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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-31 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline No. 202. 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 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 6341 15 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
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:
- Chemical analyses: Samples for analysis were taken from the control, the highest test concentration with no effect and the lowest test concentration causing 100% immobilization after 48 hours.
- Frequency of sampling: At the start (t=0h) and the end of the test (t=48h).
- Volumes of approximately 1 mL were taken for each sample.
- 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 standard test procedures required preparation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures. In this respect, pre-experiments were performed to determine the stability of the test item in test water and to select suitable methods for the preparation of a stock solution and the dosage of the test item into the test water. These pre-experiments were not performed in compliance with GLPRegulations, but the raw data of these determinations are archived under the project number of the present study (data not shown). The method of preparation of the test solutions was also based on data of the test substance supplied by the sponsor. The test sample of Nopol formed a 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-1 for the definitive test. For this purpose, 100 mg (nominal) of the test item (actual weight: 100.08 mg) were carefully added to 1 L of test water in a sealed glass vessel of 1 L capacity containing a magnetic stirring bar. The mixing was performed during 24 hours in the dark. Following mixing, the contents of the vessel was 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 the test tubes. Each prepared concentration was inverted several times before filling of test tubes to ensure adequate mixing and homogeneity. The final test solutions were all clear and colourless, and remained unchanged during the 48 hours of incubation.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass beakers containing purified drinking water (Composition (mg/L): Calcium 11.5 – Magnesium 8.0 – Sodium 11.6 – Potassium 6.2 – Silica 31.7 – Bicarbonates 71.0 – Sulfates 8.1 – Chlorides 13.5 – Nitrates 6.3; pH = 7). During the week the stock daphnids were fed daily with a suspension of freshwater algae (mix of 3 algae strains: Chlorella vulgaris = 2.5x10^6 cells/mL/day/daphnid, Desmodesmus subspicatus = 2.5x10^6 cells/mL/day/daphnid and Pseudokirchneriella subcapitata = 5x10^6 cells/mL/day/daphnid). The water was changed once per week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

ACCLIMATION
- At least 48 hours prior the start of the test, daphnids were transferred to OECD test water at the test temperature to be used in the study. Briefly, batches of at least 50 gravid daphnids were separated from the stock population and held at similar temperature and light conditions as used in the test. During this period, daphnids were fed in the same manner as that of the stock population. Only daphnids not older than 24 hours were used for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Total water hardness was approximately 250 mg/L (as CaCO3)
Test temperature:
18.8 and 19.5°C throughout the test (average value: 19.2°C)
pH:
7.70 - 7.91
requirement: pH: 6.0-9.0, not varying by more than 1.5 units
Dissolved oxygen:
8.14 - 8.38 mg O2/L
The dissolved oxygen concentration at the end of the test was >= 8.14mg.L-1 in controls and test vessels (lowest test concentration with 100% of immobilization)
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal: 10.0, 13.0, 17.0, 22.3, 29.2, 38.2 and 50.0 mg test item.L-1
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass sealed test tubes of approximately 20 mL capacity. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Test type: static
- No. of daphnids: 20 per control and test concentration, divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each containing 20 mL of test solution
- Introduction of Daphnids: Daphnids were introduced into the test medium immediately after filling of test tubes with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202 (Stock solutions: CaCl2.2H2O 11.76 g.L-1; MgSO4.7H2O 4.93 g.L-1; NaHCO3 2.59 g.L-1; KCl 0.23 g.L-1)
- Conductivity: < 10 μS/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h darkness

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 h. Immobile animals were eliminated from the vessels as soon as they were discovered. The daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of the test vessels.
- pH and dissolved O2: At the beginning and at the end of the test, for the highest test concentration and the control.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.31
- Range finding study: Daphnids were exposed to the nominal test concentrations of 0.1, 1, 10 and 100 mg/L and to a control.
- Results used to determine the conditions for the definitive study: The percent immobility was 0, 0, 0, 0 and 100 (24 h) and 0, 0, 0, 0 and 100 (48 h) at 0, 0.1, 1, 10 and 100 mg/L, respectively. Based on the results of a range-finding test, the following nominal concentrations (spaced by a factor of approximately 1.31): 10.0, 13.0, 17.0, 22.3, 29.2, 38.2 and 50.0 mg test item.L-1 were prepared.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
38.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 35.4 - 41.5 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
28.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 22.3 - 38.2 mg/L
Details on results:
- After 24 hours of exposure, immobilizations were 0% at 0, 10, 13.0, 17.0 and 22.3 mg.L-1, 15% at 29.2 mg.L-1, 30% at 38.2 mg.L-1 and 100% at 50 mg test item.L-1.
- After 48 hours of exposure, immobilizations were 0% at 0, 10, 13.0, 17.0 and 22.3 mg.L-1, 60% at 29.2 mg.L-1 and 100% at 38.2 and 50 mg test item.L-1.
Results with reference substance (positive control):
In July 2012 (most recent test), the 24h-EC50 was 1.28 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with the OECD 202 (expected 24 h-EC50: 0.6 to 2.1 mg/L) at this time.
Reported statistics and error estimates:
The evaluation of the effects was based on the nominal test item concentrations. The determination of the EC50 was performed according to the schedule below:
- When for at least two concentrations, immobilization is > 0% and < 100%, the data are analyzed by regression to determine the EC50 including the 95% confidence interval using Probit-analysis via the computer program ToxRat.
- When at only one concentration, immobilization is > 0% and < 100%, the EC50 is also calculated by Probit-analysis via ToxRat but in this case, the highest concentration causing no immobilization and the lowest concentration producing 100% immobilization are used as confidence limits.
- If at all concentrations immobilization is 0% or 100%, the EC50 corresponds to the geometric mean of the highest concentration causing no immobilization and the lowest concentration producing 100% immobilization.

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 h in the final test

 

Nominal concentration* (mg/L)

Replicate

Number of daphnids exposed

Response at 24 h

Response at 48 h

Number

Total %

Number

Total %

Control

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

10.0

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

13.0

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

17.0

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

22.3

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

29.2

1

5

1

15

1

60

2

5

1

4

3

5

0

4

4

5

1

3

38.2

1

5

2

30

5

100

2

5

1

5

3

5

1

5

4

5

2

5

50.0  1  5  5 100  5 100
 2  5  5  5
 3  5  5  5
 4  5  5  5

Water quality parameter values throughout the test:

The results of measurement of pH and oxygen concentrations (mg.L-1) remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: 3 mg.L-1 at the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.

Furthermore, the temperature of the test medium was situated between 18.8 and 19.5°C throughout the test (average value: 19.2°C), and complied with the requirements (20°C ± 2°C, constant within 1°C).

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilized nor trapped at the surface of the water or showed signs of stress.

Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 8.14 mg/L in controls and test vessels (lowest test concentration with 100% of immobilization).

Thus the validity criteria have been respected in the present study.

Validity criteria fulfilled:
yes
Conclusions:
The test substance was found to be stable over the study period. After 48 hours of experimentation, the EC50 value determined was 28.8 mg test item.L-1 based on nominal concentrations.
Executive summary:

A study was performed to assess the acute toxicity of test item Nopol to Daphnia magna. The toxic effect of the test item was investigated in a static test, according to Guideline OECD 202, referenced as Method C.2 of Commission Regulation No. 440/2008, and was performed with GLP compliance. The criterion measured was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

 

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item at the required nominal test concentrations 10.0, 13.0, 17.0, 22.3, 29.2, 38.2 and 50.0 mg test item.L-1. The immobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours. The concentrations of the test item were determined by chemical analyses at start and at the 48-hour incubation period.

 

The test item levels were found to be relatively stable throughout the test and the evaluation of the effects on daphnids was based on the nominal concentrations. After 24 hours of exposure, immobilizations were 0% at 0, 10, 13.0, 17.0 and 22.3 mg.L-1, 15% at 29.2 mg.L-1, 30% at 38.2 mg.L-1 and 100% at 50 mg test item.L-1. After 48 hours of exposure, immobilizations were 0% at 0, 10, 13.0, 17.0 and 22.3 mg.L-1, 60% at 29.2 mg.L-1 and 100% at 38.2 and 50 mg test item.L-1. Thus, the highest concentration resulting in no immobilization at 48 hours was 22.3 mg.L-1 and the lowest concentration resulting in 100% immobilization at 48 hours was 38.2 mg.L-1. The EC50 at each of the observation times was as follows:

- 24 h EC50: 38.3 mg/L (95% Confidence limits 35.4 - 41.5 mg/L)

- 48 h EC50: 28.8 mg/L (95% Confidence limits 22.3 - 38.2 mg/L)

 

Therefore, the test substance was found to be stable over the study period. After 48 hours of experimentation, the EC50 value was determined to be 28.8 mg test item.L-1 based on nominal concentrations.

Description of key information

The 48h-EC50 of Nopol to aquatic invertebrates Daphnia magna was of 28.8 mg test item.L-1 based on nominal concentrations (analytically verified).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
28.8 mg/L

Additional information

A valid study was available to assess the EC50 (Median Effective Concentration) of test item Nopol to Daphnia magna, within a period of 48 hours. The toxic effect of the test item was investigated in a static test, according to Guideline OECD 202, referenced as Method C.2 of Commission Regulation No. 440/2008, and was performed under GLP.

The test item levels were found to be relatively stable throughout the test and the evaluation of the effects on daphnids was based on the nominal concentrations. After 24 hours of exposure, immobilizations were 0% at 0, 10, 13.0, 17.0 and 22.3 mg.L-1, 15% at 29.2 mg.L-1, 30% at 38.2 mg.L-1 and 100% at 50 mg test item.L-1. After 48 hours of exposure, immobilizations were 0% at 0, 10, 13.0, 17.0 and 22.3 mg.L-1, 60% at 29.2 mg.L-1 and 100% at 38.2 and 50 mg test item.L-1. Thus, the highest concentration resulting in no immobilization at 48 hours was 22.3 mg.L-1 and the lowest concentration resulting in 100% immobilization at 48 hours was 38.2 mg.L-1.

Therefore, the EC50 value was determined to be 28.8 mg test item.L-1 based on nominal concentrations after 48 hours of experimentation.