1. Phulzet Mq.rine Biological Center Special Publicq.tion 18(1): 139-144. (1998) 139
THE EFFECT OF DIAZINON AND GLYPHOSATE (PESTICIDES) ON
OXYGEN CONSUMPTION OF THE BOX MUSSEL
SEPTIFER BILOCULARIS L.
Markus T. Lasut & Astony P. Angmalisang
Laboratory of Marine Sciences, Faculty of Fisheries & Marirue Sciences, Uniuersity of Sam
Ratulang| Fq,kultas Perikanan Unsrat. Jl. Kampus Bahu 95115 Manado, Indonesia
ABSTRACT
Oxygen consumption of box mussel Septifer bilocularis L. (0.17-0.18 g d.w) was monitored
for one hour during exposure to diazinon and glyphosate pesticides. Depletion ofdissolved
oxygen was also monitored at 10 min intervals for 2h. There were no significant differ-
ences (p>0.05) between the control and the treatments at low concentrations. At concen-
trations of 0.6, 6, and 30 ppm diazinon, the oxygen consumption rates were [mean t stand-
ard error (SE)l 193.46 * 38.84, 239.77 * 40.36, and 208.05 38.57 ml 02 h-1 g-1 respectively.
=
In sublethal concentrations of 480, 720, and 960 ppm glyphosate, the rates were
195.26+43.06, 252.28*36.06, 225.43t22.40 ml 02 h-1g-l respectively (157.27 t34.10 ml
O, h-r g-1in the control). Concentrations of 6 and 30 ppm diazinon, and 720 and 960 ppm
glyphosate were required to show a statistically significant (p<0.05) effect on the oxygen
consumption. In low concentrations, both pesticides tended to increase oxygen consump-
tion of the mussels, but oxygen consumption decreased if the concentrations increased.
INTRODUCTION
Oxygen consumption, pumping rate, and fil- break down the neurotransmitter acetylcho-
tration rate have been widely studied in line (Ach) in synapses of the nervous sys-
terms of the effects of metals on marine in- tem, thereby disrupting the nervous coordi-
vertebrates (eg Abel 1976; HoweIIet aI. 1984; nation. They may further cause deleterious
Redpath & Davenport 1988; Zanders & effects by affecting the human body (Gallo
Rojas 1992). The effect of pesticides on ma- & Lawryk 1991), increasing mortality, and
rine organisms has been studied by Hooft- inhibiting growth and reproduction in ma-
man & Vink (1980); Rompas et al. (1989); rine invertebrates (Connel & Miller 1984, p.
Kobayashi et al. (1990); Monserrat et al. 199;Persooneetul.1985;Rompasetal.!989;
(1991); Rodriguez & Monserrat (1991); Kobayashietal.l99};Monserratetal.IggT;
Rodriguez & Pisanb (f993); Lasut (f996); Rodriquez & Pisanb 1993; Lasut 1996;
Kaligis & Lasut (1997). The effect on the Kaligis & Lasut 1997).In sublethal concen-
oxygen consumption, however, has not been trations, the chemicals affect growth and
investigated. Oxygen consumption is an reproduction of the marine polychaete
important physiological parameter, because Ophryotrocha diadema (Lasut 1996). In high
it represents a measure of the energy re- concentration they cause mortality in the
quired to support and sustain life (Bayne e/ abalone Haliotis uariq, (Kaligis & Lasut
al. L985).It has commonly been used as an 1997). Glyphosate acts as a glycine mimic
indicator of the metabolic rate and damage and becomes accepted into peptides where
on organisms exposed to contaminants it blocks normal development (Alloway &
(Rodriguez & Monserrat 1991). Mussels Ayres 1993).
have been widely used as test organisms The aim of this study is to demonstrate
(Granmo 1995). the effect of pesticides (diazinon and gly-
Pesticides (especially insecticides) inac- phosate) in sublethal concentrations on the
tivate the enzyme cholinesterase (ChE) and box mussel Septifer bilocularis L. The study

2. L40 TTopical Marirue Mollusc Programme (TMMP)
is motivated by the fact that both pesticides
are still widely used in Indonesia (Sembel +CONTBOL +0.6 ppm
et aI.l99l, pers. obs.). -.r Oppm +30ppm
MATERIALS AND METHODS
Diazinon [O, O-diethyl O-(2-isopropyl-6-me-
thyl-4-pyrimidinyl) phosphorothioatel, an c
organophosphorous insecticide, and glypho- o70
o
sate [N-(phosphonomethyl)glycine], an o
organophosphorous herbicide (Gallo & Law- o
.>60
o
ryk 1991), were used as test chemicals. Both .9
o
chemicals were obtained from a pesticide
drugstore.
Box mussels S. bilocularis L. were col-
Iected on the shore ofTongkaina, northern
part of Sulawesi, Indonesia. The weight
ranged from [mean + standard error (SE)]
0.18 t 0.03 g dry weight for diazinon and 0102030405060
0.17 t 0.02 g dry weight for glyphosate ex- Time (minutes)
periments. Encrusting organisms were re- Figure 1. Relative changes of dissolved oxygen
moved and mussels held in stagnant sea when the control is compared with containers
water. They were not given food other than with S. bilocularis exposed to diazinon for one
that occurring naturally in the water sur-. hour. Each point is the mean of 3 measurements.
rounding them. The mussels were stored in
the Laboratory of Marine Sciences, Faculty
of Fisheries and Marine Sciences, Univer-
sity of Sam Ratulangi, Manado, Indonesia.
+CQNTROL +480
All water for experiments was taken from -+-720 ppm +960
ppm
ppm
the site where the specimens were collected.
Sea water was autoclave d at l2l oC and sus-
pended matter allowed to settle before use.
Distilled water was used to dilute the water
c
to obtain the salinity needed. gl 70
The experimental set-up and measure- o
ment of oxygen consumption were adapted o
:60
o
from Johnson (1973) and Bayne et al. (L985). .a
Depletion of dissolved oxygen was measured o
on groups of three mussels placed in con-
tainers with pure water (control) and water
with sublethal concentrations of diazinon
(0.6, 6, and 30 ppm) and glyphosate (480,
720, and 960 ppm). These concentrations
were chosen because preliminary studies o 'r0 20 30 40 50 60
Time (minutes)
showed that mortality occurred above the
highest concentration of each ofthe tested Figure 2. Relative changes of dissolved oxygen
chemicals. For measurement of oxygen con- in the control compared with containers with S.
sumption, groups of 3 mussels with 9 repli- bilocularis exposed to glyphosate for one hour.
cates were used. Oxygen was measured for Each point is the mean of 3 measurements.

3. Phuket Marine Biological Center Special Publication 18(1): 139-144. (7998) I47
280 280
o
I
E
zeo 8. zoo
c
8, zao 8, zao
o o
3 2zo -a 220
C
o
I 200 o 200
5 tso 5 rso
c c
o o
9 reo I 160
o o
fi
6
r+o ! r+o
tr tl 't20
120
100 100
tott^o' s60
oyonol"J"
"on""ntrutt,o'l
lppry
Fisure B oxygen ffiffi;J:,6-r g.r) or Figure 4. Oxygen consumption (ml 02 h-t g-r) of
the mussel S . bilocularis in the control compared the mussel S. bilocularis in the control compared
with indicated concentrations of diazinon during with indicated concentrations of glyphosate dur-
one hour.Vertical lines are standard errors (S.E.). ing one hour. Vertical lines are standard errors
(s.E.).
ofthe dissolved ox;r,
one hour and depletion readings during the first hour were not used
gen was measured every 10 minutes for 2h in the calculation. After the tests, the ani-
to the nearest 0.01 ppm with a Dissolved mals were dissected and soft parts were
Oxygen Meter mounted in the upper part of dried at 105 'C overnight to obtain the dry
the sealed container. Water was stirred by a weight.
magnetic stirring bar inside the containers The rate of oxygen consumption (R) for
for 1-3 minutes prior to readings. Tempera- both diazinon and glyphosate, was analysed
ture was stabilised by an air conditioner. by means of One-way ANOVA (Analysis of
Water temperature was 22.85 -+ 0.44 oC, sa- Variance) and Tukey-test (Sokal & Rohlf
linity 30.00 t 0.00 Voo, &ndpH 7.88 t 0. 24.The 1981;Fowler & Cohen 1990). Both statisti-
three variables were measured before and cal tests were applied to test whether the
after each experiment. concentrations of the two pesticides had an
The oxygen consumption was measured effect on the oxygen consumption.
as a rate ofoxygen uptake (Johnson 1973).
According to Johnson (op. cit.) the rate was RESULTS
calculated from the formula: In preceding pilot experiments, mortality
R = [(Ci-CJ'V'700]'[t'w]-1, occurred when animals were exposed to con-
where R is the rate of oxygen (O2) consump- centrations of diazinon above 30 ppm, and
tion (mlh-1 g-1 d.w.), Ci is the initial concen- above 960 ppm for glyphosate.
tration of dissolved Oz (ppm), Cl is final con- Figs. 1 & 2 show the relative depletion of
centration of dissolved 02 (ppm), 700 is a dissolved oxygen in experiments with sub-
conversion factor for 02 adapted from Iethal concentrations of diazinon and
Johnson (1973) (1 ppm = 700 mI1-1). V is the glyphosate using groups of three mussels
volume of water in the container (l), t is time with 3 replicates. The values are expressed
(h), and w is dry weight (g). as a percentage ofthe control. Both diazinon
To avoid errors due to handling, the first and glyphosate influence the ability of.the

4. r42 Tlopical Marine Mollusc Program.me (TMMP)
mussels to take up the oxygen. However, zymes. Both effects can occur separate or
there is no significant differenge (p>0.05) together.
between the control and the treatments. The concentrations ofdiazinon and gly-
Figs. 3 & 4 show the rates ofoxygen con- phosate are important for the effect on mus-
sumption during 2 h in tests at sublethal sel respiration. In diazinon, the consump-
concentrations of diazinon and glyphosate. tion of oxygen increased and reached the
In concentrations of0.6, 6, and 30 ppm dia- highest level at a concentration of 6 ppm. It
zirton, the rates were 193.46 * 38.84, decreased when the concentration was in-
239.77 * 40.36, and 208.05 t 38.57 mI 02 h-l creased (30 ppm). This was significant
g-1 respectively. In concentrations of 480, (p<0.05) compared to the control (Fig. 3). In
720, and 960 ppm glyphosate, it was glyphosate, the consumption increased and
19 5.26 * 43.06, 252.28 t 36.06, 225.43 x. 22.40 reached the highest level at a concentration
ml 02 h-1 g-1 respectively. In the control it of 720 ppm. It decreased at the concentra-
was 757.27 t 34.10 ml 02 h-1 g-1. Concen- tion of 960 ppm (Fig. a). This was signifi-
trations of6 and 30 ppm diazinon, 720 and cant (p<0.05) compared to the control. In
960 ppm glyphosate were required to show both pesticides, the effects can be explained
an effect on the oxygen consumption. The biochemically.
effect was statistically si gnifrcant (p<0. 0 5 ). Rodriguez & Monserrat (1991) have
shown the effects of parathion (insecticide)
DISCUSSION on the oxygen consumption of the marine
The oxygen uptake ofbivalves depends on crab Chasmagnathus granulata. The effect
the flow of water across the gills (J6rgensen was caused by acetylcholine (Ach) inhibition.
1990). Water is drawn into the mantle cav- Ach is widely distributed throughout the
ity through the inhalant aperture; it passes nervous system of marine animals, includ-
between the gill filaments into the ing mussels. It is acting as a neurotransmit-
suprabrachial cavity and is ejected through ter in sensory nerve fibres and in certain
the exhalant aperture (Redpath & Daven- neuromuscular junctions, such as those in-
port 1988). The water current through the nervated by the stomatogastric ganglion.
mantle cavity is generated by the lateral cilia Rodriguez & Monserrat (l-991-) showed
of the gills (Silvester & Sleigh 1984). The the effect of herbicide (2,4D) on oxygen con-
flow through the mantle cavity is laminar sumption in the marine crab C. granulata.
and then oxygen accumulated in the water This compound is a typical uncoupler of the
is taken up by diffusion process through the respiratory chain-oxydative phosphoryla-
epithelium lining of the mantle cavity tion.
(Famme & Kofoed 1980; JOrgensen et ql. Apparently no previous information ex-
1986), as well as through the tissues of the ists on the effects of pesticides on the oxy-
body; transport via the blood circulation be- gen consumption of bivalves. In relation to
ing slight (Booth & Mangum 1979) or sig- other contaminants, Brown & Newell (7972)
nificant (Famme 1981). In the latter case the found that both zinc and copper inhibited
gills are of marginal importance in the over- ciliary activity. Davenport & Manley (1978)
all oxygen consumption (Famme & Kofoed showed that Mytilus edulis responded with
1980). valve closure at a concentration of0.021 ppm
The presence of a contaminant can affect copper sulphate (CuSOa) when concentra-
the oxygen consumption in two ways. First, tions were gradually raised. Stainken (1978)
in a mechanical way by reducing the gape showed that there were significant differ-
of valves and,/or by acting directly on the cili- ences in respiratory rates in clams exposed
arypump (Jorgensen 1990). Second, in abio- to low concentrations of oil. He suggested
chemical way related to the effect on en- that the lowest concentrations of oil caused

5. Phuket Marine Biological Center Special Publication 18(1): 139-144.(L998) t43
a doubling of the respiratory rates and Davenport, J. & A. Manley. 1978. The detection
greater oil concentrations caused a depres- of heightened seawater copper concentrations
sion in rate. The respiratory rates of the by the mussel Mytilus edulis. - Journal of the
clams exposed to low oil concentrations de- Marine Biological Association of the United
creased as the hydrocarbon content of the Kingdom 58: 843-850.
water and clam tissues decreased, but re- Famme, P. 1981. Haemolymph circulation as a
mained significantly different from the con- respiratory parameter in the mussel Mytilus
trols. edulis L. - Comparative Biochemistry &
Physiology 694: 243-247 .
ACKNOWLEDGEMENTS Famme, P. & L.H. Kofoed. l-980. The ventilatory
We are much indebted to the Tropical Ma- current and ctenidal function related to oxy-
rine Mollusc Programme (TMMP) sponsored gen uptake in declining oxygen tension by the
by DANIDA for the opportunity to present mussel Mytilus edulis L. - Comparative Bio-
this paper at the Eighth ConferenceAVork- chemistry & Physiology 66A: 161-171.
shop of TMMP in Hua Hin, Thailand. We Fowler, J. & L. Cohen. 1990. Practical statistics
wish to thank Dr I.F.M. Rumengan, the head for freld biology. JohnWiley & Sons. Chiches-
of Marine Sciences Laboratory, for experi- ter,227 pp.
mental facilities, Dr L.J.L. Lumingas for dis- Gallo, M.A. & N.J. Lawryk. 1991. Organic phos-
cussions, Mr B. Pratasik for reading the frrst phorus pesticides. Pages 1049-1053 lz: Hayes,
manuscript and our colleague Mr J. Sumam- W.J.Jr., & E.R. Jr. Laws, (eds.). Handbook of
pouw for data collection. pesticide toxicology. Vol. 2. Classes of pesti-
cides. Academic press, Inc. Harcourt Brace
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