Harmful Algae 2 (2003) 235246

Toxicity of clay occulation of the toxic dinoagellate, Karenia brevis, to estuarine invertebrates and sh
Michael A. Lewis , Darrin D. Dantin, Calvin C. Walker, Janis C. Kurtz, Richard M. Greene
National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Ofce of Research and Development, Gulf Ecology Division, 1 Sabine Island Drive, Gulf Breeze, FL 32561, USA Received 19 January 2003; received in revised form 1 May 2003; accepted 6 May 2003

Abstract The environmental effects of clay occulation used to remove red tide dinoagellate blooms from the water column are relatively unknown on benthic organisms. The primary objective of this study was to determine the laboratory-derived toxicities of clay occulation of the toxic dinoagellate, Karenia brevis, for four common estuarine test species. Phosphatic clay mixed with a coagulant (polyaluminum hydroxy chloride (PAC)) was not acutely or chronically toxic in most cases to infaunal amphipods (Leptocheirus plumulosus and Ampelisca abdita), grass shrimp embryos (Palaemonetes pugio) and larval sheepshead minnows (Cyprinodon variegatus). K. brevis alone (density range = 38805060 cells ml1 ; brevetoxin (Btx) range = 19.8140.7 g l1 ) was very toxic to C. variegatus and, to a lesser extent, L. plumulosus. The addition of claycoagulant did not usually reduce this toxicity. The combination of clay, coagulant and K. brevis cells when settled over a natural sediment were usually as toxic to the benthic test species as K. brevis alone. This result suggests that clay occulation of K. brevis blooms will neither increase, nor decrease toxicity to benthic organisms relative to that attributable to an untreated bloom. Validation of this conclusion, however, is required since it is based on laboratory-derived, single species toxicity data using media collected from a simulated red tide event. The determination of environmental effects on indigenous benthic biota in near-coastal areas during a natural red tide event, prior to and after treatment with clay occulation, would provide the perspective needed for a more realistic hazard assessment of this possible control procedure. Published by Elsevier B.V.
Keywords: Clay; Coagulant; Flocculation; Karenia brevis; Toxicity; Fish; Macroinvertebrates

1. Introduction Harmful algal blooms (HABs) cause signicant environmental and human impacts and are of increasing public concern (Shumway, 1990; Anderson, 1995, 1997). As a result, several control strategies have been proposed to reduce their impacts (Anderson, 1997; Boesch et al., 1997; Perez and Martin, 1999;

Corresponding author. Tel.: +1-850-934-9382. E-mail address: lewis.michael@epa.gov (M.A. Lewis).

Kusek et al., 1999). Various chemical control methods have been evaluated, including the use of phytotoxicants such as surfactants (Kutt and Martin, 1974; Hitchcock, 1976), aerial dusting with copper sulphate (Rounsefell and Evans, 1958), and use of occulants (Anderson, 1997). Biological control methods, such as competition for nutrients by bacteria and dinoagellates (Hunter and McLaughlin, 1958; Steidinger and Joyce, 1973; Kutt and Martin, 1974) and use of pathogens (Bratback et al., 1993) have also been suggested.

1568-9883/$ see front matter. Published by Elsevier B.V. doi:10.1016/S1568-9883(03)00041-6

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The coastal waters of west Florida, as well as other regions of the Gulf of Mexico, experience recurrent red tides caused by the toxic dinoagellate, Karenia brevis (Davis) G. Hansen and Moestrup (formerly, Gymnodinium breve) (Steidinger and Williams, 1964; Steidinger and Joyce, 1973). Brevetoxins (Btx) produced by this species have affected local economies and public health and caused mortalities of sh, invertebrates, and marine mammals (Landsberg, 2002). A promising strategy to control the effects of K. brevis and other harmful algal blooms is clay occulation (Sengco et al., 2001). The effectiveness of various clays in removing red tide organisms from the water column has been investigated more frequently (Maruyama et al., 1987; Choi et al., 1988, 1989; Shirota, 1989; Yu et al., 1994a,b,c; Na et al., 1996) than their potential environmental impacts on the benthos (Howell and Shelton, 1970; Portman, 1970; McIntyre, 1983). This information, however, is needed to ensure that this type of control procedure, or any other, is less environmentally damaging to indigenous biota than the HAB event itself. To provide some initial insight on this issue, the effect of clay occulation of K. brevis on benthic organisms was determined in this study under controlled laboratory conditions. More specically, the acute and chronic toxicities of a phosphatic clay and coagulant were determined for four estuarine organisms in the presence and absence of K. brevis whole cell cultures and cell lysates.

(Ampelisca abdita and Leptocheirus plumulosus), and embryos of the epibenthic grass shrimp (Palaemonetes pugio) (Table 1). These species have been recommended for use in sediment toxicity tests due, in part, to their availability, ease of use, sensitivity, and for A. abdita and L. plumulosus, their broad salinity tolerances (ASTM, 1995). C. variegatus and P. pugio are relatively abundant and indigenous to near-coastal areas along of the Gulf of Mexico. The condition of these species, obtained from either eld collections (P. pugio) or commercial sources (L. plumulosus, C. variegatus, A. abdita), was ensured prior to use in 96 h acute toxicity tests conducted with the reference anionic surfactant, sodium dodecyl sulphate. 2.2. Test media Phosphatic clay, a by-product of phosphate mining, was collected from a tailings settling pond in central Florida. Large plant material and organic debris were removed from the clay before storage at room temperature. Clay was mixed with distilled, deionized water and diluted in the test chambers to a nal concentration of 0.25 g l1 , as recommended by Sengco et al. (2001). Natural sediment was collected from Perdido Bay, FL and used in test chambers for all species except P. pugio. Seawater collected from Santa Rosa Sound, FL with salinity between 25 and 28 psu, was used in all toxicity tests. Phosphatic clay and natural sediment were analyzed once for 25 chlorinated pesticides, 21 PCB congeners, 15 trace metals and 41 PAH analytes following USEPA (1997) techniques. The contaminant concentrations were compared to sediment quality criteria proposed for Florida near-coastal areas by MacDonald et al. (1996). The numerical, effects-based guidelines used in the comparison were the threshold effects level (TEL) and probable effects level (PEL).

2. Materials and methods 2.1. Test species Acute and chronic toxicity tests were conducted in the laboratory using larval sheepshead minnows (Cyprinodon variegatus) two infaunal amphipods
Table 1 Test species and test methodologies used in this study Test species A. abdita C. variegatus L. plumulosus P. pugio Infaunal amphipod Sheepshead minnow Infaunal amphipod Grass shrimp Size or life stage 35 mm <72 h old 14 mm Embryos

Source Commercial Commercial Commercial Field collection

Test methodology ASTM (1995), USEPA (1994a,b) USEPA (1994a,b) ASTM (1995), USEPA (2001), Emery et al. (1997) Lewis and Foss (2000)

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By denition, adverse biological effects are possible for sediments containing contaminant concentrations between the TEL and PEL guidelines but are more likely for those exceeding PEL guidelines. Total organic carbon (TOC) content and particle size distribution of the clay and natural sediment were determined using standard methods (APHA et al., 1998). The phosphatic clay was approximately 85% clay and 15% silt, whereas the natural sediment was approximately 49% clay, 46% silt and 5% sand. TOC values were 1.3% (phosphatic clay) and 4.0% (natural sediment). Polyaluminum hydroxy chloride (PAC; CAS #00132741-9), a common coagulant used for drinking water purication, was utilized in this study. PAC enhances the scavenging efciency of the phosphatic clay allowing the use of lower clay concentrations while maintaining high removal rates (Sengco et al., 2001). Although the optimal scavenging efciency of PAC occurs at 5.0 mg l1 when mixed with clay in seawater (Sengco et al., 2001), toxicity tests were conducted at nominal concentrations of 0.5, 5.0 and 50 mg l1 . The concentrations above and below the optimal were used to determine the rst-effect and no-effect toxic concentrations. The same stock solutions of PAC were used throughout the study and were stored at 4 C between applications. 2.3. K. brevis cultures K. brevis were obtained from laboratory cultures which were initiated with strains isolated from Piney Island, FL (isolate Piney Island, courtesy of K. Steidinger, Florida Marine Research Institute) and Charlotte Harbor, FL (isolate SHGB01, courtesy of S. Morton, NOAA). Cultures were grown in 20 l polycarbonate bottles containing lter-sterilized (0.22 M) Gulf of Mexico seawater (salinity range = 3234 psu) enriched with L1 nutrients and trace metals (Guillard and Hargraves, 1993). Cultures were grown without mixing or aeration in a temperature-controlled (21 0.2 C) walk-in chamber under 130 mol quanta m2 s1 for 14L:10D. Late log-phase cultures were used to provide the initial target cell concentration of approximately 5000 cells ml1 to the test chambers. Salinity was adjusted to 25 psu with sterile distilled water. Due to the tendency of K. brevis cells to accumulate at the surface

during daylight, aliquots for cell counts were collected after gentle swirling. Cell concentrations were monitored during several toxicity tests on test days 0 (test initiation), 1, 3, 7, 14, 21 and 28 using a Nikon Diaphot inverted microscope. A K. brevis lysate was frozen (20 C for 12 h), thawed and then subjected to 30 min exposures to a Brinkman Polytron tissue mixer and a Sonifer Cell Disrupter. Brevetoxin concentrations were determined for an aliquot of thawed lysate and for whole cell cultures using dichloromethane (DCM) liquidliquid partitioning, based in part on Pierce et al. (1985) and Musser et al. (1997) as described by Vogelbein et al. (2002). Btx extracts were analyzed using high pressure liquid chromatography with diode array detection (Hewlett-Packard model 1090). The method detection limit was 14 g l1 for a 250 ml sample. 2.4. Toxicity test methodology The four test species were exposed to clay, PAC, K. brevis whole cell cultures, and a K. brevis lysate either alone or in binary and ternary combinations. The toxicity test methodologies generally followed published guidelines (USEPA, 1994a,b, 2001; ASTM, 1995; Emery et al., 1997; Lewis and Foss, 2000); experimental details appear in Tables 1 and 2. Dissolved oxygen, pH, salinity and temperature of the test waters were determined during all toxicity tests in at least one replicate test chamber. The range of values were 5.57.4 mg l1 (dissolved oxygen), 8.08.2 (pH), 2526 C and 25.828.3 psu. These values were within the guidelines of the test methodologies. Not all test species and media were used in all toxicity tests. Acute toxicities of the clay and PAC were determined in the presence of seawater alone (C. variegatus and P. pugio) and seawater and natural sediment (C. variegatus, A. abdita and L. plumulosus). The amphipods require sediment to exist and the small size of the microplate wells (2 ml) used as test chambers for P. pugio embryos excluded the use of sediment. Chronic toxicities of clayPAC combinations were determined for L. plumulosus and C. variegatus; published chronic toxicity test methodologies are not available for A. abdita and P. pugio. L. plumulosus (chronic toxicity), C. variegatus (chronic toxicity) and P. pugio (acute toxicity) were exposed to clay, PAC and lysed and whole

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Table 2 Experimental conditions for the toxicity tests conducted with amphipods (L. plumulosus, A. abdita), grass shrimp embryos (P. pugio) and larval sh (C. variegatus) Parameter L. plumulosus Acute Light quality Light intensity (fc) Photoperiod Test vessel volume Volume of sediment (ml) Test water volume (ml) Replicates Organism/rep Feeding Aeration Duration Endpoint(s) Ambient 50100 16L:8D 1l 200 750 5 20 None Constant bubbling 10 days Survival Chronic Ambient 50100 16L:8D 1l 200 750 5 20 Three times a week (algae/GORP) Constant bubbling 28 days Survival and growth A. abdita Acute Ambient 500100 16L:8D 1l 200 750 5 20 None Constant bubbling 10 days Survival P. pugio Acute Darkness Darkness 16L:8D 2 m1 None 2 24 24 None None 12 days Survival C. variegatus Acute Ambient 50100 16L:8D 1l 200 800 3 10 Daily (artemia) Constant bubbling 4 days Survival Chronic Ambient 50100 16L:8D 1l 175 750 3 10 Daily (artemia) Constant Bubbling 7 days Survival and Growth

cell cultures of K. brevis in the presence of natural sediment. The sequence of addition of clay, PAC, sediment, and K. brevis (whole cell cultures or lysate) was dif-

ferent for the amphipod and sh toxicity tests (Fig. 1). Unlike in the sh toxicity tests, amphipods were added prior to addition of clay, PAC and K. brevis to allow them to burrow into the sediments (Fig. 2). For

Fig. 1. Sequence of addition of phosphatic clay, polyaluminum hydroxy chloride (PAC) and K. brevis whole cell cultures to initiate the toxicity tests.

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Fig. 2. Sediment burrow entrances of L. plumulosus prior to (A) and 1 day after addition of K. brevis whole cell cultures (B).

K. brevis treatments, whole cell cultures or the cell lysate were added to the test chambers prior to addition of clayPAC mixtures. It was important to test the effects of clay occulation using both whole cell cultures and a cell lysate because the effect of clay occulation on K. brevis cell structure, brevetoxin availability and the resultant toxicity are largely unknown under natural or simulated laboratory conditions (Sengco et al., 2001). 2.5. Statistical analysis Survival and growth of the test species were compared among treatments and among test species using a one-way analysis of variance (ANOVA) followed by Tukeys post-hoc analysis (SAS Institute, 1989). The signicance level was = 0.05 for all analyses.

3. Results 3.1. Acute toxicities of natural sediment, clay and PAC The natural sediment contained higher concentrations of several inorganic and organic contaminants than found in the phosphatic clay (Table 3). The TEL guidelines for arsenic, chromium, copper, mercury, nickel and lead were exceeded in the natural sediment but the presence of these concentrations resulted in no signicant effects on survival of L. plumulosus, A. abdita or C. variegatus, which ranged from 80 (1 standard deviation = 26) to 96 (9)% (Table 4). PEL guidelines were not exceeded in these sediments. The phosphatic clay contained contaminants that exceeded TEL (i.e. nickel) and PEL (i.e. cadmium and chromium) guidelines. Survival of the test species

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Table 3 Chemical quality of phosphatic clay and natural sediment. Concentrations in g/g dry weight except for PCBs, PAHs and DDT (ng/g dry weight) Analyte Silver Aluminum (%) Arsenic Cadmium Chromium Copper Iron (%) Mercury Manganese Nickel Lead Selenium Tin Zinc Total PAHs Total PCBs Total DDT TELa 0.73 b 7.2 0.7 52.3 18.7 0.13 15.9 30.2 124 1684 21.6 3.9 PELa 1.77 41.6 4.2 160 108 0.7 42.8 112 271 16770 189 51.7 Phosphatic clay 0.2 8.4 3.6 54 265.0 16.6 2.8 0.1 146.0 39.6 24.2 4.5 1.0 83.7 115.0 1.7 0.6 Natural sediment 0.2 6.8 12.5 0.3 96.3 22.7 4.8 0.3 589.0 26.9 33.1 1.3 2.9 116.5 606.0 4.9 2.5

iment was not signicantly different from that after their exposure to natural sediment alone (Table 4). Mean survival of the three species exposed to PAC in the presence of natural sediment ranged from 63 (1 standard deviation = 25) to 100%. Survival of A. abdita and C. variegatus following exposure to clayPAC mixtures in the presence of natural sediment ranged from a mean of 72 (6) to 100%. Although mean survival of L. plumulosus progressively declined from an average of 80 (13) to 57 (13)% with increasing PAC concentrations, the differences were not signicant relative to mean survival in natural sediment and seawater. The results of the toxicity tests conducted with P. pugio embryos, in the absence of sediment indicated no signicant affect of the clayPAC mixtures on mean survival. 3.2. Chronic toxicities of clay, natural sediment and PAC Mean survival of L. plumulosus after 28 days exposure to clayPAC mixtures in the presence of natural sediment was between 50 (1 standard deviation = 15) and 66 (15)% (Table 5). These values were not signicantly different than those after this species exposure to natural sediment (55 21%) and clay (65 36%) alone, and to PAC in the presence of natural sediment (range of mean survival: 68 (9)81 (17)%). Likewise, there were no signicant differences in mean weight of surviving organisms regardless of the toxicity test media combination. There were no signicant chronic effects of PAC alone or clayPAC mixtures on larval C. variegatus after 7 days exposure (Fig. 3). Mean survival ranged from 97 to 100%. Mean wet weights were between 1.00 (0) and 1.04 (0.16) mg for sh exposed to clayPAC mixtures relative to an average of 1.03 (0.16) and 0.97 (0.06) mg after their exposure, respectively, to natural sediment alone and clay alone. 3.3. Toxicities of clayPAC mixtures with K. brevis Initial K. brevis cell densities (time 0) for toxicity tests conducted with whole cell cultures averaged 4840 (1 standard deviation = 387; range = 45805400) cells ml1 . Brevetoxin concentrations in these whole cell cultures varied approximately two-fold averaging 119.7 (22.8), 68.1 (8.1) and

Concentrations compared to sediment quality guidelines proposed for Florida coastal areas (MacDonald et al., 1996). Values in bold exceed guidelines; double asterisks means exceeds PEL; single asterisks means exceeds TEL. a Threshold effects (TEL) and probable effects (PEL) guideline values. b No guideline available.

exposed to settled clay as substrate averaged 87 (2) and 100 (0)% for P. pugio and C. variegatus, respectively. In contrast, survival of the infaunal amphipods exposed to the same media was low, averaging 50 (5) and 58 (23)% for L. plumulosus and A. abdita, respectively. In most cases, survival of P. pugio and C. variegatus exposed to PAC dissolved in seawater was not signicantly different from their survival in seawater alone (Table 4). Larval C. variegatus were tolerant to the three test concentrations of PAC. Mean survival of P. pugio embryos was high following exposure to 0.5 and 5.0 mg l1 PAC, but was signicantly reduced (23 1 standard deviation = 3%) after exposure to 50.0 mg l1 PAC. This latter concentration exceeds by 10-fold that which, when mixed with phosphatic clay, results in the optimal removal efciency of clayPAC mixtures (Sengco et al., 2001). Survival of the test species exposed to either PAC or clayPAC mixtures in the presence of natural sed-

M.A. Lewis et al. / Harmful Algae 2 (2003) 235246 Table 4 Acute toxicities of PAC and clayPAC mixtures to the test species. Values represent mean percent survival ( 1 standard deviation) Treatments Seawater Clay and seawater Natural sediment and seawater PAC in seawater PAC (0.5) PAC (5) PAC (50) L. plumulosus (10 days) a 50 (5) 80 (26) A. abdita (10 days) 58 (23) 92 (6) 85 (9) 85 (9) 82 (8) 85 (13) 72 (6) 93 (8) P. pugio (12 days) 90 (3) 87 (2) b 85 (3) 95 (3) 23 (3) b 92 (4) 88 (5) 88 (3)

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C. variegatus (4 days) 98 (4) 100 96 (9) 100 100 100 100 98 (4) 96 (4) 100 98 (4) 96 (4)

PAC in seawater with natural sediment PAC (0.5) 88 (8) PAC (5) 88 (3) PAC (50) 63 (25) ClayPAC mixtures in seawater with natural sedimentc PAC (0.5) 80 (13) PAC (5) 73 (29) PAC (50) 57 (13)

Nominal PAC concentrations were 0.5, 5.0 and 50.0 mg l1 ; clay concentration was 0.25 g l1 ; seawater salinity was 25 psu. Values in parenthesis represent test duration. a Not conducted since these species need sediment to survive. b Not conducted. c No natural sediment used in P. pugio toxicity tests. Signicant difference (P < 0.05).

56.4 (one value) g l1 for toxicity tests conducted with L. plumulosus, P. pugio and C. variegatus, respectively. Btx-2 accounted for >90% of total Btx in these cultures. In toxicity tests conducted using
Table 5 Effects of PAC and clayPAC mixtures on L. plumulosus after 28 days exposure Treatments Natural sediment Clay Survival (%) 55 (21) 65 (36) Weight (mg) 0.50 (0.12) 0.47 (0.26) 0.57 (0.13) 0.38 (0.15) 0.37 (0.22)

PAC in seawater with natural sediment PAC (0.5) 81 (17) PAC (5) 71 (12) PAC (50) 68 (9)

ClayPAC mixtures in seawater with natural sediment PAC (0.5) 66 (15) 0.49 (0.04) PAC (5) 50 (15) 0.41 (0.46) PAC (50) 60 (11) 0.54 (0.08) Values represent mean (1 standard deviation). Nominal PAC concentrations were 0.5, 5.0 and 50.0 mg l1 ; clay concentration was 0.25 g l1 ; seawater salinity was 25 psu. Mean values are statistically similar (P > 0.05).

K. brevis cell lysates, initial mean Btx concentrations ( g l1 ) were 140.7 (1.3, L. plumulosus), 44.4 (4.2, P. pugio) and 19.8 (one value, C. variegatus). K. brevis cells were rapidly removed from the water column following addition of clayPAC mixtures (Fig. 4). For example, cell concentrations were reduced in the water column after clayPAC additions by an average of 88 (+1 standard deviation = 10) and 98 (2)% after 24 and 48 h. Water column cell concentrations were less than microscopic detection limits between 2 and 7 days. Survival of L. plumulosus and C. variegatus and, to a lesser extent P. pugio exposed to K. brevis whole cell cultures alone was considerably less compared to that after their exposure to only natural sediment (Table 6). Whole cell cultures of K. brevis alone were completely lethal to larval sh and visually reduced the number of burrow entrances of L. plumulosus (Fig. 2). The addition of clayPAC mixtures to the K. brevis whole cell cultures did not generally alter this pattern of response. The toxicities of the settled clayPACK. brevis, when settled, were similar to those for the exposures to K. brevis whole cell cultures alone, with two exceptions.

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Fig. 3. Percent survival and wet weight of larval sheepshead minnow (C. variegatus) after 7 days exposure to polyaluminum hydroxy chloride (PAC) and clayPAC mixtures. Values represent mean (1 standard deviation). There were no statistical differences among mean values P > 0.05).

First, the mean survival (31 1 standard deviation = 15%) of L. plumulosus was signicantly greater after exposure to the 50 mg l1 PAC, clay and K. brevis combination than that after exposure to K. brevis alone
Table 6 Percent survival of three test species after exposure to K. brevis whole cell cultures, cell lysate, and clayPACK. brevis mixtures Treatments Natural Sediment K. brevis whole cell cultures K. brevis cell lysate L. plumulosus 75 (17) 11 (12) 61 (12) P. pugio 93 (2) 81 (3) 85 (2) C. variegatus 100 0 0

ClayPAC mixtures with K. brevis whole cell cultures PAC (0.5) 7 (0) ND 3 (7) PAC (5) 4 (0) 49 (8) 0 PAC (50) 31 (15) 32 (2) 0 ClayPAC mixtures with K. brevis cell lysate PAC (0.5) 61 (12) ND PAC (5) 80 (11) 81 (6) PAC (50) 87 (8) 75 (3) 60 (20) 57 (13) 20 (20)

Values represent mean (1 standard deviation). Nominal PAC concentrations were 0.5, 5.0 and 50.0 mg l1 . Clay concentration was 0.25 g l1 . ND: not determined. Tests conducted using seawater and natural sediment except for P. pugio (no sediment). Signicant difference relative to K. brevis alone (P < 0.05).

(1112%). Second, this ternary combination was signicantly more toxic to P. pugio embryos particularly at the highest PAC concentration where mean survival was reduced to 32 (2)% relative to 81 (3)% after their exposure to K. brevis whole cells alone. Mean survival of L. plumulosus and P. pugio exposed to K. brevis cell lysates alone or to clayPACcell lysate was not signicantly different than that after their exposure to natural sediment alone (Table 6). Mean survival ranged from 61 (12) to 87 (8)% for L. plumulosus and 75 (3) to 85 (2)% for P. pugio. In contrast, K. brevis lysate killed all larval C. variegatus. Addition of clayPAC to the cell lysate signicantly increased sh survival (57 13 and 60 20%), except at the 50.0 mg l1 PAC concentration. There were no chronic toxicity effects of the clayPACK. brevis combinations on L. plumulosus. The total weight of L. plumulosus surviving exposure to the K. brevis whole cell cultures and cell lysates averaged 0.80 ( 0.56) and 1.12 (0.29) mg wet weight, respectively. These values were not signicantly different from mean survival of this species after the addition of clayPAC mixtures (P > 0.05). Mean weights (mg) of L. plumulosus surviving exposure to

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Fig. 4. Cell concentration of K. brevis (cells ml1 ) in test waters during the chronic toxicity tests conducted with L. plumulosus and C. variegatus. Tests conducted in presence of 0.25 mg l1 phosphatic clay and 0.5, 5.0 and 50.0 mg l1 PAC. Initial cell concentration prior to addition of clay and coagulant is shown at the left of each row. Values represent mean (1 standard deviation).

the three clayPAC mixtures in the presence of K. brevis whole cell cultures ranged from 0.88 (0.36) to 1.98 (0.34), and mean values after exposure of this species to cell lysate were between 1.08 (0.06) and 1.68 (0.06). Due to the total mortality of C. variegatus after exposure to K. brevis whole cell cultures and lysate, useful pre- and post-treatment information based on weight is unavailable.

4. Discussion The phosphatic clay and PAC used in this study have been suggested as a possible control strategy for the Florida red tide organism, K. brevis (Sengco et al.,

2001). The toxicity results indicate that their use alone would not likely result in increased toxicity to benthic life relative to that attributable to an untreated red tide event. However, the PAC and clay alone were toxic in some cases. The only signicant toxic affect of PAC alone occurred for P. pugio embryos, at 50 mg l1 which is above the 5 mg l1 concentration recommended for use by Sengco et al. (2001). The most notable effect of the clay was the low survival of the two burrowing amphipods when exposed to it alone. The reason for this result is unknown but it may be due to the presence of cadmium and chromium at concentrations exceeding the PEL effects-based guideline values and/or to the high clay content (85%). The use of L. plumulosus, for example, as a test species

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is considered more applicable in sediments where the clay content is less than 85% (USEPA, 2001). For example, Emery et al. (1997) reported that survival of L. plumulosus to a sediment containing 84% clay was signicantly reduced. The environmental signicance of the low survival in clay, regardless of the cause, is not clear since survival increased after addition of PAC and the likelihood of benthic organisms being exposed to 100% clay substrate would be less likely during a real world application due to its larger lateral dissipation and dilution over the natural sediment. Nevertheless, this nding does suggest that an important criterion for choice of a occulating material in the future should be an analysis of its chemical quality and an evaluation of its potential to cause an adverse biological impact. There was little evidence that the clayPAC mixtures were toxic prior to contact with K. brevis. Mean survival of the test species generally exceeded 80% after 428 days exposure to these binary combinations relative to an average of 8096% survival when exposed to only natural sediment. Furthermore, chronic effects on weight were not observed relative to natural sediment. In contrast, the brevetoxins contained in K. brevis were very toxic to two of the three test species and in most cases, the addition of PAC and clay did not signicantly reduce this toxicity particularly at the proposed PAC usage concentration of 5 mg l1 . However, it is important to note, that there were four signicant differences in the response of the test species after addition of clay and PAC to the K. brevis whole cell cultures and lysates and three of these were signicant increases in survival (see Table 6). The toxicities of lysed K. brevis cells alone and in combination with PAC and clay were often less than those observed for exposures initiated with intact cells, moreso for L. plumulosus and C. variegatus. The reason for this difference may be due to either the lower initial brevetoxin concentrations (C. variegatus) and/or the more rapid adsorption of the brevetoxins in the lysed cell tests to the clay, PAC or test chamber walls reducing its bioavailability for toxicity. Despite the differences in results between these two exposure scenarios, it was clear that the clay and PAC did not increase the toxicity of K. brevis cells to the test species nor, just importantly, did it usually reduce this toxicity. There were obvious differences in interspecic sensitivities to the brevetoxins contained in K. brevis.

C. variegatus was the most sensitive species which was not unexpected since brevetoxins are potent ichtyotoxins. P. pugio was the least sensitive species but this result may be attributable to the test design. The brevetoxin in the small test volumes (2 ml) may have absorbed rapidly to the glass microplate wells reducing its bioavailability. Unpublished LC50 values for brevetoxin and this species are within the range of concentrations (44.4 and 68.0 g l1 ) measured in this study during the P. pugio toxicity tests (Walker, personal communication). The effects of brevetoxins on aquatic organisms has been recently reviewed (Landsberg, 2002). However, toxic concentrations of brevetoxins for the four test species used in this study have not been previously published in the scientic literature. The available toxicity data base is limited primarily to freshwater sh, most commonly, mosquito sh, for which the reported LC50 values in terms of PbTx-2 have ranged between 0.6 and 19 g l1 (Baden et al., 1981, 1984, 1988; Lewis, 1992; Rein et al., 1994). For comparison, an initial measured concentration of 56.4 g l1 was completely lethal and 19.8 g l1 killed almost 50% of C. variegatus (sheepshead minnow) exposed in this study which is consistent with previously reported data. The inconsistent and, sometimes, low survival of L. plumulosus exposed to natural sediment and relatively high variability in survival among some replicates were noticeable in this study. The condition or health of the test species was considered normal based on the use of reference toxicant (sodium dodecyl sulfate) and water quality parameters of the test waters were consistently within test method guidelines. Therefore, the reason(s) for the low survival and variability in results is not known but their occurrence is not totally unexpected. There is limited scientic experience using this species in sediment toxicity tests and the test method and factors affecting this species response to environmental media continues to be assessed. In summary, despite some experimental anomalies, this proposed clay occulation technique will not likely increase toxicity to benthic organisms that would not be already occurring due to a red tide event alone. It is also apparent that the proposed control procedure will not likely reduce brevetoxin toxicity and may simply redistribute it. In other words, this control strategy may reduce or eliminate toxin producing

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dinoagellates in the water column and the often concomitant highly visible adverse effects (sh kills and irritating aerosols) but it may increase the rate of transfer of the toxicity to underlying substrates containing indigenous benthos. This conclusion should be considered preliminary, however, since it is unknown if the results of this study would be reective of those if derived under more natural conditions. The toxicity tests were conducted under controlled pH, temperature, salinity and light using cultured organisms and media collected from a laboratory simulated red tide event. The environmental realism of this simulation is not known as related to, among other factors, the degree of mixing, aggregate settling rates, clay deposition depth over natural sediment, K. brevis cell densities and brevetoxin fate, exposure duration and concentrations. Therefore, results for in situ toxicity tests and other biological indices of sediment quality such as benthic community composition are needed for sediments collected at different spatial and temporal scales, prior to and after clay occulation of a natural red tide event. These data would provide insight on the relevance of the results of this study as well as the information needed for an effective risk assessment.

to the agencys peer and administrative review and has been approved for publication as an EPA document. References
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Acknowledgements We gratefully acknowledge the expert assistance of the following USEPA personnel who conducted the toxicity tests, prepared test media and K. brevis cultures, and conducted statistical analyses: Steve Foss, Rebecca Hemmer, Peggy Harris, Cynthia Chancy, Jeanne Scott, James Winstead, Dragoslav Marcovich. We wish to thank Dale Coleman and James Watts (NCBA) for brevetoxin sample preparation and analysis, Peggy Rogers (NCBA) for manuscript preparation and Stephen Embry (CSC Corp.) for graphics. The authors also thank Aishao Li, Mario Sengco, and Don Anderson of the Woods Hole Oceanographic Institution for supplying clay and coagulant and reviewing the manuscript. This study was conducted in conjunction with a USEPA award (R827090) to D. Anderson (WHOI) through the Ecology and Oceanography of Harmful Algal Blooms (ECOHAB) Program. Notice: The US Environmental Protection Agency through its Ofce of Research and Development funded and managed the research described here. It has been subjected

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