Toxic effects of Gymnodinium cf. mikimotoi unsaturated fatty acids to gametes and embryos of the sea urchin Paracentrotus lividus

Article abstract of DOI:10.1016/S0043-1354(99)00181-5

The toxicity of the main Gymnodinium cf. mikimotoi polyunsaturated fatty acid, has been investigated using the sea urchin gamete and embryo bioassays. The 18:5n3 fatty acid delays or inhibits first cleavage of Paracentrotus lividus eggs and provokes abnormalities in the embryonic development. These effects were compared with those of other polyunsaturated fatty acids, 18:4n3, 20:5n3 and 22:6n3, which are also present in this alga. A classification of the different fatty acids, based on their effects on sea urchin egg cleavage and the determination of the half inhibiting concentrations (IC₅₀) is proposed. Copyright (C) 1999 Elsevier Science Ltd. The toxicity of the main Gymnodinium cf. mikitomoi polyunsaturated fatty acid, has been investigated using the sea urchin gamete and embryo bioassays. The 18:5n3 fatty acid delays or inhibits first cleavage of Paracentrotus lividus eggs and provokes abnormalities in the embryonic development. These effects were compared with those of other polyunsaturated fatty acids, 18:4n3, 20:5n3 and 22:6n3, which are also present in this alga. A classification of the different fatty acids, based on their effects on sea urchin egg cleavage and the determination of the half inhibiting concentrations (IC₅₀) is proposed.

Full text of DOI:10.1016/S0043-1354(99)00181-5

Wat. Res. Vol. 34, No. 2, pp. 550±556, 2000
# 1999 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(99)00181-5
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TOXIC EFFECTS OF GYMNODINIUM CF. MIKIMOTOI
UNSATURATED FATTY ACIDS TO GAMETES AND
EMBRYOS OF THE SEA URCHIN PARACENTROTUS
LIVIDUS
FERIEL SELLEM1, DANIELLE PESANDO2*, GUY BODENNEC3,
AMOR EL ABED1 and JEAN-PIERRE GIRARD2
1Institut National des Sciences et Technologies de la Mer, 28 rue du 2 Mars 1934, 2025, Salammbo,
2
Tunisia; LPTE, EA 2138, Faculte des Sciences, BP 71, 06108-Nice-cedex 02, France and 3Departement
Ecologie Cotiere, IFREMER Centre de Brest, BP 70, 29280-Plouzane, France
(First received 1 November 1998; accepted in revised form 1 April 1999)
AbstractÐThe toxicity of the main Gymnodinium cf. mikimotoi polyunsaturated fatty acid, has been
investigated using the sea urchin gamete and embryo bioassays. The 18:5n3 fatty acid delays or inhibits
®rst cleavage of Paracentrotus lividus eggs and provokes abnormalities in the embryonic development.
These e€ects were compared with those of other polyunsaturated fatty acids, 18:4n3, 20:5n3 and
22:6n3, which are also present in this alga. A classi®cation of the di€erent fatty acids, based on their
e€ects on sea urchin egg cleavage and the determination of the half inhibiting concentrations (IC50) is
proposed. # 1999 Elsevier Science Ltd. All rights reserved
Key wordsÐGymnodinium unsaturated fatty acids, sea urchin, embryo±larval development
INTRODUCTION
esteri®ed polyunsaturated fatty acids `PUFA' in a
glycolipid structure.
In the marine environment, microalgae represent a
Bodennec et al. (1995) showed that hexadecate-
large part of primary production. Some microalgae
traenoic acid (16:4n3), octadecapentaenoic acid
are toxic and may interfere with the environment
(18:5n3) and docosahexaenoic acid (22:6n3) were
the main PUFA in the lipid extract of the French
and with marine organisms at many stages of their
life cycles. Blooms of the marine red-tide dino¯agel-
Gymnodinium strain. 18:5n3, a fatty acid rarely
encountered in algal species has been shown to be
late Gymnodinium cf. mikimotoi (alias Gymnodinium
nagasakiense, Gyrodinium aureolum ) have frequently
more potent than saturated compounds and even
more active than the other polyunsaturated fatty
been reported during summer months along the
western coast of France (Partensky et al., 1991;
Gentien, 1998) and more recently on Tunisian
coasts, inducing massive mortality in ®sh and in-
vertebrates (Hamza and El Abed, 1994). Mortality
or abnormalities in Pecten maximus, probably at-
tributable to ingestion of algal cells are reported by
Erard-Le Denn et al. (1990). Gentien et al. (1991)
and Arzul et al. (1993) showed that a suspension in
seawater of chloroform-methanol extract of the
French isolate of Gymnodinium cells or of culture
medium having contained algal cells was toxic to
Mytilus sp. embryos and capable of stopping dia-
tom growth. Yasumoto et al. (1990) associated the
hemolytic activity of Gyrodinium aureolum, species
closely related to Gymnodinium mikimotoi, to the
presence of lipid compounds containing free or
acids in hemolytic and diatom growth inhibition
tests (Arzul et al., 1995). Fatty acid e€ects would
concern mainly cell membrane structure where they
may modify passive and active ionic transports, as
recently described by Gamberucci et al. (1997) in re-
lation to calcium conductance, with consequences
for calcium-dependent cellular events. More recently
Parrish et al. (1998) analysed glycolipids in
Gymnodinium sp. They found that monogalactosyl
diacylglycerol (MGDG) and digalactosyl diacylgly-
cerol (DGDG) which represent 35% of the total
lipids, were hemolytic and that the major unsatu-
rated fatty acid in these glycolipids was 18:5n3
Of various in vitro bioassays, the sea urchin egg
test is one of the most extensively utilized
(Kobayashi, 1980; Hose, 1985; Pagano et al., 1986;
Dinnel et al., 1988; Fusetani et al., 1989; Trie€ et
al., 1995). The sea urchin egg has many advantages
*Author to whom all correspondence should be addressed. for screening developmental toxicity. This biological
550

Toxic e€ects of fatty acids to P. lividus
551
samples of eggs were taken 5 min after fertilization and
the extent of fertilization judged by the number of el-
evations of the fertilization envelope as seen under a light
microscope. Rates of fertilization of treated eggs were
compared with those obtained, in the same conditions,
with control gametes to which only the solvent had been
added.
Determination of cleavage rates. Cleavage rates were
determined as described by Biyiti et al. (1990). Increasing
concentrations of 18:5n3 diluted in ethanol were added to
model has been shown to be an accurate and rapid
test for screening teratogenesis as a consequence of
environmental pollution (Congiu et al., 1984;
Pagano and Trie€, 1992; Graillet et al., 1993) as
well as in response to mycotoxins (Morrel and
Adams, 1993) or marine toxins isolated from mar-
ine algae (Pesando et al., 1991, 1996), sponges
(Fusetani et al., 1983; Ohta et al., 1996), star®sh
(Fusetani, 1987) tunicates (Pesando et al., 1995) the egg suspension at various times after fertilization.
Samples were taken at di€erent times during cleavage and
and anemones (Malpezzi and Freitas, 1990).
the percentage of divided cells recorded.
We therefore decided to investigate the e€ects of
18:5n3, the major PUFA in Gymnodinium cells on
Following the same procedure, the toxicity of 18:5n3
was then compared with that of the other standard
Paracentrotus lividus gametes during fertilization,
cleavage and subsequent embryonic development
and to compare them with those obtained with
three other PUFA: 18:4n3, 20:5n3 and 22:6n3.
PUFA: 18:4n3, 20:5n3 and 22:6n3. The IC50 values were
recorded for treated eggs at the time at which 90±95% of
control eggs reached the two cell stage.
Embryotoxicity assay. The toxicity of 18:5n3 was stu-
died on the developmental stages of sea urchin embryos
up to the pluteus stage. The compound was added to egg
suspensions 30 s after fertilization (20 000 eggs/ml),
a
MATERIAL AND METHODS
delay presumed to be sucient for ensuring total fertiliza-
tion (Epel, 1978). Four h after fertilization, control and
treated egg suspensions were diluted 40 times in NSW and
maintained in suspension (500 eggs/ml) by stirring with
propellers (37 rpm) for 98 h after fertilization. The ®rst
stages of cleavage and the embryonic, blastula, gastrula,
prismatic and pluteus stages were observed under a light
microscope in samples ®xed with glutaraldehyde (®nal di-
lution 0.1%) at appropriate intervals after fertilization.
Chemicals
Di€erent concentrations of 18:5n3 and 18:4n3, 20:5n3,
22:6n3, were used for carrying out toxicity tests on the
®rst stage of sea urchin development. 18:5n3 is not com-
mercially available. It has been synthesized according to
the method described by Kuklev et al. (1992) by the g-
iodolactonization of 22:6n3. Its structure (all-cis-
3,6,9,12,15-octadecapentanoic acid) was con®rmed by
GC±MS, FAB±MS, IR and H-NMR and by comparison
with a sample isolated from Gyrodinium aureolum culture
(Parrish et al., 1993). On account of the diculties
encountered in growing large volumes of this species and
the lability of 18:5n3 fatty acid, its synthesis from 22:6n3
appeared to be a bene®cial procedure to obtain this fatty
acid in sucient amount (ca. 100 mg) for biomedical tests.
GC analyses on the corresponding fatty acid methyl esters
(FAME) on polar (Supelcowax-10) and apolar (SE-54)
columns con®rmed a purity above 82%, the major impuri-
ties being 20:5n3 (5.4%), 18:4n3 (2%) and 22:n6 (1%)
fatty acids. The stock solution of 18:4n3, 20:5n3 and
22:6n3 were obtained by dissolving standard fatty acids
from Sigma chemicals in pure ethanol. All solutions were
refrigerated until use.
RESULTS
18:5n3 does not a€ect fertilization
When gametes were treated with 18:5n3 at a con-
centration of 2.5 Â 10 4 M 5 or 10 min prior to
insemination, fertilization was not a€ected: almost
98% of the eggs elevated a normal fertilization
envelope.
Fatty acids inhibit sea urchin egg cleavage
We observed that the presence of 18:5n3 reduced
the rate of progression of fertilized eggs through the
®rst cycle of cleavage (Fig. 1(A)). When added
Biological material
Paracentrotus lividus sea urchins were collected in the 30 sec after fertilization, a delay presumed sucient
bay of Villefranche-sur-Mer (France) during the breeding
season and maintained in captivity in well aerated water
percentage of ®rst cleavage decreased with the con-
at 158C in open circuit. The gonads were excised in sea-
water and the eggs maintained in 0.2 mm ®ltered natural
seawater (NSW) and dejellied by ®ve successive passages
through a 90 mm mesh nylon ®lter. With this treatment,
they could be kept in a well preserved state for 4±5 h.
Sperm were collected dry and kept at 48C. Shortly before
use they were diluted 1:50 in NSW and 10 ml of this sus-
pension were added per ml of an egg suspension adjusted
to permit total fertilization (Biyiti et al., 1990) the
centration of 18:5n3,
a total inhibition being
observed for concentrations above 1 mM.
Inhibition of the ®rst cleavage was also depen-
dent on the time at which 18:5n3 (1 mM) was
added after fertilization. Fig. 1(B) shows that inhi-
bition was total when 18:5n3 was added less than
to 20,000 eggs/ml. In order to obtain a perfect synchroni- 40 min after fertilization. When eggs were exposed
zation of cell division, eggs from a single female were used
in each experiment which was done in triplicate.
to 18:5n3 later in the cell cycle (50 and 60 min after
fertilization), the inhibition declined progressively
until almost 50% of the eggs were capable of div-
ision.
Toxicity test procedure
The highest concentration of ethanol used in the exper-
iments (0.5%) did not a€ect cell division.
Fig. 2 showed that fatty acids with di€erent
chain lengths or unsaturation numbers inhibited egg
cleavage to di€erent extents (Fig. 2(A)). For each
fatty acid 3 dose±response curves corresponding to
3 independent experiments were obtained by consid-
Fertilization. To assay the fertilizing capacity of sperm,
18:5n3 was added to diluted sperm which was added 5 min
later to an egg suspension. The e€ect of 18:5n3 on oocytes
was tested by incubating oocyte suspension with the com-
pound. After 5 or 10 min the gametes were rinsed twice
with seawater and fertilization carried out. In both cases, ering the percentage of cleavage shown in (A)

552
Feriel Sellem et al.
Fig. 1. E€ects of 18:5n3 from the Gymnodinium cf. mikimotoi on the time course of Paracentrotus livi-
dus egg cleavage. (A) Eggs were fertilized at time zero and increasing concentrations of 18:5n3 were
added 30 s after insemination. (B) Eggs were fertilized at time zero and 10 3 M 18:5n3 was added at
di€erent times after sperm contact. Percent inhibition was evaluated at the time at which 90±95% of
the control eggs had reached the two cell stage. One of three experiments giving similar results is
shown.
80 min after the fertilization. According to the bated with 18:5n3 were more or less retarded
model given by the StatSoft Statistica 5.1 (1995) depending on the concentration of the added com-
that can be used to estimate organism's responsive- pound (Fig. 3(E) and (F)). In the control group
ness to a drug, the dose response follows a sigmoid 94% of the embryos were morulae. After treatment
6
function. An example of this dose response is given with 10 or 5 Â 10 6 M 18:5n3, there were 45% or
in Fig. 2(B) for the 18:5n3. The IC50 values which 17% respectively at the morula stage. In this last
are the means of the IC50 values calculated from group, 26% of the embryos exhibited uneven num-
the dose±response curves obtained from the three bers of blastomeres and inequalities of size
experiments performed on each fatty acid, are re- suggesting that they would not continue developing
spectively 0.156 mM20.011 (18:4n3), 0.234 mM (Fig. 3(F)). During the di€erentiation stages, the
20.031 (18:5n3), 0.337 mM20.035 (20:5n3) and delay provoked by 10 6 M 18:5n3 during the prolif-
1,06 mM20;04 (22:6n3) (Fig. 2(C)).
erating stage was progressively recovered, thus the
embryos reached the pluteus stage in a synchronous
manner with the controls (Fig. 3(G) and (H)). We
observed that in presence of 5 Â 10 6 M 18:5n3,
embryos reached partially the pluteus stage, almost
40% of them remaining under the prismatic shape.
5 Â 10 6 M 18:5n3 greatly modify the appearance
of the plutei, the arms were thicker, with intersect-
ing rods and the gut wall was abnormally enlarged
(Fig. 3(I)).
Alteration of embryonic development induced by
18:5n3
The normal sequence of development of sea
urchin embryos is shown in Fig. 3(A), (D) and (G).
After the ®rst cleavage (Fig. 3(A)), within 5 h of fer-
tilization, the majority of embryos are at the morula
stage (Fig. 3(D)). Gastrulation occurs within 24 h
and after 72 h the pluteus stage characterized by an
arrowhead shape with an endoskeletal structure is
attained (Fig. 3(G)).
DISCUSSION
Fatty acid 18:5n3 was added to egg suspensions
6
at concentrations of 10
and 5 Â 10 6 M within
The results reported in this paper indicate that
30 s. from insemination, thus allowing the prolifera- 18:5n3, the major constituent of Gymnodinium tox-
tive phase to occur but with a very slight delay rela- ins (Bodennec et al., 1995) delays cleavage and
tive to the controls (see Fig. 1). At the time of the embryonic development and induces defects in sea
morula stage, 5 h after fertilization, embryos incu- urchin embryos. Our work emphasizes that 18:5n3

Toxic e€ects of fatty acids to P. lividus
553
Fig. 2. E€ects on cleavage of sea urchin eggs (Paracentrotus lividus) of 18:4n3, 18:5n3, 20:5n3 and
22:6n3 fatty acids. (A) Eggs were fertilized at time 0 and compounds added 30 s after sperm addition.
(W) 10 3 M, (*) 5 Â 10 4 M, (R) 10 4 M, (Q) control. (B) Dose±response curves were obtained from
the cleavage percentages in A at 80 min after fertilization and allowed the calculation of the IC50
values, one example is given for 18:5n3. (C) IC50 values of the four fatty acids. Values are the
means2S.E. of three independent experiments.
a€ects the ®rst steps of embryogenesis, with a dose and thus, embryonic abnormalities would ultimately
dependence, especially during the period between be observed.
fertilization and the morula stage. Two ranges of
During sea urchin embryogenesis, the vegetal
concentrations can be considered: those above hemisphere gives rise to ectodermal, endodermal
5 Â 10 4 M which inhibited cleavage and lead to and mesodermal cells (Cameron et al., 1987). In our
cell cycle arrest and those from 1 to 5 Â 10 6 M experiments, development of calci®ed spicules is
which slightly retarded cleavage and ultimately stopped or severely delayed in the presence of
caused embryonic malformations. From the latter it 5 Â 10 6 M 18:5n3 and when the calci®ed skeleton
would appear that developmental defects could be is formed, we observed a gut enlargement that
provoked without much noticeable cleavage delay could be ascribed to an abnormal development of

554
Feriel Sellem et al.
Fig. 3. The e€ects of 18:5n3 on the embryonic stages of proliferation and di€erentiation of
Paracentrotus lividus. Photos on the left represent the two cell stage (A), the morula stage, 5 h after fer-
tilization (D) and the pluteus, 72 h after fertilization (G) in control untreated embryos. Photos B, E
and H show embryos treated with 10 6 M 18:5n at the same stages and on the right, C, F and I are
embryos treated with 5 Â 10 6 M 18:5n3. (A±C, E, F) Â50, (G±I) Â100.
the endodermal territory. These results could and Photobacterium phosphoreum bioluminescence
suggest that 18:5n3 a€ects the endodermal and (Microtox test). According to these authors, 18:5n3
mesodermal territories, resulting in dose-dependent is a harmful compound, except on bioluminescence
malformations.
and surprisingly 18:4n3 is far less toxic than the 20-
A comparison between the toxicity of the syn- carbon chain PUFA (all compounds were studied
thesized 18:5n3 and that of standard fatty acids, within the micromolar range). Such a classi®cation
allow the establishment of a scale of toxicity for the suggest that the level of toxicity of PUFA could be
various PUFA studied here and a classi®cation of the hardly generalized as it varies according to the bio-
di€erent fatty acids, based on their e€ects on sea logical material used.
urchin egg cleavage and the estimation of the half
Using the sea urchin bioassay we observed that
inhibiting doses (IC50) is proposed. The statistical 18:5n3 a€ects rate of cleavage and also the ®rst
analysis of the IC50 values (ANOVA followed by stages of embryonic development when used at
Tukey test) shows that the IC50 value of 20:5n3 is lower concentrations, resulting in some larval
signi®cantly di€erent ( p < 0.01) from that of the abnormalities. A similar e€ect has already been
shorter chain PUFA 18:4n3 which shows the higher reported by Gentien et al. (1991) who found that
toxicity whereas the IC50 value of 18:5n3 is not sig- 90% of Mytilus sp. embryos showed abnormalities
ni®cantly di€erent from those of 18:4n3 and 20:5n3. in a medium containing Gymnodinium cells at a
The IC50 value of 22:6n3 is signi®cantly di€erent density exceeding 2  106 cells/l. According to
( p < 0.01) from those of the other fatty acids and Bodennec et al. (1995) who measured the intracellu-
22:6n3 presents the lower embryotoxicitiy. From lar concentration of 18:5 n3 in Gymnodinium
our results, 18:4n3 is the most toxic compound and species, a fatty acid concentration of 10 5 M might
22:6n3 the least and it can be suggested that the be expected of this red tide density.
toxicities of PUFA are inversely proportional to the
From our results and those of Arzul et al. (1995)
length of the carbon chain and the number of insa- it appears that the toxicity of PUFA on the various
turations. These conclusions can be compared with biological models occurs over the same range of
those of Arzul et al. (1995) who classi®ed PUFA concentration and 18:5n3 is generally one of the
according to their e€ects on the growth of the most toxic followed by 20:5n3 and 22:6n3.
diatom Chaetoceros gracile, hemolysis tests However, when compared with other toxins of mar-

Toxic e€ects of fatty acids to P. lividus
555
Table 1. Toxic doses of various toxins of marine origin reported in the literature dealing with Paracentrotus lividus egg cleavage
Toxins
Origin
mM used
References
Steroidal saponins
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sponge
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coral
6±15
1
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45
2.5
40
Fusetani (1987)
Fusetani et al. (1983)
Jacobs et al. (1981)
Pesando et al. (1996)
Fusetani et al. (1989)
Ettouati and Jacobs (1987)
Polyacetylene alcohol
Quinone and diterpene
Caulerpenyne
Astrogorgiadiol and astrogorgin
Pseudopterosin
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18:5n3 exhibited a lower toxicity. We found that
toxic doses for the ®rst stage of embryonic develop-
ment were around 10 4 M while previous studies
recorded toxic doses varying between 10 6 M and
4 Â 10 5 M depending on the origin of the toxins
(Table 1).
The present study on the sensitivities of eggs and
larvae of the sea urchin to these toxins con®rms the
results of previous work on the toxicities of
Gymnodinium fatty acids on other marine organ-
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marine environment must also play a potential role
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and cell proliferation will allow the assessment of
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and Dr P. Francour and Dr R. Lemee for their helpful in
statistical analyses. The authors wish to thank B. Maetz
for revising the manuscript.
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a gorgonian
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