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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 06 June 2016 and 09 July 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
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 hours in order to determine the stability of the test item under test conditions.

Definitive test:
The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 24 hours (fresh media) and at 24 and 48 hours (old media).
Samples were taken from the control and each test group from the bulk test preparation at 0 and 24 hours (fresh media) and at 24 and 48 hours (old media) from the pooled replicates for quantitative analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Storage:
The samples were analyzed on the day of receipt.
Vehicle:
no
Details on test solutions:
Test water:
Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined under 'Any other information on materials and methods incl. tables'.

Preliminary media preparation trial:
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. The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 9.88 mg/L could be obtained using a saturated solution method of preparation.

Range-finding Test
A nominal amount of test item (1100 mg) was dispersed in 11 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.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 3.0, 7.2, 17.3, 41.5 and 100% (v/v) saturated solution. A nominal amount of test item (1100 mg) was dispersed in 11 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 100% (v/v) saturated solution test concentration. A series of dilutions was made from this saturated solution to give further test concentrations of 3.0, 7.2, 17.3 and 41.5% (v/v) saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 24 hours (fresh media) and at 24 and 48 hours (old media).


Test organisms (species):
Daphnia magna
Details on test organisms:
Test System and Supporting Information
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 (see under 'Any other information on materials and methods incl. tables') in a temperature controlled room maintaining the water temperature at 18 to 22 °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.

Definitive test:
The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
Temperature was maintained at 21 °C to 22 °C throughout the test.
pH:
Fresh media: 7.6 - 8.1
Old media: 7.6 - 8.1
Dissolved oxygen:
Fresh media: 8.6 - 9.0 mg O2/L
Old media: 8.5 - 8.8 mg O2/L
Nominal and measured concentrations:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 3.0, 7.2, 17.3, 41.5 and 100% v/v saturated solution.
Chemical analysis of the freshly prepared test preparations at 0 and 24 hours ( see "Any other information on results incl. tables.") showed measured test concentrations to range from 0.24 to 11.2 mg/L. There was no significant change in the measured concentrations at 24 and 48 hours and so the results are based on the average freshly prepared measured test concentrations only.
Details on test conditions:
Experimental Design and Study Conduct

Preliminary Media Preparation Trial
Information provided by the Sponsor indicated that the test item was insoluble in water. 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:

Saturated Solution Preparation
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
- Centrifugation at 10000 g for 30 minutes
- Centrifugation at 40000 g for 30 minutes
- Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre-condition the filter)
- Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)

Information on measured concentrations in these trials are shown under ‘Any other information on results incl. tables’.

Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 9.88 mg/L.

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 9.88 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. A nominal amount of test item (1100 mg) was dispersed in 11 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °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 100 mL test and control vessel contained 100 mL of test media and was completely filled and sealed to reduce losses due to volatilization. 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. Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media. 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.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 3.0, 7.2, 17.3, 41.5 and 100% (v/v) saturated solution.
A nominal amount of test item (1100 mg) was dispersed in 11 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 100% (v/v) saturated solution test concentration. A series of dilutions was made from this saturated solution to give further test concentrations of 3.0, 7.2, 17.3 and 41.5% (v/v) saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 24 hours (fresh media) and at 24 and 48 hours (old media).

Exposure Conditions
As in the range-finding test 100 mL glass stoppered conical flasks containing approximately 100 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 completely filled and sealed in order to reduce losses through volatilization and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 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. Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.

Assessments

Test Organism Observations
Any immobilization or adverse reactions to exposure were recorded at 2, 4, 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 within 15 seconds after gentle agitation.

Water Quality Criteria
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter.

Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk test preparation at 0 and 24 hours (fresh media) and at 24 and 48 hours (old media) from the pooled replicates for quantitative analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Data Evaluation

Statistical Analysis
The EC50 values at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
- No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
- The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.





Reference substance (positive control):
yes
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Results are based on average freshly prepared measured test concentrations (0 and 24 h)
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Results are based on average freshly prepared measured test concentrations (0 and 24 h)
Details on results:
Range-finding Test
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given under ‘Any other information on results incl. tables’. No immobilization was observed at the test concentrations of 0.10 and 1.0% (v/v) saturated solution, however, immobilization was observed at 10 and 100% (v/v) saturated solution.
Based on this information test concentrations of 3.0, 7.2, 17.3, 41.5 and 100% (v/v) saturated solution were selected for the definitive test. Chemical analysis of the freshly prepared test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0089 mg/L, to 8.2 mg/L. Analysis of the old of expired media at 48 hours showed that measured concentrations of between less than the LOQ and 0.66 mg/L were obtained. No sample from the 100% v/v saturated solution test group after 48 hours was analyzed as 100% immobilization occurred after 24 hour exposure resulting in the test media not being renewed. Analysis of the test media suggested that the test item was unstable over the 24 hours periods between media renewals.

Definitive Test

Verification of Test Concentrations
Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 0.24 to 11.2 mg/L. There was no significant change in the measured concentrations at 24 and 48 hours and so the results are based on the average freshly prepared measured test concentrations only. The average freshly prepared measured test concentrations were determined (refer to ‘Any other information on results incl. tables’).

Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given under ‘Any other information on results incl. tables’. Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the average freshly prepared test concentrations gave the results shown under ‘Any other information on results incl. tables’.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed in the 17.3 and 41.5% (v/v) saturated solution test groups. This response was reduced movement (see under ‘Any other information on results incl. tables’).

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 equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
The results of the water quality measurements are shown under ‘Any other information on results incl. tables’. Temperature was maintained at 21 °C to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 476 to 640 Lux.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.


Results with reference substance (positive control):
A positive control (Envigo Study Number MS29MC) 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 results from the positive control with potassium dichromate were within the normal range for this reference item.

Preliminary Media Preparation Trial

Stirring Period and Treatment

Concentration Found (mg/L)

24 Hours Centrifuged 10000g

14.5

24 Hours Centrifuged 40000g

9.16

24 Hours Filtered ~ 1 liter discarded

7.42

24 Hours Filtered ~ 2 liters discarded

9.88

48 Hours Centrifuged 10000g

9.82

48 Hours Centrifuged 40000g

16.4

48 Hours Filtered ~ 1 liter discarded

8.37

48 Hours Filtered ~ 2 liters discarded

9.41

Positive Control

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

0.83

0.70

-

0.98

0.56

1.0

48

0.64

Not possible to determine

0.56

1.0

The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.

Analytical Method - Linearity Data

 

Concentration of Test Item

[mg/L]

 

Mean Area

[counts]

0

0

0.496

4.782 x 104

0.992

1.237 x 105

2.48

3.591 x 105

4.96

7.387 x 105

4.86

7.115 x 105

9.92

1.619 x 106

14.9

2.619 x 106

19.8

3.512 x 106

Analytical Method - Results for Range-Finding Samples

Time Point

Nominal Concentration of
Test Item in Range-Finding Sample

cnom

Sample Preparation Factor



F

Determined Concentration of Test Item in Range-Finding Sample


c

[hours]

[% v/v Saturated Solution]

 

[mg/L]

0

0.10

0.10

<LOQ

 

1.0

0.10

<LOQ

 

10

0.10

0.900

 

100

0.10

8.18

48*

0.10

0.10

<LOQ

 

1.0

0.10

<LOQ

 

10

0.10

0.661

* No sample from the 100% (v/v) saturated solution test group after 48 hours as 100% immobilization occurred after 24 hours exposure resulting in the test media not being renewed.

LOQ=Limit of Quantification

Analytical Method - Results for Spiked Recovery Samples

Nominal Concentra­tion of
Test Item


cnom

Fortified Concentra­tion of Test Item in the Spiked Sample


cfort

Sample Preparation Factor



F

Determined Concentration of Test Item in the Spiked Sample


c

Mean Analytical Recovery
Rate



R

Precision (Relative Standard Deviation of Recovery)

[mg/L]

[mg/L]

 

[mg/L]

[%]

[%]

 

 

0.10

0.505

99

1.9

 

 

0.10

0.503

0.500

0.513

0.10

0.522

 

 

0.10

0.495

 

 

0.10

0.504

Acceptance Target

 

 

80-120

<10

Analytical Method - Results for Test Samples

Time Point

Nominal Concentration of
Test Item in Test Sample

cnom

Sample Preparation Factor



F

Determined Concentration of Test Item in Test Sample

 


c

[hours]

[% v/v Saturated Solution]

 

[mg/L]

0

Control

0.10

<LOQ

(Fresh)

3.0

0.10

0.298

 

7.2

0.10

0.751

 

17.3

0.10

1.86

 

41.5

0.10

4.92

 

100

0.10

11.2

24

Control

0.10

<LOQ

(Old)

3.0

0.10

0.237

 

7.2

0.10

0.639

 

17.3

0.10

1.55

 

41.5

0.10

3.93

 

100

0.10

9.30

24

Control

0.10

<LOQ

(Fresh)

3.0

0.10

0.240

 

7.2

0.10

0.612

 

17.3

0.10

1.49

 

41.5

0.10

3.82

 

100

0.10

8.12

48

Control

0.10

<LOQ

(Old)

3.0

0.10

0.200

 

7.2

0.10

0.545

 

17.3

0.10

1.39

 

41.5

0.10

3.44

 

100

0.10

7.36

LOQ = Limit of Quantification

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal
Concentration
(% v/v Saturated Solution)

Observations (Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Cumulative Immobilized Daphnia

Observations

Cumulative Immobilized Daphnia

Observations

Control

0

10 N

0

10 N

0.10

0

10 N

0

10 N

1.0

0

10 N

0

10 N

10

2

8 N

2

8 N

100

10

A/I

10

A/I

N = No sub-lethal effects observed

A/I = All daphnia immobilized

The average freshly prepared measured test concentrations were determined to be:

Nominal Test Concentration
(% v/v Saturated Solution)

Average Freshly Prepared Measured Test Concentration

(mg/L)

3.0

0.27

7.2

0.68

17.3

1.68

41.5

4.37

100

9.66

Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the average freshly prepared test concentrations gave the following results:

Time (h)

EC50(mg/L)

95% Confidence limits
(mg/L)

24

4.0

Not determined

48

1.5

Not determined

The slopes and their standard errors of the response curves at 24 and 48 hours were 13 (SE = 0.26) and 17 (SE = 3.0) respectively.

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal
Concentration
(% v/v Saturated Solution)

2 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5N

5 N

5 N

5 N

3.0

0

0

0

0

0

0

5 N

5 N

5 N

5 N

7.2

0

0

0

0

0

0

5 N

5 N

5 N

5 N

17.3

0

0

0

0

0

0

5 N

5 N

5 N

5 N

41.5

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

N = Normal

R1– R4= Replicates 1 to 4

Nominal
Concentration
(% v/v Saturated Solution)

4 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

3.0

0

0

0

0

0

0

5 N

5 N

5 N

5 N

7.2

0

0

0

0

0

0

5 N

5 N

5 N

5 N

17.3

0

0

0

0

0

0

5 N

5 N

5 N

5 N

41.5

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

N = Normal

R1– R4= Replicates 1 to 4

Nominal
Concentration
(% v/v Saturated Solution)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

3.0

0

0

0

0

0

0

5 N

5 N

5 N

5 N

7.2

0

0

0

0

0

0

5 N

5 N

5 N

5 N

17.3

0

0

0

0

0

0

5 N

5 N

5 N

5 N

41.5

3

5

4

2

14

70

2 R

A/I

1 R

3 R

100

5

5

5

5

20

100

A/I

A/I

A/I

A/I

R1– R4= Replicates 1 to 4

R = Reduced movement

N = No sub-lethal effects observed

A/I = All daphnia immobilized

Nominal
Concentration
(% v/v Saturated Solution)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

1

0

0

0

1

5

4 N

5 N

5 N

5 N

3.0

0

0

0

0

0

0

5 N

5 N

5 N

5 N

7.2

0

0

0

0

0

0

5 N

5 N

5 N

5 N

17.3

5

3

5

4

17

85

A/I

2 R

A/I

1 R

41.5

5

5

5

5

20

100

A/I

A/I

A/I

A/I

100

5

5

5

5

20

100

A/I

A/I

A/I

A/I

R1– R4= Replicates 1 to 4

R = Reduced movement

N = No sub-lethal effects observed

A/I = All daphnia immobilized

Water Quality Measurements

Nominal
Concentration
(% v/v Saturated Solution)

0 Hours
(Fresh Media)

24 Hours
(Old Media)

pH

mg O2/L

T °C

pH

mg O2/L

T °C

Control

R1

8.0

8.7

21

8.1

8.6

22

3.0

R1

7.9

8.9

21

7.9

8.5

22

7.2

R1

7.9

8.7

22

7.9

8.5

22

17.3

R1

7.9

8.7

22

7.8

8.5

22

41.5

R1

7.9

8.8

22

7.8

8.6

22

100

R1

8.0

9.0

22

7.9

8.7

22

R1= Replicate 1

Nominal
Concentration
(% v/v Saturated Solution)

24 Hours
(Fresh Media)

48 Hours
(Old Media)

pH

mg O2/L

T °C

pH

mg O2/L

T °C

Control

R1

8.1

8.6

22

7.6

8.7

22

3.0

R1

8.0

8.7

22

7.7

8.6

22

7.2

R1

7.9

8.8

22

7.7

8.5

22

17.3

R1

7.6

8.7

22

7.7

8.6

22

41.5

R1

7.9

8.8

22

7.8

8.5

22

100

R1

7.9

8.9

22

7.8

8.8

22

R1= Replicate 1

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of this study the 48 h EC50 value with Daphnia magna is 1.5 mg/L.
Executive summary:

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 TG 202 and the study was conducted in compliance with GLP. 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 3.0, 7.2, 17.3, 41.5 and 100% v/v saturated solution for 48 hours at a temperature of 21 °C to 22 °C under semi-static test conditions. Immobilization and any adverse reactions to exposure were recorded after 2, 4, 24 and 48 hours. Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 0.24 to 11.2 mg/L, showing measured concentrations about 1/10 of the saturated solutions (SS) (3% SS gave ca 0.3 mg/l test concentrations). There was no significant change in the measured concentrations at 24 and 48 hours and so the results are based on the average freshly prepared measured test concentrations only, which were determined to be in the range of 0.27 to 9.66 mg/L. Exposure of Daphnia magna to the test item resulted in a 48 h EC50 of 1.5 mg/L, based on the average freshly prepared measured test concentrations, because test concentrations were stable during these 24 h (> 80% from the initial measured values).

Description of key information

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 TG 202 and the study was conducted in compliance with GLP. 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 3.0, 7.2, 17.3, 41.5 and 100% v/v saturated solution for 48 hours at a temperature of 21 °C to 22 °C under semi-static test conditions. Immobilization and any adverse reactions to exposure were recorded after 2, 4, 24 and 48 hours. Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 0.24 to 11.2 mg/L, showing measured concentrations about 1/10 of the saturated solutions (SS) (3% SS gave ca 0.3 mg/l test concentrations). There was no significant change in the measured concentrations at 24 and 48 hours and so the results are based on the average freshly prepared measured test concentrations only, which were determined to be in the range of 0.27 to 9.66 mg/L. Exposure of Daphnia magna to the test item resulted in a 48 h EC50 of 1.5 mg/L, based on the average freshly prepared measured test concentrations, because test concentrations were stable during these 24 h (> 80% from the initial measured values).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.5 mg/L

Additional information