Ethylene dibenzoate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Chemical name: Ethylene glycol dibenzoate
CAS No.: 94-49-5
Chemical formula: C16H1404
Manufacturer of Test Item: TCI (Shanghai) Development Co., Ltd.
Batch number: 60LJG
Appearance: White solid crystalline powder
Purity: 100%
Re-certification date: 2023.11.20

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

Preparation of test item mixtures:
The test concentrations included 10, 100, 300 and 1000 mg/L. The test concentrations of 10, 100 and 300 mg/L were tested with 1 replicate each, and 1000 mg/L was tested with triplicate and 1 abiotic control at concentration of 1000 mg/L was performed.
To prepare test item mixtures, the test item was directly weighed into the test vessel and distilled water was added.

Preparation of the inoculums blank controls:
The inoculums blank controls contained an equal volume of activated sludge and synthetic medium but not test or reference substance. They were diluted with water to the same volume as- the test and reference mixtures. Inoculums blank controls were performed with 4 replicates.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Source of activated sludge:
The activated sludge was derived from a treatment plant receiving predominantly domestic sewage. A fresh sample of activated sludge was collected from the aeration tank of Shenyang North Sewage Treatment Plant 1 day before the test was started.

Treatment of activated sludge:
On the day of activated sludge collection, coarse particles were removed by settling for 10 minutes and by decanting the upper 1ay_er_of finer solids. The sludge was washed as followed: the sludge was first centrifuged for 5 minutes at ca. 2500 rpm to produce a clear supernatant and pellet of sewage solids. The supernatant liquid was discarded and the sludge was re—suspended in chlorine-free tap water by shaking, and the wash-water was removed by re—centrifuging and discarding again. After re-suspension of the sludge pellet in chlorine-free tap water, the solids dry weight was determined by drying 5 replicates of 10 mL suspended sludge at 105°C for 1 hour and
30 minutes. From the measured results, the sludge was diluted further in chlorine-free tap water to obtain the required sludge solids concentration of 3 g/L.

Cultivation and preparation of activated sludge:
The activated sludge was fed with the synthetic sewage feed (50 mL synthetic sewage feed/L activated sludge) and continuously aerated at the test temperature (19.8~21.0°C) overnight prior to use in the test.
Before the activated sludge was used as the inoculum, the concentration of the sludge was determined again by the drying method, and the concentration was adjusted to be 3 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19.9-20.6°C

pH:

6.99-7.51

Nominal and measured concentrations:

Nominal: 10, 100, 300 and 1000 mg/L
Only a range finding study was performed because no effect was found up to 1000 mg/L and it was not needed to perform a final test at lower or higher test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: no data
- No. of vessels per concentration (replicates): 1 and 3 for 1000 mg/L
- No. of vessels per control (replicates): 4
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 1.5 g/L
- Nutrients provided for bacteria:
peptone 16 g/L
meat extract 11 g/L
urea 3 g/L
NaCl 0.7 g/L
CaCl2.2H2O 0.4 g/L
MgSO4.7H2O 0.2 g/L
K2HPO4 2.8 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic sewage feed (composition given above)
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Only a range finding study was performed because no effect was found up to 1000 mg/L and it was not needed to perform a final test at lower or higher test concentrations.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen uptake

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Based on the results of the test item test, there was no oxygen uptake in the abiotic control. In the test item mixture, the average percentage inhibition was 2.4% at the concentration of 1000 mg/L and the test item had no significant inhibition effect.

Results with reference substance (positive control):

3h-EC50 = 4.9 mg/L

Reported statistics and error estimates:

Probit analysis was used to calculate EC50 of reference item including their 95% confidence limits.

Validity criteria fulfilled:

yes

Remarks:

The specific oxygen uptake rate of the blank controls (without the test- or reference item) was: R_TBS = 22.8 mg/(g*h SS) oxygen. The coefficient of variation of oxygen uptake rate in control replicates was 10.7% at the end of test.

Conclusions:

The test item is not toxic to microorganisms.

Executive summary:

The study was conducted to determine the inhibition effect of Ethylene glycol dibenzoate on micro-organisms from activated sludge (largely bacteria) by measuring their total respiration rate. The contact time for exposure was 3 hours.
In the preliminary test, the test concentrations included 10, 100, 300 and 1000 mg/L. The test concentrations of 10, 100 and 300 mg/L were tested with 1 replicate each, and 1000 mg/L was tested with triplicate and 1 abiotic control at concentration of 1000 mg/L was performed. Inoculum blank controls were performed with 4 replicates at the beginning and at the end of the exposure period. The rate of respiration of each incubation mixture was determined after 3 hours contact time.
3,5-dichlorophenol was used as a reference item in order to check the sensitivity of the activated sludge used as microbial inoculum on the day of exposure. The reference item was tested at 0.5, 1.3, 3.5, 9.4 and 25 mg/L with one replicate.
The specific oxygen uptake rate of the blank controls (without the test item or reference item) was 22.8 mg/(g*h SS) oxygen. The coefficient of variation of oxygen uptake rate in control replicates was 10.7% at the end of the test. The validity criteria have therefore been satisfied.
Based on the results of the reference item test, the EC50 (3 hours) of 3,5-dichlorophenol for total respiration is as follows:

3h-EC50  = 4.9 mg/L; 95% confidence limits 3.7 - 6.5 mg/L

The validity criteria for the reference item EC50 values were also satisfied.
Based on the results of the test item test, there was no oxygen uptake in the abiotic control. In the test item mixture, the average percentage inhibition was 2.4% at the concentration of 1000 mg/L and the test item had no significant inhibition effect. The definitive test at higher or lower concentrations was unnecessary.
Therefore, the EC50 (3 hours) of the test item for total respiration was greater than 1000 mg/L under the conditions of the study.

Reference 2

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

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. QA statement)

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.

This conclusion is supported by a GLP OECD 209 study performed in China where no effect was found at 1000 mg/L (the highest test concentration). 

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 280 mg/L