Porewater Toxicity Testing: a Novel Approach for Assessing Contaminant Impacts in the Vicinity of Coral Reefs

Coral reef communities can be deleteriously affected by exposure to low levels of anthropogenic contaminants. Sediments in the vicinity of coral reefs serve as a sink (and when resuspended, as a source) for contaminants and provide an integrative measure of low and intermittent exposure. Sediment porewater toxicity tests using gametes and embryos of the sea urchin Arbacia punctulata were employed to provide a measure of the presence of bioavailable contaminants. As sediments are patchy and composed predominately of sand in reef areas, sediment samples were collected by divers using hand cores. The utility of this approach was assessed at coral reef sites in Hawaii and southeastern Mexico. Toxicity was observed at several reef sites off Waikiki Beach in Honolulu, Hawaii and at the Sian Ka’an Biosphere Reserve in Quintana Roo, Mexico. The results of these studies have demonstrated that porewater toxicity tests are sensitive enough to differentiate among sediments with low to moderate levels of contamination and would be a valuable tool for assessing and monitoring contaminant exposure in coral reef ecosystems. Regional anthropogenic impacts on coral reefs, exacerbated by global climatic changes, can increase rates of local species extinction (Chadwick-Furman, 1996). Coral bleaching events have been associated with chemical contamination in addition to the well-studied bleaching effects resulting from temperature increases, UV radiation, and sedimentation (Meehan and Ostrander, 1997). Domestic and industrial effluents, fertilizers, oil pollution, drilling muds, sedimentation, and disease have been identified as some of the main problems affecting coral populations (Goenaga, 1988; Fishelson, 1995; Richardson, 1998; Dustan, 1999; Harvell et al., 1999). Several authors (Brown and Howard, 1985; Dubinsky and Stambler, 1996; Meehan and Ostrander, 1997; Dustan, 1999), have reviewed the effects of stress and of marine pollution on coral reefs. Richardson (1998) emphasized that an important aspect of coral disease and overall reef degradation is the effect of anthropogenic influence, and possibly of terrestrial runoff, suggesting that this area demands focused research. Dustan (1999) reported that in the Florida Keys coral diseases seem to be prevalent in areas close to centers of human habitation, and the potential influence of pollutants on coral reefs as facilitators of disease outbreaks was discussed by Harvell et al. (1999). Different methods have been used to assess the presence and impacts of contaminants in coral reef areas. Chemical measurements were made to identify the presence of a variety of contaminants in coral annual growth bands (Law et al., 1994; Deslarzes et al., 1995; Guzman and Jarvis, 1996; Readman et al., 1996; Buesseler, 1997; Scott and Davies, 1997), in tissues of soft corals (Denton and Burdon-Jones, 1986), hard corals (Snedaker et al., 1999), and in sediments collected from the vicinity of coral reefs (Reichelt and Jones, 1994; Snedaker et al., 1995). Field assessments showed significant reduction in biodiversity in reefs subject to land-based pollution (sewage, industrial effluent, agricultural and aquacultural runoff and sedimentation) relative to unpolluted coral reefs (Edinger 408 BULLETIN OF MARINE SCIENCE, VOL. 69, NO. 2, 2001 et al., 1998). Toxicity tests with different life stages of coral species, and with other phyla from the coral reef community, have also been developed (Kusmaro et al., 1994; Davies, 1995; Peachey and Crosby, 1995; Te, 1998). The assessment of bioindicator organisms, e.g., stomatopod and amphipod crustaceans, butterfly fish, and sea urchins, has been suggested for the analysis of the health of coral reefs (Erdmann, 1998; Thomas, 1993; Risk, 1994; Reese, 1995). The sensitivity of both amphipods and echinoids to chemical contamination is well known, particularly for sediment and porewater toxicity assessments (Chapman, 1998; Carr and Chapman, 1992, 1995; Carr et al., 1996a,b; Nipper et al., 1998). Several studies have been conducted integrating pollution in sediments with uptake or impact on the coral reef biota. In Panama, concentrations of hydrocarbons in sediments were significantly correlated with coral injury and reduced growth up to 5 yrs after a major oil spill (Guzman et al., 1994). The mobilization of metals, caused by dredging activities in the vicinity of coral reefs in Australia, was analyzed by Reichelt and Jones (1994). High uranium concentrations were found not only in sediments, but also in corals, algae and seagrass of polluted sites from the Gulf of Aqaba (Abu-Hilal, 1994). Also in Florida, pesticides could be measured in sediments and in coral reef biota (Glynn et al., 1995). Sea-surface microlayer samples from the Florida Keys region exhibited adverse effects in toxicity tests with sea urchin (Lytechinus variegatus) and spotted seatrout (Cynoscion nebulosus) embryos (Rumbold and Snedaker, 1999). Likewise, the use of sensitive toxicity tests, e.g., echinoid early-life stage tests, with sediments from the vicinity of coral reefs could be applied to assess the presence of contaminants in toxic amounts in coral reef areas. Sediments serve as a sink (and when resuspended, as a source) for contaminants and provide an integrative measure of low and intermittent exposure. It has been suggested that the bioavailable fraction of contaminants in sediments is primarily associated with the porewater, and is controlled by a variety of factors which include total organic carbon (TOC) and acid volatile sulfides (AVS) (DiToro et al., 1991; Ankley et al., 1996). Sandy sediments typically contain low levels of organic carbon and therefore contaminants contained in them would be readily bioavailable for the benthic biota and would be easily dispersed into the water column in events of sediment resuspension by both, natural phenomena and dredging activities. Sediment toxicity can be assessed by a variety of methods, among which solid phase and porewater tests seem to be the most commonly employed. Porewater toxicity tests with sensitive life stages of marine species, e.g., echinoid gametes and embryos, were shown to be significantly more sensitive that the most common solid phase test using amphipods (Carr and Chapman, 1992; Carr et al., 1996a,b). In the current study, sediment porewater toxicity tests using gametes and embryos of the sea urchin Arbacia punctulata were employed with the objective of providing a measure of the presence of bioavailable contaminants. The use of sensitive life stages of echinoids for toxicity tests is considered particularly relevant in coral reef studies, given the importance of the sea urchin Diadema for the maintenance of healthy coral reef systems. The utility of this approach was assessed at coral reef sites in Hawaii and southeastern Mexico. The sampled areas were subject to contaminant inputs from sewage outfalls, and urban and agricultural storm water runoff. 409 NIPPER AND CARR: PORE WATER TOXICITY TESTING IN THE VICINITY OF CORAL REEFS MATERIALS AND METHODS SEDIMENT SAMPLING.—Surficial sediment samples were collected from six stations (M1–M6) in the Sian Ka’an Biosphere Reserve, Quintana Roo, Mexico (Fig. 1), in May 1998, and from 14 stations in Hawaii (H1–H14); 12 stations were located in Honolulu, Oahu, and two on the Kona coast, Island of Hawaii (Fig. 2), during June 1998. Site H14, on the Kona coast, was selected as a reference site on an apparently pristine location, while site 13 was adjacent to the Kona Airport. Sediment cores (10 cm deep) were collected by divers using polycarbonate corers (6 cm diameter ¥ 20 cm length), placed in presoaked half-gallon high density polyethylene containers, chilled, and shipped to the U. S. Geological Survey (USGS) Marine Ecotoxicology Research Station (MERS) in Corpus Christi, Texas, in insulated coolers with blue ice. Samples were received by the USGS in Corpus Christi, Texas, 2 d after shipment and were processed immediately upon receipt. SEDIMENT POREWATER EXTRACTION PROCEDURE AND QUALITY MEASUREMENTS.—Pore water was extracted from the sediments using a pneumatic extraction device (Carr, 1998). This extractor is made of polyvinyl chloride (PVC) and uses a 5 mm polyester filter, and has been used successfully in numerous previous sediment quality assessment surveys (Carr and Chapman, 1992, 1995; Carr et al., 1996a,b). After extraction, the porewater samples were centrifuged in polycarbonate bottles at 1200 ¥ g for 20 min to remove any suspended particulate material; the supernatant was collected and frozen at -20∞C. Prior to toxicity tests, the samples were thawed in a tepid (20∞C) water bath. Sample salinity was measured with a Reichert refractometer and, if necessary, adjusted to 30 ± 1 ‰ using purified deionized water or concentrated brine prepared by slow evaporation of seawater. Temperature and dissolved oxygen (D.O.) were measured with YSI meters, and D.O. was adjusted by gently stirring the sample if the measured concentration was <80% saturation. The water quality adjusted samples were used to prepare further test dilutions. Sulfide (as S), total ammonia (expressed as nitrogen), and pH were measured with Orion meters and their respective probes. Un-ionized ammonia (expressed as nitrogen) concentrations were calculated for each sample based on the respective pH and total ammonia values, and on test salinity (30‰) and temperature (20∞C). POREWATER TOXICITY TESTING WITH SEA URCHINS.—Porewater toxicity was assessed using the sea urchin (Arbacia punctulata) fertilization and embryo development tests, following the methodology described in Carr and Chapman (1992) and Carr et al. (1996a). The sea urchin fertilization test involved exposure of the sperm to test media for 30 min, followed by the addition of a predetermined number of eggs. After an additional 30-min incubation period, the test was terminated by the addition of 10% buffered formalin and the percentage of fertilized eggs was determined. The sea urchin embryological development test was executed concurrently with the fertilization test. Eggs were pre-fertilized and then inserted into the exposure vials. The embryos were exposed to the test solutions for 48 h, after which time the test was terminated by the addition of 10% buffered formalin. Aliquots from each of the five replicates were examined microscopically to determine the percentage of embryos that developed normally to the echinopluteus stage. A. punctulata urchins used in this study were obtained from Gulf Specimen Company, Inc. (Panacea, Florida). Each of the porewater samples was tested in a dilution series design at 100, 50, and 25%, with five replicates per treatment. Dilutions were made with 0.45 mm filtered seawater. A reference porewater sample collected from Redfish Bay, Texas, which had been handled identically to the test samples, was included with each toxicity test as a negative control. This site is far removed from any known sources of contamination and has been used previously as a reference site (Carr and Chapman, 1992, 1995; Carr et al., 1996a,b). In addition, a dilution series test with sodium dodecyl sulfate (SDS) was included as a positive control. SEA URCHIN TOXICITY TESTING DATA ANALYSIS.—Statistical comparisons among treatments were made using ANOVA and Dunnett’s one-tailed t-test (which controls the experiment-wise error rate) on the arcsine square root transformed data with the aid of SAS (SAS, 1989). Prior to statistical analysis, the transformed data sets were screened for outliers (SAS, 1992). Outliers were detected 410 BULLETIN OF MARINE SCIENCE, VOL. 69, NO. 2, 2001 Figure 1. Sample sites in Mexico. Color and pattern differentiation of symbol indicates that toxic effects in those stations were significantly different from the reference in the sea urchin (Arbacia punctulata) fertilization and embryological development tests (Dunnett’s t-test, a £ 0.05 and a £ 0.01, and detectable significance criteria applied). 411 NIPPER AND CARR: PORE WATER TOXICITY TESTING IN THE VICINITY OF CORAL REEFS by comparing the Studentized residuals to a critical value from a t-distribution chosen using a Bonferroni-type adjustment. The adjustment is based on the number of observations, n, so that the overall probability of a type I error is at most 5%. The critical value, cv, is given by the following equation: cv = t(df Error , 0.05/(2 ¥ n)). After omitting outliers but prior to further analysis, the transformed data sets were tested for normality and for homogeneity of variance using SAS/LAB Software (SAS, 1992). A second criterion was also used to compare test means to reference means. Detectable significance criteria (DSC) were developed to determine the 95% confidence values based on power analysis of all similar tests performed by our lab (Carr and Biedenbach, 1999). This value is the percent minimum significant difference from the reference that is necessary to accurately detect a difference from the reference. The DSC value for the sea urchin fertilization assay is 15.5% at a = 0.05, and 19% at a = 0.01. For the embryo development assay, the DSC value is 16.4% at a = 0.05, and 20.6% at a = 0.01. Only results that were significantly different from the reference sample and were below DSC were considered significantly toxic. The EC 50 (effective concentration to 50% of the organisms) of the reference toxicant (SDS) tests was calculated using the trimmed Spearman-Karber method (Hamilton et al., 1977) with Abbott’s correction (Morgan, 1992).