A mixture of: propan-2-one-O,O'-(methoxymethylsilandiyl)dioxime; propan-2-one-O-(dimethoxymethylsilyl)oxime; propan-2-one-O,O',O''-(methylsilantriyl)trioxime

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:

October 5, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD Guideline 209, with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

- Concentrations: 7.6, 19.2, 48.0, 120.0 and 300.0 mg of the test substance/L
- Sampling method: The appropriate amounts of the test substance were added to the sludge samples at the beginning of the incubation
- Sample storage conditions before analysis: to assure that the non altered test substance was tested as far as possible, no stock solution was prepared.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was poorly soluble in water and hydrolyses and polymerises quickly when in contact with water. To assure that the non altered test substance was tested as far as possible, no stock solution/suspension was prepared but the appropriate amounts of the test substance were added to the sludge samples at the beginning of the incubation. This treatment is in accordance with the EPA-Guideline OPPTS 850.6800.
- Controls: Positive control: A stock solution of 3,5-dichloraphenol (nominally 0.5 g/L) was prepared by dissolving 50 mg of the substance in 100 mL of deionised water. Three concentrations, nominally from 5 to 30 mg/L. No NaOH was used to increase the solubility of dichloraphenol, therefore no adjustment of the pH was necessary.
- Controls: Negative control

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: A sample of activated sludge was collected one day before the test. The container for transportation was only about 2/3 filled in order to maintain the contact with air. At arrival in the laboratory (less than 1 hour after sample collection), the supernatant over the sludge was decanted, the sludge was diluted with tap water and aerated by means of compressed air. 50 mL of synthetic sewage concentrate were added per litre each day. On the day of the test the sludge was allowed to sediment and the supernatant was decanted.
- Initial biomass concentration: Concentration of suspended solids was adjusted to 4 g/L with tap water
- Other: The inoculum was aerated until use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

18 ºC

pH:

8.31-8.42

Nominal and measured concentrations:

Nominal concentrations test item: 0 (control), 7.7, 19.2, 48, 120, and 300 mg/mL
Nominal concentration positive control (3,5-dichlorophenol): 5, 12.2 and 30 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: Glass beakers, nominal volume of 1000 mL
- Aeration: flow rate of 0.7 L/min using oil free compressed air and Pasteur-pipette as aeration device.
- No. of organisms per vessel: 4 g/L
- No. of vessels per concentration (replicates): 1 (5 concentration)
- No. of vessels per positive control (replicates): 3
- No. of vessels per negative control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
All incubation mixtures were prepared by mixing 200 mL of microbial inoculum and 200 mL of synthetic sewage solution with 100 mL of tap water and/or the appropriate amount of the test or control substance in glass beakers with a nominal volume of 1000 mL.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes
- Photoperiod:
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The oxygen concentration was determined with a DO meter "OXI Level 2" and an oxygen electrode. Respiration rates (in mg O2.L-1.h-1) were determined in aliquots of the incubation mixtures. Bottles with narrow neck and a volume of about 100 mL were filled with the sample and positioned on a magnetic stirrer. Then the oxygen electrode (equipped with a magnetic stirring wheel) was inserted in a way that no air remained in the bottle and that the electrode sealed the bottle neck to avoid contact of the sample with the atmosphere. The decline of oxygen concentration was measured by recording the concentrations of oxygen at various times. pH was determined with a pH-meter in the standardised microbial inoculum before the test and in each sample at the end of the incubation and aeration. The test was performed in a temperature controlled room with automatic temperature registration. The temperature inside the samples was measured with a mercury thermometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: The highest concentration of the test substance corresponds to the scheme given in the guidelines. The spacing between the concentrations of 2.5 is within the permitted range of 'not more than 3.2'. The amount of concentrations tested is in agreement with the guidelines which require 'at least 5'. The concentrations of 3,5-dichlorophenol were set around the expected EC50 of 5 to 30 mg/L.
- Test concentrations: Test item: 0 (control), 7.7, 19.2, 48, 120 and 300 mg/L. Positive control: 5, 12.2 and 30 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: (95% CL: 35-125 mg/L)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 300 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: EC80

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:

The test substance inhibited the respiration of the activated sludge only at higher concentrations and only slightly. The highest obtained inhibition was 27.2 % at 300 mg/L.
Usually occurring foam formation during incubation was suppressed by the test substance.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The ECso of 3,5-diehlorophenol was in the accepted range of 5 to 30 mg/L. The actual values of the EC20, EC50 and EC80 for 3,5-diehlorophenol were: EC20= 6.2 mg/L; EC50= 12.8 mg/L; EC80= 26.3 mg/L

Reported statistics and error estimates:

For each sample a linear regression was calculated from the linear part of the graph time versus oxygen content. From the oxygen content at the begin and at the end of this regression line the respiration rates (expressed in mg O2.L-1.h-1) were calculated.
The concentrations of the test substance around 50 % inhibition (if such data exist) and the concentrations of the positive control are converted to log10. These values and the corresponding percentage of inhibition are subjected to linear regression analysis. These analyses are used to obtain the regression coefficients and the respective EC20, EC50 and EC80 values.

Respiration rate and inhibition of the samples

Negative values indicate enhanced respiration compared to the control cultures.

sample	final concentr. (mg/L)	linear range (minutes)	mg O2/ L at begin	mg O2/ L at end	respiration rate (mg O2. L^-1. h^-1)	per cent respiration inhibition
neg. control 1	0.0	8.00	7.00	2.67	32.48	3.53
test substance 1	7.6	9.48	7.23	2.59	29.37	12.76
test substance 2	19.2	8.48	7.11	2.72	31.06	7.73
test substance 3	48.0	8.44	6.64	2.80	27.30	18.91
test substance 4	120.0	8.60	7.18	3.54	25.40	24.56
test substance 5	300.0	8.52	7.32	3.84	24.51	27.20
neg. control 2	0.0	8.04	7.06	2.39	34.85	-3.53
DCP 1	4.9	8.40	6.80	2.38	31.57	6.21
DCP 2	11.9	9.52	7.68	5.25	15.32	54.50
DCP 3	29.2	7.08	8.02	8.02	6.53	80.62

DCP: 3,5-dichlorophenol

Validity criteria fulfilled:

yes

Remarks:

(The respiration rates of the two negative control samples were within 15 % of each other; the EC50 of 3,5-diehlorophenol was in the accepted range of 5 to 30 mg/L)

Conclusions:

EC50 value (based on respiration inhibition) on activated sludge at 3 hours exposure period was > 300 mg/l.

Executive summary:

The study was performed to estimate possible effects of WASOX-MMAC2 on aerobic microbial sewage treatment plants. The test was performed according to the OECD Guideline 209, using activated sludge from a sewage treatment plant treating predominantly domestic sewage. Five concentrations of the test substance (7.6, 19.2, 48.0, 120.0 and 300.0 mg/L) were tested versus two negative controls (tap water). As positive control substance 3,5-dichlorophenol was used and tested in three concentrations (4.9, 11.9 and 29.2 mg/L). The microbial inoculum was a preparation of activated sludge collected one day before the test. The concentration of the microbial inoculum was adjusted to 4 g of dry weight per litre which gives a final amount of 1.6 g dry weight per litre. The respiration rates of the two negative control samples were within 15 % of each other (± 3.5 ) and the EC50 of 3,5-dichloraphenol was in the accepted range of 5 to 30 mg/L (12.8 mg/L) . The test substance inhibited the respiration of the activated sludge only at higher concentrations and only slightly. The highest obtained inhibition was 27.2 %. The obtained EC-3h for Wasox-MMAC2 were: EC20: 80 mg/L; EC50: > 300 mg/L and EC80: > 300 mg/L.

Description of key information

Key study: OECD 209 and GLP. EC50 value (based on respiration inhibition) on activated sludge at 3 hours exposure period was > 300 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:

300 mg/L

Additional information

Key study: A study to estimate possible effects of WASOX-MMAC2 on aerobic microbial sewage treatment plants was performed according to OECD Guideline 209 and GLP. EC50 value (based on respiration inhibition) on activated sludge (predominately domestic) at 3 hours exposure period was determined to be > 300 mg/l.