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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the first treatment period (Day 0) and at a treatment period in the second and third week (Days 10 and 17).
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the renewal periods, duplicate samples of the respective aged test media and controls were sampled at Day 3, 12 and 19.
One of these three stability control treatments lasted for 72 hours (weekend), two for 48 hours, corresponding to the different test medium renewal periods.
From the stock solution (= filtrate) duplicate samples were taken at days -2, test start, days 3, 10 and 17. At day 17 duplicate samples were taken of the filtrate before and after an additional filtration step.
- Sample storage conditions before analysis: The samples collected at the test start and day 12 were stored in a freezer (≤ - 10 °C), protected from light until analysis was performed, whereas all further samples were analysed directly at the day of sampling.
-Analyses: The concentrations of the test item were measured in the stock solution samples and in all duplicate samples of the test medium.
From the control samples only one of the duplicate samples was analysed from each sampling date.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Pre-Experiments: Pre-experiments were performed to determine the solubility of the test item in test water and to select suitable methods for the preparation of a stock solution and the dosage of the test item into the test media.
- Dosage of Test Item:: Before test start, a stock solution of 100 mg/L was prepared by dissolving 1011.2 mg test item into 10112 mL test water by intense stirring for 48 hours and ultrasonic treatment for 10 minutes. Then, the undissolved test item was separated by filtration (0.45 μm cellulose nitrate filter). The content of the test item was analytically determined and the dilution steps for the preparation of the test media were adjusted accordingly. The same stock solution was used for the preparation of the test solutions throughout the 21 day exposure period. The stability was regularly confirmed by chemical analyses.
Since few particles were visible, the stock solution was filtered again on day 3 and 17 of the test. The dilution steps were again adjusted according to the results of the analytical determination. The stock solution was stored at room temperature during the test to minimise precipitation of the test item. On day 17 the test substance was determined in a filtered and unfiltered sample of the stock solution to confirm that no relevant precipitation has taken place. The test media for each test interval were prepared just before introduction of Daphnia (= start of the test and each test medium renewal) by dilution of the stock solution.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Daphnia magna (Straus), clone 5
- Source: The Daphnia introduced in the test were taken from the IBACON inhouse laboratory culture.
- Breeding Conditions: The Daphnia were bred in the laboratories of IBACON under similar temperature and light conditions as in the test. The cultivation of the parental Daphnia was performed in reconstituted water of a similar quality regarding to pH, components of the main ions and total hardness as the test water used in the test (see below). The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of IBACON.
- Feeding during test: The Daphnia were fed daily with green algae (Desmodesmus subspicatus), freshly grown in the laboratories of IBACON. The amount of food was based on the concentration of total organic carbon (TOC) in the algal food suspension.
The approximate daily amounts of algal TOC/Daphnia were as follows:
Days 0-3: 0.1 mg
Days 4-7: 0.15 mg
Days 8-20: 0.2 mg

ACCLIMATION
Is not necessary since the breeding is performed in the same kind of test water.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
2.5 mmol/L (= 250 mg/L) as CaCO3
Test temperature:
20 - 21 °C in the freshly prepared media;
19 - 20 °C in the aged media;
pH:
7.8 to 8.0 in the freshly prepared media;
6.7 to 9.0 in the aged media. The pH-value did vary by more than 1.5 units during the test. However, this was not considered to have an effect on the results, since this deviation was caused by one single value (day 3, aged test medium of the Control) and the control did meet all validity criteria.
Dissolved oxygen:
8.1 to 9.1 mg/L in the freshly prepared media;
7.5 to 11.4 mg/L in the aged media
Nominal and measured concentrations:
Nominal: 7.8, 2.8, 1.0, 0.36, 0.13 mg test item/L
Measured (time-weighted mean): 7.354, 2.606, 0.924, 0.340,
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers of 100 mL volume with approximately 60 mL test medium.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Test Medium Renewal and Feeding of Daphnia: A semi-static test was performed.
The test media of all test concentrations and of the control were renewed on Days 3, 5, 7, 10, 12, 14, 17 and 19 of the exposure period (every Monday, Wednesday and Friday). At these dates the test animals were carefully transferred from the old test vessels into the freshly prepared test media of the corresponding concentrations. The handling and the allocation of the treatments was randomised on every test media renewal.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (Elendt ″M4″):
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH-values, dissolved oxygen concentrations and water temperatures were measured in all test concentrations and the control in all freshly prepared and aged test media.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark.
- Light intensity: 770 - 110 lux. The light intensity was measured once during the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The mortality of the test animals and the number of young daphnids were recorded for each day. Dead animals and offspring were removed at the same times.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
2.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
1.75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
1.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
2.21 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
7.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Analytical results:
Under the test conditions the concentration of the test item was generally stable during the test medium renewal periods of 48 and 72 hours with only a slight tendency of decrease. Therefore, all reported results are related to nominal concentrations of the test item, but the time weighted mean values are reported additionally (see above)..
Validity criteria fulfilled:
yes
Conclusions:
The influence of the test item on the survival and reproduction of Daphnia magna was determined during an exposure period of 21 days.
Based on nominal concentrations, the 21-day NOEC was determined to be 1.0 mg test item/L for the reproduction and 2.8 mg test item/L for the survival of the adult test animals, respectively. The EC50 for the reproduction was 2.21 mg test item/L.

Description of key information

The study provided was conducted to recognised testing guidelines and with GLP certification.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
1 mg/L

Additional information

The chronic toxicity to aquatic invertebrates was tested in a GLP guideline study according OECD 211 (IBACON 2013a). Daphnia magna were exposed for 21 d to the test item in a semi-static test system. The effects on survival and reproduction were measured. Based on nominal concentrations, the 21-day NOEC was determined to be 1.0 mg test item/L for the reproduction and 2.8 mg test item/L for the survival of the adult test animals, respectively. The EC50 for the reproduction was 2.21 mg test item/L.