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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:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
At the start of the test, two samples (50 mL) were taken from freshly prepared control and test media.
After 48 hours, mid-vessel samples taken from each group were pooled and replicate samples (50 mL) were taken.
Samples were stored in a freezer until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAFs)
The test substance was dispersed in dilution medium in a glass vessel (10.4 mg/5L, 9.10 mg/2L or 10 mg/L). The contents of each vessel were stirred for approximately 24 hours and then left to stand for approximately 24 hours. Aliquots (either 0.8 or 2 L) were then removed mid vessel to provide the Water Accomodated Fractions (WAF) at nominal loading rates of 2.07, 4.55, and 10 mg/L. It was considered impracticable to prepare WAFs at lower nominal loading rates because the weight of test substance required would be too small. Thus, the WAF at nominal loading rate of 2.07 mg/L was diluted to provide test concentrations at the lower levels.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: in-house culture originating from a strain supplied by the National Institute for Applied Chemical Research (IRCHA), France
- Age at study initiation: < 24 h
- Method of breeding: Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.5 to 0.8 litres of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2°C. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. The culture medium was renewed three times each week.
Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given. Culture conditions ensure that the stock animals reproduce by parthenogenesis.
The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel.
- Feeding during test: none

ACCLIMATION
- Acclimation period: not relevant, conditions were the same for maintenance and testing
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
258 mg/L as CaCO3
Test temperature:
18-20 °C
pH:
8.2-7.9
Dissolved oxygen:
94-99 % air saturation value
Nominal and measured concentrations:
nominal loading rates: 0.194, 0.427, 0.939, 2.07, 4.55 and 10 mg/L as WAF
mean measured TOC levels (after background correction):
- loading rates of 0.194, 0.427 and 0.939 mg/L: only slightly above or below the limit of quantification; the TOC could not be reliably determined by carbon analysis due to variability in the results
- loading rates of 2.07, 4.55 and 10 mg/L:
test start: 1.97, 1.76 and 3.61mg C/L;
after 48 hours: 0.55, 1.4 and 3.98 mg C/L
geometric mean: 1.0, 1.6, 3.8 mg C/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass dishes containing approx. 120 mL of medium, covered with lids leaving no headspace
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 24 mL medium per organism

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium (according to guideline), prepared in deionised, reverse osmosis water
- Total organic carbon: 1.3 mg C/L
- Alkalinity: 50 mg/L as CaCO3
- Ca/Mg ratio: 3.7:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light : 8 hours dark with periods of subdued lighting at the beginning and end of each light phase

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : numbers of mobile, immobile and floating Daphnia counted approx. 24 and 48 hours after the start of the study

RANGE FINDING STUDY
- Test concentrations: nominal loading rates of 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 100% Immobility after 48 hours at all concentrations.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
0.74 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 0.58-0.94 mg/L; results are expressed in terms of loading rates
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
0.43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: results are expressed in terms of loading rates
Duration:
48 h
Dose descriptor:
LL100
Effect conc.:
4.55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: results are expressed in terms of loading rates
Details on results:
- Immobilised Daphnia: see attached background material (Table Cumulative immobilisation (GAH0079).pdf)
- Behavioural abnormalities: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none

The test media were colourless dispersions.

Results with reference substance (positive control):
- 48-hour EC50: 1.3 mg/L (within the range typically obtained in this laboratory)
Reported statistics and error estimates:
Statistical analysis was performed using the SAFEStat LD50 application (version 1.3), SAS 9.1.3 (SAS Institute, 2002).
The “no observed effect loading rate” (NOELR) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant.
Validity criteria fulfilled:
yes
Remarks:
In the control, no Daphnia have been immobilised or trapped at the surface of the water. The dissolved oxygen concentration at the end of the test was ≥60% of the air saturation value at the temperature used.

Description of key information

48-h EL50 (based on loading rates) 0.74 mg/L for Daphnia magna (OECD 202, EU C.2)

Key value for chemical safety assessment

Additional information

EL50 for freshwater invertebrates: 0.74 mg/L

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