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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:
2017-08-21 to 2018-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: duplicate samples were taken from the test medium with the nominal test item concentration of 6.0 μg/L and from the solvent control at the start of the test (Day 0) and at nine further sampling dates distributed over the test period. The analytical samples were taken from the different replicates of each treatment.
For verification of the test item concentration in the application solution, duplicate samples were taken at the start and the end of all application solution renewal periods.
- Sample storage conditions before analysis: To each 10-mL test medium sample, a volume of 10 mL of acetonitrile was added immediately after sampling. Then, the samples were stored frozen (at about –20 °C) until analysis. In preexperiments for investigation of the storage stability of the test medium samples, the test item proved to be stable under these storage conditions.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Chemical name of vehicle: N,N-Dimethylformamide (DMF)
- Concentration of vehicle in test medium: Refer to section "Any other information on materials and methods incl. tables"
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Straus
- Age at study initiation: less than 24 hours old
- Method of breeding: under identical conditions to the test conditions
- Source: in-house culture (origin: Daphnia Collection of the University of Basel/Switzerland)
- Age of parental stock: at least 14 days old
- Feeding during test : yes
- Food type: suspension of green algae of the species Desmodesmus subspicatus
- Amount: 0.3 mg C/L at the start of the test, As the parental daphnids were growing during the test, the concentration was adjusted to 0.4 mg C/L at Day 7 and to 0.5 mg C/L at Day 14. Automatically dosed in volumes of 200 μL to each aliquot of test
water.
- Frequency: daily

ACCLIMATION
- Acclimation period: No acclimation needed, an in-house culture bred under identical conditions to those of the tests were used for this study.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
2.5 mmol/L (= 250 mg/L as CaCO3)
Test temperature:
20 °C
pH:
7.8 - 8.1
Dissolved oxygen:
9.0 - 9.7 mg/L
Nominal and measured concentrations:
Nominal concentration: 6 µg/L
Mean measured concentration: 3 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: flow-through glass beaker
- Material, size, headspace, fill volume: 50 mL fill volume
- Type of flow-through: computer-controlled automated flow-through dosing system
- Renewal rate of test solution (frequency/flow rate): Flow rate - approximately 19-fold test medium exchange rate per day
The application solution was renewed three times during the test period (Days 4, 11 and 18).
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 20
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 Medium, prepared according to OECD 211
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: no, the same

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 h light : 8 h dark
- Light intensity: approximately 3 μE m-2 s-1.

EFFECT PARAMETERS MEASURED:
- daily for immobility of adults or other visible abnormalities
- body length at the end of the test on the surviving adults
- reproduction rate at the end of the test

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: Based on a series of a stirring and rande-finding experiments, it was determined that the test item has a very low solubility in the test media and the test concentration could not be maintained stable during the exposure period without using a solubilising agent. Thus, the solvent DMF was use to achieve a relatively stable test concentration in the flow-through system.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: nominal: 6 µg/L
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: nominal: 6 µg/L
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: nominal: 6 µg/L
Details on results:
The 21-day EC10 values for the mortality, inhibition of the reproduction rate and body length could not be calculated, since no inhibitory effect of the test item on daphnia at the test concentration of nominal 6.0 μg/L was observed.

Analytical Results

The measured test item concentration in the freshly prepared application solutions (DMF/water) was between 83 and 95 % of the nominal value. In the aged application solutions, the test item concentration was between 81 and 91 % of nominal. Thus, the stability of the test item in the application solution during its use for 7 days could be demonstrated.

The concentrations of the test item Butyl methoxydibenzoyl methane measured in the test medium with the nominal test concentration of 6.0 μg/L were between 40 and 66 % of the nominal value. The mean measured concentration during the exposure period was calculated as arithmetic mean over all measurements and was 3.0 μg/L ± 0.47 μg/L, corresponding to 50 % of the nominal value. This recovery is in the range of what was expected according to dosing pre-experiments. The losses could be attributed to intrinsic properties of the difficult test item with low water solubility. The measured concentration of 3.0 μg/L corresponded to the solubility limit of the test item in the test medium of approximately 2 μg/L. All reported biological results are related to the mean measured concentration of 3.0 μg/L.

The method of analysis was validated and proven to be suitable for use. The mean measured test item concentration per sampling ranged from 2.40 to 3.98 μg/L during the test period.

 

Biological Results

In the control, the solvent control and the mean measured test item concentration of 3.0 μg/L, all parental daphnids survived until the end of the test. Therefore, the NOEC for mortality was determined to be 3.0 μg/L, corresponding to the solubility limit of the test item in the test water.

The first offspring released from their parent daphnids were recorded in both the controls and at the mean measured test item concentration of 3.0 μg/L on Day 8. Thus, the time of the first brood was not affected by the test item. The mean reproduction rate of the daphnids in the control and solvent control was 158 ± 22 (mean ± standard deviation) and 151 ± 22 living offspring per surviving adult, respectively. At the mean measured test item concentration of 3.0 μg/L the mean reproduction rate was 157 ± 26, corresponding to 104 % of the solvent control value. Therefore, the NOEC for inhibition of reproduction rate was determined to be 3.0 μg/L (solubility limit).

The mean body length of the daphnids in the control and solvent control was 4.9 ± 0.10 mm (mean ± standard deviation) and 4.8 ± 0.14 mm, respectively. At the mean measured test item concentration of 3.0 μg/L the mean body length was 4.9 ± 0.10 mm, corresponding to 101 % of the solvent control value. Therefore, the NOEC for body length was determined to be >= 3.0 μg/L (solubility limit in test medium).

The 21-day EC10 value for mortality, inhibition of the reproduction rate and body length could not be calculated, since no toxic effects of the test item on daphnia at the mean measured test concentration of 3.0 μg/L were observed. No visible abnormalities were observed in the test animals during the test.

Table 1: Biological endpoints

Biological Endpoints

Nominal Test Item Concentration
(Mean Measured Test Item Concentration)

Control

Solvent
Control

6.0 µg/L
(3.0 µg/L)

Survival [%] after
21 Days of Exposure

100

100

100

Mean Reproduction Rate

158

151

157

Mean Reproduction Rate in % of Solvent Control

104

-

104

Mean Body Length of the Adults [mm]

4.86

4.84

4.88

Mean Body Length in % of Solvent Control

100

-

101

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the test item Butyl methoxydibenzoyl methane had no toxic effects on survival, growth and reproduction of Daphnia magna after the exposure period of 21 days at the mean measured concentration of 3.0 μg/L, corresponding to the highest concentration of test item that could be reached in test water. Thus, the 21-day NOEC of the test item was determined to be >= 3.0 μg/L (mean measured), representing the water solubility limit of the test item. The 21-day LOEC and the 21-day EC10 values for the different endpoints were > 3.0 μg/L.
Executive summary:

The impact of the test item Butyl methoxydibenzoyl methane on the survival, growth (body length) and reproduction rate of Daphnia magna was investigated in a flow-through test over 21 days following the OECD 211 and the Commission Regulation (EC) No 440/2008, C.20 (amended by Commission Regulation (EC) No. 2017/735).

In this flow-through test, 10 single-housed daphnids were exposed to untreated test water (control) and 20 single-housed daphnids each were exposed to untreated test water with solvent (solvent control) or to treated test water (test medium) with a test item concentration of nominal 6.0 μg/L. This test item concentration was chosen to maintain a constant exposure concentration at the solubility limit of the test item in test water, i.e. approximately 2 μg/L, as determined in a pre-experiment.

A limit test was performed to demonstrate that the test item Butyl methoxydibenzoyl methane has no toxic effects on Daphnia magna at the highest concentration of the test item that could be reached in test water. The concentrations of the test item Butyl methoxydibenzoyl methane measured in the test medium with the nominal test concentration of 6.0 μg/L were between 40 and 66 % of the nominal value during the test period. The mean measured concentration during the test period was 3.0 μg/L ± 0.47 μg/L (arithmetic mean ± standard deviation), corresponding to 50 % of the nominal value. The losses are attributed to intrinsic properties of the difficult test item with low water solubility. All reported biological results are related to the mean measured concentration of 3.0 μg/L which represented the maximum solubility in the test medium.

All parental daphnids in the control, the solvent control and test item treatment group survived until the end of the test. The first brood was recorded at the same time in both controls and the treatment group. The mean reproduction rate and the mean body length for the test item treatment were not significantly different than the control and the solvent control.

In conclusion, the test item Butyl methoxydibenzoyl methane had no toxic effects on survival, growth and reproduction of Daphnia magna after the exposure period of 21 days at the mean measured concentration of 3.0 μg/L corresponding to the highest concentration of the test item that could be reached in test water.

Thus, the 21-day NOEC of the test item was determined to be >= 3.0 μg/L (mean measured) representing the water solubility limit of the test item. The 21-day LOEC and the 21-day EC10 values for the different endpoints were > 3.0 μg/L.

Description of key information

The test item Butyl methoxydibenzoyl methane had no toxic effects on survival, growth and reproduction of Daphnia magna after the exposure period of 21 days at the mean measured concentration of 3.0 μg/L, corresponding to the highest concentration of test item that could be reached in test water. Thus, the 21-day NOEC of the test item was determined to be >= 3.0 μg/L (mean measured), representing the water solubility limit of the test item in test medium. The 21-day LOEC and the 21-day EC10 values for the different endpoints were > 3.0 μg/L.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
3 µg/L

Additional information

The impact of the test item Butyl methoxydibenzoyl methane on the survival, growth (body length) and reproduction rate of Daphnia magna was investigated in a flow-through test over 21 days following the OECD 211 and the Commission Regulation (EC) No 440/2008, C.20 (amended by Commission Regulation (EC) No. 2017/735).

In this flow-through test, 10 single-housed daphnids were exposed to untreated test water (control) and 20 single-housed daphnids each were exposed to untreated test water with solvent (solvent control) or to treated test water (test medium) with a test item concentration of nominal 6.0 μg/L. This test item concentration was chosen to maintain a constant exposure concentration at the solubility limit of the test item in test water, i.e. approximately 2 μg/L, as determined in a pre-experiment.

A limit test was performed to demonstrate that the test item Butyl methoxydibenzoyl methane has no toxic effects on Daphnia magna at the highest concentration of the test item that could be reached in test water. The concentrations of the test item Butyl methoxydibenzoyl methane measured in the test medium with the nominal test concentration of 6.0 μg/L were between 40 and 66 % of the nominal value during the test period. The mean measured concentration during the test period was 3.0 μg/L ± 0.47 μg/L (arithmetic mean ± standard deviation), corresponding to 50 % of the nominal value. The losses are attributed to intrinsic properties of the difficult test item with low water solubility. All reported biological results are related to the mean measured concentration of 3.0 μg/L which corresponds to the maximum solubility of the test item in the test medium.

All parental daphnids in the control, the solvent control and test item treatment group survived until the end of the test. The first brood was recorded at the same time in both controls and the treatment group. The mean reproduction rate and the mean body length for the test item treatment were not significantly different than the control and the solvent control.

In conclusion, the test item Butyl methoxydibenzoyl methane had no toxic effects on survival, growth and reproduction of Daphnia magna after the exposure period of 21 days at the mean measured concentration of 3.0 μg/L corresponding to the highest concentration of the test item that could be reached in test water.

Thus, the 21-day NOEC of the test item was determined to be >= 3.0 μg/L (mean measured) representing the water solubility limit of the test item in the test medium. The 21-day LOEC and the 21-day EC10 values for the different endpoints were > 3.0 μg/L.