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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:
16 - 19 July 2014
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 with GLP certificate. All validity criteria were fulfilled.
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on June 03-05, 2013 / signed on November 05, 2013
Analytical monitoring:
yes
Details on sampling:
- Determination of the test item: All concentration levels and the control were analytically verified via GCMS at the start (0 and 24 h) and at the end of both exposure intervals (24 and 48 h).
- Sampling for the analytical monitoring: 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 with test media, but without daphnids, which were incubated under test conditions until sampling.
- Quality criteria for the analytical monitoring: Recoveries of the test item should be within ± 20% of the nominal concentrations. In this case, the effect levels can be based on the nominal concentrations.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution (80.0 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 treated with ultrasound for 15 minutes at room temperature and stirred thereafter with a magnetic stirrer at approximately 1100 rpm for 1 h at room temperature. Further test concentrations were prepared by diluting the stock solution of 80.0 mg/L with dilution water. The tested solutions were clear throughout the exposure period.
- 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 für Wasser-, Boden- und Lufthygiene (WaBoLu).
- Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany.
- Age at study initiation: 2 to 24 hours old daphnids from a healthy stock were used for the study. They were obtained by removing the juvenile daphnids from the culture vessels 22 h before the start of the exposure. The juveniles born within the 22 h preceding the exposure were used for the test 2 hours after separation from the culture vessels. No first brood progeny was used for the test.
- 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: The culture daphnids are fed 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.
- Feeding during test: The 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
Post exposure observation period:
None
Hardness:
265-271 mg/L as CaCo3
Test temperature:
20.6 °C
pH:
7.60-7.70
Dissolved oxygen:
8.96-9.04 mg/L
Salinity:
None
Conductivity:
649-658 μs/cm
Nominal and measured concentrations:
Nominal concentrations: 2.5, 5, 10, 20, 40 and 80 mg/L
All concentration levels and the control were analytically verified via GC-MS at the start (0 and 24h) and at the end of both exposure intervals (24 and 48h)
Details on test conditions:
TEST METHOD:
Semi-static (with a renewal of the test solutions after 24 h)

TEST SYSTEM
- Test vessels: Glass beakers (4 cm lD x 7 cm H), 50 mL capacity, loosely covered with watch glasses
- Test volume: 20 mL
- Renewal of 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: 5 (control and test material groups)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- 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
- 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, is used.
- Conductivity: 649-658 μs/cm

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

EFFECT PARAMETERS MEASURED:
Biological parameters:
- Immobilisation and other observations: 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.
Water Quality Parameters: Dilution water: Prior to the start of the exposure (0 h) and the water renewal (24 h), the water quality parameters (i.e, pH value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured.
Test media: At the start of the exposure intervals (0 and 24 h), the water quality parameters of the fresh media (i.e. pH value, dissolved oxygen concentration) were measured in one additional replicate per concentration level and control. At the end of the exposure intervals (24 and 48 h), the water parameters of the old media were measured in all replicates per concentration level and control.
Temperature: The incubator temperature was recorded throughout the period of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Factor 2
- Range finding study: Test concentrations were selected based on the results of a non GLP preliminary range finding test. This test was conducted under semi-static conditions with three concentrations of the test item of 1, 10 and 100 mg/L and two replicates per concentration level with 10 daphnids each were tested. The percentage immobility was determined in all concentrations and control groups after 24 and 48 h of exposure.
- Results used to determine the conditions for the definitive study: Immobilisation percentage were 0, 15, 20 and 100% (24 h) and 0, 20, 30 and 100% at 0, 1, 10 and 100 mg/L, respectively.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
29.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI: 24.8 - 35.2 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
21.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI: 19.5 - 28.5 mg/L
Key result
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Biological data: Immobilization of daphnids for the definitive test was 0, 0, 10, 5, 15, 75 and 85 % (24 h); 0, 0, 20, 25, 40, 100 and 100 % (48 h) at 0, 2.5, 5, 10, 20, 40 and 80 mg/L, respectively.
- Measured Exposure Concentrations during the Definitive Test: The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 90 to 107 % 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 88 to 108 % of the nominal values.
- Water Quality Parameters: The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.
Results with reference substance (positive control):
The percentage immobility for the reference item was determined after 24 h. The EC50-value with 95 % confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.82 mg/L (Cl 1.66-2.15 mg/L)
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.
Reported statistics and error estimates:
The EC50-values after 24 and 48 h were calculated by sigmoidal dose response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism 5. The concentration effect relationships are shown graphically. The EC50-value for the reference item and its confidence limits were calculated accordingly.
The EC100-values after 24 and 48 h were estimated directly from the observation data, without mathematical calculation.

Table 6.1.3/1:  Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

 

Nominal

test item

concentration

[mg/L]

IMMOBILISATION [%]

24 h

48 h

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

80

80

80

100

80

85

100

100

100

100

100

40

80

80

80

60

75

100

100

100

100

100

20

20

0

0

40

15

40

40

40

40

40

10

20

0

0

0

5

20

20

20

40

25

5.0

0

20

0

20

10

20

20

0

40

20

2.5

0

0

0

0

0

0

0

0

0

0

Control

0

0

0

0

0

0

0

0

0

0

 

n = 20, divided into 4 replicates with 5 daphnids each

 

Validity criteria:

- In the control group, no daphnids were immobilised or showed any signs of disease or stress, e.g. discoloration or unusual behaviour such as trapping on the surface of the water, during the 48-hour test period (required: not more than 10 percent of the daphnids in the control).

- The dissolved O2concentration at the end of the exposure intervals was ≥ 8.65 mg/L (required: ≥ 3 mg/L at the test end of the exposure intervals) in the test vessels of all tested concentration levels and the control.

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions and based on the analytically confirmed nominal concentrations of the test item, the 48 h-EC50 for Daphnia magna was 21.7 mg/L (95 % confidence limits: 19.5 - 28.5 mg/L).
Executive summary:

Study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the of the test substance to Daphnia magna, under semi-static conditions.

Test item was exposed to daphnids at the nominal concentrations of 2.5, 5, 10, 20, 40 and 80 mg/L for 48 hours. The no-treatment control daphnids were exposed to dilution water only. There were four replicates per treatment with five daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. All test solutions were prepared in dilution water. The test temperature was 20.6 °C. The tested solutions were clear throughout the exposure period. Before definitive test, a 48-hour static range-finding test was conducted and the percent immobilization was 0, 20, 30 and 100% at 0, 1, 10 and 100 mg/L, respectively. 

 

The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 90 to 107 % 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 88 to 108 % of the nominal values.

 

Immobilization of daphnids for the definitive test was 0, 0, 10, 5, 15, 75 and 85 % (24 h); 0, 0, 20, 25, 40, 100 and 100 % (48 h) at 0, 2.5, 5, 10, 20, 40 and 80 mg/L, respectively.

The EC50 -value of the reference item, potassium dichromate at 1.82 mg/L (Cl 1.66 - 2.15 mg/L) after 24 h was within the prescribed concentration range of 0.6 - 2.1 mg/L.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

 

Under the test conditions and based on the analytically confirmed nominal concentrations of the test item, the 48 h-EC50 for Daphnia magna was 21.7 mg/L (95 % confidence limits: 19.5 - 28.5 mg/L).

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

48 h-EC50 (Daphnia magna) = 21.7 mg/L (95 % confidence limits: 19.5 - 28.5 mg/L) based on the analytically confirmed nominal concentrations of the test item.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
21.7 mg/L

Additional information

One key study is available (NOACK, 2014) to assess the 48h acute toxicity of the registered substance to Daphnia magna, under semi-static conditions, according to OECD Guideline 202 and EU Method C.2 with GLP statement. 

Daphnids were exposed to six nominal test substance concentrations: 2.5, 5, 10, 20, 40 and 80 mg/L for 48 hours. The no-treatment control daphnids were exposed to dilution water only. There were four replicates per treatment with five daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. All test solutions were prepared in dilution water. The test temperature was 20.6 °C. The tested solutions were clear throughout the exposure period. Before definitive test, a 48-hour static range-finding test was conducted and the percent immobilization was 0, 20, 30 and 100% at 0, 1, 10 and 100 mg/L, respectively. 

The concentrations of the test substance were analytically verified via GC-MS at the start of the exposure intervals (0 and 24 h) and at the end of the exposure intervals (24 and 48 h) in all concentration levels and the control.The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 90 to 107 % 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 88 to 108 % of the nominal values.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Immobilization of daphnids for the definitive test was 0, 0, 10, 5, 15, 75 and 85 % (24 h); 0, 0, 20, 25, 40, 100 and 100 % (48 h) at 0, 2.5, 5, 10, 20, 40 and 80 mg/L, respectively.

The EC50 -value of the reference item, potassium dichromate at 1.82 mg/L (Cl 1.66 - 2.15 mg/L) after 24 h was within the prescribed concentration range of 0.6 - 2.1 mg/L.

Under the test conditions and based on the analytically confirmed nominal concentrations of the test item, the 48 h-EC50 for Daphnia magna was 21.7 mg/L (95 % confidence limits: 19.5 - 28.5 mg/L).