N1,N3-diallylpropane-1,3-diamine dihydrochloride

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:

2020-03-11 to 2020-04-28

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:

July 2010

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

- Concentrations: 10, 100 and 1000 mg/L
- Sampling method: After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer.
This determination was repeated for each vessel at 15-min intervals, ensuring that the contact time in each vessel was 3 h.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 g/L was prepared and added to 500 ml test vessels at 0.5, 5 and 50 ml volumes to achieve the desired test item concentrations. The pH of the test solution was measured before addition of inoculum.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: municipal wastewater treatment plant Breisgauer Bucht
- Preparation of inoculum for exposure: The activated sludge was kept aerated until use at test temperature. The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in chlorine-free tap water. Activated sludge was dried at 105 °C. Before using, the activated sludge was diluted to 3 g/L dry solids with chlorine-free tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.
- Initial biomass concentration: 4.9 g/L (dry solids)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19.9-21.3°C

pH:

7.1 - 7.4

Nominal and measured concentrations:

Nominal concentrations: 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 600 mL glass serum bottles with 500 mL test medium volume were used.
- No. of vessels per concentration (replicates): 3 for 10 and 100 mg/L, 5 for 1000 mg/L
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 4.9 g/L (final concentration = 1.5 g/L)
- Weight of dry solids per volume of reaction mixture per unit of time:
- Nutrients provided for bacteria: Synthetic sewage feed
Peptone 16 g
Meat extract 11 g
Urea 3 g
NaCl 0.7 g
CaCl2 * 2 H2O 0.4 g
MgSO4 * 7 H2O 0.2 g
K2HPO4 2.8 g
The ingredients listed in the table above are dissolved in demineralised water and made up to 1 litre. The pH of the synthetic sewage feed was 7.2.

- Nitrification inhibitor used: N-allylthiourea

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate (oxygen concentrations)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

- Range finding study : yes
- Test concentrations: 10, 100, 1000 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

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 heterotrophic respiration

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

Results with reference substance (positive control):

- Results with reference substance valid? Yes
The EC50 of 3,5-dichlorphenol was calculated as 4.22 mg/l for the total respiration rate, 28.99 mg/L for the heterotrophic respiration rate and 0.68 mg/L for the nitrification respiration rate

Reported statistics and error estimates:

The EC50 calculation for the reference item was performed with the ToxRat software by probit analysis using linear max. likelihood regression (ToxRat Professional Version 3.3.0, © ToxRat Solutions GmbH). The EC50 is calculated from the slope of the curve.

Total respiration:

There was no inhibition observed in the test vessels with a concentration of 10 and 100 mg/L. In the test vessels with a concentration of 1000 mg/L the mean inhibition of five replicates was 23.4% and therefore below 50%.

Heterotrophic respiration:

There was no inhibition observed in the test vessels with 1000 mg/L. In the test vessels with 10 and 100 mg/L a mean inhibition of about 12% was observed, this is probably caused by a relatively high variation of the respiration in the blank controls and the lower test concentrations.

Nitrification

In the test vessels with a concentration of 10 and 100 mg/L no inhibition of the nitrification respiration could be observed. In the test concentration of 1000 mg/L the mean inhibition observed was 53%. This shows that the main part of the inhibition of total respiration at a concentration of 1000 mg/L is due to the inhibition of nitrifiers.

Blanks

The mean total respiration rate of the six blank controls was 28.0 mg per one gram activated sludge. The coefficient of variation (CV) was 14% and thereforeless than 30%.

Total respiration rate of activated sludge after application of test item:

Vessel	O2 concentration in mg/L		Consumption rate [mg/(l*h)]	Consumption rate [mg/(g sludge*h)]	Inhibition [%]
	First measurement	Last measurement
Blank 1	7.38	5.5	45.1	30.1	NA
Blank 2	7.64	5.86	42.7	28.5	NA
Blank 3	7.77	6.53	29.8	19.8	NA
Blank 4	7.64	5.66	47.5	31.7	NA
Blank 5	7.71	5.99	41.3	27.5	NA
Blank 6	7.59	5.69	45.6	30.4	NA
Test 10 mg/L 1	7.62	5.88	41.8	27.8	1
Test 10 mg/L 2	7.77	6.03	41.8	27.8	1
Test 10 mg/L 3	7.77	6.22	37.2	24.8	11
Test 100 mg/L 1	7.55	5.63	46.1	30.7	-10
Test 100 mg/L 2	7.72	6.05	40.1	26.7	5
Test 100 mg/L 3	7.55	5.93	38.9	25.9	7
Test 1000 mg/L 1	7.8	6.26	37.0	24.6	12
Test 1000 mg/L 2	8.06	6.91	27.6	18.4	34
Test 1000 mg/L 3	8.02	6.75	30.5	20.3	27
Test 1000 mg/L 4	8	6.63	32.9	21.9	22
Test 1000 mg/L 5	8.15	6.78	32.9	21.9	22
Coefficient of variation of blanks				0.14
Mean consumption rate blanks				28.0

Heterotrophic respiration rate of activated sludge after application of the test item:

Vessel	O2 concentration in mg/L		Consumption rate [mg/(l*h)]	Consumption rate [mg/(g sludge*h)]	Inhibition [%]
	First measurement	Last measurement
Blank 1	8.08	6.93	27.6	18.4	NA
Blank 2	7.99	7.07	22.1	14.7	NA
Blank 3	8.16	6.78	33.1	22.1	NA
Blank 4	8.2	7.28	22.1	14.7	NA
Blank 5	8.03	7.33	16.8	11.2	NA
Blank 6	8.38	7.52	20.6	13.8	NA
Test 10 mg/L 1	8.15	7.07	25.9	17.3	-9
Test 10 mg/L 2	8.25	7.44	19.4	13.0	18
Test 10 mg/L 3	8.31	7.59	17.3	11.5	27
Test 100 mg/L 1	8.28	7.28	24.0	16.0	-1
Test 100 mg/L 2	8.3	7.43	20.9	13.9	12
Test 100 mg/L 3	8.32	7.57	18.0	12.0	24
Test 1000 mg/L 1	8.09	6.87	29.3	19.5	-23
Test 1000 mg/L 2	8.16	7.19	23.3	15.5	2
Test 1000 mg/L 3	8.25	7.21	25.0	16.6	-5
Test 1000 mg/L 4	7.86	6.81	25.2	16.8	-6
Test 1000 mg/L 5	7.44	6.81	15.1	10.1	36

Validity criteria fulfilled:

yes

Conclusions:

The 3-h EC50 for inhibition of total and heterotrophic respiration of sewage sludge microorgansims is > 1000 mg/L.

Executive summary:

To investigate potential harmful effects of N1,N3 -diallylpropane-1,3 -diamine dihydrochloride on microorganisms, an activated sludge respiration inhibition test was performed with domestic sewage sludge according to OECD guideline 209 and under GLP. All validity criteria were met for this test. Inhibition of total respiration and heterotrophic respiration were investigated by using N-allylthiourea as inhibitor of nitrification. An inhibition was observed for total respiration at 1000 mg/L test item concentration (12% - 34%, mean of five replicates = 23.4%). Heterotrophic respiration was not inhibited at the tested concentrations (10, 100, 1000 mg/L). Therefore, the inhibition of total respiration at a concentration of 1000 mg/L is due to the inhibition of nitrifiers. Hence, the following theshold concentrations were determined:

Total respiration:

EC50 (3h, nominal) > 1000 mg/L

NOEC (3h, nominal) >= 100 mg/L

Heterotrophic repiration:

EC50 (3h, nominal) > 1000 mg/L

NOEC (3h, nominal) >= 1000 mg/L

Description of key information

To investigate potential harmful effects of N1,N3-diallylpropane-1,3-diamine dihydrochloride on microorganisms, an activated sludge respiration inhibition test was performed with domestic sewage sludge according to OECD guideline 209 and under GLP. All validity criteria were met for this test. Inhibition of total respiration and heterotrophic respiration were investigated by using N-allylthiourea as inhibitor of nitrification. An inhibition was observed for total respiration at 1000 mg/L test item concentration (12% - 34%, mean of five replicates = 23.4%). Heterotrophic respiration was not inhibited at the tested concentrations (10, 100, 1000 mg/L). Therefore, the inhibition of total respiration at a concentration of 1000 mg/L is due to the inhibition of nitrifiers. Hence, the following threshold concentrations were determined:

Total respiration:

EC50 (3h, nominal) > 1000 mg/L

NOEC (3h, nominal) >= 100 mg/L

Heterotrophic respiration:

EC50 (3h, nominal) > 1000 mg/L

NOEC (3h, nominal) >= 1000 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L