Fatty acids, C16-18 and C18-unsatd., branched and linear, reaction products with diethanolamine

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 23 May 2012 Experimental Completion Date: 24 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols/ Current OECD Guidelines and to GLP standards, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Isostearamide DEA

IUPAC Name:

Isostearamide DEA

Test material form:

other: amber coloured liquid

Details on test material:

The integrity of the supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

Identification: Isostearamide DEA
Description: amber coloured liquid
Batch: OE11124 (6/202593/00)
Expiry / retest date: not given
Storage conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximize the dissolved test
item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and
water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
The pH of the test item dispersions was measured after stirring using a WTW pH/Oxi 340I pH and dissolved oxygen meter (see attached Table 1) and adjusted to between pH 7.0 and 8.0 if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 23 May 2012 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The
mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the
reference item being added. The test item vessels were prepared as described in Section 3.5.1. Finally two further control vessels were prepared.
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 approximately 7.0 mg O2/L and 2.0 mg O2/L). In the case of a rapid
oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the
respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

The test was conducted under normal laboratory lighting in a temperature controlled room at 20±2 ºC.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight
prior to use in the test. On the day of collection the activated sewage sludge (10 liters) was fed synthetic sewage sludge (450 mL). The pH of the
sample on the day of the test was 7.8 measured using a WTW pH/Oxi 340I pH and dissolved oxygen 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 prior to use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

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

Test temperature:

The test was conducted under normal laboratory lighting in a temperature controlled room at 20±2 ºC.

pH:

The pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.0 and the pH of the synthetic sewage stock used for the test was
pH 6.8 and adjusted to 7.0 using 1.0 M NaOH. The pH values were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
The pH values of the test preparations at the start and end of the exposure period are given in the attached Table 4

Dissolved oxygen:

The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in the attached Table 2. Oxygen consumption rates and
percentage inhibition values for the control, test and reference items are given in the attached Table 3.

Salinity:

Freshwater used

Nominal and measured concentrations:

Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).

Details on test conditions:

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The
mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the
reference item being added. Finally two further control vessels were prepared.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

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

Remarks:

respiration rate

Effect concentrations 2

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

Remarks:

respiration rate

Effect concentrations 3

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 4

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

Remarks:

respiration rate

Effect concentrations 5

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Reference item: 3,5-dichlorophenol

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 6

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Reference item: 3,5-dichlorophenol

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 8.7 - 14 95% confidence limits (mg/L)

Effect concentrations 7

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Reference item: 3,5-dichlorophenol

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

RESULTS
Range-Finding Test
The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 2. Oxygen consumption rates and percentage
inhibition values for the control, test and reference items are given in Table 3. The pH values of the test preparations at the start and end of the
exposure period are given in Table 4. Observations made on the test preparations throughout the study are given in Table 5.

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.

Percentage inhibition is plotted against concentration for the reference item, 3,5 dichlorophenol (see attached Figure 1).

The following results were derived:

Test Item 3,5-dichlorophenol
ECx (3 Hours)(mg/L) 95% Confidence Limits (mg/L) ECx (3 Hours)(mg/L) 95% Confidence Limits (mg/L)
EC20 >1000 - 4.0 -
EC50 >1000 - 11 8.7-14
EC80 >1000 - 31 -
NOEC 1000 - - -

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 1.8% and the specific respiration rate of the controls was 26.60 mg oxygen
per gram dry weight of sludge per hour at the end of the test. The EC50 for the 3,5-dichlorophenol was between 2 mg/L and 25 mg/L.
The validation criteria have therefore been satisfied.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg O2/L and
2.0 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen
consumption rate was determined over the linear portion of the oxygen consumption trace.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of
8.9 mg O2/L with the exception of Control replicate 1 which showed 47% saturation and the 10 mg/L, 100 mg/L, and 1000 mg/L R1 test item vessels showing between 45% to 57% saturation. This deviation was considered to have had no adverse effect on the study given that all oxygen
consumption values were measured/calculated over the linear portion of the traces.

Results with reference substance (positive control):

None

Reported statistics and error estimates:

None

Any other information on results incl. tables

Tables

Table 1     pH Values of the Test Item Preparations after Stirring and Prior to the Addition of Inoculum in the Range-Finding Test

Nominal Concentration (mg/L)		pH*
		Prior to Adjustment	After Adjustment
Test Item	10	7.4	-
	100	6.8	8.0
	1000 R1	6.9	7.4
	1000 R2	6.9	7.3
	1000 R3	6.9	7.4

 

*       Measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter

 R₁– R₃= Replicates 1 to 3

-        no adjustment required

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)*
Control	R1	4.2
	R2	5.6
	R3	5.4
	R4	6.0
Test Item	10	4.7
	100	4.0
	1000 R1	5.1
	1000 R2	5.6
	1000 R3	5.5
3,5-dichlorophenol	3.2	6.5
	10	7.4
	32	8.4

 

*Measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter

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 O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	4.7	4	2.0	40.50	-
	R2	4.8	4	2.2	39.00	-
	R3	4.8	5	1.5	39.60	-
	R4	4.7	4	2.0	40.50	-
Test Item	10	5.3	5	2.0	39.60	1
	100	4.5	4	1.8	40.50	[2]
	1000 R1	4.5	5	1.6	34.80	13
	1000 R2	3.5	2	2.1	42.00	[5]
	1000 R3	5.2	4	2.6	39.00	2
3,5-dichlorophenol	3.2	6.0	7	1.9	35.14	12
	10	7.3	10	4.0	19.80	50
	32	7.9	10	6.5	8.40	79

 

[Increase in respiration rate as compared to controls]

R₁– R₄= Replicates 1 to 4

Table 4     pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-finding Test

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Control	R1	7.6	7.5
	R2	7.6	7.7
	R3	7.4	7.7
	R4	7.4	7.6
Test Item	10	7.1	7.8
	100	7.3	7.8
	1000 R1	7.3	7.8
	1000 R2	7.4	7.8
	1000 R3	7.4	7.8
3,5-dichlorophenol	3.2	7.6	7.7
	10	7.6	7.8
	32	7.6	7.9

 

R₁– R₄= Replicates 1 to 4

Table 5     Observations on the Test Preparations throughout the Test Period in the Range-Finding Test

Nominal Concentration (mg/L)		Observations on Test Preparations
		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R3	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R4	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Item	10	Pale yellow/brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	100	Pale yellow/brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	1000 R1	Pale yellow/brown dispersion, few small globules of test item on surface	Dark brown dispersion, few small globules of test item on surface	Dark brown dispersion, few small globules of test item on surface
	1000 R2	Pale yellow/brown dispersion, few small globules of test item on surface	Dark brown dispersion, few small globules of test item on surface	Dark brown dispersion, few small globules of test item on surface
	1000 R3	Pale yellow/brown dispersion, few small globules of test item on surface	Dark brown dispersion, few small globules of test item on surface	Dark brown dispersion, few small globules of test item on surface
3,5-dichlorophenol	3.2	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	10	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	32	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible

 

*Observations made prior to the addition of activated sewage sludge

R₁– R₄= Replicates 1 to 4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave 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.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) ".

 

 

Methods

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L

(3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of 20 ± 2°C with the addition of a synthetic sewage as a respiratory substrate.

 

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item,

3,5-dichlorophenol.

 

 

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was1000mg/L.

 

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

 

The reference item gave a 3-Hour EC₅₀value of 11 mg/L, 95% confidence limits 8.7 - 14 mg/L.