Anisole

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:

From 19 April 2013 to 30 september 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in compliance with international standard guidelines and under GLP conditions.The study report was conclusive and well enough documented with all relevant information included.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

Two test flasks at 100 mg/L were carried out during the range-finding test instead of one test flask as indicated in the study plan. This deviation has no impact on the validity of the results.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

No analytical monitoring

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For the preparation of the blank controls, 16 mL synthetic sewage were added to water and activated sludge inoculum in order to obtain a final volume of 500 mL containing 1.5 g/L of suspended solids. The reference and test mixtures were prepared by adding 16 mL synthetic sewage to the appropriate amounts of reference item or test item and water followed by the addition of inoculum to obtain a final volume of 500 mL containing 1.5 g/L of suspended solids and different nominal concentrations of the test and reference items.
Stock solutions were carried out, for each test, in deionised water and used for carrying out the test concentrations (from 10 to 300 mg/L). For test concentrations at 600 and 1000 mg/L, 300 and 500 mg, respectively, of test item were directly incorporated into each flask concerned.
- Controls: yes, test water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- The inoculum was prepared from activated sludge sampled in the aeration tank treatment plant receiving predominantly domestic sewage (Montataire - (60) France).

- Preparation of inoculum for exposure:
After settling, the supernatant of activated sludge was discarded and the sludge re-suspended in chlorine-free tap water. The wash-water was removed after settling of sludge and activated sludge were re-suspended in chlorine-free tap water.
The inoculum was continuously aerated and 50 mL of synthetic sewage feed/L activated sludge were added each day until use.
Sampling of the 5th June 2013:
Test carried out 6 June 2013: dry matter of inoculum: 3130 mg/L
239.6 mL of inoculum were added per test flask, except Abiotic control at 1000 mg/L, corresponding to a concentration of suspended solids (SS) in the test mixture of 1.5 g/L.
Sampling of the 1st July 2013:
Test carried out 2 July 2013: dry matter of inoculum: 3267 mg/L
229.6 mL of inoculum were added per test flask corresponding to a concentration of suspended solids (SS) in the test mixture of 1.5 g/L.
Test carried out 3 July 2013: dry matter of inoculum: 3500 mg/L
214.3 mL of inoculum were added per test flask corresponding to a concentration of suspended solids (SS) in the test mixture of 1.5 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable

Hardness:

No data

Test temperature:

between 20.5 and 22.0°C (definitive test); between 19.5 and 20.5°C (range-finding test)

pH:

range-finding : 7.4-7.9 (test item); 7.4-7.8 (reference item); 7.5-7.6 (blank control)
definitive study: between 7.2 and 7.8 (test item); between 7.2 and 7.9 (reference item); 7.1-7.6 (blank controls)
No pH adjustment was carried out before addition of the inoculum.

Dissolved oxygen:

see result tables

Salinity:

Not applicable

Nominal and measured concentrations:

nominal concentrations: 150, 300 and 600 mg/L (definitive test); 10, 100 and 1000 mg/L (range-finding test)

Details on test conditions:

During the first test conducted in accordance with the requirements of paragraphs 39 and 40 of the test guideline 209, a significant inhibitory effect (approximately 30%) on the respiration of micro-organisms was observed at the highest concentration tested (1000 mg/L of test item). This first test is named "range-finding test" through the test report.
Range-finding test: Inoculum collected on: 05 June 2013
Test carried out 06 June 2013
In agreement with the sponsor representative a new test was carried out, named "definitive test" through the test report, to determine a no observed effect concentration (NOEC).
Definitive test: Inoculum collected on: 01 July 2013
Reference item: test carried out 02 July 2013
Test item: test carried out 03 July 2013
According to the results of the first test (i.e. " range finding test") and the objective of the second one (i.e. "definitive test"), only 3 concentrations were performed.

TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: 1 litre glass beakers
- Aeration: Yes. Air supply: clean, oil-free. Air flow 0.5 to 1 litre / min
- No. of vessels per concentration (replicates):
Range-finding test: For the substance : 1 test flask at 10 mg/L, 2 test flasks at 100 mg/L, 3 test flasks at 1000 mg/L, 3 test flasks for abiotic control at 1000 mg/L and 3 test flasks for blank controls. For the reference (3,5-DCP): 1 test flask (3.13, 10 and 32 mg/L).
Definitive test: 5 test flasks for the substance (150, 300 and 600 mg/L) and 6 test flasks for blank controls. 1 test flask for the reference (3.13, 10 and 32 mg/L) and 3 test flasks for blank controls.
- No. of vessels per control (replicates): 6
- Biomass loading rate: 1.5 g/L suspended solid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water containing less than 1 mg/L DOC
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod:Not applicable
- Light intensity: No data

EFFECT PARAMETERS MEASURED : Measurements after 3 hours of contact: Dissolved oxygen concentration, pH at the beginning and after 3 hours of contact. Apparatus: Carbon analyzer: SHIMADZU TOC-VCSN

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 in range-finding test, 2 in definitive test
- Range finding study: Yes
- Test concentrations: nominal concentrations: 150, 300 and 600 mg/L (definitive test); 10, 100 and 1000 mg/L (range-finding test) and 3.13, 10 and 32 mg/L for reference substance (range-finding test and definitive test).

Reference substance (positive control):

yes

Remarks:

3-5 dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

300 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Range-finding test: The results show that under the test conditions the test item: Anisole has no significant toxic effect on respiration of activated sludge at concentrations 10 and 100 mg/L and that the EC50 is greater than 1000 mg/L after a contact time of 3 hours. No oxygen consumption was observed for abiotic controls during this range-finding test, showing a lack of reducing properties of the test item. This control has therefore not been renewed in the definitive test. For the definitive test, test concentrations selected were: 150, 300 and 600 mg/L.

The mean results of the definitive test are presented in Table 1.

In the condition of the test, a NOEC of 300 mg/L has been determined for the test item ANISOLE, after a contact time of 3 hours.

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was graphically determined between 6.0 and 7.0 mg/L in the definitive study . The 3-hour EC50 is within the guideline-recommended range of 2 - 25 mg/L, confirming suitability of the activated sludge used.

Reported statistics and error estimates:

Statistical tests (Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming) were performed to determine the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) in the test conditions.
As the assumption of the homogeneity of variance was not verified with the data obtained during this study, a non-parametric test was carried out (Kruskal-Wallis) and showed a statistically significant effect at the highest concentration tested is, for this study, 600 mg/L.
Therefore, the first test concentration without effect observed is the test concentration 300 mg/L of test item.

Table 1: Definitive Test

Test item: Anisole

Identification	Q1 (mg/L)	Q2 (mg/L)	Dt	R mg O2/Lh	Rs mg O2/gh	IT (%)	pH
							T0	T3h
Blank control 1	6.6	3.8	5.5	30.5	20.4	/	7.2	7.5
Blank control 2	5.8	2.9	5.5	31.6	21.1		7.2	7.5
Blank control 3	5.3	2.1	5.5	34.9	23.3		7.2	7.3
Blank control 4	5.8	2.7	5.5	33.8	22.5		7.1	7.5
Blank control 5	6.2	3.0	5.5	34.9	23.3		7.2	7.5
Blank control 6	5.8	3.0	5.5	30.5	20.4		7.2	7.6
Blank controls	Mean			32.7	21.8	/
ANISOLE 150 mg/L (1)	6.5	4.0	5.5	27.3	18.2	16.5	7.2	7.7
ANISOLE 150 mg/L (2)	5.3	2.4	5.5	31.6	21.1	3.4	7.2	7.7
ANISOLE 150 mg/L (3)	5.7	3.3	5.5	26.2	17.5	19.9	7.2	7.7
ANISOLE 150 mg/L (4)	6.4	3.8	5.5	28.4	18.9	13.1	7.2	7.8
ANISOLE 150 mg/L (5)	6.6	3.8	5.5	30.5	20.4	6.7	7.2	7.7
ANISOLE 300 mg/L (1)	6.5	4.0	5.5	27.3	18.2	16.5	7.2	7.8
ANISOLE 300 mg/L (2)	5.4	2.8	5.5	28.4	18.9	13.1	7.2	7.7
ANISOLE 300 mg/L (3)	5.3	2.1	5.5	34.9	23.3	- 6.7	7.2	7.7
ANISOLE 300 mg/L (4)	5.7	3.4	5.5	25.1	16.7	23.2	7.2	7.8
ANISOLE 300 mg/L (5)	6.5	3.9	5.5	28.4	18.9	13.1	7.2	7.8
ANISOLE 600 mg/L (1)	6.4	4.4	5.5	21.8	14.5	33.3	7.2	7.8
ANISOLE 600 mg/L (2)	6.6	4.3	5.5	25.1	16.7	23.2	7.2	7.8
ANISOLE 600 mg/L (3)	5.1	2.7	5.5	26.2	17.5	19.9	7.2	7.6
ANISOLE 600 mg/L (4)	5.7	3.4	5.5	25.1	16.7	23.2	7.2	7.8
ANISOLE 600 mg/L (5)	6.3	3.9	5.5	26.2	17.5	19.9	7.3	7.7

Reference item: 3,5 -dichlorophenol

Identification	Q1 (mg/L)	Q2 (mg/L)	Dt	R mg O2/Lh	Rs mg O2/gh	IT (%)	pH
							T0	T3h
Blank control 1	6.9	3.4	7.0	30.0	20.0	/	7.2	7.6
Blank control 2	6.9	3.3	7.0	30.9	20.6		7.2	7.6
Blank control 3	7.0	3.2	7.0	32.6	21.7		7.2	7.6
Blank controls	Mean			31.1	20.8		/
3,5–DCP 3.13 mg/L	7.8	5.6	7.0	18.9	12.6	39.4	7.2	7.9
3,5–DCP 10.0 mg/L	7.9	6.3	7.0	13.7	9.1	56.1	7.2	7.6
3,5–DCP 32.0 mg/L	8.6	8.3	7.0	2.6	1.7	91.7	7.3	7.4

The EC₅₀3,5-DCP was graphically determined between 6.0 and 7.0 mg/L.

Q1 is the oxygen concentration at the beginning of the selected section of the linear phase (mg/L)

Q2 is the oxygen concentration at the end of the selected section of the linear phase (mg/L)

D_tis the time interval (minutes) between these two measurements

R (mg O₂/Lh) is the oxygen consumption rate or respiration rate.

Rs is the specific respiration rate.

I_T is the percentage inhibition of oxygen consumption for each concentration.

Validity criteria fulfilled:

yes

Remarks:

The blank controls oxygen uptake rate remained above 20 mg O2/g h during all tests and the coefficient of variation did notexceed 30% at the end of tests. EC50(3 hours) of 3,5-dichlorophenol were in the range 2 mg/L and 25 mg/L.

Conclusions:

In the condition of the test, a NOEC of 300 mg/L has been determined for the test item ANISOLE, after a contact time of 3 hours.

Executive summary:

In a reliable study, the inhibitory effect of the test item anisole on the respiration rate of the activated sludge was investigated in a 3 -hour respiration inhibition test according to OECD guideline for Testing of Chemicals, n°209.

The nominal test item concentrations were 150, 300 and 600 mg/L.

In addition, 6 controls and 3 different concentrations of the reference item 3,5-dichlorophenol (3.13; 10.0 and 32.0 mg/L) were tested in parallel.

The criterion measured was the inhibition of the respiration rate of the activated sludge, exposed to different concentrations of the test item, and expressed as a percentage of the control.

From these values, the EC50 (concentration at which respiration rate is 50% of that in the control) was calculated.

The 3h EC50 of the reference item was between 6 and 7 mg/L in this study. Therefore, the criterion for validation of the inoculum is fulfilled.

 

The highest tested concentration of the test item in the definitive study, i.e. 600 mg/L, showed a statistically significant effect. The highest tested concentration without effect observed , i.e. the NOEC, was 300 mg/L.

Description of key information

In an activated sludge respiration inhibition test, the NOEC of anisole has been determined at 300 mg/L. 

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

300 mg/L

Additional information

On reliable study is available for this endpoint. The objective of the study was to assess the effect of the test item anisole on the respiration of activated sewage sludge, over a period of 3 hours.

The criterion measured was the inhibition of the respiration rate of the activated sludge, exposed to different concentrations of the test item, and expressed as a percentage of the control.

From these values, the EC50 (concentration at which respiration rate is 50% of that in the control) was calculated.

The 3h EC50 of the reference item was between 6 and 7 mg/L in this study. Therefore, the criterion for validation of the inoculum is fulfilled.

 

The highest tested concentration of the test item in the definitive study, i.e. 600 mg/L, showed a statistically significant effect. The highest tested concentration without effect observed , i.e. the NOEC, was 300 mg/L.