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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study done under GLP, well documented
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not required
Details on sampling:
not applicable
Details on test solutions:
Reference substances and chemicals
3,5-Dichlorophenol (p.a. quality 99.7% Aldrich Lot no MKBP6894V) used as a reference substance in the activated sludge respiration inhibition test was purchased from Sigma Aldrich. All other chemicals used were of reagent grade quality.

De-ionised water
The de-ionised water used in the study contained less than 10 μg/L of copper (ISO 17025 certified analysis), a conductivity of less than 5 μS/cm and less than 2.0 mg/L NPOC.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge
Unadapted secondary activated sludge was obtained (21-10-2014) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater.Prior to use the activated sludge was homogenized with a syringe. The suspended solids (dry weight)
content of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration.

The activated sludge was aerated and fed overnight with OECD synthetic sewage concentrate at 50 mL/L prior to use on the day following collection. The suspended solids concentration of the activated sludge in the incubation vessels was 1.5 g dry weight/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.1 to 20.7°C.
pH:
7.0 to 7.2
Nominal and measured concentrations:
Nominal: 1.25, 2.5, 5, 10, 20 and 40 mg/L and 70.0, 144.0, 284.0, 562.0, and 1126.0 mg/L
Details on test conditions:
Test conditions
The test was performed in 300 mL Erlenmeyer flasks with total working volumes of 50 mL, respectively. The homogenized activated sludge was incubated in a shaking water bath (100 rpm, 20°C) for 3 hours with various concentrations of the test compound and synthetic sewage.

Synthetic sewage feed and stocks
The synthetic sewage feed was made by dissolving the following amounts of substances in 1 liter of deionized water: 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. In the incubation vessels 1.6 mL of the synthetic sewage feed is added in a total volume of 50 mL. Reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-npropyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane was weighed directly in the incubation vessels. 3,5-Dichlorophenol was added to the incubation vessels using a stock solution of 1.0 g/L. The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1003 g of 3,5-dichlorophenol in approximately 40 mL of deionized water with the addition of a few drops of 1 M NaOH. Finally the pH was corrected with 1M H2SO4 to approximately pH 7 and the solution was diluted further to 100 mL with deionized water

Test procedures
The activated sludge respiration inhibition test was performed according to the study plan. The study plan was developed from an OECD Test Guideline (OECD 209, 2010). In this test the inhibition of oxygen uptake by micro-organisms oxidizing organic carbon was not separately expressed from the
respiration by micro-organisms oxidizing ammonium. The total respiration rate of activated sludge fed with a standard amount of synthetic sewage was measured. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under otherwise identical conditions was also measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. Some aspects of the test are listed below:

• Prior to use the activated sludge was homogenized with a syringe.
• The activated sludge with test substances was incubated in a shaking bath (100 rpm) for 3 hours.
• The test substance and the reference compound (3,5-dichlorophenol) concentrations were spaced by a factor two. The tested nominal concentrations of the reference substance were 1.25, 2.5, 5, 10, 20 and 40 mg/L and 70.0, 144.0, 284.0, 562.0, and 1126.0 mg/L for the test substance. An abiotic control was not performed because neither reduction of oxygen by the test substance nor formation of oxygen due to chemical breakdown of the test substance is expected.
• The test substance was pipetted into the incubation vessels and the added weight was directly measured on an analytical balance.
Since the test substance was weighed directly into the incubation vessel and was considered unlikely to be air-stripped during incubation, no specific analyses were performed.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 126 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The inhibitory effect of reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane at a particularnominal concentration is expressed as a percentage of the two controls. From these results EC values were calculated. The calculated respiration rates and inhibition percentages of reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane are presented in Table I and the results of the graphical probit analysis are shown in Figure 1. Computation of the EC confidence limits, on the basis of Fieller's theorem (Zerbe, 1987), was not possible for the obtained dose response curve.

The EC50 of reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane for activated sludge after 3 hours contact time is > 1126 mg/L. Inhibition of the activated sludge respiration by reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane did not follow a classical dose-response curve
and varied from 2% to 6% over the measured concentration range
Results with reference substance (positive control):
The EC50 of the reference compound for activated sludge after 3 hours contact time is 13 mg/L, which is within the prescribed range of 2 to 25 mg/L
Validity criteria fulfilled:
yes
Remarks:
average oxygen uptake rate for the control replicates was 20.2 mg O2/g dry weight/hour; coefficient of variation of the replicates of the control oxygen uptake rates was 4%, EC50 of the reference compound after 3 hours contact time is 13 mg/L
Conclusions:
The 3h EC50 of reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane to activated sludge is > 1126 mg/L.
Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice. The test is valid as shown by the EC50 of the reference substance, 3,5-dichlorophenol (13 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (4%). The average oxygen uptake rate for the control replicates is 20.2 mg O2/g dry weight/hour this is just above the prescribed rate (>20 mg O2/g dry weight /hour). The toxicity of reaction mass of 3,6,9-triethyl-3,6,9- trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl- 1,2,4,5,7,8-hexoxonane to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of reaction mass of 3,6,9-triethyl-3,6,9- trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl- 1,2,4,5,7,8-hexoxonane at a particular nominal concentration is expressed as a percentage of the two controls. The EC50 of reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6- diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl- 1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane for activated sludge after 3 hours contact time is > 1126 mg/L. Inhibition of the activated sludge respiration by reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9- n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane did not follow a classical dose-response curve and varied from 2% to 6% over the measured concentration range

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
9-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: done under GLP, study is complete
Qualifier:
according to
Guideline:
other: OECD 209 and EEC directive 87/302/EEC
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 1 000 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Validity criteria fulfilled:
yes
Conclusions:
study is valid, no effect observed

Description of key information

One OECD 209 GLP study is available with an 3 hr EC50 > 1126 mg/L (nominal)

Key value for chemical safety assessment

EC50 for microorganisms:
1 126 mg/L

Additional information

An activated sludge respiration inhibition test was performed in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice. The test is valid as shown by the EC50 of the reference substance, 3,5-dichlorophenol (13 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (4%). The toxicity of reaction mass of 3,6,9-triethyl-3,6,9- trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl- 1,2,4,5,7,8-hexoxonane to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of reaction mass of 3,6,9-triethyl-3,6,9- trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl- 1,2,4,5,7,8-hexoxonane at a particular nominal concentration is expressed as a percentage of the two controls.

The EC50 of reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6- diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl- 1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane for activated sludge after 3 hours contact time is > 1126 mg/L.