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Investigating the effect of ocean aciditification on sea monkeys (Artemia nyos
Investigating the effect of ocean acidification on Sea Monkeys (Artemia nyos)
Mia Vesey
Barker College
Purpose: This paper aims to investigate the effect of ocean acidification on marine invertebrates by studying the effect of differing pH on the hatching of Artemia Nyos (sea monkeys). Methodology: The methodology used consisted of adding 0.25 g of A. nyos eggs throughout 5 substances, each with a differing pH varying from 4-10. The samples were incubated over the course of a week, and the number of A. nyos hatched was counted. Findings: The findings of this experiment were in support of the hypothesis, that hatching of A. nyos was highest at a pH of 8/8.1 but decreases as solution becomes too acidic or alkaline. Research Limitations: Time constraints caused a reduction from initially intended sample size, making it harder to determine whether data is reliable. Difficulties with equipment and materials occurred, including with eggs, bases and incubators used. Practical/Social implications: Possible practical implications include a change in understanding and thereby anthropogenic activities, in an effort to reduce the effects of Ocean acidification on marine organisms Originality: To the author’s knowledge, this is the first time A. nyos has been tested for the sole impact of acidic conditions, measured by the number of hatched A. nyos. Keywords: Ocean acidification, Artemia Nyos, pH Paper Type: Research paper
Literature Review
Ocean acidification is a rising problem that needs to be taken into consideration in our world, where humans have a growing negative impact on ecosystems.
As more fossil fuels are burnt, and methane is released from agricultural production to provide for a growing population, these greenhouse gases are released into our atmosphere, becoming trapped and increasing global temperature. Carbon sinks such as the oceans absorb carbon dioxide and trap it. When the ocean absorbs carbon dioxide, as explored (Doney, Fabry et al, 2009), it forms carbonic acid (H2CO3), which in turn decreases the oceans’ pH. Research by Caldeira and Wickett (2003) used geological records to predict that change in the oceans’ pH will occur more rapidly over the next centuries than it has in the past 300 million years. This is gradually creating an environment that is uninhabitable for many species. A study done by Bhadury (2014), on the effects of ocean acidification on marine invertebrates, found that it reduces the ability of crustaceans and corals to form hard shells, while also causing a decrease in fertilization success, growth and larvae size in a variety of marine invertebrates. Such changes could have further serious consequences for the animals that feed on these animals. It has also disrupted breeding patterns of species of fish, as they rely on changes in the ocean to indicate breeding seasons, as found by studies from Milazzo et al, (2016). There are also consequential social implications, outlined by America’s official climate website (Dahlman and Lindsey, 2018), explaining that the degradation of our oceans will negatively affect “people who depend upon marine fisheries for food and jobs”, and that they “may face negative impacts from the warming ocean”.
Waiwood & Beamish (1978) investigated the effect of many variables on the growth of rainbow trout and found that growth was slower in lower pH. The experiment measured factors including water hardness, growth and recovery rate as well as pH. Their method would require much more time, equipment and technical skill than is available.
Lastly, a study by Salma and Lee into the Effects of pH Change by CO 2 Induction and Salinity on the Hatching Rate of Artemia franciscan (2012), found that as pH became more acidic, hatching rates decreased, regardless of salinity. Similarly, this experiment will measure hatching by counting the number of organisms that have hatched after a period. Alike Artemia franciscan, Artemia nyos are a species
of brine shrimp, but a hybrid, developed to withstand the conditions of a fish tank, requiring minimal care. They grow larger and live longer than normal brine shrimp. Artemia nyos were selected to be used in this experiment, as they are cheap and much more accessible than any other marine creatures. They also require much less maintenance and care due to their ability to withstand conditions. They may also be set up on a small scale with ease. They are invertebrates, which eliminates any ethical concerns. Similar papers from Doyle and McMahon (1995) provided informative research into the larval stages that would be witnessed within experimentation (see Figure one). It also found corresponding results; that exposure to lower pH’s caused a significant decrease in hatching success and survival.
Figure 1: Larval stages of the brine shrimp (Source: Doyle & McMahon, 1995)
The design of the experimental method was heavily shaped by Gao et al (2011), whose research into the effects of pH on fertilization and hatching rate of far eastern catfish displayed several variables which have since been taken into consideration for this research project. Aspects such as monitoring pH at multiple stages throughout experimentation, have influenced the methodology within this report.
Further inspiration has been taken from Hinga, (2002), who investigated pH, and its effects on coastal marine phytoplankton. The report consisted of using 8 differing pH treatments, in increments of 0.5, as well as performing fine tuning of pH buffers before use. Consequently, a digital pH probe will be used in this project, to create accurate buffer solutions for the A. nyos. Research from Sorgeloos, (1973) and Snab biology (2017), revealed that temperature should be considered within the experimental design, and optimum temperatures lay between 22-30°C. This led to the planning to incubate A. nyos at 26 °C.
Project rationale
Thought was required as to how pH could be altered within this experiment, as standard buffers could not be used due to their lack of range of pH values, as well as their possible harmful effect on aquatic life such as A. nyos. Hargreaves and Whitton, (1976) achieved alterations of pH using HCl, however whilst this would be readily available, it would have a harmful effect on A. nyos, as further research found it would most likely be harmful for organisms, as it is corrosive and inorganic, as found from Hannigan and Coffey (2019). Other options included bubbling CO2 through the water, done by Byrne, et al (2009), however maintaining pH across many samples made this method unsuitable. A paper published by Dach, (1943) listed a very large variety of chemicals used to raise or lower acidity, (see Figure 2).
Figure 2: Table from Dach (1943) showing buffering agents used in their investigation.
After further research into each chemical, sodium bicarbonate was found to be used as an agent to reduce the acidity of a solution (raise its pH). It is not considered harmful to aquatic life as found in research from Noss (2021) and is extremely accessible and inexpensive. This was selected as an alkalinizing agent to be used within this experiment to raise the pH, as alkaline substances such as this have a high pH. To lower the pH however, a buffer of sulfuric acid was selected as acids have a low pH, including Sulfuric acid which has a pH of 0.5 at a concentration of 33.5%. This was in line with research from Roth-Schulze et al (2018) and Sheath et al (1982) which both utilised sulfuric acid in experiments involving bacteria and algal communities. The agent has acute short-term toxicity on aquatic life, as found by National pollutant inventory (2019), but will not alter hatching or survival within the scope of this experiment. It is readily available, and therefore was selected as a second buffering agent within this report.
Overall, this investigation into the effects of ocean acidification, reflected by the changing of pH, and its effects on the overall hatching of Artemia nyos, hopes to provide a pathway into furthering understanding of
how ocean acidification is impacting the oceans and marine life.
Scientific Research Question
How does ocean acidification tested by different pH levels effect the number of hatched Artemia nyos?
Scientific Hypothesis
That as the pH increases from a minimal pH of 4, the number of hatched A. nyos will increase to an optimal pH (8), followed by a decrease.
Methodology
Decided measurement of A. nyos
An experimental study on the effect of alkaline water on the dynamics of amphibian larval development, (Fominykh, 2008), found animals in alkaline conditions grew more slowly. The report considered the stages of larval development in different acidities. This would be difficult to monitor within a school environment due to time and availability constraints. Likewise, their experiment tested a range of amphibians. This would be problematic for this report due to limited accessibility, time constraints, high cost, high maintenance, and ethical considerations. Thus, this methodology is highly unsuitable for a school investigation and so testing was limited to one invertebrate species, the sea monkey (A. nyos).
Decided measurement of single independent variable (pH)
Research from Waiwood & Beamish (1978) previously mentioned which tested water hardness, growth and recovery rate as well as pH, informed the decision to test the sole effect of differing pH on the number of hatched A. nyos.
Preparation of buffers
A 2 L beaker was filled with 1500 mL of distilled water and 6 packets of ‘water purifier’(supplied with the packets of sea monkey eggs) was added and stirred for two minutes. The water was split equally across five beakers, each containing 300 mL using a 500 mL measuring cylinder and pipette. The pH probe was calibrated using standard pH buffers of 4 and 7 before testing any solutions. Prior to testing the pH of a solution, the pH electrode was rinsed with distilled water and dried. Bicarbonate soda was added to decrease acidity (raise pH) and sulfuric acid was added to increase acidity (lower pH). Each was added across treatments until the desired pH of 4, 6, 8 and 10 were achieved. The pH of each beaker’s solution was checked using digital pH probe. The fifth treatment had no alteration of pH later measured to be a pH of 8.1, despite being close to the pH 8 treatment, it acts as an experimental control as to any unaccounted-for effects of the ‘water purifier’. Beakers were put aside for 24 hours, after which the pH was checked again using digital pH probe.
Preparation of samples
Five test tubes were each filled with 20 mL of solution in treatment of pH 4 using a 50 mL measuring cylinder and pipette. This was repeated, producing five additional samples from each pH solution. The 25 test tubes were individually placed on digital scale, and after taring the scale, 0.25 grams of ‘sea monkey egg packet’ was added to each test tube. Each test tube was stirred for one minute. Samples were placed in incubator at 26 °C
Gathering results
After two days, a scoop of ‘sea monkey food’ was added to each test tube, using the provided measuring scoop. Seven days after the samples were set up, all samples were removed. Contents of test tubes were individually poured into a petri dish and visible A. nyos were counted and results recorded. All samples were disposed of in aquarium tank for future recreational use.
Data analysis
Data for each pH treatment was averaged. If any outliers were found, they were removed as a part of data cleansing. A statistical one-way ANOVA test was used to test if results were significantly different or not.
Results
After results collected data was placed in results table. Appendix one shows raw data taken from A. nyos counted in each test tube. Table one shows averages of collected data. Figure three is a graphed representation of the average number of A. nyos hatched in each solution. Table two and three reflect the results from one-way ANOVA test done using Post Hoc Tukey HSD test calculator, (2022), to check for significant difference in hatching of A. nyos in different pH solutions.
Table 1: average # of hatched A. nyos in pH 4, 6, 8 and 10. Key – *: experimental control in which pH was not altered
Solution pH
Standard deviation of treatment
Table 2: results of one-way ANOVA testing, working that the f ratio value is 8.46667. The p-value is 0.000363. The results are significant as p<0.05. Key – SS: sum of squares due to source, DF: the degree of freedom in the source, MS: the mean sum of squares due to the source.
Source SS DF MS
Between treatments 40.64 4 10.16 F = 8.46667 Within treatments 24 20 1.2
Total 64.64 24
Table 3: Post Hoc tests for one-way ANOVA testing, different pH solutions of hatching of A. nyos. Key – results highlighted in bold: individual results that were significantly different [p<0.05], T: treatment.
Pairwise comparison Q statistic Pvalue Inference
T1:T2 1.22 .90592 P > 0.05
T1:T3 5.72 .00513 P < 0.05
T2:T3 4.49 .03420 P < 0.05
T2:T4 0.41 .99835 P > 0.05
T2:T5 5.31 .00978 P < 0.05
T3:T4 4.08 .06194 P > 0.05
T3:T5 0.82 .97693 P > 0.05
T4:T5 4.90 .01844 P < 0.05
Discussion
Trends showed that in low pH’s (4 and 6) the number of hatched A. nyos was significantly lower (average of 0.4 and 1 hatched A. nyos) than that of pH 8 (an average of 3.2 A. nyos hatched). The unaltered samples, which had no bases added or pH changed, had a pH of 8.1 found some of the highest hatching numbers of A. nyos, of 3.6. As expected, the hatching number increased steadily as pH rose, however it decreased after reaching the estimated optimum pH of 8/8.1. The shape of the curve is reminiscent of that of enzyme activity, reflecting possible denaturing of proteins at a high pH, causing the curve to drop as hatching decreases.
The initial hypothesis being that, following the results of Salma et al (2012), as the pH increases, the number of hatched A. nyos will increase to an optimal pH (8), followed by a decrease, was supported. This is supported by solutions producing the highest average number of hatched A. nyos being solutions with a pH of 8 and 8.1, with average hatching counts of 3.2 and 3.6. Whilst the optimum was not the predicted pH 8, it was very close. This was followed by a predicted decrease to a hatching average of 1.2 in solution with pH of 10. The support of the hypothesis is furthered, in that the one-way ANOVA test was used to see whether the difference between these results is significantly different. It was found that there was in fact a significant difference, with a p value of 0.000363, being less than 0.05. Therefore, the null hypothesis can be rejected, and the alternate hypothesis that the mean number of hatching in each pH is significantly different can be accepted.
Similarly, the graph in Figure 3 demonstrates the trends in data, which can be explained by the fact that species are unable to survive in solutions that are too acidic or too alkaline, supported by decreased number of hatchings at low pH’s of 4 and 6, as well as another rapid decrease at pH 10. This increasing acidity in solution with pH’s of 4 and 6 is representative of Ocean acidification due to the uptake of carbon dioxide produced by anthropogenic activities, forming carbonic acid (Doney, Fabry et al, 2009), creating an unsuitable environment for A. nyos. This is because when pH of oceans lower, the mucus around the organism’s gills thickens, restricting oxygen that can enter, lowering an organism’s metabolic rate, therefore reducing hatching/survival (Smaller, 2010). Hatching numbers were highest in pH solutions closest to the ocean’s pH of 8.1, because this would be their ideal level of acidity, and one that they are well suited to.
There were no unexpected results within this experiment, perhaps due to the inability to recognise outliers due to limited sample size. However, all averages had a similar standard deviation, differing in no more than 0.95 of each other, which suggests all results were consistent.
Within this experiment there were several issues experienced. One of these being limited sample size due time constraints. It was initially planned that 15 samples of each solution would be tested, producing 75 data points altogether, however due to time constraints, this was not possible. Individually weighing out 0.25 g of powder from ‘egg sachet’ and adding into each test tube was found to be a slow process, especially with only one individual doing all experimental work. Because of this, the reliability of the data and conclusions were impacted, reducing the overall reliability of this report. For future experimentation, more time and participants would be recommended to overcome this experimental issue.
Another issue was overall low numbers of hatchings counted that decreases the reliability of the data collected. For this reason, future experimentation should consider increasing the weight of sea monkey eggs added so that the number of hatched sea monkeys counted will be higher, as well as proportionally increasing the amount of solution so that they can hatch and survive.
The assumption was made that the same mass of eggs weighed into the test tube had the same number of eggs. This was because a physical count of the initial number of eggs was not possible with the equipment available. This meant that any trends in results were not completely reliable due to the reduced sample size being unable to eliminate any outliers of varying numbers of eggs in each sample. By controlling the weight of eggs added to each sample, it would best reduce the chance of random variations in the numbers of eggs in a way that was simplistic and achievable within this experiment.
A final issue was problems with altering alkalinity of solutions using bicarbonate soda. It was originally intended to be used as it was accessible and safe for aquatic life, and used in many other studies, as explained previously. However, it was found that after reaching a point of roughly 8.3, it would not increase pH any further. This meant that a different base had to be used to create a solution with a pH of 10. Sodium hydroxide was used instead, as it is readily available and safe for A. nyos. The sodium hydroxide was very effective in increasing alkalinity to a pH of 10.
The results found were consistent with other studies including research by Fominykh (2008), Waiwood and Beamish (1978), Salma and Lee (2012) and Doyle and McMahon (1995). All of which conducted varying tests with different indicators measuring response to pH change on differing organisms, all of which found that in environments too acidic or too alkaline, growth, hatching rates, hatching success, and survival decreased. Most studies similarly found that the optimum pH occurred around 8-8.2
Future research should consist of testing the same research question with a larger sample size, as well as testing with closer increments, e.g., 7.4, 7.6, 7.8 and 8.0 etc, to gain a more accurate depiction of real ocean changes in pH and the effects that would have. This was not possible within this experiment due to limited access to suitably precise equipment.
Another suggestion for future directions of research includes exploring different methods of measuring the effect of differing pH on A. nyos, such as growth rate, size, or hatching speed. These would provide a more reliable conclusion to be drawn in addition to these results.
Conclusion
This experiment has studied and recorded data regarding the effect of differing pH solutions on the number of hatched A. nyos, and has found a significant difference in data, supporting the initial hypothesis, that, as following the results of Salma et al (2012), as the pH increases, the number of hatched A. nyos will increase to an optimal pH (8). Five different treatments were tested, of pH 4, 6, 8, 10 as well as one unaltered treatment using a pH probe. It was found that the highest average hatching occurred in pH treatments of 8, and in the experimental control solution, which had a pH of 8.1, each of which found an average hatching of 3.2 and 3.6. Hatching decreased in pH treatments with higher or lower pH, including pH 4 producing a mere 0.4 average hatchings. These results create an image of and strengthen the understanding of the effects of the negative impacts of Ocean acidification on the organisms within it. It also acts as a reflection towards the usefulness and effectiveness of using A. nyos to demonstrate these impacts. Whilst further research is needed to solidify the conclusion drawn, that Ocean acidification will have a harmful effect on the number of hatchings of A. nyos, the findings align with countless other studies, and shall hopefully contribute to igniting future research and action towards minimising the anthropogenic contributions to the increasing acidity of the oceans.
Acknowledgements
I would like to thank Dr Terena Holdaway-Clarke for helping with the formation of this investigation, in providing suggestions and feedback leading to the development of my final investigation. I would also like to thank her for being a supportive and dedicated mentor throughout the experimental and writing process. I would also like to thank Dr Matthew Hill for being an informative and helpful source in understanding the decisions and processes behind statistical analysis.
References
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Appendix
Hills, NWT, Canada. Canadian Journal of Botany, 60(1), 58-72.
Smaller. (2010). Smaller fish cope better with acidic water › News in Science (ABC Science). Abc.net.au. https://doi.org/https://www.abc.net.au/science/articles/201 0/03/11/2841714.htm
Snab Biology. (2017). The Effect Of Temperature On The Hatching Success Of Brine Shrimp. [online] Available at: https://snabbiology.co.uk/the-effect-of-temperature-onthe-hatching-success-of-brineshrimp/#Results_Calculations [Accessed 3 Nov. 2021].
[Sorgeloos, P. (1973). First report on the triggering effect of light on the hatching mechanism of Artemia salina dry cysts. Marine Biology, 22(1), 75-76.] Sulfuric acid | National Pollutant Inventory. (2019). Npi.gov.au. http://www.npi.gov.au/resource/sulfuric-acid
Number of A. nyos counted in test tube (#)
Solution pH Trial 1 Trial 2 Trial 3 Trial 4
Trial 5 average
4 0.0 0.0 1.0 0.0 1.0 0.4 6 1.0 0.0 2.0 1.0 1.0 1.0 8 4.0 3.0 5.0 3.0 1.0 3.2 *8.1 4.0 3.0 3.0 4.0 2.0 3.6 10 2.0 0.0 1.0 3.0 0.0 1.2 Appendix one – # of hatched A. nyos in pH 4, 6, 8 and 10. Key: * (experimental control in which pH was not altered)
Physics
Physics underpins all that we observe, and it was rocketry, sport science, and 3D printing that were the focus of this year’s student research reports. Only 3D printed materials were harmed in the production of this research.
Two students with particular passions for applying scientific thinking to sports undertook Science Extension this year. Jessica, an elite water polo player was curious about the effect of curvature of (3D printed) swimming paddles on performance. Swimmers use these paddles to increase resistance during training, so she proposed optimum curvature for maximum resistance. Claire and her family are heavily invested in the Ice Hockey community in Australia and the USA. Claire identified a gap in the literature in Ice Hockey helmet testing and built a contraption launching hockey pucks at 3D printed dummy heads to investigate the internal force on the brain. Two students successfully navigated theoretical rocketry involving complex calculations and predictions. Rosco compared the efficacy of re-usable rocket boosters that either return vertically to the launchpad, or horizontally landing as a plane or glider would. William recognised the current needs of rocketry surrounding small payloads (for applications such as CubeSats) and compared a coilpropelled projectile with traditional rockets. For 3D printing to be used in high-strength applications, the printed product needs to undergo a process called annealing. Jack investigated whether the extended time process taken for annealing could be reduced by microwaving the products. His results indicate a potential improved process resulting in similar tensile strength. We hope you enjoy reading about this research as much as these students have enjoyed producing it.
