Toxicity of Third Generation

Dispersants and Dispersed Egyptian

Crude Oil on Red Sea Coral Larvae
N. EPSTEIN*, R. P. M. BAK and B. RINKEVICH
National Institute of Oceanography, Tel Shikmona, P.O. Box 8030, Haifa 31080, Israel
1
Institute of Systematics and Ecology, University of Amsterdam, 1090, Amsterdam, The Netherlands
Netherlands Institute for Sea Research (NIOZ), P.O. Box 59, 1790 AB, Den Burg, The Netherlands
Harmful eects of ve third-generation oil dispersants
(Inipol IP-90, Petrotech PTI-25, Bioreico R-93, Biosolve
and Emulgal C-100) on planula larvae of the Red Sea
stony coral Stylophora pistillata and the soft coral
Heteroxenia fuscescense were evaluated in short-term (2
96 h) bioassays. Larvae were exposed to Egyptian oil
water soluble fractions (WSFs), dispersed oil water ac-
commodated fractions (WAFs) and dispersants dissolved
in seawater, in dierent concentrations. Mortality, set-
tlement rates and the appearance of morphological and
behavioural deformations were measured. While oil WSF
treatments resulted in reductions in planulae settlement
only, treatments by all dispersants tested revealed a fur-
ther decrease in settlement rates and additional high
toxicity. Dispersed oil exposures resulted in a dramatic
increase in toxicity to both coral larvae species. Further-
more, dispersants and WAFs treatments caused larval
morphology deformations, loss of normal swimming be-
haviour and rapid tissue degeneration. Out of the ve
tested dispersion agents, the chemical Petrotech PTI-25
displayed the least toxicity to coral larvae. We suggest
avoidance of the use of chemical dispersion in cases of oil
spills near or within coral reef habitats. 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: coral reefs; Eilat; Heteroxenia fuscescense; oil
dispersants; planula larvae; Stylophora pistillata.
Oil dispersants are mixtures of surfactants and solvents
which eectively disseminate oil in the water column,
creating small oil droplets (GESAMP, 1993). Treatment
of oil spills with dispersants in temperate marine envi-
ronments has become a common practice since many
years. A major reasoning for using these chemicals is to
prevent spilled oil from arriving ashore. However, an
increased toxicity of dispersed oil to marine life as
compared to untreated oil is expected as a result of
surfactants detrimental eects and elevated hydrocar-
bon dissolution. Thus, benthic and pelagic organisms
may also be exposed to both oil and dispersant harmful
impacts (Singer et al., 1996; Wolfe et al., 1999). In the
last few years, earlier generations of oil dispersants were
replaced by newly developed, environmental-friendly
third generation-compounds which are claimed to be
less toxic. With regard to tropical organisms such as reef
corals, in contrast to studies on previous generations
which documented increased toxicity of dispersed oil
(Knap et al., 1983; Dodge et al., 1984; Wyers et al.,
1986), little is known about possible negative impacts of
the newly developed compounds. The utilitarian value
of dispersants in the marine habitat as alternatives to
mechanical oil removal methodologies is therefore still
in question (Loya and Rinkevich, 1980).
More than 10 brands of dispersants are ocially ap-
proved for use in Israel. Their application along the
Israeli Mediterranean coast has been certied by the
Ministry of the Environment under CEDRE guidelines
(Anon, 1999). The possible application of these materi-
als in Eilat's coral reef (northern Red Sea), however, has
not yet been approved, waiting for an additional critical
examination of their impacts. This stems from the belief
that tropical near-shore ecosystems are rated lower on
recovery processes than temperate habitats due to their
high vulnerability to pollutants (Thorhaug, 1989). Ad-
ditionally, with regard to marine pollutants eects,
standards should be determined on tropical organisms
directly, and not on their temperate counterparts, which
are probably less sensitive (Thorhaug, 1989).
The northern Gulf of Eilat was subjected to frequent
oil spills during the 1970s and the 1980s. The harmful
impacts of oils and their water soluble fractions (WSFs)
on reef corals were then studied on the whole coral
community (Loya, 1975, 1976), on the model sclerac-
tinian coral Stylophora pistillata (Loya and Rinkevich,
1979, 1980; Rinkevich and Loya, 1977, 1983) and on the
Marine Pollution Bulletin Vol. 40, No. 6, pp. 497503, 2000
2000 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0025-326X/00 $ - see front matter PII: S0025-326X(99)00232-5
*Corresponding author. Tel.: +972-4-8515202; fax: +972-4-8511911.
E-mail address: nimrod@ocean.org.il (N. Epstein).
1
Correspondence address.
497
alcyonarian Heteroxenia fuscescense (Cohen et al.,
1977). These studies further emphasized that early de-
velopmental stages of corals are particularly vulnerable.
For example, Rinkevich and Loya (1977) recorded de-
creased viability and reduced settlement rates of S. pi-
stillata planulae exposed to increasing WSFs of oil.
Loya and Rinkevich (1979) further demonstrated the
abortion of immature planula larvae by gravid colonies
in response to low WSFs exposure. Using this scientic
background, the purpose of the present study was to test
possible acute eects of chemically dispersed Egyptian
crude oil (the major oil type imported to Israel through
Eilat) by ve approved-to-use dispersants, on planula
larvae of S. pistillata and H. fuscescense. Short-term
assays (up to 96 h) monitored planulae survivorship,
settlement rates, morphological and behavioural ab-
normalities in order to rank the dispersants in accor-
dance to their relative negative impacts on the coral
larvae.
Materials and Methods
Planulae collection
Planulae of S. pistillata were collected during two
consecutive reproductive seasons (JanuaryJune 1998,
1999) in situ in front of the H. Steinitz Marine Biology
Laboratory (MBL) at the northern Gulf of Eilat (Red
Sea). During the reproductive season, mature colonies
release planula larvae daily, about two hours after
sunset (Rinkevich and Loya, 1979). Gravid colonies
were enclosed before dark with plankton nets (45 lm),
each armed apically with a plastic ask. Nets with re-
leased larvae were collected 46 h after sunset by
SCUBA diving. Planulae of H. fuscescense are released
from gravid colonies throughout the year (Benayahu,
1991). They were collected by overnight ex situ main-
tenance of mature, eld collected colonies in aerated
aquaria at 24C. All larvae were transferred to aerated
aquaria at room temperature (24C) and were used for
experiments following the next 24 h.
Materials and experimental procedure
Short-term bioassays (296 h) were performed at
controlled room temperature (24C) and under natural
dark/light regime. Sets of 10 freshly collected planulae
were introduced, each into tissue culture dishes (Greiner,
35 10 mm) containing natural seawater and a tested
solution in a nal volume of 5 ml. The tested solutions
included oil WSFs, dispersed oil (chemically enhanced
water-accommodated fractions, WAFs, sensu Singer et
al., 1998) and dispersants dissolved in natural seawater.
Solutions were freshly prepared (in dierent concentra-
tions) and applied immediately. Acute eects studied
were planulae survivorship and settlement. Successful
settlement has been dened as complete metamorphosis
to the polyp stage with actively moving tentacles (in S.
pistillata settlement is associated with deposition of
calcium carbonate). Planulae mortality was dened as
movement arrest followed by gastrovascular laments
release and tissue degeneration. Additionally, larvae
were histologically examined in paran (Rinkevich and
Loya, 1977, 1979) and JB4 embedding media (following
manufacturer guidelines) for possible alterations on the
cellular level.
Egyptian crude oil was supplied with the courtesy of
the Eilat-Ashkelon Pipe-Line, Israel. The ve disper-
sants used were: Inipol IP-90 (CECA S.A, France),
Petrotech PTI-25 (Petrotech, USA), Biosolve (Westford
Chemicals, USA), Bioreico R-93 (Reico, France) and
Emulgal C-100 (Amgal Chemicals, Israel). For conve-
nience, trademark axes will be omitted in the follow-
ing text. The stock oil WSF solution was prepared
(Rinkevich and Loya, 1977; Loya and Rinkevich, 1979)
by overnight shaking (12 h, 80 rpm) of 5 ml oil in 995 ml
seawater (1:200 ratio). The stock WAF solution was
based on 1:10 dispersant: oil ratio (Thorhaug, 1988), by
mixing the above oil: water ratio solution with 0.5 ml of
one of the tested dispersants (applied by pipetting). An
overnight shaking procedure was also employed.
Shaking was gentle enough to mix the solution thor-
oughly without foam production. The dispersant: oil
ratio employed was sucient to accommodate most of
the oil into small droplets. A 3 h standing period was
then allowed for large oil droplets to resurface. Stock
WSF and WAF solutions were isolated by a vacuum
pipette and designated as 100% solutions. Dispersant
solutions (0.5 ml in 999.5 ml seawater, 500 ppm) were
also designated as 100% stock solutions. Stock solu-
tions were prepared and diluted with fresh ltered
(10 lm) seawater (50%, 10%, 1% and 0.1%) directly
upon application. A total of 2980 S. pistillata planula
larvae were used in 12 sets of assays (four WSFs, four
dispersants, four dispersed oil WAFs and controls, all in
triplicates). H. fuscescense tests included 480 planulae in
one set of dispersed oil WAFs assay and seawater
control (all in triplicates).
Results
S. pistillata planulae: eects of Egyptian crude oil WSFs
No mortality has been recorded in seawater control
dishes and all oil WSFs applications along the 96 h
period of observations. However, while on the average
58% of S. pistillata planulae settled in the seawater
control dishes, signicantly fewer settlements (530%,
p <0.05, Duncans multiple range test) were recorded in
1000.1% WSF solutions respectively (Fig. 1). More-
over, while after 12 h no single settlement in either one
of the WSF solutions was observed, 22% of the planulae
in the control dishes already settled at that time (Fig. 1).
There were no visible alterations in larvae and settled
polyp morphologies or larvae swimming behaviour.
Although settlement rates were reduced signicantly,
WSFs at the concentrations applied and in the time
frame observed were not lethal to S. pistillata young
stages.
498
Marine Pollution Bulletin
S. pistillata planulae: eects of dispersants
All ve dispersants at the concentrations applied and
over the short exposure periods were toxic to coral lar-
vae. Four dispersants (Bioreico, Emulgal, Biosolve and
Inipol) were highly toxic, while the material Petrotech
displayed lesser toxicity, expressed in higher survivor-
ship gures compared to the other four materials. The
most striking dierences may be seen at the 12 and 96 h
points of observation respectively (Table 1). While at the
Petrotech 100% stock solution treatment full survivor-
ship was recorded after 12 h, complete mortality ap-
peared at that point at all other stock solutions. After 96
h, still 80% and 90% survivorship was recorded at the
Petrotech 100% stock solution and 10% dilution, re-
spectively. No planulae survived at the four concurrent
10% solutions. All larvae survived the 0.1% and 1.0%
treatments of all dispersants, except a single dead
planulae (97% survivorship) at 48 h Biosolve 0.1% di-
lution (Table 1). Planulae settlements in all tested dis-
persants were signicantly fewer than in seawater
controls (p <0.05, Duncans multiple range test,
Table 2). When compared to the oil WSFs treatments,
settlement, although in some cases higher (Inipol 0.1%
dilution) did not dier signicantly (p >0.05).
Within the 96 h of observations, deformations in
planula morphologies were also observed at all dis-
persant solutions, excluding 0.1% solutions. Larvae
subjected to dispersants tended to shrink in the middle
section of the body and lost normal swimming and
substratum search behaviour within several hours of
exposure. Instead, they exhibited a spin movement or
a disoriented circled swimming pattern. Larvae in
advanced stress condition released gastrovascular la-
ments throughout the posterior end, a phenomenon
rarely observed in undisturbed S. pistillata planulae
(unpub. data). Deformed planulae never settled and
eventually died. All third generation dispersants at all
concentrations, therefore, exhibit detrimental eects to
S. pistillata planula larvae, in many cases exceeding
WSFs impacts.
S. pistillata planulae: eects of dispersed oil
Dispersed oil was highly toxic to S. pistillata larvae
(Table 3) and the marked increase in toxicity as com-
pared to dispersant treatments may be seen by com-
paring Tables 1 and 3. Further, a toxicity ranking of the
TABLE 1
Toxicity of ve dispersants tested on S. pistillata planula larvae (meanS.D.).
Dispersant tested Dispersant concentrations (%) Survivorship (%) at (h)
12 24 48 72 96
Bioreico 0.1 100 100 100 100 100
1 100 100 100 100 100
10 100 100 100 90 3.3 0
100 0
Petrotech 0.1 100 100 100 100 100
1 100 100 100 100 100
10 100 100 100 100 90 3.1
100 100 90 2.7 90 2.7 80 3.8 80 3.8
Emulgal 0.1 100 100 100 100 100
1 100 100 100 100 100
10 100 100 100 100 0
100 0
Biosolve 0.1 100 100 97 0.6 97 0.6 97 0.6
1 100 100 100 100 100
10 100 90 1.6 55 5.6 26 4.2 0
100 0
Inipol 0.1 100 100 100 100 100
1 100 100 100 100 100
10 100 100 100 100 30 5.5
100 0
Fig. 1 Settlement rates of S. pistillata planulae in seawater control
dishes and in the Egyptian crude oil WSFs treatments.
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Volume 40/Number 6/June 2000
5 compounds, similar to the ranking in the dispersant
assays emerged out of the dispersed oil assays. While all
larvae died within 2 h at the 100% solutions of Bioreico,
Emulgal, Biosolve and Inipol, complete survivorship
was recorded at the same Petrotech solution. After 96 h
almost all larvae died at the 10% solutions of all the
four materials whereas 47% of the planulae in Petrotech
10% solution survived, representing the best survival
gure in the dispersed oil assays (Table 3). No single
successful settlement was recorded within the 96 h ob-
servation period in either one of the tested dispersed oils
at neither concentrations. Many specimens exhibited
behavioural anomalies and major structural deforma-
tions at all concentrations. Release of small spherical
bodies (probably lipid droplets) through the outer layer
of deformed larvae was observed under inverted light
microscopy (Fig. 2a). Up to half of the larvae that
survived at the lower concentrations (10% solutions) for
several days formed half ball shapes and tended to ad-
here to the petri-dish bottoms or walls. This was not a
real settlement process since a continuous spin move-
ment of the shapes was documented. Specimen contin-
ued to spin or swim until death commenced. Some of the
half ball shaped planulae developed 12 pairs of septa
instead of the normal number of 6 (Fig. 2d, Rinkevich
and Loya, 1979). In a single case, an attachment of a
planula to the substratum did take place, but a de-
formed primary polyp was then developed, with no
mouth and tentacles (Fig. 2c). Larvae that survived the
96-h period at 10% WAFs dilutions were transferred to
fresh seawater for recovery. However, no settlement has
been recorded and all larvae eventually disintegrated
and died following the next 23 days. Histological sec-
tions made from few larvae at the 96-h time-point re-
vealed a deteriorated state of the ectodermal outer layer
in comparison to undamaged, intact layers of control
planulae (Fig. 2ef).
H. fuscescense: eects of dispersed oil
We assayed the same WAF concentrations of the ve
studied dispersants on H. fuscescense planulae, for up to
96 h (Table 4) and documented again high toxicity and
major anomalies. All larvae died in the 100% and the
50% solutions of Bioreico, Emulgal and Inipol within 6
h. Complete mortality was further recorded in Petrotech
and Biosolve 100% and 50% solutions within 48 and 72
h respectively. The material Biosolve displayed similar
eects as Petrotech also at the 10% solution, with
TABLE 3
Toxicity of dispersed Egyptian crude oil to S. pistillata planula larvae (meanS.D.)
Dispersant tested WAFs concentrations (%) Survivorship (%) at (h)
2 6 12 24 48 72 96
Bioreico 10 100 100 100 100 40 1.0 17 1.2 3 0.6
50 0
100 0
Petrotech 10 100 100 100 100 100 83 1.6 47 1.6
50 100 100 83 2.0 30 3.0 0
100 100 0
Emulgal 10 100 100 50 1.0 3 0.6 0
50 30 1.0 0
100 0
Biosolve 10 100 100 100 100 40 1.0 17 1.2 3 0.6
50 97 1.7 0
100 0
Inipol 10 100 100 100 100 83 2.9 27 2.9 7 1.2
50 13 2.3 0
100 0
TABLE 2
Average settlement (%) after 96 h of S. pistillata planulae subjected to dierent dispersant concentrations (meanS.D.).
Dispersant tested Settlement (%) at dispersant concentration
0.1% 1.0% 10.0% 100%
Bioreico 16.0 2.9 16.0 3.1 0 0
Petrotech 12.5 2.7 10.0 3.2 15.0 5.0 0
Emulgal 17.5 4.2 20.0 3.6 0 0
Biosolve 25.0 4.4 20.0 3.6 0 0
Inipol 40.0 5.1 25.0 3.7 10.0 0 0
500
Marine Pollution Bulletin
complete larvae survivorship after 96 h. All larvae sur-
vived in the control dishes (Table 4).
Approximately half of the surviving planulae in the
10% solutions retained their normal elongated shape.
Others exhibited a ball-like deformed structure, and
died the following day. Many of the elongated plan-
ulae assumed a vertical position within the dishes,
anterior end up and a slightly swollen posterior end in
contact with the bottom. No settlement was however,
observed.
Discussion
The results documented an increased toxicity of dis-
persed oil as compared to untreated oil. The third gen-
eration dispersants tested are harmful to early life stages
of two reef corals, exhibiting high toxicity and reduced
settlement rates at low concentrations. The oil WSF
treatments however were less toxic as measured by
the rates of settlement and by the absence of death
or morphological and behavioural alterations. The
Fig. 2 Eects of dispersed oil on S. pistillata planula larvae. (a) Dis-
integration of a planula (Emulgal 100% WAF treatment, 2 h,
40). Release of small spherical bodies through the ectodermal
layer is seen (arrow); (b) A control planula larva (40); (c) A
deformed primary polyp with no mouth and tentacles (Inipol
10% treatment, 96 h, 40); (d) A deformed, unattached planula
(Petrotech 10% WAF treatment, 96 h, 40) with 12 pairs of
septa instead of the six pairs characteristic to this species; (e)
and (f) Histological section of planula larvae (200) in JB4
embedding media, (Petrotech 10% treatment, 96 h, (e), and
from a control planula (f). Arrows show the outer ectodermal
layer which is damaged (e)).
501
Volume 40/Number 6/June 2000
dispersed oil revealed synergistic detrimental impacts
expressed as the highest mortality gures, no settlements
and signicant alterations in behaviour and morphology
in larvae of both species. Evidently, the low oil: seawater
ratio 1:200, (Rinkevich and Loya, 1977; Loya and
Rinkevich, 1979) that was not lethal to S. pistillata
planula larvae in the WSF bioassays, became highly
toxic after dispersion.
The primary function of a dispersant is to enhance the
solution of oil in the water column (Singer et al., 1998).
The degree to which each dispersant facilitates solution
of petroleum hydrocarbons into the water and the rel-
ative toxicity of the dispersant (as well as the oil), con-
tribute to the resultant level of toxicity and to other
detrimental eects such as morphological abnormalities
and reduced settlement rates recorded here. The planu-
lae of both coral species studied here revealed similar
toxic eects when exposed to the dispersed oil. The ve
types of dispersed oils may therefore be ranked in ac-
cordance to their relative toxicity to coral planulae, from
the least toxic compound, as follows: Petrotech <Bio-
solve <Emulgal <Bioreico Inipol.
Planulae abortion by adult colonies is a direct re-
sponse to contamination by petroleum hydrocarbons
(Loya and Rinkevich, 1979). Applications of dispersants
lead to the dissolution of more hydrocarbons, poten-
tially augmenting abortion of planula larvae. The
practice of third generation dispersants in oil spills in or
near coral reef habitats carries therefore substantial
negative impacts to coral planulae.
A variety of factors must be weighed when consider-
ing the use of chemical dispersants during an oil spill.
With regard to coral reefs, distance from the reef, wind
velocities and directions and amount and type of spilled
oil may all inuence eectiveness of dispersion, thus
making the decision a complicated one (GESAMP,
1993). Nonetheless, bearing in mind that the prime ob-
jective of oil dispersion is to prevent spilled oil from
arriving ashore, our results do not support the applica-
tion of chemical dispersants in the reef, or when sea
conditions may drift dispersed oil directly into coral
reefs.
This study is part of the research performed in the Minerva centre for
Marine Invertebrates Immunology and Developmental Biology and
was also supported by a grant from the Israeli Ministry of the Envi-
ronment. Thanks are due to the MBL (Eilat) personal for their hos-
pitality, to Ms. Yael Mann for image processing and to participating
companies for oil and dispersants donations.
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TABLE 4
Toxicity of dispersed Egyptian crude oil to H. fuscescens planula larvae (meanS.D.)
Dispersant tested WAFs concentrations (%) Survivorship (%) at (h)
2 6 24 48 72 96
Bioreico 10 100 90 5.7 87 23 44 32 0
50 100 0
100 100 0
Petrotech 10 100 100 100 100 100 100
50 100 100 100 0
100 100 100 7 11.5 0
Emulgal 10 100 100 60 20 0
50 100 0
100 100 0
Biosolve 10 100 100 100 100 100 100
50 100 100 100 40 10 0
100 100 100 100 23 21 0
Inipol 10 100 100 100 37 35 20 17 0
50 100 0
100 100 0
502
Marine Pollution Bulletin
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