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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:
27 August to 01 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline No. 203. All validity criteria were fulfilled.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
11 January 2013
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples for analysis were taken from the control and all test concentrations at the start (t=0 h) and every 24 h thereafter in old and new solutions.
- Storage: Samples were stored in a freezer until analysis or analysed directly after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test sample of Nopol formed clear colourless solution and appeared to be completely soluble and stable when mixed with test water at the concentrations prepared.
A stock solution was prepared with a nominal concentration of 100 mg/L for the definitive test. For this purpose, 500 mg (nominal) of the test item were carefully added to 5 L of test water in a sealed glass vessel of approximately 5 L capacity containing a magnetic stirring bar. The mixing was performed during 24 hours in the dark. Following mixing, the contents of the vessel were allowed to stand undisturbed for 1 hour at room temperature. Then, the stock solution was diluted with test water to obtain the required test concentrations and filled into aquaria. Each prepared concentration was inverted several times before filling of aquaria to ensure adequate mixing and homogeneity. The final test solutions were all clear and colourless.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Elevage de la Grande Rivière - B. et C. Romano - La Fond Garel – 69490 SAINT FORGEUX
- Length at study initiation: Range-finding test: 2.80 cm (range: 2.49 to 2.99 cm); Final test: 2.37 cm (range: 2.16 to 2.56 cm)
- Feeding during test: No feeding from 24 hours prior to the test and during the total test period.

ACCLIMATION
- Acclimation period: At least 12 days after delivery under similar temperature and light conditions as used in the test.
- Validity of batch: In the batch of fish which was used for the test, mortality during the seven days prior to the start of the test was less than 5%.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
Water hardness was approximately 250 mg/L (as CaCO3)
Test temperature:
23 ± 2°C, constant within 2 °C
pH:
6 to 8.5; Preferably, should not vary by more than 0.2 units at the end of the test in any solution.
Dissolved oxygen:
> 60% of air saturation value
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal concentrations: 1.0, 1.7, 2.9, 5.0, 8.5, 14.6 and 25.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass aquaria, of 5 L capacity filled with minimum headspace (approximately 1 cm) and closed by placing a glass plate on the aquarium and sealed with vaseline.
- Aeration: No aeration of the test solutions occurred throughout the test.
- A semi-static test was performed, with daily renewal of test solutions and the control in order to maintain the dissolved oxygen concentration more than 60 % of the air-saturation value throughout the test because the test was performed in closed vessels.
- No. of organisms per vessel: 7 fish per vessel with a loading of fish not exceeding 1.0 g/L
- Introduction of Fish: Fish were introduced into the test medium immediately after filling of test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water, as prescribed by the OECD Guideline 203
- Conductivity: < 10 μS/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark
- pH, nitrate and nitrite concentration, ammonia concentration, total water hardness and dissolved oxygen: once a week.
- Temperature: every day.

EFFECT PARAMETERS MEASURED:
- Mortality: Fish were inspected at least 3, 6, 24, 48, 72 and 96 hours following the start of exposure. Dead fish recorded were removed when observed.
- pH and Dissolved O2: Measured daily in all vessels with surviving fish.
- Temperature of Medium: Measured daily in control vessel and in the highest concentration vessel with surviving fish.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.71
- Range finding study: In the range-finding test, three fish per concentration were exposed to the concentrations of 0.10, 1.0, 10 and 100 mg/L of test item and to a control.
- Results used to determine the conditions for the definitive study: 0, 0, 0, 0 and 100% mortality were observed at 0, 0.10, 1.0, 10 and 100 mg/L, respectively. Based on the results of the range-finding test, the effect (mortality) of the test item on Danio rerio was definitively tested using seven concentrations spaced by a factor of 1.71: 1.0, 1.7, 2.9, 5.0, 8.5, 14.6 and 25.0 mg/L.

OTHERS
Euthanasia: At the end of the test the surviving fish were rapidly euthanised by exposing them to benzocaine at a concentration of 200 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
11.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Overall, the fish exposed to the test concentrations were slower than fish in the control after start of the exposure. After the end of the test (t=96 h), mortality was 0% at 0, 1.0, 1.7, 2.9, 5.0 and 8.5 mg/L and 100% at 14.6 and 25.0 mg/L (nominal). Hence, the highest concentration resulting in no mortality at 96 hours was 8.5 mg/L and the lowest concentration resulting in 100% mortality at 96 hours was 14.6 mg/L. The LC50 was estimated to correspond to the geometric mean of the highest concentration causing no mortality and the lowest concentration producing 100% mortality. Thus, the LC50 determined at each time of the experiment was 11.1 mg/L based on nominal concentrations.
Results with reference substance (positive control):
ln July 2012 (most recent test), the 24h-LC50 was 179.39 mg/L. Hence, the sensitivity of this batch of Danio rerio was in agreement with internally expected 24 h-LC50.
Reported statistics and error estimates:
The evaluation of the effects was based on the nominal test item concentrations.

Analytical results:

Overall, the analytical results of the final semi-static test showed that preparation of test solutions was reproducible. Concentrations measured at the start of the test and every 24 hours thereafter in old and new solutions were generally comparable to their respective initial concentrations. Only one concentration significantly dropped below the 80% of the initial value but this concentration was not used in the LC50 calculation. Concerning the nominal test concentration of 8.5 mg/L between t=48 h and t=72 h, the relative loss to initial value was slightly above the ± 20% (23.21%). However, the initial concentration measured at t=48 h was higher than the nominal and the final concentration measured at t=72 h was lower than the nominal and did not deviate to less than 80% of the nominal. Moreover, test item stability in test water was also confirmed by the results of the simultaneously performed test in Daphnia magna (LDP Project D12-002). Thus, it was decided to evaluate the biological effects on the nominal values for this study.

 

Table 6.1.1/3: Incidence of mortality and total mortality during the final test

Cumulative Mortality

Nominal Concentrations (mg/L)

Control

1.0

1.7

2.9

5.0

8.5

14.6

25.0

M

S

M

S

M

S

M

S

M

S

M

S

M

S

M

S

t = 3 h

0

0

0

0

0

0

0

0

0

0

0

2

6

1

7

0

t = 6 h

0

0

0

0

0

0

0

0

0

0

0

2

7

0

7

0

t = 24 h

0

0

0

0

0

0

0

0

0

0

0

1

7

0

7

0

24 h Total Mortality (%)

0

0

0

0

0

0

100

100

t=48 h

0

0

0

0

0

0

0

0

0

0

0

3

7

0

7

0

48 h Total Mortality (%)

0

0

0

0

0

0

100

100

t=72 h

0

0

0

0

0

0

0

0

0

0

0

3

7

0

7

0

72 h Total Mortality (%)

0

0

0

0

0

0

100

100

t=96 h

0

0

0

0

0

0

0

0

0

0

0

2

7

0

7

0

96 h Total Mortality (%)

0

0

0

0

0

0

100

100

 

*At the start of the test, fish were dead rapidly (< 10 minutes) at the concentration of 25.0 mg/L

M: Number of dead fish; S: Number of surviving fish but with sub-lethal effects (lack of balance / swimming on the back)

The initial number of fish in each test vessel was of 7.

Validity criteria fulfilled:
yes
Conclusions:
Based on nominal concentrations, the 96-hour LC50 for Nopol is 11.1 mg/L.
Executive summary:

A study was performed to assess the acute toxicity of test item Nopol to Danio rerio, under semi-static conditions according to Guideline OECD

203 with GLP compliance.

 

Following a preliminary range-finding test, seven fish were exposed to an aqueous solution of the test item at the required nominal test concentrations: 1.0, 1.7, 2.9, 5.0, 8.5, 14.6 and 25.0 mg/L. The total test period was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined in a semi-static 96-hour test by visual observation after 24, 48, 72 and 96 hours. Actual exposure concentrations were determined by chemical analyses at the start of the test and every 24 hours thereafter in old and new solutions.

 

The test item levels were found to be relatively stable throughout the test and the evaluation of the effects on fish was based on the nominal concentrations. After the end of the test (t=96 h), mortality was 0% at 0, 1.0, 1.7, 2.9, 5.0 and 8.5 mg/L and 100% at 14.6 and 25.0 mg/L. Hence, the highest concentration resulting in no mortality at 96 hours was 8.5 mg/L and the lowest concentration resulting in 100% mortality at 96 hours was 14.6 mg/L. The LC50 was estimated to correspond to the geometric mean of the highest concentration causing no mortality and the lowest concentration producing 100% mortality. Thus, the LC50 determined at each time of the experiment was 11.1 mg/L.

 

Therefore, the 96-hour LC50 for Nopol is 11.1 mg/L based on nominal concentrations.

Description of key information

The 96-hour LC50 for Nopol to fish Danio rerio is 11.1 mg/L based on nominal concentrations (analytically verified).

Key value for chemical safety assessment

LC50 for freshwater fish:
11.1 mg/L

Additional information

A valid acute study was available to assess the acute toxicity of test item Nopol to Danio rerio, under semi-static conditions according to Guideline OECD 203 and GLP

The test item levels were found to be relatively stable throughout the test and the evaluation of the effects on fish was based on the nominal concentrations. After the end of the test (t=96 h), mortality was 0% at 0, 1.0, 1.7, 2.9, 5.0 and 8.5 mg/L and 100% at 14.6 and 25.0 mg/L. Hence, the highest concentration resulting in no mortality at 96 hours was 8.5 mg/L and the lowest concentration resulting in 100% mortality at 96 hours was 14.6 mg/L. The LC50 was estimated to correspond to the geometric mean of the highest concentration causing no mortality and the lowest concentration producing 100% mortality. Thus, the 96-hour LC50 for Nopol is 11.1 mg/L based on nominal concentrations, under the experimental conditions.