A quantitative review of research on the influence of the carbon dioxide concentration and temperature on the toxin content of the dinoflagellate Alexandrium to see if there is a general trend
Supervisors:
Preface
In this thesis a review of the existing articles about the influence of climate change on the toxicity of harmful algal blooms and especially the dinoflagellate Alexandrium is created. Moreover,the data from several research articles about the influence of rising temperature and carbon dioxide (CO2) concentrations on the toxin content of the dinoflagellate Alexandrium will be combined in one overview. So far, various studies on this subject have performed, but a comprehensive overview combining all data from these studies is lacking. It is therefore difficult to make any general statement about rising temperatures and CO2 concentration on the toxin content of the dinoflagellate Alexandrium. To this end, I combined all available information and data on this topic in this thesis. At the end of this thesis there will be an answer to the question: ‘What is the influence of rising temperatures and the CO2 concentrations on the toxin content of Alexandrium?’. It is expected that not C-rich toxins, like N-rich toxins which will be the subject of the research, will increase with increasing CO2 concentration and rising temperatures, because there will be more energy available for phytoplankton and thus also for producing energetically costly toxins. Besides answering the research question there will also be some knowledge gaps pointed out that need more research in the future.
Introduction
Chapter 1: Climate Change
At this moment we are experiencing the consequences of global change. Global change includes, amongst others, higher atmospheric carbon dioxide (CO2) concentrations, higher temperatures, eutrophication, and changing ocean currents. Part of these various changing conditions involve a change in our climate, and clearly are related to increasing CO2 concentrations and an associated increase in global mean temperature. In this thesis the focus will be on the impacts of these climate change factors.
The chemical background of climate change
Due to human activities the CO2 concentration increased substantially over the last 250 years. Since pre-industrial times atmospheric CO2 levels have increased by nearly 40%. Today, CO2 concentrations have even reached 400 ppm, which is higher than ever over the last 800,000 years, and is a result of fossil fuel burning and deforestation (Doney, Fabry, Feely, & Kleypas, 2009; Ipcc, 2013; Moore et al., 2008). If nothing changes, atmospheric CO2 concentrations are expected to reach 900 ppm by the end of this century (Ipcc, 2013). The rate of this increase in the CO2 levels is unprecedented. In fact, over the last ten years atmospheric CO2 levels increased more than has been measured since the beginning of continuous direct atmospheric measurements (figure 1) (Hegerl et al., 2007). This increase is not only visible in the atmosphere, as 93% (=39,100 gigatonnes) of CO2 present in the atmosphere will eventually diffuse into the ocean (Doney et al., 2009; Hallegraeff, 2010). The oceans have already taken up one third of the anthropogenic produced CO2 concentration since the start of the industrial revolution. Due to this oceanic uptake, the atmospheric CO2 concentration will increase less than would be expected as result of human impact (Doney et al., 2009; Hallegraeff, 2010).
Figure 1: Atmospheric CO2 concentrations at Mauna Loa in ppm (red), surface ocean pH (cyan), and pCO2 in (��atm) (tan) at Ocean Station ALOHA in the subtropical North Pacific Ocean (Doney et al., 2009)
Phytoplankton accounts for approximately 50% of the global primary production (Longhurst et al., 1995). Each year the ocean takes up 2 gigatonnes of carbon by abiotic absorption and 1.8 gigatonnes by photosynthesis (Hallegraeff, 2010). The CO2 is taken up by phytoplankton for photosynthesis and turned into organic matter. Part of this organic matter will sink out, and ultimately leads to partial loss to the deep ocean. This transport of carbon is called the biological pump (Hallegraeff, 2010). Inter alia this biological pump causes CO2 uptake by the ocean, which is thus removed from the atmosphere (Moore et al., 2008). If this biological pump is reduced for some reason, either because of shifts in ocean circulation or reduced phytoplankton growth, it could lead to a more rapid increase in the atmospheric CO2 concentration (Heinze, 2004; Munhoven, 2007).
If the CO2 concentrations increase, the temperature of the atmosphere will rise, which causes the temperature of the sea surface water to rise. This causes stratification, which makes the nutrient availability go down, because of a lack of convection. The decrease of nutrient availability in the upper mixed layer of the ocean may result in a reduction of the primary production (Behrenfeld et al., 2006). In the end, this may weaken the biological pump and thereby increase the CO2 concentration in the ocean and thus in the atmosphere (Frouin & Iacobellis, 2002). Some researches state that his might have occurred at the end of the ice ages (Frouin & Iacobellis, 2002).
A rise of atmospheric CO2 concentrations has a range of adverse effects on the oceans, both direct and indirect, including ocean acidification, increasing sea surface temperatures, changes in the density structure of the upper ocean which leads to a change in the vertical mixing of the water (i.e. stratification), change in upwelling winds, and changes in timing and volume of freshwater runoff into coastal marine waters (Moore et al., 2008). Here, I will further focus on the impacts of ocean acidification and rising sea surface temperatures.
Results of climate change
Ocean acidification
An increase in oceanic CO2 concentration causes a drop of the surface ocean pH and thus leads to acidification of the ocean. If carbon dioxide (CO2) reacts with the water of the ocean (H2O) this will eventually lead to an increase in hydrogen ions (H+), which means the pH will drop and the ocean will get more acidic (figure 2). This drop in pH also causes a shift in the chemical carbonate balances of the ocean, resulting in more carbon dioxide (CO2) and bicarbonate (HCO3) and less carbonate (CO32-) (figure 3) (Caldeira & Wickett, 2003; Wolf-gladrow, Riebesell, & Burkhardt, 1999). This lower carbonate ion concentrations in the surface waters is the result of the uptake of hydrogen ions, which increased in concentration, by carbonate and thus the formation of HCO3-. This leads to a decrease in the availability of carbonate for calcifying organisms (Doney et al., 2009; Hallegraeff, 2010). In addition to this decrease in calcium carbonate availability, calcium carbonate will get more soluble at a lower pH, which may cause calcium carbonate shells to dissolve. It seems, however, that the decrease of the available carbonate is the biggest problem of ocean acidification for calcifying organisms. The response of different calcifying organisms varies widely, because they all differ in degree of sensitivity to acidification (Doney et al., 2009).
Due to anthropogenic forcing the pH of the surface water already dropped by 0.1 unit and will decrease even more over the next decades. It is expected that the pH will have dropped by 0.4 units by the year 2100 if there is no change in the current CO2 emissions (Caldeira & Wickett, 2003; Orr et al., 2005). This pH will be lower than experienced for millennia on this planet and the rate of change is even 100 times faster than ever experienced (figure 1) (Hallegraeff, 2010).
Phytoplankton species differ in their relative consumption of bicarbonate and CO2, and changes in the availability of these compounds can have a strong influence on phytoplankton growth (Rost, Zondervan, & Wolf-Gladrow, 2008). Various phytoplankton species were shown to possess carbon concentrating mechanisms (CCM). Species that have a CCM can take up HCO3- as well, making these species less dependent on only CO2. Species with a limited CCM, however, take advantage of an increasing CO2 concentration, because they depend entirely on CO2. In contrast, however, species that possess CCMs may downregulate their CCM and re-allocate the energy towards other cellular processes, which provides these species an additional advantage (Eberlein, Van de Waal, & Rost, 2014; Rost et al., 2008).
Rising temperature
Due to increasing CO2 emissions, the Earth’s temperature is also expected to rise, since CO2 acts as a greenhouse gas. Additionally, emissions of other greenhouse gasses, such as methane and nitrous oxide, are also expected to rise, strengthening global warming even more. The increase of these gasses are the result of agriculture and fossil fuel burning by humans. Together, these cause the atmospheric temperature to rise and hence the ocean temperature to increase (figure 4) (Hegerl et al., 2007). The oceans mediate global warming by acting like a buffer, and already have taken up more than 90% of the heat since 1961 (Beardall et al., 2004).
Figure 4: The global average temperature of the surface water from 1850 until 2000.The left axis shows the difference from 1964 till 1990, right axis the temperature in degrees Celsius (Hegerl et al., 2007).
Although the earth’s climate can change naturally and cause oscillations of tens of years as result of volcanic activity, variation in the orbit of the sun and the earth, continental drift and the internal fluctuations in the atmosphere, hydrosphere, and cryosphere, and although our planet has experienced higher and lower temperatures and CO2 concentrations than we are experiencing now, the rate in which this is happening is much higher than we saw in the past (Beardall et al., 2004). Consequently, Hereby it is very likely that the warming climate in the early 20th century is due to anthropogenic forcing (Hegerl et al., 2007). Even if the radiative forcing levels are kept the same as in the year 2000, global temperatures will increase with a rate of 0.1 in degrees Celsius per decade for the next two decades. The rate at which global temperatures will increase depends on the future magnitude of greenhouse gas emissions (Hegerl et al., 2007; Ipcc, 2013).
If the temperature of the surface water will increase this will ultimately lead to enhanced stratification, which reduces mixing of the water and thereby causes a decrease in nutrient input from deeper waters into the upper mixed layer (Moore et al., 2008). This may have large consequences for all sort of organisms in the upper layer of the ocean, notably phytoplankton that require nutrients for their growth.
Chapter 2: Harmful Algal Blooms
Harmful algal blooms (HABs) can be formed by autotrophic algae and some heterotrophic protists in aquatic environments, especially coastal areas, and can have deleterious physiological and environmental effects (Landsberg, 2002; Sellner, Doucette, & Kirkpatrick, 2003). Some HAB species also produce toxins, which can be responsible for deaths and illnesses of fish, marine mammals and seabirds and in some cases also humans. HAB species are only a small part of all known phytoplankton, namely 300 out of the 5000 known phytoplankton species (Stoecker, Tillmann, & Graneli, 2006).
Although the occurrence of HABs are a natural phenomenon and have been registered throughout history, there is an increase in frequency, intensity, and duration of HABs recently on global scale due to anthropogenic impacts (Graham, 2006; Laabir et al., 2013; Landsberg, 2002; Moore et al., 2008). Therefore, more research concerning the far-reaching effects of HABs is being done, since they can have detrimental ecological and economic consequences (Laabir et al., 2013; Landsberg, 2002).
The formation of HABs can cause a decrease in water and fish quality and a poisoning of both aquatic and terrestrial organisms (Graham, 2006). HABs can form dense scums, cover the beach with exudates or biomass, and can cause a depletion of oxygen levels due to respiration and decomposition, which can cause mass mortality of all kind of organisms (Hallegraeff, 2010; Sellner et al., 2003). This is an example of a direct effect of both non-toxic as toxic blooms (Stoecker et al., 2006).
Because of the toxins toxic algal blooms produce there is more monitoring and public advisory services needed. These costs add to economic losses, and the loss of all sorts of resources. Studies estimated the HABs cost the United States about 49 million US dollars over a 5 year study period (1987-1992) (Sellner et al., 2003).
Because of this growing interest in HABs, the Intergovernmental Panel on Climate Change (IPCC) started in 2008 to forecast the risk of HABs under a range of climate change scenarios (Moore et al., 2008). So far, however, the interactions of environmental factors are often lacking in climate simulation scenarios, or experiments used for these simulations are often incomplete and do not cover the diversity of microalgal taxa. Thus, there is much uncertainty about the influence of climate change on HABs, which makes it hard to make a predictions. Furthermore, climate change will not only have an influence on phytoplankton, but also affect all kinds of ecological interactions that may be important for phytoplankton succession (Moore et al., 2008; Peperzak, 2003). It is hard to make predictions for HAB development, because there are few long-term records of algal blooms of one single location. Yet, large-scale observations or model exercises may provide reliable information and allow careful predictions (Hallegraeff, 2010; Moore et al., 2008). These predictions suggest that if the surface temperature rises, causing stratification of the water column, growth of HAB species will be affected. It may cause a reduction in the overall phytoplankton growth and biomass, but in this regions with strong stratification it may also promote species that can migrate to layers with more nutrients, or species that have a lower nutrient requirement. Dinoflagellates have two flagella which allow them to migrate through the water column from the sunlit surface water to deeper parts where more nutrients are available. Thus, particularly dinoflagellates may be favored by warming (Moore et al., 2008).
Causes for increasing frequency of HABs
As mentioned before, HABs are increasing in frequency and it is thought that this increase is a response to changing environmental variables. Although it is strongly believed that there are more HABs than ever before, some authors, however, argue that the HABs are also the result of climate variability and not only of human impact. The increase in frequency of HABs may also be due to increased observation (Etheridge & Roesler, 2005). It is therefore important to correct for this increase in observation. Estuarine and oceanic circulation may also have a great impact on the abundance and distribution of HABs forming organisms and therefore authors are not quite sure yet what causes these HABs, although it has been demonstrated that nutrients, light, temperature, and salinity affect the photosynthesis, growth and toxicity and that there are some examples that show the relation between the frequency of HABs and anthropogenic activities (Edwards et al., 2006; Etheridge & Roesler, 2005; Sellner et al., 2003). Moreover, it is still not clear if these environmental factors affect the toxicity directly or if the toxicity is an indirect function of photosynthesis or growth rates (Etheridge & Roesler, 2005)
A higher level of atmospheric CO2 may affect the algal growth or may affect the competition between different phytoplankton groups and the algal community composition as well (Fu, Place, Garcia, & Hutchins, 2010; Hallegraeff, 2010).
Besides an increasing CO2 concentration, the temperature has a great influence on where HABs do occur as well. The growth rates of phytoplankton, and thus also of HABs, are usually higher at a higher temperature, but there is an optimum beyond which the growth rate will be lower (Eplley, 1972). Besides this, temperature can also be an important factor driving seasonal variations of phytoplankton abundances (Laabir et al., 2013). Because of the growth rate optimum it is thought that phytoplankton always occur at habitat at a temperature just below the optimum to avoid a sudden sharp drop in growth rate with small rises in temperature (Hallegraeff, 2010) (Eplley, 1972).
The change of temperature has the biggest influence on shallow waters at the coast, because these regions experience bigger fluctuations than the deeper ocean. If the temperature of the coastal water rises, the community may shift towards species that are adapted to these higher temperatures. In deeper waters, rising temperature may cause stratification which results in less supply of nutrients to the surface where the HABs are present. This may lead to a change in the composition of phytoplankton communities, because nano- and picoplankton cells with higher surface area to volume ratios have a competitive advantage in nutrient depleted environments (Hallegraeff, 2010).
All in all, the changing CO2 concentration, temperature and the eutrophication may provide ideal conditions for HABs and the production of toxins (Fu et al., 2010). It is therefore of imperative importance to acquire more knowledge on response of HABs on certain environmental conditions , in order to predict if HABs will become more prominent and more toxic in response to the changing climate. The species that may be of great importance to study are the species belonging to the dinoflagellate genus Alexandrium. These species are notorious formers of HABs and are prominent in many coastal regions around the world (Anderson et al., 2012). Alexandrium usually does not form a dense biomass that lasts throughout the whole year, but form seasonal blooms that seem to be limited to the period in which their cysts hatch. This depends on favorable environmental conditions. The cysts are able to withstand to all kinds of conditions and because of this they are able to colonize a wide range of habitats (Hallegraeff, 2010).
Alexandrium, among others, is also responsible for outbreaks of paralytic shellfish poisoning (PSP) in humans through its production of PSP toxins (Laabir et al., 2013). Because Alexandrium is so widespread and it can cause the most severe shellfish poisoning syndrome observed in humans, this genus will the main focus of this thesis.
Alexandrium species possesses a high genetic and phenotypic variability between and among species. This genetic variability is especially important in handling environmental changes, because genetically different strains may all react in a different way on changing temperature and CO2 concentration with regard to the growth rate and the toxin content (Kremp et al., 2012).
Due to slow climate changes in the past, organisms were possibly able to adapt well to the changing environment. Phytoplankton species have relatively short generation times and are therefore expected to adapt to a changing climate in a relative short time (Hallegraeff, 2010). Additionally, they can move to areas with the right environmental conditions. There is still a lot of uncertainty about this possibility of the phytoplankton to adapt at the same rate as the rate of the climate change (Hallegraeff, 2010). Moreover, it is very species-specific how different phytoplankton species will respond to the physical and chemical changes. A moderate increase in temperature might enhance photosynthesis and the growth of phytoplankton, because metabolic processes run faster at higher temperatures. Higher CO2 concentrations are expected to enhance the growth of phytoplankton as well, because at today’s CO2 concentration, Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is undersaturated. RubisCO is the carboxylating enzyme that fixes CO2 in photosynthetic organisms (Kremp et al., 2012). Under present-day CO2 levels, RubisCO is under-saturated with CO2. However, most phytoplankton species developed mechanisms to cope with carbon limitation, the so-called carbon dioxide-concentrating mechanisms (CCMs) (Giordano, 2005). One of these mechanisms is the ability of phytoplankton to switch from the uptake of CO2 to HCO3- or to reduce the CO2 leakage (Giordano, 2005; Rost, Richter, Riebesell, & Hansen, 2006). Moreover, CCMs may also implicate carbonic anhydrase (CA). CA is an enzyme that makes the transformation between CO2 and HCO3- go faster than it would without this enzyme. The presence of CCMs has a big influence on the sensitivity of phytoplankton to ocean acidification (Reinfelder, 2011; Rost et al., 2008). These mechanisms can be very different in regulation and efficiency between different species. Different species also differ in the inorganic carbon source molecules they use and in saturation levels for CO2. These preferences from each species may be a reason for different sensibilities observed to higher CO2 concentrations (Kremp et al., 2012).
It has been proven that harmful warm water species benefit from a bit higher temperature, while species that naturally occur at the same place at intermediate temperature are not or negatively affected (Fu et al., 2008; Peperzak, 2003). Species that occur in cold climates with narrow temperature tolerances are usually more affected by an increasing temperature than species that occur in warmer climates species, because there is a bigger chance the temperature will exceed their tolerance. An increased oceanic temperature can also lead to an expansion of the spatial and seasonal distribution of tropical and temperate warm water phytoplankton (Kremp et al., 2012). In general, it is stated that both photosynthesis and phytoplankton growth will double if the temperature increases by 10 degrees Celsius (Q10=2). This, however, does not apply to all phytoplankton species, because many phytoplankton species only grow in a certain temperature range, but it can be used as average response (Eppley, 1972; Raven & Geider, 1988).
Chapter 3: Toxins
Harmful algal species can produce all kind of toxins. The main syndromes in humans caused by these toxins are paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), diarrheic shellfish poisoning (DSP), azaspiracid shellfish poisoning (AZP), ciguatera fish poisoning (CFP), and cyanobacteria toxin poisoning (CTP) (Sellner et al., 2003). Poisoning can take place through direct contact with the HABs, but also by consuming filter-feeding bivalves or planktivorous fish in which the toxins can accumulate. Different types of toxins cause different poisoning symptoms, and the severity depends on the type of toxin and on dosage (Sellner et al., 2003). The reason HABs produce toxins, however, remains obscure, because it is not clear yet which role the phytoplankton toxins play in the ecology of toxin producing algae. It is often thought that those toxins are produced to discourage potential grazers, but they do not always seem to be affected by the produced toxins. So far, this is still unclear (Stoecker et al., 2006).
By passing through the food chain these toxins can accumulate in shellfish and can cause all kind of gastrointestinal and neurological illnesses by altering cellular processes, but can also cause the loss of all kind of resources (Hallegraeff, 2010; W.M. Indrasena & Gill, 2000; Laabir et al., 2013; Sellner et al., 2003). Due to the accumulation of the toxins in all steps of the food chain, even bigger oceanic or terrestrial organisms can be poised by the toxins produced by HABs or humans by consuming contaminated shellfish (indirect intoxication (Landsberg, 2002) (Hallegraeff, 2010; Laabir et al., 2013). This is the reason the toxins can even be found in higher trophic levels (Landsberg, 2002).
About 2,000 people worldwide are yearly poisoned by consuming contaminated shellfish and about 15% of these poisonings are fatal (Hallegraeff, 2010). The poisoned people were mainly poisoned by consuming toxic bivalves, sometimes by toxic gastropods and crustaceans, and rarely by toxic fish. All known (PSP) of humans so far are caused by toxic dinoflagellates, especially by Alexandrium tamarense and Alexandrium catanella, although it has been shown that numerous microalgal species produce paralytic shellfish toxins as well (Landsberg, 2002).
Besides the human health, these toxins can also cause problems in the shellfish industry (Indrasena & Gill, 2000). Wild fishes are sometimes able to detect and move away from HABs, but for fishes in the industry this is not the case, so they have a greater chance to be poisoned (direct intoxication (Landsberg, 2002)). This is why these HABs form a big problem for the fish industry (Hallegraeff, 2010; Landsberg, 2002). Some zooplankton species, however, just act as vectors in the food chain to accumulate the paralytic shellfish toxins which makes the toxins available for other organisms that consume this zooplankton species.
PSP toxins
PSP toxins are derivatives of imidazoline and a family of potent neurotoxins (W.M. Indrasena & Gill, 2000). They can be produced by dinoflagellates, like Alexandrium, and some cyanobacteria, and are highly lethal (LD50 of 10 ��g/kg i.p. in mice). These PSP toxins bind on the one side of the voltage-dependent sodium channels, which makes it impossible for sodium to bind to these channels and thus to transmit signals between neurons (Landsberg, 2002). Saxitoxins are a part of these paralytic shellfish toxins, which also includes at least 21 derivatives (Landsberg, 2002). Saxitoxin and its derivatives can be divided into three categories: the carbamate compounds, the N-sulfocarbomyl compounds, and the decarbomyl compounds. Differences among these categories are due to slightly different molecular structures , causing differences in their toxic potency as well (Laabir et al., 2013).
Each Alexandrium species or strain has a characteristic toxin profile and thus does not necessarily possess all possible PSP toxin derivatives. Some of these derivatives are highly toxic like carbamate toxins, saxitoxins (STX), neosaxitoxins (NEO), and gonyautoxins (GTX1-4). Other derivatives are less toxic, such as the decarbomyl analogues (dcSTX, dcNEO, dcGTX1-4) and the deoxycarbomyl analogues (doSTX, doNEO, doGTX1-4). The least toxic are the N-sulfocarbomyl toxins like B1 (GTX5), B2 (GTX6), and C1-C4 (Landsberg, 2002).
The toxicity of a species may be highly variable, due to differences in toxin composition (i.e. the type of saxitoxin) or toxin content (i.e. molar content per cell). These factors depend on the environmental conditions, like nutrient availability, and the geographical location (Landsberg, 2002; Stoecker et al., 2006). The toxin content is usually higher in exponential growth of the population and is reduced when the population reaches the stationary state, but this strongly depends on which nutrient becomes limited when it reaches the stationary state (Hamasaki, Horie, Tokimitsu, Toda, & Taguchi, 2001; Navarro, Mu��oz, & Contreras, 2006; Sellner et al., 2003). This toxin content is a parameter that highly depends on all sort of factors, like the losses of toxins due to catabolism, leakage into the medium, and toxin transfer to new cells during division (Sellner et al., 2003).
Effects of environment on toxicity and toxin content
Due to mainly low pH the toxins could possibly be converted to compounds with a lower toxicity, carbomyl-N-sulfo paralytic shellfish toxin (Indrasena & Gill, 2000).
Next to the influence of the pH on HABs, temperature also seems to have an effect on the toxin content. Researchers found an negative correlation between the toxin content of Alexandrium catenella and the temperature (Navarro et al., 2006). This means that if the atmospheric and oceanic temperature rise, the toxin content of Alexandrium catenella will decrease. This is consistent with something that was concluded earlier, namely that the paralytic shellfish poison level per cell is high when the temperature is low (Navarro et al., 2006). The toxicity, however, is at its lowest at the optimum temperature and becomes higher if the temperature becomes higher or lower than this optimum (Etheridge & Roesler, 2005). Additionally, temperature also has a great influence on the photosynthetic rate. If the temperature rises, the rate of photosynthesis will increase as well. This is because the rate of the electron transport chain is influenced by temperature (Etheridge & Roesler, 2005). The growth rate, however, will be lower if the temperature is high (Etheridge & Roesler, 2005). This photosynthesis and the resulting growth rate may also influence the toxin content, because the growth rate is inverse correlated with the toxin content. As indicated earlier, it is not certain if the lower temperature directly causes a higher toxin content or if the higher toxin content is an indirect effect of a lower growth rate which causes the toxin content to increase. For now it seems the increase in toxicity is the result of the effect that a low temperature has on the metabolic processes, which regulates the toxin production (Navarro et al., 2006).
Since HABs are thought to become more prominent in response to changing climate conditions, we looked at the effects rising CO2 concentrations and temperature have on the toxin content of Alexandrium cells using a meta-analysis approach. In order to make better predictions about the toxicity of Alexandrium blooms in the future.
Material & methods
In order to determine the effects of rising CO2 concentrations and temperature on the toxin content of the dinoflagellate Alexandrium, data hereof was collected from different culturing experiments using different Alexandrium species and/or strains. For this research, papers about the influence of the CO2 concentration or the temperature on toxin contents in the dinoflagellate Alexandrium were collected (table 1). Articles were searches for via Google Scholar and Web of Science, using combinations of the search terms using the conjunction ”OR”: ”harmful algal blooms (HABs)”, ”ocean acidification”, ”climate change”, ”global change”, ”eutrophication”, ”toxins”, ”phytoplankton”, ”Alexandrium”, ”Alexandrium fundyense”, ”Alexandrium catenella”, ”Alexandrium tamarense”, ”Alexandrium ostenfeldii”, ”increasing CO2 concentration”, ”decreasing pH”, and ”rising temperatures”. Data that was collected from the articles included species, strain, , toxin content, standard deviation of the toxin content, CO2 concentration, temperature, and number of replicates.
Data was subsequently categorized in high, medium or low, with high representing CO2 values of 700-1000 ppm and temperatures of 25-20 ‘C, medium representing CO2 values of 300-700 ppm and temperatures of 14-25 ‘C, and low representing CO2 values of 100-300 ppm and temperatures of 10-14 ‘C.
Then, the natural logarithm of the response ratio (lnRR) was calculated by dividing the toxin content at the high temperature/CO2 concentration by the toxin content at the medium temperature/CO2 concentration, and taking the natural logarithm of the outcome. An lnRR equal to zero means that there was no response to the climate change factor (i.e. the CO2 concentration or the temperature), while a negative or positive response ratio indicate a decrease or increase in response to the climate change factor.
Thereafter, the variance and the standard deviation were calculated and based on this the upper and lower limit of the 95% confidence intervals (95% CI). If this 95% CI does not cross zero, the response was considered significant.
Data analysis was performed in Excel.
Tabel 1: Species and strains with corresponding references
Species and strain Reference
Alexandrium catenella A-11c Tatters, Flewelling, Fu, Granholm, & Hutchins, 2013
Alexandrium catenella ACC02 Navarro et al., 2006
Alexandrium fundyense Etheridge & Roesler, 2005
Alexandrium ostenfeldii AO01 Kremp et al., 2012
Alexandrium ostenfeldii AO02 Kremp et al., 2012
Alexandrium ostenfeldii AO03 Kremp et al., 2012
Alexandrium ostenfeldii AO04 Kremp et al., 2012
Alexandrium ostenfeldii AO05 Kremp et al., 2012
Alexandrium ostenfeldii AO06 Kremp et al., 2012
Alexandrium ostenfeldii AO07 Kremp et al., 2012
Alexandrium ostenfeldii AO08 Kremp et al., 2012
Alexandrium tamarense Alex2 Van de Waal, Eberlein, John, Wohlrab, & Rost, 2014
Alexandrium tamarense Alex5 Van de Waal et al., 2014
Results
The collected data from all collected studies is combined in the two figures below.
For each strain of Alexandrium the response ratio with related 95% CI (if applicable) was calculated for higher CO2 concentrations (figure 5) and for higher temperatures (figure 6). Most of the collected strains used for the experiment at higher CO2 concentration were of A. ostenfeldii, amounting to a total of eight strains, while data on only two strains was found for both A. tamarense and A. cantenella. The strains used for the experiment at higher temperatures were three A. catenella and one A. fundyense next to most A. ostenfeldii. The average of the lnRR to an increasing CO2 concentration is approximately 0.068 (i.e. an increase of approximately 7%), and to a rising temperature -0.026 (i.e. a decrease of approximately 3%) , and respectively range from -0.525 (i.e. a decrease of approximately 41 %) to 0.957 (i.e. an increase of approximately 160%) and from -0.792 (i.e. a decrease of approximately 55 %) to 0.545 (i.e. an increase of approximately 72%).
The responses of PSP toxin content to higher CO2 concentrations and temperatures in most of the strains remained largely unaltered. A. catenella, however, seems to increase a little in response to high CO2 concentrations, although there are no standard deviations. The same applies to A. fundyense.
Figure 5: The RR of toxin content in different Alexandrium strains to CO2 concentrations
Figure 6: The RR of toxin content in different Alexandrium strains to higher temperatures
Discussion and conclusion
General trend
Figure 5 and 6 show a largely unaltered response to higher CO2 concentrations and higher temperatures for almost all Alexandrium strains. Some of the response ratios are just above and some are just below value zero, but nearly all confidence intervals (if applicable) include zero, for some strains, including A. catenella and A. fundyense, a confidence interval could not be calculated, which makes it hard to make any conclusion about these strains.
These findings indicate there is no significant change in toxin content in response to higher CO2 concentrations and temperatures. A response ratio of 0.67, however, means a duplication of toxin content, so the mean may be high, but they are not significant. Moreover, I also did not see significant differences between different Alexandrium species or similarities in RR between strains within one species either.
The fact that there is such a strong variation and no general trend in response to these changing climate variables makes it hard to predict what will happen in the future. The response we will see in the future strongly depends on which species and strain predominates in certain areas at that point, but in this case it is likely that each combination of species results is more or less a mean response ratio of 0. Variation in response within a species, however, do suggest a large genetic and phenotypic variability which can aid to their survival when environmental conditions change.
CO2 concentration
The data that has been collected for responses of toxin content to higher CO2 concentrations, may not always be reliable. When a culture grows, they make use of the available CO2 through photosynthesis, which makes the CO2 concentration go down. The supporting information of the experiment from Kremp et al. (2012) shows that only at the beginning of the experiment, the high CO2 treatment had actual high CO2 concentrations, namely above 700 ppm. After day four, there is a sharp decline in the CO2 concentration and at day 10, the concentration even decreased to approximately 400 ppm, which can be considered ambient CO2 concentrations. After day 10, the CO2 concentration slightly increases again, but remains below 400 ppm until day 16. Therefore, a large part of the cells did not experience the high CO2 and were actually grown under ambient conditions. Nevertheless, differences between the treatments were observed as the CO2 concentrations of the ambient treatments dropped down to ‘ ppm. The data thus, indicates a response of the Alexandrium species to an increasing CO2 concentration.
Toxin content
As mentioned before, there is no significant response in toxin content to higher CO2 concentrations and temperatures for all of the Alexandrium strains used in this review, however, our findings are based on only one type of toxin, namely saxitoxin. It is possible, however, that Alexandrium can switch towards different toxin analogues in response to changing environmental conditions. For example, Van de Waal et al. (2014) show that there is a switch from non-sulfated towards sulfated PSP toxin analogues at increasing CO2 concentrations in A. tamarense. In addition, A. ostenfeldii produces relatively more STX (compared to GTX) if the CO2 concentration increases (Kremp et al., 2012). This means that the toxin content of a certain toxin may not increase with increasing CO2 concentration and temperature, but there might be a shift towards other toxin analogues, which may possibly be more toxic. This possible response is not included in this thesis, so even though we showed that toxin content does not increase in response to the two climate variables considered here, the overall cellular toxicity might still actually increase.
Combined factors
In this thesis only the separate effects of higher CO2 concentration and temperature on toxin content were analyzed. The effects of these two factors combined, however, is to consider in future research, since these effects might differ from the effects these factors have on toxin content separately.
Furthermore, global change includes more factors than only increasing CO2 concentrations and rising temperatures, such as eutrophication and nutrient limitation. These factors influence the toxin content of Alexandrium as well, which may change the overall response of this species to a changing climate. Eutrophication for example stimulates the growth of harmful algal species, but the toxin production depends on which nutrient is sufficient and which one is insufficient (Anderson, Glibert, & Burkholder, 2002).
Other species
Although multiple Alexandrium species and strains were included in this study, it is not possible to make general statements about the response of toxin production to changing environmental conditions for harmful algal species, since there are many more HAB species, which may respond differently to changing environmental factors. It would be of importance to assess the effects of global change on more HAB species, as more accurate predictions concerning an increase or decrease in HABs and their toxicity can be made for the future.
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