N,N'-bis{N-[N-(4-chlorophenyl)carbamimidoyl]carbamimidoyl}hexane-1,6-bis(aminium) diundec-10-enoate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August-19 October, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

yes

Remarks:

Temperature range was 19.8 – 21.5 °C instead of 20.0 – 24.0 °C. As degradation of the positive control was in the normal range this is considered as uncritical concerning the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: CHUA
Batch no.: 001/CHUA/0116
Appearance: A WHITE TO OFF-WHITE POWDER
Composition: 10-Undecenoic acid, comp. with N1,N14-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediimidamide (2:1)
CAS No.: 1884575-91-0
EINECS-No.: unknown
Purity: > 98.0% BY HPLC
Homogeneity 100 %
Stability H2O: unknown; EtOH: unknown; acetone: unknown;
CH3CN: unknown; DMSO: unknown
Solubility H2O: < 10mg/L EtOH: 0.1 - 1 g/L; acetone: 0.1 - 1 g/L;
CH3CN: 0.1 - 1 g/L; DMSO: 0.1 - 1 g/L
Production date Jan. 2016
Expiry date Dec. 2017
Storage Room Temperature (20 ± 5°C)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, adapted

Details on inoculum:

Activated sludge from a biologic sewage treatment plant was used as inoculum. The chosen plant is treating mostly domestic sewage.
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf. Date of collection: 15. Sep. 2016, batch no: 20160915.
The sludge was filtrated, washed with tap water (2x), then washed with and re-suspended in test medium. It was then aerated until use. The dry matter was determined as 4600 mg suspended solids/L.

Duration of test (contact time):

28 d

Initial conc.:

33 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

The medium was prepared from the stock solutions. The stock solution of the positive control was pr epared and its DOC was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined. The test vessels were filled with medium and inoculum. Then, all flasks were aerated for 96 hours with purified, CO2-free, moistened air to purge the system of CO2.

Flask volume: 1500 mL
Apparatus blanks: 2, containing mineral medium only
Blank Controls: 2, containing mineral medium and inoculum
Positive control flasks: 2, containing positive control, mineral medium and inoculum
Test flasks: 2, containing test item, mineral medium and inoculum
Abiotic control: 1, containing test item, mineral medium and HgCl2
Toxicity control .1, containing test item, positive control, mineral medium and inoculum
Inoculum concentration: 25.0 mg/L
Temperature: 19.8 – 21.5 °C
Duration.: 28 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L.

The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels. Magnetic stirrers were used to prevent deposition of inoculum. The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was
separately determined in each flask.

Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena. Each sample was measured in duplicate or triplicate, respectively (depending on the variation between the measured values). The carbon analyser was calibrated with freshly prepared reference solutions containing potassium hydrogen phthalate (TC), sodium hydrogen carbonate and sodium carbonate (IC) every month. After every start, quality control samples were measured.

Reference substance:

aniline

Preliminary study:

The test item CHUA is considered as “not readily biodegradable“under laboratory condition.
-The degree of biodegradation reached 28 % after 28 days.
- The 10-day-window began on day 7, at its end 27 % degradation was reached, missing the pass level of 60 % given in the OECD guideline.
- Because the test item is a mixture, the 10-day window has not to be taken into account. Degradation missed 60% within 28 days, either. Therefore, the test item is considered as “not readily biodegradable within 28 days” under laboratory condition.
- The abiotic degradation reached 0.2 %.
- The biodegradation of a chemical in environment depends on its interaction with microorganism (strain related), soil, light, water etc. The study has been carried out under stringent laboratory conditions of dark and low temperature (19.8 – 21.5 °C). The low values of biodegradation do not mean that the test item is not biodegradable under environment conditions.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

60

Sampling time:

24 d

Results with reference substance:

All validity criteria were met. Degradation of the positive control was 65 % after 9 days.

Validity

Parameter	Criterion	Found	Assessment
IC content of test item solution in medium	≤5% of TC	0%	valid
CO2 emitted by the controls	< 70 mg/L	9.5 mg/L	valid
Difference within replicates	≤20%	3 %	valid
Degradation of positive control > 60%	≤14 days	9 days	valid
Degradation in the toxicity flask on day 14	> 25%	4 %	see discussion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item CHUA was tested using a concentration of nominally 20 mg organic carbon/L (corresponding to 33.0 mg CHUA/L) in test medium following OECD 301B and EU-Method C.4-C. Aniline was chosen as positive control. Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L). The test was left running for 28 days. All validity criteria were met. Degradation of the positive control was 65 % after 9 days.
If degradation in the toxicity flask is below 25% after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 4% after 14 days, the test item must be stated as “toxic” towards the inoculum in a concentration of 33.0 mg/L The following data were determined for the test item CHUA: 10-day-window: day 7 – 17 degradation at the end of 10-day-window 27 % degradation at the end of the test 28 % pass level following guideline: 60% at the end of 10-day-window for pure substances respective 60 % at the end of the test for mixtures
Therefore, regardless of the 10-day-window, CHUA is not readily biodegradable following OECD 301B/EU C.4-C. The biodegradation of a chemical in environment depends on its interaction with microorganism (strain related), soil, light, water etc. The study has been carried out under stringent laboratory conditions of dark and low temperature (19.8 – 21.5 °C). The low values of biodegradation do not mean that the test item is not biodegradable under environment conditions.

Executive summary:

Degradation behaviour of positive control was normal. Abiotic degradation reached 0.2 %. Both replicates of the test item showed very good correspondence. For pure substances ready degradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10%. Because the test item is a mixture, the 10-day window has not to be taken into account. If degradation in the toxicity flask is below 25% after 14 days, the test item can be considered as toxic towards the inoculum.

As degradation in the toxicity flask was 4% after 14 days, the test item must be stated as “toxic towards the inoculum in a concentration of 33.0 mg/L”. However from day 15 until the end of the test a slight increase of degradation in the toxicity flask was observed. The test item CHUA was tested using a concentration of nominally 20 mg organic carbon/ L.

According to the guideline, if toxicity could be observed, the test should be repeated using a lower test item concentration. In the test flasks a plateau of degradation around 27 % was reached on day 15. Therefore it must be stated as improbable, to reach the pass level of 60 % degradation in a test with a lower test item concentration. No observations were made which might cause doubts concerning the validity of the study outcome.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Degradation behaviour of positive control was normal. Abiotic degradation reached 0.2 %. Both replicates of the test item showed very good correspondence.

For pure substances ready degradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10%. Because the test item is a mixture, the 10-day window has not to be taken into account.

If degradation in the toxicity flask is below 25% after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 4% after 14 days, the test item must be stated as “toxic towards the inoculum in a concentration of 33.0 mg/L”.

However from day 15 until the end of the test a slight increase of degradation in the toxicity flask was observed. The test item CHUA was tested using a concentration of nominally 20 mg organic carbon/L. According to the guideline, if toxicity could be observed, the test should be repeated using a lower test item concentration.

In the test flasks a plateau of degradation around 27 % was reached on day 15. Therefore it must be stated as improbable, to reach the pass level of 60 % degradation in a test with

a lower test item concentration.

No observations were made which might cause doubts concerning the validity of the study

outcome.