Amides, tall-oil fatty, N,N'-(iminodi-2,1-ethanediyl)bis-, phosphates

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

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:

Dec 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item amounts of 5.02, 50.23, 500.34, 500.13 and 499.99 mg were weighed onto glass slides by means of an analytical balance and transferred into the designated test vessels. Glass beads (2 mm diameter) were used in order to enhance the distribution of the test item in the incubation mixture. Therefore, approximately 5 g of glass beads and 3 mL of water were added to the test item into the test vessels and the vessels were shaken for 30 minutes. Then, 231 mL water was added to each vessel and the content was intensely stirred for 60 minutes at room temperature to dissolve and finely disperse the maximum practical amount of the test item. No emulsifiers or solvents were used.
After stirring, all preparations were clearly above the water solubility limit of the test item under the test conditions. Test item lumps were still present for the nominal test item concentrations of 100 and 1000 mg/L.
The pH of the test dispersions was in the range 7.7-7.9.
- Controls: containing an equal volume of activated sludge and synthetic medium but no test or reference item.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: local wastewater treatment plant, ARA Birs (Birsfelden / Switzerland)
- Method of cultivation / Preparation of inoculum for exposure: The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in chlorine free tap water. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined.
Based on this determination, a calculated amount of wet sludge was suspended in chlorine free tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of one day prior to use, the sludge was fed once with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again and readjusted to the level of 3 g dry weight per liter in the inoculum used for the test, equivalent to 1.5 g/L in the final incubation mixture. The pH of the activated sludge inoculum was 7.3.
- Initial biomass concentration: 3.0 g/L (dry weight)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 - 22 °C

pH:

test start: 7.3 - 7.4
test end: 7.5 - 7.9

Dissolved oxygen:

test start: 8.0 - 8.4 mg/L
test end: 6.6 - 7.7 mg/L

Nominal and measured concentrations:

nominal: 10, 100, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 2 L beakers, contents 500 mL
- Type (delete if not applicable): open
- Aeration: intense stirring with magnetic followers
- No. of vessels per concentration (replicates): 10 and 100 mg/L: 1; 1000 mg/L: 3
- No. of vessels per control (replicates): 4
- Sludge concentration (weight of dry solids per volume): 1.5 g/L (dry weight)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water (<1 mg/L DOC)

OTHER TEST CONDITIONS
- Adjustment of pH: no
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : dissolved oxygen concentration after 3 hours contact time, continuously recorded for a period of up to 10 minutes or until the oxygen concentration fell below 2 mg/L

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: in conformity with the guidelines, a definitive test was not necessary since in the range-finding test the test item showed no relevant inhibition of the respiration rate at the highest tested concentration

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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

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:

- Blank controls oxygen uptake rate: 24 - 26 mg O2/gh
- Coefficient of variation of oxygen uptake rate in control replicates: 5 %

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h): 6 mg/L (within the guidelines-recommended range of 2–25 mg/L)

Reported statistics and error estimates:

The percentage inhibition of the respiration was plotted against logarithm of the test substance concentration using the software ToxRat® Professional (ToxRat® Solutions GmbH, Version 3.2.1).
The NOEC was derived by per-vessel statistics using the Student t-test. The three replicates of the highest test item concentration were compared with the four blank control replicates using one-tailed (smaller) hypothesis and α = 0.05.
The 3-hour EC10, EC20, EC50 and EC80 values of the test item and their 95 %-confidence limits could not be calculated because of the absence of a significant toxic effect of the test item on the respiration rate of activated sludge at the tested concentrations.
The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated by Probit Analysis.
All test results were based on nominal concentrations.

At all tested concentrations up to and including 1000 mg/L, the inhibition of the respiration rate of activated sludge after the incubation period of three hours was in the range -0.8 to 11.4 % compared to the control (see attached document "Table Oxygen Consumption of Activated Sludge.pdf"). For the three replicates of the highest test concentration of 1000 mg/L the mean inhibition of the respiration rate was calculated to be 4.6 %, compared to the control. According to the results of a Student t-test (one sided smaller, α = 0.05), the test item had no statistically significant inhibitory effect on the respiration rate of up to and including the highest test item concentration of 1000 mg/L. Concentrations exceeding 1000 mg/L were not tested in accordance with the guidelines.

Validity criteria fulfilled:

yes

Remarks:

The specific respiration rates in the controls were ≥20 mg O2/g/h and the coefficient of variation was ≤ 30 %.

Description of key information

Toxicity to microorganisms: not toxic to microorganisms: EC50 > 1000 mg/L, NOEC ≥ 1000 mg/L (OECD 209, EU C.11)

Additional information

EC50 for microorganisms > 1000 mg/L

NOEC for microorgansims ≥ 1000 mg/L