2-Methoxy-4-methylphenyl methyl carbonate

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:

11-01-2012 to 25-01-2012

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:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2011; signature: August 2011

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 5, 16, 50, 160, 500 mg of test item were each separately dispersed in approximately 200 mL dionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 house in order to maximise the dissolved test item concentration. All test vessels were shielded from light during mixing. Synthetic sewage (16 mL), activated sludge (250 mL) and water were added to a final volume of 500 ml to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At concentrations of 10 and 32 mg/L, no undissolved test item was visible. At concentrations of 100 mg/L few small particles of test item was visible on surface. At concentrations of 320 mg/L and 1000 mg/L, particles of test item were visible on surface and dispersed throughout.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: A mixed population of activated sewage sludge microorganisms was obtained on 12 January 2012 for the range-finding test and on 24 January 2012 for the definitive test from the aeration stage of the sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21°C overnight prior to use in the test. On the day of collection, the activated sludge (10 litres) was fed synthetic sewage sludge (500 mL). The pH of the sample on the day of the test was 7.2.
- Pretreatment: The test item was dispersed in reverse osmosis water and subjected to ultrasonication for 15 minutes and magnetic stirring for 24 hours to maximise dissolved test item concentrations. All vessels were shielded from light. The reference item was similarly ultrasonicated and dispersed for 20 minutes prior to exposure.
- Initial biomass concentration: The suspended solids concentration was determined to be 3.0 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

As each vessel reached 3 hours contact time, an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 7.0 mg O2/L and 2.0 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of the low oxygen consumption, the rate was determined over an approximate 10 minute period.

Test temperature:

20 ± 2 °C

pH:

0 hours: control: 6.7 - 7.1 and test groups: 6.7 - 6.8 and reference item: 6.8 - 6.9
3 hours: control: 7.0 - 7.2 and test groups: 7.3 - 7.6 and reference item: 7.2 - 7.5

Dissolved oxygen:

30 minutes hours: control: 5.0 - 7.0 mgO2/L and test groups: 4.4 - 6.5 mgO2/L and reference item: 5.8 - 8.2 mgO2/L
In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (7.0 mg 02/L and 2.0 mg 02/L respectively). This was considered to have had no adverse effect on the results of the study.

Nominal and measured concentrations:

Range-finder: nominal: 0 (control) 10, 100, 1000 mg/L (in single vessels, three replicates for 1000 mg/L and four for control).
Definitive test: nominal: 0 (control) 10, 32, 100, 320 and 1000 mg/L (in three replicates; six for control)
Reference item was completed: nominal: 3.2, 10, 32 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL darkened glass conical BOD flasks
- Type (delete if not applicable): Open, vessel continuously aerated with seal.
- Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute.
- 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): Three
- No. of vessels per control (replicates): Six (negative) and three (reference item)
- Nitrification inhibitor used (delete if not applicable): Not applicable.
- - 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 Dissolved Organic Carbon (DOC). 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 30 minutes after incubation had initiated.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test item dispersions was measured and adjusted to between pH 7.0 and 8.0, if necessary. Note was not reported as necessary.
- 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) 10, 32, 100, 320, 1000 mg/L (nominal)
- Range finding study: 0, (control) 10, 100, 1000 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes. No statistically significant toxic effects were shown at the test concentration of 10 mg/L, however, statistically significant toxic effects were shown at the test concentrations of 100 and 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

270 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.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (7.0 mg O2/L and 2.0 mg O2/L, respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Validity Criteria:
1. The mean specific respiration rate of the blank controls was 29.05 mg oxygen per gram dry weight of sludge per hour (i.e. was > 20 mg oxygen per gram dry weight of sludge per hour).
2. The coefficient of variation of the oxygen uptake rate in the control replicates was 5.0% (i.e. < 30%).
3. The reference substance results were valid, the EC50 for 3,5-dichlorophenol was: total respiration: actual 8.8 (C.I. 7.0 – 11.0) 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 8.8 (C.I. 7.0 – 11.0) 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 the method of Litchfield and Wilcoxon.
Analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analysis were performed using the SAS computer software package.

Table 1: Range-finding test: Control, reference substance and test substance oxygen concentration, oxygen consumption rate and percentage inhibition of the respiration rate.

Flask	Loading rate (mg/L)	Oxygen consumption (mg O2/L/h)	% Inhibition respiration rate
Control R1	-	36.00	-
Control R2	-	36.00	-
Control R3	-	38.00	-
Control R4	-	34.50	-
Test item	10	36.00	0
Test item	100	18.00	50
Test item R1	1000	14.40	60
Test item R2	1000	13.80	62
Test item R3	1000	12.00	67
Reference	3.2	27.00	25
Reference	10	15.60	57
Reference	32	4.80	87

Table 2: Definitive test: Control, reference substance and test substance oxygen concentration, oxygen consumption rate and percentage inhibition of the respiration rate

Flask	Loading rate (mg/L)	Oxygen consumption (mg O2/L/h)	% Inhibition respiration rate
Control R1	-	43.50	-
Control R2	-	43.50	-
Control R3	-	40.50	-
Control R4	-	42.00	-
Control R5	-	46.00	-
Control R6	-	46.00	-
Test item R1	10	38.40	12
Test item R2	10	37.00	15
Test item R3	10	39.00	11
Test item R1	32	30.00	31
Test item R2	32	28.50	35
Test item R3	32	27.00	38
Test item R1	100	26.40	39
Test item R2	100	22.80	48
Test item R3	100	25.20	42
Test item R1	320	22.20	49
Test item R2	320	22.80	48
Test item R3	320	22.80	48
Test item R1	1000	20.40	53
Test item R2	1000	18.67	57
Test item R3	1000	16.80	61
Reference	3.2	34.50	21
Reference	10	20.67	53
Reference	32	5.40	88

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 3 hour EC50 for total inhibition was 270 mg/L. The EC20 was 14 mg/L. 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 OECD TG 209 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 test item concentrations were 10, 32, 100, 320, 1000 mg/L (three replicates per test concentration) for a period of three hours at a temperature of 20 ± 2 °C with the addition of synthetic sewage as respiratory substrate. 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 5.0% and the specific respiration rate of the controls was 29.05 mg oxygen per gram dry weight of sludge per hour. In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (7.0 mg 02/L and 2.0 mg 02/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 8.8 (C.I. 7.0 – 11) mg/L. All validity criteria were considered to have been satisfied. The test item total respiration EC20 was 14 mg/L, EC50 was 270 mg/L. The EC80 was determined to be > 1000 mg/L (the highest concentration tested). All effect levels were based on nominal test item concentrations.

Description of key information

ASRIT: EC50 = 270 mg/L (nominal), 20 ± 2°C °C, OECD TG 209, 2012

ASRIT: EC80 = > 1000 mg/L (nominal, 20 ± 2°C °C, OECD TG 209, 2012

ASRIT: EC20 = 14 mg/L (nominal), 20 ± 2°C °C, OECD TG 209, 2012

Key value for chemical safety assessment

EC50 for microorganisms:

270 mg/L

Additional information

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 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 test item concentrations were 10, 32, 100, 320, 1000 mg/L (three replicates per test concentration) for a period of three hours at a temperature of 20 ± 2 °C with the addition of synthetic sewage as respiratory substrate. 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 5.0% and the specific respiration rate of the controls was 29.05 mg oxygen per gram dry weight of sludge per hour. In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (7.0 mg 02/L and 2.0 mg 02/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 8.8 (C.I. 7.0 – 11) mg/L. All validity criteria were considered to have been satisfied. The test item total respiration EC20 was 14 mg/L, EC50 was 270 mg/L. The EC80 was determined to be > 1000 mg/L (the highest concentration tested). All effect levels were based on nominal test item concentrations.