Castor Oil, reaction product with Soybean Oil

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:

2011-03-10 till 2011-04-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

(1984)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Direct weighings were prepared to give the different test item concentrations (10, 100, 1000 mg/L). The substance was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 16 hours.
For the reference compound 3,5-Dichlorophenol a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg reference compound in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water. The pH was adjusted to pH 7 ± 0.5 with HCl.

Test organisms (species):

activated sludge

Details on inoculum:

- Type: mixed population of aquatic microorganisms
- Origin: aeration tank of a domestic sewage treatment plant (Municipal STP Cologne-Stammheim)
- Date of collection: 2011-03-14
- Microbial inoculum: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 4500 rpm and 20 °C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
- Storage of sludge: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- pH of the suspension before application: 7.7

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

no data

Test temperature:

19.6 - 20.6 °C (including all test concentrations, controls and reference compound)

pH:

7.5 - 7.9 (including all test concentrations, controls and reference compound)

Dissolved oxygen:

4.1 - 8.1 mg O2/L at the beginning of the test (including all test concentrations, controls and reference compound)
2.5 - 8.1 mg O2/L at the end of the test (including all test concentrations, controls and reference compound)

Nominal and measured concentrations:

10, 100 and 1000 mg/L (nominal)

Details on test conditions:

Exposure conditions
- Test item concentration/s: 10, 100 and 1000 mg/L
- Test item concentration in physico-chemical oxygen consumption control: 1000 mg/L
- Concentration of reference compound 3,5-Dichlorophenol: 5, 10 and 20 mg/L

The test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations.

- Test vessels: 300 mL glass Erlenmeyer flasks
- Method of application: direct weighing
- Test concentration of the activated sludge: 800 mg/L suspended solids
- Test temperature: 20 ± 2 °C
- Stirring period of the test item before start of incubation time: 16 hours
- Incubation time: 3 hours with permanent aeration

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (purity: 99.6 %; batch no.: 04621CJ)

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

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The respiration rate for each concentration was determined graphically from the linear part of the curve of the oxygen content versus time. The inhibitory effect of the test item at a particular concentration is expressed as a percentage of the mean of the respiration rates of two controls. An EC50 value was calculated from the respiration rates at different test item concentrations using the statistics programme ToxRatPro Version 2.10 (released 2010-02-20).

Validity criteria fulfilled:

yes

Remarks:

Respiratory rates of the 2 controls differ less than 15 % from each other; EC50 of the reference compound 3,5-Dichlorophenol is in the range 5 – 30 mg/L

Conclusions:

The toxicity to aquatic microorgenisms of the substance showed 0.0 % respiration inhibition of activated sludge at the highest test item concentration of 1000 mg/L.

Executive summary:

The purpose of this test was to provide a rapid screening method whereby substances which may adversely affect aerobic microbial treatment plants can be identified, and to indicate suitable non-inhibitory concentrations of test items to be used in biodegradability tests.

The method assessed the effect of the substance on microorganisms by measuring the respiration rate under defined conditions in the presence of different concentrations of the test item.

To measure the oxygen consumption, 250 mL of sludge with the test item (or control or reference compound) was incubated for 3 h in 300 mL closed Erlenmeyer flasks (with air inlet and outlet) and aerated through a glass tube at 50-100 L/h with clean oil-free air. For the measurement, the content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles and oxygen content was measured with an oxygen meter (redox electrode) with writer.

Two controls without the test item were included in the test design, one at the start and the other at the end of the test series. Each batch of activated sludge was checked using 3,5-Dichlorophenol as a reference compound.

Since some substances may consume oxygen by chemical reactivity, a physico-chemical oxygen consumption control was carried out additionally. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was tested without activated sludge.

In order to test the toxicity to microorganism of the substance, activated sludge from predominantly domestic sewage plant was exposed to the test solution of three nominal concentrations of the substance (10, 100 and 1000 mg/L), blank control solution and solution with positive reference substance for a period of 3 hours. Dissolved oxygen was recorded and respiration rates were calculated. No physico-chemical oxygen consumption was observed at the highest concentration of test substance. No respiration inhibition was observed at the highest test item concentration of 1000 mg/L.

Description of key information

No effect regarding the toxicity to aquatic microorganisms of the substance, was observed at the highest test item concentration of 1000 mg/L (0.0 % respiration inhibition of activated sludge).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

The key value should read EC50 > 1000 mg/L as there was no effect in this limit test.