Toxicity of Third GenerationDispersants and Dispersed EgyptianCrude Oil on Red Sea Coral LarvaeN. EPSTEIN à*, R. P. M. BAKৠand B. RINKEVICH  National Institute of Oceanography, Tel Shikmona, P.O. Box 8030, Haifa 31080, Israel 1

àInstitute 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 e�ects of ®ve third-generation oil dispersants(Inipol IP-90, Petrotech PTI-25, Bioreico R-93, Biosolveand Emulgal C-100) on planula larvae of the Red Seastony coral Stylophora pistillata and the soft coralHeteroxenia fuscescense were evaluated in short-term (2±96 h) bioassays. Larvae were exposed to Egyptian oilwater soluble fractions (WSFs), dispersed oil water ac-commodated fractions (WAFs) and dispersants dissolvedin seawater, in di�erent concentrations. Mortality, set-tlement rates and the appearance of morphological andbehavioural deformations were measured. While oil WSFtreatments resulted in reductions in planulae settlementonly, treatments by all dispersants tested revealed a fur-ther decrease in settlement rates and additional hightoxicity. Dispersed oil exposures resulted in a dramaticincrease in toxicity to both coral larvae species. Further-more, dispersants and WAFs treatments caused larvalmorphology deformations, loss of normal swimming be-haviour and rapid tissue degeneration. Out of the ®vetested dispersion agents, the chemical Petrotech PTI-25displayed the least toxicity to coral larvae. We suggestavoidance of the use of chemical dispersion in cases of oilspills near or within coral reef habitats. Ó 2000 ElsevierScience Ltd. All rights reserved.

Keywords: coral reefs; Eilat; Heteroxenia fuscescense; oildispersants; planula larvae; Stylophora pistillata.

Oil dispersants are mixtures of surfactants and solventswhich e�ectively disseminate oil in the water column,creating small oil droplets (GESAMP, 1993). Treatmentof oil spills with dispersants in temperate marine envi-ronments has become a common practice since manyyears. A major reasoning for using these chemicals is toprevent spilled oil from arriving ashore. However, an

increased toxicity of dispersed oil to marine life ascompared to untreated oil is expected as a result ofsurfactants detrimental e�ects and elevated hydrocar-bon dissolution. Thus, benthic and pelagic organismsmay also be exposed to both oil and dispersant harmfulimpacts (Singer et al., 1996; Wolfe et al., 1999). In thelast few years, earlier generations of oil dispersants werereplaced by newly developed, environmental-friendlythird generation-compounds which are claimed to beless toxic. With regard to tropical organisms such as reefcorals, in contrast to studies on previous generationswhich 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 ofthe newly developed compounds. The utilitarian valueof dispersants in the marine habitat as alternatives tomechanical oil removal methodologies is therefore stillin question (Loya and Rinkevich, 1980).

More than 10 brands of dispersants are o�cially ap-proved for use in Israel. Their application along theIsraeli Mediterranean coast has been certi®ed by theMinistry of the Environment under CEDRE guidelines(Anon, 1999). The possible application of these materi-als in Eilat's coral reef (northern Red Sea), however, hasnot yet been approved, waiting for an additional criticalexamination of their impacts. This stems from the beliefthat tropical near-shore ecosystems are rated lower onrecovery processes than temperate habitats due to theirhigh vulnerability to pollutants (Thorhaug, 1989). Ad-ditionally, with regard to marine pollutants e�ects,standards should be determined on tropical organismsdirectly, and not on their temperate counterparts, whichare probably less sensitive (Thorhaug, 1989).

The northern Gulf of Eilat was subjected to frequentoil spills during the 1970s and the 1980s. The harmfulimpacts of oils and their water soluble fractions (WSFs)on reef corals were then studied on the whole coralcommunity (Loya, 1975, 1976), on the model sclerac-tinian coral Stylophora pistillata (Loya and Rinkevich,1979, 1980; Rinkevich and Loya, 1977, 1983) and on the

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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 theabortion of immature planula larvae by gravid coloniesin response to low WSFs exposure. Using this scienti®cbackground, the purpose of the present study was to testpossible acute e�ects of chemically dispersed Egyptiancrude oil (the major oil type imported to Israel throughEilat) by ®ve approved-to-use dispersants, on planulalarvae of S. pistillata and H. fuscescense. Short-termassays (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 corallarvae.

Materials and Methods

Planulae collectionPlanulae of S. pistillata were collected during two

consecutive reproductive seasons (January±June 1998,1999) in situ in front of the H. Steinitz Marine BiologyLaboratory (MBL) at the northern Gulf of Eilat (RedSea). During the reproductive season, mature coloniesrelease planula larvae daily, about two hours aftersunset (Rinkevich and Loya, 1979). Gravid colonieswere enclosed before dark with plankton nets (45 lm),each armed apically with a plastic ¯ask. Nets with re-leased larvae were collected 4±6 h after sunset bySCUBA diving. Planulae of H. fuscescense are releasedfrom gravid colonies throughout the year (Benayahu,1991). They were collected by overnight ex situ main-tenance of mature, ®eld collected colonies in aeratedaquaria at 24°C. All larvae were transferred to aeratedaquaria at room temperature (24°C) and were used forexperiments following the next 24 h.

Materials and experimental procedureShort-term bioassays (2±96 h) were performed at

controlled room temperature (24°C) and under naturaldark/light regime. Sets of 10 freshly collected planulaewere introduced, each into tissue culture dishes (Greiner,35 ´ 10 mm) containing natural seawater and a testedsolution in a ®nal volume of 5 ml. The tested solutionsincluded oil WSFs, dispersed oil (chemically enhancedwater-accommodated fractions, WAFs, sensu Singer etal., 1998) and dispersants dissolved in natural seawater.Solutions were freshly prepared (in di�erent concentra-tions) and applied immediately. Acute e�ects studiedwere planulae survivorship and settlement. Successfulsettlement has been de®ned as complete metamorphosisto the polyp stage with actively moving tentacles (in S.pistillata settlement is associated with deposition ofcalcium carbonate). Planulae mortality was de®ned as

movement arrest followed by gastrovascular ®lamentsrelease and tissue degeneration. Additionally, larvaewere histologically examined in para�n (Rinkevich andLoya, 1977, 1979) and JB4 embedding media (followingmanufacturer guidelines) for possible alterations on thecellular level.

Egyptian crude oil was supplied with the courtesy ofthe Eilat-Ashkelon Pipe-Line, Israel. The ®ve disper-sants used were: Inipol IP-90 (CECA S.A, France),Petrotech PTI-25 (Petrotech, USA), Biosolve (WestfordChemicals, USA), Bioreico R-93 (Reico, France) andEmulgal C-100 (Amgal Chemicals, Israel). For conve-nience, trademark a�xes 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 mlseawater (1:200 ratio). The stock WAF solution wasbased on 1:10 dispersant: oil ratio (Thorhaug, 1988), bymixing the above oil: water ratio solution with 0.5 ml ofone of the tested dispersants (applied by pipetting). Anovernight shaking procedure was also employed.Shaking was gentle enough to mix the solution thor-oughly without foam production. The dispersant: oilratio employed was su�cient to accommodate most ofthe oil into small droplets. A 3 h standing period wasthen allowed for large oil droplets to resurface. StockWSF and WAF solutions were isolated by a vacuumpipette and designated as 100% solutions. Dispersantsolutions (0.5 ml in 999.5 ml seawater, 500 ppm) werealso designated as 100% stock solutions. Stock solu-tions were prepared and diluted with fresh ®ltered(10 lm) seawater (50%, 10%, 1% and 0.1%) directlyupon application. A total of 2980 S. pistillata planulalarvae were used in 12 sets of assays (four WSFs, fourdispersants, four dispersed oil WAFs and controls, all intriplicates). H. fuscescense tests included 480 planulae inone set of dispersed oil WAFs assay and seawatercontrol (all in triplicates).

Results

S. pistillata planulae: e�ects of Egyptian crude oil WSFsNo mortality has been recorded in seawater control

dishes and all oil WSFs applications along the 96 hperiod of observations. However, while on the average58% of S. pistillata planulae settled in the seawatercontrol dishes, signi®cantly fewer settlements (5±30%,p<0.05, DuncanÕs multiple range test) were recorded in100±0.1% WSF solutions respectively (Fig. 1). More-over, while after 12 h no single settlement in either oneof the WSF solutions was observed, 22% of the planulaein the control dishes already settled at that time (Fig. 1).There were no visible alterations in larvae and settledpolyp morphologies or larvae swimming behaviour.Although settlement rates were reduced signi®cantly,WSFs at the concentrations applied and in the timeframe observed were not lethal to S. pistillata youngstages.

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S. pistillata planulae: e�ects of dispersantsAll ®ve dispersants at the concentrations applied and

over the short exposure periods were toxic to coral lar-vae. Four dispersants (Bioreico, Emulgal, Biosolve andInipol) were highly toxic, while the material Petrotechdisplayed lesser toxicity, expressed in higher survivor-ship ®gures compared to the other four materials. Themost striking di�erences may be seen at the 12 and 96 hpoints of observation respectively (Table 1). While at thePetrotech 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 96h, still 80% and 90% survivorship was recorded at the

Petrotech 100% stock solution and 10% dilution, re-spectively. No planulae survived at the four concurrent10% solutions. All larvae survived the 0.1% and 1.0%treatments of all dispersants, except a single deadplanulae (97% survivorship) at 48 h Biosolve 0.1% di-lution (Table 1). Planulae settlements in all tested dis-persants were signi®cantly fewer than in seawatercontrols (p<0.05, DuncanÕs multiple range test,Table 2). When compared to the oil WSFs treatments,settlement, although in some cases higher (Inipol 0.1%dilution) did not di�er signi®cantly (p>0.05).

Within the 96 h of observations, deformations inplanula morphologies were also observed at all dis-persant solutions, excluding 0.1% solutions. Larvaesubjected to dispersants tended to shrink in the middlesection of the body and lost normal swimming andsubstratum search behaviour within several hours ofexposure. Instead, they exhibited a spin movement ora disoriented circled swimming pattern. Larvae inadvanced stress condition released gastrovascular ®la-ments throughout the posterior end, a phenomenonrarely observed in undisturbed S. pistillata planulae(unpub. data). Deformed planulae never settled andeventually died. All third generation dispersants at allconcentrations, therefore, exhibit detrimental e�ects toS. pistillata planula larvae, in many cases exceedingWSFs impacts.

S. pistillata planulae: e�ects of dispersed oilDispersed 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 (mean�S.D.).

Dispersant tested Dispersant concentrations (%) Survivorship (%) at (h)

12 24 48 72 96

Bioreico 0.1 100 100 100 100 1001 100 100 100 100 10010 100 100 100 90�3.3 0100 0 ± ± ± ±

Petrotech 0.1 100 100 100 100 1001 100 100 100 100 10010 100 100 100 100 90�3.1100 100 90�2.7 90�2.7 80�3.8 80�3.8

Emulgal 0.1 100 100 100 100 1001 100 100 100 100 10010 100 100 100 100 0100 0 ± ± ± ±

Biosolve 0.1 100 100 97�0.6 97�0.6 97�0.61 100 100 100 100 10010 100 90�1.6 55�5.6 26�4.2 0100 0 ± ± ± ±

Inipol 0.1 100 100 100 100 1001 100 100 100 100 10010 100 100 100 100 30�5.5100 0 ± ± ± ±

Fig. 1 Settlement rates of S. pistillata planulae in seawater controldishes and in the Egyptian crude oil WSFs treatments.

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5 compounds, similar to the ranking in the dispersantassays emerged out of the dispersed oil assays. While alllarvae died within 2 h at the 100% solutions of Bioreico,Emulgal, Biosolve and Inipol, complete survivorshipwas recorded at the same Petrotech solution. After 96 halmost all larvae died at the 10% solutions of all thefour materials whereas 47% of the planulae in Petrotech10% solution survived, representing the best survival®gure in the dispersed oil assays (Table 3). No singlesuccessful settlement was recorded within the 96 h ob-servation period in either one of the tested dispersed oilsat neither concentrations. Many specimens exhibitedbehavioural anomalies and major structural deforma-tions at all concentrations. Release of small sphericalbodies (probably lipid droplets) through the outer layerof deformed larvae was observed under inverted lightmicroscopy (Fig. 2a). Up to half of the larvae thatsurvived at the lower concentrations (10% solutions) forseveral days formed half ball shapes and tended to ad-here to the petri-dish bottoms or walls. This was not areal 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 thehalf ball shaped planulae developed 12 pairs of septa

instead of the normal number of 6 (Fig. 2d, Rinkevichand Loya, 1979). In a single case, an attachment of aplanula to the substratum did take place, but a de-formed primary polyp was then developed, with nomouth and tentacles (Fig. 2c). Larvae that survived the96-h period at 10% WAFs dilutions were transferred tofresh seawater for recovery. However, no settlement hasbeen recorded and all larvae eventually disintegratedand died following the next 2±3 days. Histological sec-tions made from few larvae at the 96-h time-point re-vealed a deteriorated state of the ectodermal outer layerin comparison to undamaged, intact layers of controlplanulae (Fig. 2e±f).

H. fuscescense: e�ects of dispersed oilWe assayed the same WAF concentrations of the ®ve

studied dispersants on H. fuscescense planulae, for up to96 h (Table 4) and documented again high toxicity andmajor anomalies. All larvae died in the 100% and the50% solutions of Bioreico, Emulgal and Inipol within 6h. Complete mortality was further recorded in Petrotechand Biosolve 100% and 50% solutions within 48 and 72h respectively. The material Biosolve displayed similare�ects as Petrotech also at the 10% solution, with

TABLE 3

Toxicity of dispersed Egyptian crude oil to S. pistillata planula larvae (mean�S.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�0.650 0 ± ± ± ± ± ±100 0 ± ± ± ± ± ±

Petrotech 10 100 100 100 100 100 83�1.6 47�1.650 100 100 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�0.650 97�1.7 0 ± ± ± ± ±100 0 ± ± ± ± ± ±

Inipol 10 100 100 100 100 83�2.9 27�2.9 7�1.250 13�2.3 0 ± ± ± ± ±100 0 ± ± ± ± ± ±

TABLE 2

Average settlement (%) after 96 h of S. pistillata planulae subjected to di�erent dispersant concentrations (mean�S.D.).

Dispersant tested Settlement (%) at dispersant concentration

0.1% 1.0% 10.0% 100%

Bioreico 16.0�2.9 16.0�3.1 0 0Petrotech 12.5�2.7 10.0�3.2 15.0�5.0 0Emulgal 17.5�4.2 20.0�3.6 0 0Biosolve 25.0�4.4 20.0�3.6 0 0Inipol 40.0�5.1 25.0�3.7 10.0�0 0

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complete larvae survivorship after 96 h. All larvae sur-vived in the control dishes (Table 4).

Approximately half of the surviving planulae in the10% solutions retained their normal elongated shape.Others exhibited a ball-like deformed structure, anddied 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 incontact 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 stagesof two reef corals, exhibiting high toxicity and reducedsettlement rates at low concentrations. The oil WSFtreatments however were less toxic as measured bythe rates of settlement and by the absence of deathor morphological and behavioural alterations. The

Fig. 2 E�ects 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 ectodermallayer is seen (arrow); (b) A control planula larva (40´); (c) Adeformed primary polyp with no mouth and tentacles (Inipol10% treatment, 96 h, 40´); (d) A deformed, unattached planula(Petrotech 10% WAF treatment, 96 h, 40´) with 12 pairs ofsepta instead of the six pairs characteristic to this species; (e)and (f) Histological section of planula larvae (200´) in JB4embedding media, (Petrotech 10% treatment, 96 h, (e), andfrom a control planula (f). Arrows show the outer ectodermallayer which is damaged (e)).

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dispersed oil revealed synergistic detrimental impactsexpressed as the highest mortality ®gures, no settlementsand signi®cant alterations in behaviour and morphologyin larvae of both species. Evidently, the low oil: seawaterratio 1:200, (Rinkevich and Loya, 1977; Loya andRinkevich, 1979) that was not lethal to S. pistillataplanula larvae in the WSF bioassays, became highlytoxic after dispersion.

The primary function of a dispersant is to enhance thesolution of oil in the water column (Singer et al., 1998).The degree to which each dispersant facilitates solutionof 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 otherdetrimental e�ects such as morphological abnormalitiesand reduced settlement rates recorded here. The planu-lae of both coral species studied here revealed similartoxic e�ects when exposed to the dispersed oil. The ®vetypes of dispersed oils may therefore be ranked in ac-cordance to their relative toxicity to coral planulae, fromthe 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 dispersantslead to the dissolution of more hydrocarbons, poten-tially augmenting abortion of planula larvae. Thepractice of third generation dispersants in oil spills in ornear coral reef habitats carries therefore substantialnegative 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, windvelocities and directions and amount and type of spilledoil may all in¯uence e�ectiveness of dispersion, thusmaking the decision a complicated one (GESAMP,1993). Nonetheless, bearing in mind that the prime ob-

jective of oil dispersion is to prevent spilled oil fromarriving ashore, our results do not support the applica-tion of chemical dispersants in the reef, or when seaconditions may drift dispersed oil directly into coralreefs.

This study is part of the research performed in the Minerva centre forMarine Invertebrates Immunology and Developmental Biology andwas 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 participatingcompanies for oil and dispersants donations.

Anon (1999) National contingency plan for preparedness and responseto oil spill at sea. State of Israel, Ministry of the Environment,Marine and Coastal Environment Division.

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TABLE 4

Toxicity of dispersed Egyptian crude oil to H. fuscescens planula larvae (mean�S.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 10050 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 10050 100 100 100 40�10 0 ±100 100 100 100 23�21 0 ±

Inipol 10 100 100 100 37�35 20�17 050 100 0 ± ± ± ±100 100 0 ± ± ± ±

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Rinkevich, B., Loya, Y. (1979) The reproduction of the Red Sea coralStylophora pistillata. II. Synchronization in breeding and season-ality of planulae shedding. Marine Ecology Progress Series 1, 145±152.

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Wyers, S. C., Firth, H. R., Dodge, R. E., Smith, S. R., Knap, A. H.,Sleeter, T. D. (1986) Behavioural e�ects of chemically dispersed oiland subsequent recovery in Diploria strigosa (Dana). MarineBiology 7, 23±42.

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