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Toxicity of Third Generation Dispersants and Dispersed Egyptian Crude Oil on Red Sea Coral Larvae
PII: S0025-326X(99)00232-5
Marine Pollution Bulletin Vol. 40, No. 6, pp. 497±503, 2000 Ó 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0025-326X/00 $ - see front matter
Toxicity of Third Generation Dispersants and Dispersed Egyptian Crude Oil on Red Sea Coral Larvae N. 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 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 accommodated fractions (WAFs) and dispersants dissolved in seawater, in dierent concentrations. Mortality, settlement 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 further decrease in settlement rates and additional high toxicity. Dispersed oil exposures resulted in a dramatic increase in toxicity to both coral larvae species. Furthermore, dispersants and WAFs treatments caused larval morphology deformations, loss of normal swimming behaviour 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. Ó 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 environments 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 *Corresponding author. Tel.: +972-4-8515202; fax: +972-4-8511911. E-mail address: [email protected] (N. Epstein). 1 Correspondence address.
increased toxicity of dispersed oil to marine life as compared to untreated oil is expected as a result of surfactants detrimental eects and elevated hydrocarbon 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 approved for use in Israel. Their application along the Israeli Mediterranean coast has been certi®ed by the Ministry of the Environment under CEDRE guidelines (Anon, 1999). The possible application of these materials 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). Additionally, 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 scleractinian coral Stylophora pistillata (Loya and Rinkevich, 1979, 1980; Rinkevich and Loya, 1977, 1983) and on the 497
Marine Pollution Bulletin
alcyonarian Heteroxenia fuscescense (Cohen et al., 1977). These studies further emphasized that early developmental stages of corals are particularly vulnerable. For example, Rinkevich and Loya (1977) recorded decreased viability and reduced settlement rates of S. pistillata 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 scienti®c 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 abnormalities in order to rank the dispersants in accordance 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 (January±June 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 released larvae were collected 4±6 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 maintenance of mature, ®eld collected colonies in aerated aquaria at 24°C. All larvae were transferred to aerated aquaria at room temperature (24°C) and were used for experiments following the next 24 h. Materials and experimental procedure Short-term bioassays (2±96 h) were performed at controlled room temperature (24°C) 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 concentrations) and applied immediately. Acute eects studied were planulae survivorship and settlement. Successful settlement has been de®ned 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 de®ned as 498
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 dispersants 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 convenience, trademark axes will be omitted in the following 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 thoroughly 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 solutions 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, signi®cantly fewer settlements (5±30%, p < 0.05, DuncanÕs multiple range test) were recorded in 100±0.1% WSF solutions respectively (Fig. 1). Moreover, 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 signi®cantly, WSFs at the concentrations applied and in the time frame observed were not lethal to S. pistillata young stages.
Volume 40/Number 6/June 2000
Fig. 1 Settlement rates of S. pistillata planulae in seawater control dishes and in the Egyptian crude oil WSFs treatments.
S. pistillata planulae: eects of dispersants All ®ve dispersants at the concentrations applied and over the short exposure periods were toxic to coral larvae. Four dispersants (Bioreico, Emulgal, Biosolve and Inipol) were highly toxic, while the material Petrotech displayed lesser toxicity, expressed in higher survivorship ®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 survivorship was recorded after 12 h, complete mortality appeared 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, respectively. 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% dilution (Table 1). Planulae settlements in all tested dispersants were signi®cantly fewer than in seawater controls (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 dier signi®cantly (p > 0.05). Within the 96 h of observations, deformations in planula morphologies were also observed at all dispersant 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 ®laments 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 compared to dispersant treatments may be seen by comparing 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 1 10 100
100 100 100 0
100 100 100 ±
100 100 100 ±
100 100 90 3.3 ±
100 100 0 ±
Petrotech
0.1 1 10 100
100 100 100 100
100 100 100 90 2.7
100 100 100 90 2.7
100 100 100 80 3.8
100 100 90 3.1 80 3.8
Emulgal
0.1 1 10 100
100 100 100 0
100 100 100 ±
100 100 100 ±
100 100 100 ±
100 100 0 ±
Biosolve
0.1 1 10 100
100 100 100 0
100 100 90 1.6 ±
97 0.6 100 55 5.6 ±
97 0.6 100 26 4.2 ±
97 0.6 100 0 ±
Inipol
0.1 1 10 100
100 100 100 0
100 100 100 ±
100 100 100 ±
100 100 100 ±
100 100 30 5.5 ±
499
Marine Pollution Bulletin TABLE 2 Average settlement (%) after 96 h of S. pistillata planulae subjected to dierent dispersant concentrations (mean S.D.). Settlement (%) at dispersant concentration
Dispersant tested
Bioreico Petrotech Emulgal Biosolve Inipol
0.1%
1.0%
10.0%
100%
16.0 2.9 12.5 2.7 17.5 4.2 25.0 4.4 40.0 5.1
16.0 3.1 10.0 3.2 20.0 3.6 20.0 3.6 25.0 3.7
0 15.0 5.0 0 0 10.0 0
0 0 0 0 0
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 50 100
100 0 0
100 ± ±
100 ± ±
100 ± ±
40 1.0 ± ±
17 1.2 ± ±
3 0.6 ± ±
Petrotech
10 50 100
100 100 100
100 100 0
100 83 2.0 ±
100 30 3.0 ±
100 0 ±
83 1.6 ± ±
47 1.6 ± ±
Emulgal
10 50 100
100 30 1.0 0
100 0 ±
50 1.0 ± ±
3 0.6 ± ±
0 ± ±
± ± ±
± ± ±
Biosolve
10 50 100
100 97 1.7 0
100 0 ±
100 ± ±
100 ± ±
40 1.0 ± ±
17 1.2 ± ±
3 0.6 ± ±
Inipol
10 50 100
100 13 2.3 0
100 0 ±
100 ± ±
100 ± ±
83 2.9 ± ±
27 2.9 ± ±
7 1.2 ± ±
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 observation period in either one of the tested dispersed oils at neither concentrations. Many specimens exhibited behavioural anomalies and major structural deformations 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 adhere to the petri-dish bottoms or walls. This was not a real settlement process since a continuous spin movement of the shapes was documented. Specimen continued to spin or swim until death commenced. Some of the half ball shaped planulae developed 12 pairs of septa 500
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 deformed 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 2±3 days. Histological sections made from few larvae at the 96-h time-point revealed a deteriorated state of the ectodermal outer layer in comparison to undamaged, intact layers of control planulae (Fig. 2e±f). 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
Volume 40/Number 6/June 2000
Fig. 2 Eects of dispersed oil on S. pistillata planula larvae. (a) Disintegration 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)).
complete larvae survivorship after 96 h. All larvae survived 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 planulae 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 dispersed oil as compared to untreated oil. The third generation 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 501
Marine Pollution Bulletin 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 50 100
100 100 100
90 5.7 0 0
87 23 ± ±
44 32 ± ±
0 ± ±
± ± ±
Petrotech
10 50 100
100 100 100
100 100 100
100 100 7 11.5
100 0 0
100 ± ±
100 ± ±
Emulgal
10 50 100
100 100 100
100 0 0
60 20 ± ±
0 ± ±
± ± ±
± ± ±
Biosolve
10 50 100
100 100 100
100 100 100
100 100 100
100 40 10 23 21
100 0 0
100 ± ±
Inipol
10 50 100
100 100 100
100 0 0
100 ± ±
37 35 ± ±
20 17 ± ±
0 ± ±
dispersed oil revealed synergistic detrimental impacts expressed as the highest mortality ®gures, no settlements and signi®cant 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 relative toxicity of the dispersant (as well as the oil), contribute to the resultant level of toxicity and to other detrimental eects such as morphological abnormalities and reduced settlement rates recorded here. The planulae 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 accordance to their relative toxicity to coral planulae, from the least toxic compound, as follows: Petrotech < Biosolve < Emulgal < Bioreico Inipol. Planulae abortion by adult colonies is a direct response to contamination by petroleum hydrocarbons (Loya and Rinkevich, 1979). Applications of dispersants lead to the dissolution of more hydrocarbons, potentially 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 considering 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 in¯uence eectiveness of dispersion, thus making the decision a complicated one (GESAMP, 1993). Nonetheless, bearing in mind that the prime ob502
jective of oil dispersion is to prevent spilled oil from arriving ashore, our results do not support the application 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 Environment. Thanks are due to the MBL (Eilat) personal for their hospitality, to Ms. Yael Mann for image processing and to participating companies for oil and dispersants donations. Anon (1999) National contingency plan for preparedness and response to oil spill at sea. State of Israel, Ministry of the Environment, Marine and Coastal Environment Division. Benayahu, Y. (1991) Reproduction and developmental pathways of Red Sea Xeniidae (Octocorallia, Alcyonacea). Hydrobiologia 216/ 217, 125±130. Cohen, Y., Nissenbaum, A., Eisler, R. (1977) Eects of Iranian crude oil on the Red Sea octocoral Heteroxenis fuscescense. Environmental Pollution 12,173±186. Dodge, R. E., Wyers, S. C., Firth, H. R., Knap, A. H., Smith, S. R., Sleeter, T. D. (1984) The eects of oil and oil dispersants on the skeletal growth of the hermatypic coral Diploria strigosa. Coral Reefs 3, 191±198. GESAMP (IMO/FAO/UNESCO/WMO/IAEA/UN/UNEP) Joint Group of Experts on the Scienti®c Aspects of Marine Pollution (1993) Impacts of oil and related chemicals and wastes on the marine environment. GESAMP 50, p. 180. Knap, A. H., Sleeter, T. D., Dodge, R. E., Wyers, S. C., Frith, H. R., Smith, S. R. (1983) The eects of oil spills and dispersants use on corals. A review and multidisciplinary experimental approach. Oil and Petrochemical Pollution 1, 157±169. Loya, Y. (1975) Possible eects of water pollution on the community structure of Red Sea corals. Marine Biology 29, 177±185. Loya, Y. (1976) Recolonization of the Red Sea corals aected by natural catastrophes and man-made perturbations. Ecology 57, 278±289. Loya, Y., Rinkevich, B. (1979) Abortion eects in corals induced by oil pollution. Marine Ecology Progress Series 1, 77±80. Loya, Y., Rinkevich, B. (1980) Eects of oil pollution on coral reef communities. Marine Ecology Progress Series 3, 167±180. Rinkevich, B., Loya, Y. (1977) Harmful eects of chronic oil pollution on a Red Sea scleractinian coral population. In Proceedings of the Third International Coral Reef Symposium, vol. 3 (Geology), pp. 586±591. Miami, Florida.
Volume 40/Number 6/June 2000 Rinkevich, B., Loya, Y. (1979) The reproduction of the Red Sea coral Stylophora pistillata. II. Synchronization in breeding and seasonality of planulae shedding. Marine Ecology Progress Series 1, 145± 152. Rinkevich, B., Loya, Y. (1983) Response of zooxanthellae photosynthesis to low concentrations of petroleum hydrocarbons. Bulletin of the Institute of Oceanography and Fisheries 9, 109±115. Singer, M. M., Goerge, S., Jacobson, S., Lee, I., Weetman, L. L., Tjeerdema, R. S., Sowby, M. L. (1996) Comparison of acute aquatic eects of the oil dispersant Corexit 9500 with those of other Corexit series dispersants. Ecotoxicology and Environmental safety 35, 183±189. Singer, M. M., Goerge, S., Lee, I., Jacobson, S., Weetman, L. L., Blondina, G., Tjeerdema, R. S., Aurand, D., Sowby, M. L. (1998) Eects of dispersant treatment on the acute toxicity of petroleum hydrocarbons. Archives of Environmental Contamination and Toxicology 34, 177±187.
Thorhaug, A. (1988) Dispersed oil eects on mangroves, seagrasses, and corals of the wider Caribbean. In Proceedings of the Sixth International Coral Reef Symposium, vol. 2, pp. 337±339. Australia. Thorhaug, A. (1989) Dispersed oil eects on tropical near shore ecosystems. In Oil Dispersants: New Ecological Approaches, ed. L. M. Flaherty, pp. 257±273. American Society for Testing and Materials, Philadelphia. Wyers, S. C., Firth, H. R., Dodge, R. E., Smith, S. R., Knap, A. H., Sleeter, T. D. (1986) Behavioural eects of chemically dispersed oil and subsequent recovery in Diploria strigosa (Dana). Marine Biology 7, 23±42. Wolfe, M. F., Olsen, H. E., Gausad, K. A., Tjeerdema, R. S., Sowby, M. L. (1999) Induction of heat shock protein (hsp) 60 in Isochrysis galbana exposed to sublethal preparations of dispersant and Prudhoe Bay crude oil. Marine Environmental Research 47, 473± 489.
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Marine Pollution Bulletin Vol. 40, No. 6, pp. 497±503, 2000 Ó 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0025-326X/00 $ - see front matter
Toxicity of Third Generation Dispersants and Dispersed Egyptian Crude Oil on Red Sea Coral Larvae N. 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 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 accommodated fractions (WAFs) and dispersants dissolved in seawater, in dierent concentrations. Mortality, settlement 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 further decrease in settlement rates and additional high toxicity. Dispersed oil exposures resulted in a dramatic increase in toxicity to both coral larvae species. Furthermore, dispersants and WAFs treatments caused larval morphology deformations, loss of normal swimming behaviour 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. Ó 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 environments 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 *Corresponding author. Tel.: +972-4-8515202; fax: +972-4-8511911. E-mail address: [email protected] (N. Epstein). 1 Correspondence address.
increased toxicity of dispersed oil to marine life as compared to untreated oil is expected as a result of surfactants detrimental eects and elevated hydrocarbon 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 approved for use in Israel. Their application along the Israeli Mediterranean coast has been certi®ed by the Ministry of the Environment under CEDRE guidelines (Anon, 1999). The possible application of these materials 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). Additionally, 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 scleractinian coral Stylophora pistillata (Loya and Rinkevich, 1979, 1980; Rinkevich and Loya, 1977, 1983) and on the 497
Marine Pollution Bulletin
alcyonarian Heteroxenia fuscescense (Cohen et al., 1977). These studies further emphasized that early developmental stages of corals are particularly vulnerable. For example, Rinkevich and Loya (1977) recorded decreased viability and reduced settlement rates of S. pistillata 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 scienti®c 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 abnormalities in order to rank the dispersants in accordance 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 (January±June 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 released larvae were collected 4±6 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 maintenance of mature, ®eld collected colonies in aerated aquaria at 24°C. All larvae were transferred to aerated aquaria at room temperature (24°C) and were used for experiments following the next 24 h. Materials and experimental procedure Short-term bioassays (2±96 h) were performed at controlled room temperature (24°C) 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 concentrations) and applied immediately. Acute eects studied were planulae survivorship and settlement. Successful settlement has been de®ned 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 de®ned as 498
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 dispersants 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 convenience, trademark axes will be omitted in the following 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 thoroughly 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 solutions 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, signi®cantly fewer settlements (5±30%, p < 0.05, DuncanÕs multiple range test) were recorded in 100±0.1% WSF solutions respectively (Fig. 1). Moreover, 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 signi®cantly, WSFs at the concentrations applied and in the time frame observed were not lethal to S. pistillata young stages.
Volume 40/Number 6/June 2000
Fig. 1 Settlement rates of S. pistillata planulae in seawater control dishes and in the Egyptian crude oil WSFs treatments.
S. pistillata planulae: eects of dispersants All ®ve dispersants at the concentrations applied and over the short exposure periods were toxic to coral larvae. Four dispersants (Bioreico, Emulgal, Biosolve and Inipol) were highly toxic, while the material Petrotech displayed lesser toxicity, expressed in higher survivorship ®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 survivorship was recorded after 12 h, complete mortality appeared 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, respectively. 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% dilution (Table 1). Planulae settlements in all tested dispersants were signi®cantly fewer than in seawater controls (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 dier signi®cantly (p > 0.05). Within the 96 h of observations, deformations in planula morphologies were also observed at all dispersant 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 ®laments 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 compared to dispersant treatments may be seen by comparing 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 1 10 100
100 100 100 0
100 100 100 ±
100 100 100 ±
100 100 90 3.3 ±
100 100 0 ±
Petrotech
0.1 1 10 100
100 100 100 100
100 100 100 90 2.7
100 100 100 90 2.7
100 100 100 80 3.8
100 100 90 3.1 80 3.8
Emulgal
0.1 1 10 100
100 100 100 0
100 100 100 ±
100 100 100 ±
100 100 100 ±
100 100 0 ±
Biosolve
0.1 1 10 100
100 100 100 0
100 100 90 1.6 ±
97 0.6 100 55 5.6 ±
97 0.6 100 26 4.2 ±
97 0.6 100 0 ±
Inipol
0.1 1 10 100
100 100 100 0
100 100 100 ±
100 100 100 ±
100 100 100 ±
100 100 30 5.5 ±
499
Marine Pollution Bulletin TABLE 2 Average settlement (%) after 96 h of S. pistillata planulae subjected to dierent dispersant concentrations (mean S.D.). Settlement (%) at dispersant concentration
Dispersant tested
Bioreico Petrotech Emulgal Biosolve Inipol
0.1%
1.0%
10.0%
100%
16.0 2.9 12.5 2.7 17.5 4.2 25.0 4.4 40.0 5.1
16.0 3.1 10.0 3.2 20.0 3.6 20.0 3.6 25.0 3.7
0 15.0 5.0 0 0 10.0 0
0 0 0 0 0
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 50 100
100 0 0
100 ± ±
100 ± ±
100 ± ±
40 1.0 ± ±
17 1.2 ± ±
3 0.6 ± ±
Petrotech
10 50 100
100 100 100
100 100 0
100 83 2.0 ±
100 30 3.0 ±
100 0 ±
83 1.6 ± ±
47 1.6 ± ±
Emulgal
10 50 100
100 30 1.0 0
100 0 ±
50 1.0 ± ±
3 0.6 ± ±
0 ± ±
± ± ±
± ± ±
Biosolve
10 50 100
100 97 1.7 0
100 0 ±
100 ± ±
100 ± ±
40 1.0 ± ±
17 1.2 ± ±
3 0.6 ± ±
Inipol
10 50 100
100 13 2.3 0
100 0 ±
100 ± ±
100 ± ±
83 2.9 ± ±
27 2.9 ± ±
7 1.2 ± ±
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 observation period in either one of the tested dispersed oils at neither concentrations. Many specimens exhibited behavioural anomalies and major structural deformations 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 adhere to the petri-dish bottoms or walls. This was not a real settlement process since a continuous spin movement of the shapes was documented. Specimen continued to spin or swim until death commenced. Some of the half ball shaped planulae developed 12 pairs of septa 500
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 deformed 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 2±3 days. Histological sections made from few larvae at the 96-h time-point revealed a deteriorated state of the ectodermal outer layer in comparison to undamaged, intact layers of control planulae (Fig. 2e±f). 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
Volume 40/Number 6/June 2000
Fig. 2 Eects of dispersed oil on S. pistillata planula larvae. (a) Disintegration 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)).
complete larvae survivorship after 96 h. All larvae survived 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 planulae 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 dispersed oil as compared to untreated oil. The third generation 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 501
Marine Pollution Bulletin 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 50 100
100 100 100
90 5.7 0 0
87 23 ± ±
44 32 ± ±
0 ± ±
± ± ±
Petrotech
10 50 100
100 100 100
100 100 100
100 100 7 11.5
100 0 0
100 ± ±
100 ± ±
Emulgal
10 50 100
100 100 100
100 0 0
60 20 ± ±
0 ± ±
± ± ±
± ± ±
Biosolve
10 50 100
100 100 100
100 100 100
100 100 100
100 40 10 23 21
100 0 0
100 ± ±
Inipol
10 50 100
100 100 100
100 0 0
100 ± ±
37 35 ± ±
20 17 ± ±
0 ± ±
dispersed oil revealed synergistic detrimental impacts expressed as the highest mortality ®gures, no settlements and signi®cant 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 relative toxicity of the dispersant (as well as the oil), contribute to the resultant level of toxicity and to other detrimental eects such as morphological abnormalities and reduced settlement rates recorded here. The planulae 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 accordance to their relative toxicity to coral planulae, from the least toxic compound, as follows: Petrotech < Biosolve < Emulgal < Bioreico Inipol. Planulae abortion by adult colonies is a direct response to contamination by petroleum hydrocarbons (Loya and Rinkevich, 1979). Applications of dispersants lead to the dissolution of more hydrocarbons, potentially 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 considering 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 in¯uence eectiveness of dispersion, thus making the decision a complicated one (GESAMP, 1993). Nonetheless, bearing in mind that the prime ob502
jective of oil dispersion is to prevent spilled oil from arriving ashore, our results do not support the application 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 Environment. Thanks are due to the MBL (Eilat) personal for their hospitality, to Ms. Yael Mann for image processing and to participating companies for oil and dispersants donations. Anon (1999) National contingency plan for preparedness and response to oil spill at sea. State of Israel, Ministry of the Environment, Marine and Coastal Environment Division. Benayahu, Y. (1991) Reproduction and developmental pathways of Red Sea Xeniidae (Octocorallia, Alcyonacea). Hydrobiologia 216/ 217, 125±130. Cohen, Y., Nissenbaum, A., Eisler, R. (1977) Eects of Iranian crude oil on the Red Sea octocoral Heteroxenis fuscescense. Environmental Pollution 12,173±186. Dodge, R. E., Wyers, S. C., Firth, H. R., Knap, A. H., Smith, S. R., Sleeter, T. D. (1984) The eects of oil and oil dispersants on the skeletal growth of the hermatypic coral Diploria strigosa. Coral Reefs 3, 191±198. GESAMP (IMO/FAO/UNESCO/WMO/IAEA/UN/UNEP) Joint Group of Experts on the Scienti®c Aspects of Marine Pollution (1993) Impacts of oil and related chemicals and wastes on the marine environment. GESAMP 50, p. 180. Knap, A. H., Sleeter, T. D., Dodge, R. E., Wyers, S. C., Frith, H. R., Smith, S. R. (1983) The eects of oil spills and dispersants use on corals. A review and multidisciplinary experimental approach. Oil and Petrochemical Pollution 1, 157±169. Loya, Y. (1975) Possible eects of water pollution on the community structure of Red Sea corals. Marine Biology 29, 177±185. Loya, Y. (1976) Recolonization of the Red Sea corals aected by natural catastrophes and man-made perturbations. Ecology 57, 278±289. Loya, Y., Rinkevich, B. (1979) Abortion eects in corals induced by oil pollution. Marine Ecology Progress Series 1, 77±80. Loya, Y., Rinkevich, B. (1980) Eects of oil pollution on coral reef communities. Marine Ecology Progress Series 3, 167±180. Rinkevich, B., Loya, Y. (1977) Harmful eects of chronic oil pollution on a Red Sea scleractinian coral population. In Proceedings of the Third International Coral Reef Symposium, vol. 3 (Geology), pp. 586±591. Miami, Florida.
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