Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada

Deep-Sea Research II ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada Jennifer L. Martin n, Murielle M. LeGresley, Alex R. Hanke Fisheries and Oceans Canada, Biological Station, 531 Brandy Cove Road, St. Andrews, NB, Canada E5B 2L9

art ic l e i nf o

Keywords: Alexandrium fundyense Bay of Fundy Harmful algal blooms Cysts

a b s t r a c t Sediment and water samples were collected for Alexandrium fundyense spatial and temporal distribution and abundance at more than 120 locations throughout the Bay of Fundy during the summers and winters of 1980–1984. These broad surveys have been repeated at various times through the past 30 years, with more regular sampling since 2004. In addition, A. fundyense abundance has been monitored at several locations within the Bay of Fundy at weekly intervals from April to November and monthly during the remaining months since 1988. Paralytic Shellfish Poisoning (PSP) toxins in shellfish (notably Mya arenaria) have also been monitored at multiple locations in the Bay of Fundy since 1943. The datasets were examined to determine relationships and roles between overwintering resting cysts, bloom initiation, bloom decline, motile cell dispersal and A. fundyense motile populations and resulting shellfish toxicity since 1980. Cysts are widely dispersed throughout the Bay of Fundy in the offshore, inshore and intertidal zones with the largest deposits located in the offshore in silt/clay sediments to the east and north of Grand Manan Island at depths of 60–180 m. Results show that there is a constant stable source of cysts in the Bay of Fundy with highest concentrations of cysts (9780 cysts cm 3) observed in 2010 and highest concentrations of A. fundyense motile cells (18  106 cells L 1) observed in 1980. Interannual changes in abundance in A. fundyense populations, resting cysts and the temporal trends in M. arenaria toxicity are discussed. Results show that there was no relationship between the abundance of overwintering cysts and the magnitude of A. fundyense blooms. The offshore seed beds appear to be relatively constant in cyst density among most years and serve as an important source for the motile cells that lead to initiation of major blooms and resulting shellfish toxicity throughout the Bay of Fundy. Crown Copyright & 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction The Bay of Fundy has a long history of annual Alexandrium fundyense1 blooms producing paralytic shellfish poisoning (PSP) toxins and resulting shellfish harvesting area closures due to unsafe levels of toxins in shellfish tissues (Prakash et al., 1971; Martin and Richard, 1996; Martin et al., 2006a). A. fundyense blooms tend to occur during the months of April through September and result in shellfish harvesting closures, generally during spring/summer months. These closures can last for several weeks, but during some years, they can last for several months and extend into the fall and winter (Martin and White, 1988; Martin et al., 1995, 1999, 2001, 2006a; Page et al., 2006). In addition to affecting the shellfish industry in the Bay of Fundy, herring mortalities (Clupea harengus harengus) occurred in 1976 and 1979 and mortalities of caged Atlantic salmon (Salmo salar) occurred in n

Corresponding author. E-mail address: [email protected] (J.L. Martin). 1 A. fundyense was formerly called Gonyaulax excavata or Gonyaulax tamarensis in the Bay of Fundy.

September 2003, June 2004 and July 2006 and A. fundyense was determined to be the cause (White, 1977, 1980, 1981a, 1981b; Page et al., 2005; Martin et al., 2006b, 2008, unpublished; Burridge et al., 2010). Researchers are increasingly recognizing the value of long-term phytoplankton (including harmful algal bloom, or HAB) datasets to determine the status of coastal oceans, assess the importance of environmental, anthropogenic and climate change influences on coastal ecosystems, and identify species composition, abundance, timing of blooms and life cycles (Borkman et al., 2009). Understanding the behavior and population dynamics of PSP producers such as A. fundyense through long term datasets is important to the prediction of their movement, occurrence, dispersal, toxicity and ultimately the management of HABs and their impacts. A key part of the life cycle of A. fundyense for bloom initiation and seeding is its overwintering resting stage or cyst. Anderson et al. (2005, this issue) have described in detail the factors and conditions important to cyst germination and growth in the Gulf of Maine waters adjacent to the Bay of Fundy. Earlier studies of A. fundyense resting cysts in sediments throughout the bay during the winter of 1980–1981 showed that A. fundyense cysts were

0967-0645/$ - see front matter Crown Copyright & 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dsr2.2013.08.004

Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i

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widely distributed both inshore and in the intertidal regions along the coast of New Brunswick and offshore within the central Bay of Fundy. Rich deposits of cysts (2000–8000 cysts cm 3) were detected in the offshore sediments suggesting that the offshore seed beds were the primary source of the motile cells in the region (White and Lewis, 1982). In addition, broad sampling surveys for A. fundyense motile cell concentrations conducted during the five summers of 1980–1984 showed significant variations in cell abundances among years and suggested that the blooms behave as a large population with the majority of cells observed between the mouth of the Bay of Fundy and an area between Saint John, New Brunswick and Digby, Nova Scotia (Martin and White, 1988; Martin and Wildish, 1994). Surface and bottom ocean circulation in the bay were identified as playing an important role in the distribution of cysts, their germination into motile cells and the retention of cells within the system from year to year (Martin and White, 1988). Following the sediment study in the winter of 1980–1981, the survey was repeated during the winters of 1981–1982, 1982–1983, 1983–1984, 1991–1992, 2007–2009, as well as during the early stages of the blooms in May/June 1982 and March 2009, and immediately following the summer blooms in 2009 and 2010. Summertime A. fundyense populations were not only sampled in 1980–1984 but also during 1992 and 2007–2010. These cyst and motile cell studies were originally initiated in order to determine: where the highest concentrations of cysts tended to be located, the annual variability in these locations, where the major spring/ summer blooms of A. fundyense were initiated, and whether wintertime cyst abundance in sediments could be used as a predictor for the summertime motile cell populations. A long term time series phytoplankton monitoring program in the region was initiated in 1988 and continues to the present (Martin et al., 2006a, unpublished). This time series provides the opportunity to capture a weekly picture of the A. fundyense blooms each year from five locations and fill gaps in the annual or snapshot broad surveys. This paper examines distributions and abundances of A. fundyense summertime motile cells and A. fundyense cysts from surveys conducted in the inshore and offshore waters of the Bay of Fundy since 1980 and attempts to relate these to resulting shellfish toxicity.

Fig. 1. Sediment sampling locations (). Phytoplankton monitoring stations (Δ); (BC = Brandy Cove, PB = Passamaquoddy Bay, LKB = Lime Kiln Bay, DH = Deadmans Harbour, W = the Wolves Islands). Results from stations within box were used for statistical analyses.

2. Materials and methods

preparation and analyses are described in detail by White and Lewis (1982). The entire sediment sample was stirred in the plastic container and a 1.0 cm3 subsample was removed and placed in a 15 mL graduated glass centrifuge tube with cold salt water that had been filtered through a Whatman glass fiber filter. The subsample was then sonicated gently with a Millipore 40-W 55 kHz cleaning water bath. It was then sieved and rinsed with cold filtered sea water onto a 20 mm mesh sieve. The material remaining on the sieve was washed back into the centrifuge tube and centrifuged at 750 X g for 5 min. After centrifugation, the volume in the tube was reduced to 3.0 mL, with the cysts in a pellet at the bottom. The sample was then shaken and live A. fundyense resting cysts were counted in 0.1 mL capacity Palmer Maloney counting chambers with a compound microscope at 150X magnification. Each sample was counted in triplicate with mean counts used to calculate A. fundyense resting cysts cm 3 wet sediment. Empty A. fundyense cysts were not counted. This method was kept consistent over time for comparison purposes. Duplicate subsamples were also dried in an oven at 60 1C for 72 h to determine the concentration of cysts g 1 dry wt. sediment.

2.1. Overwintering cysts

2.2. Motile cells

Broad surveys collecting samples from sediments throughout the Bay of Fundy, including inshore and offshore, from as many as 140 sampling locations were conducted during January–April 1981; September 1981–January 1982; May–June 1982; November–December 1982; October–November 1983; January–February 1992; September–November 2008; March 2009; August 2009; May 2010 and August 2010 (Fig. 1). Sample locations from the January 1981 survey (White and Lewis, 1982) are shown in Fig. 2A. Following 1981, the geographic extent of the surveys was reduced to smaller areas on a more regular grid (Figs. 2B–F and 3A–D). During some of the early years the duration of the sediment sampling sometimes extended over a longer period due to scheduling of ship time as well as unsuitable winter weather conditions. Sediment samples were collected with a 0.1 m2 Hunter Simpson grab with a cover plate (Hunter and Simpson, 1976). From the sediment, a subsample 4.5 cm in diameter and 2.0 cm deep was taken and the sample was stored in a 100 mL plastic container which was capped and stored in the dark at 2–4 1C until analysis. Sample

Phytoplankton samples were collected for determination of A. fundyense distribution and abundance throughout the Bay of Fundy for 5 consecutive years on the following dates: August 14–20, 1980; July 31–August 10, 1981; August 3–12, 1982; July 26– 29, 1983; and July 10–26, 1984 (Martin and White, 1988; Fig. 4). Offshore smaller scale Bay of Fundy surveys were conducted on July 25, 2007; July 4, 2008; June 25, 2009; and June 28–29, 2010 (Fig. 5). Additionally, samples have been collected weekly in the southwest New Brunswick portion of the Bay of Fundy as part of a phytoplankton monitoring program making up a time series that was initiated in 1988. Four stations have continuous data since 1988: Brandy Cove (a brackish site influenced by the Saint Croix River estuary), Lime Kiln Bay (where a number of aquaculture sites are located), Deadmans Harbor (an open bay with offshore influence), and the Wolves Islands (an offshore indicator site) (Fig. 1). An extra sampling site was added in mid-Passamaquoddy Bay in 1999. Samples were collected and analyzed as described in Martin and White (1988) and Martin et al. (2006a). Only data from the Wolves station are presented.

Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i

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Fig. 2. Alexandrium fundyense resting cysts 1981–1992.

Water samples (250 mL) were collected from the surface and immediately preserved with 5 mL formalin:acetic acid. Later, 50-mL subsamples were settled in Zeiss counting chambers for 16 h and all phytoplankton cells 45 mm were identified and enumerated (as organisms L 1) with the Utermöhl technique using a Nikon inverted microscope (Sournia, 1978). For species identification purposes, A. fundyense cells included all its life cycle stages, and when different stages in its life cycle were observed, they were recorded separately. Life cycle stages included: duplets, triplets or quadruplets (asexually dividing cells), fusing (sexual division where two cells fuse together), planozygotes (large cells formed from the fusing cells) and temporary cysts (Anderson et al., 1983). 2.3. Statistical analyses The data from cyst and motile cell A. fundyense sampling programs conducted between 1980 and 2010 were grouped annually into seasons. Quarters 2 and 3 were aggregated into a spring/summer season whereas quarter 4 and quarter 1 of the following year were aggregated into a fall/winter season. Observations were averaged for duplicate locations and log transformed (base 10) after adding a value of one to each observation. Variograms were estimated automatically using the R package automap (Hiemstra et al., accepted for publication) and the average cell and cyst concentration, and its variance were predicted across the domain by ordinary

kriging, while the block kriging method was used to estimate the average cell concentration within a common reference area in each of the seasonal maps (R package gstat; Pebesma, 2004). Assumptions of this approach include (1) normality, (2) the variance of the difference between observations is a function of distance and direction (not absolute location), and (3) the average value across the domain is constant with patterns as a result of autocorrelation among the errors. Variograms were largely from the Matern family of models (Minasny and McBratney, 2005). with some good fits to the Gaussian and the Exponential models. The reference area is indicated by the box in Fig. 1 and represents an area that was consistently sampled and is believed to be the epicenter of the cyst bed where consistently high densities have been observed. Block kriged concentrations of cysts within the reference area were compared to cell concentrations from (a) the same season, (b) the previous season and (c) the following season (R package ggplot2; Wickham, 2009). 2.4. Shellfish toxicity A PSP shellfish monitoring program was initiated by the government of Canada in the Bay of Fundy in 1943 through the Atlantic Biological Station (Fisheries Research Board of Canada), the Department of National Health and Welfare and the Department of Fisheries. Today the Canadian Food Inspection Agency (CFIA) is responsible for toxin analyses and the Department of

Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i

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Fig. 3. A. fundyense resting cysts 2008–2010.

Fisheries and Oceans is responsible for enforcement. The mouse bioassay method was used for sample analysis from 1943 to 2010 (Prakash et al., 1971; AOAC, 1990) when analysis of samples for toxins changed to High Performance Liquid Chromatography (HPLC) (Todd, 1997; van de Riet et al., 2011; DeGrasse et al., 2011). The dataset includes PSP samples from as many as 233 sampling sites with 34,000 records and samples from a variety of species with 84.24% of the samples from soft-shell clams (Mya arenaria). When testing M. arenaria samples for PSP, sampling priority changed from year to year to accommodate both current closures and industry interests. This is reflected in the variability in the number of samples taken per year. Variability in sampling effort between locations was high, with some areas being sampled consistently and some other areas with very low sampling frequency. Because of this variability, this manuscript focuses on the samples taken from Lepreau Basin (Fig. 1). This dataset is continuous, has more than 4600 records from 1943 to 2010, and is used as an example of trends in interannual shellfish toxicity over time.

3. Results 3.1. Distributions of cysts A. fundyense resting cysts were detected at many of the sampling sites in the region and were widely dispersed between Saint John, New Brunswick and Digby, Nova Scotia and the mouth of the Bay of Fundy and Passamaquoddy Bay (Figs. 2 and 3). Although cysts were found in various sediment types, including gravel and sand, highest concentrations were located in the fine brown mud, much of which is located in the central Bay of Fundy and the region east and north of Grand Manan Island. Results from the cyst surveys from January–April, 1981; September 1981–January 1982; May–June, 1982; November– December, 1982; October–November, 1983; and January–February 1992 (Fig. 2) show that lowest concentrations of cysts were found in inshore areas of the New Brunswick coast and areas with rock

or gravel bottom such as those along the Nova Scotia coast and portions at the head of the Bay of Fundy. Inshore intertidal sampling was done more frequently than the offshore samplings and yielded consistently low cyst concentrations (Table 1). Results from January 1981 reported previously by White and Lewis (1982) show that the highest cyst abundance detected during that particular survey was 7440 cysts cm 3 in the region east of Grand Manan Island (Fig. 2A). During the same sampling period and in the same area, there were 5 sites that had more than 6000 cysts cm 3. Results from the survey conducted the following winter between September 1981 and January 1982 showed greatest concentrations of cysts in the same region as the previous winter – 5040 cysts cm 3 at Station #66 and concentrations exceeding 4000 cysts cm 3 at several nearby stations (Fig. 2B). The May–June 1982 survey (Fig. 2C) was smaller, and showed similarly high concentrations with the greatest concentration (4550 cysts cm 3) measured in the same general area, near Station #65 (Fig. 1). The November–December 1982 survey also showed rich deposits east and northeast of Grand Manan Island (42000 cysts cm 3), with the highest concentrations (4960 cysts cm 3) found in the fine mud at station #18 closer to Saint John (Fig. 2D). When the survey was repeated in October–November 1983, the greatest numbers were again observed east and northeast of Grand Manan Island with 3330 cysts cm 3 at Station #65 and 1530 cysts cm 3 at Station #18 (Fig. 2E). A survey conducted in January–February 1992 to determine whether the pattern and cyst deposits from the early 1980s had changed showed that the rich deposits were still present in the central Bay of Fundy and the highest concentration observed was 3015 cysts cm 3 at station #65 (Fig. 2F). Sediment sampling for cysts resumed in 2008. During the September–November 2008 survey, 8860 cysts cm 3 were detected at Station #57 and 3320 cysts cm 3 at Station #19 (Fig. 3A) indicating the continued presence of the rich deposits near Grand Manan Island and the slightly smaller deposit near Saint John. In March 2009 a survey prior to the spring bloom showed rich deposits persisting in the same areas. Additional sediment sampling stations were added near stations #17 and #18

Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i

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Fig. 4. A. fundyense motile cells 1980–1984.

(Stations #129–135, Fig. 1) to determine the footprint of the seed source; these stations also showed high cyst concentrations (4 1000 cysts cm 3) although less than the concentrations (4 2500 cysts cm 3) in the Grand Manan region (Fig. 3B). Immediately following the 2009 A. fundyense bloom, an August survey of sediments yielded large deposits east of Grand Manan Island with 4440 cysts cm 3 observed at Station #57 and the greatest number (5710 cysts cm 3) observed at Station #18 near Saint John (Fig. 3C). In 2010, after the decline of the A. fundyense bloom, another broad survey was conducted and revealed cyst densities at 5 locations (#18, #47, #58, #76, and #70) in excess of 5000 cysts cm 3 (Figs. 1 and 3D). Highest concentrations (9780 cysts cm 3) were observed at station #58 – this value was the highest observed from all the studies conducted in the Bay of Fundy between 1981 and 2010. 3.2. Distributions of surface vegetative cells Results from the 1980 to 1984 surveys for A. fundyense distribution and abundance were published previously (Martin and White, 1988). However, for ease of discussion, the distribution maps are presented in Fig. 4. The greatest numbers of A. fundyense documented from the Bay of Fundy were 2.0  106 cells L 1 with accompanying red water discoloration in 1980 in an area close to the coast of Nova Scotia (Fig. 4A). The following year, 1981, very

few cells were detected throughout the bay and the greatest concentration observed was 1060 cells L 1 (Fig. 4B). Martin and White (1988) show that the survey fell well within the A. fundyense bloom period in 1981 indicating that the bloom actually was very small that particular year. From 1982 to 1984 the blooms had highest concentrations of 1.65  105, 1.57  105, and 2.17  105 cells L 1 observed in 1982, 1983 and 1984, respectively (Fig. 4C–E). Results from smaller Bay of Fundy surveys conducted during the A. fundyense blooms of 2008–2010 show these years to be large bloom years as well (Fig. 5). The greatest concentrations observed in 2007, 2008, 2009 and 2010 were 3.42  105, 6.58  105, 2.4  105 and 9.45  105 cells L 1, respectively (Fig. 5A–D). Fig. 6 shows A. fundyense concentrations at The Wolves Islands in the southwest New Brunswick portion of the Bay of Fundy from weekly phytoplankton monitoring initiated at four sites in 1988 (Fig. 1). Sampling at this location and 4 other sites (data not shown) was more frequent (weekly) than the ‘snapshot’ obtained from the broad surveys of the Bay of Fundy and therefore captures a better picture of the annual A. fundyense blooms – from first detection of cells in waters in a particular year, to increase and peak followed by decline. This aids in better selection of maximum peak abundance and timing for the broad surveys in 2008–2010. It also provides an opportunity to capture a better picture of what was happening with A. fundyense populations on a weekly basis.

Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i

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Fig. 5. A. fundyense motile cells 2007–2010.

Table 1 Inshore cyst sampling stations with cyst concentrations (cysts cm

3

).

Inshore Location

# of samplings

Years

Minimum

Maximum

Mean

Crow Harbor Lepreau Basin Lepreau Harbor Passamaquodddy Baya (PB) Pocologan Lime Kiln Bay (LKB)

16 24 23 20 23 22

1981–1985 1981–1985 1981–1985 2008–2009 1981–1985 1981–1985

0 0 0 10 45 0

345 225 315 260 1110 345

74.4 87.3 98.5 104.0 377.6 75.0

a

Combination of results from 3 sites.

200,000 180,000 160,000 140,000

cells L-1

120,000 100,000 80,000 60,000 40,000 20,000 0 1988

1990

1992

1994

1996

1998

2000

2002

2004

2006

2008 5

Fig. 6. A. fundyense concentrations at the Wolves Islands (1988–2010). The highest concentration (3.19  10 cells L

2010 1

) was observed in 2004.

Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i

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Table 2 Date of maximum A. fundyense cell concentrations at Wolves Islands and date of maximum Mya arenaria toxicity detected at Lepreau Basin from 1988 to 2010.

1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

Wolves Islands

4

Lepreau Basin 1

Date

Cells L

July 12 September 20 July 16 July 16 August 31 July 13 July 11 July 19 August 20 June 24 June 9 July 27 June 27 July 17 July 30 September 9 August 3 July 19 July 25 July 31 July 2 June 23 June 29

7460 70 180 68 540 60 380 8880 40 800 52 360 14 200 6600 2280 2600 2600 1840 31 880 4000 90 746 319 056 10 240 46 127 24 336 136 408 57 800 58 089

SS83 W8384 SS80 W8081

SS84 W8485 SS08 W0809

Date

μg STX equiv 100 g

August 8 July 31 July 4 July 15 September 11 July 19 July 11 July 18 July 2 June 24 June 26 July 16 July 4 July 16 July 23 July 22 August 4 July 13 June 19 July 23 July 7 July 7 July 7

140 1530 1040 304 1800 510 800 720 220 280 82 350 58 828 200 137 422 219 111 139 1056 573 1049

1

Cells (log10)

Year

SS82 W8283

3

2

SS81 W8182

1 2.4

2.6

2.8 3.0 Cyst (log10)

3.2

3.4

Fig. 8. Block kriged estimates of the lagged mean log (cysts cm 3) versus mean log10 (cells L 1) of A. fundyense cells collected prior to cyst sampling in the reference area. Summer/spring cell concentrations are paired with the winter cyst concentrations. Labels indicate the season and year of sampling where SS refers to spring/summer, W refers to winter and the years range from 1980 to 2011.

3.5 SS82

SS83

SS10

SS80

4

SS84 SS08

3.0

Cells (log10)

Mean (log10)

SS09

2.5

3

2

2.0 0.000

0.025

0.050 Variance (log10)

0.075

0.100

Fig. 7. Block kriged estimates of the mean and variance of log (cysts cm 3) of A. fundyense in the reference area. Labels indicate the season and year of sampling where SS refers to spring/summer, W refers to Winter and the years range from 1980 to 2011.

Fig. 6 shows the significant interannual variability in A. fundyense concentrations between 1988 and 2010 and also shows periods of overall high and low cell abundances. Maximum A. fundyense cell densities and the date when these were observed at the Wolves Islands each year from 1988 to 2010 are presented in Table 2. Years where cell abundances o3.0  104 cells L 1 were observed include 1988, 1992, 1995–2000, 2002, 2005 and 2007 and are considered to be “small” bloom years. Although a broad Bay of Fundy survey was not conducted in any of those small

SS81

1 2.5

3.0 Cyst (log10)

Fig. 9. Block kriged estimates of mean log (cysts cm 3) versus mean log10 (cells L 1) of A. fundyense cells collected in the reference area. Summer/spring cyst concentrations are paired with contemporaneous summer/spring cell concentrations. Labels indicate the season and year of sampling where SS refers to spring/ summer and the years range from 1980 to 2011.

bloom years to confirm these low A. fundyense densities in the offshore region east of Grand Manan Island, results from the other 4 weekly sampling sites showed similar low A. fundyense numbers for the same 7 years (Martin et al., 1995; Martin, unpublished) suggesting the lack of high cell densities in other regions.

Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i

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ranged from 300 to 1800 cysts cm 3. The variances of these estimates were consistent except for 3 of the samples with values40.05. The winter cyst density estimates were not correlated with the motile cell concentration estimates of the preceding spring/summer season (Fig. 8) nor was there a relationship between the cyst density and motile cell concentration estimates in the same spring/summer season (Fig. 9). However, there was an apparent negative relationship that was observed between winter cyst density estimates and motile cell concentrations in the following spring/summer season (Fig. 10). The variance of the estimate of cyst abundance (Fig. 7) is a reflection of the spatial distribution of cysts in the bed. In some years/seasons the cysts appeared to be more spatially heterogeneous in their density. It may be that oceanographic processes were responsible for this feature of the beds. This added variation translated into wider confidence intervals on the estimates of mean cyst density. For example, the back transformed mean and 95% confidence interval for estimates with a mean of 2.5 and a variance of 0.01 was 316 with 95% CI of 251–398 while estimates with a mean of 2.5 and variance of 0.1 were 316 with 95% CI of 74–1356. A mean of 3.25 with a variance of 0.01 had a back transformed mean of 1778 with 95% CI of 1122–2818.

3.3. Statistical analyses Block kriged estimates of cyst and motile cell densities within the reference area (as indicated by the box in Fig. 1) were used to determine if a relationship existed between: (1) spring/summer motile cell densities and subsequent winter cysts densities (post-bloom relationship); (2) spring/summer cell densities and previous winter cysts densities (pre-bloom relationship); or (3) spring/summer cell densities and spring/summer cyst densities (concurrent relationship). The reference area is known to be a depositional region for cysts as well as an area with a high density of motile A. fundyense cells. The block kriged estimates of mean A. fundyense cyst density (Fig. 7)

W8182 SS82

W8283 SS83

4

W8384 SS84

Cells (log10)

W0809 SS09

3.4. Shellfish toxicity

3

When dealing with the Bay of Fundy shellfish toxicity data one needs to be cognitive and cautious that the intent of the time series was not for research purposes, but to safeguard humans from consumption of shellfish when the product is not safe. As such, it has inherent limitations, a major factor being the high temporal and spatial variability in sampling effort. Examples include where a particular site was monitored for a number of years and sampling priority switched to another location resulting in the first location being sampled less frequently. During some years regular toxicity sampling ceased for a period of time after toxicity values increased above the regulatory level thereby closing shellfish harvesting areas. As sampling was for human health purposes and the area was closed to harvesting, it was not deemed important to continue to sample to determine the shellfish toxicity maximum. Regular sampling resumed after a period to check for continued elevated toxicity or when toxicities were declining. When the area was closed to harvesting, the shellfish were deemed unsafe and signs were posted to indicate that harvesting

2

W8081 SS81

1 2.4

2.6

2.8 3.0 Cyst (log10)

3.2

3.4

Fig. 10. Block kriged estimates of the lagged mean log10(cysts cm 3) versus mean log10(cells L 1) of A. fundyense collected following cyst sampling in the reference area. Winter cyst concentrations are paired with the Summer/Spring cell concentrations. Labels indicate the season and year of sampling where SS refers to Spring/ Summer, W refers to Winter and the years range from 1980 to 2011.

10,000 9,000

µg per 100 g Saxitoxin equiv.

8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 43

48

53

58

63

68

73

78 Year

83

88

93

98

03

08

Fig. 11. PSP toxin values from soft shell-clams from Lepreau Basin (1943–2010).

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Table 3 Date of first presence of A. fundyense cells at Wolves Islands and start in M. arenaria PSP toxicity increase at Lepreau Basin (1988–2010). In 2000, there was no obvious increase in shellfish toxicity observed. Year

Wolves Islands

Lepreau Basin

A. fundyense first detected

Date A. fundyense 4 40 cells L 1

1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

May 25 April 25 May 22 May 14 May 5 February 12 May 24 April 17 May 8 April 21 May 12 June 1 May 11

June 1 May 10 May 22 May 14 May 5 May 12 Data not available May 23 May 8 May 13 May 12 June 1 May 16

2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

May 7 February 12 April 29 May 11 January 4 March 14 May 7 April 24 May 5 April 13

May 7 April 16 May 13 May 11 January 4 April 25 May 22 April 24 May 5 April 13

May 25 May 10 July 2 June 4 July 21 May 18 June 20 June 29 July 2 June 16 May 27 June 16 No detected increase June 11 June 23 July 15 May 19 January 6 June 19 July 16 June 9 May 1 April 26

was prohibited. In some years, this did not reflect the maximum toxicity levels and additionally, in some years toxicity values were reported as 43000 μg saxitoxin equivalents (STX eq) 100 g 1 meat. A more detailed description and analyses of the dataset can be found in Hamer et al. (2012). M. arenaria toxicity results from one of the continuously monitored Bay of Fundy sites, Lepreau Basin, show that shellfish toxicity has been an annual event in the Bay of Fundy with most peaks during the summer months (Fig. 11 and Table 2). There were periods of higher toxicity years such as the mid-late 1940s, the early 1960s, the late 1970s–1980, and the late 1980s to early 1990s and in recent years since 2008 (White, 1987; Martin, unpublished). In some years shellfish accumulated or retained low levels of toxins through the winter months. Lepreau Basin has been closed to harvesting for 52% of the total time from 1943 to 2010. Shellfish toxicities tended to increase at Lepreau Basin in late May, generally followed by peak toxicity in mid-late July (Hamer et al., 2012). Highest M. arenaria toxicity measured since 1943 was 9100 μg STX eq 100 g 1, on July 19, 1976. During that year, toxicities increased from 360 μg STX eq 100 g 1 on July 5 to 1600 μg STX eq 100 g 1 on July 14 and four days later, on July 19, toxicities were as high as 9100 μg STX eq 100 g 1. Unfortunately there were no A. fundyense surveys during 1976, but herring mortalities occurred in the Bay of Fundy that were linked to PSP toxins accumulated through the food chain and the very high shellfish toxicities suggest a high-intensity A. fundyense bloom year (White, 1977). Table 2 shows maximum shellfish toxicity results at Lepreau Basin and dates for highest A. fundyense values observed at the Wolves Islands from 1988 to 2010. Although these two sites are not adjacent, the Wolves Islands is used as an ‘offshore’ indicator site for inshore shellfish toxicity along the southwest coast of New Brunswick and it is one of the closest regular phytoplankton monitoring sites to Lepreau Basin. Presence of cells at the Wolves Islands can give an indication of increasing shellfish toxicity in southwest New Brunswick and provide advice to CFIA to increase

Fig. 12. Circulation patterns in the Bay of Fundy (Godin, 1968).

toxin sampling frequency. A. fundyense cells are observed every year and toxicity above the regulatory limit is also an annual occurrence. Dates of occurrence and maximum cell densities from surface waters at the Wolves Islands and maximum shellfish toxicity measured at Lepreau Basin from 1988 to 2010 (Table 2) show no correlation between the two variables. High A. fundyense concentration did not always correspond with high toxicity nor did low A. fundyense concentrations correspond with low toxicity. During some years (but not all) timing of the maximum cell density for the year was observed before the maximum toxicity value. Table 3 shows the date when A. fundyense was first detected in each year from 1988 to 2010, the date at which more than 40 cells L 1 were observed at The Wolves Islands and the date when M. arenaria toxicity began to increase. The first observation of A. fundyense cells in the water occurred between January and June prior to the increase in shellfish toxicity, except in 1988 when it was the same day, and in 2009 when it was four days prior. The date at which more than 40 cells L 1 were detected was in some cases the same day and at other times was closer to the date when toxin levels increased. The only year that cells and shellfish toxicity increased in January was in 2005.

4. Discussion 4.1. Cysts and cell abundance The Bay of Fundy is a unique, dynamic water system with the largest tides in the world. The tidal range can sometimes exceed 16 m at the head of the bay and 8 m at the mouth (Garrett, 1972; Greenberg, 1983; Daborn, 1986; F.H. Page, personal communication). In addition to the tidal flux, there is a pattern of surface and bottom circulations in the Bay (Godin, 1968) that appears to play an important role in the distribution of cysts, the dispersal of motile cells, and the retention of the cells within the system from year to year (Fig. 12). Surface inflow from the Gulf of Maine occurs along the southern entrance to the Bay of Fundy along the Nova Scotia coast (Bumpus, 1960; Bumpus and Lauzier, 1965). Northeast of Digby, Nova Scotia, the waters tend to cross the Bay of Fundy to the north side to the Saint John, New Brunswick area (Godin, 1968; Iles, 1975; Greenberg, 1983). Outflow waters go south and west along the New Brunswick coast around Grand Manan Island in a counterclockwise direction and flow either along the coast of Maine or cross the Bay of Fundy and remain within the Bay of

Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i

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Fundy system. Ketchum and Keen (1953) calculated the “flushing time” for the Bay of Fundy to be about 76 days, which would provide a natural retention and incubation zone for A. fundyense cells and their cysts. There is a counterclockwise movement of surface waters that encompasses most of the bay, as well as a counterclockwise gyre at both the surface and the bottom located east of Grand Manan Island (Fig. 12). Aretxabaleta et al. (2008) have described the factors controlling the gyre and the fact that it is stronger in May/June and relatively constant during the summer and emphasize its importance in the understanding and prediction of A. fundyense blooms both in the Bay of Fundy and Gulf of Maine. There is also a depositional zone for sediments, referred to as the LaHave clay zone, at depths of 80–160 m in the vicinity of the counterclockwise gyre (Fig. 12) as well as to its north, west and south (Fader et al., 1977), which seems to serve as a consolidation zone for resting A. fundyense cysts. Although cysts tended to be dispersed through much of the Bay of Fundy, lowest concentrations were found in inshore areas on the New Brunswick coast and areas that had rock, sandy or gravelly bottom such as those on the Nova Scotia coast and portions of the head of the bay. This could be as a result of the scouring effect due to strong tides and currents and the inability of many of the cysts to remain in these substrates. Highest concentrations tend to occur in areas of clay/ mud. The Bay of Fundy thus seems to retain cells in the system and act as an A. fundyense incubator. The consistently high concentrations of cysts in offshore waters (Figs. 2 and 3) throughout the years suggest a stable rich deposit and large seed bed in the central Bay of Fundy east and northeast of Grand Manan Island. Although sediment sampling at anytime during the year and during any of the years yielded persistently high concentrations, the highest concentrations ( 47000 cysts cm 3) detected from all the completed cyst surveys were January 1981 (7740 cysts cm 3); July 1984 (9270 cysts cm 3); October 2008 (8860 cysts cm 3) and August 2010 (9780 cysts cm 3). A. fundyense blooms in years immediately following these “highest” cyst count years did not reflect the high cyst concentrations in sediments. An obvious example was when a massive A. fundyense bloom with 41.8  106 cells L 1, accompanied by red water discoloration, was observed in 1980. High cyst concentrations (Fig. 2A) were observed in the winter of 1980–1981 immediately following the large bloom of 1980 (Fig. 4A). The following summer, 1981, the A. fundyense bloom was the lowest observed since our sampling began in the late 1970s – the highest concentration detected was 1060 cells L 1 (Fig. 4B). Martin and White (1988) described that the timing of the sampling for the broad survey in 1981 was within the A. fundyense bloom period for that year. When samples were collected from sediments in the winter of 1981–1982 immediately following the low cell densities in 1981, there were several sites with 44000 cysts cm 3 and the stations, #55 and #56 (Fig. 1) had 5025 and 5040 cysts cm 3, respectively. This clearly showed the persistently high cyst concentrations in sediments despite the negligible bloom in 1981. The bloom in the summer of 1982 was a more “normal” one for the Bay of Fundy with 8.78  104 cells L 1 detected east of Grand Manan Island (Fig. 4C). The cyst survey during the May/June part of the bloom in 1982 (Fig. 2C) showed the persistence of high cyst deposits as did the November/December 1982 results (Fig. 2D). All cyst surveys and mapping through the years showed the persistence of massive deposits and no link between cyst concentrations and the magnitude of A. fundyense bloom in the Bay of Fundy – a very different scenario from the Gulf of Maine (Anderson et al., this issue). For the Bay of Fundy, cysts appear to be important for bloom initiation but high cyst densities have as yet not been shown to result in high motile cell concentrations. Block kriged estimates showed that average cyst densities in the beds were not related to those of motile cell densities (Figs. 8–10). When results

from various seasons and samplings were examined in more detail, block kriged estimates showed that average motile cell concentrations in the spring and summer prior to winter were not related to subsequent winter cyst density (Fig. 8), nor was there a positive relationship between average summer/spring cyst densities and contemporaneous spring/summer cell concentrations (Fig. 9), nor a positive relationship between winter cyst abundance and the following average spring–summer cell abundance (Fig. 10). In fact, it appeared that the opposite was true, i.e., there was an apparent negative relationship between winter cyst abundance and the following average spring–summer cell abundance which deserves further exploration with a more controlled sampling program. Such a relationship would be possible if scouring transported cysts into the water column prior to cyst bed sampling. The greater abundance of cysts in the water column would presumably increase the probability of a large bloom that summer provided all other conditions were optimal. It appeared that following the massive bloom of 1980 there was an inverse relationship between cyst density in the winter of 1980–1981 and cell abundance in 1981. As these surveys only dealt with actual cyst and cell abundances and did not focus on oceanographic processes, it would be interesting to follow up with further integration of cell and cyst densities over a longer period of time and a larger area. Storms may have been responsible for moving the cysts or cells to other areas or even exporting them to the Gulf of Maine. We also recognize that the times for winter samplings for our surveys were not standardized. Earlier work from Bay of Fundy sediments has shown that high cyst numbers and viable cysts have been detected to a depth greater than 20 cm (Martin, unpublished). The resuspension of cysts within the seed bed areas may contribute to the constant elevated numbers in surface sediments, especially in years with low motile cell numbers and low cyst deposits. We have not discussed the nephloid layer in this paper, nor its cysts and the oceanographic processes associated with resuspension and dispersion and their availability for seeding blooms. Pilskaln et al. (this issue) show the rich cyst deposits or reservoir in the nephloid layers in the Bay of Fundy and discuss its importance in bloom generation via sediment resuspension through transport from the Bay of Fundy to the central coast of Maine to the south central Gulf of Maine. This could be a potential source of cysts that could fuel the Georges Bank region. Results from summertime A. fundyense bloom surveys show continued high concentrations of motile cells in most years in the central Bay of Fundy as well as a wider spread of high concentrations suggesting a continued source and seed for the large summer blooms near the gyre and dispersion through wind-driven water transport or currents (Figs. 4–6). Although the relationship between cyst concentration and bloom size is not clear, we believe that cyst numbers in the inshore do not appear to be of a large enough magnitude to be responsible for the larger scale events that can occur in the inshore. As samples are collected and analyzed from the coastal areas on a weekly basis, a high A. fundyense inshore “bloom” would be observed before it was transported and seeded the entire Bay of Fundy suggesting that the alternative (more rapid vegetation growth in coastal areas) is not happening. Therefore, transport from the offshore blooms could be important to the inshore A. fundyense abundances and resulting shellfish toxicities. Although A. fundyense surveys of cysts and motile cells show a ‘snap-shot’ of A. fundyense populations, there are limitations to the broad surveys for motile cells that show up in the more regular weekly monitoring. Cell distributions and abundances are patchy in space and time. Therefore the more intense and frequent the sampling, the less chance of missing an event. Through weekly monitoring, it is possible to capture bloom initiation, development and decline. For example, when the 2004 event occurred, A. fundyense numbers increased at the

Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i

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Wolves Islands (Fig. 6) from 1.6  104 cells L 1 on July 19 to 1.69  105 cells L 1 on July 27 and 3.19  105 cells L 1 on August 3. It may not have shown the highest value for the Bay of Fundy, but did provide information on bloom progression. Another example of an extremely unusual rapid increase and decline occurred at Brandy Cove in Passamaquoddy Bay in 2009. On June 23, a concentration of 5.6  103 cells L 1 was observed. The following week, on June 30, there were 2.79  105 cells L 1 and on July 7 the concentration had declined to 2.4  103 cells L 1 (McGillicuddy et al., this issue). Without weekly phytoplankton monitoring, this exceptional event would have been missed. In fact it was so abnormal at the time that additional samples were obtained for verification. Previous studies in the Bay of Fundy have shown that A. fundyense can behave very differently between years and locations. First appearance of A. fundyense has been observed to occur between January 4 and June 27 and can vary greatly between years and locations (Page et al., 2005, 2006). Blooms can last between 50 and 200 days and there can be between 1 and 3 blooms in a year. The mechanisms underlying bloom development and the triggers influencing the rapid increase in cell densities are still poorly understood, as is the knowledge of how many cysts or motile cells are required to form a bloom. As the number of cysts is somewhat constant in the Bay of Fundy and with the constant massive seed beds perhaps it may not be so much about the cysts, as they are always there – it may be related to ideal climate and local weather conditions that enable cells to divide rapidly and form high biomass concentrations (Martin, unpublished). Various factors such as air and water temperature, chlorophyll a, nutrients, salinity, river run-off, and rainfall have been explored to determine linkages with A. fundyense growth. Factors that we have observed to be linked to A. fundyense high concentrations include: extended periods of low winds, calm waters, fog (and associated weather conditions) and low nitrate. The relationship between low nitrate and high A. fundyense concentrations was only evident in years when A. fundyense concentrations exceeded 9.0  104 cells L 1 (Martin et al., 2009). Years with prolonged sunshine and little rainfall seem to be conducive to an extended A. fundyense bloom period with persistent low cell densities, but not high A. fundyense cell concentrations. Horecka et al. (this issue) have suggested a link between increased toxicity in eastern Maine and weather conditions characterized by clear skies and drier air. As A. fundyense is often not the major component of the phytoplankton community it can make determining linkages more complicated. 4.2. Shellfish toxicity Fig. 8 illustrates that there are patterns of higher and lower periods of shellfish toxicity at Lepreau Basin (see also Martin and Richard, 1996; Hamer et al., 2012). Results from A. fundyense motile cell surveys and continued weekly monitoring of phytoplankton show, however, that although periods of elevated shellfish toxicity are associated with high cell density, the shellfish toxicity data does not always capture the complete A. fundyense population distribution and abundance ‘story’. Our studies have shown that there can sometimes be high-density A. fundyense localized populations, either in the offshore or just a short distance (such as a few hundred meters) from the intertidal shellfish beds that are not always reflected in the shellfish toxicity values. Onshore advection of cells does not always occur. For example, in September 2003, A. fundyense concentrations 48.8  105 cells L 1 were detected near Grand Manan Island in early September and resulted in mortalities of Atlantic salmon (Salmo salar) at aquaculture operations. In 2004 43  106A. fundyense cells L 1 were detected in Lime Kiln Bay and further along the inshore areas towards Saint John that resulted in further red water and salmon mortalities at aquaculture operations

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(Martin et al., 2006b, 2008; Burridge et al., 2010). The Lepreau Basin shellfish toxicity (Fig. 11) data do not reflect these 2003–2004 high values and the high shellfish toxicities that were detected in other parts of the Bay of Fundy in these two years (data not shown). A vector such as wind-driven current could have been responsible for transporting the cells to the inshore where they began to thrive and formed a more localized bloom affecting fisheries in that particular location. During the salmon mortality events of 2003, M. arenaria from one nearby intertidal area on Grand Manan Island reached a maximum concentration of 2752 μg STX eq. 100 g 1. At another nearby site on Grand Manan Island, however, M. arenaria toxicity only exceeded the threshold level of 80 μg STX eq. 100 g 1 in one instance and only reached 219 µg STX eq. 100 g 1. This 2003 event seemed to be very localized and most likely cells were transported from the high abundance area in the central Bay of Fundy by currents, perhaps driven by the wind. Although shellfish toxicity occurred elsewhere in the Bay of Fundy at that time, no other areas experienced comparably high levels. When the red water occurred in 2004 and 3  106 cells L 1 were detected, salmon mortalities occurred again, but this event occurred in southwest New Brunswick – where the cells seemed to have been transported inshore from the offshore via wind-driven currents. Table 2 shows that there is no correlation between maximum A. fundyense cell abundance and maximum shellfish toxicity at the Wolves Islands and Lepreau Basin. This may be due to a number of factors such as the distance between the two locations, the need for a mechanism for cells to be transported inshore, or because the results from A. fundyense surveys presented in this paper are from surface waters (past surveys at discrete depths have shown that A. fundyense cells often concentrate at the surface, but sometimes, such as periods of high winds, the cells can be dispersed downward). Another factor is that A. fundyense is not always the dominant phytoplankton species in the community and shellfish can selectively feed on certain species which could affect toxin uptake and retention (Ward and Shumway, 2004; Mafra et al., 2009). Bloom duration and the density and cell toxicity of A. fundyense cells can also affect toxicity in shellfish (Bricelj and Shumway, 1998). All M. arenaria sampled by CFIA are intertidal, and therefore feeding on A. fundyense will also depend on height in the intertidal and thus immersion time. However, it is not uncommon in the Bay of Fundy for maximum cell abundance to show no correlation with shellfish toxicity. In the Bay of Fundy, it is most important to determine the early bloom presence since a concentration as low as 500 cells L 1 can result in an increase in shellfish toxicity above threshold levels. The ability to predict bloom magnitude and duration is important to the shellfish industry as this will determine the length of closures. There have been years in the Bay of Fundy with persistent low shellfish toxicity in winter months – this may be due to low cyst concentrations in sediments and resuspension, or reduced detoxification rate at low temperatures. Additionally, in years when the A. fundyense blooms have occurred late into September and October, low levels of toxins have persisted as temperatures decrease and there is less opportunity for the shellfish to feed and depurate.

5. Conclusions The Bay of Fundy has some of the richest A. fundyense cyst deposits in the world and this has been consistent through time. However, cyst concentrations in sediments do not appear to be a predictor for A. fundyense cell density or shellfish toxicity in the following year. Years with the highest A. fundyense cyst numbers have not been followed by abnormally high years and in some instances, were followed by extremely low A. fundyense bloom

Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i

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densities. Through comparison of A. fundyense motile populations with various environmental, physical and chemical characteristics, we found linkages with low nitrate values only at very high cell densities and when A. fundyense was a major component of the phytoplankton community (Martin et al., 2009). Results from our long term dataset on A. fundyense and various other phytoplankton species show that parameters for growth and bloom formation will vary among species. The Bay of Fundy, with its rich deposits of A. fundyense cysts can have large scale and high density blooms during many years and resulting high shellfish toxicity. These cyst beds have been implicated as a key source of A. fundyense cells not only to the Bay of Fundy but also to the Gulf of Maine (He et al., 2008). Aretxabaleta et al. (2008) have also shown that the Bay of Fundy gyre is important to retaining cells within the Bay of Fundy system, but the interannual variability or leakiness in the retentiveness of the gyre can influence regional bloom dynamics (Aretxabaleta et al., 2009). Through regular monitoring of A. fundyense populations in the Bay of Fundy it is possible to give industry and regulatory agencies short-term (days and in some cases weeks) early warning of upcoming shellfish closures, but it is difficult to predict cell abundance and locations affected, as weather appears to play an important role in bloom dynamics and can be very variable. However, weather forecasts have improved significantly in recent years, and continue to improve. Regular monitoring and long time series can be very important in providing an early warning as in the case in Passamaquoddy Bay, an area not previously known to have high shellfish toxicities, where A. fundyense populations increased to extremely high levels in less than a week in 2009. These results raise further questions for the future. Cysts are important to the A. fundyense blooms as they provide a seed population, but they do not seem to play a significant role in predicting bloom intensity from year to year in the Bay of Fundy. As a result, future studies in the Bay of Fundy should increasingly focus on determining the concentration of viable cysts required to initiate a bloom, concentrations of motile cells required to propagate and form a large bloom, the conditions that favor vegetative growth, mechanisms for the dispersion and transport of cells, how different life cycle stages behave in the dispersion and retention of motile cells within the Bay of Fundy, and the importance of climate, oceanographic conditions, and weather.

Acknowledgments We thank the captains and crews of the Research Vessels J.L. Hart, E.E. Prince, Pandalus III, and Viola M. Davidson, and the fishing vessel Bon-Tri-Kei for their cooperation. A number of people have helped with sample collection and counts over the years – E. Doon, G. Forbes, K. Mackenzie, J. Power, S. Regan, M. Ringuette, A. Wilson, S. Corey and E. Gao. B. Chang provided the maps. We are also grateful to B. Chang, M. Lyons, and two anonymous reviewers for providing comments to the manuscript.

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Please cite this article as: Martin, J.L., et al., Thirty years – Alexandrium fundyense cyst, bloom dynamics and shellfish toxicity in the Bay of Fundy, eastern Canada. Deep-Sea Res. II (2013), http://dx.doi.org/10.1016/j.dsr2.2013.08.004i