1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt

Endpoint summary

Additional information

Biodegradation in water (screening test):

Ready biodegradation:

Key study:

Conducted under GLP conditions, ZMB2attained 27% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301B (Safepharm Laboratories, 2003).

Supporting study:

In accordance with OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test), during the test period of 28 days ZMB2 provides a breakdown rate of 0%. ZMB2 is considered as "not readily biodegradable" (Bayer AG, 1992).

Biodegradation in water and sediments (simulation test):

This study is concerned with the aerobic mineralisation of ZMB2 in natural surface water under controlled laboratory conditions. It was carried out according to the OECD-Guideline 309 "Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test”. The incubation was performed using a 14C-labelled test item [phenyl-U-14C]zinc-4- and 5-methyl-2-mercaptobenzimidazole under aerobic conditions at 12 °C in the dark.

The study was conducted using Biggesee lake (North-Rhine-Westphalia) natural water amended with 15 mg/L suspended of its own sediment.

The biological activity of filtered lake water was characterised by the addition of the easily degradable compound 14C-sodium benzoate to control samples (blank controls). Concentration of 14C-sodium benzoate was 10 µg/L. Control samples were handled in analogy to the treated samples. The formation of 14CO2 was followed until the mineralisation level of 75 % of the applied radioactivity [% AR] was reached. This was at 13 DAT  (days after treatment). NaOH-containing traps were analysed at 1, 2, 3, 6, 9 and 13 DAT. 14CO2 quantification was performed by liquid scintillation counting (LSC). To investigate the influence of acetonitrile, which was used as solubilizer, further solvent control samples (lake water plus solvent plus 14C-sodium benzoate) were incubated under identical conditions.

Surface water was collected on 30 October 2018. Prior to filtering through 100 µm mesh it was stored for 9 days. During storage the samples were kept at ca. 4°C in the dark. An exchange of air with the environment was ensured.

Test system

Incubation of subsamples was performed in a flow-through system which was placed in a dark, temperature controlled lab-room. A constant stream of water saturated surrounding air was passed over the subsamples in order to maintain aerobic conditions during the experiment. The outgoing gas was bubbled by means of a vacuum pump through three absorption traps in sequence containing ethylene glycol and two times 1 M NaOH in order to determine volatile products and the rate of mineralization (trapping of 14CO2).

For information on the abiotic transformation of the test substance additional sterilized samples (duplicates) were taken, processed and analysed at 7 days after treatment (DAT), and 63 DAT.

Treatment

Vessels (250 mL) for low concentration (9.5 µg/L) and high concentration (65 µg/L) were spiked with 27.90 µL (low) and 28.10 µL (high) of [phenyl-U-14C]ZMB2 application solution. The applications were made directly into the test samples by a pipette.

Intended nominal treatment rate at low concentration was 12.26 kBq (9.5 µg/L) and at high concentration 83.85 kBq (65 µg/L), respectively. Based on the mass balance of the 0d of incubation samples the actual concentration confirms generally to the indented concentration and a correction was not accomplished.

Sampling and Processing

Sampling was performed after the following incubation times: 0 d (immediately after application), 2 d, 6 d, 9 d, 15 d, 20 d, 28 d, 36 d, 57 d and 64 d after application (DAT). Additionally sterile samples were sampled after 7 d and 63 d. After sampling, the water samples were extracted and worked-up. In addition, the corresponding absorption traps were removed and the trapping solutions were analysed.

Test system parameter

Test system parameters, pH and dissolved oxygen were measured throughout the study. The dissolved oxygen values were between 5.37 and 9.25 mg/L. The pH values were between pH 7.79 and pH 8.37

The biological activity of filtered lake water was characterised by the addition of the easily degradable compound 14C-sodium benzoate to control samples (blank controls, and blank control plus solvent methanol ) and following the formation of 14CO2. At 6 DAT, formation of 14CO2 exceeded 60 % AR showing the viability of the test system in both, the control samples and solvent control samples.

Results

Mass balance

The mass balances in all non-sterilised individual samples (both radiolabels) at low and high concentration ranged from 72.9 to 135.9 % AR, with an overall mean of 94.0 % AR. The mean mass balances in all sterilised samples at low and high concentration ranged from 91.0 to 94.3 % AR.

Non-extractable residues

At 0 DAT the radioactive residues on suspended matter are very low with only 0.2% AR (low test concentration) and 0.1% AR (high test concentration). The NER levels are raising over the incubation. At incubation end (64 DAT) the residues were 7.9 and 9.8% AR (low test concentration) and 5.8 and 6.2% AR (high test concentration).

Volatile degradation products

In the most of the ethylenglycol samples (24 samples) the radioactive residues were on trace level amount (<0.1% AR). At irregular intervals radioactivity between 0.1 and 9.6 % AR were found but not confirmed by the second replicate. Therefore these amounts of radioactivity should be contaminations during incubation or sample work up.

Very low amounts of applied radioactivity were found in the 1 M sodium hydroxide absorption solutions. At incubation end the amounts of radioactivity were 0.3% AR and 5.5% AR (low test concentration) and 0.1% AR (high test concentration). The value of 5.5% AR is quite high and seems to be a contamination. A second contamination was found in the absorption trap of sample 20 DAT, high test concentration, replicate A (19.7% AR).

Radioactive residues in surface water extracts (non-sterile samples)

The test substance ZMB2 itself was never found in any samples. Obviously in the presence of water it rapidly decomposes or dissociates forming MB2 and zinc species.

In the 0 DAT samples, the primary metabolite MB2 was found at mean levels of 89.4% AR (low test concentration) and 91.5% AR (high test concentration). MB2 degraded continuously over time. At 64 days of incubation the MB2 degraded to mean levels of 10.4% AR (low test concentration) and 13.9% AR (high test concentration). Two major (>10% AR) secondary degradation products were formed during incubation in low and high test concentration samples. They were reaching maximum main levels of 34,4% AR (Desthio-MB2, low test concentration) after 36 days of incubation, 50.1% AR (Desthio-MB2, high test concentration) after 64 days of incubation, 31,0% AR (MB2 sulfonic acid, low test concentration) after 28 days of incubation and 20.3% AR (MB2 sulfonic acid, high test concentration) after 57 days of incubation.

A further (third) major degradation product (MB2-acetic acid) was only found for the low test concentration samples reaching a maximum main level of 10.0% AR after 64 days of incubation. In the high test concentration samples the maximum main level of MB2 acetic acid was 5.9% AR after 64 days of incubation.

Three further unknown degradation products were detected (unknown 1, 2 and 3) not exceeding a main level of 7.3% AR.

Radioactive traces on the TLC base line (origin) were quite low not exceeding a main level of 3.5% AR.

Radioactive residues in surface water extracts (sterile samples)

For the sterilised samples, the level of MB2 was 83.4% AR (low test concentration) and 87.4% AR (high test concentration) in the 7 DAT samples. At 63 days of incubation the MB2 degraded to mean levels of 43.6% AR (low test concentration) and 65.4% AR (high test concentration).

At 63 days of incubation the degradation product Desthio-MB2 was formed to mean levels of 39.5% AR (low test concentration) and 17.6% AR (high test concentration).

All other degradation products formed only in small amounts after 63 days of incubation under sterile conditions not exceeding 6.0% AR.

Biotic degradation is generally an important factor for the degradation of MB2. However especially Desthio-MB2 is also formed in higher amounts under sterile conditions. Therefore abiotic degradation of MB2 must be considered as relevant.

Degradation products

MB2: ZMB2 decomposes to MB2 and zinc ion in water within seconds. Thus, the degradation of MB2 is described.

Desthio-MB2, MB2 sulfonic acid, MB2 acetic acid, Hydrox-MB2 and three unknown degradation products were detected.

Result

After 64 DAT 10.4 % of applied radioactivity were found assigned to MB2 in the low concentration sample and 13.9 % in the high concentration sample.The DegT50 value of MB2 (12° C) is 12.9 days (low concentration) and 24.6 days (high concentration). The geometric mean DT50 of the recommended optimisation was found to be 17.8 days for the parent compound.

Biodegradation in soil:

Based on the use pattern of the substance, intentional releases to the soil compartment are not expected.  In accordance with Column 2 of Annex IX of the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) legislation, if direct and indirect exposure of soil is unlikely, a biodegradation in soil study does not need to be conducted, therefore the endpoint is being waived.

ZMB2 is not considered readily biodegradable (Safepharm, 2003).