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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
A 2.00 mg a.i./mL primary stock solution of methyl isoamyl ketone was prepared by placing 2.0110 g of the test substance (2.0007 g as active ingredient) in a 1000-mL volumetric flask and bringing it to volume with purified reagent water. This primary stock solution was used to prepare test solutions for the definitive test. A 500 mg a.i./L stock solution of the reference substance 3,5-dichlorophenol was prepared by first placing 0.2526 g (0.2498 g as active ingredient) in 5 mL of 1 N sodium hydroxide. The solution was then brought to a volume of 10 mL with purified reagent water and stirred using a Teflon®-coated stir bar and magnetic stirrer. While the solution was stirring, 2.0 mL of 1 N sulfuric acid was added slowly to the solution until a precipitate was observed. The solution was then brought to 500 mL with purified reagent water. The pH of the resultant 3,5 dichlorophenol stock solution was 7.8 and required no adjustment.

At time zero, 16 mL of synthetic sewage feed was added to a 500-mL graduated cylinder and then brought to a volume of 300 mL with water. Inoculum was added until the solution was at full volume (500 mL). This mixture was added to a 1-L beaker and aeration was immediately initiated at 0.56 L/minute. This flask was identified as Control 1 (C1). The reference substance (3,5 dichlorophenol) sludge mixtures (1.0, 3.0, 10 and 30 mg a.i./L) were prepared by adding appropriate volumes of the 3,5-dichlorophenol stock solution to the sludge mixture containing 16 mL of synthetic sewage feed, laboratory water to 300 mL and 200 mL of inoculum for a final volume of 500 mL. In addition, an abiotic control was prepared by adding 250 mL of the 2.00 mg a.i./mL primary stock solution to the test system along with 16 mL of synthetic sewage feed and bringing to a final volume of 500 mL with water for a final concentration of 500 mg a.i./L This flask did not contain inoculum. Control 2 (C2) was prepared in the same manner as Control 1 fifteen minutes after the last reference substance test system was prepared. Lastly, the test substance test systems were initiated following the same procedure as reference substance mixtures with the exception that the appropriate amount of the test substance (rather than reference substance) was added to the sludge mixture. All test and reference substance concentrations were based on nominal levels.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
On the day prior to test initiation, approximately 8 L of activated sludge was obtained from the Wareham Waste Water Treatment Plant (WWTP), Wareham, Massachusetts, which receives primarily domestic sewage. Before use, the sludge solution was passed through a 2-mm stainless steel sieve, concentrated by centrifuging for 10 minutes at 1000 rpm, and the resulting supernatant poured off. The sludge was washed with unchlorinated tap water and centrifuged at 1000 rpm for 10 minutes, for a total of four times, with the supernatant being discarded each time. A small amount of the washed sludge was weighed and dried to calculate the quantity of wet sludge required to be suspended in 2.5 L of water to obtain an activated sludge containing a solid level of four grams per liter (± 10%). The inoculum received 125 mL of a 1/100 dilution of the synthetic sewage feed, was aerated overnight, and used for testing the next day. The pH of the sludge inoculum used in testing was 6.2 and the solids content was determined to be 4.1 g/L.
Limit test:
no
Test temperature:
20.5C
pH:
Controls: 6.77 and 7.00
MIAK: 6.97-7.19
Reference substance: 6.94-7.13
Nominal and measured concentrations:
Nominal only: 0.1, 1.0, 10, 100 and 1000 mg a.i./L
Details on test conditions:
Following three hours of aeration, a 23-mL aliquot of each sludge mixture was transferred to a 50 mL cylinder which sat inside a 20 C water bath placed over a stir plate. The pH of the sludge mixtures were measured after the respiration measurement. The cylinders received a stir bar and were fitted with a dissolved oxygen probe after receiving the sample. The probes were attached to a Strathkelvin Instruments Model 928 oxygen system and a computer which ran the Strathkelvin software. The Strathkelvin system was used to monitor the “SI Multisample Activated Sludge Respirometer”. Immediately after the dissolved oxygen probes were attached to the Strathkelvin system, the computer program was initiated so that oxygen depletion rates were measured starting at time zero. The oxygen depletion rate was terminated after a 10 minute period. The sludge mixtures were stirred continuously while the oxygen measurements were taken. The temperature of the environmental chamber in which the test was conducted was monitored using a minimum/maximum thermometer. Solution pH was measured with a Yellow Springs Instrument (YSI) pH100 pH meter. An SI Multisample Activated Sludge Respirometer was used for determination of oxygen consumption. The oxygen depletion rate was measured in a single sample from each test system such that there was one replicate per test system with zero headspace. The oxygen consumption rate was calculated for each control, test substance, and reference substance concentration by using the dissolved oxygen concentration versus time curves. The percent inhibition was calculated at each concentration of test substance and reference substance. The nominal concentrations of methyl isoamyl ketone (MIAK) or 3,5-dichlorophenol and the corresponding measurements of oxygen consumption were used to estimate a median effect concentration (EC50). The EC50 is the estimated concentration of the test substance or reference substance which inhibits the respiration of the microorganisms present in activated sludge by 50%, as compared to the respiration in the control vessels.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
At test termination, the respiration rate for the two control vessels was determined to be 21.8 and 20.3 mg O2/L/Hr, which met the acceptability criterion (i.e., within 15% of each other during the test). Respiration rate in the 0.1, 1.0, 10, 100 and 1000 mg a.i./L test substance vessels was 17.6, 19.3, 24.1, 19.2 and 19.2 mg O2/L/hr, respectively. Compared to the controls, respiratory inhibition for the 0.1, 1.0, 10, 100 and 1000 mg a.i./L test substance vessels was 17.1, 9.0, 0.0, 9.5 and 9.4%, respectively. Based on these results, the EC50 value was calculated by the Strathkelvin program and determined to be > 1000 mg a.i./L. The EC15 (equivalent to a No-Observed-Effect-Concentration) was determined by linear regression to be > 1000 mg a.i./L.
Results with reference substance (positive control):
Respiration rate in the 3,5-dichlorophenol test vessels (1.0, 3.0, 10 and 30 mg a.i./L) was 12.1, 13.8, 6.4 and 2.1 mg O2/L/hr, respectively. Compared to the controls, respiratory inhibition for the 3,5 dichlorophenol test vessels was 42.4, 34.6, 69.6 and 89.9%, respectively. Based on these results, the EC50 value for 3,5 dichlorophenol was calculated by the Microsoft Excel program and determined to be 5.1 mg a.i./L, which was within the acceptable limits (5 to 30 mg a.i./L) as specified in OECD 209 Guideline for the reference material.
Validity criteria fulfilled:
yes
Executive summary:

An activated sludge respiration inhibition test following OECD Guideline 209 was conducted on methyl isoamyl ketone (CAS No.110-12-3). The test was conducted using nominal concentrations of 0.1, 1.0, 10, 100 and 1000 mg a.i./L. Following three hours of aeration the oxygen depletion rate was determined during a 10-minute period. The percent inhibition was calculated at each concentration of test substance and reference substance. Based on these results, the EC50 value for methyl isoamyl ketone was determined to be greater than 1000 mg a.i./L. The EC15 (NOEC) value was calculated by linear regression in Excel and determined to be greater than 1000 mg a.i./L. The EC50 value for the reference substance, 3,5dichlorophenol, was calculated to be 5.1 mg a.i./L, which was within the acceptable limits (5 to 30 mg a.i./L) as specified in the OECD 209 Guideline. 

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

An activated sludge respiration inhibition test following OECD Guideline 209 was conducted on methyl isoamyl ketone (CAS No.110-12-3). The test was conducted using nominal concentrations of 0.1, 1.0, 10, 100 and 1000 mg a.i./L. Following three hours of aeration the oxygen depletion rate was determined during a 10 minute period. The percent inhibition was calculated at each concentration of test substance and reference substance. Based on these results, the EC50 value for methyl isoamyl ketone was determined to be greater than 1000 mg a.i./L. The EC15 (NOEC) value was calculated by linear regression in Excel and determined to be greater than 1000 mg a.i./L. The EC50 value for the reference substance, 3,5-dichlorophenol, was calculated to be 5.1 mg a.i./L, which was within the acceptable limits (5 to 30 mg a.i./L) as specified in the OECD 209 Guideline.