However, at the end of 7 day-incubation none of the eight autoclaved samples containing 0.1% peptone showed decreased mutagenicity of 1-NP and
1,6-diNP. The periods required for a 50% decrease in mutagenicity of NPs in the water samples and the soil suspensions are shown in Table 2. In all samples except for sea water, the period for a 50% decrease in the mutagenicity of 1-NP at the
1O0
113
low concentration was shorter than that at the high concentration. The decrease in mutagenicity of 1-NP (3/~g/ml) in the Tamiya River water was the fastest among the water samples containing 0.1% peptone (Table 2). By the 3rd day more than 90% of the mutagenicity of 1-NP in the Tamiya River water was abolished, although in the pond water, the Yoshino River water and the sea water 27.8, 38.0 and 68.9%, respectively, of the mutagenicity of 1-NP remained (Fig. 2). Furthermore, the decrease in mutagenicity of NPs was faster in the water sample containing a large number of microorganisms than in the water samples containing a small number of microorganisms (Fig. 4). After incubation of the test sample and 1-NP, a major product was detected by HPLC as shown in Fig. 5A. A major peak, at a retention time of 11.63 min, was identified as 1-AP by its HPLC retention time and UV/visible spectrum. The radioactivity of the fraction corresponding to 1-AP increased from 10% of the total injected radioac-
100~ []
g
0 1 2 3 4 5 6 7 01 234567 Incubation time (days)
Fig. 2. Decrease of mutagenicity of 1-NP and 1,6-diNP in the autoclaved (open symbols) and untreated (closed symbols) water samples (A: sea water; B: Yoshino River water; C: Tamiya River water; D: pond water) containing 0.1% of peptone. © and e: 3 /zg/ml of 1-NP; zx and A: 1/xg/ml of 1-NP; v and v : 0.2 p.g/ml of 1-NP; [] and I1:10 ng/ml of 1,6-diNP. 100% mutagenicity (revertants/plate) of the untreated and autoclaved sea water was 2,704 and 2,704 for 3 /xg/ml of 1-NP, 2529 and 2352 for 1 p,g/ml of 1-NP, and 1111 and 1225 for 0.2 /~g/ml of 1-NP and 1303 and 1566 for 10 ng/ml of 1,6-diNP. 100% mutagenicity of the untreated and autoclaved Yoshino River water was 2940 and 3035 for 3 ~ g / m l of 1-NP, 2298 and 2489 for 1/zg/ml of 1-NP, and 1254 and 1198 for 0.2 p.g/ml of 1-NP and 709 and 1174 for 10 ng/ml of 1,6-diNP. 100% mutagenicity of the untreated and autoclaved Tamiya River water was 3056 and 2884 for 3 /zg/ml of 1-NP, 2478 and 2663 for 1 /xg/ml of 1-NP, and 1284 and 1427 for 0.2 /zg/ml of 1-NP and 1062 and 1503 for 10 ng/ml of 1,6-diNP. 100% mutagenicity of the untreated and autoclaved pond water was 2903 and 2613 for 3 /~g/ml of 1-NP, 2491 and 2321 for 1/zg/ml of 1-NP, and 1270 and 1223 for 0.2 p.g/ml of 1-NP and 1421 and 1301 for 10 ng/ml of 1,6-diNP. The mutagenicity was assayed at 0.75 /xg, 0.625 /xg and 0.4/zg equivalent/plate for 3/zg, 1 ~g and 0.2/zg/ml of 1-NP reaction mixture, respectively, and at 1.94 ng equivalent/plate for 1,6-diNP.
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L Tahara et al. / Mutation Research 343 (1995) 109-119
Table 2 The period (days) required for 50% decrease in mutagenicity of 1-nitropyrene and 1,6-dinitropyrene in water samples and soil suspensions containing 0.1% peptone Sample
Period for 50% decrease in mutagenicity (days) 1-NP
Water sample Sea water Yoshino River water Tamiya River water Pond water Filtrate of soil suspension Red soil Sandy soil Black soil Sludge of Tamiya River
100A~
3 p~g/ml
1 p.g/ml
0.2 p,g/ml
10 n g / m l
3.55 2.47 1.95 2.27
1.83 1.56 1.37 2.02
2.13 0.68 0.63 1.55
2.15 0.56 0.53 0.54
2.37 2.03 1.88 0.56
2.12 1.61 1.32 0.53
1.36 0.74 0.53 0.51
0.61 0.51 0.51 0.50
tivity a f t e r o n e - d a y i n c u b a t i o n t o 6 1 . 3 % a f t e r seven-day incubation of 1-NP in the Tamiya River w a t e r c o n t a i n i g 0 . 1 % p e p t o n e (Fig. 5B).
B O0
^
50
01
0 0 1 2 3 4 5 6 7 234567 Incubation time (days)
100
O01
100,
2 3 45
6 7
1,6-diNP
Oq
234567
Incubation time (days)
6
Fig. 3. Decrease of mutagenicity of 1-NP and 1,6-diNP in the autoclaved (open symbols) and untreated (closed symbols) filtrates of soil-suspensions (A: Red soil; B: Sand soil; C: Black soil; D: Sludge of the Tamiya River) containing 0.1% of peptone. © and o: 3 ~ g / m l of 1-NP; ,x and A: 1 ~ g / m l of 1-NP; v and • : 0.2/zg/ml of 1-NP; [] and • : 10 n g / m l of 1,6-diNP. 100% mutagenicity (revertants/plate) of the untreated and autoclaved filtrate of Red soil was 2817 and 3094 for 3 /zg/ml of 1-NP, 2492 and 2525 for 1 /zg/ml of 1-NP, and 1610 and 1,306 for 0.2/zg/ml of 1-NP and 1215 and 1324 for 10 n g / m l of 1,6-diNP. 100% mutagenicity of the untreated and autoclaved filtrate of sand soil was 2826 and 2943 for 3 p.g/ml of 1-NP, 2437 and 2450 for 1/~g/ml of 1-NP, and 1388 and 1217 for 0.2 /~g/ml of 1-NP and 1216 and 1169 for 10 ng/ml of 1,6-diNP. 100% mutagenicity of the untreated and autoclaved filtrate of Black soil was 2927 and 2459 for 3 p,g/ml of 1-NP, 2314 and 2343 for 1 t~g/ml of 1-NP, and 1464 and 1352 for 0.2 /zg/ml of 1-NP and 1298 and 1261 for 10 n g / m l of 1,6-diNP. 100% mutagenicity of the untreated and autoclaved the filtrate of sludge was 2829 and 2970 for 3 /xg/ml of 1-NP, 2141 and 2666 for 1/zg/ml of 1-NP, and 1177 and 1167 for 0.2 /xg/ml of 1-NP and 1,376 and 1101 for 10 n g / m l of 1,6-diNP. The mutagenicity was assayed at 0.75/zg, 0.625/xg and 0.4 p.g equivalent/plate for 3/zg, 1 /zg and 0.2 /~g/ml of 1-NP reaction mixture, respectively, and at 1.94 ng equivalent/plate for 1,6-diNP.
L Tahara et al./Mutation Research 343 (1995) 109-119
4
o
Yoshino River water / I Pond water
Sea water /
¢0
115
Tamiya River water
"0
I~Np 3/Jg/rnl
.N
0
I
I
I
I
I
I
I I I
102
I
I
I
I
I
I
I I I
103
I
I
I
I
104 CFU/ml
Fig. 4. Relationship b e t w e e n the number of colony-forming units of aerobic microorganisms/ml and the period required for 50% decrease in mutagenicity of NPs in the w a t e r samples containing 0.1% peptone.
Table 3 shows the decrease in mutagenicity of NPs in the untreated water samples containing no peptone during incubation at 30°C. The decrease in mutagenicity of 1,6-diNP (10 ng/ml)
was much faster than that of 1-NP (0.2 /zg/ml). After 30 days, the percentages (spontaneous revertants were subtracted) of mutagenicity of 1-NP and 1,6-diNP remaining in the sea water, the
25
o. "o v
"7,
t0oo
E
o
J
E
,,1l
c 0
N
10
0 25
--_
2'0 -
i
_
-
- !
30
4O
B
Q
0 o. <
2000 .~ "7,
Ill
,ooo o
/
0
10
20
30
0
tr
40
Retention time (rain)
Fig. 5. HPLC patterns of 1-NP metabolites in the Tamiya River water containing 0.1% peptone after one-day (A) and seven-day (B) incubation. Absorbance and radioactivity were shown as solid lines and histograms, respectively.
116
I. Tahara et al. /Mutation Research 343 (1995) 109-119
Table 3 Remaining mutagenicity of 1-nitropyrene and 1,6-dinitropyrene in various water samples containing no peptone Sample
Incubation time (days)
Revertants/plate (Remaining mutagenicity, %) 1-NP (0.2/xg/ml) Untreated
1,6-diNP (10 ng/ml) Autoclaved
Untreated
Autoclaved
Sea water
0 1 3 5 10 15 20 30 1540
967 (100) 971 (100.4) 893 (92.3) 916 (94.7) 872 (90.2) 904 (93.5) 879 (90.9) 762 (78.8) 430 (44.5)
1013 (100) N.D. N.D. 1173 (115.8) 1213 (119.7) N.D. 1294 (127.7) 1183 (116.8) 534 (52.7)
2000 (100) 1147 (57.4) 236 (11.8) 736 (36.9) 367 (18.4) 268 (13.4) 219 (11.0) 315 (15.8) 59 (3.0)
1832 (100) N.D. N.D. 1755 (95.8) 1633 (89.1) N.D. 1544 (84.3) 1510 (82.4) 864(47.2)
Yoshino River water
0 1 3 5 10 15 20 30 1540
1002 (100) 1001 (99.9) 785 (78.3) 1038 (103.6) 916 (91.4) 900 (89.8) 965 (96.3) 871 (86.9) 593 (59.2)
1116 (100) N.D. N.D. 1095 (98.1) 1070 (95.9) N.D. 1242 (111.3) 1048 (93.9) 664 (59.5)
1956 (100) 1466 (74.9) 410 (21.0) 1249 (63.9) 797 (40.7) 762 (39.0) 827 (42.3) 900 (46.0) 214 (10.9)
2061 (100) N.D. N.D. 2012 (97.6) 1623 (78.7) N.D. 1797 (87.2) 2008 (97.4) 2101 (101.9)
Tamiya River water
0 1 3 5 10 15 20 30 1540
1037 (100) 1131 (109.1) 903 (87.1) 869 (83.8) 784 (75.6) 1021 (98.5) 871 (84.0) 814 (78.5) 277(26.7)
1014 (100) N.D. N.D. 1185 (116.9) 698 (68.8) N.D. 1204 (118.7) 1173 (115.7) 762(75.1)
1840 (100) 1115 (60.6) 76 (4.1) 431 (23.4) 310 (16.8) 344 (18.7) 205 (11.1) 212 (11.5) 55 (3.0)
1527 (100) N.D. N.D. 1385 (90.7) 1286 (84.2) N.D. 1601(104.8) 1652 (108.7) 1506 (98.6)
Pond water
0 1 3 5 10 15 20 30 1540
1039 (100) 1117 (107.5) 770 (74.1) 919 (88.5) 896 (86.2) 860 (82.8) 780 (75.1) 609(58.6) 118 (11.4)
1074 (100) N.D. N.D. 1090(101.5) 1167 (108.7) N.D. 1275 (118.7) 1266 (117.9) 318 (26.9)
1856 (100) 778 (41.9) 113 (6.1) 354 (19.1) 214 (11.5) 188 (10.1) 161 (8.7) 169 (9.1) 52 (2.8)
1578 (100) N.D. N.D. 1722(109.1) 1527 (96.8) N.D. 1578 (100) 1372 (86.9) 1684(106.7)
N.D., Not done. Spontaneous revertants and AF-2 (0.1 ~zg/plate)-induced revertants were 45 _+9 and 499 _+41, respectively. The numbers of spontaneous revertants were not subtracted from all the mutagenicity values. The mutagenicity was assayed at 0.4/xg equivalent/plate for 1-NP and 1.94 ng equivalent/plate for 1,6-diNP.
Yoshino River water, the Tamiya River water a n d t h e p o n d w a t e r w e r e 77.6 a n d 14.0, 86.2 a n d 44.9, 77.4 a n d 9.5, a n d 56.5 a n d 7.1, r e s p e c t i v e l y . The mutagenicity of both 1-NP and 1,6-diNP r e m a i n e d at m o r e t h a n 8 0 % in t h e a u t o c l a v e d w a t e r s a m p l e s a f t e r 30 days o f i n c u b a t i o n .
W e m e a s u r e d t h e m u t a g e n i c i t y in t h e unt r e a t e d w a t e r s a m p l e s c o n t a i n i n g n o p e p t o n e aft e r 1540 days o f i n c u b a t i o n ( T a b l e 3). T h e m u t a g e n i c i t y o f 1 , 6 - d i N P was c o m p l e t e l y a b o l i s h e d in t h e w a t e r s a m p l e s a f t e r 1540 days. T h e m u t a g e n i c i t y o f 1 - N P a n d 1 , 6 - d i N P in t h e a u t o c l a v e d
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water samples was also measured (Table 3). 1,6diNP was very stable and almost all mutagenicity was retained in the autoclaved water samples except for the sea water.
4. Discussion
Microbial degradation of organic chemicals is generally recognized to be an important removal mechanism in natural environments (Colwell and Walker, 1977; Higson, 1992; Jones and Alexander, 1986; Leahy and Colwell, 1990). Biodegradability is often estimated by screening tests in the laboratory. A simple and rapid biodegradation test method was developed by Kondo et al. (1988) for the screening of chemicals that are unable to be degraded by microorganisms in the natural water. We slightly modified this method for measuring the remaining mutagenicity of 1-NP and 1,6-diNP in the test solutions after incubation at 30°C. We also used a mixture of benzene/ethanol (4:1) for extraction of NPs from the test solutions because benzeneethanol is the most effective solvent for extraction of NPs from diesel engine particulates (Tahara et al., 1994) and from airborne particulates (Goto et al., 1981) The number of CFU in the water samples after aerobic incubation was higher than that after anaerobic incubation, suggesting that the microflora of these water samples is dominated by obligate aerobic bacteria such as P s e u d o m o n a s as reported previously (Nasu et al., 1993a,b). The DO values in the pond water was the highest in the all water samples. This may be due to the production of oxygen by the photosynthetic reaction in green algae growing in the pond. The mutagenicity of 1-NP and 1,6-diNP decreased in all samples containing 0.1% peptone, but not in all autoclaved samples containing 0.1% peptone (Figs. 2 and 3). This finding suggests that the microflora in the water samples and the soil suspensions played an important role in degradation of 1-NP and 1,6-diNP. The decrease in mutagenicity of NPs was faster in the water sample containing a large number of microorganisms than in the water samples containing a small number
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of microorganisms (Fig. 4), suggesting that the rate of decrease in the mutagenicity of NPs was dependent on the number of microorganisms in the water samples (Fig. 4). The 1-AP in the reaction mixture was also increased (Fig. 5B). These results suggest that the decrease in mutagenicity was due to the enzymatic reduction of 1-NP to 1-AP by the microflora of the water samples and the soil suspensions. This is due to the biotransformation of nitro groups of NPs to less mutagenic amino-derivatives (Kinouchi et al., 1982; Fifer et al., 1986; Howard et al., 1987) by microbial nitroreductase(s). Almost all of the reactions involved in biodegradation can be classified as oxidative, reductive, hydrolytic or conjugative (Scow, 1981). The decrease in mutagenicity of NPs in the water samples containing no peptone may also be due to nitroreduction of NPs by microorganisms in the water samples. The mutagenicity of 1,6-diNP in the Tamiya River water decreased faster than in the Yoshino River water because of its large number of CFU. In the pond water and the sea water, the mutagenicity of 1,6-diNP also decreased faster than in the Yoshino River water. This may be due to the high content of suspended solids (Table 1) a n d / o r the presence of different kinds of microorganisms that degrade diNP. However, in the autoclaved water samples, the decrease in mutagenicity of 1-NP and 1,6-diNP was different. Although the concentration of 1,6diNP was lower than that of 1-NP, the mutagenicity of 1,6-diNP was more stable than that of 1-NP in the autoclaved water samples (Table 3). This may be due to a difference in sensitivity to oxidation by oxygen dissolved in the autoclaved water samples. The pyrene-ring oxidative metabolites of 1-NP have been detected in the reaction mixture of 1-NP and hepatic microsomes of various animals (EI-Bayoumy and Hecht, 1983; Kataoka et al., 1991; Ohnishi et al., 1991) and by photoreaction (Benson et al., 1985; Stark et al., 1985). The sites C-3, C-6 and C-8 of the pyrene ring of 1-NP are reactive. Therefore, 1-NP is metabolized by both reductive and oxidative pathways. However, none of the oxidative metabolites of diNP are detected (Bryant et al., 1984; Tee et al., 1988)
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because the active sites are already occupied by nitro groups and cannot react with dissolved oxygen (Kinouchi et al., 1994). The importance of sediment in aquatic systems as a source of nutrients and as microhabitats for microbial populations has been indicated (Scow, 1981). In the biodegradation study of 2,4-dichlorophenoxyacetic acid in river water, the compound was degraded 50% during 40 days in the sample mixed with sediment, while only 10% was degraded during the same time in the sample without sediment. Another important property of sediment is its absorption of NPs. Only 0.15% of the radioactivity of 1-NP and 1.94% of that of 1,6-diNP was recovered just after mixing sludge and NPs (data not shown). If unchanged NPs were present in the river water they would be adsorbed to the sludge of the river and changed to compounds that are less mutagenic and possibly less hazardous to humans. These results suggest that other process(es) of degrading NPs, especially 1-NP, occur in the water samples although microorganisms play an important role in the primary degradation of NPs.
Acknowledgements This work was supported by cancer research grants-in-aid from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare of Japan.
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