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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:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Oct 2012
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
February 2017 (REG (EC) No. 440/2008 amended by REG (EC) No. 2017/735
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken from the control and from the freshly prepared WAF with the loading rate of 10 mg/L (without food) at the start of the test and at two further dates thereafter (Day 7 and Day 16).
Stability samples (with food) were taken by combining the contents of all replicate test beakers at the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19).
All samples were frozen (at about –20 °C) immediately after sampling.
Vehicle:
no
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, 2018.
- Method: A supersaturated solution was prepared by dispersing the test item into test water (Elendt M7 medium) at a nominal concentration of 10 mg/L*. Ultrasonic treatment was applied for 15 minutes and thereafter, the dispersions were stirred for 23 hours in the dark to dissolve a maximum of test item in test water. After stirring, the mixture was allowed to stand for 1 hour to separate undissolved test material from the test medium. The middle layer of the aqueous phase was a clear solution and was separated and tested as single WAF with the loading rate of 10 mg/L. The test medium was prepared at the start of the test and before each test medium renewal. Media preparation was conducted under laboratory safety lighting.
- Control: test water (Elendt M7 medium) without addition of test item

* the maximum required test concentration for chronic effects testing
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flee
- Strain/clone: STRAUSS
- Age at study initiation (mean and range, SD): ≤ 24 hours
- Method of breeding:
- Source: in-house culture
- Age of parental stock: between 14 days and 4 weeks, not first brood progeny
- 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: not necessary (see below)
- Acclimation conditions (same as test or not): same
- 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)
- Feeding frequency: three times a week
- Health during acclimation (any mortality observed): no signs of stress such as high mortality, presence of males or ephippia, discoloured animals, etc. were observed and the brood stock was healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Each stock animal was maintained separately in a 100 mL glass beaker filled with about 80 mL Elendt M7 medium and was transferred twice a week to fresh medium. The condition of the stock animals was frequently checked
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
2.5 mmol/L (250 mg CaCO3/L)
Test temperature:
20 - 21°C
pH:
7.6 - 7.8
Dissolved oxygen:
≥ 8.2 mg O2/L
Nominal and measured concentrations:
nominal loading rate: 10 mg/L (WAF).
measured concentration (loading rate 10 mg/L):
freshly prepared media (Day 0, 7 and 16): 49.3, 101 and 30.6 µg/L
aged media (Day 2, 9 and 19): 9.53, 67.1 and 16.6 µg/L
average exposure concentrations (the time-weighted mean over all measurements): 40 µg/L

The test item is a multi-constituent substance consisting of three different sparingly soluble compounds. The analytical method detected only two of them. Thus, the absolute amount of test item could not be analytically determined and the measured test item concentrations should be regarded as approximated values. Therefore, all reported biological results are based on the loading rate of the test item (in accordance with the OECD Guidance Document No. 23 on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2018).
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
- 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 17 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: n.a., limit test
- Range finding study
- Results used to determine the conditions for the definitive study:
12-day range finding study at nominal loading rates of 0.10, 1.0 and 10 mg/L.
- adult mortality: none
- inhibition of reproduction: no adverse effects up to 10 mg/L (above the water solubility limit of the test item and the maximum test concentration recommended by the Guidelines).
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. (dissolved fraction)
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. (dissolved fraction)
Basis for effect:
growth
Remarks on result:
other:
Remarks:
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. (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
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 at the loading rate of 10 mg/L was 99% compared to the control.
- Other biological observations: A mortality of 10% at the loading rate 10 mg/L was regarded as natural and not considered to be due to a toxic effect of the test item as according to the test guideline a mortality of up to 20% in the control is accepted for the test to be valid. Moreover, neither the reproduction nor the length of the daphnids were significantly reduced at this loading rate.
- Mortality of control: No mortality was observed in the blank control.
- Other adverse effects control: none
- Abnormal responses: none
For detailed biological results obtained for reproduction and parental mortality and body length see Attachment "S-900 - Tables_long-term toxicity to daphnia (IES 20180241).pdf".
Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional®, Version 3.2.1 (ToxRat® Solutions GmbH).
For the test item treatment an adult mortality of 10 % (regarded as natural) was observed. Therefore, the reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test, according to the OECD Guideline No. 211, 2012. The mean reproduction rate and the mean body length of the daphnids at the loading rate of 10 mg/L were compared with the control by a two-sample t-test procedure and by two-sample Welch t-test, respectively.
The NOEL for mortality was determined directly from the raw data. No EL values could be calculated since no toxic effect of the test item was determined on the reproduction and the body length of the daphnids at the loading rate of 10 mg/L.
Validity criteria fulfilled:
yes
Remarks:
validity criteria for the control: mortality of the parent animals was 0% at the end of the test (≤20%), mean surviving offspring per parent animal was 204 (≥60) and the coefficient of variation was 9% (<25%)
Conclusions:
The test item showed no adverse effects on survival, growth and reproduction rate the maximum required test concentration for chronic effects testing, nominal 10 mg/L and thus up to the limits of its water solubility.

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