C,C'-azodi(formamide)

Biodegradation in water: screening tests

Description of key information

Three out of four available screening studies indicate that ADCA is not readily biodegradable. It is therefore concluded, on weight of evidence, and based on screening studies, that the ADCA is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Available studies

To assess the biodegradability of Azodicarbonamide (ADCA) two studies have been performed according to OECD Guideline 301B and one according to OECD 301A, all in compliance with GLP (OECD 301B: Safepharm report, 1992 and Huntingdon Life Sciences reports 2010; OECD 301A: Huntingdon Life Sciences report, 2013). An additional study, realised under the same conditions, is not described here (Bayer, 1991).

In the first study, performed by Safepharm, ADCA was tested in medium and activated sludge bacteria from a predominantly domestic sewage at concentrations of 10 and 20 mg/l during 28 days. Cultures vessels were sealed and CO2-free air was bubbled through solutions; the released CO2 was evaluated daily, indicative of the degradation percentage of ADCA. ADCA attained 124% degradation at a concentration of 10 mg/l and 70% at 20 mg/l and so, therefore, can be considered as ready biodegradable under the strict terms and conditions of OECD Guideline No. 301 B.

In the second study, performed by Huntingdon Life Sciences in 2010 to OECD Guideline 301B, test, control and reference mixtures were aerated for 29 days with air that had been treated to remove carbon dioxide

(CO2). The CO2 produced by each culture was trapped in a series of Dreschel bottles containing barium hydroxide, which were connected to the outlet from each test vessel. The residual barium hydroxide was determined at intervals by titration.

Substances are considered to be readily biodegradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. The mean cumulative CO2 production by mixtures containing ADCA at 10 mg[C]/L was equivalent to 10% of the TCO2 after approximately 9 days and 32% by the end of the test on Day 29. Therefore, ADCA was not considered to be readily biodegradable under the conditions of this test. However, ADCA has shown evidence of primary degradation.

 

In the third study, performed by Huntingdon Life Sciences in 2013, a different test system was selected (OECD 301A), because in the previous Huntingdon study CO2 evolution was slow throughout the test. The test was designed to measure the dissolved organic carbon (DOC) removal using ADCA within its limit of water solubility at a test concentration of 5 mg[C]/l. Mean DOC removal was, at most, 6% of the initial measured values on Day 28. Therefore, ADCA was not considered to be readily biodegradable under the conditions of this test.

In the Bayer test (not completely described here), a biodegradation of 21 % after 30 day was found (Bayer, 1991), supporting the “non-readily biodegradability” properties of ADCA.

Available studies give contradictory results. The differences can be explained by many factors:

Differences in guideline/10-days window criterion:

The first study has been realised in the early 90’s and the guideline for the evaluation of the ready biodegradability potential was different. The 10-days window criterion was not included in the former version of the OECD 301B guideline. Results of the Safepharm study would have failed this 10-days window criterion, but it is not a criterion used for all the biodegradability assessments (e.g. MITI test).

Huntingdon Life Sciences’ results also fail this criterion.

 

Differences in the inoculums (STP) used for the assay:

Inoculums from different sources have been known to give differing results. The level of organisms in the first sludge tested, apparently capable of degrading ADCA, may have been higher than in the inoculums employed in the other tests.

 

Differences in concentrations tested:

Safepharm report tested 10 and 20 mg/l, which correspond to a very low concentration of Carbon (2.07 and 4.14 mg[C]/l). These concentrations are below the recommended concentrations in the current guideline (10 and 20 mg[C]/l). Using a concentration of the test substance of 20 mg/l, a final biodegradation rate of 70 % after 28 days is obtained.

 

Higher analytical errors due to very low concentrations induced a very high experimental variability in the results, as indicated by the non-relevant result of 124% of degradation at the low dose-level. The report mentioned: “As a result of the low carbon content any small increase in CO2 production or variations in the production of CO2 by the control vessels will produce large variations in the percentage degradation values”.

As stated in the SIDS 2000: “With an azodicarboxamide concentration of 10 mg/l a biodegradation of even 124 % after 28 days was found. However, the authors of the study consider this value to be the result of experimental variation due to analytical error and the low carbon content of the test material. As a result of the low carbon content any small increase in CO₂production or variations in the production of CO₂by the control vessels will produce large variations in the percentage degradation values”.

Therefore, the result of the test using the concentration of 10 mg/l seems not to be plausible and is not considered here. We can only consider the 20mg/l results, because they show a lower variability and because the concentration is closer to those recommended by the guideline.

The OECD 301A test performed by Huntingdon Life Sciences also used a concentration below the recommended concentrations in the guideline (10 – 40 mg [C]/l) but the method of quantitation on this occasion was considered sufficiently reliable at the level tested .

Problem of solubility of ADCA:

In the Huntingdon Life Sciences’ report for the OECD 301B study, a concentration of 10 mg[C]/l has been tested – as recommended in the guideline –, which corresponds to a concentration of approximately 48 mg/l. This value is above the measured limit of solubility of ADCA (33mg/l).

Water solubility is an essential parameter in ecotoxicological testing. Testing above the solubility limit can cause misinterpretation of the results and possibly alter the final result, by underestimating the final biodegradation percentage or by delaying the degradation process.

 

Conclusion on ADCA’s biodegradation properties:

Three out of four available screening studies indicate that ADCA is not readily biodegradable. It is therefore concluded, on weight of evidence, and based on screening studies, that ADCA is not readily biodegradable