Soybean oil, maleated, ester with triethanolamine

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:

Experimental start date 13 September 2016 Experimental completion date 14 September 2016

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 (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Test Item Preparation range-finding test
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.

Nominal 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, at temperatures of between 20 C and 22 C, 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 Hach HQ40d Flexi handheld meter (see Table 1) and adjusted to between pH 7.0 and 8.0 if necessary.

The exposure conditions for each flask were as described in Section “Preparation of Inoculum”.

As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

The control group was maintained under identical conditions but not exposed to the test item.

Reference Item Preparation
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. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 12 minutes. The pH of this stock solution was measured to be pH 5.6 and was adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

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 and was used on the day of collection. The pH of the sample was 7.8 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.
* rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven

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 0.5 to 1.0 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 "Test Item Preparation". Finally two further control vessels were prepared.

The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 19 and 21 ºC.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not reported

Test temperature:

20 °C

pH:

The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.

pH Values of the Test Item Preparations after Stirring and Prior to the Addition of Inoculum in the Range-Finding Test (see table 1)
Nominal Concentration (mg/L Prior to Adjustment After Adjustment
10 6.9 7.3
100 7.5 -
1000 R1 8.2 7.7
1000 R2 8.2 7.9
1000 R3 8.2 7.8
R1 – R3 = Replicates 1 to 3
- no adjustment required



pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-Finding Test (see table 4)

pH
Nominal Concentration (mg/L 0 Hours 3 Hours
10 7.8 7.8
100 7.8 7.7
1000 R1 7.6 7.5
1000 R2 7.6 7.5
1000 R3 7.6 7.5


R1 – R4 = Replicates 1 to 4

Dissolved oxygen:

Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test (see table 2)
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
Test Item 10 6.3 71
100 6.4 72
1000 R1 5.9 66
1000 R2 5.6 63
1000 R3 6.3 71
R1 – R4 = Replicates 1 to 4

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test (for test item, for full data see table 3)
Nominal Initial O2 Measurement Final O2 O2 %
Concentration Reading Period Reading Consumption Rates Inhibition
(mg/L) (mg O2/L) (minutes) (mg O2/L) (mg O2/L/hour)
10 5.7 6 2.0 37.00 [1]
100 4.8 4 2.1 40.50 [10]
1000 R1 4.3 4 1.6 40.50 [10]
1000 R2 4.5 4 1.9 39.00 [6]
1000 R3 4.2 4 1.5 40.50 [10]
[Increase in respiration rate as compared to controls] R1 – R4 = Replicates 1 to 4

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Nominal Concentrations 10, 100, 1000 R1, 1000 R2, 1000 R3
R1 – R4 = Replicates 1 to 3

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

Remarks on result:

other: 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.

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

Remarks on result:

other: see Remarks The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a No Observed Effect Concentration (NOEC) after 3 hours exposure of 1000 mg/L.

Details on 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 item after 3 hours contact time 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 range-finding test are given in the attached Table 5.
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.
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.
Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol (see attached Figure 1).

Validation Criteria
The coefficient of variation of oxygen uptake in the control vessels was 4.7% and the specific respiration rate of the controls was 24.45 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

Inhibition of Respiration Rate
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)
EC10 >1000 - 1.8 -
EC20 >1000 - 2.5 -
EC50 >1000 - 7.6 6.0 – 9.7
EC80 >1000 - 23 -
NOEC 1000 - - -

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 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.

Results with reference substance (positive control):

The reference item gave a 3-Hour EC50 value of 7.6 mg/L, 95% confidence limits 6.0 to 9.7 mg/L.

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:

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 measured temperatures of between 19 and 21 Deg 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 EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after

3 hours exposure was 1000 mg/L.

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

The reference item gave a 3-Hour EC50 value of 7.6 mg/L, 95% confidence limits 6.0 to 9.7 mg/L.

Description of key information

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.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

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 measured temperatures of between 19 and 21 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 EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after

3 hours exposure was 1000 mg/L.

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

The reference item gave a 3-Hour EC50 value of 7.6 mg/L, 95% confidence limits 6.0 to 9.7 mg/L.