Mt lasut 1996-tesis-aarhus univ-dk
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Mt lasut 1996-tesis-aarhus univ-dk



Thesis of Master Degree at Aarhus University, Aarhus, Denmark (1999)

Thesis of Master Degree at Aarhus University, Aarhus, Denmark (1999)



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Mt lasut 1996-tesis-aarhus univ-dk Mt lasut 1996-tesis-aarhus univ-dk Document Transcript

  • Toxic effects of ethyl parathion and polluted seawater on the polychaete Ophryotrocha diadent a (Dorvilleidae) M.Sc. Thesis Markus Talintukan Lasut tl ilr 1! .International M.Sc. Programme in Marine SciencesInstitute of Biological Sciencesat University of Aarhus, Denmark, 1996.
  • University of Aarhus Institute of Biological Sciences rnternational Programme in Marine sciences Toxic effects of ethyl parathion and polluted seawater on the polychaete ophryotrocha diaderua (Dorvilleidae) .r-r cr _ ,Ttl_ - _! , - Ivl.Dc. tltgsls Markus Thlintukan LasutInstitute of Biological SciencesDepartment of Ecology and GeneticsUniversity of AarhusDK-8000 Arhus CDenmarkPermanent address:Fakultas PerikananUniversitas Sam RatulangiJL. Kampus BahuManado 95115IndonesiaDefended at Department of Ecoldiy and Genetics,Institute of Biological Sciences, University ofAarhus, 23 January 1996, 09.00 AM.Cover illustration: Larvae of Ophryotrocha diadema, and egg mass.
  • Practical Research education (PRE)PRE (Practical Research Education) concludes the last 9 months of the curriculum(second year) of the M.Sc. Programme in Marine Sciences at University of Aarhus.PRE constitutes the implementation of the research proposal submitted at the endof the preceding three months course (Theoretical Research Education referred to asTRE).PRE encompasses two courses (1-2) and submission of a thesis (3):1. Scientific Drawing TechniquesTeacher: Dr. Tomas Cedhagen. Literature: Compendium written specifically for thecourse. fime: 12 hours.2.Application of the Internet in researchTeacher: Dr. Tomas Cedhagen. Literature: Compendium written specifically for thecourse. Time: 12 hours.3. Submission of a thesis describing independent and original research carried outduring a 6 months study period. The printed thesis must be submitted as a manu-script prepared according to guidelines in international journals, e.g., Ophelia. Thecontents of the thesis must be presented in the form of a public lecture. The lengthof this lecture must be 20 minutes in accordance with the time generally availablefor presentations at international meetings. Examination (questions from the boardof examiners and the audience) concludes the PRE. With the exception of language corrections and technical guidance, the contri-butions appear in print as when they were handed over to the M.Sc. programme forevaluation. The research work has been carried out by the student and has notpreviously formed the basis for the award of degree, diploma or other similar titles. I wish to acknowledge the staffof the Institute of Biological Sciences, the Geo-logical Institute, and the International M.Sc. Programme at University ofAarhusfor guidance of individual students. 1.Rtlrr Hylleberg Course Director The International Masters Programme in Marine Sciences, University of Aarhus, Denmark
  • 1 TOXIC EFFECTS OF ETHYL PARATHION AND POLLUTED SEAWATER ON THE POLYCHAETB OPHRYOTROCHA DADEMA (DORVILLEIDAE) by Markus Talintukan lasut ABSTRACT The dorvilleid polychaete , Ophryotocha diadema,including its egg-eating behaviour was..used to study the toxic effects of ethyl parathion *C poffiirO sea;yater. The studies were conducted in experiments of acuti (lethal) exposure for 96 hours (short- term) and chronic (sublethal) for 2A and 30 days (long+erm). The lethal and sublethal aspects studied were mortality, growth ani reproduction. The larval development was also studied and described. The observations g-n developmcni showed that iiie eggs grew from irochophore larval through meratrochophore to tarvar sage-inside the eggliuri io results of the toxicity tests showeo ttrat the LCro ror id hours, t; tl;ys. The for both tarvae and adults, were 1.23 and 4.47 mgll, respectively.- Obuiously, the larvae were more susceptible than the adults. In the sublethal tests, mortality OiO not occur ir, replicate and the parameter used gave a very incomplete picture "u"ry of the eifects of ethyl parathion. Growth retardation was signihcant at a concentration of 0,g t gll (p 9.8 rren (l) < 0.05). The estimated naarc values mostly concentrations of a,.2 tg ranged between 0.1 a1d 0.8 pgll. The water quality test showed that the warer from Aarhus harbor was of better quality than the *.tofrom off Aakrogen village.I discovered egg-eating behaviour in o. diadema. This aspet may influence theinteqpreation of previous tests made with this species.Y alorfu: Polychaeta (Dorvilleidae), Ophryotrocha diadema,polychaetelarval development, (lethal) bioassay, acute toxicity, ihronic (sublethal)t*irity, short- andlong-term tests, mortality, growth and reproduction, Ltrtyr p*utttion, r water quality,MATC, egg-eating behaviour. INTRODUCTIONMany environmental contaminants have a toxic effect on marine organisms. Theydiminish the number of survivors, influence metabolism, breeding efficiency, alterbehavioural patterns, and affect structure and form (Reish 1974; Rosenthal &Alderdice 1976). Since the concern of this study is the deleterious effects ofcontaminants on marine organisms, the first step was to find a method for measuringsuch effects. Hitherto, biological methods provide the most appropriate way of
  • 2assessing toxicity effects (Stebbing et al. 1930). One of these methods is thebioassay, also called the aquatic toxicity test. In this test, by using some parameters,the relative potency of a substance is evaluated by comparing living organismsexposed to the substance with unexposed organisms of the same type (Bliss 1957;Stebbing 1979; Chapman & Iong 1983; Rand & Petrocelli 1985; Govindarajulu1988).In the bioassay procedure, the selection of test species is a significant problem,because the test species should be representative of the ecosystem to which theconsequential parameters are applied. Therefore, they should be selected with regardto their sensitivity, availability, ild position in the food chain (Anderson &DApollonia 1978). Several authors 1e.g. Akesson 1970, 1975a,1980; Reish rg73,1984; Stebbing et al. 1980; Rand & Petrocelli 1985; Pocklington & Wells 1992)have discussed the properties of good test species for a laboratory bioassay. Theyagreed that the use of easily cultivated laboratory animals has many advantages.Polychaete bioassays are among the most sensitive tools for acquiring data on short-term (acute) and long-term (chronic) effects of pollutants in marine environments(water and sediment) when using lethal and sublethal response parameters (appliedto mortality, growth and reproduction), but particularly when applied toreproduction, which is a parameter of direct ecological importance (Brown &Ahsanullah l97l; Reish 1974; Rossi & Anderson 1976, 1978; Hooftman & vink1980; Roed 1980; carr & curran 1986; Moore & Dillon 1993; Harrison &Anderson 1994).Dorvilleid polychaetes of the genus Ophryotrochahave been found to be among themost useful invertebrate species for bioassay purposes. In this bioassay, O. diodemawas selected because it is sensitive to environmental perturbations, it ishermaphroditic with litfle intra-specific aggression, ild because its reproductiveevents can be easily recorded lAkesson 1975b; 1980). The species is small(maximum length, 4.6 mm), easily cultivated, and has a high reproductive capacity.
  • 3The transparent egg masses contain a fairly low number of eggs, which facilitate thestudy of the fate of individual eggs. The short life cycle of about 4 weeks at roomtemperature (Hooftman & Vink 1980) is also advantageous. Furthermore, O.diadzma has been used for determining the toxic effects of some pollutants, includingheavy metals (Klockner 1979; Reish & carr 1978; Hooftman & vink 1980; Reish1978, 1984; Parker 1984).The chronic toxicity test is a kind of bioassay that permits the evaluation of anyadverse effects of a chemical after long-term exposure at sublethal concentrations.In this test, the organism is exposed for an entire reproductive life-cycle period toat least five concentrations of the test substance (Rand & Petrocelli 1985). Theresults often are considered to predict the potential environmental effects of apollutant better than those of an acute-lethal bioassay. By using such a test, thesafe environmental concentration of the toxicant (MATC: Maximum AcceptableToxicant Concentration) can be established; this value will be found between thevalues of NOEC (No Observed Effect Concentration) and LOEC (Lowest ObservedEffect Concentration) (Rand & Petrocelli 1985).The chemical substance (ethyl parathion) used in the present study is anorganophosphorous insecticide. Like other organophosphorous insecticides, itinactivates the enzyme cholinesterase (ChE) and can break down the neurotransmitteracetylcholine (ACh) in a synapse of the nervous system and thereby disrupt thenervous coordination. It may cause deleterious effects by way of increasing mortalityand inhibiting growth and reproduction in marine invertebrates (Persoone et al.1985; Rompas et al. 1989; Kobayashi et a|.1990; Monserrat et al. 199I; Rodr(guez& Pisand 1993; connel & Miller 1984, p. 199). rn 1982, ethyl parathion was foundin concentration of 0.2to0.7 p,gll in water from a glasshouse in the Netherlands(Iristra et al. 1984). .The effects of the dangerous contaminant ethyl parathion in aquatic ecosystem canbe found in the entire ecosystem which is indicated by changes in species
  • 4composition and population number. Typically, these changes follow a sequence ofdynamic events as outlined below (Pimentel t971,; Pimentel & Goodman 7974; andBrown 1978 in Connel & Miller 1984).1. If lethal or sublethal concentrations of pesticides are dispersed in an ecosystem, the number of species in the ecosystem becomes reduced.2. If the reduction in number of species is sufficient, this may lead to instability of the ecosystem and subsequently to population outbreaks in some nontarget species. Outbreaks result from a breakdown in the normal feed-back of the system.3. When a pesticide disappears from the affected ecosystemn species in the lower trophic levels usually increase to outbreak levels.4. Predators and parasites existing at the higher trophic levels become susceptible to loss of a species or large scale fluctuations in numbers of species in the lower parts of the food chain upon which they depend.The aims of this study, from a general point of view, were to study the lethal(mortality) and sublethal (growth and reproduction) aspects of O. diadema, andtheiruse in marine ecotoxicological studies. Particularly, to determine the toxic effects ofethyl parathion in very low concentrations, to establish its MATC value for a marineenvironment, and to apply the method used to water quality testing.The studies were also made in order to improve the polychaete bioassay method, andwere motivated by the fact that still very little is known about the toxic effects ofsimilar chemicals on marine invertebrates. Furthermore, the studies were alsomotivated by the fact that ethyl parathion is still widely used as a biocide (Haskoningt994).Therefore, the studies were designed to answer the two questions: 1. If ethyl parathion, in a very low concentrations, and mildly polluted seawater
  • 5 has sublethal effects on the growth and reproduction of O. diadema. 2. Can the egg-eating behaviour of adults affect the results of the toxicity tests. In the present report, I first figure out the larval development of O. diademain order to have a reference point. Then I investigate the sublethal effects of ethylparathion on O. diadema in terms of mortality, growth and reproduction. I also testthe effects of polluted seawater on O. diodema. Finally, I describe the egg-eatingbehaviour of O. diadema as a factor that can affect the results of the toxicity tests. MATERIALS AND METHODSAnimsfu and compounds testedThe marine polychaete Ophryotrocha diadema (Dorvilleidae: Polychaeta) was usedas test species. The animals were supplied by Prof. B. Akesson (Department ofZooIogy, University of Gothenburg, Sweden) in May 1995.The two kinds of seawater solutions used were:- Seawater containing etlryl parathion. Ethyl parathion (0,O-diethyl 0-4-nitrophenylphosphorothioate) (NIOSH 1990; Haskoning 1994),an organophosphorus insecticide,was used. The water solubility and persistence in water of ethyl parathion are 24mg/l at 25 "C and 108 days (at pH7.4 and20 "C) and 2 to 6 days (under naturalconditions), respectively (Haskoning 1994).- Polluted seawater. Water samples were collecteA at three selected sites in AarhusBay, Denmark (Fig. 1), and tested. The first sample was taken off the Aakrogenvillage, the second within the Aarhus harbor, and the third off the Moesgaard forest.Cultivation proceduresThe stock and test animals were cultivated in the laboratory at the Department ofGenetics and Ecology, Institute of Biological Sciences, University of Aarhus,Denmark. The procedures for cultivation were integrated from those of the AmericanPublic Health Association (1980), Hooftman & Vink (1980), Ward & Parrish (1982),Akesson (1983), and Parker (1984). The animals were kept in 80 ml and20 ml glassbowls, respectively. The volume of the water medium for the tests was 10 ml in
  • 6each bowl.The bowls were placed in a bucket with water and lid to prevent evaporation.Water culture and foodAll water used for cultivation, including dilutions and controls, was collected fromGullmarsfiorden, Sweden. It was filtered (0.45 pm) and heated (to 80"C) before use.Distilled water was used to dilute the water to obtain the salinity needed. Theanimals, both larvae and adults, were fed with a seawater suspension of frozen andfragmented spinach.Figure 1. Sampling localities. (1) off the Aakrogen village, (2) inside the Aarhusharbor, (3) off the Moesgaard forest.Environmental conditionsThe environmental conditions in the laboratory (temperature, salinity, and pH) werecontrolled. All experiments were conducted at a water temperature of 24.4 + 0.2oC (except Experiment C, which was conducted at 2I.0 + 0.1 "C). The salinity andpH were 32.7 + 0.1%o and 8.0 + 0.0 (mean J I S.E.), respectively.
  • 7ExperimentsLarval developmentAdult animals from a single stock culture were kept in a 80 ml glass bowl until theyhad produced egg masses. The development of the egg masses and the eggs wereobserved daily until the larval stage. The various stages were documented with aphotographic microscope.A living egg mass was placed onto a modified object glass (see Appendix 1). Theobject glass was constructed so that it allowed me to observe the egg masses andeggs alive under a microscope.Tests of toxicityL e t h a I t o x i c i t y. Five concentrations of ethyl parathion were tested: 0.01,0.L, 1, 10, and 100 mg/l and compared with the control. They were chosen on thebasis of the results of a preliminary study that showed no effects of ethyl parathionon the mortality of O. diadema in concentrations less than 0.01 mg/l after 96 hoursof exposure.For both larvae and adults, four 20 ml semispherical glass bowls, each containingseven animals and 10 ml of prepared seawater of each concentration, were used. Thewater and the bowls were renewed every 24 hours, and dead animals weresimultaneously counted. The tests were terminated after 96 hours and LCrocalculated.S ub leth a1 toxicity. Theparameters measured werethoseof Klockner(1977) and Hooftman & Vink (1980) viz., growth (using the count of setigers (setae-carrying segments)), mortality, time of the first egg mass deposit, number of eggmasses per animal, number of eggs per egg mass, number of larvae per egg mass,mortality in the egg masses, and reproductive potential. (The reproductive potentialwas calculated by dividing the final number of larvae of each concentration by thefinal number of larvae in the control, and is expressed as a percentage of the controlvalue (Hooftman & Vink 1980).
  • 8The tests were ilrranged in two parallel series (one beginning with larvae and theother with adults) according to Akesson (1983).Experiment A: larvaeLarvae of the same age were used Q to 3 days, starting to feed). Five concentrationsof the test solution were chosen (0.05, 0J,A.2,0.4, and 0.8 pgll) on thebasis ofthe allowed concentration proposed by the European Community (0.1, p,g/I) for freshand marine water (Haskoning 1994).Four 20 ml semispherical glass bowls, each containing ten animals and 10 mlprepared seawater of each concentration, were used. The time of exposure was 30days. Every third day, the animals were fed (with suspended spinach during the firsttwo weeks and thereafter with fragmented frozen spinach), the water solution and thebowls were renewed, and the parameters were observed.Experiment B: adultsF 0 I e v e I (E x p. B - I). A11 the adult animals were 4 weeks. The preparations,concentrations, time intervals of renewals of solutions, and times of duration of theexperiments were as described in Exp. A.F1 level (Exp. B-II). Thetestanimalswerepreparedbyexposing twocouples of l-week-old juveniles from the same F0 stock culture in each concentrationuntil they reached the adult stage of the first generation (F1). The experiment startedwith 4-week-old adults of Fl animals, and ran for 20 days. The test was carried outin the same way as in the F0 Exp. (B-I), except that 4 animals were used in eachbowl.Experiment C: polluted seawaterSeawater samples from three selected sites of Aarhus Bay were used. The testanimals (2 to 3 days old larvae, starting to feed) were exposed for 30 days.Preparations of water, the number of test animals, renewals, and observedparameters were as described for Exp. A.
  • 9Egg-eating behaviourA11 experiments ran for 48 hours, and the number of missing eggs were counted.Four 20 ml cylindrical plastic bowls containing 10 ml seawater and ten adult animals(Exp. 1), different numbers of adult couples (Exp. 2), and one couple (Exp. 3) ineach were used for four kinds of experiment arrangements, viz.: 1) eggs, noanimals, no food; 2) eggs, no animals, food; 3) eggs, animals, no food; and 4) eggs,animals, food.Analyses of dataThe median lethal concentrations (LCr) of ethyl parathion after 96 hours of exposurewere calculated by using probit analysis according to Finney (197I). One-wayANOVA (Analyses of variance) and Tukey test were applied to test whether theconcentrations of ethyl parathion and water of bad quality affect the mortality,growth and reproduction. The Mann-Whitney U-test and the two-way ANOVA wereapplied in the behavioural studies. The tests were computed using the Minitab@computer program and the manual for the Tukey test (Fowler & Cohen 1990; Sokal& Rohlf 1981), respectively. REST]LTSLarval developmentThe study of larval development of O. diadema verified the results of Akesson(1976) and was used as a reference point. O. diodema is a simultaneoushermaphrodite with internal fertilization. It produced egg masses about 15 days afterhatching. The eggs are yellow and oval-shaped, and deposited in mucous masses thatare attached to the wall of the culture bowls. The egg mass is contained in atransparent fusiform membrane; the eggs and the fate of the individual egg can beeasily observed through the membrane. The total number of eggs per egg massranged from 16 to 18 in a water temperature of 24.4 + 0.2 oC, & salinity of 32.7+ 0.1%o, and a pH of 8.0 + 0.0 (mean + 1 S.E). The time from incipient eggdevelopment to released larvae is about 7 to 9 days under the conditions.
  • l_os sffi &w ffi "": ;"" :.$ r Figure 2. Larval development of O. diadema. (1-8) The development of larvae in the egg mass from the first to the eighth day. (Scale bar : 1 prm).
  • 11Table 1. Experiment A: effects of ethyl parathion on growth, mortalityand reproduction of O. diadema. The experiment started with2- to 3-day-old larvae;duration 30 days. The values show mean and standard emor (S.E.). ETHYL PARATHION CONCENTRATIONS (p,ell) 0,00. ::0i05 ii:lO:iiil0 .t.:..i0.i90 0.i:80 Growth (number of r6.6 16.0 15.9 16.1 15.9 15.5 setiger, 30 days) 0.1 0.2 0.2 0.2 0.2 0.2 First egg deposit 15 t6 t6 17 18 19 (day after start) 0.0 0.8 0.6 t.3 1.0 1.5 Mortality o-o 2.5 0.0 5.0 5.0 28.0 (%, after 30 days) Mean number of eggs 16.2 12.6 16.5 t6.6 t2.7 12.6 per egg mass 0.9 1.3 0.5 1.0 1,.4 1.0 Mean number of egg 1.6 1..7 r.2 t.3 1.4 1.4 masses per animal 0.2 0.2 0.2 0.2 0.2 0.1 Mean number of larvae 8.6 4.9 6.3 5.5 4.4 3.9 per egg mass 0.9 0.5 t.2 1,.2 1.5 1.0 Mortality in egg 44 60 59 66 68 66 masses (%) 4.3 4.5 7.0 6.6 8.0 6.0 Reproductive potential 100 66 51 67 50 37 (%, control : 100) 0.0 18.8 9.4 26.4 2t.4 6.3In the egg mass, the eggs developed to trochophore larvae in the fourth day afterhatching (Fig. 2). The trochophores have a ciliary band for locomotion around thebody and swim by rotation. The trochophores were yolky and non-feeding, so-calledlecithotrophic (see also Barnes (1987, p. 305); Brusca & Brusca (1990, p. a2fl).They reached the metatrochophore stage in the sixth day. The first pair of parapodiawith setae developed on the first setigerous segment at this stage, and continued untilit reached four pairs as a complete larva on the eighth day. At this time, the larvaebroke the egg mass and escaped. The larvae started to feed 2 to 3 days after release.If the development failed, dead eggs were found among alive in the same egg mass.They were distinguished from the life ones by their white opaque colour.
  • L2Table 2. Experiment B-I: Long-term effects of ethyl parathion on mortality andreproductionof O. diadema. The experiment started with 4-week-old adults; duration30 days. The values show mean and standard error (S.E). ETIIYL PARATHION P.t T,ERS CONCENTRATIONS (pell) 0,,.OCI 0:05 i ,:$:i::Ql;;,;;1 j0iii20 Mortality 0.0 0.0 2.5 ,: 5.0 5.0 (%, after 30 days) Mean number of eggs 1,8.7 16.5 T7.T t4.9 13.9 t4.9 per egg mass 1.0 t.2 0.8 0.9 0.5 0.8 Mean number of egg 3.4 4.8 3.9 4.0 3.9 3.1 masses per animal 0.4 0.2 0.2 0.1 0.2 0.2 Mean number of larvae 13.8 TI.4 11.8 9.1, 7.6 6.9 per egg mass 0.9 0.9 0.8 0.8 1.1 0.7 Mortality in egg 26 30 31 40 49 52 masses (%) 2.9 0.7 3.5 1.3 5.2 4.2 Reproductive potential 100 118 101 79 67 46 (Vo, eontrol :100) 0.0 t3.7 16.0 12.8 17.0 5.1I also observed that the larval stage, especially the trochophore, was critical. Mostof the larvae that failed to survive did it at this stage.Tests of toxicityEfficts of etlryl parathionLethal to xicity. The mortalityin theacute (lethal) exposure for96hourswas 7, 39, 43, and l00Vo for the experiment with larvae, and 4, 29, 36, and lNVofor the experiment with adults at concentrations of 0.01, 0.1, 1, and 10 mg/l. In bothtests, no mortality was observed at the lowest concentration (0.01 mg/l), but it was1,A0% at the highest (10 mg/l).The LCro at 96 hours for larvae and adults, were I.23 and 4.47 mgll, respectively.The larvae were more susceptible than adults. Unfortunately, because of therestricted duration of these tests, threshold values for constant LC5o could not bederived.
  • l_3Table 3. Experiment B-II: Long-term effects of ethyl parathion on mortality andreproduction of O. diadema. The experiment started with 4-week-old adults F1-generation; exposed to ethyl parathion at l-week-old juveniles (F0-generation);duration 20 days. The values show mean and standard enor (S.E.). ETTTYL PARATIIION CONCENTRATIONS (pell) .,.,,,.,0.::00 .0l,.1,0 0120.....,. ......0.i.40 0;.80....,..l Mortality o-o 0.0 0.0 0.0 0.0 12.5 (Vo, after 30 days) Mean number of eggs 18.1 T6.I 17.4 14.8 14.2 t2.5 per egg mass 0.8 t.9 1.1 2.3 1.7 0.6 Mean number of egg 4.3 4.5 4.0 3.6 3.3 3.7 masses per animal 0.4 0.4 0.5 0.4 0.7 4.4 Mean number of larvae 10.9 9.6 8.7 7.4 4.7 3.4 per egg mass 0.8 t.3 1.1 2.6 1.2 0.6 Mortality in egg 39 42 51 55 6I 73 masses (%) 5.5 2.0 6.2 9.9 4.5 6.3 Reproductive potential 1m 103 93 51 37 26 (%, control : 100) 0.0 30.8 38.s 15.0 11.5 2.5S ub lethal tox icity. Thelong-termeffectsof ethylparathion onthegrowth of O. diadema (Exp. A) along a complete life-cycle (30 days) are shown inTable 1. There were small temporary growth retardations at concentrations of 0.05to 0.4 p,gll, bnt the big permanent retardation occurred at the highest concentration,0.8 1tglI, P < 0.05 (Fig. 3).The reproductive potential decreased at concentrations from 0.05 to 0.8 p,gl (Exp.A) in Table 1 , 0.2 to 0. 8 pgll (Exp. B-I) in Table 2, and 0. 1 to 0.8 lt glI (Exp. B-II)in Table 3. It was caused mainly by a reduced number of larvae, in combination witha relatively high mortality in egg masses in all experiments, and also a delayed timeof hatching. However, such effects were not significant (P > 0.05) in larvae and F0-adults, but it was in experiments with Fl-adults (P < 0.05).
  • L4 20 1a 16 t U 14 (9 --€- frt2 a O.OO ppb O.O5 ppb b 10 -+K- d O.1 O ppb UA m --*- O.2O ppb = Zt) O.4O ppb -+<- O.AO ppb 12 15 16 21 24 27 50 TIME (DAY)Figure 3. Growth of O. diad.emd exposed to ethyl parathion in different concentra-tions tested for 30 days (ppb : trgll). Each point represents 20 animals.Table 4. Results of estimated MATC of ethyl parathion on long-term tests with O.diadema. ) The values are NOEC and LOEC, and the MATC value is lying betweenthose values, *) No significant effect of treatment. NSD) No significant differencebetween the control with the other treatments. ESTIMATED MATC (pg/l)" ,.EXP.i...,fi First egg deposit Mean number of eggs per egg mass NSD 0.1-0.2 :r Mean number of egg masses per animal *NSD* Mean number of larvae per egg mass :r. 0.1-0.2 0.4-0.8 Mortality in egg masses rc 0.2-0.4 0.4-0.8 Growth 0.4-0.8 Reproductive potential * NSD 0.1-0.2
  • 15Table 5. Experiment C: Growth, mortality and reproduction of O. diademc exposedto polluted seawater from different locations. The experiment started with 2- to 3-day-old larvae; duration 30 days. The values show mean and standard error (S.E.). WATER SAMPLES LOCATIONS a, i.iCO"N.TROL x Growth (number of 15.15 14.85 15.15 14.85 setiger, 30 days) 0.13 0.23 0.2s 0.15 Mortality o-o ,: 5.0 10.0 (%, after 30 days) Mean number of eggs 7.6 6.2 7.1 7.4 per egg mass 0.62 0.54 0.44 0.66 Mean number of egg 1.2 1.3 1.4 1.2 masses per animal 0.10 0.08 0.09 0.26 Mean number of larvae 5.4 2.8 4.7 5.1 per egg mass 0.52 0.41 0.34 0.30 Mortality in egg 31 58 38 29 masses (%) 5.29 5.62 6.70 4.46 Reproductive potential 100 59 104 96 (%, control : 100) 13.8 23.52 24.2The mortality increased clearly only at the highest concentration (0.8 pgll) for allthe tests, while no mortality was observed in the control. This high mortalityoccurred mostly during the first week (larval-juvenile stage) of the experiments. Nostatistical tests were carried out for this parameter, because it did not even occur inevery replicate. It was therefore considered as a slightly sensitive parameter in thesublethal toxicity tests.The mortality of eggs in egg masses tended to increase at the highest concentration(0.8 pgll) in all experiments. It mostly occurred at 3 to 4 days (trochophore stage)after hatching. This parameter is therefore considered as a susceptible test.Maximum Acceptable Toxicant Concentration (MATC)The estimated MATC values, including NOEC and LOEC, of ethyl parathion using
  • 16O. diadema were mostly between 0.1 and 0.8 pgll (Table 4).Effects of polluted seawaterAnimals cultured in water from off Aakrogen village have a markedly reducednumber of larvae per egg mass and a high mortality in the egg masses (Table 5).The effects were followed by those in the water from the harbor and off Moesgaardforest, respectively.Table 6. Egg-eating behaviour of O. diadema; duration 48 hours. EXP. 1: (food & no food) Eggs & no animals 0&0 Eggs & animals 22.8 & 54.8 EXP. 2: (food & no food) 1 Couple r9.0 & 36.3 2 Couples 30.3 & 50.0 3 Couples 23.3 & 46.3 EXP. 3: (no food) The eggs did not belong to the animals 11 The eggs belonged to the animals 33.8Egg-eating behaviourStatistically, no significant differences were observed in three behavioural (egg-eating) experiments on O. diadema. The eggs were eaten by the adults and the egg-eating was not influenced by food supply and/or animal densities (Exp. 1 & 2). Theyate not only their own eggs, but also from other parents (Exp. 3) (Table 6). DISCUSSIONSusceptibility of O. diodcmaO. diadema has been proven as a sensitive test species to a range of chemicals
  • L7(Parker 1984). Its sublethal responses used in the present study were found tothoroughly reflect the toxicity of ethyl parathion. Previously Hooftman & Vink(1980) reported that the effects of pesticides (Pentachlorophenol (PCP), 3,4-Dichloroaniline (DCA), Dieldrinn and I ,1,2-Tichloroethane) on the mortality of O.diadema provided only a very limited picture of toxic actions. They pointed out thatO. diadema is a moderately or slightly sensitive test species. However, if thereproductive potential and all other life-cycle stages are studied, O. diadema appe*lrto be very sensitive. This as found occurred in the present tests with lowconcentrations of ethyl parathion.Hooftman & Vink (1980) further pointed out that the reproductive potential of O.diadema was the most sensitive parameter. This parameter was also significant (seeExp. Fl-adults) in my study. Ehrenstrom (1979) reported that the reproductivepotential of larvae and adults decreased when exposed to a second generationdispersant, BP 1100 WD alone and mixed with diesel oil.Hooftman & Vink (1980) showed that there was no significant suppression of growthof O. diadema by the pesticides tested. My study shows that the growth wassignificantly suppressed. It therefore provides a good picture of the toxic action ofethyl parathion (Exp. FO-adults), and it can also be used to establish the estimatedMATC values (Table 4).Gametes, embryos and larvae are generally the most critical stages in an organismslife cycle (Akesson 1983). When pollution occurs at levels which are lethal to adultanimals, the larvae may have little chance to survive. Pollution at sublethal levelsmay leave the adults seemingly unaffected but nevertheless this stress can causecomplete inhibition of the reproduction. Ehrenstrom (1979) and Hooftman & Vink(1980) have used O. diadema in toxicity tests and they showed that larvae were moresusceptible than adults.
  • L8Most of the larvae that died failed to develop after the trochophore stage. This isprobably the most critical stage for larvae to survive. Seemingly, once they can passthrough this stage, they can probably succeed to survive to other stages in their lifecycle. Thus, mortality in egg masses could be considered as one of the mostsensitive parameters. The parameter was statistically significant with ethyl parathion(Exp. B-I & B-II). Hooftman & Vink (1980) pointed out that the early larval stagesof O. diadema were affected at lower PCB and Dieldrin concentrations than theadults.Effects of ethyl parathion in aquatic ecosystems.Mulla et al. (1981, p. 4l); Connel & Miller (1984, p. 2W) reported that the effectsof insecticides can be followed from a single individual organism to an entireecosystem, and the effects are always found in non target species, like marineinvertebrate populations. Lethal and sublethal concentrations of the chemicals affectgrowth and reproduction of an organism and may directly or indirectly lead toreduced survival, and changes in abundance, composition, and productivity of thepopulations in marine ecosystems (Ken & Vass 1973 in Mulla et al. t98l).Regarding the effects on the invertebrates (especially on polychaetes), few relevantcomparative studies have been done. The comparison of those data must be handledwith caution because of the variability of exposure periods, test conditions, as wellas response differences between the species used.Ethyl parathion in concentrations of 0.01 to 0.1 mg/l (acute exposure) did not causeany mortality of the two oligochaetes, Tubifex sp. and Limnodrilus sp., whilecomplete mortality was obtained at 5.2 mg/l (Whitten & Goodnight 1966 in Mullaet al. 1981). Persoone et al. (1985) have shown that LCn (24-96 hrs) of thissubstance on crustaceans and mollusks are 0.00004-5.6 and 0.8-10 mgll,respectively. In my study, 1.23 and 4.47 mgll, the LCro 96-hrs values for larvae andadults are of the same order of magnitude.In low concentrations and chronic exposure, ethyl parathion inhibited growth and
  • 19reproduction of O. diadema (Exp. A, B-I, & B-II). Rodriguez & Pisan6 (1993)reported that this substance reduced the number of larvae hatched per female of thecrab, Chasmagnathus granulata (Decapoda: Brachyura), ild at L p,gll it causesmorphological abnormalities (hydropsy and atrophy of eyes) in the larvae. AlsoPersoone et al. (1985) reported that the NOEC for crustaceans and mollusks of suchchemical is 0.@004-0.0025 and > 0.2 mglI, respectively. Rodriguez & Pisan6(1993) have calculated ECro (EC: concentration required to immobilize 50% of testanimals) values for clutch loss in egg incubations and hatching larvae of the crab,C. granulata. They found it to be 34 p,glI. The estimated that MATC of ethylparathion on O. diadema, fall within the interval 0.1 - 0.8 p,gllby using larvae andadults (Table 4).My results show that the LOEC value of ethyl parathion is around 0.8 p,glI, and itssolubility limit in water is 24 mglI, as well as its partition coefficient (K*) is 6,430(Haque et al. 1977 in Mulla et al. l98l); this coefficient is directly correlated withits bioaccumulation in the food chain. These clearly indicate that ethyl parathion ishighly dangerous to marine organisms.Unfortunately, it is difficult to generalize for all invertebrates or even polychaetes.This is due to differences in the sensitivity of responses to the pesticides betweendifferent species or even individuals (Connel & Miller 1984). This variation inresponse means that a pesticides can eliminate susceptible individuals from apopulation or an entire susceptible species from a community of organisms (Pimentel& Goodman, 1974 inMulla et al. l98l).Water quality studyIt was strikingly surprising that the water from the harbor had a higher quality thanthe water from off Aakrogen village. However, no obvious reason was found toexplain these results. It may be a normal reaction of O. diademc when transferredto water from uncustomary sources, and is not necessarily attributable to thepresence of unnatural contaminants. But, it can also be interpreted as an indicationon a reduced water quality.
  • 20Egg-eating behaviour of O. diademaThe influence of the egg-eating behaviour on the results was recorded by thebehaviour experiments (Exp. C). This behaviour occurred regularly when the eggswere 1 to 2 days old (pers. obs.). According to B. Akesson (pers. comm.), non-developing eggs are often eaten by the parents in order to clean out the egg massbecause they can otherwise poison the viable ones. But, it was surprising to observethat some egg masses were found empty after two days. Moreover, no eggsdisappeared later in the sequence of development. It seems that the animals did notselect the un-developing eggs only. This behaviour may particularly cause a bias onthe results of the sublethal toxicity tests. It may further affect final conclusions.Therefore, I suggest that the parents and their egg masses should be separated as aprecaution when toxic effects are measured using O. diadema.I have not found anypublished reports about the egg-eating behaviour in dorvilleids, especially not inrelation to toxicity tests. However, S. Mattson (pers. comm.) frequently found anOphryotrocha species in egg masses of other polychaetes from Gullmars{orden, andhe believes that they may be egg-eaters in the nature. CONCLUSIONSI have used the polychaete O. diademc in bioassay where I studied the lethal andsublethal effects of a biocide, ethyl parathion. I also applied it in the determinationof polluted seawater. My conclusions are that the method is sensitive and useful inmarine ecotoxicological studies. It can be used to determine toxic effects ofchemicals at low concentrations, and to investigate the quality of seawater (Table 5).The method can also be used to find out the LCro and the MATC values (Table 4)of chemicals such as biocides.The present studies have shown several long-term (chronic) effects of ethyl parathionon O. diadema. The deleterious effects may cause not only growth retardation, butalso inhibition of reproduction (Tables l, 2, and 3). Because of these effects, Iconclude that ethyl parathion is highly dangerous to the marine environment.
  • 2LI also conclude that the egg-eating behaviour of O. diadcma may affect the resultsof toxicity tests. So I suggest that precautions are made in future investigations toavoid bias caused by this behaviour. ACKNOWLEDGMENTSI am very indebted to DANIDA for scholarship and Aarhus University through theFaculty of Natural Sciences and the Institute of Biological Sciences for studyfacilities. I am particularly grateful to my supervisors Prof. J. Hylleberg and Ass.Prof. T. Cedhagen for help and advice. I also want to thank Prof. B. Akesson fromGothenborg University, Sweden, for supplying the brood stock of O. diodema andas a good contact person. I benefited from Dr. S. Mattson who gave comments onmy manuscript. I am particularly grateful to Mr. H. Jalk who always help and solvepractical problems during my work. I am grateful to laboran Mrs. A. H. Jensen whoprovided the laboratory equipments, and Mrs. K. Petersen who provided thechemical solution.
  • 22 REFERENCESAkesson, B. 1970. Ophryotocha labronica as test animal for the study of marine pollution. -HelgoliinderWissenschaftlicheMeeresuntersuchungen20:293-303..Akesson, B. 1975a. Bioassay studies with polychaetes of the Ophryotrocho as test animals. Page t21.-I35 in Koeman & Strik (eds.). Sublethal effects of toxic chemicals on aquatic animals. - Elsevier.Akesson, B. 1975b. Reproduction in the genus Ophryotrocha (Polychaeta: Dorvilleidae). - Pubbl. Staz. Zool. Napoli 39:377-398.Akesson, B. 1976. Morfology and life cycli of Ophryotrocha diodema, a new polychaete species from California. - Ophelia l5(l):23-35."Akesson, B. 1980. The use of certain polychaetes in bioassay studies. - Rapports et Proces-Verbaux des Rdunions Conseil International pour lExploration de la Mer 179:315-321.Akesson, B. 1983. Methods for assessing the effects of chemicals on reproduction in marine worms. Page 459-482 in V. B. Vouk & P. J. Sheehan (eds.). Methods for assessing the effects of chemicals on reproductive functions. - Scope.Anderson, P. D. & S. DApollonia. 1978. Aquatic animals. Page 187-221 in G. C. Buttler (ed.). Principles of ecotoxicology. Scope 12. - John Wiley & Sons.APHA-AWWA-WPCF. 1980. Standard methods for the examination of water and waste-water. Fifteenth edition. Page615-743. Part 800. Bioassay methods for aquatic organisms.Barnes, R. D. l9ST.Invertebratezoology. Fifthedition. Page263-341,. Theannelids (Chapter 10). - Saunders college publishing.Bliss, C. I. 1957. Some principles of bioassay. - American Scientist 45:449-466.Brown, B. & M. Ahsanullah . 1971. Effect of heavy metals on mortality and growth. - Marine Pollution Bulletin 2:182-187.Bruscao R. C. & G. J. Brusca. 1990. Invertebrates. Page 381,-436. Chapter thirteen. Phylum Annelida: the segmented worms. - Sunderland, Massachusetts.Carr, R. S. & M. D. Curran. 1986. Evaluation of the archiannelid, Dinophilus gyrociliatus for use in short-term life-cycle toxicity tests. - Environmental Toxicology and Chemistry 5:703-7 12.Chapman, P. M. & E. R. Long. 1983. The use of bioassay as part of a comprehensive approach to marine pollution assessment. - Marine Pollution Bulletin 14(3):81-8a.Connell, D. W. & G. J. Miller. 1984. Chemistry and ecotoxicology of pollution. Page 162-223. Pesticides (Chapter 7). - John Wiley & Sons.Ehrenstrcim , F . 1979. De biologiska effekterna av oljedispergerings-medlet BP 1100 WD separat och i kombination med dieselolja, studerade med Ophryotrocha diadema (Polychaeta: Dorvilleidae) som testdjur. - Mimeographed report. Department of Zoology, University of Goteborg, Sweden (in Swedish).Finney, D. I. 1971.. Probit analysis. Third edition. - Cambridge university press. 333 pp.Fowler, J. &L. Cohen. 1990. Practical statistics for field biology. - John Wiley & Sons. Chichester. 227 pp.Govindarajtlu, Z. 1988. Statistical techniques in bioassay. Karger. Basel. 166 pp.
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  • 24Reish, D. J. 1973. Laboratory populations for long-term toxicity tests. - Marine Pollution Bulletin 4:46.Reish, D. J. 1974. The sublethal effects of environmental variables on polychaetous annelids. - Review International Oc6anogr. M6d. Tome XXXItr:83-90.Reish, D. J. 1978. The effects of heavy metals on polychaetous annelids. - Review of International Oceanogr. Med. Tome XLIX:99-105.Reish, D. J. & R. S. Can. 1978. The effect of heavy metals on the survival, reproduction, development, and life cycles for two species of polychaetous annelids. - Marine Pollution Bulletin 9(l):24-27.Reish, D. J. 1984. Marine ecotoxicological tests with polychaetous annelids. Page 427-454 in G. Persoone, E. Jaspers & C. Claus (eds.). Ecotoxicological testing for the marine environment. State University Ghent and Institute of Marine Scient. Research, Bredene, Belgium. Vol. L. 798 pp.Rodriguez, E. M. & A. Pisand. 1993. Effects of parathion and 2,4-d, to eggs incubation and larvae hatching in Chasmagnathus granulata (Decapoda: Brachyura). - Comparative Biochemistry and Physiology 104C(l):71-78.Rompas, R. M., K. Kobayashi, Y. Oshima, N. Imada, K. Yamato & Y. Mitsuyasu. 1989. Relationship between toxicity and acetylcholinesterase inhibition of some thiono- and oxo-form organophosphate in tiger shrimp larvae at different stages. - Nippon Suisan Gakkaishi 55@):669-673.Rosenthal, H. & D. F. Alderdice. 1976. Sublethal effects of environmental stressors, natural and pollutional, on marine fish eggs and larvae. - Journal of Fishery Research Board Canada 33:2047-2065.Rossi, S. S. & J. W. Anderson. 1976. Toxicity of water-soluble fractions of No.2 fuel oil and south louisiana crude oil to selected stages in the life history of the polychaete, Neanthes arenaceodentata. - Bulletin of Environmental Contamination & Toxicology 16(1): 18-25.Rossi, S. S. & J. W. Anderson. 1978. Effects of No. 2 fuel oil water-soluble- fractions on growth and reproduction in Neanthes arenaceodentata (Polychaete: Annelida). - Water, Air, and Soil Pollution 9:155-170.Roed, K. H. 1980. Effects of salinity and cadmium interaction on reproduction and growth during three successive generations of Ophryotrocha labronica (Polychaete). - Helgoliinder Meeresuntersuchungen 33:47-58.Sokal, R. R. & F. J. Rohlf. 1981. Biometry. The principles and practice of statistics in biological research. Second Edition. - W. H. Freeman and Company. New York. 859 pp.Stebbing, A. R. D. 1979. An experimental approach to determinants of biological water quality. - Philosophical Transactions of the Royal Society, I-ondon, Series B 286:465-481..Stebbing, A. R. D., B. Akesson, A. Calabrese, J. H. Gentile, A. Jensen & R. Lloyd. 1980. The role of bioassay in marine pollution monitoring. - Rapports et Proces-Verbaux des Rdunions Conseil International pour IExploration de la Mer L79:322-332.Ward, G. S. & P. R. Parrish. 1982. Manual of methods in aquatic environment research. Part 6. Toxicity Tests. - Food and Agriculture Organization of The United Nations. Rome. 23 pp.
  • 26 APPENDX 2 HOMOGENEITY (F-MAX), ANALYSIS OF VARIANCE (ONE-WAY), AND TUKEY TESTS OF THE DATA IN EXPERIMENT A PARAMETERS TESTS t::r:!::::: .....A .......B c D E F............. ::::6. ,, ,,tjI 6.25 7.94 7.68 7.58 3.48 2.92 17.71 F-max TEST .i.lrH 62.0 62.4 62.0 62.0 62.0 3.94 62.W iiig.x"OS H* H* H* H* H* H* H* iiii.F 1.86 3.95 1.33 2.51 2.42 3.26 t.72 ANOVA F*l 2.77 2.77 2.77 2.77 2.77 2.30 2.77 ..,..CEGS NS s* NS NS NS s* NS 4.7t 0.81 4.49 TTJKEY TEST SD- with NSD 0.8 ttgllNOTE: A. Time of first egg deposit B. Mean number of eggs per egg mass C. Mean number of egg masses per animal D. Mean number of larvae per egg mass E. mortality in egg masses F. Growth G. Reproductive potential CLCS Conclusion H Homogeny NS Non significant S Significant (95% Confidence) HS Highly significant (99Vo Confidence) SD Signif. different, compared to Control (0.00 pgll). NSD Non significant different :F 95/o confrdence cal. Calculation tab. table
  • 27 APPENDIX 3 HOMOGENEITY (F-MAX), ANALYSIS OF VARIANCE (ONE-WAY), AND TUKEY TESTS OF THE DATA IN EXPERIMENT B-I PARAMETERS TESTS .,... tl....E E.., *4.... 4.95 16.10 3.03 55.67 1 1.18 F-max 62.0 62.0 62.0 62.4 62.0 TEST ,F6,,;.., cLgs H* H* H- H- H* ru:i:ii:i:lil 4.27 5.82 9.38 10.2 4.41 F,l*....,,.,. 4.25 4.25 4.25 4.25 4.25 ANOVA c[€s. HS HS HS HS HS T; 3.85 1.03 3.93 t5.23 55.58 T;:I 4.49 4.49 4.49 4.49 4.49 TTJKEY TEST SD- SD- sD- sD- with with with with 0.2,0. 0.05 0.2, 0.4,& NSD 4r& ttgll 0.4,& 0.8 0.8 0.8 ttgll p,gll tr,glINOTE: A. Mean number of eggs per egg mass B. Mean number of egg masses per animal C. Mean number of larvae per egg mass D. mortality in egg masses E. Reproductive potential CLCS Conclusion H Homogeny NS Non significant S Significant (95 % Conftdence) HS Highly significant (99Vo Confidence) SD Signif. different, compared to Control (0.00 pgll). NSD Non significant different * 95Vo confrdence cal. Calculation tab. table
  • 28 APPEIIDX 4 HOMOGENETTY (F-MAX), ANALYSTS OF VARTANCE (ONE-WAY), AND TUKEY TESTS OF THE DATA IN EXPERIMENT B-II PARAMETERS TESTS A B::::::::l rr,::F$..,.,, 14.42 3.12 20.r2 23.92 29.27 F-max ix1$ 62.0 62.0 62.0 62.0 62.O TEST cLcs H* H* H" H* H" ru t.9t 0.92 4.21 4.05 43.58 2.77 2.77 2.77 2.77 4.25 ANOVA NS NS s S HS 6.32 27.86 0.51 4.49 4.49 4.49 TI,JKEY TEST sD- sD. SD- with with with 0.8 0.8 0.05, ttEll pglr 0.2,0.4 0.8prg/1NOW: A. Mean number of eggs per egg mass B. Mean number of egg masses per animal C. Mean number of larvae per egg mass D. mortality in egg masses E. Reproductive potential CLCS Conclusion H Homogeny NS Non significant s Significant (95 Vo Confrdence) HS Highly significant (99Vo Confidence) SD Signif. different, compared to Control (0.00 pgll) NSD Non significant different * 95% confidence cal. Calculation tab. table T Logarithmic transformation
  • 29 APPFAIDD( 5 HOMOGENEITY (F-MAX), ANALYSIS OF VARIANCE (ONE-WAY), AND TUKEY TESTS OF THE DATA IN EXPERIMENT C PARAMETERS TESTS :ii:l::u F::.., F*...t 2.2r 0.53 3.22 2.04 3.25 3.10 F-max F.m 39.2 39.2 39.2 39.2 39.43 39.2 TEST .....CLGS H- H- H* H* H* H- :.:iFan..r.r.r 1.11 0.26 8.72 5.47 0.76 1.27 3.49 3.49 5.95 3.49 2.74 3.49 AIOYA NS NS HS S NS NS t.70 23.48 4.20 4.20 TIJKEY TEST SD- SD- Contr. Contr. GL€S. +2, *2, 2+3, 2+4 2+4NOTE: A. Mean number of eggs per egg mass B. Mean number of egg masses per animal C. Mean number of larvae per egg mass D. Mortality in egg masses E. Growth F. Reproductive potential CLCS Conclusion H Homogeny NS Non significant S Significant (95 Vo Confidence) HS Highly significant (99% Confidence) SD Signif. different. NSD Non significant different * 95% conftdence cal. Calculation tab. table