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Accumulation of domoic acid by the sea scallop (Placopecten magellanicus) fed cultured cells of toxic Pseudo-nitzschia multiseries1 Donald J. Douglas, Ellen R. Kenchington, Carolyn J. Bird, Roger Pocklington, Brenda Bradford, and William Silvert
Abstract: Sea scallops (Placopecten magellanicus) were fed Pseudo-nitzschia multiseries (formerly P. pungens f. multiseries, Nitzschia pungens f. multiseries) cells of high domoic acid (DA) content (4.0–6.7 pg DA⋅cell–1) for 22 days, followed by 14 days of feeding with nontoxic microalgae. DA was incorporated within 24 h by the scallops, with increased uptake after 6 days, and was concentrated in tissues in the following order: digestive gland >> remaining soft tissue >> adductor muscle. A maximum DA concentration of 3108 µg⋅g–1 was recorded in the digestive gland, approximately 150 times the regulatory limit (20 µg DA⋅g–1) and among the highest levels observed in bivalve molluscs; however, only trace amounts, 0.7–1.5 µg⋅g–1, were found concomitantly in the adductor muscle. At the end of the exposure period, 50.9% of the DA that had been supplied to the scallops had been incorporated into the tissues. Concentrations in the digestive gland 14 days after termination of the toxic diet remained high, 752 µg DA⋅g–1. Throughout the experiment, there was no sign of illness or mortality attributable to high DA loading, although the destructive sampling of animals did not allow us to assess the effects of the toxin in the longer term. Résumé : Des pétoncles géants (Placopecten magellanicus) ont été alimentés avec des cellules de Pseudo-nitzschia multiseries (anciennement P. pungens f. multiseries, Nitzschia pungens f. multiseries) à teneur élevée en acide domoïque (AD) (4,0–6,7 pg AD⋅cellule–1) pendant 22 jours, après quoi ils ont été nourris avec des algues microscopiques non toxiques pendant 14 jours. L’AD a été incorporé en moins de 24 h par les pétoncles, une absorption accrue étant observée après 6 jours, et l’acide a été concentré dans les tissus dans l’ordre décroissant suivant : glande digestive >> tissus mous restants >> muscle adducteur. Une concentration maximale d’AD de 3 108 µg⋅g–1 a été enregistrée dans la glande digestive, soit environ 150 fois la limite fixée par la réglementation (20 µg AD⋅g–1) et cette concentration figure parmi les valeurs les plus élevées jamais enregistrées chez un mollusque bivalve; toutefois, seules des quantités traces, 0,7–1,5 µg⋅g–1, ont été observées en même temps dans le muscle adducteur. À la fin de la période d’exposition, 50,9% de l’AD qui avait été fourni aux pétoncles avaient été incorporés dans les tissus. La concentration dans la glande digestive 14 jours après la fin du régime alimentaire toxique demeurait élevée, soit 752 µg DA⋅g–1). Pendant toute la durée de l’expérience, on n’a observé aucun signe de maladie ou de mortalité attribuable à l’exposition à des concentrations élevées d’AD, bien que l’échantillonnage destructif des animaux ne nous ait pas permis d’évaluer les effets de la toxine à plus long terme. [Traduit par la Rédaction]
Introduction In the autumn of 1993, sea scallop (Placopecten magellanicus) fishers in the lower Bay of Fundy reported a mass mortality of scallops on German Bank (43°20′N, 66°25′W) at approximately 100 m depth. Mass scallop mortalities have occurred previously in this area (Kenchington and Lundy 1991; Robinson et al. 1992), but the cause of death has remained elusive. Scallops were collected from German Bank, with the assistance of
the fishers, and a number of tests were performed on them. Disease and parasite diagnostics were completed by the Fish Health Service Unit of the Department of Fisheries and Oceans, Canada. Nine of the 50 animals had Rickettsia-like inclusions in the digestive tubes and 3 had abscesses in the kidney connective tissue, which contained normal-looking haemocytes. However, this level of infection is not abnormal, and the histopathological examination did not identify significant tissue damage or host response to insult amongst the 50
Received October 6, 1995. Accepted September 27, 1996. J13104 D.J. Douglas2 and C.J. Bird.3 Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, NS B3H 3Z1, Canada. E.R. Kenchington and B. Bradford. Department of Fisheries and Oceans, Science Branch, P.O. Box 550, Halifax, NS B3J 2S7, Canada. R. Pocklington and W. Silvert. Department of Fisheries and Oceans, Bedford Institute of Oceanography, P.O. Box 1006, Dartmouth, NS B2Y 4A2, Canada. 1 2 3
NRCC contribution No. 38111. Present address: Industrial Research Assistance Program, National Research Council of Canada, Bedford Institute of Oceanography, P.O. Box 1006, Dartmouth, NS B2Y 4A2, Canada. Author to whom all correspondence should be addressed. e-mail:
[email protected]
Can. J. Fish. Aquat. Sci. 54: 907–913 (1997)
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animals. Specific disease agents that have been previously identified with shellfish mortality were absent, including the protistan parasite Perkinsus and Ostracoblabe, the agent of “shell disease.” One hypothesis favoured by the fishers was that the scallops were starving. However, the digestive glands of the 25 scallops in the sample were full, indicating that the animals were ingesting food. Analysis of the gut contents revealed an assortment of centric and pennate diatoms, including cells and fragments of Pseudo-nitzschia pungens (formerly P. pungens f. pungens, Nitzschia pungens f. pungens; see Hasle 1995). There were also a large number of sponge spicules, which indicated recent storm activity. The presence of the diatom Pseudo-nitzschia, albeit a nontoxic species, prompted an assessment of the domoic acid (DA) content of the digestive gland. High-performance liquid chromatography (HPLC) revealed a DA content of 93.4 µg⋅g tissue–1 in a blended sample of the scallop digestive glands. This level is well above the Canadian regulatory limit of 20 µg DA⋅g tissue–1 and was considerably higher than the highest levels of DA previously recorded in other bivalves from the Bay of Fundy, 74 µg⋅g–1 in Mytilus edulis and 53 µg⋅g–1 in Mya arenaria (Martin et al. 1993). Although some bivalve molluscs have been reported to contain high concentrations of DA and not show any symptoms (e.g., Bates et al. 1988), the spiny scallop (Chlamys hastata) has been observed to die rapidly after being exposed to cultures of toxic Pseudo-nitzschia multiseries (Dr. J.N.C. Whyte, Department of Fisheries and Oceans, Pacific Biological Station, Nanaimo, BC V9R 5K6, personal communication). Uptake, partitioning among the body tissues, and elimination of some of the marine microalgal toxins, e.g., those responsible for paralytic shellfish poisoning (PSP), have been investigated in several shellfish species (Cembella et al. 1993, 1994; Waiwood et al. 1995). Compared with some other bivalve molluscs, sea scallops appear to concentrate and retain PSP toxins readily. There have been few published investigations of the uptake, localization, and elimination of DA in the sea scallop, a species of considerable commercial importance, especially in the Atlantic provinces of Canada. In one preliminary study, Scarratt (1991) reported that bay scallops (Argopecten irradians) took up DA to concentrations of 60 µg⋅g–1 in the digestive gland after approximately 84 h of exposure to toxic P. multiseries. DA concentrations were observed to fall to 5 µg⋅g–1 after 48 h of depuration. In his report, Scarratt noted that longer exposure and depuration periods, with adequate supplies of toxic algae, were needed to gain information more comparable with natural conditions. On the basis of the unexplained deaths of sea scallops carrying a significant toxin load and the paucity of information on DA uptake and clearance, we designed an experiment to determine whether sea scallops graze effectively on toxic cells of P. multiseries. We expected that if the toxic cells were ingested and the toxin retained, it would be possible to assess whether this resulted rapidly in illness and (or) mortality in the sea scallops as seen in the spiny scallop. By feeding sea scallops with highly toxic, cultured cells of P. multiseries and following the incorporation of the toxin in the tissues, we were able to further the understanding of the kinetics of DA in this commercially important shellfish species.
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Materials and methods Cultures of P. multiseries The source material of DA for the scallop-feeding experiment was a nonaxenic strain of P. multiseries (KP59) isolated from New London Bay (46°30′N, 63°27′W), Prince Edward Island, Canada, by K. Pauley and kindly provided by J.C. Smith, Department of Fisheries and Oceans, Gulf Fisheries Centre, Moncton, N.B., Canada. The culture stock was maintained at 15°C in F/2 medium (Guillard 1975) with a 12 h light : 12 h dark cycle. Cells for feeding were grown in glass multiport carboys equipped with teflon stirring paddles driven by overhead motors (Bellco Glass Inc., Vineland, N.J.). The flasks were filled with filtered seawater and sterilized in an autoclave. After the seawater had cooled, nutrients (F/2 Algae Food, Fritz Chemical Co., Dallas, Tex.) were added (2.0 mL per 10 L of solutions A and B; 20 mL of a 30 g⋅L–1 working solution of sodium metasilicate) using sterile filtration. The flasks were aerated with sterile-filtered air and adjusted, with stirring, to an initial pH of approximately 8.0 with sterile-filtered CO2. Flask cultures were grown at 18°C at a photon flux density of 150 µmol⋅m–2⋅s–1 provided by Cool-White fluorescent lights, with a 12 h light : 12 h dark period. A 15-L stirred flask was used to grow the inoculum for the initial 35-L flask culture; 35-L flasks were used for all of the remaining cultures used in the feeding experiment. The 15-L flask was inoculated with 1000 mL of a P. multiseries culture grown in a 2800-mL Fernbach flask on a shaker. The first 35-L stirred flask was inoculated with 2.5 L of culture from the 15-L flask using sterilized tubing and a peristaltic pump. Every 3–4 days, another 35-L flask was prepared with sterile culture medium and inoculated with 2.5 L of the previous, i.e., 3- to 4-day-old, culture. During the feeding experiment, seven stirred flasks were prepared sequentially to provide a continuous supply of toxic P. multiseries cells for 22 days. Daily samples were withdrawn from the stirred flask cultures for measurement of pH, DA content, and optical density. When the pH increased to >8.2, sterile-filtered CO2 was added to the culture to reduce the pH to approximately 8.0. Samples (20 mL) for analysis of DA were frozen immediately and stored at –20°C. Optical density (at 750 nm) was measured with a colorimeter probe (model PC 800, Brinkmann Instruments, Westbury, N.J.; Douglas and Bates 1992). The cell concentration of P. multiseries in the cultures was calculated from the regression of a dilution series of microscopic cell counts versus optical density (Sorokin 1973). To ensure a relatively high DA content in the cells, cultures were used for the feeding experiment after they had been in stationary growth phase for at least 3 days. Scallop feeding The feeding experiment took place in the Quarantine Unit of the Department of Fisheries and Oceans Halifax Fisheries Laboratory. The facility is equipped with a pumped supply of seawater and a reservoir where returning water is treated with chlorine to prevent live cells from being released into the receiving waters. Sea scallops were collected in early March 1994 from the Bay of Fundy, using a conventional scallop drag, and were maintained in standard holding tanks at the Department of Fisheries and Oceans for several months prior to the start of the experiment. Sixty individuals with a mean shell height of 79 ± 14 mm were randomly divided into two equal groups and placed in two 40-L tanks. A continuous flow of sand-filtered seawater was supplied to the tanks at ambient temperature (7.5–8°C). The scallops were acclimated for 4 days in the tanks before beginning the feeding experiment with toxic P. multiseries. During this period, they were fed a maintenance diet of cultures of Chaetoceros muelleri and Thalassiosira pseudonana on alternate days. The tanks were equipped with several aerators to maintain the algae in suspension. Immediately before the feeding experiment, three scallops were collected to provide an initial sample (T = 0). On each day of the feeding experiment, beginning at day 0, 10 L of the culture was gently © 1997 NRC Canada
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Douglas et al. siphoned by sterile silicone tubing from the 35-L flask into a clean 12-L carboy for delivery to the scallops. This carboy was placed above the scallop tanks and aerated to maintain the cells in suspension while its contents were transferred gradually to the tanks. For the first 3 days of the feeding experiment, the P. multiseries cells were supplied by a gravity drip tube. After the 3rd day, a peristaltic pump was used to obtain a more constant delivery rate, approximately 5 mL culture⋅min–1⋅tank–1. The scallops were thus supplied for 22 days with a continuous flow of P. multiseries cells at relatively uniform density and toxicity. Earlier trials with this method of delivery indicated that there was no appreciable disruption of chains or cells. From day 22 through day 36, the scallops were fed again with C. muelleri and T. pseudonana, alternating daily between the two species. The general health and feeding states of the scallops were observed daily. Feces were also examined to provide evidence of ingestion and digestion of P. multiseries. Initially, water samples were collected from the feeding tanks in an attempt to estimate the ambient concentration of P. multiseries. However, owing to the apparently high ingestion rates of the scallops, these samples were found to contain virtually no P. multiseries cells and cell counts in the tanks were discontinued. Sampling of scallops To allow for variability among individuals, three scallops (sampled randomly, two from one tank and one from the other tank, alternating between samplings) were collected and the various tissue types pooled for each sample. This sampling protocol was patterned on the standard method of shellfish sampling to monitor DA content and was intended to show the average response of the scallops as far as the available number of individuals would allow. During the feeding period with toxic P. multiseries, the scallops were sampled daily for the first 9 days and then every 2nd day for an additional 12 days. After 22 days, when the diet was changed to nontoxic algae, the scallops were sampled daily for 2 days and then at days 27, 31, and 36 (end of the experiment). Immediately after each sampling, the three scallops were dissected. Shell height, total organic tissue weight, and the weights of the digestive gland, adductor muscle, and remaining soft tissue (mantle, gills, and immature gonad) were measured and recorded. The pooled sample of each tissue type was then frozen (–85°C) pending analysis for DA.
weight of the digestive gland, which are highly correlated. The results did not differ greatly between the two, and we chose the assumption that the ingestion rate was proportional to the weight of the digestive gland. Because slightly over 50% of the total DA added to the scallop tanks over the 22-day feeding period was later found in the digestive glands of the scallops, we assumed that a fixed fraction of available DA would be ingested and that the ingestion rate of each scallop would be given by ai = FAwi /W where ai is the amount of DA ingested per day by the ith scallop (all of which is assumed to be in the digestive gland), F is a constant fraction, A is the amount of DA added per day to the scallop tank, wi is the weight of the digestive gland of the ith scallop, and W = Σwi is the total weight of all the digestive glands of scallops still present. The value of W decreased with time, since three scallops were removed on each sampling date, and therefore the amount of DA available to and ingested by each scallop increased steadily during the course of the experiment until day 22, at which point no more DA was introduced. So far as we could tell, the percentage of available DA ingested did not decrease as the supply per scallop increased, indicating that feeding was always food limited and the scallops never approached satiation. During the first 3 weeks of the experiment the concentration (C) of DA in the scallops was therefore governed by the equation dC/dt = (ingestion) – (detoxification) = ai /wi – kC where k is the depuration rate. After this period, no more DA was added and the equation became dC/dt = –kC which is a standard clearance model.
Results
DA analyses Whole-culture concentrations of DA were determined from frozen 20-mL daily samples of algal culture from the 35-L carboys. Samples were thawed, sonicated, filtered, and then analyzed with the FMOC (Pocklington et al. 1990) or UV diode array (Quilliam et al. 1989) method. In addition, cellular content of DA (as opposed to that present in the liquid medium) was determined daily from the suspension of P. multiseries being fed to the scallops. A 10-mL aliquot from the 12-L feeder carboy was filtered onto a polycarbonate membrane with 3.0-µm pore size (Nuclepore Corp., Pleasanton, Calif.) and both the cells and filtrate were collected. The cells were then resuspended in 10 mL of fresh F/2 medium, and the suspensions and filtrates were frozen separately until analyzed as above for DA. Scallop tissue samples (digestive gland, other soft tissue, and adductor muscle) were analyzed for DA concentration using a UV diode array method (Quilliam et al. 1989).
Growth and DA production of the P. multiseries cultures The growth rate of the algal cultures in 35-L flasks was exponential for 6–9 days and then decreased over the next few days as the cultures approached stationary phase (Fig. 1a). Cell concentration at stationary phase in the seven cultures varied from 2.66 × 105 to 3.30 × 105 cells⋅mL–1 (average = 2.85 × 105 cells⋅mL–1). Production of DA began as the cultures approached stationary phase and continued for several days (Fig. 1b). DA concentration reached 1200 –1700 ng⋅mL–1 in the whole culture, or 3.97– 6.67 pg DA⋅cell–1 (average = 5.04 pg DA⋅cell–1). Separate analyses of the cells and their culture medium indicated that 61.6–100% (average = 87.0%) of the total DA in the culture was located in the cells when the cultures were fed to the scallops (Fig. 2). There was a decline in the percentage of cellular DA over the 3–4 days that each culture was used from an average of 92% on the first day of harvest (culture age 12–14 days) to an average of 79.8% on the final day of harvest (age 14–17 days). To demonstrate this trend further, two stirred cultures that were not used in feeding experiments were maintained for 27 days; these showed a marked decrease in the proportion of toxin in the cells to levels below 50% (Fig. 2).
Modelling For analysis of these data, we used a model similar to that used by Silvert and Subba Rao (1992) in a study of the uptake of DA by mussels, modified to deal with a variable rate of uptake. As in the earlier work, the uptake rate was assumed to be proportional to the algal concentration in the water column. The quantity of algae ingested per day by individual scallops was assumed to be proportional to the size of the scallop. We considered both total wet weight and the
Accumulation and depuration of DA by the scallops The concentration of DA in the various scallop tissues over time is presented in Fig. 3. Assays of the digestive gland showed clearly that the scallops began to feed on P. multiseries immediately upon its introduction into the tanks (Fig. 3a). The vast majority of the toxin accumulated by the scallops was concentrated in the digestive gland (note changes of scale in © 1997 NRC Canada
910 Fig. 1. (a) Growth and (b) whole-culture DA production of P. multiseries in 35-L stirred flasks.
Figs. 3a, 3b, and 3c). There was a nearly linear and relatively low uptake rate into the digestive gland for the first 6 days of the toxic diet (Fig. 3a), followed by a substantially greater rate of accumulation until (and apparently for a short time after) the supply of toxic algae was terminated. The concentration of DA in the digestive gland was maximum, 3108 µg⋅g–1, on day 24 and then declined rapidly (Fig. 3a). It is notable, however, that DA concentration in the digestive gland remained relatively high (752 µg⋅g–1) after 14 days on a nontoxic diet (Fig. 3a). Virtually no DA was incorporated by the adductor muscle or “meat” of the scallops (Fig. 3b). The only two samples containing detectable amounts of DA in the adductor muscles were those taken towards the end of the exposure period (days 18 and 22), when DA concentrations in the digestive gland were at or approaching maximum. Exceedingly low levels of toxin, 1.5 and 0.7 µg DA⋅g–1, were observed in the muscle and were lost in less than a day after termination of the toxic diet (Fig. 3b). Small but significant amounts of DA were also found in the soft tissue other than the digestive gland. The DA content of this tissue increased during the exposure period to a maximum
Can. J. Fish. Aquat. Sci. Vol. 54, 1997 Fig. 2. Percentage of DA in the cellular fraction of P. multiseries cultures versus age of culture at time of harvest.
of 36.7 µg⋅g–1 (Fig. 3c), although concentrations varied considerably more among the samples over time than did those in the digestive gland. Also, unlike the pattern observed for the digestive gland, DA levels in this tissue declined almost immediately after the supply of toxic algae ceased but then remained relatively stable at approximately 10–12 µg⋅g–1 until the end of the experiment (Fig. 3c). Calculation of the budget of DA, i.e., the relationship between DA supplied by the algae and that recovered in the various scallop tissue types, is presented in Table 1. Over the course of feeding with toxic algae, approximately 25–73% of the total DA fed was present in the scallop digestive glands. Only 5–13% of the supplied DA was located in the other soft tissue, and the adductor muscle contained less than 0.1% on the only 2 days that enough DA was present to be detected (Table 1). At the end of the feeding period with toxic algae, 45.9 and 5.0% (total of 50.9%) of the DA that had been fed was contained in the digestive gland and other viscera, respectively. Concentrations of DA in the digestive gland and remaining soft tissue varied considerably, particularly after the initial 6 days of the toxic diet (Fig. 3). This variability cannot be explained by analytical error, as the precision of the analytical methods was on the order of 3%. Thus, the scatter in the data represents either variations in the collective feeding behaviour of the scallops or variation among individual scallops. We believe that the latter is more likely, as other studies have reported large differences in the toxin content of individuals (White et al. 1993). Although we attempted to establish trends and reduce the scatter by pooling the tissue types from three scallops at each sampling, this clearly did not fully resolve the problem. It must be noted that the variability we observed does not allow fine-scale, i.e., daily, rates of grazing or depuration to be determined accurately. It was for this reason that we chose to model the data to search for a pattern independent of the natural variability. Modelling Initial attempts to fit an uptake–clearance model to the data were unsuccessful because the initial rate of increase of the toxin load was much lower than that observed later in the experiment, and this type of model does not describe any such © 1997 NRC Canada
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Douglas et al. Fig. 3. Uptake and depuration of DA in sea scallop (a) digestive gland, (b) adductor muscle, and (c) remaining soft tissue.
Table 1. Cumulative domoic acid (DA) supplied to scallops and the percentage of the DA supplied that was present in the three tissue compartments.
Time (days) 0 1 2 3 4 5 6 7 8 10 12 14 16 18 22 23 24 27 31 36
Percentage of DA supplied present in:
Cumulative DA supplied (mg)
digestive gland
other soft tissue
adductor muscle
0.0 12.2 24.2 35.2 48.5 60.0 71.0 81.7 98.0 130.8 155.6 178.8 202.2 228.0 272.7 272.7 272.7 272.7 272.7 272.7
0.0 33.8 25.6 30.1 28.7 25.2 34.8 37.2 43.7 73.3 58.7 58.2 46.2 57.8 45.9 nd 44.0 37.0 nd 34.4
0.0 0.0 0.0 0.0 12.0 9.6 4.9 6.8 8.3 13.0 9.5 6.8 5.3 7.1 5.0 4.1 4.2 4.1 4.2 4.2
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
