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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
REG (EC) No. 440/2008 amended by REG (EC) No. 2017/735
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Triplicate samples were taken from all test concentrations and the solvent control from freshly prepared and aged (48 and 72 hours) test media.
Samples were taken from the freshly prepared test media without food.
Aged samples were taken from the actual test by combining the contents of all replicate test beakers after the end of the test medium renewal periods (with food). On the same dates, samples incubated under the test conditions, however without food and daphnids were sampled.
All samples were frozen (at about –20 °C) immediately after sampling and stored until analysis.
Vehicle:
yes
Remarks:
acetone
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The preparation of the test media is based on the OECD series on testing and assessment No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
- Method: The application solution for the dosage of the highest loading rate of 10 mg/L was prepared by completely dissolving 100 mg of the test material in 10 mL of acetone by intense stirring for 5 minutes at room temperature. This application solution was diluted with acetone to prepare the application solutions used for the dosage of the lower test concentrations.
For the preparation of the test media, 1.5 mL of each application solution were spiked into the empty stirring vessels. The acetone was evaporated to dryness under a nitrogen stream. Thereafter, 1500 mL test water, respectively, were added into the vessels in order to disperse the test item, using ultrasonic treatment for 15 minutes and intense stirring for 96 hours at room temperature in the dark. The stirring period was chosen since in the analytical pre-experiments (non-GLP) the DT50 of the main component of the test item was found to be <1 hour. Therefore, the hydrolysis product was tested according to the OECD guideline No. 23.
After stirring, the dispersions were filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 µm) just before the start of the test. The filter was pre conditioned with at least 200 mL of the respectively dispersion. The filtrates of the equilibrated dispersions were tested as WAFs. The test media were prepared just before the start of the test and prior to each test medium renewal. After filtration, the test media were clear solutions.
- Controls:
Blank control: test water without addition of test item, nor solvent.
Solvent control: 1.5 mL acetone without test item were spiked into the empty stirring vessel. The further procedure was the same as used for preparation of the WAFs.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flee
- Strain/clone: STRAUSS
- Source: in-house culture originally obtained from the Daphnia Collection of the University of Basel/Switzerland in 2015
- Feeding during test
- Food type: suspension of freshly grown green algae (Desmodesmus subspicatus) and a commercial fish diet (Tetra Min® Hauptfutter, TETRA-Werke, 49304 Melle / Germany)
- Amount: 0.20 mg total organic carbon/animal/day
- Frequency: daily

ACCLIMATION
- Acclimation period: culture medium, temperature and light conditions were identical to those in the test.
- Type and amount of food: green algae suspension (Desmodesmus subspicatus) or a mixture of this suspension and a commercial fish diet (TETRA MIN Hauptfutter, TETRA-Werke, 49304 Melle / Germany), three times a week
- Health during acclimation (any mortality observed): no mortality or signs of stress were observed and the brood stock was healthy.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Stock animals were maintained separately in a 100 mL glass beaker filled with about 80 mL culture medium and were transferred twice a week to fresh medium. The condition of the stock animals was frequently checked.
The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals were less than 24 hours old, by removing the offspring <24 hours before start of the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
2.5 mmol/L (250 mg CaCO3/L)
Test temperature:
20- 22°C
pH:
7.6 - 8.4
Dissolved oxygen:
≥ 7.9 mg O2/L
Nominal and measured concentrations:
Samples from all sampling dates (with and without food) of the highest loading rate of 10 mg/L and from the solvent control were analyzed. The lower loading rates were not analyzed since they were below the 21-day NOELR therefore not relevant for the interpretation of the biological results.

nominal loading rates: 0.32, 0.1, 3.2, 32 and 10 mg/L (WAFs).
measured concentration (loading rate 10 mg/L): 7.4 to 8.8 mg/L in the freshly prepared test media. In aged samples, 94–103 % of the initially measured values were found. No main component was detected

Note: Due to the instability of the main component (DT50 < 1 hour), the hydrolysis product was tested and analysed see "Details on analytical methods").
Details on test conditions:
TEST SYSTEM
- Test vessel: 100-mL glass beakers containing 80 mL test medium, covered with glass plates
- Aeration: none
- Renewal rate of test solution: three times a week every 48 or 72 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- Biomass loading rate: 80 mL/animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (Elendt M7 medium) according to the OECD Guideline No. 211. Analytical grade salts and additives were dissolved in ultrapure water.
- Alkalinity: 0.9 mmol/L
- Ca/Mg ratio: 4:1
- 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 each test medium renewal period. At the same dates the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period
- Lighting: between approximately 15 and 19 µE m-2 s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- daily: immobility of adults, number of living and dead offspring and presence of aborted eggs.
- test end: body length of surviving adults (measuring the daphnids from the top of the head to the basis of the spina with the use of a binocular microscope).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Results used to determine the conditions for the definitive study:
13-day range finding study at nominal loading rates of 0.10, 1.0 and 10 mg/L.
- adult mortality: none;
- inhibition of reproduction: 10%, 22% and 36% at 0.10, 1.0 and 10 mg/L
Reference substance (positive control):
not required
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: results are expressed in terms of loading rate
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks on result:
other: results are expressed in terms of loading rate
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211} (migrated information)
Remarks on result:
other: results are expressed in terms of loading rate
Details on results:
- Behavioural abnormalities: none
- Observations on body length: no detrimental effect on the body length was observed. The mean body length of the daphnids in the controls and in the treatments up to and including the loading rate of 10 mg/L was in the range of 4.77-4.83 mm (corresponding to 99-100% compared to the blank control).
- Other biological observations: Mortality of adult daphnids: No biological relevant mortality (0-10%) was observed at up to and including the loading rate of 10 mg/L.
- Mortality of control: No biological relevant mortality (0-10%) was observed in the blank control and the solvent control.
- Other adverse effects control: none
- Abnormal responses: none

For detailed biological results obtained for reproduction and parental mortality and body length see Attachment "Tables & Figures_long-term toxicity to daphnia WS400104 (IES 20180011).pdf".
Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional®, Version 3.2.1 (ToxRat® Solutions GmbH).
According to OECD Guideline No. 211 (2012), if mortality was lower or equal 10% the reproduction rate was calculated as the total number of living offspring produced per surviving parent female. For higher (test substance dependent) mortality the reproduction rate was calculated as the total number of living offspring produced per parent female introduced in the test.
The mean reproduction rate and the body length of the daphnids in the control and in the solvent control were not significantly different (Student t-test,  = 0.05, two-sided). Thus, statistical evaluations were performed using averaged values of the controls (pooled control).
The mean reproduction rates and the mean body lengths of the daphnids at the loading rates were compared with the pooled control by Williams t-test (α = 0.05, one-sided).
The NOEL for mortality was determined directly from the raw data.
The EL10 and EL50 of the mean reproduction rates and the body length after 21 days could not be calculated due to the low toxic effect of the test item on the these endpoints up to the highest loading rate.
Validity criteria fulfilled:
yes
Remarks:
validity criteria for the controls: mortality of the parent animals was 0% at the end of the test (≤20%), mean surviving offspring per parent animal was 178-179 (≥60) and the coefficient of variation was 10-12% (<25%)

Description of key information

Overall 21-d NOELR (based on loading rates) ≥ 10 mg/L for Daphnia magna (OECD 211, EU C.20)

Key value for chemical safety assessment

Additional information

NOELR for freshwater invertebrates: 10 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.