mancozeb (ISO); manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt  

Hydrolysis

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Validity criteria fulfilled:

yes

Conclusions:

The identity of CPIII was proposed in Cardinali 2007 and 2016. However, it could be confirmed that CPIII did not occure in the Dobson (2018) study.

Executive summary:

The summary below is taken from the updated RAR Volume 3 CA B8 version December 2018

The hydrolysis of [14C]-mancozeb radiolabelled in the ethylenediamine position was investigated in the dark in sterile aqueous solutions at pH 4, 7 and 9. Three temperatures were investigated in the test (10°C, 20°C and 50°C). Normal ‘tier 1’ preliminary tests were not conducted as mancozeb is known to be hydrolytically unstable. The rate of hydrolysis of mancozeb as well as the formation of hydrolysis products were investigated for 30 days.

[Ethylenediamine-14C]-mancozeb with a radiopurity of 88.2% and a specific activity of 3.24 MBq/mg was used in this study. The study author noted that whilst the radiochemical purity was lower than would normally be expected for radio-labelled studies, the purity was typical for mancozeb. During the renewal evaluation the RMS  agreed with this observation.
The hydrolysis test was performed at three different pH-values. The following buffers were used: pH4 Citrate buffer (potassium citrate/sodium hydroxide, 0.05M) pH7 1.3 g of imidazole was diluted in 960 mL purified water and adjusted to pH 7.0 with 1M HCl. The solution was made up to 1000 mL (0.02M). pH9 Borate buffer solution (borate/potassium chloride/sodium hydroxide, 0.05M).
Buffer solutions were sterilised by filtration using sterile 0.2 μm filters and purged with nitrogen to remove oxygen. Sterility checks indicated that the solutions remained sterile during the course of the study.
Application suspensions of [14C]-mancozeb (0.14 mg/ml for suspension-1and 0.20 mg/mL for suspension-2) were prepared in extra dry acetonitrile. 0.07 mL aliquots of suspension-1 and 0.055 mL aliquots of suspension-2 were added to 7 mL of the appropriate buffer solution and continuously stirred (magnetic stirrer). Average test concentration was 1.54 mg/L which is lower than the reported water solubility of mancozeb, i.e. 6.2 mg/L. In order to aid metabolite identification, some suspensions were prepared with an exaggerated 10x dose.
The experiment was conducted at temperatures of 10, 20 and 50°C and pH 4, 7 and 9. The experiment was conducted by incubating samples of 7 mL sterile buffer solution containing the test item in screw capped amber glass sterile vessels under magnetic stirring (duplicates for 20°C pH7). The incubations were performed in the dark. For each pH and temperature, seven sampling intervals were taken (0, 0.06, 1, 2, 7, 25 and 30 days). There was no collection of volatile substances.
Duplicate samples were taken at each sample time. For the sample times until less than 5% AR was recovered as ethylene bis-dithiocarbamate (EBDC), one specific sample sampling method was used with a second method being used for subsequent sample times.
In the first sample method, 3 mL of an alkaline (approximately pH 9) ‘work-up’ buffer containing sodium hydroxide, EDTA and L-cysteine was added to the samples. The intention of the work-up buffer was to depolymerise mancozeb in order to release EBDC and maintain it in a stable form for HPLC analysis. HPLC analysis was performed immediately using two HPLC methods (1 and 4). Samples generated by the first sampling method were stored at -80°C.
In the second sampling method, 3 mL of pure water was added to the samples and HPLC analysis was performed ‘promptly’ using HPLC method 1. Storage of samples generated using sampling method 2 was at -20°C.

For comparability with previous studies where no work-up buffer (cysteine/EDTA) was used, and to investigate the effect of the work-up buffer on the pattern of metabolite formation in early interval samples, a limited number of additional early samples (0.06d and 1d at pH 4 and 25°C; 1d at pH 7, 25°C; 1d at pH9, 25°C) were prepared and amended with pure water rather than the work-up buffer at the time of sampling.
Sample analysis was by HPLC and TLC with UV and radiochemical [14C] detection. The study author considered that in view of the degradation products of mancozeb being of low molecular weight, no single analytical method provided good resolution for the full range of degradates as well as good detection options to allow for confirmation of identity of known degradates and the identification of unknown degradates. As a consequence, a number of different HPLC methods were used to help identification of degradates, methods 1 and 4 being the primary methods. LC was also conducted on some samples but specific details were not given of methodology, equipment and solvents used.

Mancozeb/EBDC, EBIS, ETU, EU, N-formyl ETU, TRCIT, TDIT, Jaffe’s Base and ethylene diamine were used as unlabelled reference standards in the analysis.
The study author conducted a kinetic analysis of the decline rates of mancozeb using CAKE v.3.3.

The temperature was constant throughout the incubation period (10 ±0.5°C; 25 ± 0.4 °C; 50 ± 0.5 °C with the exception of one short duration drop to 48.3°C). No significant variation of the pH value was observed in the buffer solutions and the solutions were sterile during the whole incubation period. Some microbial growth in sterility checks for two sampling points was seen, but as the negative sterility control also showed microbial growth, this was considered to be a procedural error with these two sterility checks rather than cases of microbial contamination. In addition, results of these two sample points were considered consistent within the trends seen in the experiment.

Maximum observed occurrences of mancozeb degradates in the Dobson 2018 hydrolysis study

Degradate ID	Sample ID	% ROI of Degradate	%AR in sample	%AR of Degradate*
Ethylene Diamine (EDA) RT, ca. 42 min on HPLC method 4**	S97, 1d, pH4, 50°C	68.4	96.0	65.7
Ethylene Urea (EU) RT, ca. 4.3 min on HPLC method 1 (subject to co-elutions in some samples***)	S125, 30d, pH 9, 50°C	36.7	100.4	37.0
2-(aminoethyl)-carbamodithioic acid (and/or isomers) RT, ca. 25 min on HPLC method 4**	S49, 1.5h, pH 4, 25°C	53.5	98.6	52.8
Ethylene Thiourea (ETU)	pH 7, 50°C, 30d	88.3	99.9	88.2
Unknown at 5.8min (primarily pH 9 10°C and 25°C samples, RT, 5.8 min on HPLC method 1)****	S72, pH 9, 25°C, 7d	25.2	99.1	25.0
N-Formyl-ETU (7.5-8.5min)	2d, pH 7, 25C	27.3	106.7	29.2
Unknown at 10-10.5min	S29,7d, pH9, 10°C	15.8	100.3	15.8
TCIT	S15, 1d, pH 7, 10°C	34.9	98.1	34.2
Unknown at 19.5min	S22, 2d, pH 7, 10°C	22.6	98.5	22.3
EBDC-Dimer (M11)	S155, Alternate work-up, pH 9, 1d, 25°C	9.6	97.7	9.4*****
Unknown at 40-42 min (probable dimer, treatment with cysteine/EDTA yields degradate at 19.5 min on HPLC method 1)	25d pH7 10°C	14.5	99.6	14.4
Terminal residue at pH 4 and 50°C degradate at 14.9 min on HPLC method 4**	S121,pH 4, 30d, 50°C	11.2	98.8	11.1
Terminal residue at pH 4 and 50°C degradate at 25.9 min on HPLC method 4**	S121,pH 4, 30d, 50°C	20.7	98.8	20.5
Terminal residue at pH 4 and 50°C degradate at 28.0 min on HPLC method 4**	S121, pH 4, 30d, 50°C	46.0	98.8	45.4

* Calculated manually from the % ROI and % AR values tabulated (% AR deg.= % AR samp × (% ROI/100), may show minor differences to values shown elsewhere due to rounding.

** Quantitation of this degradation was only possible on HPLC method 4 due to co-elution with other degradates on the primary HPLC method (Method 1). Not all samples were analysed by method 4, as such the maximum shown here is the maximum of the samples analysed, but possibly not of all samples.

*** Maximum observed in late phase pH 9 samples where the ID was confirmed by co-elution with the reference standard on TLC method 1, higher levels of activity at 4.3 min on HPLC method 1 (e.g. 1d, pH 4, 25°C) are due to EDA and 2-(aminoethyl)carbamodithioic acid (and isomers) which co-elute with EU on HPLC method 1.

**** This is probably an overestimate as it is based on HPLC method 1 where this degradate elutes on the tail of the much larger ETU peak, actual levels may be lower, further analysis may be required.

***** This degradate was only quantified in a single sample where an alternate work-up method was used.

The results shown are based primarily on HPLC method 1, however some chromatographic regions of interest determined by HPLC method 1 were found to contain multiple degradates when analysed by the alternative chromatographic methods e.g. HPLC method 4 (HILIC) and normal phase TLC.

The original proposed structure for CPII reported in the Agria study in the RAR (Cardinali (2007&2016) is considered to be incorrect based on new accurate mass LC-FTMS in a repeat hydrolysis study (Dobson, 2018).
It is highly likely that the two different studies used different types of MS and the accuracy of the method used in Dobson 2018 appears to be greater. The RMS considered in Renewal Process that the proposed identity of CPII as N-formyl ETU is plausible and reasonable.
The metabolite allocated the name CPIII was stated to have a molecular mass of 159 with a proposed molecular formula of C6H13N3S. Based on this, the study author of Dobson 2018 performed an investigation by LC-FTMS to look for fractions in chromatography from selected samples that would have corresponding theoretical exact mass m/z based on the proposed formula.

Reference 2

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

July 6, 1988 - October 31,1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)

Deviations:

no

Principles of method if other than guideline:

Work performed prior to implementation of GLP requirements

GLP compliance:

not specified

Radiolabelling:

yes

Analytical monitoring:

not specified

Details on sampling:

Samples (3 ml) were taken at 0, 1, 4, 8, 24, 30, 48, 54, 72; and 96 hours. It was noted that because the actual day 0 sample time varied between 0.25 and 1.58 hours after dosing, the 0 time sample was taken as the radiochemical purity of the test substance and 1 hour sample was actually 0.25 hours at pH 5, 1 hour at pH 7 and 1.58 hours at pH 9.

Buffers:

pH 5 buffer: 146 mL 0.1N acetic acid + 100 mL 0.1N NaOH + water qsp. 1L; pH adjusted to 5 with 0.1N NaOH.

pH 7 buffer: 12.9 mL 0.1M disodium hydrogen phosphate + 11.2 mL 0.1M potassium dihydrogen phosphate + water qsp. 500 mL adjusted to pH 7 with 0.1 N NaOH

pH 9 buffer: 56 mL 0.04 N HCl + 500 mL 0.01 M sodium borate + water qsp. 1 L; pH adjusted to 9 with 0.1 N NaOH.

Details on test conditions:

187 µL of a homogeneous suspension (it was commented by RMS that the term ‘homogeneous suspension’ was used in the original DAR, but reference to the study report indicates that mass balance in the study was very variable and attributed by the study authors to inhomogeneity of the dosing suspension) containing 12.7 mg of [14C] Mancozeb in 2.12 mL acetone were added to 100 ml of the respective buffer solutions in 250 mL Erlenmeyer flasks. The flasks are stoppered and incubated in a water bath at 25.4 ± 1.1 °C

Duration:

96 h

pH:

5

Temp.:

25.4 °C

Initial conc. measured:

8.6 other: ppm

Duration:

96 h

pH:

9

Temp.:

25.4 °C

Initial conc. measured:

8.6 other: ppm

Duration:

96 h

pH:

7

Temp.:

25.4 °C

Initial conc. measured:

8.6 other: ppm

Number of replicates:

1

Positive controls:

not specified

Negative controls:

not specified

Test performance:

Material balance: The mean recovery from the different buffer solutions was 104.8 ± 27.6 %, 101.0 ± 8.6%, and 119.0 ± 21.8%, at pH 5, 7, and 9. Variability in recovery was attributed to the fact that the test substance was applied as a suspension rather than a solution, i.e. it is likely that there would be inhomogeneity in a suspension. The actual range in recovery over the study was 43-188% where all day 0 samples were treated as 100%; of the 80 results, 45 fell outside the range 90-110%.

Hydrolysis rates: The hydrolysis rates of Mancozeb to more polar compounds was relatively fast at all 3 pH's. Half-lives of degradation were 2.2, 5.5, 14.1 hours at pH 5, 7, and 9, respectively.

Transformation products:

yes

No.:

#2

No.:

#1

Details on hydrolysis and appearance of transformation product(s):

Degradation profile: At pH 5, Mancozeb almost exclusively degraded to Ethylenethiourea (ETU). Only traces of ethyleneurea (EU) were observed. Slight amount (4%) of unknown compounds were observed at 96 h. TLC analysis confirmed this degradation profile and show the presence of ethylenebisisothyocyanatesulfide (EBIS).

At pH 7, ETU is the major degradation product of Mancozeb. At 96 h, only traces of EU were observed. TLC analysis show evidence of the presence of ETU, EU, and EBIS. EBIS concentration ranged from 18.4 to 44.5% of the applied dose throughout the study.

Conclusions:

Half-lives of degradation were 2.2, 5.5, 14.1 hours at pH 5, 7, and 9, respectively.

The proposed degradation pathway of Mancozeb can be described as follows: at pH 5 and 7, Mancozeb degraded mainly to ETU and then small amount of EU while at pH 9, it degraded first to EBIS and then to ETU and small amount of EU.

Executive summary:

The summary below is taken from the updated RAR Volume 3 CA B8 version December 2018

Materials and methods:

[¹⁴C] Mancozeb (specific activity: 9.61 mCi/g, purity: 92.5 %) was used together with 3 buffer solutions.

 

pH 5 buffer: 146 mL 0.1N acetic acid + 100 mL 0.1N NaOH + water qsp. 1 L; pH adjusted to 5 with 0.1N NaOH.

 

pH 7 buffer: 12.9 mL 0.1M disodium hydrogen phosphate + 11.2 mL 0.1M potassium dihydrogen phosphate + water qsp. 500 mL adjusted to pH 7 with 0.1 N NaOH

 

pH 9 buffer: 56 mL 0.04 N HCl + 500 mL 0.01 M sodium borate + water qsp. 1 L; pH adjusted to 9 with 0.1 N NaOH.

 

187 µL of a homogeneous suspension containing 12.7 mg of [¹⁴C] Mancozeb in 2.12 mL acetone were added to 100 mL of the respective buffer solutions in 250 mL Erlenmeyer flasks. The flasks are stoppered and incubated in a water bath at 25.4 ± 1.1 °C. Samples (3 ml) were taken at 0, 1, 4, 8, 24, 30, 48, 54, 72; and 96 hours.  It was noted that because the actual day 0 sample time varied between 0.25 and 1.58 hours after dosing, the 0 time sample was taken as the radiochemical purity of the test substance and 1 hour sample was actually 0.25 hours at pH 5, 1 hour at pH 7 and 1.58 hours at pH 9.  Different aliquots of the samples were respectively quantified by Liquid Scintillation Counting (LSC), analysed by HPLC and TLC with an ethanol:ethyl acetate:ammonium hydroxide (3:1:1) development system.

 

For HPLC analysis, samples were analysed within four hours of collection.  Two systems were used.  In the first, samples were fortified with a solution of reference standard of mancozeb with 0.5 N EDTA.  The HPLC mobile phase consisted of differing ratios of methanol/tetrabutylammonium phosphate and water/tetrabutylammonium phosphate/0.1M EDTA.  Whilst not specifically explained in the report, the use of EDTA is presumed to allow for solubilisation of mancozeb to facilitate HPLC by complexing manganese and zinc in the active substance.  The study authors indicated that a second HPLC method was required as the first system did not provide adequate separation of degradation products (data from HPLC system 1 gave concentrations for mancozeb, ‘degradates’ and ‘unknowns’).  For the second HPLC system, samples were fortified with a solution of reference standards of ETU, EU, hydantoin and EBIS.  No EDTA was added and the mobile phase was water.  The study authors stated that this second HPLC system retained mancozeb.

 

Findings:

Material balance: The mean recovery from the different buffer solutions was 104.8 ± 27.6 %, 101.0 ± 8.6%, and 119.0 ± 21.8%, at pH 5, 7, and 9.  Variability in recovery was attributed to the fact that the test substance was applied as a suspension rather than a solution, i.e. it is likely that there would be inhomogeneity in a suspension.  The actual range in recovery over the study was 43-188% where all day 0 samples were treated as 100%;  of the 80 results, 45 fell outside the range 90-110%.

 

Hydrolysis rates: The hydrolysis rates of Mancozeb to more polar compounds was relatively fast at all 3 pH's. Half-lives of degradation were 2.2, 5.5, 14.1 hours at pH 5, 7, and 9, respectively.

 

Degradation profile: At pH 5, Mancozeb almost exclusively degraded to Ethylenethiourea (ETU). Only traces of ethyleneurea (EU) were observed. Slight amount (4%) of unknown compounds were observed at 96 h. TLC analysis confirmed this degradation profile and show the presence of ethylenebisisothyocyanatesulfide (EBIS).

 

At pH 7, ETU is the major degradation product of Mancozeb. At 96 h, only traces of EU were observed. TLC analysis show evidence of the presence of ETU, EU, and EBIS. EBIS concentration ranged from 18.4 to 44.5% of the applied dose throughout the study.

 

At pH 9, Mancozeb is rapidly degraded to an intermediate (EBIS) that quickly degraded to ETU, then some EU.

 

Conclusions:

Half-lives of degradation were 2.2, 5.5, 14.1 hours at pH 5, 7, and 9, respectively.

The proposed degradation pathway of Mancozeb can be described as follows: at pH 5 and 7, Mancozeb degraded mainly to ETU and then small amount of EU while at pH 9, it degraded first to EBIS and then to ETU and small amount of EU.

Reference 3

Endpoint:

hydrolysis

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

other information

Study period:

completed 30th June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

FOCUS Degradation Kinetics (2006)

Estimation method (if used):

The hydrolytic degradation of mancozeb has been investigated in the laboratory in two studies at pH5, 7 and 9 (Lawrence et al. 1988 and Wang 1991).

The purpose of this evaluation was to analyse the degradation kinetics observed in the studies, taking into account the current guidance of the FOCUS workgroup on degradation kinetics and using a compartment modelling approach. Kinetic evaluation was performed in order to derive:

i) Degradation parameters as triggers for additional work (trigger endpoints)
ii) Degradation parameters for environmental fate models (modelling endpoints).

The calculated degradation parameters and kinetic endpoints of mancozeb for use as triggers for additional work and for modelling are summarised in the following tables. For the evaluated degradation experiments, the visual assessment and the goodness-of-fit statistics show plausible fit. The low t-test values of the degradation rate constants (p<0.05) indicate that the parameters were estimated significantly different from zero. Therefore, the resulting DegT50 values can be considered reliable. Kinetic modelling following the appropriate FOCUS Kinetics (2006) flowchart was carried out using CAKE 3.1.

Details on results:

The calculated trigger and modelling endpoint DT50 values are summarised in the table shown as "overall remarks"

Mancozeb trigger DT₅₀ and DT₉₀ endpoints – hydrolysis at pH5, 7 and 9

System	Best-fit model	Chi^2 (%)	Trigger endpoints
			DegT50 (hours)	DegT90 (hours)
pH5 Lawrence et al. (1988)	DFOP	12.1	0.85	9.2
pH7, Lawrence et al. (1988)	DFOP	10.2	1.7	18.3
pH9 Lawrence et al. (1988)	SFO	15.4	12.9	42.9

Mancozeb modelling DT₅₀ and DT₉₀ endpoints – hydrolysis at pH5, 7 and 9

System	Best-fit model	Chi^2 (%)	t-test (-)	Modelling endpoints
				DegT50 (hours)	DegT90 (hours)
pH5 Lawrence et al. (1988)	DFOP DT90/3.32	12.1	-	2.8	9.2
pH7, Lawrence et al. (1988)	DFOP DT90/3.32	10.2	-	5.5	18.3
pH9 Lawrence et al. (1988)	SFO	15.4	4.85E-05	12.9	42.9

Validity criteria fulfilled:

yes

Conclusions:

With the kinetic re-assessment according to FOCUS (2006) appropriate DT50 values could be derived for modelling and persistence endpoints. The DegT50 derived as trigger endpoints for pH 5, pH 7 and pH 9 where 0.85, 1.7 and 12.9 h, respectively.
The DegT50 derived as modelling endpoints for pH5, 7 and 9 are 2.8, 5.5 and 12.9 h respectively.

Executive summary:

The hydrolytic degradation of mancozeb has been investigated in the laboratory in two studies at pH5, 7 and 9 [Lawrence et al. (1988) and Wang (1991)].

 

The purpose of this evaluation was to analyse the degradation kinetics observed in the studies, taking into account the current guidance of the FOCUS workgroup on degradation kinetics and using a compartment modelling approach.

Kinetic evaluation was performed in order to derive

1. i) Degradation parameters as triggers for additional work (trigger endpoints)


2. ii) Degradation parameters for environmental fate models (modelling endpoints).

For the evaluated degradation experiments, the visual assessment and the goodness-of-fit statistics show plausible fit. The low t-test values of the degradation rate constants (p<0.05) indicate that the parameters were estimated significantly different from zero. Therefore, the resulting DegT₅₀ values can be considered reliable.

Reference 4

Endpoint:

hydrolysis

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

other information

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Estimation method (if used):

Kinetic modelling following the appropriate FOCUS Kinetics (2006) flowchart was carried out using CAKE 3.1.
The purpose of this evaluation was to analyse the degradation kinetics observed in the studies, taking into account the current guidance of the FOCUS workgroup on degradation kinetics and using a compartment modelling approach.
Kinetic evaluation was performed in order to derive
i) Degradation parameters as triggers for additional work (trigger endpoints)
ii) Degradation parameters for environmental fate models (modelling endpoints).
The calculated degradation parameters and kinetic endpoints of mancozeb for use as triggers for additional work and for modelling are summarised in the following tables. For the evaluated degradation experiments, the visual assessment and the goodness-of-fit statistics show plausible fit. The low t-test values of the degradation rate constants (p<0.05) indicate that the parameters were estimated significantly different from zero. Therefore, the resulting DegT50 values can be considered reliable.

Details on results:

The calculated trigger and modelling endpoint DT50 values are summarised in the table shown under "other information on results"

Mancozeb trigger and modelling DT₅₀ and DT₉₀ endpoints – hydrolysis at pH4, 7 and 9

System	Model	Chi^2 (%)	t-test (-)	Trigger/modelling endpoints
				DegT50 (hours)	DegT90 (hours)
pH4 20^oC	SFO	0.2	0.001905	0.39	1.3
pH4 35^oC	SFO	*	*	0.15	0.51
pH7 20^oC	SFO	15.8	7.79E-04	0.53	1.8
pH7 35^oC	SFO	6.2	0.006769	0.52	1.7
pH9 20^oC	SFO	0.2	0.002344	0.34	1.1
pH9 35^oC	SFO	0.2	0.003254	0.30	1.0

                               * not calculated, only 2 datapoints

Validity criteria fulfilled:

yes

Conclusions:

The derived endpoints DegT50 from the kinetic re-evaluation according to FOCUS (2006) that can be used as trigger and modelling endpoints for pH 4,7 and 9 at 20°C are 0.39, 0.53 and 0.34 hours, respectivelly.

Executive summary:

The hydrolytic degradation of mancozeb has been investigated in the laboratory in one study at pH4, 7 and 9 [Volkel (2001b)].

 

The purpose of this evaluation was to analyse the degradation kinetics observed in the studies, taking into account the current guidance of the FOCUS workgroup on degradation kinetics and using a compartment modelling approach.

Kinetic evaluation was performed in order to derive

1. i) Degradation parameters as triggers for additional work (trigger endpoints)


2. ii) Degradation parameters for environmental fate models (modelling endpoints).

For the evaluated degradation experiments, the visual assessment and the goodness-of-fit statistics show plausible fit. The low t-test values of the degradation rate constants (p<0.05) indicate that the parameters were estimated significantly different from zero. Therefore, the resulting DegT₅₀ values can be considered reliable.

Reference 5

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 30, 2000 Study plan - January 25, 2001 Final Report

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Analytical monitoring:

yes

Details on sampling:

The hydrolysis of [14C]-mancozeb i.e. manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt was investigated in aqueous solutions at pH 4, 7 and 9. Two temperatures were investigated in the main test (20°C and 35°C) since a pre-test at 50°C showed rapid hydrolysis of the test item. The rate of hydrolysis of mancozeb as well as the formation of hydrolysis products were investigated for up to 66 days.
The pre-test was performed at a temperature of 50°C. Three series of samples (pH 4, 7 and 9) were incubated. For each pH, four sampling intervals were taken (0 hours and at days 1, 4, and 14).

The main experiment was conducted at temperatures of 20 and 35°C for pH 4, 7 and 9.

For each pH and temperature, eight sampling intervals were taken (0 hours and at days 1, 2, 4, 7, 18, 32 and 66).

Buffers:

The hydrolysis test was performed at three different pH-values. The following buffers were used:
pH4: Citrate buffer (sodium citrate/hydrochloric acid, 0.05M)
pH7: 1.3 g of imidazole was diluted in 960 mL purified water and adjusted to pH 7.0 with 1M HCl. The solution was made up to 1000 mL (0.02M).
pH9: Borate buffer solution (borate/potassium chloride/sodium hydroxide, 0.05M).

Details on test conditions:

Buffer solutions were sterilised by autoclaving at 120°C for 30 minutes.

Application solutions (0.36 mg/mL) were prepared in acetonitrile. 1 mL aliquots of the application solution were added to 100 mL of the appropriate buffer solution and continuously stirred (magnetic stirrer). Measured test concentrations ranged from 2.44 - 3.76 mg/L.

The pre-test was performed at a temperature of 50°C. Three series of samples (pH 4, 7 and 9) were incubated. For each pH, four sampling intervals were taken (0 hours and at days 1, 4, and 14).

The main experiment was conducted at temperatures of 20 and 35°C for pH 4, 7 and 9.
The main experiment was conducted by incubating samples of 100 mL sterile buffer solution containing the test item in tightly closed sterile vessels under magnetic stirring (duplicates for 20°C pH7). The flasks were equipped with a gas inlet and outlet. The system was periodically ventilated with nitrogen. The outcoming gas was passed through a trapping system, equipped with absorption traps containing 50 mL of ethylene glycol and 50 mL of 2N NaOH, to trap organic volatiles and 14CO2, respectively. The incubation vessels (i.d. 10.6 cm, height 20 cm) were made of glass. The different series of samples were set-up in an incubation room at 20°C and in an incubator at about 35°C, respectively. In order to avoid photolytic effects, the incubations were performed in the dark.

Duration:

66 d

pH:

9

Temp.:

35 °C

Initial conc. measured:

3.2 mg/L

Duration:

66 d

pH:

9

Temp.:

20 °C

Initial conc. measured:

3.11 mg/L

Duration:

14 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

3.7 mg/L

Remarks:

Pre-test

Duration:

66 d

pH:

7

Temp.:

20 °C

Initial conc. measured:

2.99 mg/L

Duration:

66 d

pH:

7

Temp.:

35 °C

Initial conc. measured:

3 mg/L

Duration:

14 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

3.63 mg/L

Remarks:

Pre-test

Duration:

14 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

3.76 mg/L

Remarks:

Pre-test

Duration:

66 d

pH:

4

Temp.:

35 °C

Initial conc. measured:

2.92 mg/L

Duration:

66 d

pH:

4

Temp.:

20 °C

Initial conc. measured:

2.44 mg/L

Number of replicates:

1

Positive controls:

yes

Negative controls:

not specified

Preliminary study:

Except to confirm the rapid hydrolysis of mancozeb, the pre-test at 50°C (pH 4, 7 and 9) served to adapt the analytical methods to the rapidly appearing degradation products. After four days of incubation, the main end products were ETU and EU. Then after 14 days of incubation, the solutions were re-analysed and this result was confirmed.

Test performance:

Since the results of the pre-test at 50°C showed rapid hydrolysis of the test item mancozeb, the main test samples were therefore incubated at 20°C and 35°C.
The temperature was constant throughout the incubation period (20 ± 0.5 °C, 35 ± 0.5 °C and 50 ± 0.5 °C). No significant variation of the pH value was observed in the buffer solutions and the solutions were sterile during the whole incubation period.

The mean recoveries of total radioactivity were in the range of 99.4% to 104.5% of initial radioactivity for all pH values and temperatures during the main test.
[14C]-mancozeb and radioactive fractions were characterised by comparison of the retention times of reference items by HPLC and by co-chromatography using TLC analyses. The quantitative determination was carried out based on the results of the HPLC-analysis.

Transformation products:

yes

Remarks:

Apart from the test item mancozeb up to 19 additional radioactive fractions were detected during the course of the hydrolyses at the three pHs. 3 of them were identified as EU (ethylene urea), ETU (ethylene thiourea) and EBIS (ethylenebis isothcyanate).

No.:

#3

No.:

#2

No.:

#1

Details on hydrolysis and appearance of transformation product(s):

Mancozeb was hydrolysed very rapidly under acidic, basic as well as neutral conditions The hydrolysis of mancozeb passes through different transient intermediates. They are probably unstable ionic substances or organo-metallic complexes.
The number of intermediates and the duration of their existence depended on the pH of the solution. However, in all pH solutions, the final products were always ETU and EU (91.6-98.6%). This means that all intermediates are also transformed to these two substances. At pH 9, more EU was formed after 66 days of incubation than at pH 4 and 7. EU and ETU were shown to be stable to hydrolysis at all pH values tested (terminal residues).
The formation of the final products ETU and EU are influenced by pH. At pH 9 more EU is formed after 66 days of incubation than at the other pHs at 20°C as well as at 35°C. EU could be formed from ETU, but this reaction showed to be very slow even at higher temperature.
Apart from the test item mancozeb up to 19 additional radioactive fractions were detected during the course of the hydrolyses at the three pHs. Three of them were identified as EU (ethylene urea), ETU (ethylene thiourea) and EBIS (ethylenebis isothiocyanate sulfide). Radioactive fractions M4, M6, M7, M8, M12 and M14 were attempted to be characterised by LC/MS. All other degradates were not exceeding 10% of the applied radioactivity at any interval.
Mancozeb is quickly degraded under hydrolytic conditions (DT50 <<1 day) at pH 4, 7 and 9, with extensive degradation to EBIS, ETU and EU, as well as other intermediate components. ETU and EU, as terminal hydrolytically stable residues at all pH values, represent 91.6-98.6% and thus the vast majority of other components must be intermediates in their formation.
At pH 4, the terminal residues are ETU (85.7%) and EU (5.9%), with EBIS reaching a maximum of 33.4%.
At pH 7, the major metabolites are ETU (87.3%), EBIS (41.3%) and EU (13.1%).
At pH 9, mancozeb rapidly degraded to EBIS (up to 30.6%), with then further degradation to ETU (71.3%) and EU (31.2%).

% Recovery:

>= 91.3 - <= 104.4

pH:

9

Temp.:

35 °C

Duration:

66 d

% Recovery:

>= 90.1 - <= 107.3

pH:

9

Temp.:

20 °C

Duration:

66 d

% Recovery:

>= 91.4 - <= 104

pH:

7

Temp.:

35 °C

Duration:

66 d

% Recovery:

>= 98.5 - <= 104.7

pH:

7

Temp.:

20 °C

Duration:

66 d

% Recovery:

>= 92.3 - <= 108

pH:

4

Temp.:

35 °C

Duration:

66 d

% Recovery:

>= 92.3 - <= 114.2

pH:

4

Temp.:

20 °C

Duration:

66 d

pH:

9

Temp.:

35 °C

DT50:

<= 1.5 h

Type:

(pseudo-)first order (= half-life)

pH:

7

Temp.:

35 °C

DT50:

0.5 h

Type:

(pseudo-)first order (= half-life)

pH:

4

Temp.:

35 °C

DT50:

< 1.5 h

Type:

(pseudo-)first order (= half-life)

Key result

pH:

9

Temp.:

20 °C

DT50:

< 1.5 h

Type:

(pseudo-)first order (= half-life)

Key result

pH:

4

Temp.:

20 °C

DT50:

< 1.5 h

Type:

(pseudo-)first order (= half-life)

Key result

pH:

7

Temp.:

20 °C

DT50:

0.5 h

Type:

(pseudo-)first order (= half-life)

Other kinetic parameters:

A re-evaluation of DT50 values according to FOCUS (2006) was done by Hardy, I (2015)

Details on results:

The temperature was constant throughout the incubation period (20 ± 0.5 °C, 35 ± 0.5 °C and 50 ± 0.5 °C). No significant variation of the pH value was observed in the buffer solutions and the solutions were sterile during the whole incubation period.
The mean recoveries of total radioactivity were in the range of 99.4% to 104.5% of initial radioactivity for all pH values and temperatures during the main test.

The purity of mancozeb was determined on several occasions: in the application solution of the pre-test (92.1%), in the stock solution of the main test (91.0%), in the application solution of the main test (92.3%) and in the second application solution of the main test 92.0%.

Due to the relatively low specific activity of mancozeb, the hydrolysis was performed at a concentration of about 3 mg/L in order to be able to analyse aliquots of the test solutions directly without further working-up. Due to the rapid reactions and the presence of transient hydrolysis products, concentrating the solutions was not possible.
The quantitative determination was carried out based on the results of the HPLC-analysis using HPLC methods I and III, while mancozeb was still present, and thereafter by using HPLC method II. Methods I and III were run in parallel at the same time.

Pre-test:
Except to confirm the rapid hydrolysis of mancozeb, the pre-test at 50°C (pH 4, 7 and 9) served to adapt the analytical methods to the rapidly appearing degradation products. After four days of incubation, the main end products were ETU and EU. Then after 14 days of incubation, the solutions were re-analysed and this result was confirmed.
Main test:
Since the results of the pre-test at 50°C showed rapid hydrolysis of the test item mancozeb, the main test samples were therefore incubated at 20°C and 35°C.
The temperature was constant throughout the incubation period (20 ± 0.5 °C, 35 ± 0.5 °C and 50 ± 0.5 °C). No significant variation of the pH value was observed in the buffer solutions and the solutions were sterile during the whole incubation period.

The mean recoveries of total radioactivity were in the range of 99.4% to 104.5% of initial radioactivity for all pH values and temperatures during the main test.
[14C]-mancozeb and radioactive fractions were characterised by comparison of the retention times of reference items by HPLC and by co-chromatography using TLC analyses. The quantitative determination was carried out based on the results of the HPLC-analysis.

Apart from the test item mancozeb up to 19 additional radioactive fractions were detected during the course of the hydrolyses at the three pHs. Three of them were identified as EU (ethylene urea), ETU (ethylene thiourea) and EBIS (ethylenebis isothiocyanate sulfide). Radioactive fractions M4, M6, M7, M8, M12 and M14 were attempted to be characterised by LC/MS. All other degradates were not exceeding 10% of the applied radioactivity at any interval.
Volatile substances did not exceed 0.1% of the applied radioactivity.

Mancozeb was hydrolysed very rapidly under acidic, basic as well as neutral conditions. Its longest half-life (DT50) was observed at pH 7 (0.5 hours).

The hydrolysis of mancozeb passes through different transient intermediates. They are probably unstable ionic substances or organo-metallic complexes.
The number of intermediates and the duration of their existence depended on the pH of the solution. However, in all pH solutions, the final products were always ETU and EU (91.6-98.6%). This means that all intermediates are also transformed to these two substances. At pH 9, more EU was formed after 66 days of incubation than at pH 4 and 7. EU and ETU were shown to be stable to hydrolysis at all pH values tested (terminal residues).

The formation of the final products ETU and EU are influenced by pH. At pH 9 more EU is formed after 66 days of incubation than at the other pHs at 20°C as well as at 35°C. EU could be formed from ETU, but this reaction showed to be very slow even at higher temperature.

Mancozeb is quickly degraded under hydrolytic conditions (DT50 <<1 day) at pH 4, 7 and 9, with extensive degradation to EBIS, ETU and EU, as well as other intermediate components. ETU and EU, as terminal hydrolytically stable residues at all pH values, represent 91.6-98.6% and thus the vast majority of other components must be intermediates in their formation.
At pH 4, the terminal residues are ETU (85.7%) and EU (5.9%), with EBIS reaching a maximum of 33.4%.
At pH 7, the major metabolites are ETU (87.3%), EBIS (41.3%) and EU (13.1%).
At pH 9, mancozeb rapidly degraded to EBIS (up to 30.6%), with then further degradation to ETU (71.3%) and EU (31.2%).

At pH4, two transient unknowns measured after 1.5 hours at 44.8% (M12) and 36.0% (M14) but
LC/MS characterisation of unknowns:
Attempts were made to identify the transient unknown metabolites (M4, M6, M7, M8, M11, M12 and M14 by LC/MS. For the LC/MS evaluations, a higher concentration solution was prepared at 88 mg/L [above water solubility] and pH9.
EBIS, ETU, EU and Jaffe’s Base were used as reference substances.
Positive ion APCI mode was used in full-scan over the range m/z 80-500.

M4 was not found in the high dose pH 9 samples, thus no MS characterisation was possible.

M6 LC/MS analysis indicated mass ions of 147, 115 and 87, with 147 being attributed as [M+H]+. Based on these data a tentative structure was proposed as shown below.

M7 and M8 are very polar intermediates, that elute very fast (within 2 minutes) of the HPLC column. It was impossible to detect any specific mass in the total ion current, because the buffer medium showed very strong signals at the retention times of M7 and M8. Therefore, it was not possible to detect the masses of M7 and M8 by LC/MS in the buffer solution where they were generated.

M11 shows a similar chromatographic behaviour as EBIS using HPLC method II or TLC with solvent system 6. In none of the samples analysed by LC/MS could the degradate M11 be ionised. A further test was run with GC/MS, also without success.

M14 showed in the HPLC methods II and III extremely different retention times. HPLC method II has an acidic mobile phase and method III an alkaline mobile phase. The elution behaviour of M14 is similar to that of a weak carboxylic acid. In the acidic medium it protonated and in the alkaline medium dissociated and therefore eluted earlier.
The LC/MS spectra for M14 show an [M+H]+ molecular ion at m/z 195 with fragment ions at m/z 163, 131, 103, 99 and 86. MS/MS fragmentation of the m/z 131 ion resulted in ions of m/z 103, 99 and 72. Based on these data a tentative structure was proposed as shown below.

M12 has a mass spectra pattern very similar to those of M14, but in several fragments two mass units less. M12 was detected in the high dose pH9 buffer solution.
The LC/MS spectra for M12 show an [M+H]+ molecular ion at m/z 193 with fragment ions at m/z 161, 133, 129, 103, 101 and 87. MS/MS fragmentation of the m/z 193 ion resulted in ions of m/z 161, 133, 129 and 101. Based on these data a tentative structure was proposed as shown below.


The proposed structures are only tentative assignments based on the MS data and were not subsequently confirmed against synthesised standards. Their transient nature would anyhow most likely preclude synthesis.
At pH 7, M4, M12 and M14 all appear to be transient intermediates in the pathway between EBIS and ETU based on temporal formation. The tentative molecular weight of M14 of 194 g/mol is consistent as being a hydrolysis product of EBIS (mw 176 g/mol).
At pH 7 day 32, 99.9% applied radioactivity is accounted for by ETU (86.8%) and EU (13.1%) indicating that all other metabolites would appear to transients in the pathway between EBIS and ETU.
At pH 4, EBIS is somewhat more stabilised and M12 and M14 appear to be transformation products between mancozeb and EBIS/ETU. M11 appears to be transient between mancozeb/EBIS and ETU.
At pH 4 day 32, ETU (90.7%) and EU (3.6%) are the terminal residues accounting for 94.3%AR (102.3% including EBIS).
At pH 9, M7 and M11 appear to be transformation products between mancozeb and EBIS/ETU, M8 between EBIS and ETU and M6 between ETU and EU.
At pH 9 day 32, ETU (65.7%) and EU (31.2%) are the terminal residues accounting for 96.9%AR.

Thus overall, the key hydrolytic transformation pathways are formation EBIS, ETU and EU via various transient intermediates, resulting in terminal hydrolytically stable residues of ETU and EU.

Distribution of [¹⁴C]-mancozeb and formation of hydrolysis products during incubation in buffer solution at pH 4 (at 20°C and 35 °C)

20^oC / pH4	Percent applied radioactivity
Radioactive	Sampling interval (days)
Fractions	0	0.06	1	2	7	18	32	66
Mancozeb	92.3	6.2	*	*	*	*	*	*
M1 (EBIS)	*	2.1	26.6	33.4	31.4	9.1	8.0	2.4
M2 (ETU)	*	4.8	23.4	32.5	60.7	72.8	90.7	85.7
M3 (EU)	*	*	*	*	3.9	2.3	3.6	5.9
M4	*	*	*	*	*	3.6	*	2.6
M5	*	*	*	*	*	3.7	*	*
M6	*	*	*	*	2.5	*	*	*
M7	*	0.3	7.5	2.7	7.4	*	*	*
M8	*	*	*	*	*	*	*	2.6
M11	*	5.3	46.1	37.3	4.7	7.0	*	*
M12	*	44.8	*	*	*	*	*	*
M14	*	36.0	*	1.3	3.5	4.1	3.8	2.6
M15	*	3.2	*	1.5	*	*	*	*
M16	*	*	*	*	*	2.3	*	1.2
M17	*	*	*	0.7	*	*	*	*
^14CO2	np	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Total	92.3	102.7	103.6	109.4	114.2	104.9	106.0	102.9

 

35^oC / pH4	Percent applied radioactivity
Radioactive	Sampling interval (days)
Fractions	0	0.06	1	2	7	18	32	66
Mancozeb	92.3	*	*	*	*	*	*	*
M1 (EBIS)	*	44.4	27.6	22.8	6.0	*	*	*
M2 (ETU)	*	5.1	40.6	58.9	84.4	90.3	83.9	81.3
M3 (EU)	*	*	*	*	8.9	11.2	17.2	14.9
M4	*	*	*	*	*	*	*	1.7
M5	*	*	*	2.3	*	4.2	4.3	*
M6	*	*	*	3.0	*	*	*	*
M7	*	8.4	*	0.8	*	*	*	0.8
M8	*	*	*	*	*	*	*	4.1
M11	*	40.1	33.9	7.4	2.4	*	*	*
M14	*	*	*	2.6	2.5	*	1.5	0.8
M15	*	4.3	*	1.2	*	*	*	*
M16	*	*	*	*	*	*	*	1.2
M17	*	*	*	0.6	*	*	*	*
M19	*	*	*	*	*	*	1.1	*
^14CO2	np	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Total	92.3	102.2	102.1	99.4	104.2	105.8	108.0	104.9

np Not performed

* Not detected

 

 

 

 

Distribution of [¹⁴C]-mancozeb and formation of hydrolysis products during incubation in buffer solution at pH 7 (at 20°C and 35 °C)

20^oC / pH7	Percent applied radioactivity
Radioactive	Sampling interval (days)
Fractions	0	0.06	1	2	7	18	32	66
Mancozeb	86.3	13.1	11.3	*	*	*	*	*
M1 (EBIS)	14.0	41.3	8.0	5.7	*	*	*	*
M2 (ETU)	*	37.0	34.8	40.5	65.9	87.3	86.8	85.6
M3 (EU)	*	*	*	2.4	11.7	6.9	13.1	9.9
M4	*	*	18.2	13.4	*	*	*	*
M5	*	*	*	*	2.1	0.8	1.5	2.0
M6	*	*	*	*	2.4	4.7	*	0.4
M7	*	*	*	3.4	3.9	1.5	1.4	3.1
M8	*	*	*	1.5	3.2	0.4	*	0.9
M9	*	*	*	*	*	*	*	0.9
M10	*	*	*	*	7.8	*	*	*
M11	*	4.1	4.1	5.4	2.6	0.6	*	*
M12	*	1.6	9.4	5.4	*	*	*	*
M14	*	1.4	14.0	18.7	1.5	0.9	*	*
M15	*	*	0.3	0.4	1.1	1.0	1.8	1.2
M16	*	*	*	1.8	*	*	*	*
M17	*	*	*	1.1	*	*	*	*
M18	*	*	*	1.1	*	*	*	*
M19	*	*	*	0.8	*	*	*	*
^14CO2	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Total	100.3	98.5	100.0	101.6	102.0	104.2	104.7	103.9

 

35^oC / pH7	Percent applied radioactivity
Radioactive	Sampling interval (days)
Fractions	0	0.06	1	2	7	18	32	66
Mancozeb	86.3	12.4	4.1	*	*	*	*	*
M1 (EBIS)	14.0	26.5	0.9	*	*	*	*	*
M2 (ETU)	*	52.1	68.9	75.7	84.7	89.5	93.8	83.9
M3 (EU)	*	*	5.9	3.7	6.7	8.5	10.2	8.8
M4	*	*	*	3.1	*	*	*	*
M5	*	*	*	*	*	1.7	*	*
M6	*	*	*	*	*	*	*	2.6
M7	*	*	2.8	2.4	*	0.7	*	3.6
M8	*	*	*	2.5	*	*	*	1.2
M9	*	*	*	*	*	*	*	0.8
M10	*	*	*	*	*	*	*	*
M11	*	2.3	2.5	3.9	*	*	*	*
M12	*	6.2	*	2.3	*	*	*	*
M14	*	*	15.7	4.8	*	*	*	*
M15	*	*	*	*	*	2.2	*	0.3
M16	*	*	*	1.1	*	*	*	*
M17	*	*	*	0.9	*	*	*	*
^14CO2	n.p.	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Total	100.3	99.4	100.9	100.5	91.4	102.6	104.0	101.4

np Not performed

* Not detected

 

 

 

 Distribution of [¹⁴C]-mancozeb and formation of hydrolysis products during incubation in buffer solution at pH 9 (at 20°C and 35 °C)

20^oC / pH9	Percent applied radioactivity
Radioactive	Sampling interval (days)
Fractions	0	0.06	1	2	7	18	32	66
Mancozeb	66.5	4.5	*	*	*	*	*	*
M1 (EBIS)	*	30.6	2.5	0.8	*	*	*	*
M2 (ETU)	8.6	19.3	57.2	57.4	63.4	57.6	65.7	71.3
M3 (EU)	*	*	*	8.8	14.1	27.0	31.2	27.3
M5	*	*	*	*	*	5.3	*	*
M6	*	*	9.8	12.0	16.0	5.5	3.6	1.5
M7	8.4	12.3	13.4	4.5	5.8	1.3	1.9	2.7
M8	*	14.7	10.0	7.0	2.8	5.1	1.9	2.0
M9	*	*	*	*	*	*	*	1.2
M11	10.6	8.6	2.9	1.1	*	*	*	*
M12	*	*	*	0.8	*	*	*	*
M15	*	*	1.7	3.0	1.1	*	1.5	1.2
^14CO2	np	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Total	94.1	90.1	97.5	95.3	103.3	101.8	105.8	107.3

 

35^oC / pH9	Percent applied radioactivity
Radioactive	Sampling interval (days)
Fractions	0	0.06	1	2	7	18	32	66
Mancozeb	66.5	2.8	*	*	*	*	*	*
M1 (EBIS)	*	21.6	*	*	*	*	*	*
M2 (ETU)	8.6	20.4	60.2	66.4	64.5	62.3	67.1	60.5
M3 (EU)	*	*	*	19.7	27.7	37.6	34.5	35.8
M6	*	*	16.1	8.5	2.8	*	*	*
M7	8.4	46.9	8.2	3.5	0.8	0.7	1.4	2.7
M8	*	*	6.8	3.1	0.9	1.6	1.4	3.4
M9	*	*	*	*	*	*	*	1.3
M11	10.6	5.9	*	*	*	*	*	*
M12	*	*	*	*	*	0.7	*	*
M15	*	*	*	2.4	*	1.2	*	*
^14CO2	np	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Total	94.1	97.6	91.3	103.7	96.6	104.2	104.4	103.8

np Not performed

* Not detected

 

Validity criteria fulfilled:

yes

Conclusions:

Mancozeb is quickly degraded under hydrolytic conditions (DT50 <<1 day) at pH 4, 7 and 9, with extensive degradation to EBIS, ETU and EU, as well as other intermediate components.

Executive summary:

The summary below taken from the updated RAR Volume 3 CA B8 version December 2018

The hydrolysis of [14C]-mancozeb i.e. manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt was investigated in aqueous solutions at pH 4, 7 and 9. Two temperatures were investigated in the main test (20°C and 35°C) since a pre-test at 50°C showed rapid hydrolysis of the test item. The rate of hydrolysis of mancozeb as well as the formation of hydrolysis products were investigated for up to 66 days.

The experiment was set up by incubating for each temperature about 100 mL of sterile buffer solution containing the test item in closed vessels placed in an incubator (35°C) or a temperature controlled incubation room (20°C) using a magnetic stirrer for mixing. The vessels were equipped with an air inlet and outlet. The system was periodically ventilated with nitrogen. The outcoming gas was passed through a trapping system, equipped with absorption traces containing ethylene glycol and 2N NaOH, in this order, to trap organic volatiles and CO₂, respectively.

During the incubation, for each pH and temperature, 8 samples (duplicates for pH 7 / 20°C only) were taken and analysed by HPLC. Selected samples were also analysed by TLC. Due to the unstable nature of the test item mancozeb, it was stabilised before analysis by adding EDTA solution (4%, ethylenediamine tetraacetic acid disodium salt).

The temperature was constant throughout the incubation period (20 ± 0.5 °C, 35 ± 0.5 °C and 50 ± 0.5 °C).  No significant variation of the pH value was observed in the buffer solutions and the solutions were sterile during the whole incubation period.

The mean recoveries of total radioactivity were in the range of 99.4% to 104.5% of initial radioactivity for all pH values and temperatures during the main test.

[14C]-mancozeb and radioactive fractions were characterised by comparison of the retention times of reference items by HPLC and by co-chromatography using TLC analyses. The quantitative determination was carried out based on the results of the HPLC-analysis. Apart from the test item, mancozeb, up to 19 additional radioactive components were detected. Three of them were characterised as EU (ethylene urea), ETU (ethylene thiourea) and EBIS (ethylenebis isothiocyanate sulfide). Radioactive fractions M6, M12 and M14 were characterised by LC/MS.

 

Mancozeb was hydrolysed very rapidly under acidic, basic as well as neutral conditions. Its longest half-life (DT₅₀) was observed at pH 7 (0.5 hours).

 

The hydrolysis of mancozeb passes through different transient intermediates. They are probably unstable ionic substances or organo-metallic complexes. 

The number of intermediates and the duration of their existence depended on the pH of the solution. However, in all pH solutions, the final products were always ETU and EU (91.6-98.6%).  This means that all intermediates are also transformed to these two substances. At pH 9, more EU was formed after 66 days of incubation than at pH 4 and 7.  EU and ETU were shown to be stable to hydrolysis at all pH values tested (terminal residues).

 

The formation of the final products ETU and EU are influenced by pH.  At pH 9 more EU is formed after 66 days of incubation than at the other pHs at 20°C as well as at 35°C.  EU could be formed from ETU, but this reaction showed to be very slow even at higher temperature.

Mancozeb is quickly degraded under hydrolytic conditions (DT₅₀ <<1 day) at pH 4, 7 and 9, with extensive degradation to EBIS, ETU and EU, as well as other intermediate components.  ETU and EU, as terminal hydrolytically stable residues at all pH values, represent 91.6-98.6% and thus the vast majority of other components must be intermediates in their formation).  At pH 4, the terminal residues are ETU (85.7%) and EU (5.9%), with EBIS reaching a maximum of 33.4%.  At pH 7, the major metabolites are ETU (87.3%), EBIS (41.3%) and EU (13.1%).  At pH 9, mancozeb rapidly degraded to EBIS (up to 30.6%), with then further degradation to ETU (71.3%) and EU (31.2%).  At pH4, two transient unknowns measured after 1.5 hours at 44.8% (M12) and 36.0% (M14) but <LOD by 1 day were tentatively identified by LC/MS.  A third unknown (M11) reached 46.1% at 1 day before dropping to <10% by day 7 [M11 could not be ionised by LC/MS or GC/MS and may be the same as the highly transient unknown in the aerobic soil study].  At pH7, M12 (9.4%) and M14 (18.7%) were found at lower levels.  At pH9, intermediate unknowns were measured at 16.0% (M6), 13.4% (M7), 14.7% (M8) and 10.3% (M11), with M6 being analysed by LC/MS.

Description of key information

Mancozeb is quickly degraded under hydrolytic conditions (DT50 <<1 day) at pH 4, 7 and 9, with extensive degradation to EBIS, ETU and EU, as well as other intermediate components.

 

The summary below taken from the updated RAR Volume 3 CA B8 version December 2018:

The derived endpoints DegT50 from the kinetic re-evaluation according to FOCUS (2006) that can be used as trigger and modelling endpoints for pH 4, 7 and 9 at 20°C are 0.39, 0.53 and 0.34 hours, respectivelly.

Key value for chemical safety assessment

Half-life for hydrolysis:

0.53 h

at the temperature of:

20 °C

Additional information

Völkel, 2001

Mancozeb was hydrolysed very rapidly under acidic, basic as well as neutral conditions. Its longest half-life (DT₅₀) was observed at pH 7 (0.5 hours).

The number of intermediates and the duration of their existence depended on the pH of the solution. However, in all pH solutions, the final products were always ETU and EU (91.6-98.6%).  This means that all intermediates are also transformed to these two substances. At pH 9, more EU was formed after 66 days of incubation than at pH 4 and 7.  EU and ETU were shown to be stable to hydrolysis at all pH values tested (terminal residues).

 

Re-assessment of Völkel, 2001

Mancozeb trigger and modelling DT₅₀ and DT₉₀ endpoints – hydrolysis at pH4, 7 and 9

System	Model	Chi^2 (%)	t-test (-)	Trigger/modelling endpoints
				DegT50 (hours)	DegT90 (hours)
pH4 20^oC	SFO	0.2	0.001905	0.39	1.3
pH4 35^oC	SFO	*	*	0.15	0.51
pH7 20^oC	SFO	15.8	7.79E-04	0.53	1.8
pH7 35^oC	SFO	6.2	0.006769	0.52	1.7
pH9 20^oC	SFO	0.2	0.002344	0.34	1.1
pH9 35^oC	SFO	0.2	0.003254	0.30	1.0

                               * not calculated, only 2 datapoints

 

Lawrence, 1988

Materials and methods:

[¹⁴C] Mancozeb (specific activity: 9.61 mCi/g, purity: 92.5 %) was used together with 3 buffer solutions.

 

pH 5 buffer: 146 mL 0.1N acetic acid + 100 mL 0.1N NaOH + water qsp. 1 L; pH adjusted to 5 with 0.1N NaOH.

 

pH 7 buffer: 12.9 mL 0.1M disodium hydrogen phosphate + 11.2 mL 0.1M potassium dihydrogen phosphate + water qsp. 500 mL adjusted to pH 7 with 0.1 N NaOH

 

pH 9 buffer: 56 mL 0.04 N HCl + 500 mL 0.01 M sodium borate + water qsp. 1 L; pH adjusted to 9 with 0.1 N NaOH.

 

187 µL of a homogeneous suspension containing 12.7 mg of [¹⁴C] Mancozeb in 2.12 mL acetone were added to 100 mL of the respective buffer solutions in 250 mL Erlenmeyer flasks. The flasks are stoppered and incubated in a water bath at 25.4 ± 1.1 °C. Samples (3 ml) were taken at 0, 1, 4, 8, 24, 30, 48, 54, 72; and 96 hours.  It was noted that because the actual day 0 sample time varied between 0.25 and 1.58 hours after dosing, the 0 time sample was taken as the radiochemical purity of the test substance and 1 hour sample was actually 0.25 hours at pH 5, 1 hour at pH 7 and 1.58 hours at pH 9.  Different aliquots of the samples were respectively quantified by Liquid Scintillation Counting (LSC), analysed by HPLC and TLC with an ethanol:ethyl acetate:ammonium hydroxide (3:1:1) development system.

 

For HPLC analysis, samples were analysed within four hours of collection.  Two systems were used.  In the first, samples were fortified with a solution of reference standard of mancozeb with 0.5 N EDTA.  The HPLC mobile phase consisted of differing ratios of methanol/tetrabutylammonium phosphate and water/tetrabutylammonium phosphate/0.1M EDTA.  Whilst not specifically explained in the report, the use of EDTA is presumed to allow for solubilisation of mancozeb to facilitate HPLC by complexing manganese and zinc in the active substance.  The study authors indicated that a second HPLC method was required as the first system did not provide adequate separation of degradation products (data from HPLC system 1 gave concentrations for mancozeb, ‘degradates’ and ‘unknowns’).  For the second HPLC system, samples were fortified with a solution of reference standards of ETU, EU, hydantoin and EBIS.  No EDTA was added and the mobile phase was water.  The study authors stated that this second HPLC system retained mancozeb.

 

Findings:

Material balance: The mean recovery from the different buffer solutions was 104.8 ± 27.6 %, 101.0 ± 8.6%, and 119.0 ± 21.8%, at pH 5, 7, and 9.  Variability in recovery was attributed to the fact that the test substance was applied as a suspension rather than a solution, i.e. it is likely that there would be inhomogeneity in a suspension.  The actual range in recovery over the study was 43-188% where all day 0 samples were treated as 100%;  of the 80 results, 45 fell outside the range 90-110%.

 

Hydrolysis rates: The hydrolysis rates of Mancozeb to more polar compounds was relatively fast at all 3 pH's. Half-lives of degradation were 2.2, 5.5, 14.1 hours at pH 5, 7, and 9, respectively.

 

Degradation profile: At pH 5, Mancozeb almost exclusively degraded to Ethylenethiourea (ETU). Only traces of ethyleneurea (EU) were observed. Slight amount (4%) of unknown compounds were observed at 96 h. TLC analysis confirmed this degradation profile and show the presence of ethylenebisisothyocyanatesulfide (EBIS).

 

At pH 7, ETU is the major degradation product of Mancozeb. At 96 h, only traces of EU were observed. TLC analysis show evidence of the presence of ETU, EU, and EBIS. EBIS concentration ranged from 18.4 to 44.5% of the applied dose throughout the study.

 

At pH 9, Mancozeb is rapidly degraded to an intermediate (EBIS) that quickly degraded to ETU, then some EU.

 

Conclusions:

Half-lives of degradation were 2.2, 5.5, 14.1 hours at pH 5, 7, and 9, respectively.

The proposed degradation pathway of Mancozeb can be described as follows: at pH 5 and 7, Mancozeb degraded mainly to ETU and then small amount of EU while at pH 9, it degraded first to EBIS and then to ETU and small amount of EU.

 

Re-assessment of Lawrence, 1988

The summary below taken from the updated RAR Volume 3 CA B8 version December 2018

With the kinetic re-assessment according to FOCUS (2006) appropriate DT50 values could be derived for modelling and persistence endpoints. The DegT50 derived as trigger endpoints for pH 5, pH 7 and pH 9 where 0.85, 1.7 and 12.9 h respectively.
The DegT50 derived as modelling endpoints for pH5, 7 and 9 are 2.8, 5.5 and 12.9 h, respectively.

 

Cardinali, 2007 & 2016

The summary below is taken from the updated RAR Volume 3 CA B8 version December 2018

The study reported half-lives for mancozeb at pH 4, pH7 and pH 9 were less than one day at 25, 35 and 50°C, respectively. 

 

Dobson, 2018

As a result, the identity or presence of CPIII in Dobson 2018 could not be confirmed. Therefore the identity of CPIII as proposed in Cardinali 2007 and 2016 is still doubtful.