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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:
23 Jan 2017 to 17 Feb 2017
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:
2004
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis; with the exception of the 10 mg/L test concentration at 48 hours due to 100% immobilization observed. All samples were taken for analysis on the day of sampling. Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST 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, as a precautionary measure, 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 mg/L test preparation. Dilutions were made from this stock solution to obtain the desired test concentrations. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: in-house laboratory cultures
- Breeding conditions: Adult daphnids were maintained in 150 mL glass beakers containing Elendt M7 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.
- Food during culture: 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.
- Feeding during test: no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
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
Test temperature:
19 - 22 ºC
pH:
7.6 - 8.1
Dissolved oxygen:
9.0 - 9.5 mg O2/L
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 0.10, 0.32, 1.0, 3.2 and 10 mg/L (based on range-finding test)
- Geometric mean measured test concentrations: 0 (control), 0.055, 0.10, 0.38, 2.38 and 9.11 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL ground glass stoppered conical flasks completely filled with test preparation
- Type: closed; the test vessels were sealed after addition of test organisms
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Aeration: no
- Feeding during tests: no
- Renewal rate of test solution: 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
- Test water: Reconstituted water (Elendt M7)
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start, pre and post media renewal and termination of the test.

OTHER TEST CONDITIONS
- Photoperiod:16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 540 to 688 Lux

EFFECT PARAMETERS MEASURED:
- Immobility: 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.
- Additional parameters measured: water temperature (daily), dissolved oxygen concentraitons and pH (test start, pre- and post media renewal and at termination of the test), appearance of test media (daily).

RANGE-FINDING STUDY
- Test concentrations: nominal test concentrations of 0 (control), 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No immobilization was observed at the test concentrations of 0.10 and 1.0 mg/L. At 10 and 100 mg/L, all daphnids were immobile after 24 hours. Furthermore, all daphnids in the 1.0 mg/L test group were observed to have reduced movement after 48 hours exposure.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.L.: 0.29 - 0.68 mg/L
Details on results:
After 24 hours of exposure, immobilisation was observed from the measured concentration (geometric mean) of 0.38 mg/L (nominal 1 mg/L) and increased in a dose-dependent manner up to 100% immobility at the highest concentration. After 48 hours of exposure, 5 - 10% immobilisation was observed in control and the two lowest test concentrations, 35% immobility was observed at the measured concentration (geometric mean) of 0.38 mg/L (nominal 1 mg/L) and 100% immobility at the two highest concentrations. For details, see 'Any other information on results incl. tables'.
Results with reference substance (positive control):
The most recent test conducted between 20 October 2016 and 22 October 2016, determined a 24h-EC50 of 1.3 mg/L. The results from the positive control with potassium dichromate were within the normal range for this reference item.
Reported statistics and error estimates:
Probit analysis using Linear Maximum-Likelihood regression and Step- down Cochran-Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Tables: immobility of daphnids after 24 and 48 hours exposure

Nominal

Concentration

(mg/L)

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

0.10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.32

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1.0

0

1

0

0

1

5

5 N

4 N

5 N

5 N

3.2

5

5

5

4

19

95

A/I

A/I

A/I

1 R

10

5

5

5

5

20

100

A/I

A/I

A/I

A/I

Nominal

Concentration

(mg/L)

48 Hours

Cumulative Immobilized Daphnia

(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

1

1

2

10

5 N

5 N

4 N

4 N

0.10

1

0

0

0

1

5

4 N

5 N

5 N

5 N

0.32

1

0

0

0

1

5

4 N

5 N

5 N

5 N

1.0

2

2

2

1

7

35

3 N

3 N

3 N

4 N

3.2

5

5

5

5

20

100

A/I

A/I

A/I

A/I

10

5

5

5

5

20

100

A/I

A/I

A/I

A/I

R1– R4= Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced mobility

A/I = All daphnia immobilized

Validity criteria fulfilled:
yes
Remarks:
see 'Any other information on materials and methods incl. table's
Conclusions:
The 48-h Daphnia magna EC50 value is 0.43 mg/L in aquatic invertebrates
Executive summary:

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study daphnids (D. magna, 20 per concentration) were exposed to nominal concentrations of 0 (control), 0.10, 0.32, 1.0, 3.2 and 10 mg/L which were based on a range-finding study for 48 hours under semi-static conditions. Immobility and abnormal behaviour were determined after 24 and 48 hours exposure. Analytical confirmation of nominal test concentrations showed that the deviation of the exposure concentrations of the test substance was greater than ±20% of the nominal concentrations (according to the measured concentrations of 24h old solutions), and therefore the results are expressed in terms of geometric means of the exposure concentration. The average exposure concentrations (geometric means) were calculated to correspond to: 0 (control), 0.055, 0.10, 0.38, 2.38 and 9.11 mg/L. After 48 hours of exposure, immobilisations were 10% at the control, 5% at 0.055 and 0.10 mg/L, 35% at 0.38 mg/L and 100% at 2.38 and 9.11 mg/L. Thus, immobilisation occurred in a dose-dependent manner. Based on these data, the 48-h EC50 value is determined at 0.43 mg/L (95% C.L. 0.29 - 0.68 mg/L).

Description of key information

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study daphnids (D. magna, 20 per concentration) were exposed to nominal concentrations of 0 (control), 0.10, 0.32, 1.0, 3.2 and 10 mg/L which were based on a range-finding study for 48 hours under semi-static conditions. Immobility and abnormal behaviour were determined after 24 and 48 hours exposure. Analytical confirmation of nominal test concentrations showed that the deviation of the exposure concentrations of the test substance was greater than ±20% of the nominal concentrations (according to the measured concentrations of 24h old solutions), and therefore the results are expressed in terms of geometric means of the exposure concentration. The average exposure concentrations (geometric means) were calculated to correspond to: 0 (control), 0.055, 0.10, 0.38, 2.38 and 9.11 mg/L. After 48 hours of exposure, immobilisations were 10% at the control, 5% at 0.055 and 0.10 mg/L, 35% at 0.38 mg/L and 100% at 2.38 and 9.11 mg/L. Based on these data, the 48-h EC50 value is determined at 0.43 mg/L (95% C.L. 0.29 – 0.68 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.43 mg/L

Additional information