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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 8, 2001 till October 10, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD guideline 209 (Activated Sludge, Respiration Inhibition Test) under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: ZP-TIX 1014
Batch No.: NW-01-028
Purity: >99%
Appearance: white solid
Storage: At room temperature at about 20°C (in the dark)

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
The nominal final test item concentrations tested were 10, 32, 100, 320 and 1000 mg/L. Concentrations exceeding 1000 mg test item per liter were not tested.
According to the results of a pre-test, no stock solution could be prepared due to the low water solubility of ZP-TIX 1014.Therefore, test item amounts of 5.1, 16.2, 50.3, 162 and 501 mg were directly weighed (by means of an analytical balance) into the test flasks. Then 284 mL of tap water were added to each flask. The test item was mixed into the tap water by ultrasonic treatment over 15 minutes and intense stirring over 24 hours at room temperature in the dark. Then, 16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added .
The test item is a poorly soluble mixture of isocyanates and it is generally known, that these compounds degrade fast in water. Therefore, the degradation products were tested in addition to the undissolved fraction of the test item dispersion. A long stirring period of 24 hours was chosen, to enrich the test water with the maximum concentration of degradation products within a practically acceptable time period.
In addition to the test media, 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 test conditions.
The test was performed in 1000-mL glass flasks. The test flasks were labelled with the necessary information to assure unmistakable identification.
At the start of the test 200 mL activated sludge inoculum (see Section 2.3) with a sludge concentration of 2.5 9 dry weight/L (corresponding to about 1.0 9 dry material per liter test medium) was added. 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 liter 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 6.6 mg/L. The temperature in the test media, measured in one control, was 21°C at the start and 20°C at the end of the incubation period.
For test media containing the reference item a stock solution of 3,5-dichlorophenol was prepared according to the test guidelines: 0.5 9 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 neutralized with 0.5 mol/L H2S04 to the point of incipient precipitation. Thereafter, the mixture was made up to one liter with purified water. The pH was determined to be 7.8 and the final concentration 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 pre-test to the solubility of the test item was not performed in compliance with the GLP. Regulations and therefore are excluded from the Statement of Compliance. However, the raw data, respectively copies of raw data will be archived under the RCC study number of the present study.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was washed once with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliqout of the final sludge supsension was weighed, thereafter dried and the ration of wet to dry weight was calculated.
Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a cconcentrations equivalent to 3 g dry material per liter.
During the holding of two and a half days prior to use, the sludge was fed with 50 ml synthetic wastewater /liter / day and was kept at room temperature under continuous aeration until use. Immediately 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 adjusted from 8.4 to 7.6 with a diluted sulfuric acid solution.

Synthetic wastewater:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCI
0.4 g CaCI2 * 2H20
0.2 g MgS04 * 7H20
2.8 g K2HP04
was filled up to a final volume of 1 liter with deionized water.

Study design

Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h

Test conditions

Test temperature:
The temperature in the test media, measured in one control, was 21 °C at the start and 20 °C at the end of the incubation period.
pH:
The pH of the activated sludge inoculum was adjusted from 8.4 to 7.6 with a diluted sulfuric acid solution
Dissolved oxygen:
The concentration of dissolved oxygen did not drop 2.5 mg/L during the incubation period, and just before the measurements of the respiration rates the dissolved oxygen concentrations were at leat 6.6 mg/L.
Salinity:
Synthetic wastewater:
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
Nominal and measured concentrations:
The nominal concentrations tested were 10, 32, 100, 320 and 1000 mg/l.
Concentrations exceeding 1000 mg/l were not tested.
Down to the lowest test concentration of 10 mg/l a part of the test item was suspended in the test media
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks:
< 15 %
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Conc. based on:
test mat.
Details on results:
Up to and including the concentration of 1000 mg/l the test item and its degradation products, respectively, had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours.
The 3-hour NOEC (EC15) of the substance to activated sludge microorganisms was at least 1000 mg/l or higher. The 3-hour EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/l.




Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 16 mg/L (the 95 % confidence limits were not calculable). The 3-hour EC50 is witin the guideline-recommended range of 5 - 30 mg/L, confirming the suitability of the activated sludge used.

Any other information on results incl. tables

Table: Influence of ZP-TIX 1014 on oxygen consumption of activated sludge
Flask Test chemical Nominal Oxygen Inhibition pH values Oxygen
concentration consumption concentration
of test rate (mg O)
chemical
(mg/I) (mg 02/l min-1) (%) start end start end
1 Control 0 0.968 8.0 8.7 6.8 6.9
10 Control 0 1.098 8.0 8.7 7.9 7.5
Mean 1.033
Deviation (%) 13.4
5 ZP-TIX 1014 10 1.002 3.0 7.9 8.7 7.9 7.2
6 ZP-TIX 1014 32 0.988 4.4 8.0 8.7 8.0 7.9
7 ZP-TIX 1014 100 1.026 0.7 8.0 8.7 7.2 8.0
8 ZP-TIX 1014 320 1.063 -2.9 8.0 8.7 7.9 7.6
9 ZP-TIX 1014 1000 1.155 -11.8 8.0 8.7 7.9 6.6

Table: Influence of 3,5-dichlorophenol on oxygen consumption of activated sludge
Flask Test chemical Nominal Oxygen Inhibition pH values Oxygen
concentration consumption concentration
of test rate (mg O)
chemical
(mg/I) (mg 02/l min-1) (%) start end start end
1 Control 0 0.968 8.0 8.7 6.8 6.9
10 Control 0 1.098 8.0 8.7 7.9 7.5
Mean 1.033
Deviation (%) 13.4
2 3,5-dichlorophenol 5 0.812 21.4 7.7 8.5 7.0 7.7
3 3,5-dichiorophenol 16 0.590 42.9 7.8 8.1 7.5 7.5
4 3.5-dichlorophenol 50 0.179 82.7 7.8 8.4 7.8 8.3

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Up to and including the concentration of 1000 mg/l the test item and its degradation products, respectively, had no significant inhibitory effect (< 15 %) on the respiration rate of activated sludge after the incubation period of three hours. The 3-hour NOEC (EC15) of the substance to activated sludge microorganisms was at least 1000 mg/l or higher.
Executive summary:

The inhibitory effect of the test item on the registration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3 -hour respiration inhibition test according to the EU Commission Directive 87/302/EEC, and the OECD Guideline for testing of chemicals, No. 209.

The nominal concentrations tested were 10, 32, 100, 320 and 1000 mg/l. Concentrations exceeding 1000 mg/l were not tested. Down to the lowest test concentration of 10 mg/l a part of the test item was suspended in the test media.

Two controls (tap water, synthetic wastewater and activated sludge inoculum, without test item) and three different concentrations of the reference item 3,5 -dichlorophenol (5, 16 and 50 mg/l) were tested in parallel. The results of these treatments confirmed the suitability of the activated sludge and the method used.

Up to and including the concentration of 1000 mg/l the test item and its degradation products, respectively, had no significant inhibitory effect (< 15 %) on the respiration rate of activated sludge after the incubation period of three hours. The 3-hour NOEC (EC15) of the substance to activated sludge microorganisms was at least 1000 mg/l or higher. The 3-hour EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/l.

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