Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-11-05 to 2002-01-25
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 87/302lEEC
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): T002113
- Physical state: pale yellow solid/powder
- Analytical purity: 92.9%
- Lot/batch No.: RT002113G3A231
- Expiration date of the lot/batch: 2001-12-01
- Stability under test conditions: results from a hydrolysis pre-experiment (without GLP), indicated that, at a pH of 7, the test item concentration decreased rapidly within two hours by about 50% and thereafter stayed constant for at least five days (RCC Study No. 825265).
- Storage condition of test material: at room temperature, 20°C, away from direct sunlight
Other:
* Solubility in water: 120 g/L
* pH in aqueous solution: 3.8 at a concentration of 120 g/L
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RT00211G3A231
- Expiration date of the lot/batch: 2001-12-01 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature at about 20°C, away from direct sunlight
- Stability under test conditions: Determined in RCC study 825265
- Solubility and stability of the test substance in the solvent/vehicle: in water 120 g/L (non-GLP, sponsor's information; determined in RCC study 825030)

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: no stock solution could be prepared due to insufficient water solubility. Test item amounts were directly weighed into the test flasks. Then, 284 mL of tap water and 16 mL of synthetic wastewater were added to each flask. The test item was mixed by intense stirring for ten minutes at room temperature. Then, 200 mL of activated sludge inoculum was added. The stirring was kept short because according to the results of a pre-test the test item was not stable in water.
- Controls: two controls (tap water, synthetic wastewater and activated sludge inoculum, without test item) and the reference item 3,5-dichlorophenol (positive control) at nominal concentrations of 5, 16 and 50 mg/L were tested in parallel under otherwise identical test conditions.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: predominantly domestic wastewater treatment plant, ARA Ergolz II, Füllinsdorf, Switzerland
- Preparation of inoculum for exposure: washed once with tap water by centrifugation, supernatant liquid phase decanted, homogenized aliquot of final sludge suspension weighed, dried, and ratio wet to dry weight calculated.
- Pretreatment: an aliquot of washed sludge was suspended in tap water at 3 g dw/L, holding 3 days prior to testing, fed daily with 50 mL synthetic wastewater per L, kept at room temperature and continuous aeration.
- Initial biomass concentration: 2.3 g dw/L test medium
- The pH of the activated sludge inoculum was adjusted from 8.4 to 6.9 with a diluted sulfuric acid solution.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Hardness:
not applicable
Test temperature:
21-22°C
pH:
8.0-8.6
Dissolved oxygen:
The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period, and just before measurement of the respiration rates the dissolved oxygen concentrations were at least
7.2 mg/L.
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: Control, 11, 34, 100, 320, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL glass flasks, 500 mL fill-volume
- Aeration: continuously aerated by intense stirring on magnetic stirrers.
- Number of replicates: 5
- Number of control replicates: 2
- Biomass loading rate: initial loading ca. 2.3 g dw/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water + synthetic wastewater, as prescribed by guidelines

OTHER TEST CONDITIONS
- Adjustment of pH: the pH of the activated sludge inoculum was adjusted from 8.4 to 6.9 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
- Spacing factor for test concentrations: x 3.2
- Range finding study
- Test concentrations: 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
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
696 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.L.: 398-1902 mg/L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
325 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 47-529
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: no data
- The 3h-EC20 = 375 mg T002113/L (95% CL 77-607 mg T002113/L)
- The 3h-EC80 = 1293 mg T002113/L (95% CL 766-16012 mg T002113/L - The upper CL should be valued critically since the inhibition of the respiratory rate was about 70% at the highest test concentration)
Results with reference substance (positive control):
3-h EC50 = 20 mg/L (95% CI = 3-449 mg/L).
This is within the recommended range of 5-30 mg/L confirming suitability of the activated sludge used.
Reported statistics and error estimates:
The 3-h EC50 values and their 95% confidence intervals were as far as possible calculated by Probit Analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
An activated sludge respiration inhibition test (according OECD guideline 209) with activated sludge from a predominantly domestic wastewater treatment plant resulted in a 3-h EC50 of 696 mg T002113/L. The results of the study can be considered reliable without restriction.