The use of coral nubbins in coral reef ecotoxicology testing

The use of coral nubbins in coral reef ecotoxicology testing

Biomolecular Engineering 20 (2003) 401 /406 www.elsevier.com/locate/geneanabioeng The use of coral nubbins in coral reef ecotoxicology testing Shai ...

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Biomolecular Engineering 20 (2003) 401 /406 www.elsevier.com/locate/geneanabioeng

The use of coral nubbins in coral reef ecotoxicology testing Shai Shafir a,b,*, Jaap Van Rijn b, Baruch Rinkevich a a b

Israel Oceanographic and Limnological Research, National Institute of Oceanography, Tel Shikmona, P.O. Box 8030, Haifa 31080, Israel Food and Environmental Quality Sciences, Faculty of Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

Abstract While there is an urgent demand to establish reliable ecotoxicological assays for reef corals, there has not been yet an available source material that can supply the high number of colony replicates needed for reliable tests. In past experiments, the major obstacle to obtaining as many fragments as possible had been the damage inflicted to donor colonies by pruning. In this paper, we present the application of coral nubbins, a novel source material for coral ecotoxicology assays. Nubbins from the branching Red Sea coral Stylophora pistillata (n /450) were used for evaluating the impacts of water soluble fractions from a crude oil, an oil dispersant and dispersed oil. Coral nubbins (minute coral fragments in the size of one to several polyps) harvested from a single colony are genetically identical to each other, may be obtained in any quantity needed and whenever research activities demand their use. Several dozens of nubbins can be obtained from a single small branch in branching coral species, a procedure that has minimal impact on donor genotypes. Nubbins production is a low cost procedure and requires limited maintenance space. Results of short and long-term acute ecotoxicological tests are revealed and discussed here, indicating the advantageous use of nubbins as ubiquitous coral material for toxicology assays and physiological studies. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Coral; Dispersant; Ecotoxicology; Nubbin; Oil pollution; Stylophora pistillata

1. Introduction Upon exposure to anthropogenic stress factors, marine organisms, (at all levels of biological organizations: molecular, sub cellular, cellular, organ, whole organism, behavioral, etc.) start to manifest a number of symptoms. When integrated, these symptoms may culminate in the organism’s death, and may at a later stage indirectly affect higher levels of organization, such as the whole population leading to reduced reproductive success, etc. In many cases, the anticipated symptoms have been reproducibly induced in laboratory experiments by employing a variety of biological assays, cumulatively called ecotoxicological assays [1]. Two general issues are associated with the topic of ecotoxicological assays: (1) acute versus chronic responses to various contaminants, and (2) the criteria for model species selection [2]. When considering the species to be used, tests are, in many cases, conducted on

* Corresponding author. Tel.: /972-4-856-5275; fax: /972-4-8511911. E-mail addresses: [email protected], [email protected] (S. Shafir).

organisms that are readily obtained, easily cultured, and sensitive enough to be assayed and reveal repeatable results. The ecological significance of test species, in many ecotoxicological assays, is of secondary consideration and major efforts are invested to develop and use either common model organisms or micro-scale toxicity tests (including tests in the marine environment; reviewed in [3,4]). Ecotoxicological tests that constitute a hazard assessment and are conducted under controlled laboratory conditions, often fail to match or relate laboratory tests and outcomes closely with field situations [5,6]. One such example is the evaluation of dispersed crude-oil toxicity on coral larvae as a predictor to whole reef outcomes [7]. The above discussion is particularly relevant to coral reef ecotoxicological studies. A variety of toxicants and other man-made perturbations are continuously affecting coral reefs worldwide [6,8]. Different biological parameters such as coral reproduction [9 /11], growth and calcification rates [12] coral metabolism [13 /15] and gene transcription levels [16] are frequently used criteria for evaluating reef health and anthropogenic impacts. A major obstacle in many studies is the availability of

1389-0344/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1389-0344(03)00062-5

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coral colonies for the ecotoxicological studies [17]. In many cases, corals are not readily available and the number of coral colonies that are required for a detailed study may exceed the collection permit quota. Moreover, the high variation recorded for any physiological parameter tested [18] usually blurs the outcomes, emphasizing the need for even larger coral sampling operations. Coral nubbins [19,20] may supply the need for coral material used in ecotoxicological studies [6] without severely impacting natural reefs during collections. Nubbins are the established minute fragments taken from coral colonies down to the size of a single or few polyps [20]. When comparing them to the traditional used coral material, such as planula larvae [7,21], branch fragments [14,16] and whole colonies [15], nubbins have several potential advantages as source material for ecotoxicological tests: (1) They are easily pruned from any part of the coral colony. A set of about 50 nubbins per a 5 cm branch length can be established within a short period of 30 min (personal observation), thus reducing the damage to donor colonies to a minimum. (2) Approved protocols reveal survivorship exceeding 90% [20]. (3) All nubbins from a single colony are genetically identical, an outcome that reduces variation within and among tests. (4) Pruning, attaching nubbins to substrata and exposing them to toxicants materials is an easy, simple and quick procedure. (5) The use of nubbins enables the researchers to simultaneously test, several levels of biological organization, including the whole organism level (survivorship), the physiological (growth rates), biochemical (protein expression) and the DNA levels (gene transcription). Assays may be preformed as short-term tests (few hours), intermediate (few days) or as long-term tests (several weeks to months), revealing different facets of coral stress responses. When following the fate of coral nubbins taken from branching forms (such as Stylophora , Acropora and Pocillopora species; [20,22] and our unpublished observation), several developmental stages were characterized. The first stage (14 /21 days; Fig. 1a) is manifested by a tissue regeneration process to cover exposed skeletons. This regeneration process further enables nubbins to firmly attach their tissue to the substratum. Following this regenerative phase, nubbins grow horizontally on substrata forming a thin layer of tissue and secreted skeleton (the second stage lasting 1/3 months; Fig. 1b1, b2,). This allows an easy and accurate way to measure growth rates. Four to six months after pruning, the nubbins start to grow vertically (the third stage) and form small branching colonies (Fig. 1c). The present study aims to demonstrate the usefulness of coral nubbins as an improved source material for short and long-term ecotoxicology assays. Tests are employed on nubbins during their first and second

stages of development and reveal toxicological reactions on survivorship, growth rates and stress proteins gene expression. Tested stressors were water-soluble fractions (WSFs) of Egyptian crude oil, oil dispersant and dispersed oil.

2. Materials and methods The experiments conducted here were carried out on three Stylophora pistillata colonies that were collected in shallow water (4 /6 m depth) in front of the H. Steinitz Marine Biology Laboratory, the Red Sea, Eilat. The colonies were transported immersed in seawater within insulated containers to the National Institute of Oceanography, Haifa. Maintenance conditions are as described [20]. Nubbins (average aerial area 31.19/9.7 mm2, approximately 5 /10 polyps each) were pruned from the above colonies using an electrician’s wire cutter and immediately immersed in seawater in order to minimize stress [20]. The exposed skeletal surfaces of the freshly cut nubbins were dried and glued with a drop of cyanoacrylate glue (Super Glue 3, Loctite, Ireland) on dry glass slides. A stock solution of WSFs was prepared from Egyptian crude oil by adding 5 ml of crude oil to 995 ml filtered seawater (1:200 ratio) and shaking the mixture for 24 h at 80 rpm. A stock solution of water accommodated fractions (WAFs) from dispersed oil was prepared according to the recommended use of 1:10 dispesant:oil volume ratio, by adding 0.5 ml of the dispersant Emulgal C-100 (Amgal Chemicals, Israel) to the above oil/seawater solution and by shaking the solution for 24 h at 80 rpm. The dispersant stock solution was based on 0.5 ml of Emulgal C-100 in 999.5 ml filtered seawater (500 ppm; [7]). Concentrations of all three stock solutions were termed as 100% concentrations. For each pollutant, nubbins were introduced to six different concentrations: 100, 80, 60, 40, 20, 0% WSFs; 100, 10, 1, 0.1, 0.01 and 0% dispersant and WAFs. The assays performed here tested short and long-term acute responses to oil, dispersed oil and dispersant. Seventy-five nubbins (25 from each colony), were subjected for 24 h to different concentrations of pollutants. The nubbins were then washed carefully under fresh filtered sea-water for several minutes and placed in new aquaria with running filtered seawater. Survivorship was checked daily. Nubbins that started to spread on substrates were followed and photographed digitally. Photographs were analyzed with image-analysis software (TINA 2.07) to obtain spreading tissue area. Five nubbins from each colony (total 15 nubbins per pollutant concentration) were taken for evaluating HSP70 expression in tissues 1 h after placing them in the

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Fig. 1. Stages in the development of a nubbin to a coral colony: (a) First stage (14 /21 days) characterized by tissue regeneration that covers exposed skeleton, (b1) early second stage (30 days) that started with the of nubbin’s horizontal growth on the substrate, (b2) late second stage (75 days); mature phase of tissue growth on the substrate, including a well developed calcified plate, (c) early third stage (120 days) characterized by the vertical growth of the nubbin, forming a small colony.

pollutant solutions. The nubbins were then immersed in RNAzol-B and total RNA was extracted for 2 h to ensure coral tissue removal. RT was performed in two steps [23]: a volume of 16.75 ml aqueous solutions containing 100 pmol of the reverse primer CAACACGTTC TTTTCACCG (designated HSP4R) and 3 mg RNA from each sample was heated to 70 8C for 5 min and chilled on ice. RT was carried out in 25 ml solution, after adding to the primer-RNA solution: 5 ml of 5/ reverse transcriptase buffer (Promega), 1.25 ml of 10 mM dNTP mix, 25 units of rRNasin, and 200 units of MMLV-reverse transcriptase (Promega). The RT reaction was carried out for 1 h at 42 8C followed by 3 min at 94 8C. Two ml of each RT solution served as the PCR templates. PCR amplifications of the RT products were designed, each, for 35 cycles at 94 8C (1 min), 60 8C (1 min), and 72 8C, using the reverse primer HSP4R and the forward primer HSP4F (ATGGTAAAGT TGAGATCATC GC).

3. Results The survivorship of nubbins exposed to WSFs of Egyptian crude oil in all five oil concentrations was not different from the controls (P /0.05, one way ANOVA; Fig. 2a). Even 43 days after, the 24 h exposure period to the water soluble fractions of the oil, nubbins survivorship at the high concentrations (80 /100% of stock solution) average 82/76% as compared with 75/77% at the intermediate WSF concentrations (40 /60%) or 83% survivorship in the controls (Fig. 2a). However, the oil dispersant (Emulgal) was highly toxic (Fig. 2b). Within 1 day following the exposure, all nubbins (n /60 per each concentration) at 100, 10 and 1% of the stock solutions died. A delayed enhanced mortality was

Fig. 2. Survivorship of S. pistillata nubbins following 43 days of acute, 24 h exposure period to different WSF (a), dispersant (b) and dispersed oil (c) concentrations.

recorded in the 0.1% concentration of the stock solution as from day 6 after exposure (849/10% survivorship). At day 43 only 629/20% of the nubbins at this concentra-

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tion survived as compared with 909/4% in the controls (P /0.05; t-test). Survivorship of nubbins that were exposed to WAFs of dispersed oil, were similar to the values recorded for nubbins exposed to the dispersant alone (P /0.05, ttest). The nubbins at 100, 10 and 1% of the stock solutions died after 1 day following the treatments and exposure to a concentration of 0.1% WAFs revealed reduced survivorship (599/16%) as compared with the control (909/4%; P B/0.05, t-test) (Fig. 2c). The 0.01% concentration for both pollutants was not different from controls (P /0.05, t-test). Three weeks after exposure, nubbins that survived the Egyptian crude oil treatment have started to grow horizontally on the substrate. We followed this growth for 65 days following the treatments. The WSF concentration had no effect on the average horizontal growth area on substrata (Fig. 3), nor was there any statistical difference (P /0.05 one way Anova) between the colonies. Abnormal growth patterns appeared in some of the nubbins that were exposed to the high WSFs concentrations (100 /80%). Polyps with double mouth openings (Fig. 4) or with 18 tentacles appeared in the newly formed tissue spreading on substrate. The number of nubbins that started to grow on the substrate differed significantly between the three S. pistillata colonies (P B/0.05; one way ANOVA). At day 66 post treatment, only 309/16% of the nubbins from colony A spread, as compared with 619/33 and 639/9% in colonies B and C, respectively (Fig. 5). There was, however, no statistical difference (P /0.05) within colonies analyses. RT-PCR of total extracted RNA from nubbins exposed to WSFs of crude oil, revealed S. pistillata HSP-70 induction at all studied concentrations whereas in the control nubbins the PCR reading failed to amplify the HSP-70 transcript (Fig. 6).

Fig. 3. Average horizontal growth (percent of original aerial size) of S. pistillata nubbins exposed to different WSFs concentrations.

Fig. 4. An abnormal double-mouth polyp developed 35 days following the exposure of a nubbin to Egyptian crude oil 100% WSF concentration. Arrows point to the double-mouth formation.

Fig. 5. Percentages of the spreading nubbins on substrates as revealed by colony specific values (S. pistillata genotypes A /C) following the exposure to WSFs of Egyptian crude oil.

4. Discussion This study demonstrates the applicability of using coral nubbins in ecotoxicology assays, employed on the molecular (HSP-70 expression), physiological (growth rates) and whole organism (survivorship) levels. More than 450 nubbins pruned from each one of the three coral colonies served to analyze short and long term impacts invoked by exposing corals to water soluble fractions of Egyptian crude oil, to oil dispersant and to dispersed oil solutions. Each type of toxicant was analyzed through six different concentrations, revealing the spectrum of impacts for the tested criteria. The same donor coral-colonies could provide nubbins for testing the three pollutants studied in the research, without being harmed by the pruning procedure ([20,22]; unpublished results). These numbers are large enough not only to allow good statistical analyses of several toxicants and toxicant concentrations, simultaneously, but were also enough to detect both lethal and sub lethal effects in a single set of experiments. Furthermore, the nubbin’s method also enabled evaluation of the influence of a tested toxicant within a single coral genotype

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conjunctively with oil pollution; [7]) and revealing a wide confidence intervals for ecological predictions, as has been done for temperate ecosystems [26]. Coral nubbins are further being employed in coral physiological studies [19,24,27 /30] and as potential source supply for the marine ornamental trade [19,20]. It is, therefore, an ideal source material that should be considered as a ubiquitous scientific tool in variety of biological disciplines.

Acknowledgements This study was supported by the BARD (no IS 331902 R) and by a grant from the Israeli Ministry of the Environment.

References Fig. 6. HSP-70 PCR results after 1 h exposure to crude oil WSFs. 1, size marker, 2 /7 different WSF stock solution concentrations (100, 80, 60, 40, 20% and 0, control, respectively); arrow indicates the deduced HSP-70 band.

and at the same time revealed the differences evoked from natural variation between different coral genotypes. While bioassays that use the responses of an organism as ecotoxicant detectors (to either acute or chronic situations) were developed and established for temperate marine indicator species (by the US Environmental Protection Agency, the American Society for Testing and Material, and by others [6]), very little is known on how well these species are suitable in characterizing the responses of tropical marine species to the same toxicants or how well they may be tested under tropical conditions. Specially prepared uniformly sized nubbins taken from suitable coral species provide an excellent and far better source material for field [22] and laboratory ([24]; this study) ecotoxicology tests than irregularly shaped whole colonies or large ramet [6]. Ecotoxicology assays should also be designed to study the impacts of pollutants on different levels of organization, taking into account the normal variation characteristic to corals, within and between colonies of the same species [25]. The employment of nubbins as the preferential source material in coral ecotoxicology studies may also improve the capability of simultaneously testing multiple species responses and comparing the relative sensitivities of each species. It will also elucidate the toxicant concentrations for each particularly tested adverse effect. This will enable the extrapolation of the results to natural reef conditions, unveiling the uncertainties in ecological risk assessment (such as from the use of oil dispersant

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