Reaction mass of Benzenepropanal, 4-ethyl-α,α-dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal

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:

10 May 2016 to 11 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This is a range-finding study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test item was dispersed directly in water. Nominal amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures of between 20 °C and 21 °C, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).

Test organisms (species):

activated sludge

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: A mixed population of activated sewage sludge micro-organisms was obtained on 11 May 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Preparation of inoculum: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.0 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L.
- Preparation of test system: At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. Finally two further control vessels were prepared.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 - 21 °C

pH:

7.2 - 7.9 (after pH adjustment)

Dissolved oxygen:

- After 30 minutes contact time: 5.8 - 5.9 (test item vessels; the dissolved oxygen in all vessels (including controls) were above 60 % of the dissolved oxygen saturation level of 8.9 mg O2/L).

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 10, 100 and 1000 mg/L
- Test concentrations were not analytically verified.

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask, final volume 500 mL
- Measurement vessel: Biological Oxygen Demand (BOD) bottle, 250 mL, darkened glass
- No. of vessels per concentration (replicates): 3 replicates for test concentration 1000 mg/L; 1 replicate for test concentrations 10 and 100 mg/L, respectively.
- No. of vessels per control (replicates): 4
- Sludge concentration (weight of dry solids per volume): 3.0 g/L

TEST WATER
The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC). Synthetic sewage was added to each test vessel to act as a respiratory substrate. See 'Any other information on materials and methods incl. tables' for its composition.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0.
- Lighting: normal laboratory lighting

EFFECT PARAMETERS MEASURED
- Respiration rate: 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 7 mg O2/L and 2 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 low oxygen consumption, the rate was determined over an approximate 10 minute period.
- Oxygen concentration: The oxygen concentrations in all vessels were measured after 30 minutes contact time.
- Observations: Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.
- pH measurements: The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 - < 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 10 - < 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

No statistically significantly toxic effects were shown at the test concentration of 10 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentrations of 100 and 1000 mg/L.

Results with reference substance (positive control):

The 3-h EC50 value for the reference substance was determined to be 8.7 mg/L (95% C.L.: 6.9 to 11 mg/L).

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line. 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949). One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal
and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data for the range-finding test after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups to see where the NOEC lies. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table: oxygen consumption rates and percentage inhibition values after 3 hours contact time

Nominal concentration (mg/L)		Initial O2 reading (mg O2/L)	Measurement period (minutes)	Final O2 reading (mg O2/L)	O2 consumption rates (mg O2/L/hour)	% inhibition
Control	R1	5.5	5	2.2	38.40	-
	R2	5.0	5	1.9	37.20	-
	R3	5.1	5	1.9	38.40	-
	R4	5.5	6	1.9	36.00	-
Test Item	10	6.2	6	2.4	38.00	[1]
	100	5.4	7	2.0	29.14	22
	1000 R1	7.7	10	6.7	6.00	84
	1000 R2	8.5	10	7.7	4.80	87
	1000 R3	8.1	10	7.2	5.40	86
3,5-dichlorophenol	3.2	5.8	7	2.2	30.86	18
	10	7.9	10	5.5	14.40	62
	32	8.2	10	7.1	6.60	8.2

[Increase in respiration rate as compared to controls] 

R1 - R4 = replicates 1 to 4

Validity criteria fulfilled:

yes

Conclusions:

The 3-h EC50 value for microorganisms in activated sludge is between 100 and 1000 mg/L.

Executive summary:

The toxicity of the test substance to microorganisms was investigated in a range-finding activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria. In this study activated sludge from predominantly domestic sewage was exposed to nominal test substance concentrations of 0 (control), 10, 100 and 1000 mg/L for 3 hours. Test concentrations were not analytically verified. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol. No inhibition was observed at 10 mg/L, whereas slight negative inhibition was determined at 100 mg/L (22%). At 1000 mg/L, the respiration rate was significantly inhibited (86%). Based on these results, the 3-h EC50 value was determined to be between 100 and 1000 mg/L and the 3-h EC10 between 10 - 100 mg/L, respectively. A conservative value of 10 mg/l will be used for risk assessment purposes.

Description of key information

The toxicity of the test substance to microorganisms was investigated in a range-finding activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria. In this study activated sludge from predominantly domestic sewage was exposed to nominal test substance concentrations of 0 (control), 10, 100 and 1000 mg/L for 3 hours. Test concentrations were not analytically verified. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol. No inhibition was observed at 10 mg/L, whereas slight negative inhibition was determined at 100 mg/L (22%). At 1000 mg/L, the respiration rate was significantly inhibited (86%). Based on these results, the 3-h EC50 value was determined to be between 100 and 1000 mg/L and the 3-h EC10 between 10 - 100 mg/L, respectively. A conservative value of 10 mg/l will be used for risk assessment purposes.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

EC10 or NOEC for microorganisms:

10 mg/L