Dimethyl(octyl)amine

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:

14 Oct 2015 - 03 Nov 2015

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

To the test mixtures (250 mL) containing deionised water, synthetic sewage feed (16 mL) and the appropriate test/reference item concentration and the inoculum of activated sludge (250 mL) was added to the final volume of 500 mL. The test item was applied directly into the respective test vessels and was dispersed by intense aeration. The reference assay with the concentrations 0.2, 1, 5, and 25 mg/L was set up by dilution of a stock solution. The ATH assay was set up by adding 2.5 mL ATH-stock solution to the respective vessel. The abiotic control was set up without the inoculum of activated sludge. The blank control was set up in the same way as the test item concentrations but without test item.
The abiotic control was prepared with the highest test item concentration, water and synthetic sewage feed.
The main test was performed with the following concentrations: 9.54, 30.5, 97.7, 312.5 and 1000 mg/L. The test item amounts were weighed directly onto a Teflon tape and given into the test vessels.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant of Pforzheim, Germany, collected from the aeration tank, was used as the microbial inoculum for the test. This plant was predominantly treating domestic sewage.
The sludge was used one day after collection. It was settled for about 10 minutes and the upper layer with finer solids was decanted. Before starting the test, it was washed three times with chlorine free tap water by centrifugation (10 minutes at 3000 rpm). After centrifuging, the supernatant was decanted and discarded and the sludge was resuspended in chlorine free tap water.
The mixed liquor suspended solids (MLSS) were adjusted to a concentration of 3.0 g/L (± 10 %). The activated sludge was continuously aerated at the test temperature, the solids did not settle down.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

none

Hardness:

no data

Test temperature:

18.3 – 21.0 °C

pH:

7.20 – 7.97

Dissolved oxygen:

The test solutions without microbial inoculum were aerated between 15 min and 3 hours (due to the test performance) to saturate the solution with O2 above 60 %

Salinity:

no data

Nominal and measured concentrations:

The main test was performed with the following concentrations: 9.54, 30.5, 97.7, 312.5 and 1000 mg/L. All test item concentrations were performed with three replicates whereas the control was performed with six replicates. The test item amount were weighed directly into the test vessels.

Details on test conditions:

1 litre glass beakers with test volume of 500 mL.; Measurements were taken 3 hours after test start (t0) which was defined as the initial contact of the activated sludge inoculum with the other constituents of the final mixture. The test solution was filled into a BOD-flask and stirred, and the oxygen concentration was measured and recorded over a period of about 5 minutes. All treatments were measured in the same way at approximately 5-minute intervals.
Aeration was performed during the whole test period but not during the measurements.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol (DCP)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

355 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: total oxygen uptake

Remarks on result:

other: 272 - 473 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

893 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: heterotrophic oxygen uptake

Remarks on result:

other: 751 - 1110 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

30.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: oxygen uptake due to nitrification

Remarks on result:

other: 15.9 – 53.1 mg/L

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

< 9.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: total oxygen uptake and for the oxygen uptake due to nitrification

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

9.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: total oxygen uptake and for the oxygen uptake due to nitrification

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

97.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: heterotrophic oxygen uptake

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

312.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: heterotrophic oxygen uptake

Details on results:

After 3 hours, statistically significant effects were observed at all concentration levels for the total oxygen uptake and oxygen uptake due to nitrification. For heterotrophic oxygen uptake statistically significant effects were observed at 312.5 and 1000 mg/L (within the dose response of 97.7 to 1000 mg/L).
After 3 hours, an inhibitory effect of 72.5 % was determined in the highest test item concentration. For the heterotrophic oxygen uptake, after 3 hours, an average inhibitory effect of 54.0% was determined at the highest test item concentration. For the oxygen uptake due to nitrification an inhibitory effect of 100 % was determined at the highest test item concentration after 3 hours.
The EC50 of the total oxygen uptake was determined to be 355 mg/L (95 % fiducial limits: 272 - 473) and 893 mg/L (95 % fiducial limits: 751 - 1110) for the heterotrophic oxygen uptake. The EC50 for the oxygen uptake due to nitrification was 30.9 mg/L (95 % fiducial limits: 15.9 – 53.1).

Results with reference substance (positive control):

The EC50 of the total oxygen uptake for DCP was estimated between 5-25 mg/L after 3 hours. This is within the accepted range of 2 to 25 mg/L for total respiration. For the heterotrophic oxygen uptake for DCP, the EC50 was estimated between 5-25 mg/L after 3 hours. This is within the recommended range of 5 to 40 mg/L. For the oxygen uptake due to nitrification the EC50 was estimated between 1 - 5 mg/L after 3 hours. This value was also within the recommended range of 0.1 to 10 mg/L.

Reported statistics and error estimates:

The statistical evaluation was performed for specific respiration rate of the control and all test item concentrations using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was performed by calculating the Levene Test. The NOEC and LOEC were determined by using Dunnett`s t-test and Welch Bonferroni-Holms corrected. The calculation of the EC50 values for the total oxygen uptake, heterotrophic oxygen uptake and oxygen uptake due to nitrifiers was calculated using probit analysis following logistic or gompertz procedure. Only test concentrations with a dose-response were used. Test concentrations with negative inhibition values were set to zero, inhibition values above 100 were set to 100.

Table 5:        Results of the main test, specific respiration rates

	Specific respiration rates [mg O2/(g × h)]
Test assay	Total	Heterotrophic	Due to nitrification
	3 h	3 h	3 h
Control 1	31.35	19.22	11.55*
Control 2	30.99	18.07
Control 3	30.95	20.14
Control 4	33.03	19.67
Control 5	29.90	21.29
Control 6	32.81	21.36
9.54 mg/L 1	29.57	20.73	9.6*
9.54 mg/L 2	30.05	20.27
9.54 mg/L 3	30.46	20.30
30.5 mg/L 1	25.93	20.25	6.2*
30.5 mg/L 2	25.25	19.29
30.5 mg/L 3	26.21	19.27
97.7 mg/L 1	23.13	20.73	1.24*
97.7 mg/L 2	18.20	19.37
97.7 mg/L 3	22.52	20.03
312.5 mg/L 1	18.43	17.71	0.71*
312.5 mg/L 2	17.28	17.05
312.5 mg/L 3	18.23	16.39
1000 mg/L 1	8.15	7.42	-0.51*
1000 mg/L 2	8.86	11.49
1000 mg/L 3	9.04	8.65
DCP 0.2 mg/L	31.65	22.70	8.95
DCP 1 mg/L	27.57	17.08	10.49
DCP 5 mg/L	25.04	26.45	-1.41
DCP 25 mg/L	6.25	6.36	-0.11

* the nitrification values were determined by calculating the difference between the mean of the total specific respiration rate and the mean of the heterotrophic respiration rate

Table 6:       Results, inhibition

Test assay [mg/L]	Inhibition [%]
	Total	Heterotrophic	Due to Nitrification
	3 h	3 h	3 h
9.54 mg/L (mean)	4.7*	-2.4^1)	16.9*
30.5 mg/L (mean)	18.1*	1.8	46.3*
97.7 mg/L (mean)	32.5*	-0.4^1)	89.3*
312.5 mg/L (mean)	42.9*	14.6*	91.9*
1000 mg/L (mean)	72.5*	54.0*	104.4*
DCP 0.2 mg/L	-0.4^1)	-13.7^1)	22.5
DCP 1 mg/L	12.5	14.4	9.2
DCP 5 mg/L	20.5	-32.5^1)	112.2
DCP 25 mg/L	80.2	68.1	101.0

*statistically significant different to the control.

¹⁾negative value means stimulating effects

Table 7: Endpoints for AD-1

Endpoint	AD-1	95% confidential limits
Total EC50	355 mg/L	272 -473 mg/L
Heterotrophic EC50	893 mg/L	751 - 1110 mg/L
Due to Nitrification EC50	30.9 mg/L	15.9 - 53.1 mg/L
Total NOEC	< 9.54 mg/L	-
Heterotrophic NOEC	97.7 mg/L	-
Due to Nitrification NOEC	< 9.54 mg/L	-
Total LOEC	9.54 mg/L	-
Heterotrophic LOEC	312.5 mg/L	-
Due to nitrification LOEC	9.54 mg/L	-

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on microorganisms of an aerobic microbial treatment plant was tested using the activated sludge respiration inhibition test.
The EC50 of the total oxygen uptake was determined to be 355 mg/L (95 % fiducial limits: 272 - 473) and 893 mg/L (95 % fiducial limits: 751 - 1110) for the heterotrophic oxygen uptake. The EC50 for the oxygen uptake due to nitrification was 30.9 mg/L (95 % fiducial limits: 15.9 – 53.1).
The NOEC (3h) for the total oxygen uptake and for the oxygen uptake due to nitrification could not be determined since the lowest test item concentration of 9.54 mg/L already showed statistically significant differences. The LOEC was determined at 9.54 mg/L. For the heterotrophic oxygen uptake the NOEC (3h) was observed at 97.7 mg/L and the LOEC at 312.5 mg/L.
All validity criteria according to OECD Guideline for Testing Chemicals 209 were fulfilled.

Executive summary:

Report:	Falk, S. (2016): AD 1: Toxicity to Microorganisms, Activated Sludge, Respiration Inhibition Test
Source:	Eurofins Agroscience Services EcoChem GmbH/ Eurofins Agroscience Services Ecotox GmbH,Eutinger Str. 24 75223; unpublished report no.: S15-04907; Issued: 26 Jan 2016
Guidelines:	OECD guideline 209
Deviations:	No major
GLP:	Yes, certified laboratory
Objective:	The respiration rate of an activated sludge, fed with a standard amount of synthetic sewage feed, was measured 3 hours after application of the test item. The inhibitory effects of AD 1 at particular concentrations were expressed as a percentage of the mean respiration rate of the controls.
Materials and methods:	Test item: AD 1, Batch No.: 13602808. Chemical name: N,N-dimethyloctylamine, Purity (nominal): >95 % (w/w). Activated sludge from the municipal wastewater treatment plant of Pforzheim, Germany, was used as microbial inoculum for the test. This plant was predominantly treating domestic sewage. The sludge was adjusted to a content of 1.5 g/L dry matter and was exposed to the test item under continuous aeration. Thereafter, the respiration rate (O2 consumption) of the sludge microorganisms was measured for approx. 5 minutes. The slope of the O2 consumption straight line is an indication for toxic effects on respiration activity of microorganisms. Following a range-finding test with nominal test item concentrations of 10, 100 and 1000 mg/L a main test was performed with concentrations of 9.54, 30.5, 97.7, 312.5 and 1000 mg/L test item. Six replicates were used for the control and three for each test item concentration. Four concentrations of 3,5-dichlorophenol (DCP) as toxic reference item were tested in parallel to demonstrate the sensitivity of the test system. The oxygen uptake rate was determined after 3 hours.
Date of work:	14 Oct 2015 – 03 Nov 2015
Findings:	The results of the main test are presented below:

 

	Inhibition [%] *
Test assay	Total	Heterotrophic	Nitrification
	3 h
9.54 mg/L (mean)	4.7*	-2.4^1)	16.9*
30.5 mg/L (mean)	18.1*	1.8	46.3*
97.7 mg/L (mean)	32.5*	-0.4^1)	89.3*
312.5 mg/L (mean)	42.9*	14.6*	91.9*
1000 mg/L (mean)	72.5*	54.0*	104.4*
DCP 0.2 mg/L	-0.4^1)	-13.7^1)	22.5
DCP 1 mg/L	12.5	14.4	9.2
DCP 5 mg/L	20.5	-32.5^1)	112.2
DCP 25 mg/L	80.2	68.1	101.0

^*statistically significantly different to the control

¹⁾  negative values mean stimulating effects

 

Conclusions:

After 3 hours, statistically significant effects were observed at all concentration levels for the total oxygen uptake and oxygen uptake due to nitrification. For heterotrophic oxygen uptake statistically significant effects were observed at 312.5 and 1000 mg/L (within the dose response of 97.7 to 1000 mg/L). After 3 hours, an inhibitory effect of 72.5 % was determined in the highest test item concentration. For the heterotrophic oxygen uptake, after 3 hours, an average inhibitory effect of 54.0 % was determined at the highest test item concentration. For the oxygen uptake due to nitrification an inhibitory effect of 100 % was determined at the highest test item concentration after 3 hours. The EC50 of the total oxygen uptake was determined to be 355 mg/L (95 % fiducial limits: 272 - 473) and 893 mg/L (95 % fiducial limits: 751 - 1110) for the heterotrophic oxygen uptake. The EC50 for the oxygen uptake due to nitrification was 30.9 mg/L (95 % fiducial limits: 15.9 – 53.1). All validity criteria of OECD Guideline for Testing of Chemicals No. 209 were fulfilled: •  The mean oxygen uptake rate of the blank control were determined to be 32 mg O2/(g × h) dry matter after 3 hours. Hence the rate was not less than 20 mg oxygen uptake per g of activated sludge (dry weight of suspended solids) per hour. •  The coefficients of variation of the control for total oxygen uptake were 4 % after 3 hours and did not exceed 30 % at the end of the test. •  The EC50 of the total oxygen uptake for DCP was estimated between 5-25 mg/L after 3 hours.This is within the accepted range of 2 to 25 mg/L for total respiration. For the heterotrophicoxygen uptake for DCP, the EC50 was estimated between 5-25 mg/L after 3 hours. This is within the recommended range of 5 to 40 mg/L. For the oxygen uptake due to nitrification the EC50 was estimated between 1 - 5 mg/L after 3 hours. This value was also within the recommended range of 0.1 to 10 mg/L.

Description of key information

After 3 hours, an inhibitory effect of 72.5% was determined in the highest test item concentration. For the heterotrophic oxygen uptake, after 3 hours, an average inhibitory effect of 54.0 % was determined at the highest test item concentration. For the oxygen uptake due to nitrification an inhibitory effect of 100 % was determined at the highest test item concentration after 3 hours.

The EC50 of the total oxygen uptake was determined to be 355 mg/L (95% fiducial limits: 272 - 473) and 893 mg/L (95% fiducial limits: 751 - 1110) for the heterotrophic oxygen uptake. The EC50 for the oxygen uptake due to nitrification was 30.9 mg/L (95% fiducial limits: 15.9 – 53.1).

Key value for chemical safety assessment

EC50 for microorganisms:

355 mg/L