FALMOUTH MARINE SCHOOL

An Investigation into Ocean Acidification and the effects on Artemia’s mortality, growth rate and

behavior.

Project

Caroline Nelson

5/3/2012

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An Investigation into Ocean Acidification and the effects on Artemia salina’s mortality, growth rate and behaviour.

Caroline Nelson1

1 Wet Laboratory, Falmouth Marine School, Cornwall, England.

Received 3rd May 2012.

A R T I C L E I N F O A B S T R A C T Article history: Received 3rd May 2012 Keywords: Ocean Acidification Artemia salina pH Decalcification Calcium carbonate

1. Introduction Artemia salina is a zooplankton, which a major food source for many marine species. It is

also the bases of the food web. As Artemia is highly tolerable to poor water conditions and stated by Daintith 1996 and Lewan1992, it is thought to be the best organism to test the effects on declining pH of the earth’s has on its survival, growth, hatching rate and behaviour. Although test and previous experiments have been carried out it is yet to be sure whether Artemia will be affect by 0.1 units. Extreme conditions would be carried out on the organism whilst in its dormant cysts form. Calculating the hatching rate in a solution of6 (pH) in contrast to that of its optimum hatching pH of 8. Experiments in the past have been carried out , monitoring the breading rate and it has been concluded that; although the Artemia do breed to cysts and juveniles produced are much smaller which a thinner skeletal shell and appendages missing. Artemia salina comes from the Kingdom Animalia, Phylum Arthropoda, Subphylum Crustacea, Class Branchiopoda, Order Anostraca, Family Artemiidae, genus Artemia and Species Artemia salina . Artemia have been dated back to 982 and today there has been little change to the external anatomy of Artemia since the Triassic period (Asem 2008). The distribution of Artemia is vast, populating in lakes inland. The species can tolerate very poor water quality and can survive in waters with extreme salinities (Daintith 1996), this aids their survival against predators. Their resilience to survive in poor conditions makes them ideal subject to test biological toxicity assays and is now one of the standard organisms for testing the toxicity of chemicals (Lewan1992). As stated in The Biology of Invertebrates, the anatomy of an Artemia is

The ocean’s water chemistry have been relatively stable for centuries, however, due to an increase in fossil fuel emissions and other climate change factors, our oceans are beginning to change (Canadell 2007). 91% of humanities CO2 emissions can from fossil fuels, equivalent 33.4 billion metric tonnes. At present our atmospheric carbon dioxide contains 390.1 ppm (SIO 2011); it is thought that levels will continue to rise till the next century or longer (The Royal Society 2005). Artemia salinas are very important to the marine food web as they are zooplanktons which are at the bottom of the food chain. Testing a theory that, by decreasing a pH and hatching cysts of Artemia salina in them one can predict when levels are too low and no zooplankton can survive and bread therefore disrupting the whole food web. The experiment conducted showed that cysts hatch in a pH of 6 had the slowest growth rate. By observation it also showed that many species were missing appendages and had other disfigurations to their body structure.

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similar to that of a primitive arthropod as it has large appendages which are connected to its segmented body, which has 19 segments. The segments near to the head of the organisms usually has the appendages whereas the back, hold the reproductive organs. The body of Artemia are completely covered in an exoskeleton made out of chitin, which in females they shed every ovulation. The exoskeleton is thin and mobile. The average length of a female Artemia is 12mm whereas in males its 10mm. The Local nervous system ganglia control’s the locomotion, reproduction and digestion which would usually be controlled by the brain of the specie. Ocean water covers seventy percent of the earth’s surface. With a vast area covered, oceans play a major role in the regulating the earth’s climate and other factors (Abbasi et al., 2011). Oceans have been widely seen as a vital natural resource for humanity (Costanza 1998) but also crucial for marine species. The ocean’s water chemistry have been relatively stable for centuries, however, due to an increase in fossil fuel emissions and other climate change factors, our oceans are beginning to change (Canadell 2007). 91% of humanities CO2 emissions can from fossil fuels, equivalent 33.4 billion metric tonnes. At present our atmospheric carbon dioxide contains 390.1 ppm (SIO 2011); it is thought that levels will continue to rise till the next century or longer (The Royal Society 2005). Like other gases, carbon dioxide follows Henry’s Law which states that ‘the amount of a given gas that dissolves in a given type and volume of liquid is directly proportional to the partial pressure of that gas in equilibrium with that liquid.’ With this theory, an increase of atmospheric carbon dioxide it increases the concentration of carbon dioxide on the ocean’s surface (Revelle et al., 1957). 25 million tonnes of carbon dioxide gets absorbed into the world’s oceans daily (Porzio et al., 2011). As a consequence of the absorption of CO2, water quality will be altered; decrease in calcium carbonate saturation, increase of water temp due to global warming and a decrease in pH. The lowering of the oceans pH is known as ‘Ocean Acidification’ (Caldeira et al., 2003). The ocean’s surface is relatively alkaline, with an average pH of 8.2 (RSC 2005). Carbonic acid is produced when carbon dioxide is dissolved into the water which alters the ratio of pH maintaining carbonate and bicarbonate buffers as creating a weak acid (Feely et al., 2004; Raven et al., 2005). The release of hydrogen ions is due to the formation of other types of dissolved inorganic carbon, when a weak acid is present. Caldeira and Wickett (2003) stated that with this reaction, the average pH has been lowered by 0.1 units from pre-industrial levels and in the last two million years pH levels have never been this low (Honisch et al., 2009). With pH levels decreasing further, there will consequently be irreversible changes to the marine habitat and the biodiversity that inhabit the area. Reason for this result is to investigate whether the sudden decrease of pH affects the growth of Artemia salina and in doing so affected the whole marine food web.

2. Materials and method. 2.1 Study site.

The study site is located in the south coast of Cornwall in the Fal estuary. From the entrance between Pendennis Point and St Anthony Head lies the Fal, it extends 18kn inland to Tresillian’s north most tidal point. The total length of the Estuary is 127km. The areas surrounding the Fal were once populated with mines. In 1992, there was an incident where a mine, Wheel Jane flooded causing toxic heavy metals pour in the estuary(Bryan et al., 1992; Langston et al., 2006). This had a dramatic effect on that of the ecosystem and the economy(Adams, 1976). Ten years later and heavy metals still preside in the estuary and affecting the water quality. Further up the estuary by King Harry the pH levels are lower than that at the opening of the Fal, this is due to the flooding at the mine. The northern section of the estuary consists of six main tributaries and twenty-eight rivers and minor creeks which all lead into Carrick Roads. The estuary inhabits a variety of species and habitats, some of which are of high economic and ecological importance; species such eel

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grass (Zostera) and Maerl (Lithothamnion corallioide. Many juvenile species inhabit these beds as they provide a great source of protection and have a high supply of food (Langston et al., 2003). Eel grass beds are deemed to have an important role within habitat; many species use these beds as breeding grounds (JNCC, 2010). Not only do they provide shelter for many juvenile species they produce 64% of the Estuaries primary production (Adams, 1976). Like Eel grass bed, Maerl are an important habitat for many species which can live amongst or in the gravel. It also plays an important role in sustainable fisheries, both commercial fish as well as shellfish. (Challinor et al., 2009). By King Harry Ferry is a Mussel Farm and further up the Fal are oyster beds, both of which help the economy greatly. Both marine and freshwater mussels are filter feeders; they feed on plankton and other microscopic sea creatures which are free-floating in seawater. A mussel draws water in through its incurrent siphon. The water is then brought into the branchial chamber by the actions of the cilia located on the gills for ciliary-mucus feeding.

2.2. Experimental design and sampling.

It is important to set up the correct tank for the A. salina to hatch in to ensure optimum yield is achieved. A cone shaped jar is typically used whilst hatching; it is placed upside down on a stand so that the base is facing upwards. This shape is usually used due to nature of the larvae is once hatched. This shape ensures that the larvae are free swimming in the middle of the water column. An oxygen supply is given not only for aeration but so that the larvae so stay stationary for long periods of time. The oxygen bubbles also push the Artemia salina around the cone shape jar, this also makes sure they don’t attach themselves to one another and minimises the chances of them attaching themselves to the walls of the hatching jar. Each cone is filled with approximately 1.75l of sea water, each hatching jar containing natural sea water but with different pH levels. A heater is added to bring the temperature up to the preferred heat. Once each cone has the optimum temperature and aeration than the cysts can be added. When weighing out cysts in relation to the volume of water it is expected to add slightly more than the original number. This is because, it is inevitable that some cysts are not submerged completely at all times and therefore do not get hatched. Once the weighed out cysts are added to each jar, it would take approximately 24 hours for the cysts to completely hatch. Measurements are taken of each hatching jar daily. This is done by collecting a sample of larvae, placing a droplet onto a microscope slide and examining the specimens under the microscope at a magnification of x10. Using a Micrometer Eyepiece, this provides the means of measurement. The length (top of the head to the end of the sphincter muscle), head width and appendages length are all measured. This process is repeated, measuring ten specimens in each hatching jar each day for five working days.

2.3. Requirements 2.3.1. Biotic Requirements

Biotic requirement for cultured Artemia salina are limited. When culturing the species stock density is vital to ensure species don’t over compete with one another. If the stock density is too high than the total hatch yield will be lower. With cultured species, disease is not usually an issue, especially if the salt mixture is artificial.

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2.3.2. Abiotic Requirements

Abiotic requirement are those which can be controlled. To hatch Artemia salina you must first ensure that the salinity, pH, temperature, light, aeration and stock density are at its optimum.

Although Artemia can survive in high concentrations of salt and have done so in the wild. When hatching Artemia in a controlled environment for aquacultural use, it is best to lower the salinity, allowing the cysts to become un-dormant. Salinity of the solution they are hatch in should have a salt content of 25 parts per thousand (p.p.t) per litre of water. PH should be looked at closely as Artemia do not usually hatch in low pH, with a pH of 7 or lower it is advised that either magnesium sulphate or Epson salt be added to the solution. This acts as a buffer allowing the Artemia cysts to hatch as well as survive. A pH of 8.0 (minimum) or higher is recommended to start the cysts hatching.

If you are want your cysts to hatch within a 24 hour period than the temperature you need to set you hatching jars to be between 26-28ᵒC. Below the temperature of 26 degrees centigrade, the hatching rate tends to be longer. In contrast when temperatures exceed 30 degrees centigrade the cysts genially don’t hatch and stay domain. With an increase of temperature, it also increases the chances of the protein molecules inside the cysts to denature, disabling them to hatch.

Light is important as it triggers a chemical mechanism within the embryo. During the first few hours of the cysts being incubated illumination prompts the cysts to hatch. With continuous light exposure, it also regulates the temperature of the solution. When exposed to light, Artemia increase their locomotion, however in darkness they stay still. Aeration is vital as it keeps the cysts suspended and adequate levels of oxygen are supplied to them. The minimum dissolved oxygen that cysts can hatch in is 3 parts per million. Artemia will start producing haemoglobin (and thus turn red in colour) only if they experience a shortage in oxygen levels. However, increasing the D.O will not harm the cysts. When calculating stock density it is important not to over calculate. Although, 100% survival rate is unrealistic in any circumstance, a high stock density will cause a lower hatch and survival rate, as species will be competing for not only food but space. In a litre of water 1 gram of Artemia cyst is required.

2.3.4. Nutritional requirements

Artemia are filter feeders, however there have been documental evidence that some species have been known to be carnivores. (Emslie 2003). The way in which Artemia move their phyllopods, it encourages the water current to move into the food groves. Food grooves lie between the gnathobases of the phyllopods, appendages near the mouth. From this pressure created by the gnathobases the food particles are prohibited from leaving the grooves. The phyllopodia move in the rhythmic pattern guiding the food particles to the mouth. The main diet of A. salina is green algae. (Hall 2011) They either filter feed with the aid of their appendages or they graze on the benthic floor, removing algae off rocks.

2.3.5. Feeding Cultured Species.

If you’re using natural sea water than after the Artemia have hatched, they should not be fed for a few days, between 2 to 5 days. The water they were hatched would have had phytoplankton or algae the Artemia would feed on. Adding extra food would feed the bacteria in the water and would than multiply quickly, consuming more oxygen.

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2.3.6. Life support requirements

Artemia are relatively easy to look after. They require the basic equipment to survive. To hatch Artemia from cysts, a hatching jar which can hold between 2 to 4 litres of water dependant on how large your yield is. An oxygen supply is needed as well as a heater. Heater is required as cysts stay dormant unless it is in temperatures of between 24 to 28 degrees. The final requirement cysts need is light, place the hatching jar in a well lit area.

3. Results

Water quality of the each water sample was tested first before used in the experiment. It was deduced that the seawater with a pH of 6 had less calcium than that of the water sample with a pH of 7.2. It was also discovered that copper levels were extremely high with the Tank 2 (pH6). These may have had an adverse affect on the data. During the collection of data, 450 specimens were measured. There were three hatching jars all which were running for five consecutive days. This experiment was repeated three times in total, to ensure that the date was acute and reliable. Repeating the experiment also mean that the number of anomalies would be reduced and would not affect the results. 3.1 Survival During the hatching process, all abiotic factors were the same apart from the pH. It was important to do so to ensure that the data collected would be reliable as well as accurate. When there is a presence of acid in the water, the natural instinct for the species is to not hatch. In Tank 2 (pH 6) there was a high level of dormant cysts. Figure 1The graph shows the percentage of hatched cysts and the percentage of cysts that stayed dormant.

Tank 3 indicates the highest hatch rate, with 95% of the specimens hatching (Figure1) and with Tank 2 having the lowest. It was important that survival rate was recorded as it indicates whether the species can breed and survive if ocean pH were to reach the level of tank 2. The percentage survival rate was recorded by observation, looking at a 5cm by 5cm area and counting the number of cysts present, as well as the number of hatched Artemia. From the data collected it was possible to put it into percentages. This was repeated for the 5 days of the experiment.

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3.2 Growth Measuring the growth of the species gives an accurate and reliable source of data when analysis is required. The length of the species, the length of the appendages and the head width are all measured.

Figure 2 Graph to Show the Average Length Growth of A.salina over 5 days.

The average length of A.salina was recorded by adding up all the samples from Tank 1 together (30 sets of data for Tank 1, Day1) and dividing it by the number of data collected. This was then repeated for Tank2 and Tank3 for days 2 till 5. Looking primarily at the length of the species, it shows a clear indication of the actual growth of each specimen.

Figure 3 Scatter Graph to show the average length growth for A. salina on Day 1.

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Figure 4 A scatter Graph to show the length growth of A. salina on Day 5.

Day 1 and day 5 Length growth graphs were used as it clearly illustrates the comparison between the initial size from hatching to when they are adults. It shows the total growth over the 5 day experimental period.

Figure 5 The growth rate over the 5 day period of all three tanks (pH6, 7.2 and 8.6) This graph clearly shows the comparison in growth between all three hatching jars. It also illustrates the differences in growth between length, head width and appendages length in each tank.

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4. Discussion This study provides a detailed account of the full range of Artemia salina growth when put under different pH conditions and collated it over a five day period. From when it was a cyst to hatching. The reaction encompasses a succession of stress behaviour in the tank with the lowest pH and as a result many didn’t hatch and stayed dormant (figure 1). Artemia salina are able to stay dormant in cysts for over 10 years, unless they are put into a solution with the correct water quality than they will hatch. Such occurrence is common in the species as in the wild they are found in high salinity and in poor water quality (Lewan 1992) Artemia are fast growing zooplankton and in conducting this experiment has shown that with the increase of dissolved CO2 adverse affect on the behaviour and growth of the species. In Figure 1, the percentage of specimens that hatch and stayed dormant is clearly shown in a bar graph. From this, the theory that A. salina prefer higher pH is proven, with a 95% hatch rate with a pH of 8.6. However, on the other hand, in Tank 2 (pH 6) the percentage of specimens hatched were only 60%. With a decrease of pH by 2.6 units there is decrease in hatch rate by 35%. It is inevitable when cysts are added to the water solution to expect a small percentage not to hatch; this is due to several reasons; poor water quality, expired cysts or attachment to the side of the hatching jar of other appliance in the jar. Whilst conducting the experiment, daily observation of the hatching jars and of the behaviour was taken. It was recorded that a number of cysts were in fact attached to appliances in the hatching jar. This may account for the dormant cysts percentage in all three tanks. Taking this into account and predicting the number of cysts that would of hatch if this factor didn’t occur. Tank 3 would have had a higher hatch rate, close to 100%. Just looking at this graph it can be deduced that the optimum pH for hatching Artemia salina cysts is 8.6. By observation it is apparent that species which are hatched in a pH solution 6 have some deformities. Many of specimens examined we missing appendages, others the shape of their body was disfigured. It can be thought that the lowering of the pH not only slows down the growth rate and hatch rate but changes the biology of the species (Anderson 2002) In Figure 5, illustrates the average growth of all three hatching jars. There is a clear comparison between all three jars and looking at figure 4 and figure 3 Tank 1 and Tank 3 are very similar in growth rate throughout the 5 days. However, one day 1 of recording the growth, there is a distinct difference. Due to the conditions in which the cysts were hatched in (tank 3) they would have had longer to grow and were in a settled environment. Tank 1, the pH was lower therefore took longer for the cysts to hatch resulting in reduced growth rate.. However, the growth rate soon increased resulting in similar size of Artemia at the end of 5 days. A possible reason for Tank 3, reducing its growth rate may be because the hatching jar was next to a kettle. The factor may have resulted in the data being flawed. The heat which was expelled from the kettle whilst it was boiling would have caused an increase of water temperature in hatching jar no.3. This sudden increase in temperature every few hour would cause the growth rate to decline, slowing down the growth rate meant that when it came to measurement it would have been inaccurate and unreliable. Unfortunately, this occurred throughout the experimental period. However, as there was only the 1.4 unit difference between the two tanks there probably was a more scientific sound reason. As Croghan stated, the preferred pH for Artemia salina to be hatched is 8.0, 0.8 more than that of Tank 1. This and the increase of water temperature in Tank 3 could result in very similar growth rates. More research is needed to predict when pH levels in the sea will be too low for zooplankton to bread and hatch. As they are at the bottom of the food chain and the marine food chain requires them survive in order for top predators to survive. Artemia salina are found mainly in salt water lakes. Future research would be based on zooplankton found in the ocean. From this scientist can exactly predict when levels are too low and predict a date when this would occur.

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