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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 March - 07 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
July 17, 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Date of production: 30 November 2016
Expiration date: 30 November 2021
Analytical monitoring:
yes
Details on sampling:
The test solution used in the test was prepared by mechanical dispersion. 0.2 g of test item was dissolved in 2000 mL dilution water (ISO medium) in order to obtain the concentration of 100 mg/L. Based on the results of the non-GLP preliminary range-finding test, single concentration of 100 mg/L (limit concentration) was tested in a limit test.
Vehicle:
no
Details on test solutions:
The test item solution used in the test was prepared by mechanical dispersion. Firstly, ca. 0.5 g test item was added into 2000 mL test medium (ISO medium; see 5.3) and was placed into the ultrasonic bath for approx. 15 minutes. Thereafter, this solution was completed up to 5000 mL with ISO medium in order to obtain the concentration of 100 mg product/L.
The test solution was freshly prepared in the testing laboratory just before introduction of fish.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish (Danio rerio)
- Source: Akvárium Magazin Kft. (Pasaréti Gyula), 1222 Budapest, Dévény u. 36
- Age at study initiation: Juveniles
- Length at study initiation (length definition, mean, range and SD): 2.0 ± 1 cm.

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions: same conditions as used during exposure period
- Type and amount of food during acclimation: During holding, fish are fed with appropriate, commercial diet for fish at least three times per week until 24 hours before the test start.
- Health during acclimation: The health of the breeding is continuously monitored and any mortality or abnormal behaviour is recorded.

FEEDING DURING TEST
- The fish were not fed during the test.
Test type:
static
Water media type:
other: reconstituted water (ISO medium)
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
Fish were exposed to aqueous test media containing the test item for 96 hours test period in a static test.
Hardness:
The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).
Test temperature:
21-25 °C, constant within ± 1 °C.
pH:
6.0 - 8.5. Test carried out without adjustment of pH.
Nominal and measured concentrations:
Based on the results of the non-GLP preliminary range-finding test, single concentration of 100 mg/L (limit concentration) was tested in a limit test.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr. (total fraction)
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr. (total fraction)
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr. (total fraction)
Basis for effect:
mortality (fish)
Details on results:
Validity of the Study

There was no mortality observed in control or in the treatment group during the study.
The dissolved oxygen concentration in the test solutions did not fall below 60 % of air saturation value during the study. The dissolved oxygen concentration in the test solutions were in the range of 74.3-96.1 %.
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Test Conditions
The water temperature, pH value and dissolved oxygen concentration were determined at the start of the test and on the subsequent days (once daily) in the test concentration and the control.
Temperature: Aquariums were kept in a climatic chamber, the measured temperature was in the range of 21.6-22.7 °C during the study.
pH: The range of pH for control was between 7.59 and 7.84. The test was carried out without adjustment of pH.
Oxygen concentration: The range of oxygen concentration was between 74.3-96.1 % of the air saturation value. Test solutions were not aerated.

Analytical Results
A single concentration of 100 mg/L (limit test) and a concurrent control were tested in the main test. The concentration of the test item was analytically determined in the test item solution at the start and at the end of the experiment. The ISO Medium control samples show a small absorbance. The amount of any interfering component was less than 20 % of the lowest concentration of the calibration curve. The measured concentration of Solubilised Sulphur Black 1 was 114 mg/L at the start and 116 mg/L at the end of the test. The measured test item concentration remained within ± 20 % of the nominal during the test period of 96 hours. Therefore, the biological results are based on the nominal test item concentration.
Reported statistics and error estimates:
Due to lack of mortality and sublethal effects, no statistical analysis was performed.
Sublethal observations / clinical signs:

Cumulative mortality data in the definitive test

Test Group

Cumulative mortality
(initial population = 10 fish / test group)

24h

48h

72h

96h

Control

0

0

0

0

100 mg/L

0

0

0

0

Note: No sublethal effects occurred throughout the test in the control and the treatment groups

 

Measured and calculated data of body weight

Test Group

Measured weight
of 10 fish (g)

Calculated mean
weight of 1 fish (g)

Loading of testing aquarium
(g fish/L testing liquid)

Control

1.88

0.188

0.38

100 mg/L

2.09

0.209

0.42

 

Bodylength of test animals

Test Group

Body length [cm]

Control

2.40

2.05

2.15

2.25

2.65

2.55

2.40

2.55

2.40

2.40

100 mg/L

2.25

2.50

2.45

2.55

2.20

2.40

2.45

2.60

2.25

2.40

 

pH-values measured during the experiment

Test Group

pH

0h

24h

48h

72h

96h

Control

7.84

7.59

7.71

7.62

7.69

100 mg/L

7.75

7.70

7.73

7.71

7.66

 

Dissolved Oxygen Concentrations measured during the experiment

Test Group

Dissolved Oxygen Concentrations[%]

0h

24h

48h

72h

96h

Control

96.1

80.9

74.3

77.1

77.0

100 mg/L

95.7

83.1

80.5

84.6

83.0

Temperatures measured during the experiment

Test Group

Temperature[°C]

0h

24h

48h

72h

96h

Control

21.6

22.3

22.4

22.5

22.1

100 mg/L

21.9

22.5

22.4

22.7

22.0

Results of the Method Validation (Study No.: 805-100-2718)

Measurement repeatability
(7 parallels)

CV% < 1 %

Repeatability of the Preparation (n=5)

CV% < 2 %

Linear range

0.5 - 30 µg/mL

Correlation coefficients

> 0.999

Limit of Quantification

0.5 µg/mL

Limit of Detection

0.2 µg/mL

Recovery from distilled water
(concentration levels: 1-200 mg/mL)

94 – 116 %

Recovery from ISO medium, (5-100 mg/L)

103-106 %

Recovery from 20X AAP medium (5-100 mg/L)

107-108 %

Recovery from ultra-pure water, 350 g/L

101 %

Recovery from n-Octanol (~4 and ~40 mg/L)

89-100  %

Accuracy

≤ 4 % (samples used for calibration)

Precision

≤ 5 % (samples used for calibration;
12% at LOQ)

Stabilityindistilled water

(concentration levels: 1-200 mg/mL)
at room temperature at least for 24 hours
and at least for 3 days in refrigerator

Stable

Stability of the test item inISO mediumat the temperature 18-22°C for 2 days

Stable under test conditions

Stability of the test item in ISO medium at the temperature 18-25°C for 4 days

Stable under test conditions

Stability of the test item in 20X AAP medium at the temperature 22-26°C for 7 days

Stable under test conditions

Stock solution stability (1 mg/mL)

for at least 7 days at 5±3 °C

 

Validity criteria fulfilled:
yes
Conclusions:
In this 96-hour acute toxicity test on Zebrafish (Danio rerio) the test item Solubilised Sulphur Black 1 had no toxic effect on fish up to a concentration of 100 mg/L. Accordingly, the 96-h LC50 value was determined to be >100 mg/L. The 96-h NOEC was determined to be 100 mg total product/L, based on nominal test concentration.
Executive summary:

The acute toxicity of the test item Solubilised Sulphur Black 1 to zebrafish was determined according to OECD Guideline 203. A limit test was performed using a single concentration of 100 mg test item/L. ISO medium was used as dilution water. Fish were exposed to aqueous test media containing the test item for 96 hours in a static test. Ten fish were exposed to the test concentration and control. Fish were observed at approximately 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality.

No mortality was observed in fishes of the control group or test concentration (100 mg/L). The 96-h NOEC was determined to be 100 mg/L and the LC50 was determined to be >100 mg/L, based on nominal test concentration.

Description of key information

No mortality was observed in fishes of the control group or test concentration (100 mg/L). The 96-h NOEC was determined to be 100 mg/L and the LC50 was determined to be > 100 mg/L, based on nominal test concentration.

Key value for chemical safety assessment

Additional information

The acute toxicity of the test item Solubilised Sulphur Black 1 to zebrafish was determined according to OECD Guideline 203. A limit test was performed using a single concentration of 100 mg test item/L. ISO medium was used as dilution water. Fish were exposed to aqueous test media containing the test item for 96 hours in a static test. Ten fish were exposed to the test concentration and control. Fish were observed at approximately 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality.

No mortality was observed in fishes of the control group or test concentration (100 mg/L). The 96-h NOEC was determined to be 100 mg/L and the LC50 was determined to be > 100 mg/L, based on nominal test concentration.