Genotoxic effects in the Eastern mudminnow (Umbra pygmaea) after prolonged exposure to River Rhine water, as assessed by use of the in vivo SCE and Comet assays

Chemicals

All chemicals were of proanalysis quality. Ethyl methanesulphonate (EMS) was obtained from Fluka (Buchs, Switserland). Sodium N-lauroylsarcosine, collagenase, bovine serum albumin (BSA), phosphate-buffered saline (PBS), normal melting point agarose (NMP), low-melting point agarose (LMP), HEPES, ethidium bromide, bromodeoxyuridine (BrdU), colchicine, Hoechst 33528, and Giemsa were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Potassium chloride (KCl), sodium chloride (NaCl), acetic acid, EDTA, sodium hydroxide (NaOH), phosphate and citrate salts for buffers, Triton-X-100, methanol, and DMSO were obtained from VWR International B.V. (Amsterdam, The Netherlands). Tris was obtained from Invitrogen (Breda, The Netherlands).

Experimental Setup and Exposure to River Rhine Water

After permission of the Animal Welfare Committee of Wageningen University and in collaboration with the Dutch Forest Service, 90 Eastern mudminnows (Umbra pygmaea) were collected from small ponds in the National Park “De Groote Peel,” a nature reserve near Ospel in The Netherlands. The fish were transported to the intake station for the city of Amsterdam water works “Waternet” at Nieuwegein, located at the River Rhine. To prevent stress, the fish were adapted gradually to the conditions of Rhine water and control water. During a period of 14 days, De Groote Peel water was slowly diluted with River Rhine water or ground water for the control. Fish were fed daily with frozen red mosquito larvae (chironomids) until the end of the exposure.

Fish were exposed to Rhine or control water in the same manner and at the same location as in the previous experiment in 2005 [Alink et al.,2007]. The control water was natural groundwater of drinking-water quality. The groundwater has been retained in deep aquifers for over 100 years, thus considered to be free of contaminants. Before distribution to the community as drinking water, this water is aerated and rapidly filtered through sand without treatment with chlorine or any other disinfectants. This water is ideal as control water due to lack of influence from infiltrating river water. For comparison with previous studies, sodium chloride (NaCl) was added at the exposure site to the control water to increase the conductivity to the same level as River Rhine water (∼ 700 μS/cm). The pH of both waters was around 8. There were no other relations between the control water and the Rhine water; thus, the ground water control can be considered as a laboratory blank.

Starting on December 4, 2007, 8 fish for the Comet assay and 12 fish for the SCE test were exposed to Rhine water for 42 days. Starting on January 4, 2008, four fish for the Comet assay and six fish for the SCE test were exposed to Rhine water for 11 days. Two groups of fish, 8 fish for the Comet and 12 fish for the SCE test, were kept in control water for 42 days.

The fish were kept in 100 l all-glass flow-through aquariums (Fig. 1), with a flow rate of 800 l per day and continuous aeration. Silt was removed from the Rhine water, using a sedimentation tank and filtration unit with four serially interconnected cotton candle filters (30, 10, 3, and 1 μm pore size, respectively). Only dissolved substances and substances absorbed to particles smaller than 1 μm are considered to be able to pass through the filters. A temperature of 12°C was maintained by heating the incoming Rhine and control water.

As a positive control, six fish for the SCE test and four fish for the Comet assay were exposed in separate 5 l aquariums to ethyl methanesulfonate (25 mg/l) for 3 days.

Sister Chromatid Exchange Test

The sister chromatid differentiation technique in vivo, as described by Kligerman and Bloom [1976] was used with slight modifications. For the experiments, nine fish were used for the control group, five fish were used for the group with 11 days exposure to River Rhine water, and, for the River Rhine group exposed for 42 days, eight fish were used. All the fish for the SCE test were injected intraperitoneally (i.p.) with 0.5 mg BrdU/g fish and exposed to this DNA base analog for 10 days (two cell cycles). At the end of the exposure period, the fish were injected i.p. with 0.25 mg colchicine/g fish. Ten to twelve hours later, the fish were decapitated, and the gills were removed and placed in a 0.4% hypotonic solution of KCl for 30 min. The tissues were then fixed in a methanol-acetic acid (3:1) solution. Cell preparations were made by the solid-tissue technique [Kligerman and Bloom,1977]. The cells were dried for at least 24 hr and then stained according to a modified fluorescence-plus-Giemsa method [Perry and Wolff,1974]. The preparations were first treated with Hoechst 33528 (50 μg/ml) in Sorensen's buffer (pH 7.0) for 10 min in the dark, rinsed in distilled water, and then exposed to UV radiation (HPW 125 W-T, Philips, Belgium) for 4 hr in a phosphate-citrate buffer (pH 7.0). Subsequently, the preparations were heated in 2× saline-sodium citrate buffer at 60°C for 40 min and stained in 5% Giemsa in Sorensen's buffer (pH 6.8) for 10 min. The preparations were dried for at least 48 hr, and the SCEs were scored. Scoring was done in a double-blind fashion.

Comet Assay

The alkaline Comet assay was a modification according to a procedure described for zebrafish (Danio rerio) [Schnurstein and Braunbeck,2001] of the standard method for zebra mussels (Dreissena polymorpha) [Osman et al.,2004]. After the exposure period, four fish were decapitated. For the Comet assay, the number of fish used was according to the recommendation described by Hartmann et al. [2003]. The gills of the fish and, subsequently, the cell suspensions and comet slides were kept away from strong lights to avoid photolysis. The gills were removed and placed in cold PBS buffer with 5 mM HEPES and 0.65 mM EDTA. A cell suspension of the gills was obtained by treatment with a collagenase solution for 15 min. After filtration (150-μm pore size) and centrifugation (2,000 rpm, 5°C, 5 min), the pellet was resuspended in cold PBS buffer with 5 mM HEPES, 0.65 mM EDTA, and 0.1% BSA. The cell suspension was mixed with 1% LMP agarose (37°C) and transferred to a slide precoated with 1% NMP agarose. A top layer of LMP agarose (1:1 diluted with PBS and 0.1% BSA) was added to the slide to fill in any residual holes in the second agarose layer. Per fish four slides were prepared.

The slides were placed for at least 1 hr in cold lysis buffer (2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris, 1% sodium N-lauroylsarcosine, 1% Triton-X-100, and 10% DMSO, pH 10). To produce single-stranded DNA, the slides were placed on a horizontal electrophoresis unit and covered with electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, and pH 13). After 30-min incubation time, electrophoresis was performed for 20 min at 25 V and 400 mA (Hoeffer Supersub, Pharmacia biotech). Subsequently, the slides were rinsed using neutralization buffer (0.4 M Tris, pH 7.5, and 5 min), and agarose gels were dehydrated by immersing the slides in absolute ethanol for 1–2 min. The DNA was stained for 10 min using ethidium bromide solution (20 μg/ml). The equipment used for scoring was an Olympus BH-2 fluorescence microscope (excitation wavelength 515–560 nm) with image-analysis software Comet II (Perceptive Instruments, Haverhill, UK). The software provides information about the Comet like tail length, percentage DNA in tail, and tail moment (product of the tail length and the fraction of total DNA in the tail). When very low levels of DNA damage are present, tail length is most informative [Collins et al.,2008]. Per slide, the tail length of 50 comets was measured. Scoring was done in a double-blind fashion.

Chemical Analysis

The chemical analysis of water samples from the sampling location Nieuwegein was performed at Het Waterlaboratorium, Haarlem, the Netherlands. Using solid-phase extraction, HPLC, and GCMS, a large array of organics and other micropollutants were measured. Specific information about the performed analysis can be obtained from the RIWA Annual Reports 2007 and 2008, available at www.riwa.org under Publications or from the first author.

Statistics

Mean values and standard errors of SCEs and tail lengths were determined. Each fish was considered as a test unit as described by others [Tice et al.,2000; Hartmann et al.,2003]. Within each test, the differences between groups were studied using the Student's t-test with significance levels at P < 0.05.

Water Conditions

The temperature of the water in both groups (control and Rhine group) was adjusted to 12°C during the exposure time (Fig. 2). The conductivity of the water for the control group was adjusted frequently depending on the change in conductivity of the River Rhine water. The conductivity of the River Rhine water decreased with increasing discharge of the River Rhine. The pH value remained constant over the entire period. The total hardness of the control water was 202 mg/l CaCO₃. The control water has been monitored for chemical contamination during the exposure period. The chemicals analyzed in control water in this study were all below the detection limits (data not presented).

Physical Chemical Quality Parameters of River Rhine Water

The general physical chemical quality parameters of the River Rhine water in December 2007 and January 2008 are presented in Table I together with annual means of 2007 and 2008.

In addition to the regular chemical analyses within the framework of the RIWA monitoring program, additional analyses were conducted under the surveillance screening program in operation at the Nieuwegein intake for drinking water production. A selected set of results for quantification of organics from this program is presented in Table II, in which the reporting level is 0.010 μg/l. As far as the analyzed compounds are concerned, no significant change in the overall water quality between the 42 days exposure group and the 11 days exposure group was observed. Table II also indicates which compounds analyzed are compounds listed as carcinogenic or mutagenic [Anonymous,2011] as well as which compounds are listed as carcinogenic, genotoxic, or reprotoxic [Pflaumbaum,2010]. From this, it follows that River Rhine water used for the study contains a variety of compounds of concern because of genotoxicity albeit in low concentrations.

Sister Chromatid Exchange Test

All fish showed sister chromatid differentiation. As an example, in Figure 3, a metaphase image of a gill cell of the Eastern mudminnow with sister chromatide exchange events is presented. As shown in Figure 4, after 11 days of exposure to River Rhine water, there was a significant increase in the number of SCEs per chromosome compared to the control (P = 0.007). A significant increase in the number of SCEs per chromosome was also present in gill cells of fish exposed to River Rhine water during 42 days compared to the control (P = 0.005). No significant difference in numbers of SCEs was observed between the 11 and 42 days groups (P = 0.92). From this, it was concluded that no increase of SCEs occurred after a further prolonged exposure to River Rhine water.

Comet Assay

Using the trypan blue assay, the viability of the gill-cell suspension varied between 80 and 98% for all fish used. The cell suspensions used were therefore considered acceptable for use in the Comet assay.

As shown in Figure 5, there was a tendency toward an increase in the tail length for the fish exposed to River Rhine water during 11 (P = 0.21) or 42 days (P = 0.43) compared to the groundwater 42 days, but these effects were not statistically significant. No significant difference was found between the 11 and 42 days groups (P = 0.29).