4'-methoxyacetophenone

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:

2017-05-09 to 2017-05-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

adopted 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Sampling method for measurement of the respiration rate: well-mixed sample of each incubation mixture poured into a 250 mL glass BOD bottle after three hours incubation time (not further aerated)
- Sample storage conditions before analysis: the dissolved oxygen (DO) concentration was measured subsequently after sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the test, 16 mL synthetic sewage feed were made up to 250 mL with deionized water and then 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L (dry weight) were added to this first control vessel. Thereafter, in time intervals of about fifteen minutes (an arbitrary but convenient interval) the procedure was repeated, except that the 16 mL synthetic sewage feed were added to the appropriate amounts of reference item or test item in deionized water to obtain a volume of 250 mL, followed by the addition of 250 mL inoculum. The test item was mixed into the deionized water by intense stirring for 15 minutes at room temperature to finely disperse and dissolve the maximum practical amount of the test item. No emulsifiers or solvents were used. Then, 16 mL of synthetic sewage feed and 250 mL of the activated sludge inoculum were added.

- Controls: Negative control, containing activated sludge and synthetic medium but no test or reference item; positive control, containing 3.2, 10 and 32 mg/L 3,5-dichlorophenol, activated sludge and synthetic medium

- Evidence of undissolved material: The test media for the final test concentrations up to and including 100 mg/L appeared as clear solutions. For the higher loading rates the test item was not completely dissolved under the test conditions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: ARA Birs (Birsfelden / Switzerland); aeration stage
- Preparation of inoculum for exposure: washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in chlorine free tap water, dried thereafter

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not performed.

Hardness:

Not measured.

Test temperature:

20 - 22 ºC.

pH:

- Negative control: 7.3 - 8.0
- Test item: 7.3 - 8.1
- Positive control: 7.3 - 8.6

Dissolved oxygen:

- Negative control: 7.3 - 8.5 mg/L
- Test item: 6.9 - 8.4 mg/L
- Positive control: 7.2 - 8.6 mg/L

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

- Nominal: 10, 32, 100, 320 and 1000 mg/L
- Measured: not performed

Details on test conditions:

TEST SYSTEM
- Test vessel: 2-L beakers
- Fill volume: 500 mL
- Aeration: yes, maintained by magnetic stirrers

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1 for every concentration in positive control, 6 replicates for blank control
- Sludge concentration (weight of dry solids per volume): 3 g dry material per liter
- Nutrients provided for bacteria: peptone, meat extract, urea, NaCl, CaCl2, MgSO4 , K2HPO4
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water, containing less than 1 mg/L DOC (dissolved organic carbon) and chlorine free tap water (for preparation of inoculum)
OTHER TEST CONDITIONS
- Adjustment of pH: prior the test from pH 6.9 to pH 7.5 with a sodium hydroxide solution
- Light intensity: normal laboratory lighting
EFFECT PARAMETERS MEASURED:
TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 10, 32, 100, 320 and 1000 mg/L,

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

403 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CI: 270-660 mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CI: 11-83 mg/L

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

VALIDITY OF THE TEST

- The specific respiration rate of the blank controls was in the range of 37 to 46 mg oxygen per gram dry weight of sludge per hour. (Validity criterion: at least 20 mg O2/gh.)
- The coefficient of variation of oxygen uptake rate in control replicates was 7 %. (Validity criterion: less than 30 %).
- The 3-hour EC50 of the reference item 3,5-dichlorophenol was 9 mg/L. (Validity criterion:
2 to 25 mg/L).

The test is regarded as valid.

DOSE-RESPONSE RELATIONSHIP

A clear concentration effect relationship on the inhibition of the respiration rate was observed.

Results with reference substance (positive control):

- Results with reference substance: valid
- Relevant effect levels: 3-hour EC50 = 9 mg/L (the 95 % confidence limits could not be calculated due to mathematical reasons)

Reported statistics and error estimates:

The percentage inhibition of the respiration was plotted against logarithm of the test substance concentration using suitable software ToxRat® Professional. The NOEC was derived by per-vessel statistics using the Williams t-test (one sided smaller, α = 0.05).
The 3-hour ECx values and their 95 % confidence limits were calculated, as far as possible, by Probit Analysis. The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated by Probit Analysis, too.
All test results were based on nominal concentrations of the test and reference item.

Table 1: Influence of the test item and 3,5-Dichlorophenol (Reference Item) on the Oxygen Consumption of Activated Sludge.

Vessel no.	Nominal concentration of test chemical	Oxygen consumption rate (R)	Specific respiration rate (Rs)	Inhibition		pH values		Oxygenconcentration [mg O2/L]
	[mg/L]	[mg O2 /Lh]	[mg O2 /gh]	%		start*	end*	start*	end*
Control
1	0	66.93	44.62			7.6	8.0	8.5	7.5
2	0	63.87	42.58			7.4	7.9	8.5	7.3
6	0	68.67	45.78			7.3	7.8	8.3	7.3
7	0	68.40	45.60			7.3	8.0	8.4	7.3
23	0	56.07	37.38			7.3	7.9	8.2	7.4
24	0	68.25	45.50			7.3	7.8	8.2	7.3
Mean		65.36	43.58
Standard Deveation		4.89	3.26
Coefficient Variation		7.48%	7.48%
Test item					Mean
8	10	64.80	43.20	0.9	0.4	7.3	8.0	8.4	7.5
9	10	67.80	45.20	-3.7		7.4	7.7	8.3	6.9
10	10	62.64	41.76	4.2		7.3	8.0	8.4	7.6
11	32	52.91	35.27	19.1	13.4	7.3	8.0	8.4	7.5
12	32	61.20	40.80	6.4		7.3	7.9	8.4	7.4
13	32	55.64	37.09	14.9		7.4	7.9	8.3	7.4
14	100	54.00	36.00	17.4	18.9	7.5	7.9	8.3	6.9
15	100	52.50	35.00	19.7		7.4	8.0	8.3	7.5
16	100	52.47	34.98	19.7		7.3	8.0	8.2	7.0
17	320	37.13	24.76	43.2	42.0	7.3	8.0	8.4	8.1
18	320	36.57	24.38	44.1		7.3	8.0	8.2	7.9
19	320	40.05	26.70	38.7		7.4	8.0	8.2	7.6
20	1000	17.95	11.96	72.5	72.6	7.4	8.0	8.2	8.3
21	1000	17.51	11.67	73.2		7.4	8.1	8.2	8.4
22	1000	18.27	12.18	72.0		7.3	8.1	8.2	8.1
Reference item
3	3.2	55.65	37.10	14.9		7.5	7.9	8.6	7.2
4	10	28.20	18.80	56.9		7.3	7.9	8.5	7.8
5	32	8.62	5.75	86.8		7.5	8.0	8.6	8.2

*: Start and end of the 3-hour incubation period.

- % inhibition: Negative value due to increased oxygen consumption rate relative to the control.

n.a.: Not applicable

Table 2: The 3-hour NOEC and ECX-values and their 95 % confidence limits

Endpoint after 3 Hours	[mg/L]
NOEC	10
EC10 95 % confidence limits	43 11 – 83
EC20 95 % confidence limits	93 37 – 152
EC50 95 % confidence limits	403 270 - 660
EC80	>1000

Validity criteria fulfilled:

yes

Conclusions:

The test item had a clear concentration-effect relationship on the inhibition of the respiration rate of activated sludge after the incubation period of three hours. The 3-hour EC50 value was 403 mg/L.

Executive summary:

The inhibitory effect of the test item on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated under GLP-conditions in a 3-hour respiration inhibition test according to the OECD Guidelines for Testing of Chemicals, No. 209, adopted 2010 and to the Commission Regulation (EU) No. 2016/266, C.11. (2015). Based on the results of range-finding test a definitive test with the following nominal test item concentrations was performed: 10, 32, 100, 320 and 1000 mg/L, with three replicates per concentration. As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates). Six blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item, were included in the definitive test. The vessels were prepared in the following order: two blank controls, the reference item group (with ascending concentrations), two further blank controls, the test item group (with ascending concentrations) and finally again two blank control vessels. A clear concentration-effect relationship on the inhibition of the respiration rate was observed. The 3-hour NOEC was 10 mg/L and the EC50 value resulted in 403 mg/L (95% CI: 270 - 660).

The results of the blank and positive control treatments confirmed the suitability of the activated sludge and the method used. All the validity criteria were fulfilled.

Description of key information

The toxicity of the test item to microorganisms was determined in a test according to OECD guideline 209 and Commission Regulation (EU) No. 2016/266, C.11.

The test determined an EC50 value of 403 mg/L and a NOEC of 10 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

403 mg/L

EC10 or NOEC for microorganisms:

43 mg/L

Additional information

The inhibitory effect of the test item on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated under GLP-conditions in a 3-hour respiration inhibition test according to the OECD Guidelines for Testing of Chemicals, No. 209, adopted 2010 and to the Commission Regulation (EU) No. 2016/266, C.11. (2015). Based on the results of range-finding test a definitive test with the following nominal test item concentrations was performed: 10, 32, 100, 320 and 1000 mg/L, with three replicates per concentration. As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates). Six blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item, were included in the definitive test. The vessels were prepared in the following order: two blank controls, the reference item group (with ascending concentrations), two further blank controls, the test item group (with ascending concentrations) and finally again two blank control vessels. A clear concentration-effect relationship on the inhibition of the respiration rate was observed. The 3-hour NOEC was 10 mg/L and the EC50 value resulted in 403 mg/L (95% CI: 270 - 660). The results of the blank and positive control treatments confirmed the suitability of the activated sludge and the method used. All validity criteria were fulfilled.