Amines, (2-ethylhexyl)(hydrogenated tallow alkyl)methyl

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 27, 2016 to September 16, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

1078712-76-1

Molecular formula:

C23-27H49-59N (Substance is a UVCB)

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge collected from the Easton Wastewater Treatment Facility, Easton, Maryland on
July 27, 2016 was utilized as the inoculum for the test. The Easton facility receives wastes from
predominately domestic sources. The sludge was sieved using a 2 mm screen and allowed to settle for
30 minutes. The supernatant above the settled solids was removed and the total suspended solids (TSS)
concentration of the settled sludge was determined. Total suspended solids in the settled sludge were
adjusted to a nominal concentration of approximately 3000 mg/L by dilution with municipal water. Fifty
mL of synthetic sewage (Appendix I of Protocol) was added to each liter of adjusted sludge. The sludge
was maintained at a temperature of 20  2C and continuously aerated overnight. The pH and total
suspended solids concentration of the activated sludge were determined on the day of use in the study.

Study design

Test type:

static

Limit test:

no

Total exposure duration:

3 h

Test conditions

Details on test conditions:

Test mixtures were prepared at 15 minute intervals starting with the first control. The control
contained 16.0 mL of synthetic sewage, 234 mL of municipal water, and 250 mL of inoculum for a total
volume of 500 mL. The mixture was promptly aerated at a rate sufficient to keep the solids in suspension.
Subsequent reference and test substance dosed biotic mixtures were prepared by combining 16.0 mL of
synthetic sewage, the appropriate volume of reference substance stock solution or appropriate amount of
test substance, and enough municipal water needed to achieve a volume of 250 mL was added. After the
municipal water was added, 250 mL of inoculum was added to bring the total volume up to 500 mL.
The abiotic mixture contained 16.0 mL of synthetic sewage, 0.5000 g of test substance, and 484 mL
of municipal water for a final volume of 500 mL. Finally, a second control was prepared in the same
manner as the first control. All mixtures were aerated for three hours.

Results and discussion

Effect concentrations

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

Results with reference substance (positive control):

EC50 of 22.6 mg/L with 3,5-dichlorophenol

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The potential effect of Amines, (2-ethylhexyl)(hydrogenated tallow alkyl)methyl (from this point

on referred to Amines MHTEH) on activated sludge microorganisms maintained in an aerobic

environment was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline

209). The test contained control, reference and treatment groups. The control replicates were used to

determine the background respiration rate of the sludge and were not dosed with the test or reference

substance. The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at

concentrations of 3, 15 and 50 mg/L. The treatment group was dosed with Amines MHTEH at

concentrations of 10, 100, and 1000 mg/L. The 1000 mg/L treatment was tested in triplicate. An abiotic

control was dosed with the test substance at a concentration of 1000 mg/L to discriminate between abiotic

uptake by the test substance and microbial respiration. After an exposure period of three hours, the

respiration rates of the test solutions were measured using an YSI Model 5000 Dissolved Oxygen Meter.

The respiration rates in the two controls were 29.6 and 27.2 mg O2/L/hr. Both controls had

respiration rates above 26.87 mg O2/L/hr, which is the low limit based on the validity criteria for mixtures

with 1.34 g dry weight of suspended solids per liter. The coefficient of variation of the two control

respiration rates was 5.9%, and was within the 30% limit established for the test. The validity of the test

was further supported by the results from the 3,5-dichlorophenol reference group, which resulted in an

EC50 value of 22.6 mg/L, with 95 percent confidence limits of 15 mg/L and 50 mg/L. The EC50 for the

reference substance was within the 2 to 25 mg/L range considered acceptable for the test.

An inhibitory dose response effect was not observed for the 10 mg/L, 100 mg/L, or 1000 mg/L

treatment groups. The EC50 value for Amines MHTEH is greater than 1000 mg/L, the highest

concentration tested. The abiotic treatment mixture dosed with 1000 mg/L of Amines MHTEH had a

respiration rate of 0.0 mg O2/L/hr showing there was no significant uptake or release of oxygen resulting

from abiotic reactions of the test substance.