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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:
2014-07-09 to 2014-07-15
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:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on 03-05 June 2013 / signed on 05 November 2013
Specific details on test material used for the study:
- Stability under test conditions: Not specified, assumed to be stable
- Storage condition of test material: Keep in the dark at 0 - 10 °C, under nitrogen
Analytical monitoring:
yes
Details on sampling:
- Determination of the test item: 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 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 or initially measured concentrations. The effect levels can be given based on the nominal test item concentrations as this is fulfilled for the biologically relevant test concentrations of 25.0 to 200 mg/L.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution (200 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.
- 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
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
269 mg/L as CaCo3
Test temperature:
20.9-21.0 °C
pH:
7.66-7.81
Dissolved oxygen:
8.49-8.73 mg/L
Salinity:
None
Conductivity:
652-669 µs/cm
Nominal and measured concentrations:
Nominal concentrations: 12.5, 25, 50, 100 and 200 mg/L.
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 lb x 7 cm H), 50 ml capacity, loosely covered with watch glasses
- Test volume: 20 mL
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- 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: 652-669 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 was 0, 0, 10 and 15% (24 h) and 0, 0, 20 and 40% at 0, 1, 10 and 100 mg/L, respectively.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
188 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% Cl: 104- >200 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
108 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% Cl: 85.2-145 mg/L
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 200 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, 0, 0, 30 and 50 % (24 h); 0, 0, 0, 10, 45 and 80 % (48 h) at 0, 12.5, 25, 50, 100 and 200 mg/L, respectively.

- Measured Exposure Concentrations during the Definitive Test: The concentrations of the test item ST 11 C 13 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 106 % 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 76 to 94 % of the nominal values. The measured test item concentrations at the biologically relevant test item concentrations of 25 to 200 mg/L inducing mean immobilisation rates of 0 - 80 % were within ± 20 % of the nominal concentrations. This indicates that the test item concentrations in this relevant range were successfully maintained for the duration of the test.

- 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 prism5. The concentration effect relationships are shown graphically. The EC50-value for the reference item and its confidence limits were calculated accordingly.

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

 

 

IMMOBILISATION [%]

Nominal

test item

concentration

[mg/L]

24 h

48 h

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

200

60

60

40

40

50

100

80

60

80

80

100

60

40

0

20

30

80

60

20

20

45

50

   0

   0

   0

   0

   0

20

0

20

0

10

25

   0

   0

   0

   0

   0

   0

   0

   0

   0

   0

12.5

   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

 

Table 6.1.3/2: Measured Concentrations of the Test Item during the Definitive Test

 

Sampling date

2014-07-09

Start of the exposure, 0 hours

2014-07-10

End of the exposure, 24 hours

2014-07-10

Start of the exposure, 24 hours

2014-07-11

End of the exposure, 48 hours

Date of analysis

2014-07-09

2014-07-10

2014-07-10

2014-07-11

Nominal

test item

concentration

[mg/L]

ST 11 C 13

Meas. conc.

[mg/L]

%

Meas. conc.

[mg/L]

%

Meas. conc.

[mg/L]

%

Meas. conc.

[mg/L]

%

200

190

95

178

89

196

98

161*

81

100

99.1

99

87.0

87

96.8

97

86.0

86

50

49.3

99

42.7

85

44.9

90

46.1

92

25

26.0

104

22.2

89

22.9

92

23.5

94

12.5

12.4

99

11.2

90

13.2

106

9.51**

76

Control

< SYSQL

 

< SYSQL

 

< SYSQL

 

< SYSQL

 

 Meas. conc. = measured concentration of the test item, single injection, dilution factors taken into account

% = percent of the nominal concentration of the test item

SYSQL = System quantification limit (4 µg/L)

*Reanalysis, mean value of 2 replicates each with a single injection, dilution factor taken into account

**Reanalysis, mean value of 3 replicates each with a single injection, dilution factor taken into account
Validity criteria fulfilled:
yes
Conclusions:
Based on the analytically confirmed nominal concentrations of the test item ST 11 C 13, the 48 h-EC50 to Daphnia magna was 108 mg/L (95 % Cl: 85.2 - 145 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 toDaphnia magna, under semi-static conditions.

 

Test item was exposed to daphnids at the concentrations of 12.5, 25, 50, 100 and 200 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 20.9-21.0 °C. The tested solutions were clear throughout the exposure period. Before definitive test, a 48-hour semi-static range-finding test was conducted and the percent immobilization was 0, 0, 20 and 40% at 0, 1, 10 and 100 mg/L, respectively.

 

The concentrations of the test item ST 11 C 13 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 106 % 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 76 to 94 % of the nominal values. The measured test item concentrations at the biologically relevant test item concentrations of 25 to 200 mg/L inducing mean immobilisation rates of 0-80 % were within ± 20 % of the nominal concentrations. This indicates that the test item concentrations in this relevant range were successfully maintained for the duration of the test. 

 

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 guidelines were fulfilled.

 

Immobilization of daphnids for the definitive test was 0, 0, 0, 0, 30 and 50 % (24 h); 0, 0, 0, 10, 45 and 80 % (48 h) at 0, 12.5, 25, 50, 100 and 200 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.

 

Based on the analytically confirmed nominal concentrations of the test item ST 11 C 13, the 48 h-EC50 to Daphnia magna was 108 mg/L (95 % Cl: 85.2 - 145 mg/L).

Description of key information

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

48h-EC50 (Daphnia magna) = 108 mg/L (95% CI: 85.2 -145 mg/L) based on analytically confirmed nominal concentration

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
108 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 five nominal test substance concentrations: 12.5, 25, 50, 100 and 200 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 20.9-21.0 °C. The tested solutions were clear throughout the exposure period. Before definitive test, a 48-hour semi-static range-finding test was conducted and the percent immobilization was 0, 0, 20 and 40% 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 106 % 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 76 to 94 % of the nominal values. The measured test item concentrations at the biologically relevant test item concentrations of 25 to 200 mg/L inducing mean immobilisation rates of 0-80 % were within ± 20 % of the nominal concentrations. This indicates that the test substance concentrations in this relevant range were successfully maintained for the duration of the test. 

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 guidelines were fulfilled. Immobilization of daphnids for the definitive test was 0, 0, 0, 0, 30 and 50 % (24 h); 0, 0, 0, 10, 45 and 80 % (48 h) at 0, 12.5, 25, 50, 100 and 200 mg/L, respectively.

Based on the analytically confirmed nominal concentrations of the test substance, the 48 h-EC50 to Daphnia magna was 108 mg/L (95 % Cl: 85.2 - 145 mg/L).