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Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-Mar-15 - 2021-Apr-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Due to UCVB substance with constituents of different water solubility, guidance documents OECD 23 and ASTM d6081-19 were used

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

02. Oct. 2012

Deviations:

yes

Remarks:

test item is a poorly soluble UVCB substance with constituents of different water solubility, according to OECD guidance document no. 23 and ASTM D6081–19, water-accommodated fractions (WAF) were prepared for the test

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: information provided by sponsor, Batch no. 0320SA017,
- Purity, including information on contaminants, isomers, etc.: not applicable, UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (20± 5 °C) in tightly closed vessel under dry, dark conditions
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: homogeneous, stable under storage conditions
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): preparation of water-accommodated fractions: nominal loading (2 / 1 mg/L)added to cellulose ester filter. Filters were put on decanting bottles filled with algal medium. Stirring for 24 ± 1h on magnetic stirrer under closed conditions. Settling period of 1 h ± 5 min, lower phase was used without filtration/centrifugation, first few mL were discarded.

Analytical monitoring:

yes

Details on sampling:

750 µL aqueous sample were mixed with 250 µL THF in a vial. If necessary, further dilutions were performed using THF/ water 25/75 (% v/v) with a maximum dilution factor of 10667.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct application via cellulose ester filter and 24 h stirring under closed conditions. After a settling period of 1 hour± 5 min the lower phase was used without filtration or cen-trifugation as test solutions. The first few mL was discarded.
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):-
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): -
- Test concentration separation factor: default, nominal loadings 1 and 2 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):
- Other relevant information:

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Berlin
- Age at study initiation (mean and range, SD): 0 - 24h
- Length at study initiation (length definition, mean, range and SD):
- Method of breeding/holding: similar to method in OECD guideline, following SOP 115 002 01 (“Zucht und Hälterung von Daphnia magna STRAUS“), edition 13, adopted 11. Nov. 2019.
- Source: bred in LAUS GmbH, originally obtained from Umweltbundesamt, Berlin
- Age of parental stock (mean and range, SD): not specified
- Feeding during test: yes
- Food type: unicellar Desmodesmus subspicatus
- Amount: 0.20mg org. carbon/animal/day, based on photometric measurement
- Frequency: daily

ACCLIMATION
- Acclimation period: yes, time not specified
- Acclimation conditions (same as test or not): not specified
- Type and amount of food: see above
- Feeding frequency: see above
- Health during acclimation (any mortality observed): not specified, only healthy animals were selected

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
- 19h45min before start of test, adults were separated from young daphnia. 30min before start, newborn daphnia were sieved (age <24h) and placed into beaker with dilution water

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

see Table 1,
Deviation: In the last week no determination of hardness was performed. This can be stated as non-critical for the outcome of the study.

Test temperature:

19.4 – 23.2°C,
Deviation: The temperature and the temperature difference were not within the range state in the study plan (19.4 – 32.2 °C instead of 18.0 - 22.0 °C and a temperature difference of highest 2°C). Because the temperature deviation was only for a short period of time and all validity criteria were fulfilled, this deviation was stated as non-critical.

pH:

see Table 1

Dissolved oxygen:

see Table 1

Nominal and measured concentrations:

nominal: 1 and 2 mg/L ;
time-weighted mean: 0.399 and 0.709 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers, 100 mL. Glass tubes for transfer
- Material, size, headspace, fill volume: glass, filled with 80 mL/animal
- Aeration: none
- No. of organisms per vessel: animals were kept individually
- No. of vessels per concentration (replicates): 10/treatment
- No. of vessels per control (replicates): 10/treatment
- No. of vessels for analytical determination (replicates): 1 each

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water with an enrichment of certain minerals was used in the test. The minerals which are added comply with the composition M4 (see Table 4) derived by ELENDT as stated in the OECD-Guideline.
- Intervals of water quality measurement: once/week

OTHER TEST CONDITIONS
- Photoperiod: 16/8h
- Light intensity: 1015 – 1089 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
reproductive capacity expressed as total number of neonates per adult daphnid, counted daily

TEST CONCENTRATIONS
- Range finding study: yes, Non-GLP
- Test concentrations: 1 and 5 mg/L
- Results used to determine the conditions for the definitive study: 1mg/L had no significant inhibition on reproduction, whereas with 5 mg/L, all daphnia died until day 5

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

> 2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Behavioural abnormalities: none
- Mortality of control: 10%
- Other adverse effects control: none
- Immobilisation of control: not indicated
- Abnormal responses: Abnormalities as ephippia, presence of males, presence of aborted eggs and dead off-spring were not observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none

Reported statistics and error estimates:

no statistical evaluation was performed due to absence of toxicity

Validity criteria fulfilled:

yes

Remarks:

The mortality of parent animals in the control was below 20 % (found: 10 %) and the mean surviving offspring in the control was above 60 (found: 123).

Conclusions:

The valid experiment with two concentrations showed no toxicity of the test item concerning the reproduction of Daphnia magna. The mean number of offspring per concentration was higher than in the control. Therefore, no statistical evaluation was performed. The content of the test item was measured weekly in the same batch of the freshly prepared and in the used solutions using LC-MS-MS. The measured concentrations lay between 25 % and 61 % of the nominal concentrations, resulting in a calculated mean measured concentration of 0.399 mg/L and 0.709 mg/L. As the test item is a UVCB substance, according to OECD guidance document No. 23 (2019), the results are presented, based on the nominal WAF loading rates (see OECD guidance document no 23, § 177).

Executive summary:

Because the test item is a poorly soluble UVCB substance with constituents of different water solubility, in accordance with the OECD guidance document no. 23 and ASTM D6081 – 19, water-accommodated fractions (WAF) were prepared for the test. The WAFs were used as test solutions without filtration or centrifugation.

In a non-GLP pre-test, no significant inhibition on the reproduction of Daphnia magna was observed at the loading rate 1 mg/L whereas at the loading rate 5 mg/L all daphnia were dead on day 5. Therefore, the two concentrations 1 mg/L and 2 mg/L were used in the main test. 

Because during validation, the test item showed insufficient stability at low concentrations, the study was performed under semi-static conditions and daily medium renewal to guarantee a maximum exposure concentration.

The offspring of the animals was counted each medium renewal. The basis of effect was reproductive capacity expressed as the total number of neonates per adult daphnid at the end of the test. Decrease in the reproduction of the daphnia could not be observed in any treatment.

The mean number of offspring in both concentrations was higher than in the control. Therefore, no statistical evaluation was performed.

The content of the test item was measured weekly in the same batch of the freshly prepared and in the used solutions using LC-MS-MS. The measured concentrations lay between 25 % and 61 % of the nominal concentrations, resulting in a calculated mean measured concentration of 0.399 mg/L and 0.709 mg/L.

As the test item is a UVCB substance, according to OECD guidance document No. 23 (2019), the results are presented, based on the nominal WAF loading rates (see OECD guidance document no 23, § 177).

The following results for inhibition of reproduction based on introduced parent animal were determined for the test item:

NOEL ≥ 2 mg/L
LOEL > 2 mg/L

Description of key information

The test item is a poorly soluble UVCB substance with constituents of different water solubility. Therefore, water-accommodated fractions (WAF) were prepared for the test.

The study was performed in accordance with OECD guideline 211 under semi-static conditions and daily medium renewal to guarantee a maximum exposure concentration and is considered reliable without restriction (Klimisch 1). The offspring of the animals was counted each medium renewal. The basis of effect was reproductive capacity expressed as the total number of neonates per adult daphnid at the end of the test. Decrease in the reproduction of the daphnia could not be observed in any treatment. The following results for inhibition of reproduction based on introduced parent animal were determined for the test item: NOEL ≥ 2 mg/L, LOEL > 2 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 2 mg/L