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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:
Tis study was conducted between 26 September 2016 and 12 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study conducted according to OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
Aptil 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
EC No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: FRET 10-0367
Physical state/Appearance: Clear colorless liquid
Batch: RDGM38757
Purity: 98.3%
Expiry Date: 01 June 2018
Storage Conditions: Approximately 4 ºC in the dark
Intended use/Application: Fragrance ingredient for use in cleaners, detergents, fabric softeners and air fresheners
Analytical monitoring:
yes
Details on sampling:
Test concentrations were verified by chemical analysis. Water samples were taken from the control and each exposure level at 0 and 48 hours
Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
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 (See additional information, below).

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 29 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 1.0, 10 and 100% v/v saturated solution.
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for approximately 24 hours. After stirring 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 10 and 1.0% 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: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
A nominal amount of test item (550 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 32, 10, 3.2 and 1.0% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain:
- Source:
- Age at study initiation (mean and range, SD): Gravid adults were isolated 24 hours prior to initiation of the test. Young daphnids produced
overnight were used for testing.

Test type:
static
Water media type:
freshwater
Remarks:
Reconstituted water (Elendt M7) used for both the range-finding and definitive test. The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC
Limit test:
no
Total exposure duration:
48 h
Hardness:
Not reported
Test temperature:
22 ±1 °C
pH:
7.9±0.3
Dissolved oxygen:
Not reported
Nominal and measured concentrations:
Range-finding Test
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.43 to 27 mg/L. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations at 0 and 48 hours (see Annex 6) showed measured test concentrations to range from 0.39 to 41 mg/L and 0.41 to 36 mg/L respectively. Given the slight variability in results it was considered appropriate to calculate the results based on the geometric mean measured test concentrations.
The geometric mean measured test concentrations were determined to be:
Nominal Test Concentration
(% v/v Saturated Solution) Measured Concentration at 0 hours (mg/L) Measured Concentration at 48 hours (mg/L) Geometric Mean Measured Test Concentration (mg/L)
1.0 0.388 0.408 0.40
3.2 1.36 1.27 1.3
10 4.15 3.98 4.1
32 13.2 12.8 13
100 41.3 35.5 38
Details on test conditions:
ange-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 29 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 1.0, 10 and 100% v/v saturated solution.
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for approximately 24 hours. After stirring 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 10 and 1.0% 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 was completely filled with test media and sealed 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 immediate 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: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
A nominal amount of test item (550 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 32, 10, 3.2 and 1.0% 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 48 hours (see Annex 6).

Exposure Conditions
As in the range-finding test 100 mL glass conical flasks each completely filled 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 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.

Statistical Analysis
The EC50 values and associated confidence limits 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. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Step-down Cochran-Armitage Test. 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
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
8.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL 6.0-11 mg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
4.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Test
No significant immobilization was observed at the test concentrations of 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 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.43 to 27 mg/L. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 0.39 to 41 mg/L and 0.41 to 36 mg/L respectively. Given the slight variability in results it was considered appropriate to calculate the results based on the geometric mean measured test concentrations.
The geometric mean measured test concentrations were determined (see Table 3 below)

Immobilization Data
Inspection of the data at 24 hours and analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 48 hours gave the following results based on the geometric mean measured test concentrations:
Time (h) EC50 (mg/L) 95% Confidence limits (mg/L)
24 18 Not determined
48 8.2 6.2 - 11

The No Observed Effect Concentration after 24 and 48 hours exposure was 1.3 mg/L. The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 4.1 mg/L.
The slopes and their standard errors of the response curve at 24 and 48 hours were3.4 (SE = 0.063) and 4.3 (SE = 0.056).

Sub-Lethal Effects
A sub-lethal effect of exposure was observed in the 13 mg/L test concentration. This response was reduced mobility.

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.

Water Quality Criteria
Temperature was maintained at 20 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 486 to 496 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):
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.
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 3 Verification of Test Concentrations

 Nominal Test Concentration

(% v/v Saturated Solution)

 Measured Concentration at 0 hours (mg/L)  Measured Concentration at 48 hours (mg/L)  Geometric Mean Measured Test Concentration (mg/L)
 1.0  0.388  0.408  0.40
 3.2  1.36  1.27  1.3
 10  4.15  3.98  4.1
 32  13.2  12.8  13
 100  41.3  35.5  38
Validity criteria fulfilled:
yes
Remarks:
The test was considered to be valid in 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
Conclusions:
The 48 h EC50 (immobilisation) value with Daphnia magna is 8.2 mg/I.
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

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.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 29 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 20 C to 22 C under static test conditions.  The test item solutions were prepared by stirring an excess (50 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 filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item.  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

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 0.39 to 41 mg/L and 0.41 to 36 mg/L respectively.  Given the slight variability in results it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.40, 1.3, 4.1, 13 and 38 mg/L.  

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

 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  8.2  6.2 - 11  1.3  4.1

                         

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.

Results

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 0.39 to 41 mg/L and 0.41 to 36 mg/L respectively.  Given the slight variability in results it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.40, 1.3, 4.1, 13 and 38 mg/L.  

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

 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  8.2  6.2 - 11  1.3  4.1

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
8.2 mg/L

Additional information