Pseudo-nitzschia and Domoic Acid Along the U.S. West Coast

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Harmful Algal Blooms: The Variability of

Pseudo-nitzschia and Domoic Acid Along the

U.S. West Coast

Steffaney M. Wood1, David Backus1, Brian D. Bill2, Vera L. Trainer2

1Davidson College Department of Environmental Studies, Davidson, NC; 2NOAA

Northwest Fisheries Science Center, Seattle, WA

[email protected]

ABSTRACT: Since the occurrence of a massive, destructive harmful algal bloom (HAB)

along the U.S. West Coast in 2015, the variability in the occurrence of toxigenic planktonic

diatom Pseudo-nitzschia has posed a threat to fisheries, marine mammal survival, and

public health. Certain species of the genus Pseudo-nitzschia produce the neurotoxin

domoic acid (DA), which can cause amnesic shellfish poisoning (ASP) in humans who

consume shellfish contaminated with this toxin. The objectives of the continued

assessment of abundance and toxicity of Pseudo-nitzschia, include the identification of

emerging hotspots, the investigation of Pseudo-nitzschia and DA occurrence relative to

environmental phenomena such as El Niño and the Blob, and the synthesis of data that are

incorporated into the HAB early-warning system. Seawater samples collected on the

NOAA research vessel Bell M. Shimada in May 2017 were analyzed in the laboratory using

the following methods: 1. indirect cBASI Enzyme-linked Immunosorbent Assays

(ELISAs) to determine DA concentration; 2. light microscopy to determine abundance

of Pseudo-nitzschia; and 3. scanning electron microscopy to determine Pseudo-

nitzschia species composition at select locations. Available data from 2015-17 was

analyzed using RStudio. Results indicated the existence of emerging HAB hotspot off the

coast of Trinidad, CA and that freshwater plumes can affect the location of Pseudo-

nitzschia at the HAB hotspot Heceta Bank, OR. For future analysis, nutrient and

chlorophyll data will be examined. This research will add to the time series data used to

determine the influence El Niño, La Niña and Pacific Decadal Oscillation on the increasing

frequency of HABs along the U.S. West coast.

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INTRODUCTION

The massive, destructive harmful algal bloom (HAB) along the U.S. west coast in 2015

illustrated the threat posed to fisheries, marine mammal survival, and public health by toxigenic

diatom Pseudo-nitzschia. Certain species of the genus Pseudo-nitzschia produce domoic acid

(DA), a neurotoxin that leads to amnesic shellfish poisoning causing gastrointestinal and

neurological problems in humans, marine mammals, and sea birds (Jeffrey et al., 2004; Lelong et

al., 2012; Trainer et al., 2012; McKibben et al., 2017). Following a deadly bloom in 1987,

Pseudo-nitzschia and DA have been identified as public health threats, and consequently DA

levels have been monitored in shellfish since 1991 (Van Dolah, 2000; Mos et al., 2001;

McKibben et al., 2017). Since this first outbreak, the prevalence of harmful algal blooms,

including Pseudo-nitzschia, continues to grow along the west coast (Van Dolah, 2000; Lewitus

et al., 2012; McKibben et al., 2017; Zalzal, 2017). These disruptive, toxic blooms have led to

unprecedented economic deficit within the fishery industry, food safety risks, mass sea lion and

pelican deaths, and diagnosis of shellfish poisoning in humans (Trainer et al., 2012). Although

the changing climate has allowed these harmful algae species to realize and exploit new habitats,

the specific environmental conditions producing toxic algal blooms remain largely unclear.

To this end, the objectives of my capstone project are to: 1. analyze samples collected

along the U.S. west coast during May, 2017; 2. assess and compare the abundance and toxicity of

harmful algal species of the genus Pseudo-nitzschia along the U.S. west coast between 2015-

2017; 3. identify existing and emerging hotspots; 4. determine potential environmental and

oceanographic regulators of toxicity and abundance; 5. investigate the role of the Columbia

River at hotspot location Heceta Bank, Oregon. Overall, this research adds to the time series

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data needed to determine the relative influence of various drivers of Pseudo-nitzschia blooms,

including local chemical and physical oceanographic factors such as temperature and salinity, as

well as climatic patterns such as El Niño, La Niña, and the Pacific Decadal Oscillation over the

three years of available data.

BACKGROUND

2015 Bloom conditions

Over the past couple decades, published research on harmful algal blooms has grown

approximately by twenty-fold per year (Lewis, 2017), which is postulated to be the result of the

increasing frequency of major harmful algal bloom events (Van Dolah, 2000; Lewitus et al.,

2012; McKibben et al., 2017; Zalzal, 2017). One such event took place along the U.S. west

coast in the spring and summer of 2015, causing the poisoning of shellfish, marine mammals,

and sea birds as well as widespread fisheries closures (Trainer et al., 2007). In fact, estimates

suggest that the Dungeness crab fishery closures alone caused approximately $100 million-dollar

loss (Fisheries of the United States, 2015). At its peak, the bloom spanned from southern

California to the Aleutian Islands of Alaska and is largely regarded as the largest toxic bloom on

record in the region (Du et al., 2016). Such outbreaks are associated with a variety of factors,

including coastal upwelling zones, nutrient availability, wind patterns, precipitation,

eutrophication, and anomalously warm waters (Barth et al., 2005; Trainer et al., 2007; Anderson

et al., 2008; Du et al., 2016). Most recently, researchers hypothesize that the causes of the 2015

bloom included upwelling prevalence, anomalously warm sea-surface temperatures, presence of

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spring storms, and nutrient stress during the time leading up to the bloom (Du et al., 2016;

McCabe et al., 2016).

Normal conditions: Geographical distributions and species along the west coast

Typically, Pseudo-nitzschia accounts for less than 20% of any major phytoplankton

bloom along the west coast between May and July (Trainer et al. 2009; Trainer et al. 2012).

However, there are also known “hotspots” off the coast, regarded as initiation sites for toxic

blooms where Pseudo-nitzschia blooms occur annually. “Hotspot” sites for Pseudo-nitzschia

along the Pacific coast include the Juan de Fuca eddy region, Washington; Heceta Bank, Oregon;

Monterey Bay, San Luis Obispo, Santa Barbara Basin, and Point Conception, California (Fig. 1;

Trainer et al., 2000; Tweddle et al., 2010; Trainer et al., 2012).

Figure 1. Global distribution of toxic species of the genus Pseudo-nitzschia (Trainer et al., 2012).

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To date, researchers have identified less than fifteen species of Pseudo-nitzschia that produce

DA and roughly ten of those proliferate in the west coast environment, with P. australis and P.

multiseries posing the greatest toxigenic threats (Horner et al., 1973; Anderson et al. 2008;

Trainer et al. 2012).

Overall, Pseudo-nitzschia pungens is less toxic than P. australis and P. multiseries, and

P. pungens is considered the “cosmopolitan diatom species” with two distinct populations

inhabiting the Juan de Fuca eddy area (Trainer et al., 2002; Adams et al., 2009). At Heceta

Bank, Oregon as well as Monterey Bay, Santa Barbara, and San Diego, California, P. australis

and P. multiseries tend to predominate and lend to their hotspot status (Busse et al., 2006; Barron

et al., 2013; Barron et al., 2013; Trainer et al., 2012; Wood et al., 2017). Historically, toxic

species have been less common in southern California; however, many studies show an increase

in toxic species in the region (Busse et al., 2006; Barron et al., 2013; Barron et al. 2013).

Amnesic shellfish poisoning incidence in humans

As previously stated, some species of the genus Pseudo-nitzschia produce DA, a

neurotoxin that causes amnesic shellfish poisoning (Jeffrey et al., 2004; Lelong et al., 2012;

Trainer et al., 2012; McKibben et al., 2017). Acute poisoning initiates gastrointestinal and

neurological problems in humans, marine mammals, and sea birds. Since a deadly bloom in

1987, Pseudo-nitzschia and DA have been identified as public health threats, and the FDA has

monitored DA levels shellfish in the United States since 1991 (Van Dolah, 2000; Mos et al.,

2001; McKibben et al., 2017).

The FDA and EPA mandated safety level of DA in all fish is 20 parts per million (ppm),

with the omission of the Dungeness crab where 30 ppm remains permissible (“FDA Fish and

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Fishery Products Hazards and Controls Guidance,” 2011). This safety level, or action limit,

refers to the maximum concentration before the official close the fishery, and the government

advises against human consumption (“FDA Fish and Fishery Products Hazards and Controls

Guidance,” 2011). After the first known Pseudo-nitzschia bloom in the United States occurred

off the coast of Washington state in 1991, primarily infecting razor clams, the FDA has

monitored levels of DA in all seafood. However, the first documented case of a toxic Pseudo-

nitzschia bloom and related human deaths due to DA took place off the coast of Prince Edward

Island, Canada in 1987. A total of three people died and over 100 people showed symptoms of

amnesic shellfish poisoning, including confusion, nausea, and short-term memory loss (Mos,

2001). Since both initial outbreaks, DA has been detected on the northwest and northeast coasts

of the United States, as well as off the western coast of Florida (Parsons et al., 2002; Thessen et

al., 2005).

When humans eat shellfish poisoned with DA, the DA enters the bloodstream and targets

the limbic system in the brain, which is largely responsible for emotions and memories. DA

mimics glutamate, which is an important excitatory neurotransmitter in the brain, activating

signals between neurons. This allows it to target the central nervous system and interact with

glutamate receptors on nerve cell endings. Most studies have shown that DA binds to glutamate

receptors with such stringency that it causes receptors to become over-activated. With

continuous activation of the glutamate receptor, cations—primarily calcium (Ca2+)—

continuously flow into the post-synaptic neuron. Eventually, this leads to swelling, damage, and

cell death, resulting in symptoms associated with neuronal degeneration (Pulido, 2008).

The majority of clinical data characterizing amnesic shellfish poisoning in humans stems

from the Canadian outbreak in 1987. These data suggest that if someone ingests a concentration

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of DA of between 0.9-2.0 milligrams DA per kilograms body weight (mg/kg DA) they are likely

to show mild clinical signs of toxicity (Iverson et al., 1989; Lefebvre and Robertson, 2010).

These symptoms can include abdominal pain, diarrhea, headache, nausea, and confusion.

Greater concentrations of between 1.9-4.2 mg/kg DA cause more severe neurological symptoms

of amnesic shellfish poisoning such as disorientation, vomiting, seizures, and memory loss

(Mulido, 2008). While the 1987 case study was successful in prompting the 20 ppm food safety

limit, the relevance of the knowing effect of trace amounts of DA on humans has increased with

the growing frequency of toxic Pseudo-nitzschia blooms and lack of known medical treatment

for amnesic shellfish poisoning (Tiedecken and Ramsdell, 2013; Funk et al., 2014; Ramsdell and

Gulland, 2014; Lefebvre et al., 2017).

Since 1987, research surrounding DA poisoning has focused on the acute, short-term

neurological effects. More recently, studies have examined its effect on the renal system as well

as the effects of chronic, low-dose exposure (Funk et al., 2014; Lefebvre et al., 2017). Funk et

al. (2014) found that more DA accumulates in the kidney than the hippocampus, or any other

organ known to have glutamate receptors. With mice as a model species, the accumulation of

DA in the kidneys at doses of less than or equal to 0.1 mg/kg DA causes glutamate receptor

over-activation, which damages the cells required for the filtration function of the kidney.

Ideally, these cells would filter out DA and other toxic substances. The inhibition of the

filtration function by greater exposure to DA can lead to more serious symptoms of amnesic

shellfish poisoning (Funk et al., 2014). In addition, Lefebvre et al. (2017) tested chronic low-

level DA exposure in mice to detect neurological symptoms, giving the mice doses of

approximately 0.75 mg/kg DA weekly for several months. Their results showed “significant

spatial learning impairment and hyperactivity after 25 weeks of exposure.” These symptoms

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were reversible after a nine-week recovery period without exposure to DA (Lefebvre et al.,

2017). Overall, these analyses suggest that smaller doses have the potential to cause permanent

kidney damage and should dictate a new conversation surrounding the levels of DA permissible

in food.

A recently designated long-term side effect of DA poisoning is known as DA epileptic

disease, often found in humans and sea lions long after initial exposure to the toxin. It is

diagnosed based on the development of recurrent spontaneous seizures and behavioral

abnormalities weeks to months after acute poisoning and symptoms of amnesic shellfish

poisoning have subsided. There has been only one case in humans, but it reflects a similar

condition that is common among sea lions after toxic Pseudo-nitzschia outbreaks. The single

human case study contracted amnesic shellfish poisoning during the 1987 outbreak, a year later

developed temporal lobe epilepsy, and eighteen months afterwards died of pneumonia (Ramsdell

and Gulland, 2014). Using rats as a model species, studies revealed that DA epileptic disease

progression paralleled the advancement of temporal lobe epilepsy in three stages: the early

biological effect of DA poisoning, the structural damage during latent period of toxicity, and

then radical damage in the form of epileptic disease (Tiedecken and Ramsdell, 2013). Although

the human case was more susceptible to the disease because of old age, altogether this

individual’s case, the rat model, and prevalence of DA poisoning in sea lions show the potential

for long-term damage as a result of accidental ingestion of DA.

A major gap in the research surrounding Pseudo-nitzschia, DA, and amnesic shellfish

poisoning is epidemiological evidence that links the agent to the outcome. This is likely due to

the documentation of only one major outbreak. Perl et al. (1990) holistically investigated the

1987 outbreak and determined the cause to be DA, by analyzing both public health

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questionnaires taken by those who showed symptoms and available clinical information. Of the

250 reports of the sickness related to the ingestion of mussels from Prince Edward Island, 107

met the case definition of developing gastrointestinal symptoms within 24 hours and

neurological symptoms within 48 hours. In one of the earliest descriptions of amnesic shellfish

poisoning, these researchers ultimately determined that “the clinical syndrome caused by DA is

unlike two other intoxications that are associated with the consumption of mollusks and affect

the nervous system—paralytic shellfish poisoning and neurotoxic shellfish poisoning” (Perl et

al., 1990). Their study was, however, limited by a lack of analytical investigation (Perl et al.,

1990).

Physical and chemical properties of DA

DA is an excitatory non-protein amino acid, meaning that it acts as a neurotransmitter

critical for synaptic transmission, often found as colorless crystalline needles (Clayden et. al

2005). Structurally similar to kainic acid, glutamic acid, and aspartic acid, it contains three

carboxyl groups, a proline ring, and two conjugated double bonds in the side chain (Kizer, 1994).

Its geometry is vital to both its toxicity and ability to interact with glutamate receptors (Lefebvre

and Robertson, 2010). Due to the presence of multiple carboxyl groups and single amino group,

it is both polar and water-soluble, which permits it to thrive in the ocean environment. While its

low octanal-water partition coefficient value of -0.89 shows that DA cannot typically

bioaccumulate in aquatic organisms (Table 1). Unlike many other intoxicants that dissipate

when the shellfish is cooked, steaming or freezing shellfish tissue known to contain DA does not

significantly decrease the concentration (Lefebvre and Robertson, 2010).

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Domoic Acid

Molecular formula C15H21NO6

Molecular weight 311.334 g/mol

Structure

Density 1.27 g/cm3

Solubility In water at 8 mg/mL

LogKow -0.89

Table 1. Properties of DA (“Domoic Acid | C15H21NO6”).

The function of DA production in the fourteen known strains of DA-producing Pseudo-

nitzschia remains largely unknown (Lelong et al., 2012). However, these strains tend to prefer

warm, low salinity, nutrient-rich ocean water. Detection of DA and Pseudo-nitzschia are often

one in the same: scientists and governmental agencies generate prediction forecasts for toxic

species of Pseudo-nitzschia and monitor outbreaks for toxicity. Identification of the specific

toxin-producing species requires comprehensive inspection by electron microscopy. Therefore,

DA detection is the primary method of monitoring and forecasting toxic blooms. Such

monitoring and identification programs exist along the U.S. west coast, Canada, and parts of

Florida (Mos, 2001). In addition to monitoring and species identification, shellfish are tested for

the concentration of DA as a matter of food safety via high-performance liquid chromatography

followed by ultraviolet detection and mass spectrometry (Papiol, 2015). Recent studies have

recommended that new analytical methods also be used to detect smaller amounts of the toxin

that fall below the official action limit (Rossi et al., 2016).

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DA poisoning ecosystem effects

Diatoms are classified as a type of phytoplankton, meaning that they act as the primary

producers in many marine environments. In order for DA to progress up trophic levels of the

food chain, secondary consumers must consume prey with high concentrations of DA, which

occurs most frequently during the springtime and summertime bloom seasons. DA does not

bioaccumulate because it is a hydrophilic molecule; rather, it persists in digestive tissue and is

biologically filtered out at various rates, depending on the organism (Lefebvre and Robertson,

2010; Trainer et al., 2012). Large scale poisoning events have only gone up three trophic levels:

(1) the diatom, (2) the filter-feeding fish, and (3) a marine mammal, seabird, or human (Sekula-

Wood et al., 2011; Trainer et al. 2012).

Exposure to DA and risk of poisoning in third tier trophic levels occurs most commonly

as a result of eating fish and shellfish, particularly filter-feeding marine organisms (Kizer, 1994;

Trainer et al., 2012). These filter-feeding organisms, such as mussels, crabs, clams, anchovies,

and sardines, passively consume organic matter that is present in the water column.

Additionally, DA can be present in carnivorous fish, typically as a result of biomagnification

(eating poisoned prey) as well as incidental, direct consumption (Lefebvre et al., 2002). Feeding

in the proximity of a toxic Pseudo-nitzschia bloom, therefore makes surrounding marine

organisms susceptible to ingesting the toxigenic diatom or DA itself, causing the toxin to build

up in their digestive tissue (Mos, 2001). Although DA is not toxic to shellfish or finfish, the

accumulation in their digestive tissues still renders them unsafe for consumption by humans and

other aquatic animals. However, this buildup poses a particular threat to marine mammals and

sea birds, for which DA negatively affects their health in ways similar to humans (Mos, 2001;

Lefebvre et al., 2002; Trainer et al., 2012).

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Important case studies for DA poisoning have been conducted on charismatic fauna, such

as sea lions and whales (Scholin et al., 2000; Lefebvre et al., 2002; Fire et al., 2010). As the

frequency of toxigenic species of Pseudo-nitzschia, particularly P. australis, has increased

southward down the California coast, California sea lions have shown increasing toxicity and

mortality during bloom events (Lewitus et al., 2012; Du et al., 2016; Zalzal, 2017). Signs of

intoxication, usually via the ingestion of poisoned anchovies and sardines, include aggressive

behavior, neurological dysfunction, seizures, and death (Scholin et al., 2000; Tiedecken et al.,

2013; Ramsdell et al., 2014; Trainer et al., 2012). In an extreme, but not necessarily uncommon,

example, Scholin et al. (2000) found that over 400 California sea lions died as a result of DA

poisoning during May and June 1998. Different from sea lions that become ill due to

biomagnification, baleen whales such as Minke, Humpback, and Blue whales often consume the

toxin via filter-feeding of the diatom, toxin, and small finfish (Scholin et al., 2000; Lefebvre et

al., 2002; Trainer et al. 2012). Researchers often analyze beached whale fluids and feces to

detect levels of DA (Scholen et al., 2000; Lefebreve et al., 2002).

Methods for DA detection

Many forms of enzyme-linked immunosorbent assays (ELISAs) have been developed for

purposes of analyzing environmental samples and shellfish tissue. The Biosense ELISA method

is often the most universal method and has been validated as an effective method (Klievdal et al.

2007, Litaker et al. 2008, Eberhart et al. 2012). Traditionally, in situ biological samples have not

been preserved for analysis, making time series data for harmful algal blooms nearly impossible.

In order to detect DA in remote seawater samples, Eberhart et al. (2012) developed a method

using formalin to preserve the seawater samples for ELISA analysis. In this study, authors used

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0.5% formalin in the place of Lugol’s iodine or glutaraldehyde to preserve phytoplankton for

microplate analysis, which they use for the “PNW ELISA” in place of the Biosense ELISA to

detect particulate and dissolved DA (Eberhart et al., 2012). The NOAA/MSI ELISA, created to

detect DA in phytoplankton seawater samples and shellfish tissue with greater efficiency, was

found to be as equally accurate as high-liquid chromatography and liquid chromatography-mass

spectrometry methods (Litaker et al., 2008).

Environmental factors

The biogeochemical factors leading to outbreaks of Pseudo-nitzschia and DA can

indicate the potential of a bloom to cause a variety of negative impacts, such as those that are

location-specific, those related to public health, ecosystem health, and the economic success of

fisheries. However, the study of the environmental dynamics that can induce a bloom and

contribute to the increasing global frequency of Pseudo-nitzschia remain important, because they

factor into the development of a harmful algal bloom monitoring system. The building body of

literature surrounding the precursors to major, toxic algal blooms typically aims to aid in the

eventual creation of a more effective harmful algal bloom monitoring system (Lefebvre et al.,

2002; Trainer et al., 2007; McCabe et al., 2016; McKibben et al., 2017; Zalzal, 2017).

The California Current

Part of the North Pacific Gyre, the California Current runs southward along the North

American west coast (Fig. 2, Smayda et al., 1989; Checkley and Barth, 2009; Sekula-Wood et

al., 2011; Barron et al., 2013; Reimer et al., 2015; Seo et al., 2016; Moore et al., 2017). The

North Pacific Gyre Oscillation, Pacific Decadal Oscillation, El Niño/Southern Oscillation, North

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Atlantic Oscillation, and other climatic wind patterns combine to drive the ocean currents along

the west coast including the current itself, the North Pacific Current, the Coastal jet, California

Undercurrent, Davidson Current, and Southern California Eddy (Fig. 2; Checkley and Barth,

2009).

Coastal upwelling brings cold, nutrient-rich water from the depths to the surface. It is

driven by north-to-south-moving coastal winds, which in the Northern hemisphere moves water

perpendicular to this wind movement—referred to as Eckman Transport—and away from the

coast. As the water moves westward, cold water comes up to replace it, resulting in upwelling

(Trainer et al., 2000; Goodman et al., 2012; Schnetzer et al., 2013). Pseudo-nitzschia has been

known to flourish during the spring-time transition between summertime upwelling and winter

downwelling, related to changing coastal wind patterns (Tweddle et al., 2010; Du et al., 2015).

Figure 2. Diagram of the dynamics of the California Current (Checkley and Barth, 2009).

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In comparison with the past two centuries, the warming of the California Current at the

recent turn of the century marked an upward trend in the frequency of diatoms and toxigenic

species of Pseudo-nitzschia, particularly in Southern California and the Santa Barbara Basin

(Barron et al. 2013). Many researchers attribute this trend to a shift in the North Pacific Gyre

Oscillation (NPGO), pressure changes over the North Pacific that drive wind patterns that, in

turn, regulate nutrient availability along the Pacific coast; though, there remains debate

surrounding whether or not the NPGO can be classified as natural variability or is instead a result

of global climate change (Sekula-Wood et al., 2011; Barron et al., 2013; Wells et al., 2015).

This potential shift in the NPGO, its origins, and effects have been recommended for further

study (Wells et al., 2015).

However, based on current understandings of the effects of climate change in the North

Pacific, Pseudo-nitzschia appears to respond both positively and negatively to increased

temperature and ocean acidification (Tatters et al., 2012; Wells et al., 2015). Yet, species of

Pseudo-nitzschia tended to respond generally negatively to augmented anthropogenic

eutrophication and temperature stratification by decreasing in cellular abundance (Tatters et al.,

2012; Wells et al., 2015).

Effects of the Columbia River

The Columbia River is located along the border between Oregon and Washington State

and is regarded as the largest river on the U.S. west coast (Liu et al., 2009). Generally,

freshwater plumes can affect the salinity, nutrient availability, temperature stratification, gross

primary productivity, and energy flux in its reciprocating ocean basin (Hickey et al., 2005;

Thessen et al., 2005; Liu et al., 2009; Hickey et al., 2013; Fouilland et al., 2017). The

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summertime freshwater plume originating from the melting snowpack of the Columbia River,

frequently described as eddy-like, is bi-directional as it moves primarily southward toward

Heceta Bank but also northward along the Washington coast depending on the fluctuations in of

seasonal winds, the California Undercurrent, and the Davidson Current (Fig. 3; Hickey et al.,

2005; Liu et al., 2009; Hickey at al., 2013).

Figure 3. Seasonal direction of the Columbia River plume during the summer and fall months, with varying weather

models (Hickey et al., 2005; Hickey et al., 2013).

During the summer, winds turn to favor coastal upwelling conditions, which can move a portion

of the plume hundreds of meters offshore. Consequently, the portion of the Columbia River

plume that gets transported offshore can take some of the Pseudo-nitzschia outbreak that occurs

before upwelling conditions far offshore, as well (Liu et al., 2009). This can be seen from data

collected from May 2017, with high abundance of minimally toxic P. pungens hundreds of

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meters offshore south of the Columbia River and just north of Heceta Bank (Hickey et al., 2013;

Wood et al., 2017).

Pacific Decadal Oscillation and El Niño

The Pacific Decadal Oscillation (PDO) and El Niño Southern Oscillation (ENSO) regulate

patterns in the North Pacific Ocean and thereby the California Current, as well (Chekley and

Barth, 2009; Moore et al., 2010; Barron et al., 2013; McKibben et al., 2017). The most recent

phenomena stemming from these climatic regulators are the 2014-2016 El Niño and warm phase

anomaly of the PDO known as “The Blob,” that together created abnormally warm waters along

the U.S. west coast, sparking the most toxic harmful algal bloom on record (Rao and Ren, 2017;

Zhu et al., 2017). Accordingly, DA events over the past 20 years are strongly correlated with El

Niño and warm phases of PDO (Moore et al., 2010; McKibben et al., 2017).

The interannual El Niño occurrence typically causes the entire Pacific Ocean to become

warmer, flattening the thermocline, and interrupts upwelling and Eckman transport along the

U.S. West Coast every two to seven years. The 2014-2016 El Niño is often regarded as the

strongest such event recorded in the central Pacific (Moore et al., 2010; McCabe et al., 2016;

L'heureux et al., 2017; Lim et al., 2017; McKibben et al., 2017; Rao and Ren, 2017).

“The Blob” developed in fall 2013 in the northeast Pacific Ocean and reached the west coasts

of the U.S. and Canada in spring 2015 (Sekula-Wood et al., 2011; McCabe et al., 2016;

McKibben et al., 2017; Zhu et al., 2017). Caused by the expansion of an atmospheric high-

pressure cell, this extensive body of anomalously nutrient-poor, warm water—roughly 2.5˚C

above the long-term average—spanned approximately 500km and reached up to 100m in depth.

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The coastal movement of “The Blob” supplied it with sufficient nutrients to prompt a major

phytoplankton bloom (McCabe et al., 2016).

In addition, studies have connected increased metabolic DA production by Pseudo-nitzschia

with warm phases of the California Current (McCabe et al., 2016; McKibben et al., 2017). As

the frequency of widespread, anomalous warm sea-surface temperature ocean events increase,

harmful algal blooms and particularly the typically warm-water favoring toxigenic Pseudo-

nitzschia pose greater risks to coastal communities around the world (Engel et al., 2011; Trainer

et al., 2012; McCabe et al., 2016; McKibben et al., 2017).

While local conditions such as upwelling and freshwater plumes can characterize harmful

algal bloom “hotspots,” larger-scale factors often determine coast-wide events (Sekula-Wood et

al., 2011; McKibben et al., 2017; Zhu et al., 2017). Although many causes of the “The Blob”

remain largely debated among scientists, it acted as a substantial positive temperature anomaly

water mass—greatest in the region since the 1980s—that invaded the west coast in 2015 (Sekula-

Wood et al., 2011; McKibben et al., 2017; Zhu et al., 2017).

Overall, in 2015, a very strong El Niño combined with the appearance of “The Blob” created

the largest, most toxic harmful algal bloom along the U.S. west coast (McCabe et al., 2016;

McKibben et al., 2017; Zhe et al., 2017).

METHODS

Whole water and net water samples were collected aboard NOAA research vessel Bell M.

Shimada from 63 sampling sites between California and Oregon during May 2017.

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Crewmembers filtered samples for particulate DA (DA) analysis, froze samples for dissolved

DA analysis, and added formalin (<1%) for cell counts.

Cells were counted using a Zeiss Axiovert 135 inverted light microscope. Preparing the

seawater samples for cell counting included allowing the 154 original 20mL samples to settle in

test tubes for one week, and aspirating the top 18mL out to leave a concentrated 2mL sample.

Cells were counted on a 100-box, 1mL hemocytometer. Each concentrated 2mL sample was

individually mixed, 1mL was put on the hemocytometer, and cells were counted on half of the

hemocytometer (every other row).

To prepare particulate DA samples, 47-mm diameter nitrocellulose frozen filters (0.45µm

pore size, Millipore MF-Membrane filters) in 15mL conical tubes were removed from -20˚C and

4mL of MilliQ dH2O was added to each tube. Filters were mashed in the 4mL of MilliQ dH2O

using a spatula. Samples were vortexed for approximately 10 seconds, placed in sonicator bath

for 20 minutes, again vortexed for approximately 10 seconds, and placed in refrigerator

overnight (4˚C). Lastly, samples were centrifuged at the highest setting (8500 x g) for 10

minutes.

To prepare dissolved DA samples, frozen samples were thawed from -20˚C to room

temperature, diluted at 25X into 2mL micro-centrifuge tubes, and placed in refrigerator (4˚C) for

short-term storage.

Scanning electron microscopy (SEM) was used to determine relative abundance of species at

selected locations. To prepare samples for SEM, net tow samples were filtered using a

polycarbonate isopore membrane filter (13mm diameter, 0.6µm pore size, Millipore Corp.).

Cells present on the filter oxidized using potassium permanganate, cleaned with concentrated

hydrochloric acid (37%), and rinsed with distilled water. After drying, filters were glued to

20

aluminum stubs with conductive adhesive, coated with gold-palladium, and analyzed using

JEOL model 6360 LV SEM. Specific species were determined using published morphological

characteristics.

DA analysis was done via the PNW ELISA method, as developed and prescribed by the

National Oceanic and Atmospheric Administration (NOAA) Northwest Fisheries Science Center

and Eberhart et al. (2012). First, 50µL of sample or standard was placed into duplicate wells of

96-well plate pre-coated with DA-carboxyl-linked bovine serum albumin conjugate (Beacon

Analytical Systems Inc., Portland, ME), and then 50µL of anti-DA antibody diluted 1:500 in

blocking buffer (3% non-fat dry milk/0.01 M PBS). The plate was covered, mixed for 1-2

minutes, and placed in a dark environment for one hour. After the hour incubation period, wells

were washed three times with phosphate-buffered saline plus tween (PBST; 0.01 M PBS/0.05%

Tween 20). Next, goat anti-rabbit secondary antibody (P-488 DakoCytomation, Denmark A/S)

horseradish peroxidase (HRP, 100µL of 1:3000 dilution) was added to each well, the plate was

covered, mixed for 1-2 minutes, and placed in a dark environment for 30 minutes. After the 30-

minute incubation period, the wells were washed in an identical manner with PBST, followed by

the addition of 100µL of tetramethylbenzidine (TMB) substrate (BioFX, Owings Mills, MD) to

all wells and the placement of the plate in a dark environment for 15 minutes. The addition

100µL of 0.1 N hydrochloric acid (HCl) to each well ceased the reaction, and absorbance was

measured at 450nm using a micro-plate reader (Versamax, MDS, Sunnyvale, CA). The assay has

a linear range from 0.1 to 3 ppb.

Unknown DA concentrations were estimated via interpolation from a standard curve

generated with a 4-parameter curve-fit algorithm. The PNW ELISA method has a minimum

detection limit of 250 ng DA L-1 in seawater (Eberhart et al., 2012).

21

The NOAA Northwest Fisheries Science Center provided additional data including: Pseudo-

nitzschia abundance, particulate DA concentration, temperature, and salinity for the years 2015-

2017. Columbia River discharge data was accessed on the USGS National Water Information

System. Coastal upwelling index data, courtesy of National Marine Fisheries Service (NMFS),

Pacific Fisheries Environmental Laboratory (PFEL), was accessed on the University of

Washington School of Aquatic and Fishery Sciences, Columbia Basin Research DART (Data

Access in Real Time) website (“DART Pacific Ocean Coastal Upwelling Index Graphics &

Text”). These data were analyzed using linear regressions and analysis of variance tests using

RStudio.

RESULTS

Pseudo-nitzschia spp. cells were present at roughly at approximately 65% of stations

sampled in 2017, in comparison with 87% in 2015 and 83% in 2016. At approximately 9%

percent of sampling stations, abundance was greater or equal to 100,000 cells/L in both 2016 and

2017. Areas of such great abundance included Heceta Bank, Oregon off the coast of Newport

Oregon and Trinidad, California near the California-Oregon state border. Between 2015 and

2017, abundance was greater closer to the shore, with the exception of an area of high abundance

hundreds of meters off shore Heceta Bank, Oregon in 2017 (Figs. 4, 11, and 12). The date of

sampling for this area of high abundance also corresponded with above average Columbia River

discharge (Figs. 4 and 5).

22

Figure 4. 2017 Cellular abundance of Pseudo-nitzschia spp. (cells/L) and concentration of particulate domoic acid

(ng/L) at corresponding sampling locations from May 2017 Bell M. Shimada cruise.

Figure 5. Provisional Columbia River discharge (ft3/s) data from USGS Port Westward station between May 1 to

August 30, 2017. Source USGS Current Conditions for the Nation; https://waterdata.usgs.gov/nwis/uv.

Trinidad Trinidad

Heceta

Bank

Heceta

Bank

https://waterdata.usgs.gov/nwis/uv

23

This offshore location, however, was not highly toxic—predominately characterized by

72% minimally toxic species P. pungens and 28% highly toxic species P. australis. In 2017,

SEM analysis was conducted on samples from this location as well as the Heceta Bank and

Trinidad localities (Table 2; Figs. 6-8). The inshore region of Heceta Bank had a roughly even

amount of P. pungens (59%) and P. australis (41%) (Table 2). On the other hand, Trinidad, CA

was dominated by highly toxic species 75% P. australis and 22% P. multiseries, as well as 3%

P. pungens (Table 2). Linear regression analysis revealed that Columbia River discharge had

minimal direct impact on cellular abundance (Fig. 10; R2 = 0.04785, p = 1.576 x 10-5). In

addition, cellular abundance did not significantly correlate with water temperature or salinity

(Table 3).

In 2016, SEM analysis was also conducted at the Heceta Bank and Trinidad locations

(Figs. 6-8). Heceta Bank was characterized by primarily P. pungens (84%) as well as P.

multiseries (16%). The species P. australis (72%) dominated Trinidad, and P. multiseries was

also present (28%). In 2015, only P. australis was present in the Trinidad mid-July sample, and

only P. fraudulenta was present just south of the Columbia River (Table 3).

Location

Percent (%)

prevalence of

Pseudo-nitzschia

australis

Percent (%)

prevalence of

Pseudo-nitzschia

multiseries

Percent (%)

prevalence of

Pseudo-nitzschia

pungens

Offshore location

near Heceta Bank,

Oregon 2017

72

0

28

2015 2016 2017 2015 2016 2017 2015 2016 2017

Heceta Bank, Oregon n/a 0 41 n/a 16 0 n/a 84 59

Trinidad, California 100 72 75 0 0 22 0 28 3

Table 2. Results from scanning electron microscopy (SEM) at select locations between 2015-2017.

24

Figure 6. Pseudo-nitzschia australis, enlarged 10,000X (left) and 1,000X (right) using scanning electron

microscopy.

Figure 7. Pseudo-nitzschia multiseries, enlarged 10,000X (left) and 1,000X (right) using scanning electron

microscopy.

Figure 8. Pseudo-nitzschia pungens, enlarged 10,000X (left) and 1,000X (right) using scanning electron

microscopy.

25

Particulate domoic acid was present at all but one station in May 2017, with maxima at

Heceta Bank (5,669 ng/L) and Trinidad (4,984 ng/L). During May to June of 2016, domoic acid

was observed at 63% of stations, also with maxima at Trinidad. Similarly, particulate domoic

acid concentration was shown to be greater inshore across all three bloom seasons, even in the

case of the area of high abundance offshore in May 2017 (Fig. 4). Concentration of particulate

domoic acid also did not correlate significantly with water temperature or salinity (Table 3).

2015

Abundance

(n = 349)

2015

Toxicity

(n = 349)

2016

Abundance

(n = 203)

2016

Toxicity

(n = 203)

2017

Abundance

(n = 63)

2017

Toxicity

(n = 63)

R2 Salinity 0.0007 0.0004 0.008 0.007 0.01 0.02

R2

Temperature

0.0006 0.0004 0.02 0.002 0.03 9E-06

P-value

Salinity

0.6326 0.7115 n/a n/a 0.4043 0.2173

P-value

Temperature

0.6517 0.7231 n/a n/a 0.6519 0.9815

Table 3. Results from linear regression analysis between salinity, temperature, cellular abundance, and

concentration of particulate domoic acid from 2015-2017 summertime bloom sampling. See figures 15 to 24 in the

appendix for graphs of this data.

Correlation between salinity and cellular abundance as well as concentration of

particulate domoic acid increased annually between 2015 and 2017 (Table 3). This was also the

case for the correlation between temperature and cellular abundance as well as concentration of

particulate domoic acid, with the exception of a considerably lower correlation between

temperature and concentration of domoic acid in 2017 (Table 3). However, the discrepancy

between these correlations among all three years could be due to varying sample size, as sample

size decreased annually (Table 3).

26

Water temperature at time of sampling during 2015, 2016, and 2017 cruises were

significantly similar (Welch Two Sample t-test; t = 1.694, df = 351, p-value = 0.09116).

Upwelling near Trinidad (42N, 125W), identified as an emerging Pseudo-nitzschia hotspot in

2015, has decreased between 2015 and 2017 during the months of April to September (Fig. 9).

Upwelling near Trinidad during bloom season was significantly similar in 2016 and 2017 (p =

4.29 x 108), but not between 2017 and 2015 (p = 0.127).

Figure 9. Coastal upwelling index (m3/s/100m) between April 1 and September 30 during the years 2015-2017 near

Trinidad, California (42N, 125W). Data courtesy of “DART Pacific Ocean Coastal Upwelling Index Graphics &

Text.”

27

DISCUSSION

2015 mega-bloom abundance greater than 2016 and 2017 blooms

The scale and intensity of the Pseudo-nitzschia summer bloom in 2015 exceeded that of

2016 and 2017. In both 2016 and 2017, abundance was greater than or equal to 100,000 cells/L

at approximately 9% percent of sampling stations. In contrast, out of the more than 300 stations

sampled in 2015, at least 27% exceeded 100,000 cells/L (McCabe et al., 2016). On the other

hand, identified hotspots, including Heceta Bank, Oregon and Trinidad, California, had similar

quantities of Pseudo-nitzschia cells among the three years.

In 2017, an anomalous area of offshore abundance existed near Heceta Bank, Oregon,

just southwest of the Columbia River. At over 350 km offshore Newport, Oregon, the whole

water sample from this station had an abundance of approximately 16,500 cells/mL—the sixth

most abundant May 2017 sample. However, without a strong correlation between Columbia

River discharge and cellular abundance (Fig. 10), it is most likely that the location of this

Pseudo-nitzschia bloom was due to the above average discharge from the Columbia River in

early May (Fig. 5). In the absence of severe storms in the summer, the Columbia River plume

generally moves southwest towards Heceta Bank (Fig. 3; Hickey et al., 2005; Hickey et al.,

2013). In the summer months, the increased force of the plume in combination with the turn of

summertime upwelling—during which winds move existing warmer water offshore—causes the

offshore movement of blooms that originated inshore. This data reaffirms the influence of the

Columbia River plume on the location and distribution of Pseudo-nitzschia, but does not directly

impact cellular abundance.

28

In 2015, the dominant species along the west coast was P. australis. Due to a series of

storms that transported the species up the coast, P. australis exceeded its typical range off the

coast of California and persisted because of the anomalously warm ocean temperatures (Fig. 11;

McCabe et al., 2016). Yet, in 2016 and 2017, P. australis was the dominant species only at

Trinidad, California (Fig. 4 and 12). Species variation at Heceta Bank was similar among the

three years with primarily P. pungens, accompanied by slightly less than 50% P. australis (Table

3).

Current research indicates that the 2013-2015 North Pacific Ocean warm anomaly,

commonly referred to as “The Blob,” was the result of a high-pressure cell that decreased ocean

cooling and was transported via easterly winds. This caused sea surface water temperatures to

increase more than 2.5˚C above their longstanding mean (McCabe et al., 2016). In addition, the

2014-2016 El Niño also increased sea surface temperatures, though not as significantly as “The

Blob in 2015 (McCabe et al., 2016; McKibben et al., 2017; Zhu et al., 2017). Despite this,

recorded temperatures at sampling locations in 2015 never exceeded 20.9˚C. The difference

between literature temperature ranges and the recorded values from NOAA research vessels

between 2015 and 2017 was likely caused by the timing and inconsistency of sampling and lack

of upwelling index data at specific sampling locations. Additionally, this disparity potentially

accounts for the insignificant correlation between temperature and abundance across all three

years.

Abundance and toxicity not necessarily correlated

Due to species variation, areas of high Pseudo-nitzschia abundance did not necessitate

that the same location was also an area of high domoic acid concentration. In 2017, three species

29

of Pseudo-nitzschia were identified via scanning electron microscope: Pseudo-nitzschia

australis, Pseudo-nitzschia multiseries, Pseudo-nitzschia pungens (Figs. 6-8; Table 3). Of these

three distinct species, P. australis is known to be the most toxic, followed by P. multiseries, and

P. pungens is largely regarded as minimally toxic (Trainer et al., 2000; Trainer et al., 2012). In

2017, the area of offshore abundance near Heceta Bank was marginally toxic, despite high

cellular abundance, because P. pungens was the most prevalent species at that location (Table 3;

Fig. 4). The inshore sampling station at Heceta Bank was highly toxic due to the presence of

roughly half P. pungens and P. australis and high cellular abundance of both species (Table 3;

Fig. 4). Sampling station off the shore of Trinidad, California was identified to be an area of

high toxicity but low cellular abundance, which was due to the prevalence of approximately 75%

P. australis, and 22.03% P. multiseries, and 3.39% P. pungens (Table 3; Fig. 4). The high

concentration of particulate domoic acid at this location can be attributed to the cellular toxicity

of P. australis. In 2016, these distributions at each given location were similar (Table 3).

The summer blooms decreased in average toxicity by year. The May 2015 bloom was

the most domoic acid-producing bloom on record (Fig. 11; McCabe 2016). The 2015, 2016, and

2017 average particulate domoic acid concentrations among all sampling stations were 778.96

ng/L, 525.00 ng/L, and 516.62 ng/L, respectively. This is likely due to the decreased prevalence

of P. australis, because in 2016-2017 storms did not transport the species up the coast as in 2015.

Emerging hotspot Trinidad, California and upwelling

Dominant species at Trinidad, California was P. australis among all three years (Table 3;

Figs. 11 and 12). P. australis responds rapidly to excess nutrients (McCabe et al., 2016; Howard

et al., 2007; Cochlan et al., 2008), as often provided by upwelling. This could explain the

30

significant correlation between abundance (R2 = 0.6064, p = 0.00356), toxicity (R2 = 0.6527, p =

0.035) and upwelling at Trinidad, California during May 2017 (Figs. 14 and 15). Further

research at this location is crucial due its status as the major fishing zone, which could result in

major economic loss because of Pseudo-nitzschia related closures.

CONCLUSIONS

Overall, the Pseudo-nitzschia was less prevalent in 2017 and 2016 than 2015, as seen

through the extent and toxicity of the 2015 mega-bloom. The combination of anomalous events,

including the El Niño, “The Blob,” and severe storms, allowed for increased sea-surface

temperatures and for toxic species to persist along the coast, accounting for the devastation

caused in 2015 along the California Current. While these factors were not present in 2017 or

2016, sampling data from those years showed the persistence of an emerging hotspot off the

coast of Trinidad, California, which is possibly due to each location’s proximity to upwelling

zones. In addition, the May 2017 data indicated that the Columbia River plume can affect the

location and distribution of Pseudo-nitzschia blooms, particularly near Heceta Bank. Results

from this analysis of three years of sampling data suggest that further research needs to focus on

the relationship between toxic algal blooms and coastal upwelling zones. Ultimately, these

conclusions can be added to NOAA’s harmful algal bloom monitoring system to better predict

toxic algal bloom outbreaks and mitigate the threat posed by Pseudo-nitzschia to the fisheries

industry and public health.

31

ACKNOWLEDGMENTS

I would like to thank David Backus, Brian Bill, Vera Trainer, Annie Merrill, Bradley

Johnson, Bich-Thuy Eberhart, and Nick Adams for their guidance and mentorship. This project

was made possible because of the support and efforts of the NOAA Ernest F. Hollings

Scholarship Program team (Silver Spring, MD), the Marine Biotoxins Program at NOAA

Northwest Fisheries Science Center (Seattle, WA), and the Davidson College Department of

Environmental Studies (Davidson, NC).

32

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APPENDIX

Figure 10. Results from linear regression analysis between Pseudo-nitzschia abundance (cells/L)

and mean Columbia River discharge (ft3/s) from May 2017 (R2 = 0.04785, p = 1.576 x 10-5, n =

20).

R² = 0.0479

0

5000

10000

15000

20000

25000

30000

490,000 500,000 510,000 520,000 530,000 540,000 550,000 560,000

Cel

ls p

er L

iter

Discharge (ft^3/s, mean)

Pseudo-nitzschiaAbundance and Average Columbia River

Discharge near Heceta Bank

42

Figure 11. (a) Particulate domoic acid concentration and (b) Pseudo-nitzschia cellular

abundance seawater samples collected aboard the NOAA Ship Bell M. Shimada from 2015. (c) A

net tow sample off Point Conception, California on 24 June (concentrated sample, top panel;

microscopic image of diluted sample at 200X magnification, bottom panel). Source McCabe et

al., 2016.

43

Figure 12. (right) Particulate domoic acid concentration and (left) Pseudo-nitzschia cellular

abundance seawater samples collected May-June 2016. Source Showalter, 2016.

44

Figure 13. Variation in cellular abundance (cells/L) and Coastal Upwelling Index (m3/s/100m)

near Trinidad, California from May 2017, (R2 = 0.60638, p = 0.00356, n = 7).

R² = 0.6064

-5000

0

5000

10000

15000

20000

0 20 40 60 80 100 120 140 160 180

Cel

ls p

er L

iter

Coastal Upwelling Index (m^3/s/100m)

Pseudo-nitzschiaAbundance and Coastal Upwelling Index at

Trinidad, CA May 2017

45

Figure 14. Variation in domoic acid concentration (ng/L) and Coastal Upwelling Index

(m3/s/100m) near Trinidad, California from May 2017, (R2 = 0.62224, p = 0.035, n = 7).

R² = 0.6222

-1

0

1

2

3

4

5

6

0 20 40 60 80 100 120 140 160 180

Dom

oic

aci

d c

once

ntr

atio

n (

ng/L

)

Coastal Upwelling Index (m^3/s/100m)

Domoic Acid Concentration and Coastal Uwelling Index at Trindad,

CA May 2017

46

Figure 15. Variation in cellular abundance (cells/L) and sample water temperature (˚C) from

May 2017, (R2 = 0.00336, p = 0.6519, n = 63).

R² = 0.0034

0

5000

10000

15000

20000

25000

30000

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0

Cel

ls p

er L

iter

Temperature (˚C)

Pseudo-nitzschiaAbundance and Water Temperature, May 2017

47

Figure 16. Variation in cellular abundance (cells/L) and salinity (ppm) from May 2017, (R2 =

0.02488, p = 0.2173, n = 63).

R² = 0.0115

0

5000

10000

15000

20000

25000

30000

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

Cel

ls p

er L

iter

Salinity (ppm)

Pseudo-nitzschiaAbundance and Salinity, May 2017

48

Figure 17. Variation in particulate domoic acid concentration (ng/L) and temperature (˚C) from

May 2017, (R2 = 8.9e-06, p = 0.9815, n = 63).

R² = 9E-06

0

1

2

3

4

5

6

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0

pD

A (

ng/L

)

Temperature (˚C)

Domoic Acid Concentration and Water Temperature, May 2017

49

Figure 18. Variation in particulate domoic acid concentration (ng/L) and salinity (ppm) from

May 2017, (R2 = 0.02488, p = 0.2173, n = 63).

R² = 0.0249

-1

0

1

2

3

4

5

6

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

pD

A (

ng/L

)

Salinity (ppm)

Domoic Acid Concentration and Salinity, May 2017

50

Figure 19. Variation in cells of Pseudo-nitzschia spp. per liter given temperature (˚C) from May

to June 2016, (R2 = 0.017, n = 118) . Source Showalter, 2016.

10 12 14 16 18

0

1000

2000

3000

4000

5000

6000

Cells per Liter with Temperature

Temperature(C)

Cells

per

Lite

r

R2 =0.017

51

Figure 20. Variation in cells of Pseudo-nitzschia spp. per liter given salinity (ppm) from May to

June 2016, (R2 = 0.008, n = 118). Source Showalter, 2016.

24 26 28 30 32 34

0

1000

2000

3000

4000

5000

6000

Cells per Liter with Salinity

Salinity(ppm)

Cells

per

Lite

r

R2 =0.008

52

Figure 21. Variation in cellular toxicity (pgDA/cell) given salinity (ppm) from May to June

2016, (R2 = 0.007, n = 118). Source Showalter, 2016.

24 26 28 30 32 34

0

10

20

30

40

Cellular Toxicity with Salinity

Salinity(ppm)

Ce

llula

r T

oxic

ity (

pg d

om

oic

acid

/ce

ll)

R2 =0.007

53

Figure 22. Variation in cellular toxicity (pgDA/cell) given temperature (˚C) May to June 2016,

(R2 = 0.002, n = 118). Source Showalter, 2016.

10 12 14 16 18

0

10

20

30

40

Cellular Toxicity with Temperature

Temperature(C)

Ce

llula

r T

oxic

ity (

pg d

om

oic

acid

/cell)

R2 =0.002

54

Figure 23. Variation in particulate domoic acid concentration (ng/L) and temperature (˚C) from

June to August 2015, (R2 = 0.0004, p = 0.7231, n = 349).

R² = 0.0418

-5000

0

5000

10000

15000

20000

25000

0 5 10 15 20 25

pD

A (

ng/L

)

Temperature (˚C)

Domoic Acid Concentration and Temperature 2015

55

Figure 24. Variation in particulate domoic acid concentration (ng/L) and salinity (ppm) from

June to August 2015, (R2 = 0.0004, p = 0.7115, n = 349).

R² = 0.063

-5000

0

5000

10000

15000

20000

25000

0 5 10 15 20 25 30 35 40

pD

A (

ng/L

)

Salinity (ppm)

Domoic Acid Concentration and Salinity 2015

56

Figure 24. Variation in cellular abundance (cells/mL) and temperature (˚C) from June to August

2015, (R2 = 0.006, p = 0.6517, n = 349).

R² = 0.0029

0

500

1000

1500

2000

2500

3000

3500

0 5 10 15 20 25

Cel

ls p

er m

L

Temperature (˚C)

Pseudo-nitzschiaAbundance and Temperature 2015

57

Figure 24. Variation in cellular abundance (cells/mL) and salinity (ppm) from June to August

2015, (R2 = 0.0007, p = 0.6326, n = 349).

R² = 0.003

0

500

1000

1500

2000

2500

3000

3500

0 5 10 15 20 25 30 35 40

Cel

ls p

er m

L

Salinity (ppm)

Pseudo-nitzschiaAbundance and Salinity 2015