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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 May 2011 - 09 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant. Guideline study. Acceptable without restriction.
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
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and control were sampled..
- Sampling method: Duplicate samples were taken from the test media of all test concentrations at the start and end of each test medium renewal period. For sampling from the aged test medium , the test medium of the treatment replicates were pooled.
- Sample storage condition before analysis: Samples were deep-frozen immediately after sampling and stored at about -20˚C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test item in test water, a dispersion with the loading rate of 100 mg/L was prepared at the start of each test period by dispersing 150.0 mg of the test item in 1500 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 96 hours at room temperature in the dark, to dissolve a maximum amount of the test item in the dispersion. After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (pore size 0.45 μm). The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. The test media were prepared just before the start of the test (= addition of daphnids) and before each test medium renewal.
- Controls: Reconstituted test water (ISO 6341 with hardness lowered by factor of 1.7 of the normal hardness). Potassium dichromate is tested as a positive control twice a year.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported after filtration.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain: Clone 5
- Source: In lab culture
- Age at study initiation (mean and range, SD): 6-24 hours and were not first brood progeny
- Feeding during test: No

ACCLIMATION
- Acclimation period: The parental daphnids were maintained in test water for at least 48 hours prior to the start of the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
150 mg CaCO3/L
Test temperature:
20-21°C
pH:
Test start range ( 0 hours) = 7.7 - 7.8
After 24 hours (old) = 7.9 - 8.0
After 24 hours (new) = 7.6 - 7.7
At end of test (old) = 7.7
Dissolved oxygen:
Test start range ( 0 hours) = 8.6 - 8.9 mg/L
After 24 hours (old) = 8.7 - 8.9 mg/L
After 24 hours (new) = 8.1 - 8.6 mg/L
At end of test (olds) = 8.2 - 8.6 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
The following concentrations of test substance were tested: undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2.2, 1:4.6, 1:10 and 1:22 of the undiluted filtrate with the loading rate of 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. Measured concentrations in the fresh test medium at 0 and 24 hours were: 5.29 and 4.86 mg test substance/L for the dilution 1:2.2 and 13.3 and 10.4 mg test substance/L for the undiluted filtrate. Measured concentrations in the old test medium at 24 and 48 hours were: 4.66 and 4.82 mg test substance/L for the dilution 1:2.2 and 8.92 and 10.2 mg test substance/L for the undiluted filtrate. The samples from the dilutions 1:4.6, 1:10 and 1:22 were not analyzed. The biological results were based on the mean measured concentrations calculated as geometric means of the test item concentrations for each test medium renewal and the average of these geometric means (see table below).
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers
- Type (delete if not applicable): closed (covered with glass plates)
- Material, size, headspace, fill volume: 250 mL glass beakers filled with 125 mL of test medium
- Aeration: Prior to test until oxygen saturation but not during test.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 25mL test medium/daphnid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (analytical grade salts dissolved in purified water) according to ISO 6341, with hardness lowered to meet OECD and EPA guideline requirements.
- Alkalinity: 0.47 mmol/L
- Ca/Mg ratio: 4:1
- Intervals of water quality measurement: At the start and end of each test medium renewal period for pH, dissolved oxygen and water temperature. The appearance of the test media was visually recorded at the same time.

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: 16:8 light/dark cycle with 30 minute transition period.
- Light intensity: Between approximately 485-640 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Daphnids were considered immobile if they do not swim after 15 seconds of gentle agitation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: Yes
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate, tested twice a year
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: The 48-hour EC50 value is above the solubility limit of the test substance.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
4.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: None
- Other adverse effects control: None observed
- Abnormal responses: After 48 hours of exposure, no immobilized test organisms were determined in the control and up to and including the mean measured test item concentration of 4.9 mg/L. At the highest mean measured test concentration of 11 mg/L, 25% of the test organisms were found to be immobile.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None observed
- Effect concentrations exceeding solubility of substance in test medium: Yes for 48-hour EC50. The 48-hour EC50 of the test item could not be calculated because none of the responses exceeded 50%, and thus, was above the practical limit of solubility of the test substance in the test water.

Also see table below.
Results with reference substance (positive control):
Most recent test (April 2011, Harland Laboratories Study D25745) ) reported an EC50 of 0.62 mg potassium dichromate/L. This demonstrates the sensitivity of the test organisms and is within the internal historical range (48-hour EC50 from 2000 to 2011: 0.43 - 1.1 mg/L).
Reported statistics and error estimates:
The 48-hour EC50 of the test item could not be calculated because none of the responses exceeded 50%, and thus, was above the practical limit of solubility of the test substance in the test water.
NOEC determined directly from the raw data.

The biological results were based on the mean measured concentrations calculated as geometric means of the test item concentrations for each test medium renewal and the average of these geometric means.
  Mean measured concentration of the test item (geometric mean)
Treatment/Dilution mg test substance /L
Dilution 1:22 ---
Dilution 1:10 ---
Dilution 1:4.6 ---
Dilution 1:2.2 4.9
Undiluted filtrate (loading rate 100/mg test substance/L) 11
---: not analysed 

Effect of Test Substance/L on the Mobility of Daphnia magna
Treatment/Dilutions Mean measured concentration No. of daphnids tested Immobilized daphnids after 24 hours Immobilized daphnids after 48 hours
   (mg test substance/L No. % No. %
    20 0 0 0 0
Control --- 20 0 0 0 0
01:22 --- 20 0 0 0 0
01:10 --- 20 0 0 0 0
01:04.6 --- 20 0 0 0 0
1.2.2 4.9 20 0 0 0 0
Undiluted filtrate (loading rate 100 mg/L)  1.1

20

0

0 5 25
Validity criteria fulfilled:
yes
Remarks:
Mortality in controls <10%; dissolved oxygen levels at end of test ≥ 3 mg/L.
Conclusions:
The 48 hour-EC50 for Daphnia magna exposed to the test substance could only be estimated as > 11 mg test substance/L, because none of the responses exceeded 50%, and thus, was above the practical limit of solubility of the test substance in the test water. The 48-hour NOEC was determined to be 4.9 mg test substance/L. The biological results were based on the mean measured concentrations calculated as geometric means of the test substance concentrations for each test medium renewal and the average of these geometric means.
Executive summary:

The sensitivity of Daphnia magna to the test substance was determined in a GLP-compliant test performed to standard guidelines. The 48 hour-EC50 for Daphnia magna exposed to the test substance could only be estimated as > 11 mg test substance/L, because none of the responses exceeded 50%, and thus, was above the practical limit of solubility of the test substance in the test water. The 48-hour NOEC was determined to be 4.9 mg test substance /L. The Daphnia were exposed for up to 48 hours to 5 concentrations of the test substance. As the test substance has low solubility in the test media a dispersion was formed by mixing the test substance in the test media for a prolonged period of time. After filtration this stock solution was used undiluted as the maximum exposure concentration and as a series of dilutions to form the other test concentrations. Analytical monitoring of the exposure concentrations was undertaken and the results are based on the mean measured concentrations calculated as geometric means of the test substance concentrations for each test medium renewal and the average of these geometric means.

Description of key information

48-h EC50 > 11 mg/l, OECD 202, Semi-static, Daphnia magna, Liedtke 2011

The 48 hour-EC50 for Daphnia magna exposed to the test substance could only be estimated as > 11 mg test substance/L, because none of the responses exceeded 50%, and thus, was above the practical limit of solubility of the test substance in the test water. The 48-hour NOEC was determined to be 4.9 mg test substance/L. The biological results were based on the mean measured concentrations calculated as geometric means of the test substance concentrations for each test medium renewal and the average of these geometric means.

Key value for chemical safety assessment

Additional information

The acute toxicity of the test substance to Daphnia magna was determined. Daphnia were exposed to the test item at concentrations of 4.9 and 11mg/l alongside a water control. The 48h EC50 was found to be above the highest concentration tested, which in itself was limited by the water solubility of the test substance.  The study is considered reliable and relevant for risk assessment. As no EC50 could be determined the result was not considered adequate for classification and labeling purposes.