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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:
This study was conducted between 27 August 2017 and December 2017
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
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:
Information as provided by the Sponsor.
Identification: FAT 65004/F TE
Physical state/Appearance: Beige Solid
Batch: 15060201 (China)
Purity: 99.9 %
Expiry Date: 25 August 2020
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 M7medium)
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.

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.63 mg/L could be obtained using a saturated solution method of preparation.
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 % 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 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution.

Definitive Test
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at highest attainable test concentration no immobilization or adverse reactions to exposure were observed.
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 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g to give a 100% v/v saturated solution.


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 2) 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:
no
Total exposure duration:
48 h
Test temperature:
22”C
pH:
The reconstituted water had a pH of 7.9 ± 0.3
Dissolved oxygen:
aerated until the dissolved oxygen concentration was 8.4 - 9.7 mg O2/L
Salinity:
Not reported
Conductivity:
Not reported
Details on test conditions:
Experimental Design and Study Conduct
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

Preliminary Media Preparation Trial
Saturated Solution Preparation
A nominal amount of test item (550 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis purified 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)

Results
Stirring Period and Treatment Concentration Found (mg/L)
24 Hours Centrifuged 10000 g 0.255
24 Hours Centrifuged 40000 g 0 .499
24 Hours Filtered ~ 1 liter discarded 24 Hours Filtered ~ 2 liters discarded 48 Hours Centrifuged 10000 g 0.314
48 Hours Centrifuged 40000 g 0.633
48 Hours Filtered ~ 1 liter discarded 48 Hours Filtered ~ 2 liters discarded
Discussion
It is evident from these results that filtration was not appropriate as measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method suggest that the test item was adsorbing to the filter matrices.
The 48-Hour preparation gave the highest dissolved concentration after centrifugation at 40000 g (0.63 mg/L). Based on these results, for the purposes of the study, the test item was prepared as a saturated solution at an initial loading rate of 50 mg/L, stirred via propeller stirrer for 48 hours prior to the removal of any undissolved test item by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution with a nominal test concentration of approximately 0.63 mg/L

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.63 mg/L could be obtained using a saturated solution method of preparation.
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 % 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 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution.
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 250 mL test and control vessel contained 200 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 a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at highest attainable test concentration no immobilization or adverse reactions to exposure were observed.
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 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g to give a 100% v/v saturated solution.
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 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.
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. Concentrations at which 100% immobilization was observed after 24 hours exposure were not renewed.

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
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.
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 freshly prepared bulk test preparations at 0 and 24 hours and from the aged pooled replicates at 24 and 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.


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

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.:
> 0.63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.93 mg/L
Nominal / measured:
meas. (geom. mean)
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 immobilization was observed in any the test concentrations.
A sub-lethal effect of exposure was observed in the 100% v/v saturated solution test preparation. This response was reduced mobility (see Table 1).
Based on this information, a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at highest attainable test concentration, no immobilization or adverse reactions to exposure were observed.
Chemical analysis of the test preparations at 0 hours showed a measured test concentration of 4 % of nominal concentration. There was a significant decline in the measured concentrations at 48 hours indicating that the test item not stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the freshly prepared test media at 0 and 24 hours) showed measured test concentrations to range from 1.3 to 4.4 mg/L. Analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations had declined, to range from 0.16 and 0.46 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.
The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration
(% v/v Saturated Solution) Mean Measured Test Concentration (mg/L) Expressed as a percentage of the 0-Hour Measured Test Concentration
100 0.93 72

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 0.93 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 >0.93 Not determined
48 >0.93 Not determined

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.93 mg/L.

Sub-Lethal Effects
A sub-lethal effect of exposure was observed in one daphnid in the control group. This response was trapping at the surface (see Table 2).

Validation Criteria
The test was considered to be valid given that none 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 773 to 834 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
(% v/v Saturated Solution)

Observations (Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Cumulative Immobilized Daphnia

Observations

Cumulative Immobilized Daphnia

Observations

R1

R2

R1

R2

R1

R2

R1

R2

Control

0

0

5N

5N

0

0

5N

5N

0.10

0

0

5N

5N

0

0

5N

5N

1.0

0

0

5N

5N

0

0

5N

5N

10

0

0

5N

5N

0

0

5N

5N

100

0

0

5N

5N

0

0

5N

4N, 1R

R1– R2= Replicates 1 to 2

N = No sub-lethal effects observed

T = Trapped at surface

R = Reduced mobility

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

24 Hours

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

5N

5N

5N

5N

100

0

0

0

0

0

0

5N

5N

5N

5N

48 Hours

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

0

0

0

0

0

0

5N

5N

4N, 1T

5N

100

0

0

0

0

0

0

5N

5N

5N

5N

R1 – R4 = Replicates 1 to 4

N = No sub-lethal effects observed

T = Trappd at surface

Table 3: 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

7.7

8.8

22

7.9

9.7

22

100

R1

7.6

8.5

22

7.9

9.0

22


Validity criteria fulfilled:
yes
Remarks:
Considered to be valid - 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 based on the mean measured test concentrations gave a 48-Hour EC50 value of greater than 0.93 mg/L. The No Observed Effect Concentration was 0.93 mg/L.
This study showed that there were no toxic effects at saturation.
Executive summary:

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, "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 0.63 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 a concentration of 100 % v/v saturated solution for 48 hours at a temperature of approximately 22°C under semi static test conditions. The test item solution was prepared by stirring an excess (550 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by centrifugation at 40000g for 30 minutes to produce 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 fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 1.3 to 4.4 mg/L. Analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.16 and 0.46 mg/L (10% to 12% of the 0-Hour measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Exposure ofDaphnia magnato the test item gave EC50values based on the geometric mean measured test concentrations of greater than 0.93 mg/L. The No Observed Effect Concentration was 0.93 mg/L.

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

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the mean measured test concentrations gave a 48-Hour EC50 value of greater than 0.93 mg/L. The No Observed Effect Concentration was 0.93 mg/L (sturation concentration). This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

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. The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the mean measured test concentrations gave a 48-Hour EC50 value of greater than 0.93 mg/L. The No Observed Effect Concentration was 0.93 mg/L. This study showed that there were no toxic effects at saturation.