ANALYSIS OF DITHIOCARBAMATES AND THIURAM

DISULPHIDES USED IN AGRICULTURE.* 1.-Collaborative

Study of Macro-methods available for the Evaluation of Technical
and Formulated Products
By A. STEVENSON

The paper details results found in a recent collaborative study of methods of analysis
for the dithiocarbamates and thiuram disulphides.
In order to ensure maximum accuracy, reproducibility, and simplicity of apparatus
and operation, preference is given t o macro-volumetric methods for determining the pro-
ducts of acid hydrolysis, i.e., carbon disulphide and, where possible, the combined amine.
Shortcomings of previously published methods are illustrated, and alternative procedures
given which are now accepted as official methods for zineb, maneb and thiram in technical
products and simple formulations.
More complex formulations, including admixtures with copper and mercxry salts, will
be dealt with in a later paper.

Introduction
In 1960, the Collaborative Pesticides Analytical Committee of Europe (CPAC)l allocated
to Germany the task of providing a method of analysis for thiuram (tetramethylthiuram
disulphide), while in 1961 the Netherlands were made responsible for a method for zineb (zinc
ethylene-r,z-bis-dithiocarbamate). However, as these compounds are finding increased use in
agriculture, in January 1962, it was considered desirable, by the Pesticides Analysis Advisory
Committee (PAC) of the Ministry of Agriculture, to set up a Dithiocarbamates Panel to examine
critically methods in common use in Great Britain.. The Reports of this Panel were submitted
to PAC and finally to CPAC. As a consequence of interest shown in other European countries,
CPAC enlarged the original terms of reference and made Great Britain, Belgium, Germany,
the Netherlands and Scandinavia jointly responsible for providing agreed methods. All are
represented on the Dithiocarbamates Panel of PAC.
In view of the wide interest shown it was thought desirable to publish an account of the
Panel’s work which has led to the adoption by CPAC of Methods for thiram, maneb and zineb.
CPAC further decided that Scandinavia should be responsible for setting up a Panel
including Belgium, the Netherlands, and Great Britain to consider colorimetric methods of
analysis for dithiocarbamates.
At the first meeting of the British Panel, it was agreed that any method recommended
to CPAC should fulfil the following conditions:
(I) The active ingredient content must, wherever possible, be evaluated via a deter-
mination of its functional group, -CS-S- in the dithiocarbamates, and the disulphide link,
-CS*S-S-CS- in thiram, since antifungal activity has been correlated with these functional
groups.
(2) The accuracy obtainable, and the reproducibility between different laboratories, must
be of the highest order possible so that the method could be used as the basis of a purchase
specification, often having close acceptance limits. Thus, the precision must be well within
the acceptance limits (usually 98% minimum in the case of technical thiram for example) if
unnecessary disputes are to be avoided between user and supplier.
(3) As the method may be used for routine analysis of bulk consignments it should, if
possible, be capable of speedy operation by semi-skilled or junior staff and, preferably, involve
the use of simple, safe and inexpensive reagents and apparatus.
(4) Interference from likely impurities must be minimised where possible.
With these aims in mind it was decided that macro-methods, involving the volumetric
determination of the products of acid hydrolysis, were preferable to spectrophotometric or
polarographic methods-although it was acknowledged that the latter are more adaptable for
the micro-determination of crop residues.
* This paper is the basis of a lecture given before the Pesticides Group on 16 December, 1963

J. Sci. Fd Agric., 1964, Vol. 15, August

I1
510 STEVENSON-DITHIOCARBAMATES A N D T H I U R A M DISULPHIDES. I
Hence an intensive study of available methods was made, but before describing the
collaborative work it is fitting to consider, very briefly here, the state of the literature to date
and the chemical reactions involved in analysis.
Literature survey
Conditions for the acid digestion have been investigated by a number of workers with
particular attention being given to the acid concentration. Callan & StraffordZmust be credited
with having first devised the method involving hydrolysis with 7-5~-sulphuricacid.
Later workers found that much lower acid concentrations were preferable to minimise
interference from impurities. Clarke et al., employed I.IN-acid, but acknowledged that much
longer reaction times for maneb, zineb and ferbam (ferric dimethyldithiocarbamate) were needed.
Other worker^^-^ used orthophosphoric acid, but admitted interference from such com-
pounds as ethylenethiourea.
In 1932, Matuszak' made an exhaustive study of the iodometric titration conditions following
the absorption of carbon disulphide in alcoholic potassium hydroxide. He stressed the import-
ance of keeping the xanthate solution cool, and neutralising to the phenolphthalein end-point
before titration. Pattersons agreed with these points, which seem to have been ignored in later
published methods. Eusuf & Khundkarg specified an optimum pH of 5.4-5'8 for the iodine
titration and the use of an acetate buffer.
Halllo reported on a collaborative study using the methods of Rosenthal and Clarke and,
finding that the results for maneb, zineb and ferbam were inconsistent, proposed that a further
study of analytical methods was needed. Bontoyanll reported on such a study, but again
concluded that further investigation was necessary, and also suggested that a survey of other
methods should be made.
These constitute some of the reasons for the formation of the present Panel.
Experimental
In general, the dithiocarbamates are smoothly decomposed by hot dilute mineral acid to
yield carbon disulphide and the corresponding amine, either of which may be determined.
However, usually carbon disulphide is selected and determined iodometrically or colorimetrically.
The compounds used in agriculture can be divided broadly into two classes :
(a) The mono-dithiocarbamates based on methylamine and dimethylamine, such com-
pounds include ziram (zinc dimethyldithiocarbamate), ferbam, cumate (copper
dimethyldithiocarbamate) and metham-sodium (sodium N-methyldithiocarbamate).
( b ) The bis-dithiocarbamates, based on the bi-functional primary amine, ethylenediamine-
including nabam (disodium ethylene-1,z-bisdithiocarbamate),zineb and maneb.
Group ( a ) in the presence of dilute acid decomposes thus:
R,N*CS.S--X+ --f R,NH + CS, + X+
where R = CH, or H and X is a uni- or poly-valent cation-usually Na, Zn or Fe. Thus the
method is concerned only with the accurate determination of carbon disulphide or arnine liber-
ated. Any hydrogen sulphide evolved must have come from the breakdown of impurities, e.g.,
sulphides or trithiocarbonates, present in the original sample.
The ease of decomposition of the metal salts in dilute acid is known to fall off in the order
Na+, Zn2+, Fe3+, Cu2+, but all follow the above route.
GYO@ (b) ethylene bis-dithiocarbamates, as pointed out by Clarke, but not generally appre-
ciated, can follow at least two decomposition routes, which can produce misleading results, as
illustrated :

J. Sci. Fd Agric., 1964, Vol. 15, August

 STEVENSON-DITHIOCARBAMATES A N D T H I U R A M DIS ULPHIDES. I 511

Route A is said to be quantitative when boiling dilute sulphuric acid is used, and yields the
anticipated 2 moles of carbon disulphide per mole of ethylene bisdithiocarbamate.
Route B is a slower reaction favoured by the use of cooler acid. If this route is taken
50% of the available sulphur, which would otherwise have been evolved as carbon disulphide,
is fixed as ethylenethiourea and hydrogen sulphide, and a low result is obtained. Furthermore,
it might be wrongly inferred that, because of the evolution of hydrogen sulphide, sulphidic
impurities are present.
Tautomeric changes in structure make feasible several similar reactions in which the net
result, as with Route B, is that low recoveries of carbon disulphide are found.
Thus, the aim must be to use conditions of acid digestion such that not only are these side
reactions eliminated, but also that interference from likely impurities is kept at a minimum.
Such impurities include the bis-thiuram mono- and poly-sulphides which do not hydrolyse
quantitatively under conditions of normal acid digestion (see mechanism proposed for thiram
later).
Owing to the low volatility of ethylene diamine, dithiocarbamates based on this are not
easily determined from the amine residue.

(I) Survey of methods proposed by other countries

Because of the reservations given earlier, a number of methods proposed by other CPAC
countries were rejected for the time being. The only two methods which seemed promising
were those due to Roth12 and Clarke.3 The latter method has, however, in the considerable
manufacturing experience of one of the Panel members, been shown to lack precision and repro-
ducibility, and this has entailed modification of the method. This modified method is described
later.
The Roth method, unlike that of Clarke, is claimed as a general procedure for dithio-
earbamates and thiuram disulphides, and its scope is said to include technical and formulated
products, except when large amounts of copper are present. The sample is digested with a
mixture of pyridine and formic acid, and the carbon disulphide evolved is determined in the
usual manner by absorption in alcoholic potassium hydroxide and titration, with iodine, of
the xanthate formed.
As a result of a critical practical survey of the Roth method by the author, it was found
that certain substances afford abnormal results of poor reproducibility.
(a) Dithiocarbanzates
When zineb and maneb formulations were tested by the Roth and modified Clarke methods
respectively, the former gave lower and poorly reproducible results.
The function of the various reagents, as employed by Roth, was investigated and that of
the pyridine seems questionable, as this causes lower results, the depression from the result by
the Clarke method being proportional to the volume used. The method specifies 3 ml. of pyri-
dine, but the degree of accuracy in measurement is not stated. Some results are shown in
Table I.

Table I
Comparison of Roth and Clarke methods
M a n e b 65 % formulation Zineb 65% formulation
_ _ . _ ~
Roth ~ ~_ Clarke
___ Roth -
Clarke
F'yridine used, Maneb found, Maneb found, Pyridine used, Zineb found, Zineb found,
ml. % % ml. % %
nil 66.7 66.7 nil 65.2 65.6
0.5 65'5 66.5 3 61.8
3 60.8, 62.5, 61-5 66.4

The lower result could be due to the pyridine (which is added prior to the acid) promoting
the slow reaction B, referred to earlier, which will also be favoured by the low temperature a t
which the formic acid is added. In the Clarke method this is minimised by adding the acid hot.
J. Sci. Fd Agric., 1964, Vol. 15, August
512 STE VENSON-DITHIOCARBAMATES A N D T H I URAM DISULPHIDES. I
In an addendum to his method Roth proposed that the maneb sample should be dissolved
in 2 ml. of wsodium hydroxide instead of pyridine and that the running time be extended to
2 h.
This method was examined, and the results by the Roth and modified Clarke methods are
compared in Table 11, for two different maneb formulations.
Table I1
Analysis of maneb formulataons by Roth and modtjied Claike methods
Formulation Maneb found, yo
~ ~~

Roth Modified
Clarke
Technical 82.5 85.1
84.0 85.1
70% wettable 67.0 72.1
70'5 ( 0 . 2 9.) 72-5
76.0 (0.9 g ) 72'3

The figures in brackets refer to the sample weight used, and the results indicate that the
method is susceptible to the size of the sample taken.
Again, the recoveries are lower than by the Clarke method, and this is undoubtedly due
to oxidation side reactions taking place while pre-dissolving the sample in alkali, producing
ethylene bis-thiuram mono- and poly-sulphides.
(b) Thiuram disulphides
In early trials with the method, it was suspected that the breakdown in acid solution was
much more complex than was suggested by Roth. A number of results were obtained in excess
of I O O ~ ~even
, ranging up to 105-110% for technical samples of thiram and tetraethylthiuram
disulphide (T.E.T.). With pure, recrystallised T.E.T. it was demonstrated that the decomposi-
tion involved the formation of both carbon disulphide and oxysulphide, and also of sulphides
via side reactions. As T.E.T. finds no use in agriculture, detailed information will not be given
here, but was included in the Panel's first report to CPAC and clearly shows that these features,
and insufficient specifying of the test details are, at least in part, responsible for the anomalous
results found.
For thiram much more realistic and reproducible results were obtained in general, but in
attempting to explain the occasional high result it was found, unexpectedly, that the assay
returned varied with the weight of sample taken. This point was checked with pure recrystallised
thiram (Table 111).
These results follow a smooth curve when plotted and were obtained using the conditions
exactly as specified for thiram, namely : reagents : formic acid 20 ml., pyridine 3 ml, zinc
sulphate 0.2 g. A second series was then run with omission of the pyridine with the results
shown in Table IV.
Thus, again the results obtained decrease with an increasing weight of sample, but, in this
case, to such a slight degree that for all practical purposes the accuracy given is ample.
In view of these results, the Panel could not recommend the use of the Roth method as
published, but did acknowledge that it could be satisfactorily applied to thiram if the pyridine
were omitted. One remaining objection in this case is to the use of formic acid, which is
expensive and unpleasant to handle.
Table I11
Results wzth daffeevent wezghts of sample (Roth method)
Sample weight taken, Thiram found,
g. %
0'1021 107.2
0.2335 104.8
0.4290 101'0
0'5270 gs.0
1.0980 93'0
Note: Roth spwifies that 0.1-0.3 g. of sample should be used
J. Sci. Fd Agric., 1964, Vol. 15, August
 STEVENSON-DITHIOCARBAMATES A N D THIURAM DISULPHIDES. I 513
Table IV
Analyses by Roth method but pyvidine omitted
Sample weight taken, Thiram found,
g. %
0.1150
0.2850
0.5000
0.8290
1'0370

(2) Methods proposed by the British Panel

(a) Determination of maneb and zineb in technical and formulated products
The procedure described is the modified Clarke method referred to earlier, and, while some
of the modifications have received scattered reporting in the previous literature, they are
incorporated in this method, along with other features which to the best of our knowledge are
being stressed for the first time. Although the method is essentially the same as that of Clarke,
it is considered that more emphasis has been placed on elimination of interfering side reactions.
Following collaborative study the method has now been accepted as an official CPAC
method for maneb and zineb, and has also been shown to be suitable for the dithiocarbamates
in general, with the exception of admixtures with copper salts.
The method is not specific for any one of these compounds, and separate characterisation
tests are needed.
Outline of method.-After dispersion in tetrasodium EDTA,l33 l4 the maneb sample is
decomposed by boiling sulphuric acid to give the corresponding amine and carbon disulphide.
The latter is swept by an air stream through cadmium sulphate solution to remove hydrogen
sulphide and then into an absorption train containing methanolic potassium hydroxide. The
xanthate solution formed is neutralised, titrated with standard aqueous iodine, and then the
maneb content of the original sample calculated.
Reagents
Sodium diethyl dithiocarbamate trihydrate : pure standard (Note I )
Sulphuric acid : approximately 4~
Tetrasodium EDTA : 34% w/v solution
Potassium hydroxide : 2N-methanolic solution-this reagent should contain less than I p.p,m. of copper
and iron, and no water must be used in the preparation
Cadmium sulphate solution o f : 18.5 g. CdS0,,8H20 in IOO ml. of distilled water
Acetic acid : 10% w/v solution
Phenolphthalein indicator. Starch indicator
Iodine : o.m-solution, standardised against arsenious oxide.
Apparatus: This is shown in Fig. I.

FIG.I .-A@aratus f o r determination of maneb and aineb

All parts, unless otherwise stated, are standard ground glass fittings
NS = non-standard. Dimensions in cm.

J. Sci. Fd Agric., 1964, Vol. 15, August

514 STEVENSON-DITHIOCARBAMATES A N D T H I U R A M DIS ULPHIDES. I
Procedure
Assemble the apparatus as shown (Note 2 ) with 30 ml. of cadmium sulphate solution in
the first absorber, and 25 ml. of methanolic potassium hydroxide in the second absorber (Note 3 )
and 5 ml. in each of the bubblers. Turn on the condenser water and heat the water-bath to,
and maintain at, 70-80" throughout the test.
Weigh accurately a sample containing 0.4-0.5 g. of maneb (Note 4) and transfer to the
digestion flask. Assemble the dropping funnel and air bleed (Note 5 ) and add the tetrasodium
EDTA (20 ml.) through the funnel (Note 6). Connect the absorption train to a controlled vacuum
supply (Note 7) so that approximately three bubbles of air per second pass through the system
(the actual aspiration rate is -50 ml./min.). Allow I min. for the dispersion of the sample
(Note 8), which can be assisted by gentle swirling of the flask, and then, through the dropping
funnel, add quickly 50 ml. of boiling sulphuric acid and begin heating the flask immediately
with a naked flame as shown.
Maintain a brisk rate of reflux until the sample is fully decomposed (about 25-30 min. are
needed) and then turn off the condenser cooling water for 2-3 min. In this way the condenser
and first absorber are flushed with steam to remove any entrapped carbon disulphide.
Remove the burner, disconnect the absorption train, and carefully wash the contents of the
methanolic potassium hydroxide absorber and bubblers into a 600-ml. beaker .with 300-400 ml.
of water. Add one or two drops of phenolphthalein indicator, just neutralise with acetic acid
added from a burette, and then add three drops in excess. Next, with continuous stirring by
a glass rod, titrate the solution immediately (Note 9) with iodine to the usual starch end-point
(titre t ml.).
Periodically, carry out a blank test, omitting the sample (titre b ml.). (See note 10.)
Calculations
I ml. of oem-iodine = 0.01326 g. of maneb or 0.01377 g. of zineb.
1*326(t-- b )
Then yo maneb = ~~~~

weight taken
Note r : The apparatus should be checked periodically for absorber efficiency and apparatus leaks, using
the pure standard, when recoveries of 99-101 yo should be found. An independent check on the purity
of the standard can be made by dissolving 0.5 g., accurately weighed, in -roo ml. of water and titrating
directly with iodine with starch as indicator.
Note 2: All joints must be absolutely gas-tight. Phosphoric acid may be used for this purpose, but Vaseline
or silicone grease is equally effective if applied in small quantities.
Note 3 :The main methanolic potassium hydroxide absorber must be kept below 2 5 O by immersion in a beaker
of cold water throughout the test. The absorbers must be dry before charging with the potassium
hydroxide solution, which should contain less than I p.p.m. of copper or iron. These requirements
reduce the possibility of decomposition of the alkaline methanolic xanthate solution in such a way as
t o produce sulphides, thus :
CH,O'CS'SK --+CH;OH COS + + KSH
KSH + KOH s H,S + H,O
also COS + KOH -+CO, + KSH
and several side reactions. Any sulphide formed, of course, will consume 2 equivalents of iodine per mole,
as opposed t o the I equivalent required by the xanthate and hence will cause the results to be falsely high.
Note 4 : To avoid the possibility of erratic results being returned due t o sample deterioration i t is advised
that all laboratory samples of maneb should be left in full containers and at a temperature below 25'.
Note 5: The air bleed must reach almost to the bottom of the digestion flask.
Note 6 :For dithiocarbamates other than maneb or zineb, tetrasodium EDTA need not be used. With com-
pounds such as ferbam, having some water repellency, however, the addition of a trace of wetting agent
(Teepol) is permissible.
When no tetrasodium EDTA is used the amount of acid is reduced to 30 ml. of q~-sulphuricacid
which, with the addition of approx. 25 ml. of wash water, results in the required working concentration
of acid of approx. PN a t the digestion flask.
Note 7 :Alternatively, supply compressed air from a small electric pump to the tube at the top of the dropping
funnel.
Note 8: Because of the possibility of oxidation side reactions occurring under alkaline conditions it is essential
that the contact time with the tetrasodium EDTA does not exceed the I min. specified, and also that no
heat is applied before addition of the acid.
J. Sci. Fd Agric., 1964, Vol. 15, August
 S T E VENSON-DITHIOCARBAMATES A N D T H I U R A M DISULPHIDES. I 515
Note g :After neutralisation, free xanthic acid can be regarded as being present, and as this has only a transient
existence under these conditions, undergoing rapid oxidation, it is necessary t o complete the titration
with iodine before any measurable secondary oxidation can proceed. Otherwise, the iodine titre will be
correspondingly low, and the result inaccurate.
For maximum accuracy, it is important that the iodine titration is completed within r min. from
the time of neutralisation, and for normal routine working the time taken should not exceed z min.
An experimental study of the actual deterioration rate has shown it t o represent a loss of 0.3% per
min. as related t o the assay of the sample during the first 5 min. following neutralisation.
Not? ro: It is necessary t o perform a blank test on the reagents under test conditions. Normally, the
value found with methanolic potassium hydroxide will be of the order of 0.1-03 ml. of o-IN-iodine,
and will not alter appreciably on storage of the reagent for several weeks. Potassium hydroxide solutions
made with ethanol are markedly less stable, and yield appreciable and increasing blanks within a few
days of preparation.

Interfering substances
Ethylenethiourea and the ethylenebis-thiuram mono- and poly-sulphides will amost cer-
tainly be found as impurities in commercial zineb or maneb, and have been credited previously
with causing interference in acid digestion methods.
That they have little effect in the method proposed is illustrated by the results in Table V
which compares results found for maneb alone with repeat analyses containing 20% of added
impurity, i.e., an impurity level far higher than would be found in practice.
Table V
Effect of impurities on results of determination of maneb by profiosed method
Sample Active strength found
by CS,. %
Maneb 79'9-80.1
Maneb + 20% E.T.U. 80.5-80.3
Maneb + 20% E.T.M./E.T.D. 8I .5-8 1'7
Note. E.T.U. = ethylenethiourea
E.T.M./E.T.D. = mixed ethylenebis-thiuram mono- and di-sulphides

Thus, for all practical purposes, the interference shown is negligible.

With admixtures of the dithiocarbamates and the lower thiuram disulphides, such as
thiram, some partial interference can be expected, and special techniques, to be studied late].,
have then to be applied.
Results
Table VI shows results of collaborative analyses of various products.
(3) Determination of thiram in technical products and simple formulations
The following method uses substantially the same apparatus and follows a similar pattern
to that described for the dithiocarbamates. The reagents, however, and the conditions in the
reaction flask are different. The method was developed during the work of the Panel and follow-
ing a collaborative study was accepted as an official method for thiram by CPAC. Its scope
includes technical products and simple formulations, with the exception of those containing
copper salts. From the mode of action proposed under the discussion, it will be realised that
the method is not specific for thiram, and that a separate characterisation test is needed.
Outline of method
The thiram is decomposed by boiling with mixed mineral acids to the corresponding amine,
carbon disulphide and carbonyl sulphide. The gaseous mixture is carried by an airstream
through a cadmium sulphate scrubber to remove hydrogen sulphide and then into a further
absorption system containing methanolic potassium hydroxide. The mixed xanthate/mono-
thiocarbonate solution is then neutralised and titrated with standard aqueous iodine. From
the titre the thiram content of the sample is calculated.
As a check analysis the dimethylamine content of the residue in the digestion flask can be
determined.
J. Sci. Fd Agric., 1964, Vol. 15, August
516 STE VEWSON-DITHIOCARBAMATES A N D T H I U R A M DISULPHIDES. I
Table VI
Collaborative ataalyses of dithiocarbamates by member labovatovies uszng the recommended method
Laboratory yo Maneb yo Zineb yo Ziram yo
Ferbam
in_sample
_ in sample in sample in sample
-
K
~ ~ ~

1
J Q- R 0 1
I 83.9 92'5 70'4 100'0 95.8
83.3 92'7 70'7 100'0 9545
83.5 99'5
83'7
I1 83.8 85.5 91.6 70.1 99.6 95'0
83.8 86.8 92'4 69.9 97'1 94'7
83.5 86. j

IV 84.8 87'5 90.0 70.2 98.3 9743

84.7 87.6 90.6 69.7 98.3 94.8
84.9
8 j.0

V 85." 92'3 71'4 103 9.7.8

87.2 87.2 92.3. 71.2 102 95.6
87.2 85.6 92.1 71.3 102 9 j.8
87.4 87.1
86.1

VI 83.9 83.2 89.3 69.9 97'9 96.5

84.1 82.6 90.8 70.0 98.1 99'9
90.1 69.8 98.4
VII 84.6 85.8 91.6 70.6 100.1 97'3
92.0 71.0 I 00.3

VIII 83'4 84.4 103'7 97'9

82.3 84.7 102'0 97'0
83.3 84.8 97'3
IS 85'4 88.5 92'7 70'5 99'4
84.9 90'7 89.9 70'5 99'4
86.9 90.6 99.6
89.2

x 84.3 88.3 98.9

84.9 88.0 98.:
84.6 88.1 98.7
84.8 87.9
84'3 87.9
84'5 88.9

Mean 84.6 86.8 91'7 70'7 99.8 96.2

Standard
deviation 1'2 1'9 1.3 0.6 1.6 1'0
o/, Coefficient
of variation 1'4 2'2 1'3 0.8 1.6 1'1

KEY t o Table VI
J Maneb water disperdible powder K Maneb technical
Q Zineb technical R Zineb 70% wettable powder
0 Ziram technical Id Ferbam technical
~. -_ - -~
Heageizts
( a ) Acid mixture: an aqueous solution containing
49% conc. hydrochloric acid
i:/o conc. sulphuric acid
by volume
0 . 1 % Teepol
5% w / v zinc sulphate
(b) Cadmium sulphate solution, 1576 w/v
(c) Methanolic potassium hydroxide, 2x
( d ) Acetic acid, 10% w/v
( e ) o.IN-iodine
Indicators : Phenolphthalein and starch
A p p a r a t u s : as in Fig. 2 .
J. Sci. Fd Agric., 1964, Vol. 15, August
 STEVENSON-DITHIOCARBAMATES A N D T H l URAM DISULPHIDES. I 517

JointJ

FIG.~.--4pj3aratus for determznation of thiram

lrgend as In Fig, I

Procedure
(i) Vetermination of carbon disulphide
Assemble the apparatus as shown in Fig. 2, with the same amount of reagents in the
respective absorbers as in the dithiocarbamate method (Xote I). Maintain the water-bath
surrounding the cadmium sulphate scrubber at 70-80" throughout the test, and turn on the
condenser water.
Weigh accurately about 0.3 g. of thiram and transfer to the digestion flask (Kote 2).
Assemble the air bleed and dropping funnel as shown, and add the acid mixture (20 ml.) via
the funnel. Connect the apparatus with some form of controlled aspiration (vacuum or com-
pressed air) so that approximately 3 bubbles per second pass through the absorbers. After the
sample is evenly dispersed, raise the temperature to boiling point and maintain a moderate
rate of reflux for 30 min. Turn off the cooling water and allow the condenser and first absorber
to be flushed with steam for not more than I min., and then remove the burner and disconnect
the train : retain the digestion flask residue for (ii) below.
Carefully wash the contents of the potassium hydroxide absorbers into a 600-ml. beaker
with 300-400 ml. wash water, add phenolphthalein, just neutralise with acetic acid from a burette,
and add three drops in excess. Then, with stirring by glass rod, titrate the solution immediately
with iodine (Note 3 ) with starch as indicator (titre t ml.).
Carry out a blank determination in the same manner, but omitting the sample (titre b ml.).

Calculation
I ml. of o.ir;-iodine r O ~ O T Z O Z g. of thiram
% thiram I.zoz(t - b ) / ~ t ~
=1

where w sample weight taken, g.

7

Note r :All joints must be gas-tight, and the apparatus should be checked for absorber leaks and efficiency,
periodically, using pure sodium diethyl dithiocarbamate.
Note 2 : If necessary, the sample can be washed into the digestion flask with a little water, provided that the
volume used is not such as t o cause priming of the condenser.
Note 3 :The iodine titration must be performed Lmmedzately, and preferably within I min., after neutralisation,
as decomposition of the mixed xanth;tte/monothiocarbonate solution is extremely rapid under acidic
conditions.
The actual decomposition rate, found by experiment, amounts to 0.4';(, per min. as related t o the
assay of the sample.

See also Notes 3, 5 and 10 as under Dithiocarbamates.

(ii) Determination of dimethylamine
Reagents
Sodium hydroxide : 7oo/u w/v solution
Boric acid. saturated solution

J. Sci. Fd Agric., 1964, Vol. 15, August

518 STEVENSON--DITHIOCARBAMATES A N D T H I U R A M DISULPHIDES. I
Bromo-cresol green/methyl red mixed indicator
Sulphuric acid : O'IN
Red litmus paper.
Apparatus: as shown in Fig. 3, or any conventional Kjeldahl apparatus.

500 ml.

turated boric
d solution
n / \ w ----- -
7///////,,,/1111/,///1111111/1/ ,./,//,///,,-

FIG.3.-Apparatus f o r determination of combined dimethylamine in thirum

Procedure
Wash the residue from (i) into the distillation flask with approx. 300 ml. of distilled water.
Add a little pumice and assemble the apparatus as shown, making sure that the tip of the con-
denser adaptor is well under the surface of the boric acid.
Add 50 ml. of the sodium hydroxide solution slowly via the dropping funnel.
Boil the contents of the flask and collect the distillate in the boric acid solution. After
about 20 min., test the distillate with red litmus paper, and if it is neutral, discontinue the
distillation. Remove the burner and wash down the condenser walls and adaptor with a little
distilled water into the boric acid solution.
Titrate the solution with the o-IN-sulphuric acid, using the mixed indicator to the first
pink colour (v ml.).
yo Thiram = 1.202 v / w
Note
This simple procedure affords an easy means of checking the result found under (i) and also gives an
indication of the composition of the sample under test. Thus, if the sample contains only thiram as active
ingredient, then the results from sections (i) and (ii) should agree t o within & I % .
If the results do not agree, this indicates that either (a) the decomposition has not proceeded as proposed
later or (b) some carbon disulphide has been lost physically, or via decomposition of the xanthate (which
can also produce sulphides, leading to a falsely high result), or (c) other thiuram disulphides or dithiocarbamates
are present, possibly based on amines other than dimethylamine.
It will be realised that any dithiocarbamate based on a volatile amine will give the above coincidence
between amine and carbon disulphide found, and hence will interfere with the method. Normally, however,
only trace amounts, if any, of these substances will be present as impurities in technical thiram and any inter-
ference from this source can be discounted.
If by any chance a sample of technical ziram, for example, was analysed in mistake for thiram, this would
give the agreement between methods (i) and (ii) and a misleading assay of -79% calculated as thiram would
be obtained. This illustrates the need for a separate and supplemental characterisation test for thiram,
and such methods are now under review.

Results
Collaborative results for thiram analyses are shown in Table VII.
Discussion and suggested mechanism of the method for thiram
A study of the early literature will reveal that, in general, the hydrolysis of the thiurams
in acid solution has always been thought to be non-quantitative. Hence most of the pre-
viously published methods have involved the use of a conventional reducing agent either prior
J. Sci. Fd Agric., 1964, Vol. 15, August
 STEVENSON-DITHIOCAKBAMATES A N D THIURAM DISULPHIDES. I 519

Table VII
Collaborative analyses of thiram samples obtained by Laboratorzes I-VIII using the recommended Method
(yo Thiram in sample)
Laboratory F . - __ _.
G Ii
-
by CS, by CS, by am& b y cs, by aminc
I 93'9 78.3 15.1 15'3
79'3 14.8 15'3
80.0 '4'5 14'7
79.0 14'9 15'3

I1 98.5 81.5 15.1 15'4

98.4 81.4 15.2 15.5
98.3 81.2
98.8
98.2

111 97'4 81.2 15.1

97.6 82.4 15.1
96.1 81.8 15.r
96.2 80.5 15.1
97'7
96.0

IV 99.1 79'9 14.6

98.0 80.4 '4'5
99'2
v 96.2 85.2 15.4 15'7
96.7 85.1 I 5.6 15.8
96.8

VI 98.7 81.8 15.2

97'7
VII 77'6 14.6
77'3 14.6
78.7 14.6
VIII 79'0 15.3
84'4 '4'5
Mean 97'5 80.8 15.0 15'4
Standard
deviation
yo Coefficient
''3 2.2 0'3 0'3

of variation 1.3 2'7 2'0 2.0

Sample F Thiram technical H I jyo Thiram dust

G 80% Thiram water-dispersible powder

to, or simultaneously with, the acid digestion. Such agents have included metallic zinc or
magnesium, zinc amalgam, sodium sulphide, bisulphite and thiosulphate. In our experience
these tend t o make the methods cumbersome and messy, and very prone to interference from
gaseous products formed when excess of the reagent has to be destroyed.
Hence, simpler methods were sought and, during early experiments, it was noted that
partial hydrolysis of thiram occurs even in water, and that the recovery is improved by addition
of acids of increasing strength. Finally, working with pure samples of thiram, it was found
that quantitative results were given using the mixed mineral acid/zinc sulphate solution shown.
The composition of the mixture is quite critical as higher proportions of hydrochloric acid than
quoted will tend to give slightly high results and, conversely, higher proportions of sulphuric
acid will produce rather low results.
An attempt was made to elucidate the mechanism involved, and that proposed below,
which assumes two simultaneously occurring routes, seems best to fit the observed facts.
Reaction I is believed to predominate and assumes initial breakdown of the thiram, when
in the molten state in suspension in the hot acid, into free radicals. (A similar mechanism
has been postulated previously to explain the thermochroism of thiram, i.e., although colourless
J. Sci. Fd Agric., 1964, Vol. 15, August
520 STEVENSON-DITHIOCARBAMATES A N D THIURAM DISULPHIDES. I

Ma\ ,N-o--S-S-C-N /Me

Me ‘Me

I+ ZnZC
t
2(Md2NH +
acid
hydrolysis

COS

acid
hydrolysis

when in pure solid form it becomes yellow when melted and reverts to a colourless form again
on resolidification.) Under the influence of the zinc ions, the free radicals will pass, as
dithiocarbamyl ions, into the acid phase and there decompose to carbon disulphide and
dimethylamine.
That the decomposition is much more complex, and hence that this cannot be the sole
mechanism, is shown by the facts that, even when analysing pure thiram,
(a) a small undigested residue remains in the flask which analysis has proved to be pure
sulphur (Schoniger method) ;
(b) by interposing a scrubber containing either silver nitrate or lead acetate* solution,
immediately before the methanolic potassium hydroxide absorber, a lower final iodine
titre is found, and a precipitate, which includes some silver or lead sulphide, forms in
the scrubber. Furthermore, from some limited work done, the extent to which the
final iodine titre is reduced, calculated as sulphur, seems to approximate closely to
the weight of sulphur residue remaining in the flask.
These facts support the existence of some such side mechanism as Route (11) in which
any sulphur which is lost by deposition is replaced by an equivalent amount of some iodine-
reducing body (probably carbonyl sulphide) which is finally estimated as carbon disulphide.
The dimethylamine liberated will, of course, be available for evaluation, by the method given,
along with that from Route (I), and the total recovered should agree to within &I% with the
results found for carbon disulphide. Collaborative results support this.
Again from some limited work, it has been found that the extent to which the decom-
position proceeds via Route (11) varies widely between - 2 and 12%. The reason for such
variation is not known, but is possibly associated with the rate of heating and agitation (by the
air stream) given in the early stages of digestion.
Certainly, the presence of zinc ions is needed to promote Route (I) as upwards of 25% of
the decomposition occurs via Route (11)if zinc sulphate is omitted, and the final assay is slightly
low. This latter feature can possibly be explained by the fact that more carbonyl sulphide is
liberated than in the recommended method, and hence the ‘ xanthate ’ solution formed contains
a higher proportion of monothiocarbonate and is more liable to deterioration under the acidic
titration conditions.
I t is believed that the mechanism involved in the Roth method proceeds in a similarly
* It was confirmed, by separate experiment, that neither of these additional scrubbers will absorb carbon
disulphide.

J. Sci. Fd Agric., 1964, Vol. 15, August

 S T E VENSON-DITHIOCARBAMATES A N D T H I U R A M DISULPHIDES. I 521

complex manner and that the pyridine reagent, which has been shown to cause anomalous results,
in some way promotes Route (11).
Having considered the above it will be appreciated that interfering substances must include
sodium dimethyldithiocarbamate and tetramethylthiuram monosulphide, both of which are
likely trace impurities in technical thiram. The former, if present, can be readily detected by
addition of a dilute copper sulphate solution to a cold aqueous suspension of the thiram, when
a dark brown precipitate of cupric dimethyldithiocarbamate will form immediately.
As regards tetramethylthiuram monosulphide, a separate, supplemental method is now under
consideration for quantitative estimation of this impurity.
Sulphur, another likely trace contaminant, does not interfere, as has been confirmed from
results obtained with mixtures of pure thiram and 10% added sulphur.

Conclusions
The collaborative results given, which represent a cross-section of those submitted to CPAC,
show that the methods recommended by the Panel are suitable for the estimation of dithio-
carbamates and thiram in technical products and simple formulations.
The methods in themselves are not sufficiently specific to allow of full characterisation of
the compounds, and, as part of its further work, the Panel is considering supplemental methods
for identification of the combined metals and/or amines.
Also being studied collaboratively are methods for the evaluation of more complex formula-
tions such as mixtures of dithiocarbamates and thiram, and dithiocarbamate and thiram formula-
tions containing copper and mercury salts.
The results obtained will be published, in due course, as Part I1 of this series.

Acknowledgments
The Dithiocarbamates Panel of the Pesticides Analysis Advisory Committee responsible
for the collaborative work described in this paper has included the following :

Boots Pure Drug Co. Ltd. Mr. N. Nix

Fisons Pest Control Ltd. Mr. K. Gardner
F. TN. Berk & Co. Ltd. Mr. J. B. Szymanski
Lennig Chemicals Ltd. Mr. C. Jackson
Pan Britannica Industries Ltd. Mr. G. A. Lloyd
Mr. M. E. Rose
Plant Pathology Laboratory, Harpenden Dr. R. de B. Ashworth
Mr. J. F. Lovett (Secretary)
Plant Protection Ltd. Mr. J. H. Dunn
Robinson Brothers Ltd. Mr. C. W. Attwood
(now deceased)
Mr. A. Stevenson
The Mirvale Chemical Co. Ltd. Mr. M. Karvot
The Murphy Chemical Co. Ltd. Mr. H. Crossley (Chairman)
Vitax Ltd. Mr. D. J. Dugdale

Overseas members
Belgium (Ministkre de I’Agriculture, Station de
Phytopharmacie de l’Etat, Gembloux) Dr. H. Henriet
Denmark (Kemikaliekontrollen, Strandvejen 740, Spring-
forbi) Dr. H. Poulsen
Germany (Biologische Bundesanstalt fur Land und
Forstwirtschaft, (zob) Braunschweig, Messeweg 11/12) Prof. Dr. N. Zeumer
Netherlands (Plantenziektenkundige Dienst, Wageningen) Dr. L. Westenburg
The author is greatly indebted to the above members for their keenness and enthusiasm
J. Sci. Fd Agric., 1964, Vol. 15, August
522 TRAMER C FOWLER-NISIN I N FOODS
shown in the collaborative work, and for the many helpful suggestions made. Special thanks
are also due to Mr. R. J. Hadley for his assistance throughout the work.

Robinson Brothers Ltd

West Bromwich
Staffs.
Received 14 February, 1964

References
Ashworth, R. de B., F A 0 Plant Protection Bull.,
1960,9, (No. 2 )
Callan, T., & Strafford, N. J., J . SOC.chem. I n d . ,
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Clarke, D. G., Baum, H., Stanley, E. L., & Hester,
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Rosenthal, I., Carlsen, R. L., & Stanley, E. L., J .
Ass. off. agric. Chem., 1953,36, 1 1 7 0
6 Hilton, C. L., & Newell, J. E., Rubb. Age, 1958,
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6 Roth, H., & Beck, W., Mikrochim A d a , 1957,
P. 844
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Patterson, J. D., J . A s s . off. agric. Chem., 1950. 33,
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Eusuf, M.,& Khundkar, M. H., A n a l . chim. Ada,
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Bontoyan, W.R., J . Ass. off. agric. Chem., 1963,
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ESTIMATION OF NISIN IN FOODS

B y J. TRAMER and G. G. FOWLER

Methods! for the estimation of nisin in solution and in foods are described and the
nisin unit is redefined. The presence of interfering substances in certain food extracts
has been demonstrated. In the foods examined compensation for the effect on the plate
assay could be made by inactivating the nisin by an alkali treatment, and using these
treated extracts as a basis for controls.
For quantitative determination of suspensions of nisin alone and in food extracts,
a large plate diffusion method requiring no prediffusion and using M . f l a w s as test
organism is described. For semi-quantitative estimations, or for sorting purposes, a simple
reverse-phase disk assay technique based on B. stearothermophilus spores, with overnight
incubation a t 55", is proposed.

Introduction
The antibiotic nisin consists of more than one polypeptide and has a molecular weight
of approx. 7500. It is only slightly soluble in neutral solutions but is much more soluble a t
lower pH values. These properties make diffusion of nisin through agar a very slow process
so that a few years ago some workers went so far as to say that nisin could not be assayed by
agar diffusion techniques. It is not surprising therefore that nisin was, and still is, mainly
estimated by dilution techniques, using the inhibition of reductase activity, or acid production
by bacteria in milk, as a measure of nisin potency.lr Tests have also been devised which
measure the optical density developed in broth by a suitable test organism under standard
conditions. While the dilution methods are reasonably satisfactory as to speed, the critical
timing of the culture propagation, addition of indicators and interpretation of colour changes
in the indicators and low sensitivity are serious limitations of these procedures. The inter-
pretation of colour changes is even more difficult when dealing with food extracts. However,
dilution techniques are very useful in circumstances where nisin concentrations have to be
quickly estimated.
J. Sci. Fd Agric., 1964, Vol. 15, August