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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:
Experimental start date: 28 August 2019, Experimental completion date: 25 October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: LESA
Batch: 8901525
Purity: 98.6%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 20 March 2022
Storage Conditions: Room temperature in the dark
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 and 24 hours and from the pooled replicates at 24 and 48 hours for quantitative analysis. All samples were taken for immediate analysis. Duplicate sets of samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.
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. A media preparation trial was conducted in order to determine the solubility of the test item under test conditions
Based on this trial the test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 4.7 mg/L.

Range-finding Test:
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 4.7 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 0.10, 1.0, 10 and 100% v/v saturated solution.

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 (1100 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 Gelman Acrocap filter (the first approximate 2 liters used to pre condition the filter was discarded), 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:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 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 (Raphidocelis subcapitata) and GEMMA Micro 300 fish 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:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Water harndess: 250 mg/L as CaCO3
Test temperature:
Temperature was maintained at 20 °C to 21°C throughout the test
pH:
pH in control and test samples ranged from 8.1 - 8.4 at 0, 24 and 48 hrs.
No treatment related differences for pH.
Dissolved oxygen:
Dissolved oxygen in control and test samples ranged from 8.6 - 9.1 at 0, 24 and 48 hrs
No treatment related differences for oxygen concentration .
Nominal and measured concentrations:
Definitive Test:
Nominal: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
Geometric mean measured: 0.080, 0.21, 0.33, 1.5 and 3.1 mg/L
Details on test conditions:
TEST SYSTEM
120 mL glass vessels 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 and 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.


TEST MEDIUM / WATER PARAMETERS
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.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.


Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Test:
No immobilization was observed at the test concentrations of control, 0.10 and 1.0% v/v saturated solution, however; immobilization was observed at 10 and 100% v/v saturated solution.
Sub lethal effects of exposure were observed in 0.10 and 10% v/v saturated solution test concentrations. These responses were reduced mobility and trapped at the surface.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solutions were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.011 to 4.9 mg/L. There was a significant decline in the measured concentrations at 48 hours ranging from 0.0045 to 1.4 mg/L indicating that the test item was not stable under test conditions.

Definitive Test:
Verifcation of Test Concentrations:
Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 0.060 to 4.2 mg/L. Analysis of the test preparations at 24 and 48 hours showed measured test concentrations had declined, to range from 0.040 to 2.4 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: 0.080, 0.21, 0.33, 1.5 and 3.1 mg/L

Immobilization Data:
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given Table 'Cumulative Immoblilization Data and Observations in the Definitive Test' (see any other information on results incl. tables section).

Analysis of the immobilization data by Logit analysis using Linear Maximum Likelihood regression method at 24 and 48 hours based on the geometric mean measured test concentrations gave the following results:
24 hour EC50: 1.5 mg/L (95% confidence limits: 1.2 - 1.7 mg/L).
48 hour EC50: 0.60 mg/L (95% confidence limits: 0.22 - 1.6 mg/L)

The No Observed Effect Concentrations (NOEC) after 24 and 48 hours exposure was 0.33 mg/L. The Lowest Observed Effect Concentrations (LOEC) after 24 and 48 hours exposure was 1.5 mg/L.
The slopes and their standard errors of the response curves at 24 and 48 hours were 10 (standard error = 0.040) and 11 (standard error = 0.088) respectively.

Sub-lethal Effects:
Sub lethal effects of exposure were observed in 0.33 and 1.5 mg/L. These responses were reduced mobility.

Water Qualitiy Criteria:
Temperature was maintained at 20 °C to 21 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 878 to 1041 Lux.

Observation on Test Item Solubility:
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

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.









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.
Analysis of the immobilization data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package. Accordingly the following results were obtained:
24 hr EC50: 1.2 mg/L
24 hr NOEC: 0.56 mg/L
24 hr LOEC: 1.0 mg/L
48 hr EC50: 0.71 mg/L
48 hr NOEC: 0.56 mg/L
48 hr LOEC: 1.0 mg/L
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.

Verification of Test Concentrations:

The geometic mean measured concentrations were determined to be:

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

Expressed as a percentage of the 0‑Hour Measured Test Concentration

1.0

0.080

133%

3.2

0.21

136%

10

0.33

75%

32

1.5

101%

100

3.1

73%

Cumulative Immobilization Data and Observations in the Definitive Test

Geometric Mean Measured Concentration (mg/L)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5N

5 N

5 N

5 N

0.080

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.21

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.33

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1.5

1

3

4

2

10

50

4 R

2 R

1 R

3 R

3.1

5

5

5

5

20

100

A.I

A.I

A.I

A.I


Geometric Mean Measured Concentration (mg/L)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.080

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.21

0

0

0

1

1

5

5 N

5 N

5 N

4 N

0.33

0

0

0

0

0

0

5 N

5 N

3 N 2 R

5 N

1.5

5

5

5

5

5

100

A.I

A.I

A.I

A.I

3.1

5

5

5

5

5

100

A.I

A.I

A.I

A.I

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item has been investigated and gave the following results based on the geometric mean measured test concentrations:48 hr EC50: 0.60; 48 hr NOEC: 0.33 mg/L
Executive summary:

Introduction:

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 4.7 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, 20 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 21 °C under semi‑static test conditions. The test item solutions were prepared by stirring an excess (100 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 through a 0.2 µm Gelman Acrocap filter (the first approximate 2 liters used to pre‑condition the filter was discarded), to give 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 fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.060 to 4.2 mg/L. Analysis of the old/expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.040 and 2.4 mg/L 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. The geometric mean measured test concentrations were determined to be 0.080, 0.21, 0.33, 1.5 and 3.1 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)

NOEC
(mg/L)

LOEC
(mg/L)

48

0.60

0.22

-

1.6

0.33

1.5


NOEC=No observedeffect concentration

LOEC=Lowest observed effect concentration

Description of key information

Key study (Covance 2020):

Introduction:

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 4.7 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, 20 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 21 °C under semi‑static test conditions. The test item solutions were prepared by stirring an excess (100 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 through a 0.2 µm Gelman Acrocap filter (the first approximate 2 liters used to pre‑condition the filter was discarded), to give 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 fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.060 to 4.2 mg/L. Analysis of the old/expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.040 and 2.4 mg/L 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. The geometric mean measured test concentrations were determined to be 0.080, 0.21, 0.33, 1.5 and 3.1 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)

NOEC
(mg/L)

LOEC
(mg/L)

48

0.60

0.22

-

1.6

0.33

1.5

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.6 mg/L

Additional information