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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 11 to 20, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Complete read across justification is attached in section 13. Source study has reliability 1.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on test solutions:
No stock solution could be prepared due to the low water solubility. Therefore, test item amounts of 5.02, 16.06, 50, 160 and 500 mg were directly weighed into the test flasks. Then 284 ml of deionized water was added to each flask. The test item was mixed into the deionized water by ultrasonic treatment for 15 minutes and intense stirring for 24 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. Then, 16 ml of synthetic wastewater and activated sludge inoculum were added.

A stock solution of 3,5-dichlorophenol was prepared according to the test guidelines: 0.5 g of 3,5-dichlorophenol was dissolved in 10 ml 1 M NaOH and diluted to about 30 ml with purified water. Excess of NaOH was neutralized with 0.5 mol/l H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to one liter with purified water. The pH was determined to be pH 7.7 and the final concentration amounted to 500 mg/l. Aliquots of this 3,5-dichlorophenol stock solution were mixed with synthetic wastewater and deionized water in the respective test flasks to obtain the desired concentrations.


Synthetic wastewater:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2 × 2H2O
0.2 g MgSO4 × 7H2O
2.8 g K2HPO4

filled up to a final volume of 1 liter with deionized water
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater.
The sludge was washed once with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During holding, the sludge was fed daily for two days with 50 ml synthetic wastewater per liter. On the third day of holding immediately prior to use, the sludge was fed once again with 25 ml synthetic wastewater per liter. During the entire holding period, the sludge was kept at room temperature under continuous aeration. Immediately before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge was adjusted from 8.4 to 6.3 with a diluted sulfuric acid solution.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21 °C
pH:
7.6 - 8.4
Dissolved oxygen:
7.2 - 8.4 mg/l
Nominal and measured concentrations:
The final nominal test item concentrations were 10, 32, 100, 320 and 1000 mg/l. Concentrations in excess of 1000 mg test item per liter were not tested.
Additionally, two controls (deionized 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.
Details on test conditions:
Test system
- Test vessel: 1000-ml glass flasks
- Aeration: continous aeration with compressed air at a flow of ca. 1 liter per minute
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 0.7 g per liter test medium

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
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:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: down to the lowest test concentration of nominal 10 mg/l part of the test item was suspended in the test medium. Thus, all test concentrations were clearly above the water solubility limit of test substance.
- Blank controls oxygen uptake rate: oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 6.6 % (guideline-recommended maximum variation: 15 %).


Results with reference substance (positive control):
The 3-hour EC50 of the reference item) was calculated to be 17.9 mg/l (the 95 % confidence limits were not calculable). The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/l, confirming suitability of the used activated sludge.

Up to and including the highest concentration of nominal 1000 mg/l the test item had no significant inhibitory effect (< 15 %) on the respiration rate of activated sludge after the incubation period of 3 hours. Thus, the 3-hour EC50 is clearly higher than the limit of water solubility of test substance under the present test conditions, or higher than nominal 1000 mg/l. The 3-hour NOEC to activated sludge microorganisms is at least at the limit of water solubility, or at least nominal 1000 mg/l.

test item nominal conc. mg/l O2 consumption rate % inhibition pH values mg O2/l
start end start end
control 0 0.666 7.6 8.4 7.8 7.6
0 0.710 7.8 8.4 7.8 7.4
test substance 10 0.756 -9.9 7.7 8.4 7.9 7.6
32 0.807 -17.3 7.8 8.4 7.8 7.2
100 0.792 -15.1 7.8 8.4 8.0 7.4
320 0.693 -0.7 7.8 8.4 7.9 7.2
1000 0.766 -11.3 7.8 8.4 8.0 7.2
3,5-dichlorophenol 5 0.644 6.4 7.6 8.4 7.9 7.5
16 0.326 52.6 7.6 8.4 8.0 7.9
50 0.121 82.4 7.6 8.3 7.7 8.4
Conclusions:
NOEC (3h) = limit of water solubility under test conditions
EC50 (3h) > limit of water solubility under test conditions
Executive summary:

Method

Aerobic wastewater microorganisms of activated sludge exposed to test substance in a 3 -hour respiration inhibition test according to OECD guideline 209. Nominal tested concentrations were: 10, 32, 100, 320 and 1000 mg/l. Concentrations in excess of nominal 1000 mg/l were not tested. In addition, 2 controls and 3 different concentrations of the reference item 3,5-dichlorophenol (5, 16 and 50 mg/l) were tested in parallel.

Results

The results of these treatments confirmed suitability of the activated sludge and the method used. Up to and including the highest concentration of nominal 1000 mg/l, test substance had no significant inhibitory effect (< 15 %) on the respiration rate of activated sludge after the incubation period of 3 hours. Based on the physical appearance of test suspensions, the saturation concentration (equilibrium) under present test conditions was reached and resulted in less than 15 % inhibition after 3 hours contact time. Thus, the 3-hour EC50 is clearly higher than the limit of water solubility of test substance, or higher than nominal 1000 mg/l. The 3-hour NOEC to activated sludge microorganisms is at least at the limit of water solubility, or at least nominal 1000 mg/l. This value might be even higher, but concentrations in excess of nominal 1000 mg/l were not tested. The 3-hour EC20 and EC80 could not be calculated, but are clearly higher than the limit of water solubility.

Description of key information

No inhibition at the water solubility limit, after 3 h.

NOEC (3h) = water solubility limit (or highest test concentration of 1000 mg/l).

EC50 (3h) > water solubility limit.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information