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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 nov 2010 - 30 nov 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the REACH, C.4-D, Manometric Respirometry Test.
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Acid Yellow RN 2903
IUPAC Name:
Acid Yellow RN 2903
Details on test material:
Identity: Acid Yellow RN 2903
Batch No.: Vers. Kilo 6, 26.05.2009
Purity: Approx. 65%
Expiration Date: 01-Aug-2015
Storage Conditions: At room temperature at about 20 °C, away from direct sunlight.

Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period of one day prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a final concentration of 30 mg dry material per liter.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
101 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST WATER
The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:

1) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 x 2H2O 33.40 g/L
NH4Cl 0.50 g/L

The pH of this solution was 7.4.

2) MgSO4 x 7H2O 22.50 g/L

3) CaCl2 x 2H2O 36.40 g/L

4) FeCl3 x 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter

To obtain the final test water, 10 mL of stock solution No. 1 and 1 mL each of stock solution Nos. 2, 3 and 4 were combined and made up to 1000 mL with purified water. The pH was adjusted from 7.8 to 7.4 with a diluted hydrochloric acid solution.

TEST CONCENTRATIONS
The test item was added to the designated test flasks with test water. No emulsifiers or solvents were used but ultrasound dispersion was employed for 5 minutes to obtain a suspension of the test item as homogeneous as possible.

The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 2.5 g sodium benzoate per liter test water was prepared by completely dissolving 250 mg sodium benzoate in 100 mL test water. From this stock solution, 10 mL aliquots were added to the corresponding test flasks containing test water.

Finally, activated sludge was added to each test flask . The final test volume was 250 mL per test flask.

TEST CONDITIONS
Apparatus: The test flasks (500 mL reaction vessels, labeled with the necessary information to ensure unmistakable identification) were incubated under continuous stirring in a SAPROMAT D12 (Voith GmbH, Heidenheim, Germany). Oxygen consumption was recorded manually by taking a daily reading at least on each working day.

Principle: Electro-chemical analysis process:
The biodegradation process consumes the dissolved oxygen in the test medium and generates CO2. The CO2 is adsorbed by soda lime, which results in a decrease of the total pressure in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate solution.

Test duration: 28 days

Light conditions: Darkness

Test temperature: 22 °C, maintained with a built-in thermostat and checked once per week.

pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum (see any other information on results section). At the end of incubation, the pH was measured again in each test flask.

DETERMINATION OF THE CHEMICAL OXYGEN DEMAND
Due to the complex structure (composition) of the test item, its chemical oxygen demand (COD) was determined according to the Commission Directive 92/69/EEC, C.6 following DIN 38414-S9.

The COD was determined at BMG Engineering AG, Ifangstrasse 11, 8952 Schlieren / Switzerland. The COD determination was not performed in compliance with the GLP regulations. Therefore, it is excluded from the Statement of Compliance in this report. However, it was performed in compliance with ISO/IEC 17025. The results are archived under this study number.





Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
None
Test performance:
None
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
7
Sampling time:
28 d
Details on results:
Due to the complex structure (composition) of the test item, its chemical oxygen demand (COD) was determined according to the Commission Directive 92/69/EEC, C.6 following DIN 38414-S9.
The COD was determined at BMG Engineering AG, Ifangstrasse 11, 8952 Schlieren / Switzerland. The COD determination was not performed in compliance with the GLP regulations. Therefore, it is excluded from the Statement of Compliance in this report. However, it was performed in compliance with ISO/IEC 17025.

For the results calculations were based on see attached tables 1(Oxygen consumption) and 2 (Biodegradation in Test Flasks)

The percent biodegradation of the test item was calculated based on the chemical oxygen demand (COD) of 1.14 mg O2/mg test item(for calculations used see attachement)

The mean biochemical oxygen demand (BOD) of the test item Acid Yellow RN 2903 in the test media was in the normal range found for the inoculum controls throughout the study period of 28 days.

Consequently, Acid Yellow RN 2903 was not biodegradable under the test conditions within 28 days.

BOD5 / COD results

BOD5 / COD
Parameter:
COD
Value:
1.14 g O2/g test mat.
Results with reference substance:
The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg

In the procedure control, the reference item was degraded by 91% by Exposure Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 97%.

Any other information on results incl. tables

TOXICITY CONTROL

The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the COD of the test item and the ThOD of the reference item.

 

In the toxicity control, the run of the curve of the oxygen consumption over the 28-day exposure period was similar to the two procedure controls, containing only the reference item. Within 14 days of exposure, biodegradation amounted to 54%.

 

Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 100 mg/L because biodegradation in the toxicity control was >25% within 14 days.

CHEMICAL OXYGEN DEMAND

The chemical oxygen demand (COD) of the test item Acid Yellow RN 2903 was determined according to the Commission Directive 92/69/EEC, Part C.6 followingDIN 38414-S9.

 

The duplicate values were 1.16 and 1.11 mg O2/mg test item. The mean COD was 1.14 mg O2/mg test item.

PH MEASUREMENT

The pH measured in all flasks at the start of the test was 7.4. At the end of exposure (Day 28), pH values of 7.5 – 7.9 were measured.

Replicate

Identification

pH

No.

 

Start

End

1

Test item

7.4

7.5

2

Test item

7.4

7.5

1

Inoculum control

7.4

7.5

2

Inoculum control

7.4

7.5

1

Procedure control

7.4

7.9

1

Toxicity control

7.4

7.8

 


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The biochemical oxygen demand (BOD) of the test item Acid Yellow RN 2903 in the test media was in the normal range found for the inoculum controls.
Consequently, Acid Yellow RN 2903 was not biodegradable under the test conditions within 28 days.
Executive summary:

The test item Acid Yellow RN 2903 was investigated for its ready biodegradability in a manometric respirometry test over 28 days according to the Commission Directive 92/69EEC, C.4-D, Commission Regulation (EC) No 440/2008, C.4-D and the OECD Guideline for Testing of Chemicals No. 301 F.

 

The biochemical oxygen demand (BOD) of the test item Acid Yellow RN 2903 in the test media was in the normal range found for the inoculum controls.

 

Consequently, Acid Yellow RN 2903 was not biodegradable under the test conditions within 28 days.

 

In the toxicity control, containing both Acid Yellow RN 2903 and the reference item sodium benzoate, Acid Yellow RN 2903 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 100 mg/L.

 

In the procedure control, the reference item sodium benzoate was degraded by 91% by Exposure Day 14, and reached an biodegradation of 97% by the end of the test (Day 28), thus confirming suitability of the activated sludge.

 

The chemical oxygen demand (COD) of the test item Acid Yellow RN 2903 was determined according to the Commission Directive 92/69EEC, C.6 following DIN38414-S9 and was found to be 1.14 mg O2/mg test item.