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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:
01 April 2011 - 29 April 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 the test. For sampling at the end of test, 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 the test by dispersing 149.7 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 3 hours in the dark, to dissolve a maximum amount of the test item in the dispersion. After the 3-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, 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.
- 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
- Strain: Clone 5
- Age at study initiation (mean and range, SD): 6-24 hours
- Feeding during test: No

ACCLIMATION
- Acclimation period: None
Test type:
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 = 7.7 - 7.8
End test range = 7.7
Dissolved oxygen:
Test start range = 8.9 mg/L
End test range = 7.8 - 8.3 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
The following concentrations of test material 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 at day 0 were: 0, 0.385, 0.783 and 1.74 mg/L and at day 4 they were: 0, 0.326, 0.692 and 1.67 mg/L for the control and dilutions 1:4.6, 1:2.2 and the undiluted filtrate with a loading rate of 100 mg/L, respectively. The samples from the dilutions 1:22 and 1:10 were not analyzed, since these concentrations were below the NOEC determined in the test. The biological results were related to the mean measured test item concentration calculated as the geometric means of the concentrations measured at the start and end of the test (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: Beginning and end of test for pH, dissolved oxygen and water temperature. The appearance of test media visually inspected at the start of the test and after 24 and 48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: 16:8 light/dark cycle with 30 minute transition period.
- Light intensity: Approximately between 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 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.:
> 1.7 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.:
0.35 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: During the first 24 hours one daphnid at 0.74 mg/L was observed to be immobile. This immobilization was not estimated as a toxic effect of the test item because in the highest test concentration of 1.7 mg/L no immobilization was observed.
- 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 item in the test water.

Also see table below.
Results with reference substance (positive control):
Most recent test (October 2010, study C97643) reported an EC50 of 0.56 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 2010: 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 item iin the test water.
NOEC determined directly from the raw data.

The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and end of the test:

Mean measured concentration of the test item (geometric mean)
Treatment/Dilution mg/L % of initially measured
Dilution 1:22 --- ---
Dilution 1:10 --- ---
Dilution 1:4.6 0.35 92
Dilution 1:2.2 0.74 94
Undiluted filtrate 1.7 98
---: not analysed (see above)

Table 1. Effect 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/L No. % No. %
20 0 0 0 0
Control --- 20 0 0 0 0
1:22 --- 20 0 0 0 0
1:10 --- 20 0 0 0 0
1:4.6 0.35 20 0 0 0 0
1.2.2 0.74 20 1 5 1 5
Undiluted filtrate (loading rate 100 mg/L)  1.7 20 0 0 2 10
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 material could only be estimated as > 1.7 mg/L, because none of the responses exceeded 50%, and thus, was above the practical limit of solubility of the test item in the test water. The 48-hour NOEC was determined to be 0.35 mg/L. The results are based on the mean measured test item concentration calculated as the geometric means of the concentrations measured at the start and end of the test.
Executive summary:

The sensitivity of Daphnia magna to the test material was determined in a GLP-compliant test performed to standard guidelines. The 48 hour-EC50 for Daphnia magna exposed to the test material could only be estimated as > 1.7 mg/L, because none of the responses exceeded 50%, and thus, was above the practical limit of solubility of the test item in the test water. The 48-hour NOEC was determined to be 0.35 mg/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 test item concentration calculated as the geometric means of the concentrations measured at the start and end of the test.

Description of key information

The 48 hour-EC50 for Daphnia magna exposed to the test material could only be estimated as > 1.7 mg/L, because none of the responses exceeded 50%, and thus, was above the practical limit of solubility of the test item in the test water. The 48-hour NOEC was determined to be 0.35 mg/L. The results are based on the mean measured test item concentration calculated as the geometric means of the concentrations measured at the start and end of the test.

Key value for chemical safety assessment

Additional information

The sensitivity of Daphnia magna the test material was determined in a GLP-compliant test performed to standard guidelines. The 48 hour-EC50 forDaphnia magnaexposed to the test material could only be estimated as > 1.7 mg/L, because none of the responses exceeded 50%, and thus, was above the practical limit of solubility of the test item in the test water. The 48-hour NOEC was determined to be 0.35 mg/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 test item concentration calculated as the geometric means of the concentrations measured at the start and end of the test.

The study is considered reliable and relevant for risk assessment. As no 96h EC50 could be determined for the growth rate the result was not considered adequate for classification and labeling purposes.