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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.1.-17.3.1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study approved by authority in ELINCS notification

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to guideline
Guideline:
other: Directive 87/302/EEC Part C: Biodegradation: Activated sludge respiration inhibition test
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(phenylmethoxy)naphthalene
EC Number:
405-490-3
EC Name:
2-(phenylmethoxy)naphthalene
Cas Number:
613-62-7
Molecular formula:
C17H14O
IUPAC Name:
2-(benzyloxy)naphthalene
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): BNE
- Substance type: organic
- Physical state: white powder
- Expiration date of the lot/batch: August 31, 1994
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
- PREPARATION OF TEST SUBSTANCE CONCENTRATIONS
Since BNE was hardly soluble in water, weighed amounts were added directly to a total of 500 ml medium, i.e. 21.3, 31.B, 50.2 and 101.0 mg.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
Test System: Activated sludge
Source: Municipal sewage treatment plant, Waterschap de Aa, Schijndel, The Netherlands.
Number of micro-organisms: determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
Rationale: Recognized by international guidelines as the recommended test system.

PREPARATION OF THE SLUDGE
The sludge was coarsely sieved and washed with tap-water.
A small amount of the sludge (250 ml) was weighed and dried at 100-105°C.
Then the dry weight was determined. From this result the amount of wet sludge was calculated: the level was 5.82704 g/l MLSS.
The batch of sludge was used on the next day, therefore 50 ml of synthetic sewage feed (composition acc. to guidelines) was added to each litre of activated sludge at the end of the day. The sludge was kept aerated at 20 ± 2°C until use. Before use the pH was 7.2.

- Initial biomass concentration: 5.82704 g/l MLSS Mixed Liquor Suspended Solids (MLSS) per litre test medium.

Study design

Test type:
static
Water media type:
not specified
Limit test:
no
Total exposure duration:
30 min

Test conditions

Test temperature:
20 ± 2 °C
pH:
7.4 (starting pH)
Nominal and measured concentrations:
nominal: 42.6, 63.6, 100.4 and 202.0 mg/l
Details on test conditions:
TEST PROCEDURE AND CONDITIONS
- Test vessel: all glass, 500 ml beakers and 300 ml oxygen bottles
- Milli-Q water Tap-water purified by reverse osmosis and sbsequently passed over activated carbon and ion-exchange cartridges
(Millipore Corp., Bedford, Mass., USA).
- Oxygen meter: Oxygen electrode (Tri Ox EO 200, WTW, FRG) supplied with a recorder (Kipp B040).
- Sodium bicarbonate solution: 70 g NaHC03 dissolved per litre Milli-Q water
- Synthetic sewage feed: 16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaC12.2H20
0.2 g MgS04 .7H20
2.8 g K2HP04
Dissolved in 1 1 Milli-Q water and filtered: the pH was 7.1.
The nutrient solution could be used for one week if stored at 4°C.
Before use, it was mixed thoroughly and the pH adjusted.
- Temperature of medium 20 ± 2°C

PERFORMANCE OF THE TEST:
The activated sludge was diluted at a ratio of 1:1 with tap water providing a suspended solids content of 3 g/l MLSS.
The synthetic sewage feed (16 ml) and an adequate amount of the test substance solution were mixed and made up to 300 ml with Milli-Q water.
A volume of 200 ml of the diluted sludge was added and the mixture was aerated in a 500 ml beaker during contact time using a Pasteur pipette as an aeration device.
Then a well mixed sample of the contents was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder forcing the air not to remain in the vessel. Oxygen consumption was measured and recorded for approximately 10 min. During measurement the sample was not aerated but continuously stirred on a magnetic stirrer.
This procedure was repeated for all concentrations of the test substance.
Each batch of activated sludge was checked for susceptivity by testing the reference substance 3,5-dichlorophenol.
Five controls without test substance were tested, i.e. one at the start, two between the reference and the test substance series and two at the end of the test.
The flasks were filled up to 500 ml final volume.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrations
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 202 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: At a concentration of the reference substance of 1 mg/l the inhibition of the respiration rate was 22.5%.
Inhibition increased to 83.B% at 50 mg/l.
The EC50 determined by probit analysis was found to be 6.4 mg/l. (95% fiducial limits: 3.9 to 9.9 mg/l)

Any other information on results incl. tables

 Flask  Concentration test substance mg/l  Oxagen consumption mgO2/l/h  Inhibition %
 Control 2  39.7  0
 Control 3  0  40.4  0
 T1 42.6  38.9  2.9 
 T2  63.6  39.8  0.5
 T3  100.4  39.4  1.6
 T4  202.0  38.3  4.2

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The mean respiration rate of all controls was 39.3 ± 1.7. The difference between two controls was < 9%. The EC50 of 3,5-dichlorophenol was 6.4 mg/l (95% Confidential limits: 3.9-9.9 mg/l).
Conclusions:
The testing of the inhibitory effect of 2-(phenylmethoxy)naphthalene on aerobic waste-water bacteria of activated sludge showed that the EC50 for 2-(phenylmethoxy)naphthalene exceeded 202 mg/l. In conclusion, 2-(phenylmethoxy)naphthalene was found to be not toxic to aerobic waste-water bacteria.
Executive summary:

The inhibitory effect of 2-(phenylmethoxy)naphthalene on aerobic waste-water bacteria of activated sludge was investigated in a respiration test. 2-(phenylmethoxy)naphthalene did not inhibit the respiration rate at concentrations up to and including 202 mg/l. Hence, the EC50 for 2-(phenylmethoxy)naphthalene exceeded 202 mg/l. The EC50 for the reference substance 3,5-dichlorophenol tested under the same conditions was 6.4 mg/l (95% c.i.: 3.9-9.9 mg/l). In conclusion: under the conditions of this test, 2-(phenylmethoxy)naphthalene was not toxic to aerobic waste-water bacteria.

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