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years and seasonality. In general, summer samples were more genotoxic than winter samples. In the vast majority of the 75 water samples, all four para...
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Research A 2.5-Year Genotoxicity Profile for a Partially Restored Polluted River NANTHAWAN AVISHAI, CLAUDETTE RABINOWITZ, AND BARUCH RINKEVICH* National Institute of Oceanography, Israel Oceanographic & Limnological Research, Tel-Shikmona, P.O. Box 8030, Haifa 31080, Israel

This study evaluates water genotoxicity of the Kishon River, the most polluted river in Israel that is under restoration. Water samples were collected every other month (January 2001-May 2003) from five sites, and genotoxicity was assayed by the alkaline comet assay using a fish hepatoma cell line (RTH-149). Genotoxicity in the Kishon River was reduced during 2002 as compared to the previous year. The results further revealed fluctuations in genotoxicity levels at all sites throughout the studied period with variations for the same month during consecutive years and seasonality. In general, summer samples were more genotoxic than winter samples. In the vast majority of the 75 water samples, all four parameters for genotoxicity that were employed revealed significant higher genotoxic levels than the controls. Comet percentage values in Kishon River samples were, on average, 1.8-2.4 times higher than controls. Damage score, comet tail length, and cumulative tail length values were 2.2-3.1, 2.4-3.7, and 2.4-3.7 times higher than controls, respectively. The Histadrut Bridge and Haifa fishing harbor (3.0 m depth) emerged as the most polluted sites, whereas Kiryat Haroshet was the least contaminated. Results call for a longterm genotoxicity follow-up plan at the Kishon River in order to assess the possibly evolving chronic genotoxicity state.

Introduction The Kishon River is regarded as the most polluted river system in Israel (1, 2). The river drains an area of approximately 1100 km2 and flows through agricultural, domestic, and industrial districts before joining the Mediterranean Sea near the city of Haifa. The Kishon’s water is contaminated by agricultural runoff, various types of industrial effluents, and domestic sewage (2); by heavy metals and a mixture of organic materials (3-5) including polycyclic aromatic hydrocarbons (PAHs), alkylated benzenes, halogenated alkanes, and chlorinated aromatic organic compounds; and some radionucleotides (3). As a result, the lower part of the Kishon River has been denuded for years of its multicellular life forms (3). Moreover, tissue analyses of fishes, crustaceans, and mollusks from the shallow waters of Haifa Bay at the mouth of Kishon River revealed high levels of heavy metals and deviations in oxidizing enzyme activities (6, 7). The Kishon River has also elicited major public concern for its potential long-term * Corresponding author phone: +972-4-8565275; fax: +972-48511911; e-mail: [email protected]. 3482

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cancer risks to fisherman working in its vicinity and to navy soldiers who used to dive in the river (3). In response to public commotion, the Kishon River Authority (KRA) was founded in December 1994; (www.kishon.org.il) aiming to stop all sources of pollution and to rehabilitate the river by the end of the year 2010, by means of restoring its biological ecosystems. The KRA action plan for the rehabilitation of the river is based on four parallel modes of operation: (i) creation of a database for water and sediment quality; (ii) monitoring, inspection, and enforcement of measures to stop-up all pollution sources; (iii) restoration of the river and its environment; and (iv) establishment of parks at specific locations. The master plan also involves the possible construction of a pipeline to divert treated industrial wastes directly into Haifa Bay (8, 9). Following the first 8 yr of activities, the KRA revealed a significant improvement of water quality in the Kishon River (www.kishon.org.il). Given the unsolved concerns for the genotoxic impacts of the river water, we have independently developed a monitoring plan using the comet assay as a biological test (10, 11). The Kishon River can be divided into two main subsystems, the upper system, which is supplied by freshwater sources and is flowing through agricultural and domestic areas, and the lower system, which reveals estuary characteristics and is running through an industrial area. About 3 yr ago (10), we started a program for monitoring the genotoxicity in the Kishon River at several sampling sites denoting different levels of river pollution. The Kiryat Haroshet site (Figure 1) represents the upper system conditions while the other sampling sites (Figure 1) are located in the lower system. This study summarizes the genotoxicity levels of the Kishon River during the 2.5-yr follow-up program to evaluate the efficacy of rehabilitation activities on water genotoxicity. Here, we analyze data obtained from 15 sampling dates at five sampling sites.

Experimental Section Sampling Procedures. Five water-sampling sites were designated in four localities along a gradient of water genotoxicity (11) in the Kishon River, Israel (Figure 1). Two were sited at the highly polluted Haifa fishing harbor (32°48.5′ N, 35°01.5′ E)sat the surface and at ∼3.0 m deep. Additional water samples were taken from Yigael Yadin Bridge (32°48.0′ N, 35°03.0′ E), Histadrut Bridge (32°47.5′ N, 35°03.0′ E) and from the relatively “clean” area of Kiryat Haroshet (32°42.0′ N, 35°05.0′ E) at ∼11 km upstream. Samples were collected in disposable plastic bottles as described (10) and brought to the laboratory at the National Institute of Oceanography, Haifa. Water was adjusted to pH 7 using 1-3% (v/v) of 1 M HEPES buffer. Samples were then filtered through 0.22-µm disposable filters. Water samples from Kiryat Haroshet (freshwater osmolarity) were mixed with double-strength Dulbecco’s minimal essential medium (DMEM, 2×) to correct the osmolarity to ∼220 mOsM/kg. The other samples were mixed with DMEM containing twice the concentration of supplements. The final concentration of all samples were supplemented with 5% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM HEPES buffer, and 1% penillin-streptomycin-amphotericin B (PSA; stock solution: 10 000 units/mL penicillin, 10 000 µg/mL streptomycin, and 25 mg/mL amphotericin B). Each sample was coded, stored overnight at 4 °C, and tested the following day. Artificial Kishon water (AKW: [1×], pH 7.3: 467 mM NaCl, 11.05 mM 10.1021/es035264a CCC: $27.50

 2004 American Chemical Society Published on Web 05/26/2004

FIGURE 1. Area map of the Kishon River, depicting sampling sites. KCl, 9.81 mM CaCl2‚2H2O, 45.19 mM MgSO4‚7H2O, and 30 mM MgCl2‚6H2O; stock solution of AKW [2× (ref 12)] was diluted with DMEM) and DMEM, which have the same pH and salinity as the Kishon water samples, were used as

controls. Water samples were taken every other month from January 2001 untill May 2003. Cell Preparations and Exposure. We used the fish hepatoma cell line RTH-149, originated from an alfatoxininduced hepatoma in an adult rainbow trout (Oncorhynchus mykiss) (13). Cells were cultured in 25-cm2 flasks using DMEM supplemented as above, at 20 °C and 5% CO2 atmosphere. Three days before the experiment, confluent cultures were dislodged using 0.25% trypsin-EDTA in calcium-magnesium-free PBS and plated in 24-well plates (Nunc) at a concentration of 2 × 104 cells/well in 0.5 mL of medium. Cultures usually reached 95% confluency at the day of experiment (∼2 × 105 cells/well). The medium was changed 2 h before the experiment. Experiments began by exposing cells, in independent triplicates, to 50% diluted Kishon water samples (2 h, 20 °C). Thereafter, tissue culture plates were placed on a tray over ice. The medium was removed; the cells were washed with 0.5 mL of calcium-magnesium-free PBS and then dislodged with 0.25 mL of 0.25% trypsin-EDTA solution. After trypsinization, cells were washed with cold PBS (×2). Cell viability was determined using trypan blue dye exclusion assay. Wells representing cell survival >90% were processed for the comet assay. Comet Assay. Kishon water samples are characteristic of variable estuary conditions. Previously we established the methodology of using RTH-149 cells for the comet assay (11). This methodology follows the protocol of Singh et al. (14), with slight modifications including pH and salinity adjustments, as described (11). In brief, 10 µL of cell suspension (∼2 × 105 cells) was embedded in 90 µL of 0.65% low-melting agarose layer on a Star-frost microscope slide precoated with 0.65% normal melting agarose. After 20 min of solidification at 4 °C, a third layer of 120 µL of 0.65% low-melting agarose was placed on top and left at 4 °C for an additional period of 20 min to allow solidification. The cells were then lysed by immersing the slides overnight in a freshly prepared lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton

FIGURE 2. Comet assay characteristics showing different stages of cell damage. VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Evaluating genotoxicity of the Kishon River (January 2001-May 2003) with the four comet assay parameters: a, comet percentage; b, comet score; c, average comet tail length (µm); d, cumulative tail length (µm). DMEM, Dulbecco’s minimal essential medium (control); AKW, artificial Kishon water (control); FHS, fishing harbor at surface; FHD, fishing harbor at 3.0 m depth; YB, Yigael Yadin Bridge; HB, Histadrut Bridge; KH, Kiryat Haroshet. X-100, 10% DMSO, pH 10.0) at 4 °C. The slides were then washed in cold water (×3) for 5 min and placed on a horizontal gel electrophoresis tray containing freshly prepared alkaline electrophoresis buffer (1 mM EDTA, 300 mM NaOH, pH 13.0) for 20 min to allow DNA unwinding. Electrophoresis was carried out at 20 V (1 V/cm) and at a 3484

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starting current of 300 mA for 20 min at 4 °C. Thereafter, the slides were neutralized with 0.4 M Tris at pH 7.5 for 5 min (×3), dehydrated with ethanol, and left to dry. The slides were stained with 60 µL of 20 µg/mL ethidium bromide solution and viewed under a fluorescent microscope using a U-MNG filter (Olympus). All steps were

TABLE 1. Genotoxicity of Kishon River Samples Measured by the Parameter of Percent Cometa sampling date

DMEM

AKW

FHS

Jan 2001 Mar 2001 May 2001 Jul 2001 Sep 2001 Nov 2001 Jan 2002 Mar 2002 May 2002 Jul 2002 Sep 2002 Nov 2002 Jan 2003 Mar 2003 May 2003

28.67 ( 1.16 not done 34.67 ( 1.16 32.67 ( 1.15 28.00 ( 3.46 21.33 ( 4.16 27.33 ( 4.16 24.00 ( 3.46 20.00 ( 7.21 26.67 ( 3.06 24.00 ( 2.00 28.67 ( 1.15 22.67 ( 1.15 19.33 ( 4.16 27.33 ( 6.43

28.67 ( 3.06 43.33 ( 9.24 33.33 ( 4.16 32.00 ( 2.00 29.33 ( 1.15 21.33 ( 4.06 26.67 ( 4.16 23.33 ( 1.15 16.00 ( 2.00 25.33 ( 2.31 19.33 ( 1.15 30.33 ( 5.29 18.00 ( 3.46 18.67 ( 3.06 26.00 ( 7.21

54.00 ( 2.00* 58.67 ( 1.16* 53.33 ( 6.11* 50.67 ( 7.02* 70.67 ( 9.45* 54.67 ( 5.03* 45.33 ( 3.06* 53.33 ( 2.31* 45.33 ( 8.08* 54.67 ( 2.31* 44.00 ( 8.72* 58.67( 11.02* 50.67 ( 7.02* 50.67 ( 10.07* 72.00 ( 2.00*

% comet at FHD 64.67 ( 3.06* 85.33 ( 6.43* 69.33 ( 2.31* 73.33 ( 1.15* 80.00 ( 14.42* 50.67 ( 5.15* 53.33 ( 5.03* 55.33 ( 3.06 * 46.67 ( 9.24* 49.33 ( 14.47* 38.00 ( 5.29* 62.67 ( 6.11* 60.00 ( 9.17* 66.67 ( 9.45* 80.00 ( 14.00*

YB

HB

KH

59.33 ( 6.43* 69.33 ( 2.31* 58.00 ( 3.46* 53.33 ( 11.02* 88.00 ( 4.00* 50.00 ( 2.00* 49.33 ( 6.11* 53.33 ( 2.31* 39.33 ( 8.08* 39.33 ( 5.03* 33.33 ( 11.02* 60.67 ( 1.15* 41.33 ( 5.77* 33.33 ( 5.03* 71.33 ( 18.04*

68.00 ( 2.00* 80.00 ( 9.17* 75.33 ( 6.43* 75.33 ( 3.06* 96.67 ( 4.16* 57.33 ( 3.06* 45.33 ( 8.08* 52.67 ( 2.31* 36.00 ( 4.00* 60.67 ( 8.08* 39.33 ( 3.06* 88.00 ( 17.44* 41.33 ( 13.61* 44.00 ( 6.93* 77.33 ( 22.03*

46.67 ( 5.03* 60.00 ( 8.72* 58.00 ( 2.00* 49.33 ( 1.15* 70.67 ( 18.58* 27.33 ( 4.62 40.00 ( 8.72* 34.00 ( 9.17* 30.67 ( 4.16* 38.00 ( 4.00* 27.33 ( 6.11 89.33 ( 6.11* 32.00 ( 6.93* 30.00 ( 12.17* 65.33 ( 20.43*

a Mean ( SD. For locality codes, see legend to Figure 3. An asterisk (*) indicates significant difference from controls at p < 0.05 using Duncan’s test.

TABLE 2. Genotoxicity of Kishon River Samples Measured by the Parameter of Damage Scorea sampling date

DMEM

AKW

Jan 2001 Mar 2001 May 2001 Jul 2001 Sep 2001 Nov 2001 Jan 2002 Mar 2002 May 2002 Jul 2002 Sep 2002 Nov 2002 Jan 2003 Mar 2003 May 2003

19.67 ( 2.52 not done 32.67 ( 4.73 23.67 ( 2.08 30.33 ( 6.81 16.00 ( 4.58 22.67 ( 8.14 19.33 ( 3.06 17.67 ( 1.53 23.67 ( 2.89 17.33 ( 4.04 26.33 ( 2.08 16.67 ( 2.08 15.67 ( 3.06 16.00 ( 4.36

28.33 ( 4.04 31.67 ( 5.68 31.00 ( 2.65 21.67 ( 2.89 33.33 ( 4.51 17.00 ( 2.00 22.33 ( 3.51 19.67 ( 2.31 18.00 ( 4.48 23.00 ( 2.65 13.33 ( 1.20 20.67 ( 5.13 14.33 ( 0.58 14.33 ( 2.31 16.67 ( 3.51

FHS

damage score at FHD

YB

HB

54.00 ( 0.00* 72.00 ( 1.73* 64.33 ( 3.51* 71.67 ( 3.06* 51.33 ( 5.51* 90.33 ( 11.59* 74.00 ( 10.58* 74.67 ( 7.57* 55.67 ( 9.298* 72.33 ( 8.50* 59.67 ( 3.51* 80.67 ( 5.13* 46.00 ( 8.54* 79.33 ( 1.15* 51.67 ( 11.02* 82.00 ( 2.00* 91.00 ( 16.82* 101.67 ( 19.50* 119.33 ( 5.51* 131.33 ( 11.15* 56.67 ( 8.96* 59.00 ( 1.73* 60.00 ( 7.00* 75.67 ( 6.66* 43.00 ( 6.24* 54.00 ( 1.00* 47.00 ( 7.81* 43.00 ( 8.66* 58.67 ( 1.53* 59.33 ( 7.51* 62.67 ( 4.62* 53.67 ( 3.21* 47.67 ( 8.62* 47.00 ( 10.15* 34.00 ( 11.00* 31.00 ( 4.58* 57.33 ( 4.04* 50.33 ( 13.05* 46.00 ( 7.00* 63.00 ( 6.00* 39.33 ( 3.96* 31.33 ( 2.19* 24.33 ( 4.63* 30.33 ( 1.93* 57.33 ( 11.02* 59.33 ( 6.00* 62.33 ( 2.89* 95.00 ( 18.52* 50.33 ( 8.50* 73.67 ( 21.13* 44.33 ( 6.81* 42.00 ( 17.58* 44.00 ( 13.08* 56.67 ( 8.14* 30.00 ( 5.29* 41.33 ( 8.05* 59.00 ( 7.21* 83.00 ( 10.39* 66.67 ( 19.22* 80.00 ( 28.00*

KH 46.00 ( 7.94* 47.00 ( 19.31* 61.00 ( 8.89* 48.33 ( 2.89* 97.00 ( 24.58* 26.00 ( 4.58 41.00 ( 10.58* 39.33 ( 16.65* 26.33 ( 4.62* 33.67 ( 4.16* 21.00 ( 1.99 87.33 ( 13.65* 31.00 ( 8.66* 31.00 ( 14.93* 59.33 ( 25.70*

a Mean ( SD. For locality codes, see legend to Figure 3. An asterisk (*) indicates significant difference from controls at p < 0.05 using Duncan’s test.

conducted in dimlight to prevent nonspecific additional DNA breakage. Comet Evaluation. Slides were examined at 400× magnification by visual analysis (15-17). A total of 150 randomly chosen cells from triplicate slides were examined for each water sample (50 cells per slide). Four highly scorable (15) visual parameters for genotoxicity were taken. In the first two parameters (comet stage and comet score), each cell was scored as belonging to either one of five specific damage stages based on the relative intensity of the head and tail fluorescence (from undamaged DNA stage 0 to maximal damage stage D, Figure 2). Undamaged DNA stage 0 has no tail; damaged stage A has a tail length equal to or shorter than the length of the nucleus head diameter; stage B has a tail length 1.1-3.5 times longer than the head diameter, stage C has a tail length greater than 3.5 times the head diameter; stage D has no “head” since all DNA migrated to the tail. Stages A-D received numerical values of 1-4, respectively. The percentages of cells in each stage were counted. The “comet score” for the 50 cells examined on a slide ranged therefore between 0 (where DNA was intact in all cells) and 200 (where the DNA in all cells migrated into tails). The other two parameters are average tail length and cumulative tail length. The head diameter and total tail length (in mm) of each nucleus were measured. Tail length was calculated as total comet length subtracted by head diameter.

The parameter of mean average tail length was calculated from three independent samples. The parameter of cumulative tail length was determined by the sum of 50 tail length values in each independent sample. Slides were coded, and a single investigator analyzed all slides throughout this study to minimize scoring variation. Statistical Analyses. Average and standard deviation values for each treatment were calculated from three independent samples. A statistical program, SPSS 11.0 for Windows, was used for data analyses. ANOVA or a nonparametric Krushal-Wallis test was performed according to the distribution and variance of the data.

Results and Discussion Samples were taken at 15 different dates over a 2.5-yr study (January 2001-May 2003); 75 samples of Kishon water were taken and 15 600 comet assays were analyzed (Figure 3; Tables 1-4). Most control cells (DMEM and AKW) did not show any DNA damage (comet stage 0) or low DNA damage (comet stage A) whereas exposures to Kishon samples have resulted, in most samples, in increasing levels of DNA damage (stages B-D). Samples from the fishing harbor (at ∼3.0 m depth) and the Histadrut Bridge have continuously manifested the highest percentages of severe DNA damage stages (comet stages C and D) during the entire sampling period (Figure VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. Genotoxicity of Kishon River Samples Measured by the Parameter of Comet Tail Lengtha sampling date

DMEM

AKW

FHS

Jan 2001 Mar 2001 May 2001 Jul 2001 Sep 2001 Nov 2001 Jan 2002 Mar 2002 May 2002 Jul 2002 Sep 2002 Nov 2002 Jan 2003 Mar 2003 May 2003

4.37 ( 0.25 not done 10.83 ( 2.70 6.53 ( 0.88 11.53 ( 3.19 4.43 ( 1.43 6.77 ( 3.92 6.15 ( 1.57 4.55 ( 0.56 7.40 ( 1.61 4.87 ( 2.21 7.92 ( 0.34 4.72 ( 0.79 4.02 ( 0.86 3.73 ( 1.20

9.36 ( 1.79 8.21 ( 2.38 11.05 ( 1.27 5.81 ( 1.24 11.75 ( 2.72 4.68 ( 0.80 6.60 ( 1.38 6.65 ( 0.91 5.67 ( 1.91 6.50 ( 1.33 3.53 ( 1.20 4.85 ( 1.03 3.83 ( 0.60 4.18 ( 0.90 4.32 ( 0.86

15.33 ( 1.03* 16.55 ( 3.06* 20.28 ( 4.68* 15.15 ( 3.10* 36.85 ( 9.07* 19.33 ( 5.15* 12.57 ( 2.51* 20.78 ( 0.35* 14.67 ( 1.40* 19.35 ( 0.90* 12.42 ( 3.96* 16.77 ( 5.40* 15.95 ( 1.95* 15.07 ( 0.32* 15.63 ( 2.00*

comet tail length at FHD 27.70 ( 1.92* 31.20 ( 5.98* 25.88 ( 5.38* 28.33 ( 1.42* 38.32 ( 9.41* 21.90 ( 0.62* 17.98 ( 2.00* 22.18 ( 3.26* 15.83 ( 4.09* 19.35 ( 3.26* 9.85 ( 2.19* 18.88 ( 2.32* 24.00 ( 3.64* 21.23 ( 3.41* 24.95 ( 2.98*

YB

HB

KH

22.73 ( 1.02* 23.35 ( 3.56* 21.52 ( 2.62* 18.35 ( 3.24* 49.15 ( 2.90* 21.58 ( 3.72* 13.63 ( 2.29* 23.87 ( 1.40* 9.50 ( 3.12* 15.28 ( 2.10* 7.22 ( 4.63 20.85 ( 1.89* 14.92 ( 2.24* 9.47 ( 1.45* 19.50 ( 6.87*

26.57 ( 3.26* 20.28 ( 2.15* 29.55 ( 0.95* 30.12 ( 1.94* 52.37 ( 4.17* 28.23 ( 1.60* 12.75 ( 3.12* 19.73 ( 1.50* 9.32 ( 2.08* 22.10 ( 1.53* 8.52 ( 1.93* 34.78 ( 6.86* 14.63 ( 7.11* 11.97 ( 3.91* 24.23 ( 13.19*

11.58 ( 2.46* 15.32 ( 7.58* 22.98 ( 6.11* 15.02 ( 15.02* 38.68 ( 0.73* 8.72 ( 2.59 13.42 ( 4.48* 14.97 ( 7.68* 8.22 ( 1.75* 10.50 ( 2.63* 5.88 ( 1.99 23.73 ( 6.35* 10.55 ( 3.38* 10.68 ( 5.86* 17.33 ( 9.95*

a (µm). Mean ( SD. For locality codes, see legend to Figure 3. An asterisk (*) indicates significant difference from controls at p < 0.05 using Duncan’s test.

3b, Table 2). As compared to other sites, the Yigael Yadin Bridge samples revealed fluctuations in genotoxicity levels, which were high in some sampling dates (such as September 2001, March 2002) and intermediate (such as May 2001, November 2002) or low in other sampling dates (May 2002, March 2003; Figure 3, Tables 1-4). However, all four parameters employed in this study pointed to the state of continuous genotoxicity of the Kishon water. While background levels of comet percentages the controls (DMEM and AKW) were 26.1 ( 4.5 and 26.1 ( 7.2, respectively, all Kishon samples exhibited increased comet percentages of up to 54.5 ( 8.1, 62.4 ( 13.7, 53.3 ( 15.3, 62.5 ( 19.2, and 46.6 ( 18.9 (for FHS, FHD, YB, HB, and KH, respectively; Table 1, Figure 3A). Average damage scores in controls were 21.3 ( 5.5 and 21.7 ( 6.6, respectively, while values in Kishon samples increased to 54.1 ( 12.0, 70.0 ( 18.2, 56.4 ( 22.6, 66.4 ( 27.2, and 46.4 ( 22.0 (for FHS, FHD, YB, HB, and KH, respectively; Table 2, Figure 3B). The average comet tail lengths in controls were 6.3 ( 2.5 and 6.4 ( 2.6 µm, respectively, whereas the Kishon samples showed values up to 3-4 times higher (17.8 ( 5.7, 23.2 ( 6.8, 19.4 ( 9.9, 23.0 ( 11.4, and 15.2 ( 8.2 µm for FHS, FHD, YB, HB, and KH sites, respectively; Table 3, Figure 3C). The cumulative tail lengths in the Kishon samples also resulted in values 3-4 times higher than the controls (313.6 ( 123.0 and 321.4 ( 128.8 µm in controls, respectively, as compared to 889.0 ( 292.8, 1158.7 ( 340.6, 969.0 ( 493.0, 1150.5 ( 574.6, and 774.2 ( 432.6 µm for FHS, FHD, YB, HB, and KH sites, respectively; Table 4, Figure 3D). The vast majority of the 75 Kishon water samples, except two KH samples (November 2001 and September 2002) and a single YB sample (September 2002) revealed, for the four parameters studied, significantly higher genotoxic levels than the controls (p < 0.05 ANOVA using Duncan’s test; Tables 1-4). While the most polluted sites were the Histadrut Bridge and Haifa fishing harbor (at ∼3.0 m depth), Kiryat Haroshet emerged as the least contaminated site (p < 0.001 ANOVA using Duncan’s test). The Histadrut Bridge sample of September 2001 also exhibited the highest genotoxicity values measured during this study in all four parameters studied. The correlation among the four parameters was extremely high (0.945 at p ) 0.01, two-tailed Pearson correlation). This higher genotoxicity level recorded from the Histadrut Bridge site downstream was the result of ongoing industrial discharges (3, 8, 9) and residues of previous discharges. It is documented (8, 9) that the Kishon River is still receiving sewage discharges from Haifa municipal wastewater treatment plant and waste discharges from six major industrial sources. At least part of Kishon River genotoxicity is prob3486

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ably caused by agricultural runoff and domestic waste. Variation in river genotoxicity between the different years has also been recorded. For example, during September 2001, all five sampling sites exhibited the highest genotoxicity levels in all four parameters studied (probably resulted from the floods of the first rain of this year) and then showed the lowest levels 1 yr later (September 2002, Figure 3). Genotoxic values in May 2002 were lower than in May 2001 and May 2003 values. When evaluating seasonality, the four parameters depicted minimal genotoxic levels at all Kishon sites during winter (January) months. Genotoxicity of the water samples increased to moderate levels during MarchJuly and reached the maximum levels during SeptemberNovember (2001-2002; Figure 3). This seasonal fluctuation is probably due to a combination of natural (floods caused by rain, seawater penetration during storms) and manmade (variation in the discharge rates of industrial effluents, etc) conditions. A significant decrease of genotoxic levels in the Kishon River was recorded in all 2002-2003 samples as compared to 2001 (p < 0.05 using Duncan’s test). The comet percentages during 2002 were reduced by 11.9-28.9% to 2001 values. Percentages of damage score in 2002 were 14.5-38.8% less than 2001 values whereas the average tail lengths and cumulative tail lengths in 2002 were reduced by 21.8-42.3%. During 2001, the pH of the samples from Histadrut and Yigael Yadin Bridges were extremely low, down to a pH 2.3 (10). The Kishon Authority has started to regulate the acidic waste discharges as from the end of 2001, bringing their pH to brine level. As a result, Histadrut and Yigael Yadin Bridges water samples became neutral as from January 2002. While pH is not regarded as a genotoxic agent (18), the persistence, mobility, chemical reactivity, and sorption dynamics of many genotoxic contaminants are governed by water pH (19). For example, low pH levels inhibit precipitation and settling of metals in sediment and sludge (20). Most heavy metals (Cd, Cu, Pb, Hg, and Zn) and organic chemicals (such as PAHs) tend to bind to suspended particulate matter within the water body, often with high affinity and then they rapidly precipitate, accumulating in the sediment (21). It is therefore interesting to note that the Kishon Authority reported higher concentrations of heavy metals in the Kishon sediments during the fiscal years 2001, 2002 ,and 2003 as compared to previous values (www.kishon.org.il). These chemicals are a potential source for chronic genotoxic pollution in the Kishon River, a threat that should be considered during and following the river rehabitation plans.

579.17 ( 122.92* 765.83 ( 379.22* 1149.17 ( 305.51* 750.83 ( 750.83* 1934.17 ( 36.68* 435.83 ( 129.33 670.83 ( 224.07* 748.33 ( 383.77* 410.83 ( 87.55* 525.00 ( 131.27* 294.17 ( 99.29 1420.83 ( 317.28* 527.50 ( 168.76* 534.17 ( 272.79* 866.67 ( 497.37*

We are grateful to J. Douek and G. Paz for their assistance and helpful advice. This work was supported by a grant from the Ministry of the Environment, Israel, and by Hal’ha- the Environmental Fund, Israel.

Literature Cited

a

(µm) Mean ( SD. For locality codes, see legend to Figure 3. An asterisk (*) indicates significant difference from controls at p < 0.05 using Duncan’s test.

1328.33 ( 163.14* 1014.17 ( 107.77* 1477.50 ( 47.7* 1505.83 ( 97.03* 2618.33 ( 208.78* 1411.67 ( 80.01* 637.50 ( 155.88* 986.67 ( 75.06* 465.83 ( 104.11* 1105.00 ( 76.53* 425.83 ( 96.38* 1739.17 ( 342.90* 731.67 ( 355.32* 598.33 ( 199.44* 1211.67 ( 659.42* 1136.67 ( 51.07* 1167.67 ( 177.79* 1075.83 ( 131.11* 917.50 ( 162.25* 2457.50 ( 145.11* 1078.83 ( 186.35* 681.67 ( 114.46* 1193.33 ( 70.06* 475.00 ( 115.96* 764.17 ( 105.07* 360.83 ( 231.28 1042.50 ( 94.37* 745.83 ( 111.92* 470.00 ( 69.69* 967.50 ( 337.81* 1385.00 ( 95.88* 1560.00 ( 298.82* 1294.17 ( 268.75* 1416.67 ( 70.90* 1915.83 ( 470.37* 1095.00 ( 31.22* 899.17 ( 99.82* 1109.17 ( 163.14* 791.67 ( 204.13* 967.50 ( 162.96* 492.50 ( 109.66* 944.17 ( 115.77* 1200.00 ( 182.02* 1061.50 ( 170.25* 1247.50 ( 149.08* 468.17 ( 89.93 410.83 ( 119.30 552.50 ( 63.79 290.83 ( 62.07 587.50 ( 136.11 234.17 ( 40.49 300.00 ( 68.78 332.50 ( 45.62 283.33 ( 95.27 325.00 ( 66.66 176.67 ( 60.23 242.50 ( 51.66 191.67 ( 29.83 209.17 ( 44.88 215.83 ( 42.89 218.33 ( 12.58 not done 541.67 ( 135.12 326.67 ( 44.18 576.67 ( 159.73 221.67 ( 71.43 338.33 ( 196.09 307.50 ( 78.58 227.50 ( 28.17 370.00 ( 80.47 243.33 ( 110.46 395.33 ( 17.02 235.83 ( 39.55 200.83 ( 43.04 186.67 ( 60.02 Jan 2001 Mar 2001 May 2001 Jul 2001 Sep 2001 Nov 2001 Jan 2002 Mar 2002 May 2002 Jul 2002 Sep 2002 Nov 2002 Jan 2003 Mar 2003 May 2003

766.67 ( 51.26* 827.50 ( 152.87* 1014.17 ( 233.88* 757.50 ( 155.06* 1842.50 ( 453.35* 966.67 ( 257.59* 628.33 ( 215.36* 1039.17 ( 17.74* 733.33 ( 70.06* 967.50 ( 45.21* 620.83 ( 197.88* 838.33 ( 270.19* 797.50 ( 97.44* 753.33 ( 16.07* 781.67 ( 99.89*

cumulative tail length at FHD FHS AKW DMEM sampling date

TABLE 4. Genotoxicity of Kishon River Samples Measured by the Parameter of Cumulative Tail Lengtha

YB

HB

KH

Acknowledgments

(1) Herut, B.; Kress, N.; Hornung, H. Nutrient pollution at the lower reaches of Mediterranean coastal rivers in Israel. Water Sci. Technol. 2000, 42, 147-152. (2) Herut, B.; Kress, N. Particulate metals contamination in the Kishon River estuary, Israel. Mar. Pollut. Bull. 1997, 34, 706711. (3) Richter, E. D.; Friedman, L. S.; Tamir, Y.; Berman, T.; Levy, O.; Westin, J. B.; Peretz, T. Cancer risks in naval divers with multiple exposures to carcinogen. Environ. Health Perspect. 2003, 111, 609-617. (4) Herut, B.; Hornung, H.; Kress, N. Mercury, lead, copper, zinc and iron in shallow sediments of Haifa Bay, Israel. Fresenius Environ. Bull. 1994, 3, 147-151. (5) Krumgalz, B. “Fingerprints” approach to the identification of anthropogenic trace metal sources in the nearshore and estuarine environments. Estuaries 1993, 16, 488-495. (6) Kress, N.; Herut, B.; Shefer, E.; Hornung, H. Trace element levels in fish from clean and polluted coastal marine sites in the Mediterranean Sea, Red Sea and North Sea. Helgoland Mar. Res. 1999, 53, 163-170. (7) Herut, B.; Kress, N.; Shefer, E.; Hornung, H. Trace element levels in mollusks from clean and polluted coastal marine sites in the Mediterranean, Red and North Seas. Helgoland Mar. Res. 1999, 53, 154-162. (8) Ministry of the Environment (MoE), State of Israel. Kishon River Masterplan Report; 2001; pp 1-21. (9) Brigden, K.; Stringer, R. In Greenpeace. A Critical Assessment of the Kishon River Masterplan Report Published by the State of Israel Ministry of Environment, July 2001; Technical Note 03/ 2002; Exeter, UK, 2002; pp 1-23. (10) Avishai, N.; Rabinowitz, C.; Moiseeva, E.; Rinkevich, B. Genotoxicity of the Kishon River, Israel: the application of an in vitro cellular assay. Mutat. Res. 2002, 399, 135-147. (11) Kamer, I.; Rinkevich, B. In vitro application of the comet assay for aquatic genotoxicity: considering a primary culture versus a cell line. Toxicol. in Vitro 2002, 16, 177-184. (12) Rinkevich, B.; Rabinowitz, C. In vitro culture of blood cells from the colonial protochordate Botryllus schlosseri. In Vitro Cell. Dev. Biol. 1993, 29A, 79-85. (13) Fryer, J. L.; Maccaine, B. B.; Leong, J. C. A cell line derived from Rainbow trout (Salmo gairdneri) hepatoma. Fish Pathol. 1981, 15, 193-200. (14) Singh, N. P.; McCoy, M. T.; Tice, R. R.; Scheider, E. L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 1988, 175, 184-191. (15) Avishai, N.; Rabinowitz, C.; Rinkevich, B. Use of the comet assay for studying environmental genotoxicity: Comparisons between visual and image analyses. Environ. Mol. Mutagen. 2003, 42, 155-165. (16) Tice, R. R.; Agurell, E.; Anderson, D.; Burlinson, B.; Hartmann, A.; Kobayashi, K.; Miyamae, E.; Rojas, E.; Ryu, J. C.; Sasaki, Y. F. Single cell gel/ comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 2000, 35, 206-221. (17) Hartmann, A.; Agurell, E.; Beevers, C.; Brendler-Schwaab, S.; Burlinson, B.; Clay, P.; Collins, A.; Smith, A.; Speit, G.; Thybaud, V.; Tice, R. R. Recommendations for conducting the in vivo alkaline comet assay. Mutagenesis. 2003, 18, 45-51. (18) Pick, C.; Fomin, A.; Paschke, A. Use of Tradescantia-bioassays to detect the genotoxicity of pH-unmodified environmental samples Mutat. Res. 1997, 379 (Suppl. 1), S158. (19) Witters, H. E. Chemical speciation dynamics and toxicity assessment in aquatic systems. Ecotoxicol. Environ. Saf. 1998, 41, 90-95. (20) Cohen, Y.; Kress, N.; Hornung, H. Organic and trace metal pollution in the sediments of the Kishon River (Israel) and possible influence on the marine environment. Water Sci. Technol. 1993, 27, 7-8. (21) ASTDR (Agency for Toxic Substances and Disease Registry). Toxicological profiles on CD-ROM Version 3.1; U.S. Public Health Service: Washington, DC, 2000.

Received for review November 13, 2003. Revised manuscript received April 13, 2004. Accepted April 21, 2004. ES035264A VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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