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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares LA 300 (phenol, methylstyrenated)
- Lot/batch No.: 28166
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water extract as Water Accommodated Fraction (WAF)
- Eluate: The WAF for the Activated Sludge, Respiration Inhibition Test as Limit Test was prepared by stirring an amount of 100 mg/L
of the test item in tap water for 24 h with a magnetic stirrer.
The test loading rate of the test substance was weighed on a weighing scoop that afterwards was placed into the glass beaker.
The beaker was then filled with tap water. The mixing was carried out at a speed that was slow enough not to cause dispersion
or emulsification of the undissolved fraction of the test item. To ensure this, the vortex developed at the surface by stirring was set
at ~ 10 % of the water depth. After stirring for 24 h the WAF was allowed to stand for 1 h before use to facilitate phase separation.
The extract gained with this method was clear.
- Differential loading: yes, only one loading tested
- Controls: yes without test substance
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
activated sludge
Details on inoculum:
- Source: Activated sludge from a sewage treatment plant (Kläranlage Oberes Echaztal, Pfullingen)
- Pretreatment: The activated sludge was washed three times with tap water.
- Initial biomass concentration: suspended solids (dry weight) ~ 4 g/L
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
10 min for determination of respiration inhibition:
After 3 hours of aeration the contents of the test vessels were transferred into a measuring apparatus,
and the respiration rate was measured and recorded over a period of up to 10 minutes.
Test temperature:
19.6 - 20.5 °C (20 +-2 °C)
no data
Dissolved oxygen:
> 6.5 mg/L
Nominal and measured concentrations:
100 mg/L (nominal)
Details on test conditions:
- Test vessel:
- Type (delete if not applicable): open
- Material, size, fill volume: 2L glass beaker, test volume 0.5 L
- Aeration: yes
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: dry weight of suspended solids 4 g/L (+-10 %)
- Conditioning: The test vessel was conditioned with the WAF for 0.5 h in order to saturate the potential adsorptive capacity of the glass walls.
The spent medium was exchanged for fresh WAF-containing test medium.

- Source/preparation of dilution water: Tap water (blank controls) or WAF/tapwater (test) supplemented with "synthetic sludge feed"
(cocktail of peptone, meat extract urea an minerals) in deionised water.

- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: Inhibition of O2-consumption (respiration)

Reference substance (positive control):
Key result
3 h
Dose descriptor:
other: NOELR (No Observed Effect Loading Rate)
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
other: WAF of 100 mg test material/L
Basis for effect:
inhibition of total respiration
respiration rate
Key result
3 h
Dose descriptor:
other: EL50 (Effect Loading at 50 % inhibition)
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
other: WAF of 100 mg test material/L
Basis for effect:
inhibition of total respiration
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 14.4 mg/L

The EC50 (EC = Effective Concentration) of the Reference item 3,5-dichlorophenol was determined byusing Probit-analysis (Finney-method, log-normal distribution; confidence limit 95 %, significance level: 0.05)
Concentr. Respiration Inhibition (%)
(mg/L) [mg O2/(L x h)
3 61.5 11.3
10 45.7 34.1
30 18.0 74.1
Reported statistics and error estimates:
not applicable

Results of the Activated Sludge, Respiration Inhibition Test of Novares LA 300

Loading rate (mg/L)

Respiration rate

[mg O2/(L x h)]

Inhibition (%)

Control 1




Control 2




Test item




Remark: * With reference to the validity criterion of OECD-Guideline 209 that

recommends a deviation of respiration rates of the two controls of ≤15 %,

the result of inhibition of theTest Item of 6.5 % was not regarded as a significant inhibition.

Validity criteria fulfilled:
- Deviation between respiration rates of the controls: 12.2 % (nominal value ≤ 15 %); - EC50 (3 h) of 3,5-dichlorophenol: 14.4 mg/L (nominal value 5 - 30 mg/L); - Dissolved oxygen in all vessels: >= 6.5 mg/L (nominal value >=3 mg/L).
Water Accommodated Fractions (WAFs) of Novares LA 300 caused no acute toxic effects on bacteria in an Activated Sludge, Respiration Inhibition Test as Limit Test according to OECD-Guideline 209. A 3h EL50 of > 100 mg/L and a NOELR of 100 mg/L have been determined.

Description of key information

Due to substance properties, toxicity to microorganisms is determined based on loadings. The effective loading resulting in no observable effect (NOELR) or in 50 % inhibition of total respiration (EL50) was identified to be 100 mg/L or > 100 mg/L test substance, respectively.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

The substance 'Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol' (OAPP) (previous name phenol, methylstyrenated) is a UVCB substance with a complex and within limits variable composition. Water solubility is low and not clearly defined due to the differing solubility of individual components. Depending on the quantity used, concentration of material dissolved in saturated water samples is variable. Therefore, water accommodated fractions (WAFs) have been used in aquatic toxicity tests. Thus, results are presented as loadings (EL50, NOELR).

Toxicity to microorganisms has been tested in a valid GLP guideline study (OECD TG 209) with one concentration (loading) of OAPP (LA 300) (limit test). The NOELR was determined to be 100 mg/L while the EL50 was > 100 mg/L based on inhibition of total respiration.