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Long-term toxicity to fish

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Endpoint:
fish, juvenile growth test
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose for cross-reference:
read-across source
Post exposure observation period:
No
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
37.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
lethal and non lethal effects
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 37.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
lethal and non lethal effects
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 37.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
tank average
Remarks on result:
other: not applicable
Details on results:
- Fish weights (individual and mean values) on day 0 and 28: see Table under Results
- Tank-average or pseudo-specific growth rates for periods 0-28 or 0-14 and 0-28 (if possible): see Table under Results
- Type of and number with morphological abnormalities: 0
- Type of and number with behavioural abnormalities: 0
- Other biological observations: No biological observation
- Effect concentrations exceeding solubility of substance in test medium: No
- Incidents in the course of the test which might have influenced the results: No
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
Mortality rate: the mortality rate was indicated for all test concentrations.

Regression analysis: no regression curve of the growth rate was calculated since no dose-related effects were found.

EC50 determination: no EC50-values by probit analysis were calculated since no dose-realted effects were found.

Statistical significance: one way analysis of variance (ANOVA) was carried out with the pseudo-specific growth rates, body weights and length for the determination of statistically significant differences compared to control replicates. P-values for normality and equal variance test were 0.05. The α-value for ANOVA (acceptable probability of incorrectly concluding that there is a difference) was α =0.05.

Lethal Effects

Throughout the study no mortality was observed

Non Lethal Effects

No lethal or non lethal effects were observed throughout the study.

Body Weight and Growth Rates

The mean body weight of the control fish increased from 75.5 mg to 176.5 mg (0-28 days). No statistically significant differences among the pseudo-specific growth rates were found.

Mean Body Weight [mg] of the Fish, Day 0            

 

 

Test item concentration (mean)

 

control

3.82 mg/L

12.2 mg/L

37.7 mg/L

Mean of 16 animals

75.5

73.4

75.5

70.4

coefficient of variation

14.1

13.1

12.3

13.7

           

Mean Body Weight [mg] of the Fish, Day 28          

 

 

Test item concentration (mean)

 

control

3.82 mg/L

12.2 mg/L

37.7 mg/L

Mean of 16 animals

176.5

168.5

166.5

164.7

coefficient of variation

12.9

16.0

13.0

11.1

                                                                                                                                       

Pseudo-specific Growth Rate (0 - 28 d) of the Test Fish          

 

Test item concentration (mean)

control

3.82 mg/L

12.2 mg/L

37.7 mg/L

3.41

3.50

3.11

2.95

3.70

3.58

3.67

2.83

2.72

3.28

3.53

2.64

2.81

2.73

2.92

3.24

2.79

3.09

2.30

3.84

2.89

2.49

3.18

2.73

3.17

2.59

2.20

2.80

3.07

2.72

2.19

3.09

2.63

3.32

2.35

3.66

2.25

3.06

3.21

3.19

3.79

1.96

2.63

3.05

3.53

2.27

3.10

2.49

3.48

3.41

2.85

3.23

2.75

3.41

3.03

2.55

3.40

1.82

2.36

3.58

2.25

3.93

3.83

2.85

-

No statistically significant difference to control

Validity criteria fulfilled:
yes
Conclusions:
In this study Pigment Yellow 191 did not cause any effects to Zebrafish (growth rates, lethal and non lethal effects), at the highest concentration level tested. Therefore the NOEC after 28 days of exposure was 37.7 mg/L (mean, measured).
Executive summary:

The effects of the test item on the growth rate of juvenile Zebrafish were determined according to OECD-Guideline 215 at Dr.U.Noack-Laboratorien, Sarstedt, Germany. A semi-static test with 3 renewals of the test medium respectively (3.82, 12.2, 37.7 mg/L) per week was carried out. Duration of the test was 28 days. 16 fish were exposed to the test solutions and the control respectively. No vehicles were used for dissolving the test item. Daily observation of the fish was carried out with respect to lethal and non lethal effects. Body length and weight of the fish were determined on days 0 and 28. Water quality parameters temperature, pH value, oxygen saturation were determined to be within the acceptable limits. The test item concentrations [mg/L] were measured at days 0, 7, 12, 21 (fresh medium, 0 h) and days 2, 9, 14, 23 (old medium, 48 h).

No effects were observed up to the highest concentration level tested. Therefore the NOEC = 37.7 mg/L.

Endpoint:
fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD 215 and GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 215 (Fish, Juvenile Growth Test)
Version / remarks:
revised draft document dated January 1999
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The test item concentrations [mg/L] were measured at days 0, 7, 12, 21 (fresh medium, 0 h) and days 2, 9, 14, 23 (old medium, 48 h):

day 0 day 2 day 7 day 9 day 12 day 14 day 21 day 23 mean
39.5 39.4 38.1 37.7 37.4 38.1 36.3 34.9 37.7
12.6 12.5 12.3 12.3 12.1 12.4 11.6 11.7 12.2
3.94 3.91 3.93 3.88 3.83 3.86 3.59 3.60 3.82

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: The stock solution (53 mg/L test item was weighed out) was prepared with dilution water. The stock solution (2 L) was intensively stirred (20500 rpm) for 5 min and filtrated with 0.45 µm (RC, Schleicher & Schuell) to get a saturated solution.

Dilutions of the saturated solution: 1:10, 1:3.16, 1:1
Mean measured concentrations: 3.82, 12.2, 37.7 mg/L
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Aquarium am Aegi, 30175 Hannover, Germany
- Weight at study initiation (mean and range, SD): 0.05 - 0.1 g
- Method of breeding: Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1-10 µmol photons x m-2 x s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within the last 7 days before the study started was used in the test. No disease treatment was administered throughout holding and testing.



FEEDING DURING TEST
- Food type: Brine shrimp nauplii Artemia salina eggs were purchased from Silverstar Artemia Brine Shrimp Ex, Inter Ryba GmbH Germany. Fresh cultures were prepared with salt water (NaCl 40 g/L, 2 g eggs to 1 L salt water, gentle aeration). 24 h old brine shrimp nauplii were harvested and washed with a stainless steel mesh and resuspended in dechlorinated tap water.

- Amount: The fish were fed daily ad libitum. The food was given in two feeds per day separated by at least 5 hours (workdays). One feed was applied on weekends.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Post exposure observation period:
No
Hardness:
Total Hardness [mg/L] as CaCO3

Control Water

day 0 day 7 day 14 day 21
55 68 66 60
Test temperature:
Water Parameter Temperature [°C]

. new medium old medium

Control 21.1 – 22.1 21.2 – 21.9
3.82 mg/L 21.1 – 22.1 21.0 – 22.0
12.2 mg/L 21.1 – 22.1 21.0 – 22.0
37.7 mg/L 21.2 – 22.1 21.2 – 22.1



pH:
Water Parameter pH

. new medium old medium

Control 7.07 – 7.46 7.07 – 7.46
3.82 mg/L 7.09 – 7.45 6.94 – 7.33
12.2 mg/L 7.08 – 7.45 7.01 – 7.33
37.7 mg/L 7.07 – 7.46 6.98 – 7.32
Dissolved oxygen:
Water Parameter Oxygen-saturation [%]

. new medium old medium

Control 96 – 100 % 91 – 100 %
3.82 mg/L 96 – 100 % 86 – 100 %
12.2 mg/L 97 – 100 % 83 – 100 %
37.7 mg/L 97 – 100 % 84 – 100 %



Salinity:
Not measured, freshwater
Nominal and measured concentrations:
measured concentrations see "details on sampling"
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria, loosely covered by glass tops; for days 0 - 14: 6.5 L; for days 14 to 28: 15 L
- Type (delete if not applicable): open
- Aeration: The test aquaria were gently aerated
- Renewal rate of test solution (frequency/flow rate): 3 times a week
- No. of organisms per vessel: 16 fish
- No. of vessels per concentration (replicates): one replicate
- No. of vessels per control (replicates): one replicate
- Biomass loading rate: 0.2 - 1.0 g/L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
The water is analysed biannual acc. to German tap water regulation.
- Intervals of water quality measurement: pH, temperature and oxygen saturation were measured in old and fresh media whenever a renewal took plase. Total hardness was determined weekly out of the control tank.

- Culture medium different from test medium: No



OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A daily 12 h photoperiod
- Light intensity: 0.1 - 10 µmol photonsxm-2xs-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Lethal effects: a fish was presumed to be dead if no respiratory movement and no reaction to a slight mechanical stimulus has been detected. Dead fish were removed and the feeding rate was adjusted. Non lethal effects: these include all effects on appearance, size and behaviour of the fish that made them clearly distinguishable from the control animals, e.g. different swimming behaviour, different reaction to external stimuli, changes in appearance of the fish, reduction or cessation of food intake, changes in length or body weight.
The fish were weighed to the nearest mg and measured to the nearest mm. The weighing and length measuring procedure was carried out on days 0 and 28. The fish were anaesthetised on day 0 to determine the initial weight and length. All fish were weighed as wet weights (blotted dry). They were not fed 24 h prior to test start. Only fish with weights within the range of 0.05 and 0.10 g were randomly introduced to the test vessels.

RANGE-FINDING STUDY
- Test concentrations: a preliminary test was carried out over a duration of 11 days with the saturated solution of 37.2 mg/L. the test media were renewed 3 times. No mortalities were ovserved. Based on the preliminary study three dosage levels were chosen.



Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
37.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
lethal and non lethal effects
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 37.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
lethal and non lethal effects
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 37.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
tank average
Remarks on result:
other: not applicable
Details on results:
- Fish weights (individual and mean values) on day 0 and 28: see Table under Results
- Tank-average or pseudo-specific growth rates for periods 0-28 or 0-14 and 0-28 (if possible): see Table under Results
- Type of and number with morphological abnormalities: 0
- Type of and number with behavioural abnormalities: 0
- Other biological observations: No biological observation
- Effect concentrations exceeding solubility of substance in test medium: No
- Incidents in the course of the test which might have influenced the results: No
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
Mortality rate: the mortality rate was indicated for all test concentrations.

Regression analysis: no regression curve of the growth rate was calculated since no dose-related effects were found.

EC50 determination: no EC50-values by probit analysis were calculated since no dose-realted effects were found.

Statistical significance: one way analysis of variance (ANOVA) was carried out with the pseudo-specific growth rates, body weights and length for the determination of statistically significant differences compared to control replicates. P-values for normality and equal variance test were 0.05. The α-value for ANOVA (acceptable probability of incorrectly concluding that there is a difference) was α =0.05.

Lethal Effects

Throughout the study no mortality was observed

Non Lethal Effects

No lethal or non lethal effects were observed throughout the study.

Body Weight and Growth Rates

The mean body weight of the control fish increased from 75.5 mg to 176.5 mg (0-28 days). No statistically significant differences among the pseudo-specific growth rates were found.

Mean Body Weight [mg] of the Fish, Day 0            

 

 

Test item concentration (mean)

 

control

3.82 mg/L

12.2 mg/L

37.7 mg/L

Mean of 16 animals

75.5

73.4

75.5

70.4

coefficient of variation

14.1

13.1

12.3

13.7

           

Mean Body Weight [mg] of the Fish, Day 28          

 

 

Test item concentration (mean)

 

control

3.82 mg/L

12.2 mg/L

37.7 mg/L

Mean of 16 animals

176.5

168.5

166.5

164.7

coefficient of variation

12.9

16.0

13.0

11.1

                                                                                                                                       

Pseudo-specific Growth Rate (0 - 28 d) of the Test Fish          

 

Test item concentration (mean)

control

3.82 mg/L

12.2 mg/L

37.7 mg/L

3.41

3.50

3.11

2.95

3.70

3.58

3.67

2.83

2.72

3.28

3.53

2.64

2.81

2.73

2.92

3.24

2.79

3.09

2.30

3.84

2.89

2.49

3.18

2.73

3.17

2.59

2.20

2.80

3.07

2.72

2.19

3.09

2.63

3.32

2.35

3.66

2.25

3.06

3.21

3.19

3.79

1.96

2.63

3.05

3.53

2.27

3.10

2.49

3.48

3.41

2.85

3.23

2.75

3.41

3.03

2.55

3.40

1.82

2.36

3.58

2.25

3.93

3.83

2.85

-

No statistically significant difference to control

Validity criteria fulfilled:
yes
Conclusions:
In this study Pigment Yellow 191 did not cause any effects to Zebrafish (growth rates, lethal and non lethal effects), at the highest concentration level tested. Therefore the NOEC after 28 days of exposure was 37.7 mg/L (mean, measured).
Executive summary:

The effects of the test item on the growth rate of juvenile Zebrafish were determined according to OECD-Guideline 215 at Dr.U.Noack-Laboratorien, Sarstedt, Germany. A semi-static test with 3 renewals of the test medium respectively (3.82, 12.2, 37.7 mg/L) per week was carried out. Duration of the test was 28 days. 16 fish were exposed to the test solutions and the control respectively. No vehicles were used for dissolving the test item. Daily observation of the fish was carried out with respect to lethal and non lethal effects. Body length and weight of the fish were determined on days 0 and 28. Water quality parameters temperature, pH value, oxygen saturation were determined to be within the acceptable limits. The test item concentrations [mg/L] were measured at days 0, 7, 12, 21 (fresh medium, 0 h) and days 2, 9, 14, 23 (old medium, 48 h).

No effects were observed up to the highest concentration level tested. Therefore the NOEC = 37.7 mg/L.

Description of key information

The effects of the test item on the growth rate of juvenile zebra fish were determined according to OECD-Guideline 215. A 28 d semi-static test was carried out. No effects were observed up to the highest concentration level tested. Therefore the NOEC = 37.7 mg/L (measured concentrations) 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
37.7 mg/L

Additional information