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Aquatic Osteoporosis
- 1. AQUATIC OSTEOPOROSIS: Remediating the emerging problem of lake calcium decline Isabella O’Brien Grade 10 Westmount Secondary School Hamilton, Ontario, Canada
- 2. BACKGROUND1
- 3. LAKE CALCIUM DECLINE - A LEGACY OF ACID RAIN • Lake calcium decline is an emerging environmental issue currently impacting softwater shield lakes in Canada, North-Eastern United States and in Scandinavia. • The harmful effects of acid rain were recognized in the 1980s as acid rain lowered lake pH, upsetting the ecological balance and killing aquatic life. • These acids, however, also caused the calcium in soil to leach at a rapid pace and it occurred at a faster rate than it could be replaced by mineral weathering or atmospheric deposits. • In the late 1980s, aggressive environmental policies were put in place to reduce the harmful carbon dioxide emissions and these measures succeeded in reducing acid rain. • Since then, lake pH levels have mostly recovered, however, it has recently been discovered that lake calcium levels have not been restored and are continuing to decrease, with a steep decline occurring after 1991 (Jeziorski et al., 2012). • Other environmental stressors that have contributed to this decline include: [1] increase of shoreline residential development; [2] forest clearing and regrowth, and [3] climate change (Hadley, 2012).
- 4. (a) Before acid rain the weathering of minerals and atmospheric deposits of calcium-rich dust (from ocean spray, forest fires, wind erosion of soils, agriculture, unpaved roads, etc.) all added to the available pool of calcium nutrients for both soil and aquatic requirements. (b ) During the early stages of acid rain, the acids caused the calcium in soil to leach at a rapid pace into the surrounding lakes. The calcium levels in these lakes rose very quickly, especially in softwater lakes in shield regions which have thin layers of soil laying on top of weathering resistant bedrock. (c) Eventually, with continued acidic rain, the available calcium pool in the shield regions diminished to the point that calcium leaching is greatly reduced and occurred at a faster rate than it could be replaced. In addition, the effects of multiple stressors have further diminished the calcium supply. (Smol, 2010) NATURAL CONDITIONS EARLY STAGES OF ACID DEPOSITION MULTIPLE STRESSORS LEADING TO AQUEOUS CALCIUM DECLINE Figure 1: Calcium Cycle in Forest Ecosystems [Source: USGS, 1999] Root Uptake Forest Floor Adsorption to Surfaces Desorption from Surfaces Calcium in Rocks Weathering Mineral Soil Calcium in Soil Water Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Exchangeable Calcium Ca CaCa Ca CaCa Ca Ca Ca Rain and Dust Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Ca Wet Deposition (rain, snow, sleet) Sulphuric Acid [H2SO4] Nitric Acid [HNO3] SO2 NOx Dry Deposition (particulates and gases) Sulphur Dioxide [SO2] NOx Nitrogen Oxide [NOx] Figure 2: Acid Rain Cycle [Source: Dowdey, 2007]
- 5. SERIOUS THREAT TO VITAL ZOOPLANKTON & LAKE BIODIVERSITY • Lake calcium decline is a serious issue for Daphnia pulex which are an important component of freshwater lakes and very sensitive to declining calcium levels. For Daphnia pulex, lake calcium decline has meant that their calcium rich exoskeletons are smaller and softer making them more vulnerable to prey (Riessen et al., 2012). • Invasive species such as the spiny water flea are hunting the daphnia, allowing population explosions of Holopedium, plankton competitors of Daphnia pulex (Jeziorski et al., 2015). • This increased jellification of lakes prevents vital nutrients from being passed up the food chain to fish stocks and also clogs filtration systems that help the lakes provide drinking water to residents in the area (Ibid., 2015). • In laboratory studies, it has been determined that Daphnia pulex are unable to reproduce at calcium levels below 1.5 mg/L and currently ⅓ of Canadian Shield lakes are below this level (Ashforth & Yan, 2008).
- 6. • “Calcium-rich daphniids are some of the most abundant zooplankton in many lake systems, and their loss will substantially affect food webs” (Jeziorski & Yan, 2008: 1377). • Due to their larger body size, Daphnia pulex are important herbivores in freshwater systems as they are able to filter food particles at a much faster rate and they graze a wider size and range of algae compared to other species (Korosi et al., 2012). • As Daphnia pulex numbers decline, the rate of softwater lake algae blooms are increasing and the entire biodiversity of lakes is changing (Ibid., 2012). ECOSYSTEM IMPLICATIONS Photo credits: 1. Reuters/Stringer 2. Paul Herbert 3. Michael Lencioni 4. Isabella O’Brien 5. ontariofishspecies.com Algae Daphnia Pulex Waterfowl Invertebrate Predators Fish Figure 3: Ecosystem Implications [Source: Smol, 2014]
- 7. REMEDIATION BY RECYCLING • Current research on lake calcium decline is focused on the extent of the problem and its implications, rather than on remediation. However, existing research on remediating acidity (low pH) in lakes and oceans may suggest a way forward. • Lime treatment of inland waters in Sweden to neutralize acidity (Olem, 1991) and small-scale oyster shell recycling programs in various U.S. coastal areas to restore oyster beds damaged by ocean acidification (NOAA MPA Centre, 2015) , are suggestive of a possible method for remediating lake calcium decline. • Both lime and waste shells are a rich source of calcium carbonate, with waste shells composed of 95 to 98% calcium carbonate (Hamester, 2012). • With an estimated six million metric tons of shell waste produced globally each year based on worldwide statistics of aquaculture and commercial catches of mussels, clams and oysters and about ½ a million metric tons just in the U.S. and Canada (Food and Agriculture Organization of the UN, 2013) , the potential exists to have this shell waste recycled instead of being sent to landfill, the disposal of which has become a worldwide issue (Fisheries and Oceans Canada, 2011; Yan & Chen, 2015).
- 9. PURPOSE: • The purpose of this experiment was to determine whether the addition of pulverized recycled shell waste to calcium-deficient lake water would increase the calcium concentration and to examine what effect it would have on the survival and reproductive output of Daphnia pulex exposed to the calcium-augmented waters. • The information gained from this experiment will hopefully aid in developing a process to help restore calcium-deficient lakes, which will be beneficial in maintaining Daphnia pulex populations as well as help protect lake biodiversity, minimize lake algae blooms and divert shell waste from landfill. HYPOTHESIS: It is hypothesized that if calcium carbonate waste shells are introduced to calcium-deficient lake water, then the absorption of the calcium carbonate will increase lake calcium levels as well as the survivorship and reproductive capabilities of Daphnia pulex.
- 11. EXPERIMENTAL DESIGN: • The two trials (3.4 mgCa/L and 1.89 mgCa/L, representing low and critical levels of lake water calcium, respectively) with differing treatments (0 mg, 10 mg, and 50 mg shell powder added) were run for 21 days with ten replicates each. • Each test vessel (250 ml polyethylene terephthalate cups) contained 200 ml of the appropriate treatment plus .03 ml of algae food source ( Nannochloropsis ) and was populated with one juvenile (less than 1 day old) daphnia, as follows: TRIAL 1 Control = 200 ml lake water @ Ca 3.4 mg/L + 0 mg shell powder + one <1-day old daphnia x 10 replicates Treatment 1 = 200 ml lake water @ Ca 3.4 mg/L + 10 mg shell powder + one <1-day old daphnia x 10 replicates Treatment 2 = 200 ml lake water @ Ca 3.4 mg/L + 50 mg shell powder + one <1-day old daphnia x 10 replicates TRIAL 2 Control = 200 ml diluted lake water @ Ca 1.89 mg/L + 0 mg shell powder + one <1-day old daphnia x 10 replicates Treatment 1 = 200 ml diluted lake water @ Ca 1.89 mg/L + 10 mg shell powder + one <1-day old daphnia x 10 replicates Treatment 2 = 200 ml diluted lake water @ Ca 1.89 mg/L + 50 mg shell powder + one <1-day old daphnia x 10 replicates
- 12. 12 2 Trials of 1 Control and 2 Treatments TRIAL 1 (Low Calcium Level @ Ca 3.4 mg/L ) TRIAL 2 (Critical Calcium Level @ Ca 1.89 mg/L) 10 Replicates per Control & Treatment per Trial 1 juvenile Daphnia pulex < 1-day old per vessel Control + 0 mg shell powder Treatment 1 + 10 mg shell powder Treatment 2 + 10 mg shell powder 12 12 12 12 12 Sampling Rate: daily for 21 days – treatments were refreshed every third day Controls: Light/Dark ratio (18/6 hr), temperature (20 – 22oC), feed (.03 ml algae per day) Control + 0 mg shell powder Treatment 1 + 10 mg shell powder Treatment 2 + 10 mg shell powder
- 13. EXPERIMENTAL DESIGN: QUANTITY MATERIALS USED 1 Coffee grinder 1 Box of wax paper 1 Box of scientific wipes 1 Box of nitrile-free disposable gloves 1 Plastic bucket for collecting lake water 1 Fine mesh water filter (200-micron) 1 Calibration fluids (pH 4.7, 7.0 and 10.0) 1 Digital pH Meter – Accuracy +/- .01 1 Digital scale 1 Wash bottle 1 Metal spoon 1 1 L bottle algae food source (Nannochloropsis) 2 1 cup measuring cups 2 1 large and 1 medium funnel 2 Cool fluorescent lamps 2 Light timers 3 Living Daphnia pulex cultures 3 20 L water storage jugs 4 50 mL beakers 4 1.5 L plastic holding tubs for Daphnia pulex cultures 6 10 L jug containers 9 Blue Mussels (Mytilus edulis – from P.E.I.) 9 Littleneck Clams (Venerupis philippinarum – from B.C.) 9 Malpeque Oysters (Carassostrea Virginia – from B.C.) 9 1 L bottles of spring water 10 1 mg and 10 mg pipettes 20 10 mL vials with lids 50 Litres of deionized water 500 200 mL plastic cups INDEPENDENT, DEPENDENT & CONTROLLED VARIABLES: Question Can using pulverized calcium carbonate shells in calcium –deficient lake water increase the calcium concentration without being detrimental to Daphnia pulex? Independent Variable The independent variable was the test solutions [Trial 1 and Trial 2] and the amount of shell powder added [the treatments: +0 mg, +10 mg and +50 mg] Trial 1- Low calcium level lake water @ 3.4 mg/L Trial 2 - Critical calcium level lake water @ 1.89 mg/L Dependent Variables • The percentage increase of calcium concentration. • The survivability of the Daphnia pulex. • The reproduction rate of the Daphnia pulex. • The number of broods over a 21 day period. Controlled Variables • The amount of each treatment (200 ml). • The amount of time each baby daphnia remained in the solution (21 days). • The exposure to light / dark during the testing period (16/8 hours). • The temperature of the room where the solutions were stored (kept at 20- 22oC). • The amount of daphnia algae feed per day (.03 ml).
- 14. SET-UP and PROCEDURE: WATER SAMPLE: • Water used in this experiment was collected from a long-term monitoring site (Plastic Lake, Dorset region, Ontario, Canada; 5 10'47" North and 78 49'16" West). • The site is near the southern edge of the Precambrian Shield and the boundary of the Boreal eco-zone and was chosen based on its last reported critical calcium level of 1.2 mg/L. • Test results using a university lab Dionex Ion Chromatography system revealed the actual calcium level of the sample was 3.4 mg/L (probably due to several factors: rain earlier in the week, only being able to obtain the water sample near the shore, and time of year). • The water samples were filtered into clean containers using a 200-micron water filter and stored in the dark at 5 C until required for preparing the test treatments at which time they were brought to the test room temperature (20 – 22 C). ° ° ° °
- 15. DAPHNIA CULTURE: • Three separate cultures of Daphnia pulex were obtained and reared in spring water. The daphnia cultures were maintained for 60 days prior to use in the experimental trials. MEDIA PREPARATION: • Two trials were run for this experiment: [1] low calcium level lake water at 3.4 mg/L and [2] critical calcium level lake water (lake water diluted with deionized water) to bring the calcium level closer to the Daphnia pulex calcium threshold of 1.5 mg/L. The Ion Chromatography test result for the prepared diluted batch returned a calcium reading of 1.89 mg/L. SHELL POWDER PREPARATION: • The shells of mussels, clams, and oysters were used to prepare the shell powder. The mollusks were shucked, cleaned of all organic material and heat treated to remove any bacteria. Each shell type was ground separately to a fine powder, sifted using a fine mesh sieve and stored separately. For the purposes of testing, 38 grams of each shell powder type was measured and combined in a single container for testing use.
- 16. TESTING: • During the 21 day testing period, each test cup was examined daily for daphnia survivorship and reproduction. • Typical observations included daphnia viability, the number of broods produced, and how many offspring per brood were present. • All offspring were counted and removed each day. • The temperature (20 – 22 C), light exposure (16 hours light / 8 hours dark), and length of time in solution (21 days) were held constant throughout the experiment. • Additionally, the pH level was measured each day for every treatment, using a calibrated pH meter. • To prevent stagnation and bacteria buildup in the test cups, daphnia were transferred via pipette to a new unused cup containing a fresh treatment solution every three days. °
- 17. Image 1: Plastic Lake, Dorset, Ontario Images 2 & 3: Collecting & filtering lake water Image 4: Preparing samples for calcium testing Images 5 & 6: Shell grinding & measuring shell powder treatments Images 7, 8 & 9: Daphnia culture tanks; harvesting adult daphnia with eggs; adult daphnia with eggs Image 10: Basement testing lab set-up Photos: I. O’Brien / A. Ceccato 1 2 3 4 5 6 7 8 9 10
- 18. RESULTS4
- 19. CALCIUM LEVEL RESULTS Trial 1 – Low calcium level @ 3.4 mg/L The calcium level results for Treatment 1 and Treatment 2 significantly differed from the control (p=0.043 and p=0.01 respectively) but the two treatments did not differ significantly from each other. This result indicates that the shell powder had a direct effect on increasing calcium levels in the water, but this effect was not dose dependent. In Treatment 1, the calcium level increased from 3.4 mg/L to 13.2 mg/L, an increase of 288% and in Treatment 2 the calcium level increased from 1.89 to 12.92 mg/L, an increase of 280%. Trial 2 - Critical calcium level @ 1.89 mg/L Treatment 2 significantly differed from the control (p=0.01), but not from Treatment 1 (p=0.54) and Treatment 1 and 2 did not differ significantly from each other . This result indicates that the shell powder had an effect on increasing calcium levels in the lake water, but this effect was more significant when the higher level of shell powder was added. In Treatment 1, the calcium level increased from 1.89 mg/L to 14.18 mg/L, an increase of 650% and in Treatment 2 the calcium level increased from 1.89 mg/L to 13.33 mg/L, an increase of 605%. DAY OF TRIAL CALCIUMLEVEL(mg/L) Day 1 Day 2 Day 3 Day 4 16 14 12 10 8 6 4 2 0 3.40 3.40 3.40 3.40 3.40 4.31 8.88 11.33 13.2 8.83 12.83 11.51 12.92 Calcium Level Trend Over Renewal Period [Trial 1- Low Calcium Level @ 3.4 mg/L] Control Treatment 1 Treatment 2 DAY OF TRIAL CALCIUMLEVEL(mg/L) Day 1 Day 2 Day 3 Day 4 0 2 4 6 8 10 12 14 16 1.89 1.89 1.89 1.89 3.57 7.21 9.43 14.18 1.89 7.48 11.9 13.03 13.33 Calcium Level Trend Over Renewal Period [Trial 2 - Critical Level Calcium @ 1.89 mg/L] Control Treatment 1 Treatment 2
- 20. SURVIVORSHIP For both trials, Treatment 1 and Treatment 2 significantly differed from the control treatment with (p<0.001), however, Treatment 1 and Treatment 2 did not differ significantly from each other (p=1.0). This result indicates that the shell powder had a direct effect on increasing the survivorship of the daphnia, but this effect was not dose dependent. For both trials, the treatments that had shell powder added resulted in 100% survivorship. For the low calcium level control, 80% of the daphnia survived over a 21 day period and for the critical calcium level control, only 40% survived. DAY OF TRIAL NUMBER OF LIVING DAPHNIA REPLICATES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 0 2 4 6 8 10 12 Survivorship Over 21 Day Trial Period [Trial 2 - Critical Calcium Level @ 1.89 mg/L] Control Treatment 1 Treatment 2 DAY OF TRIAL NUMBER OF LIVING DAPHNIA REPLICATES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 0 2 4 6 8 10 12 Survivorship Over 21 Day Trial Period [Trial 1 - Low Calcium Level @ 3.4 mg/L] Control Treatment 1 Treatment 2
- 21. REPRODUCTION Time to First Brood: For both Treatment 1 and 2 in Trial 1, there was no significant difference in the number of days before the first broods were produced and the control (p>0.05). Therefore the addition of shell powder, regardless of the dosage, had no effect on the number of days to first brood in the low level calcium trial. For both treatments in Trial 2, the average number of days before the first brood appears was significantly different from the control (p=0.020 and p=0.011 respectively) but the two treatments did not differ significantly from each other. This indicates that the added shell powder had a direct effect on decreasing the number of days to first brood in critical calcium level water, but was not dose dependent. TRIALS AVERAGENUMBEROFDAYS Trial 1 Trial 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 12.6 15.2 11.8 12.2 11.4 11.9 Average Number of Days Before First Brood Control Treatment 1 Treatment 2
- 22. REPRODUCTION (cont’d) Mean Number of Broods Produced & Offspring Born: In Trial 1, the mean for both the number of broods produced and the number of offspring (p=0.193 and p=0.060 respectively) was not significantly different between groups. However, in Trial 2 the mean for both the number of broods and the number offspring in both Treatments 1 and 2, was significantly different from the control (p=0.21, p= 0.004, and p=0.036, p=0.005 respectively). These results indicate that the added shell powder had a significant effect on increasing the reproductive output, but only in Trial 2 and this effect was not dose dependent. In Trial 2, the mean number of broods for Treatment 1 was 125% greater than the control and 158% greater in Treatment 2. Also in Trial 2, the mean number of offspring for Treatment 1 was 143% greater than the control and 195% greater in Treatment 2. TRIALS MEANNUMBEROFBROODS Trial 1 Trial 2 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 2.3 1.2 3.5 2.7 3.2 3.1 Mean Number of Broods Produced Over 21 Day Trial Period Control Treatment 1 Treatment 2 TRIALS MEANNUMBEROFOFFSPRING Trial 1 Trial 2 20 18 16 14 12 10 8 6 4 2 0 7.9 4.0 15.3 9.710.0 11.8 Mean Number of Offspring Born Over 21 Day Trial Period Control Treatment 1 Treatment 2
- 23. OBSERVATIONS: • The juvenile daphnia in the treatments with shell powder added, visually grew larger than the daphnia in treatments with no shell powder added. • During the testing period, the pH of the treatments was measured. The pH levels increased significantly in all test cups after the shell powder was added to the lake water. In both treatments, the pH level significantly differed from the controls. However, the pH level in Treatment 1 (10 mg) and Treatment 2 (50 mg) did not differ significantly from each other. SOURCES OF ERROR: • The pH meter used had an accuracy of +/- 0.1 which may have affected the readings. • With regard to the reproduction and viability of the daphnia, it should be noted that these results only reflect controlled lab conditions and in a real world scenario, the daphnia would have been subjected to other stressors which would have altered the results in terms of viability, reproduction, brood size, etc. • The process of transferring the daphnia, either at the less than one-day old stage or during each treatment solution refresh period is also another possible source of error, as the transferring process may have caused a level of stress in the Daphnia leading to either death or impacting the number of broods and offspring.
- 25. CONCLUSION • The experiment’s hypothesis was supported when the addition of the shell powder to calcium-deficient lake water increased the calcium levels as well as increased daphnia survivorship and the number of broods and offspring. The results were most significant in the critical calcium level lake water. • As a result, the addition of pulverized recycled shell waste to the independent variable [the treatments] has a direct and significant effect on the dependent variables [the calcium concentration and daphnia survival and reproduction]. • The results clearly show that the addition of shell powder to increase calcium levels can be effective both for remediation of critical lake calcium levels, which proved to be very beneficial for daphnia survivorship and reproduction and it can also be used as a preventative measure in low calcium level lakes to prevent lake calcium levels from decreasing any further and to maintain existing daphnia populations. • The decline of Daphnia pulex populations, the increase of lake algae blooms and the appearance of invasive species are all consequences of this emerging environmental problem which is causing widespread transformations of aquatic food webs in softwater shield lakes in North America and in other acid-sensitive regions of the globe. • This experiment identifies a method to remediate lake calcium decline and has the potential to be beneficial in maintaining Daphnia pulex populations, which will protect lake biodiversity and help mitigate algae blooms while at the same time contribute toward addressing the global issue of shell waste disposal.
- 26. DISCUSSION • The inspiration for this project came from my previous work on ocean acidification where waste shells in pulverized form were used to buffer ocean acidity. The positive results of my previous project inspired me to find additional ways in which waste shells could be used as waste shells are a readily available and abundant reusable resource. • Globally, the disposal of the millions of metric tons of waste shells has become an increasing problem just due to the sheer volume and weight of waste. Recycling of these shells from large volume producers such as canneries, processors, and even seafood restaurants is a potentially highly cost-effective solution. • The disposal costs of the waste shells are high because it is done by the ton. If a recycling program is put in place for large volume producers, the cost of recycling and processing the waste shells could be recovered by selling the pulverized shells for other uses as well such as construction, soil treatment, pharmaceuticals, etc. Processing costs could be reduced by allowing the shells to age in the sun for about a year in order to dry and sanitize them and also by using renewable energy for the shell grinding process. • Furthermore, based on my previous research into ocean acidification, the shell powder could also be used in marine protected areas to increase pH levels and mitigate the effects of ocean acidification. • In terms of the application of the shell powder to the lakes, the costs would be dependent on the method of application. For accessible lakes, the shell powder could be applied by boat or on the watershed and in winter, it could be applied on the ice. Where there is limited access to a lake, aerial methods would have to be used to apply the shell powder in the same manner as limestone was applied to lakes in Sweden to combat lake acidity. • The dosage of shell powder would need to be stoichiometrically calculated taking into consideration the volume of the lake, the current pH, and calcium level, the desired calcium level, the flow through rate, etc. Also, constant monitoring of the lake calcium level would be required to determine if and when additional applications of shell powder would be required. • Further investigation could be conducted in using this method as a watershed soil treatment to see if leaching from the soil to the lake, instead of direct application to the lake, would be another approach to not only increase lake calcium levels but also restore soil calcium levels which have been having a damaging effect on tree growth.