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Toxicity to microorganisms

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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:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Principles of method if other than guideline:
The activated sludge respiration inhibition test was performed according to the study plan.
The study plan was developed from an OECD Test Guideline (OECD, 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/min) for 3 hours.
• The test substance and the reference compound (3,5-dichlorophenol) concentrations were spaced by a factor two.
• 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.
• No specific analyses were performed.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch/lot No.: 10000005282
Appearance: White particles
Solubility: Not soluble in water
Volatile: Expected non-volatile (vapour pressure 0.005 Pa at 25°C)
Analytical monitoring:
no
Details on sampling:
N.A.
Vehicle:
no
Details on test solutions:
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. Ethylene glycol dibenzoate was weighed directly in the incubation vessels. 3,5-Dichlorophenol was added to the incubation vessels using a 1.0 g/L stock solution.
The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1008 g of 3,5-dichlorophenol (99.7%) in approximately 40 mL of deionized water with the addition of a few drops of 2 M NaOH. Finally the pH was corrected with 2M H2SO4 to approximately pH 7 and the solution was diluted further to 100 mL with deionized water.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Unadapted secondary activated sludge was obtained (04-04-2017) 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:
19.9 to 20.3 °C
pH:
6.8 to 7.1
Nominal and measured concentrations:
Nominal test concentrations: control, 20.4, 42.2, 80.0, 161.4, 323.0, 641.6 and 1280.4 mg/L
Details on test conditions:
The test was performed in 300 mL Erlenmeyer flasks with a total working volume of 50 mL. 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.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 280 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.:
> 1 280 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
the EC50 of the reference compound for activated sludge after 3 hours contact time is 10 mg/L, which is within the prescribed range of 2 to 25 mg/L.
Reported statistics and error estimates:
The EC values were computed from the best-fitted line (linear maximum likelihood regression) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the substance. All computations were performed with the
program ToxRatPro version 2.1 (ToxRat Solutions GmbH 2001-2010).

Respiration rates of the activated sludge, inhibition percentages at various concentrations of ethylene glycol dibenzoate after 3 hours contact time.

Concentration (mg/L)  Activity   
(mg O2/g/min)
Inhibition (%)
Control 0.2571 -
Control  0.2583 -
Control  0.2583 -
Control  0.2333 -
20.4 0.2571 0
42.2 0.2524 2
80.0 0.2667 -3
161.4 0.2778 -8
323.0 0.2571 0
641.6 0.2524 -3
1280.4 0.2714 -10

 

Validity criteria fulfilled:
no
Remarks:
The inoculum has a low oxygen uptake rate; the average oxygen uptake rate for the control replicates was 16 mg O2/g dw/h, which is only slightly lower than prescribed. It does not influence sensitivity as shown by the result of the reference substance.
Conclusions:
Ethylene glycol dibenzoate is 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 EC50 of the reference compound 3,5-dichlorophenol (10 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (7%). 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 ethylene glycol dibenzoate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of ethylene glycol dibenzoate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC10, EC20, EC50 and EC80 values of ethylene glycol dibenzoate for activated sludge after 3 hours contact time are all >1280 mg/L , the highest concentration tested. Ethylene glycol dibenzoate is therefore considered not harmful to activated sludge.

Description of key information

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 toxicity of ethylene glycol dibenzoate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of ethylene glycol dibenzoate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC10, EC20, EC50 and EC80 values of ethylene glycol dibenzoate for activated sludge after 3 hours contact time are all >1280 mg/L , the highest concentration tested. Ethylene glycol dibenzoate is therefore considered not harmful to activated sludge.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 280 mg/L

Additional information