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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-10-16 to 2018-01-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.43, 0.94, 2.07, 4.55 and 10.00 mg/L.
- Sampling method: For determination of the test item concentrations, three replicate samples (20 mL/replicate) were taken from each test concentration at the start and at the end of each water renewal period and from the control at the start of the renewal periods. The 10.00 and 4.55 mg/L concentrations were analysed only at the start and end of the first renewal period as all fish died during the first day of the test.
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 solution. For preparation of test solutions (at each renewal period) a stock solution of 50 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.04 g test item in 800 mL ISO medium using approx. 10 min ultrasonic bath to obtain clear solution. The test solutions of chosen test concentrations were prepared by appropriate dilution of this stock solution.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Akvárium magazin Kft. (Pasaréti Gyula) 1222 Budapest, Dévény u. 36.
- Age at study initiation: Juveniles were used.
- Length at study initiation: The body length of each fish used was within the range of 2 ± 1 cm.
- Weight at study initiation: The body weight of fish was measured and recorded: first the weight of a vessel filled with water was determined. Then the fish were selected randomly and placed into this vessel. The weight of the group of fish was registered and the average body weight was calculated for 1 fish. The calculated mean weight of 1 fish was beteen 0.17 and 0.21 g.

ACCLIMATION
- Acclimation period: at least 12 days before test initiation.
- Acclimation conditions: Same conditins as used during to exposure period.
- Type and amount of food during acclimation: Fish were fed with appropriate, commercial diet for fish.
- Feeding frequency during acclimation: At least three times per week until one day before the test start.
- Health during acclimation: The health of the breeding was continuously monitored and any mortality or abnormal behaviour recorded. No significant mortality (less than 5 % of population) occurred in seven days before the start of the experiment, therefore the batch was considered to be acceptable for testing.

FEEDING DURING TEST: The fish were not fed during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
249 mg/L (as CaCO3)
Test temperature:
21.4 – 22.0 °C (the maximum deviation did not exceed ± 2 °C)
pH:
7.32 – 7.89
Dissolved oxygen:
70.0 – 95.3 %
Salinity:
Not applicable
Conductivity:
no data
Nominal and measured concentrations:
Nominal concentrations: 0.43, 0.94, 2.07, 4.55 and 10.00 mg/L.
Geometric mean concentrations: 0.26, 0.52, 1.38, 2.62 and 7.63 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquariums with 2 litre test liquid.
- Aeration: Test solutions were not aerated during the test.
- Renewal rate of test solution: Daily water renewal periods
- No. of organisms per vessel: 7
- No. of vessels per concentration/control (replicates): 1
- Biomass loading rate: The loading of the test aquariums were in the range of 0.59 – 0.74 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test was performed using ISO medium, prepared according to recommendation of Annex 2 of the OECD 203 guideline.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light: 8 h dark

EFFECT PARAMETERS MEASURED: The fish were observed at approximately 3, 6, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality.

TEST CONCENTRATIONS
- Range finding study:
In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item.
A stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.2 g test item in 2000 mL ISO medium using approx. 10 min ultrasonic bath to obtain clear solution. The test solutions of subsequent lower concentrations (50, 20, 10 and 1 mg/L) were prepared by appropriate dilution of this stock solution. Mortality was observed within few hours after the treatment in the concentrations of 10, 20, 50 and 100 mg/L, therefore a second series of test solutions was prepared including lower concentrations (0.5, 0.1 and 0.01 mg/L). For these concentrations a stock solution of 5 mg/L (nominal concentration) was prepared by dissolving an amount of 0.005 g test item in 1000 mL ISO medium using approx. 5 min ultrasonic bath to obtain clear solution. The test solutions (0.5, 0.1 and 0.01 mg/L) were prepared by appropriate dilution of this stock solution.
- Test concentrations (range-finding test): 0.01, 0.1, 0.5, 1, 10, 20, 50 and 100 mg/L
- Results used to determine the conditions for the definitive study: All animals died in the four highest test item concentrations of 10, 20, 50 and 100 mg/L. No mortality was observed in the remaining concentrations.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.89 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95 % conf. limit: 1.35 – 2.70
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.38 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
2.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Mortality: There was no mortality observed in the control group and in the three lowest test concentrations (0.26, 0.52, 1.38 mg/L measured) during the 96-h test period.
All fish were observed as dead at the 3-h observation period in the 7.63 mg/L (measured) concentration and at the 24-h observation period in the 2.62 mg/L (measured) concentration.The observed mortalities are detailed in in the section 'any other information on results incl. tables' (Table 2).
- Sub-lethal effects: No signs of abnormalities were observed in the control group and in the two lowest concentrations (0.26 and 0.52 mg/L measured) during the whole test period. In the concentration of 1.38 mg/L (measured) reduced activity, slight off-balance and staying at the bottom of aquarium was observed after approximately 3 and 6 hours of treatment. However, by the 24-h observation period all fish recovered and showed no sign of intoxication during the test. In the concentration of 2.62 mg/L (measured) reduced activity, staying mostly close to the water surface and slight off-balance was observed at the 3-h and 6-h observation periods (at 24 h 100 % mortality was noticed). In the highest concentration 7.63 mg/L (measured) observation of sub-lethal effects was not possible as all fish were dead 3 hours after treatment.
Reported statistics and error estimates:
The LC50 value was calculated by Probit analysis using SPSS software. Other endpoints were determined directly from the raw data.
Sublethal observations / clinical signs:

Analytical results:

Five different concentrations arranged in a geometric series with a spacing factor of 2.2 were tested in the main experiment. The nominal test item concentrations were 0.43, 0.94, 2.07, 4.55 and 10.00 mg/L.

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 (see Table 1 below).

The measured concentrations of the test item were in the range of 63 – 103 % of the nominal at the start and 30 – 70 % at the end of the renewal periods.

Table 1: Calculation of exposure concentrations

Nominal concentration

[mg/L]

Measured concentration [mg/L]

1st renewal period

2nd renewal period

3rd renewal period

4th renewal period

Geometric mean

start

end

start

end

start

end

start

end

Control

n.d.

-

n.d.

-

n.d.

-

n.d.

-

NA

0.43

0.32

0.24

0.33

0.25

0.38

0.13

0.38

0.19

0.26

0.94

0.81

0.56

0.59

0.34

0.82

0.34

0.78

0.28

0.52

2.07

1.74

0.92

1.95

0.84

2.12

1.17

1.82

1.12

1.38

4.55

3.70

1.85

-

-

-

-

-

-

2.62

10.00

8.32

7.00

-

-

-

-

-

-

7.63

Table 2: Cumulative mortality data

Concentration

Cumulative mortality (initial population = 7 fish / group)

Nominal

Measured

3h

6h

24h

26h

48h

72h

96h

Control

not detected

0

0

0

0

0

0

0

0.43

0.26

0

0

0

0

0

0

0

0.94

0.52

0

0

0

0

0

0

0

2.07

1.38

0

0

0

0

0

0

0

4.55

2.62

0

4

7

7

7

7

7

10.00

7.63

7

7

7

7

7

7

7

Validity criteria fulfilled:
yes
Conclusions:
In this 96-hour semi-static acute toxicity test on Zebrafish (Brachydanio rerio) the test item Methyl-isobutyl-ketone-peroxide (MIKP) had significant toxic effects on fish. The 96-h LC50 value was determined to be 1.89 mg/L. The 96-h NOEC and LOEC values were determined to be 1.38 mg/L and 2.62 mg/L, respectively. The results are based on measured test item concentrations.
Executive summary:

The influence of the test item Methyl-isobutyl-ketone-peroxide (MIKP) to zebrafish (Brachydanio rerio) was evaluated in a short-term toxicity test according to OECD guideline 203 and EU method C.1. Young Zebrafish were exposed to aqueous test media containing the test item for 96 hours at a range of concentration in a semi-static test system (daily renewal periods). Based on the results of the preliminary experiment, nominal concentrations of 0.43, 0.94, 2.07, 4.55 and 10.00 mg/L were investigated in the main study. The corresponding measured test item concentrations (calculated as the geometric mean of the measured start and end values of each renewal periods) were: 0.26, 0.52, 1.38, 2.62 and 7.63 mg/L. The measured concentrations of the test item were in the range of 63 – 103 % of the nominal at the start and 30 – 70 % at the end of the renewal periods. Thus, all biological results are based on the measured geometric mean concentrations.

In this 96-hour semi-static acute toxicity test on Zebrafish the test item had significant toxic effects on fish. The 96-h LC50 value was determined to be 1.89 mg/L. The 96-h NOEC and LOEC vaues were was determined to be 1.38 mg/L and 2.62 mg/L, respectively.

Description of key information

In this 96-hour semi-static acute toxicity test on Zebrafish (Brachydanio rerio) the test item Methyl-isobutyl-ketone-peroxide (MIKP) had significant toxic effects on fish. The 96-h LC50 value was determined to be 1.89 mg/L. The 96-h NOEC and LOEC values were determined to be 1.38 mg/L and 2.62 mg/L, respectively. The results are based on measured test item concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
1.89 mg/L

Additional information

The influence of the test item Methyl-isobutyl-ketone-peroxide (MIKP) to zebrafish (Brachydanio rerio) was evaluated in a short-term toxicity test according to OECD guideline 203 and EU method C.1. Young Zebrafish were exposed to aqueous test media containing the test item for 96 hours at a range of concentration in a semi-static test system (daily renewal periods). Based on the results of the preliminary experiment, nominal concentrations of 0.43, 0.94, 2.07, 4.55 and 10.00 mg/L were investigated in the main study. The corresponding measured test item concentrations (calculated as the geometric mean of the measured start and end values of each renewal periods) were: 0.26, 0.52, 1.38, 2.62 and 7.63 mg/L. The measured concentrations of the test item were in the range of 63 – 103 % of the nominal at the start and 30 – 70 % at the end of the renewal periods. Thus, all biological results are based on the measured geometric mean concentrations.

In this 96-hour semi-static acute toxicity test on Zebrafish the test item had significant toxic effects on fish. The 96-h LC50 value was determined to be 1.89 mg/L. The 96-h NOEC and LOEC vaues were was determined to be 1.38 mg/L and 2.62 mg/L, respectively.