(2E)-2-methyl-3-(4-methylphenyl)prop-2-en-1-ol

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:

20-10-2011 to 25-11-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item tested was a white waxy solid at temperature <23°C and a clear colourless liquid at temperature >23°C. To liquefy the test substance, it was heated up to a temperature of maximum 50°C before use. The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 mL of test substance mixtures in RO water (tap water purified by reverse osmosis) with initial loading rates of 2.5 times the final loading rate. The test substance was pipetted to the test bottles. Due to possible volatility of the test item, the mixtures were stirred in closed dark brown bottles for approximately 2 hours. Subsequently, 16 ml synthetic sewage feed, 250 ml sludge and RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring. The deployed concentrations were: Range Finder concentrations: 10, 100, 1000 mg ; Definitive Test concentrations: 1.0, 3.2, 10, 32, 100, 320 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None in the prepared solutions. See comments above.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: aerobic activated sludge from sewage treatment plant at Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, which treats predominantly domestic sewage (dates provided in the full study report).
- Preparation of inoculum for exposure: The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.12 g/L of sludge, as used for the test).
- Initial biomass concentration: Determination of the suspended solids level of the activated sewage sludge was carried out. The suspended solids concentration was equal to 3.0 g/L prior to use.
- Other: Synthetic waste water was prepared according to OECD 209. The pH value of the activated sludge was determined prior to test start. The pH was 7.8 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

During 3 hour exposure aeration and stirring took place.

Post exposure observation period:

The synthetic sewage feed (16 mL) and an adequate amount of the test substance loading were mixed and made up to 250 mL with RO water in a 1 litre bottle. The pH was determined. Thereafter 250 mL activated sludge was added. This was the start of the test. The mixture was then aerated during the contact time, using a pipette as an aeration device. After the 3-hour contact time, the oxygen consumption was recorded for a period of 10-17 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls.

Test temperature:

20 ± 2 °C

pH:

0 hours: control: 7.3 - 7.4 and test groups: 7.2 - 7.3 and reference item: 7.3
3 hours: control: 8.0 - 8.3 and test groups: 8.1 - 8.3 and reference item: 8.3

Dissolved oxygen:

0 minutes: control: 6.5 - 8.1 mgO2/L and test groups: 5.8 - 8.8 and reference item: 7.2 - 7.4 mgO2/L (all dissolved oxygen concentrations were above 60% saturation i.e. > 5 mgO2/L).

Nominal and measured concentrations:

Range Finder concentrations: 10, 100, 1000 mg/L (in single vessels, three replicates for 1000 mg/L and two for control).
Definitive Test concentrations: 1.0, 3.2, 10, 32, 100, 320 mg/L (in five replicates; six for control)
Reference item was completed: nominal: 5, 12, 32 mg/L (in three replicates)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 L glass BOD bottle
- Type (delete if not applicable): Open, vessel continuously aerated with seal.
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start is above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) and to maintain the sludge flocs in suspension.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): None.
- No. of vessels per concentration (replicates): Five
- No. of vessels per control (replicates): Six (negative) and three (reference item)
- Nitrification inhibitor used (delete if not applicable): The final test was standard test without nitrification inhibitor and abiotic conditions.
- Biomass loading rate: See table.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the range-finding and definitive tests was deionised reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC). Synthetic sewage was subsequently prepared.
- Culture medium different from test medium: Not applicable.
- Intervals of water quality measurement: pH and temperature were determined in all test media and controls; prior to and at the end of the 3-hour incubation period. Dissolved oxygen values were determined in all vessels.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Light intensity: The test was conducted under normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Biological Oxygen Demand. Monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase.

TEST CONCENTRATIONS
- Test concentrations: Definitive study: 0 (control), 1.0, 3.2, 10, 32, 100, 320 mg/L (nominal) (in five replicates; six for control)
- Range finding study: 0, (control) 10, 100, 1000 mg/L (nominal) (in single vessels, three replicates for 1000 mg/L and two for control).
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

140 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

C.I. 52 - 400 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

C.I. 20 - 140 mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

C.I. 14 - 100 mg/L

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Validity Criteria:
1. The controls oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.
2. The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test.
3. The reference substance results were valid, the EC50 for 3,5-dichlorophenol was: total respiration: actual 9.8 (C.I. 4.0 – 23.7) mg/L. This was within the expected range: 2 to 25 mg/L.
Therefore, the validity criteria was met.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Relevant effect levels: the EC50 for 3,5-dichlorophenol was: total respiration: actual 9.8 (C.I. 4.0 – 23.7) mg/L. This was within the expected range: 2 to 25 mg/L. Full information is provided in the full study report.

Reported statistics and error estimates:

95% confidence limits were calculated for the reference item EC50 value using linear regression analysis. The NOECD was based on statistical analysis using TOXSTAT Release 3.5 (1996).

Table 1. Definitive Test – summarised study results

Flask	Loading Rate (mg/L)	Temperature	pH before sludge addition	pH after 3 h contact time	Oxygen concentration at the start (mg O2/L)	Respiration Rate (mg O2/L h)	Respiration Rate (mg O2/g h) #	% Inhibition respiration relative to the control (mean value)
Control	0	18.9 –22.8	7.3 – 7.4	8.0 – 8.3	6.5 – 8.1	35	22
T1	1.0	19.8 – 21.1	7.3	8.1 – 8.2	6.8 – 7.8	33	21	6
T2	3.2	20.4 – 21.2	7.3	8.1	6.7 – 7.4	35	22	1
T3	10	19.8 – 20.7	7.3	8.1 – 8.2	7.0 – 7.6	32	20	10
T4	32	19.7 – 20.1	7.3	8.1 – 8.2	7.1 – 7.5	31	20	12
T5	100	19.8 - 21.9	7.3	8.0 – 8.2	5.8 – 7.4	26	17	25
T6	320	19.7 – 20.7	7.2 – 7.3	8.1 – 8.2	8.0 – 8.8	7	4	81

#: The amount of suspended solids in the final test mixture was 1.56 g/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 3-hour EC50 for total inhibition was 140 (C.I. 52 - 400) mg/L. The EC20 was 53 (C.I. 20 – 140) mg/L, the EC10 was 38 (C.I. 14 – 100) mg/L. The NOEC was 32 mg/L with no significant inhibition of respiration rates below this concentration determined under the conditions of the study. All effect levels were based on nominal test item concentrations.

Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to EU Method C.11, OECD TG 209 and ISO 8192 (2007) in accordance with GLP. Following a preliminary range finding test at 10, 100 and 1000 mg/L concentration, a definitive test was conducted examining total respiration only was carried out under static conditions. The Definitive Test concentrations were 0 (control), 1.0, 3.2, 10, 32, 100, 320 mg/L (in five replicates per test item concentration and six for control) for a period of three hours at a temperature of 20 ± 2 °C with the addition of synthetic sewage as respiratory substrate. The test item was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in RO water with initial loading rates of 2.5 times the final loading rate. The was pipetted to the test bottles. Due to possible volatility of test item, the mixtures were stirred in closed dark brown bottles for approximately 2 hours. Subsequently, 16 ml synthetic sewage feed, 250 ml sludge and RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for 10-17 minutes. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The coefficient of variation of oxygen uptake in the control vessels was < 30 % and the specific respiration rate of the controls was 22 mg oxygen per gram dry weight of sludge per hour. The reference substance results were valid, the EC50 for 3,5-dichlorophenol was: total respiration: actual 9.8 (C.I. 4.0 – 23.7) mg/L. This was within the expected range: 2 to 25 mg/L. All validity criteria were considered to have been satisfied. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 32 mg/L. At higher loading rates the inhibitory effect of test item on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 25% inhibition at a loading rate of 100 mg/L to 81% at a loading rate of 320 mg/L (mean values). Under the conditions of the study, the 3-hour EC50 for total inhibition was 140 (C.I. 52 - 400) mg/L. The EC20 was 53 (C.I. 20 – 140) mg/L, the EC10 was 38 (C.I. 14 – 100) mg/L. The NOEC was 32 mg/L with no significant inhibition of respiration rates below this concentration determined under the conditions of the study. All effect levels were based on nominal test item concentrations.

Description of key information

ASRIT: EC50-3h = 140 (C.I. 52 - 400) mg/L (nominal), 20 °C, EU Method C.11, 2012

ASRIT: EC20-3h = 53 (C.I. 20 - 140) mg/L (nominal), 20°C, EU Method C.11, 2012

ASRIT: EC10-3h = 38 (C.I. 14 - 100) mg/L (nominal), 20°C, EU Method C.11, 2012

ASRIT: NOEC-3h = 32 mg/L (nominal), 20 °C, EU Method C.11, 2012

Key value for chemical safety assessment

EC50 for microorganisms:

140 mg/L

EC10 or NOEC for microorganisms:

32 mg/L

Additional information

Key data: OECD TG 209, 2018: The effect on respiration rate of activate sludge was examined using a method according to EU Method C.11, OECD TG 209 and ISO 8192 (2007) in accordance with GLP. Following a preliminary range finding test at 10, 100 and 1000 mg/L concentration, a definitive test was conducted examining total respiration only was carried out under static conditions. The Definitive Test concentrations were 0 (control), 1.0, 3.2, 10, 32, 100, 320 mg/L (in five replicates per test item concentration and six for control) for a period of three hours at a temperature of 20 ± 2 °C with the addition of synthetic sewage as respiratory substrate. The test item was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in RO water with initial loading rates of 2.5 times the final loading rate. The was pipetted to the test bottles. Due to possible volatility of test item, the mixtures were stirred in closed dark brown bottles for approximately 2 hours. Subsequently, 16 ml synthetic sewage feed, 250 ml sludge and RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for 10-17 minutes. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The coefficient of variation of oxygen uptake in the control vessels was < 30 % and the specific respiration rate of the controls was 22 mg oxygen per gram dry weight of sludge per hour. The reference substance results were valid, the EC50 for 3,5-dichlorophenol was: total respiration: actual 9.8 (C.I. 4.0 – 23.7) mg/L. This was within the expected range: 2 to 25 mg/L. All validity criteria were considered to have been satisfied. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 32 mg/L. At higher loading rates the inhibitory effect of test item on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 25% inhibition at a loading rate of 100 mg/L to 81% at a loading rate of 320 mg/L (mean values). Under the conditions of the study, the 3-hour EC50 for total inhibition was 140 (C.I. 52 - 400) mg/L. The EC20 was 53 (C.I. 20 – 140) mg/L, the EC10 was 38 (C.I. 14 – 100) mg/L. The NOEC was 32 mg/L with no significant inhibition of respiration rates below this concentration determined under the conditions of the study. All effect levels were based on nominal test item concentrations.