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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:
The study was conducted between 17 August 2015 and 17 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
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:
Details on properties of test surrogate or analogue material (migrated information):Not applicable
Analytical monitoring:
yes
Details on sampling:
Verification of Test ConcentrationsSamples were taken from the control and each test group from the bulk test preparation at 0 and 24 hours (fresh media) and from the pooled replicates at 24 and 48 hours (old media) 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.
Vehicle:
yes
Details on test solutions:
Range-finding Test 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 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 0.10, 1.0 and 10% v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.Definitive TestA 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 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 6.25, 12.5, 25 and 50% 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 1st instar Daphnia magna derived from in-house laboratory cultures.Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minutes 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:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
The water temperature was 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 temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Temperature was maintained at 20 to 22 °C throughout the test.
pH:
The pH was 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 was measured using a Hach Flexi handheld meter. There were no treatment related differences for pH (Range pH 7.9 - 9.0).
Dissolved oxygen:
The 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 dissolved oxygen concentration were measured using a Hach Flexi handheld meter . There were no treatment related differences for oxygen concentration (range 8.6 - 8.8 mg O2/L).
Salinity:
No data
Nominal and measured concentrations:
Range-finding Test: Nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.Definitive Test: Nominal test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
Details on test conditions:
Test WaterReconstituted water (ISO medium) was used for both the range-finding and definitive test.ProcedurePreliminary Media Preparation TrialPreliminary 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 testThe results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item degradation product concentration of approximately 86 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. 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 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 0.10, 1.0 and 10% 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 at approximately 20 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minutes dawn and dusk transition periods. 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 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 degradation product 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 TestBased on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100% v/v saturated solution.Experimental PreparationA 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 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 6.25, 12.5, 25 and 50% v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.The concentration and stability of freshly prepared test media was verified by chemical analysis at 0 and 24 hours and of the old media at 24 and 48 hours.Exposure ConditionsAs in the range-finding 150 mL glass jars containing approximately 100 mL of test preparation 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 covered to reduce evaporation and maintained in a temperature controlled room at 20 to 22 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minutes 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 test item degradation product 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. EvaluationsTest Organism ObservationsAny 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 CriteriaThe light intensity during the light period was measured using an ATP Instruments Lux meter.Geometric Mean Measured Test ConcentrationsThe geometric mean measured degradation product concentrations of the samples were calculated as follows using the measured test concentrations of the bulk test preparations at 0 and 24 hours (fresh media) and from replicates R1 – R4 pooled at 24 and 48 hours (old media):GM = √(C0 x C1)Where:GM = geometric mean measured degradation product concentration (mg/L)C0 = measured concentration at the start of the renewal period, 0 and 24 hours (mg/L)C1 = measured concentration at the end of the renewal period, 24 and 48 hours (mg/L)The geometric mean measured concentrations were determined for each exposure period (0-Hour fresh to 24-Hour old and 24-Hour fresh to 48-Hour old) and then arithmetic average calculated of these values.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits of 35 - 39 mg/L.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
50 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 0.10, 1.0 and 10% v/v saturated solution, however, immobilization was observed at 100% v/v saturated solution. Sub-lethal effects were observed; these include reduced mobility, pale daphnia and trapping at the surface.Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.Chemical analysis of the test item degradation product test preparations at 0 hours showed measured test concentrations to range from 7.42 to 81.4 mg/L and 1.51 to 8.58 mg/L at 48 hours. There was a significant decline in the measured concentrations at 48 hours indicating that the test item degradation product was not stable under test conditions.Definitive TestImmobilization DataThe 24-Hour results were by inspection of the data. Analysis of the immobilization data by trimmed Spearman- Karber at 48 hours based on the geometric mean measured test concentrations gave the following results:The 24 h EC50 was > 50 mg/L.The 48 h EC50 was 37 mg/L with 95% confidence limits of 35 - 39 mg/L.The No Observed Effect Concentrations after 24 and 48 hours exposure were 50 and 25 mg/L respectively. The Lowest Observed Effect Concentration after 48 hours exposure was 50 mg/L.Due to the nature of the data it was not possible to calculate the slope and error of response curve at 24 and 48 hours.Sub-Lethal EffectsSub-lethal effects of exposure were observed in the 100% v/v saturated solution test concentration at 24 hours and in the 12.5 and 50% v/v saturated solution test concentrations at 48 hours. These responses were reduced mobility and trapping at the surface
Results with reference substance (positive control):
A positive control (Study Number 41500252) 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 by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:The 24 h EC50 was 1.1 mg/L. It was not possible to determine the 95 % confidence limits.The 49 h EC50 was 0.75 mg/L with 95 % confidence limits of 0.69 - 0.82 mg/L.The NOEC was 0.56 mg/L and the LOEC was 1.0 mg/L.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.
Reported statistics and error estimates:
The EC50 values and associated confidence limits at 48 hours were calculated by trimmed Spearman Karber. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxCalc and ToxRat Professional computer software package (ToxCalc and ToxRat).

Verification of Test Concentrations

Chemical analysis of the freshly prepared test concentrations at 0 and 24 hours showed measured test concentrations of the degradation product to range from less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 1.0 mg/L, to 83.9 mg/L and less than the LOQ to 29.5 mg/L respectively. Analysis of the degradation product in the old test preparations from 24 to 48 hours showed measured test concentrations had declined, to less than the LOQ to 101 mg/L and less than the LOQ to 25.2 mg/L. Given this, 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:

 

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration of degradation product (mg/L)

50

25

100

50

 

Validation Criteria

The test was considered to be valid given 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

Throughout the test the light intensity was observed to be in the range 673 to 717 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.

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

Control

0

10 N

0

10 N

0.10

0

9 N, 1 R

0

10 N

1.0

0

8 N, 2 P

0

9 N, 1 T

10

0

9 N, 1 P

0

9 N, 1 P

100

4

4 N, 2 R

8

2 R

N = No sub-lethal effects observed

T = Trapped at surface

R = Reduced mobility

P = Pale

Cumulative Immobilization Data and Observations in the Definitive Test

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

5 N

5 N

5 N

5 N

6.25

0

0

0

0

0

0

5 N

5 N

5 N

5 N

12.5

0

0

0

0

0

0

5 N

5 N

5 N

5 N

25

0

0

0

0

0

0

5 N

5 N

5 N

5 N

50

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

2

0

0

0

2

10

2 N, 1 R

5 N

5 N

5 N

 

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

5 N

5 N

5 N

5 N

6.25

0

0

0

0

0

0

5 N

5 N

5 N

5 N

12.5

0

0

0

0

0

0

5 N

5 N

4 N, 1T*

5 N

25

0

0

0

0

0

0

5 N

5 N

5 N

5 N

50

0

0

0

0

0

0

1 N, 4 T

2 N, 3 R

2 N, 3 R

5 N

100

5

5

4

4

18

90

A/I

A/I

1 N

1 N

*Single daphnia trapped at 12.5% v/v considered to be insignificant as no other sub lethal effects observed at this concentration

R1– R4= Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced mobility

A/I = All daphnia immobilized 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

8.1

8.8

21

8.2

8.8

21

6.25

R1

8.2

8.8

21

8.1

8.6

21

12.5

R1

8.3

8.7

21

8.1

8.6

21

25

R1

8.5

8.7

21

8.1

8.6

21

50

R1

8.7

8.7

22

8.1

8.6

21

100

R1

9.0

8.6

22

8.2

8.6

21

 

Nominal
Concentration
(% v/v Saturated Solution)

24 Hours
(Fresh Media)

48 Hours
(Old Media)

pH

mg O2/L

T°C

pH

mg O2/L

T°C

Control

R1

8.1

8.7

20

7.9

8.8

21

6.25

R1

8.1

8.6

21

8.0

8.7

22

12.5

R1

8.1

8.6

21

8.1

8.8

21

25

R1

8.3

8.6

21

8.1

8.8

21

50

R1

8.6

8.7

21

8.1

8.8

21

100

R1

8.9

8.7

20

8.2

8.8

21

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item has been investigated and gave the following results based on the mean measured test item degradation product concentrations:The 48 h EC50 was 37 mg/L with 95% confidence limits of 35 - 39 mg/L. The NOEC was 25 mg/L and the LOEC was 50 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.

 

The test item was known to rapidly degrade in water. Given this, and in accordance with current regulatory guidance, the concentration of the degradation product was determined. A preliminary media preparation trial indicated that a dissolved degradation product concentration of approximately 86 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of the degradation product 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 degradation product at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution for 48 hours at a temperature of 20 to 22°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 24hours. 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 degradation product. 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 freshly prepared test concentrations at 0 and 24 hours showed measured test concentrations of the degradation product to range from less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 1.0 mg/L, to 83.9 mg/L and less than the LOQ to 29.5 mg/L respectively. Analysis of the degradation product in the old test preparations from 24 to 48 hours showed measured test concentrations had declined, to less than the LOQ to 101 mg/L and less than the LOQ to 25.2 mg/L. Given this, 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 of Daphnia magna to the test item gave the following results based on the mean measured test item degradation product 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

37

35

-

39

25

50

 

Description of key information

A study was performed to assess the acute toxicity of the test item to the aquatic invertebrate Daphnia magna The method followed was designed to be compatible with the OECD 202 Guideline for Testing of Chemicals.

Based on mean measured concentrations the 48 -hour EC50 was determined to be 37 mg/L and the NOEC was 25 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
37 mg/L

Additional information

The test substance is not toxic to Daphnia magna.