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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
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Oct 2012
Deviations:
no
Qualifier:
according to guideline
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. QA statement)
Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken from the control and from the freshly prepared WAF (without food) and the corresponding aged test solutions (stability samples; with food) at each renewal period.
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, 2000.
- Method: A supersaturated solution was prepared by dispersing the test item into test water (Elendt M7 medium) at a nominal concentration of 10 mg a.i./L*. Based on pre-experiments, ultrasonic treatment was applied for 15 minutes followed by stirring for 48 hours in the dark at room temperature.
After stirring, the dispersion was filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 µm) just before use. The filter was pre conditioned with 200 mL of the dispersion. After filtration, the test media were clear solutions. The filtrate of the equilibrated dispersion was tested as a single WAF. The test media were prepared just before the start of the test and prior to each test medium renewal.
- 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:
7.5 -8.9
Dissolved oxygen:
≥ 7.0 mg O2/L
Nominal and measured concentrations:
One of the duplicate samples from each sampling date was analyzed.
freshly prepared media: 8.4 – 9.7 mg a.i./L
aged media: 8.1 – 9.4 mg a.i./L; 94 – 111% of the initially measured concentrations

The test item is a mixture of several constituents with different solubility, and the analytical method is based on one peak (m/z 528). Thus, the absolute amount of active ingredient could not be analytically determined and the measured active ingredient concentrations should be regarded as approximated values. Therefore and since the test item is a 50 % suspension in water, all reported biological results were based on the loading rate of the active ingredient. This procedure of evaluation of data of WAFs is based on recommendations of 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: approximately 16 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 a.i./L.
- adult mortality: none
- inhibition of reproduction: no adverse effects up to 10 mg a.i./L (above the water solubility limit of the a.i. 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:
act. ingr. (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:
act. ingr. (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:
act. ingr. (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 a.i./L was 97% compared to the control.
- Other biological observations: none
- 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, parental mortality and body length see Attachment "WS400123 - Tables_long-term toxicity to daphnia (IES 20180199).pdf".
Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional®, Version 3.3.0 (ToxRat® Solutions GmbH).
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 exposed daphnids were compared with the control by a two-sample t-test procedure.

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.
Validity criteria fulfilled:
yes
Remarks:
validity criteria for the control (target): mortality of the parent animals was 0% at the end of the test (≤20%), mean surviving offspring per parent animal was 200 (≥60) and the coefficient of variation was 11% (<25%)
Conclusions:
The test item showed no adverse effects on survival, growth and reproduction rate at 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

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.

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