Calcium 4-chloro-2-(5-hydroxy-3-methyl-1-(3-sulfonatophenyl)pyrazol-4-ylazo)-5-methylbenzenesulfonate

Biodegradation in water: screening tests

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Administrative data

Endpoint:

biodegradation in water: screening test, other

Remarks:

inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD 302 C and compliant to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: bulk

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Inoculum of the aqueous phase of non adapted activated sludge. Municipal sewage treatment plant, D-31137 Hildesheim and D-31157 Sarstedt. Activated sludge from the sewage plants at Hildesheim and Sarstedt are well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Pretreatment: Same volumes of each source were filled together. The activated sludge was washed twice with autoclaved tap water before. This mixture was allowed to settle and the supernatant was decanted. The inoculum was stirred and aerated. It was fed every working day. therefore the inoculum was allowed to settle and on third was decanted and filled up with nutrient solution accord. to the guideline.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 302C
- Test temperature: 25.0 - 25.5 °C
- pH:
pH-Values in the Test Vessels at Test Start and Test End
pH-Value
Start End
Inoculum Control 7.32 1) 7.70
2) 7.66
Functional Control 7.36 1) 9.17
2) 9.15
Test Item 7.04 1) 7.29
2) 7.28
3) 7.22
Blank 6.38 1) 8.72

- pH adjusted: yes
- Aeration of dilution water: Continuous stirring
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Brown glass bottles, volume 500 mL
- Number of culture flasks/concentration: Triplicates
- Method used to create aerobic conditions: Continuous stirring
- Measuring equipment: The oxygen consumption was determined in the incubation vessels by the OxiTop measuring system at 360 measuring points during the 28 d incubation period.
- Details of trap for CO2 and volatile organics if used: A rubber sleeve with soda lime was hung into the opening of the bottles. The bottles were closed with OxiTop measuring heads and the measuring system was activated.


SAMPLING
- Sampling frequency: The temperature in the incubator was documented continuously throughout the test with a hygrothermograph.
The colony forming units and concentration of suspended solids of the inoculum were determined at test start.
The pH of the test item solution was determined in a separate replicate at test start. pH was adjusted from 7.46 to 7.04 with 4 % phosphoric acid.
The pH of the test solutions was determined at test end.
The oxygen consumption was determined in the incubation vessels by the OxiTop measuring system at 360 measuring points during the 28 d incubation period.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 30 mg/L test item in aqua demin.
- Functional control: sodium acetate, 100 mg/L
- Inoculum control: inoculum and mineral nutrient solution

Reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

puriss. Fluka

Results and discussion

Test performance:

Based on the calculated oxygen demand (ThOD), the test concentration of 30 mg/L corresponding to an oxygen demand of
44.7 mg O2/L in the test vessel was selected.

The test solutions were prepared in measuring flasks and given into brown glass bottles as incubation vessels (inoculum control, functional control) or were prepared directly in brown glass
bottles (test item, blank):
• 3 incubation vessels for the test item (P1, P2, P3)
• 2 incubation vessels for the functional control (R1, R2)
• 1 incubation vessel for the blank (B1)
• 2 incubation vessels for the inoculum control (C1, C2)

A separate test item replicate was prepared for pH measurement at test start:
• 1 incubation vessel for the test item (PpH).

The test item was weighed out and was transferred directly into the brown glass bottles with 250 mL test medium (consisting of the appropriate volumes of mineral medium stock solutions, de-mineralised water and inoculum) for the test item replicates and with 250 mL demineralised water for the blank, respectively.

For the functional control the reference item was weighed out and was transferred into a 500 mL measuring flask with demineralised water. The appropriate volumes of mineral medium stock solutions and inoculum were added, then the flask was filled up with demineralised water.

The inoculum control, consisting of mineral medium stock solutions, demineralised water and inoculum, was prepared in a 500 mL measuring flask.
The test solutions for reference item and inoculum control were divided using 250 mL measuring flasks before being filled into the brown glass bottles.
A rubber sleeve with soda lime was hung into the opening of the bottles. The bottles were closed with OxiTop measuring heads and the measuring system was activated.

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

3

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

The functional control reached the pass level of > 65 % after 4 days. After 14 days a biodegradation rate of 69 % (mean) was reached.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The percentage degradation of the functional control reached the pass level > 65 % after 14 days. The difference of extremes of replicate values of biodegradation of the test item at the end of the test was less than 20 %.

Interpretation of results:

not inherently biodegradable

Conclusions:

Under our test conditions, the test item was not inherently biodegradable within 28 days.

Executive summary:

The inherent biodegradability of the test item was determined in the Modified MITI Test (II) with non adapted activated sludge for a period of 28 days. The study was conducted according to OECD Guideline 302 C. The test item concentration selected as appropriate was 30 mg/L, corresponding to a ThOD of 44.7 mg O₂/L. The oxygen was depleted by respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation is therefore expressed as the percentage BOD depletion and was calculated for each study day.

The mean oxygen depletion in the inoculum control came to 19.7 mg O₂/L after 28 days.

In order to check the activity of the test system sodium acetate was used as functional control. The functional control reached the pass level of > 65 % after 4 days. It came to a biodegradation rate of 69 % after 28 days.

The 10 % level (beginning of biodegradation) was reached only by one test item replicate on day 28. The mean biodegradation came to 3 % after 28 days. No elimination/abiotic degradation of the test item occured in the blank.

The validity criteria of the guideline are fulfilled.

Under our test conditions, the test item was not inherently biodegradable within 28 days.