4-methylcyclohexanone

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-25 to 2016-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant study, performed according to guideline, fully adequate for assessment

Remarks:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exemption is considered not to affect the purpose or integrity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

The appearance of the mixtures, pH and oxygen concentration were recorded. Respiration rate measurements were adapted where oxygen consumption was outside the normal range. Deviations were not considered to have an adverse effect.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Nominal amounts of test item (250, 450, 800, 1400 and 2500 mg) were dissolved separately in water with the aid of ultrasonication for approximately 10 minutes and the volumes adjusted to 1 litre to give 250, 450, 800, 1400 and 2500 mg/L stock solutions. An aliquot (200 mL) of the 250 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 100 mg/L. Similarly, aliquots (200 mL) of the 450, 800, 1400 and 2500 mg/L stock solutions were used to prepare the test concentrations of 180, 320, 560 and 1000 mg/L. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
There were 5 replicates for all test solutions.
- Controls: Blank control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No undissolved test item visible throughout test for all test concentrations

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Method of cultivation: A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection, the activated sewage sludge (10 litres) was fed synthetic sewage (500 mL) as a respiratory substrate.The pH of the sample on the day of test was 7.8.
- Initial biomass concentration: 3.0 g/L suspended solids

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 - 21 ºC

pH:

Start of test: 7.1 - 7.6
End of test: 7.4 - 7.9

Dissolved oxygen:

5.4 - 6.6 mg O2/L (test item after 30 minutes of exposure)
6.1 - 8.2 mg O2/L (reference item after 30 minutes of exposure)

Nominal and measured concentrations:

100, 180, 320, 560 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Type (open / closed): Not reported
- Material, size, headspace, fill volume: 500 mL flask containing 16 mL of synthetic sewage, 250 mL test solution and 250 mL of inoculum
- Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute during the preparation of the test system
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Biomass loading rate: 3.0 g/L suspended solids

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized reverse osmosis water
- Total organic carbon: < 1 mg/L Dissolved Organic Carbon (DOC)
- Intervals of water quality measurement: Changes in the appearance of the mixtures in the vessels before the addition of activated sludge and during the exposure period were recorded. The pH of the test preparations were measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-hour incubation period. The oxygen concentrations in all vessels were measured after 30 minutes contact time.

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted to between pH 7.2 and 7.7 prior to addition of inoculum
- Photoperiod: Normal laboratory lighting
- Light intensity: Normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): For measurement of the respiration rate, a well-mixed sample of each test vessel was poured into a 250 mL darkened glass BOD flask after three hours incubation time, and was not further aerated. The dissolved oxygen concentration was measured with an oxygen probe attached to an oxygen meter and was continuously recorded for a period of up to 10 minutes or until the oxygen concentration fell below 2 mg/L. During measurement, the samples were continuously stirred.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

220 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

1 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: Value obtained by extrapolation of the concentration response curve as no concentration employed resulted in greater than 50 % inhibition

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No undissolved reference item visible at the test start (prior to the addition of activated sewage sludge), or at 30 minutes and 3 hours of exposure

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 3-hour EC50 = 7.0 mg/L (95 % confidence limits = 5.4 - 9.0 mg/L)

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration and a line was fitted using the Xlfit software package (IDBS).The EC10 and EC20 values were determined from the equation for the fitted line. The EC50 and EC80 values were determined by inspection of the inhibition of respiration rate data. The NOEC value was determined using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 1. Oxygen consumption rates and percentage inhibition values after 3 hours contact time

Nominal concentration (mg/L)	Replicate	Initial O2 reading (mg O2/L)	Measurement period (minutes)	Final O2 reading (mg O2/L)	O2 consumption rate (mg O2/L/hour)	% inhibition
Control	1	5.2	4	2.4	42.00	-
Control	2	5.1	5	1.7	40.80	-
Control	3	4.5	3	2.2	46.00	-
Control	4	4.6	3	2.4	44.00	-
Control	5	5.0	4	2.2	42.00	-
Control	6	5.6	5	2.1	42.00	-
100	1	4.6	4	1.9	40.50	5
100	2	5.4	5	2.1	39.60	7
100	3	3.6	2	2.3	39.00	9
100	4	4.5	4	1.9	39.00	9
100	5	4.9	4	2.3	39.00	9
180	1	4.6	4	2.2	36.00	16
180	2	5.0	5	2.0	36.00	16
180	3	5.0	5	2.0	36.00	16
180	4	4.7	4	2.3	36.00	16
180	5	4.6	4	2.2	36.00	16
320	1	4.8	5	2.2	31.20	27
320	2	4.6	5	1.9	32.40	24
320	3	5.3	6	2.1	32.00	25
320	4	5.4	6	2.1	33.00	23
320	5	5.2	6	2.0	32.00	25
560	1	5.6	8	2.0	27.00	37
560	2	5.9	9	2.0	26.00	39
560	3	5.4	7	2.0	29.14	32
560	4	5.8	8	2.3	26.25	39
560	5	5.9	8	2.2	27.75	35
1000	1	5.9	10	2.2	22.20	48
1000	2	5.8	10	2.1	22.20	48
1000	3	6.0	10	2.3	22.20	48
1000	4	6.5	10	2.8	22.20	48
1000	5	6.6	10	3.0	21.60	50

Validity criteria fulfilled:

yes

Remarks:

The coefficient of variation of oxygen uptake in the control vessels was 4.4 % and the specific respiration rate of the controls was 28.53 mg oxygen per gram dry weight of sludge per hour. The reference item 3-hour EC50 value was 7.0 mg/L.

Conclusions:

The 3-hour EC10, EC20, EC50 and EC80 were determined to be 120, 220, 1200 and > 1000 mg/L, respectively. The EC50 value was obtained by extrapolation of the concentration response curve as no concentration employed resulted in greater than 50 % inhibition. The 3-hour NOEC was 100 mg/L.

Executive summary:

The toxicity of the test item to activated sewage sludge was assessed according to OECD Guideline 209. The study is reliable without restrictions, as it is GLP-compliant and was conducted according to a standard guideline. Following a preliminary range-finding test, activated sewage sludge was inoculated with the test item at nominal concentrations of 100, 180, 320, 560 and 1000 mg/L for 3 hours, with the addition of a synthetic sewage as a respiratory substrate. A blank control and positive control were also included in the study. The 3-hour EC10, EC20, EC50 and EC80 were determined to be 120, 220, 1200 and > 1000 mg/L, respectively. The EC50 value was obtained by extrapolation of the concentration response curve as no concentration employed resulted in greater than 50 % inhibition. The 3-hour NOEC was 100 mg/L.

Description of key information

The toxicity of the test item to activated sewage sludge was assessed according to OECD Guideline 209. The study is reliable without restrictions, as it is GLP-compliant and was conducted according to a standard guideline. Following a preliminary range-finding test, activated sewage sludge was inoculated with the test item at nominal concentrations of 100, 180, 320, 560 and 1000 mg/L for 3 hours, with the addition of a synthetic sewage as a respiratory substrate. A blank control and positive control were also included in the study. The 3-hour EC10, EC20, EC50 and EC80 were determined to be 120, 220, 1200 and > 1000 mg/L, respectively. The EC50 value was obtained by extrapolation of the concentration response curve as no concentration employed resulted in greater than 50 % inhibition. The 3-hour NOEC was 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

120 mg/L