Physiological effects and biotransformation of PSP toxins in the New Zealand scallop, Pecten novaezelandiae.
KEY WORDS: scallop, Pecten novaezelandiae, Alexandrium tamarense, Alexandrium margalefi, paralytic shellfish poisoning, clearance rate
Toxic algal blooms have been increasing worldwide, with environmental impacts and ongoing effects on ocean ecosystems, fisheries, aquaculture, and tourism. Shellfish feed readily on algae containing neurotoxins, accumulate these in their tissues, and, when eaten by humans, can cause gastrointestinal and neurological illnesses. Scallops can accumulate paralytic shellfish poisoning (PSP) toxins in their tissues and can be unsuitable for human consumption (White 1993a, White 1993b) even when algal blooms are not evident (Shumway & Cerebella (1993), and Bricelj & Shumway (1998), and references therein).
In some parts of the world, such as New Zealand, where scallops represent a valuable commercial product, it is particularly important to prevent the sale of potentially toxic shellfish and it is necessary to understand the ability of particular species to sequester and biotransform algal toxins. During the past 40 y, scallop aquaculture in New Zealand has grown from very small beginnings to a significant primary industry (Marsden & Bull 2006), currently estimated to be worth in excess of NZS360 million annum, with a target of reaching S1 billion annum in sales by 2025 (New Zealand Aquaculture farm facts 2011). New Zealand shellfish have been monitored for marine biotoxins since January 1993, when shellfish toxicity was first detected in New Zealand. Scallops are included regularly in the New Zealand biotoxin monitoring program; however, there have been few records of toxic scallops.
Similar to other suspension-feeding bivalve molluses, scallops accumulate various toxins from harmful algal species. Transformation of paralytic shellfish toxins occurs after consumption of dinoflagellates by bivalve species, and research suggests that the bivalves usually have lower proportions of N-sulfocarbomyl toxins and higher proportions of carbamoyl toxins than the toxigenic dinoflagellates that were ingested (Oshima et al. 1990, Bricelj et al. 1991, Cembella et al. 1993, Choi et al. 2003). Several kinds of transformation have been documented for PSP toxins, such as epimerization, reduction, and hydrolysis (Cembella et al. 1993, Cembella et al. 1994, Oshima 1995, Murakami et al. 1999a, Murakami et al. 1999b). These transformations are species specific and are dependent on the algal-bivalve concentrations. For example, Choi et al. (2003) demonstrated that the scallop Chlamys nobilis and the green-lipped mussel Perna viridis exposed to Alexandrium tamarense differ in their depuration kinetics, biotransformation, and tissue distribution of PSP toxins. In both species, the toxin profile differed from the toxic algae that had been consumed. Some scallops such as Placopecten magellanicus and Patinopecten yessoensis bind the toxins in different tissues for extended periods and release them slowly (Bricelj & Shumway 1998). The study of detoxification kinetics may help to determine the time required to reduce PSP toxicity in shellfish tissues to levels that are safe for human consumption.
It is well known that toxic algal blooms can affect the physiology and behavior of bivalves, including scallops, and they are taxon and species specific (Shumway & Cucci 1987, Gainey & Shumway 1988, Marsden & Shumway 1992). The responses also vary according to the algal toxin strain and geographical location, as well as previous history of exposure to algal toxins (see Shumway (1990) and Bricelj and Shumway (1998), and references therein). When the scallop Placopecten magellanicus was exposed to the toxic dinoflagellate Alexandrium tamarense, the bivalves immediately closed their shell then exhibited vigorous swimming and clapping activity accompanied by copious amount of mucus and pseudofeces production (Shumway & Cucci 1987). These movements, which reflect a typical escape response from predators, may also result in accelerated stroke volume, increased heart rate and cardiac output, together with a shift to anaerobic metabolism (Thompson et al. 1980).
Measurement of physiological responses to environmental stressors regularly include the oxygen uptake, excretion, and clearance rates. As with behavioral responses, these physiological and metabolic responses are species specific and produce variable results. Some bivalves such as mussels, clams, and oysters, exposed to algal toxins, appear to adapt readily to the presence of toxic algae in their diet and are able to maintain their normal clearance rates. There is less information available for scallops, and although some studies suggest that the clearance rate can be maintained in the presence of toxic algae (Shumway & Cucci 1987), other studies suggest that clearance rates are inhibited in the presence of algal blooms (White et al. 1993b). For example, Leverone et al. (2007) found that among 4 species of bivalves exposed to the dinoflagellate Karenia brevis, the bay scallop Argopecten irradians was the most sensitive species, showing reduction of its clearance rate. These species-specific responses may depend on a variety of factors, such as algal toxicities (Bricelj et al. 1991, Bardouil et al. 1993, Bricelj et al. 1996, Lassus et al. 1996, Li & Wang 2001), algal concentration (Li et al. 2002), and history of exposure to toxic algae (Shumway & Cucci 1987, Bricelj et al. 2005).
Very little research has been undertaken on the physiological effects of toxic algae on the New Zealand scallop. In a recent study, Contreras et al. (2012) found a reduced clearance rate over 4 days of exposure to concentrations of [10.sup.6] cells/day/scallop of the toxic dinoflagellate Alexandrium tamarense. In the current study, adult scallops Pecten novaezelandiae were exposed to lower concentrations of the same dinoflagellate over a 10-day period to establish the effects of longer term exposure to the toxic algae. The exposure period was followed by 8 days of depuration (detoxification), when scallops were fed with nontoxic Alexandrium margalefi. At intervals, the scallop tissues were tested for PSP toxins, and clearance rate, excretion rate, and oxygen uptake were measured. The research will therefore provide a preliminary indication of the accumulation and depuration kinetics of PSP toxins in New Zealand scallop tissues.
Collection of scallops and culture of microaigae
Individuals of Pecten novaezelandiae (n = 125) were collected by dredging at Ketu Bay, Malborough Sounds, New Zealand (40[degrees]59'6" S, 173[degrees]59'15" E) in February 2009. Scallops were transported to the laboratory holding aquarium at the University of Canterbury. Three microalgae, supplied by the Cawthron Institute (Nelson, New Zealand), were cultured for feeding the scallops: Tetraselmis sueeica (CAWPR01), a nontoxic green marine flagellate commonly used as live food for bivalves, crustaceans, and rotifers in hatcheries and cultured in a nutrient-enriched filtered seawater medium (f/2) (Guillard 1975) at 19[degrees]C with constant light and aerated conditions in 10-L plastic bottles; the toxic dinoflagellate Alexandrium tamarense (CAWD 121; diameter, 34 [micro]m); and the nontoxic dinoflagellate Alexandrium margalefi (CAWD10; diameter, 40 [micro]m). Both species of dinoflagellates were isolated from Marsden Point in 1997 and 1993, respectively (MacKenzie et al. 2004). Both Alexandrium species were grown in a nutrient-enriched filtered seawater medium (GP) (Loeblich & Smith 1968) under a 12-h light/dark cycle at 19[degrees]C and with no aeration in 2-L flasks with 1.5 L culture medium.
The scallops were acclimated for 2 wk to laboratory conditions in circulating, unfiltered seawater (120 scallops distributed into 3 aquaria of an 800-L capacity; salinity, 30; temperature, 15[degrees]C), and each tank was supplied with a calculated volume of concentrated algal culture ration of Tetraselmis suecica of approximately 25 x [10.sup.7] cells twice a day. A subsample of 48 scallops (dry tissue weight, 3.7 [+ or -] 0.7 g; shell length, 94.3 [+ or -] 7.0 mm) was split randomly into 2 groups: the PSP group, which was fed toxic dinoflagellates, and the control group, which was fed nontoxic dinoflagellates. Each treatment had 4 replicates using independent aquaria containing 6 scallops, each in 50 L unfiltered seawater (salinity, 30; temperature, 15[degrees]C) with constant aeration (noncirculating flow system). The seawater was renewed every 48 h. During the first 2 days, both treatment groups were standardized by feeding with Tetraselmis suecica and the nontoxic dinoflagellate Alexandrium margalefi (for concentrations, see Table 1).
After the 2-day standardization period, for the next 10 days the PSP group was supplied with Tetraselmis suecica and the PSP toxic dinoflagellate Alexandrium tamarense, and the control group continued to be provided with similar quantities of T. suecica and Alexandrium margalefi. At each feeding time, samples from the concentrated algal cultures were combined into 1 vessel and the cell numbers were assessed. A standard quantity of algal food was supplied to each replicate twice each day, once in the morning and again in the evening. The quantities of algal culture added were calculated so that each scallop was exposed to 5 x [10.sup.5] cells/day (Table 1). Twice a day the scallops were exposed to low concentrations of toxic and nontoxic dinoflagellates below bloom conditions. At these concentrations, no pseudofeces were produced. For the next 8 days (detoxification period), both the PSP group and the control group were fed T. suecica and A. margalefi using the procedures described earlier.
Clearance and Excretion Rate Measurements
The clearance and excretion rates were measured on the same individuals (PSP group, n = 4; control group, n = 4) on days 0, 3, 6, and 10 (intoxication period), and days 14 and 18 (detoxification period).
Although this is a small number of replicates, our preliminary studies demonstrated a low coefficient of variation for the results such that this number was sufficient to detect differences in physiological responses of this species. To measure clearance rate, a known quantity of algae was supplied to the scallop (initial concentration). The decrease in the concentration of algae in the water as a result of the filtration by the scallops was determined using an AquaFluor handheld fluorometer (linear range chlorophyll in vivo, 0-300 [micro]g/L; Turner Designers, CA) calibrated previously with a Sedgwich-Rafter chamber. To test for any sedimentation of the cells during the feeding measurements, a control vessel without a scallop present was also maintained. As an initial concentration, the PSP group was fed 5 x [10.sup.6] cells/L Tetraselmis suecica and 2.5 x [10.sup.5] cells/L Alexandrium tamarense; the control group was fed 5 x [10.sup.6] cells/L T. suecica and 2.5 x [10.sup.5] cells/L Alexandrium margalefi. No pseudofeces were produced under these cell concentrations. Clearance rates were measured on the same individual from each replicate every 30 min for 2 h and were calculated using the equation by Coughlan (1969);
CR = (ln [C.sub.1] - ln [C.sub.2]) V/t
where CR is the clearance rate (measured in liters per hour), [C.sub.1] is the number of cells at time 0, [C.sub.2] is the number of cells at time 1, V is the experimental volume (measured in liters), and t is the time between measures [C.sub.1] and [C.sub.2] (measured in hours).
At the end of clearance rate measurements, the scallops were transferred to vessels containing 0.5 L filtered seawater. Scallops were maintained individually, and a control vessel without a scallop was also used. Scallops were left undisturbed for 1 h, then seawater samples from each vessel were analyzed to measure ammonia concentration by the phenol hypochlorite method (Solorzano 1969).
Extraction of PSP Toxins from the Dinoflagellates and Scallop Tissue
The PSP toxin profiles of the scallop tissues were determined from 1 individual from each replicate (n = 4) collected on days 0, 3, 6, and 10 (intoxication period), and days 14 and 18 (detoxification period). Samples of the culture of the toxic dinoflagellates Alexandrium tamarense used in the diet were analyzed in triplicate. The dissected scallop tissue was divided into digestive gland as a separate group and the other tissues, comprising the gonad, gills, mantle, and muscle together. There were insufficient resources to undertake separate analyses of each tissue and because, in New Zealand, whole tissues are consumed, a decision was made to combine them.
The PSP toxins from the dinoflagellates and the tissues were extracted following the procedure described by Rourke et al. (2008). The final filtrate was analyzed using high-performance liquid chromatography (HPLC; Shimadzu Corporation, Kyoto, Japan) with fluorescence detection. The total amount of toxin in the dinoflagellates and the tissues was determined as the sum of all toxins quantified in the run and is expressed as pictograms STX di-HCL equivalents/cell and micrograms STX di-HC1 equivalents/100 g wet tissue, respectively.
Quantification of Toxins by HPLC
A rapid liquid chromatography assay with postcolumn oxidation was used for quantification of PSP toxin profiles following the methods in Rourke et al. (2008). The system was calibrated using the following standards from the Certified References Materials Program operated by the National Research Council of Canada: C1, C2, dcGTX2, dcGTX3, dcSTX, dcNeo, GTX1, GTX2, GTX3, GTX4, GTX5, Neo, and STX. For quantification calculations, the same procedure as described in Rourke et al. (2008) were used.
In March 2009, a separate experiment was undertaken to measure the effects of PSP exposure on oxygen consumption of the New Zealand scallop Pecten novaezelandiae. The individuals used (n = 8; dry weight, 3.4 [+ or -] 0.4 g; shell length, 90.8 [+ or -] 2.8 mm) had been acclimated in the aquarium and fed daily with Tetraselmis suecica (capacity, 800 L; salinity, 30; temperature, 15[degrees]C). Scallops were placed individually in vessels containing 8 L unfiltered seawater (salinity, 30; temperature, 15[degrees]C) with constant aeration. They were separated into the PSP group (n = 4), exposed to a low concentration ([10.sup.6] cell/day/scallop) of the toxic dinoflagellate Alexandrium tamarense (35 nmol STX di-HCl equivalent/day/scallop), and the control group (n = 4) was exposed to [10.sup.6] cell/day/scallop of the nontoxic dinoflagellate, for a period of 10 days.
On days 0, 3, 6, and 10, the oxygen consumption from each replicate was measured in a closed respirometric chamber where the scallop was isolated in a fixed volume (0.5 L) of filtered seawater with constant stirring. Scallops were allowed to settle in the chamber for 1 h, and 100% oxygenated seawater was passed through the system. The temperature was controlled using a double-chamber system in which the outside part of the chamber contained recirculating freshwater at 15[degrees]C. At the start, each chamber was closed and a 1-mL water sample was taken and injected into the oxygen unicell electrode that measures the oxygen partial pressure of the water sample ([P.sub.water][O.sub.2]). The same procedure was repeated every 20 min for 2 h. The oxygen consumption of the scallop ([C.sub.scallop] [O.sub.2]) was calculated from the decrease in [P.sub.water][O.sub.2] in the chamber. A chamber without a scallop was used as control for recording the decrease in oxygen over the same time interval. The following equation was applied to calculate oxygen consumption:
=[([DELTA][P.sub.water][O.sub.2](sample)- [DELTA][P.sub.water][O.sub.2](control))a x V 60]/[(T x W)],
where [C.sub.scallop][O.sub.2] in measured in micromoles per gram per hour, a is the solubility of oxygen in seawater at 15[degrees]C (measured in micromoles per liter per torr), V is the volume of the experimental chambers (measured in liters), T is the experimental temperature (measured in degrees Kelvin), and W is the weight of the animal (measured in grams). The results were then transformed to milliliters [O.sub.2] using the following equivalence: 1 mol [O.sub.2] = 22.4 L [O.sub.2].
Statistical Analysis and Standardization of Data
The physiological responses were measured on the same individual over a period of time and the results were compared using repeated-measures ANOVA to test the effects of the different treatments (within tests = PSP and control group) and the effect of exposure time on the response of the scallops (between tests = days). All measurements were corrected to 1 g dry tissue weight. Data were first examined for normality (Kolmogorov-Smirnov test) and homogeneity of variance (Levene's test). When the data did not conform to normality or homogeneity, they were transformed, either by natural log or square root. When there were significant differences within or among means, a post hoc Tukey's multiple-comparison test was performed (Snedecor & Cochran 1989). All analyses were carried out using STATISTICA 8 (StatSoft, OK).
Throughout the course of the experiment, the average clearance rates for the PSP group were between 0.07 L/h/g and 0.37 L/h/g and between 0.45 L/h/g and 0.55 L/h/g for the control group (Fig. 1A). On days 0, 3, and 6, the PSP group had significantly lower clearance rates than the control group ([F.sub.1,6] = 7.87, P = 0.03), with an average value of 0.11 [+ or -] 0.10 L/h/g. By day 10, despite continued exposure to the toxic algae, the clearance rate of the PSP group was similar to the controls and this continued up to day 18. The clearance rate of the control group remained constant from day 0-18 (0.51 [+ or -] 0.10 L/h/g) and there were no significant differences during the intoxication and detoxification period (post hoc Tukey test).
Average excretion rates ranged from 31.5-66.9 [micro]g N[H.sub.4]-N/h/g. Scallops had similar excretion rates during the intoxication and detoxification periods (Fig. 1B). Overall, the excretion rate of the PSP group was 48.1 [+ or -] 3.9 [micro]g N[H.sub.4]-N/h/g and the control group was 50.5 [+ or -] 3.8 [micro]g N[H.sub.4]-N/h/g. No significant difference in the excretion rate between PSP and control groups ([F.sub.1,6] = 0.22, P = 0.66) was found. In contrast, there was a significant effect of time on the excretion rate ([F.sub.5,30] = 18.62, P < 0.001), which was explained by the higher excretion rates in the PSP and control group on day 10 (post hoc Tukey test).
PSP Toxin Profile of Alexandrium tamarense
The toxin profile of the dietary dinoflagellate species Alexandrium tamarense was dominated by the N-sulfocarbomyl toxin C2 (91.5%). The other toxins identified were dcNeo + Neo (5.3%) and GTX4 (1.2%), accompanied by trace amounts of dcSTX, dcGTX3 + GTX5, STX, C1, GTX1, and GTX3. Although the total toxin content was 353 [+ or -] 97 fmol/cell, the predominance of C2 resulted in a cell-specific toxicity of 35 [+ or -] 9 fmol STX di-HC1 equivalent/cell or 16 [+ or -] 4 pg STX di-HC1 equivalent/cell (Table 2).
PSP Toxin Profile of the Digestive Gland
After 3 days of exposure to the toxic alga, the toxin profile of the scallop digestive gland contained high quantities of the N-sulfocarbomyl C2 (91%), which is similar to the toxin profile of the toxic dinoflagellate Alexandrium tamarense (Table 3). By day 6, the toxin profile had changed and GTX1 became dominant and remained high throughout the experiment. The toxins dcGTX3 + GTX5 and GTX2 were detected in the digestive gland, but were not present in the toxin profile of A. tamarense. The digestive gland accumulated most of the total PSP toxin burden of Pecten novaezelandiae. The highest concentrations of PSP toxins accumulated in this tissue were measured during the depuration period (day 3, 47%; day 6, 54%; day 10, 79%; day 14, 98%; day 18, 97%).
PSP Toxin Profile in Other Tissues
The main toxin detected in the remaining tissues was GTX4, which dominated (>92%) the toxin profile throughout the intoxication period (Table 3). The proportions of C1 and C2 increased during the detoxification period, and small amounts of dcGTX3 + GTX5 and GTX2 were detected in the other tissues, but were not present in the toxin profile of Alexandrium tamarense. The other tissues accumulated lower concentrations of PSP toxins than the digestive gland. These concentrations decreased during the experiment, with lowest values during the detoxification period (day 3, 53%; day 6, 46%; day 10, 21%; day 14, 2%; day 18, 3%).
Total Toxin Burden of PSP Toxins in the Tissues
The total toxin burden of PSP toxins in the tissues of Pecten novaezelandiae was calculated by adding the concentration of toxins detected in the digestive gland and in the homogenate of the other tissues (gonads + gills + mantle + muscle). After 3 days of exposure to the toxic Alexandrium tamarense diet, scallops accumulated toxins quickly in a linear manner up to 10 days, at which point they had accumulated 297 [micro]g STX di-HCl equivalents/100 g tissue (Fig. 2). During this time, scallops may not have reached the intoxication plateau. When the toxic diet was replaced by a nontoxic equivalent, the tissue toxins decreased at a slightly slower rate than the previous intoxication rate, such that after 8 days of exposure to nontoxic conditions (detoxification period), the total body burden was about 150 [micro]g STX di-HC1 equivalents/100 g.
The oxygen consumption of scallops fed with the toxic dinoflagellate Alexandrium tarnarense was constant throughout the experiment and the average was 0.21 [+ or -] 0.03 mL/h/g (Fig. 3). The oxygen consumption of the control group fed with the nontoxic dinoflagellate Alexandrium margalefi also remained unchanged during the experiment and the average was 0.19 [+ or -] 0.03 mL/h/g.
Previous research on the effects of toxic algae on bivalves has shown that the behavioral and physiological responses are species specific and depend on many factors, including the exposure levels, the toxicity of the algal species, and the previous history of exposure (Shumway & Cucci 1987, Gainey & Shumway 1988, Bricelj et al. 1990, Bricelj et al. 2005, May et al. 2010). The results from the current study illustrate that the feeding rate of the New Zealand species decreases in the presence of toxic dinoflagellates, the physiological responses fall into the general patterns found for other scallops, and the species has a moderate to slow detoxification rate.
Numerous studies have measured clearance rates in bivalves, and the ability to accumulate toxins depends on the feeding characteristics of species. Some groups such as mussels feed readily on toxic dinoflagellates, and toxin concentrations increase rapidly in their tissues (Shumway & Cucci 1987, Marsden & Shumway 1993). In contrast with these results, some clams, such as the northern quahog, close their shells in the presence of toxic algae and may accumulate low concentrations of toxins in their tissues (Bricelj et al. 1991). For scallops, variable responses in clearance rates have been reported for shellfish provided with a diet containing toxic algal cells. For example, Lesser and Shumway (1993), exposed juveniles of Argopecten irradians and Placopecten magellanicus continuously to concentrations of [10.sup.5] cells/L Alexandrium tamarense and [10.sup.6] cells/L Gyrodinium aureolum for a week. It was noted that exposure of P. magellanicus to G. aureolum resulted in a cessation of feeding together with production of copious amounts of mucus.
The New Zealand scallop, P. novaezelandiae, adapted to the presence of lower concentrations of toxic algae in its diet, and between 6 and 10 days of exposure, clearance rates had returned to values that were similar to controls. The rates remained similar throughout the detoxification period, suggesting no long-term inhibition in clearance rates.
Scallops, as in other bivalve groups, are expected to have species-specific feeding responses to particular toxic algae, a feature confirmed by Hegaret et al. (2007), who found that the valves of the scallop Argopecten irradians closed initially in response to Alexandrium fundyense whereas no such closure was found on exposure to Heterosigma akashiwo and Prorocentrum minimum. That study found that the clearance rates of the bivalves were not inhibited by the toxic algae; indeed, the bivalves appeared to ingest the toxic algae preferentially over the control alga Rhodomonas. Several explanations were provided for this, including the experimental design, in which clearance rates were recorded after only 1 h of exposure to the toxic algae. Others (Gainey & Shumway 1988, Bardouil et al. 1993) have suggested that the responses of bivalves to Alexandrium tamarense could take up to an hour to readjust and affect the clearance rate.
The toxic dinoflagellates did not affect the excretion rate of Pecten novaezelandiae (70 [micro]g N[H.sub.4]-N/h/g), but rates were higher than those measured for other scallops, such as Placopecten magellanicus (MacDonald et al. 1998) and Argopectenpurpuratus (Navarro et al. 2000), in which the excretion rates were from 18-- 35 [micro]g N[H.sub.4]-N/h/g. When the mussel Mytilus chilensis was exposed experimentally to the toxic dinoflagellate Alexandrium catenella, the rate of ammonia excretion increased after 8 days of exposure and was correlated with the toxin levels accumulated in the tissues (Navarro & Contreras 2010). The authors attributed this response to increased feeding, and high excretion rates due to the degradation of the toxin, which is high in nitrogen.
For bivalves exposed to toxic algae, the relationship between oxygen uptake and other physiological measurements is often inconsistent. Studies by Shumway et al. (1985) found that when the scallop Plaeopecten magellanicus was exposed to Alexandrium (GT429), there was decreased oxygen uptake, an increase in valve closure, but no change in clearance rate. Oxygen consumption of Pecten novaezelandiae was unaffected by PSP toxins, which has been found in some bivalves, including mussels (Li et al. 2002, Navarro & Contreras 2010). Similarly, Marsden and Shumway (1993) studied 5 species of juvenile filter-feeding bivalves (including the scallop P. magellanieus) and concluded that oxygen consumption was unaffected after 1 h of exposure to Alexandrium tamarense.
The toxin profile of Alexandrium tamarense used in our experiments had a large proportion of low-potency C toxins (N-sulfocarbamoyl), as described by MacKenzie et al. (2004). In contrast, the toxin profile of the New Zealand scallop digestive tissues was dominated by carbamoyl toxins, confirming biotransformation of PSP toxin into more toxic compounds. The accumulation and transformation of PSP toxins from the causative dinoflagellates is well known, as reported from field collections and experimental studies of scallops (Shimizu & Yoshioka 1981, Band-Schmidt et al. 2005, Jaime et al. 2007, Wang et al. 2011).
The bioconversions within the tissues of bivalves most likely result from chemical reactions such as reduction, epimerization, hydrolysis, and decarbamoylation, during which the molecule changes its original structure (Oshima 1995). Epimerization is the most common bioconversion found in the tissues of bivalves; the [beta]-epimers (C2, C4, GTX3, and GTX4) primarily produced by dinoflagellates are usually transformed to the more thermodynamically stable [alpha]-epimers (C1, C3, GTX2, and GTX1) after uptake by shellfish (Oshima 1995, Bricelj & Shumway 1998). Epimerization of PSP toxins was observed in the digestive gland of Pecten novaezelandiae during both the intoxication and detoxification periods. After 3 days of exposure to PSP toxins, the toxin profile of the digestive gland of P. novaezelandiae was similar to the toxin profile found in Alexandrium tamarense. This proportion, however, changed during the experiment (from day 6) and was characterized by an increase in GTX1 with no detection of GTX4, indicating that epimerization had taken placed. Because GTX4 was present in the dinoflagellate, we assume that this toxin was transformed immediately to GTX1 without being detected in the toxin profile of the digestive gland.
The toxin profile of the combined other tissues (gonad, gills, mantle, and muscle) of Pecten novaezelandiae contained high proportions of GTX4; however, it was noted that epimerization from GTX4 to GTX1 did not take place. During the detoxification period, the proportion of GTX4 decreased, but the absence of GTX1 indicated that scallops had eliminated GTX4 from these tissues rather than convert it to GTXI, as was clearly observed in the digestive gland. Thus, the metabolism of PSP toxins in this bivalve is more complex than the transformation of toxin components, which has been found in other bivalves, including scallops (Cembella et al. 1993, Cembella et al. 1994).
The current study confirmed that the tissues of the New Zealand scallop can exceed the limit for human consumption within 6 days of exposure to very low concentrations of toxic algae. The values reported are much lower and also less variable than was found in previous studies on field-collected scallops from Georges Bank (White et al. 1993b). After 8 days of detoxification in Pecten novaezelandiae, the toxin values in the digestive tissues and in the combined whole tissue were similar (>130 [micro]g STX di-HCl equivalents/100 g), and both were above the regulatory safe limit of 80 gg STX di-HC1 equivalents/100 g for human consumption. Bivalve species differ markedly in their capacity to eliminate PSP toxins, falling into 2 slow or fast detoxifiers based on their detoxification kinetics (Bricelj & Shumway 1998). Two scallop species were included as slow detoxifiers Placopecten magellanicus and Patinopeeten yessoensis. Both species accumulated high peak toxicity levels in the digestive gland (2,720-340,000 lag STX di-HC1 equivalents/ 100 g tissue) and required more than 9 wk to more than 52 wk to reduce to the regulatory level of 80 [micro]g STX di-HCl equivalents/ 100 g tissue. Bougrier et al. (2001) also assessed the king scallop Pecten maximus as a slow detoxifier, because after 14 days of nontoxic conditions, the scallop did not meet the safety threshold for consumption. We suggest that P. novaezelandiae may be considered similar to a moderate-to-slow detoxifier. This is, however, a cautionary statement because there is scant complementary evidence of toxicities from field collections, and also the depuration kinetics are likely to be influenced by the peak toxicity and the maximum toxin body burden. In addition, other environmental factors such as temperature may or may not influence toxin kinetics (Shumway & Cembella 1993, Cembella et al. 1994).
Although the toxic dinoflagellate Alexandrium tamarense affected the clearance rate of Pecten novaezelandiae, there was accumulation of algal toxins in the tissues. The ability to maintain oxygen uptake and nitrogen excretion in the presence of the toxic dinoflagellate suggests that, at the levels of exposure used in our experiments, the scallop can either adjust to these environmental conditions or that these conditions did not represent a significant stressor to these physiological processes.
Although the majority of the PSP toxins were located within the scallop digestive gland, low levels of toxins occurred in other tissues, including gills, mantle, gonad, and muscle. Many studies on scallops indicate that, although adductor muscle tissue is generally low or nonexistent in toxicity, this is not the case for gonad tissue (Cembella et al. 1993, Cembella et al. 1994) which can be high in PSP toxins, or appear toxic because the digestive gland goes through the gonad. Unfortunately, because of limited resources, we were unable to analyze separately the adductor muscle, but there is a clear need to do this in the future.
The results of this study could have implications for scallop aquaculture in New Zealand if or when PSP blooms occur. Scallops would be expected to become toxic more slowly than mussels (this study and Contreras et al. (2012)), and therefore it may be possible to harvest uncontaminated scallops from the seafloor immediately or relocate them by boat to nearby uncontaminated areas. Some studies suggest that the toxicity of shellfish on lines can occur faster than those grown in bottom culture (Hallegraeff & Sumner 1986). Therefore, it may be useful to establish mussel lines adjacent to the scallop beds to provide shellfish samples for monitoring as an early warning of shellfish toxicity.
Last, the effects of toxic algal blooms on the biology and ecology of scallops in New Zealand are still largely unknown. Anecdotal evidence from the North Island of New Zealand suggests that reduced spawning success occurs in areas of toxic algal blooms, and reduced recruitment has been attributed to toxic algae (Williams 2005). Elsewhere, Yan et al. (2001) reported reduced hatching success of the scallop Chlamysfarreri in the presence of Alexandrium Tamarense, and in a later study (Yan et al. 2003) there was reduced larval survival and growth. In addition, several studies have reported increased sensitivity in juvenile scallops compared with adult scallops when exposed to PSP toxins (Estrada et al. 2007, Leverone et al. 2007). Irregular recruitment together with a short life span is characteristic of many scallops (Vause et al. 2007) and this is a feature of New Zealand populations (Marsden & Bull 2006). It is clear that, in addition to the size of the spawning stock, the effects of environment, water conditions, nutrient levels, and food supply may all play a part in establishing and maintaining aquaculture and wild fisheries. For this reason, it is important to understand and model the risks associated with the establishment of toxic algal blooms, and to investigate how other factors affect the ability of scallops to withstand toxic blooms.
We thank Lincoln MacKenzie and Veronica Beuzenberg from Cawthron Institute, Nelson, for initial training and advice in setting up the HPLC for the PSP toxin analyses in the Chemistry Department, University of Canterbury. We thank Rennie Bishop, Jan McKenzie, and Gavin Robinson from the School of Biological Sciences for collecting scallops and setting up experiments. We thank the University of Canterbury for the doctoral scholarship awarded to A. M. C. Scallops were collected under permit from the New Zealand Ministry of Fisheries.
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ANDREA M. CONTRERAS, (1, 2) ISLAY D. MARSDEN (1) * AND MURRAY H. G. MUNRO (2)
(1) School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8021, New Zealand;
(2) Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch 8021, New Zealand
* Corresponding author. E-mail: firstname.lastname@example.org
TABLE 1. Composition of the diets provided to individual Pecten novaezelandiae during the experiment. Periods Treatments TETR (cells/day) AT (cells/day) Standardization PSP group 2.5 x [10.sup.7] 0 to Alexandrium Control group 2.5 x [10.sup.7] 0 (2 days) Intoxication (10 PSP group 2.5 x [10.sup.7] 5 x [10.sup.5] days) Control group 2.5 x [10.sup.7] 0 Detoxification PSP group 2.5 x [10.sup.7] 0 (8 days) Control group 2.5 x [10.sup.7] 0 Toxicity (nmol STX Periods AM (cells/day) equivalents/day) Standardization 5 x [10.sup.5] 0 to Alexandrium 5 x [10.sup.5] 0 (2 days) Intoxication (10 0 17 days) 5 x [10.sup.5] 0 Detoxification 5 x [10.sup.5] 0 (8 days) 5 x [10.sup.5] 0 AM, Alexandrium margalefi; AT, Alexandrium tamarense; PSP, paralytic shellfish poisoning; TETR, = Tetraselmis suecica. TABLE 2. Paralytic shellfish poison (PSP) toxin profile of Alexandrium tamarense expressed in femtomoles per cell, femtomoles STX di-HCI equivalent per cell, and as a proportion of the total PSP content per cell (measured as a percentage). Specific toxicity Toxic content (fmol STX di-HCI Proportions of PST toxin (fmol/cell) equivalent/cell) PSP toxins (%) GTX4 0.6 0.4 1.2 GTX1 0.1 0.1 0.2 dcGTX3 + GTX5 0.4 0.1 0.4 GTX3 0.1 0.05 0.1 dcNeo + Neo 2.0 1.9 5.3 dcSTX 0.4 0.2 0.5 STX 0.2 0.2 0.4 C1 17.6 0.1 0.3 C2 332.1 32.0 91.5 Total 353 35 100 GTX, gonyautotoxins; dc, decarbamoyl; Neo, neosaxitoxin; STX, saxitoxin; C1 and C2, C toxins. Table 3. Paralytic shellfish poisoning toxin profile of the digestive gland and other tissues from scallops exposed to intoxication and detoxification periods. Day 3 Day 6 Day 10 Tissue Type T % T % T % Digestive gland GTX l -- -- 33 53 155 66 dcGTX3 + GTX5 0.4 1.8 -- -- 0.7 0.3 dcGTX2 0.3 1.4 1.0 1.6 2.4 1.0 GTX3 0.4 1.7 1.0 1.6 2.4 1.0 GTX2 0.3 1.3 0.5 0.9 1.0 0.4 dcNeo + Neo 0.5 2.2 0.8 1.3 3.1 1.3 dcSTX -- -- -- -- 0.2 0.1 STX -- -- 0.2 0.3 3.6 1.5 Cl 0.2 0.8 0.3 0.5 1.5 0.6 C2 1.9 91 25 40 65 28 Total 21 100 63 100 235 100 Other tissues GTX4 22 92 53 98 58 93 GTXI -- -- -- -- 0.6 1.0 dcGTX3 + GTX5 0.01 0.06 0.02 0.04 0.1 0.2 GTX3 0.03 0.1 0.1 0.2 0.4 0.6 GTX2 0.1 0.4 -- -- 0.03 0.04 dcNeo + Neo 0.04 0.2 -- -- -- -- Cl 0.04 0.16 0.1 0.1 0.05 0.1 C2 1.6 6.8 0.9 1.6 2.9 4.7 Total 24 100 54 100 62 100 Day 14 Day 18 Tissue Type T % T % Digestive gland GTX1 140 58 93 70 dcGTX3 + GTX5 1.0 0.4 0.8 0.6 dcGTX2 2,0 0.8 1.3 1.0 GTX3 2.0 0.8 1.3 1.0 GTX2 0.6 0.3 0.4 0.3 dcNeo + Neo 2.9 1.2 1.6 1.2 dcSTX 0.3 0.1 0.2 0.1 STX 3.9 1.6 2.4 1.8 C1 2.1 0.9 1.1 0.8 C2 88 36 30 23 Total 242 100 132 100 Other tissues GTX4 2.1 42 2.0 49 GTX1 -- -- -- -- dcGTX3 + GTX5 0.1 1.6 0.1 1.3 GTX3 0.2 3.4 0.1 3.4 GTX2 -- -- -- -- dcNeo + Neo -- -- -- -- C1 0.1 1.9 0.1 2.2 C2 2.5 50.7 1.8 43.8 Total 5 100 4 100 Values are expressed in micrograms STX di-HCl equivalents/100 g (T) and as a proportion of the total content of toxins in this tissue (percent). On each day, the compound dominating the toxin profiles is shown in bold type. GTX, gonyautotoxins; dc, decarbamoyl; Neo, neosaxitoxin; STX, saxitoxin; C1 and C2, C toxins.
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|Author:||Contreras, Andrea M.; Marsden, Islay D.; Munro, Murray H.G.|
|Publication:||Journal of Shellfish Research|
|Date:||Dec 1, 2012|
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