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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from 08 NOV 2005 to 24 JAN 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to an international guideline and according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

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

Constituent 1
Reference substance name:
Dicerium tricarbonate
EC Number:
208-655-6
EC Name:
Dicerium tricarbonate
Cas Number:
537-01-9
IUPAC Name:
dicerium tricarbonate

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
No analytical monitoring

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was not soluble in tap water at the required concentration and no stock solution could be prepared. Therefore, 500.2 mg of the test item was weighed by means of an analytical balance and transferred to the test flask with 284 mL of tap water. The test item was mixed into the tap water by ultrasonic treatment for ten minutes and intense stirring for 24 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used. After the stirring period, 16 ml of synthetic wastewater and 200 ml of the activated sludge inoculum were added.
- Differential loadings: No (limit test)
- Controls: two controls containing only tap water, synthetic wastewater and inoculum were tested in parallel to the limit test concentration of the test item, under identical test conditions.
- Chemical name of vehicle : none
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the limit test concentration of 1000 mg/L, the test item was unevenly suspended in the turbid test medium after the 24-hour stirring period. Thus, the test concentration was clearly above the water solubility limit of cerium carbonate under the test conditions.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: no, aerobic activated sludge came from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, CH)
- Method of cultivation: During the holding period of three days prior to use, the sludge was fed daily with 50 mL synthetic wastewater per liter and was kept at room temperature under continuous aeration until use. The pH of the activated sludge inoculum was 7.1.
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. An aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 4 g dry material per liter.
- Pretreatment: no
- Initial biomass concentration: 4 g dry material per liter

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
none

Test conditions

Hardness:
no data
Test temperature:
the temperature measured in test media in one control was 19°C at the start and 20°C at the end of the incubation period
pH:
7.3 to 8.2
Dissolved oxygen:
7.1 to 8.6 mg/L
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentration: 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: The test was performed in 2000-mL glass beakers, filled with around 500 mL of test medium and sludge
- Aeration: During the incubation period of exactly 3 hours, all test media and the controls were continuously aerated by intense stirring on magnetic stirrers, to avoid possible foaming and/or stripping of the test item.
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: sludge concentration = 1.6 g dry material / L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH values and dissolved oxygen concentrations were determined after the addition of synthetic wastewater and activated sludge, in all test media and the controls at the start and the end of the 3-hour incubation period. The water temperature was measured in one control at the start and at the end of the incubation period. Before the addition of activated sludge and synthetic wastewater, the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Lighting: no data

EFFECT PARAMETERS MEASURED : 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-1 minute-1) was determined from the linear part of the respiration curve in the range of 6.5-2.5 mg O2/L. I n 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 ten minutes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Justification for using less concentrations than requested by guideline: a limit test has been performed to demonstrate that the test item has no toxic effect on activated sludge up to the nominal concentration of 1000 mg/L.
- Range finding study none
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol tested at 5, 16 and 50 mg/L

Results and discussion

Effect concentrationsopen allclose all
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
Remarks:
respiration rate
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
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the limit test concentration of 1000 mg/L, the test item was unevenly suspended in the turbid test medium after the 24-hour stirring period. Thus, the test concentration was clearly above the water solubility limit of cerium carbonate under the test conditions.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 12 mg/L with 95% confidence limits from 6 to 20 mg/L . The 3-hour EC50 is within the guideline-recommended range of 5 - 30 mg/L, confirming suitability of the activated sludge used.
Reported statistics and error estimates:
The 3h-EC50, -EC20 and -EC80 of the test item and their 95% confidence limits could not be calculated due to the absence of a toxic effect. For the reference substance, the probit analysis was used (slope b: 2.059, intercept a: 2.782).

Any other information on results incl. tables

Table 1 : Influence of cerium carbonate humide and 3, 5-dichlorophenol on the oxygen consumption of activated sludge

 

Vessel

No.

Test chemical

Nominal concentration of test chemical

(mg/L)

Oxygen consumption rate

(mg O2/L min-1)

Inhibition

(%)

1

11

Control

Control

0

0

0.973

0.978

 

Mean

Deviation (%)

 

 

0.975

0.6

 

2

3

4

5

3,5-dichlorophenol

3,5-dichlorophenol

3,5-dichlorophenol

Cerium carbonate humide

5

16

50

1000

0.756

0.402

0.092

1.037

22.5

58.8

90.6

-6.3

 

-% inhibition: increased oxygen consumption rate relative to control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The EC50 of the reference substance is in the acceptance range and the oxygen consumption rates of the 2 controls differed by less than 15% (0.6%).
Conclusions:
The test item has no toxic effect on the respiration rate of activated sludge microorganisms at the limit test concentration of 1000 mg/L.
Executive summary:

The inhibitory effect of the test item dicerium tricarbonate on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, and the OECD Guideline for Testing of Chemicals, No. 209

In accordance with the EU Commission Directive 92/69/EEC (C.1, C.2, and C.3) and the OECD Series on Testing and Assessment No. 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000), a limit test was performed with one concentration of dicerium tricarbonate of nominal 1000 mg/L, to demonstrate that the test item has no toxic effect on activated sludge microorganisms up to and including this concentration.

In addition, two controls 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 reference treatments (EC50 = 12 mg/L within the guideline-recommended range of 5 - 30 mg/L) confirmed suitability of the activated sludge and the method used.

The test item, dicerium tricarbonate, had no significant inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at the limit test concentration of 1000 mg/L. Thus, the 3-hour NOEC of dicerium tricarbonate to activated sludge microorganisms was at the limit of water solubility of dicerium tricarbonate under the test conditions, or at 1000 mg/L. The 3-hour EC20, EC50 and EC80 were clearly higher than the limit of water solubility of dicerium tricarbonate under the test conditions, or higher than 1000 mg/L.