Genotoxicity study of photolytically treated 2-chloropyridine aqueous solutions

Journal of Hazardous Materials 177 (2010) 892–898

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Genotoxicity study of photolytically treated 2-chloropyridine aqueous solutions Dimitris Vlastos a,∗ , Charalambos G. Skoutelis a , Ioannis T. Theodoridis a , David R. Stapleton b , Maria I. Papadaki a,b a b

Department of Environmental and Natural Resources Management, University of Ioannina, Seferi 2, Agrinio 30100, Greece Chemical Engineering, IPSE, School of Process Environmental and Materials Engineering, University of Leeds, Leeds LS2 9JT, UK

a r t i c l e

i n f o

Article history: Received 4 November 2009 Received in revised form 21 December 2009 Accepted 30 December 2009 Available online 7 January 2010 Keywords: Genotoxicity Photolysis Micronucleus assay 2-Chloropyridine mineralisation Pyridines

a b s t r a c t 2-Chloropyridine (2-CPY) has been identified as a trace organic chemical in process streams, wastewater and even drinking water. Furthermore, it appears to be formed as a secondary pollutant during the decomposition of specific insecticides. As reported in our previous work, 2-CPY was readily removed and slowly mineralised when subjected to ultraviolet (UV) irradiation at 254 nm. Moreover, 2-CPY was found to be genotoxic at 100 ␮g ml−1 but it was not genotoxic at or below 50 ␮g ml−1 . In this work 2-CPY aqueous solutions were treated by means of UV irradiation at 254 nm. 2-CPY mineralisation history under different conditions is shown. 2-CPY was found to mineralise completely upon prolonged irradiation. Identified products of 2-CPY photolytic decomposition are presented. Solution genotoxicity was tested as a function of treatment time. Aqueous solution samples, taken at different photo-treatment times were tested in cultured human lymphocytes applying the cytokinesis block micronucleus (CBMN) assay. It was found that the solution was genotoxic even when 2-CPY had been practically removed. This shows that phototreatment of 2-CPY produces genotoxic products. Upon prolonged irradiation solution genotoxicity values approached the control value. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Genotoxic substances may represent a health hazard to humans but also may affect organisms in the environment. Pyridine and pyridine derivatives (PPDs) have received great attention because of their potential environmental and health threats. They may enter the environment as a consequence of their extensive use as insecticides and herbicides in agriculture and through industrial activities associated with pharmaceutical and textile manufacture and chemical synthesis [1–3]. Widespread use of such pesticides and urban, industrial effluents can result in contamination of surface and ground waters in areas around application or discharges zones. Degradation of those compounds via naturally occurring physicochemical and/or biological processes can result in significant levels of PPDs in water environments [4,5]. 2-Chloropyridine (2-CPY), in particular has been reported to be an environmental contaminant, although it has not been reported to occur naturally. According to Arch Chemicals, Inc. [6] it is a key intermediate in the manufacture of pyrithione-based biocides for use in cosmetics and various pharmaceutical products. The Dow Chemical Co. has identified it as a trace organic chemical in pro-

∗ Corresponding author. Tel.: +30 26410 74148; fax: +30 26410 74150. E-mail address: [email protected] (D. Vlastos). 0304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2009.12.117

cess streams and wastewater [7]. It has also been identified as a Rhine River pollutant in the Netherlands and a trace organic contaminant in drinking water derived from river water in Barcelona, Spain [8,9]. Furthermore, it appears to be formed as a secondary pollutant during the decomposition of specific insecticides, such as imidacloprid. All the main imidacloprid metabolites identified in mammals, plants and insects contain the 2-CPY moiety. This moiety has an important contribution in the toxic effects that those metabolites impose on target and non-target insects [10,11]. The application of photochemical advanced oxidation processes (PAOPs) for the removal of organics is gaining importance in water treatment. Among others, ultraviolet (UV) light-induced degradation processes constitute a well-established practice in water and wastewater treatment and the interest in this area has been constantly increasing in the recent past. UV-driven advanced oxidation processes (AOPs) are primarily based on the generation of powerful oxidizing species, such as OH radicals, through the photolysis of H2 O2 or through processes such as photo-Fenton reactions and semiconductor photocatalysis [10]. UV-driven photochemical treatment and TiO2 based heterogeneous photocatalysis effectively degrade many organic pollutants such as dyes, pesticides, halogenated compounds, and surfactants, e.g. [2,3,12–15]. It is worth noting that thousands of research studies on the chemicalphotodegradation of various organics have been undertaken over the years, a great number of which result on their successful pho-

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todegradation. However, the elimination of their genotoxicity has not been equally researched. Only few investigations have been carried out with the aim to evaluate the in vitro genotoxicity in humans’ cells before and after their treatment. From an environmental point of view, there are advantages if photochemical methods for the degradation of the environmental pollutants are used. On the other hand, a serious question arises, about the toxicity and/or genotoxicity fate of the photodegradation products into the environment and their side effects to humans and other organisms. In recent years, there has been intense development of bioanalytical techniques that employ live organisms as indicators. Biotests detect the presence of toxic substances in the environment, and determine their toxicity in the samples analyzed by quantitatively estimating the harm that they cause to live organisms. Until now, many microbiotests that have been developed have employed various indicator organisms such as algae, vascular plants, crustaceans, rotifers and protozoans [16]. However, the available information on the genotoxicity of the intermediates which are generated by the different photodegradation methods on several environmental pollutants is limited. Moreover, the difficulties in the interpretation of the biotest results in terms of the extrapolation from various organisms to humans cannot be underestimated. It is therefore important to combine photochemical treatment work with genotoxicity studies on human cells. Traditionally, different genotoxic endpoints in different bioassays have been used to investigate the genotoxicity of several environmental pollutants. The cytokinesis block micronucleus (CBMN) assay, first reported by Fenech and Morley [17], has been widely used in different cell types including human lymphocytes for the evaluation of the genotoxic and cytotoxic potential of various agents [18–21]. Micronuclei (MN) contain acentric chromosome fragments or whole chromosomes and they can be recognized as distinct formations that exist in daughter cells separated from the main nucleus [18]. They are the result of chromosome breakage and/or chromosome loss due to lagging chromosomes during anaphase of mitosis. The main characteristic of the assay is the use of Cytochalasin-B, an inhibitor of actin polymerization, which prevents cytokinesis, while permitting nuclear division [17,22]. As a result, binucleated (BN) cells are produced which are scored for the presence of MN. Scoring MN only in BN cells makes the method very sensitive for the analysis of lymphocytes that have undergone one division. In our previous work [23,24] it was reported that 2-CPY, which was found to be genotoxic at 100 ␮g ml−1 , is readily removed upon photolytic treatment at 254 nm. 2-CPY rate of removal was found to increase with increasing temperature, and on lowering solution volume or initial 2-CPY concentration. 2-CPY photolysis yields 2-hydroxypyridine (2-HPY) as the primary intermediate which is further removed upon prolonged photolysis. The present work shows the effect of 254 nm UV light-induced degradation on solution total organic carbon (TOC) removal and on the solution effective genotoxicity. The changes of the genotoxicity of aqueous solutions of 2-CPY irradiated by means of UV light at 254 nm, at solution natural pH, at different treatment times are studied. 2. Materials and methods 2.1. Chemicals and photo-treatment 2-CPY (C5 H4 NCl) was supplied by Fluka (product code: 26280; CAS number 109-09-1; purity ≥98.0% by GC). It was used without further purification. Aqueous solutions were made using Milli-Q purity water.

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2.1.1. Measurements employed in the purely photolytic study Unless otherwise stated, experiments were performed at initial substrate concentrations of 300 ␮g ml−1 under different conditions, i.e. 400 ml of solution was irradiated isothermally at 50 ◦ C for the time required to achieve complete TOC removal. Measurements were performed in an open or air tight system, with or without agitation, with or without air purging with or without radical scavenger tert-butanol, with or without helium purging, at solution natural pH. The basic experimental set-up used in all of the above measurements is shown in detail elsewhere [23,24]. Briefly, it consists of a glass reactor where the solution is illuminated by means of a low-pressure, 110 W, mercury lamp with 90% emittance at 254 nm. The reaction mixture was held in a dark vessel submerged in a Huber-ministat 1200 constant temperature bath and was continuously and steadily circulated through the reactor at a flow-rate of 200 ml min−1 . The photoreactor had 27 mm i.d. and an approximate height of 300 mm. Sample pH and temperature were continuously measured and logged in the computer. Two Pt-A thermometers were placed at the UV reactor entrance and exit, respectively. The arithmetic average of these two temperatures, which differed by less than 1 ◦ C, was reported as the measurement temperature. To examine the effect of stripping in the purged with air measurements, where loss of volatile compounds is more pronounced, experiments were run with the addition of a reflux system. A coil condenser was fitted in order to cool the air leaving the system and condense as much as possible from the stripped organic material. An antifreeze/water mixture was re-circulated through condenser coil at −10 ◦ C. Samples periodically drawn from the vessel were analysed by means of high performance liquid chromatography (HPLC) and 2-CPY and primary intermediate product, 2-HPY, concentration was quantified. HPLC measurements were performed using the methodology presented in our previous work [2]. 2-CPY degradation products which could not be identified and quantified with HPLC were identified via GC–MS according to the methodology presented in the same work [2]. As reported in [2], those analyses were performed on fit-for-the-purpose measurements where the entire end-volume of the solution was used for a single GC–MS analysis. The results obtained were qualitative, i.e. there was no-quantification or association of their production with phototreatment time. TOC was analysed by means of catalytic combustion/nondispersive infrared gas analysis on a Shimadzu 5050 TOC analyzer following the methodology presented in [23]. A Hanna Instruments pH211 pH meter with a Hanna Instruments HI1131 probe was used for the measurement of pH. 2.1.2. Measurements employed in the genotoxicity study Six series of experiments were performed at initial substrate concentrations of 2000 ± 100 ␮g ml−1 . 210 ml of solution was irradiated isothermally at around 35 ◦ C, in a closed but not air tight system, outlined in Section 2.1.1, under vigorous agitation, at solution natural pH. Samples were withdrawn and analysed by HPLC (Dionex P680 system) with a Dionex 1024 dio-array equipped with an Acclaim C18 5 ␮m 120 Å, 406 mm × 250 mm column using a 50:50 water:acetonitrile isocratic mobile phase at 1 ml min−1 . UV detection was at 201, 206, 210, 254 and 261 nm. Where appropriate, calibration curves were established using external standards at various concentrations and were used for quantification. HPLC analyses were run in duplicate and mean values of two separate measurements are quoted as results. The same parent samples were used for the genotoxicity study following the protocol described in Section 2.2.

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2.2. CBMN assay in human lymphocytes in vitro Blood samples were derived from two healthy individuals (22 years old, non-smokers) not undergoing any drug treatment and who did not have any viral infection or X-ray exposure for over a year. Whole blood (0.5 ml) was added to 6.5 ml Ham’s F-10 medium (Invitrogen), 1.5 ml foetal calf serum (Invitrogen) and 0.3 ml phytohaemagglutinin (Invitrogen) to stimulate cell division. The appropriate volume of 2-CPY or 2-CPY phototreated solution (the same for all samples) was added 24 h post-culture initiation to achieve a final nominal 2-CPY concentration of 100 ␮g ml−1 . The selection of this concentration is justified in Stapleton et al. [24]. 2-CPY nominal concentration (100 ␮g ml−1 ) was equal to the actual 2-CPY concentration only in the photochemically untreated samples (t = 0). For photolytically treated solutions the nominal concentration was calculated relatively to the initial solution concentration and not to the actual 2-CPY concentration at the time the sample was withdrawn. Samples were dissolved in double distilled H2 O (dd H2 O). Mitomycin-C (Sigma) at final concentration of 0.5 ␮g ml−1 served as positive control. For the MN study, 6 ␮g ml−1 Cytochalasin-B (Sigma) was added to the culture medium 44 h after its initiation and 20 h after the addition of the appropriate 2-CPY solution. Cells were harvested 72 h after the initiation of culture and were collected by centrifugation. A mild hypotonic treatment with a 1:3 solution of Ham’s medium and dd H2 O supplemented with 2% serum was given for 3 min at room temperature and was followed by 10 min fixation with a fresh 5:1 solution of methanol/acetic acid. Cells were stained with 7% Giemsa [19,20]. At least 2000 BN cells with preserved cytoplasm were scored per treatment, in order to calculate the frequency of MN. Standard criteria [18] were used for scoring MN. The Cytokinesis Block Proliferation Index (CBPI), is given by the equation: CBPI = M1 + 2M2 + 3(M3 + M4 )/N, where M1 , M2 , M3 and M4 correspond to the numbers of cells with one, two, three and four nuclei, respectively and N is the total number of cells [25]. CBPI was calculated by counting at least 2000 cells in order to determine possible cytotoxic effects. The calculation of MN size was also used as an additional parameter to indicate whether the activity of the tested substances was clastogenic or aneugenic [19,26]. Small size MN is more likely to contain acentric chromosome fragments indicating a clastogenic effect, while large size MN may possibly contain whole chromosomes thus indicating an aneugenic effect [27–29].

Fig. 1. Normalised TOC removal at 50 ◦ C and solution natural pH, without agitation (laminar-flow). (䊉) 2-CPY initial concentration 500 ␮g ml−1 , solution volume 400 ml; (+) 2-CPY initial concentration 300 ␮g ml−1 , solution volume 400 ml; () 2-CPY initial concentration 300 ␮g ml−1 , solution volume 250 ml.

Fig. 2. Normalised TOC removal: 2-CPY initial concentration 300 ␮g ml−1 , solution volume 400 ml: at solution natural pH and 50 ◦ C. () Laminar, non-aerated flow; (), swirl-flow; (䊉), swirl-flow, purging with air and overhead condenser; (+) swirlflow, purging with air. Inset: pH during photo-treatment: () laminar, non-aerated flow; () swirl-flow; (䊉) swirl-flow, purging with air and overhead condenser; (+) swirl-flow, purging with air; (*) laminar, purged with air flow.

MN size is expressed as the ratio of MN diameter to the cell nucleus diameter. MN size was characterised as small, when this ratio was ≤1/10, medium, when this ratio was in the range 1/3 to 1/9 and large when its value was ≈1/3 of nuclear diameter [19]. 2.3. Statistical analysis All results are expressed as the mean frequency ± standard error (MF ± SE). Differences of MN data among treatment and control groups were tested by the G-test for independence on 2 × 2 tables. This test is based on the general assumption of the 2 analysis, but offers theoretical and computational advantages [30]. The 2 -test was used for the analysis of CBPI among each treatment. Differences at p < 0.05 were considered significant. G-test was evaluated using the data analysis statistical software Minitab (Minitab Inc., Pennsylvania, USA). 3. Results and discussion 3.1. Photolytic treatment of 2-CPY As mentioned earlier, 2-CPY was found to be readily removed following photolytic treatment, yielding primarily 2-HPY [23]. Upon prolonged irradiation TOC removal is possible. Fig. 1 shows the effect of volume and solution initial concentration on the rate of TOC removal. As can be seen in Fig. 1, increased volume or initial concentration prolongs TOC removal. Fig. 2 shows TOC removal of 300 ␮g ml−1 , 400 ml solution at 50 ◦ C under different conditions, namely under air purging, with and without agitation in an open but not air tight system as well as with an overhead condenser. In the same figure the pH history of those measurements as well as the pH profile of a laminar-flow measurement with air purging is shown. Although 2-CPY rate of removal has been found not to depend on purging with air or agitation, TOC removal does depend on those factors as can be seen in Fig. 2. It can also be seen that TOC is faster removed in purged with air measurements. 254 nm UV irradiation results in 2-CPY fast de-halogenation, with a very rapid pH drop. Prolonged UV-treatment results in complete mineralisation, which is however faster in the measurements were oxygen is readily available. So, the fastest TOC removal is observed in the agitated, purged with air measurement. The solution pH rises in all purged with air measurements to approximately neutral where it is maintained until sample complete mineralisation. The availability of oxygen appears to affect directly how fast pH rises. In measurements purged with helium (not shown here) or measure-

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the molecular ion at m/z = 127 is also present. The degradation of the later aliphatic product could then result in the production of short-chain compounds such as, formamide (P8 ), [33–35], as well as acetic and formic acid. The latter two (products P6 and P7 , respectively) have been identified in the current study by GC–MS. Compound P11 was detected in traces and is also expected to form at the later stages of the reaction potentially through the formation of 2-carbamoylpyridine, as a result of reaction of formamide and pyridine, that could be dehydrated during the GC–MS analysis. The formation of 2-pyridinecarboxylic acid, compound P9 , can be produced by a reaction of the substrate, 2-CPY, with formic acid, which is formed in the later stages of the degradation process. 3.2. CBMN assay in human lymphocytes in vitro

Fig. 3. Outline of 2-CPY 254 nm photolytic decomposition pathways and products.

ments where oxygen is not provided, pH remains acidic throughout the measurement. 2-HPY is the first major intermediate produced by this reaction [23,24,31], the removal of which was found to be faster in a rich in oxygen solution due to photo-oxidation [3]. Purging with helium (not shown here) and addition of radical scavenger tert-butanol resulted in the same mineralisation history as the measurement performed under laminar-flow in a closed system. The presence of tert-butanol in the purged with air measurement was slower, with a respective slower pH rise than the measurement performed at the same otherwise conditions but tert-butanol addition. Purging with air, however, results in some stripping of volatile products, which macroscopically appears as TOC removal. This was verified by the measurement performed with the use of an overhead condenser which displayed a slower TOC removal and pH rise than the measurement where this condenser was absent, at the same otherwise conditions. The products obtained via GC–MS (and HPLC) analysis, their GC–MS retention time and similarity match are shown in Table 1 while Fig. 3 outlines their macroscopic formation paths. All products reported in Table 1 but formamide (P8 ) have been identified via GC–MS or/and HPLC. 2-HPY (P1 ), is directly produced from 2-CPY and it is subsequently forming Dewar pyridinone (product P2 ) as in the case of other 2-halogenated pyridines while a small amount of 2-CPY reacts directly to form Dewar pyridinone [2]. Products P2 (5-azabicyclo[2.2.0]hex2-en-6-one), P3 (1H-pyrrole-2-carboxaldehyde), P4 (6-hydroxy2(1H)-pyridinone), P5 (N-formyl-3-carbamoylpropenal) and P11 (2-pyridinecarbonitrile) have been also identified in the degradation of other 2-halogenated pyridines [2], while products P5 and P9 (6-chloro-2-pyridinecarboxylic acid) were also found to form during 2-CPY photocatalysis [32]. Moreover, products P5 and P11 were also identified during 2-fluoropyridine photocatalysis [32]. N-Formyl-3-carbamoylpropenal, product P5 , was also identified in the degradation of pyridine and its formation bares close analogy to the mechanism followed for the degradation of pyridine [33]. The mass spectrum of P5 is similar to that reported in [33], together with the analysis of the different ion fragments while

2-CPY and 2-HPY concentrations were measured as a function of time via HPLC and the profile of those two concentrations of a typical measurement used for the genotoxicity study is shown in Fig. 4. 2-CPY initial concentration employed in the photolytic measurements was ca. 2000 ␮g ml−1 . The addition of the phototreated sample to the culture inevitably resulted in sample dilution. In order to achieve consistency with previous study [24], the samples employed in the genotoxicity study where obtained from the phototreated ones after appropriate dilution to the concentration range of the ones employed in [24]. Thus, the samples employed in the genotoxicity study had an equivalent to 2-CPY initial concentration of 100 ␮g ml−1 . The embedded Fig. 4 shows the total number of large MN found in 2000 BN cells (which indicate aneuploidy) and the mean CBPI values (which indicate cytotoxicity) of each treatment. The arrows indicate the axis where each quantity is read. Table 2 shows the results obtained from peripheral blood lymphocytes cultures treated with 2-CPY before and after treatment by UV light in different degradation times. As can be seen in Table 2, 2-CPY induced a statistically significant increase (p < 0.001) in the frequency of MN and binucleated micronucleated (BNMN) cells at the concentration of 100 ␮g ml−1 , before UV light treatment, compared to the control. In the case of treatments by UV light at 20, 50, 120, 160, 240 and 300 min the frequencies of MN and BNMN were still significant (p < 0.001). In the case of treatments by UV light at 600, 900 and 1200 min our data indicated no significant statistical differences in both MN and BNMN frequencies compared to the control value. The reported control and positive control frequencies of MN shown in this work are consistent with the literature [20,36,37].

Fig. 4. Normalised concentration of 2-CPY and 2-HPY at solution natural pH, 35 ◦ C and, laminar, non-aerated flow. () [2-CPY]/[2-CPY]0 ; (䊉) [2-HPY]/[2-CPY]0 . Inset: Total number of large MN found in 2000 BN cells (left-hand axis, wide bars) and mean CBPI value (right hand axis, thin grey bars) of diluted phototreated samples.

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The 2-CPY cytotoxic effect before and after UV irradiation was evaluated by the determination of CBPI. Regarding the cytotoxic index, statistically significant differences (p < 0.001) on CBPI were detected between control and untreated or treated by UV irradiation, 2-CPY cultures. As can be seen in Table 2 and Fig. 4, solution irradiation caused a gradual drop of CBPI value for the first 240 min, thus indicating the formation of cytotoxic intermediates. The lowest CBPI value was observed at 240 min. Subsequently CBPI value was gradually increasing, reaching the control value after 1200 min of irradiation. The size ratio of MN in the in vitro CBMN assay is an alerting index as effective as the fluorescence in situ hybridization (FISH) analysis for the discrimination of clastogenic and aneugenic effects [19,26].

Data on the size ratio of MN (‰) induced by 2-CPY before and after irradiation compared to the control, are presented in Fig. 5. Compared to the control size ratio of MN, an over threefold increase in small size MN frequency was observed up to 160 min, as well as, an over twofold increase from 240 up to 600 min of irradiation of 2-CPY. In addition, three and over threefold increase in large MN frequency was observed during the first 300 min of irradiation of 2-CPY, which subsequently was decreased to reach the control value after 600 min of irradiation, as shown in Fig. 4. There are very few studies in the literature on the mutagenicity of the tested compound. The findings reported in those studies were not always consistent. 2-CPY was mutagenic when tested at concentrations up to 7500 ␮g/plate in the Salmonella typhimurium/mammalian micro-

Table 1 Products formed during 254 nm photolytic treatment of 2-CPY aqueous solutions. Compound

Name

P1

Structure

GC–MS retention time (min)

Similarity match (%)

2-Hydroxypyridine

16.559

97

P2

5-Azabicyclo[2.2.0]hex-2-en-6-one (or Dewar pyridinone)

12.644

85

P3

1H-Pyrrole-2-carboxaldehyde

11.117

80

P4

6-Hydroxy-2(1H)-pyridinone

10.348

70

P5

N-Formyl-3-carbamoylpropenal

13.907

72

P6

Acetic acid

5.158

90

P7

Formic acid

4.982

90

P8

Formamide

No direct measurement

P9

6-Chloro-2-pyridinecarboxylic acid

7.768

87

P10

2,3-Dichloro-pyridine

11.154

92

P11

2-Pyridinecarbonitrile

20.420

78

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Table 2 Frequencies of micronucleated binucleated cells (BNMN) and micronuclei (MN), during 254 nm UV photo-treatment of 2-CPY aqueous solutions. Photo-treatment time (min) Control 0 Positive control (0.5 ␮g ml−1 MMC) 0 2-CPY (100 ␮g ml−1 ) 0 20 50 120 160 240 300 600 900 1200

BNMN MF(‰) ± SE 4±0 76.0 ± 4.0* 12.0 12.0 8.5 11.0 9.0 10.5 8.0 5.0 6.0 4.0

± ± ± ± ± ± ± ± ± ±

0.0* 2.0* 0.5* 2.0* 0.0* 0.5* 1.0* 1.0 2.0 0.0

MN MF(‰) ± SE 4.0 ± 0 83.0 ± 4.0* 12.5 13.0 9.5 13.0 11.5 11.0 8.5 6.0 6.0 4.0

± ± ± ± ± ± ± ± ± ±

0.5* 1.0* 0.5* 1.0* 0.5* 0.0* 0.5* 2.0 2.0 0.0

CBPI MF ± SE 2.09 ± 0.18 1.39 ± 0.04* 1.71 1.67 1.64 1.68 1.56 1.45 1.65 1.65 1.60 1.80

± ± ± ± ± ± ± ± ± ±

0.04* 0.05* 0.10* 0.02* 0.02* 0.03* 0.16* 0.16* 0.08* 0.07*

2-CPY: 2-Chloropyridine, BNMN: micronucleated binucleated cells, MN: micronuclei, CBPI: cytokinesis block proliferation index, MMC: mitomycin-C, MF(‰) ± se: mean frequencies (‰) ±standard error, *p < 0.001 [G-test for BNMN and MN; 2 for CBPI].

some assay in strains TA97, TA98, TA100 and TA102 with metabolic activation, but non-mutagenic when tested in the same strains at concentrations up to 5000 ␮g/plate without activation [38]. Zimmermann et al. [39] reported 2-CPY to be one of a series of pyridine derivatives which induced mitotic aneuploidy in Saccharomyces cerevisiae. The ability to induce chromosomal aberrations by 2-CPY, was tested in V3 cells (an African Green monkey kidney cell line). In a dose range from 400 to 3200 ␮g ml−1 , 2-CPY was found to be noncytotoxic and non-clastogenic [40]. However, when tested in the L5178Y mouse lymphoma mammalian system, in concentrations up to 2004 ␮g ml−1 , induced gene mutations and chromosomal aberrations with and without metabolic system. 2-CPY also induced MN when tested at concentrations from 1920 to 1992 ␮g ml−1 in mouse lymphoma cells [41]. In our recent study of the potential in vitro genotoxicity of 2-CPY, statistically significant differences were observed in MN frequency between control and 100 ␮g ml−1 of 2-CPY [24]. Our results are in accordance with the findings of two of the above-referred studies in S. cerevisiae and in mouse lymphoma cells [39,41]. However, it should be highlighted here, that among all

genotoxicity studies employing 2-CPY, our work is the only one that has evaluated 2-CPY genotoxic effects on human cells. In the present study, we have quantified the concentration of 2-CPY and we have investigated its genotoxicity after solution UV photo treatment of different time lengths. As can be seen in Figs. 4 and 5, a significant increase in large MN frequencies and a drop in CBPI value were noticed up to 240 min of UV irradiation. An increase in both, small and large size MN frequencies were observed, indicating a possible clastogenic and aneugenic effect. The negative genotoxic results in our findings, the slight positive response of cytotoxic index as well as the decrease in large size MN frequency was observed after 600 min of UV irradiation. The genotoxic activity of 2-CPY decreased only after prolonged UV irradiation far beyond the required for the removal of the original substrate (i.e. 120 min). The removal of the initially loaded 2-CPY was approximately 50% at 50 min (i.e. 50 ␮g ml−1 actual 2CPY concentration), 70% at 120 min (i.e. 30 ␮g ml−1 actual 2-CPY concentration), and over 95% at 300 min. According to our previous genotoxicity findings [24], 2-CPY is not genotoxic or cytotoxic at concentrations at or below 50 ␮g ml−1 . In the current work, 2-CPY concentration was lower than 50 ␮g ml−1 in all but the first four treatments (t ≤ 120 min) employed in the genotoxicity study. Thus, the mixture genotoxicity cannot be attributed to 2-CPY. The solution genotoxicity reached its maximum at 240 min, as can be seen in Fig. 4. As can be seen in the same figure, this is near the maximum 2-HPY concentration. The rate of genotoxicity reduction was significantly slower than the rate of 2-CPY removal. This could be attributed to other genotoxic products, such as 2-HPY formed during 2-CPY photolytic treatment or due to synergistic effects between compounds present in the solution. To our best knowledge there are no genotoxic effects reported for any of 2-CPY degradation products presented in Table 1. The genotoxicity of individual products formed during 2-CPY formation is currently researched in our laboratory. 4. Conclusions

Fig. 5. Mean frequencies (‰) of micronuclei (MN) per size, during 254 nm UV phototreatment of 2-CPY aqueous solutions.

UV irradiation of aqueous solutions of 2-CPY results in 2-CPY fast de-halogenation, with a very rapid pH drop. A number of intermediate products shown in Table 1 were produced, which degraded upon solution prolonged photo-treatment thus resulting in complete TOC removal. Genotoxicity studies were performed using untreated and 254 nm UV treated solutions without air purging, at solution nat-

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ural pH. It was found that the mixture genotoxicity increases as treatment time increases, thus indicating that the photolytic treatment of the original substrate, 2-CPY, is initially producing genotoxic products. There are no genotoxic effects of any of the compounds shown in Table 1 reported, so it cannot be deduced whether specific compounds or synergistic effects are responsible for the solution genotoxicity. This study indicates that genotoxicity studies should form an essential part of research on the chemical or photochemical removal of persistent compounds to show the optimum end point of a chemical or photochemical treatment as it indicates the length of treatment required to achieve innocuous to humans water or wastewater. In the present study the rate of genotoxicity reduction was slower than the rate of 2-CPY removal. However, the solution ceased to be genotoxic when all 2-HPY was practically removed. The available data cannot reveal whether 2-HPY itself, other intermediates present in the solution at the same time or synergistic effects between co-existing compounds are responsible for the mixture genotoxicity. However, they show that from a genotoxicity point of view, the solution has been sufficiently treated and there is no need for further photo-treatment thus defining an operational optimum. These results demonstrate the promising potential of the combined use of bioassays, such as CBMN assay, with chemical treatment methods for the assessment of environmental pollutants fate and applications. Conflict of interest statement The authors declare that they have no competing financial or conflicts of interest with regard to this work. Acknowledgements The Engineering and Physical Sciences Research Council (EPSRC, UK) is greatly acknowledged for the DTA PhD studentship for D.R. Stapleton. References [1] J.H. Kuney (Ed.), Chemcyclopedia 95—The Manual of Commercially Available Chemicals, 194, The American Chemical Society, Washington, DC, 1994, p. 410. [2] D.R. Stapleton, I.K. Konstantinou, D.G. Hela, M. Papadaki, Photolytic removal and mineralisation of 2-halogenated pyridines, Water Res. 43 (2009) 3964–3973. [3] D.R. Stapleton, I.K. Konstantinou, A. Karakitsou, D.G. Hela, M. Papadaki, 2Hydroxypyridine photolytic destruction by 254 nm UV irradiation at different conditions, Chemosphere 77 (2009) 1099–1105. [4] K. Fent, A. Weston, D. Caminada, Ecotoxicology of human pharmaceuticals, Aquat. Toxicol. 76 (2006) 122–159. [5] I.K. Konstantinou, D.G. Hela, T.A. Albanis, The state of pesticide pollution in freshwater resources (rivers and lakes) of Greece. Part I. Review on occurrence and levels, Environ. Pollut. 141 (2006) 555–570. [6] Arch Chemicals, Inc., High Production Volume (HPV) Challenge Program, Test Plan for 2-Chloropyridine CAS no. 109-09-1, 2003, Available www.epa.gov/hpv/pubs/summaries/2chlorop/c14277rt.pdf (accessed 14.12.09). [7] National Institutes of Health (NIH), Summary of Data for Chemical Selection 2-Chloropyridine, CAS no. 109-09-1, 2004, Available: ntp.niehs.nih.gov/ntp/htdocs/Chem Background/ExSumPdf/2-Chloropyridine, pdf-10-06-2004 (accessed 21.12.09). [8] A.J. Hendricks, J.L. Maas-Diepeveen, A. Noordsij, M.A. van der Gaag, Monitoring response of XAD-concentrated water in the Rhine delta: a major part of the toxic compounds remains unidentified, Water Res. 28 (1994) 581–598. [9] A. Guardiola, F. Ventura, L. Matia, J. Caixach, J. Rivera, Gas chromatographicmass spectrometric characterization of volatile organic compounds in Barcelona tap water, J. Chromatogr. 562 (1991) 481–492. [10] S. Suchail, G. De Sousa, R. Rahmani, L.P. Belzunces, In vivo distribution and metabolisation of 14 C-imidacloprid in different compartments of Apis mellifera L., Pest. Manage. Sci. 60 (2004) 1056–1062.

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