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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:
The study was conducted between 07 April 2014 and 10 May 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch: SC00010054
Purity: 96.5%
Appearance / Physical Description: Clear colorless liquid
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and the all test groups (replicates R1 - R4 pooled) at 0 and 48 hours for quantitative analysis. Samples collected at 0 hours were frozen and analysed at the end of the test with the 48-Hour samples.
Duplicate samples were taken and stored frozen for further analysis if necessary.
Only samples at the No Observed Effect Concentration and above were analyzed.
Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial:
Preliminary solubility work conducted indicated that it was not possible to prepare a 100 mg/L stock solution in water using traditional methods of preparation e.g. ultrasonication.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. 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
Test temperature:
Water temperature was recorded daily throughout the test using a Hanna Instruments HI 93510 digital thermometer.
pH:
PH was recorded at the start and termination of the test using a Hach HQ30d Flexi handheld meter.
Dissolved oxygen:
Dissolved oxygen concentrations were recorded at the start and termination of the test using a Hach HQ30d Flexi handheld meter.
Nominal and measured concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10, and 100% v/v saturated solution.
The following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
Analysis of the test preparations at 0 hours showed measured test concentrations of the 10, 32 and 100 % v/v saturated solutions as 0.38, 1.2 to 3.7 mg/L respectively.
Details on test conditions:
Range-finding Test:
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 3 mg/L could be obtained using a saturated solution method of preparation.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10, and 100% v/v saturated solution.
An amount of test item (nominally 2250 mg) was dispersed in 22.5 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10 % v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
Experimental Preparation:
An amount of test item (nominally 2250 mg) was dispersed in 22.5 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give the required test concentration of 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 1.0, 3.2, 10 and 32% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Exposure Conditions:
In the definitive test 250 mL glass jars containing approximately 250 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then sealed to maintain dissolved test item concentrations and maintained in a temperature controlled room at approximately 20 °C with a photoperiod of 16 hours light (527 to 550 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period.

Test Organism Observations:
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits of 0.72 - 0.11
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.38 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Test
No immobilization was observed at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, 50% immobilization was observed at 10% v/v saturated solution and 100% immobilization was observed at 100% v/v saturated solution.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the 1.0 and 100% v/v saturated solution test preparations at 0 hours showed measured concentrations of 0.0479 and 3.80 mg/L respectively and at 48 hours showed measured concentrations of 0.0369 and 2.95 mg/L respectively indicating that the test item was not stable under test conditions, therefore, the definitive test was conducted in sealed vessels to reduce the loss of test item due to volatilization.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.380 to 3.72 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on 0-Hour measured test concentrations only.

Immobilization Data
The No Observed Effect Concentration after 24 and 48 hours exposure was 0.38 mg/L. The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 1.2 mg/L. The single immobilized daphnia recorded in each of the control, 1.0 and 3.2% v/v saturated solution test preparations was considered to be due to natural causes and have not been included in the statistical analysis.
During the range-finding test 50% immobilization was recorded in the 10% v/v saturated solution whereas 0% immobilization was recorded in the same concentration in the definitive test. As the definitive test design provides more reliable immobilization data when compared to the range-finding test, it is considered that the immobilization data for the 10% v/v saturated solution from the range-finding test may be anomalous.
Results with reference substance (positive control):
Positive Control
A positive control (Harlan Study Number 41400711) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
The 24 h EC50 was 0.87 mg/L with 95 % confidence limits of 0.75 - 1.0. The 24 h NOEC was 0.56 and the 24 h LOEC was 1.0.
The 48 h EC50 was 0.71 mg/L with 95 % confidence limits of 0.65 - 0.78. The 48 h NOEC was 0.56 and the 48 h LOEC was 1.0.

The No Observed Effect Concentration is based upon less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.
Reported statistics and error estimates:
The EC50 values and associated confidence limits at 24 and 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al, 1977) using the ToxCalc computer software package (ToxCalc, 1999).
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Validation Criteria

The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Water Quality Criteria

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

Observations on Test Item Solubility

The test item preparations were observed to be clear colorless solutions throughout the test.

Definitive TestVerification of Test Concentrations

The 0-Hour measured test concentrations were determined to be:Nominal Test Concentration (% v/v Saturated Solution)

0-Hour Measured Test Concentration (mg/L)

10

0.380

32

1.20

100

3.72

Immobilization Data

Analysis of the immobilization data by the trimmed Spearman-Karber method (Hamiltonet al, 1977) at 24 and 48 hours using 0-Hour measured test concentrations gave the following results:

Time (h)

EC50(mg/L)

95% Confidence limits (mg/L)

24

1.2

0.93 - 1.5

48

0.90

0.72 - 1.1

Cumulative Immobilization Data in the Range-finding Test

Nominal Concentration

(% v/v Saturated Solution)

Cumulative Immobilized Daphnia

(Initial Population: 10 Per Replicate)

 

24 Hours

48 Hours

Control

0

0

0.10

0

0

1.0

0

0

10

5

5

100

10

10

 

Cumulative Immobilization Data in the Definitive Test

Nominal Concentration

(% v/v Saturated Solution)

0-Hour Measured Test

Concentration

(mg/L)

Cumulative Immobilized Daphnia

(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

1

1

5

1.0

-

0

0

0

0

0

 0

0

0

1

0

1

5

3.2

-

0

0

0

0

0

 0

1

0

0

0

1

5

10

0.380

0

0

0

0

0

 0

0

0

0

0

0

0

32

1.2

2

2

3

3

10

 50

4

3

4

4

15

75

100

3.72

5

5

5

5

20

 100

5

5

5

5

20

100

  R1 – R4 = Replicates 1 to 4

- = Measured concentration not determined as test concentration less than the NOEC.

Water Quality Measurements

Nominal Concentration

(% v/v Saturated Solution)

Replicate

0 h

24 h

48 h

pH

mg/ O2/L

T°C

T°C

pH

mg/ O2/L

T°C

Control

R1

7.7

8.6

20

21

7.6

8.4

20

1.0

R1

7.8

8.6

20

21

7.7

8.5

20

3.2

R1

7.8

8.7

20

21

7.8

8.5

21

10

R1

7.9

8.6

20

21

7.8

8.6

21

32

R1

7.9

8.7

20

21

7.8

8.3

21

100

R1

7.9

8.7

21

21

7.9

8.5

21

  R1 = Replicate 1

Positive Control

Analysis of the immobilization data by the maximum-likelihood probit method (Finney, 1971) at 24 hours and by the trimmed Spearman-Karber method (Hamiltonet al1977) at 48 hours based on the nominal test concentrations gave the following results:

 

Time Point

(Hours)

EC50

(mg/L)

95% Confidence Limits

(mg/L)

No Observed Effect Concentration

(NOEC) (mg/L)

Lowest Observed Effect Concentration

(LOEC) (mg/L)

24

0.87

0.75 - 1.0

0.56

1.0

48

0.71

0.65 - 0.78

0.56

1.0

 

Results of analytical investigations

Preparation of Test Samples

Nominal Concentration of Test Item

[% v/v Saturated Solution]

Sample Volume

[mL]

Final Volume

[mL]

Sample Preparation Factor

F

Control

200

20

0.1

10

200

20

0.1

32

200

20

0.1

100

200

20

0.1

 

Linearity Data

Concentration of Test Item

[mg/L]

Mean Area

[counts]

0

0

0.197

7.137 x 104

0.494

1.544 x 105

0.987

2.949 x 105

4.94

1.564 x 106

9.87

3.088 x 106

10.0

3.182 x 106

29.6

9.280 x 106

59.2

1.884 x 107

 

Results for Range-Finding Samples

Timepoint

[hours]

Nominal Concentration of Test Item

cnom

[% v/v saturated solution]

Measured Concentration of Test Item in Sample Vial

x

[mg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item

c

[mg/L]

0

1

0.479

0.1

0.0479

100

38.0

0.1

3.80

48

1

0.369

0.1

0.0369

100

29.5

0.1

2.95

 

Results for Spiked Recovery Samples

Nominal Concentration of Test Item

cnom

[mg/L]

Fortified Concentration of Test Item in the Spiked Sample

cfort

[mg/L]

Measured Concentration of Test Item in the Sample Vial

x

[mg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item in the Spiked Sample

c

[mg/L]

Mean Analytical Recovery Rate

R

[%]

Precision (Relative Standard Deviation of Recovery)

[%]

0.1

0.100

0.987

0.1

0.0987

98

4.2

0.100

0.949

0.1

0.0949

0.100

0.948

0.1

0.0948

0.100

0.988

0.1

0.0988

0.100

1.05

0.1

0.105

3

3.01

29.7

0.1

2.97

98

1.4

3.01

29.0

0.1

2.90

3.01

29.5

0.1

2.95

3.01

30.1

0.1

3.01

3.01

29.3

0.1

2.93

Acceptance target

80 - 120

<10

For reporting purposes, x has been calculated from c retrospectively.

Results for Test Samples

 

Timepoint

[hours]

Nominal Concentration of Test Item

cnom

[% v/v Saturated Solution]

Measured Concentration of Test Item in Sample Vial

x

[mg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item

c

mg/L]

0 (fresh)

Control

<LOQ

0.1

<LOQ

10

3.80

0.1

0.380

32

12.0

0.1

1.20

100

37.2

0.1

3.72

48 (old)

Control

<LOQ

0.1

<LOQ

10

3.55

0.1

0.355

32

11.3

0.1

1.13

100

33.4

0.1

3.34

 

LOQ = Limit of Quantification

For reporting purposes, x has been calculated from c retrospectively.

 

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item has been investigated and gave the following results based on the 0-Hour measured test concentrations:
The 48 EC50 was 0.90 mg/L with 95 % confidence limits of 0.72 - 1.1 mg/L. The NOEC was 0.38 mg/L and the LOEC was 1.2 mg/L.
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to 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

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 3 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 % v/v saturated solution for 48 hours at a temperature of approximately 20 °C under static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore, first approximate 2 liters discarded in order to pre-condition the filter) produce a saturated solution of the test item with a measured concentration of 3.72 mg/L. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations of the 10, 32 and 100 % v/v saturated solution as 0.38, 1.2 to 3.7 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on 0-Hour measured test concentrations only. Exposure of Daphnia magna to the test item gave the following results based on the 0-Hour measured test concentrations:

The 48 h EC50 was 0.90 mg/L with 95 % confidence limits of 0.72 – 1.1. The 48 h NOEC was 0.38 and the 48 h LOEC was 1.2.

Description of key information

The short term toxicity to Daphnia of the test substance was assessed according to OECD Guidelines for Testing of Chemicals No. 202 using an acute immobilisation method.  The 48 EC50 was 0.90 mg/L with 95 % confidence limits of 0.72 - 1.1 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.9 mg/L

Additional information

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 3 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. The test item solutions were prepared by stirring an excess of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 % v/v saturated solution for 48 hours at a temperature of approximately 20 °C under static test conditions. The test vessels were sealed to maintain dissolved test item concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. The percentage mobility in the control and test item groups after 48 hours was respectively 5 (control), 5, 5, 0, 75% and 100%. The single immobilized daphnia recorded in each of the control, 1.0 and 3.2% v/v saturated solution test preparations was considered to be due to natural causes and have not been included in the statistical analysis.

Analysis of the test preparations at 0 hours showed measured test concentrations of the 10, 32 and 100 % v/v saturated solution as 0.38, 1.2 to 3.7 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on 0-Hour measured test concentrations only. Exposure of Daphnia magna to the test item gave the following results based on the 0-Hour measured test concentrations: The 48 h EC50 was 0.90 mg/L with 95 % confidence limits of 0.72 – 1.1. The 48 h NOEC was 0.38 and the 48 h LOEC was 1.2.