4. Material and Methods

4.1 Fish species

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The two fish species studied under laboratory conditions were the zebrafish Danio rerio and the sunbleak Leucaspius delineatus, bothof whichwere obtained from laboratory stocks.

The strain of Danio rerio that was used, was provided by Dr. Oberemm, Institute of Freshwater Ecology and Inland Fisheries (Berlin, Germany). The breeding groups consisted of about 12 wild-type females and 15 males of Danio rerio each,thatwere derived labelled as a Singapore import, from a wholesale trader. The animals were kept under standard conditions according to Westerfield (1993) and they were 160-171 days of age at the start of each experiment.

The strain of Leucaspius delineatus that was used, was provided by Dr. Jähninchen, Institute of Freshwater Ecology and Inland Fisheries (Berlin, Germany) and was laboratory reared offspring of a population from lake Malchower See (Germany). The animals were acclimated to a recirculation system at a temperature of 20°C and kept under standard conditions (adapted from Westerfield, 1993). They were 170- 185 days of age at the start of each experiment.

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For both species, 6 schools of seven adult individuals each were kept in 15-litre glass aquaria with a swimming space of 40 x 25 x 15 cm. For Danio rerio one school consisted of three females and four males. For Leucaspius delineatus the sex ratio could not be ascertained in vivo. Mean total length and mean body mass of both species are shown in Table 3.

After 3 weeks of acclimatisation to the test conditions (see 4.2), the behaviour of all fish groups was recorded under standard test conditions. Thereafter, during exposure four groups of both species were exposed to the test substances MC-LR or PCB 28 (see 4.3). New groups of animals were used for every experiment.

Tab. 3. Mean total length (TL) and mean body mass (BM) of Danio rerio and Le u caspius delineatus individuals for the tests with MC-LR and PCB 28.

 

Danio rerio

Leucaspius delineatus

 

TL [cm]

BM [g]

TL [cm]

BM [g]

MC-LR

3.45 ± 0.26

0.25 ± 0.02

4.55 ± 0.22

0.30 ± 0.02

PCB 28

3.47 ± 0.25

0.26 ± 0.03

4.57 ± 0.21

0.31 ± 0.03

4.2 Test conditions

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The experiments aimed to maintain very constant external conditions concerning water quality parameters, artificial illumination in a distinct time regime, avoidance of optical or visual perturbations and exclusion of noise and vibrations. Feeding, temperature and artificial light/dark rhythms were constant and automatically controlled. Fish were exposed to a 12:12 h light/dark rhythm (without any natural light). Six 60 W halogen lamps were used to illuminate the aquaria during the daylight phase. The light intensity on the water surface of the aquaria was 800 Lux. Infrared light (of 880 nm) was used to illuminate the arenas during the nighttime phase. This wavelength is not detectable by the visual system of fish that is limited up to a maximum of 800 nm (Douglas and Hawryshyn, 1990). Over a period of 10 minutes the light was dimmed to become gradually on or off. The animals were automatically fed with TetraMin® flakes twice a day at a ratio of 3% body mass per day, three and seven hours after light-on.

Tab. 4. Physico-chemical parameter of the aquaria water for the experiments with Danio rerio and Leucaspius delineatus under the influence of MC-LR and PCB 28.

Parameter

Unit

Measured value

Temperature

°C

26 ± 0.5 (1)

20 ± 0.5 (2)

Conductivity

µS cm-1

720 ± 10

pH- value

 

7.5-7.7

Total hardness

°dH

16.5 ± 0.5

Total organic carbon (TOC)

mg l-1

4.1 ± 0.4

Oxygen

mg l-1

7.7 ± 0.2 (1)

8.2 ± 0.3 (2)

Ammonium

mg l-1

<0.5

Iron

mg l-1

<0.03

Nitrate

mg l-1-1

5 ± 0.2

Nitrite

mg l-1

<0.03

For the experiments aerated drinking water (“Berlinwasser Holding Friedrichshagen”) was used. The physico-chemical composition of the water is listed in Table 4. Basic water quality parameters of pH, oxygen and ammonium were measured both in storage tanks and aquaria once a day. The physico-chemical parameters of the used aquarium water were constant over the exposure period and within the normal physiological ranges for fish (Schreckenbach et al., 1987, 2001; Schäperclaus, 1990).

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Tests were carried out in a flow-through system with a continuous discharge of 10 l aerated tap water per aquarium and day from storage tanks whereby the flow rates were controlled by multi-channel peristaltic pumps.

4.3 Test substances

4.3.1 Microcystin-LR

The test substance MC-LR was purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA). Purified MC-LR (10 mg) was dissolved in 1 ml of methanol and diluted in 100 ml of distilled water. To keep experimental conditions as consistent as possible, fish exposed to 0.003 % methanol in water only served as controls. A comparison of behaviour during the pre-exposure standard test conditions with behaviour of the controls during exposure revealed that the single exposition to 0.003% methanol in water had no effect on behavioural parameters.

During the exposure period both fish species were exposed to four different concentrations of MC-LR (nominal concentrations): 0.5, 5 and 15 µg l-1 for a period of 17 days each and 50 µg l-1 for a period of 6 days. The storage tank water (10 l) was renewed daily and MC-LR was added at nominal concentrations.

4.3.2 Trichlorobiphenyl

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The test substance trichlorobiphenyl was purchased from Sigma-Aldrich, Schnelldorf, Germany. PCB 28 (60 mg) was dissolved in 6 ml ethanol and diluted in 80 ml of distilled water. Fish exposed to 0.003% ethanol in water only served as controls.

During the exposure period four groups of fish were exposed to two different (nominal) concentrations of PCB 28 (duplicates): 100 and 150 µg l-1 for a period of 8 days. The storage tank water was renewed daily and PCB 28 was added at nominal concentrations. Two groups served as controls, the first group without any exposure to solvents and the second group with 0.003 % ethanol. There were no significant differences between both control groups, what revealed that the single exposition to 0.003 % ethanol in water had no effect on behavioural parameters.

4.4 Recording Procedures

Fig.4 Scheme of the equipment.

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The fish activity was monitored continuously with the automated video processing system BehavioQuant® (Spieser et al., 2000). The experimental design is shown in Figure 4. Fish were observed by infrared video cameras, one in front of each tank which were able to handle normal as well as infrared light, enabling the continuous observation even during the night. The positions of the untagged fish were recorded at a two-dimensional area, data were digitised and paths of individual fish afterwards tracked by the object recognition software. Thus it was possible to reconstruct the real movements of every fish of the school.

Fig.5 Screen shot of the movement tracks of one fish group during one measuring interval of 2 minutes. The different lines represent the single individuals.

The screen shot (Fig. 5) shows an example of the movement tracks of one fish school. Video was filmed at a frequency of 25 frames per second, and overall there were 69 measuring cycles of 2 min per day. Every picture of the experimental chamber was in real time compared point-by-point with a background reference picture. The x-y positions of recognized objects are written to disk for every measuring interval. The raw data were pre-processed and converted into tables which contained the behavioural parameter values: the motility is the swimming velocity in video-pixels per second and the turns are the number of changes of the direction per second. Motility and turns characterised the swimming activity.

4.5 Statistics and calculations

4.5.1 Analysis of mean motility and mean number of turns

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The spontaneous locomotor behaviour of Danio rerio and Leucaspius delineatus was registered 23 hours per day, and data were averaged per hour. Mean motility and mean number of turns were analysed in different ways:

over the whole measured time per day (23 h d-1) and over the whole exposure period

divided into light and dark phases over the whole exposure period

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divided into light and dark phases over intervals of the exposure period

On these bases all the results of the exposed groups were compared with those of the controls. Statistical analysis of all results was performed in SPSS 9.0. using the ANOVA procedure, as well as the Dunnett T3 post hoc test for comparison of groups with unequal variances. Generally significant differences were accepted at p < 0.05. In all figures and tables a significance level of

p < 0.05 is indicated by one asterisk,

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p < 0.01 is indicated by two asterisks, and

p < 0.005 is indicated by three asterisks.

4.5.2 Regression between motility and turns

For each exposed group and for the control a regression between motility versus number of turns was fitted. A linear model (y = a*x + b) proved to be most appropriate for the totality of evaluated relations. F-statistics were performed for testing the significance of the determination coefficients r2. Student’s t-tests were used to compare the values for slope and intercept to 0.0. Deviations between regression parameters of control and exposition in detail were evaluated by calculating and comparing their 95% confidence intervals.

4.5.3 Zeitgeber analysis

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Effects of zeitgeber (time trigger) that were calculated as a quotient of the motility during the light phase and the overall motility during the light and dark phase allows one to distinguish between diurnal and nocturnal activity rhythms of the test species. Values between 0 and 0.5 indicated that the animals were nocturnally active and values between 0.5 and 1 that they were diurnally active. Statistical evaluation was made by the Student’s t-Test to compare the group values to 0.5 and furthermore the exposed group to controls using the computer program SPSS 9.0.

4.5.4 Cosinor analysis

Oscillations are normally characterized by up and down, left and right or back and forth movements of measurable parameters (y-values) during the course of time (x-values). So they appear in a diagram as wavelike or vibration or pendulation curves.

These curves are mathematically modelled by trigonometric functions like sinus or cosinus which are adapted to the exact shape of the curve by multiplicative and additive terms for example: y = A * sin (B * t + C) (t being time, A,B,C being variables).

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In the present study circadian rhythms of locomotory activity (represented by the motility) were objectively determined by a single cosinor model using the non-linear regression procedure of the statistical program SPSS 9.0. The implementation of this method applied a simple parameterised cosinus equation to the raw data series and used an approximation by sequential quadratic optimisation:

f(x) = M + A * cos (6.283/P * (x - K)).

The variables are explained in Figure 6.

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Fig.6 Scheme of a circadian rhythm.

The acrophase is defined as phase angle corresponding to the maximal value of the rhythmic parameter studied, in the present study this was the time between local midnight and maximum peak of activity. The term inside the cosinus brackets was transformed to radians by the factor 2*π (equals 6.283) so the original time units could be used inside the equation. Calculated regression parameters were considered significant if its 95% confidence interval did not include zero.

The calculated overall solutions for exposure and control were compared by an F-test (Zar, 1996). Deviation between regression parameters of control and exposition in detail was evaluated by calculating and comparing their 95% confidence intervals.

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Polar Plots of the cosinor analysis visualise the acrophase and the amplitude with confidence intervals. The maximum value of activity is located with a 95% probability in this area of confidence. The radial length of vector is proportional to the amplitude, while its angular direction indicates the temporal localisation of acrophase in physical time.

4.5.5 Periodic frequency analysis

For quantification of the harmonic frequency structure of activity rhythms of Danio rerio and Leucaspius delineatus a power spectral analysis of the motility which is a Fourier transformed autocorrelation function was used. The calculations were performedby the mean of the program “Zeit” which application is described in Scheibe et al. (1999, 2002). Periodic frequencies which explain a significant proportion of the total variation of the original data series and which are furthermore harmonic to the circadian period were ascertained. Periods are called harmonic in a chronobiological context if their lengths are integer dividers of 24 h. All periods of the power spectra were tested for significance by the integrated function of the program “Zeit” (see Scheibe et al., 1999, 2002).

Degrees of Functional Coupling (DFCs) were used for comparison of the rhythmic structures (Sinz and Scheibe, 1976; Scheibe et al., 1999). DFCs express the percentage of the circadian component and harmonic ultradian components in relation to all significant rhythmic components of a spectrum based on the respective relative parts of their assigned variance. Therefore, the DFC describes the percentage of cyclic activity components which is synchronised with the circadian rhythm.

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The equation for the DFC is: DFC=100*SP(harm)/SP(total) with:

SP(total) being the sum of all significant periodogram ordinates (i.e. the variance assigned to significant periods) and

SP(harm) giving the sum of those periodogram ordinates which are significant and harmonic to the circadian period.

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The harmonic portion (HP) describes in contrast the percentage of the number of the circadian and their harmonic ultradian components in relation to the number of all significant periods smaller or equal to the circadian rhythm.


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