Anthracene oil

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Creosote

EC Number:

232-287-5

EC Name:

Creosote

Cas Number:

8001-58-9

Molecular formula:

not applicable

IUPAC Name:

Creosote

Test material form:

liquid

Details on test material:

- Substance type: organic

Specific details on test material used for the study:

- Name of test material (as cited in study report): Creosote
- Supplier: HGD S.A., France; Creosote WEI-Type B, containing less than 50 ppm BaP

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct addition and agitation by aeration in the presence of inoculum
- Differential loading: yes

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Cultivation: Activated sludge from the effluent of the aeration tank of the municipal WWTP at Taunusstein-Biederstadt/Germany.
The isolated sludge mixture (MLSS) (approx. 4 g/L dry mass) was pre-incubated in synthetic medium for 2 d.
The culture medium contained 8 mL synthetic sewage feed (prepared from peptone (16 g/L), beef extract (11 g/L), urea (3 g/L), mineral salts), 100 mL inoculum, and tap water for a total volume of 250 mL.
- Initial biomass concentration: approx. 1.5 g/L (dry mass).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

exposure duration corresponds to the incubation time of the test solutions (test substance + inoculum) under aeration prior to measuring

Test conditions

Test temperature:

18.0 - 18.1 °C

pH:

8.3 - 8.4

Dissolved oxygen:

parameter to be measured

Nominal and measured concentrations:

125, 250, 500, 1000, and 2000 mg/L (nominal, without separation of undissolved material) (derived from a pre-test).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed at measuring of O2 consumption
- Material, size, headspace, fill volume: 250-mL glass bottle with inlet for aeration
- Aeration: 3 h until start of determination of the respiration rate
- Measuring equipment: pH-Electrode: WTW portable; O2-Electrode: WTW OXI 530
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): Two parallel trials without test substance / one abiotic control containing 2000 mg creosote/L (without biomass)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic; the culture medium contained 8 mL synthetic sewage feed (prepared from peptone (16 g/L), beef extract (11 g/L), urea (3 g/L), mineral salts, 100 mL inoculum, and tap water for a total volume of 250 mL.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : O2-consumption

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study:
- Test concentrations/loadings: 10, 100, and 1000 mg/L
- Results used to determine the conditions for the definitive study:
10 mg/L: no inhibition, but about > 40 % stimulation
100 mg/L: ca. 26 % inhibition
1000 mg/L: ca. 50 % inhibition

Reference substance (positive control):

yes

Remarks:

3,5- dichlorophenol (5 - 30 mg/L)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

other: Non-inhibitory loading EL0

Effect conc.:

ca. 36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: derived from the dose-response curve (see att. doc.)

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

other: Inhibitory loading EL10

Effect conc.:

ca. 62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: derived from the dose-response curve (see att. doc.)

Effect concentrations 3

Key result

Duration:

3 h

Dose descriptor:

other: Inhibitory loading EL50

Effect conc.:

670 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% C.I. of the inhibition rate: approx. ± 12 %

Effect concentrations 4

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 33 mg/L

Nominal / measured:

estimated

Conc. based on:

dissolved

Remarks:

test material (WAF)

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: dispersion
- Effect concentrations exceeding solubility of substance in test medium: yes (at the EL50, the WAF is estimated to have been between 30 and 40 mg dissolved carbon/L, based on water solubility data)
- Pre-test:
- Test loadings: 10, 100, and 1000 mg/L
- Results: 10 mg/L: no inhibition, but about > 40 % stimulation
100 mg/L: ca. 26 % inhibition
1000 mg/L: ca. 50 % inhibition

Results with reference substance (positive control):

- Results with reference substance valid? yes (concentrations 5, 15, 30 mg/L)
- Relevant effect levels: EC50 approx. 10 mg/L

Reported statistics and error estimates:

Standard programme for variance and S.D.

Any other information on results incl. tables

Summary of effects data: Inhibition in % versus loadings of test substance:

Test [mg/l, nominal]	Respiration Rate [mg O2/(l*min), after 3-h incubation	Inhibition of Respiration [%]
Control [mean of 2]	0.84 0.91 av. 0.88	0
Abiotic Control [2000 mg/l]	0	--
125	0.71	18.9
250	0.54	38.4
500	0.46	47.4
1000	0.48	45.1
2000	0.22	74.9

It can be assumed that the water soluble fraction of creosote at 670 mg/L was about 5 %, which is in between the solubilities at loadings of 100 mg/L and 10,000 mg/L (2,5 - 8 mg C/L and 110 mg C/L). This gives an EC50 for the water soluble fraction of ca. 33 mg TOC/L (670 x 0.05). As a conservative approach, this value can be used as the creosote EC50 for inhibition of respiration in activated sludge.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

results of controls and with reference substance in acceptable range

Conclusions:

- Low doses appear to stimulate rather than to inhibit microbial activity under test conditions (see pre-test at 10 mg/l).
- Results of main test were in agreement with preliminary findings of the pre-test.
- The dose-response suggests no significant inhibition up to approx. 40 mg/L (nominal concentration) corresponding to a WAF concentration of 2 - 4 mg/L (not measured in the test itself)