Persistence and Effect of TMTD on
Soil Respiration and Nitrification
In Two Nursery Soils
BY
M. A. RADWAN
Abstract.
Persistence of tetramethylthiuram disulfide
(TMTD)
and its effect on
activity of soil microflora in general and on nitrifying micro-organisms in particular
were investigated in soils from two tree nurseries in western Washington. TMTD was
depleted in both soils; rate of depletion depended on initial concentration of the chemical
and action of soil micro-organisms. Addition of some commercial fertilizers enhanced
depletion. Use of these fertilizers or addition of well-decomposed organic matter in the
nursery would probably increase degradation of TMTD in soil. TMTD decreased soil
respiration.Soils responded differently to treatment, but initial depressions of C02 pro­
duction in both soils were proportional to concentration of TMTD in soil. Following
initial inhibition in two treatments, respiration was stimulated, probably due to utiliza­
tion of decomposition products of T MTD by micro-organisms. Nitrification was im­
paired by TMTD for 20 days but by 60 days had recovered completely in almost all
cases. Effects of TMTD reaching the soil during spray operations with a repellent
formulation containing the chemical do not seem serious, and even beneficial effects
seem possible under some conditions.
T ETRAMETHYLTHIURAM DISUJ,FIDE
(TMTD), in various spray formulations,
has been recommended for treatment of
planting stock before lifting from nursery
beds (Besser and Welch 1959, Duffield
and Eide 1962) to protect planted seed­
lings from damage by wild mammals,
particularly hares and rabbits. A number
of forest nurseries in the Pacific North­
west have adopted this technique, and
each year millions of seedlings are
sprayed. However, the mechanical equip­
ment used also deposits large amounts of
spray on seedbed soil (Duffield and Eide
1962).
TMTD has successfully controlled
several soil-borne diseases (McKeen 1950,
Cram and Vaartaja 1957) and, at the
rates recommended, is reported to change
soil microbiological balance (Richardson
1954) with little or no phytotoxic effects
(Kendrick and Zentmyer 1957). Yet little
is known of the chemical's persistence and
effects as related to its use as a repellent
in forest tree nurseries. Objectives of this
study were, therefore, to investigate per­
sistence of TMTD in soil and effect of the
chemical on soil respiration and nitrifi­
cation.
The author is Plant Physiologist, Division of
Timber Management Research, Pacific North­
west
Forest
and
Range
Expt. Sta.,
Forest
Service, U. S. Dept. Agric., Portland, Oreg. For
materials provided for this study, acknowledg­
ment is due to E. I. du Pont de Nemours & Co.,
Wilmington,
Del.; State Dept.
Natural
Re­
sources, Olympia, Wash.; and Industrial Forestry
Association, Portland,
Oreg.
Manuscript
re­
ceived July 7, 1964.
About This File: This file was created by scanning the printed publication. Misscans
identified by the software have been corrected; however, some mistakes may remain.
152 I Forest Science
'FABLE 1. Some characteristics of soils studied.1
Cation
Nursery
sampleJ
pH
exchange
Moisture
Organic
Clay
capacity
equivalent
matter
content
Meq/100 g
-------Percent--------
Webster
6.1
14 . 1
16.0
4.3
Greeley
5.9
19.9
25 . 0
3. 5
1
7.4
22.4
Each value is an average of two determinations.
Materials and Methods
Persistence Studies
Soils were collected at L. T. Webster and
Colonel W. B. Greeley forest nurseries in
western Washington. In each case, com­
posite soil samples of the surface 6 inches
were taken from areas where TMTD had
not been applied. Samples were then air­
dried, individually mixed, screened
through a 4 mm sieve, and stored at
room temperature in polyethylene bags.
To characterize the soils, samples were
analyzed (Table 1) by the following
methods: reaction (pH) electrometrically
in a 1:1 soil-water suspension (Peech
et al. 1947); cation exchange capacity by
a modification of the ammonium acetate
method of Schollenberger and Simon
(1945); moisture equivalent by the cen­
trifuge method of Briggs and McLane
(1907); total organic matter by the
dichromate oxidation method (Peech
et al. 1947, Jackson 1960); and clay con­
tent by the hydrometer method (Bouy­
oucos 1927).
Two experiments were carried out. In the
first, TMTD at 300 and 600 parts per
million by weight (ppmw) was thoroughly
mixed with autoclaved (15 psi for 3 hours,
121° C) and non-autoclaved 50-g ovendry
samples of the two soils in 250-ml Erlen­
meyer flasks. The autoclaved soils were
treated with TMTD under aseptic con­
ditions, and moisture of all samples was
raised to the moisture equivalent with
sterile water. Duplicate samples of each
treatment were immediately analyzed for
TMTD. All remaining flasks, lightly
stoppered with cotton plugs to permit
aeration, were incubated in a randomized
block design at 28° ± 1° C in a water­
jacketed incubator, and soil moisture was
maintained at the moisture equivalent
by periodic additions of sterile water. At
the end of 20, 60, 100, 140, and 180 days,
TMTD was analyzed in duplicate samples
of each treatment.
Determination of TMTD in Soil
TABLE 2. Recovery of TMTD from soil.
Known amounts of TMTD were added to
50-g (ovendry basis) soil samples, and
recovery was immediately determined by
a modification of Keppel's method (1959).
In each case, TMTD was extracted with
chloroform, and aliquots of extracts were
treated with cuprous iodide. Absorbance
of resulting solutions was then measured
in a spectrophotometer at 440 mu and
amount of TMTD determined from a
previously constructed calibration curve.
Recovery data are summarized in Table 2.
Percent recovery
TMTD level No. deter-----------­
(ppmw)1
ruinations
Range
Aver.
Webster nursery
300
4
91-93
92
600
4
89-95
92
Greeley nursery
300
600
1
4
4
86-92
89
88-90
89
Parts per million by weight.
volu,me11,nu,mber2,196l!
I
153
The second experiment was to deter­
mine the effect of two commercial fer­
tilizers-ammonium nitrate (33-1/2-0-0)
and ammonium phosphate-sulfate (16­
20-0)-on persistence of TMTD. In this
case, 50-g soil samples in 250-ml Erlen­
meyer flasks were either treated with
fertilizer at 1,000 ppmw or left without
treatment. All samples were then treated
with TMTD at 300 ppmw, thoroughly
mixed, and maintained at moisture
equivalent throughout the test. TMTD
was determined immediately and at 20, 60,
and 100 days following incubation at 28°
± 1° C. As in the first experiment, two
replications and a randomized block
design were adopted.
Soil Respiration and
Nitrification Experiments
Influence of TMTD on total microbial
activity was measured by determination
of C02 evolved from treated and un­
treated soil samples. In this experiment,
100-g (ovendry basis) portions of each
soil were placed in 250-ml Erlenmeyer
flasks. Soils were treated with TMTD
at 0, 180, 480, 720, and 1,200 ppmw,1
mixed, and brought to moisture equiva­
lent with water. The flasks, connected
to a manifold supplying moist, C02-free
air, were then incubated at 28° ± 1° C.
During incubation, moisture was main­
tained at moisture equivalent, and C02
liberated by respiration of soil micro­
organisms was swept out of the flasks,
trapped in 125-ml Erlenmeyer flasks con­
taining 1N-NaOH, and determined by
differential titration with IN and 0.1 N
HC1 according to Cooper's method
(1941). Measurements of C02 were made
on the same flasks at 20- to 40-day inter­
vals during a 180-day incubation period.
A randomized split plot design was used
with two replications. Soils and TMTD
1 These rates are comparable to concentra­
tions of TMTD in the surface 6 inches of soil
when 0, 15, 40, 60, and 100 percent of a 10-per­
concentrations were main plots, and time
intervals for C02 determinations were
subplots.
Effect of TMTD on nitrification was
studied using 100-g samples (ovendry
basis) of the two soils in 250-ml Erlen­
meyer flasks. TMTD at 0 and 180 ppmw
and an ammonium N source at 0 and 100
ppmw of N were thoroughly mixed with
soil. Nitrogen sources, NH40H and
(NH4)2S04, were added to the soil in
volumes of water necessary to raise soil
moisture to the moisture equivalent.
Also, with (NH4)2S04, sufficient CaCOa
was added to neutralize acids produced
during nitrification (Chandra and Bollen
1961). Flasks of all treatments, lightly
stoppered with cotton plugs to permit
aeration, were incubated in a randomized
block design at 28° ± 1° C. Moisture lost
during incubation was replaced periodi­
cally, and duplicate samples were ana­
lyzed for nitrate N by the phenoldisul­
fonic acid method (Harper 1924) 20, 40,
and 60 days following treatment.
Results and Discussion
Persistence of TMTD in Soil
recovery. Amount of TMTD
recovered immediately after addition of
the chemical averaged 92 percent in the
Webster soil and 89 percent in the much
heavier textured Greeley soil. High clay
content of Greeley soil (Table 1) appar­
ently made extraction of TMTD more
difficult. Recovery, however, was not
affected by initial concentration of
TMTD (Table 2) or soil sterilization
(Fig. 1).
Initial
Effect of soil type. TMTD was broken
down in both soils. The chemical, how­
ever, was depleted more rapidly in
Greeley soil except during the 20- to
60-day incubation period, when Webster
lost significantly2 more TMTD.
Depletion of TMTD in both soils
varied with initial concentration of
2
In this paper, "significant" is used in a statis­
tical sense and means that a relationship was
cent TMTD spray formulation reaches the soil
shown to exist at the 5-percent level of proba­
during spray operations.
bility or lower.
154
I
Forest Science
RECOVERY OF TMTD
lOOPERCENT
WEBSTER SOIL
80
---- ....
20
Autoclaved
Nonautocl aved
QL____L___J
____
---L----L-------
--------
----
100
GREELEY SOIL
60
Autoe laved
Non auto cia ved
40
20
O L---�----�-0
20
40
6Q
80
100
140
180
DAYS
PERIOD OF INCUBATION
FIGURE 1. Recovery of TMTD initially and at different time intervals after addition of the chemical at 300
and 600 ppm by weight to autodaved and non-autr,claved soils.
Recoverie.r are expres.red a.r perc ent of
TMTD originalZv added.
volume 11,number 2, 1965
1
155
RECOVERY OF TMTD
100 PERCENT
I
Soils:
Webster
Greeley
Untreated
Nitrate
Phosphate·
sulfate
80
60
40
20
0
20
100
60
DAYS
PERIOD OF INCUBATION
FIGURE 2. Recovery of TMTD initially and at different time intervals after addition of the
chemical at 300 ppm by weight to fertilizer-treated and untreated samples of the soils.
Recoveries are expressed as percent of TMTD originally added.
TMTD. Throughout the incubation
period, TMTD frorn the 600 pprnw
treatments in Webster soil was broken
down at a slower rate than that from the
300 ppmw treatments. Similar but less
marked results were observed with
Greeley soil during the first 120 days
following treatment. This trend suggests
that under conditions of this experiment
TMTD inhibited depletion factors and
higher initial concentrations increased
inhibition.
of autoclaving soil. Autoclav­
ing affected persistence of TMTD,
and the effect varied with time, soil, and
initial TMTD concentration (Fig. 1).
During early stages of incubation, rate
of TMTD decomposition was signifi­
cantly reduced by autoclaving, especially
in Webster soil, and with the 600 ppmw
treatments of each soil. However, this
effect tended to disappear during later
stages of the study.
Effect
156
1
Forest Science
Micro-organisms, or some product of
microbial activity apparently were im­
portant in decomposing TMTD in soil.
Micro-organisms capable of breaking
down TMTD were probably present in
each soil. Further, higher decomposition
rates of the chemical in Greeley soil
suggest that greater microbial activity
occurred in that soil than in Webster
soil (Fig. 3). Autoclaving eliminated most
micro-organisms present in the soil and
resulted in reduced rates of depletion of
TMTD. However, higher decomposition
rates of the chemical were soon reestab­
lished in all autoclaved samples as soils
became contaminated with air-borne
microbes and as the introduced micro­
organisms and those that had escaped
autoclaving multiplied.
Effect of fertilizers. Effect of fertili­
zation on persistence of TMTD in soil is
shown in Figure 2. The fertilizers had no
Mg C EVOLVED AS C02 PER 100 g SOIL PER DAY
1.2
1.0
WEBSTER SOIL
0.8
0.6
0.4
0.2
0 L----J-----L--�--L-
1.8
\
\
\
\
1.6
GREELEY SOIL
\\\\
1.4
1.2 0
180
480
720
1,200 ppmw TMTD
1.0
\,
0.8 __ ---._
......
'----..... ......
',
'
0.6 "''--
0.4 ""' ............ ""' ..............
"- -
.................. ;;
.... ----- -----
----
0.2
0
20
40
60
80
100
140
180 DAYS
TIME AFTER TREATMENT
FIGURE 3. Effect of TMTD on carbon dioxide evolution from two .<oils. Rates of TMTJ)
7 20, and 1,200 ppm by weight.
were:
0, 180, 4SO,
volume 11, number 2, 196"5
I
157
the small difference in organic matter
between these soils (Table 1). High
microbial activity may also explain
higher rates of depletion of TMTD in
Greeley soil (Fig. 1).
During the first 120 days, treated soils
produced less C02 than non-treated soils,
indicating that TMTD significantly
depressed soil microflora. Depressions in
both soils were proportional to TMTD
concentrations but were more pronounced
in Greeley soil. In addition, low yields of
C02 continued throughout the incubation
period except with the 180- and, to a
lesser degree, 480-ppmw treatments. With
these two treatments, respiration in both
soils increased significantly over the con­
trols at different times during incubation.
Such increases were probably due to
utilization of decomposition products of
TMTD by soil micro-organisms and sub­
sequent increases in microbial numbers
and/or activities (Chandra and Bollen
1961).
Depressive effects observed with
TMTD treatments were not due entirely
to the chemical. There was also a decline
in C02 production of controls during
incubation, suggesting depletion of avail­
able food materials required to sustain
significant effect on rate of TMTD de­
composition during the first 20 days of
incubation but significantly enhanced
depletion thereafter. Response of Greeley
soil was more pronounced, but fertilizers
did not significantly differ in their effect
on TMTD depletion in either soil. Al­
though fertilizers were applied at much
higher rates than is usual in nursery
practice, lower rates might also enhance
loss of TMTD from soil in the nursery.
Influence of TMTO on Microbial Activity
Effect on respiration. Demonstrated per­
sistence of TMTD in soil led to study of
the chemical's effect on soil microbial
activities. Evolution of C02 over 180
days, measured at intervals of 20 to 40
days, was utilized to determine effect of
different TMTD levels on overall acti­
vity of soil micro-organisms. Results
were statistically analyzed, and curves
depicting influence of TMTD with re­
spect to time were prepared (Fig. 3).
For untreated soils, C02 production
was significantly greater in Greeley soil
during all time periods. This indicates
more micro-organisms or more favorable
conditions for their activity in that soil
compared with Webster soil, in spite of
TABLE 3. Effect of TMTD on nitrification in Webster and Greeley soils.
Nitrification of added
Webster soil, by
Greeley soil, by
days after treatment
days after treatment
Treatment'
20
4-0
60
------------·
(NH.),so.
(NH4),S04 + TMTD
NH40H
NH40H + TMTD
N2
20
Percen t
40
60
-----------
38a3
95a
95a
41a
86a
0
39b
97a
0
35b
93bc
74a
42a
86a
100a
45a
89a
100b
0
47b
100a
7b
38b
90c
1
Nitrogen sources and TMTD were added to give 100 and 180 parts per million by weight, respectively.
2
Each value is the average of two replications. Values were corrected for native
produced from native
3
N
NO,
as well as that
sources during the experiment.
Means in each column followed by the same letter or letters do not differ significantly at the 5-percent
level of probability, using Tukey's test.
158
1
Forest Science
.
.
m1cro-orgamsms.
Literature Cited
Effect on nitrification. Evolution of C02
BESSER, jEROME F., and JAcK F. WELCH. 1959.
was used as an index of general activity
of soil microflora. The biocidal properties
of TMTD were not known to be limited
to harmful micro-organisms, so the possi­
bility remained that TMTD might have
some deleterious effect on beneficial soil
micro-organisms also. In this experi­
ment, therefore, effect of TMTD on
formation of nitrate, which reflects acti­
vity of nitrifying micro-organisms, was
determined over a 60-day period (Table
3).
Both N sources were nitrified in soil
indicating presence of nitrifying micro­
organisms. In the absence of TMTD,
ammonium N was rapidly transformed
into nitrate form. Approximately 90 per­
cent of added N was nitrified in 40 days,
and nitrification was complete by 60
days. In addition, differences between
soils in nitrifying ability and between N
sources in rate of nitrification were not
significant.
Nitrate formation from each N source
was completely impaired by TMTD in
both soils during the first 20 days. Part
of this impairment, however, was over­
come in both soils after 40 days, and in
Webster soil all NH40H and (NH4)2S04
was nitrified by 60 days. Nitrification of
N sources in presence of TMTD, how­
ever, was less in Greeley soil in which 60­
day nitrification of NH40H and (NH4)2
so4 was 90 and 74 percent, respectively.
Thus, TMTD at 180 ppmw strongly
depresses nitrification initially. In light
of persistence and C02 production results
(Figs. 1 and 3), it appears that duration
of this effect depends on TMTD con­
centration. Such inhibition, however, is
not considered serious in practice since
tree seedlings utilize ammonium as well
as nitrate N. In addition, interruption of
nitrification may even be beneficial in
case of Webster nursery where soil is
coarse textured and much irrigation and
rainfall cause considerable leaching of
nitrates.
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volume 11, number 2, 1965
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159