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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27-01-2017 t/m 01-02-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP according guidelines, all quality criteria were met
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Chemical name Sodium oleylamphopolycarboxyglycinate
Trade name Ampholak XO7/C
CAS No. 97659-53-5
Composition See certificate of analysis (Annex 2)
Active ingredient: 29.8% (40.2% corrected for salt content of 10.4%)
Batch/lot No. 1297986
Appearance Light yellow liquid
Stability Unknown under test conditions
Solubility Soluble in water
Volatile Expected non-volatile
Storage At ambient temperature in the dark
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
The highest sodium oleylamphopolycarboxyglycinate concentration was added to the incubation vessels using a 2 g/L stock solution and the other test substance concentrations were dosed from a 1 g/L stock solution. The 1 g/L and 2 g/L stock suspension of sodium oleylamphopolycarboxyglycinate were prepared by dissolving 0.42 g and 2.00 g Test substance (sodium oleylamphopolycarboxyglycinate) in 423 and 1000 mL deionized water, respectively.
The pH of the 1 g/L and 2 g/L stock solution were 8.9 and 9.1, respectively

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Unadapted secondary activated sludge was obtained (31-01-2017) 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 dry weight of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The dry weight of the activated sludge in the incubation vessels was 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
Temperature varied from 20.3 to 20.9°C.
pH:
The pH of the reaction mixtures after the incubation period ranged from 7.1 to 7.3.
Dissolved oxygen:
aerobic during test
Details on test conditions:
-The test was performed in 300 mL Erlenmeyers with a total working volume of 50 mL.
-Prior to use the activated sludge was homogenized with a syringe
-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 was added to the activated sludge (1.6 mL on a total volume of 50 mL).
Synthetic sewage contained per liter: 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
-Different concentrations (spaced by a factor two) of test compound and reference compound where added using stock solutions
-Tap-water was used to fill up the total test volume to 50 mL.
- The dry weight concentration of the activated sludge in the test was 1.5 g/L.
-The tested concentrations of the reference compound 3,5-dichlorophenol where: 2.5 mg/L, 5.0 mg/L,10.1 mg/L, 20.1 mg/L and 40.2 mg/L
-The tested concentrations of test substance (sodium oleylamphopolycarboxyglycinate) where: 20, 40, 79, 159, 317, 635, and 1280 mg/L
-Controls without test substance (or reference compound) are measured at the beginning and at the end of the test
-The inhibitory effect of the test substance/reference compound is expressed as a percentage of the mean of the two control respiration rates.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (p.a. quality 99.7% Aldrich Lot no MKBP6894V)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 381.44 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
50.66 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
170 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 29.8% active ingredient content
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 280 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 29.8% active ingredient content
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
474 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:

Test conditions
The pH of the reaction mixtures after the incubation period ranged from 7.1 to 7.3. Temperature varied from 20.3 to 20.9 °C. These conditions allow respiration of the activated sludge used.

Validity of the test
The validity of the test is demonstrated although only two out of three validity criteria were met according the guideline. First, the prescribed oxygen uptake rate of >20 mg O2/g dry weight/hour for the control measurements was not met. The inoculum used was obtained from a treatment plant which is operated at a low organic sludge load and as a consequence has a low oxygen uptake rate. Operating at low organic sludge loads is nowadays very common. Nonetheless the average oxygen uptake rate for the control replicates was 16 mg O2/g dry weight/hour, which is slightly lower than the prescribed rate. This does not influence the sensitivity of the activated sludge as shown by comparable EC50 values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate (AkzoNobel 2012). The lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test. Second, the coefficient of variation of the replicates of the control oxygen uptake rates was 7%, which shows a high precision and fulfills the maximum prescribed variation of <30%. Third, the EC50 of the reference compound for activated sludge after 3 hours contact time is 12 mg/L, which is within the prescribed range of 2 to 25 mg/L.

Activated sludge respiration inhibition test
The calculated respiration rates and inhibition percentages of sodium oleylamphopolycarboxyglycinate are presented in Table II and the results of the graphical probit analysis are shown in Figure 2. The inhibitory effect of sodium oleylamphopolycarboxyglycinate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of sodium oleylamphopolycarboxyglycinate for activated sludge after 3 hours contact time is >1280 mg/L. The EC80 value could not be determined and the EC10 and EC20 values were 170 and 474 mg/L, respectively. Sodium oleylamphopolycarboxyglycinate is therefore considered not harmful to activated sludge.




Results with reference substance (positive control):
The EC50 of the reference compound for activated sludge after 3 hours contact time is 12 mg/L, which is within the prescribed range of 2 to 25 mg/L
Validity criteria fulfilled:
yes
Remarks:
See validity of test in "details on results"
Conclusions:
Test performed under GLP according guidelines meeting the validity criteria of the OECD guideline.
The inhibitory effect of sodium oleylamphopolycarboxyglycinate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of sodium oleylamphopolycarboxyglycinate for activated sludge after 3 hours contact time is >1280 mg/L. The EC80 value could not be determined and the EC10 and EC20 values were 170 and 474 mg/L, respectively. Sodium oleylamphopolycarboxyglycinate is therefore considered not harmful to activated sludge.
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 EC50of the reference compound 3,5-dichlorophenol (12 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (9%). The average oxygen uptake rate for the control replicates is lower (16 mg O2/g dry weight/hour) than the prescribed rate (>20 mg O2/g dry weight /hour). This does not influence the sensitivity of the activated sludge as shown by comparable EC50 values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate (AkzoNobel 2012). The lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test.

The toxicity of sodium oleylamphopolycarboxyglycinate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of sodium oleylamphopolycarboxyglycinate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of sodium oleylamphopolycarboxyglycinate for activated sludge after 3 hours contact time is >1280 mg/L (>381.4 mg active ingredient/L). The EC80 value could not be determined and the EC10 and EC20 values were 170 (=50.7 mg active ingredient/L) and 474 mg/L, respectively. Sodium oleylamphopolycarboxyglycinate is therefore considered not harmful to activated sludge.

Description of key information

One activated sludge respiration test with Sodium oleylamphopolycarboxy glycinate is available which was performed in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice.

The toxicity of sodium oleylamphopolycarboxyglycinate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The EC50of sodium oleylamphopolycarboxyglycinate for activated sludge after 3 hours contact time is >1280 mg/L test substance. The EC10 value was 170 mg/L test substance. Sodium oleylamphopolycarboxyglycinate is therefore considered not harmful to activated sludge.

Based on active ingredient (40.2%) the 3h EC50 >381.4 mg/L

Based on active ingredient the 3h EC10 = 50.7 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
50.7 mg/L

Additional information

One activated sludge respiration test with Sodium oleylamphopolycarboxy glycinate is available.

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 compound 3,5-dichlorophenol (12 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (9%).

The toxicity of sodium oleylamphopolycarboxyglycinate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of sodium oleylamphopolycarboxyglycinate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50of sodium oleylamphopolycarboxyglycinate for activated sludge after 3 hours contact time is >1280 mg/L. The EC80 value could not be determined and the EC10 and EC20 values were 170 and 474 mg/L, respectively. Sodium oleylamphopolycarboxyglycinate is therefore considered not harmful to activated sludge.

Based on active ingredient (40.2%) the 3h EC50 >381.4 mg/L

Based on active ingredient the 3h EC10 = 50.7 mg/L