Aquatic Osteoporosis: Remediating the Emerging Problem of Lake Calcium Decline


An emerging environmental issue of lake calcium decline is affecting soft water shield lakes in Canada, the United States of America, and Scandinavia. Following a legacy of acid rain during the 1970s and 1980s, the consequent low pH caused rapid leaching of calcium from lakes and soil. Although pH has stabilised due to emission control policies, calcium levels have not, and are now approaching ecologically critical levels. Daphnia pulex, a keystone indicator of aquatic food webs and principal grazers of algae, are at risk. This project examines whether adding powdered calcium carbonate, from recycled shell waste, to calcium deficient soft water lakes will remediate calcium levels and ensure the survival and reproduction of D. pulex. Two trials, (3.4 mgCa/L and 1.89 mgCa/L – low and critical levels of calcium, respectively) were run for 21-days with differing treatments (0 mg, 10 mg, 50 mg shell powder added). Each treatment had 10 replicates with one juvenile daphnia < 24-hours old. The calcium level, survivability, and reproduction improved in all treatments with shell powder and improved significantly in the critical level trial with added shell powder. This method can restore lake calcium and therefore be beneficial in maintaining D. pulex populations which will protect lake biodiversity, help mitigate algal blooms, and will contribute towards addressing the global issue of shell waste disposal.  


Circular economy, Daphnia pulex, lake calcium decline; shell recycling; soft water lakes.


Lake Calcium Decline – A Legacy of Acid Rain

Lake calcium decline is an emerging environmental issue currently impacting soft water lakes on the Canadian Shield, the United States of America, and Scandinavia. Multiple widespread environmental stressors have contributed to this decline including:
1) acidification via atmospheric deposition (e.g. acid rain),
2) an increase of shoreline residential development,
3) forest clearing and regrowth, and
4) climate change.[1]

Prior to the prominence of acid rain, both the weathering of minerals and atmospheric deposits of calcium-rich dust all added to the available pool of calcium for both soil and aquatic requirements. As increasing sulphuric and nitric acids were deposited due to anthropogenic processes, soil calcium leached at a rapid pace into the surrounding lakes. Dissolved calcium levels in these lakes then rose very quickly, especially in soft water lakes in the shield regions which have thin layers of soil laying on top of weathering resistant bedrock.[2] However, as the leaching of calcium occurred at a faster rate than it could be replaced via mineral weathering or atmospheric deposits, less calcium was introduced to lakes over time.

Simultaneously, acid rain lowered lake pH levels: as soft water lakes typically form over granite instead of calcium carbonate-based substrate (limestone), their minimal acid-neutralising capacity therefore makes them predisposed to acidification.[3] The effects of multiple stressors further reduced the calcium supply to lakes. For example, with shoreline residential development, logging and timber removal (especially the calcium-rich bark), additional calcium is lost from the ecosystem.  Then as trees are replanted and re-grow, more demand is placed on the available calcium in the soil, leaving less to leach into the lakes. Furthermore, due to reduced precipitation and warmer air temperatures associated with climate change, lake inflow and outflow has been reduced thus contributing to calcium decline.[4]

In the late 1980s, aggressive environmental policies were put in place to reduce the harmful sulphur 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.[5]

Serious Threat to Vital Zooplankton and Lake Biodiversity

Lake calcium decline is a serious issue for D. pulex, the most common species of water flea. They are an important component of freshwater lakes and very sensitive to declining calcium levels. For D. pulex, lake calcium decline has meant that their calcium-rich exoskeletons are smaller and softer, making them more vulnerable to prey.[6] Invasive species such as Bythotrephes longimanus (the Spiny Water Flea) are hunting the daphnia, allowing population explosions of jelly-clad Holopedium, plankton competitors of D. pulex.[7] Holopedium populations require less calcium and have a jelly-coat that gives them greater protection from predators. This increased jellification of the lakes prevents vital nutrients from being passed up the food chain to fish stocks, and it also clogs filtration systems that help the lakes contribute drinking water to residents in the area.[7]

In laboratory studies, it has been determined that D. pulex are unable to reproduce at calcium levels below 1.5 mg/L and currently one-third of Canadian Shield lakes are below this level.[8] “Calcium-rich daphnids are some of the most abundant zooplankton in many lake systems, and their loss will substantially affect food webs”.[9] Due to their larger body size, D. pulex are important herbivores in freshwater systems as they can filter food particles at a much faster rate and they graze a wider size and range of algae compared to other species.[10] As D. pulex populations decline, as evidenced in dated sediment core studies[11], the rate of freshwater algal blooms is increasing, and the entire biodiversity of lakes is changing.[10]

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 neutralise acidity[12] and small-scale oyster shell recycling programs in various US coastal areas to restore oyster beds damaged by ocean acidification[13] 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.[14] It is estimated that globally there is over eight million metric tons of shell waste produced each year based on worldwide statistics of aquaculture and commercial catches of molluscs, and approximately half a million metric tons of shell waste is generated in the United States and Canada alone.[15] The potential exists to have this shell waste recycled rather than having it sent to landfill, the disposal of which has become a worldwide issue.[16][17] It, therefore, might be possible to use these waste shells in a crushed or pulverised form as a calcium supplement in soft water lakes to help remediate the emerging problem of lake calcium decline.

This work aims to explore the effectiveness of introducing pulverised calcium carbonate shells into calcium-deficient lake water and examine its impact on the survivorship and reproductive capabilities of D. pulex, as well as its effect on lake calcium levels. It was hypothesised that if calcium carbonate from pulverised recycled shell waste is added to calcium-deficient soft water lakes, then calcium levels will increase, as will the survivorship and reproduction of D. pulex.

Method and Materials


Water Sample: Water used in this experiment was collected from a long-term monitoring site (Plastic Lake, Dorset region, Ontario, Canada (Figure 1)); and the boundary of the boreal ecozone and was chosen based on its 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 collected was 3.4 mg/L. 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).

Figure 1: Plastic Lake, Ontario, Canada. Photograph by Isabella O\’Brien, October 2015.

Daphnia Culture: Three separate cultures of D. 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 deionised water) to bring the calcium level closer to the D. 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 molluscs 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, an equal mass of each shell powder type was combined in a single container for testing use.


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 0.03 mL of algae food source (Nannochloropsis). Each vessel was then populated with one juvenile daphnia less than one day old (Figure 2). To prevent stagnation and bacteria build-up in the test vessels, the medium was refreshed every three days and the daphnia were transferred via pipette to a new vessel containing the refreshed treatment.

During the 21-day testing period, each test vessel was examined daily for daphnia survivorship and reproduction. Typical observations included daphnia viability, the birth of a brood, 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.

Figure 2: Experimental Design


IBM SPSS Statistics software was used to perform univariate analyses to assess whether test groups differed significantly from each other. Independent t-tests were done to identify where the differences occurred.

Calcium level

Trial 1 – Low calcium level @ 3.4 mg (the control):
The calcium level results for Treatment 1 and 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. 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 mg/L to 12.92 mg/L, an increase of 280% (Figure 3).

Trial 2 – Critical calcium level @ 1.89 mg/L (the control):
Treatment 2 significantly differed from the control (p = 0.01), however, Treatment 1 did not significantly differ from the control (p = 0.54) but was approaching significance. Treatment 1 and 2 did not differ significantly from each other. 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% (Figure 4).

Figure 3 (left): Calcium level trend over refresh period for Trial 1 [Low Calcium Level @ 3.4 mg/L].
Figure 4 (right): Calcium level trend over refresh period for Trial 2 [Critical Calcium Level @ 1.89 mg/L].

For both Trial 1 (Figure 5) and Trial 2 (Figure 6), Treatment 1 and 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). 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.

Figure 5 (left): Survivorship over 21-day trial period for Trial 1 [Low Calcium Level @ 3.4 mg/L].
Figure 6 (right): Survivorship over 21-day trial period for Trial 2 [Critical Calcium Level @ 1.89 mg/L].

Mean Time to First Brood:
For Treatment 1 and 2 in Trial 1 (Figure 7), there was no significant difference from the control in the number of days before the first broods were produced (p > 0.05).

For both treatments in Trial 2 (Figure 7), the mean number of days before the first brood appears was significantly less from the control (p = 0.020 and p = 0.011 respectively), but the two treatments did not differ significantly from each other.

Figure 7: Mean number of days before first brood (Note: Error bars represent standard deviation).

Mean Number of Broods and Mean Number of Offspring:
In Trial 1, the mean for both the number of broods produced (Figure 8) and the number of offspring (Figure 9), was not significantly different between groups (p = 0.193 and p = 0.060 respectively).

However, in Trial 2 the mean for both the number of broods and the number offspring for both  Treatment 1 and Treatment 2 was significantly higher from the control (Broods: p = 0.021, p = 0.004; Offspring: p = 0.020, p = 0.003).

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.

Figure 8 (left): Mean number of broods produced over a 21-day trial period (Note: Error bars represent standard deviation).
Figure 9 (right): Mean number of offspring born over a 21-day trial period (Note: Error bars represent standard deviation).


The results illustrate that the addition of shell powder to calcium-deficient lake water increased the lake calcium level significantly in both trials (i.e. Trial 1 – 3.4 mgCa/L and Trial 2 – 1.89 mgCa/L, low and critical levels of calcium, respectively), regardless of dosage (i.e. Treatment 1 – 10 mg shell powder and Treatment 2 – 50 mg shell powder) and it continued to increase over time as the shell powder continued to dissolve.

With regards to survivorship, the addition of shell powder to calcium-deficient lake water had a significant effect on increasing the rate of survival in both trials and this was not dose dependent. Additionally, the addition of shell powder did increase the number of broods as well as the number of offspring of the D. pulex. This effect was most significant in the critical calcium level lake water (Trial 2) with shell powder added, regardless of the dosage. Finally, the number of days to first brood decreased significantly when the shell powder was added to the critical calcium level lake water (Trial 2), but was not dose dependent.

These findings demonstrate that pulverised calcium carbonate shells could potentially serve as a remediation approach to lake calcium decline. The 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 produced. Results were most significant in critical calcium level lake water.

As a result, the addition of pulverised recycled shell waste to the independent variable [the treatments] has a direct and significant effect on the dependent variables [the calcium concentration and D. pulex survival and reproduction].

In conducting this experiment, there were several possible sources of error. With regard to the reproduction and viability of the daphnia, it should be noted that these results only reflect controlled laboratory conditions whereas in the actual environment, the daphnia would have been subjected to other stressors which would have altered the results in terms of viability, reproduction, brood size, etc. Furthermore, 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 they produced.

This experiment’s use of waste shells and the results obtained are significant given that globally the disposal of the millions of metric tons of waste shells has become an increasing problem due to the sheer volume and mass of the waste. Recycling of this waste product 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 the cost is determined by total mass. 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 pulverised shells for other uses as well such as construction, soil treatment, pharmaceuticals, etc. Furthermore, based on previous research, the shell powder could also be used in marine protected areas to increase pH levels and mitigate the effects of ocean acidification.[18] The processing costs of the shells could be reduced by using renewable energy at processing plants for things such as heat treatment to rid the shells of bacteria and grinding to make the shells more soluble. “In a time of increased awareness of the need for a circular economy, shells should be considered a valuable biomaterial that can be reused for both environmental and economic benefit”.[19]

The application method of shell powder to lakes and the associated costs must be taken into consideration and 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. Furthermore, 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. Constant monitoring of the lake calcium level would be required to determine if and when additional applications of shell powder would be required.

This approach of using waste shells is preferable to limestone as limestone is mined in massive quantities and is one of the world’s most heavily exploited minerals[20], whereas waste shells are a readily available and abundant reusable resource. Therefore, the use of waste shells to help combat environmental problems such as lake calcium decline, lake acidification or even ocean acidification, in turn helps to alleviate the growing issue of disposing of the millions of metric tons of waste shells produced worldwide each year.

Figure 10: Example of shell recycling program at Horn Point Laboratory, Cambridge, Maryland, USA. Photograph by Isabella O\’Brien, September 2016.

Although the results of this experiment were promising, this topic requires more study and experimentation to determine if remediation using pulverised recycled shell waste has either any long-term effects or impacts on other lake organisms. Further investigation could also 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 to also restore soil calcium levels. Calcium depleted soil has resulted in forest decline in the North-Eastern United States and has had a damaging effect on tree growth (in particular the sugar maple and red spruce trees).[21] Additionally, other cations such as magnesium, ammonium, potassium, sodium and or lithium levels could also be examined. Although the university lab included these test results when measuring the calcium levels, they were not the focus of this experiment and could warrant further analysis.

With recent reports that freshwater acidification is again on the rise due to the increasing CO2 levels[22], the implications for daphnia and freshwater ecosystems make both the emerging problems of lake calcium decline and lake acidification pressing issues which have the potential of being addressed by the findings of this experiment.


This work has shown that the addition of shell powder to increase calcium levels can, therefore, be effective for remediation of critical lake calcium levels, which proved to be very beneficial for daphnia survivorship and reproduction. Furthermore, it can also be used as a preventative measure in low calcium level lakes to prevent lake calcium levels from decreasing any further and thus help maintain existing daphnia populations.

The decline of D. 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 soft water 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 D. 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.


I would like to extend my many thanks to Professor Merrin Macrae and Mr. Vito Lam at the University of Waterloo (Waterloo, Ontario, Canada) for providing me access to the Dionex Ion Chromatography system for the testing of my water samples. Thanks also to Professor Norman Yan (York University – Toronto, Ontario, Canada) and Mr. Andrew Jeziorski (Queen’s University – Kingston, Ontario, Canada) for answering my email questions and providing me with advice and access to their research papers. I would also like to acknowledge and thank Rice Engineering for their generous donation of testing supplies, to Dr. Patricia Gillis from Environment Canada for the loan of test equipment and testing supplies and to Ms. Susan Samuel-Herter for her assistance with the statistical analysis.


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About the Author

Isabella O\’Brien, Canada

Isabella O’Brien attends Westmount Secondary School in Hamilton, ON, Canada. In 2017 she was named one of Canada\’s Top 25 Environmentalists under 25. She won the gold medal & best in category at the 2016 & 2014 Canada-Wide Science Fairs. Isabella is a 2015 Google Science Fair global finalist & is the Ocean Youth Ambassador for the NGO LemonSea.

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