2-propanol and 2-butanol production, distn. residues

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:

8 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully documented study under GLP and according to international guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

The temperature in the pre-test was in a range of 21.2 – 23.1 °C instead of 20 ±2 °C. The oxygen uptake rate of the activated sludge was lower than stated in the guideline. These deviations were stated as uncritical.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The temperature in the pre-test was in a range of 21.2 – 23.1 °C instead of 20 ±2 °C. The oxygen uptake rate of the activated sludge was lower than stated in the guideline. These deviations were stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because of high volatility, the nominal load of test item was directly injected into the vessels with a suitable syringe. Then the vessels were immediately closed. The injected volume based on a mean density of test item of 0.75 g/mL.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: Upon arrival in the test facility, the sludge was washed with tap water three times, then centrifuged and resuspended in tap water.
- Pretreatment: No
- Initial biomass concentration: 3.40 g/L, corresponding to a concentration of 1.70 g/L suspended solids in the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21.2 – 23.1 °C measured in vessels

pH:

pH in the activated sludge was measured as 7.3. pH at the end of the test: see table in "any other information on results".

Nominal and measured concentrations:

Nominal concentrations of test item (mg/L): 10000, 3200, 1000, 320, 100, 32, 10, 3.2, 1

Details on test conditions:

TEST SYSTEM
- Test vessel: 2000 mL Schott flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: material: glass, fill volume: 500 mL
- Aeration: none, as the test item is volatile; in deviation from the guideline, the treatments were not aerated but stirred strongly to ensure oxygen maintenance
- No. of vessels per concentration (replicates): one replicate/treatment (positive control), one replicate/treatment (test item)
- No. of vessels per control (replicates): two replicates before and two after measuring positive control and test item, respectively


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: <1 mg/L
- Particulate matter: no data
- Metals:
antimony < 0.001 mg/L
arsenic < 0.001 mg/L
lead < 0.001 mg/L
cadmium < 0.0005 mg/L
chromium < 0.002 mg/L
copper 0.001 mg/L
nickel < 0.001 mg/L
mercury < 0.0001 mg/L
selenium < 0.001 mg/L
bor 0.017 mg/L

- Pesticides: < limit of detection
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: 189 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: no data
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen concentration

Reference substance (positive control):

yes

Remarks:

3.5-Dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 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: EC20

Effect conc.:

400 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% confid. interv.: 120 - 970 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

2 400 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% confid. interv.: 1000 - 7300 mg/L

Results with reference substance (positive control):

The calculated EC50 value for the positive control 3,5-dichlorophenole is 10 mg/L which lies in the recommended range of 2 – 25 mg/L.

Reported statistics and error estimates:

The estimation of the EC50 was accomplished using the software OriginTM. The data were evaluated using linear fit on a probability-logarithmic scale.

Study Performance – Main Study

Time Schedule

The time schedule is presented in the following table.

Time Schedule

No.	Start of Test	Begin of O2-Measurement	End of O2-Measurement
1	10:25	13:25:36	13:29:06
2	10:30	13:29:16	13:35:11
3	10:35	13:35:46	13:39:47
4	10:40	13:40:17	13:45:05
5	10:45	13:45:40	13:50:04
6	10:50	13:50:42	13:55:00
7	10:55	13:55:18	13:59:58
8	11:00	14:00:36	14:04:59
18	11:50	14:50:11	14:54:46
19	11:55	14:54:24	14:59:59
20	12:00	15:00:29	15:04:52
21	12:05	15:05:17	15:09:23
22	12:10	15:11:11	15:14:53
23	12:15	15:15:11	15:19:59
24	12:20	15:20:42	15:24:59
25	12:25	15:25:22	15:29:40
26	12:30	15:30:35	15:34:48
27	12:35	15:35:10	15:39:40
28	12:40	15:40:30	15:44:40
29	12:45	15:45:10	15:49:48
30	12:50	15:50:11	15:55:28

Annotation: Other test item was measured in vessels 9 – 17.

 

1.1.3      O₂-Consumption, Inhibition

The values of the O₂ consumption (which is a measure for the viability of the bacteria) of controls, test and positive control and the calculated inhibition are presented in the following table. The measured pH at the end of the test is also stated.

 O₂-Consumption, Inhibition

Vessel No.	Content	Concentration in mg/L	O2consumption in mg/(L*min.)	O2consumption in mg/(L*h)	Inhibition in %	pH
1	Control	0	0.4050	24.302	--	7.7
2	Control	0	0.4064	24.382	--	7.7
3	Positive Control	5	0.3143	18.858	28.6	7.5
4	Positive Control	10	0.2029	12.174	53.9	7.5
5	Positive Control	20	0.1205	7.233	72.6	7.5
6	Positive Control	40	0.0922	5.532	79.0	7.6
7	Control	0	0.4177	25.060	--	7.7
8	Control	0	0.4603	27.615	--	7.7
18	Control	0	0.4632	27.789	--	7.7
19	Control	0	0.4454	26.724	--	7.7
20	Test Item	10000	0.0982	5.894	77.7	7.6
21	Test Item	3200	0.2103	12.619	52.2	7.8
22	Test Item	1000	0.3129	18.776	28.9	7.8
23	Test Item	320	0.4425	26.553	-0.6	7.7
24	Test Item	100	0.4035	24.211	8.3	7.7
25	Test Item	32	0.4616	27.696	-4.9	7.7
26	Test Item	10	0.4591	27.545	-4.4	7.7
27	Test Item	3.2	0.4351	26.106	1.1	7.8
28	Test Item	1	0.4579	27.475	-4.1	7.7
29	Control	0	0.4420	26.517	--	7.8
30	Control	0	0.4797	28.780	--	7.8

 

Annotation: Other test item was measured in vessels 9 – 17.

The inhibition in treatment 320 mg/L was unexpectedly low. This value was considered as not valid and therefore not used for evaluation of the results.

In treatments below 100 mg/L, no inhibition could be observed. Therefore, treatments 10; 3.2 and 1 mg/L were not required for evaluation of the results.

Validity criteria fulfilled:

yes

Remarks:

Oxygen uptake rate was too low for the blank control.

Conclusions:

The following results for the test item "2-propanol and 2-butanol production, distn. residues" were determined:
3h NOEC = 32 mg/L
3h EC20 = 400 mg/L (95% confid. interv.: 120 - 970 mg/L)
3h EC50 = 2400 mg/L (95% confid. interv.: 1000 - 7300 mg/L)

Executive summary:

The Inhibition of the Respiration of Activated Sludge when exposed to "2-propanol and 2-butanol production, distn. residues" was determined according to OECD 209 resp. EU C.11

One valid experiment was performed and in the main study nine concentrations, ranging from 10000 to 1 mg/L (nominal concentrations) were tested. Due to the volatility of the test item, the study was performed in a closed system without aeration.

The dry matter of the activated sludge was determined as 3.4 g suspended solids/L, giving a concentration of 1.7 g suspended solids/L in the test. 3.5-Dichloro-phenole was used as positive control and four concentrations were tested; an EC50 of 10 mg/L (95% confidence interval: 3 – 19 mg/L) was determined, which lies within the demanded range of 2 – 25 mg/L.

The following results for the test item "2-propanol and 2-butanol production, distn. residues" were determined:

3h NOEC = 32 mg/L

3h EC20 = 400 mg/L (95% confid. interv.: 120 - 970 mg/L)

3h EC50 = 2400 mg/L (95% confid. interv.: 1000 - 7300 mg/L)

Description of key information

The following results for the test item "2-propanol and 2-butanol production, distn. residues" were determined:

3h NOEC = 32 mg/L

3h EC50 = 2400 mg/L (95% confid. interv.: 1000 - 7300 mg/L)

Key value for chemical safety assessment

EC50 for microorganisms:

2 400 mg/L

EC10 or NOEC for microorganisms:

32 mg/L

Additional information

The EC20 value, EC50 value and the NOEC for the inhibition of the respiration in activated sludge of "2-propanol and 2-butanol production, distn. residues" was measured according to OECD 209, respectively EU C.11 guideline under GLP. The following results were obtained:

3h NOEC = 32 mg/L

3h EC20 = 400 mg/L (95% confid. interv.: 120 - 970 mg/L)

3h EC50 = 2400 mg/L (95% confid. interv.: 1000 - 7300 mg/L)

These values are used for the calculation of the PNEC for microorganisms in sewage treatment plants (PNEC STP).