Phulzet Mq.rine Biological Center Special Publicq.tion 18(1): 139-144. (1998) 139

THE EFFECT OF DIAZINON AND GLYPHOSATE (PESTICIDES) ONOXYGEN CONSUMPTION OF THE BOX MUSSEL

SEPTIFER BILOCULARIS L.

Markus T. Lasut & Astony P. AngmalisangLaboratory of Marine Sciences, Faculty of Fisheries & Marirue Sciences, Uniuersity of Sam

Ratulang| Fq,kultas Perikanan Unsrat. Jl. Kampus Bahu 95115 Manado, Indonesia

ABSTRACTOxygen consumption of box mussel Septifer bilocularis L. (0.17-0.18 g d.w) was monitoredfor one hour during exposure to diazinon and glyphosate pesticides. Depletion ofdissolvedoxygen 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 were195.26+43.06, 252.28*36.06, 225.43t22.40 ml 02 h-1g-l respectively (157.27 t34.10 mlO, h-r g-1in the control). Concentrations of 6 and 30 ppm diazinon, and 720 and 960 ppmglyphosate were required to show a statistically significant (p<0.05) effect on the oxygenconsumption. In low concentrations, both pesticides tended to increase oxygen consump-tion of the mussels, but oxygen consumption decreased if the concentrations increased.

INTRODUCTIONOxygen 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 deleteriousRedpath & Davenport 1988; Zanders & effects by affecting the human body (GalloRojas 1992). The effect of pesticides on ma- & Lawryk 1991), increasing mortality, andrine 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 andinvestigated. Oxygen consumption is an reproduction of the marine polychaeteimportant physiological parameter, because Ophryotrocha diadema (Lasut 1996). In highit represents a measure of the energy re- concentration they cause mortality in thequired to support and sustain life (Bayne e/ abalone Haliotis uariq, (Kaligis & Lasutal. L985).It has commonly been used as an 1997). Glyphosate acts as a glycine mimicindicator of the metabolic rate and damage and becomes accepted into peptides whereon 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 thetivate the enzyme cholinesterase (ChE) and box mussel Septifer bilocularis L. The study

L40

is motivated by the fact that both pesticidesare still widely used in Indonesia (Sembelet aI.l99l, pers. obs.).

MATERIALS AND METHODSDiazinon [O, O-diethyl O-(2-isopropyl-6-me-thyl-4-pyrimidinyl) phosphorothioatel, anorganophosphorous insecticide, and glypho-sate [N-(phosphonomethyl)glycine], anorganophosphorous herbicide (Gallo & Law-ryk 1991), were used as test chemicals. Bothchemicals were obtained from a pesticidedrugstore.

Box mussels S. bilocularis L. were col-Iected on the shore ofTongkaina, northernpart of Sulawesi, Indonesia. The weightranged from [mean + standard error (SE)]0.18 t 0.03 g dry weight for diazinon and0.17 t 0.02 g dry weight for glyphosate ex-periments. Encrusting organisms were re-moved and mussels held in stagnant seawater. They were not given food other thanthat occurring naturally in the water sur-.rounding them. The mussels were stored inthe Laboratory of Marine Sciences, Facultyof Fisheries and Marine Sciences, Univer-sity of Sam Ratulangi, Manado, Indonesia.All water for experiments was taken fromthe 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 waterto obtain the salinity needed.

The experimental set-up and measure-ment of oxygen consumption were adaptedfrom Johnson (1973) and Bayne et al. (L985).Depletion of dissolved oxygen was measuredon groups of three mussels placed in con-tainers with pure water (control) and waterwith sublethal concentrations of diazinon(0.6, 6, and 30 ppm) and glyphosate (480,720, and 960 ppm). These concentrationswere chosen because preliminary studiesshowed that mortality occurred above thehighest concentration of each ofthe testedchemicals. For measurement of oxygen con-sumption, groups of 3 mussels with 9 repli-cates were used. Oxygen was measured for

TTopical Marirue Mollusc Programme (TMMP)

co70ooo.>60o.9o

+CONTBOL +0.6 ppm

-.r Oppm +30ppm

0102030405060Time (minutes)

Figure 1. Relative changes of dissolved oxygenwhen the control is compared with containerswith S. bilocularis exposed to diazinon for onehour. Each point is the mean of 3 measurements.

+CQNTROL +480 ppm

-+-720 ppm +960 ppm

o 'r0 20 30 40 50 60

Time (minutes)

Figure 2. Relative changes of dissolved oxygenin the control compared with containers with S.

bilocularis exposed to glyphosate for one hour.Each point is the mean of 3 measurements.

cgl 70

oo:60o.ao

Phuket Marine Biological Center Special Publication 18(1): 139-144. (7998) I47

280o

8. zoo

8, zao

o-a 220Coo 200

5 rsocoI 160o

! r+o

tl't20

100

280

I zeoEc8, zao

o3 2zo

I 200

5 tsoco9 reoo

fi r+o6tr

120

100

Fisure B oxygen ffiffi;J:,6-r g.r) orthe mussel S . bilocularis in the control comparedwith indicated concentrations of diazinon duringone hour.Vertical lines are standard errors (S.E.).

one hour and depletion ofthe dissolved ox;r,gen was measured every 10 minutes for 2hto the nearest 0.01 ppm with a DissolvedOxygen Meter mounted in the upper part ofthe sealed container. Water was stirred by amagnetic stirring bar inside the containersfor 1-3 minutes prior to readings. Tempera-ture was stabilised by an air conditioner.Water temperature was 22.85 -+ 0.44 oC, sa-linity 30.00 t 0.00 Voo, &ndpH 7.88 t 0. 24.Thethree variables were measured before andafter each experiment.

The oxygen consumption was measuredas a rate ofoxygen uptake (Johnson 1973).According to Johnson (op. cit.) the rate wascalculated from the formula:

R = [(Ci-CJ'V'700]'[t'w]-1,where R is the rate of oxygen (O2) consump-tion (mlh-1 g-1 d.w.), Ci is the initial concen-tration of dissolved Oz (ppm), Cl is final con-centration of dissolved 02 (ppm), 700 is aconversion factor for 02 adapted fromJohnson (1973) (1 ppm = 700 mI1-1). V is thevolume of water in the container (l), t is time(h), and w is dry weight (g).

To avoid errors due to handling, the first

tott^o' oyonol"J"

"on""ntrutt,o'l lppry

s60

Figure 4. Oxygen consumption (ml 02 h-t g-r) ofthe mussel S. bilocularis in the control comparedwith indicated concentrations of glyphosate dur-ing one hour. Vertical lines are standard errors(s.E.).

readings during the first hour were not usedin the calculation. After the tests, the ani-mals were dissected and soft parts weredried at 105 'C overnight to obtain the dryweight.

The rate of oxygen consumption (R) forboth diazinon and glyphosate, was analysedby means of One-way ANOVA (Analysis ofVariance) and Tukey-test (Sokal & Rohlf1981;Fowler & Cohen 1990). Both statisti-cal tests were applied to test whether theconcentrations of the two pesticides had aneffect on the oxygen consumption.

RESULTSIn preceding pilot experiments, mortalityoccurred when animals were exposed to con-centrations of diazinon above 30 ppm, andabove 960 ppm for glyphosate.

Figs. 1 & 2 show the relative depletion ofdissolved oxygen in experiments with sub-Iethal concentrations of diazinon andglyphosate using groups of three musselswith 3 replicates. The values are expressedas a percentage ofthe control. Both diazinonand glyphosate influence the ability of.the

r42

mussels to take up the oxygen. However,there is no significant differenge (p>0.05)between the control and the treatments.

Figs. 3 & 4 show the rates ofoxygen con-sumption during 2 h in tests at sublethalconcentrations of diazinon and glyphosate.In concentrations of0.6, 6, and 30 ppm dia-zirton, the rates were 193.46 * 38.84,239.77 * 40.36, and 208.05 t 38.57 mI 02 h-lg-1 respectively. In concentrations of 480,720, and 960 ppm glyphosate, it was19 5.26 * 43.06, 252.28 t 36.06, 225.43 x. 22.40ml 02 h-1 g-1 respectively. In the control itwas 757.27 t 34.10 ml 02 h-1 g-1. Concen-trations of6 and 30 ppm diazinon, 720 and960 ppm glyphosate were required to showan effect on the oxygen consumption. Theeffect was statistically si gnifrcant (p<0. 0 5 ).

DISCUSSIONThe oxygen uptake ofbivalves depends onthe flow of water across the gills (J6rgensen1990). Water is drawn into the mantle cav-ity through the inhalant aperture; it passesbetween the gill filaments into thesuprabrachial cavity and is ejected throughthe exhalant aperture (Redpath & Daven-port 1988). The water current through themantle cavity is generated by the lateral ciliaof the gills (Silvester & Sleigh 1984). Theflow through the mantle cavity is laminarand then oxygen accumulated in the wateris taken up by diffusion process through theepithelium lining of the mantle cavity(Famme & Kofoed 1980; JOrgensen et ql.1986), as well as through the tissues of thebody; transport via the blood circulation be-ing slight (Booth & Mangum 1979) or sig-nificant (Famme 1981). In the latter case thegills are of marginal importance in the over-all oxygen consumption (Famme & Kofoed1980).

The presence of a contaminant can affectthe oxygen consumption in two ways. First,in a mechanical way by reducing the gapeof valves and,/or by acting directly on the cili-arypump (Jorgensen 1990). Second, in abio-chemical way related to the effect on en-

Tlopical Marine Mollusc Program.me (TMMP)

zymes. Both effects can occur separate ortogether.

The concentrations ofdiazinon and gly-phosate are important for the effect on mus-sel respiration. In diazinon, the consump-tion of oxygen increased and reached thehighest level at a concentration of 6 ppm. Itdecreased when the concentration was in-creased (30 ppm). This was significant(p<0.05) compared to the control (Fig. 3). Inglyphosate, the consumption increased andreached the highest level at a concentrationof 720 ppm. It decreased at the concentra-tion of 960 ppm (Fig. a). This was signifi-cant (p<0.05) compared to the control. Inboth pesticides, the effects can be explainedbiochemically.

Rodriguez & Monserrat (1991) haveshown the effects of parathion (insecticide)on the oxygen consumption of the marinecrab Chasmagnathus granulata. The effectwas caused by acetylcholine (Ach) inhibition.Ach is widely distributed throughout thenervous system of marine animals, includ-ing mussels. It is acting as a neurotransmit-ter in sensory nerve fibres and in certainneuromuscular junctions, such as those in-nervated by the stomatogastric ganglion.

Rodriguez & Monserrat (l-991-) showedthe effect of herbicide (2,4D) on oxygen con-sumption in the marine crab C. granulata.This compound is a typical uncoupler of therespiratory chain-oxydative phosphoryla-tion.

Apparently no previous information ex-ists on the effects of pesticides on the oxy-gen consumption of bivalves. In relation toother contaminants, Brown & Newell (7972)found that both zinc and copper inhibitedciliary activity. Davenport & Manley (1978)showed that Mytilus edulis responded withvalve closure at a concentration of0.021 ppmcopper sulphate (CuSOa) when concentra-tions were gradually raised. Stainken (1978)showed that there were significant differ-ences in respiratory rates in clams exposedto low concentrations of oil. He suggestedthat the lowest concentrations of oil caused

Phuket Marine Biological Center Special Publication 18(1): 139-144.(L998) t43

a doubling of the respiratory rates andgreater oil concentrations caused a depres-sion in rate. The respiratory rates of theclams exposed to low oil concentrations de-creased as the hydrocarbon content of thewater and clam tissues decreased, but re-mained significantly different from the con-trols.

ACKNOWLEDGEMENTSWe are much indebted to the Tropical Ma-rine Mollusc Programme (TMMP) sponsoredby DANIDA for the opportunity to presentthis paper at the Eighth ConferenceAVork-shop of TMMP in Hua Hin, Thailand. Wewish to thank Dr I.F.M. Rumengan, the headof Marine Sciences Laboratory, for experi-mental facilities, Dr L.J.L. Lumingas for dis-cussions, Mr B. Pratasik for reading the frrstmanuscript and our colleague Mr J. Sumam-pouw for data collection.

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