2-Butanone, peroxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-01-19 to 2001-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Directive 92/69/EC, annex II, method C3 Algal inhibition test

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(4/6/84)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO/FDIS 14442: Water Quality - Guidance for algal growth inhibition tests with poorly soluble organic and inorganic solids, volatile compounds, heavy metals and waste water (1999)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

A stock solution of the test item diluted in water was prepared at the beginning of the test (time 0) by mixing 100 mg of methyl ethyl ketone peroxide in phthalate plasticizer per litre of dilution water

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Centre of Algae and Protozoa (Ambleside, UK)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 ± 1 °C

pH:

pH (T0) = 7.13 - 7.26
pH (T72h) = 7.27 - 7.55
During the test, the control pH varied by 0.29 units.

Dissolved oxygen:

Dissolved O2 (T0h) = 8.4 - 8.9 mg/L
Dissolved O2 (T72h) = 8.7 - 10.2 mg/L

Nominal and measured concentrations:

- Nominal concentrations: 1.0, 2.1, 4.7, 10.3, 22.7 and 50.0 mg/L
- Mesured in non inoculated solutions: (initial) 1.06, 2.06, 4.73, 10.64, 22.50 and 48.66 mg/L; (final) 0.87, 1.89, 4.50, 9.30, 22.08 and 48.66 mg/L
- Initial and final concentrations were equivalent to nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: The study was performed using 120 mL glass bottles with PTFE bungs and sealed with aluminium caps, and placed in the photoculture cupboard.
- Cell density of pre-culture: The cell density was about 3.53 E6 cells/mL for the preliminary test and about 4.35 E6 cells/mL for definitive test.
- The appearance of the test solutions was visually checked at the beginning and at the end of the test.
- Dissolved oxgen and pH were measured in the test solutions at the beginning and at the end of the test.

TEST MEDIUM / WATER PARAMETERS
- Water: ultra-pure water was used for the preparation of dilution water (resistivity > 18MΩ)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CI: 2.1 - 4.7 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CI: 0.91 - 2.5 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other:

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 3 - 9.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear , colourless over the period of the test. No precipitation was observed at the end of the test.
Microscopic observation confirmed that the algae is a crescent shaped cell with an average lenth of 5 - 10 µm.
During the test, the control pH varied by 0.29 units.
The final concentrations of Methyl ethyl ketone peroxide in phthalate plasticizer were maintained within the designated limit of 80 % of the initial concentrations in non-inoculated flasks.
The increase in cell density (R), measured in the control solution between the end and the beginning of the test, was greater than a factor of 16 (R = 115).

Reported statistics and error estimates:

Dunnett test was used as a statistical analytical method.

Dissolved oxygen and pH were measured in the test solutions at the beginning and at the end of the test:

Nominal concentration (mg/L)	pH*		Dissolved O2 (mg/L)*
	T0	T72 h	T0	T72 h
0	7.26	7.55	8.8	10.0
1.0	7.21	7.52	8.4	9.9
2.1	7.20	7.54	8.4	10.2
4.7	7.14	7.34	8.5	9.1
10.3	7.16	7.31	8.5	8.7
22.7	7.13	7.27	8.7	8.9
50.0	7.13	7.31	8.9	8.9

* Measurements were carried out in non-inoculated solutions at T0 and in inoculated solutions after 72 hours of incubation.

 

The average percentage inhibition of cell growth (IAi) and growth rate (Iµi) are presented in the following table:

 

Sample Reference	Nominal concentration (mg/L)	IAi (%)	Iµi (%)
T	0	0.00	0.00
f	1.0	-4.88	-0.55
e	2.1	12.32	3.64
d	4.7	88.26	46.39
c	10.3	100.09	96.15
b	22.7	100.00	97.36
a	50.0	99.64	101.46

Validity criteria fulfilled:

yes

Conclusions:

The concentration of test item causing a 50 % reduction in biomass (EbC50 = 3.2 mg/L) and in growth rate (ErC50 = 5.6 mg/L) were determined. The NOEC was 2.1 mg/L.

Executive summary:

The toxicity of methyl-ethylketone peroxide in DMP towards fresh-water green algae Pseudokirchneriella subcapitatawas assessed to according to the method C.3 of the European Directive 92/69/EEC which is in conformity with OECD guideline 201. The concentration of test item which results in a 50% reduction in either cell growth (EbC50) or growth rate (ErC50) of the culture relative to the control is, if possible, estimated over a period of time of 72 hours. The no observed effect concentration (NOEC), highest tested concentration at which no significant inhibition of growth is observed relative to the control, is also estimated. Algae were exposed to a range of concentrations of methyl-ethylketone peroxide dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours. The study was performed using 120 mL glass bottles stoppered with PTFE bungs and sealed with aluminium caps, containing 50 mL of test solution inoculated with an algal suspension so that the initial cell concentration was equal to 1x10⁴ cells/mL. Test flasks were incubated at 23 ± 0.5 °C continously shaken and constantly illuminated. The cell density was measured daily. Analytical chemistry and physico-chemical measurements were carried out at the beginning and the end of the test. The following nominal test solution concentrations were used in the definitive test: 1.0, 2.1, 4.7, 10.3, 22.7, 50.0 mg/L. Concentrations of methyl-ethylketone peroxide in DMP were measured by gas chromatography. Initial and final concentrations weren`t equivalent to nominal concentrations.

The results were as follows:

 

Effective concentration and NOEC (mg/L)
		Value	95%CI
Cell growth	EbC50-72h	3.2	2.1 – 4.7
	EbC10-72h	1.7	0.91 – 2.5
	NOECb	2.1	-
Growth rate	ErC50-72h	5.6	3 - 9.5
	ErC10-72h	2.1	0.78 - 3.7
	NOECr	2.1	-

Description of key information

The concentration of test item causing a 50 % reduction in biomass (EbC50 = 3.2 mg/L) and in growth rate (ErC50 = 5.6 mg/L) were determined. The NOEC was 2.1 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.6 mg/L

EC10 or NOEC for freshwater algae:

2.1 mg/L

Additional information

The toxicity of methyl-ethylketone peroxide in DMP towards fresh-water green algae Pseudokirchneriella subcapitatawas assessed to according to the method C.3 of the European Directive 92/69/EEC which is in conformity with OECD guideline 201. The concentration of test item which results in a 50% reduction in either cell growth (EbC50) or growth rate (ErC50) of the culture relative to the control is, if possible, estimated over a period of time of 72 hours. The no observed effect concentration (NOEC), highest tested concentration at which no significant inhibition of growth is observed relative to the control, is also estimated. Algae were exposed to a range of concentrations of methyl-ethylketone peroxide dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours. The study was performed using 120 mL glass bottles stoppered with PTFE bungs and sealed with aluminium caps, containing 50 mL of test solution inoculated with an algal suspension so that the initial cell concentration was equal to 1x10⁴ cells/mL. Test flasks were incubated at 23 ± 0.5 °C continously shaken and constantly illuminated. The cell density was measured daily. Analytical chemistry and physico-chemical measurements were carried out at the beginning and the end of the test. The following nominal test solution concentrations were used in the definitive test: 1.0, 2.1, 4.7, 10.3, 22.7, 50.0 mg/L. Concentrations of methyl-ethylketone peroxide in DMP were measured by gas chromatography. Initial and final concentrations weren`t equivalent to nominal concentrations.

The results were as follows:

 

Effective concentration and NOEC (mg/L)
		Value	95%CI
Cell growth	EbC50-72h	3.2	2.1 – 4.7
	EbC10-72h	1.7	0.91 – 2.5
	NOECb	2.1	-
Growth rate	ErC50-72h	5.6	3 - 9.5
	ErC10-72h	2.1	0.78 - 3.7
	NOECr	2.1	-