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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:
1999-02-08 to 1999-04-02
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted under GLP conditions in accordance with the official OECD guideline No 202 (1984).

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
(December 1992)
according to
other: OECD Guideline 202 (Part I: Daphnia sp., Acute Immobilisation Test) (Adopted April, 1984)
according to
ISO 6341 15 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Version / remarks:
(Third edition, 1996-04-01)
GLP compliance:

Test material

Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Mucosolvan HBr
- Physical state: beige solid
- Stability under storage: stable
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: 1.0, 3.2 and 18 mg/l and control
- Sampling method: 10 ml from the approximate center of the test vessels.
- Sampling frequency: t = 0h and t = 48h
- Sample storage conditions before analysis: the samples not analysed on the same day of sampling were stored in a deep-freeze
- Others: Additionally, reserve samples of 10 ml were taken from all concentrations. These samples were stored in a deep-freeze for possible analysis until delivery of the final report with a maximum of three months. Specification of the samples analysed.

Test solutions

Details on test solutions:
Preparation started with stock solutions at 100 mg/l (range-finding test) or 20 mg/l (final test). The stocks were magnetically stirred for 30 minutes to dissolve the test substance in the test medium. The various test concentrations were prepared by subsequent dilutions of these stocks. The final test solutions were all clear and colourless.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Strain: (Crustacea, Cladocera) (Straus, 1820)
- Age at study initiation (mean and range, SD): <24 hours
- Method of breeding: with new-born animals, i.e. less than 3 days old, by placing about 250 of them into 10 liters of medium in an all-glass culture vessel. Maximum age of cultures: 4 weeks; renewal of the cultures: after 7 days of cultivation half of the medium twice a week; temperature: 18-22°C +- 1°C; feeding: Daily, a suspension of fresh water algae; Medium: M7
- Feeding during test: no

Study design

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

Test conditions

250 mg/l expressed as CaCa3 at pH 8.0 after areation
Test temperature:
21.0 to 21.6 °C (blank control)
7.9 to 8.0
Dissolved oxygen:
7.5 to 9.0 mg O2/l
not applicable
Nominal and measured concentrations:
see table 2
Details on test conditions:
- Aeration: no
- Material, size, headspace, fill volume: 100 ml, all-glass, containing 80 ml medium
- No. of organisms per vessel: 10 per vessel
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

- ISO, prepared in milli-RO water

- Photoperiod: 16 hours photoperiod daily

- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 mg/l (geometric progression with a factor of 10).
- Results used to determine the conditions for the definitive study: yes (concentration range). Analysis of the samples taken during the range-finding test showed that the measured concentration was in agreement with nominal and remained stable during the 48-hour test period.
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
24 h
Dose descriptor:
Effect conc.:
11 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL 8.7 - 17 mg/L
48 h
Dose descriptor:
Effect conc.:
7.3 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL 6.3 - 9.2 mg/L
Results with reference substance (positive control):
The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at NOTOX. The 24h EC50 was > 1.8 mg/L. The 48 EC50 was 0.95 mg/L with 95% fiducial limits of 0.78 - 1.3 mg/L.
Reported statistics and error estimates:
The EC50 -value was calculated at 24 and 48 hours of exposure from the probits of the percentages of affected daphnia and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method (Finney, D.J, 1971: Probit analyisis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Any other information on results incl. tables

Final test:

Analysis of the samples taken during the final test showed that measured concentrations were in agreement with nominal.

The responses recorded in this test allowed for reliable determination of an EC50. The response recorded at 10 mg/l after 24 hours of exposure was less then the response recorded in the range-finding. However, at the end of the test period the response at 10 mg/l was comparable to the response recorded during the range-finding.

Table 1: Acute immobilisation of daphnia after 24 and 48 hours in the final EC50 -test

 Concentration Mucosolvan HBr (mg/L)  Vessel Number  Number Daphnia exposed     Response at 24h     Response at 48h
       number  %  number  %
 Blank-control  A  10  0  0  0  0
 Blank-control  B  10  0  0  0  0
1.0  A  10  0  0  0  0
1.0  B  10  0  0  0  0
1.8  A  10  0  0  0  0
1.8  B  10  0  0  0  0
3.2  A  10  0  0  0  0
3.2  B  10  0  0  0  0
5.6  A  10  1  10  2  20
5.6  B  10  0  0  0  0
 10  A  10  20  10  100
 10  B  10  1  10  7  70
 18  A  10  10  100  10  100
 18  B  10  10  100  10  100

Table 2: Concentration of Mucosolvan HBr in test medium (final test)

 Time of sampling (hour)  Date of sampling (dd-mm-yy)  Date of analysis (dd-mm-yy)        Concentration
       Nominal (mg/L)  Analysed* mg/L)  Relative to nominal (%)
 0  31 -03 -99  02 -04 -99**  0  n.d  n.a.
 0      1.0  0.963  96
 0      3.2  3.25  102
 0      18  18.8  104
 48  02 -04 -99  02 -04 -99  0  0.070  n.a.
 48      1.0  0.955  96
 48      3.2  3.23  101
 48      18  18.7  104

* Mean of duplicate analysis. The maximum deviation between the responses was calculated for each sample and was <10%

** Samples were frozen until analysis

n.d. not detected

n.a. not applicable

In the nominal 0 mg/L sample taken at t=48 hours, a small peak was observed at the retention time of the test substance. Because in the same sample taken at the start of the test no test substance was observed, it was assumed that the 0.070 mg/L concentration measured either does not derive from test substance or was introduced during sample pretreatment. The other samples taken at t=48 hours, were not corrected for the concentration measured in the nominal 0 mg/l sample.

Applicant's summary and conclusion

Validity criteria fulfilled:
Under the conditions of the present study Mucosolvan HBr did not induce acute immobilization of Daphnia magna at 3.2 mg/l after 48 hours of exposure (NOEC). The 24h-EC50 was 11 mg/L with a 95% confidence interval between 8.7 and 17 mg/L. The 48h-EC50 was 7.3 mg/L with a 95% confidence interval between 6.3 and 9.2 mg/L.
Executive summary:

Acute Toxicity Study in Daphnia magna with Mucosolvan HBr.

The study procedures described in this report were based on the ISO international Standard 6341: “Water quality – Determination of the inhibition of the mobility of Daphnia magna Straus - Acute toxicity test, Third edition, 1996-04-01.

Mucosolvan HBr is a beige solid with a purity of 100.4% and completely soluble at the concentrations tested. After a range-finding test, a final test was performed exposing Daphnia for maximum of 48 hours to a range of nominal concentrations from 1.0 to 18 mg/L. The test was performed in duplicate with 10 daphnia per vessel. Samples for analytical confirmation of actual test concentration were taken at the start and the end of the test period.

Analysis of the samples taken during the final test showed that the measured concentrations were in agreement with nominal.

Mucosolvan HBr did not induce acute immobilisation of Daphnia magna at 3.2 mg/L after 48 hours exposure (NOEC).

The 24h-EC50 was 11 mg/L with a 95% confidence interval between 8.7 and 17 mg/L.

The 48h-EC50 was 7.3 mg/L with a 95% confidence interval between 6.3 and 9.2 mg/L.