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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:
other: GLP study conducted according to a recognised guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken all test concentrations and the control. Fresh and expired media were sampled and analysed..
- Sampling method: Samples (approx. 50 mL) were stored in high density polyethylene bottles.
- Sample storage conditions before analysis: samples were stored deep frozen (<-10C).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: analytical and internal standard stock solutions were prepared by weighing DMC or acetone respectively into glass volumetric flasks and dissoving in and diluting to volume with water.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: not stated
- Strain/clone: STRAUS, Clone 5
- Justification for species other than prescribed by test guideline:
- Source: the Umweltbundesamt, Germany
- Age of parental stock (mean and range, SD):
- Feeding during test
- Food type: D. subspicatus
- Amount:
- Frequency: daily

Daphnia magna were bred in 1 L beakers with test medium. About 20 adults were kept per litre. The animals were fed on single cell green algae (e.g.
Desmodesmus subspicatus) about 3 times a week. Only as much food was added as the animals consumed per day.
The daphnids were reared at a temperature of 20 ± 2 DC in a RUMED chamber with 16 hours of illumination and 8 hours of darkness. The medium was changed three times per week, and the young were separated from the adults with sieves of 2 different meshes (ISO 3310-1) at the same time.



Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none
Hardness:
231 mg/L as calcium carbonate
Test temperature:
test temperatures were in the range 19.5 to 20.20.8C with a mean of 20.0C
See table 1 (Any other information on materials and methods including tables)

pH:
pH values ranged from 7.92 to 8.96
See table 2 (Any other information on materials and methods including tables)
Dissolved oxygen:
dissolved oxygen concentrations ranged from 6.9 to 11.0 mg/L
see table 3 (Any other information on materials and methods including tables)
Salinity:
not reported
Nominal and measured concentrations:
See tables 4 and 5 in section, "Any other informaytion on materials and methods including tables"
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): assume closed and flask contents were mixed by shaking
- Material, size, headspace, fill volume: glass flask
- Aeration: not stated
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static
- Renewal rate of test solution (frequency/flow rate): Test solutions were renewed on each Monday, Wednesday and Friday.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not stated


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: mix of deionised water and dechlorinated drinking water
- Total organic carbon: not stated
- Particulate matter: not stated
- Metals: not stated
- Pesticides: not stated
- Chlorine: not stated
- Alkalinity: not stated
- Ca/mg ratio: not stated
- Conductivity: 490 uS/cm
- Salinity: not stated
- Culture medium different from test medium: no
- Intervals of water quality measurement:


OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 h photoperiod daily
- Light intensity: 800 - 1400 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :


VEHICLE CONTROL PERFORMED: yes/no


RANGE-FINDING STUDY
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
No significant mortality of adult animals was seen
Details on results:
- Mortality of parent animals: no significant adult mortality
- No. of offspring produced per female: 58.1


Reported statistics and error estimates:
The mean quotient (only vessels with alive adults at the end of the test) of each treatment group was compared with the mean quotient of the control of group with a U-test (2-tailed, p = 0.05) according to Mann/Whitney. For each day of the test, the statistical evaluation was done with the non-cumulative quotients of all individual vessels at which no adult died until the end of the test. The results show an increasing variance with increasing dosage. A dosage dependent inhibition was observed at and above 50 mg/L.
Comparison of the quotients (number of all offspring per number of adults per vessel) to the control quotients show a decrease of reproduction at concentration levels at and above 50 mg/L of DMC. Based on the means of the cumulative number of offspring per adult and vessel over the entire reproduction period, significant dose dependent inhibition effects on reproduction in comparison to the control could not be calculated up to 25 mg/L. The detected statistical significance at 6.25 mg/L is out of the dose/response range and considered to be caused by the variance of the biological system. Therefore, the NOEC for reproduction was determined at a concentration of 25 mg/L ofDMC and the LOEC at 50 mg/L.

See table in "Remarks on results including tables and figures"

Mean number of all offspring (0 d - 21 d) per adult and vessel,

reduction of reproduction (%)

 

Group mg/L

Mean of cumulative number of offspring per adult

Percentage inhibition

Control

66.1

0.0

3.13

61.0

7.7

6.25

56.0

15.3

12.5

60.4

8.6

25

61.2

7.4

50

53.6

19.0

100

48.6

26.5

Validity criteria fulfilled:
yes
Conclusions:
Due to the distribution of data the 21 d EC50 (immobilisation) could not be calculated. It is however estimated to be greater than 100 mg/L.
Due to the distribution of data no EC50 value for reproduction could be calculated on the basis of the reproduction inhibition values obtained. Therefore, the EC50 for reproduction was estimated to be above 100 mg/L.
All toxicological evaluations presented in this report were performed using nominal concentrations. Significant reproduction inhibition was not observed up to 25.0 mglL. Therefore, the NOEC can be estimated to be 25.0 mgIL DMC..

Description of key information

The NOEC can be estimated to be 25.0 mg/L DMC. The reproduction rate was decreased 26.5 % at the highest test concentration of 100 mglL.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
25 mg/L

Additional information

Significant reproduction inhibition was not observed up to 25.0 mg/L. Therefore, the NOEC can be estimated to be 25.0 mg/L DMC. The reproduction rate was decreased 26.5 % at the highest test concentration of 100 mglL.

Due to the distribution of data the 21 d EC50 (immobilisation) could not be calculated. It is however estimated to be greater than 100 mg/L. Due to the distribution of data no EC50 value for reproduction could be calculated on the basis of the reproduction inhibition values obtained. Therefore, the EC50 for reproduction was estimated to be above 100 mg/L.

The concentration range for the test was chosen using information from the acute toxicity test with DMC provided by the sponsor: Six concentration levels in the range 3.13 and 100 mg/L DMC with a dilution factor of 2.0 were selected. The concurrent control group was exposed to test medium without DMC. No EC50 values on toxicity could be calculated because no mortality caused by dosage treatment was observed at the end of the test after 21 days. Due to the distribution of data no EC50 value for reproduction inhibition could be calculated. All toxicological evaluations presented in the report were performed with the nominal concentrations.