N,N-dicyclohexylbenzothiazole-2-sulphenamide

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:

1988-12-07 till 1989-01-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: basic data given

Qualifier:

according to guideline

Guideline:

EU Method C.4-F (Determination of the "Ready" Biodegradability - MITI Test)

Version / remarks:

Cited as Directive 84/449/EEC, C.7

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Reference substance:

aniline

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 2

Sampling time:

28 d

Details on results:

reference substance (aniline): 71% biodegradation in 28 days

Validity criteria fulfilled:

not specified

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

2% biodegradation of DCBS was observed in 28 days according to EU Method C.4-F (Determination of the "Ready" Biodegradability - MITI Test) (Bayer AG 1989); and hence DCBS is considered as "Not Readily Biodegradable".

Executive summary:

2% biodegradation of DCBS was observed in 28 days according to EU Method C.4-F (Determination of the "Ready" Biodegradability - MITI Test) (Bayer AG 1989); and hence DCBS is considered as "Not Readily Biodegradable".

Description of key information

Readily Biodagradation:
2% biodegradation of DCBS was observed in 28 days according to EU Method C.4-F

(Determination of the "Ready" Biodegradability - MITI Test) (Bayer AG 1989); and hence DCBS is considered as "Not Readily Biodegradable". A hydrolysis half-life of 53 hours at pH 7 and 25°C was observed for DCBS (Bayer AG, 1997) with Dicyclohexylamine and 2-mecarptobenzothiazole (MBT) as the main hydrolysis products (MITI, 2005). N,N-Dicyclohexylamine was reported as readily biodegradable with 96% biodegradation observed in 20 days according to OECD guideline 301D (Bayer AG, 1975), but MBT was not readily biodegradable with only 2.4 % biodegradation observed in 14 days (MITI, 1992). In the Appendix A of EU-Risk Assessment for CBS (CAS: 95-33-0), information regarding the biodegardabilities of the main metabolites of MBT, such as MBTS, BT, BTon and MeSBT, is also summarized and discussed. The described metabolites are also not readily biodegradable. Only when the inoculi were adapted under special conditions, certain level of degradation of MBT, BT, BTon and MeSBT was observed. Such processes are expected in biological treatment plant, but not in the environment.
Inherent biodegradation:
Report Currenta 2013a describes the inherent degradation of DCBS. Although performed under GLP and an accepted OECD guideline, there was one significant deficiency as it cannot be excluded that one of the four test vessels was contaminated with NaOH solution. Removal of the result of this test vessel shows degradation rates in the three remaining vessels of 13, 23 and 50% after 28 days (mean 29%) and 16, 64 and 85% after 60 days (mean 55%). The substance is therefore considered to be" Not Inherently Biodegradable" . The reference compound sodium benzoate showed 94 % degradation after 14 days.

Report Currenta 2014a describes the inherent degradation of DCBS with identification of metabolites.The analytical recovery of the parent compound at day 0 was found to be unsatisfactory low. Nevertheless, MBT as a primary metabolite was found to be too high. Obviously the analytical method at these low concentrations was not stable enough.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Ready biodegradation:

The MITI study (2005) was used as critical study for SIDS endpoint and assigned to be reliability of 1. However here it is only assigned to be a reliability of 2 since the original reference is not yet available for assessment and hence it is not used as key study.

Inherent biodegradation:

The test for its inherent biodegradability potential was determined according with the OECD Guideline 302C “Inherent Biodegradability: Modified MITI Test (II)” (1981) (based on standard operating procedures for OECD Guideline 301F (1992) and the Council Regulation (EC) No 440/2008, method C.4-D “Manometric Respirometry Test” (2008)) (Currenta 2013a). Deviating from the guideline OECD 302C “Inherent Biodegradability: Modified MITI Test (II)”, the test was conducted with reduced amount of activated sludge and test item. The guideline states: If the test material is not soluble at the test concentration, special measures are allowed to achieve a good dispersion. Therefore a reduced test item concentration of 10 mg/L and a reduced amount of activated sludge (33 mg/L) from three different sources were used. A suspension of the substance in a mineral medium was inoculated with 40 % sludge of each of two different municipal sewage treatment plants (STP) and 20 % sludge of an industrial STP and incubated under aerobic conditions. During this period, degradation was followed by continuous automated BOD determinations. The test was prolonged until 60 days as the biodegradation curve showed that biodegradation has started but that the plateau has not been reached. The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item. A reference compound (sodium benzoate) was run in parallel to check the operation of the procedures. The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given by the manufacturer of the equipment. At the end of incubation, the pH was measured in the flasks. The degradation rate of the test item was set in relation to its experimentally determined COD. As the COD implicitly covers the oxygen demand for the nitrification process, there was no need to take additional correction measures into account. The test item is an N-containing substance. Therefore, the concentration of nitrite and nitrate was determined at test start. The oxygen consumed by nitrification was not determined after 28 days. Therefore, the substance is considered to be "Not Inherently Biodegradable" but shows a continuous degradation up to 66 % within 60 days. Although the conditions for "inherently biodegradable" are not fulfilled, the phrase "inherently degradable but not fulfilling specific critera" was chosen in order to describe the degradation behaviour.

In their response to this study, the evaluationg member state Germany outlined: The inherent biodegradability tests

(Currenta a/c) show experimental shortcomings. One test vessel with test substance in both

studies has undesignedly and extremely higher pH value (both > pH 9.4) than all the other

test vessels with test substance and those with blank tests (all < pH 7.1). These two test

vessels show extremly high biochemical oxygen demand (BOD) values, which wrongly

indicate a biodegradation of the test substance beyond 100%. This influences the calculated

mean value, erroneously. Following the guideline, these two test vessels have to be

removed from any further assessment and conclusion. The mean value of the degradation

(%) of the remaining three test vessels for both studies was calculated following the

procedure of calculation by the author of the studies. The mean degradation reached after

28 days is 3.3 °h for Currenta 2013c and 28.7 % for Currenta 2013a. It can be therefore

concluded that DCBS is NOT inherently biodegradable.

In a second inherent biodegradation test (Currenta 2013b) DCBS was used in even lower concentrations (67 µg/L). As this is still above the water solubility, Trifluoroethanol was used as a solubiliser. Samples were taken in intervals of 7 days up to 61 days. DCBS and the metabolites MBT, DCHA, MeSBT, BT, BTon and BTSO3H were monitored with a specific LC/MS/MS method. DCBS was found to degrade by 81% within 14 days thus fulfilling the requirement for inherent biodegradability (>60%). After 61 days DCBS and metabolites have decreased to value of 0.1 to 2 µg/L.

In their response to this study, the evaluationg member state Germany outlined:

Two other screening tests on inherent biodegradability (Currenta, 2013b/d) are not reliable

because purity was unclear, the mass balance at the start of the experiment is not

confirmed, and pH values are partly implausible or not reported. Nevertheless, MET (CAS

149-30-4) and Dicyclohexylamine (CAS 101-83-7) were identified as primary degradation

products suggesting that hydrolysis, already before the start of the experiment, is the

dominant degradation process in these tests and not biological degradation.