Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-10-08 to 2002-01-18
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Commission directive 87/302/EEC, Part C, Activated Sludge Respiration Test, 1988.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate from The Swiss GLP Monitoring Authority.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study reports): T002102
- Physical state: solid
- Appearance: white (SDS)

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: According to the results of a pre-test, the test item was soluble at a concentration of at least 5 g/L in tap water. The stock solution of T002102 with a nominal concentration of 5 g/L was prepared: 1000 mg of T002102 were completely dissolved in 200 mL of tap water and thoroughly mixed by intense stirring. In this way a clear slightly yellowish solution was obtained. Adequate volumes of this stock solution were diluted with tap water to prepare the test media with the lower test item concentrations. Additionally, two controls (tap water, synthetic wastewater and activated sludge inoculum, without test substance) and the reference item 3,5-dichlorophenol (positive control) at nominal concentrations of 5, 16 and 50 mg/L were tested in parallel under identical test conditions.

Test organisms

Test organisms (species):
sewage, domestic
Details on inoculum:
- Laboratory culture: aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, FOllinsdorf, Switzerland) treating predominantly domestic wastewater.
- Preparation of inoculum for exposure: The sludge was washed once with tap water by centrifugation and the supernatant liquid phase was decanted
- Pretreatment: A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of the wet to dry weight was calculated. During the holding of two and a half days prior to use, the sludge was fed with 50 mL synthetic wastewater* / liter / day and was kept at room temperature under continuous aeration until use. Immediately before use, the dry weight of the activated sludge was measured again in the inoculum used in the test. The pH of the activated sludge inoculum was adjusted from 7.7 to 6.3 with a diluted sulfuric acid solution.
- Initial biomass concentration: 3g of dry material per liter

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
not applicable

Test conditions

Test temperature:
20 deg C
pH:
Start: 7.3 - 7.6
End: 8.1 - 8.4
Dissolved oxygen:
Start: 8.4 - 8.8
End: 7.4 - 8.5
Nominal and measured concentrations:
Nominal concentrations: 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000-mL glass flasks, 500-mL fill-volume
- Aeration: continuously with compressed air at a flow of ca. 1 L/min
- Number of replicates: 1
- Number of control replicates: 2
- Biomass loading rate: initial loading ca. 3.9 g dw/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic wastewater composition is sumarized below and was filled up to a final volume of 1 liter with deionized water:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCI
0.4 g CaCI2 . 2H2O
0.2 g MgS04 . 7H2O
2.8 g K2HP04

OTHER TEST CONDITIONS
- Adjustment of pH: pH of the activated sludge inoculum was adjusted from 7.7 to 6.3 with a diluted sulfuric acid solution.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration rate measured after 3 h of exposure (well-mixed sample poured into a BOD flask and not further aerated, dissolved oxygen concentration measured with oxygen electrode (WTW OXI 539 meter, Weilheim, Germany) and continuously recorded for 10-15 min).

TEST CONCENTRATIONS
- Range finding study: yes, results not shown
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3.5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
167 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: 93-248 mg/L 95% confidence limits
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
774 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: 515-1462 mg/L 95% confidence limits.
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium: no
- 3-h EC20 = 223 mg/L (136- 326 mg/L 95% confidence limits)
- 3 h EC80 > 1000 mg/L
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 14 mg/L with 95% confidence limits from 8.0 to 23 mg/L. The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming the suitability of the activated sludge used.
Reported statistics and error estimates:
The NOEC (3-hour EC15), the 3-hour EC20-, EC50- and EC80-values for the test item and the reference item and their 95%-confidence limits were calculated as far as possible by Probit Analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The inhibitory effect of the test item T002102 on the respiration rate of aerobic wastewater microorganisms of activated sludge resulted in a 3-h EC50 of 774 mg/L (515-1462 mg/L 95 % confidence limits). The 3-h NOEC was determined to be 167 mg/L (93-248 mg/L 95% confidence limits)