Butanone

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is found to be readily biodegradable and the applied test concentrations are in the range of concentrations that can be expected in the influent of a sewage treatment plant

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without detailed documentation.

Qualifier:

equivalent or similar to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

Deviations:

not specified

Principles of method if other than guideline:

The concentration of the bacterial suspension is measured turbidimetrically; it is expressed by the extinction of the primary light of the monochromatic radiation at 436 nm for a layer of 10 mm thickness. The concentration at which the inhibitory action of Butanone (MEK) starts will be present in that step of a dilution series of MEK having an extinction value at the end of the test period that is = 3% below the mean value of extinction for non-toxic dilutions of the test cultures.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Before preparing the test cultures neutralize the Butanone (MEK) solution having a known content in sterile double-distilled water to be tested by using the minimum volume of acid or alkaline solution. The initial concentration of the MEK solution was not reported.

From this MEK solution, prepare four parallel dilution series in 300 ml Erlenmeyer flasks, stoppered with cotton-lined plastic caps. Each of the dilutions contains 1 part v/v of MEK solution in 2E0 to 2E14 parts v/v mixture. Prepare the dilution series as follows: the first flask of each series contains 160 ml of MEK solution at the start. Starting from this flask prepare the subsequent dilution steps at a constant dilution ratio by consistently mixing 80 ml of preliminary MEK dilution and 80 ml double distilled water. Consequently, each flask contains 80 ml of culture liquid at the start. Make up each flask of the three dilution series to be inoculated to 100 ml by adding 5 ml each of stock solution I, 5 ml of stock sultion II and 10 ml each of the prepared bacterial suspension from the preliminary culture having a known adjusted extinction value.

Test organisms (species):

Pseudomonas putida

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16 h

Hardness:

not reported

Test temperature:

25ºC

pH:

not reported

Dissolved oxygen:

not reported

Salinity:

freshwater

Nominal and measured concentrations:

2E0 to 2E14 parts v/v mixture

Details on test conditions:

Following inoculation, the extinction value of the monochromatic radiation at 436 nm for a 10-mm layer of the bacterial suspension of the test cultures will correspond to the extinction value of the Formazin standard suspension TE/F/436 nm = 10.

Leave both inoculated and non-inoculated dilution series at 25ºC for 16 hours. After termination of the test period measure the extinction of the monochromatic radiation at 436 nm in a 10-mm layer in the inoculated dilution series.

Nutrient medium (for stock and preliminary cultures)
Dissolve in 1000 ml double -distilled water:
1.060 g sodium nitrate, NaNO3
0.600 g dipotassium hydrogen phosphate, K2HPO4, anhydrous
0.300 g potassium dihydrogen phosphate, KH2PO4
0.200 g magnesium sulphate, MgSO4.7 H2O
10.000 g D(+) glucose
18.00 g Difco Bacto agar
0.010 g ferrous sulphate, FeSO4.7 H2O
1.5 ml trace elements solution.

Sterilize the solution in a steam sterilizer for 1.5 hours, after which add 3 ml of vitamin solution.

Trace elements solution (in grams per liter of double-distilled water)
0.055 Al2(SO4)3.18 H2O
0.028 KI
0.028 KBr
0.055 TiO2
0.028 SnCl2.2 H2O
0.028 LiCl
0.389 MnCl2.4 H2O
0.614 H3BO3
0.055 ZnSO4.7 H2O
0.055 CuSO4.5 H2O
0.059 NiSO4.6 H2O
0.055 Co(NO3)2.6 H2O

Vitamin solution
0.2 mg biotin (as D+ biotin)
2.0 mg nicotinic acid
1.0 mg thiamine (as thiamine HCl)
1.0 mg p-aminobenzoic acid
0.5 mg panthothenic acid (as D-panthothenic acid, Na-salt)
5 mg pyridoxamine (as pyridoxamine dihydrochloride)
2.0 mg cyanocobalamin (vitamin B12)
100 ml double distilled water

Fill 6 ml each of the nutrient medium into culture tubes, sterilize the latter in a steam sterilizer by fractionated sterilization (three times) for 30 min. Let solidify in slant position.

Stock solution I
20.000 g D(+) glucose
4.240 g sodium nitrate, NaNO3
2.400 g dipotassium hydrogen phosphate, K2HPO4 anhydrous
1.200 g potassium dihydrogen phosphate, KH2PO4
30 ml trace elements solution.

Dissolve glucose and nutrient salts separately in 500 ml double-distilled water each, sterilize in a steam sterilizer for 30 min and unite solutions when cooled.

Stock solution II
Dissolve:
0.200 g ferrous sulphate, FeSO4.7 H2O
4.000 g magnesium sulphate MgSO4.7 H2O

in 1000 ml sterile double distilled water.

Saline
Dissolve:
0.500 g sodium chloride, NaCl

in 1000 ml double-distilled water. Sterilize solution in a steam sterilizer for 30 min.

Reference substance (positive control):

no

Duration:

16 h

Dose descriptor:

other: Toxicity threshold

Effect conc.:

1 150 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

other: mean extinction value

Details on results:

For evalution of the toxicological findings at the end of the test period, the mean value (A) of the extinction is calculated for all test cultures that are free from both toxic influence and stimulation of growth except for those having extinction values outside a standard deviation of <3% and also, the mean value (B) of the extinction for those test cultures having the lowest toxic pollutant concentration within the dilution series.

For mathematical evaluation, (a) (highest non-toxic pollutant concentration) is plotted against (A) and (b) (lowest toxic pollutant concentration) against (B) as coordinates. After having entered (A - 3%), the pollutant concentration at which the inhibitory action (c) begins may be obtained from the regression line between (a;A) and (b;B) if a negative deviation of the mean extinction by a 3% difference against the mean extinction value of all test cultures having a non-toxic and non-stimulating pollutant concentration is used as an indicator of the beginning of inhibitory action.

Validity criteria fulfilled:

not applicable

Executive summary:

According to the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.7b: Endpoint specific guidance section R.7.8.17.1 Laboratory data on toxicity on STP microorganisms, results of the cell multiplication inhibition test with P. putida (Bringmann and Kühn 1980) can be used for calculation of the PNECmicro-organisms.

Reference 3

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not GLP, not guideline compliant. Growth expressed as relative turbidity was determined only at the beginning and the end of the study (16 h). Procedure in accordance with generally accepted standards.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Inhibition of cell proliferation following addition of the test substance to cultures of Pseudomonas putida. Growth inhibition was measured as relative turbidity at the end of the study (16 h). Method is sufficiently described in the article.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: TS was dissolved in sterile ddH2O. Whenever necessary, pH was adjusted to 7.0.
- Initial concentration: not specified

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: yes
- Method of cultivation: 24 h incubation at 25 °C in slant (culture tube in which medium solidified with the tube held at about 30° from the surface).
- Preparation of inoculum for exposure: bacteria were diluted 1:9 in sterile 5% NaCl
- Pretreatment: bacteria were washed in in sterile 5% NaCl
- Initial biomass concentration: relative turbidity (formazin turbidity equivalents TE/F/436 nm) = 10

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16 h

Post exposure observation period:

none

Test temperature:

25 °C

pH:

7.0

Nominal and measured concentrations:

1 part TS solution (concentration not specified) in 2E0 - 2E14 parts mixture (i.e. 1:1, 1:2, 1:4, 1:8... 1:16384) (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask covered with plastic caps filled with cotton
- Type: open
- Material: glass; size: 300 mL; fill volume: 100 mL
- Initial cells density: relative turbidity (formazin turbidity equivalents TE/F/436 nm) = 10
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- No. of vessels per sterility control: 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: double distilled water
- Medium (100 mL) prepared with 5 mL Stammlösung 1 + 5 mL Stammlösung 2 + 10 mL NaCl 5% (with bacteria for the test/control and without for the sterility control) + 80 mL TS (test and control)/ddH2O (sterility control)

Stammlösung 1
20 g glucose
4.24 g NaNO3
2.4 g K2HPO4
1.2 g KH2PO4
30 mL trace elements
Glucose and nutrient salts were separately dissolved in 500 mL ddH2O, sterilized, and, when cool, mixed together.

Stammlösung 2
0.2 g FeSO4*7(H2O)
4 g MgSO4*7(H2O)
dissolved in 1 L sterile ddH2O

Trace elements (in 1 L ddH2O)
0.055 g Al2(SO4)3*18(H2O)
0.028 g KI
0.028 g KBr
0.055 mg/L TiO2
0.028 g SnCl2*2(H2O)
0.028 g LiCl
0.389 g MnCl2*4(H2O)
0.614 g H3BO3
0.055 g ZnSO4*7(H2O)
0.055 g CuSO4*5(H2O)
0.059 g NiSO4*6(H2O)
0.055 g Co(NO3)2*6(H2O)
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Adjustment of pH: none

EFFECT PARAMETERS MEASURED: turbidity at the end of the study

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Duration:

16 h

Dose descriptor:

other: TGK (Toxische Grenzkonzentration or "toxicity threshold concentration")

Effect conc.:

1 150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Validity criteria fulfilled:

not applicable

Remarks:

no guideline followed. Dilution series sufficient to determine a dose effect relationship

Conclusions:

The TGK(16 h) of MEK in the Pseudomonas putida growth inhibition test is: 1150 mg/L

Reference 4

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not GLP, not guideline compliant. Procedure in accordance with generally accepted standards.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The ß-mesosaprobic colorless bacteriovorous flagellate Entosiphon sulcatum served as model organism in a standardized mineral medium. The protozoan cultures were fed with pure cultures of Escherichia coli prepared in a standardized nutrient medium. Feeding was quantitatively controlled by turbidimetry. The test period required for determination of the TGK was 72h. An electronic cell counter (Coulter) was used for quantitative determination of the protozoa inoculated and their multiplication within the test cultures.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: TS was dissolved in sterile ddH2O. Whenever necessary, pH was adjusted to 6.9.

Test organisms (species):

Entosiphon sulcatum

Details on inoculum:

- Laboratory culture: yes
- Method of cultivation: E. sulcatum cultures (20 mL mineral medium) were incubated in 300 mL Erlenmeyer flasks, closed with metal caps, at 25 °C. Cells were fed with cultures of Escherichia coli.
- Preparation of inoculum for exposure: after 72 h at 25 °C, the pre-cultures were examined over an inverted microscope
- Pretreatment: none
- Initial biomass concentration: 2 mL of appr. 15000 cell/mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test temperature:

25 °C

pH:

6.9

Nominal and measured concentrations:

1 part TS solution (concentration not specified) in 2E0 - 2E14 parts mixture (i.e. 1:1, 1:2, 1:4, 1:8... 1:16384) (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask covered with metal caps
- Type: open
- Material: glass; size: 300 mL; fill volume: 20 mL
- Initial cells density: 2 mL (appr. 15000 cell/mL)
- No. of vessels per concentration: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: double distilled water
- Medium (20 mL) prepared with 8 mL Stammlösung 1 + 2 mL dead bacteria feed + 8 mL TS + 2 mL E. sulcatum culture

Stammlösung 1
290 mg Ca(NO3)2*4(H2O)
70 mg Mg(NO3)2*6(H2O)
40 mg KNO3
dissolved in 1 L ddH2O and sterilized through a 0.2 µm filter. pH was adjusted with KOH to 6.9.

OTHER TEST CONDITIONS
- Adjustment of pH: none

EFFECT PARAMETERS MEASURED: cell number (Coulter counter)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Duration:

72 h

Dose descriptor:

other: TGK (Toxische Grenzkonzentration or "toxicity threshold concentration")

Effect conc.:

190 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Validity criteria fulfilled:

not applicable

Remarks:

no guideline followed. Dilution series long enough (15 concentrations) to determine a dose effect relationship

Conclusions:

The TGK(72 h) of MEK in the Entosiphon sulcatum growth inhibition test is: 190 mg/L

Reference 5

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not GLP, not guideline compliant. Procedure in accordance with generally accepted standards.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The bacteriovorous ciliate protozoon Uronema parduzci served as model organism in a standardized mineral medium. The protozoan cultures were fed with pure cultures of Escherichia coli prepared in a standardized nutrient medium. Feeding was quantitatively controlled by turbidimetry. The test period required for determination of the TGK was 20h. An electronic cell counter (Coulter) was used for quantitative determination of the protozoa inoculated and their multiplication within the test cultures.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: TS was dissolved in sterile ddH2O. Whenever necessary, pH was adjusted to 6.9.

Test organisms (species):

Uronema parduzci

Details on inoculum:

- Laboratory culture: yes
- Method of cultivation: U. parduzci cultures (20 mL mineral medium) were incubated in 300 mL Erlenmeyer flasks, closed with metal caps, at 25 °C. Cells were fed with cultures of Escherichia coli.
- Preparation of inoculum for exposure: after 22 h at 25 °C, the pre-cultures were examined over an inverted microscope
- Pretreatment: none
- Initial biomass concentration: 2 mL of appr. 15000 cell/mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

20 h

Post exposure observation period:

none

Test temperature:

25 °C

pH:

6.8

Nominal and measured concentrations:

1 part TS solution (concentration not specified) in 2E0 - 2E14 parts mixture (i.e. 1:1, 1:2, 1:4, 1:8... 1:16384) (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask covered with metal caps
- Type: open
- Material: glass; size: 300 mL; fill volume: 20 mL
- Initial cells density: 2 mL (appr. 15000 cell/mL)
- No. of vessels per concentration: 2
- No. of vessels per control: 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: double distilled water
- Medium (20 mL) prepared with 8 mL Stammlösung 2 + 2 mL dead bacteria feed + 8 mL TS + 2 mL U. parduzci culture

Stammlösung 2
290 mg Ca(NO3)2*4(H2O)
70 mg Mg(NO3)2*6(H2O)
40 mg KNO3
dissolved in 1 L ddH2O and sterilized through a 0.2 µm filter. pH was adjusted with KOH to 6.8.

OTHER TEST CONDITIONS
- Adjustment of pH: none

EFFECT PARAMETERS MEASURED: cell number (Coulter counter)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Duration:

20 h

Dose descriptor:

other: TGK (Toxische Grenzkonzentration or "toxicity threshold concentration")

Effect conc.:

2 830 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Validity criteria fulfilled:

not applicable

Remarks:

no guideline followed. Dilution series long enough (15 concentrations) to determine a dose effect relationship

Conclusions:

The TGK(20 h) of MEK in the Uronema parduzci growth inhibition test is: 2830 mg/L

Reference 6

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not GLP, not guideline compliant. Procedure in accordance with generally accepted standards.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The saprozoic flagellate protozoon Chilomonas paramaecium served as model organism in a standardized medium. The test period required for determination of the TGK was 48h. An electronic cell counter (Coulter) was used for quantitative determination of the protozoa inoculated and their multiplication within the test cultures.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: TS was dissolved in sterile ddH2O. Whenever necessary, pH was adjusted to 6.9.

Details on inoculum:

- Laboratory culture: yes
- Method of cultivation: C. paramaecium cultures (20 mL organic mineral medium) were incubated in 300 mL Erlenmeyer flasks, closed with metal caps, at 20 °C with the same medium as the test culture
- Preparation of inoculum for exposure: after 72 or 96 h at 20 °C, the pre-cultures were examined over an inverted microscope
- Pretreatment: none
- Initial biomass concentration: 4 mL of appr. 15000 cell/mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Test temperature:

20 °C

pH:

6.9

Nominal and measured concentrations:

1 part TS solution (concentration not specified) in 2E0 - 2E14 parts mixture (i.e. 1:1, 1:2, 1:4, 1:8... 1:16384) (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask covered with metal caps
- Type: open
- Material: glass; size: 300 mL; fill volume: 20 mL
- Aeration: shaken
- No. of organisms per vessel: appr. 60000 cell
- No. of vessels per concentration: 2
- No. of vessels per control: 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: double distilled water
- Medium (20 mL) prepared with 8 mL Stammlösung 1 + 8 mL TS + 4 mL C. paramecium culture

Stammlösung 1
8 g glycine
290 mg Ca(NO3)2*4(H2O)
70 mg Mg(NO3)2*7(H2O)
40 mg KNO3
26 mg K2HPO4
dissolved in 1 L ddH2O, pH 6.9. To the sterile Stammlösung 1 were then added 7.5 mL pasteurized Stammlösung 2.

Stammlösung 2
0.2 mg D-biotine
2.0 mg nicotinic acid
1.0 mg thiamin HCl
1.0 mg p-aminobenzoic acid
0.5 mg sodium D-pantothenate
5 mg pyridoxamine dihydrochloride
2.0 mg cyanocobalamine
dissolved in 100 mL ddH2O. Pasteurized 30 min at 60 °C

- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: none

EFFECT PARAMETERS MEASURED: cell number (Coulter counter)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Duration:

48 h

Dose descriptor:

other: TGK (Toxische Grenzkonzentration or "toxicity threshold concentration")

Effect conc.:

2 982

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Validity criteria fulfilled:

not applicable

Remarks:

no guideline followed. Dilution series long enough (15 concentrations) to determine a dose effect relationship

Conclusions:

The TGK(48 h) of MEK in the Chilomonas paramaecium growth inhibition test is: 2982 mg/L

Description of key information

The substance is readily biodegradable.

Experimental studies give weight of evidence, that there is no hazard for microorganisms:
Toxicity Threshold = 1150 mg/L; 16 hours, Pseudomonas putida (Bringmann & Kühn, 1980)
Toxicity Threshold = 1150 mg/L; 16 hours, Pseudomonas putida (Bringmann & Kühn, 1977)
Toxicity Threshold = 190 mg/L; 72 hours, Entosiphon sulcatum (Bringmann, 1978)
Toxicity Threshold = 2830 mg/L; 20 hours Uronema parduzci (Bringmann, 1980)
Toxicity Threshold = 2982 mg/L; 48 hours Chilomonas paramaecium (Bringmann et. al., 1980)

Additional information

Please refer to the document attached to Section 13 for further discussion of the studies.

According to Annex VIII of the REACH regulation the activated sludge respiration inhibition study does not need to be conducted, as the substance is found to be readily biodegradable and the applied test concentrations are in the range of concentrations that can be expected in the influent of a sewage treatment plant.

The following supporting studies on Pseudomonas and Protozoa support the conclusion that MEK is not toxic towards microorganisms.

The toxicity of methyl ethyl ketone to Pseudomonas putida was assessed in a published non-guideline study that predates GLP requirements for ecotoxicity studies (Bringmann & Kühn 1980). The study used a static freshwater system. Cell culture was maintained on nutrient agar slants and examined for purity periodically. The inoculum was prepared by growing the microorganism on nutrient agar plates for 24 hours. The cells were washed off from the medium and resuspended in a nutrient medium. By determining the extinction of the monochromatic radiation at 436 nm for a 10 mm layer of the bacterial suspension, the final turbidity value of the bacterial suspension was adjusted, by means of sterile saline, such that it corresponded to the extinction value of a Formazin standard suspension TE/F/436 nm = 10.

 

Four parallel dilution series in 300 mL Erlenmeyer flasks were prepared of test solution. Each dilution contains 1 part v/v of test solution in 2⁰to 2¹⁴parts v/v mixture. The first flask in the series contained 160 mL of test solution at the start. Starting from this flask, the subsequent dilution steps were prepared at a constant dilution ratio by consistently mixing 80 mL of preliminary test dilution and 80 mL double distilled water. Consequently, each flask contained 80 mL of culture liquid at the start. Each flask of the three dilution series were inoculated to 100 mL by adding 5 mL each of stock solution I, 5 mL of stock solution II and 10 mL each of the prepared bacterial suspension from the preliminary culture. Both inoculated and non-inoculated flasks were incubated at 25ºC for 16 hours. After 16 hours, the extinction of the monochromatic radiation at 436 nm for a 10 mm layer of the bacterial suspension was determined.

 

The results were analyzed on a semi-logarithmic chart according to the following scheme. The calculated values (A), (A-3%), and (B) were located on the Y-axis (linear): the average (A) of all the absorbance of the non-inhibited and non-stimulated cultures (as long as those values were below a standard deviation of less than 3 %); and the average (B) of all the absorbance of the cultures showing the lowest toxic effect. The highest non-toxic concentration (a) and the lowest toxic concentration (b) were located on the X-axis.

 

The placing of (A-3 %) on the line between the coordinates (a, A) and (b, B) allowed the value (C) to be determined on the abscissa of the initial concentration at which growth inhibition was determined or Toxicity Threshold. The Bringmann & Kühn (1980) results indicate that Pseudomonas putida had a 16 hour Toxicity Threshold concentration of 1050 mg/L.

 

None of the five published studies presented documenting the toxicity of methyl ethyl ketone to microorganisms were conducted with a mixed inoculum that would asses the functioning of the entire microbial community in a sewage treatment plant, rather the tests were based on single species systems. According to the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.7b: Endpoint specific guidance section R.7.8.17.1 Laboratory data on toxicity on sewage treatment plant microorganisms, results of the cell multiplication inhibition test with P. putida (Bringmann and Kühn 1980) can be used for calculation of the PNECstp.

 

The supporting data report a lower Toxicity Threshold for Entosiphon sulcatum (flagellated protozoa) (Bringmann, 1978) and higher Toxicity Thresholds for Uronema parduzci (ciliated protozoa) (Bringmann, 1980) and Chilomonas paramaecium (flagellated protozoa) (Bringmann et. al., 1980). According to the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.7b: Endpoint specific guidance section R.7.8.16.1 Laboratory data on toxicity to STP microorganisms and its sources, no correlation exists between activated sludge and ciliated protozoa test results. The lower Toxicity Threshold is for flagellated protozoa and a contact time of 48 hours. In general in accordance with the hydraulic retention time in a sewage treatment plant, short-term toxicity measurements in the order of hours are preferred. As these three protozoan results are equal to or greater than 20 hours contact time, these results should not be used to determine the PNECstp.