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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:
06/05/2020 to 27/06/2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study undertaken to GLP certification, based on OECD guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Principles of method if other than guideline:
Guideline study
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
RM1007030 RL750/18
- Expiration date of the lot/batch:
24/10/2020
- Purity test date:
No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Closed vessel at room temperature (17.3 – 23.0 °C).
- Stability under storage conditions:
No data
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Solubility H2O: < 0.1 g/L; EtOH: > 1 g/L; acetone: > 1 g/L; CH3CN: > 1 g/L; DMSO: unknown; Stability: H2O: unknown; EtOH: 96h; acetone: 96h; CH3CN: 96h; DMSO: unknown
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):
No dat

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
No data
- Preparation of a nanomaterial dispersion (incl. dilution):
No data
- Final dilution of a dissolved solid, stock liquid or gel:
No data
- Final preparation of a solid:
No data

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
No data
- other information: No data
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Nominal concentrations were 0.46, 1, 2.2, 4.6, 10 mg/l
Time weighted mean measured concentrations were 0.40, 0.99, 2.10, 4.12, 9.84 mg/l

- Sampling method:
The content of the test item in the solutions (test solutions and controls) was measured in the freshly prepared solutions and in the used solutions at the beginning and at the end of the test and each medium renewal.
Additionally, an extra replicate for each treatment and the blank control without algae and daphnia was prepared. To avoid adsorption onto the algae cells or the test organism, the content of the test item was measured in extra replicates without algae and daphnia.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Following OECD (2019)Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, stock solutions containing 99.6 mg/L in dilution water were prepared. For this, the loading rate of 106.4 µL/L (based on a density of 0.94 g/mL, stated in the SDS) was mixed with dilution water. These solutions were treated with an ultrasonic bath for 30 minutes in order to achieve complete dissolution of the test item. The concentrations to be tested (10 / 4.6 / 2.2 / 1 / 0.46 mg/L) were prepared by dilution of this stock solution with dilution water
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

Daphnia magna is bred in the test facility throughout the year. The animals are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical. The keeping is performed similar to the method described in the OECD guideline, following SOP 115 002 01 („Zucht und Hälterung von Daphnia magna STRAUS“), version 13 from 11. Nov. 2019.

- Common name: Water flea
- Strain/clone: Berlin
- Sex: Female
- Age at study initiation (mean and range, SD): between 0 and 24 hours
- Vessels: glass beakers, nominal volume 2 L
- Medium: M4-Medium (recipe of ELENDT)
- Food type: green algae (Desmodesmus subspicatus)
- Medium renewal: twice a week
- Photo period: 16/8 hours, using neon tubes
- Temperature: 20 (+/- 2 °C)

18.5 hours before the start of the test, the adult animals are separated from the young. 45 minutes be-fore the start of the test, the adults are caught with the help of a glass tube, and the newborn daphnia, aged < 24 hours, are sieved from the medium. After a settling-in period, animals which show no apparent damage are used for the test.

Using a glass tube, the daphnia were caught and lifted from the beaker. They were put on a small sieve, and the medium surrounding the animals was sucked off using absorbent paper. Immediately after that, the animals were put into the respective test solution.

The test vessels were inspected for offspring once per working day. The newborn daphnids were count-ed and removed from the test vessel at each medium renewal. The animals were fed on each working day. On weekends, the Daphnia were not fed. On Fridays, the double amount of alga suspension was added into the test vessels.
Test type:
semi-static
Water media type:
freshwater
Remarks:
M4 Medium (Elendt)
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
Hardness was 267 mg/l CaCO3 in blank controls, and 267 - 356 mg/l CaCO3 in test items
Test temperature:
18.9 – 21.9 °C across the test concentrations, replicates and test duration.
pH:
pH ranged from 7.7 - 8.2 in blank controls, and 7.8 - 8.4 in test items
Dissolved oxygen:
Dissolved oxygen ranged from 8.7 - 10.3 mg/l in blank controls, and 8.7 - 10.9 mg/l in test items
Salinity:
Not applicable
Conductivity:
No data (M4 Medium Elendt)
Nominal and measured concentrations:
Nominal concentrations were 0.46, 1, 2.2, 4.6, 10 mg/l
Time weighted mean measured concentrations were 0.40, 0.99, 2.10, 4.12, 9.84 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (nominal volume 100 mL), 80 mL/animal, animals were kept individually
- Renewal rate of test solution (frequency/flow rate): 3 times a week, using freshly prepared test solutions
- No. of organisms per vessel: One
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 medium (Elendt)

OTHER TEST CONDITIONS
- Light intensity: 1030 – 1072 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test vessels were inspected for offspring once per working day. The newborn daphnids were count-ed and removed from the test vessel at each medium renewal.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
4.11 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
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}
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
9.84 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
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}
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
4.12 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
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}
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
4.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
9.84 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
4.12 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}
Details on results:
: The NOEC is determined by comparing the respective treatment with the blank value control using a hypothesis test. Statistically insignificant variation is considered as “no observed effect”. Whereas the EC10 is determined via regression analysis and read from the graph toxicity vs. concentration. In this study toxicity occurred only at the highest concentration tested. Therefore, the dose-response relationship was extremely steep and the EC10 which is read from the graph was slightly lower than the NOEC.- Behavioural abnormalities:
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Analysis of the NOEC/LOEC (for itntroduced parent- and surviving parent mortality) was carried out using the Williams test. Analysis of the EC10 (for itntroduced parent- and surviving parent mortality) was carried out using the Levenberg-Marquardt test with non-linear regression. All statistical analysis was carried out using the ToxRat Professional computer software package.

Biological Results (based on introduced parent animal)

Parameter

Value

NOEC

4.12 mg/L

LOEC

9.84 mg/L

EC10

4.11 mg/L

 

 Biological Results (based on surviving parent animal)

Parameter

Value

NOEC

4.12 mg/L

LOEC

9.84 mg/L

EC10

4.10 mg/L

Validity criteria fulfilled:
yes
Conclusions:
A reliable OECD 211 Guideline reproduction study on Daphnia magna reported an EC10 of 4.11 mg/l (measured TWA).
Executive summary:

A reliable OECD 211 Guideline reproduction study on Daphnia magna was undertaken to GLP standards. The study was performed using five concentrations ranging from 0.46 to 10 mg/L nominal concentration. Ten Daphnia each were exposed to the test item and the control for 21 days. The offspring of the animals was counted at each medium renewal. Medium renewal was performed three times weekly using freshly prepared test solutions.Toxicity was observed only in the highest concentrated treatment 10 mg/l (nominal). In this treatment only two parent animals survived at the end of the test.

 

Test item concentrations were measured using LC-MS determination.The content of the test item in the solutions (test solutions and controls) were measured in the freshly prepared solutions and in the used solutions at the beginning and at the end of the test and each medium renewal. The measured concentrations were not within the range of 80 % - 120 % of the nominal concentration. Therefore, the time-weighted arithmetic mean was used for determination of the biological results (see OECD guidance document No. 23, Annex 2). Evaluation was based on the total offspring per introduced parent animal and per surviving parent animal.

 

The following results (based on introduced parent animal) were determined for the test item:

 

*NOEC = 4.12 mg/L
LOEC = 9.84 mg/L
*E
C10= 4.11 mg/L

The following results (based on surviving parent animal) were determined for the test item:

*NOEC = 4.12 mg/L
LOEC = 9.84 mg/L
*E
C10= 4.10 mg/L

*Note: The NOEC is determined by comparing the respective treatment with the blank value control using a hypothesis test. Statistically insignificant variation is considered as “no observed effect”. Whereas the EC10is determined via regression analysis and read from the graph toxicity vs. concentration. In this study toxicity occurred only at the highest concentration tested. Therefore, the dose-response relationship was extremely steep and the EC10which is read from the graph was slightly lower than the NOEC value.

Description of key information

The 21 day EC10 is 4.11 mg/l, based on D. magna offspring mortality (Muckle, 2020).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
4.11 mg/L

Additional information