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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 10, 2016 to May 08, 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, tetrasodium and lithium chloride
EC Number:
Molecular formula:
C43H24ClCuN15O16S6.xLi.yNa, (x + y) = 4; 0 < (x,y) < 4 with 1341.9 < MW < 1374 g/mol (UVCB substance), and traces of NaCl and NaSO4
Reaction products of cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, tetrasodium and lithium chloride
Test material form:
solid: particulate/powder

Sampling and analysis

Details on sampling:
One test solution with a final volume of 400 mL (ratio of composition of each test mixture referring to 500 mL according to the guideline) was prepared per treatment in a glass flask.
A volume of 12.8 mL synthetic sewage and an adequate amount of the stock solution of the test item were placed into the test flask (Table 1) and filled up with deionized water to 200 mL before the start of the incubation. At the start of the test, 200 mL activated sludge inoculum with a sludge concentration of 3 g/L of suspended solids was added to all flasks, with the exception of the abiotic control flasks.
Abiotic controls in 5 parallels (A1, A2, A3, A4 and A5), and one untreated blank (B1) control was started as the first step of the test. Then at appropriate time internals of approximately 30 minutes the further test groups were started. The second series were 4 different concentrations (without replicates) of the reference item (REF1, REF2, REF3, REF4) and to the second blank control (B2). The third series was the 5 replicates of the test item concentration of 10 mg/L nominal concentration (T111, T112, T113, T114 and T115) and the third blank control (B3). The forth series was the five replicates of test item concentration 100 mg/L nominal concentration (T121m T122, T123, T124 and T125) and the fourth blank control (B4). The final series was the five replicates of test item concentration 1000 mg/L nominal concentration (T131, T132, T133, T134 and T135) and the fifth blank control (B5).
In summary, 3 test item concentration series and one abiotic control series were prepared in 5 parallels with one blank control series by series. In parallel with the above test mixtures four reference item concentrations without replicates were also prepared with one blank control.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
Source of inoculum/activated sludge: the sewage plant for domestic sewage in Veszprém, Hungary.

Study design

Total exposure duration:
3 h

Test conditions

Test temperature:
19.5 - 21.8℃
7.70 - 8.34
Nominal and measured concentrations:
The concentrations of the test item: 10, 100 and 1000 mg/L.
Details on test conditions:
- Aeration: With compressed air (about 1 L/min)
- No. of vessels per concentration (replicates): five
- No. of vessels per blank control (replicates): five
- No. of vessels per abiotic control (replicates): five
- Sludge concentration: 3 g/L
Reference substance (positive control):

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
In comparison to the inoculum the inhibition of the respiration rate in the case of the activated sludge was between -3.47% and 7.15% in the examined range of 10~1000 mg/L of CJ303 test item.
The average specific respiration rate (Rs) of the untreated (Blank) controls (B1-B5) was 22.61 mg O2/L/h/g solid.
The variation coefficient of oxygen uptake rate in above untreated replicates was 5.01%.
Results with reference substance (positive control):
The 3-hour EC50 of 3,5-Dichlorophenol was 16.94 mg/L.

Any other information on results incl. tables

Table 2. Influence of test item on oxygen consumption of activated sludge

Test group

Conc. of test item

in test mix (mg/L)

Total oxygen consumption rate

(RT) (mg O2/L/h)

Specific respiration rate (Rs) (mg O2/L/h/g solid) Inhib.(%) pH value# Temperature (℃) (measured in the Laboratory)* Variation coefficient of O2consumption rate (%)
ID Name start end
A1 Abiotic 1000 1.41 - - 7.86 7.82 19.5/21.8 -
A2 1000 0.62 - - 7.85 7.81 -
A3 1000 0.37 - - 7.84 7.79 -
A4 1000 -0.33 - - 7.83 7.77 -
A5 1000 0.99 - - 7.82 7.76 -
B1 Blank 0.00 33.30 22.20 - 7.70 8.14 19.5/21.8 5.01
B2 0.00 36.27 24.18 - 7.94 8.29
B3 0.00 32.31 21.54 - 7.76 8.34
B4 0.00 32.62 21.75 - 7.85 8.33
B5 0.00 35.10 23.40 - 7.74 8.31
REF1 Ref. Item 1.00 29.80 19.87 13.95 8.03 8.32 19.5/21.8 -
REF2 3.20 28.17 18.78 18.76 8.00 8.33 -
REF3 10.00 26.73 17.82 22.99 7.97 8.34 -
REF4 32.00 9.27 6.18 74.47 7.94 8.34 -
TI1 Test item 10 32.11 21.40 7.15 7.76 8.30 19.5/21.8 -
TI2 100 32.57 21.72 5.78 7.92 8.33 -
TI3 1000 35.71 23.81 -3.47 7.73 8.29 #NAME?

#: Individual pH values of the BOD flasks, except TI1-3, where the average values are reported

*: Minimum / Maximum temperature (℃) measured in the Laboratory, during the experiment

Applicant's summary and conclusion

Validity criteria fulfilled:
According to OECD 209, the 3-hour EC50 of CJ303 was greater than 1000 mg/L.
Executive summary:

A laboratory test was carried out with CJ303 to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

The test concentrations (10, 100 and 1000 mg/L) were chosen to permit the determination of the approximate EC50.

The reference control results showed that in this study, the inoculum and methodology used provide a goof measure of inhibition of activated sludge respiration rate.

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of CJ303 was the following:

The 3-hour EC50>1000 mg/L

The NOEC was determined to be 1000mg/L.

Based on the results of this study, CJ303 had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000mg/L. No further testing is necessary.