Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
red powder
Specific details on test material used for the study:
Fonnulation: Methylgelb
Chemical Name: ( 1-(2'-H ydroxyethy I )-amino-4-meth y 1-2-nitro-benzol)
Batch No.: B 3/89
Molecular mass: 196 g/mol
Purity: 98%
Consistency: red cristalline powder
Storage conditions: room temperature, dark
Water Solubility: ca. 0.16%
Hydrolytic Stability: stable

Test solutions

Details on test solutions:
Test Medium:
Dechlorinated tap \Vater from the SG dechlorination sytem with a hardness of 14 °d.H was used as test water.
Test article:
In the pre!iminary test, test solutions were prepared only once prior to the initiation of the test. For concentration Ievels of 100 mg/1 and below, a stock solution of 1000 mg/L of the test article Methylgelb was prepared with purified water. The solution was sonified for 30-45 min. The actual test concentrations were achieved by dilution of the stock solution with dechlorinated tap water. The highest concentration of 1000 mg/1 was prepared by dissolving 10 g of the test article in the final volume of 10 I of test water. During the main test, the preparation of fresh test solutions was done prior to the initiation of the test, and after 48 h. A stock solution containing 500 mg/1 of the test article Methylgelb was prepared. The stock solution was sonified for 30-45 min and the actual test concentrations were achieved by dilution of the stock solution with dechlorinated tap water.

Preparation of the test solutions
In the preliminary test, 9 Iiters each of dechlorinated tap water were placed in 12 I test vessels. For the concentrations of 100 mg/1 and below, 1 I of a ten-fold concentrated test solution was prepared from the test article stock solution. These solutions were prepared by using a 1000 ml volumetric cylinder and added to the test aquaria. To yield the highest concentration of 1000 mg/1, 10 g of the test article were suspended in 2000 ml of test water and added to 8 I of test water in the test aquarium. In the main test, 8 Iiters each of dechlorinated tap water were placed in the test vessels. For every concentration Ievel, 21 of a five-fold concentrated test solution were prepared from a test article stock solution of 500 mg/1. These solutions were added to the test aquaria.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The fish were obtained from the supplier on the 18th of July, 1990. Prior to the initiation of the test, the fish were acclimatized for a minimum of 14 days in 250 I glas Containers under static conditions with permanent filtration of the water. Beyond this, the fish were kept under identical conditions for their entire holding period. Daily controls regarding the temperature, the 02 concentration and the pH-value were performed. There were no mortalities during this period.
The totallength of the fish was 2 ± lern.
Holding Conditions:

Temperature: 25°C± 1.5 °C
02- Conc.: at least 60 % of air saturation (ca. 6.0 mg O:zll)
pH- Value: 7.0- 8.5
Hardness: 14°dH
Exposure to light: 16 h daily
Light intensity: 600- 800Lux
Feeding: every day with Tetra min(Tetra-Werke)

Study design

Test type:
semi-static
Water media type:
other: dechlorinated tap water
Total exposure duration:
96 h
Remarks on exposure duration:
Due to the low stability of the test article as established in the preliminary test, the main test was performedas a semistatic procedure with renewal of the test solutions at 48 h intervals.

Test conditions

Hardness:
14°dH
Test temperature:
25 °C +-1.5 °C
pH:
8.0 +-0.5
Dissolved oxygen:
ca. 6.0 mg O2/l
Details on test conditions:
Duration: 96h
Temperature: 25°C± 1.5°C
02- Conc.: > 60 % of air Saturation (ca. 6.0 mg Oyl)
pH- Value: 8.0±0.5
Number of animals: 10 fish at each concentration
Test volume: 10 I for 10 fish
Test vessels: for each concentration, one 121 glass Container
Exposure to light: 16 h daily
Light intensity: 600- 800Lux
Aeration: continuous aeration using a membrane pump system
and pasteur pipettes for contact with the test medium
Feeding: none

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
24 h
Dose descriptor:
LC0
Effect conc.:
36 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
LC0
Effect conc.:
36 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
72 h
Dose descriptor:
LC0
Effect conc.:
36 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
36 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
LC100
Effect conc.:
71 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
LC100
Effect conc.:
71 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
72 h
Dose descriptor:
LC100
Effect conc.:
51 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
51 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Table 2 shows the mortalities in % in dependence of the time of exposure for the main test.
After 24 h the LCo was around 36 mg/1 and remained at this value until the end of the test.
The LC100 was 71 mg/1 after 24 h and 48 h, and 51 mg/1 after 72 h and until the end of the
test.

Observations during the test
During the preliminary test phase,the test article formed a precipitate at the highest
concentration of 1000 mg/1 approx. 24 h after the initiation of the test.
At a concentration Ievel of 36 mg/1, two fish were swimming on their back on the last day of
the main test.

Reported statistics and error estimates:
Due to the nature of the results, the statistical calculation of the LC5o value was performed as a geometric mean from the LC0 and LC 100·

Any other information on results incl. tables

 Conc. mg/l

Mortality

 100

%

71

51

36

26

19

14

10

 control

%

 2 -4 h 100   100  0  0  0  0  0  0  0
 24 h  100  100  50  0  0  0  0  0
 48 h  100  100  90  0  0  0  0  0  0
 72 h  100  100  100  0  0  0  0  0  0
 96 h  100  100  100  0  0  0  0  0  0
                   

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute fish toxicity was determined for the test article Methylgelb. The Zebrafish "Brachydanio Rerio" was used as test fish.
The preliminary test determined the concentration range for the main test to be at a range between 100 and 10 mg/1. The dilutions were performed stepwise by a factor of 1.4. The LC50 for the test article Methylgelb was calculated as a geometric mean from the LC0 and LC100·
The NOEC was determined at a concentration of 26 mg/l.