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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. 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 TTopical Marirue Mollusc Programme (TMMP)is motivated by the fact that both pesticidesare still widely used in Indonesia (Sembel +CONTBOL +0.6 ppmet aI.l99l, pers. obs.). -.r Oppm +30ppm MATERIALS AND METHODSDiazinon [O, O-diethyl O-(2-isopropyl-6-me-thyl-4-pyrimidinyl) phosphorothioatel, an corganophosphorous insecticide, and glypho- o70 osate [N-(phosphonomethyl)glycine], an oorganophosphorous herbicide (Gallo & Law- o .>60 oryk 1991), were used as test chemicals. Both .9 ochemicals 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 and 01020304050600.17 t 0.02 g dry weight for glyphosate ex- Time (minutes)periments. Encrusting organisms were re- Figure 1. Relative changes of dissolved oxygenmoved and mussels held in stagnant sea when the control is compared with containerswater. They were not given food other than with S. bilocularis exposed to diazinon for onethat occurring naturally in the water sur-. hour. Each point is the mean of 3 measurements.rounding them. The mussels were stored inthe Laboratory of Marine Sciences, Facultyof Fisheries and Marine Sciences, Univer-sity of Sam Ratulangi, Manado, Indonesia. +CQNTROL +480All water for experiments was taken from -+-720 ppm +960 ppm ppmthe 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 cto obtain the salinity needed. gl 70 The experimental set-up and measure- oment of oxygen consumption were adapted o :60 ofrom Johnson (1973) and Bayne et al. (L985). .aDepletion of dissolved oxygen was measured oon 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 studies o r0 20 30 40 50 60 Time (minutes)showed that mortality occurred above thehighest concentration of each ofthe tested Figure 2. Relative changes of dissolved oxygenchemicals. 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.
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,ol lppryFisure B oxygen ffiffi;J:,6-r g.r) or Figure 4. Oxygen consumption (ml 02 h-t g-r) ofthe mussel S . bilocularis in the control compared the mussel S. bilocularis in the control comparedwith 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 usedgen 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 wereOxygen Meter mounted in the upper part of dried at 105 C overnight to obtain the drythe sealed container. Water was stirred by a weight.magnetic stirring bar inside the containers The rate of oxygen consumption (R) forfor 1-3 minutes prior to readings. Tempera- both diazinon and glyphosate, was analysedture was stabilised by an air conditioner. by means of One-way ANOVA (Analysis ofWater temperature was 22.85 -+ 0.44 oC, sa- Variance) and Tukey-test (Sokal & Rohlflinity 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 theafter 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 RESULTScalculated from the formula: In preceding pilot experiments, mortality R = [(Ci-CJV700][tw]-1, occurred when animals were exposed to con-where R is the rate of oxygen (O2) consump- centrations of diazinon above 30 ppm, andtion (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 ofcentration of dissolved 02 (ppm), 700 is a dissolved oxygen in experiments with sub-conversion factor for 02 adapted from Iethal concentrations of diazinon andJohnson (1973) (1 ppm = 700 mI1-1). V is the glyphosate using groups of three musselsvolume 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
r42 Tlopical Marine Mollusc Program.me (TMMP)mussels to take up the oxygen. However, zymes. Both effects can occur separate orthere 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 theIn concentrations of0.6, 6, and 30 ppm dia- highest level at a concentration of 6 ppm. Itzirton, 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 significantg-1 respectively. In concentrations of 480, (p<0.05) compared to the control (Fig. 3). In720, and 960 ppm glyphosate, it was glyphosate, the consumption increased and19 5.26 * 43.06, 252.28 t 36.06, 225.43 x. 22.40 reached the highest level at a concentrationml 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. In960 ppm glyphosate were required to show both pesticides, the effects can be explainedan 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 marineThe oxygen uptake ofbivalves depends on crab Chasmagnathus granulata. The effectthe 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 theity 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 certainthe 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-) showedof 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 theis 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 toing 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 inhibitedgills are of marginal importance in the over- ciliary activity. Davenport & Manley (1978)all oxygen consumption (Famme & Kofoed showed that Mytilus edulis responded with1980). 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 exposedarypump (Jorgensen 1990). Second, in abio- to low concentrations of oil. He suggestedchemical way related to the effect on en- that the lowest concentrations of oil caused
Phuket Marine Biological Center Special Publication 18(1): 139-144.(L998) t43a doubling of the respiratory rates and Davenport, J. & A. Manley. 1978. The detectiongreater oil concentrations caused a depres- of heightened seawater copper concentrationssion in rate. The respiratory rates of the by the mussel Mytilus edulis. - Journal of theclams exposed to low oil concentrations de- Marine Biological Association of the Unitedcreased as the hydrocarbon content of the Kingdom 58: 843-850.water and clam tissues decreased, but re- Famme, P. 1981. Haemolymph circulation as amained significantly different from the con- respiratory parameter in the mussel Mytilustrols. edulis L. - Comparative Biochemistry & Physiology 694: 243-247 . ACKNOWLEDGEMENTS Famme, P. & L.H. Kofoed. l-980. The ventilatoryWe 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 theby 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 statisticswish 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 ofpouw for data collection. pesticide toxicology. Vol. 2. Classes of pesti- cides. Academic press, Inc. Harcourt Brace REFERENCES Jovanovich, Publishers. San Diego, California.Abel, P.D. 1976. Effects of some pollutants on the Granmo, A. fggS. Mussels as a tool in impact filtration rate of Mytilzs. - Marine Pollution assessment. - Phuket Marine Biological Bulletin 7:228-231. Center Special Publication t5 215-220.Alloway, B.J. & D.C. Ayres. 1993. Chemical prin- Hooftman, R.N. & G.J. Vink. 1980. The determi- ciples of environmental pollution. Blackie Aca- nation of toxic effects of pollutants with the demic & Professional.An Imprint of Chapman marine polychaete worm Ophryotrocha dia- & HaIl,291 pp. dema. - Ecotoxicology and EnvironmentalBayne, B.L., D.A. Brown, K. Burns, D.R. Dixon, Safety 4: 252-262. A. Ivanovici, D.R. Livingstone, D.M. Lowe, Howell, R., A.M. Grant & N.E.J. MacCoy. 1984. M.N. Moore, A.R.D. Stebbing & J. Widdows. Effects of treatment with reserpine on the 1985. The effects of stress and pollution on change in filtration rate of Mytilus edulls sub- marine animals. Praeger. Praeger Special jected to dissolved copper. - Marine Pollution Studies. Praeger Scientific, 384 pp. Bulletin 15(12): 436-439.Booth, C.E. & C.P. Mangum. 7979. Oxygen up- Johnson, W.S. 1973. Respiration rates of some take and transport in the lamellibranch mol- New Zealand echinoderms (Note). - New Zea- hscModiolus demissus. - PhysiotogicalZool- land Journal of Marine & Freshwater Re- ogy 5l:!7-32. search 7(1&2):165-169.Brown, B.E. & R.C. Newell. 7972.The effects of Jgrgensen, C.B. 1990. Bivalve filter feeding: hy- copper and zinc on the metabolism of the drodynamics, bioenergetics, physiology and mussel Mytilus edulis. - Marine Biology 16: ecology. Olsen & Olsen 140 pp. 108-118. Jgrgensen, C.B., F. Mohlenberg & O. Sten-Knud-Connell, D.W. & G.J. Miller. 1984. Chemistry and sen. 1986. Nature of relation between ventila- ecotoxicology of pollution. JohnWiley & Sons, tion and oxygen consumption in frlter feeders. NewYork,444pp. - Marine Ecolory Progress Series 29: 73-88.
744 Tlopical Marine Mollusc Programme (TMMP)Kaligis, F.G. & M.T. Lasut. 1997. Effects of salin- Sembel, D.T., F. Kaseger, J. Pongoh & D. ity and diazinon on the abalone Haliotis uaria Kandowanglo. 1991. Pengkajian terhadap (Gastropoda: Haliotidae). - Phuket Marine Bio- penggunaan pestisida oleh petani di Kab. logical Center Special Publication L7 II1-I2}. Minahasa dan Bolaang Mongondow, Prop.Kobayashi, K., R.M. Rompas, T. Maekawa, N. Sulawesi Utara (in English: Study on the use Imada & Y. Oshima. 1990. Change in meta- of pesticides in the farm in Minahasa and bolic activity of tiger shrimp larvae at differ- BolaangMongondow, Prop. Sulawesi Utara). - ent stages to fenitrothion, an organophospho- Jurnal Fakultas Perikanan Unsrat 1(4): 6-18. rus insecticide. - Nippon Suisan Gakkaishi Silvester, N.R. & M.A. Sleigh. 1984. Hydrody- 56(3):489-496. namic aspects of particle capture by Mytilus.Lasut, M.T. 1996. Toxic effects of ethyl parathion - Journal of the Marine BiologicalAssociation and polluted seawater on the marine poly- of the United Kingdom 64(4):859-879. chaete Ophryotrocha diadema (Dorvilleidae). Sokal, R.R. & R.J. Rohlf. 1981. Biometry. The prin- MSc Thesis, University of Aarhus, Denmark, ciples and practice of statistics in biological 29 pp. research. Second Edition. W. H. Freeman andMonserrat, J.M., E.M. Rodriguez & R.J. Company, NewYork, 859 pp. Lombardo. 1991. Effects of salinity on the tox- Stainken, D.M. 1978. Effects of uptake and dis- icity ofparathion to the estuarine crab Chas- charge ofpetroleum hydrocarbons on the res- magnathus granulata (Decapoda, Grapsidae). piration of the soft-shellclam,Mya arenaria. - Bulletin of environmerital contamination -Journal ofFisheries Research Board Canada and toxicology 46(4): 569-57 5. 35(5):637-642.Persoone, G., P. Vanhaecke & E. Gobbers. 1985. Zanders, I.P. & W.E. Rojas. 1992. Cadmium accu- Evaluation of the impact of parathion, methyl- mulation, LC56 and oxygen consumption in parathion (Part A), fenitrothion and fenthion the tropical marine amphipod Elasmopus (Part B) on the aquatic environment. - Final rapatc. - Marine Biology 113(3): 409-478. report EEC Contract XI/7 85183, 579 pp.Redpath, K.J. & J. Davenport. 1988. The effect of cooper, zinc and cadmium on the pumping rate of My tilu s e d.ulis-L. - Aquatic Toxicology I B( 3 ) : 277-226.Rodriguez, E.M. & J.M. Monserrat. 19g1. Acute /SSN 0858-s6ss and chronic effects ofparathion and 2,4 D on the oxygen consumption of Chasmagnathus granulata (Decapoda, Brachyura). - Acta Physiologia Pharmacologica et Therapeutica Latino Americana 4L(2) : 20 7-2L0.Rodriguez, E.M. &A. Pisand. 1993. Effects of par- athion and 2, 4-D to eggs incubation and lar- vae hatching in Chasmagnathus granulata (Decapoda: Brachyura). - Comparative Bio- chemistry and Physiolo gy LO4C(L): 7 I-7 8.Rompas, R.M., K. Kobayashi, Y. Oshima, N. Imada, K. Yamato & Y. Mitsuyasu. 1989. Re- lationship between toxicity and acetylcho- linesterase inhibition of some thiono- and oxo- form organophosphate in tiger shrimp larvae at different stages. - Nippon Suisan Gakkaishi 5514):669-673.
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