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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-06-01 to 2004-06-03
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:
1984
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
1988
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Reference item:
The reference item and the following information concerning the reference item were supplied by Aldrich Chemie:

Identity: 3,5-Dichlorophenol
Product no: D7060-0
Lot no: 02611ES
Purity: 99.1% (GLC)
Expiration date: 2005-07-17
Storage conditions: In tightly closed container, at room temperature at about 20°C
Analytical monitoring:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenised aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per litre. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic wastewater* per litre and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used in the test. The pH of the activated sludge inoculum was 6.8.

*Synthetic wastewater (filled up to a final volume of 1 L with deionised 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

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
Room temperature
pH:
7.3-8.3
Nominal and measured concentrations:
The final nominal concentrations tested were 10, 32, 100, 320 and 1000 mg/L. Concentrations above 1000 mg of test item per litre were not tested
Details on test conditions:
Dosage and concentrations:
According to the results of a pre-test (without GLP), no stock solution could be prepared due to the low water solubility of the test item.

Therefore, test item amounts of 5.0, 16.1, 50.0, 160.2 and 500.2 mg were directly weighed (by means of an analytical balance) into the test flasks. Then, 284 mL of tap water was added to each flask. The test item was mixed into the tap water by intense stirring. The dispersion was stirred for 3 hours at room temperature to dissolve a maximum amount of the test item in the dispersion. Ultrasonic treatment was not applied to avoid accelerating hydrolysis.

The short stirring period of 3 hours was chosen because the components of the test item are not stable in water (results of a pre-experiment, without GLP).

After the stirring period, 16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added. Thus, the final volume was 500 mL per test flask.

Additionally, two controls (tap water, synthetic wastewater and activated sludge inoculum without test item) and the reference item 3,5-dichlorophenol (positive control) at nominal concentrations of 5, 16 and 50 mg/L were tested in parallel under otherwise identical conditions of the test.

For test media containing the reference item, a stock solution of 3,5-dichlorophenol was prepared according to the test guidelines: 0.5 g of 3,5-dichlorophenol was dissolved in 10 mL 1 M NaOH and diluted to about 30 mL with purified water. Excess of NaOH was neutralised with 0.5 mol/L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to one litre with purified water. The pH was determined to be 7.6. The final concentration of 3,5-dichlorophenol amounted to 500 mg/L. Aliquots of this 3,5-dichlorophenol stock solution were mixed with synthetic wastewater and tap water in the respective test flasks to obtain the desired concentrations.

The composition of the test media is shown in Table 1.

Experimental conditions:
The test was performed in 1000 mL glass flasks. The test flasks were labelled with the necessary information to ensure unmistakable identification.

At the start of the test (after the stirring period of 3 hours), synthetic wastewater and activated sludge inoculum were added. The sludge concentration of the inoculum was 4 g/L dry weight (corresponding to 1.6 g dry material per litre test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, then to the test solutions of the reference item, then to the test solutions of the test item, and finally to the second control.

During the incubation period of exactly 3 hours, all test media and the controls were continuously aerated with compressed air at a flow of approximately one litre per minute. The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period, and just before the measurements of the respiration rates, the dissolved oxygen concentrations were at least 6.1 mg/L (Table 2 and 3). The temperature in the test media measured in one control was 19°C at the start and at the end of the incubation period.

Measurement of the respiration rates:
For measurement of the respiration rate, a well-mixed sample of each test medium was poured into a BOD-flask after exactly three hours incubation time, and was not further aerated. Then, the dissolved oxygen concentration was measured with an oxygen electrode (WTW TriOxmatic 300 and an oxygen meter WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten, WTW, Weilheim/Germany), and was continuously recorded for about ten to fifteen minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2/L/minute) was determined from the linear part of the respiration curve in the range of 6.5-2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption, the rate was determined over a period of at least 10 minutes.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
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
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The influence of the test item on the respiration rate of activated sludge is presented in Table 2.

Down to the lowest test concentration of 10 mg/L, some of the test item was suspended in the test media. Thus, all test concentrations were clearly above the water solubility limit of the test item under the present conditions of the test.

Up to and including the concentration of 1000 mg/L, the test item had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of 3 hours (see Table 2). Concentrations above 1000 mg/L were not tested. The saturation concentration (water solubility of the test item under present test condition) was obviously reached, no inhibitory effect on the respiration rate was determined. Thus, the 3-hour EC20 and EC50 are clearly higher than the water solubility limit of the test item under the present conditions of the test or higher than 1000 mg/L.

The oxygen consumption rates of the two controls (run at the start or at the end of the test) differed by 6% (guideline recommended maximum variation of 15%).
Results with reference substance (positive control):
The results are presented in Table 3.

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 10 mg/L (98% confidence limits were not calculable). The 3-hours EC50 is within the guideline recommended range of 5-10 mg/L, confirming the suitability of the activated sludge used.

Table 2: Influence of the test item on oxygen consumption of activated sludge

Flask

Test chemical

Nominal concentration of test chemical (mg/L)

Oxygen consumption rate (mg O2/L/min)

Inhibition (%)

pH values

Oxygen concentration (mg O2/L)

Start*

End*

Start*

End*

1

Control

0

0.890

 

7.4

8.3

8.9

8.0

10

Control

0

0.942

 

7.5

8.3

9.2

7.5

Mean Deviation (%)

0.916

5.8

 

 

 

 

 

5

Test item

10

0.972

-6.1

7.4

8.3

9.0

8.2

6

Test item

32

0.993

-8.5

7.3

8.2

9.3

8.3

7

Test item

100

0.915

0.0

7.3

8.2

9.2

6.1

8

Test item

320

0.907

0.9

7.3

8.3

9.3

6.2

9

Test item

1000

0.894

2.4

7.3

8.2

9.0

8.0

-% inhibition: increased oxygen consumption rate relative to that of control

 

Table 2: Influence if 3,5-dichlorophenol on oxygen consumption of activated sludge

Flask

Test chemical

Nominal concentration of test chemical (mg/L)

Oxygen consumption rate (mg O2/L/min)

Inhibition (%)

pH values

Oxygen concentration (mg O2/L)

Start*

End*

Start*

End*

1

Control

0

0.890

 

7.4

8.3

8.9

8.0

10

Control

0

0.942

 

7.5

8.3

9.2

7.5

Mean Deviation (%)

0.916

5.8

 

 

 

 

 

2

3,5-Dichlorophenol

5

0.683

25.4

7.7

8.3

9.5

8.1

3

3,5-Dichlorophenol

16

0.282

69.2

7.3

8.3

9.7

6.7

4

3,5-Dichlorophenol

50

0.076

91.7

7.3

8.3

9.6

8.5

*Start and end of the 3-hour incubation period

 

Validity criteria fulfilled:
yes
Conclusions:
A 3-hour NOEC value of ≥1000 mg/L was determined for the substance using a relevant test method and in compliance with GLP. The result is considered to be reliable.

Description of key information

Toxicity to microorganisms: 3 hour EC50>1000 mg/L; NOEC 1000 mg/L (OECD 209)

Key value for chemical safety assessment

Additional information

An activated sludge respiration inhibition (ASRI) 3 hour EC50 >1000 mg/L and NOEC 1000 mg/L (OECD 209) were determined for the submission substance, in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.