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Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9-Apr-2018 to 9-May-2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
- Source and lot/batch No.of test material: Lot 20160912
- Purity: 99.2%
- Expiration date of the lot/batch: 1-August-2018
- Storage condition of test material: at room temperature
- Stability under test conditions: test substance stable

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: negative control (0 mg/L), 10, 18, 32, 56, and 100 mg/L
- Volume: 3.6 mL for the regular samples and 1.8 mL for quality control (QC) samples
- Sampling method: Duplicate samples for analysis, and reserve samples, were taken from each concentration before transfer to the replicate test vessels for the initial (T = 0 hours) samples. Two extra samples were taken from the 32 mg/L concentration for analytical QC, and two reserve QC samples were also taken. At test completion (T = 48 hours) test solutions were not pooled, due to the volatility of MTDID 49220, and duplicate samples were taken from at least one individual replicate.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤ -15°C) until analysis at the analytical laboratory of the test facility.

Test solutions

Details on test solutions:
- Method: The batch of MTDID 49220 tested was a clear yellow liquid with a purity of 99.2% and was completely soluble in test medium at the concentrations tested. Preparation of the test solutions started with the highest concentration of 100 mg/L, applying a short period of magnetic stirring to accelerate dissolution of the test item in medium. The pH was adjusted from 9.3 - 9.5 to 8.0 - 8.5 with 1M HCl in all tests. The lower concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none, test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water flea
- Strain/clone: Daphnia magna (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history
- Age of parental stock: > 2 weeks to < 4 weeks
- Age at study initiation: < 24 hrs
- Feeding during test: No
- Culturing: Adult daphnids were cultured in M7 medium (adjusted ISO medium plus trace elements, micronutrients, and vitamins). Adult daphnids in the cultures were held for at least 14 days, and were not more than 4 weeks old prior to collection of the juveniles for testing. During the period preceding the test, water temperatures were 18 - 22°C. Daphnids in the cultures were fed daily a suspension of freshwater algae. The adults were fed prior to test initiation, but neonates were not fed during the test.
- Health: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

180 mg/L as CaCO3 (in test medium)
Test temperature:
20 °C first test
20 - 21 °C second test
8.1 - 8.6 first test
8.0 - 8.4 second test
Dissolved oxygen:
7.3 - 8.5 mg/L first test
8.3 - 9.2 mg/L second test
Nominal and measured concentrations:
Nominal: 0 mg/L, 10 mg/L, 18 mg/L, 32 mg/L, 56 mg/L, 100 mg/L
Measured: see Table 1, 2
Results are based on nominal concentrations since analytically determined concentrations were greater than the nominal values for the first test (109 -157%), and 92-111% of nominal for the second test.
Details on test conditions:
- Test vessel: 60 mL airtight, all-glass, closed with no headspace to prevent loss of MTDID 49220 due to volatilization
- Fill volume: 60 mL volume
- Aeration: no
- No. of organisms per vessel: five
- No. of vessels per concentration (replicates): four
- No. of vessels per control (replicates): four
- Source/preparation of dilution water:adjusted ISO medium prepared using reverse-osmosis purified water (GEON Waterbehandeling, Berkel-Enschot, The Netherlands)
CaCl2.2H2O - 211.5 mg/L
MgSO4.7H2O - 88.8 mg/L
NaHCO3 - 46.7 mg/L
KCl - 4.2 mg/L
- Culture medium different from test medium: Yes, culture medium was M7 (adjusted ISO medium with addition of M7 trace elements, micronutrients, and vitamins).
- Adjustment of pH: No
- Photoperiod: 16 hours daily
EFFECT PARAMETERS MEASURED: immobilization (including mortality) observed at 24 and 48 hours
- Test concentrations: 0 mg/L (control) and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrationsopen allclose all
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
Result from the first test. 10% immobilization observed at the 56 mg/L test concentration and the 100 mg/L test concentrations at 48 hours.
Key result
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
Result from the second of two tests. 40% immobilization observed at the 100 mg/L test concentation at 48 hours.
Details on results:
Test was conducted twice since the result of the first test indicated MTDID 49220 to be less toxic than indicated by the range finding test.
See Tables 3, 4 for detailed results.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Dose-response test: Yes
- ECx: 48-hr EC50 = 0.33 mg/L (95% CI, 0.29 mg/L to 0.38 mg/L)
- Other: The sensitivity of Daphnia magna originating from the batch used in this test, for potassium dichromate fell within the expected range of sensitivity observed during previous years (n > 60; 48-hr EC50 between 0.3 and 1.0 mg/L.) The reference substance test was conducted within one month prior to initiation of the testing of MTDID 49220.

Any other information on results incl. tables

Table 3, Acute immobilization of daphnids after 24 and 48 hours - First Test

Nominal concentration (mg/L) Number of Daphnia exposed Response at 24 hours (number) Response at 24 hours (%) Response at 48 hours (number) Response at 48 hours (%)
0 20 0 0 0 0
10 20 0 0 0 0
18 20 0 0 0 0
32 20 0 0 0 0
56 20 1 5 2 10
100 20 0 0 2 10

Table 4, Acute immobilization of daphnids after 24 and 48 hours - Second Test

Nominal concentration (mg/L) Number of Daphnia exposed Response at 24 hours (number) Response at 24 hours (%) Response at 48 hours (number) Response at 48 hours (%)
0 20 0 0 0 0
10 20 0 0 0 0
18 20 0 0 0 0
32 20 2 10 2 10
56 20 0 0 6 30
100 20 5 25 8 40

Applicant's summary and conclusion

Validity criteria fulfilled:
Control immobilization was < 10% at the end of the test. The dissolved oxygen concentration was ≥ 3 mg/L in the control and test vessels at the end of the test.
The 48-hour EC50 (immobilization) of MTDID 49220 to Daphnia magna exceeded 100 mg/L. The test was conducted according to OECD TG 202.
Executive summary:

The 48-hour EC50 of MTDID 49220 to Daphnia magna was examined in a static test conducted according to OECD TG 202. Two tests were conducted in which test solutions were prepared as the control (0 mg/L), and at concentrations of 10, 18, 32, 56 and 100 mg/L (nominal). Nominal concentrations were verified with analysis. No immobilization of daphnids was observed in the controls. At 48 hours, 10% immobilization was observed at 100 mg/L in the first test, and 40% immobilization was observed at 100 mg/L in the second test. Thus, the 48-hour EC50 was > 100 mg/L for MTDID with Daphnia magna. The test was conducted according to internationally accepted test guidelines and was GLP compliant. It is reliable without restriction and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.