THE EFFECT OF SYSTOX ON IONIC FLUXES IN

EXCISED BARLEY ROOTS

J. J. OERTLI and N EMATOLLAH AHMADI
Department of Soil Science mid Agricultural Engineering
University of CaliJbrt~ia. Riverside 92502

The influence of various concentrations of the thiol isomer of Systox on ionic fluxes in excised
barley roots was investigated. For purposes of comparison, treatments with DNP alone, or in com-
bination with the Systox isomer, were included.

Rb and C1 absorption, regardless of the external sz,lt concentrations, were progressively

reduced as the Systox concentration in the external solution increased. Treatment with Systox did
not result in any appreciable increase in the efflux rates of Rb or C1 ions.

DNP treatment enhanced the efflux of Rb but not of C1. The increased efflux was too small to
account for the reduced net uptake. A comparison of individual and combined effects of Systox
and DNP on ion absorption indicated an independent action of the two inhibitors.

Systox, a tradename for an organophosphorus insecticide, is a mixture of two closely

related isomers, O,O-diethyl S-ethyl-2-mercaptoethyl phosphorothionate (thiono isomer)
and O,O-diethyl S-ethyl-2-mercaptoethyl phosphorothiolate (thiol isomer). The thiono
isomer is easily converted to the thiol isomer upon heating, and the kinetics o f the
rearrangement have been shown to be a first order reaction (Fukuto and Metcalf 1954).

Systemic properties of Systox, Le., its absorption by various plant parts as well as its
translocation within the plant system, were studied and discussed by a n u m b e r of
researchers (A.hmed et al. 1954, Dahm 1957, Fukuto 1957, M e t c a l f e t a/. 1954 and 1955).
Fukuto (1957) investigated the metabolism of Systox in cotton plants and found that soon
after absorption, both isomers were oxidized and converted to corresponding oxidation
products. It is evident that Systox has been thoroughly studied with respect to its insectici-
dal activity, overall phytotoxicity, and breakdown in plants; but we are not aware of
research on its effect on specific metabolic processes in plants. Accordingly, the main ob-
jective of the present study was to investigate the effect of Systox on the mechanism of ion
uptake by root tissues.

Overwhelming evidence links ion uptake with metabolic processes in plants, and the
dependence of salt transport on respiration is well known. Lundegardh (1958a,b) in his
anion respiration hypothesis proposes that the electron flow in the respiratory chain is
directly coupled to the m o v e m e n t of anions into the plant cells, and cations are subse-
quently carried along. This hypothesis is difficult to reconcile with some experimental evi-
dence. For example, Ordin and Jacobson (1955) found that respiratory inhibitors affected
both cation and anion uptake in a way that would not be expected from anion respiration.

Archives of Environmental Contamination 97

and Toxicology, Voi. 3, No. 2, 1975
9 1975 by Spdnger-Veflag New York Inc.
98 J. J. Oertli and N. Ahmadi

Budd and Laties (1964) observed that under anaerobic conditions, but in the presence of
ferricyanide as an electron acceptor, the absorption of chloride was similar to that of aero-
bic uptake. From this observation, they concluded that it is not electron flow per se, but
electron flow coupled with phosphorylation that is involved in ion transport across cellular
membranes. Similar conclusions were reached by Higinbotham (1959) and Weigl (1963)
who found that arsenate did not inhibit oxygen consumption, but reduced both cation and
anion absorption. Many investigators (Ordin and Jacobson 1955, Higinbotham 1959, Weigl
1963, Budd and Laties 1964, MacRobbie 1965 and 1966, Rains 1968) now believe that ac-
tive ion absorption by plant tissues is dependent on phosphorylation because it regulates
the availability of adenosine" triphosphate (ATP).

Evidence is now presented that Systox reduces ion uptake by plant tissues, possibly
through interference with the energy metabolism.

Materials and methods

Preparation of root material. The experimental procedures used were essentially pat-
terned after those of Jacobson et al. (1961). Six days after the germination of barley seeds
(Hordeum vulgate var. Atlas), the roots were excised, and then cut into approximately one-
cm lengths, washed in several changes of water, and centrifuged at 65 x g for five min.

Absorption experiments. Root samples weighing 500 mg were transferred to 500-ml

Pyrex flasks containing 400 ml of the treatment solutions. In one set of experiments (for
Figures 3 and 4) only 250 mg of roots were used in 200 ml of solution. During the entire
length of absorption, ranging from 0.5 to 6 hr, the flasks were placed on a gyratory shaker
at constant speed.

The treatment solutions consisting of RbC1 and KC1 were labeled with 86Rb and 36C1
respectively, to give approximately 0.2 to 0.4/zc/mmole of salt. The lower specific activity
was used in solutions having concentrations of up to 0.5 raM. The changes in solution con-
centration during an experiment were always less than 10%. The pH of the unbuffered
treatment solution was 6.0 4- 0.2, and the temperature was 25~ Systox treatment
levels ranged from zero to one mM, DNPlevels were 0, 0.005, and 0.01 mM. Only the thiol
isomer of Systox was used. It has been shown (Fukuto et al. 1955) that the thiono isomer,
upon entry into the plant, is rapidly changed to the thiol isomer; thus, the thiol isomer
alone suffices to give an indication of the overall effect of Systox. The thiol isomer, 99.9%
pure, was obtained through the courtesy of Chemagro Corporation.

At the end of the absorption period, the roots were washed briefly with approximately
200 ml of distilled water, followed by a one-rain rinse in 0.1 N Ca(NO3)2, and a final one-
min wash in water. After being dried at 60~ for 48 hr, the roots were weighed and
thereafter spread evenly on a planchet which had been previously lined with a layer of
double-stick Scotch tape. Each sample was counted twice for ten min with a low back-
ground thin-window GM gas-flow counter. Self-absorption, for all practical purposes, was
nonexistent. The counts were compared with a standard, and the amounts of Rb or C1 ab-
sorbed were calculated on a dry weight basis.
 Effect of Systox on Ionic Fluxes 99

Desorption experiments. In the desorption studies, roots were first placed into the ab-
sorption solution for two hr, washed as described earlier, and then transferred into perfor-
ated crucibles. Duplicate samples were kept for the analysis of the initial ion uptake. Each
perforated crucible was then placed in a larger crucible containing ten ml of the desorption
solution. At the end of each time interval the roots were transferred into ten ml of fresh
solution. Duplicate samples, 1.0 ml each, of the desorption solutions were pipetted into
aluminum planchets, evaporated under a floodlight, and the radioactivity counted. All
desorption solutions contained 0.5 mMCa(NO3)2, in addition to the treatments which were
either ten m M thiol isomer of Systox or 0.01 m M 2,4-dinitrophenol (DNP).

Results and discussion

Effects of different levels of Systox and D N P on absorption of Rb and C1 by excised

roots from 0.1 and 10 m M R b C 1 solutions are shown in Figures 1 and 2. The uptake of Rb
and CI from the two external solutions was reduced nearly linearly by increasing levels of
Systox. At low treatment levels of Systox between 0 and 0.2 raM, the effect is nonlinear for
C1 and the inhibition of the uptake is more pronounced for C1 than for Rb in this range.
DNP, at a concentration of 0.01 raM, was more effective in reducing Rb and C1 uptake

100

3
75
4
'7,
5
0
E
::::L 6
50
-o"

25

0
0 2 4 6

Time, hours

Figure 1. Influence of Systox and DNP on the time course of Rb absorption by excised roots under a
constant external concentration of 0.1 m M RbC1. Curves: (1) Control; (2, 3, 4, 5, 6) 0.2, 0.4, 0.6, 0.8, and
1.0 mMSystox, respectively; (7)0.01 mMDNP; (8) 1.0 mMSystox plus 0.01 mM DNP.
100 J. J. Oertli and N. Ahmadi

250 150 -
9 \

9 9 Rb
O - - - - - - O CI
"13
O\

"13
200 100 ~ , , , ~ P~-

~ o "~',9~
, ,oo
Q~O

150
0
E 10 mM R b C / ~'\ 0.1 mM RbCI
E \
\
\ Y,=
,.~^-- - 16.5 x +27. 2
~0~0~"~0~ ~0
100 I I , I I 0 t3 I I I I I
0.2 0.4 0.6 0.8 1.0\ 0.2 0,4 0.6 0.8 1.0
9 mM SYSTOX
Figure 2. Effect of Systox on the uptake of Rb and C1 by excised barley roots from 0.1 and 10 mM
RbC1 solutions during a six-hr absorption period.

than the highest Systox concentration (1.0 raM). This same result was also obtained for Rb
uptake from ten m M solutions and also for the chloride uptake from both solutions.
Simultaneous presence of Systox and D N P reduced Rb uptake more than either one alone.

Assuming that the dry weight represents 10% of the fresh weight, the internal Rb con-
centrations at the end of the experiment can be estimated. Such computations yield inter-
nal concentrations of 5 to 10 m M R b for samples treated with 1.0 mMSystox, and 10 and
25 m M Rb for control samples, under external salt concentrations of 0.1 and 10.0 raM,
respectively. Similar estimates for internal C1 concentrations were obtained. I t is evident
that the internal Rb and C1 concentrations, with the exception of one treatment (1.0 m M
Systox and 10.0 m M salt) is in excess of the concentration of the bathing solution.
Although a higher internal concentration does not necessarily mean higher electrochemi-
cal activity, at least one of the two ions Rb and C1 must have been transported against the
decreasing electrochemical potential gradient.

The similarity in the effect of Systox on Rb and C1 uptake is further exhibited in

Figures 3 and 4, where uptake is measured as a function of variable external salt con-
centrations. Systox is inhibitory both at low and at high substrate concentrations.
 Effect of Systox on Ionic Fluxes 101

100
4~
m,-
-I-
75
0
(3
.?
0
E
::k
50
_--43
_ ~Q)-

n 25 ~.0" ""
E:
/
?
I I I I
o 2.5 5.0 7.5 lO.
Rb, mM
Figure 3. Absorption of Rb by excised roots as a function of external RbC1 concentration in the pre-
sence (---) and absence (--) of 1.0 mMSystox; absorption time two hr.

Figure 5 shows the effect of Systox and DNP, each at 0.5 m M S y s t o x and 0.005 m M
D N P and at 1.0 m M S y s t o x and 0.01 m M D N P , as well as their c o m b i n a t i o n , o n t h e Rb and
C1 uptake from 10 m M R b C 1 . Again, the Systox treatment reduced absorption of C1 more
than Rb, in agreement with the findings shown in Figure 2. Systox, 0.5 raM, plus 0.005
m M D N P resulted in equal or more inhibition of Rb and C1 uptake than either 1.0 m M
Systox or 0.01 m M DNP, alone. If it is assumed that these inhibitors act independently
when present simultaneously in a solution, then the combined effect will be the product of
the fractional uptake in presence of Systox or DNP alone. It follows from Table I, that such
calculated uptake rates are reasonably close to observed ones. Thus, it seems that these in-
hibitors act independently.

Absorption or uptake, as used in this paper, is actually a measure of net flux (flux = in-
flux -- efflux) of individual ions. For healthy higher plant cells, the ionic efflux is probably
only a small fraction of the overall process (except at or near "equilibrium") and, therefore,
absorption or uptake is a close approximation of total influx. The reduction in net Rb and
C1 uptake could result either from a reduced influx or an enhanced efflux. In order to dis-
tinguish between these two possibilities, desorption experiments were conducted.

The results indicate that Systox does not have any appreciable effect on either Rb
(Figure 6) or C1 (Figure 7) desorptions. However, D N P increased Rb desorption with no
appreciable effect on C1 desorption. The amount of total Rb desorbed at the end of three
hr for roots with Rb concentrations of 210 ~mole/g dry weight of tissue (Figure 5), was
102 J. J. Oertli and N. Ahmadi

60

r
-r 40

o
0
E ,,.=. ~ ~ -- ~ C ~ ' ~
<3
:::L

-d
0
t
~ 2O 10
/
/
5
/0
/ oiP I I
/ 0 0.1 0.2

OI I r I I
0 2.5 5.0 7.5 10.
CI, mM

Figure 4. Absorption of C1 ion by excised roots as a function of external KC1 concentration, in the
presence (---) and absence (--) of 1.0 mMSystox; absorption time two hr.

200:
150

150 i
r r
0 o 100
E
=L =L

9o"
r
IO0 -o"

=,9,
~ 50
,1- 50

0 0
1 2 3 4 5 6 7 1 2 3 4 5 6 7

Figure 5. Effect of Systox and DNP on Rb and C1 absorption from 10.0 m M R b C I solutions; absorp-
tion time two hr. (I) Control; (2) 1.0 mMSystox; (3) 0.01 m M DNP; (4) 1.0 m M Systox plus 0.01 m M
DNP; (5) 0.5 m M Systox; (6) 0.005 m M DNP; and (7) 0.5 m M Systox plus 0.005 m M DNP.
 Effect of Systox on Ionic Fluxes 103

10.5% for the control, 13.2% for the Systox treatment, and 20.0% for the D N P treatment.
Similarly, the amount o f total C1 desorbed after three hr from roots containing 165.0/z-
mole C l / g dry weight (Figure 7) was 11:8% for the control, 10.2% for the Systox treatment,
and 11.0% for the DNP treatment. Essentially the same fractional losses were observed
after a pretreatment with a 0.i m M RbC1 solution.

These results demonstrate that Systox had very little, if any, influence on the efflux
rates of either Rb or C1 ions. DN P, on the other hand, increased efflux rates of Rb to about

Table I. Comparison of calculated and observed combined effects of systox plus DNP on the
relative uptake of Rb and Cl from 10 m M RbCI by excised barley roots. The calculation
assumed independent action of each inhibitor

Treatment levels Relative uptake

Systox DNP lnhibitors alone Systox plus DNP combined
mM mM Ion Systox DNP Calculated Observed
1.0 0.01 Rb 0.530 0.448 0.239 0.258
1.0 0.01 C1 0.575 0.072 0.041 0.046
0.5 0.005 Rb 0.701 0.609 0.427 0.387
0.5 0.005 C1 0.830 0.138 0.114 0.102

22sI

i
~ " "" .0~..... -0- 0.. ~ ~ 0 ~ ~ ..0 ~ ~ ....0 q ~ . . 0 ~ ~ ..0

175

-- " ~ O 9~ . . . . . ~ . ~ . _.E]. ~ . . . . E l , ~.....El. ~ , ~E]

150 -
r,"

125 -

I I I
0 1 2 3

T i m e , hours

F i g u r e 6. D e s o r p t i o n o f R b b y excised b a r l e y roots as a f u n c t i o n o f time. Rb: 10.0 m M d u r i n g t w o hr o f

a b s o r p t i o n time. D e s o r p t i o n solution c o n t a i n e d 0.5 m M Ca(NO3) 2 in the c o n t r o l ( - - ) , plus 1.0 m M
Systox ( - - - ) , or 0.01 m M D N P (-.-).
 104 J. J. Oertli and N. Ahmadi

200

"o:='. 150
e-

lO0-

0 1 2 3
Time, hours
Figure 7. Desorption of C1 by excised barley roots as a function of time. CI: 10.0 mMduring two hr of
absorption time. Control (closed circles), 1.0 mM Systox (open circles), and 0.01 mM DNP (squares).

twice that of the control, with no apparent effect on efflux of C1 ions. Nevertheless, even
in this case, the efflux rate is much smaller than the absorption rate. Thus, inhibitory
effects of either Systox or DNP on ion absorption cannot be accounted for by an
equivalent increase in leakage; rather, it must be a reduction in the influx. Robertson et aL,
(1951) similarly concluded that the reduction in ion absorption by xylem p a r e n c h y m a of
carrot roots, as a result of D N P treatment, could not be attributed to increased leakage.

It is common knowledge that most nutrients are transported into plant cells in the direc-
tion of their positive free energy gradient. This reaction must necessarily be coupled with
another process during which the free energy decreases. In root tissues, the m a i n energy
source stems from ATP derived from oxidative phosphorylation. In the case where the
energy supply from this source is inhibited through uncouplers such as DNP, the ion up-
take is also reduced. The interpretation of the foregoing is that active ion transport is
coupled in some way with utilization of ATP. Little conclusion can be taken about the
mechanism of interference by Systox. The parallelism--not p e r f e c t - - b e t w e e n the effects
of DNP and the insecticide Systox in root tissues might suggest that Systox is perhaps an
inhibitor of the production or utilization, or both, of high-energy phosphorylated com-
pounds, e.g., ATP. If this were the case, one would expect little or no effect o f Systox on
respiration rates. Indeed, some exploratory experiments on the respiration of roots, in pre-
sence and absence of KC1 salts, indicated that Systox had no effect on respiration as
measured by a respirometer. Whatever be the mechanism of inhibition, the possibility
must also be considered that the observed interaction is not caused directly by Systox but
by a product resulting from the oxidative breakdown of this compound.
 Effect of Systox on Ionic Fluxes 105

Acknowledgement

Grateful acknowledgement is made to the Dry Lands Research Institute of the Univer-
sity of California, for the award of a graduate fellowship to one of us (N.A.). -

References

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Manuscript received May 19, 1973; accepted June 4, 1974