Propionamide

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:

2020-08-26 to 2020-11-23

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

Version / remarks:

adopted July 22, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Version / remarks:

2007

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Appropriate amounts of test item were directly weight into the test vessels and test water was added. The composition was stirred intensively.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: municipal sewage treatment plant Bensheim, Germany

- Preparation of inoculum for exposure: The coarse particles were removed by settling for a short period (15 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed with 50 mL synthetic sewage feed per litre and kept aerated at room temperature overnight. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gives a concentration of 1.5 g/L suspended solids in the test medium. The pH of the activated sludge inoculum was 6.7 and therefore no adjustment necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

6.7 - 7.6

Dissolved oxygen:

6.9 – 8.4 mg O2/L

Nominal and measured concentrations:

Nominal conc.: 10, 32, 100, 320 and 1000 mg/L
Measured conc.: not measured

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 300 mL volume
- Aeration: Compressed air (1.017 litre per minute)
- No. of vessels per concentration: 5
- No. of vessels per control: 6
- No. of vessels per abiotic control: 3
- Sludge concentration: 3 g/L (based on dry weight)
- Nutrients provided for bacteria: synthetic sewage
- Nitrification inhibitor used: N-allylthiourea

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: activated sludge respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: 10, 32, 100, 320 and 1000 mg test item/L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

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

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Details on results:

3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Heterotrophic Respiration: No sensible ECx values could be determined. The NOEC was determined to be ≥ 1000/L.

3-hour EC10, EC20 and 3-hour EC50 and NOEC based on Nitrification Respiration: No sensible ECx values could be determined. The NOEC was determined to be < 10 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:
3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol based on Total Respiration: EC50: 2.6 mg/L (95 % confidence limits: 1.1 – 6.3 mg/L)
3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol based on Heterotrophic Respiration: EC50: 11.7 mg/L (95 % confidence limits: 3.0 – 45.0 mg/L)
3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol based on Nitrification Respiration: EC50: 0.4 mg/L (95 % confidence limits: 0.0 – 4.6 mg/L)

Validity Criteria of the Study

Inoculum Control: The oxygen uptake in the blank controls was 24.2 mg/g/h and therefore clearly above 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour. The respiration rates of the six controls differed not more than 30 %. The value was 5.6 % for the total respiration and 23.6 % for the heterotrophic respiration. The validity criterion was fulfilled.

Reference Item (Total Respiration): The 3 hour EC₅₀ of the reference item 3,5-dichlorophenol for the total respiration was 2.6 mg/L and therefore in the range of 2 to 25 mg/L for the used activated sludge batch.

Reference Item (Heterotrophic Respiration): The 3 hour EC₅₀ of the reference item 3,5-dichlorophenol for the heterotrophic respiration without nitrification was 11.7 mg/L and therefore also in the range of 5 to 40 mg/L for the used activated sludge batch.

Reference Item (Respiration based on Nitrification): The 3 hour EC₅₀ of the reference item 3,5-dichlorophenol for the oxygen uptake due to nitrification was 0.4 mg/L and therefore in the range of 0.1 to 10 mg/L for the used activated sludge batch.

 

Table 1.: Influence of the test item and the Reference Item 3,5-Dichlorophenol on the Inhibition of the Total Respiration Rate of Aerobic Waste Water Microorganisms after 3 Hours of Exposure

Flask No.	Treatment	Conc. [mg/L]	Respiration Rate [mean]^a [mg O2/g*h]	Standard deviation^a	Inhibition [mean]^a [%]	Significant effect compared to control^b
1, 2, 18, 19, 45, 46	control	---	24.2	1.4	---	---
3-7	3.5-DCP	1	15.0	0.6	38.2	---
8-12	3.5-DCP	4	12.0	0.6	50.5	---
13-17	3.5-DCP	16	4.2	0.2	82.5	---
20-24	Test item	10	21.4	1.2	11.5	*
25-29	Test item	32	21.7	0.4	10.5	*
30-34	Test item	100	22.3	1.1	7.9	*
35-39	Test item	320	22.6	1.1	6.5	*
40-44	Test item	1000	18.7	1.5	22.6	*
Coefficient of Variation of control: 5.6 %

^a: mean value of 5 replicates, 6 replicates in case of control

^b: significance according to Williams Multiple Sequential t-test, one sided smaller, a = 0.05 (* = significant; n. s.: not significant)

Inhibition [mean] % = 100-(Respiration Rate [mean] [mg O₂/g*h]*100 / mean value respiration rate in controls)

 

Table 2.: Influence of the test item and the Reference Item 3,5-Dichlorophenol on the Inhibition of the Heterotrophic Respiration Rate of Aerobic Waste Water Microorganisms after 3 Hours of Exposure

Flask No.	Treatment	Conc. [mg/L]	Respiration Rate [mean]^a,b [mg O2/g*h]	Standard Deviation^a'b	Inhibition [mean]^a, b [%]	Significant effect compared to control^c
47, 48, 64, 65, 91, 92	control	---	14.5	3.4	---	---
49-53	3.5-DCP	1	12.2	0.2	15.4	---
54-58	3.5-DCP	4	11.6	0.6	19.8	---
59-63	3.5-DCP	16	4.5	0.2	69.0	---
66-70 ^b	Test item	10	21.0	2.1	-45.2	n.s.
71-75	Test item	32	20.0	2.1	-38.2	n.s.
76-80	Test item	100	20.7	1.4	-43.2	n.s.
81-85	Test item	320	22.0	2.3	-52.4	n.s.
86-90	Test item	1000	17.9	1.5	-23.9	n.s.
Coefficient of Variation of control: 23.6 %

^a: mean value of 5 replicates, 6 replicates in case of control

^b: mean value of 4 replicates for a test item concentration of 10 mg/L

^c: significance according William’s Multiple sequential t-test Procedure (α= 0.05, one-sided smaller) (* = significant; n. s.: not significant)

Inhibition [mean] % = 100-(Respiration Rate [mean] [mg O₂/g*h]*100 / mean value respiration rate in controls)

 

Table 3. Influence of the test item and the Reference Item 3,5-Dichlorophenol on the Inhibition of the Nitrification Respiration Rate of Aerobic Waste Water Microorganisms after 3 Hours of Exposure

Treatment	Concentration [mg/L]	Respiration Rate [mean]^a [mg O2/g*h]	Standard Deviation^a	Inhibition [mean]^a	Significant effect compared to control^d
control	—	9.8	1.4	—	---
3.5-DCP	1	2.7	0.6	72.1	---
3.5-DCP	4	0.4	0.6	95.9	---
3.5-DCP	16	-0.2	0.2	102.5	---
Test item	10	0.4	1.2	95.5	*
Test item	32	1.7	0.4	82.8	*
Test item	100	1.6	1.1	83.6	*
Test item	320	0.6	1.1	93.8	*
Test item	1000	0.8	1.5	91.5	*
Coefficient of Variation of control: 14.0 %

^a: mean value of 5 replicates, 6 replicates in case of control

^d: significance according Williams Multiple Sequential t-test (α= 0.05, one-sided smaller) (* = significant; n. s.: not significant)

Inhibition [mean] % = 100-(Respiration Rate [mean] [mg O₂/g*h]*100 / mean value respiration rate in controls)

Validity criteria fulfilled:

yes

Conclusions:

Based on a low inhibition for total respiration and strong inhibiting effects on the nitrification respiration rate, the NOEC was determined to be < 10 mg/L. The EC20 for total respiration rate was determined to be > 1000 mg/L. Due to the fact that no inhibition could be measured for the heterotrophic respiration rate, the NOEC was calculated to be ≥ 1000 mg/L.

Executive summary:

The purpose of the 3 hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions in a study according to OECD TG 209 under GLP. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The test item was investigated at nominal concentrations of 10, 32, 100, 320 and 1000 mg test item/L. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control; furthermore blank (inoculum) control and nitrification controls were investigated. The test item was directly dosed into each test flask and pure water was added. The test item was dissolved into the pure water as homogeneously as possible. All validity criteria of the study were met. In comparison to the inoculum controls, the total respiration rates of the activated sludge were slightly inhibited at any test item concentration. The inhibition was 11.5 % at a test concentration of 10 mg/L and 10.5 % at a test item concentration of 32 mg/L. For the test item concentrations of 100 mg/L and 320 mg/L the inhibition rates were 7.9 %, and 6.5 %, respectively. The highest test concentration of 1000 mg/L resulted in an inhibition of 22.6 %. Concentrations exceeding 1000 mg/L nominal were not tested. The EC₁₀ and EC₂₀ were determined to be 55.8 mg/L and >1000 mg/L. due to the large confidence limits, the values have to be treated with care. The NOEC was determined to be lower than a test item concentration of 10 mg/L. In comparison to the inoculum controls the respiration was not inhibited at any test item concentration. The inhibition rates were -45.2 %, -38.2 %, -43.2 %, -52.4 % and -23.9 % at test item concentrations of 10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L and 1000 mg/L respectively. No sensible ECx values could be established, the NOEC was determined to be higher than a test item concentration of 1000 mg/L. In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification were strongly inhibited at all test item concentrations. The inhibition rates were 95.5 %, 82.8 %, 83.6 %, 93.8 % and 91.5 % at test item concentrations of 10, 32, 100, 320 and 1000 mg/L. For the oxygen uptake due to nitrification, no ECx values could be established. The NOEC was determined to be lower than a test item concentration of 10 mg/L. The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3 hour EC₅₀ for total respiration was found to be 2.6 mg/L thus lying in the range of 2 - 25 mg/L recommended by the test guidelines. The 3 hour EC₅₀ for heterotrophic respiration was found to be 11.7 mg/L thus lying in the range of 5 - 40 mg/L recommended by the test guidelines For the oxygen uptake due to nitrification the 3 hour EC₅₀ was 0.4 mg/L, lying in the range of 0.1 - 10 mg/L recommended by the test guidelines. The result confirms the suitability of the activated sludge used.

Description of key information

Based on a low inhibition for total respiration and strong inhibiting effects on the nitrification respiration rate, the NOEC was determined to be < 10 mg/L. The EC₂₀ for total respiration rate was determined to be > 1000 mg/L. The NOEC for heterotrophic respiration rate was calculated to be ≥ 1000 mg/L (reference 6.1.7-1).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The purpose of the 3 hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions in a study according to OECD TG 209 under GLP. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The test item was investigated at nominal concentrations of 10, 32, 100, 320 and 1000 mg test item/L. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control; furthermore blank (inoculum) control and nitrification controls were investigated. The test item was directly dosed into each test flask and pure water was added. The test item was dissolved into the pure water as homogeneously as possible. All validity criteria of the study were met. In comparison to the inoculum controls, the total respiration rates of the activated sludge were slightly inhibited at any test item concentration. The inhibition was 11.5 % at a test concentration of 10 mg/L and 10.5 % at a test item concentration of 32 mg/L. For the test item concentrations of 100 mg/L and 320 mg/L the inhibition rates were 7.9 %, and 6.5 %, respectively. The highest test concentration of 1000 mg/L resulted in an inhibition of 22.6 %. Concentrations exceeding 1000 mg/L nominal were not tested. The EC₁₀ and EC₂₀ were determined to be 55.8 mg/L and >1000 mg/L. due to the large confidence limits, the values have to be treated with care. The NOEC was determined to be lower than a test item concentration of 10 mg/L. In comparison to the inoculum controls the respiration was not inhibited at any test item concentration. The inhibition rates were -45.2 %, -38.2 %, -43.2 %, -52.4 % and -23.9 % at test item concentrations of 10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L and 1000 mg/L respectively. No sensible ECx values could be established, the NOEC was determined to be higher than a test item concentration of 1000 mg/L. In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification were strongly inhibited at all test item concentrations. The inhibition rates were 95.5 %, 82.8 %, 83.6 %, 93.8 % and 91.5 % at test item concentrations of 10, 32, 100, 320 and 1000 mg/L. For the oxygen uptake due to nitrification, no ECx values could be established. The NOEC was determined to be lower than a test item concentration of 10 mg/L. The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3 hour EC₅₀ for total respiration was found to be 2.6 mg/L thus lying in the range of 2 - 25 mg/L recommended by the test guidelines. The 3 hour EC₅₀ for heterotrophic respiration was found to be 11.7 mg/L thus lying in the range of 5 - 40 mg/L recommended by the test guidelines For the oxygen uptake due to nitrification the 3 hour EC₅₀ was 0.4 mg/L, lying in the range of 0.1 - 10 mg/L recommended by the test guidelines. The result confirms the suitability of the activated sludge used.