Isooctadecanoic acid, reaction products with tetraethylenepentamine

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:

21 January 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Test solutions: the test material was weighed on to a glass microscope cover slip and added directly into each test vessel at the required concentration. 16 mL of the synthetic sludge feed was added together with200 mL of sludge inoculum. Vessels were made up to 500 mL with dechlorinated tap water. Aeration began immediately and the vessel placed in a temperature controlled incubator.
- Controls: a control group was maintained under identical conditions but not exposed to the test material.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Method of cultivation: Activated sludge was obtained from the Cottonwood Subdivision Wastewater Treatment Plant located in Franklin, Tennessee, USA, on the day of testing. The activated sludge was fed with 50 mL synthetic sewage feed per litre of sludge per day and aerated with low pressure, oil-free air.

A synthetic sewage mixture was prepared containing the following per litre of solution:
16.0 g peptone,
11 g beef extract,
3.0 g urea,
0.70 g NaCl,
0.40 g CaCl2.2H2O,
0.20 g MgSO4.7H2O,
2.80 g H2HPO4.

- Triplicate 4 mL samples of the mixed sludge were dried at 100 ºC, until a constant weight was achieved. Based on these results the sludge was diluted to produce a dry weight per unit volume concentration of 4 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

10 minute period, following the 3 hour exposure duration, to measure oxygen consumption.

Test temperature:

20 ºC

Nominal and measured concentrations:

1, 10, 100, 1000, 10000 ppm (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 L glass serum bottles.
- Type: closed.
- Fill volume: 500 mL
- Aeration: The flasks were aerated throughout the exposure period.
- No. of vessels per concentration: one.
- No. of vessels per blank control: duplicate.
- No. of vessels per positive control: one.
- Final solids concentration: 4 g/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water.

OTHER TEST CONDITIONS
- Photoperiod: the test was conducted in the dark.

EFFECT PARAMETERS MEASURED: The rate of respiration was measured after 3 hours contact time. An aliquot was removed from each test vessel and transferred into the BOD bottle containing a Teflon-coated magnetic stir bar. The bottle was capped with a BOD bottle oxygen probe to eliminate air space and placed on a magnetic stirring plate. The sludge mixture was stirred at constant rate and the consumption of oxygen over time was recorded on a strip chart recorder. The oxygen consumption readings were recorded for 10 minutes or until a linear trace covering a sufficient range of oxygen concentrations was obtained.

To measure the rate of oxygen consumption, a dissolved oxygen meter was used. The oxygen meter was fitted with a BOD bottle probe, fitted with a standard sensitivity membrane and an external output to a strip chart recorder. The strip chart recorder was set at the 100 mV range and run at 1 cm per minute. To measure the consumption of oxygen, a sub-sample of the exposed sludge was placed in a clean BOD bottle, the change in oxygen consumption over time was measured with the meter and probe, and the results recorded on the strip chart recorder.

The respiration rate was measured over the linear part of the curve for five to ten minutes. The rates for oxygen uptake were expressed at mg O2/L/h. The respiration rates of the flasks dosed with the test material, or the reference substance, were expressed as percentages of the mean respiration rate of the control flasks, from which the percentage inhibition was derived:

% inhibition = (1-(Respiration rate of test flask/Mean respiration rate of control flasks)) x 100

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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:

other: NOEL

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:

- Results of control vessels: the first control vessel showed a change of 4.4 mg/L for oxygen concentration for a 5 3/6 minute period. This equates to an oxygen consumption rate of 48.0 mg O2/L per hour. The second control vessel showed a change in oxygen concentration of 4.0 mg/L for 5 1/6 minutes. The second control thus had an oxygen consumption rate of 46.5 mg O2/L per hour. The average for the two controls was 47.3 mg O2/L per hour.

Results with reference substance (positive control):

- Results with reference substance valid? The results were valid.
- Relevant effect levels: The reference substance caused substantial inhibition of the respiration rate of the activated sludge. From the results obtained, the 3 hour EC50 was estimated to be 11.0 mg/L. This is within the expected normal range of 5 to 30 mg/L.

Reported statistics and error estimates:

The percentage inhibition data were not monotonically increasing and thus the EC50 could not be calculated. The EC50 was thus expressed as a greater than value.

To determine the No Observed Effect Level (NOEL) a t test was performed following the procedures of Sokal & Rohlf (1981). The test is a comparison between a single observation and the mean of a sample.

The results of the t test revealed that none of the test concentrations expressed a statistically significant inhibition of oxygen consumption as compared to the controls.

Table 1: Test Material Results

Test concentration (mg/L)	Change in O2 (mg/L)	Change in time (mins)	O2 consumption (mg/L per hour)	Percent inhibition
1	3.0	3 5/6	47.0	0.5
10	5.0	6 2/6	47.4	-0.3
100	3.9	5	46.8	1.0
1000	5.0	7	42.6	9.8
10000	4.0	5 3/6	43.6	7.7

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, none of the test concentrations expressed a statistically significant inhibition of oxygen consumption as compared to the controls. The No Observed Effect Level (NOEL) was therefore determined to be the highest concentration tested, 10000 mg/L. The EC50 was estimated to be in excess of 10000 mg/L. The study is considered to be reliable, relevant and adequate for risk assessment purposes.

Executive summary:

The effect of the test material on the respiration rate of activated sludge was investigated in accordance with the standardised guideline OECD 209. During the study, activated sludge was exposed to nominal concentrations of test material of 1.0, 10, 100, 1000 and 10000 mg/L; duplicate blank controls and a reference substance control (3,5-dichlorophenol) were run concurrently for comparison. The rate of respiration was determined after 3 hours contact time and compared to data for the control and reference material. Under the conditions of the study, none of the test concentrations expressed a statistically significant inhibition of oxygen consumption as compared to the controls. The No Observed Effect Level (NOEL) was therefore determined to be the highest concentration tested, 10000 mg/L. The EC50 was estimated to be in excess of 10000 mg/L. The reference substance caused substantial inhibition of the respiration rate of the activated sludge. From the results obtained, the 3 hour EC50 was estimated to be 11.0 mg/L. This is within the expected normal range of 5 to 30 mg/L confirming the validity of the test methods employed.

Description of key information

3 h EC50 = > 10000 mg/L, NOEL = 10000 mg/L, OECD 209, Goodrich 1993

Key value for chemical safety assessment

EC50 for microorganisms:

10 000 mg/L

EC10 or NOEC for microorganisms:

10 000 mg/L

Additional information

The effect of the test material on the respiration rate of activated sludge was investigated in accordance with the standardised guideline OECD 209. During the study, activated sludge was exposed to nominal concentrations of test material of 1.0, 10, 100, 1000 and 10000 mg/L; duplicate media controls and a reference substance control (3,5-dichlorophenol) were run concurrently for comparison. The rate of respiration was determined after 3 hours contact time and compared to data for the control and reference material. Under the conditions of the study, none of the test concentrations expressed a statistically significant inhibition of oxygen consumption as compared to the controls. The No Observed Effect Level (NOEL) was therefore determined to be the highest concentration tested, 10000 mg/L. The EC50 was estimated to be in excess of 10000 mg/L. The reference substance caused substantial inhibition of the respiration rate of the activated sludge. From the results obtained, the 3 hour EC50 was estimated to be 11.0 mg/L. This is within the expected normal range of 5 to 30 mg/L confirming the validity of the test methods employed. The study was performed in line with GLP and an accepted standardised guideline and assigned a reliability score of 1.