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Ecotoxicological information

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:
This study was conducted between 08 May 2017 and 13 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
EC No 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification: FAT 93588/A TE
Physical state/Appearance: White Solid
Batch: 0041847900
Purity: 98.5 %
Expiry Date: 01 January 2018
Storage Conditions: Room temperature, in the dark
Analytical monitoring:
yes
Details on sampling:
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 the concentration to be used for the initial experiment was analyzed
Vehicle:
yes
Remarks:
Reconstituted water (Elendt M7 medium)
Details on test solutions:
Range-finding Test
The test concentration to be used in the definitive test was 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 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 further 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
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration.
The test 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 first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium (see Annex 1) 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.

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
Not reported
Test temperature:
18-22 ±1”C
pH:
The reconstituted water had a pH of 7.9 ± 0.3
Dissolved oxygen:
Dissolved oxygen concentration was 8.5 - 9.0 mg O2/L
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Chemical analysis of the 100 mg/L preparation at 0 and 48 hours (see Annex 3) showed near nominal concentrations were obtained, indicating that the test item was stable under test conditions.
Details on test conditions:
Range-finding Test
The test concentration to be used in the definitive test was 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 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 further 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 5 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. Two replicate test and control vessels were prepared. 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 was recorded. The control 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 a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed. A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration. The test 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 jars containing approximately 100 mL of test preparation were used. At the start of the test five 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 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. The test preparations were not renewed during the exposure period.

Assessments

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 within 15 seconds after gentle agitation.

Water Quality Criteria
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. 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. The appearance of the test media was recorded daily.

Verification of Test Concentrations
Samples were taken from the control and the 100 mg/L test group 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.

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.

Data Evaluation
An estimate of the EC50 values was given by inspection of the immobilization data.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
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
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1. No significant immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L. Based on this information, a single test concentration of four replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines no immobilization or adverse reactions to exposure were observed. Chemical analysis of the 100 mg/L preparation at 0 and 48 hours (see Annex 3) showed near nominal concentrations were obtained, indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal so the results are based on nominal test concentrations.

Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:
Time (h) EC50 (mg/L) 95% Confidence limits(mg/L)
24 >100 Not applicable
48 >100 Not applicable
The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Sub-Lethal Effects
A sub-lethal effect of exposure was observed in both the control and 100 mg/L test concentration. This response was trapping at the surface (see Table 2).

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 given in Table 3. Temperature was maintained at approximately 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 446 to 499 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 XT01HS) 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, however, throughout the positive control the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the test. Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and 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 1.3 1.0 - 1.8 1.0 1.8
48 1.2 1.1 - 1.3 0.56 1.0
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.

Table 1: Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal
Concentration
(mg/L)

Observations (Initial Population: 5 Per Replicate)

24 Hours

48 Hours

Cumulative Immobilized Daphnia

Observations

Cumulative Immobilized Daphnia

Observations

R1

R2

R1

R2

R1

R2

R1

R2

Control

0

1

5N

4N

0

1

5N

4N

0.10

0

0

5N

5N

0

0

5N

5N

1.0

0

0

1T, 4N

5N

0

0

5N

2T, 3N

10

0

0

5N

5N

0

0

5N

5N

100

0

0

1T, 4N

1T, 4N

1

0

4N

5N

R1– R2= Replicates 1 and 2

N = No sub-lethal effects observed

T = Trapped at surface

Table 2: Cumulative Immobilization Data and Observations in the Definitive Test

24 Hours

Nominal
Concentration
(mg/L)

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

1T, 4N

100

0

0

0

0

0

0

5N

5N

5N

1T, 4N

48 Hours

Nominal
Concentration
(mg/L)

48 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

100

0

0

0

0

0

0

5N

5N

5N

1T, 4N

R1– R4= Replicates 1 to 4

N = No sub-lethal effects observed

T = Trapped at surface

Table 3: 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

9.0

22

22

7.8

8.6

22

100

R1

7.8

8.9

22

22

8.0

8.5

22

R1= Replicate 1

Validity criteria fulfilled:
yes
Remarks:
Considered to be valid as 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
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of greater than 100 mg/L. The No Observed Effect Concentration was 100 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 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 the test item at a nominal concentration of 100 mg/L for 48 hours at a temperature of approximately 22 °C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.


 


Results


Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations. Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

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 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.


48-hours exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information