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Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2017-03-20 to 2017-03-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide
EC Number:
221-088-9
EC Name:
3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide
Cas Number:
3001-98-7
Molecular formula:
C7H14O6P2
IUPAC Name:
3,9-dimethyl-2,4,8,10-tetraoxa-3λ⁵,9λ⁵-diphosphaspiro[5.5]undecane-3,9-dione
Test material form:
solid: particulate/powder
Details on test material:
AFLAMMIT® PCO 962

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: At the start of the exposure intervals (0 and 72 h), sampling was carried out after preparation of the limit concentration. At the end of the exposure intervals (48 and 96 h), samples were taken directly from the test vessels.
- Sample storage conditions before analysis: All original samples were stored at room temperature until preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An appropriate amount of the test item was weighed and transferred into a test vessel filled with an appropriate volume of dilution water. The test media was mixed with an ultraturrax (1 min, 17000 rpm). This procedure was repeated for every water renewal.
- Controls: Dilution water without test item incubated under the same conditions as the limit test item concentration
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Strain: not specified
- Source: All fish used in the test was gained at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD): Average body length at the test start: 2.80 cm
- Weight at study initiation (mean and range, SD): Average body weight at the test start: 0.22 g
- Maintenance of the brood fish: Holding was performed at the test facility at 23 ± 2 °C and diffuse light (7 – 750 Lux, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value.

ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatization and mortality < 5 % within the last 7 days before the start of the exposure were used in the test.
- Acclimation conditions (same as test or not): not specified
- Type and amount of food during acclimation: The amount of food was 4 % of the fish body weight per feeding day. Sera vipan food
- Feeding frequency during acclimation: Food was provided 3 times per week.
- Health during acclimation (any mortality observed): No mortality was observed during this time. No disease treatments were administered throughout holding and testing.

FEEDING DURING TEST
Fish were not fed during the test.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
not relevant

Test conditions

Hardness:
65 mg/L as CaCO3
Test temperature:
22.9 ± 0.3°C
pH:
7.51-8.02
Dissolved oxygen:
oxygen saturation: 85-100%
Salinity:
not relevant
Conductivity:
181 µS/cm
Nominal and measured concentrations:
100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria of 13 L volume were used, filled with approx. 10 L of dilution water
- Type (delete if not applicable): Glass aquaria covered with glass lids
- Material, size, headspace, fill volume: 25.5/18/28 cm, depth of water: 20 cm
- Aeration: No aeration was provided.
- Renewal rate of test solution: A semi-static test procedure with daily renewal of the test media was performed.
- No. of organisms per vessel: 7 zebrafish (sexes not specified) were used for each test concentration and control.
- No. of vessels per concentration (replicates): One replicate per test concentration.
- No. of vessels per control (replicates): One replicate per control.
- Biomass loading rate: Fish density in the tanks was 0.16 g fish per litre test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 hours to remove chlorine
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: light / dark cycle: A natural photoperiod (approx. light / dark cycle: 16 / 8 h) was maintained during exposure.
- Light intensity: 7 – 750 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Observations were made after 3, 24, 48, 72 and 96 h.
Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behavior, respiratory function, pigmentation, etc.).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Range finding study: no The threshold concentration (TC) is based on a daphnia toxicity test of the test item (nominal concentrations): EC50 > 100 mg/L, SCHEERBAUM, 2018 and an algae toxicity test of the test item (nominal concentrations): ErC50 (0-96 h) > 100 mg/L, EyC50 (0-96 h) > 100 mg/L, SCHEERBAUM, 2018.

Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: not observed
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The LC0 and LC100 after 96 h were derived from the raw data of the limit concentration. LC50-values do not have to be calculated in a limit test.
The data for the tables in this report were computer generated and have been rounded for presentation from the full derived data. Consequently if calculated manually based on the given data minor variations may occur from these figures.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
All effect levels are given based on the nominal threshold concentration of the test item AFLAMMIT® PCO 962. The threshold concentration of the test item was found to have no effects to zebrafish.
In conclusion, the LC0 corresponds to the nominal concentration of 100 mg test item/L.