mussels (Mytilus edulis) and deep sea Atlantic scallops (Placopecten magellanicus) to elucidate conditions under which domoic acid (DA) was accumulated and ...
Journalof Applied Phycology 4: 297-310, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
297
Dynamics of the phycotoxin domoic acid: accumulation and excretion in two commercially important bivalves Gary D. Wohlgeschaffen, Ken H. Mann, D.V. Subba Rao & Roger Pocklington Department of Fisheries and Oceans, Bedford Institute of Oceanography, P.O. Box 1006, Dartmouth, NS, Canada Received 9 May 1992; revised 25 June 1992; accepted 26 June 1992
Key words: Nitzschia, neurotoxin, domoic acid, Mytilus, Placopecten Abstract Batch cultures of the toxigenic diatom Nitzschia pungens Grunow f. multiseries Hasle were fed to blue mussels (Mytilus edulis) and deep sea Atlantic scallops (Placopecten magellanicus) to elucidate conditions under which domoic acid (DA) was accumulated and excreted (depurated). Mussels accumulated the toxin to a maximum level of 13 #g g- 1, at rates of 0.21 to 3.7 ug h- 1 g- ' dry weight. Accumulation efficiency (the proportion of accumulated DA to estimated net uptake) ranged from 1-5%. The highest filtration rate of 1.7 1 h - ' occurred at concentrations of 4-8 x 106 Nitzschia cells 1- with no formation of pseudofeces. Depuration rates between fed and starved mussels over a 2 h test period were the same. The depuration rate of domoic acid was about 17% d- ' and did not account for the low uptake efficiencies, so it is suggested that most of the DA is lost from mussels in the solution during the feeding process. Domoic acid accumulation in mussels was dependent on the amount of toxin available, which in turn was a function of the density and growth phase of the Nitzschia population. Changes in filtration rate with Nitzschia concentration and depuration rate with time can account for the DA levels of mussels collected during toxic episodes in Cardigan Bay, Prince Edward Island, Canada in 1988 and 1989. Scallops accumulated DA (0.39-1.3 g h- ' g- ) more slowly than mussels, however, accumulation efficiencies ranged from 5-100%. Filtration rates remained relatively low and constant at 0.08 1h- '. Scallops retained domoic acid longer than mussels, a fact which must be considered in the marketing of whole scallops for human consumption.
Introduction During autumn 1987, amnesic shellfish poisoning in Eastern Canada, caused by domoic acid, affected over 100 people who had consumed contaminated, cultured blue mussels (Mytilus edulis) from the area of Cardigan Bay, Prince Edward Island (P.E.I.) (Addison and Stewart, 1989). The diatom Nitzschia pungens dominated the phy-
toplankton in bloom proportions (Bates etal., 1989) from mid-December (8 x 106 - 15 x 106 cells - '), through January (2 x 106 cells 1- ), to mid-February (1 x 106-5 x 106 cells - '), while domoic acid in samples of M. edulis from the area reached a peak of 790 uig g - wet weight of meat in November, which declined to zero by April. Cells of Nitzschia pungens Grunow f. multiseries isolated from Cardigan Bay water samples
298 (Subba Rao et al., 1988) yielded DA (Subba Rao et al., 1990); however, the transfer of DA from the diatom to mussels was not experimentally established at that time. The objective of the present study was to understand the process of domoic acid accumulation in blue mussels, as well as deep sea Atlantic scallops (Placopecten magellanicus) whose average annual contribution to the fishing industry on the Grand Banks and Georges Bank is Can $81 million. Both accumulation and depuration (by egestion or excretion) were studied, since it was possible that the depuration rate could equal or exceed the accumulation rate. Materials and methods Bivalves Juvenile mussels were collected from the intertidal zone of P.E.I.: Cardigan Bay on 28 Feb 1989, and Rustico Bay on 13 Dec. 1989. Cultured scallops of 1-1.5 y old supplied by the Great Maritime Scallop Trading Co. were obtained from Passamaquoddy Bay, New Brunswick. Control specimens of the bivalves maintained in running, aerated sea water at ambient temperature (410 C) at the Bedford Institute of Oceanography did not yield DA. Animals were acclimated for 24 h at 15 C before each experiment.
1 #gDA ml - 1 of extract compared to about 100 g ml- 1 for the mouse bioassay.
Phytoplankton Nitzschia pungens f. multiseries, clone NPBIO (Subba Rao et al., 1988), was maintained in FE medium at 10 C and 490-500 Amol photon m- 2 s-1 continuous fluorescent light and 130-1501 volumes of culture producing DA were grown in a vat incubator with immersion core illumination (VIICI; Wohlgeschaffen et al., 1992). Cylindrothecafusiformis var. fusiformis (Bigelow Laboratory, clone MNC), a non-DA producing pennate diatom (dimensions 3.0 + 0.60, m x 33 + 1.5 m), was chosen as a surrogate for N. pungens f. multiseries (dimensions 5.3 + 0.26 im x 51 + 2.3 m) in one experiment, and maintained similarly. Samples of phytoplankton were taken for DA determination (Pocklington et al., 1990) and cell counts. Any samples from bivalve feeding experiments that contained pseudofeces (mucus strands bearing undigested cells) were sonicated for 2-5 min (Ultrasonik, NEY, 60 W) then enumerated; this procedure did not disrupt the cell membranes.
Experimental methods Sample analysis Whole mussels and scallops were stored frozen (-25 C). The shell contents of thawed specimens were removed, rinsed with glass-distilled water, blotted dry, and weighed. The digestive gland stomach complex (henceforth simply called the digestive gland) was dissected, rinsed, blotted dry, weighed and finely chopped, then transferred to a centrifuge tube and weighed. A single-step extraction of domoic acid, followed by HPLC analysis with diode array detection was employed according to the method of Quilliam et al. (1989) who obtained good reproducibility and estimated recovery at 100%. The detection limit was
The series of experiments on DA accumulation in the bivalve digestive gland showed the necessity of long time-series of measurements requiring several tens of bivalves, and an abundant supply of Nitzschia containing DA, hence large volumes of Nitzschia were produced in a VIICI culture apparatus and batch-renewal of food was replaced by a flow-through design. Animals that had been loaded with DA were transferred to DA-free Bedford Basin water and sampled periodically to determine DA depuration (decline in the DA concentration in the digestive gland through egestion or excretion). The DA accumulation experiments are reported first, followed by the depuration experiments.
299 Experiments 1, 2 & 3: batch-renewal Experiment 1. The accumulation of DA by mussels over a short time was examined employing frequent sampling. Four litres of a culture of N. pungens in the stationary phase of growth were used to feed mussels in a 3.5 h experiment. Surface illumination was kept at 8.5-9.0 btmol photon m - 2 s-1 to prevent growth of the algae. Twenty-four mussels (35-45 mm length) were placed in 2 1 of culture. After 1 h, two mussels were taken, followed by 4 more after 1.5. At this time the grazed culture was replaced with 2 1 of fresh culture. Half an hour later, two more mussels were taken, and thereafter 3 mussels were removed every 15 min. On each occasion the mussels were combined to give the required amount of tissue for the DA analysis. Each time mussels were sampled, phytoplankton concentration of the medium was determined. Experiment 2. A much higher concentration (about 220 x 106 vs. 14 x 106 cells 1- in experiment 1) of N. pungens was used and the culture was changed every 30 min. Mussels (140 of 3545 mm length) were placed in 4.2 1 of culture. Every 30 min the grazed culture was poured off and fresh culture added (in decreasing amounts to match decreasing mussel numbers). Every hour, before renewing the medium, 20 ml culture samples were taken for cell counts and DA analysis, and 20 mussels were removed. These mussels were divided into 2 groups of 10, each of which was pooled for DA analysis. The experiment lasted 3 h. Two aspects of the results influenced subsequent experiments. The first was that in experiment 1, the amplitude of the DA signal on the HPLC-DAD from 3 pooled mussels was small compared with results from experiment 2 using 10 pooled mussels, so a standard sample size of 5 pooled mussels was decided. Secondly, it was clear that much larger volumes of diatom culture were required to present the mussels with a more constant food supply; a third experiment was done to estimate the filtration rate of mussels and calculate the rate of replenishment of culture. Experiment 3. An experimental medium was prepared by mixing 1.5 1of N. pungens culture with 60 1 of 0.4 m filtered Cardigan Bay sea water.
Ten mussels (41.1-43.6 mm length) were placed in a container with 10 1of the medium, which was gently aerated and circulated slowly with a polypropylene stirrer. Cell concentration was determined at the beginning and after one hour. The procedure was repeated 6 times using the same 10 mussels. Experiments 4 & 5: domoic acid accumulation in a flow-through system In experiment 4, seventy-five mussels (40-45 mm length) were placed on an elevated plastic grid in a 12 1 feeding chamber (Fig. 1). Carboys (10 1) served as reservoirs for Nitzschia grown in a VIICI. A peristaltic pump delivered the culture from the carboys to the feeding chamber. Carboys were refilled as required, and kept well mixed by vigorous aeration, that also kept the phytoplankton in suspension in the feeding chamber. Every 12 h, the following samples were taken from the feeding chamber: 20 ml of water for cell counts, 2 samples (20 ml each) of water for DA analysis, 3 samples of 5 mussels each for DA analysis. The experiment lasted 36 h. Experiment 5 was a parallel experiment which examined accumulation of DA in Placopecten magellanicus. Seventy-five scallops (38-43 mm shell height) were used. Depuration of domoic acid from fed and starved mussels The concentration of DA in mussel digestive glands was monitored after the cessation of feeding on Nitzschia to measure depuration. In view of its commercial application, it was of interest to know whether the process of depuration would proceed faster if mussels continued to feed on a high concentration of phytoplankton, a situation simulating a natural bloom. Cultured Cylindrothecafusiformis var. fusiformis was used as DA-free food. Eighty mussels which had accumulated DA in experiment 2 were divided into two groups. Half were placed in 1.2 1 of a dense culture (average 291 x 106 cells -1) of Cylindrotheca which was replaced every 0.5 h for 2 h, the other half in DA-free Bedford Basin sea water which was replaced every 0.5 h. After 1 h and again after 2 h,
300 line
pump
10 phyt
JW
pi; Fig. 1. Schematic diagram of flow-through system used to feed bivalves with a continuous supply of the diatom N. pungens f. multiseries, and mathematical variables used in the calculation of filtration and ingestion rates.
duplicate samples each of 10 mussels were taken from each container and analyzed for DA content. At the same time changes in the concentration of Cylindrotheca cells were monitored. Comparisons of depurationfrom mussels and scallops To examine depuration of domoic acid over 24 h, the 30 mussels and 30 scallops remaining from DA loading experiments 4 and 5 were transferred to glass containers supplied with ambient, flowing Bedford Basin sea water. Every 12 h, three samples of 5 mussels and 3 samples of 5 scallops
were taken for DA analysis. Algal cells were enumerated. Depuration rates were calculated as the percent decrease of DA per 12 h sampling interval and were normalized to 1 g dry weight of bivalve meat using the allometric function, a(W/Wo)b, where 'a' and 'b' were determined graphically, W o = 1 g, and W dry weight calculated by linear regression of dry vs. wet weight for the mussels and estimated as 15% of the wet weight for scallops. In a longer experiment, Nitzschia was supplied to mussels and scallops for 10 days, thus approaching more closely the conditions of a natu-
301 ral bloom. A continuous supply of Nitzschia was pumped to a feeding chamber (Fig. 1) containing 16 mussels (38-43 mm) and 16 scallops (4850 mm) for 10 d. Then the bivalves were held in flowing Bedford Basin sea water for 15 d. Samples for DA analysis consisted of two batches of 4 mussels each and the same for scallops, taken at the end of the 10-day loading period, and after the 15 d in running sea water. Samples of phytoplankton were taken from the feeding chamber and the carboys on days 1, 4, 8 and 9 to measure cell concentrations and DA levels.
Calculation offiltration rates To calculate filtration rates (F), bivalve feeding was approximated by a model (Fig. 1) in which the change in the ambient cell concentration of Nitzschia in a feeding chamber with time is equal to the rate of inflow of cells, minus the rate of outflow, minus the rate at which cells are filtered (cleared) from the medium: dC dt
C R - CR - CNF V
(1)
where Cr = phytoplankton concentration of inflow (cells 1- 1); R = flow rate (1 h- ); C = ambient concentration of phytoplankton in the feeding chamber (cells -I1); t = time (h); V = volume of medium in which bivalves are feeding (1); N = number of bivalves; and F = filtration rate ( h- 1 g- ). Implicit is that the algal cells were kept uniformly distributed throughout the medium (which was the purpose for the vigorous aeration), that algal growth was negligible (which is reasonable because of the dim light used) and that the pumping rate of the bivalves was constant. The last assumption underscores the need for long-term experiments. If the inflow of phytoplankton cells
(CR) is balanced by the outflow (CR) plus the amount consumed by the animals (CNF), then the phytoplankton concentration in the feeding chamber would change very little over time and an equilibrium concentration (C,) would be established; when dCt concentration '=CRoaches dan CNF '+ and concentration 'C approaches 'C,', so CrR
- R + NF'
(2)
The decrease in phytoplankton concentration resulting from grazing is an exponential function of time (Coughlan 1969), thus the ambient phytoplankton concentration in the feeding chamber at a particular time, Ct, is presumed to be equal to the equilibrium concentration, C, plus the product of the natural log function and the difference between the initial cell concentration in the chamber (CO) and C.,: C = Co*+ (Co - C,)e - k ' .
(3) The rate constant, 'k', for algal cell depletion in (3) is equal to the rate of removal of cells due to water flow plus the rate at which cells are being removed because of bivalve filtering: kR
+NF V
(4)
Hence 'kV' is substitute for '(R + NF)' in (2) and 'C, = CrR/kV' is used in equation 3. The filtration rate ( h - 1 animal - 1) derived from (4) is F- kV-R N
(5)
and is calculated with varying values for 'k' in (3) until C,t most closely matches the actual measured final Nitzschia cell concentration of the feeding chamber, then substituting this value for 'k' in (5). Batch-renewal experiments represent a special case for the feeding model in which the flow rate, R, equals zero, thus C., equals zero. The underlying assumption that all the cells filtered from the water are ingested was accounted for by including in cell counts any phytoplankton cells that
302 occurred as pseudofeces in the samples of medium. Average Nitzschia cell concentration, used in the estimation of gross uptake of domoic acid by bivalves, was calculated as Average cell concentration
=-| Cdt t o
=
Co (e-kt,
(t - t) + C(e
C
k
e-k2)
(6)
e2)
Results Domoic acid accumulation Mussels in batch-renewal experiments reduced to under 20% of the inithe cell concentrations ] tial levels within half an hour (Fig. 2a). The ex-
ponential curves describing the decrease in the concentration of Nitzschia cells in the medium were based on equation 3 (see methods). A small quantity of pseudofeces produced in experiment 2 was not measured. In the grazing trials of experiment 3, cell concentrations never fell below 8% of the initial value (Table 1)and pseudofeces were not produced. The concentration of DA in the mussels varied considerably (Fig. 2b). The level in the last 2 samples of experiment 1 was over 0.3 pg g- wet weight while a sample of mussels from the second experiment contained 3.16 ygDA g-1 wet weight, an order of magnitude higher. In the flow-through system the concentration of phytoplankton remained > 13 x 106 cells I 1 (Fig. 3a). Again the exponential curve for the decline in cell concentration was plotted using equation 3 of the methods section. By 36 h about 10% of the Nitzschia cell concentration in the chamber 100
300
a
A
250200-
I
I
o C
1 1
150a
100 N
II
50-
I L___i__
II
: " : " : " : ,, I I
: 1"
11 11 I :I 11 I I I * E .: ' I
·
Expt. 1 Expt. 2 75 4 0
II II
'j
50
I l l'
25
0
- 4~~
15
44
b
0 NO ,$
3 -
10 ._
2-
0 In
A:
-i --
io
0
a
o
5
0
E
0
o 0
2
3
Time (h)
Fig. 2. Batch-renewal experiments. (a) Changes in cell concentration in the medium. Samples in experiment 2 were taken at the beginning and end of each hour, but medium was exchanged every 0.5 h. (b) Toxin in Mytilus. Error bars = 1 S.D.
0
5
10
15
20
25
30
35
40
Time (h)
Fig. 3. Uptake of domoic acid in mussels and scallops fed continuously with Nitzschia in the flow-through system. (a) Changes in cell concentration in the feeding chamber. (b) Toxin in bivalves. Error bars = 1 S.D.
303 Table 1. Filtration rates of mussels and scallops fed on N. pungens. Initial (Co ), final (C,) and inflow (Cr) cell concentrations were measured in the feeding chamber. R = flow rate, k = rate constant. C,
CO
C,
R
Animals
(1 h-
(106 cellsMytilus edulis 0 13.2 0 0.820 0 0.863 0 1.05 0 0.949 0 1.00 0 0.80 0 1.41 158 69.8 122 19.9 138 13.0
1)
Time
Volume
(h)
(1)
k
1
')
3.18 0.750 0.0707 0.242 0.219 0.179 0.217 0.297 19.9 13.0 67.6
Placopecten magellanicus 156 80.8 52.3 162 52.3 33.3 166 33.3 18.9
0 0 0 0 0 0 0 0 2.22 2.42 2.00
18 1 10 10 10 10 10 10 75 60 45
0.25 0.25 1.0 1.0 1.0 1.0 1.0 1.0 12 12 12
3.00 1.26 0.480
75 60 45
12 12 12
with mussels was contained in pseudofeces. Scallops produced few pseudofeces. The equilibrium concentration was usually established about 1-6 h after a sample of animals had been taken from the feeding chamber (from 13-24 h and 3036 h in Fig. 3a). There was significant uptake of DA by the mussels over time (Fig. 3b), and much less variation in DA levels at each sampling time than in batch-renewal experiments. The level peaked at 13 btgDA g- 1 wet weight in one sample of mussels. The level increased more slowly in scallops than mussels reaching a maximum of 4.4 g g- 1 wet weight. Filtration rates of bivalves (Fig. 4) were calculated using the values in Table 1, and corrected to 1 g dry weight. The parameters 'a' and 'b' of the allometric function were 2.58 and 0.57 respectively, similar to those found by other authors for M. edulis (Vahl, 1973; Winter, 1973). Filtration rates of mussels < 0.5 1 h- ' were omitted because of the obvious insufficient supply of food. Corrected rates for mussels ranged from 0.7 to 1.71 h - , increasing from 1.1 1h - at algal concentrations up to 8 x 106 cells 1- ' then decreasing to 0.7 1h - . Filtration rates of scallops corrected
2.00 1.90 10 10 10 10 10 10 12 12 12
12 112 12
Filtration (1 h- ) Uncorrected
Corrected
5.698 0.357 2.501 1.472 1.466 1.718 1.398 1.560 1.471 1.890 0.336
0.63 0.68 2.5 1.5 1.5 1.7 1.4 2.0 0.21 0.34 0.045
1.7 1.2 1.2 1.2 1.2 1.2 1.2 1.2 0.69 0.69 0.69
0.748 0.512 0.355
0.080 0.081 0.084
0.085 0.084 0.082
for dry weight (a = 0.313, b = 0.69) were constant and lower (0.08 1h- 1) than mussels (Fig. 4). Depuration of domoic acid from fed and starved mussels A two-way ANOVA (domoic acid level in mussels was the dependent variable and diet and time were factors) showed significant loss of DA between sampling times (p < 0.005) but not between diets. The concentration of Cylindrothecachanged little (Fig. 5a). Domoic acid decreased from an average of 2.9 to 1.2 ug g- wet weight in 2 h (Fig. 5b). Had the experiment been extended, diet might have been a significant factor, however, the results indicated that it was unnecessary to feed the animals with a surrogate alga to achieve rapid depuration over a short time. Depuration of DA from mussels and scallops Depuration rates were corrected for dry weight of mussels (a = 2.33, b = - 0.57 in the 24 h period of experiment 4). Smaller animals appeared to depurate DA more rapidly than others. The short-term
304 500
2
a 400 -
,.
300 -
Co .a
o0 v
- ............... . ...... "i 200 -
".%.."~~~
¢-
0
C
100 -
0
·i " .0P 0
0 0
10
20
30
40
50
60
70
80
Average Nitzschia Concentration (100 cells 1-1) 2
Fig. 4. Filtration rates of Mytilus and Placopecten. Curve fit-
ted by eye.
(24 h) comparison of DA loss from mussels and scallops showed a rapid decrease of DA in mussels from about 9 to 1.2 gg - 1 wet weight (Fig. 6). This represents a weight-corrected rate of 0.8% h- 1. Over the same period there was no statistically significant loss of DA from scallops (one-way ANOVA, p>0.1) which retained the toxin at levels of 1.4 to 3.6 g g 1 wet weight (Fig. 6). The apparent retention of DA by scallops was consistent with the results of the longer-term feeding experiment. Bivalves were fed with Nitzschia culture from carboys over 10 d and then transferred to flowing sea water for 15 d. By day 10 one sample of mussels had 0.40 IgDA g- 1 wet weight. The toxin was not detected in the other sample of mussels. The two samples of scallops had 0.31 and 0.81 pgDAg - wet weight. Following the 15 d of starvation, no DA was detected in the mussels whereas the two samples of scallops had levels of 0.57 and 0.70 ipgDA gwet weight.
-4
0 M
0
1
Fig. 5. Loss of domoic acid from fed and starved mussels. (a) Changes in cell concentration of the container with fed animals. (b) Average toxin content of each group. Error bars = 1 S.D.
ditions when fed Nitzschia pungens f. multiseries containing the toxin. The low levels of DA in bivalves from the batch-renewal experiments emphasized the need of a flow-through feeding system. Some understanding of the relationship, DA 1
14 .M
12 -
* utue · Pleasopecten
10 8
-3
6-
o o
4.I
Discussion
2 Time (h)
... ------- ---- --
e -
2 ar %J
v
Domoic acid uptake, retention and loss
0
5
10
15
20
25
3C
Time (h)
Both Mytilus edulis and Placopecten magellanicus accumulated domoic acid under laboratory con-
Fig. 6. Loss of domoic acid from bivalves over 24 h. Error bars = 1 S.D.
305 Uptake - DA Loss= DA Accumulated, was based on a comparison of calculated values (Table 2). Estimated gross uptake of domoic acid was calculated as the product of filtration rate (corrected for dry weight of the animal and Nitzschia concentration), average cell concentration (equation 6, methods), intracellular toxin concentration of Nitzschia and time of the sampling interval. The percent loss for the time interval was obtained from the depuration experiments and corrected for animal dry weight. Hence the estimated net accumulation was estimated gross uptake minus the loss. Accumulation efficiency in mussels was between 1 and 5 % suggesting that a very small fraction of the ingested domoic acid is retained. Scallops accumulated the toxin more slowly, but retained it at greater efficiencies (5100 %). By comparison, the ingestion efficiency of paralytic shellfish poison (PSP) by M. edulis was
much higher--about 79% (Bricelj et al., 1990)-based on cumulative ingestion and maximum total toxin levels attained, and the estimated accumulation rates ranged from 1.5 to 2.9 #g saxitoxin equivalents h - ' g- ' in the first 50 h, comparable with the rates for DA (Table 2). The enormous difference between the estimated net accumulation and observed accumulation in mussels as reflected by the low accumulation efficiencies might be due to an overestimation of filtration rates, or to changes in feeding activity of the bivalves from exposure to the toxin. The relationship of filtration rate of M. edulis to the concentration of algae that was fed was the usual one (Winter, 1973, 1978; Schulte, 1975; Bayne et al., 1976; Morton, 1983). The filtration rates of P. magellanicus were remarkably stable over the range of Nitzschia concentrations used, consistent with the observations of Shumway & Cucci
Table 2. Comparison of estimated gross and net uptake of domoic acid (g animal - ') with observed toxin accumulation in two species of bivalve, experiments 4 and 5. A sample with a net loss of domoic acid was omitted. Observed accumulation rate was calculated as the weight-corrected increase in DA level of one sample over the average level of the previous samples. Estimated gross uptake = Filtration rate x Average cell concentration x Intracellular DA x Time, Estimated net accumulation = Estimated gross uptake - Loss, % Efficiency = Observed accumulation/Net x 100.
Filtration
Time
Average
Intracellular
Estimated
Loss
Estimated
Observed
Efficiency
Observe
rate
(h)
cell
domoic
gross
%
net
accumulation
%
accumulation
concentration (106 cell 1- )
acid (pg cell- )
uptake (pg)
accumulation (mg)
(pg)
23.8 23.8 23.8 13.9 13.9 71.8 71.8 71.8
2.0 2.0 2.0 2.3 2.3 1.6 1.6 1.6
388 388 388 268 268 973 973 973
354 354 354 245 245 887 887 887
8.98 7.79 10.2 12.9 9.12 22.5 12.3 10.6
3 2 3 5 4 3 1 1
2.8 2.4 3.4 1.0 0.20 3.7 0.68 0.21
7.47 3.07 3.05 12.0 6.95 10.9 13.5 11.3 12.9
11 5 5 63 36 57 100 85 97
1.3 0.51 0.55 1.3 0.38 1.3 0.70 0.39 0.44
(1 h- )
rate (Mg h-
Mytilus edulis
0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.69
12 12 12 12 12 12 12 12
8.7 8.7 8.7 8.8 8.8 8.8 8.8 8.8
Placopecten magellanicus
0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
12 12 12 12 12 12 12 12 12
61.3 61.3 61.3 41.7 41.7 41.7 26.4 26.4 26.4
1 1 1 0.46 0.46 0.46 0.51 0.51 0.51
62.3 62.3 62.3 19.2 19.2 19.2 13.3 13.3 13.3
0 0 0 0 0 0 0 0 0
62.3 62.3 62.3 19.2 19.2 19.2 13.3 13.3 13.3
1 g- i)
306 (1987). The pseudofeces-free cell density for M. edulis fed on N. pungens f. multiseries was estimated by the highest concentration that precedes a decline in the rate of ingestion (Winter, 1978), and was 4 to 8 x 106 cells 1- ', which is about half the pseudofeces-free concentration when Phaeodactylum tricornutum (3.24.2 ,um x 19.2-27.2 m) was used (Winter, 1978). Though this concentration was frequently exceeded and pseudofeces produced, filtration rates cannot be overestimated because cells in pseudofeces were included in the cell counts. Many authors have described effects of algal toxins on bivalve physiology; responses of different species of bivalves vary (Shumway etal.,
22
1985). We hypothesize that domoic acid inhibits its own assimilation, but in most documented cases a toxin directly or indirectly affects filtration rates and cannot explain the imbalance between estimated net accumulation and observed accumulation (Table 2) which are based on the measured reduction in phytoplankton density due to grazing. The best explanation for the discrepancy is that the method used to determine the depuration of DA did not adequately show the loss during the feeding process. The rate of domoic acid depuration in M. edulis was about 170% d-' (Table 2) compared with 8-13% d- for PSP (Chang et al., 1988). Mussels generally accumulate and lose toxins faster
A__
1 cells of Nitzschia 2 gills 3 cells of Nitzschia inpseudofeces 4 byssal threads 5 foot 6 labial palps 7 mouth 8 anterior adductor
9 major typhlosole in sorting caecum 1 0 ducts of digestive diverticuli in stomach 11 crystalline style 12 minor typhlosole 13 intestinal groove 14 intestine 15 digestive gland
16 17 18 19 20 21 22 23
pericardium ventricle auricle kidney anus posterior adductor soluble DA cells of Nitzschia in feces
Fig. 7. Digestive system of Mytilus edulis (after Morton, 1979) with arrows indicating the possible paths of domoic acid (DA).
307 than other bivalves (Quayle, 1969; Madenwald, 1985; Lassus et al., 1989) and in the case of domoic acid, which is highly soluble in water (Quilliam et al., 1989), a large fraction of the ingested DA could pass into solution in the mussel's stomach and be rapidly lost through the mouth or anus. As domoic acid is hydrophilic in contrast to most other toxins (Fisher & Teyssie, 1986; Ingebrigtsen et al.,, 1988; Bricelj et al., 1990) the process of DA transfer and the fate of the toxin in blue mussels might be described as follows. In a toxic bloom of N. pungens f. multiseries, cells filtered and ingested by a mussel are usually ruptured in the stomach by the action of the crystalline style (Morton, 1979) and most of the DA is probably lost immediately through the mouth (Fig. 7). Fine particles remain in suspension and move passively along typhlosoles to the ducts of the digestive diverticuli while large particles are conveyed to the intestine for egestion. It appears that most of the remaining DA is egested in feces along with some undigested intact cells, and only a fraction of it accumulates in the stomach, digestive diverticuli and intestine. A minute quantity of DA might be assimilated by the mussel (Madhyastha et al., 1991) and excreted from the kidneys. Only a small fraction of the DA ingested by Mytilus seems to be assimilated into the tissues. Of this, some might be converted to another form and retained, for example bound to a less active metabolite with a high tissue residence time, thus prolonging depuration. Biotransformation and differential retention of toxins is common in M. edulis (Ribera et al., 1989; Bricelj et al., 1990; Broman et al., 1990), and P. magellanicus (Shimizu & Yoshioka, 1981). Silvert & Subba Rao (1992) suggested that there is some evidence for this occurring with domoic acid in nature. In their model, known temperature variations had little effect on the rate of DA depuration, however, other researchers (Novaczek etal., 1992) have found that an increase of temperature from 6 to 10 C results in an increase in depuration rate from 39 to 63 % d- 1 for mussels 60-70 mm in length, and smaller mussels depurate faster than
large ones, consistent with our data. Complexing of domoic acid might explain residual levels found in Placopecten in the 'longer-term' experiment. Residual DA has been found in P. magellanicus and Volsella modiolus (red horse mussels) from areas of the Bay of Fundy, Canada (Gilgan et al., 1990).
Implicationsfor mariculture In Cardigan Bay, 1988, the mussels were feeding for about 2 months on a bloom of N. pungens f. multiseries at densities of 0.2 x 106 - 1.3 x 106 cells 1- and attained a peak loading of 280 /g DA g- wet weight. Experimental values for filtration rate as it varies with phytoplankton concentration (Fig. 4) were used in the model developed by Silvert and Rao (1992); the results support the authors suggestion that a decrease in depuration rate with increased time of feeding on Nitzschia, coupled with a Q1 0 of 2.2 could easily account for the accumulation of DA to levels found in mussels from Cardigan Bay in both 1988 and 1989. The apparent rapid loss of domoic acid during the feeding process suggests that a minimum concentration of Nitzschia cells and a minimum level of intracellular DA must be available before the toxin is accumulated in mussels. Field study in Cardigan Bay, P.E.I. (1988) showed a lag of 9 days between the appearance of DAloaded Nitzschia (about 2 pg cell - ) in the water column and the presence of DA in the mussel tissues. At this time the concentration of Nitzschia in the water was 0.2 x 106 - 0.4 x 106 cells 1(Smith et al. 1990), therefore the average particulate DA in the water (Nitzschia concentration x intracellular toxin concentration) was 0.4-0.8 /gDA 1- and may explain the lag in accumulation. The maximum level of 13 /gDA gin mussels in the present study represents 65 % of the current provisional legal limit of 20 Mg g- 1 set by national Health and Welfare Canada (Gilgan et al., 1990). Scallops attained a maximum level of 4.4 /g g- 1, 22% of the limit. The observation that mussels continue to lose DA once they have ceased consumption of
308 N. pungens f. multiseries, though they might feed on other phytoplankton, is confirmed by experience with DA-contaminated mussels in the field; this has commercial significance. After long feeding periods of up to several weeks in a bloom of the toxic diatom in the field, mussels have been known to purge themselves of DA if left in the same locale. Moreover, keeping mussels fed during depuration would maintain their marketability. Temporarily relocating strings of cultured mussels to an unaffected area would accelerate the depuration of DA and might prevent severe economic losses. The same is not true for the red horse mussel, V. modiolus, or the scallop, P. magellanicus which tend to retain DA in the viscera. Red horse mussels are currently not of commercial importance, but scallops are. Domoic acid accumulates in the digestive tissue and gonads of deep sea Atlantic scallops, not in the adductor muscle (Gilgan etal., 1990), which along with PSP's presents an obstacle to the establishment of markets for 'whole scallops' (Shumway et al., 1988), and a point of concern for such extant markets. Experiments to determine the utility of processes for the rapid depuration of DAintoxicated bivalves might be useful. Not all blooms of N. pungens f. multiseries are toxic. The growth stage of the diatom plays an important role in toxicity (Subba Rao et al., 1991). Gametes and zygotes of the diatom, and the first few subsequent vegetatively dividing cells (essentially in their exponential phase of growth) do not produce domoic acid. Bivalves feeding on the early stages of a bloom of Nitzschia would not become toxic. Later generations of the Nitzschia population begin to produce domoic acid, but only on entering the early stationary phase of growth. It is from this time onward that the average cellular toxin level in the water peaks, and bivalves feeding on such populations can accumulate the toxin. Thereafter the level declines upon further aging of the Nitzschia population (Smith et al., 1990). The recurrence of toxic blooms of N. pungens f. multiseries in Cardigan Bay, P.E.I. proves that they are not a singular event. Neither should the amnesic shellfish poisoning episode in P.E.I. be
regarded as strictly local, considering the ubiquity of the diatom (Subba Rao & Wohlgeschaffen 1990), the global scale of bivalve mariculture, and the fact that domoic acid has been shown to occur elsewhere.
Conclusions In the current study, mussels fed on a species of algae natural to the environment experienced shorter uptake and loss periods than those in the field. The rates of accumulation and loss of DA to some extent reflect what may have happened in Cardigan Bay, P.E.I., during the toxic mussel episodes of 1988 and 1989. Rates of filtration and loss measured in the laboratory can be used to model and account for levels of DA in mussels obtained from the field. Of the DA ingested by mussels, less than 10% is found in the tissues probably because most of the DA is rapidly lost to solution during feeding. Scallops accumulated DA more slowly and with greater efficiency than mussels, but were less able to depurate it when held in DA-free sea water. The reason for this retention was unclear.
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