Mt lasut 1996-tesis-aarhus univ-dk

Toxic effects of ethyl parathion and polluted

M.Sc. Thesis

Markus Talintukan Lasut

International M.Sc. Programme in Marine Sciences

Institute of Biological Sciencesat University of Aarhus, Denmark, 1996.

seawater on the polychaete Ophryotrochadiadent a (Dorvilleidae)

tl

ilr '.1!

University of AarhusInstitute of Biological Sciences

rnternational M.sc. Programme in Marine sciences

Toxic effects of ethyl parathion and pollutedseawater on the polychaete ophryotrocha

diaderua (Dorvilleidae)

- .r-r cr _ ,Ttl_ - _! ,Ivl.Dc. tltgsls

Markus Thlintukan Lasut

Institute of Biological SciencesDepartment of Ecology and GeneticsUniversity of AarhusDK-8000 Arhus CDenmark

Permanent address:Fakultas PerikananUniversitas Sam RatulangiJL. Kampus BahuManado 95115Indonesia

Defended 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.Rt'lrrHylleberg

Course DirectorThe International Masters Programme in Marine Sciences,

University of Aarhus, Denmark

1

TOXIC EFFECTS OF ETHYL PARATHION AND POLLUTED SEAWATERON THE POLYCHAETB OPHRYOTROCHA DADEMA (DORVILLEIDAE)

by Markus Talintukan lasut

' ABSTRACT

The dorvilleid polychaete , Ophryotocha diadema,including its egg-eating behaviourwas..used to study the toxic effects of ethyl parathion *C poffiirO sea;yater. Thestudies 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 andsublethal aspects studied were mortality, growth ani reproduction. The larvaldevelopment was also studied and described.

The observations g-n larval developmcni showed that iiie eggs grew from irochophore

through meratrochophore to tarvar sage-inside the eggliuri io t; tl;ys. Theresults of the toxicity tests showeo ttrat the LCro ror id hours, for both tarvae andadults, were 1.23 and 4.47 mgll, respectively.- Obuiously, the larvae were moresusce'ptible than the adults. In the sublethal tests, mortality OiO not occur ir,

"u"ryreplicate' and the parameter used gave a very incomplete picture of the eifects ofethyl parathion. Growth retardation was signihcant at a concentration of 0,g t gll (p

concentrations of a,.2 tg 9.8 rren (l) < 0.05). The estimated naarc values mostlyranged between 0.1 a1d 0.8 pgll. The water quality test showed that the warer fromAarhus harbor was of better quality than the *.to from 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,polychaete bioassay,larval development, acute (lethal) toxicity, ihronic (sublethal)'t*irity, short- andlong-term tests, mortality, growth and reproduction, Ltrtyr p*utttion, water quality,MATC, egg-eating behaviour. r

INTRODUCTION

Many 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

2

assessing toxicity effects (Stebbing et al. 1930). One of these methods is the

bioassay, also called the aquatic toxicity test. In this test, by using some parameters,

the relative potency of a substance is evaluated by comparing living organisms

exposed to the substance with unexposed organisms of the same type (Bliss 1957;

Stebbing 1979; Chapman & Iong 1983; Rand & Petrocelli 1985; Govindarajulu

1988).

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 the

consequential parameters are applied. Therefore, they should be selected with regard

to their sensitivity, availability, ild position in the food chain (Anderson &D'Apollonia 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. They

agreed 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 (applied

to 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 the

most useful invertebrate species for bioassay purposes. In this bioassay, O. diodema

was selected because it is sensitive to environmental perturbations, it is

hermaphroditic with litfle intra-specific aggression, ild because its reproductive

events can be easily recorded lAkesson 1975b; 1980). The species is small

(maximum length, 4.6 mm), easily cultivated, and has a high reproductive capacity.

3

The transparent egg masses contain a fairly low number of eggs, which facilitate the

study of the fate of individual eggs. The short life cycle of about 4 weeks at room

temperature (Hooftman & Vink 1980) is also advantageous. Furthermore, O.

diadzma has been used for determining the toxic effects of some pollutants, including

heavy metals (Klockner 1979; Reish & carr 1978; Hooftman & vink 1980; Reish

1978, 1984; Parker 1984).

The chronic toxicity test is a kind of bioassay that permits the evaluation of any

adverse 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 to

at least five concentrations of the test substance (Rand & Petrocelli 1985). The

results often are considered to predict the potential environmental effects of a

pollutant better than those of an 'acute-lethal' bioassay. By using such a test, the

'safe' environmental concentration of the toxicant (MATC: Maximum Acceptable

Toxicant Concentration) can be established; this value will be found between the

values of NOEC (No Observed Effect Concentration) and LOEC (Lowest Observed

Effect Concentration) (Rand & Petrocelli 1985).

The chemical substance (ethyl parathion) used in the present study is an

organophosphorous insecticide. Like other organophosphorous insecticides, itinactivates the enzyme cholinesterase (ChE) and can break down the neurotransmitter

acetylcholine (ACh) in a synapse of the nervous system and thereby disrupt the

nervous coordination. It may cause deleterious effects by way of increasing mortality

and 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 found

in 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 can

be found in the entire ecosystem which is indicated by changes in species

4

composition and population number. Typically, these changes follow a sequence of

dynamic events as outlined below (Pimentel t971,; Pimentel & Goodman 7974; and

Brown 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 ofspecies 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, andtheir

use in marine ecotoxicological studies. Particularly, to determine the toxic effects ofethyl parathion in very low concentrations, to establish its MATC value for a marine

environment, and to apply the method used to water quality testing.

The studies were also made in order to improve the polychaete bioassay method, and

were motivated by the fact that still very little is known about the toxic effects of

similar chemicals on marine invertebrates. Furthermore, the studies were also

motivated by the fact that ethyl parathion is still widely used as a biocide (Haskoning

t994).

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. diadema

in order to have a reference point. Then I investigate the sublethal effects of ethyl

parathion on O. diadema in terms of mortality, growth and reproduction. I also test

the effects of polluted seawater on O. diodema. Finally, I describe the egg-eating

behaviour of O. diadema as a factor that can affect the results of the toxicity tests.

MATERIALS AND METHODS

Animsfu and compounds tested

The marine polychaete Ophryotrocha diadema (Dorvilleidae: Polychaeta) was used

as test species. The animals were supplied by Prof. B. Akesson (Department of

ZooIogy, 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-nitrophenyl

phosphorothioate) (NIOSH 1990; Haskoning 1994),an organophosphorus insecticide,

was used. The water solubility and persistence in water of ethyl parathion are 24

mg/l at 25 "C and 108 days (at pH7.4 and20 "C) and 2 to 6 days (under natural

conditions), respectively (Haskoning 1994).

- Polluted seawater. Water samples were collecteA at three selected sites in Aarhus

Bay, Denmark (Fig. 1), and tested. The first sample was taken off the Aakrogen

village, the second within the Aarhus harbor, and the third off the Moesgaard forest.

Cultivation procedures

The stock and test animals were cultivated in the laboratory at the Department of

Genetics and Ecology, Institute of Biological Sciences, University of Aarhus,

Denmark. The procedures for cultivation were integrated from those of the American

Public Health Association (1980), Hooftman & Vink (1980), Ward & Parrish (1982),

Akesson (1983), and Parker (1984). The animals were kept in 80 ml and20 ml glass

bowls, respectively. The volume of the water medium for the tests was 10 ml in

6

each bowl.

The bowls were placed in a bucket with water and lid to prevent evaporation.

Water culture and food

All water used for cultivation, including dilutions and controls, was collected from

Gullmarsfiorden, 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. The

animals, both larvae and adults, were fed with a seawater suspension of frozen and

fragmented spinach.

Figure 1. Sampling localities. (1) off the Aakrogen village, (2) inside the Aarhusharbor, (3) off the Moesgaard forest.

Environmental conditions

The environmental conditions in the laboratory (temperature, salinity, and pH) were

controlled. 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 and

pH were 32.7 + 0.1%o and 8.0 + 0.0 (mean J I S.E.), respectively.

7

Experiments

Larval development

Adult animals from a single stock culture were kept in a 80 ml glass bowl until they

had produced egg masses. The development of the egg masses and the eggs were

observed daily until the larval stage. The various stages were documented with a

photographic microscope.

A living egg mass was placed onto a modified object glass (see Appendix 1). The

object glass was constructed so that it allowed me to observe the egg masses and

eggs alive under a microscope.

Tests of toxicity

L 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 the

basis of the results of a preliminary study that showed no effects of ethyl parathion

on the mortality of O. diadema in concentrations less than 0.01 mg/l after 96 hours

of exposure.

For both larvae and adults, four 20 ml semispherical glass bowls, each containing

seven animals and 10 ml of prepared seawater of each concentration, were used. The

water and the bowls were renewed every 24 hours, and dead animals were

simultaneously counted. The tests were terminated after 96 hours and LCro

calculated.

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 egg

masses per animal, number of eggs per egg mass, number of larvae per egg mass,

mortality in the egg masses, and reproductive potential. (The reproductive potential

was calculated by dividing the final number of larvae of each concentration by the

final number of larvae in the control, and is expressed as a percentage of the control

value (Hooftman & Vink 1980).

8

theThe tests were ilrranged in two parallel series (one beginning with larvae and

other with adults) according to Akesson (1983).

Experiment A: larvae

Larvae of the same age were used Q to 3 days, starting to feed). Five concentrations

of the test solution were chosen (0.05, 0J,A.2,0.4, and 0.8 pgll) on thebasis of

the allowed concentration proposed by the European Community (0.1, p,g/I) for fresh

and marine water (Haskoning 1994).

Four 20 ml semispherical glass bowls, each containing ten animals and 10 ml

prepared seawater of each concentration, were used. The time of exposure was 30

days. Every third day, the animals were fed (with suspended spinach during the first

two weeks and thereafter with fragmented frozen spinach), the water solution and the

bowls were renewed, and the parameters were observed.

Experiment B: adults

F 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 the

experiments were as described in Exp. A.

F 1 level (Exp. B-II). Thetestanimalswerepreparedbyexposing two

couples of l-week-old juveniles from the same F0 stock culture in each concentration

until they reached the adult stage of the first generation (F1). The experiment started

with 4-week-old adults of Fl animals, and ran for 20 days. The test was carried out

in the same way as in the F0 Exp. (B-I), except that 4 animals were used in each

bowl.

Experiment C: polluted seawater

Seawater samples from three selected sites of Aarhus Bay were used. The test

animals (2 to 3 days old larvae, starting to feed) were exposed for 30 days.

Preparations of water, the number of test animals, renewals, and observed

parameters were as described for Exp. A.

9

Egg-eating behaviour

A11 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) in

each were used for four kinds of experiment arrangements, viz.: 1) eggs, no

animals, no food; 2) eggs, no animals, food; 3) eggs, animals, no food; and 4) eggs,

animals, food.

Analyses of data

The median lethal concentrations (LCr) of ethyl parathion after 96 hours of exposure

were calculated by using probit analysis according to Finney (197I). One-way

ANOVA (Analyses of variance) and Tukey test were applied to test whether the

concentrations of ethyl parathion and water of bad quality affect the mortality,

growth and reproduction. The Mann-Whitney U-test and the two-way ANOVA were

applied 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]LTS

Larval development

The study of larval development of O. diadema verified the results of Akesson

(1976) and was used as a reference point. O. diodema is a simultaneous

hermaphrodite with internal fertilization. It produced egg masses about 15 days after

hatching. The eggs are yellow and oval-shaped, and deposited in mucous masses that

are attached to the wall of the culture bowls. The egg mass is contained in a

transparent fusiform membrane; the eggs and the fate of the individual egg can be

easily observed through the membrane. The total number of eggs per egg mass

ranged 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 egg

development to released larvae is about 7 to 9 days under the conditions.

l_o

s s

&

ffi

ffi

w

"" "":

; :.$

r'

Figure 2. Larval development of O. diadema. (1-8) The development of larvae inthe egg mass from the first to the eighth day. (Scale bar : 1 prm).

11

Table 1. Experiment A: l.ong-term 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.i'90 0.i:80

Growth (number ofsetiger, 30 days)

r6.60.1

16.00.2

15.90.2

16.1

0.215.90.2

15.50.2

First egg deposit(day after start)

15

0.0t6

0.8t6

0.617

t.318

1.019

1.5

Mortality(%, after 30 days)

o-o 2.5 0.0 5.0 5.0 28.0

Mean number of eggsper egg mass

16.20.9

12.61.3

16.50.5

t6.61.0

t2.71,.4

12.61.0

Mean number of eggmasses per animal

1.60.2

1..7

0.2r.20.2

t.30.2

1.40.2

1.40.1

Mean number of larvaeper egg mass

8.60.9

4.90.5

6.3t.2

5.51,.2

4.41.5

3.91.0

Mortality in eggmasses (%)

444.3

604.5

597.0

666.6

688.0

666.0

Reproductive potential(%, control : 100)

100

0.066

18.851

9.467

26.450

2t.4376.3

In the egg mass, the eggs developed to trochophore larvae in the fourth day after

hatching (Fig. 2). The trochophores have a ciliary band for locomotion around the

body and swim by rotation. The trochophores were yolky and non-feeding, so-called

'lecithotrophic' (see also Barnes (1987, p. 305); Brusca & Brusca (1990, p. a2fl).

They reached the metatrochophore stage in the sixth day. The first pair of parapodia

with setae developed on the first setigerous segment at this stage, and continued until

it reached four pairs as a complete larva on the eighth day. At this time, the larvae

broke 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.

L2

Table 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).

P.t T,ERSETIIYL PARATHION

CONCENTRATIONS (pell)

0,,.OCI 0:05 i,:$:i::\'Ql;;,;';1 j0iii20


0.0 0.0 2.5 ,: 5.0 5.0


1,8.7

1.016.5t.2

T7.T

0.8t4.90.9

13.90.5

t4.90.8


3.40.4

4.80.2

3.90.2

4.00.1

3.90.2

3.10.2


13.80.9

TI.40.9

11.80.8

9.1,

0.87.61.1

6.90.7


262.9

300.7

31

3.5401.3

495.2

524.2

Reproductive potential(Vo, eontrol : 100)

1000.0

118

t3.7101

16.079

12.867

17.0465.1

I also observed that the larval stage, especially the trochophore, was critical. Most

of the larvae that failed to survive did it at this stage.

Tests of toxicity

Efficts of etlryl parathion

Lethal to xicity. The mortalityin theacute (lethal) exposure for96hours

was 7, 39, 43, and l00Vo for the experiment with larvae, and 4, 29, 36, and lNVo

for the experiment with adults at concentrations of 0.01, 0.1, 1, and 10 mg/l. In both

tests, no mortality was observed at the lowest concentration (0.01 mg/l), but it was

1,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 the

restricted duration of these tests, threshold values for constant LC5o could not be

derived.

l_3

Table 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(Vo, after 30 days)

o-o 0.0 0.0 0.0 0.0 12.5


18.1

0.8T6.It.9

17.41.1

14.82.3

14.21.7

t2.50.6


4.30.4

4.50.4

4.00.5

3.60.4

3.30.7

3.74.4


10.90.8

9.6t.3

8.71.1

7.42.6

4.71.2

3.40.6


395.5

422.0

51

6.2559.9

6I4.5

736.3


1m0.0

103

30.893

38.s51

15.037

11.5262.5

S ub lethal tox icity. Thelong-termeffectsof ethylparathion onthe

growth of O. diadema (Exp. A) along a complete life-cycle (30 days) are shown in

Table 1. There were small temporary growth retardations at concentrations of 0.05

to 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 with

a relatively high mortality in egg masses in all experiments, and also a delayed time

of 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

--€-O.OO ppb

O.O5 ppb-+K-O.1 O ppb--*-O.2O ppb

O.4O ppb-+<-O.AO ppb

12 15 16 21TIME (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 depositMean number of eggs

per egg mass

Mean number of egg massesper animal


Mortality in egg masses

GrowthReproductive potential

NSD 0.1-0.2 :r

*NSD*

:r. 0.1-0.2 0.4-0.8'rc 0.2-0.4 0.4-0.8

0.4-0.8* NSD 0.1-0.2

1a

16

t 14U(9

fr't2ab 10dUAm

=Zt)

502724

15

Table 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.).

The mortality increased clearly only at the highest concentration (0.8 pgll) for all

the tests, while no mortality was observed in the control. This high mortality

occurred mostly during the first week (larval-juvenile stage) of the experiments. No

statistical tests were carried out for this parameter, because it did not even occur in

every replicate. It was therefore considered as a slightly sensitive parameter in the

sublethal 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

WATER SAMPLES

LOCATIONSi.iCO"N.TROL x a,

Growth (number ofsetiger, 30 days)

15.150.13

14.850.23

15.150.2s

14.850.15


o-o ,: 5.0 10.0


7.60.62

6.20.54

7.10.44

7.40.66


1.20.10

1.30.08

1.40.09

1.20.26


5.40.52

2.80.41

4.70.34

5.10.30


31

5.2958

5.6238

6.7029

4.46


100 5913.8

10423.52

9624.2

16

O. diadema were mostly between 0.1 and 0.8 pgll (Table 4).

Effects of polluted seawater

Animals cultured in water from off Aakrogen village have a markedly reduced

number 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 Moesgaard

forest, respectively.

Table 6. Egg-eating behaviour of O. diadema; duration 48 hours.

EXP. 1:Eggs & no animalsEggs & animals

(food & no food)0&0

22.8 & 54.8

EXP. 2:1 Couple2 Couples3 Couples

(food & no food)r9.0 & 36.330.3 & 50.023.3 & 46.3

EXP. 3:The eggs did not belong

to the animalsThe eggs belonged to

the animals

(no food)

11

33.8

Egg-eating behaviour

Statistically, 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). They

ate not only their own eggs, but also from other parents (Exp. 3) (Table 6).

DISCUSSION

Susceptibility of O. diodcma

O. 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 to

thoroughly 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 that

O. diadema is a moderately or slightly sensitive test species. However, if the

reproductive potential and all other life-cycle stages are studied, O. diadema appe*lr

to be very sensitive. This as found occurred in the present tests with low

concentrations 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 (see

Exp. Fl-adults) in my study. Ehrenstrom (1979) reported that the reproductive

potential of larvae and adults decreased when exposed to a 'second generation'

dispersant, BP 1100 WD alone and mixed with diesel oil.

Hooftman & Vink (1980) showed that there was no significant suppression of growth

of O. diadema by the pesticides tested. My study shows that the growth was

significantly suppressed. It therefore provides a good picture of the toxic action of

ethyl parathion (Exp. FO-adults), and it can also be used to establish the estimated

MATC values (Table 4).

Gametes, embryos and larvae are generally the most critical stages in an organism's

life cycle (Akesson 1983). When pollution occurs at levels which are lethal to adult

animals, the larvae may have little chance to survive. Pollution at sublethal levels

may leave the adults seemingly unaffected but nevertheless this stress can cause

complete inhibition of the reproduction. Ehrenstrom (1979) and Hooftman & Vink

(1980) have used O. diadema in toxicity tests and they showed that larvae were more

susceptible than adults.

L8

Most of the larvae that died failed to develop after the trochophore stage. This is

probably the most critical stage for larvae to survive. Seemingly, once they can pass

through this stage, they can probably succeed to survive to other stages in their life

cycle. Thus, mortality in egg masses could be considered as one of the most

sensitive parameters. The parameter was statistically significant with ethyl parathion

(Exp. B-I & B-II). Hooftman & Vink (1980) pointed out that the early larval stages

of O. diadema were affected at lower PCB and Dieldrin concentrations than the

adults.

Effects of ethyl parathion in aquatic ecosystems.

Mulla et al. (1981, p. 4l); Connel & Miller (1984, p. 2W) reported that the effects

of insecticides can be followed from a single individual organism to an entire

ecosystem, and the effects are always found in non target species, like marine

invertebrate populations. Lethal and sublethal concentrations of the chemicals affect

growth and reproduction of an organism and may directly or indirectly lead to

reduced survival, and changes in abundance, composition, and productivity of the

populations in marine ecosystems (Ken & Vass 1973 in Mulla et al. t98l).

Regarding the effects on the invertebrates (especially on polychaetes), few relevant

comparative studies have been done. The comparison of those data must be handled

with caution because of the variability of exposure periods, test conditions, as well

as response differences between the species used.

Ethyl parathion in concentrations of 0.01 to 0.1 mg/l (acute exposure) did not cause

any mortality of the two oligochaetes, Tubifex sp. and Limnodrilus sp., while

complete mortality was obtained at 5.2 mg/l (Whitten & Goodnight 1966 in Mulla

et al. 1981). Persoone et al. (1985) have shown that LCn (24-96 hrs) of this

substance 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 and

adults are of the same order of magnitude.

In low concentrations and chronic exposure, ethyl parathion inhibited growth and

19

reproduction 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 the

crab, Chasmagnathus granulata (Decapoda: Brachyura), ild at L p,gll it causes

morphological abnormalities (hydropsy and atrophy of eyes) in the larvae. Also

Persoone et al. (1985) reported that the NOEC for crustaceans and mollusks of such

chemical is [email protected] and > 0.2 mglI, respectively. Rodriguez & Pisan6

(1993) have calculated ECro (EC: concentration required to immobilize 50% of test

animals) 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 ethyl

parathion on O. diadema, fall within the interval 0.1 - 0.8 p,gllby using larvae and

adults (Table 4).

My results show that the LOEC value of ethyl parathion is around 0.8 p,glI, and its

solubility 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 with

its bioaccumulation in the food chain. These clearly indicate that ethyl parathion is

highly 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 between

different species or even individuals (Connel & Miller 1984). This variation in

response means that a pesticides can eliminate susceptible individuals from a

population or an entire susceptible species from a community of organisms (Pimentel

& Goodman, 1974 inMulla et al. l98l).

Water quality study

It was strikingly surprising that the water from the harbor had a higher quality than

the water from off Aakrogen village. However, no obvious reason was found to

explain these results. It may be a normal reaction of O. diademc when transferred

to water from uncustomary sources, and is not necessarily attributable to the

presence of unnatural contaminants. But, it can also be interpreted as an indication

on a reduced water quality.

20

Egg-eating behaviour of O. diadema

The influence of the egg-eating behaviour on the results was recorded by the

behaviour experiments (Exp. C). This behaviour occurred regularly when the eggs

were 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 mass

because they can otherwise poison the viable ones. But, it was surprising to observe

that some egg masses were found empty after two days. Moreover, no eggs

disappeared later in the sequence of development. It seems that the animals did not

select the un-developing eggs only. This behaviour may particularly cause a bias on

the 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 a

precaution when toxic effects are measured using O. diadema.I have not found any

published reports about the egg-eating behaviour in dorvilleids, especially not in

relation to toxicity tests. However, S. Mattson (pers. comm.) frequently found an

Ophryotrocha species in egg masses of other polychaetes from Gullmars{orden, and

he believes that they may be egg-eaters in the nature.

CONCLUSIONS

I have used the polychaete O. diademc in bioassay where I studied the lethal and

sublethal effects of a biocide, ethyl parathion. I also applied it in the determination

of polluted seawater. My conclusions are that the method is sensitive and useful in

marine ecotoxicological studies. It can be used to determine toxic effects of

chemicals 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 parathion

on O. diadema. The deleterious effects may cause not only growth retardation, but

also inhibition of reproduction (Tables l, 2, and 3). Because of these effects, I

conclude that ethyl parathion is highly dangerous to the marine environment.

2L

I also conclude that the egg-eating behaviour of O. diadcma may affect the results

of toxicity tests. So I suggest that precautions are made in future investigations to

avoid bias caused by this behaviour.

ACKNOWLEDGMENTS

I am very indebted to DANIDA for scholarship and Aarhus University through the

Faculty of Natural Sciences and the Institute of Biological Sciences for study

facilities. 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 from

Gothenborg University, Sweden, for supplying the brood stock of O. diodema and

as a good contact person. I benefited from Dr. S. Mattson who gave comments on

my manuscript. I am particularly grateful to Mr. H. Jalk who always help and solve

practical problems during my work. I am grateful to laboran Mrs. A. H. Jensen who

provided the laboratory equipments, and Mrs. K. Petersen who provided the

chemical solution.

22

REFERENCES

Akesson, 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 toxicchemicals 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 l'Exploration de laMer 179:315-321.

Akesson, B. 1983. Methods for assessing the effects of chemicals on reproductionin 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. D'Apollonia. 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 andwaste-water. Fifteenth edition. Page615-743. Part 800. Bioassay methods foraquatic 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. - EnvironmentalToxicology and Chemistry 5:703-7 12.

Chapman, P. M. & E. R. Long. 1983. The use of bioassay as part of acomprehensive approach to marine pollution assessment. - Marine PollutionBulletin 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 1100WD separat och i kombination med dieselolja, studerade med Ophryotrochadiadema (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.

23

Harrison, F. L. & S. L. Anderson. 1994. Effects of acute irradiance on reproductivesuccess of the polychaete worm, Neanthes arenaceodentata. - RadiationResearch 137:59-66.

Haskoning. 1994. Technical and economic aspects of measures to reduce waterpollution caused by the discharge of certain organophosphorus compounds. -ECSC-EC-EAEC, Brussels-Luxembourg. 87 pp.

Hooftman, R. N. & G. J. Vink. 1980. The determination of toxic effects ofpollutants with the marine polychaetg worm Ophryotrocha diadema. -Ecotoxicology and Environmental Safety 4:252-262.

Klockner, K. 1977. The effect of cadmium on mortality, growth and reproductionof Ophryotrocha diadema (Polychaeta). In Annual Reports of the BiologicalInstitute Helgoland, Hamburg.

Klciuckner, K. 1979.Uptake and accumulation of cadmium by Ophryotrocha diadema(Polychaeta). - Marine Ecology Progress Series l:71-76.

Kobayashi, K., R. M. Rompas, T. Maekawa, N. Imada & Y. Oshima. 1990.Changes in metabolic activity of tiger shrimp larvae at different stages tofenitrothiotr, ffi organophosphorus insecticide. - Nippon Suisan Gakkaishi56Q):a89-a96.

Leistrao M., L. G. M. Th. Tuinstra, A. M. M. van der Burg & S. J. H. Crum.1984. Contribution of leaching of diazinon, parathion, tetrachlorvinphos andtriazophos from glasshouse soils to their concentrations in water courses. -Chemosphere 13(3):403-413.

Monserrat, J. M., E. M. Rodriguez & R. J. Lombardo. 199I. Effects of salinity onthe toxicity of parathion to the estuarine crab Chasmagnathus granulata(Decapoda: Grapsidae). -Bulletin of Environmental Contamination &Toxicology 46:569-57 5.

Moore, D. W. & T. M. Dillon. 1993. The relationship between growth andreproduction in the marine polychaete Nereis (Neanthes) arenaceodentata(Moore): implications for chronic sublethal sediment bioassay. - Journal ofExperimental Marine Biology and Ecology 173:231-246.

Mulla, M. S., L. S. Mian & J. A. Kawecki. 1981. Distribution, transport, and fateof the insecticides malathion and parathion in the environment. Page l-L37inF. A. Gunther & J. D. Gunther. Residue Reviews. Residues of pesticidesand other contaminants in the total environment. - Springer-Verlag. NewYork.

MOSH. 1990. Pocket guide to chemical hazards. - U.S. Department of Health andHuman Services. Public health service. 245 pp.

Parker, J. G. 1984. The effects of selected chemicals and water quality on themarine polychaete Ophryotrocha diadema. - Water Research 18(?:865-868.

Persoone, et al. 1985. Evaluation of the impact of parathion, methyl-parathion (PartA), fenitrothion and fenthion (Part B) on the aquatic environment. Finalreport to EC/DG XI, part B; no. XIl785l83 (41841120)

Pocklington, P. & P. G. Wells. 1992. Polychaetes: Key taxa for marineenvironmental quality monitoring. Review. - Marine Pollution Bulletin24(12):593-598.

Rand, G. M. & S. R. Petrocelli. 1985. Fundamentals of aquatic toxicology. -Hemisphere Publishing Corporation. New York. 666 pp.

24

Reish, D. J. 1973. Laboratory populations for long-term toxicity tests. - MarinePollution Bulletin 4:46.

Reish, D. J. 1974. The sublethal effects of environmental variables on polychaetousannelids. - Review International Oc6anogr. M6d. Tome XXXItr:83-90.

Reish, D. J. 1978. The effects of heavy metals on polychaetous annelids. - Reviewof 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 polychaetousannelids. - Marine Pollution Bulletin 9(l):24-27.

Reish, D. J. 1984. Marine ecotoxicological tests with polychaetous annelids. Page427-454 in G. Persoone, E. Jaspers & C. Claus (eds.). Ecotoxicologicaltesting for the marine environment. State University Ghent and Institute ofMarine Scient. Research, Bredene, Belgium. Vol. L. 798 pp.

Rodriguez, E. M. & A. Pisand. 1993. Effects of parathion and 2,4-d, to eggsincubation 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 ofsome thiono- and oxo-form organophosphate in tiger shrimp larvae atdifferent 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 FisheryResearch Board Canada 33:2047-2065.

Rossi, S. S. & J. W. Anderson. 1976. Toxicity of water-soluble fractions of No.2fuel oil and south louisiana crude oil to selected stages in the life history ofthe polychaete, Neanthes arenaceodentata. - Bulletin of EnvironmentalContamination & 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 andgrowth 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 statisticsin biological research. Second Edition. - W. H. Freeman and Company. NewYork. 859 pp.

Stebbing, A. R. D. 1979. An experimental approach to determinants of biologicalwater 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. - Rapportset Proces-Verbaux des Rdunions Conseil International pour I'Exploration dela Mer L79:322-332.

Ward, G. S. & P. R. Parrish. 1982. Manual of methods in aquatic environmentresearch. Part 6. Toxicity Tests. - Food and Agriculture Organization of TheUnited Nations. Rome. 23 pp.

25

APPENDIX 1

MODIFIED OBJECT GLASS FOR PHOTO-GRAPHIC MICROSCOPEPREPARATIONS. (A) Modified object glass, (B) Cover glass.

26

APPENDX 2HOMOGENEITY (F-MAX), ANALYSIS OF VARIANCE (ONE-WAY),

AND TUKEY TESTS OF THE DATA IN EXPERIMENT A

NOTE: A. Time of first egg depositB. Mean number of eggs per egg mass

C. Mean number of egg masses per animalD. Mean number of larvae per egg mass

E. mortality in egg masses

F. GrowthG. Reproductive potential

CLCSHNSS

HSSDNSD:F

cal.tab.

ConclusionHomogenyNon significantSignificant (95% Confidence)Highly significant (99Vo Confidence)Signif. different, compared to Control (0.00 pgll).Non significant different95/o confrdenceCalculationtable

TESTSPARAMETERS

.....A .......B c D Et::r:!:::::

F.............'::::'6.,, ,,tjI

F-maxTEST

6.25 7.94 7.68 7.58 3.48 2.92 17.71

.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*

ANOVA

iiii.F 1.86 3.95 1.33 2.51 2.42 3.26 t.72

F*l 2.77 2.77 2.77 2.77 2.77 2.30 2.77

..,..CEGS NS s* NS NS NS s* NS

TTJKEYTEST

4.7t 0.81

4.49 4.lt

NSD

SD-with0.8ttgll

27

APPENDIX 3HOMOGENEITY (F-MAX), ANALYSIS OF VARIANCE (ONE-WAY),

AND TUKEY TESTS OF THE DATA IN EXPERIMENT B-I

TESTSPARAMETERS

.,... tl....E

F-maxTEST

E..',*4.... 4.95 16.10 3.03 55.67 1 1.18

',F6,,;..,62.0 62.0 62.0 62.4 62.0

cLgs H* H* H- H- H*

ANOVA

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

c[€s. HS HS HS HS HS

TTJKEYTEST

T; 3.85 1.03 3.93 t5.23 55.58

T;:I 4.49 4.49 4.49 4.49 4.49

SD-with0.2,0.4r&0.8p,gll

SD-with0.05

ttgll

sD-with0.2,0.4,&0.8tr,glI

sD-with0.4,&0.8ttgll

NSD

NOTE: A. Mean number of eggs per egg massB. Mean number of egg masses per animalC. Mean number of larvae per egg mass

D. mortality in egg masses

E. Reproductive potential

CLCSHNSS

HSSDNSD*cal.tab.

ConclusionHomogenyNon significantSignificant (95 % Conftdence)Highly significant (99Vo Confidence)Signif. different, compared to Control (0.00 pgll).Non significant different95Vo confrdenceCalculationtable

28

APPEIIDX 4HOMOGENETTY (F-MAX), ANALYSTS OF VARTANCE (ONE-WAY),

AND TUKEY TESTS OF THE DATA IN EXPERIMENT B-II

TESTSPARAMETERS

A B::::::::l

F-maxTEST

rr,::F$.'.,.',, 14.42 3.12 20.r2 23.92 29.27

ix1'$ 62.0 62.0 62.0 62.0 62.O

cLcs H* H* H" H* H"

ANOVA

ru t.9t 0.92 4.21 4.05 43.58

2.77 2.77 2.77 2.77 4.25

NS NS s S HS

TI,JKEYTEST

6.32 27.86 0.51

4.49 4.49 4.49

sD-with0.8ttEll

sD.with0.8pglr

SD-with0.05,0.2,0.40.8prg/1

NOW: A. Mean number of eggs per egg massB. Mean number of egg masses per animalC. Mean number of larvae per egg massD. mortality in egg masses

E. Reproductive potential

CLCSHNSsHSSDNSD*cal.tab.T

ConclusionHomogenyNon significantSignificant (95 Vo Confrdence)Highly significant (99Vo Confidence)Signif. different, compared to Control (0.00 pgll)Non significant different95% confidenceCalculationtableLogarithmic transformation

29

APPFAIDD( 5HOMOGENEITY (F-MAX), ANALYSIS OF VARIANCE (ONE-WAY),

AND TUKEY TESTS OF THE DATA IN EXPERIMENT C

NOTE: A. Mean number of eggs per egg mass

B. Mean number of egg masses per animalC. Mean number of larvae per egg mass

D. Mortality in egg masses

E. GrowthF. Reproductive potential

CLCSHNSS

HSSDNSD*cal.tab.

ConclusionHomogenyNon significantSignificant (95 Vo Confidence)Highly significant (99% Confidence)Signif. different.Non significant different95% conftdenceCalculationtable

TESTSPARAMETERS

:ii:l::u F::..,

F-maxTEST

F*..'.t 2.2r 0.53 3.22 2.04 3.25 3.10

F.m 39.2 39.2 39.2 39.2 39.43 39.2

.....CLGS H- H- H* H* H* H-

AI\OYA

:.: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

NS NS HS S NS NS

TIJKEYTEST

t.70 23.48

4.20 4.20

GL€S.

SD-Contr.+2,2+3,2+4

SD-Contr.*2,2+4

Mt lasut 1996-tesis-aarhus univ-dk

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