Tar bases, coal, lutidine fraction

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:

Experimental start: 16 December 2015, Experimental termination: 12 January 2016, Report date: 07 March 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))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Nominal amounts of test substance were dissolved in water and the volumes adjusted to 2 liters to give stock solutions of appropriate concentrations from which dilutions were made to produce further stock solutions. An aliquot of 200 mL of each stock solution was then mixed with appropriate volumes of synthetic sewage, activated sludge and water to 500 mL to obtain the appropriate test concentrations.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: not applicable
- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water Plc sewage treatment plant, Loughborough, Leicestershire, United Kingdom
- Method of cultivation: activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of about 21 °C overnight prior to use in the test. On the day of collection the sludge (10 L) was fed synthetic sewage (500 mL)
- Preparation of inoculum for exposure: not applicable
- Pretreatment: not applicable
- Initial biomass concentration: 3.0 g/L prior to use

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable

Hardness:

not reported

Test temperature:

ca. 20 °C

pH:

Activated sewage sludge sample: 7.5
Stock solutions of test substance: 7.4 to 7.9 (after pH adjustment)
Test vessels at start: 7.1 to 7.7
Test vessels at end: 7.8 to 8.2

Dissolved oxygen:

Oxygen concentrations in all vessels were measured after 30 minutes contact time.
Controls: 5.4 to 5.8 mg/L
Test item: 5.4 to 6.9 mg/L

Salinity:

not applicable

Conductivity:

not reported

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Material, size, headspace, fill volume: 500 mL; the test item was prepared by dispersing an aliquot of 200 mL of the appropriate stock solution 16 mL of synthetic sewage, 250 mL of activated sewage sludge and dilution water to obtain a final volume of 500 mL to give the required test concentrations
- Aeration: the mixtures were aerated with clean, oil-free compressed air via a narrow bore glass tube at a rate of 0.5 to 1.0 liter per minute
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable
- No. of vessels per abiotic control (replicates): not applicable
- Sludge concentration (weight of dry solids per volume): 3.0 g/L
- Nutrients provided for bacteria: a synthetic sewage was prepared by dissolving the following constituents in 1 liter of water: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g sodium chloride, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the range-finding and definitive tests was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
- Particulate matter: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: the pH of stock solutions was adjusted to a value between 7.0 and 8.0, if necessary
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study: a range finder was conducted before the definitive test was carried out
- Test concentrations: range finder concentrations were 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: range finder results were used to select appropriate test concentrations in the definitive test

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

6.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- No undissolved test substance visible
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption (e.g. of test material to the walls of the test container): no
- Blank controls oxygen uptake rate: 33.0 mg oxygen per gram dry weight of sludge per hour
- Coefficient of variation of oxygen uptake rate in control replicates: 4.19% of the mean

Results with reference substance (positive control):

The following effect concentrations were obtained with the positive control substance 3,5-dichlorophenol: 3-hour EC50 = 9.2 mg/L (7.1-12 mg/L); 3-hour EC10 = 2.0 mg/L; 3-hour EC80 = 29 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The test substance inhibited the respiration of activated sewage slude. The 3-hour EC50 value was 75 mg/L and the 3-hour NOEC was 3.2 mg/L

Executive summary:

The effect of the test substance on the activity of micro-organisms in activated sewage sludge was tested in an Activated sludge respiration inhibition test in accordance to OECD TG 209 (version 2010) under GLP. The experiment is considered relevant, adequate and conclusive.

Sewage sludge from a public sewage treatment plant processing predominantly domestic waste water was used in the study. The activated sewage sludge was sampled on the day before the tests and aerated and fed with synthetic sewage after sampling. On the day of the tests, activated sewage sludge was mixed with appropriate amounts of synthetic sewage, freshly prepared stock solutions of test item and water to a final volume of 500 mL to achieve appropriate test concentrations. In a preliminary test, test concentrations of 10, 100 and 1000 mg/L were applied and a significant inhibiting effect on the respiration of the activated sewage sludge was found at all test concentrations after a 3-hour exposure period. A definitive test with the test substance was then conducted at about 20 °C over a period of 3 hours at nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L, and also the positive control substance 3,5-dichlorophenol was tested at concentrations of 3.2, 10 and 32 mg/L. The dissolved oxygen concentrations in all vessels after 30 minutes ranged from 5.4 to 5.8 mg/L in the controls and from 5.4 to 6.9 mg/L in the vessels containing the test substance, and all oxygen concentrations were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/liter. Oxygen consumption rates and percentage inhibition values were determined after 3 hours of exposure, and the specific respiration rate of the blank controls was 33 mg oxygen per gram dry weight of sludge per hour and the coefficient of variation of oxygen uptake rate in the control replicates was 4.2%. A 3-hour EC50 value of 75 mg/L and a 3-hour NOEC of 3.2 mg/L were found for the test substance, and the 3-hour EC50 value for the positive control substance was 9.2 mg/L (7.1 - 12 mg/L 95% confidence limit). All validation criteria were met, as the EC50 value of the positive control was in the range from 2 to 25 mg/L, the specific respiration rate of the blank controls was not less than 20 mg oxygen per gram dry weight of sludge per hour, and the coefficient of variation of oxygen uptake rate in the control replicates was less than 30%.

Description of key information

3-hour EC50 = 75.0 mg/L, activated sewage sludge, Best 2016 (OECD TG 209, GLP)

3-hour NOEC = 6.1 mg/L, activated sewage sludge, Best 2016 (OECD TG 209, GLP)

Key value for chemical safety assessment

EC50 for microorganisms:

75 mg/L

EC10 or NOEC for microorganisms:

6.1 mg/L