Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
22 July 2010
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Appearance: colourless or light yellow viscous liquid
Storage conditions: at room temperature, container tightly closed, flushed with nitrogen.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the test sample (0.40, 2.0, 10, 50 and 250 mg), synthetic sewage (8 mL), purified water (117 mL) and the activated sludge (125 mL) were added to each test vessel.
- Controls: a blank control, containing only synthetic sewage, purified water and activated sludge, was used.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Kurume central sewage treatment center (Kurume-shi, Fukuoka, Japan)
- Method of collection: sludge was collected from an aeration reactor on the day before testing in accordance with Japanese Industrial Standards (JIS), K 0102:2016 Section 14.1
- Preparation of inoculum for exposure: the activated sludge (5L) was fed with synthetic sewage (250 mL) and cultivated for 20.7 hours at 20 ± 2 °C under aerobic conditions in order to acclimate to the test conditions. The sludge was not acclimated to the test item.
- Pretreatment: no
- Initial biomass concentration: 3300 mg/L (concentration of suspended solids)

Study design

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

Test conditions

Test temperature:
20 ± 2 °C
Nominal and measured concentrations:
Nominal concentrations (test substance): 1.6, 8.0, 40, 200 and 1000 mg/L
Nominal concentrations (reference substance): 4.0, 8.0, 16 and 32 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: approx. 300 mL glass vessels, fill volume: 250 mL
- Aeration: by air pump at a flow rate of 0.5-1 L/min
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (3 replicates at the beginning and 3 at the end of the exposure period each)
- No. of vessels per reference substance (replicates): 1 per concentration
- Sludge concentration (weight of dry solids per volume): 105,6 mg/L
- Weight of dry solids per volume of reaction mixture per unit of time:
- Nutrients provided for bacteria: yes, synthetic sewage, according to OECD 209
- Nitrification inhibitor used: none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified water containing synthetic sewage

OTHER TEST CONDITIONS
- Adjustment of pH: medium was not adjusted since the pH was in the acceptable range, the pH of the test solution was adjusted to 7.5 with 1 mol/L sodium hydroxide

EFFECT PARAMETERS MEASURED:
Directly after the 3-hour exposure period, the measurement bottle (102 mL glass incubator bottle) was gently filled with the test solution to avoid foaming and the concentration of dissolved oxygen was measured continuously for 10 minutes under continuous stirring.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes, percentages of respiration inhibition were 13%, 24% and 38%, respectively. Based on these results, the EC50 was expected to be > 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (purity: 99.7%)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- The average percentage of inhibition for the 3 replicates per concentration were 6, 1, 14, 20 and 47% for 1.6, 8.0, 40, 200 and 1000 mg/L, respectively. The percentage of inhibition found in the highest test concentration was significantly different from the blank control (p<0.01).
- Blank controls oxygen uptake rate: 47.8 mg O2/L/h (n=6)
- The variation in inhibition of respiration between replicates may be caused by differences in dissolution of the test material en differences in degradation products between test bottles.
- The effect concentrations were based on nominal loading rates because the test substance was poorly water soluble.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Concentration range tested: 4.0, 8.0, 16 and 32 mg/L
- Relevant effect levels: EC50 = 10 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks, attachments'
Conclusions:
The EC50 and the NOEC for inhibition of respiration by STABiO D-376N were determined to be >1000 mg/L and 200 mg/L, respectively.
Executive summary:

In an activated sludge respiration inhibition test of STABiO D-376N, performed according to OECD test guideline 209 and GLP principles, activated sludge from a municipal sewage treatment receiving predominantly domestic sewage, was exposed to the test substance for 3 hours. The test substance was weighed and added directly to the test vessels together with synthetic sewage, purified water and activated sludge. The following nominal concentrations were used: 1.6, 8.0, 40, 200 and 1000 mg/L. Three replicates per test concentration were used next to one replicate of the reference item (3,5 -dichlorophenol) and six replicates of the blank control.

After 3 hours of exposure, the concentration of dissolved oxygen was measured for 10 minutes, with continuous stirring of the test solution, in measurement bottles. The percentage of inhibition of respiration, compared to the blank controls, was determined by calculation. Statistical analysis was used to determine a significant difference between the test solutions and blank controls. The percentage of inhibition found in the highest test concentration was significantly different from the blank control (p<0.01).

The average percentage of inhibition for the 3 replicates per concentration were 6, 1, 14, 20 and 47% for 1.6, 8.0, 40, 200 and 1000 mg/L, respectively.

The EC50 and the NOEC for inhibition of respiration by STABiO D-376N were determined to be >1000 mg/L and 200 mg/L, respectively.

All acceptability criteria were met and the study was considered to be valid.