Ethanol, 2-(ethenylthio)-

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:

2016-05-17 to 2016-05-31

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

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 230-2015
- Expiration date of the lot/batch: 17 Nov 2017

Analytical monitoring:

no

Details on sampling:

No analytical analysis was done.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Test substance preparations: The test substance was added in the required amounts according to the test concentrations directly to the test vessels with 234 mL deionized water. 16 mL synthetic medium were added.

- Reference substance preparations: 1000.0 mg of the reference substance was added to about 800 mL of deionized water and stirred at room temperature until the reference substance was completely dissolved. The pH value of the stock solution was measured and adjusted to 7.1 with 1 M sodium hydroxide solution. Following this the stock solution was made up to 1L with deionized water. The stock solution appeared yellowish and no undissolved reference substance was visible. Aliquots of the reference substance stock solution were dosed to the test vessels and made up with deionized water to a volume of 234 mL. 16 mL synthetic medium were added.

- Control preparations: To prepare the blank control assays 234 mL of deionized water and 16 mL synthetic medium were mixed.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: aeration tank of the wastewater treatment plant of Mannheim, Germany
- Method of cultivation: No cultivation on site. The sludge was used on the day after collection.
- Preparation of inoculum for exposure: After arrival of the activated sludge suspension in the test facility the suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L dw.
- Pretreatment: No pretreatment was done.
- Initial biomass concentration: 3 g/L dw

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19.0 – 19.9 °C

pH:

7.5

Dissolved oxygen:

start of measurement: > 7 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

1000, 500, 250, 125, 62.5 mg/L as nominal concentration without correction of purity and blank controls
100, 10, 1 mg/L as nominal concentration based on reference substance

Details on test conditions:

TEST SYSTEM
- Test vessel: Glas-beakers (nominal volume 1L)
- Fill volume: 500 mL
- Aeration: Oxygen concentration during aeration: > 2 mg/L; Oxygen concentration immediately before measurement: > 7 mg/L; Duration of the measurement of oxygen consumption: About 8 - 10 min
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per reference control (replicates): 2
- Sludge concentration: 1.5 g/L dw
- Nutrients provided for bacteria: None
- Nitrification inhibitor used: None

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: An adjustment was not necessary.

EFFECT PARAMETERS MEASURED:
The consumption rate (mg O2/L*x minutes) of each test concentration and the blank controls were determined after 3 hours of exposure. The consumption rates (mg O2/L*h) were used to calculate the inhibition effects at particular test concentrations compared to the mean consumption rate of all controls.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ≤ 3.2
- Range finding study: Not conducted.
- Test concentrations selection: According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor of ≤ 3.2 should be used, preferably encompassing the range from 10 – 80 % inhibition.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

< 62.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

210 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations that might cause a difference between measured and nominal values: No observations reported.
- Effect concentrations exceeding solubility of substance in test medium: No.
- Adsorption: No.
- Blank controls oxygen uptake rate: 25 mg/g*h
- Coefficient of variation of oxygen uptake rate in control replicates: 2.2 % O2 consumption
- Outliers: The inhibition in the test assays TS4 – TS6 (test concentration 125 mg/L) was about 50 % and higher than the test concentration 250, 500 and 1000 mg/L. This is not possible. Therefore the test assays TS4 – TS6 were set as outliers. The statistical evaluation were done without the oxygen consumption values of the the test assays TS4 – TS6.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- Relevant effect levels: The EC50 of 3,5-dichlorophenol was 7.0 mg/L (OECD 209 range: 2-25 mg/L after 3 hours of incubation)

Reported statistics and error estimates:

The consumption rates were used for the determination of the ECx by the probit method based on Finney with the software TOXRAT Professional 2.10. The effect concentrations were given with an accuracy of 2 significant digits.

Measured data of oxygen content of blank control and reference substance assays at the start and end of the evaluation time

Assay idenification	Blank Control						Reference Substance
Replicate	1	2	3	4	5	6	1	2	3	4	5	6
O2 conc. Start [mg/L]	8.4	7.5	7.5	8.0	8.0	8.0	7.6	8.0	9.0	8.9	8.9	9.1
O2 conc. End [mg/L]	4.5	3.9	3.9	4.4	4.4	4.5	4.0	4.5	7.7	7.5	8.6	8.8
O2 consumption rate [mg/L]	3.9	3.6	3.6	3.6	3.6	3.5	3.6	3.5	1.3	1.4	0.3	0.3
O2 consumption in minutes	6.2	6.0	5.9	5.9	6.0	5.9	5.9	6.0	6.0	6.0	6.0	5.9
O2 consumption rate [mg/L×h], RT	38	36	37	37	36	36	37	35	13	14	3	3
Specific O2 consumption rate [mg/g×h]	25	24	25	25	24	24	25	23	9	9	2	2
Calculation of inhibition respiration [%], IT	-						-1	5	65	62	92	92

The respiration rate was calculated in mg O2/L×h. The oxygen consumption was

measured over a period of 8-10 minutes. 5.9 - 6.2 minutes from the plotted track within a linear range of the graph was used for the determination of the respiration rate.

 

Measured data of oxygen content of the test assays at the begin and end of the

evaluation time

Assay idenification	Test Assay
Replicate	1	2	3	4*	5*	6*	7	8	9	10	11	12	13	14	15
O2 conc. Start [mg/L]	8.0	7.8	7.5	8.5	8.5	8.2	7.5	7.5	7.7	7.9	7.5	8.0	7.7	7.5	7.3
O2 conc. End [mg/L]	5.0	4.5	4.4	6.5	6.6	6.3	4.5	4.6	4.5	5.3	4.8	5.4	5.2	4.9	4.7
O2 consumption rate [mg/L]	6.2	6.0	5.9	6.5	6.1	6.1	6.0	6.0	6.1	6.1	6.1	6.0	6.0	6.0	6.0
O2 consumption in minutes	3.0	3.3	3.1	2.0	1.9	1.9	3.0	2.9	3.2	2.6	2.7	2.6	2.5	2.6	2.6
O2 consumption rate [mg/L×h], RT	29	33	32	18	19	19	30	29	31	26	27	26	25	26	26
Specific O2 consumption rate [mg/g×h]	19	22	21	12	13	13	20	19	21	17	18	17	17	17	17
Calculation of inhibition respiration [%], IT	21	10	13	51	48	48	18	21	15	29	26	29	32	29	29

The respiration rate was calculated in mg O2/L×h. The oxygen consumption was

measured over a period of 8-10 minutes. 6 - 6.1 minutes from the plotted track within a linear range of the graph was used for the determination of the respiration rate.

 

Equation1: IT=(1-(RT/RTB)*100

For calculating the inhibition the results of the fractions in eq.1 were rounded to

two decimal places.

 

IT= inhibition of the total respiration [%]

RT = oxygen consumption in the test assay [mg O2/L×h]

RTB= mean value oxygen consumption of all blank controls [mg O2/L×h]

Validity criteria fulfilled:

yes

Description of key information

The toxic effects of the test substance on the oxygen consumption rate of aerobic microorganisms (activated sludge) after short-term exposure of 3 h were determined according to the OECD 209 (2010). The EC50 was determined to be > 1000 mg/L (nominal), the EC20 was determinded to be 210mg/L (nominal)

and the EC10 was determined to be < 62.5 mg/L(nominal).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

The study according to OECD 209 (2010) was conducted to determine the toxic effects of the test substance on the oxygen consumption rate of aerobic microorganisms (activated sludge). The exposure duration was 3 h and the test concentrations were 1000, 500, 250, 125, 62.5 mg/L (nominal, without correction of the purity). No measurements of the test concentrations were done. The activated sludge was derived from a municipal waste treatment plant. The sludge concentration was 1.5 g/L and a synthetic medium according to the guideline was used. A reference substance (3,5-dichlorphenol) was used as positive control. The EC50 value of the reference substance 3,5-dichlorphenol was determined to be 7.0 mg/L, which was within the acceptable limits (2 to 25 mg/L), as specified in OECD 209 Guideline. In addition the coefficient of variation of the six replicates of blank control was determined to be 2.2 % O2 consumption. The mean oxygen uptake of the blank controls was determined to be 25 mg/g*h. Based on these results it can be stated that the test was valid. For the test substance the EC50 was determined to be > 1000 mg/L (nominal), the EC20 was determinded to be 210mg/L (nominal) and the EC10 was determined to be < 62.5 mg/L (nominal).