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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Commission Directive 92/69/EEC, 1992
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
1984
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal 100 mg/L and control
- Sampling intervals: start (0 h), and at test end (48 h)
- Sampling method:
At test start: unfiltrated and filtrated (maximum pore size approx. 1.2 µm) samples from the freshly prepared undiluted supersaturated suspension.

At test end: the contents of the test beakers were pooled and filtered (maximum pore size approx. 1.2 μm). In addition, undiluted supersaturated suspensions, incubated in parallel without daphnids under the same conditions as in the actual test were collected (collecting of representative suspension-samples from the actual test itself was not possible since the test item was not homogeneously distributed in the test medium).
Control samples were taken directly from the test beakers.
Samples were taken in duplicate.
- Sample storage conditions before analysis: All samples were deep-frozen immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item the supersaturated suspension with a nominal concentration of 100 mg/I was prepared as follows: 262 mg of the test item were suspended in a small volume of test water by using a homogenisator. Then the mixture was transferred quantitatively into an adequate volume of test water to obtain a final concentration of nominal 100 mg/L. This suspension was treated ultrasonically for 15 minutes. Then, the supersaturated suspension was stirred by a magnetic stirrer at room temperature in the dark over 96 hours to dissolve a maximum concentration of the test item in the suspension. No auxiliary solvent or emulsifier was used. The stirring period of 96 hours was chosen according to the results of a pre-test, which showed that during this time no significant loss of the test item was to be expected. After stirring and after another 15 minutes ultrasonic treatment prior to the start of the test, a homogeneous suspension was obtained. The undiluted supersaturated suspension with the nominal concentration of 100 mg/L dissolved and suspended test item was used as test medium.
- Controls: Test water without addition of the test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the test start, the test substance was very finely and homogeneously suspended in the test medium. After about one hour, test substance particles were observed at the water surface. After one day, substance particles were observed at the water surface and the bottom of the beaker.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flee
- Strain/clone: STRAUSS
- Source: in-house culture
- Feeding during test: none

ACCLIMATION
- Acclimation period: none. Breeding was performed in reconstituted water of identical quality (regarding pH, main ions and total hardness) and under identical temperature and light conditions as in the tests.
- Type and amount of food: not reported
- Health during acclimation (any mortality observed): not reported

The daphnids used for the test were 6 - 24 hours old and were not first brood progeny.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
2.5 mmol/l as CaCO3
Test temperature:
20-22°C
pH:
7.8 - 8.2
Dissolved oxygen:
>= 8.5 mg/l
Nominal and measured concentrations:
nominal loading rate: 100 mg/L
measured test concentrations
- unfiltered sample of the suspensions: 88% and 83% of the nominal value (total mean 85%)
- dissolved and very finely suspended test item (measured in the filtrate of the samples): below the limit of detection (LOD) of approx. 1.0 mg/L.

The biological results are based on the loading rate (nominal concentration) of the test item.


Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass beakers containing 50 mL test medium
- Aeration: none
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 5 mL/animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water acc. to ISO 6341. Analytical grade salts and additives were dissolved in purified water.
- Ca/Mg ratio: 4:1
- Alkalinity: 0.8 mM
- Culture medium different from test medium: no
- Intervals of water quality measurement: water temperature, oxygen concentration and pH were measured at the start and at the end of the test. The appearance of the test media was recorded at test start and after 24 and 48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: not necessary
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity: between 200 and 1200 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Daphnids were inspected for immobility, mortality and other signs of intoxication after 24 and 48 hours of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: the test concentration was based on the results of a range-finding test and the results of pre-experiments to the solubility of the test material.
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: mobility and sub-lethal effects
Details on results:
Behavioural abnormalities:
- Other biological observations: none; no immobility or mortality or other signs of intoxication were observed.
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Effect concentrations exceeding solubility of substance in test medium: Yes, the solubility of the test item in test water was below the LOQ of the analytical method (< 1 mg/L).
Results with reference substance (positive control):
not reported
Reported statistics and error estimates:
not applicable
Validity criteria fulfilled:
yes
Conclusions:
The results indicate that the test material is not toxic to daphnids up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.

Since no classification criteria according to GHS/CLP (Regulation (EC) No. 1272/2008) are fulfilled, the substance is not classified for environmental hazards.
Executive summary:

Due to the low water solubilty of the test item, an unfiltrated supersaturated suspension with a nominal concentration of 100 mg/l was used as the test medium, containing a max. concentration of the test substance.


During the test period the total mean measured concentration of dissolved and suspended test substance in the unfiltrated duplicate samples of the suspension was 85% of the nominal value. Under the test conditions, the concentration was sufficiently constant during the 48-hours test period. The concentration of dissolved and very finely suspended test substance was below the detection limit of approx. 1.0 mg/l.


As no moratlity was observed, the 48-hour EL10 (NOELR) and thus a PNEC cannot be determined.

Description of key information

effect value for Daphnia magna, based on loading rates (OECD 202, EU C.2):
48-h EL50: >100 mg/L (solubility in test water < 1 mg/L)

Key value for chemical safety assessment

Additional information

EL50 for Daphnia magna >100 mg/L


The results indicate that the test material is not toxic to daphnia up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.