The Calcium Carbonate Content of White to Brown Eggshells

ABSTRACTDuring my experiment, I have been investigating my research question: To what extent do white and brown eggshells differ in respect to percentage by mass of calcium carbonate content?Calcium carbonate is a substance found in the eggshells giving them hardness and strength. It is essential to the commercial egg industry that the biggest possible amount of eggs reaches the market. Hence, as calcium carbonate reacts steadily with hydrochloric acid to produce carbon dioxide as well as two other substances, I investigated which type of an eggshell contains more calcium carbonate. This reaction cannot be used directly to titrate the calcium carbonate because it is very slow when the reaction is close to the endpoint. Hence, I carried out back titration by adding an excess of hydrochloric acid to dissolve all of the calcium carbonate and then titrating the remaining H3O+ with sodium hydroxide solution. This way I could determine the amount of acid which has reacted with the calcium carbonate, hence, the calcium carbonate content of a sample of an eggshell.

The results of the investigation proved my hypothesis that brown eggshells contain more calcium carbonate than white eggshells do. This trend comes from the difference in the hens dietary. The average mass percent of calcium carbonate in brown eggshell is 91% while in the white eggshell it is 87.6 %. The difference between these means and the theoretical value of 95% might have come from such methodological flaws as the calcium carbonate that failed to dissolve or the remnants of the organic inner membrane of an egg. Further suggestions for the experiment might include broader eggshells sampling and investigation into more features of an eggshell, not only the mineral content.

1 INTRODUCTIONCalcium carbonate, CaCO3, is found in the nature giving hardness and strength to things such as seashells, rocks, and eggshells . Much information has been learned about eggshell quality during the past fifty years. During this period of time, the genetics of the chicken diets, house design and management practices have changed dramatically. In the future it is very likely that additional changes will have to be made by the commercial egg industry. In my investigation I am particularly focused in determining the amount of calcium carbonate present in white (chicken) eggshells and brown eggshells. The reason for choosing this topic is that no matter what changes occur, the eggshell needs to be as strong as possible to maximize the number of eggs reaching the market, thus, this topic might be treated as a really relevant issue nowadays.

As hard as calcium carbonate substance is , it reacts readily with hydrochloric acid to yield carbon dioxide gas (and two other products). During the experiment I will investigate the reaction between 1 M hydrochloric acid solution and calcium carbonate in eggshells in order to compare the calcium carbonate composition of white to brown eggshells.

RESEARCH QUESTIONTo what extent do white and brown eggshells differ in respect to percentage by mass of calcium carbonate content?2 BACKGROUND INFORMATION2.1 The concepts of eggshell qualityA good quality eggshell should contain, on average, 2.2 grams of calcium in the form of calcium carbonate. Approximately 95% of a dry eggshell is calcium carbonate and has a typical mass of 5.5 grams, although these values can differ depending on sources. The remaining mass is composed largely of phosphorus and magnesium, and trace amounts of sodium, potassium, zinc, manganese, iron, and copper.

If the calcium from the shell is removed, the organic matrix material is left behind. This organic material has calcium binding properties and its organization during shell formation influences the strength of the shell. The organic material must be deposited so that the size and organization of the crystalline components (calcium carbonate mostly) are ideal, thus leading to a strong shell. The majority of the true shell is composed of long columns of calcium carbonate. There are other zones that are involved in the self-organization giving the eggshell its strength properties. Thus, shell thickness is the main factor but not the only factor that determines strength. Presently, dietary manipulation is the primary means of trying to correct eggshell quality problems. However, the shell to organic membrane relationship is also critical to good shell quality and should be considered.

2.2 Structure of an eggshellFig. 1: Structure of the egg shellThe small amount of organic matter mostly consists of matrix proteins (mixture of proteins and polysaccharides rich in sulphated molecules) and shell pigment. The matrix proteins are critically important in determining the egg shell structure and serves as foundation for the deposition of calcium carbonate.

There are about 8000 microscopic pores on the shell. The outer surface of the shell itself consists of a mucous coating (cuticle) which is deposited on the egg just prior to the lay. This proteinous covering helps to protect the interior content of the egg from bacterial penetration through the shell.

2.3 How calcium influences shell quality?Both excess and deficiency of calcium will negatively affect the shell quality. An egg contains almost 2 grams of calcium; hence, an average of 4 grams of calcium intake per day is required by a layer to maintain a good shell quality, since only 50 - 60% of dietary calcium is actually used in shell formation. Calcium requirement of a laying hen is 4 - 6 times that of a non-laying hen. The egg enters the shell-gland region of the oviduct (the uterus) 19 hours prior to oviposition, and the shell does not store calcium ions to attach on protein matrix. During the last 15 hours of shell formation, calcium movement across the shell gland reaches a rate of 100-150 mg/hr. This process draws calcium from two sources: diet and bone. Normal blood calcium level is about 20 - 30 mg/dl with a normal layer ration of 3.56% calcium or higher, while layers on a 2% calcium diet, 30- 40% of the calcium is derived from bone. It is therefore important to have pullets, prior to lay, on a high level of calcium to store it on body. Intestinal absorption of calcium in the diet is about 40% when the shell gland is inactive, but reaches 72% when active. This time closely coincides with late afternoon or the dark hours for the layer. Having higher calcium levels in the gut during this time is important to ensure calcium is being taken from the diet and not bone. Large particle sizes of calcium sources allow calcium to be metered throughout this time. In growers, most importantly, high calcium levels during the growth period will interfere with the proper development of the parathyroid gland by increasing gut pH, which will decrease absorption. The damage to the parathyroid would be permanent and would affect the bird 's laying cycle afterwards.

2.4 Differences in white and brown eggshellsThe color of the eggs is nothing more than a result of a different breed; and the quality, nutritional value, and taste are identical between white and brown eggs, though two notable differences are size and price. Brown eggs are usually larger and slightly more expensive. The reason for the price increase is because brown eggs come from larger hens, which need to be fed more food daily. With a larger intake of calcium each day, one might expect the produced egg to have higher calcium carbonate content. However, large sized eggs will usually break more easily than small ones. The main reason for this is that the hen is genetically capable of placing only a finite amount of calcium in the shell. As the hen ages and the eggs get bigger a similar amount of calcium has to be spread over a larger surface. Therefore, controlling the rate of egg weight change can influence eggshell quality as the hen ages. A brown eggshell has increased tendency to break, when compared to white, is often attributed to this thinning out of calcium during deposition.

2.5 Eggshell quality and economicsPoor egg shell quality is a huge hidden cost to the egg producer. Estimates are that more than 10% of eggs produced in the hen house are uncollectible or break before intended use. The first 2-5 percent is lost simply due to form which may be shell less, cracked or broken to the extent that they are not suitable for collection. Another 3-8 percent is lost during collection, moving through the belts, cleaning, packing and transportation to the end user. Because the first 2-5% loss is due to uncollectible eggs, most egg producers often estimate their egg loss due to poor shell quality at only this percentage, which is most likely an underestimation. Even a 5 percent loss could be as much as about 2.7 million/year for 100 thousand layer house. The economic losses for the breeders will be even more due to reduced hatchability and chick livability. Therefore, every effort must be directed towards improving shell quality and reducing egg breakage.

3 METHOD¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬3.1 DesignThe major component of eggshells is calcium carbonate (CaCO3). The analysis of quality of the eggshells will be done volumetrically by using a characteristic reaction of carbonate compounds, namely their reaction with hydrochloric acid. Calcium carbonate (limestone) is very insoluble in pure water but will readily dissolve in acid according to the reaction:2HCl(aq) + CaCO3(s) → CaCl2(aq) + CO2(g) + H2O (l)This reaction cannot be used directly to titrate the calcium carbonate because it is very slow when the reaction is close to the endpoint. Hence, a procedure, called a back titration must be carried out. This is achieved by adding an excess of hydrochloric acid to dissolve all of the calcium carbonate and then titrating the remaining H3O+ with sodium hydroxide solution to determine the amount of acid which has not reacted with the calcium carbonate. The difference between amounts of the acid (HCl) initially added and the amount left over after the reaction is equal to the amount used by the calcium carbonate. The reaction used to determine the leftover acid is:HCl(aq) + NaOH(aq) → H2O (l) + NaCl(aq)Aim: to determine the mass percent of calcium carbonate (CaCO3) in different types of the eggshells (white and brown).

Hypothesis: the amount of calcium carbonate in the brown eggshell should be bigger due to different hen feeding diet.

Variables: the mass percent of calcium carbonate (dependent variable) will be altered by changing the type of an eggshell (white and brown; independent variable).

3.2 Materials and equipment2 different eggs(I.e. 1-white & 1-brown)Distilled waterFilter paper40 mL of 1 M HCl0.100 M NaOHEthanolPhenolphthaleinMortar & pestleFunnelBurette ± 0.05 mLStandard lab scales ± 0.01 gPipette (20 mL) ± 0.1 mLVolumetric flasks (100 mL) ± 0.1 mLBeakersHot platesThermometersPaper towelOvenStop-watch3.3 ProcedureOrigins of the method have come from several textbooks ; however, the method was altered in order to make it more applicable in the school laboratory.

1.The white egg is broken into a beaker and after water is added to the egg, contents are stirred. Then, the drain is poured down using filter paper.

2.The eggshell is washed with distilled water and all the protein membranes from the inside of the shell are peeled off.

3.Shell is dried with a paper towel and put into a labeled beaker in the oven at about 110°C for 20 minutes to obtain the result than any unnecessary water is lost, and, in this way, trigger a more precise outcome of an experiment.

4.Shell is grind to a fine powder in a mortar.

5.The amount of 0.5 g of dried shell is accurately weighted into labeled 100 mL volumetric flask.

6.Several drops of ethanol are added to the flask. This acts as a wetting agent and helps the hydrochloric acid dissolve the calcium carbonate.

7.20.00 mL of 1.0 M hydrochloric acid solution is put into a flask by using a pipette. The flask is swirled to wet the entire solid. Any excess of hydrochloric acid should be disposed of in the sink by diluting with distilled water.

8.The solution is heated in the flask until it begins to boil and then is allowed to cool. The wall of the flask is rinsed with the water from the distilled wash bottle.

9.3-4 drops of phenolphthalein (a.k.a PhPL) is added to the flask.

10.Using filter paper, solid particles are removed from the solution.

11.The contents of the flask are mixed with the distilled water so that the final volume of a solution is 100 mL.

12.The 100 mL solution is divided into 10 equal solutions into 10 different beakers, each containing 10 mL of solution.

13.By using a funnel, a clean burette is partly filled with 0.100 M sodium hydroxide solution to rinse it. The burette is emptied into the sink. Then, the burette is filled with the same 0.100 M sodium hydroxide solution. Some solution is run out to remove all bubbles from the tip. The initial volume is read and recorded to ± 0.05 mL.

14.One sample is titrated to the first persistent pink color. When it is close to the endpoint the color will fade slowly. The remaining sodium hydroxide is dropped until the color remains for at least 30 seconds. The final volume is read and recorded to ± 0.05 mL (making uncertainty of burette recordings ± 0.05 x 2 = ± 0.1 mL)15.The titration is repeated for the other nine samples.

16.The whole experiment is repeated with a brown eggshell.

17.The percentage of calcium carbonate in each kind of eggshell is calculated.

3.4 Safety precautionsReactants, risk, and safety data:1.Calcium carbonate. Dust may cause irritation to eyes, respiratory system, and skin. In case of contact with eyes, they must be rinsed immediately with plenty of water and medical advice must be sought.

2.Hydrochloric acid. Extremely corrosive; inhalation of vapor can cause serious injury; ingestion may be fatal. Liquid can cause severe damage to skin and eyes.

3.Sodium hydroxide. Very corrosive; causes severe burns; may cause serious permanent eye damage; very harmful by ingestion; harmful by skin contact or by inhalation of dust. Suitable gloves must be worn (Neoprene or PVC).

Products, risk, and safety data:4.Calcium chloride (anhydrous). Irritating to eyes. Dust must not be inhaled and contact with skin must be avoided.

5.Carbon dioxide. In high concentration acts as an asphyxiant; respiratory stimulant. If working with carbon dioxide in confined spaces where the concentration of gas may build up, adequate ventilation must be ensured.

6.Sodium chloride. May cause skin, eye or respiratory irritation. Not believed to present a significant hazard to health.

Others, risk, and safety data:7.Ethyl alcohol. Causes skin, eye and respiratory system irritation. Ingestion can cause nausea, vomiting and inebriation; chronic use can cause serious liver damage. Highly flammable. Container must be kept tightly closed.

8.Phenolphthalein. Irritating to eyes, respiratory system and skin.

4 RESULTS4.1 Raw dataTable 1 Volume of 0.1M sodium hydroxide titrated in respect to brown eggshell in 10 trials.

TrialsInitial V(0.1M NaOH) /mL ±0.05Final V(0.1M NaOH) /mL ±0.05V (0.1M NaOH) titrated /mL ± 0.101st0.0010.9010.902nd0.0011.5011.503rd0.0010.6510.654th0.0010.5010.505th0.0010.9510.956th0.0011.0511.057th0.0011.3011.308th0.0010.9010.909th0.0010.8010.8010th0.0010.4010.40Average 10.90Standard deviation 0.34 Table 2 Volume of 0.1M sodium hydroxide titrated in respect to white eggshell in 10 trials.

TrialsInitial V(0.1M NaOH) /mL ±0.05Final V(0.1M NaOH) /mL ±0.05V (0.1M NaOH) titrated /mL ± 0.101st0.0011.4011.402nd0.0011.5511.553rd0.0011.4511.454th0.0011.2011.205th0.0010.9510.956th0.0011.3011.307th0.0011.0511.058th0.0011.7011.709th0.0011.5011.5010th0.0011.1511.15Average 11.33Standard deviation 0.244.2 Data AnalysisFrom tables 1 and 2 it could be clearly seen that average amount of 0.1M sodium hydroxide used in back titration is 10.90 mL (± 0.10) to brown eggshell and 11.33 mL (± 0.10) to white eggshell.

In order to obtain the percentage of calcium carbonate in the different kind of eggshells, following calculations have to be made:1.According to the equation HCl(aq) + NaOH(aq) → H2O (l) + NaCl(aq)one mole of hydrochloric acid reacts with one mole of sodium hydroxide, so, we have to calculate the amount of moles (formula c=n/V) of sodium hydroxide used in the back titration and it will be equal to the moles of hydrochloric acid that reacted in the back titration:Brown eggshell:V(NaOH) titrated = 10.90 mL = 1.09 x 10-2 L;c(NaOH) = 0.1 M;n(NaOH) = c x V = 0.1 x 1.09 x 10-2 = 1.09 x 10-3 mol;→n(HCl) reacted with NaOH = 1.09 x 10-3 mol;White eggshell:V(NaOH) titrated = 11.33 mL = 1.133 x 10-2 L;c(NaOH) = 0.1 M;n(NaOH) = c x V = 0.1 x 1.133 x 10-2 = 1.133 x 10-3 mol;→n(HCl) reacted with NaOH = 1.133 x 10-3 mol;2.If we want to determine how many moles of hydrochloric acid reacted with the calcium carbonate in the eggshell, first we have to find out how many moles of hydrochloric acid were put onto the eggshells and then subtract the moles from the [1] part from these initial moles.

Initial moles of hydrochloric acid:V(HCl) poured = 20.00 mL = 0.02 L;c(HCl) = 1 M;n(HCl) = c x V = 1 x 0.02 = 0.02 mol;However, solution was divided into 10 equal parts, so the amount of moles of hydrochloric acid in each of the beakers is ten times smaller than 0.02, which is then 2 x 10-3 mol.

Hydrochloric acid reacted with the calcium carbonate in the brown eggshell:n(HCl) initial n(HCl) reacted with NaOH = 2 x 10-3 1.09 x 10-3 = 9.1 x 10-4 mol;Hydrochloric acid reacted with the calcium carbonate in the white eggshell:n(HCl) initial n(HCl) reacted with NaOH = 2 x 10-3 1.133 x 10-3 = 8.67 x 10-4 mol;3.As we already have the moles of hydrochloric acid, then we can calculate the moles of calcium carbonate reacted according to the equation:2HCl(aq) + CaCO3(s) → CaCl(aq) + CO2(g) + H2O (l)As 2 moles of hydrochloric acid react to 1 mole of calcium carbonate, the moles of calcium carbonate could be determined by dividing hydrochloric acid moles by 2:Brown eggshell:n(CaCO3) reacted with HCl = 9.1 x 10-4 / 2 = 4.55 x 10-4 mol;White eggshell:n(CaCO3) reacted with HCl = 8.67 x 10-4 / 2 = 4.335 x 10-4 mol;4.After that, mass of the calcium carbonate content of 0.5 g crushed eggshell sample could be calculated according to the formula m = n x M, when M(CaCO3) = 100 g mol-1. However, at first we can determine only the mass of the calcium carbonate in the 0.05 g eggshell sample, as 0.5 g sample was divided into 10 beakers equally.

Brown eggshell:m(CaCO3) in 0.05 g sample = 4.55 x 10-4 x 100 = 4.55 x 10-2 g;m(CaCO3) in 0.5 g sample = 4.55 x 10-2 x 10 = 4.55 x 10-1 g;White eggshell:m(CaCO3) in 0.05 g sample = 4.335 x 10-4 x 100 = 4.335 x 10-2 g;m(CaCO3) in 0.5 g sample = 4.335 x 10-2 x 10 = 4.335 x 10-1 g;5.Finally, if we want to determine the mass of the calcium carbonate in the sample of 100 grams we have to multiply the mass of the calcium carbonate of 0.5 g sample by 200 (as 100 is 200 times bigger than 0.5). Also, the outcome would be equal to percentage calcium carbonate content.

Brown eggshell:m(CaCO3) in 100 g sample = 4.55 x 10-1 x 200 = 91 g = 91 %;White eggshell:m(CaCO3) in 100 g sample = 4.335 x 10-1 x 200 = 86.7 g = 86.7 %;4.3 Percentage uncertaintiesThe equipment with uncertainties is:Burette ± 0.05 mLStandard lab scales ± 0.01 gPipette (20 mL) ± 0.10 mLVolumetric flasks (100 mL) ± 0.10 mLUncertainty of V(NaOH) with brown eggshell = (0.1 x 100) / 10.9 = 0.92 %;Uncertainty of V(NaOH) with white eggshell = (0.1 x 100) / 11.33 = 0.88 %;Uncertainty of m(eggshell) = (0.01 x 100) / 0.5 = 2 %;Uncertainty of V(HCl) = (0.1 x 100) / 20 = 0.5 %;Uncertainty of solution = (0.1 x 100) / 100 = 0.1 %;Overall uncertainty of m(CaCO3) in brown eggshell = 0.92 + 2 + 0.5 + 0.1 = 3.52 %;Overall uncertainty of m(CaCO3) in white eggshell = 0.88 + 2 + 0.5 + 0.1 = 3.52 %;4.4 Percentage yield calculationsPercentage yield is calculated according to the formula:Percentage yield = (Experimental yield / Theoretical yield) x 100Theoretical value of the percentage percent of calcium carbonate in a dry eggshell is 95%.

Hence, percentage yield in respect to brown eggshell is: yield = (91 / 95) x 100 = 95.8 %;Percentage yield in respect to white eggshell is: yield = (86.7 / 95) x 100 = 91.3 %;4.5 Final results of an investigationBrown eggshell:% of CaCO3 in 100 g sample = 91.0 ± 3.52 %;White eggshell:% of (CaCO3) in 100 g sample = 86.7 ± 3.48 %;5 CONCLUSIONIn this part I am going to discuss the results that are summarized in the charts 1 and 2 of the previous chapter. Raw data reveals that bigger amount of sodium hydroxide (11.33 mL) was used to titrate hydrochloric acid that was left from reaction with calcium carbonate in the white eggshell than in the case of brown eggshell (10.90 mL). The standard deviation of ten trials with white eggshells is lessen (SD = 0.24) than with brown eggshells (SD = 0.34), which could mean that the final result of white eggshell calcium carbonate content is also more reliable.

After analysis of the raw data, it turned out that average mass percent of calcium carbonate in brown eggshell is 91.0 ± 3.52 % while in the white eggshell it is 87.6 ± 3.48 %. If we compare these values to the theoretical value of 95%, there is a systematic error in the results of the experiment that will be considered in the evaluation part.

In general, I might say that my experiment proved my hypothesis: the calcium carbonate content of brown eggshell is slightly bigger than of white eggshell. This, therefore, states that it is harder to break brown eggs shell as calcium carbonate is the source of hardness and strength. Though precise statistics are not available, the economic loss due to poor eggshell quality is estimated to be about six billion a year. Hence, calcium carbonate content of an eggshell is a very important factor that should concern egg industry as it is more likely that more brown eggs not white eggs will reach the market unbroken.

6 EVALUATIONThe percentage yield for brown eggshells was found to be 95.8 % and for white eggshells is 91.3 %. There may be some logical explanations for such an error, thus I will try to discuss them in the evaluation part of my investigation.

6.1 UncertaintiesThe uncertainties of tools and measurements were taken into the account; it made finally ± 3.52 % error for brown eggshell and 3.48 % error for white eggshell. So, the usage of the equipment is one of the sources of obtaining reliable results. However, as these were ten trials for each eggshell, these uncertainties shouldnt be considered as the main error source.

Bigger standard deviation of brown eggshells (SD = 0.34) might have arrived from the fact that brown eggshells did not solve that easily in the hydrochloric acid as the white eggshells did (SD = 0.24). This might be due to brown eggshells are harder at the origins, and it was harder to crush them into the fine powder than white eggshells.

6.2 MethodologySources of error due to the methodology of the experiment might suggest that some of organic egg inner membrane may still have remained on the shell. It was impossible to peel every piece of it very clearly. Any membrane remnants would have reacted with the sodium hydroxide titrant; therefore, not all of the titrant would have reacted directly with the hydrochloric acid. One would have to consider that the volumes of the titrant measured would also include the amount of titrant reacted with the membranes. To avoid this problem, more analytical techniques of peeling of the membranes would help.

Another source of error might be that not all calcium carbonate has dissolved in hydrochloric acid. It is important that the entire ground eggshell dissolves because it contains the calcium carbonate that is being analyzed in the experiment. The reaction between the calcium carbonate and the hydrochloric acid might not have been completed if the flasks were removed from hotplates too early, since the higher temperature is beneficial in increasing the rate of said reaction. If that is the case, there would have been a greater volume of hydrochloric acid remaining in the flasks before the titrations. Therefore, the calculated mass percent calcium carbonate in the eggshell would be lower than it would have been had all of the calcium carbonate dissolved.

What is more, factor that might increase error could be that the eggshell contains also other chemical substances (phosphorus 0.3%; magnesium 0.2%; sodium, potassium, manganese, iron, copper traces; organic matter <2%) that could also have reacted with the hydrochloric acid, and, hence, alter the outcome results.

6.3 Reliability of sourcesThe books are always more reliable sources of information than internet. However, I used books only for methodological issues and websites for journals and other texts about investigations into the eggshells. Everything that I found about my topic in the internet articles shouldnt be taken as a bare truth; nevertheless, some of them are treated as a primary sources and, thus, are quite reliable.

6.4 Further suggestionsWhile analyzing my experiment results I thought of some improvements that I would embody if I replicate the experiment once more. Firstly, I would take not only white and brown eggs shells, but also other animals eggs (such as ostrich and quail). Secondly, I would also try to determine the difference of calcium carbonate in the content of eggshells in respect to whether a hen was living in the wild (country-side) or in the industry farm. Thirdly, I would also try to apply some other methods to measure shell quality . On farm methods and sophisticated equipments are available for accessing shell quality parameters. The egg shell strength is the main, but not the only factor that determines shell quality. Egg shell quality can be measured as11: egg size and visual shell defects, specific gravity, shell color, shell breaking strength, percentage shell ((shell weight x 100) / egg weight), shell thickness (mm), ultra structure of the shell. The specific gravity of an object equals the weight of its volume relative to the weight of an equal volume of water, when both are at the same temperature. Since the specific gravity of a shell is more than two times higher than the other parts of the egg, the percentage of the shell has major influence on the specific gravity of whole egg. As the amount of shell increases, the specific gravity of the egg increases. Egg specific gravity, therefore, is a good indicator of percentage shell and shell quality.

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Bibliography: ¬¬¬¬Brewer, Warren B. Eggshell Titration;URL: http://chem.lapeer.org/Chem1Docs/EggshellTitration.html; [Accessed: April 12, 2009]Butcher, G. D. & Miles, R. D. Concepts of Eggshell Quality;URL: http://www.afn.org/poultry/flkman4.htm; [Accessed: April 12, 2009]Gupta, Lokesh Dr. How to improve the eggshell quality;URL: http://www.thepoultrysite.com/articles/1004/how-to-improve-shell-quality;[Accessed: July 17, 2009]Hunton, Peter A research on eggshell structure and quality: A historical overview;Brazilian Journal on Poultry Science, ISSN 1516-635X, April - June 2005 / v.7 / n.2 / 67-71International Programme on Chemical Safety;URL: http://www.inchem.org/; [Accessed: October 10, 2009]Lechtanski, V. L. Inquiry Based Experiments in Chemistry; Oxford: New York, 2000Patnaik, Pradyot Ph. D. Handbook of Inorganic Chemical Compounds, (2003),ISBN 0-07-049439-8