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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:
07 July 2000 - 31 July 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Stable in water for at least 96 hours.
Analytical monitoring:
yes
Details on sampling:
Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 and t=48
Volume: 10 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.

Additionally, reserve samples of 10 mL were taken. These samples were stored in a freezer for possible analysis until delivery of the final report with a maximum of three months.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: the test substance was practically insoluble in water (in an earlier study at NOTOX determined to be < 9 µg/L). A test solution was prepared at nominally 100 mg/L, treated with ultrasonic waves and subsequently stirred for three days to achieve maximum saturation. Subsequently, part of this solution was filtered through a paper filter (5 µm) to remove the larger undissolved test substance particles. The lower concentrations tested in the final test were prepared by subsequent dilutions of the filtrate in test medium. Note that a sufficient amount of filtrate was present after filtration to prepare all the dilutions. However, an extra amount of test solution was filtered through the same paper filter to apply for the test vessels containing the undiluted filtrated.
- Controls: test medium without test item or other additives (blank-control).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera)
- Strain: Straus, 1820
- Source: not indicated
- Age at study initiation: < 24 hours
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel. After 7 days of cultivation half of the medium was renewed twice a week.
- Medium other than test medium: yes, M7
- Feeding: daily, a suspension of fresh water algae.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20.3 - 21.0 °C
pH:
8.0 - 8.3 throughout the test period
Dissolved oxygen:
8.7 - 8.8 throughout the test period
Nominal and measured concentrations:
Nominal test concentrations: 10, 18, 32, 56 and 100% of a filtered solution prepared at 100 mg/L
Mean measured test concentrations (0-48 h): 0.008, 0.009, 0.011, 0.117 and 0.048 mg/L respectively
Effect concentrations were based on geometric mean measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass, open, fill volume: 80 mL
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, ISO medium
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Culture medium different from test medium: yes, M7

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours, daily

EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning and at the end of the test, for all concentrations and the controls; temperature of the medium: daily in one control vessel beginning at the start of the test.

LIMIT STUDY
- Test concentrations: 0 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (May 2000)
Key result
Duration:
48 h
Dose descriptor:
EC50
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: The 48h-EC50 was above the solubility limit in water.
Details on results:
- Effect concentrations exceeding solubility of substance in water: yes
- Analysis of the samples taken from all test concentrations showed mean measured concentrations of 0.008, 0.009, 0.011, 0.117 and 0.048 mg/L, respectively for increasing nominal concentrations.
- Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.
- On the filter (which was used to filter the nominal 100 mg/L stock solution) an absolute amount of 43.6 mg test substance was analysed.

- When exposed to detectable concentrations above the water solubility limit, daphnids become affected without a clear dose-response (see attached illustration). The variations between concentrations measured at the end of the test confirmed the presence of undissolved fraction of test substance and de-homogenisation of the test solutions during the 48-hour test period. This indicates that likely other effects than the toxic potential of the test substance play a role (see 'any other information on results, incl. tables').
For detailed results, see attached background information.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 48h-EC50: 0.60 mg/L, 95% confidence interval: 0.52 - 0.79 mg/L.
- Other: results fell within the historical range.

In a previous risk assessment performed by the Netherlands, the following was stated:

''Since measured concentrations in the 5 µm filtrates at the start of the test exceeded the limit water solubility by a factor of 6 -17, it is evident that Daphnia were exposed to undissolved substance. After 48 hours actual concentrations remained in the range of (three lower concentrations) or above (two higher concentrations) the water solubility limit. Hence, effects on Daphnia (between 10 and 80% immonbilisation) were considered related to undissolved substance rather than a dissolved fraction. The conclusion of the working group was that for Daphnia as well al fish and algae KY-AF induces no toxic effects up to the water solubility limit and therefore E(L)C50 values are considered above the maximum water solubility.''

This statement was agreed upon by the applicant and the same approach will be followed for the recent risk assessment.

Validity criteria fulfilled:
yes
Remarks:
For details on validity criteria, see 'Overall remarks' section.
Conclusions:
The effects seen with the daphnia are considered to be related to undissolved test material, rather than the toxic potential of the (dissolved) test material. Therefore, the 48h-EC50 for Daphnia magna was above the solubility limit of the test substance in water, i.e. above 9 µg/L.
Executive summary:

In a 48 h acute toxicity study conducted according to OECD guideline 202 and GLP principles, water fleas (Daphnia magna) were exposed to the test substance under static conditions at the following nominal concentrations: 0 (blank-control), 10, 18, 32, 56 and 100% of a filtered solution prepared at a nominal concentration of 100 mg/L. Analysis of samples taken at all test concentrations showed mean measured concentrations of 0.008, 0.009, 0.011, 0.117 and 0.048 mg/L respectively for increasing nominal concentrations. The incidence of immobilisation was recorded at 24 and 48 hours. Daphnids became affected at detectable concentrations above water solubility. However, there was no dose-response relationship with the measured concentrations. The variations in the effects seen with daphnia are considered to be related to undissolved test material, rather than the toxic potential of the (dissolved) test material. Therefore, the 48h-EC50 for Daphnia magna was above the solubility limit of the test substance in water, i.e. above 9 µg/L.

Description of key information

In a reliable study, performed according to OECD guideline 202 and GLP principles, the 48h-EC50 was determined to be above the solubility limit in water.

Key value for chemical safety assessment

Additional information

In a 48 h acute toxicity study conducted according to OECD guideline 202 and GLP principles, water fleas (Daphnia magna) were exposed to the test substance under static conditions at the following nominal concentrations: 0 (blank-control), 10, 18, 32, 56 and 100% of a filtered solution prepared at a nominal concentration of 100 mg/L. Analysis of samples taken at all test concentrations showed mean measured concentrations of 0.008, 0.009, 0.011, 0.117 and 0.048 mg/L respectively for increasing nominal concentrations. The incidence of immobilization was recorded at 24 and 48 hours. Daphnids became affected at detectable concentrations above water solubility. However, there was no dose-response relationship with the measured concentrations. The variations in the effects seen with daphnia are considered to be related to physical effects due to undissolved test material, rather than the toxic potential of the (dissolved) test material. Therefore, the 48h-EC50 for Daphnia magna was above the detection limit of the test substance in water, i.e. above 9 µg/L; note that the actual maximum solubility of the test substance in water is < 9 µg/L, as no test substance could be detected in the water samples during the water solubility test.

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