Indian Journal of Experimental Biology
Vol. 42, July 2004, pp.732-735
Toxicological studies of pesticides on cytoplasmic streaming in Nitella
Sudhir Kumar Pandey & Tasneem Fatma*
Department of Biosciences, Jamia MiIIia Islamia,
New Delhi 110025, India.
Received 15 May 2003; revised 22 April 2004
In the present study, changes in velocity of cytoplasmic
streaming in the giant internodal cells of Nitella for varying concentration of the pesticides, 2,4-0, dieldrin, malathion, methyl
parathion and endosulfan, were measured. Marked decrease in the
velocity of cytopl asmic streaming was found at the concentrations
of 0.01,0.1, \, 10 and 100mM. Dieldrin was the most toxic to all
the pesticides investigated, followed by methyl parathion, endosulfan, malathion and 2,4-0. Threshold values for dieldrin, methylparathion, endosulfan, malathion and 2,4-0 as indicated by the
onset of decrease in the normal cytoplasmic streaming velocity
were less than 6.25x 10 -6, 2.5x 10-5, 5x 10-5, 5x 10-5 and 1.25x
1O-5M respectively. Cessation of streaming ' was noticed above
ImM in dieldrin and above lOmM when exposed to methylparathion and endosulfan. Cessation of streaming was not seen up to
l00mM concentration of 2,4-0 and malathion.
Keywords: Aquatic pollution, Biomonitoring, Cytoplasmic
streaming, Nitella , Pesticides
Frequent uses of pesticides pose a serious threat to
aquatic ecosystems. Since their removal from the
aquatic system is difficult, effects of pesticides on
non-target species have become important. Effects of
pesticides on various algal species have been studied
earlier with respect to growth, photosynthesis and nitrogen fixation I. None of these studies exploited cytoplastmic streaming in the Characean alga, Nitella,
while studying toxicological effects of pesticides.
Pesticides reach water either directly or indirectly
through run off from agricultural fields, spray drifts,
rain water, sewage and effluent from industries manufacturing pesticides or using them in their processes 2 .
Toxic chemicals such as pesticides, heavy metals etc.
may affect the metabolic or physiological processes
common to life in general and can inhibit or stimulate
vital process detectable at the molecular, cellular, tissue, organism or population level 3 . Thus, pollution
monitoring (biomonitoring) has become an essential
*Correspondent author
E-mail: [email protected]
component for pollution assessment as it acts as
backbone for future line of action for remediation
strategies.
Numerous methods for toxicity testing with plants
have been devised, end points of the most common
are based on rate of population growth (in microalagae),
sexual
reproduction/zoospore
germination/vegetative growth of sporophyte and germ tube
growth (in macroalgae), rate of germination/ rate of
root elongation/rate of growth in hydroponic culture
and rate of growth in sediments in non- vascular
plants 4. Apart from these, measuring the inhibition of
test algae by 14C assimilations. Reduction in the velocity of cytoplasmic streaming has been also used for
measuring toxicity of natural and wastewater for
heavy metals, industrial effluents and sewage for the
first time by US 6-7 . The objective of present study was
to prove use of reduction in cytoplasmic streaming
velocity in Nitella as a new biomonitoring tool for
pesticide pollution in aquatic bodies .
Nitella sp. with giant algal cells (2-5 cm long
multinucleate internodal cells), exists as sub-aquatic
meadows, was collected from a freshwater pond at
Budkal, Faridabad, India and maintained in glass
aquaria (75 x 40 x 30 cm) containing wet soil mixed
with humus (5 cm thick layer). Growth of phytoplanktons over the cells was prevented by co-culture
of zooplanktons like Cyclops and Daphnia. Healthy
culture of Nitella could be established in a few
months. Prior to experiment, internodal cells of medium size (about 4 cm long and 1 mm in diam.) were
separated from the main stem and kept in artificial
pond water (APW) 8 containing O.lmM each of KCI,
CaCh and NaCl. Pesticides (malathion, methylparathion and endosulfan) used were of commercial grade
and obtained from Bayer (India) Ltd. available in the
form of emulsifying concentrate or wettable powder
except 2,4-D (99% pure) that was obtained from
Sigma Aldrich Corp. USA. Different concentrations
(0.01, 0.1, 1.0, 10 and l00mM) of stock solutions of
pesticides were made using artificial pond water
(APW). We used perfusion chamber method in order
to avoid shock that otherwise leads to the cessation of
streaming during change from one concentration to
another. The perfusion chamber was made up of a
plain glass plate (8 x 4 cm) with all the four sides
NOTES
glued by glass rods (8 mm diam.). Another glass rod
was glued asymmetrically so as to partition the whole
inside area into two compartments in ratio, 4: 1
(Fig. 1).
Cytoplasmic streaming velocity can be measured
by exposing a giant algal cell to APW and lor test solution(s) in perfusion chamber (Fig. 1) followed by
light microscopic observations (under 100x magnification) and recording of the time taken by cytoplasmic particles to move a fixed ocular distance (with a
stopwatch). In lieu of one of the eyepiece, an ocular
with scale was inserted with a stage micrometer and
found that one scale of the ocular corresponded to
115 p,m. Observations made to record the time (~t)
taken for medium size cytoplasmic particles to cross
six (centrally located) s~ales of the ocular. The velocity was, therefore, 690 p,m1~t. The internodal cell of
Nitella (preconditioned in APW for 24 hr) was kept in
the large chamber and brought to position under objective of the microscope. Initial readings were taken
in APW. After this 1 ml APW was taken out from the
smaller chamber with the help of pipette and discarded. It was followed by addition of 1 ml of test
solution (pesticides solution) in smaller chamber. The
cotton bridge was kept over the barrier of two chambers which led to the equilibrium of effective concentration. Observations were made after 15 min as it
--J-I-,-F_
--J_ _ _
C
Fig. I-Perfusion chamber [A-glass rod; B-barrier; C-Iarge
chamber; D-cotton bridge; E-smal\ chamber and F-Nitella internode
733
was found that time taken by test solution to get
equilibrated between the chambers was approximately
15 min. In order to eliminate the inherent though
small differences in the streaming velocity in APW
(V APW) in internodal cells arising from age, season
and other ungovernable parameters we have used the
percentage relative decrement in streaming velocity
(V PRO)6 for quantitative analyses. Formula used was
-v PRO =~ APwl ~ t x 100, where ~ APW is average
Recorded time in artifical pond water and ~ t is recorded time in different test solutions taken by parti- .
cles to traverse the fixed distance (ie, 690 p,m). For
each averaged value of t, 10 recordings were taken on
one internodal cell. Each sequence was repeated on
three internodal cells. The three values of relative percentage variation in streaming velocity for the three
internodes were again averaged and were represented
in the experimental data.
The gradual increase in pesticides concentrations
(0.01, 0.1, 1, 10 and 100 mM) had shown visible
quantitative changes in cytoplasmic streaming
velocity expressed as V PRO (Table 1). The decrease in
VPRO values at different concentrations of pesticides
test solutions in comparison to control indicate extent
of toxicity. For the first time, cessation of streaming
was observed at 10 mM concentration of dieldrin
indicating its highest tOXICity followed by
methylparathion (50.7%), endosulfan (63.1 %), 2,4-D
(83 .6%) and mal athion (87 .1%). At 100 mM
concentration of pesticides cessation of cytoplasmiC
steaming could be observed with methylparathion and
endosulfan. Surprisingly, in 2,4-D and malathion (l00
mM)cytoplasmic streaming continued (77.9 and 77.2
% respectively) indicating their lesser toxicity. Order
of toxicity for minimum used concentration (0.01mM)
of pesticides was 93.2% for dieldrin, 94.8% for 2,4-D,
97.1% ' for endosulfan, 98% for methylparathion and
99% for malathion (Table 1). At this concentration it
Table I-Effect of different concentrations of pesticides on V PRD values
[Values are mean ± SD of 10 replicatesl
V PRO (%)
Cone, (mM)
0.00
0.01
0.1
I
10
100
Dieldrin
Methyl Parathion
100
93.2 ± .03
83.1 ± .03
79.3 ± .03
CEASED
CEASED
100
98 .2 ± .41
91.8 ± .30
88.5 ± .26
50.7±1.6
CEASED
-
Endosulfan
Malathion
2,4-D
100
97.1 ±1.l5
84.4 ±1.4
74.1 ±1.26
63.1 ±1.21
CEASED
100
99 ±2.1
98 ±2.1
96.2 ±2.0
87.1±2.1
77.2 +2.0
100
94.8 ± .57
91 ± .65
86.7 ±.43
83.6 ± .62
7.9 + .38
734
INDIAN J EXP BIOL, JULY 2004
was interesting to observe a decrement of about 6% in
VPRD for 2,4-0, which was only next to dieldrin
(about 7%) that may be the organomercury
compound. Besides, cessation of cytoplasmic
streaming velocity, threshold concentration was also
studied for these pesticides. Threshold concentration
(below O.OlmM), at which no effect or response was
observed, for the pesticides was 6.25x 10-6 for
dieldrin; 2.5 x 10-5 for methylparthion; 5 x 10-5 for
endosulfan; 5 x 10-5 for malathion and 1.25 x 10-6 for
2,4-0.
Any correlation between changes in cytoplasmic
streaming and pesticides is not known so far in terms
of mode and site of action. However, at molecular
level cytoplasmic streaming is known to be regulated
as a result of actin-myosin interactions, so anything
that affects streaming either directly or indirectly affects the actin-myosin interaction in moving cytoplasm. The organelles in moving cytoplasm are attached to actin filaments by myosin molecules, which
use the energy of ATP hydrolysis to slide along the
actin filaments pulling the organelles with them.
Streaming is inhibited if depletion of free Mg2 and
ATP occurs. Cytoplasmic streaming in Nitella is susceptible to change in pH, temperature, mechanical
shock, extracellular concentration of potassium and
calcium and viscosity of bathing medium9- 1O • These
changes occur at interface of the ectoplasm where
actin filaments are spread and the endoplasm containing the dispersed myosin exists. These changes
are also affected by change in membrane potential by
.
.
.
11-12 Th
means of acto-myosm
mteractlOns
.
e cy t op Iasmic streaming velocity changes reflects the cumulative effects of all the toxicants/stresses present in the
aquatic habitat.
Organo-mercury compound, 2,4-0, is an auxin
analog that stimulates destructive growth in higher
plants. Algae do not have auxin growth regulation,
but react to inhibition of photosynthesis and energy
transport process 13. Similarly, chlorinated insecticide
(dieldrin) has been reported to inhibit enzyme activity
and photosynthesis and alters the cell membrane permeability. Endosulfan is known to cause hypomagnesemia
and
hypocalcemia.
Organophosphates
(malathion and methylparathion) are reported to decrease the activity of glucose dehydrogenase. They
also decrease the activity of mitochondrial enzymes
such as isocitrates, succinate, malate dehydrogenase,
cytochrome-C-oxidase and Mg++ ATPase which
would be due to the masking of active sites or due to
blocking of sulphydril groups 13 . The gradual decrease
in streaming with exposure to the studied pesticides
might be associated with the depletion of ATP and
magnesium ion directly or indirectly, which are required for actin-myosin interaction regulated movement of the cell organelles.
Biological monitoring of water pollution is conducted to determine the deleterious effect of toxic
substances and is preferred over chemical monitoring
because- (1) the list of chemicals to be monitored is
unending, (2) biological effect often occur at concentrations below analytical capabilities. Many of the
pollutants are present at such a low concentration that
instrument sensitivity is too poor to determine the microquantity of pollutants, (3).the toxicants and other
traces may act quite differently in mixture than individually. Such toxicants affect the ecosystem in a
synergistic manner which cannot be detected by
chemical analysis alone, and (4) chemical nature of
toxicant is highly dynamic in environment with time
and space: whereas biological system can integrate all
environmental variables over a long period of time in
terms of effects can be easily measured and quantified. Scientists have used several methods (microplate
technique I4 - 15 , algistatic method and standard bottle
test technique l6 , EC50 value tests 17 -21 ) for bioassay .
These methods are too laborious and time-consuming
for routine tests. Our observations suggested that
change in cytoplasmic streaming velocity was the first
visible (though microscopic) metabolic signal of
cell's response against the environmental stress and in
comparison to other referred techniques was easy,
quick and cheap.
The order of toxicity obtained for pesticides from
our study (dieldrin > methy lparathion > endosulfan >
malathion> 2,4-0). From the findings of our experiment, during present study and earlier lab studies 6-7
and their comparison with established ADI and LD50
values 22 of these pesticides as well as with values
~iven by WHO (1996), USEPA (1979), BIS (1991)235, it appears that the techniques based on the alterations in the cytoplasmic streaming velocity in Nitella
with reference to aquatic pollutants could be possibly
introduced as a new biomonitoring technique for
aquatic pollution.
This work was supported by the Department of Environment, New Delhi, India. Dieldrin (commercial
grade) was a kind gift of Professor N P Agnihotri,
IARI, New Delhi, India.
NOTES
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