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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 07 January 2013 to 21 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 202 and EU Method C.2 in compliance with GLP. All validity criteria were fulfilled and no deviations were observed.
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2012-11-30
Specific details on test material used for the study:
- Storage condition of test material: Room temperature in the dark. Dry area, unopened containers.
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and each test group (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis. Samples were stored at approximately -20 °C prior to analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
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, a media preparation trial was conducted in order to determine the solubility of the test item under test conditions. The test item was prepared using a saturated solution method of preparation at an initial loading rate of 15 mg/L, stirred for a period of 48 hours prior to removal of any undissolved test item by centrifugation (40000 g for 30 min) to produce a 100% v/v saturated solution (corresponding to 3.1 mg/L in the pre-study media preparation trial).

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
An amount of test item (165 mg) was added to 11 litres of reconstituted water and stirred using a propeller stirrer at approximately 1500 rpm for 48 hours. After stirring, any undissolved test item was removed by centrifugation (40000 g for 30 minutes) to give the 100% v/v test concentration. Aliquots (20, 64, 200 and 640 mL) of the 100% v/v saturated solution test concentration were added to a final volume of 2 litres to give the remaining test concentrations of 1.0, 3.2, 10 and 32% v/v saturated solution. Each stock 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: no data
- Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): 1st instar (less than 24 hours old)
- Weight at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): no data
- Valve height at study initiation, for shell deposition study (mean and range, SD): no data
- Peripheral shell growth removed prior to test initiation: no data
- Method of breeding: 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 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.
- Feeding during test: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
No data
Test temperature:
19 to 22°C
pH:
8.0 to 8.2
Dissolved oxygen:
80-101% air saturation value (ASV).
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentrations: Control, 1.0, 3.2, 10, 32 and 100% v/v saturation solution
- Measured concentrations: See table 6.1.3/1 in "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars containing approximately 200 mL of test preparation. The test vessels were covered to reduce evaporation.
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water Elendt M7 Medium.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light and 8h darkness with 20 minute dawn and dusk transition periods
- Light intensity: 622 to 641 lux

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 for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Range finding study: yes (0.10, 1.0, 10 and 100% v/v saturation solution).
The test item was prepared using a saturated solution method of preparation at an initial loading rate of 15 mg/L, stirred for a period of 48 hours prior to removal of any undissolved test item by centrifugation (40000 g for 30 min) to produce a 100% v/v saturated solution test concentration from which serial dilutions were prepared to give the remaining test concentrations.
- Results used to determine the conditions for the definitive study: 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. Chemical analysis of the 10 and 100% v/v saturated test solutions at 0h showed that measured concentrations of 0.349 and 3.48 mg/L were obtained respectively, and at 48 hours showed that measured concentrations of 0.305 and 3.01 mg/L were obtained respectively indicating that the test item was stable under test conditions.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 1.0-3.3 mg/L. Geometric mean between EC0 (1.0 mg/L) and EC100 (3.3 mg/L).
Duration:
48 h
Dose descriptor:
other: The highest concentration without observed effect
Effect conc.:
1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
See table 6.1.3/2 in "Any other information on results incl. tables".
- Behavioural abnormalities: not reported
- Observations on body length and weight: not reported
- Other biological observations: not reported
- Mortality of control: none (0%)
- Other adverse effects control: none
- Abnormal responses: not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: No. Throughout the test, the test preparations were observed to be clear colourless solutions.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 24h EC50 = 0.75 mg/L (95% CL: 0.56-1.0 mg/L).
- 48h EC50 = 0.45 mg/L (95% CL: 0.42-0.48 mg/L).
Reported statistics and error estimates:
None

Table 6.1.3/1: Measured concentrations during the test

Nominal Concentration (% v/v Saturated Solution)

0h Measured Concentration (mg/L)

48h Measured Concentration (mg/L)

Mean Measured Concentration (mg/L)

32

1.05

0.97

1.0

100

3.39

3.11

3.3

Table 6.1.3/2: Cumulative immobilization data in the definitive test

Nominal Concentration (% v/v Saturated Solution)

Mean Measured Concentration (mg/L)

Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)

24h

48h

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Total

%

Control

1.0

3.2

10

32

100

-

-

-

-

1.0

3.3

0

0

0

0

0

5

0

0

0

0

0

5

0

0

0

0

0

5

0

0

0

0

0

5

0

0

0

0

0

20

0

0

0

0

0

100

0

0

0

0

0

5

0

0

0

0

0

5

0

0

0

0

0

5

0

0

0

0

0

5

0

0

0

0

0

20

0

0

0

0

0

100

Validity criteria fulfilled:
yes
Conclusions:
The 48-Hour EC50 for the test item to Daphnia magna based on mean measured test concentrations was 1.8 mg/L with 95% confidence limits of 1.0 – 3.3 mg/L.
Executive summary:

This study was performed, according to OECD Guideline 202 and EU Method C.2 with GLP statement, to assess the acute toxicity of the substance to Daphnia magna.

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation. A pre-study media preparation trial indicated that a dissolved test item concentration of approximately 3.1 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of the test 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 concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution for 48 hours at a temperature of 19 °C to 22 °C under static test conditions. The test item solution was prepared by stirring an excess (15 mg/L) of test item in test medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by centrifugation (40000 g for 30 minutes) to produce a 100% v/v saturated solution of the test item from which dilutions were made to give the remainder of the test concentrations. The number of immobilized Daphnia magna were recorded after 24 and 48 hours.

Analysis of the 32 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations of 1.05 and 3.39 mg/L were obtained respectively and at 48 hours showed measured test concentrations of 0.967 and 3.11 mg/L were obtained respectively. Accordingly, the results were based on mean measured test concentrations.

The 48-Hour EC50 for the test item to Daphnia magna based on mean measured test concentrations was 1.8 mg/L with 95% confidence limits of 1.0 – 3.3 mg/L. The lowest concentration with observed effect was 3.3 mg/L and the highest concentration without observed effect was 1.0 mg/L.

All validity criteria were fulfilled and no deviations were observed.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2014-08-20 to 2014-08-22 and from 2014-10-28 to 2014-10-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study summarised two non-GLP range finding tests, performed according to OECD Guideline 202 and EU Method C.2. As the results obtained are in accordance with a previous GLP study (Harlan, 2013), the definitive test was not considered necessary.
Reason / purpose:
reference to other 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
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentration levels and the control were analytically verified via GC-MS at the start (0 and 24 h) and at the end of both exposure intervals (24 and 48 h)
- Sampling method: At the start of the exposure intervals (0 and 24 h), sampling was carried out after preparation of the test concentrations.
At the end of the exposure intervals (24 and 48 h) samples were taken from additional replicates prepared with test media, but without daphnids, because volumes of the test replicates were insufficient for the analytical determination of the test concentrations. These additionally prepared replicates were incubated under test conditions until sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of the Test Solutions in the 1st Test
- Method: The stock solution (5 mg/L of the test item were weighed out) was freshly prepared with dilution water before the start of both exposure intervals (at 0 and 24 h).
- The stock solution was stirred with approximately 1100 rpm for 2 h at room temperature and treated with ultrasound for 5 minutes at room temperature until the stock solution was visually clear.
- Controls: Dilution water without test item tested under the same conditions as the test groups.

Preparation of the Test Solutions in the 2nd Test
- Method: The stock solution (2.5 mg/L of the test item were weighed out) was freshly prepared with dilution water before the start of both exposure intervals (at 0 and 24 h).
- The stock solution was stirred with approximately 1100 rpm for 2 h at room temperature and treated with ultrasound for 5 minutes at room temperature until the stock solution was visually clear.
- Controls: Dilution water without test item tested under the same conditions as the test groups.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna STRAUS (Clone 5)
- Source: Institut fur Wasser-, Boden- und Lufthygiene (WaBoLu)
- Culture: In glass vessels (2-3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination; light intensity of max. 20 µE/m2/s
- Culture feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10^6 cells/mL
- Age at study initiation: 2 to 24 h old daphnids from a healthy stock were used for the study
- Method of breeding: 2 to 24 h old daphnids from a healthy stock were used for the study. They were obtained by removing the juvenile daphnids from the culture vessels latest 22 h before the start of the exposure. The juveniles born within the 22 h preceding the exposure were used for the test after an acclimatisation phase of 2 h in the dilution water. No first brood progeny was used for the test.
- Feeding during test: Daphnids were not fed during the study

ACCLIMATION
- Acclimation period: At least 2 h in dilution water
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
160 to 180 mg CaCO3/L
Test temperature:
18-22 °C
pH:
5 mg/L: 7.65 (0 h); 7.61 (24 h)
2.5 mg/L: 7.58 (0 h); 7.67 (24 h)
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
1st Test: 5 mg/L
2nd Test: 0.025, 0.25 and 2.5 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm) 50 mL capacity, loosely covered with watch glasses
- Type (delete if not applicable): Closed
- Test volume: 20 mL
- Renewal of the test solutions: The test solutions were renewed after 24 h. For this purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by a pipette.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Application: Per replicate, 20 g test solution was weighed out into the test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by a pipette.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202
- Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L

OTHER TEST CONDITIONS
- Photoperiod: 16 h/8 h light/dark cycle
- Light intensity: Diffuse light; light intensity of max. 20 µE/m2/s

EFFECT PARAMETERS MEASURED:
Immobilisation was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other relevant observations were not made.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
2nd test: 3 test item concentrations in a geometric series with a separation factor of 10, prepared by diluting the stock solution of 2.5 mg/L with dilution water were tested as follows: 0.025, 0.25 and 2.5 mg/L.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.67 - 3.58 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Based on the nominal concentrations, the EC50 is expected to be between 2.5 mg/L and 5 mg/L.
Details on results:
At 5 mg/L, 90 and 100 % immobilisation was observed at 24 and 48 h observation, respectively.
At 2.5 mg/L, 20 and 25 % immobilisation was observed at 24 and 48 h observation, respectively.
At 0.25 and 0.025 mg/L, no immobilisation was observed.

The measured concentrations of the test item ST 15 C 12 at the start of the exposure intervals (0 and 24 h) were in the range of 73 to 85 % of the nominal values. At the end of the exposure intervals (24 and 48 h) the measured concentrations of the test item ST 15 C 12 were in the range of 57 to 68 % of the nominal values. The geometric mean measured concentrations were calculated to be 3.58 mg/L (1st range finding test) and 0.0185, 0.175 and 1.67 mg/L (2nd range finding test).
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
None

Table 6.1.3/1: Immobilisation Rates

Nominal

test item

concentration

[mg/L]

IMMOBILISATION [%]

24 h

48 h

Replicates

Replicates

1st Range Finding Test (non GLP)

 

1

2

MV

1

2

MV

5

80

100

90

100

100

100

Control

0

0

0

0

0

0

2nd Range Finding Test (non GLP)

2.5

20

20

20

20

30

25

0.25

0

0

0

10

0

5

0.025

0

0

0

0

0

0

Control

0

0

0

0

0

0

 

MV = mean value

Validity criteria fulfilled:
yes
Conclusions:
The biological effects have to be expected between nominal test item concentrations of 0.25 mg/L and 5 mg/L, which corresponds to geometric mean measured concentrations of 0.175 to 3.58 mg/L. Based on the nominal concentrations, the EC50 is expected to be between 2.5 mg/L and 5 mg/L. Based on the geometric mean measured concentrations, the EC50 is expected to be between 1.67 mg/L and 3.58 mg/L.
Executive summary:

Two non GLP acute immobilisation tests with Daphnia magna (STRAUS) were performed as range finding tests to determine the effects of the test item according to OECD 202 and EU Method C.2 guidelines. 

The range finding tests were conducted under semi-static conditions over a period of 48 h. A nominal test item concentration of 5 mg/L in the 1st range finding test. In the 2nd range finding test, 3 concentrations of the test item were tested, which were prepared with dilution water in a geometric series with a separation factor of 10. The test concentrations in the 2nd range finding test were 0.025, 0.25 and 2.5 mg/L. Twenty daphnids were exposed to each concentration level and the control. All test solutions were prepared in dilution water. The test temperature was 18-22 °C.

 

The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 73 to 85 % of the nominal values. At the end of the exposure intervals (24 and 48 h) the measured concentrations of the test item were in the range of 57 to 68 % of the nominal values. The geometric mean measured concentrations were calculated to be 3.58 mg/L (1st range finding test) and 0.0185, 0.175 and 1.67 mg/L (2nd range finding test).

 

At 5 mg/L, 90 and 100 % immobilisation was observed at 24 and 48 h observation, respectively. At 2.5 mg/L, 20 and 25 % immobilisation was observed at 24 and 48 h observation, respectively. At 0.25 and 0.025 mg/L, no immobilisation was observed.

 

The biological effects have to be expected between nominal test item concentrations of 0.25 mg/L and 5 mg/L, which corresponds to geometric mean measured concentrations of 0.175 to 3.58 mg/L. Based on the nominal concentrations, the EC50 is expected to be between 2.5 mg/L and 5 mg/L. Based on the geometric mean measured concentrations, the EC50 is expected to be between 1.67 mg/L and 3.58 mg/L.

Description of key information

OECD Guideline 202, EU Method C.2, GLP, key study, validity 1:

48h-EC50 (D. magna) = 1.8 mg/L (95% CL = 1.0-3.3 mg/L), based on mean measured concentrations.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.8 mg/L

Additional information

To assess the short-term toxicity of the registered substance to aquatic invertebrates, two experimental studies are available.

The first study (Harlan, 2013) was assessed as the key study and was performed on the registered substance according to OECD Guideline 202 and EU Method C.2 in compliance with GLP, on Daphnia magna. Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test substance using traditional methods of preparation. A pre-study media preparation trial indicated that a dissolved test substance concentration of approximately 3.1 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of the test 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 substance at concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution for 48 hours at a temperature of 19 °C to 22 °C under static test conditions. Analysis of the 32 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations of 1.05 and 3.39 mg/L respectively, and at 48 hours showed measured test concentrations of 0.967 and 3.11 mg/L respectively. Accordingly, the mean measured concentration between the start and the end of the test were retained. For the 32 and 100% v/v saturated solution test preparations, the mean measured concentrations were 1.0 and 3.3 mg/L, respectively. At 1.0 mg/L, 0% immobilisation was observed after 24 and 48 hours, and at 3.3 mg/L, 100% immobilisation was observed after 24 and 48 hours. Therefore, the 48h-EC50 value was determined to be 1.8 mg/L, which correspond to the geometric mean between 1.0 and 3.3 mg/L.

The second study (Noack, 2015) was assessed as the supporting study and was performed on the registered substance according to OECD Guideline 202 and EU Method C.2. This study summarised two non-GLP range-finding tests on Daphnia magna, performed under semi-static conditions over a period of 48 hours. In the 1st range finding test, a nominal test substance concentration of 5 mg/L was tested. In the 2nd range finding test, 3 concentrations of the test substance were tested: 0.025, 0.25 and 2.5 mg/L. Twenty daphnids were exposed to each concentration level and the control. All test solutions were prepared in dilution water. The test temperature was 18-22 °C. The measured concentrations of the test substance at the start of the exposure intervals (0 and 24 h) were in the range of 73 to 85 % of the nominal values. At the end of the exposure intervals (24 and 48 h) the measured concentrations of the test item were in the range of 57 to 68 % of the nominal values. The geometric mean measured concentrations were calculated to be 3.58 mg/L (1st range finding test) and 0.0185, 0.175 and 1.67 mg/L (2nd range finding test). In these tests, the limit of water solubility obtained under test conditions was 3.58 mg/L. At 3.58 mg/L, 90 and 100 % immobilisation were observed at 24 and 48 h, respectively. At 1.67 mg/L, 20 and 25 % immobilisation were observed at 24 and 48 h, respectively. At 0.175 and 0.0185 mg/L, no immobilisation was observed. Therefore, the 48h-EC50 is expected to be between geometric mean measured concentrations of 1.67 and 3.58 mg/L. As the results of these non-GLP range finding tests were in accordance with the previous GLP study (Harlan, 2013), the definitive study was not considered necessary.

In conclusion, based on the key study (Harlan, 2013), the 48h-EC50 value based on mean measured concentration was 1.8 mg/L with 95% CL of 1.0 -3.3 mg/L.