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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 December, 2009 through 07 January 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
NECTARYL :
Lot No. : VE00026199
Purity : 95.4%
Date of Expiry : 22 February, 2010
Analytical monitoring:
no
Details on test solutions:
Control C1: at time "0", in a 500-ml scaled cylinder, 16 ml of the synthetic sewage feed and 5 ml ethanol was made up to 300 ml with water. 200 ml of sludge were added and the mixture was aerated continuously for 3 hours by bubbling air into it.

Test S1: at time "15 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 5 ml of test substance stock solution A1 were made up to 300 ml with water. 200 ml of sludge were added and the mixture was aerated. Final concentration: 100.0 mg/l.

Test S2: at time "30 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 5 ml of test substance stock solution A2 were made up to 300 ml with water. 200 ml of sludge were added and the mixture was aerated. Final concentration: 31.6 mg/l.

Test S3: at time "45 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 5 ml of test substance stock solution A3 were made up to 300 ml with water. 200 ml of sludge were added and the mixture was aerated. Final concentration: 10.0 mg/l.

Test S4: at time "60 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 5 ml of test substance stock solution A4 were made up to 300 ml with water. 200 ml of sludge were added and the mixture was aerated. Final concentration: 3.16 mg/l.

Test S5: at time "75 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 5 ml of test substance stock solution A5 were made up to 300 ml with water. 200 ml of sludge were added and the mixture was aerated. Final concentration: 1.00 mg/l.

Test R1: at time "90 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 5 ml of 3,5-dichlorophenol stock solution + 5ml ethanol were made up to 300 ml with water. 200 ml of sludge were added and the mixture was aerated. Final concentration: 5.00 mg/l

Test R2: at time "105 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 30 ml of 3,5-dichlorophenol stock solution + 5 ml ethanol were made up to 300 ml with water. 200 ml of sludge were added and the mixture was aerated. Final concentration: 30.0 mg/l
Control TR: at time "120 minutes", the same as for control C1 was done. This flask serves only as “spacer” between the rather toxic R2 and the final Control C2.

Control C2: at time "120 minutes", the same as for control C1 was done.

Measurements of the oxygen consumption: at time "3 hours", the pH of the first cylinder C1 was measured. The content was used to fill up the BOD flask which was closed with the O2-electrode and the measurement of the oxygen consumption was started for about 10 minutes. The same procedure was repeated every 15 minutes for each cylinder in the order they had been prepared, so that the contact time is three hours for each of them.

The whole experiment was carried out at 20 ± 1 °C (temperature controlled water bath).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated Sludge :
Activated sludge was obtained from a water treatment plant treating predominantly domestic sewage (Bois-de Bay, 1242 Satigny, Switzerland). The sludge was kept aerobic until being used on the same day. 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: 3.058 g/l.

By adding 200 ml of sludge to the 500-ml test vessels, the final concentration of suspended solids was 1.22 g/l, i.e. in the range 0.8 to 1.6 g/l as recommended in ISO 8192-1986.

Synthetic Sewage Feed :
A synthetic sewage feed was made by dissolving the following amounts of substances in 1 litre of tap water:

- 16.00 g peptone (from casein trypsin-digested)

- 11.00 g yeast extract powder

- 3.00 g urea

- 0.71 g NaCl

- 0.40 g CaCl2 · 2 H2O

- 0.30 g MgSO4 · 7 H2O

- 2.81 g K2HPO4

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 +/- 1 °C
pH:
The pH in the samples tested containing NECTARYL was, as follows :

S1 100 mg/L pH7.89
S2 31.60 mg/L pH 7.83
S3 10.00 mg/L pH 8.01
S4 3.16 mg/L pH 8.11
S5 1.00 mg/L pH 7.96

In the Control samples :
C1 pH 7.98
C2 pH 8.12

In the Reference Substances samples :
R1 5.0 mg/L of 3,5-DichloroPhenol pH 8.11
R2 30.00 mg/L of 3,5-DichloroPhenol pH 8.04
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
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 on result:
other: 26.0% inhibition of respiration observed at the highest tested test concentration of 100 mg/L NECTARYL (nominal).
Duration:
3 h
Dose descriptor:
EC0
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: No inhibition of respiration (-3.3%) with respect to Control observed at 10 mg/L.
Details on results:
Sample NECTARYL Concn. (mg/L) % Inhibition
S1 100 26.0
S2 31.60 12.1
S3 10.00 -3.3
S4 3.16 -0.4
S5 1.00 0.2
Results with reference substance (positive control):
Sample 3,5-DichloroPhenol Concn. (mg/L) % Inhibition
R1 5.00 13.7
R2 30.00 80.3

3,5-dichlorophenol was used as a reference substance to check the activity of the sludge: its EC50 ( 3 hours) should be within the range 5 to 30 mg/L.
Validity criteria fulfilled:
yes
Conclusions:
All tested concentrations (1 to 100 mg/L) produced inhibitions below 50%.

The EC50 of NECTARYL is > 100 mg/L

The EC0 of NECTARYL is > or = 10 mg/L.
Executive summary:

All tested concentrations (1 to 100 mg/L) produced inhibitions below 50%.

The EC50 of NECTARYL is > 100 mg/L

The EC0 of NECTARYL is > or = 10 mg/L.

Description of key information

All tested concentrations (1 to 100 mg/L) produced inhibitions below 50%.

The EC50 of NECTARYL is > 100 mg/L

The EC0 of NECTARYL is > or = 10 mg/L.

Key value for chemical safety assessment

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

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