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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8th June 2011 to 10th June 2011
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
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1-phenylethyl acetate
- Physical state: colourless to pale yellow liquid
- Name of test material (as cited in study report): Gardenol
- Lot/batch No.: VE00123242
- Expiration date of the lot/batch: 14th January 2013
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: The test substance is not soluble enough in water to allow the preparation of an aqueous stock solution. Different solutions were prepared by dissolution of 1-phenylethyl acetate in ethanol in order to always add the same volume (i.e. 5 mL) of ethanol in each cylinder.The following solutions were prepared:A1: 100 mg 1-phenylethyl acetate in 10 mL ethanolA2: 31.6 mg 1-phenylethyl acetate in 10 mL ethanolA3: 10.0 mg 1-phenylethyl acetate in 10 mL ethanolA4: 3.2 mg 1-phenylethyl acetate in 10 mL ethanolA4: 1.0 mg 1-phenylethyl acetate in 10 mL ethanol
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge was obtained from a water treatment plant treating predominantly domestic sewage (Bois0de Bay, 1242 Satigny, Switzerland). The sludge was kept aerobic until being used on the same day and the pH was between 7 and 8.The suspended solids content was determined by drying and weighing 50 mL of the sludge: dry weight of suspended solids was 5.28 g/L. After dilution and addition of synthetic effluent, a concentration of 2.64 g/L was obtained.By adding 200 mL of sludge to the 500 mL test vessels, the final concentration of the suspended solids was 1.06 g/L (in the range 0.8 to 1.6 g/L as recommended in ISO 8192-1986).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20 ± 2 ºC
pH:
8.5 - 8.6
Nominal and measured concentrations:
100 mg/L nominal
Details on test conditions:
TEST SYSTEM- Test vessel: 500 mL measuring cylinders- Aeration: AeatedSYNTHETIC SEWAGE FEED- A synthetic sewage feed was made by dissolving the following in 1 L tap water:16 g peptone (from casein trypsin-digested)11 g yeast extract powder3 g urea0.7 g NaCl0.4 g CaCl2.2H2O0.3 g MgSO4.7H2O2.8 g K2HPO4TEST PROCEDURE- Control C1: at time '0', 16 mL of synthetic sewage feed and 5 mL ethanol was made up to 300 mL with water. 200 mL of sludge was added and the mixture aerated continuously for 3 hours by bubbling air into it.- Test S1: at time '15 min', 16 mL of synthetic sewage feed and 5 mL test substance stock solution A1 was made up to 300 mL with water. 200 mL of sludge was added and the mixture aerated. Final concentration 100 mg/L.- Test S2: at time '30 min', 16 mL of synthetic sewage feed and 5 mL test substance stock solution A2 was made up to 300 mL with water. 200 mL of sludge was added and the mixture aerated. Final concentration 31.6 mg/L.- Test S3: at time '45 min', 16 mL of synthetic sewage feed and 5 mL test substance stock solution A3 was made up to 300 mL with water. 200 mL of sludge was added and the mixture aerated. Final concentration 10.0 mg/L.- Test S4: at time '60 min', 16 mL of synthetic sewage feed and 5 mL test substance stock solution A4 was made up to 300 mL with water. 200 mL of sludge was added and the mixture aerated. Final concentration 3.2 mg/L.- Test S5: at time '75 min', 16 mL of synthetic sewage feed and 5 mL test substance stock solution A5 was made up to 300 mL with water. 200 mL of sludge was added and the mixture aerated. Final concentration 1.0 mg/L.- Test R1: at time '90 min', 16 mL of synthetic sewage feed, 5 mL 3,5-dichlorphenol stock solution and 5 mL ethanol was made up to 300 mL with water. 200 mL of sludge was added and the mixture aerated. Final concentration 5.0 mg/L.- Test R2: at time '105 min', 16 mL of synthetic sewage feed, 30 mL 3,5-dichlorphenol stock solution and 30 mL ethanol was made up to 300 mL with water. 200 mL of sludge was added and the mixture aerated. Final concentration 30.0 mg/L.- Control TR: at time '120 min', the same as control C1 was performed to act as a 'spacer' between the toxic R2 and the final control.MEASUREMENT OF OXYGEN CONSUMTPTION- At time '3 hours', the pH of control C1 was measured. The content was used to fill up the BOD flask which was closed with the O2-electrode and the measurement of oxygen consumption was started for about 10 minutes. The same procedure was repeated every 15 minutes for the test vessels in the order in which they had been prepared so that the contact time for each test vessel was 3 hours.- Control C2: at time '135 min', the same as control C1 was performed.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 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:
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:
All tested concentrations produced inhibitions below 50%.
Results with reference substance (positive control):
The percentage inhibition of 3,5-dichlorophenoll was 0.6% (R1) and 63.6% (R2).

Table 1: Results

 Test vessel  pH  Δ O2 (mg/L)  Time (min)  Rate  % inhibition  Conc. final (mg/L)  Log conc.
 C1  8.5  3.85  5.00  0.77  -  -  -
 C2  8.5  2.20  2.50  0.88  -  -  -
 R1  8.6  4.10  5.00  0.82  0.6  5.0  0.70
 R2  8.5  1.50  5.00  0.30  63.6  30.0  1.48
 S1  8.6  3.65  5.00  0.73  11.5  100.0  2.00
 S2  8.6  3.90  5.00  0.78  5.5  31.6  1.50
 S3  8.6  4.15  5.00  0.83  -0.6  10.0  1.00
 S4  8.6  3.30  5.00  0.66  20.0  3.2  0.50
 S5  8.6  4.05  5.00  0.81  1.8  1.0  0.00

None of the five test concentrations (1.0; 3.2; 10.0; 31.6; 100 mg/L; see Table 1) produced significant inhibition of respiration.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the inhibition of the test substance to sludge micro-organism respiration was determined to be EC50 (3 h) >100 mg/L with a NOEC of 100 mg/L.
Executive summary:

The activated sludge respiration inhibition test according to OECD guideline 209 was performed with 1-phenylethyl acetate. Five concentrations in the range 1 to 100 mg/L were tested. The result can be expressed as EC50 (3 hours) > 100 mg/L with a NOEC of 100 mg/L.

Description of key information

The EC50 of 1-phentlyethyl acetate was determined to be >100 mg/L and the NOEC to be 100 mg/L according to a study performed in line with OECD Guideline 209, EU Method C.11 and ISO 8192-1986.

Key value for chemical safety assessment

EC50 for microorganisms:
729 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information

Two activated sludge respiration inhibition studies, performed according to standardised guideline OECD 209, are available for 1-phenylethyl acetate. In the key study, five concentrations in the range 1 to 100 mg/L were tested. None of the five test concentrations produced significant inhibition of respiration. The NOEC was determined to be 100 mg/L and the 3 hour EC50 to be >100 mg/L. In the supporting study the 3 hour EC50 was determined to be 729 mg/L.

The NOEC for this endpoint of 100 mg/L is supported by results of the inhibition control in a standardised ready biodegradability test (Givaudan, 1996b). At the concentration used in the test (100 mg/L), 1-phenylethyl acetate is not inhibitory to the micro-organisms.

An activated sludge respiration inhibition study for the structurally closed related analogue benzyl acetate is also included as relevant supporting information to support the rationale for the use of read-across to benzyl acetate (source substance) in order to address the aquatic toxicity endpoints for 1-phenylethyl acetate (target substance). The 3 hour EC50 of benzyl acetate was determined to be 855 mg/L.