5. Results

5.1 Effects of Microcystin-LR

5.1.1 Mean motility over the whole exposure period

▼ 54 (continued)

A rapid decrease of mean motility of Danio rerio as well as of Leucaspius d e lineatus was noted at the highest MC-LR concentration of 50 µg l-1 which was statistically significant (Fig. 7). In contrast, lower MC-LR concentrations (5 µg l1, 15 µg l1) tended to increase the motility of Danio rerio but this was not statistical relevant. However, the overall motility of Leucaspius delineatus increased significantly at concentrations of 0.5 µg l-1, 5 µg l-1 and 15 µg l-1.

Fig.7 Average motility of Danio rerio and Leucaspius delineatus exposed to MC-LR over the whole measuring time per day (23 h per day) and the whole exposure period. Means and 95% confidence intervals are shown.

5.1.2 Mean motility during light and dark phases

▼ 55 

With Danio rerio the lowest MC-LR exposure concentration of 0.5 µg l-1 led to a significant increase in motility during the light phases and a significant decrease during the dark phases. Elevated MC-LR exposure (50 µg l-1) led to significantlydecreased motility during the light as well as dark phase (Fig. 8). The overall influence of MC-LR is also illustrated by the daily activity curves (Fig. 9). It is shown that the significant MC-LR induced changes of activity of Danio rerio occurred especially shortly after the onset of light which is the period of mating and spawning behaviour for Danio rerio. The changes in the time interval of three hours after the onset of light were statistically significant for the lowest test concentration of 0.5 µg l-1 which led to an increase of motilityand for the highest MC-LR concentration of 50 µg l-1 which led to a decrease of motility (Fig. 10).

Fig.8 Average motility of Danio rerio and Leucaspius delineatus divided in light (white bars) and dark phases (grey bars) over the whole period of exposure to MC-LR. Means and 95% confidence intervals are shown.

With Leucaspius delineatus the lowest exposure concentration of 0.5 µg l-1 caused a significant increase in motility during both the light and dark phase. During the dark phases, the motility of Leucaspius delineatus also increased significantly at 5 µg l-1 and15 µg l-1 (Fig. 8).

▼ 56 

Fig.9 Smoothed curve of average motility of Danio rerio and Leucaspius delineatus over the whole period of exposure to MC-LR.

The concentration of 15 µg l-1 led to a significant decrease of motility during light phases. At the highest concentration (50 µg l-1) the motility during light phases decreased significantly, as was shown for Danio rerio. In contrast to Danio rerio, the motility of Leucaspius delineatus increased significantly during dark phases. Also for Leucaspius delineatus it is shown that significant MC-LR induced changes of activity occurred shortly after the onset of light (Fig. 9).

The changes in this time interval of three hours after the onset of light were statistically significant for the lowest test concentration of 0.5 µg l-1 which led to an increase of motilityand for the higher MC-LR concentration of 15 and
50 µg l-1 which led to a decrease of motility (Fig. 10).

▼ 57 

Fig.10 Average motility of Danio rerio and Leucaspius delineatus during the time interval of 3 hours after the onset of light over the whole period of exposure to MC-LR. Means and 95% confidence intervals are shown.

5.1.3 Temporal development of mean motilities

For analysing the temporal development of mean motilities the whole period of exposure to MC-LR (17 days) was divided into five time intervals of three days each and one interval of two days. The mean motility of every exposure group was calculated for each of the 6 intervals, distinguished between daylight and nighttime activity. With Danio rerio during light phases, the observed increase in motility caused by the lowest concentrations (0.5 µg l-1) occurred from the fourth exposure interval on and did not disappear up to the last exposure interval (Fig. 11). During dark phases, the motility at the exposure concentration of 0.5 µg l-1 decreased significantly already in the second exposure interval whereas significant changes did not exist in the last exposure interval. During the light phase MC-LR in a concentration of 5 µg l-1 caused also an increase in motility from the fourth exposure interval on which continued up to the last exposure interval, whereas during the dark phase there were only significant differences in the fourth exposure period. During the light phases, the MC-LR exposure with 15 µg l-1 led to a decrease of motility in the second and third interval followed by an increase of motility in the fourth and fifth interval. During the dark phases, a significant change of motility at the exposure concentration of 15 µg l-1 occurred only in the last interval, whereby the motility increased significantly. The highest MC-LR concentration (50 µg l-1)caused a significant decrease in motility in the second and third exposure interval during the light phases and a significant decrease of motility in the first and second time interval during the dark phases. After MC-LR exposure (with 50 µg l-1 the exposure was only 6 days, see 4.3.1), motility returned to control values in the fourth exposure interval.

During the light phases, the motility of Leucaspius delineatus at the concentration of 0.5 µg l-1 significantly increased from the third exposure interval up to the last interval, whereas during the dark phases the motility significantly increased over all exposure intervals (Fig. 11). The MC-LR exposure concentration of 5 µg l-1 led to a significant increase of daytime motility only in the fifth exposure interval, whereas the motility during the night was significantly increased over the whole exposure time. The MC-LR exposure concentration of 15 µg l-1 caused a significant increase in motility during the dark phases from the first to the fifth exposure interval, while during the light phases the motility was not significantly changed.

▼ 58 

At the highest exposure concentration (50 µg l1), the motility significantly decreased from the second to the fifth exposure interval during light phases, whereas

Fig.11 Differences in motility between the different exposure groups and the controls for Danio rerio and Leucaspius delineatus, divided into light (white bars) and dark phases (grey bars). The whole period of exposure to MC-LR was divided into five time intervals of 3 days each and one interval of two days. Means and 95% confidence intervals are shown.

during the dark phases the motility significantly increased only in the third time interval.At the highest exposure concentration which was terminated after 6 days, motility values returned to control levels during the last exposure interval.

5.1.4 Number of turns over the whole exposure period

▼ 59 

With Danio rerio, the number of turns was significantly decreased at the highest MC-LR concentration (50 µg l-1) during light phases, whereas during dark phases they rose statistically significant at concentrations of 5 µg l-1, 15 µg l-1 and50 µg l1 (Fig. 12).

With Leucaspius delineatus, the number of turns increased at concentrations between 0.5 µg l-1, 5 µg l-1and15 µg l-1 during light phases. In contrast to this increase, the highest exposure concentration (50 µg l-1) caused a significant decrease of the number of turns. During the dark phases, the number of turns of Leucaspius delineatus was significantly increased at all exposure concentrations (Fig. 12).

Fig.12 Average number of turns of Danio rerio and Leucaspius delineatus divided in light (white bars) and dark phases (grey bars) over the whole period of exposure to MC-LR. Means and 95% confidence intervals are shown.

5.1.5 Regression between motilities and turns

▼ 60 

The swimming mode of both species was analysed by regression between motility and turns. The relationship between motility and number of turns was best described by a linear function for both fish species (Tab. 5; Fig. 13).

Fig.13 Number of turns over motility with fitted linear function: y = a*x + b for Danio rerio and Leucaspius delineatus exposed by MC-LR.

For Danio rerio under unexposed conditions, the number of turns increased with increasing motility with r2 = 0.602 as determination coefficient. Elevated concentrations of MC-LR led to a decrease of the overall determination coefficient compared to the control for Danio rerio. At the highest MC-LR concentration of 50 µg l-1 a determination coefficient of r2 = 0.171 was found. Although this regression was still statistically significant (F = 83.7; p < 0.005) this fact indicated that the statistic relation between the increasing number of turns and increasing motility was weaker under exposure for Danio rerio. Furthermore the slope of the regression was significantly decreased at the highest MC-LR concentration compared to the control.

▼ 61 

For Leucaspius delineatus under unexposed conditions, with increasing motility, number of turns increased with a determination coefficient of r2 = 0.489 which was statistically significant (Tab. 5; Fig. 13). In contrast to Danio rerio elevated concentrations of MC-LR led to an increase of the overall determination coefficient compared to the control for Leucaspius delineatus indicating that the statistic relation between increase of number of turns and increasing motility was stronger under exposure. In contrast to Danio rerio the exposure to MC-LR (0.5, 5, 15 and 50 µg l-1) led to significant increased slopes of the regressions.

Tab. 5. Number of turns versus motility with fitted linear function: y = a*x + b
for Danio rerio and Leucaspius delineatus exposed by MC-LR;
Variables of the function: a (slope), b (intercept), r2 (determination coefficient).

  

MC-LR concentration (µg l -1 )

Species

Parameter

0.5

5

15

50

Control

Danio rerio

a (slope)

0.018

0.016

0.022

0.010

0.019

p-value

***

***

***

***

***

b (intercept)

0.259

0.294

0.192

0.443

0.217

p-value

***

***

***

***

***

r2

0.696

0.566

0.578

0.171

0.602

F-value

928.105

528.886

556.516

83.702

612.829

p-value

***

***

***

***

***

Leucaspius

delineatus

a (slope)

0.027

0.059

0.062

0.041

0.018

p-value

***

***

***

***

***

b (intercept)

0.250

-0.336

-0.302

-0.098

0.229

p-value

***

***

***

***

***

r2

0.276

0.657

0.563

0.666

0.489

F-value

139.284

702.415

471.898

730.570

349.551

p-value

***

***

***

***

***

5.1.6 
Effects of zeitgeber

Both Danio rerio and Leucaspius delineatus revealed a significant diurnal activity under unexposed conditions, since the effects of zeitgeber (which were calculated as a quotient of the motility during the light phase and the overall motility during the light and dark phase) had a value which was significantly > 0.5 (Fig. 14).

▼ 62 

Influenced by long-term exposure of MC-LR, Danio rerio remained significantly diurnally active (effects of zeitgeber > 0.5), whereas Leucaspius delineatus remained only diurnal active at the lowest MC-LR concentration of 0.5 µg l-1 (effects of zeitgeber > 0.5). At elevated concentrations of MC-LR (5 µg l-1, 15 µg l-1 and 50 µg l-1) Leucaspius delineatus reversed their prominent diurnal activity and the fish became significantly nocturnal (effects of zeitgeber < 0.5) (Fig. 14).

Fig.14 Effects of zeitgeber with 95% confidence intervals for Danio rerio and Le u caspius delineatus exposed by MC-LR. Asterisks indicate significant differences to the value of 0.5. At values between 0 and 0.5, the animals are nocturnally active and between 0.5 and 1 they are diurnally active. Means and 95% confidence intervals are shown.


5.1.7 Cosinor analysis and Polar Plots

▼ 63 

Figures 15 and 16 show the fit of cosinor regression to the original data series. F-statistics and rhythmical parameters of cosinor analysis: MESOR, amplitude, acrophase and period length, are shown in Table 6.

Obviously the exposure to MC-LR altered the motility rhythm compared to the control. For Danio rerio all tested MC-LR concentrations as well as the control showed clearly single peaks of motility (Fig. 15). In contrast, for Leucaspius d e lineatus MC-LR concentrations of 5 µg l-1, 15 µg l-1 and 50 µg l-1 led to clear double peaks of motility, whereby the control and the lowest MC-LR concentration of 0.5 µg l-1 showed single peaks of motility (Fig. 16).

For Danio rerio the lowest MC-LR concentration of 0.5 µg l-1 led to a significant increase in the circadian motility amplitude by 2.96 pix s-1 (Tab. 6). Elevated MC-LR concentration of 5 µg l-1 significantly increased the MESOR by 2.23 pix s-1. The highest test concentration of 50 µg l-1 led to a significant decrease of MESOR (by 1.83 pix s-1) as well as amplitude (by 2.2 pix s-1). The period of the activity cycles was stable in all cases without any significant differences and showed a pronounced circadian rhythm (Tab. 6).

▼ 64 

For Leucaspius delineatus an MC-LR concentration of 0.5 µg l-1 significantly increased the MESOR by 2.98 pix s-1 (Tab. 4). Elevated concentrations of 5 µg l-1 and 15 µg l-1 significantly increased the MESOR by 2.68 pix s-1 and 1.69 pix s-1, respectively. At the highest MC-LR concentration of 50 µg l-1, the MESOR was significantly smaller than that in the controls by 2.03 pix s-1. At this concentration of 50 µg l-1 theamplitude tended to decrease, but this was not statistically relevant.

The circadian period of the activity cycles was stable only at the lowest test concentration of 0.5 µg l-1. All elevated MC-LR concentrations of 5 µg l-1, 15 µg l-1 and 50 µg l-1 reduced the period length significantly and clearly revealed an ultradian rhythm with a cycle length < 20h (Tab. 6).


▼ 65 

Fig.15 Circadian motility rhythms of Danio rerio determined by the cosinor method. Comparison of control and MC-LR exposed groups. Average motilities per hour (grey line) and cosinor fitted curves (black line).

Fig.16 Circadian motility rhythms of Leucaspius delineatus determined by the cosinor method. Comparison of control and MC-LR exposed groups. Average motilities per hour (grey line) and cosinor fitted curves (black line).

Fig.17 Polarogram of cosinor analysis for Danio rerio exposed by MC-LR. Angular axis represents the amplitude [pix s-1]. Radial axis represents the time of the day [h]. Confidence ellipse covers the 95% interval.

▼ 66 

The rhythmical parameters amplitude and acrophase are graphically represented in the polar plots (Fig. 17 and Fig. 18).

For Danio rerio the acrophase delayed significantly at MC-LR concentrations of 0.5 µg l-1, 15 µg l-1 and 50 µg l-1 compared to the control group. At an MC-LR concentration of 0.5 µg l-1 the acrophase delayed for 51 min, at a concentration of 15 µg l-1 it delayed for 58 min and at a concentration of 50 µg l-1 the acrophase delayed for 2 h and 58 min.

Fig.18 Polarogram of cosinor analysis for Leucaspius delineatus exposed by MC-LR. Angular axis represents the amplitude [pix s-1]. Radial axis represents the time of the day [h]. Confidence ellipse covers the 95% interval.

▼ 67 

For Leucaspius delineatus in contrast, the acrophase advanced significantly at MC-LR concentrations of 5 µg l-1, 15 µg l-1 and 50 µg l-1. For the MC-LR concentration of 5 µg l-1 the acrophase advanced for 6 h and 53 min, for the concentration of 15 µg l-1 it advanced for 9 h and 57 min and for the concentration of 50 µg l-1 the acrophase advanced for 6 h and 12 min.

Tab. 6. Rhythmical parameters of cosinor analysis: MESOR (M), amplitude (A), acrophase (K) and period length (P) for Danio rerio and Leucaspius delineatus exposed by microcystin-LR (* > means that the value of the exposure group is significantly higher and * < means that the value of the exposure group is significantly lower compared to the control at the 0.05% level; n.s. means that there are no significant differences between the exposure group and the control group). The F- and p-values indicate the overall difference between the exposure group and the control.

  

MC-LR concentration (µg l -1 )

          

Species

Parameter

0.5

5

15

50

Contr.

      

Danio r e rio

F-value

2087.2

2739.7

2729.7

1441.6

2032.2

      

p-value

0.000

0.000

0.000

0.000

0.000

      

M (pix s-1)

19.73

n.s.

21.27

* >

19.25

n.s.

17.21

* <

19.04

  

A (pix s-1)

10.38

* >

7.59

n.s.

5.67

n.s.

5.22

* <

7.42

  

K (hours)

12:03

* >

11:22

n.s.

12:10

* >

14:10

* >

11.12

  

P (hours)

22:55

n.s.

22:03

n.s.

26:21

n.s.

23:21

n.s.

25.14

  

Leucaspius

delineatus

F-value

3385.5

4188.63

3908.6

1912.8

2929.1

      

p-value

0.000

0.000

0.000

0.000

0.000

      

M (pix s-1)

18.14

* >

17.84

* >

17.12

* >

13.13

* <

15.16

  

A (pix s-1)

2.34

n.s.

2.69

n.s.

1.60

* <

2.53

n.s.

3.24

  

K (hours)

9:29

n.s.

3:41

* <

0:37

* <

4:22

* <

10.34

  

P (hours)

24:55

n.s.

15:00

* <

17:29

* <

15:34

* <

24.23

  

5.1.8 Periodic frequency analysis

Like the cosinor analysis the power spectral analysis (Fig. 19) revealed that there is a significant circadian component (of 24 hours) which determined the swimming activity of Danio rerio and Leucaspius delineatus under unexposed conditions.

▼ 68 

For Danio rerio the periods of 24 hours, 12 hours, 8 hours and 6 hours explained a significant proportion of the total variation under unexposed conditions. For all exposed groups of Danio rerio, the significance of these rhythms remained, whereby there was additionally a significant period of 4.8 hours compared to the control. For Danio rerio the explained proportion of the 24 hours period increased at lower MC-LR concentrations of 0.5 and 5 µg l-1 and decreased at elevated MC-LR concentrations of 15 and 50 µg l-1 (Fig. 19). The power spectrum of MC-LR concentrations of 15 µg l-1 isequal to that of the control for Danio rerio.

For Leucaspius delineatus the periods of 24 hours, 12 hours, 8 hours, 6 hours and 4.8 hours explained a significant proportion of the total variation under unexposed conditions. The significance of the rhythm components of 24 hours and 12 hours remained for all exposed groups. However the amplitude of circadian rhythm decreased under exposure and furthermore at elevated MC-LR concentrations of 5 µg l-1, 15 µg l-1 and 50 µg l-1 the dominance of circadian rhythms (τ = 24 h) was clearly reduced and simultaneously the proportion of a harmonic oscillation with a 12 hours rhythm increased.

Fig.19 Power spectrum of motility of Danio rerio and Leucaspius delineatus exposed to MC-LR.

▼ 69 

For the MC-LR experiment with Danio rerio the DFC (Degree of Functional Coupling) was 90.4% and the harmonic portion was 68.4% for the controls (Tab. 7). As the explained proportion of the 24 hours period the DFC and the harmonic portion increased at lower MC-LR concentrations of 0.5 and 5 µg l-1 and decreased at elevated MC-LR concentrations of 15 and 50 µg l-1.

For Leucaspius delineatus the DFC was 76.8% and the harmonic portion was 39.2% for the control. In all MC-LR exposed groups both DFC value and harmonic portion were decreased (Tab. 7).

Tab. 7. Degree of Functional Coupling (DFC) and the harmonic portion (HP) of Danio rerio and Leucaspius delineatus exposed by MC-LR.

  

MC- LR concentration (µg l -1 )

Species

Parameter

0.5

5

15

50

Control

Danio r e rio

DFC [%]

98.37

93.54

89.43

67.52

90.44

HP [%]

85.25

70.54

64.57

37.82

68.40

Leucaspius

delineatus

DFC [%]

49.53

52.93

69.15

59.26

76.78

HP [%]

20.16

20.24

16.31

23.05

39.25

5.2 
Effects of Trichlorobiphenyl

5.2.1 Mean motility over the whole exposure period

▼ 70 

The mean motility of Danio rerio was significantly decreased when exposed to PCB 28 at concentrations of 100 µg l-1 and 150 µg l-1 (Fig. 20).

Fig.20 Average motility of Danio rerio and Leucaspius delineatus exposed by PCB 28 over the whole measuring time per day (23 h per day) and the whole exposure period. Means and 95% confidence are shown.

For Leucaspius delineatus the highest test concentration of 150 µg l-1 ledto asignificant decrease of motility, too, whereas the lower PCB 28 concentration of 150 µg l-1 did not change the motility significantly (Fig. 20). Comparing the effects of PCB 28 between the duplicates of both fish species there were no significant differences at the 0.05 level between each duplicates of 100 µg l-1 and 150 µg l-1 and between the control groups.

5.2.2 Mean motility during light and dark phases

▼ 71 

With Danio rerio the lower PCB 28 exposure concentration of 100 µg l-1 led to a significant decrease in motility during the light phase and dark phase over the whole exposure period (Fig. 21). The elevated PCB 28 exposure of 150 µg l-1 led to a significantly decreased motility during the light phase. As it is shown in the daily activity curves (Fig. 23) the reaction of Danio rerio to the onset of light indicated the influence of PCB 28 very clearly. For three hours after the onset of light the motility of Danio rerio was significantly reduced under the influence of PCB 28 for both test concentrations of 100 µg l-1 and 150 µg l1 (Fig. 22).

With Leucaspius delineatus a PCB 28 concentration of 100 µg l-1 caused a significant decrease in motility during the light phase, whereas the motility was significantly increased during the dark phase. At the concentration of 150 µg l-1 the motility of the light as well as dark phase was significantly decreased (Fig. 22). As it was shown for Danio rerio the reaction of Leucaspius delineatus to the onset of light was significantly reduced under PCB 28 exposure (Fig. 22, Fig. 23).

Fig.21 Average motility of Danio rerio and Leucaspius delineatus divided in light (white bars) and dark phases (grey bars) over the whole period of exposure to PCB 28. Means and 95% confidence intervals are shown.

▼ 72 

Fig.22 Average motility of Danio rerio and Leucaspius delineatus during the time interval of 3 hours after the onset of light over the whole period of exposure to PCB 28. Means and 95% confidence intervals are shown.

Fig.23 Smoothed curve of average motility of Danio rerio and Leucaspius delineatus over the whole period of exposure to PCB 28.

5.2.3 
Temporal development of mean motilities

For analysing the temporal development of mean motilities the whole period of exposure to PCB 28 was divided into four time intervals of two days each. The mean motility of every exposure group was calculated for each of the four intervals, distinguished between daylight and nighttime activity.

▼ 73 

Fig.24 Differences in motility between the different exposure groups and the controls for Danio rerio and Leucaspius delineatus, divided into light (white bars) and dark phases (grey bars). The whole period of exposure to PCB 28 was divided into four time intervals of 2 days each. Means and 95% confidence intervals are shown.

With Danio rerio during light phases, the decrease in motility caused by the PCB 28 concentration of 100 µg l-1 occurred from the first to the third exposure interval and disappeared in the last exposure interval. During dark phases, the motility at the exposure concentration of 100 µg l-1 decreased significantly only in the second exposure interval. During the light phase a PCB 28 concentration of 150 µg l1 caused a decrease in motility from the first exposure interval which continued up to the last exposure interval, whereas during the dark phase there were no significant differences in any exposure period (Fig. 24).

During the light phase, the motility of Leucaspius delineatus at the concentration of 100 µg l-1 significantly decreased only in the second exposure interval and only for this second interval the motility during the dark phase was significantly increased. The PCB 28 exposure concentration of 150 µg l-1 led to a significant decrease of daytime motility in all four exposure intervals, whereas the motility during the night was significantly decreased in the first, third and last exposure interval (Fig. 24).

5.2.4 Number of turns over the whole exposure period

▼ 74 

With Danio rerio, the number of turns was significantly decreased at the PCB 28 concentrations of 100 µg l-1 and of 150 µg l-1 during the light phases (Fig. 25). Only at the higher PCB 28 concentration of 150 µg l-1 the number of turns was significantly increased during the dark phase.

With Leucaspius delineatus, the number of turns did not change significantly at the lower PCB 28 concentration of 100 µg l-1 (Fig. 25). In contrast, the higher exposure concentration of 150 µg l-1 caused a significant decrease of the number of turns during both the light and dark phases.

Fig.25 Average number of turns of Danio rerio and Leucaspius delineatus divided in light (white bars) and dark phases (grey bars) over the whole period of exposure to PCB 28. Means and 95% confidence intervals are shown.

5.2.5 Regression between motilities and turns

▼ 75 

For analysing the swimming mode a regression between motility versus number of turns was fitted. With increasing motility the number of turns increased in all controls and PCB 28 exposed test groups of both species, described by a linear function (Tab. 8, Fig. 26). For Danio rerio under unexposed conditions, the number of turns increased with increasing motility with an determination coefficient of r2 = 0.939. The determination coefficient, the slope and the intercept did not change significantly under exposure of PCB 28 for Danio rerio. This indicated a similar statistic relation between the increasing number of turns and increasing motility of the exposed groups compared with the control.

For Leucaspius delineatus under unexposed conditions, with increasing motility, number of turns increased with an determination coefficient of 0.382 (Tab. 8, Fig. 26). In contrast to Danio rerio elevated concentrations of PCB 28 led to an increase of the overall determination coefficient compared to the control for Le u caspius delineatus indicating that the statistic relation between increase of number of turns and increasing motility was stronger under exposure. The slopes of the regressions were significantly increased under PCB 28 exposure.

Tab. 8. Number of turns over motility with fitted linear function: y = a*x + b for Danio rerio and Leucaspius delineatus exposed by PCB 28.

 

PCB 28 concentration (µg l -1 )

Species

Parameter

100

150

Control

Danio rerio

a (slope)

0.052

0.051

0.053

p-value

***

***

***

b (intercept)

-0.144

-0.106

-0.142

p-value

***

***

***

r2

0.951

0.845

0.939

F-value

3513.222

995.004

2801.652

p-value

***

***

***

Leucaspius

delineatus

a (slope)

0.040

0.050

0.022

p-value

***

***

***

b (intercept)

0.033

-0.121

0.283

p-value

 

*

***

r2

0.424

0.500

0.382

F-value

134,020

182,020

112,405

p-value

***

***

***

▼ 76 

Fig.26 Number of turns over motility with fitted linear function: y = a*x + b for Danio rerio and Leucaspius delineatus exposed by PCB 28.


5.2.6 Effects of zeitgeber

Both Danio rerio and Leucaspius delineatus revealed a significant diurnal activity under unexposed conditions since the effects of zeitgeber (which were calculated as a quotient of the motility during the light phase and the overall motility during the light and dark phase) had a value which is clearly higher than 0.5 (Fig. 27). Influenced by PCB 28 Danio rerio and Leucaspius delineatus remained significantly diurnal active. For Danio rerio and Le u caspius delineatus the values of the effects of zeitgeber were significantly reduced for the exposure groups (100 and 150 µg l-1) compared to the control groups.

▼ 77 

Fig.27 Effects of zeitgeber with 95% confidence intervals for Danio rerio and Le u caspius delineatus exposed by PCB 28. Asterisks indicate significant differences to the value of 0.5. At values between 0 and 0.5, the animals are nocturnal active and between 0.5 and 1 they are diurnal active. Means and 95% confidence intervals are shown.


5.2.7 Cosinor analysis and Polar Plots

The rhythmical parameters of cosinor analysis: MESOR, amplitude, acrophase and period length, are shown in Table 9. Figure 28 and Figure 29 show the fit of cosinor regression to the original data series of the PCB 28 test. The rhythmical parameter amplitude and acrophase are graphically represented in the polar plots (Fig. 30).

▼ 78 

The exposure to PCB 28 altered the expression of the motility rhythm of both fish species compared with the control values.

Tab. 9. Rhythmical parameters of cosinor analysis: MESOR (M), amplitude (A), acrophase (K) and period length (P) for Danio rerio and Leucaspius delineatus exposed by PCB 28. (* > means that the value of the exposure group is significantly higher and * < means that the value of the exposure group is significantly lower compared to the control at the 0.05 % level; n.s. means that there are no significant differences between the exposure group and the control group). The F- and p-values indicate the overall difference between the exposure group and the control.

  

PCB 28 concentration (µg l -1 )

       

Species

Parameter

100

150

Contr.

     

Danio r e rio

F-value

2822.4

3098.6

2662.1

     

p-value

0.000

0.000

0.000

     

M (pix s-1)

14.79

* <

13.88

* <

16.71

   

A (pix s-1)

4.75

* <

2.71

* <

5.68

   

K (hours)

9:10

n.s.

8:38

n.s.

9:27

   

P (hours)

22:55

n.s.

28:19

n.s.

23:05

   

Leucaspius

delineatus

F-value

2578.8

3673.37

1470.1

     

p-value

0.000

0.000

0.000

     

M (pix s-1)

14.72

n.s.

12.59

* <

14.87

   

A (pix s-1)

1.15

* <.

1.34

* <

3.13

   

K (hours)

8:09

n.s.

6:52

* <

8:28

   

P (hours)

23:33

n.s.

23:58

n.s.

22:46

   

Fig.28 Circadian motility rhythms of Danio rerio determined by the cosinor method. Comparison of control and PCB 28 exposed groups. Average motilities per hour (grey line) and cosinor fitted curves (black line).

▼ 79 

For Danio rerio a PCB 28 concentration of 100 µg l-1 led to a significant decrease in the MESOR by 1.93 pix s-1 as well as the circadian motility amplitude by 0.93 pix s-1 (Tab. 9).#SYMBOL##SYMBOL#The elevated PCB 28 concentration of 150 µg l-1 led to a significant decrease of MESOR by 2.83 pix s-1 and amplitude by 2.97 pix s-1. The period of the activity cycles was stable in all cases without any significant differences and showed a pronounced circadian rhythm. Furthermore the rhythmical parameter acrophase did not change significantly influenced by PCB 28.


Fig.29 Circadian motility rhythms of Leucaspius delineatus determined by the cosinor method. Comparison of control and PCB 28 exposed groups. Average motilities per hour (grey line) and cosinor fitted curves (black line).

▼ 80 

For Leucaspius delineatus a PCB 28 concentrations of 100 µg l-1 significantly decreased the amplitude by 1.98 pix s-1 whereas MESOR, acrophase and period length did not change significantly. A PCB 28 concentration of 150 µg l-1 significantly decreased both MESOR (by 2.28 pix s-1) and amplitude (by 1.79 pix s-1). The acrophase advanced significantly for 1 h and 36 min at the concentration of 150 µg l-1. Furthermore at this concentration the period length tended to increase with a difference to the control group of 1 h and 12 min, but this change was not significant.


Fig.30 Polarogram of cosinor analysis for Danio rerio and Leucaspius delineatus exposed by PCB 28. Angular axis represents the amplitude [pix s-1]. Radial axis represents the time of the day [h]. Confidence ellipse covers the 95% interval.

5.2.8 
Periodic frequency analysis

▼ 81 

The activity of Danio rerio as well as Leucaspius delineatus was dominated by the 24 hours period under unexposed conditions. The 24 hours periodicity continued also under PCB 28 exposure, but the spectral power of the 24 hours component was dose related reduced compared to the control group for both fish species (Fig. 31).

For Danio rerio the complex of ultradian components (12, 8 and 4.8 hours period) did not change under PCB 28. In contrast, for Leucaspius delineatus the activity structure of ultradian components changed from formerly two significant periods of 12 and 8 hours to a single significant 12 hours period at a PCB 28 concentration of 100 µg l-1 and to significant 12 and 6 hours periods at a PCB 28 concentration of 150 µg l-1 (Fig. 31).

Fig.31 Power spectrum of motility Danio rerio and Leucaspius delineatus exposed to PCB 28.

▼ 82 


Tab. 10. Degree of Functional Coupling (DFC) and the harmonic portion (HP) of
Danio rerio and Leucaspius delineatus exposed by PCB 28.

  

3-PCB 28 concentration (µg l -1 )

Species

Parameter

100

150

Control

 

Danio r e rio

DFC [%]

97.30

94.76

98.60

HP [%]

80.31

69.48

83.59

Leucaspius

delineatus

DFC [%]

37.98

71.90

82.76

HP [%]

11.81

30.25

48.40

For the PCB 28 experiment with Danio rerio the DFC was 98.6% and the harmonic portion was 83.6% for the control. The DFC values of activities and the harmonic portion decreased under exposure100 µg l-1and 150 µg l-1 (Tab. 10).

▼ 83 

For Leucaspius delineatus the DFC was 82.8% and the harmonic portion was 48.4% for the control. Also for this species the DFC´s and the values for the harmonic portion of the exposed groups were lower compared to the control (Tab. 10).


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