Reaction products of cyclohexylamine and 4-alkylaniline and 1,1'-methanediylbis(4-isocyanatobenzene)

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:

From 2005-03-01 to 2005-03-08

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Substance type: Organic
- Physical state: off-white powder
- Composition of test material, percentage of components: component 1 ca 50%, component 2 ca 30%, component 3 ca 20%
- Lot/batch No.: 050210A
- Expiration date of the lot/batch: February 2007
- Storage condition of test material: room temperature in the dark

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

See "any other information on materials and methods"

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works, which treats predominantly domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required.

The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.

On the day of collection, aliquots (25 mL) of the activated sludge were filtered through dried and preweighed Whatman GF/C filter papers which were then dried again at approximately 105°C for one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the sample of activated sludge and the mixture aerated overnight.

On the day of the test, the MLSS content of the sludge was detennined and adjusted to 4 g/L by the addition of dechlorinated tap water. The pH of the sludge was also measured.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

See table 2 attached background information

pH:

3,5-DCP stock solution 7.6
Activated sludge 7.3

See table 2 attached background information

Dissolved oxygen:

See table 1 attached background material

Nominal and measured concentrations:

Nominal: 1, 10, 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / test beakers covered with loosely fitting aluminium foil
- Material, size, headspace, fill volume: one-litre size, fill volume approximately 600mL
- Aeration: yes for 3 hours
- No. of vessels per concentration (replicates): 1 at 1 and 10 mg/L; 3 at 100mg/L
- No. of vessels per control (replicates): 1 each for positive control, 2 for antifoam-treated control, 1 control without antifoaming agent
- Biomass loading rate: 4 g/L


TEST MEDIUM / WATER PARAMETERS
Synthetic sewage feed for activated sludge was prepared by dissolving the following in one litre of ultrapure water (nominal resistivity, >= 0: 18 MegOhm.cm):

peptone 16.0 g
meat extract 11.0 g
urea 3.0 g
sodium chloride 0.7 g
calcium chloride dihydrate 0.4 g
magnesium sulphate heptahydrate 0.2 g
di-potassium hydrogen phosphate 2.8 g

OTHER TEST CONDITIONS
- Adjustment of pH: none reported
- Photoperiod: none
- Light intensity: not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The rate of oxygen consumption was measured, over a period of approximately ten minutes or until the dissolved oxygen concentration fell below 2 mgO2/L, using a Yellow Springs Instruments (YSI) dissolved oxygen meter, with temperature probe and self-stirring bottle probe, connected to a chart recorder. The pH and temperature of the samples were measured at the start and end of the test.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The test item was considered to have had no biologically significant inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. Sludge respiration was decreased by 1% of the mean control rate in one mixtures containing test item at 100 mg/L. The EC20, EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 100 mg/L, the highest level tested.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 5.5 mg/L
- Other:
Specific respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The three-hour 50% effect concentration (EC50) for 3,5-DCP was to be 5.5 mg/L (95% confidence limits 3.6 - 8.2 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

The test item was considered to have had no inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. The EC20 , EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 100 mg/L, the highest level tested.

Executive summary:

The effect of test item on the respiration rate of activated sludge was assessed by the methods detailed in EC Directive 88/302 and OECD Test Guideline 209. Samples of activated sludge (suspended solids 1.6 g/litre), fed with synthetic sewage, were exposed to the test substance at nominal concentrations of 1, 10 and 100 mg/litre for three hours. Single mixtures were prepared at 1 and 10 mg/litre and the highest level was prepared in triplicate. Their rates of oxygen consumption were determined and compared with those of controls, containing activated sludge and synthetic sewage alone, which were established at the beginning and end of the culture series. The reference inhibitor 3,5-dichlorophenol (3,5-DCP) was employed at 3, 10 and 32 mg/litre, as a positive control. An antifoaming agent (silicone based, oil in water emulsion) was added to selected sludge mixtures to assist dispersal of the test substance and suppress foam development which may have caused the test substance to deposit above the surface of the liquors. The specific respiration rate of the antifoam-treated control cultures, established at the beginning and end of the test series (25.4 mgO2/g/h), and the control without antifoaming agent (25.4 mgO2/g/h) confirmed that the test precision was acceptable and that the antifoam had had no effect on the activity of the sludge inoculum. The three-hour 50% effect concentration (ECso) for 3,5-DCP was calculated to be 5.5 mg/L (95% confidence limits 3.6 - 8.2 mg/L). These results show that the test was valid and that the sample of activated sludge employed was sensitive to inhibition. The test item was considered to have had no biologically significant inhibitory effect on the respiration rate of activated sludge in the test. The specific respiration rate was decreased by 1% of the mean control value in one mixture containing the test substance at 100 mg/litre. The EC20, EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 100 mg/L, the highest level tested.

Description of key information

The effect of test item on the respiration rate of activated sludge was assessed by the methods detailed in EC Directive 88/302 and OECD Test Guideline 209. The test item was considered to have had no biologically significant inhibitory effect on the respiration rate of activated sludge in the test. The specific respiration rate was decreased by 1% of the mean control value in one mixture containing the test substance at 100 mg/litre. The EC20, EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 100 mg/L, the highest level tested.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

Additional information

The EC20, EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 100 mg/L, the highest level tested.