Fatty acids, tallow, compds. with triethanolamine

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:

26 January 2016 to 19 August 2016

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 (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test material was deemed insufficiently soluble to permit addition via aqueous solution. Individually weighed quantities of test substance were added to the appropriate test vessels.
- Controls: The reference substance and nitrification inhibitor were added to the test system in aqueous solution.
- Chemical name of vehicle: Not applicable
- Concentration of vehicle in test medium: For the range-finder test, the vessels were prepared at concentrations of 1, 10, 100 and 1000 mg/L. For the definitive test, the vessels were prepared at concentrations of 3.05, 9.77, 31.25, 100 and 320 mg/L.
- Evidence of undissolved material: None specified.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Collected from the sludge return line at Burley Menston sewage treatment works (West Yorkshire, UK), which has a predominantly domestic catchment. The point of collection was to ensure that the activated sludge sample was relatively free of exogenous material.
- Method of cultivation: The sludge was transported to the test facility in a closed container with an adequate headspace to prevent the sample becoming anoxic. On arrival at the test facility, the sludge was aerated with a compressed air supply delivered through a suitable distribution device.
The suspended solids concentration was determined gravimetrically following homogenisation and adjusted using dechlorinated tap water to 3 g/L (±0.3 g/L). The sludge was maintained at 20 ± 2 °C with aeration. During storage it was maintained at 20 ± 2 °C and fed with synthetic sewage concentrate at a rate of 50 mL/L.
The sludge for the range-finder test was collected on 03 February 2016. The sludge for the definitive test was collected on 01 March 2016.
- Preparation of inoculum for exposure: The suspended solids concentration was determined before the range-finder test. The concentration was 3 g/L after adjustment with dechlorinated tap water and was within the nominal range of 3 ± 0.3 g/L. The pH of the sludge was 7.37 and was within the acceptable range of 7.5 ± 0.5. In the definitive test, the suspended solids concentration was 3 g/L after adjustment and was within nominal range of 3 ± 0.3 g/L. The pH of the sludge was 7.52 within the range of 7.5 ± 0.5.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

7.5 ± 0.5

Nominal and measured concentrations:

The nominal concentrations used for the range-finder test were 1, 10, 100 and 1000 mg/L
The nominal concentrations used for the definitive test were 3.05, 9.77, 31.25, 100 and 320 mg/L.
Each vessel containing the nominal concentration was used as an individual replicate for respiration assessments.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Material, size, headspace, fill volume: 250 mL
- Aeration: Each culture was aerated for about 3 hours by bubbling air through the test system. The rate of aeration was sufficient to keep the test samples adequately mixed.
- No. of vessels per concentration: 1 replicate
- No. of vessels per control: Single preparations were tested containing the reference substance at 0.1, 2.0 and 40 mg/L.
- No. of vessels per vehicle control: 6 blank controls were prepared to enable measurement in eight vessels during the test series.
- Nutrients provided for bacteria: A synthetic sewage concentrate was used to feed the activated sludge during storage and to provide a uniform respiration substrate in the test cultures. The packets of pre-made synthetic sewage are added to water at 1 packet per 250 mL of water.
- Sludge concentration: The suspended solids concentration was 3 g/L after adjustment with dechlorinated tap water and was within the nominal range of 3 ± 0.3 g/L. After addition of the test substance, inoculum, and additional water, the nominal suspended solids concentration was 1.5 g/L.
- Nitrification inhibitor used: N-allylthiourea was added to appropriate test and reference vessels to give a final concentration of ca 11.6 mg/L ATU (actual 11.7 mg/L) in the test system.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water.
- Particulate matter: Not specified

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the sludge was 7.52 within the range of 7.5 ± 0.5.

EFFECT PARAMETERS MEASURED: At the end of the 3-hour incubation period, a portion (20 mL) of each test preparation was transferred to an appropriate sample tube containing a PTFE stirrer. For those samples requiring amendment with ATU, this was undertaken at least 10 minutes prior to oxygen consumption measurement. The DO (dissolved oxygen) electrode was sealed in the neck of the flask, ensuring air was completely excluded. The flask contents were stirred at a constant rate during DO measurements. Oxygen consumption was measured over a period of up to 10 minutes.
Respiration rates (mg O2/L/h) were determined by measuring the gradient of the linear portion of the graph. Measurements at high (>7.0 mg/L) or low (<2.0 mg/L) DO levels were avoided, where possible. The Strathtox instrumentation generated both graphical data as well as calculated results.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Based on range-finding study.
- Range finding study: Yes
- Test concentrations: 1, 10, 100 and 1 000 mg/L
- Results used to determine the conditions for the definitive study: The total and nitrification respiration rates for the test material concentrations 10, 100 and 1 000 mg/L were below the range observed for the blank control samples. Inhibition of respiration relative to the blank controls was therefore observed at these concentrations. There was negative inhibition noted for heterotrophic respiration at the 10 and 1 000 mg/L concentration levels.
The validity criteria (blank control respiration rate and the coefficient of variation between replicates) were met; the results of the range-finder test can therefore be considered to be valid.

Reference substance (positive control):

yes

Remarks:

3,5-DCP at nominal concentrations of 0.1, 2.0 and 40 mg/L.

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: NOAEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

There were no significant effects on total, heterotrophic or nitrification respiration at any concentration in the definitive test. For nitrification respiration, there was 34 % inhibition observed at 31.25 mg/L and 37 % inhibition observed at 320 mg/L. However, neither of these results was determined to be statistically significant and, due to the negative inhibition values seen for the other concentrations tested, were considered to be indicative of the intrinsic variability of data for this study type.

As there was <50 % inhibition observed in the definitive test, the EC50 for total, heterotrophic and nitrification respiration could not be determined statistically and is classed as > 320 mg/L, which is the highest concentration level used in this test.

The NOAEC (No Observed Adverse Effect Concentration) were therefore determined to be:
Total Respiration: 320 mg/L
Heterotrophic Respiration 320 mg/L
Nitrification Respiration: 320 mg/L

The validity criteria (blank control respiration rate and the coefficient of variation between replicates) were also satisfied; the results of the definitive test can therefore be considered to be valid.

Results with reference substance (positive control):

- Results with reference substance valid? Yes. Reference substance (3,5-DCP) inhibition was observed to be within the validity criteria:
Total Respiration: 4.9 mg/L
Nitrification Respiration: 0.7 mg/L

Reported statistics and error estimates:

ESTIMATION OF NO-EFFECT AND EFFECT CONCENTRATIONS
Reference substance
Estimation of the EC50 for the reference substance was calculated from concentration versus effect for total and nitrification respiration for the range-finder test as well as the definitive test. The EC50 was therefore based on a statistical analysis (linear interpolation) of concentration versus effect in total respiration and nitrification respiration.
Test substance
The NOAEC for the test substance was based on both visual assessment of the data as well as from calculated data. Estimation of the EC50 for the test substance was derived using statistical analysis (linear interpolation) of concentration versus effect.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, there were no statistically significant effects on respiration observed during the definitive test. The 3 hour EC50 for the test material was greater than 320 mg/L. The NOAEC value was determined to be 320 mg/L, the highest concentration tested.

Executive summary:

The potential of the test material to cause toxic effects to microorganisms was investigated in accordance with the standardised guideline OECD 209 under GLP conditions.

The activated sludge inoculum was collected from a sludge return line which has a predominantly domestic sewage catchment. The point of collection was to ensure that the activated sludge sample was relatively free of exogenous material. A range-finder test, employing nominal test material concentrations of 1, 10, 100 and 1 000 mg/L, was undertaken to determine the appropriate concentration levels for a definitive test. The range-finding test showed limited inhibition of total or heterotrophic respiration but did indicate inhibition of nitrification respiration.

Based on the results of the range-finding test, a 3 hour definitive toxicity test was conducted at concentrations of 3.05, 9.77, 31.25, 100 and 320 mg/L. Reference substance inhibition, blank control respiration rate and the coefficient of variation between replicates were observed to be within the validity criteria. The results of the definitive test can therefore be considered to be valid. As enhancement effects were noted (observed as negative inhibition), the endpoints are reported as EC50 and No Observed Adverse Effect Concentration (NOAEC).

Under the conditions of the test, there were no statistically significant effects in heterotrophic, nitrification or total respiration inhibition observed during the definitive test, thus determination of EC50 was not possible. The 3 hour EC50 for the test material was therefore greater than 320 mg/L. The NOAEC value was determined to be 320 mg/L, the highest concentration tested.

Description of key information

Under the conditions of the test, there were no statistically significant effects in heterotrophic, nitrification or total respiration inhibition observed during the definitive test, thus determination of EC50 was not possible. The 3 hour EC50 for the test material was greater than 320 mg/L. The NOAEC value was determined to be 320 mg/L, the highest concentration tested.

Key value for chemical safety assessment

EC50 for microorganisms:

320 mg/L

Additional information

The potential of the test material to cause toxic effects to microorganisms was investigated in accordance with the standardised guideline OECD 209 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The activated sludge inoculum was collected from a sludge return line which has a predominantly domestic sewage catchment. The point of collection was to ensure that the activated sludge sample was relatively free of exogenous material. A range-finder test, employing nominal test material concentrations of 1, 10, 100 and 1 000 mg/L, was undertaken to determine the appropriate concentration levels for a definitive test. The range-finding test showed limited inhibition of total or heterotrophic respiration but did indicate inhibition of nitrification respiration.

Based on the results of the range-finding test, a 3 hour definitive toxicity test was conducted at concentrations of 3.05, 9.77, 31.25, 100 and 320 mg/L. Reference substance inhibition, blank control respiration rate and the coefficient of variation between replicates were observed to be within the validity criteria. The results of the definitive test can therefore be considered to be valid. As enhancement effects were noted (observed as negative inhibition), the endpoints are reported as EC50 and No Observed Adverse Effect Concentration (NOAEC).

Under the conditions of the test, there were no statistically significant effects in heterotrophic, nitrification or total respiration inhibition observed during the definitive test, thus determination of EC50 was not possible. The 3 hour EC50 for the test material was greater than 320 mg/L. The NOAEC value was determined to be 320 mg/L, the highest concentration tested.