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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-10-17 to 2018-04-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13th April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.0256, 0.064, 0.16, 0.4, 1.0, 2.5, 6.25 and 15.6 mg/L
- Sampling method: For determination of the test item concentrations, samples were taken from each test concentration at the start and at the end of each water renewal period and from the controls at the start of the renewal periods.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. After the formulation procedure the test animals were immediately introduced into the test solutions.
Main Experiment:
For preparation of test solutions (at each renewal period) a stock solution of 10 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.006 g test item in 600 mL ISO medium using 10 min ultrasonic bath. The test solutions of subsequent concentrations were prepared by appropriate dilution of this stock solution.
Complementary Experiment:
For preparation of test solutions (at each renewal period) a stock solution of 15.6 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.0094 g (at the start of the test) and 0.0096 g (at the first renewal period) test item in 600 mL ISO medium using ultrasonic bath. The test solutions of subsequent concentrations were prepared by appropriate dilution of this stock solution.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Daphnia magna (Straus)
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Age of parental stock: Less than 24 hours old
- Sex: Female
- Feeding during test: None

ACCLIMATION
Test animals were bred under similar conditions as that used during the exposure period, therefore additional acclimatisation before the test was not necessary.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
249 mg/L (as CaCO3)
Test temperature:
19.8 – 20.8 °C (measured in the test vessels)
19.4 – 21.7 °C (within the climate chamber )
pH:
7.95 – 8.20
Dissolved oxygen:
7.80 – 8.42 mg/L
Salinity:
not applicable
Conductivity:
no data
Nominal and measured concentrations:
Main Experiment:
Nominal concentrations: 0.0256, 0.064, 0.16, 0.4 and 1.0 mg/L
Calculated geometric mean measured concentrations: 0.03, 0.04, 0.09, 0.21 and 0.36 mg/L.

Complementary Experiment:
Nominal concentrations: 2.5, 6.25 and 15.6 mg/L
Calculated geometric mean measured concentrations: 2.00, 5.11 and 14.04 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers of approximately 100 mL test medium
- Renewal rate of test solution (frequency/flow rate): 24-h renewal periods
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD guideline.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light (artificial illumination) and 8 hours darkness

EFFECT PARAMETERS MEASURED:
The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.

RANGE-FINDING STUDY:
- Test concentrations (Range-Finding Test): A non-GLP preliminary test was performed at the nominal concentations of 100, 10, 1, 0.1, and 0.01 mg/L in order to check the toxicity of the test item on the daphnids.
- Results used to determine the conditions for the definitive study:
In the Preliminary Range-Finding Test ten daphnids in the test concentrations and control were exposed for 48 hours (respectively). 0/10 daphnids were immobilised at the end of the test in the three highest test item concentrations (1, 10, 100 mg/L) and no immobilised daphnids were observed in the two lowest test concentrations (0.1 and 0.01 mg/L).
Based on the results of the non-GLP Preliminary Range-Finding Test five test concentrations in a geometric series (with a spacing factor of 2.5) were used in the main test. As just 5% immobility was observed in the test concentrations of 0.064 and 1 mg/L in this test the toxicity of test item was further investigated at higher concentrations in a complementary experiment to determine the EC50 value.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
2.19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % conf. limits: 1.152 – 2.930
Duration:
48 h
Dose descriptor:
other: EC20
Effect conc.:
2.98 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % conf. limits: 2.151 – 3.726
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
4.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % conf. limits: 3.729 – 5.581
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
5.11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
The 24h EC50: 1.53 mg/L (95% conf. limits: 1.30 – 1.78 mg/L)
Reported statistics and error estimates:
The 48-h ECx values were calculated by Probit analysis using SPSS software. The ECx values were calculated from the results of the following concentrations (nominal): 0.16, 0.4, 1.0, 2.5, 6.25 and 15.6 mg/L. The concentrations of 0.0256, 0.064 mg/L were excluded from the statistical evaluation as these concentration levels were not validated.
For the determination of the LOEC and NOEC, Dunnett t-Test (2-sided, α= 0.05) was used.

Analytical results

Concentrations of the test item were analytically determined at the start and at the end of each water renewal period.

The measured concentrations deviated more than 20 % from the nominal therefore the exposure concentrations were calculated as the geometric mean of the concentrations measured at the start and end of the water renewal periods


Table 1: Calculation of exposure concentrations

Nominal concentration
[mg/L]

Measured concentration[mg/L]

1strenewal period

2ndrenewal period

Geometric mean

start

end

start

end

Main Experiment

Control

not detected

not measured

not detected

not measured

Not Applicable

0.0256*

0.02

0.05

0.03

0.04

0.03

0.064*

0.04

0.04

0.04

0.03

0.04

0.16

0.14

0.06

0.15

0.06

0.09

0.4

0.27

0.17

0.26

0.16

0.21

1.0

0.71

0.11

0.71

0.30

0.36

Complementary

Experiment

Control

not detected

not measured

not detected

not measured

Not Applicable

2.5

2.48

1.66

2.34

1.65

2.00

6.25

5.95

4.46

6.02

4.27

5.11

15.6

16.1

12.0

16.2

12.4

14.04

*this concentration level is not validated

In the Main Experiment:
The measured concentrations of the test item were in the range of 56 – 95 % of the nominal at the start and 11 – 68 % at the end of the renewal periods.


In the Complementary Experiment:
The measured concentrations of the test item were in the range of 94 – 104 % of the nominal at the start and 66 – 79 % at the end of the renewal periods.

Biological results:

Biological results are based on the measured test item concentrations.

Table 2: Immobilisation of the test animals (summary table)

Measured concentration
[mg/L]

Number of
Daphnia
tested

Number of immobilised
Daphnia after

% of immobilised
Daphnia after

24 h

48 h

24 h

48 h

Main Experiment

Control

20

0

0

0

0

0.03

20

0

0

0

0

0.04

20

1

1

5

5

0.09

20

0

0

0

0

0.21

20

0

0

0

0

0.36

20

1

1

5

5

Complementary Experiment

Control

20

0

0

0

0

2.00

20

0

1

0

5

5.11

20

0

13

0

65

14.04

20

0

20

0

100


Validity criteria fulfilled:
yes
Conclusions:
In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item Methyl-isobutyl-ketone-peroxide (MIKP) had significant toxic effects on the mobility of Daphnia. The 48-h EC50 value was determined to be 4.48 mg/L. The 48-h NOEC was determined to be 2.00 mg/L and the 48-h LOEC was determined to be 5.11 mg/L. The results are based on measured test item concentrations.
Executive summary:


The influence of the test item Methyl-isobutyl-ketone-peroxide (MIKP) on the mobility and survival of Daphnia magna was determined in an acute toxicity study according to OECD guideline 202 and EU method C.2. 
Young Daphnia were exposed to aqueous test medium containing the test item at concentrations of 0.0256, 0.064, 0.16, 0.4 and 1.0 mg/L. The percentage of Daphnia being no longer capable of swimming at the end of the test or being dead was recorded for each test concentration. As just 5% immobility was observed in the test concentrations of 0.064 and 1 mg/L in this test the toxicity of test item was further investigated at higher concentrations of 2.5, 6.25 and 15.6 mg/L in a complementary experiment to determine the EC50 value.
The measured concentrations deviated more than 20 % from the nominal during the experiment therefore the geometric mean of the concentrations measured at the start and end of the water renewal periods were calculated to determine exposure concentrations. The corresponding calculated geometric mean test item concentrations were: 0.03, 0.04, 0.09, 0.21, 0.36, 2.00, 5.11 and 14.04 mg/L.
In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item had significant toxic effects on the mobility of Daphnia. The 48-h EC50 value was determined to be 4.48 mg/L. The 48-h NOEC was determined to be 2.00 mg/L and the 48-h LOEC was determined to be 5.11 mg/L.

Description of key information

In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item Methyl-isobutyl-ketone-peroxide (MIKP) had significant toxic effects on the mobility of Daphnia. The 48-h EC50 value was determined to be 4.48 mg/L. The 48-h NOEC was determined to be 2.00 mg/L and the 48-h LOEC was determined to be 5.11 mg/L. The results are based on measured test item concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
4.48 mg/L

Additional information

The influence of the test item Methyl-isobutyl-ketone-peroxide (MIKP) on the mobility and survival of Daphnia magna was determined in an acute toxicity study according to OECD guideline 202 and EU method C.2. 
Young Daphnia were exposed to aqueous test medium containing the test item at concentrations of 0.0256, 0.064, 0.16, 0.4 and 1.0 mg/L. The percentage of Daphnia being no longer capable of swimming at the end of the test or being dead was recorded for each test concentration. As just 5% immobility was observed in the test concentrations of 0.064 and 1 mg/L in this test the toxicity of test item was further investigated at higher concentrations of 2.5, 6.25 and 15.6 mg/L in a complementary experiment to determine the EC50 value.
The measured concentrations deviated more than 20 % from the nominal during the experiment therefore the geometric mean of the concentrations measured at the start and end of the water renewal periods were calculated to determine exposure concentrations. The corresponding calculated geometric mean test item concentrations were: 0.03, 0.04, 0.09, 0.21, 0.36, 2.00, 5.11 and 14.04 mg/L.
In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item had significant toxic effects on the mobility of Daphnia. The 48-h EC50 value was determined to be 4.48 mg/L. The 48-h NOEC was determined to be 2.00 mg/L and the 48-h LOEC was determined to be 5.11 mg/L.