Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

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:
10 February 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:
The test material was insufficiently soluble to permit addition via aqueous solution. Individually weighed quantities of test substance were added to the appropriate test vessels.
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 predominatly domestic catchment. The point of collection was to ensure that the activated sludge samples was relatively free of exogenous material.
- Date of collection: Sludge for the range-finder test was collected on 25 February 2016 and sludge for the definitive test was collected on 15 March 2016.
- Preparation and maintenance of inoculum: 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 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 7.61 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 with dechlorinated tap water and was within nominal range of 3 ± 0.3 g/L. The pH of the sludge was 7.48, 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 1 000 mg/L
The nominal concentrations used for the definitive test were 9.77, 31.25, 100, 320 and 1 000 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: Test vessels were 250 mL glass conical flasks.
- Material, size, headspace, fill volume: 250 mL
- Aeration: At appropriate intervals, test samples were inoculated and aerated immediately. 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: No replicates
- No. of vessels per vehicle control: 6 replicates
- Suspended solid concentration of activated sludge: After addition of the test substance, inoculum and additional water, the nominal suspended solids concentration was 1.5 g/L.
- Nutrients provided for bacteria: A synthetic sewage concentrate concentrate at a rate of 50 mL/L 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.
- Preparation of test vessels: Each vessel contained a synthetic sewage concentrate, dechlorinated tap water, test substance or reference substance and nitrification inhibitor, as appropriate. The reference substance and nitrification inhibitor were added to the test system in aqueous solution. Each vessel contained surplus test preparation than required for the number of samples assessed.
The test substance was insufficiently soluble to permit addition via aqueous solution. Individually weighed quantities of test substance were added to the appropriate test vessels. For the range-finder test, the vessels were prepared at concentrations of 1, 10, 100 and 1 000 mg/L. For the definitive test the vessels were prepared at concentrations of 9.77, 31.25, 100, 320 and 1 000 mg/L. Each vessel was used as an individual replicate for respiration assessments. Immediately after preparation, the pH of each vessel was determined.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water
- Culture medium different from test medium: No
- Water quality measurement: Immediately after preparation, the pH of each vessel was determined.

OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment
- Nitrification Inhibitor: The nitrification inhibitor, N-allylthiourea (ATU), 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.

EFFECT PARAMETERS MEASURED:
- Measurement of Respiration Rates: 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.
- Calculation of Percentage of Inhibition: The percentage inhibition, IT, of total oxygen consumption at each concentration of test substance, was calculated as follows:

IT = [1 – (RT – RTA)/RTB] x 100 %

The percentage of heterotrophic oxygen uptake, IH, at each concentration of test substance was calculated where appropriate, as follows:

IH = [1 – (RH – RHA/RHB)] x 100 %

The inhibition of oxygen uptake due to nitrification, IN, at each concentration was calculated, where appropriate, as follows:

IN = [1 – (RT – RH)/(RTB – RBH)] x 100 %

TEST CONCENTRATIONS
Range finding study:
- Test concentrations: 1, 10, 100 and 1 000 mg/L
- Results used to determine the conditions for the definitive study: Yes. Total, heterotrophic and nitrification respiration rates were similar to blank controls across treatment levels up to, and including 100 mg/L. At the top concentration of 1 000 mg/L, respiration rates were noticeably higher. There was an indication of a slight effect (≤ 8 % for total respiration, ≤ 18 % for heterotrophic respiration but negligible nitrification respiration (≤ 1 %)) at the 10 and 100 mg/L test concentrations, but negative inhibition was observed at 1 and 1 000 mg/L.
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:
Single preparations were tested containing the reference substance (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.:
> 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
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 heterotrophic respiration
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 respiration due to nitrification
Details on results:
Inhibition of total and nitrification respiration was observed at the lower test concentrations (54 % inhibition of total respiration and 108 % inhibition of nitrification respiration were observed for the 9.77 mg/L test concentration, for example) but these effects were confirmed not to be statistically significant. In addition, it was noted that the degree of inhibition reduced with increasing test concentration. Statistical analysis confirmed there were no significant differences between the controls and any treatment level, for any respiration type.
The EC50 for total, heterotrophic and nitrification respiration could not be determined statistically and is therefore classed as > 1 000 mg/L, which is the highest concentration used in this test.
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: 13.5 mg/L
Nitrification Respiration: 0.1 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 NOEC for the test substance was based on both visual assessment of the data as well as from calculated data. The EC50 for total, heterotrophic and nitrification respiration could not be determined statistically due to the inhibition observed decreasing with increasing test concentration and was reported as greater than the highest test concentration.

Validity Criteria

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.

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 1 000 mg/L. The NOEC value was determined to be 1 000 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 (≤ 8 % for total respiration, ≤18 % for heterotrophic respiration) and negligible nitrification respiration (≤ 1 %). Negative inhibition was observed at 1 and 1 000 mg/L for both total and nitrification respiration. Based on the results of the range-finding test, a 3 hour definitive toxicity test was conducted at concentrations of 9.77, 31.25, 100, 320 and 1 000 mg/L.

The validity criteria applied to this study type were met and therefore the data are considered valid.

In the definitive test, inhibition of total and nitrification respiration was observed at the lower test concentrations (54 % inhibition of total respiration and 108 % inhibition of nitrification respiration were observed for the 9.77 mg/L test concentration, for example) but these effects were confirmed not to be statistically significant. Furthermore, the degree of inhibition reduced with increasing test concentration. As there was no statistically significant difference between the controls and all treatment levels, determination of EC50 was not possible.

Under the conditions of the test, the EC50 is therefore reported to be greater than 1 000 mg/L (the highest concentration of test material under these conditions) and the NOEC, based on statistical analysis, is reported to be 1 000 mg/L.

Description of key information

The EC50 is therefore reported to be greater than 1 000 mg/L (the highest concentration of test material under these conditions) and the NOEC, based on statistical analysis, is reported to be 1 000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 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 (≤ 8 % for total respiration, ≤18 % for heterotrophic respiration) and negligible nitrification respiration (≤ 1 %). Negative inhibition was observed at 1 and 1 000 mg/L for both total and nitrification respiration. Based on the results of the range-finding test, a 3 hour definitive toxicity test was conducted at concentrations of 9.77, 31.25, 100, 320 and 1 000 mg/L.

The validity criteria applied to this study type were met and therefore the data are considered valid.

In the definitive test, inhibition of total and nitrification respiration was observed at the lower test concentrations (54 % inhibition of total respiration and 108 % inhibition of nitrification respiration were observed for the 9.77 mg/L test concentration, for example) but these effects were confirmed not to be statistically significant. Furthermore, the degree of inhibition reduced with increasing test concentration. As there was no statistically significant difference between the controls and all treatment levels, determination of EC50 was not possible.

Under the conditions of the test, the EC50 is therefore reported to be greater than 1 000 mg/L (the highest concentration of test material under these conditions) and the NOEC, based on statistical analysis, is reported to be 1 000 mg/L.

Categories Display