This thesis examines the toxic effects of ethyl parathion and polluted seawater on the polychaete worm Ophryotrocha diadema. Short and long term experiments were conducted to study lethal and sublethal impacts on mortality, growth, and reproduction. The larval development of O. diadema was also described. Results showed larvae were more susceptible than adults to ethyl parathion. Growth was significantly reduced at concentrations of 0.9 ug/L. The thesis also discovered the egg-eating behavior of O. diadema, which may influence interpretation of previous toxicity tests using this species.

1. 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 Sciences
Institute of Biological Sciences
at University of Aarhus, Denmark, 1996.

2. University of Aarhus
Institute of Biological Sciences
rnternational M.sc. 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 Lasut
Institute of Biological Sciences
Department of Ecology and Genetics
University of Aarhus
DK-8000 Arhus C
Denmark
Permanent address:
Fakultas Perikanan
Universitas Sam Ratulangi
JL. Kampus Bahu
Manado 95115
Indonesia
Defended at Department of Ecoldiy and Genetics,Institute of Biological Sciences, University of
Aarhus, 23 January 1996, 09.00 AM.
Cover illustration: Larvae of Ophryotrocha diadema, and egg mass.

3. 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 end
of the preceding three months course (Theoretical Research Education referred to as
TRE).
PRE encompasses two courses (1-2) and submission of a thesis (3):
1. Scientific Drawing Techniques
Teacher: Dr. Tomas Cedhagen. Literature: Compendium written specifically for the
course. fime: 12 hours.
2.Application of the Internet in research
Teacher: Dr. Tomas Cedhagen. Literature: Compendium written specifically for the
course. Time: 12 hours.
3. Submission of a thesis describing independent and original research carried out
during 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. The
contents of the thesis must be presented in the form of a public lecture. The length
of this lecture must be 20 minutes in accordance with the time generally available
for presentations at international meetings. Examination (questions from the board
of 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 for
evaluation. The research work has been carried out by the student and has not
previously 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 ofAarhus
for guidance of individual students.
1.Rt'lrr
Hylleberg
Course Director
The International Masters Programme in Marine Sciences,
University of Aarhus, Denmark

4. 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
susce'ptible 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 the
inteqpreation of previous tests made with this species.
Y alorfu: Polychaeta (Dorvilleidae), Ophryotrocha diadema,polychaete
larval development, (lethal)
bioassay,
acute toxicity, ihronic (sublethal)'t*irity, short- and
long-term tests, mortality, growth and reproduction, Ltrtyr p*utttion,
r water quality,
MATC, egg-eating behaviour.
INTRODUCTION
Many environmental contaminants have a toxic effect on marine
organisms. They
diminish the number of survivors, influence metabolism, breeding
efficiency, alter
behavioural patterns, and affect structure and form (Reish 1974;
Rosenthal &
Alderdice 1976). Since the concern of this study is the deleterious
effects of
contaminants on marine organisms, the first step was to find
a method for measuring
such effects. Hitherto, biological methods provide the most
appropriate way of

5. 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 to
reproduction, which is a parameter of direct ecological importance (Brown &
Ahsanullah l97l; Reish 1974; Rossi & Anderson 1976, 1978; Hooftman & vink
1980; 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.

6. 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, it
inactivates 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

7. 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 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, andtheir
use in marine ecotoxicological studies. Particularly, to determine the toxic effects of
ethyl 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

8. 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

9. 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 Aarhus
harbor, (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 + 0.2
oC (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.

10. 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).

11. 8
The tests were ilrranged in two parallel series (one beginning with larvae and the
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.
F1 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.

12. 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.

13. l_o
s s
ffi &
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).

14. 11
Table 1. Experiment A: l.ong-term effects of ethyl parathion on growth, mortality
and 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 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 6.3
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.

15. L2
Table 2. Experiment B-I: Long-term effects of ethyl parathion on mortality and
reproductionof O. diadema. The experiment started with 4-week-old adults; duration
30 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 5.1
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.

16. l_3
Table 3. Experiment B-II: Long-term effects of ethyl parathion on mortality and
reproduction 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 2.5
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).

17. L4
20
1a
16
t
U 14
(9 --€-
fr't2
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 between
those values, *) No significant effect of treatment. NSD) No significant difference
between 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

18. 15
Table 5. Experiment C: Growth, mortality and reproduction of O. diademc exposed
to 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 24.2
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

19. 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: (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 33.8
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

20. 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.

21. 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

22. 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 0.@004-0.0025 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.

23. 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.

24. 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.

25. 22
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26. 23
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28. 25
APPENDIX 1
MODIFIED OBJECT GLASS FOR PHOTO-GRAPHIC MICROSCOPE
PREPARATIONS. (A) Modified object glass, (B) Cover glass.

29. 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 4.lt
TTJKEY
TEST SD-
with
NSD 0.8
ttgll
NOTE: 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

30. 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,glI
NOTE: 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

31. 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/1
NOW: 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

32. 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+4
NOTE: 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