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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 29 April 2015 and 22 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
Samples were taken from the control and each of the test groups from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Range-finding test
A nominal amount of test item (50 mg) was dissolved in test water with the aid of high shear mixing at 7500 rpm for 30 minutes and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give the required test concentrations of 0.10, 1.0, and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive test
A nominal amount of test item (200 mg) was dissolved in test water with the aid of high shear mixing at 7500 rpm for 30 minutes and the volume adjusted to 2 L to give the 100 mg/L test solution from which a series of dilutions was made to give further test concentrations of 4.5, 10, 22 and 45 mg/L.
Each test solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
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
Total exposure duration:
48 h
Test temperature:
Water temperature was recorded daily throughout the test. Temperature was maintained at approximately 20 to 21 °C throughout the test
pH:
pH was recorded at the start and termination of the test, measured using a Hach Flexi handheld meter. There were no treatment related differences.
Dissolved oxygen:
Dissolved oxygen concentrations were recorded at the start and termination of the test measured using a Hach Flexi handheld meter. There were no treatment related differences.
Nominal and measured concentrations:
RANGE-FINDING TEST
Nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.

DEFINITIVE TEST:
- Nominal test concentrations of 4.5, 10, 22, 45 and 100 mg/L.
- Measured concentrations at t=0h: 3.30, 7.73, 16.8, 34.0 and 78.3 mg/L (73-78% of nominal)
- Measured concentrations at t=48h: 2.89, 6.83, 15.3, 29.9 and 69.2 mg/L (88-91% of initial)
Details on test conditions:
Range-finding Test
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 mg/L.

A nominal amount of test item (50 mg) was dissolved in test water with the aid of high shear mixing at 7500 rpm for 30 minutes and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give the required test concentrations of 0.10, 1.0, and 10 mg/L.

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 to 21 °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 150 mL test and control vessel contained 100 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: 4.5, 10, 22, 45 and 100 mg/L.

Experimental Preparation
A nominal amount of test item (200 mg) was dissolved in test water with the aid of high shear mixing at 7500 rpm for 30 minutes and the volume adjusted to 2 L to give the 100 mg/L test solution from which a series of dilutions was made to give further test concentrations of 4.5, 10, 22 and 45 mg/L.

Each test solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours

Exposure Conditions
As in the range-finding test 150 mL glass beakers 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 then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20 °C to 21 °C with a photoperiod of 16 hours light (561 to 624 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.

Evaluations
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.:
24 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.L: 21 - 27 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
17 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
34 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, 1.0 and 10 mg/L, however, immobilization was observed at 100 mg/L.

Based on this information test concentrations of 4.5, 10, 22, 45 and 100 mg/L were selected for the definitive test.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 80% to 88% of nominal concentrations. Analysis of the test preparations at 48 hours showed measured test concentrations to range from 78% to 83% of the 0-Hour measured concentrations.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 73% to 78% of nominal concentrations. Analysis of the test preparations at 48 hours showed measured test concentrations to range from 88% to 91% of the 0-Hour measured concentrations and so it was considered justifiable to calculate the EC50 values in terms of the 0-Hour measured test concentrations.

Immobilization Data
Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the 0-Hour measured test concentrations gave the following results:
The 24 h EC50 was > 78 mg/L. It was not possible to calculate confidence limits.
The 48 h EC50 was 24 mg/L with 95 % confidence limits of 21 - 27 mg/L.

The No Observed Effect Concentrations after 24 and 48 hours exposure were 78 and 17 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were greater than 78 and 34 mg/L respectively.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed at 24 hours at nominal test concentrations of 45 mg/L and above. These responses were reduced mobility.
Results with reference substance (positive control):
A positive control (Study Number 41500252) 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.
Analysis of the immobilization data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:

The 24 h EC50 was 1.1 mg/L. It was not possible to determine 95 % confidence limits.
The 48 h EC50 was 0.75 mg/L with 95 % confidence limits of 0.69 - 0.82 mg/L.
The NOEC was 0.56 mg/L and the LOEC was 1.0 mg/L.

The No Observed Effect Concentration is based upon equal to or 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 Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (ToxRat).

Validation Criteria

The test was considered to be valid given that none 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.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Cumulative Immobilization Data and Observations in the in the Range-finding Test

Nominal
Concentration
(mg/L)

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

0

10 N

0

10 R

100

10

A/I

10

A/I

N = No sub-lethal effects observed

R = Reduced mobility

A/I = All daphnia immobilized

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal
Concentration
(mg/L)

24 Hours

Cumulative ImmobilizedDaphnia
(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

4.5

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

22

0

0

0

0

0

0

5 N

5 N

5 N

5 N

45

0

0

0

0

0

0

5R

1N, 4R

1N, 4R

3N, 2R

100

5

0

0

0

5

25

A/I

5R

5R

5R

Nominal
Concentration
(mg/L)

48 Hours

Cumulative ImmobilizedDaphnia
(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

4.5

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

22

0

0

0

0

0

0

5 N

5 N

5 N

5 N

45

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

N = No sub-lethal effects observed

R = Reduced mobility

A/I = All daphnia immobilized

Water Quality Measurements

Nominal
Concentration
(mg/L)

0 Hours

24 Hours

48 Hours

pH

mg O2/L

T°C

T ºC

pH

mg O2/L

T°C

Control

R1

7.9

9.6

20

21

8.2

9.4

21

4.5

R1

7.9

9.4

20

21

8.3

9.4

21

10

R1

8.0

9.2

20

21

8.2

9.3

20

22

R1

8.0

9.3

20

21

8.2

9.1

20

45

R1

8.0

9.1

20

21

8.2

9.3

20

100

R1

8.0

8.6

20

21

8.2

9.3

20

R1= Replicate 1

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item has been investigated and gave the following results:
The 48 h EC50 was 24 mg/L with 95 % confidence limits of 21 - 27 mg/L.
The NOEC was 17 mg/L and the LOEC was 24 mg/L.
Executive summary:

Introduction

A study was performeed to assess the acute toxicity of the test item to Daphnia magna. The methoc followed was designed to be 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, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of te test item at concentrations of 4.5, 10, 22, 45 and 100 mg/L foe 48 hours at a temperature of approximately 20 °C to 21 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 73% to 78% of nominal concentrations. Analysis of the test preparations at 48 housr showed measured test concentrations to range from 88% to 91% of the 0 -Hour measufred concentrations and so it was considered justifiable to calculate the EC50 values in terms of the 0 -Hour measured test concentrations.

Exposure to Daphnia magna to the test item gave the follwoing 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)

48

24

21-27

17

34

Description of key information

For aquatic invertebrates the 48-h EC50 value is 24 mg/L as determined from a study according to OECD TG 202 and in compliance with GLP criteria.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
24 mg/L

Additional information

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study groups of daphnids (D. magna, 4 replicates of 5 animals per concentration) were exposed to nominal concentrations of 0 (control), 4.5, 10, 22, 45 and 100 mg/L for 48 hours under static conditions. Nominal test concentrations were analytically verified at the start and at the end of the test. At the start measured concentrations were <LOQ, 3.30, 7.73, 16.8, 34.0 and 78.3 mg/L. Analysis at 48 hours showed measured test concentrations to range from 88% to 91% of the initial measured concentrations and therefore effect values are expressed as initial measured concentrations. Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. All OECD validity criteria were met.

No effects on mobility were observed up to the nominal test concentration of 22 mg/L. After 24 hours exposure a cumulative immobility of 25% was observed at the test concentration of 100 mg/L. All other daphnids at 100 mg/L as well as 15 of 20 daphnids at the 45 mg/L test concentration showed signs of reduced mobility. After 48 hours exposure, cumulative immobility reached 100% at test concentrations of 45 and 100 mg/L. Based on these findings the 48 -h EC50 value was determined at 24 mg/L.