Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Creosote
Creosote WEI-Type B, containing less than 50 ppm BaP
- Substance type: organic
- Physical state: liquid
- Composition

Name CAS-No. Concentration [w%]
RUETGERS Chemicals // Biochem. Inst.Grimmer
=================================================
Naphthalene 91-20-3 5.07 5.39
Fluorene 86-73-7 3.06 3.07
Acenaphthylene 208-96-8 -- 0.09
Acenaphthene 83-32-9 4.09 4.04
Phenanthrene 85-01-8 8.95 8.56
Anthracene 120-12-7 0.45 0.38
Fluoranthene 206-44-0 3.70 3.84
Pyrene 129-00-0 2.49 2.08
Benz[a]anthracene 56-55-3 0.04 0.04
Chrysene 218-01-9 0.03 0.03
Benzo(a)pyrene 50-32-8 <10 ppm ca. 6. ppm
----------------------------------------------------------------

Total of 5 remaining EPA-PAH: --- < 20 pp
----------------------------------------------------------------
(Benzo[b]fluoranthene 205-99-2
Benzo[k]fluoranthene 207-08-9
Indeno[1,2,3-cd]pyrene 193-39-5
Dibenzo[a,h]anthracene 53-70-3
Benzo[ghi]perylene) 191-24-2
===========================================

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
--

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:
= incubation time of the test solutions (test substance + inoculum) under aeration prior to measuring
Post exposure observation period:
--

Test conditions

Hardness:
--
Test temperature:
18.0 - 18.1 °C
pH:
8.3 - 8.4
Dissolved oxygen:
parameter to be measured
Salinity:
--
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

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


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
yes
Remarks:
3,5- dichlorophenol (5 - 30 mg/L)

Results and discussion

Effect concentrations
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 %
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: No inhibition, but about >40-% stimulation (10 mg/L);
26-% inhibition (100 mg/L);
50-% inhibition (1000 mg/L)
Results with reference substance (positive control):
- Results with reference substance valid? yes, at 5, 15, 30 mg/L
- Relevant effect levels: EC50 approx. 10 mg/L

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

Applicant's summary and conclusion