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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
Study period:
22-10-2018 to 23-10-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
The test item was added to the incubation vessels using a
was added to the incubation vessels using a 1 – 2 g/L stock suspension and 3,5-dichlorophenol was added from a 1 g/L stock solution. The 1 g/L stock suspension of the test item was prepared by dissolving 0.38 in approximately 100 g deionized water, and adjusting the pH from 6.2 to ~7 with 1 M NaOH, and finally diluting the suspension further to a total mass of 379.24 g with deionized water. The 2 g/L stock suspension of tetradecyl ethoxylated glycidylether was prepared by dissolving 0.15052 g in a small volume of deionized water, adjusting the pH from 6.0 to ~7 with 1 M NaOH, and finally diluting the suspension further to a total mass of 75.11 g with deionized water.
The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1032 g 3,5-dichlorophenol in approximately 40 mL of deionized water with the addition of a few drops of 1 M NaOH. Subsequently the pH was checked 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 (22-10-2018) 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 measured dry weight of the homogenized activated sludge was 2.9 g/L and was concentrated by settlement to 6.3 g/L dry weight. Subsequently, the concentrated activated sludge was diluted in the incubation vessels to an activated sludge dry weight concentration of 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
Temperatures measured in the Erlenmeyer flasks at the end of the incubation period varied from 21.0 to 21.3 °C.
pH:
The pH of the reaction mixtures after the incubation period ranged from 7.0 to 7.3
Dissolved oxygen:
aerobic during test
Nominal and measured concentrations:
Concentrationst
Nominal: 0, 20, 40, 80, 160, 320, 640 and 1280 mg/L
Details on test conditions:
Test conditions
The test was performed in 300 and 1000 mL Erlenmeyer flasks with a total working volume of 50 and 200 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 and 6.4 mL of the synthetic sewage feed is added in a total volume of 50 and 200 mL, respectively.
Tetradecyl ethoxylated glycidylether was added to the incubation vessels using a 1 – 2 g/L stock suspension and 3,5-dichlorophenol was added from a 1 g/L stock solution. The 1 g/L stock suspension of tetradecyl ethoxylated glycidylether was prepared by dissolving 0.38 in approximately 100 g deionized water, and adjusting the pH from 6.2 to ~7 with 1 M NaOH, and finally diluting the suspension further to a total mass of 379.24 g with deionized water. The 2 g/L stock suspension of tetradecyl ethoxylated glycidylether was prepared by dissolving 0.15052 g in a small volume of deionized water, adjusting the pH from 6.0 to ~7 with 1 M NaOH, and finally diluting the suspension further to a total mass of 75.11 g with deionized water.
The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1032 g 3,5-dichlorophenol in approximately 40 mL of deionized water with the addition of a few drops of 1 M NaOH. Subsequently the pH was checked 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, 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.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (p.a. quality 99.7% Aldrich Lot no MKBP6894V)
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
27 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:
EC50
Effect conc.:
271 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:158 - 507 mg/L
Details on results:
Test conditions
The pH of the reaction mixtures after the incubation period ranged from 7.0 to 7.3. The temperature of the BOM thermostated vessel measured at the start and end of the test was 20.0 °C. The temperature of the water bath, used for the incubation of the activated sludge, at the beginning and end of the test was 21.3 °C and 21.1 °C, respectively. Temperatures measured in the Erlenmeyer flasks at the end of the incubation period varied from 21.0 to 21.3 °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 18 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 12%, 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 4 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 tetradecyl ethoxylated glycidylether are presented in Table II and the results of the graphical probit analysis are shown in Figure 2. The inhibitory effect of tetradecyl ethoxylated glycidylether at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of tetradecyl ethoxylated glycidylether for activated sludge after 3 hours contact time is 271 mg/L with 95% confidence limits of 158 and 507 mg/L. The EC10, EC20 and EC80 values were 27, 59 and 1243 mg/L, respectively. Tetradecyl ethoxylated glycidylether 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 4 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 EC50 of the test item for activated sludge after 3 hours contact time is 271 mg/L. The substance 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 (4 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (12%). The average oxygen uptake rate for the control replicates is lower (18 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 EC50values 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 tetradecyl ethoxylated glycidylether to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of tetradecyl ethoxylated glycidylether at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50of tetradecyl ethoxylated glycidylether for activated sludge after 3 hours contact time is 271 mg/L with 95% confidence limits of 158 and 507 mg/L. The EC10, EC20and EC80values were 27, 59 and 1243 mg/L, respectively. Tetradecyl ethoxylated glycidylether is therefore considered not harmful to activated sludge.

Description of key information

In a test according to OECD Test Guideline 209 the toxicity of the test item to activated sludge was determined. The EC50 for activated sludge after 3 hours contact time is 271 mg/L. The substance is therefore considered not harmful to activated sludge.

Key value for chemical safety assessment

EC50 for microorganisms:
271 mg/L
EC10 or NOEC for microorganisms:
27 mg/L

Additional information