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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
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)
Specific details on test material used for the study:
Identification: 2,2'-[propane-1,3-diylbis(oxy)]bis(3",5,5"-tri-tert-butyl-5'-methyl-1,1':3',1"-terphenyl-2’-ol)
Batch: 1502501005
CAS number: 1042662-40-7
Purity: 98.1%
Physical state/Appearance: Sponsor - solid powder, white, odorless
Envigo - white powder
Expiry Date: 01 February 2025
Storage conditions: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken for quantitative analysis from the control and the test group, from the freshly prepared bulk test preparation at 0 hours and from old or expired pooled replicates at 48 hours. 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:
The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a magnetic stirrer at a speed such that a dimple is formed at the water surface for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (the first approximate 1 liter discarded in order to pre condition the filter) to give a 100% v/v saturated solution of the test item.
Test organisms (species):
Daphnia magna
Details on test organisms:
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium 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
Test temperature:
Temperature was maintained at 21 °C to 22 °C throughout the test
Nominal and measured concentrations:
Range-finding Tests: 0.10, 1.0, 10 and 100% v/v saturated solution (nominal)

Definitive Test: 100% v/v saturated solution (nominal)
Details on test conditions:
Range-finding Tests
In the initial 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.
In the initial range-finding test five 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 24 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 hours the number of immobilized daphnids were recorded.
As an inconsistent pattern of immobilization was observed, testing was terminated after 24 hours exposure and the test repeated as detailed above. The number of immobilized daphnids in the second test were recorded after 24 and 48 hours. The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test
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 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.
Static test conditions were employed in the test.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Tests
No immobilization was observed at the test concentration of 100% v/v saturated solution.
No sub-lethal effects of exposure were observed throughout the test.
Based on this information a limit test at 100% v/v saturated solution was selected for the definitive test.
Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained.

Definitive Test
Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained.

Immobilization Data
Two daphnids were immobilized in one replicate in the test concentration during the test.
The NOEC after 48 hours exposure was 100% v/v saturated solution.

Sub-Lethal Effects
One daphnid was pale in the test concentration.

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.
Results with reference substance (positive control):
A positive control 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 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: 24 hours. EC50 (mg/L):0.79. 95% Confidence Limits (mg/L): 0.73 - 0.86. NOEC (mg/L): 0.56. LOEC (mg/L): 1.0
Time Point: 48 hours. EC50 (mg/L):0.75. 95% Confidence Limits (mg/L): 0.56 - 1.0. NOEC (mg/L): 0.56. LOEC (mg/L): 1.0

The NOEC 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.

Nominal Concentration (% v/v)

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

5N

5N

5N

5N

100

2

0

0

0

2

10

3N

1P, 4N

5N

5N

N = No sub lethal effects observed

P = Pale

The analytical results: 

Nominal Conc

(% v/v)

Sat Sol

0 hrs

48 hrs

Measured Concentration (mg/L)

Measured Concentration (mg/L)

Control

<LOQ

<LOQ

100

<LOQ

<LOQ

LOQ = 0.00053 mg/L

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the nominal test concentration gave a 48 hour EC50 value of greater than 100% v/v saturated solution. The NOEC was greater than or equal to 100% v/v saturated solution.
This study showed that there were no toxic effects at saturation.
Executive summary:

Introduction                  

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

Method              

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 and high shear mixing.

Following a preliminary range‑finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a magnetic stirrer at a speed such that a dimple is formed at the water surface for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (the first approximate 1 liter discarded in order to pre‑condition the filter) to give a 100% v/v saturated solution of the test item. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results              

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.00053 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Exposure of Daphnia magna to the test item gave EC50values based on the nominal test concentrations of greater than 100% v/v saturated solution. The No Observed Effect Concentration (NOEC) was 100% v/v saturated solution.

This study showed that there were no toxic effects at saturation.

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.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.00053 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Given this it was considered appropriate to calculate results based on the nominal test concentrations only.

Exposure of Daphnia magna the test item gave EC50values based on the nominal test concentrations of greater than100% v/v saturated solution. The NOEC was determined to be100% v/v saturated solution.

This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

Additional information