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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-23 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: tert-Butyl glycidyl ether
CAS No.: 7665-72-7
Batch No.: ZMG-195668
Appearance: Clear colourless liquid
Molecular formula: C7H14O2
Content: 99.8% (area)
Storage conditions: Cool, protected from light
Expiry date: 07 March 2019
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test item
545 µl of the test item were added directly into the test vessels with a test volume of 500 mL to obtain a final concentration of 1000 mg/L. The pH of the test solution was measured before addition of inoculum.
Reference item
25.1 mg of 3,5-dichlorophenol were dissolved in deionised water and filled up to 25 mL using a magnetic stirrer to get a stock solution with 1.0 g/L. 0.5, 5 and 15 mL of the stock solution were added to the reference vessels in order to obtain final concentrations of 1, 10 and 30 mg/L.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from the municipal wastewater treatment plant Staufener Bucht was used as test system. The treatment plant has a capacity of 140 000 inhabitant equivalents and clarifies predominantly domestic wastewater. Sampling date of activated sludge was 22 November 2017.
- Method of cultivation: The activated sludge was kept aerated until use.
- Preparation of inoculum for exposure: Dry solid of the activated sludge was determined as 4.3 g/L by weight measurements before and after 3.5 h drying at 105°C (mean of triplicate measurements). The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in aerated tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.
- Initial biomass concentration: 1.5 g/l dry solids in the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
19.9 - 20.3 °C
pH:
test item: 7.4
reference item: 7.3
blank: 7.3
Dissolved oxygen:
kept above 60 - 70% saturation through stirring and aeration
Nominal and measured concentrations:
0, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 600 mL serum bottles, Gerätebau Ochs, Bovenden-Lenglern, Germany; 250 - 300 ml BOD bottles with adapter for oxygen electrode, Germany
- Aeration: All vessels were stirred and aerated throughout the contact time with room air to keep the dissolved oxygen concentration above 60 – 70 % saturation. Before starting the test, the aeration rate was measured in all test vessels. After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 3 g/L dry solids
- Nutrients provided for bacteria: yes, synthetic sewage feed, 16 mL added to each test vessel
(1 L synthetic sewage feed contains: 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)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralized water

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: not reported
- Light intensity: not reported
- Details on termination of incubation: After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer. This determination was repeated for each vessel at 15-min intervals, ensuring that the contact time in each vessel was 3 h.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test
- Test concentrations: 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (97 % purity)
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
Remarks:
respiration rate
Remarks on result:
other: the mean respiration rate in the treated test vessels was only slightly lower than in the control
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: no inhibition observed
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption (e.g. of test material to the walls of the test container): not reported
- Blank controls oxygen uptake rate: The blank controls oxygen uptake rate was 20 mg oxygen per one gram of activated sludge per hour.
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation (CV) was 10 % and therefore less than 30 %.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 of 3,5-dichlorphenol was calculated as 3.78 mg/l and was therefore within the acceptable range of 2 to 25 mg/l.
Reported statistics and error estimates:
The EC50 calculation was performed with the ToxRat software by probit analysis using linear max. likelihood regression (ToxRat Professional Version 3.2.1, © ToxRat Solutions GmbH). The EC50 is calculated from the slope of the curve.
Validity criteria fulfilled:
yes
Conclusions:
There was no inhibition observed in the test vessels with 1000 mg/L test concentration (inhibition range from -7 to 33%). The respiration rates in the test vessels were not significant lower than the respiration rate of the controls (STUDENT t-test).
EC50 > 1000 mg/L
LOEC > 1000 mg/L
NOEC ≥ 1000 mg/L
Executive summary:

The influence of tert-butyl glycidyl ether on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study was conducted according to the OECD test guideline 209 (July 22nd, 2010).

A concentration of 1000 mg/L was tested. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring.

No significant inhibition of respiration rate of the sludge was recorded at 1000 mg Tert-butyl glycidyl ether per liter. No further testing was needed.

Since all criteria for acceptability of the test were met, this study was considered to be valid:

> The coefficient of variation (CV) of the blank respiration rates was less than 30 %.

> The blank controls oxygen uptake rate was 20 mg oxygen per one gram of activated sludge per hour.

> The EC50 of the reference item was within the acceptable range of 2 to 25 mg/L

The result is therefore:

EC50 > 1000 mg/L           

LOEC > 1000 mg/L         

NOEC ≥ 1000 mg/L

In conclusion, under the conditions of the present test, tert-butyl glycidyl was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 1000 mg/L.

Description of key information

A limit test with the concentration of 1000 mg/L was performed according to OECD 209 and under GLP. No inhibition was observed in the test vessels with 1000 mg/L test concentration (inhibition ranged from –7 to 33 %).

The result is therefore:

EC50> 1000 mg/L

NOEC ≥ 1000 mg/L

This indicates that no hazard has been identified.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information