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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:
25-01-2012 to 13-02-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
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)
Remarks:
inspected: July 2011; signature: August 2011
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L. Samples from control replicates and test group replicates of equal nominal concentration were pooled for analysis. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.
- Sampling method: Duplicate samples were taken at 0 h and 48 h for quantitative analysis. Samples were analysed following addition of formic acid (0.1 mL per 100 mL of sample). If required, samples were diluted to 10 mg/L with 0.1% formic acid in test medium.
- Sample storage conditions before analysis: stored at approximately -20ºC.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dissolution of test item in culture medium to prepare stock; serial dilution of stock to prepare test media.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No precipitate reported.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: not reported
- Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): < 24 hours
- Method of breeding: Parthenogenesis
- Feeding during test: No. The daphnids were not fed during the study. During culture: The culture daphnids are fed daily ad libitum with a mix of algae suspension, e.g. Desmodesmus subspicatus and flake food suspension.
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: None reported. Can be presumed to be < 2 hours in dilution water.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Dilution water quality parameters: 0 hours: 2.5 mmol/L, 250 mg CaCO3/L
Test temperature:
Target: 19 - 21ºC (actual: 21ºC)
pH:
Control: 0 hours: pH 8.0 - 8.2 and 48 hours: 8.1 - 8.4; test groups: 0 hours: pH 7.9 - 8.0 and 48 hours: 8.0 - 8.1
Dissolved oxygen:
Control: 0 hours: 9.5 - 9.6 mg O2/L (100-105% ASV) and 48 hours: 8.9 - 9.0 mg O2/L (100-101% ASV); test groups: 0 hours: 9.5 - 9.6 mg O2/L (100-105% ASV) and 48 hours: 9.0 mg O2/L (101% ASV)
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.
Salinity:
Not applicable.
Nominal and measured concentrations:
Range finding tests were performed prior to the definitive test: 0.1, 1.0, 10 mg/L and 100 mg/L
Chemical analysis verified that the test item was maintained at 89-96% of nominal at 0 hours and 82-86% of nominal at the 48 hours. Therefore concentrations are presented as nominal.
Definitive test: nominal test item concentrations: 0 (control), 1.0, 1.8, 3.2, 5.6, 10.0 mg/L.
Chemical analysis verified that the test item was maintained at 88-94% of nominal at 0 hours and 81-87% of nominal at the 48 hours. Therefore concentrations are presented as nominal.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass; 200 mL fill volume, headspace ca. 50 mL.
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel (divided into 4 replicates)
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: equivalent to 40 mL test media per animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water - analytical grade salts dissolved in purified water equivalent or similar to ISO Test water, according to OECD 202, Annex 3: composition (mg/L): KCl 5.8; NaHCO3: 64.8; CaCl2.2H2O: 294 ; MgSO4.7H2O: 123.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: 0.8 mmol/l
- Ca/mg ratio: 4 (molar terms; based on 2.0 mmol CaCl2.2H2O; 0.5 mmol MgSO4.7H2O)
- Conductivity: <5 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0 hours and 48 hours, (dissolved oxygen, pH and temperature)

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light / 8 hours dark, 20 minute dawn and dusk transition periods.
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility (or adverse effects including mortality), 24 hours and at 48 hours. An organism 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study:
- Test concentrations: 0 (control), 0.10, 1.0, 10, 100 mg/L ; nominal test item concentration.
- Results used to determine the conditions for the definitive study: Yes. No immobilisation observed at 0.10 and 1.0 mg/L. However, immobilisation was observed at 10 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
4.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (95% CL. 3.9-4.3 mg/L)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: None reported.
- Observations on body length and weight: Not applicable.
- Other biological observations:
- Mortality of control: No mortalities in control.
- Other adverse effects control: None reported.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. Presence of undissolved test item during preparation and during the test was not reported.
- Effect concentrations exceeding solubility of substance in test medium: No.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes. Conducted under the same conditions as the test item definitive test.
- Mortality: None. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 1.30 mg/L (C.I. 1.00 – 1.80) and 48-h EC50: 1.10 mg/L (C.I. 1.00 – 1.30) and 48-h NOEC: 0.56 mg/L
- Other: The results from the positive control with potassium dichromate were reportedly within the normal range.
Applicant assessment indicates: The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD Guideline TG 202
Reported statistics and error estimates:
The EC50 value and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method ((Hamilton et al. 1977)

Table 1.0: Cumulative immobilisation data in the definitive test

Nominal concentration (mg/L)

Cumulative immobilised Daphnia (initial population: 5 per replicate)

24 h:

Total

%

48 h:

Total

%

Control

Replicate #1

0

0

0

0

0

0

 

Replicate #2

0

0

 

Replicate #3

0

0

 

Replicate #4

0

0

1.0 mg/L

Replicate #1

0

0

0

0

0

0

 

Replicate #2

0

0

 

Replicate #3

0

0

 

Replicate #4

0

0

1.8 mg/L

Replicate #1

0

0

0

0

0

0

 

Replicate #2

0

0

 

Replicate #3

0

0

 

Replicate #4

0

0

3.2 mg/L

Replicate #1

0

0

0

1

1*

5

 

Replicate #2

0

0

 

Replicate #3

0

0

 

Replicate #4

0

0

5.6 mg/L

Replicate #1

0

0

0

5

20

100

 

Replicate #2

0

5

 

Replicate #3

0

5

 

Replicate #4

0

5

10 mg/L

Replicate #1

0

6

30

5

20

100

 

Replicate #2

1

5

 

Replicate #3

1

5

 

Replicate #4

4

5

* single immobilised daphnid attributed within the study to natural causes rather than a true toxic effect, given that <10% immobilisation was observed to have occurred.

Validity criteria fulfilled:
yes
Conclusions:
The test item 48h-EC50 was 4.1 mg/L (C.I: 3.9 – 4.3 mg/L) based on analytically confirmed nominal concentrations.
Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines. Following preliminary range finding test in the 0.1 to 100 mg/L an identically conducted definitive study was completed in 250 mL glass flasks with a 200 mL fill volume under static conditions. This was over a period of 48 hours with five concentration levels of the test item in the nominal concentration range of 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L prepared in a geometric series with a separation factor of 1.8. A stock solution of 100.0 mg/L was prepared with reconstituted water (consistent with OECD 202, Annex 3) and then serial dilutions made to the required test item concentrations. Twenty daphnids were exposed to each concentration level and the control in four replicates, respectively. The concentrations of the test item were analytically verified from pooled water samples from each control and test group via HPLC-DAD at the start (0 hours) and at the end of the exposure (48 hours) in all relevant concentration levels and the control. Samples were stored at -20°C prior to analysis. Physicochemical measurements on water quality were made throughout the test including: temperature, pH and oxygen concentration. The temperature was maintained at 19 to 21°C and the pH was 7.9 to 8.4 and the dissolved oxygen concentrations were 8.9 to 9.0 mgO2/L. The validity criteria of the test guideline were fulfilled. The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 89 to 96% and at the end of the exposure (48 hours) in the range of 82 to 86% of the nominal concentrations. Based on the analytical recoveries and the measured test item concentrations remaining within ± 20% of the nominal concentrations during the exposure, the EC50-values are based upon the nominal concentrations. Under the conditions of this study, the 48h-EC50 was 4.1 mg/L (C.I: 3.9 – 4.3 mg/L) based on analytically confirmed nominal concentrations.

Description of key information

EC50 (invertebrates) = 4.1 mg/L (C.I: 3.9 – 4.3 mg/L) mg/L based on nominal concentrations, 48hour-freshwater, OECD TG 202, 2012

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
4.1 mg/L

Additional information

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines. Following preliminary range finding test in the 0.1 to 100 mg/L an identically conducted definitive study was completed in 250 mL glass flasks with a 200 mL fill volume under static conditions. This was over a period of 48 hours with five concentration levels of the test item in the nominal concentration range of 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L prepared in a geometric series with a separation factor of 1.8. A stock solution of 100.0 mg/L was prepared with reconstituted water (consistent with OECD 202, Annex 3) and then serial dilutions made to the required test item concentrations. Twenty daphnids were exposed to each concentration level and the control in four replicates, respectively. The concentrations of the test item were analytically verified from pooled water samples from each control and test group via HPLC-DAD at the start (0 hours) and at the end of the exposure (48 hours) in all relevant concentration levels and the control. Samples were stored at -20°C prior to analysis. Physicochemical measurements on water quality were made throughout the test including: temperature, pH and oxygen concentration. The temperature was maintained at 19 to 21°C and the pH was 7.9 to 8.4 and the dissolved oxygen concentrations were 8.9 to 9.0 mgO2/L. The validity criteria of the test guideline were fulfilled. The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 89 to 96% and at the end of the exposure (48 hours) in the range of 82 to 86% of the nominal concentrations. Based on the analytical recoveries and the measured test item concentrations remaining within ± 20% of the nominal concentrations during the exposure, the EC50-values are based upon the nominal concentrations. Under the conditions of this study, the 48h-EC50 was 4.1 mg/L (C.I: 3.9 – 4.3 mg/L) based on analytically confirmed nominal concentrations.

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