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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not applicable
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLPs
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):Not applicable
Analytical monitoring:
no
Details on sampling:
The contents of the first control vessel were transferred to a BOD bottle and dissolved oxygen concentrations were measured every 10 seconds over a 10 minute period, or until the DO measurement dropped below 1.0 mg O2/L using a YSI Model 50B-115 Dissolved Oxygen Meter. The respiration rate in subsequent vessels was determined in an identical manner at 15 minute intervals. The contact time for each concentration of reference or test substance with the activated sludge was three hours.
Vehicle:
yes
Details on test solutions:
The test substance was dosed by volumetric addition using a stock solution of the test substance prepared in NANOpure® water at a nominal concentration of 2000 mg/L. This method was deemed appropriate based on the solubility of the test substance in water.Test mixtures were prepared at 15 minute intervals starting with the first control. The control contained 16.0 mL of synthetic sewage, 200 mL of inoculum, and enough municipal water to bring the total volume up to 500 mL. The mixture was promptly aerated at a rate sufficient to provide aerobicconditions and keep the solids in suspension. Subsequent mixtures contained 16.0 mL of synthetic sewage, 200 mL of inoculum, the appropriate amount of test substance stock or reference substance stock solution, and enough municipal water to bring the total volume up to 500 mL. Finally, a second control was prepared. All mixtures were aerated for three hours.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge collected from the Denton Wastewater Treatment Facility, Denton, Maryland USA on 15 February 2010 was utilized as the inoculum for the test. The Denton facility receives wastes from predominately domestic sources. The sludge was sieved using a 2 mm screen and allowed to settle for approximately 30 minutes. The supernatant above the settled solids was removed and the total suspended solids (TSS) concentration of the settled sludge was determined. Total suspended solids in the settled sludge were adjusted to a nominal concentration of approximately 4000 mg/L by dilution with municipal water. 50 mL of synthetic sewage was added to each liter of adjusted sludge. The sludge was maintained at a temperature ranging from 21.5 to 22.3 °C and continuously aerated overnight. The pH and total suspended solids concentration of the activated sludge were determined on the day of use in the study.
Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h
Post exposure observation period:
Not applicable
Test temperature:
21.5 to 22.3 °C
pH:
7.8
Nominal and measured concentrations:
10, 30, 100, 300 and 1000 mg/L.
Details on test conditions:
Test SystemThe biological test system was a consortium of microorganisms common to the activated sludge treatment process. The organisms responsible for the decomposition of organic materials are principally aerobic and facultative aerobic bacteria. The test system was chosen because it is representative of a treatment process that may receive the test substance.Test Conditions and ApparatusControl, reference, and treatment test mixtures were incubated at temperatures ranging from 21.6 to 22.2 °C and aerated for three hours at a rate sufficient to provide aerobic conditions and maintain solids in suspension. The mixtures were prepared and aerated in 1000 mL Erlenmeyer flasks and then transferred to fill 300 mL biochemical oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements. Test mixtures were identified by project number, test substance identification, test concentration, and bottle number.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol; Lot Number: 15809KI; Physical Description: Solid; Purity (%): 97; Storage Conditions: Ambient
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
342.9 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% CL
Details on results:
The EC50 value for AMP (2-Amino-1-Methylpropanol) was determined to be 342.9 mg/L. No inhibition was observed at a test concentration of 100 mg/L. Thus, the no observed effect level was ≥ 100 mg/L.
Results with reference substance (positive control):
The EC50 value for the reference substance (3,5-dichlorophenol) was 11.8 mg/L and was within the 5 to 30 mg/L range considered acceptable for the test.
Reported statistics and error estimates:
When the dose response pattern (percent inhibition versus test substance concentration) allowed for the calculation of an EC50 value, the data were analyzed using the computer program of C.E. Stephan (Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal Communication.). The program was designed to calculate the EC50 value and the 95% confidence interval by probit analysis, the moving average, or binomial probability with nonlinear interpolation (Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London; Thompson, W.R. 1947. Biological Reviews. Vol. II, No. 2. Pp. 115-145; Stephan, C.E. 1977. “Methods for Calculating an LC50,” Aquatic Toxicology and HazardEvaluations. American Society for Testing and Materials. Publication Number STP 634, pp 65-84.). Confidence limits (95%) for the EC50 and the EC50 value itself (when applicable) were determined using binomial probability with nonlinear interpolation only.

Temperatures measured throughout the period of sludge maintenance and during the test ranged from 21.5 – 22.3°C. The initial total suspended solids (TSS) concentration of the activated sludge after removal of the supernatant layer measured 13,780 mg/L. The measured TSS concentration of the sludge on the day of testing was 3813 mg/L. The measured pH of the sludge on the day of testing was 7.8.

The respiration rates observed in the two controls were 86.4 and 86.4 mg O2/L/hr, or a difference of 0.0%. The EC50 value for the reference substance was 11.8 mg/L and was within the 5 to 30 mg/L range considered acceptable for the test. Confidence limits (95%) for the EC50 and the EC50 value itself, were calculated using binomial probability with nonlinear interpolation. The observed percent inhibitions for AMP (Amino Methyl Propanol) ranged from –29.6 to 85.9%

Validity criteria fulfilled:
yes
Conclusions:
Inhibitory effects upon respiration by the test substance at the concentrations evaluated in this study were observed, and exhibited a concentration dependent dose response pattern. The EC50 value for AMP (Amino Methyl Propanol) was determined to be 342.9 mg/L, with 95 percent confidence limits of 100 mg/L and 1000 mg/L. No inhibition was observed at a test concentration of 100 mg/L. Thus, the no observed effect level was ≥100 mg/L.
Executive summary:

The effect of AMP (Amino Methyl Propanol) on activated sludge microorganisms maintained in an

aerobic environment was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD

Guideline 209). The test contained control, reference and treatment groups. The control replicates were

used to determine the background respiration rate of the sludge and were not dosed with the test or

reference substance. The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of

respiration, at concentrations of 3, 15 and 50 mg/L. The treatment group was dosed with AMP (Amino

Methyl Propanol) at concentrations of 10, 30, 100, 300 and 1000 mg/L. After an exposure period of three

hours, the respiration rates of the test solutions were measured using a YSI Model 50B-115 Dissolved

Oxygen Meter. The respiration rates in the two controls were 86.4 and 86.4 mg O2/L/hr. The difference

between the two control respiration rates was 0.0 %, and was within the 15% difference limit established

for the test. The validity of the test was further supported by the results from the 3,5-dichlorophenol

reference group, which resulted in an EC50 value of 11.8 mg/L, with 95 percent confidence limits of

3 mg/L and 15 mg/L. The EC50 for the reference substance was within the 5 to 30 mg/L range considered

acceptable for the test. An inhibitory dose response effect was observed for the treatment group. The

EC50 value for AMP (Amino Methyl Propanol) was determined to be 342.9 mg/L. The following is a

summary of the results:

Test Substance

Nominal Concentrations mg/L

Respiration Rate mg O2/L/hr1

Percent Respiration Inhibition1

Control 1

0

86.4

NA

Control 2

0

86.4

NA

3,5-dichlorophenol

3

180.0

-108.3%

3,5-dichlorophenol

15

32.0

63.0%

3,5-dichlorophenol

50

14.0

83.8%

AMP (Amino Methyl Propanol)

10

94.2

-9.0%

AMP (Amino Methyl Propanol)

30

112.0

-29.6%

AMP (Amino Methyl Propanol)

100

112.0

-29.6%

AMP (Amino Methyl Propanol)

300

47.2

45.4%

AMP (Amino Methyl Propanol)

1000

12.2

85.9%

 

 

 

 

1Calculations performed using MS Excel 2000 in full precision mode. Manual calculations may differ slightly.

NA - Not Applicable

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Similar to Guideline but some test condition details missing
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
effect on growth measured
GLP compliance:
not specified
Analytical monitoring:
no
Details on sampling:
- Concentrations: 100, 180, 320, 580, 1000 mg/L
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Pretreatment: After 2nd sedimentation, washed with tap dechlorinated water
- Initial biomass concentration: 2 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21C
Nominal and measured concentrations:
nominal - 100, 180, 320, 580, 1000 mg/L
Details on test conditions:
no data
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
240 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
580 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
3h EC10 ca 240 mg/L
3h EC20 ca 650 mg/L
3h EC30 ca >1000 mg/L
3h EC80 ca >1000 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 47% inhibition at 60 mg/L
Reported statistics and error estimates:
Statistical Method - Ausgewahite Methoden der Wasseruntersuchung, part 2, p 379-382, VEB Fisher Verlang
EC determinations made using probability grid

Test Concentration - Control #1, Control #2, Positive Control, and 100, 180, 320, 580, 1000 mg/L

Respiratory Rate (mg O2/L/h) - 29.2, 33.6, 16.8, 28.8, 28.4, 28.4, 27.6, 23.2

Inhibition (%) - 0, 0, 47, 8, 10, 10, 12, 26

Validity criteria fulfilled:
yes
Conclusions:
The 3h EC10 for the test substance was 240 mg/L which is equivalent to 156 mg/L active substance. The NOEC based on growth inhibition was 580 mg/L which is equivalent to 377 mg/L active substance.

Description of key information

Both the studies on AMP and PTSA show that no effects are expected at 100 mg/L. Therefore the NOEC is set at 100 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

The rationale for read-across can be found in the document attached in section 13.