Glycerides, C16-18 (even numbered) and their amidation products with diethylenetriamine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

An OECD 209 GLP study for a similar substance is available and is used within a read-across approach. The composition of source and target substance is described and discussed in detail in the attached supporting information as well as the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Target substance:

Amidoamine 2 (UVCB, low nitrogen):
Former chemical name: Amides, from diethylenetriamine and hydrogenated palm oil
CAS No.: 1618093-67-6
New chemical name: Glycerides, C16-18 (even numbered) and their amidation products with diethylenetriamine
Physical state: pale yellowish solid at 20 °C
Batch No.: PU50070067
Purity: 100 % (UVCB)
Storage condition of test material: Room temperature, protected from light
Stability: stable under test conditions

Analytical monitoring:

no

Details on sampling:

As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L).

Vehicle:

no

Details on test solutions:

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.6 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 26 August 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

The test water used for the test was deionized reverse osmosis water containing less than
1 mg/L Dissolved Organic Carbon (DOC).

Test temperature:

18 - 22 °C

pH:

The pH of the test solution was measured at the start and the end of the 3 hour incubation period. pH was betwenn 7.0 - 8.0.

Dissolved oxygen:

The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Reference substance (positive control):

yes

Remarks:

A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum.

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 total respiration

Details on results:

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above
60% of the dissolved oxygen saturation level of 8.9 mg O2/L. No statistically significant toxic effects were shown at all of the test concentrations employed.
It was therefore considered justifiable not to perform a definitive test.

Results with reference substance (positive control):

The EC50 (3 h) of the reference item 3,5-dichlorphenol was in the range of 2 to 25 mg/l for total respiration.

Range-Finding Test

The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 2. The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 11.25% and the specific respiration rate of the controls was 24.60 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied. The validation criterion for the reference item EC50 value was also satisfied.

Table 1: Inhibition of Respiration Rate

	Amidoamine (UVCB)		3,5-dichlorophenol
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	>1000	-	2.0	-
EC20	>1000	-	2.9	-
EC50	>1000	-	9.8	7.5 - 13
EC80	>1000	-	33	-
NOEC	1000	-	-	-

 

Table 2: Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Control	R1	6.3	71
	R2	6.5	73
	R3	6.2	70
	R4	6.6	74
Test Item	10	5.8	65
	100	6.3	71
	1000 R1	6.2	70
	1000 R1	5.6	63
	1000 R1	6.4	72
3,5-dichlorophenol	3.2	5.7	64
	10	7.1	80
	32	8.3	93

R1 – R4 = Replicates 1 to 4

 

Table 3: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	6.0	6	2.0	40.00	-
	R2	5.7	6	2.0	37.00	-
	R3	5.2	5	1.9	39.60	-
	R4	4.9	6	1.8	31.00	-
Test Item	10	4.6	4	2.0	39.00	[6]
	100	5.5	5	2.2	39.60	[7]
	1000 R1	5.2	5	1.9	39.60	[7]
	1000 R1	5.8	6	1.7	41.00	[11]
	1000 R1	5.3	5	2.0	39.60	25
3,5- dichlorophenol	3.2	6.7	10	2.1	27.60	45
	10	7.6	10	4.2	20.40	82
	32	8.4	10	7.3	6.60

[Increase in respiration rate as compared to controls]
R1 – R4 = Replicates 1 to 4

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration rates of STP organisms has been investigated and gave a NOEC value of 1000 mg/L, i.e. the substance is not toxic to STP microorganisms. It is considered likely, that the target substance Amidoamine 2 (UVCB, low nitrogen) will show a comparable toxicity to microorganisms as Amidoamine (UVCB) based on the similar chemical composition.

Executive summary:

In a valid GLP study according to OECD 209 the test item was aerobically exposed to standard OECD mineral medium and activated sludge (3 g/l) obtained from a STP (non-adapted, domestic). Exposure was for 3 hours under stirring conditions between 18 and 22°C. Glass reactors were constantly aerated with CO2 free air. Respiration rates were measured by pouring a well-mixed sample of each test vessel into a 250 mL darkened glass BOD flask after 3 hours incubation time without further aeration. The dissolved oxygen concentration was measured with an oxygen probe attached to an oxygen meter.

The effect of the test item on the respiration of activated sewage sludge micro-organismsgave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. The substance is not toxic to STP microorganisms, since NOEC for total respiratory inhibition was 1000 mg/l.

It is considered likely, that the target substance Amidoamine 2 (UVCB, low nitrogen) will show a comparable toxicity to microorganisms as Amidoamine (UVCB) based on the similar chemical composition. It is expected that the higher concentration of saturated mono- and diglycerides and the presence of saturated triglycerides in Amidoamine 2 (UVCB, low nitrogen) will have no adverse effect on inoculum microorganisms.

 

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Source substance:

Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Former chemical name: Amides, from diethylenetriamine and hydrogenated palm oil
Former CAS No.: 1618093-67-6
New chemical name: Triglycerides, C16-18 (even-numbered), reaction products with diethylenetriamine
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2018
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Target substance:

Amidoamine 2 (UVCB, low nitrogen):
Former chemical name: Amides, from diethylenetriamine and hydrogenated palm oil
CAS No.: 1618093-67-6
New chemical name: Glycerides, C16-18 (even numbered) and their amidation products with diethylenetriamine
Physical state: pale yellowish solid at 20 °C
Batch No.: PU50070067
Purity: 100 % (UVCB)
Storage condition of test material: Room temperature, protected from light
Stability: stable under test conditions

Analytical monitoring:

no

Details on sampling:

As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L).

Vehicle:

no

Details on test solutions:

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.6 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 26 August 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

The test water used for the test was deionized reverse osmosis water containing less than
1 mg/L Dissolved Organic Carbon (DOC).

Test temperature:

18 - 22 °C

pH:

The pH of the test solution was measured at the start and the end of the 3 hour incubation period. pH was betwenn 7.0 - 8.0.

Dissolved oxygen:

The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Reference substance (positive control):

yes

Remarks:

A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum.

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 total respiration

Details on results:

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above
60% of the dissolved oxygen saturation level of 8.9 mg O2/L. No statistically significant toxic effects were shown at all of the test concentrations employed.
It was therefore considered justifiable not to perform a definitive test.

Results with reference substance (positive control):

The EC50 (3 h) of the reference item 3,5-dichlorphenol was in the range of 2 to 25 mg/l for total respiration.

Range-Finding Test

The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 2. The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 11.25% and the specific respiration rate of the controls was 24.60 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied. The validation criterion for the reference item EC50 value was also satisfied.

Table 1: Inhibition of Respiration Rate

	Amidoamine (UVCB)		3,5-dichlorophenol
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	>1000	-	2.0	-
EC20	>1000	-	2.9	-
EC50	>1000	-	9.8	7.5 - 13
EC80	>1000	-	33	-
NOEC	1000	-	-	-

 

Table 2: Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Control	R1	6.3	71
	R2	6.5	73
	R3	6.2	70
	R4	6.6	74
Test Item	10	5.8	65
	100	6.3	71
	1000 R1	6.2	70
	1000 R1	5.6	63
	1000 R1	6.4	72
3,5-dichlorophenol	3.2	5.7	64
	10	7.1	80
	32	8.3	93

R1 – R4 = Replicates 1 to 4

 

Table 3: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	6.0	6	2.0	40.00	-
	R2	5.7	6	2.0	37.00	-
	R3	5.2	5	1.9	39.60	-
	R4	4.9	6	1.8	31.00	-
Test Item	10	4.6	4	2.0	39.00	[6]
	100	5.5	5	2.2	39.60	[7]
	1000 R1	5.2	5	1.9	39.60	[7]
	1000 R1	5.8	6	1.7	41.00	[11]
	1000 R1	5.3	5	2.0	39.60	25
3,5- dichlorophenol	3.2	6.7	10	2.1	27.60	45
	10	7.6	10	4.2	20.40	82
	32	8.4	10	7.3	6.60

[Increase in respiration rate as compared to controls]
R1 – R4 = Replicates 1 to 4

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration rates of STP organisms has been investigated and gave a NOEC value of 1000 mg/L, i.e. the substance is not toxic to STP microorganisms.

Executive summary:

In a valid GLP study according to OECD 209 the test item was aerobically exposed to standard OECD mineral medium and activated sludge (3 g/l) obtained from a STP (non-adapted, domestic). Exposure was for 3 hours under stirring conditions between 18 and 22°C. Glass reactors were constantly aerated with CO2 free air. Respiration rates were measured by pouring a well-mixed sample of each test vessel into a 250 mL darkened glass BOD flask after 3 hours incubation time without further aeration. The dissolved oxygen concentration was measured with an oxygen probe attached to an oxygen meter.

The effect of the test item on the respiration of activated sewage sludge micro-organismsgave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. The substance is not toxic to STP microorganisms, since NOEC for total respiratory inhibition was 1000 mg/l.

 

Description of key information

The effect of the test item on the respiration rates of STP organisms has been investigated and gave a NOEC value of 1000 mg/L, i.e. the substance is not toxic to STP microorganisms. 

It is considered likely, that the target substance Amidoamine 2 (UVCB, low nitrogen) will show a comparable toxicity to microorganisms as Amidoamine (UVCB) based on the similar chemical composition. It is expected that the higher concentration of saturated mono- and diglycerides and the presence of saturated triglycerides in Amidoamine 2 (UVCB, low nitrogen) will have no adverse effect on inoculum microorganisms.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L