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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:
Experimental stating date: 13 March 2012. Experimental completion date: 14 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
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
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Range-finding test: a sample of each test concentration was taken for chemical analysis at 0 and 48 h in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20 °C prior to analysis. Only concentrations within the range to be used for the definitive test were analysed.

Definitive test: the concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 h. Water samples were taken from the control and the 10, 18, 32, 56 and 100 mg/L test groups (replicates R1- R4 pooled) at 0 and 48 h for quantitative analysis. Samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary. Samples at the NOEC and above only were analysed.
Vehicle:
no
Details on test solutions:
Range-finding test: an amount of test item was dissolved in reconstituted water with the aid of high shear mixing for 5 min and the volume adjusted to 1 L to give the 100 mg/L test concentration. Serial dilutions were prepared from the 100 mg/L test concentration to give thr reminder of the test series of 10, 1.0 and 0.10 mg/L. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. Ten daphnids were placed in each test and control vessel. Each 300 mL of test and control glass stoppered conical flask contained 300 mL of test media and was sealed to reduce evaporation and to prevent any loss of the test item through volatilisation. After 24 and 48 h the number of immobilised Daphnia magna were recorded.

Definitive test: an amount of the test item was dissolved in reconstituted water with the aid of high shear mixing at approximately 7500 rpm for 5 min and the volume adjusted to 2 L to give the 100 mg/L test concentration. Aliquots of the 100 mg/L test concentration were separately added to a final volume of 1 L of reconstituted water to give the 10, 18, 32 and 56 mg/L test concentrations respectively. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Age at study initiation (mean and range, SD): 1st instar
- Method of breeding: culture conditions ensured that reproduction was by parthenogenesis
- Feeding during test
- Food type: a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Frequency: Daily

ACCLIMATION
- Acclimation period: gravid adults were isolated the day before initiation of the test, such taht the young daphnids produced overnight were less than 24 h old. These young were removed from the cultures and used for testing
- Type and amount of food: a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Feeding frequency: daily

OTHER
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 h after the start of exposure. The criterion of effect used that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 sec after gentle agitation.
Hardness:
The reconstituted water had an approximate theoretical hardness of 250 mg/L as CaCO3
Test temperature:
The test vessels were maintained in a temperature controlled room at 20- 23 °C
pH:
7.8 ± 0.2
Dissolved oxygen:
Reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass stoppered conical flasks
- Type (delete if not applicable): closed to reduced evaporation and possible loss of the test item through volatilisation
- Aeration: test vessles were not aerated
- Renewal rate of test solution (frequency/flow rate): the test preparations were not renewed during the exposure period
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Other: the diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted (if necessary) with NaOH or HCl
- Photoperiod: 16 h light and 8 h darkness with 20 min dawn and dusk transition periods
- Light intensity: 359- 363 lux

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Ten daphnids were placed in each test and control vessel. Each 300 mL of test and control glass stoppered conical flask contained 300 mL of test media and was sealed to reduce evaporation and to prevent any loss of the test item through volatilisation. After 24 and 48 h the number of immobilised Daphnia magna were recorded. No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/L. However, immobilisation was observed at 100 mg/L. Therefore, test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding test: no immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/L. However, immobilisation was observed at 100 mg/L.

Chemical analysis of the test preparations at 0 and 48 h showed measured concentrations to range from 76- 79 % of nominal indicating that the test item was stable under test conditions.

Definitive test
-Immobilisation data: there was no significant immobilisation in 20 daphnids exposed to a range of test concentrations of 10, 18, 32, 56 and 100 mg/L for a period of 48 h. The No Observed Effect Concentration after 24 and 48 h exposure was 100 mg/L. Two immobilised daphnids were observed at the 18 mg/L test concentration after 48 h exposure. This immobilisation was considered to be due to natural causes rather an toxic effect given that only 10 % immobilisation was observed and no immobilisation was observed at the higher test concentrations of 32, 56 and 100 mg/L. The results of the definitive test differed slightly from those of the range-finding test in that 20 % immobilisation was observed at a test concentration of 100 mg/L in the range-finding test whereas zero immobilisation was observed at 100 mg/L in the definitive test. It was therefore considered that the immobilisation observed in the range-finding test was due to natural causes rather than a toxic effect given the low numbers of immobilised daphnids observed.
-Observations on test item solubility: the test preparations were observed to be clear, colourless solutions throughout the duration do the test.

Cumulative immobilisation data in the range-finding test

Nominal concentration (mg/L)

Cumulative immobiliseddaphnia

(initial population: 10 per replicate)

24 Hours

48 Hours

Control

0

0

0.10

0

0

1.0

0

0

10

0

0

100

2*

2*

*Immobilisation considered to be due to natural causes rather than a toxic effect given the low number of immobilised daphnids and results from the definitive test

Cumulative immobilisation data in the definitive test

Nominal concentration (mg/L)

Cumulative immobiliseddaphnia

(initial population: 5 per replicate)

24 Hours

48 Hours

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Total

%

Control

0

0

0

0

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

0

0

0

0

18

0

0

0

0

0

0

0

0

2*

0

2*

10*

32

0

0

0

0

0

0

0

0

0

0

0

0

56

0

0

0

0

0

0

0

0

0

0

0

0

100

0

0

0

0

0

0

0

0

0

0

0

0

R1-R4= Replicates 1 to 4

*Immobilisation considered to be due to natural causes rather than a toxic effect given that only 10 % immobilisation was observed and no immobilisation was observed and no immobilisation was observed at the higher loading rates of 32, 56 and 100 mg/L

Physico-chemical measurements

Temperature was maintained at 20- 23 °C throughout the test, while there was no treatment related differences for oxygen concentration or pH.

Some of the temperatures were measured slightly in excess of the 18- 22 °C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test. In addition, the temperatures deviated by more than ±1 °C during the test. This deviation was considered not to affect the integrity of the test given the temperature did not deviate by more than ±1 °C in the individual test vessels.

Verification of test concentrations

Analysis of the test preparations at 0 and 48 h showed measured test concentrations to be 103 % to 95 % of nominal respectively and so it was considered justifiable to estimate the EC50values in terms of nominal test concentrations only.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to Daphnia magna was assessed according to OECD guideline 202. The 48-h EC50 was found to be > 100 mg/L. The NOEC was 100 mg/L.
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, “Daphnia sp., Acute Immobilisation Test” referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L for 48 h at a temperature of 20 °C to 23 °C under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 h.

Results

The 48-Hour EC50for the test item to Daphnia magna based on nominal test concentrations was > 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Analysis of the test preparations at 0 and 48 h showed measured test concentrations to be 103 % and 95 % of nominal respectively and so the results are based on nominal test concentrations only.

Description of key information

The acute toxicity of the test item to Daphnia magna was assessed according to OECD guideline 202. The 48-h EC50 was found to be > 100 mg/L. The NOEC was 100 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

Introduction

A study was performed to assess the acute toxicity of the test item Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, “Daphnia sp., Acute Immobilisation Test” referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L for 48 h at a temperature of 20 °C to 23 °C under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 h.

Results

The 48-Hour EC50for the test item to Daphnia magna based on nominal test concentrations was > 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Analysis of the test preparations at 0 and 48 h showed measured test concentrations to be 103 % and 95 % of nominal respectively and so the results are based on nominal test concentrations only.