Feasibility of increasing calcium content of drinking tap water following quality regulations to improve calcium intake at population level

Background Calcium intake is below recommendations in several parts of the world. Improving calcium intake has benefits not only for bone health but also helps to prevent pregnancy hypertension disorders. Calcium concentration of tap water is usually low The aim of the present study was to determine the maximum amount of calcium that can be added to tap water while complying with drinking water Argentine regulations. Methods Tap water samples were collected from the Province of Buenos Aires (Argentina). Physicochemical properties and saturation index were measured. Different incremental concentrations of calcium chloride were added to the experimental aliquots. Results Baseline water had a mean calcium concentration of 22.00 ± 2.54 mg/L, water hardness of 89.9 ± 6.4 mg/L CaCO 3, and a saturation index of -1.50 ± 0.11. After the addition of 0.4554 ± 0.0071 g of salt, water hard-ness reached 355.0 ± 7.1 mg/L CaCO 3, a calcium concentration of 140.50 ± 2.12 mg/L, and a saturation index -0.53 ± 0.02. Conclusions This study shows that at laboratory level it is feasible to increase calcium concentration of drinking water by adding calcium chloride while complying with national standards. Calcium concentration of drinking tap water could be evaluated and minimum calcium concentration of tap water regulated so as to improve calcium intake in populations with low calcium intake.


Introduction
Calcium is one of the inorganic elements in the human body involved in many vital functions, influencing many extracellular and intracellular processes [1][2][3] .Calcium is essential for development and growth.In addition, optimal calcium intake is necessary for bone health at all stages of life 2,4,5 .Furthermore, appropriate calcium intake has shown many other health benefits such as those related to reduction of hypertensive disorders of pregnancy 3,[6][7][8] .Despite the benefits of calcium intake, intake values are well below recommendation in many parts of the world 3,[9][10][11] .Thus, it is imperative to consider other sources of calcium that may contribute to calcium recommendations.
Calcium intake is usually associated with dairy products intake; however, the impact that these foods have on total calcium intake depends on food consumption patterns 12,13 .Drinking water naturally contains calcium, however, the contribution of water minerals to total intake isseldom considering calcium bioavailability is comparable to calcium from dairy products 2,14,15 .
Studies have reported that calcium-rich water significantly contributes to daily calcium intake in adults [16][17][18][19][20] .Calcium in water may be an efficient way to provide and improve calcium intake in countries that report low calcium intake 15 .There are studies of the use of water as a fortification vehicle for different micronutrients such as fluoride [21][22][23][24] , iodine 25 , iron 26 , and ascorbic acid 27 .The literature showed beneficial effects of calcium-rich water on biochemical parameters of bone metabolism 28,29 .Moreover, some studies have shown that cooking with high calcium water could avoid calcium loss from foods and increase calcium concentration of cooked foods 30,31 .A simulation analysis performed in 62 Low-and Middle-Income Countries (LMICs) revealed that if calcium concentration in tap and bottled water was increased at levels of 100 mg/L and 400 mg/L, respectively, an extra nine countries could have calcium availability at a level that would provide enough calcium to satisfy the needs of their populations 32,33 .
Calcium concentrations in water vary significantly according to the water source and geographic areas.For instance, calcium concentration of tap water varies from 1 to 135 mg/L across the US 16 .The average calcium concentration of tap water is 50.6 ± 29.4 mg/L that is similar to most bottled spring waters 16 .On the other hand, bottled mineral waters have much higher calcium concentrations, an average of 208 mg/L and could reach 500 mg/L 16 .
In a previous study, using a national representative dietary intake data from Argentina (Second Health and Nutrition National Survey, ENNyS 2), we reported a high prevalence of low calcium intake, that reaches 88% of adult population 13 .With this data we simulated increasing tap and bottled water concentrations and showed its contribution to recommended calcium intake 33 .In a triangle test, we found that the sensory detection threshold of bottled water with added calcium salts allows the increase of calcium concentration of water up to a level of 500 mg of calcium/L 34 .However, bottled water intake in Argentina seems to be low; thus, efforts to improve calcium content of other sources, such as tap water, is relevant.The calcium concentration of tap water in some cities of Argentina is low with an average calcium concentration between 12.9-19.0mg/L depending on the region, and increasing this concentration could be explored to help an improvement in calcium intake 32 .
There is a lack of knowledge about the maximum calcium level that could be added to tap water while complying with local regulations.Therefore, the objective of this study is to determine the maximum calcium that can be added to tap water complying with drinking water local regulations.

Methods
This study was performed at a laboratory level using tap water directly obtained from the treatment water plant Aguas Bonaerenses S.A (ABSA) Donato Gerardi.This water plant has a capacity of 15,000 m3/h and supplies tap water to around 800,000 inhabitants of the Buenos Aires Province, Argentina.This conventional water plant uses surface water from Rio de La Plata that is treated with a combined process of coagulation, flocculation, sedimentation, filtration, and disinfection.It treats water in a central location and then distributes water via distribution networks.

Physicochemical characterization of tap water
Drinking water quality in Argentina is regulated by the Food Codex of Argentina (Código Alimentario Argentino, CAA).This local regulation defines the maximum level of physicochemical parameters, inorganic, organic, biological, and radioactive substances for drinking water 35 .
To characterise the tap water we collected from the treatment water plant a duplicate water sample of 1.5 L daily over 15 consecutive days.The purpose of this sampling strategy was to collect a representative sample following the general recommended collection of samples for water analysis by American Public Health Association (APHA) 36 .Samples were taken from existing sampling locations at the final stage in the treatment plant where the variation in the parameters is expected to be minimal.Samples were obtained following

Amendments from Version 1
We have reviewed all the manuscript.In this new version, we have improved the introduction section adding a new suitable background.Then, we modified the method section, in particular, the description of sample water preparation.Also, we have improved the discussion section referencing previous studies following the reviewer's suggestions.Finally, we have modified the conclusion section to a better key results summary.
Any further responses from the reviewers can be found at the end of the article standardised methodologies and physicochemical parameters of each water sample were assessed following analytical standardized methodologies 36 .Calcium concentration was measured by an atomic absorption spectrometer at 422.7 nm (Varian AA 240 FS) by direct air-acetylene flame method.Turbidity was obtained using a portable turbidimeter WGZ-2A (Shanghai Xinrui Instruments Co., Ltd).Colour was determined by visual comparison method and pH, water temperature (degrees Celsius, °C), electrical conductivity, and total dissolved solids (TDS) were measured with Sper Scientific Water Quality Meter (Model 850081).Total water hardness was assessed according to the titrimetric method with ethylenediaminetetraacetic acid (EDTA).Chloride ion concentration was analysed by titration (Mohr's Method) and total alkalinity, carbonates and bicarbonates were measured by titration method.TDS, pH, and conductivity were measured at the time of sampling while the other parameters were analysed within 24 hours after collection 36 .Corrosive and scaling properties were estimated using the Langelier method.The saturation index provides precipitation or solubility tendency of calcium carbonate that defines if a water sample is over-saturated, saturated, or unsaturated 37,38 .

Preparation of water sample with added calcium
To increase calcium concentration of water we used calcium chloride dihydrate that is a highly soluble inorganic salt (74.5 g per 100 ml at 20 °C) 26 .We obtained the salt from Sigma-Aldrich (Cat#223506, Germany) that meets the analytical specifications of The European Pharmacopoeia (Ph.Eur.), Pharmaceutical Reference Standards (USP), FCC, E509.
We first theoretically estimated the amount of calcium that could be added to water using the results obtained in the water physicochemical characterization and the maximum total tap water hardness allowed by the CAA 35 .With the estimated mean water hardness, we determined the theoretical amount of calcium chloride dihydrate to be added to the water samples to obtain solutions with hardness between 50 ppm up and 400 ppm of calcium carbonate, the maximum total water hardness allowed by the CAA 35 .
We estimated that 4 L of sample water was required to prepare solutions with added calcium and perform duplicate analytic measurements.Of this volume, we took 3.5 L that was required.The 4 L of water were then divided into 14 aliquots of 250 ml each (C0, control duplicated sample) and the rest were used to add calcium chloride (C1 to C6, duplicated at each sample).Afterwards we determined all the physicochemical parameters and using the saturation index, we calculated again the corrosive scaling properties of each sample of water 38 .

Statistical analysis
The data analysis was carried out using SPSS statistical software package version 22.0 (SPSS Inc., USA).

Results
The average physicochemical characterization of tap water samples obtained from the treatment water plant during 15 consecutive days is shown in Table 1.The mean calcium concentration was 22.00 ± 2.54 mg/L, the mean water hardness was 89.9 ± 6.4 mg/L CaCO3, and the saturation index was-1.50± 0.11 (Table 1).
Table 2 shows the different physicochemical parameters of the sample water before and after adding calcium chloride dihydrate.The maximum calcium concentration obtained was 140.50 ± 2.12 mg/L when we added 0.4554±0.0071g of salt.This water had a final water hardness of 355.0 ± 7.1 mg/L CaCO 3 complying with CAA and a saturation index of -0.53 ± 0.02.We also obtained water with a calcium concentration of 67.30 ± 3.18 and a saturation index of -0.80 ± 0.01 when we added 0.1656 ± 0.0023 g of salt.This water had a hardness of 197.0 ± 1.4 mg/L CaCO 3 (Table 2) complying both with CAA.

Discussion
In our research we used tap water obtained from the treatment central water plant to determine the maximum amount of calcium that can be added.This study shows that, at laboratory level, it is feasible to increase the calcium concentration of a drinking tap water that originally had a calcium concentration of around 22 mg/L.This baseline level concentration was improved by adding calcium chloride; the calcium concentration of this tap water could reach 140 mg/L complying with the CAA which is the national tap water regulation in the country 35 .We defined the maximum calcium level to be added as that level which complied with the local regulations of physicochemical parameters.This information would be required as a first step to start thinking in proposing the addition of a minimum standard of calcium concentration in tap water to local regulations.The current knowledge about calcium to be added to tap water comes from simulation studies and fortification of bottled water in other water sources 39,40 .Our research brings to light the maximun calcium that could be added to tap water while complying regulations in Argentina.
Water hardness and TDS increased with the addition of calcium chloride, but still complied with the tap water hardness and other physicochemical parameters regulated by the CAA 35 .
The CAA regulation allows a maximum total drinking water hardness of 400 mg/L, a maximum turbidity of 3 NTU, a maximum TDS of 1500 mg/L, a maximum chloride ion of 350 mg/L Cl, and maximum colour of 5 scale Pt-Co 35 .On the other hand, the maximum level of chloride ion permitted by the Province of Buenos Aires is 250 mg/L, which is lower to the 350 mg/L permitted at national level.This regulation would limit the addition of calcium chloride, allowing increasing the calcium concentration of water from 22 to 100 mg/L 41 .All physicochemical parameters measured before and after adding the salts indicate that the water complies with the water standards regulated by CAA 35 .
Our results were obtained under controlled laboratory conditions to be able to perform all analytical tests and all water samples analysed were collected from the same point in the water treatment plant.The mean baseline water hardness was almost 90 mg/L CaCO 3 indicating that the water is soft and had a general corrosive tendency with a saturation index of -1.5 (Table 1).Corrosive tendency depends on physical and chemical characteristics of the water such as pH, total alkalinity, and hardness.The Langelier method is the widest method to determine whether water tends to precipitate CaCO 3 .Hence, this saturation index is not related directly to corrosion, but to the deposition of a calcium carbonate film or scale 42 .After adding calcium chloride, the Langelier Index was maintained and the water samples were under-saturated.
The baseline water calcium concentration we report in this study is similar to the concentrations we previously reported from different areas of Argentina 20 .Drinking water from water distribution plants tend to have low calcium concentrations as national regulations usually focus on the maximum hardness level to avoid scaling, but not on the minimum hardness level to avoid corrosion 35 .In this way, water that complies with regulatory standards can have very low calcium concentration and be corrosive.Although previous epidemiologic studies have suggested an inverse relationship between water hardness and cardiovascular mortality, mainly determined by concentrations of calcium and magnesium, often drinking water guidelines do not base recommended hardness or TDS on health outcomes [43][44][45][46][47][48] .
According to World Health Organization (WHO) guidelines, calcium ion threshold taste is around 100-300 mg/L, depending on the associated anion; however, consumers could tolerate water hardness more than 500 mg/L 42 .A study using water samples with similar characteristics to the sample used in this study showed that the sensory detection threshold of water with added calcium chloride dihydrate allowed an increase of calcium concentration of water up to a level of 291 ± 73 mg/L 34 .TDS also contributes to palatability of drinking-water, and at TDS levels greater than about 1000 mg/L the water becomes significantly and increasingly unpalatable 42 .
Even though water guidelines usually do not specifically refer to calcium concentration values, drinking-water can be a contributor to calcium intake and could be important for those who are marginal for calcium 42 .In LMICs, including Argentina, the simulated strategy of increasing water with 500 mg of calcium/L showed that the prevalence of low calcium intake in all age groups could decrease without exceeding the recommended upper levels of calcium intake 13 .Also, we have shown that the intake of one litre of drinking water from Argentina could represent on average between 1.2 and 8.0% of the calcium daily values for an adult 20 .
In this study we propose that 140 mg of calcium per liter could be a maximum level; however in a previous study, we simulated adding 100 mg of calcium per liter of tap water considering real water consumption reported in the ENNyS 2.
Using this data, we showed that the addition of 100 mg of calcium per liter of tap water represented a reduction of 11.3% percentage points in the prevalence of low calcium intake (from 91.0% to 79.7%) in women aged 19 to <31 years old and a reduction of 12.9% percentage points (from 89.4% to 76.5%) in women aged 31 to <51 years of age.In men, represented a reduction of 13.3% percentage points in the prevalence of low calcium intake (from 80.6% to 67.3%) in men aged 19 to <31, and a reduction of 15.6% percentage points (from 88.4% to 72.8%) in those aged 19 to <51 years 33 .The reductions found in our simulations could be slightly increased taking into consideration the maximum level of 140 mg of calcium per liter that we assessed in this study 33 .
In this study, we analyzed calcium content of drinking water obtained from a centralized water treatment plant.Although our study was performed with water samples from only one treatment water plant, this plant provides tap water to the second most important agglomerate of Argentina.Our samples were taken at the final stage, before dedicated distribution networks.It is known that water characteristics could vary throughout the supply system.Thus, future research into the feasibility of water with added calcium should consider these potential changes.The inclusion of water quality analysis into distribution systems could apply developed sensor systems 49 .A second approach could be to analyze the optimum stage of the water treatment process to add calcium.For instance, this procedure could include a detailed analysis of the water treatment process besides storage temperature, seasonality changes and pressure.Also, it could analyse characteristics of pipeline materials in the treatment and distribution systems.Furthermore, research into the feasibility of water with added calcium deserves to be considered since different water supplies and different baseline waters.
The chemical composition of drinking water, including calcium concentration, is varied depending on the origin, treatment received, and distribution system 50 .
In addition, we suggest that a possible approach to be implemented as large-scale addition of calcium chloride could be a point-of-use (POU) mineralization unit under the kitchen sink with a separate tap or a separate high calcium water line to the kitchen sink.However, adding calcium chloride after the POU installations or to naturally soft waters in a home presents some technical difficulties that must be resolved.Thus, this approach should be part of an implementation study.
The drinking water quality data is limited in LMICs 51-53 .
Drinking-water quality refers to physicochemical, organoleptic (taste-related), and biological characteristics of water based on standards 42 .Water quality is one of four distinct types of health-based targets defined by WHO to protect human health 42 .Drinking water services coverage has improved in all regions, however inequalities varied widely between LMICs 52 .In Argentina, almost 90% of 31 urban agglomerates, including the area of our study, have accessibility to safe drinking water services 54 , suggesting universal access is still not achieved.Moreover, in almost all LMICs the coverage gap between urban and rural areas can be seen.For instance, in Latin America significant disparities were estimated in coverage of safely managed drinking water between urban (81%) and rural (53 %) areas 52 .National averages often mask significant inequalities in service levels within countries 42,55 .It would be valuable to count with disaggregated information about water composition and types of water supply systems in regions with low calcium intake to improve water quality characteristics to enhance calcium availability.
Calcium concentration of tap water could be increased if water is boiled.This higher concentration would not be affected by tap water regulations if boiling occurs after leaving the pipe system.Boiling water for at least 20 minutes effectively kills or inactivates most protozoa, bacteria, and viruses and minerals such as calcium get more concentrated as the water evaporates 42,56,57 .
Considering the potential negative effects of drinking water with low calcium levels on the cardiovascular system setting a minimum calcium concentration of tap water should be contemplated.

Conclusions
Calcium and water are essential elements for life, and their adequate intake is, therefore, vital for the maintenance of the body's homeostasis.Calcium deficiency is high in many LMICs and multiple strategies should be developed in conjunction, to aid these populations attain an adequate calcium intake.Also, adequate dietary calcium intake is necessary for the maintenance of bone health, and calcium-rich drinking tap waters in accordance with national standards can represent a valid strategy to reach this purpose.
The current research results could be a valuable and feasible resource to cast light on the discussion on standards for minimum calcium concentration in drinking-tap water.The research helps our scientific community to know about the maximum calcium level that could be added to tap water while complying with local regulations.Considering that calcium in water has a good bioavailability and that water is universally consumed, water with added calcium could help improve calcium intake in our country.Further assessments of the amount of calcium that could be added could be performed in each area of the country where it is demonstrated that calcium intake and calcium concentration of water are low as this could help improve calcium intake and prevent health risks.Calcium accounts for 1 to 2 percent of adult human body weight.Over 99 percent of total body calcium is found in teeth and bones.Say that is most abundant elements makes no sense.Rephrase it.

METHODS:
4L dived in 14 aliquots makes 3.5L, can you clearly specify where is the rest?It is unclear.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Food fortification, mineral ingredients, calcium fortification, I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Response: Thanks for your kindly comments and suggestions.
We have added the following paragraph: Discussion: In our research we used tap water obtained from the treatment central water plant to determine the maximum amount of calcium that can be added.This study shows that, at laboratory level, it is feasible to increase the calcium concentration of a drinking tap water that originally had a calcium concentration of around 22 mg/L.This baseline level concentration was improved by adding calcium chloride; the calcium concentration of this tap water could reach 140 mg/L complying with the CAA which is the national tap water regulation in the country 35 .We defined the maximum calcium level to be added as that level which complied with the local regulations of physicochemical parameters.This information would be required as a first step to start thinking in proposing the addition of a minimum standard of calcium concentration in tap water to local regulations.The current knowledge about calcium to be added to tap water comes from simulation studies and fortification of bottled water in other water sources 39,40 .However, little is known about calcium added to tap drinking water.Thus, this research brings to light the real calcium added to tap water complying Argentine regulations.
We have modified the conclusion section: Conclusions: Calcium and water are essential elements for life, and their adequate intake is, therefore, vital for the maintenance of the body's homeostasis.Calcium deficiency is high in many LMICs and multiple strategies should be developed in conjunction, to aid these populations attain an adequate calcium intake.Also, adequate dietary calcium intake is necessary for the maintenance of bone health, and calcium-rich drinking tap waters in accordance with national standards can represent a valid strategy to reach this purpose.The current research results could be a valuable and feasible resource to cast light on the discussion on standards for minimum calcium concentration in drinking-tap water.The research helps our scientific community to know about the maximum calcium level that could be added to tap water while complying with local regulations.Considering that calcium in water has a good bioavailability and that water is universally consumed, water with added calcium could help improve calcium intake in our country.Further assessments of the amount of calcium that could be added could be performed in each area of the country where it is demonstrated that calcium intake and calcium concentration of water are low as this could help improve calcium intake and prevent health risks.
Moreover, there is a lack on how fortification can be implemented at large scale with sustainable source of calcium chloride.

Response:
Our research shows that at the laboratory level, it is feasible to increase calcium concentration in drinking water while complying with national standards, as the first step to proposing a large-scale study, adding calcium chloride.
We discuss about this topic in the following paragraph: Thus, future research into the feasibility of water with added calcium should consider these potential changes.The inclusion of water quality analysis into distribution systems could apply developed sensor systems 49 .A second approach could be to analyze the optimum stage of the water treatment process to add calcium.For instance, this procedure could include a detailed analysis of the water treatment process besides storage temperature, seasonality changes and pressure.Also, it could analyse characteristics of pipeline materials in the treatment and distribution systems.Furthermore, research into the feasibility of water with added calcium deserves to be considered since different water supplies and different baseline waters.
We have now added more options that require research.
We have added the following paragraph to the discussion section: Discussion: In addition, we suggest that a possible approach to be implemented as largescale addition of calcium chloride could be a point-of-use (POU) mineralization unit under the kitchen sink with a separate tap or a separate high calcium water line to the kitchen sink.However, adding calcium chloride after the POU installations or to naturally soft waters in a home presents some technical difficulties that must be resolved.Thus, this approach should be part of an implementation study.
Besides, it would be interesting to investigate the available calcium after heat-treatment; as heat processing of water is mandatory in area where water are not microbiologically pure.

Response:
For this study we used water from one treatment plant that supplies tap water to around 800,000 inhabitants of the Buenos Aires Province.Argentina is highly urbanized; 28 water treatment plants provide tap water to other districts of the Buenos Aires Province and three surface water treatment plants supply drinking tap water to more than 11 million inhabitants in Greater Buenos Aires, mostly from surface water.Thus, most homes in Buenos Aires have tap drinking water.
We have added the following paragraph in the discussion section in red color: Calcium concentration of tap water could be increased if water is boiled.This higher concentration would not be affected by tap water regulations if boiling occurs after leaving the pipe system.Boiling water for at least 20 minutes effectively kills or inactivates most protozoa, bacteria, and viruses and minerals such as calcium get more concentrated as the water evaporates 42, 56,57 .

Background:
A full stop is missing between 'low The' Response: we have added a full stop (red color).
Local regulation of where?It is very broad and too generic Response: we have added the word Argentine in red color; the final version of the background is: Calcium intake is below recommendations in several parts of the world.Improving calcium intake has benefits not only for bone health but also helps to prevent pregnancy hypertension disorders.Calcium concentration of tap water is usually low.The aim of the present study was to determine the maximum amount of calcium that can be added to tap water while complying with drinking water Argentine regulations.

INTRODUCTION:
Calcium accounts for 1 to 2 percent of adult human body weight.Over 99 percent of total body calcium is found in teeth and bones.Say that is most abundant elements makes no sense.Rephrase it.
Response: We have changed the sentence as follows: Calcium is one of the inorganic elements in the human body involved in many vital functions, influencing many extracellular and intracellular processes.

METHODS:
4L dived in 14 aliquots makes 3.5L, can you clearly specify where is the rest?It is unclear.
Response: It is known that during laboratory experiments, it is well established that the sample volume collected should be higher than the calculated volume.
In particular, to prepare solutions with added calcium and perform duplicate analytical measurements, we estimated that 3.5 liters of water were theoretically needed.We planned to take the water sample with a plus volume of 0.5 L to ensure the volume required for preparing the solutions in the laboratory.In this way, we obtained a 4 L of volume sample.We changed the paragraph as follows: Methods: We estimated that 4 L of sample water was required to prepare solutions with added calcium and perform duplicate analytic measurements.Of this volume, we took 3.5 L of water that was then divided into 14 aliquots of 250 ml each (C0, control duplicated sample) and the rest were used to add calcium chloride (C1 to C6, duplicated at each sample).Afterwards, we determined all the physicochemical parameters and using the saturation index, we calculated again the corrosive scaling properties of each sample of water 38 .
We have modified the reference section.
Competing Interests: No competing interests were disclosed.

Isidro Vitoria
Hospital Universitari i Politècnic la Fe, Valencia, Spain The calcium in water has a bioavailability similar to the calcium in dairy products.The calcium intake of the population often does not reach the nutritional recommendations, especially at the present time when there is a lower consumption of dairy products.I think this article is very opportune in proposing calcium in drinking water as an adequate supplementary source of calcium.The study proposes to enrich the calcium content of drinking water in Buenos Aires, with a low hardness.The authors show that they can reach 140 mg/l of calcium and that these figures do not pose technical problems of taste or precipitation and saturation.The limitation of added calcium in relation to the water distribution system is also well stated.I think it would gain a lot if the authors would add a table indicating the percentage of the recommended calcium intake by age that could be achieved with waters of 100 and 150 mg/l of calcium.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Inborn errors of metabolism I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Reviewer Report 14
May 2024 https://doi.org/10.21956/gatesopenres.16527.r36153© 2024 Vitoria I.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Table 2 . Physicochemical parameters of water samples before (C0) and after adding calcium chloride dihydrate in increased amounts (C1- C6).
Data are expressed in mean ± SD.NTU: nephelometric turbidity units

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