Soft Tissue Fossilization

Author’s Note: Fossilization is a topic that can be used near the end of a high school chemistry course to review and integrate topics and to promote discussion on worldview issues. This paper includes a chart of common fossilizing minerals and an experiment that demonstrates the effect of pH on the precipitation of apatite.

Introduction

The process of fossilization has been thought of as being a long drawn out process of mineral replacement taking millions of years. In 1989 David Martill challenged this belief in his journal article, “The Medusa Effect: Instantaneous Fossilization.”1 He said that for a few fish whose gills were mineralized in apatite, “lithification was instantaneous and fossilization may have even been the cause of death.”2 The fossilization process can be quick, and often must be. The time required for fossilization fits well within the biblical time frame of an approximately 6000-year-old earth.

Are the conditions necessary for soft tissue fossilization those that would be expected as a consequence of the worldwide Flood? What happens to a creature to bring about the fossilization of its soft tissue? The processes that affect an organism from its death to its discovery in the fossil record are called its taphonomy. An understanding of taphonomy requires knowledge from biology, chemistry, and the earth sciences. Decay experiments are done using the carcasses of creatures living today that are similar to fossilized ones. Some animal tissues, such as bones and teeth, are chemically less susceptible to decay. Other tissues, muscle for example, decay very quickly, yet their form can be found preserved in intricate detail in rock. The results of these decay experiments show how a carcass changes through various stages of deterioration. Paleontologists use this information to interpret the fossil record. To understand the taphonomy of these exquisite fossils, one must also understand the chemical and biological processes that quickly precipitated minerals in and around soft tissue. Experiments try to duplicate this fossilization process to determine the controls on tissue mineralization. The results give clues about the conditions when the fossils were formed. Some taphonomic studies read like a forensic medicine report, giving evidence of the circumstances and cause of death of the fossilized creature.

This paper will review the mineralization process in general and then focus on the lithification of soft animal tissues in apatite. A report on the taphonomy of a group of squid-like creatures preserved in apatite will be abstracted for the conditions and circumstances that are believed to have brought about the exceptional preservation of these coleoids. Similar conditions may have been present to fossilize microscopic gelatinous embryonic cells entombed in rock. The conditions required to bring about the rapid fossilization of soft tissue would likely have occurred during the global Flood in which

. . . all flesh died that moved upon the earth, both of fowl, and of cattle, and of beast, and of every creeping thing that creepeth upon the earth, and every man: All in whose nostrils was the breath of life, of all that was in the dry land, died. And every living substance was destroyed which was upon the face of the ground, both man, and cattle, and the creeping things, and the fowl of the heaven; and they were destroyed from the earth: and Noah only remained alive, and they that were with him in the ark. And the waters prevailed upon the earth an hundred and fifty days (Genesis 7:21–23).

The Mineralization Process

Inside an oval concretion of grey limestone found in the Santana formation of Brazil are the buff-colored remains of a fish whose gills have been fossilized in apatite.3 The discovery of many intricately preserved specimens like these has spurred research into the mysterious fossilization process. Over the past two decades we have learned much about fossilization. “The Role of Decay and Mineralization in the Preservation of Soft-bodied Fossils,” published in the Annual Review of Earth and Planetary Sciences4 is a good resource for summarizing the current understanding of the fossilization of soft tissues. There is much more to learn about this process.

The Role of Decay

Fossilization is a race between decay and preservation of tissue. Whatever the process of preservation may be, it must occur before the tissues deteriorate completely. Tissues have varying susceptibilities to decay. Biomineralized tissues such as shells, bones, and teeth are least prone to decay. Organic tissues normally decay much more quickly, but even they have a varying tendency to decay. Proteins are among the first biomolecules to decay; however, structural tissues surrounding them can protect even them from deterioration. At times fossilized arthropod cuticles—like fossil crab shells—retain traces of protein.5 Decay-prone organic tissues can also be preserved by attachment to mineral surfaces.6

The decay process plays an essential role in biogeochemical cycling. The microbes responsible for the quick decay of tissues release ions as they metabolize the organic matter. These ions are then available to precipitate in and around the nearby tissues, fossilizing them. Microbes play a role in both destroying the soft tissue and mineralizing it.

The Minerals Involved

The most common minerals involved in fossilization are forms of silica, aragonite, calcite, dolomite, illite, pyrite, and apatite. A table with their chemical compositions and some properties relevant to fossilization can be found in Appendix A. Quartz silica, a transparent to translucent crystal, is usually colorless or white, but can be any color, depending on impurities. Chalcedony is the fibrous microcrystalline form of quartz, and opal is the amorphous form. Their colors may be found in layers, as in banded agates. Aragonite is the calcium carbonate with a pearly luster that is found in clams and oysters. Calcite has the same chemical composition as aragonite, but has a different crystal structure. It is colorless and transparent, and like quartz, can be colored by impurities. Calcium carbonate can be identified by the acid test. It will bubble rapidly under a drop of dilute acid. It can also be dissolved in an acidic solution like vinegar. Dolomite has a chemical composition similar to calcite, but some of the calcium ions are replaced with magnesium ions. This makes the mineral less apt to effervesce in the acid test, and it is less soluble in an acidic solution. Illite is a clay mineral, a hydrous alumino-silicate, that has a chemical composition similar to a mica called muscovite, but it has more silica and less potassium. Pyrite is a pale, brass-yellow mineral composed of iron and sulfur that can form in granular masses as well as in cubic crystals. Apatite, a major component of vertebrate bones, is a variously colored mineral composed primarily of calcium and phosphate. When fossilizing soft tissue it is often a buff color. It has a hydroxyl group in its chemical structure that can be replaced by iodine or fluorine. Apatite can be found in several forms: amorphous, slightly crystalline, and crystalline.

A Chemical Process

The precipitation of these fossilizing minerals from solution will depend on factors that include temperature, ion concentration, pressure, pH, and the tendency of a particular mineral to crystallize or nucleate on a specific substance. Minerals will precipitate quickly under the right conditions. Quick changes in pressure, temperature, pH, or ion concentration can cause rapid precipitation. The process of fossilization was typically thought of as a prolonged process. Time alone, however, will not drive a chemical reaction. Processes that change the temperature, pressure, concentration of mineral ions, or pH can drive a chemical process toward the precipitation of preserving minerals.

Temperature—a measure of the average kinetic energy of matter—affects rates of diffusion, solubility of substances, and rates of the metabolic activity of the microbes. Temperatures for quick fossilization in minerals need to be above freezing; lower temperatures would promote a totally different form of preservation. In general, solids have a greater solubility in water at higher temperatures and gasses are more soluble in colder water. The minerals calcite and aragonite are exceptions. Warmer water favors their precipitation because the gas CO₂ is involved. A quick change in temperature can cause sudden precipitation of a mineral. Warm temperatures can also increase the metabolic activity of microbes, which in turn can increase the concentration of mineral ions necessary to lithify certain tissues.

In order to precipitate, the mineral ions have to locally reach or be beyond saturation point. This can happen through changes in temperature, changes in mineral ion concentration, and changes in pH. The acidity can determine which mineral will precipitate. The clay mineral, illite, is stable in a very acidic environment, one in which the aragonite in shells and the apatite in bones will actually dissolve. Apatite will precipitate at a pH in which the carbonates stay in solution. As the pH gets more basic, the magnesium and calcium carbonates will precipitate. A change in the pH can cause the precipitation of apatite or calcite in the vicinity. A simple experiment written for high school chemistry students that demonstrates the effect of pH on the precipitation of apatite is included in Appendix B.

Bacteria can set up steep chemical concentration gradients that drastically affect the conditions in their immediate vicinity. Tooth enamel, for example, can be affected by the presence of bacteria in plaque. As the microbes decompose food left on the teeth, they release CO₂, which goes into solution, making the water more acidic by increasing the concentration of H₃O⁺ ions. This can be seen by applying Le Châtelier’s principle to the following equilibrium equation: CO₂ + 2H₂O ↔ HCO₃^-+ H₃O +. The dissolved CO₂, along with other acidic byproducts of decay, make the immediate area so acidic that if the plaque is not removed from the tooth’s surface, the biomineralized apatite goes into solution, leaving permanent white scars on the tooth.

The fossilization of soft tissues with authigenic minerals—minerals that precipitate on location—requires the immediate presence of decay microbes. These microbes are anaerobic. Any oxygen present is quickly used up and conditions become anoxic, so that anaerobic bacteria do the metabolizing of organic matter. The microbes induce steep chemical gradients within a carcass that are necessary for maintaining the fossilizing mineral in solution and subsequently precipitating the mineral. The microbes also release mineral ions. Phosphate ions can be supplied by the acidic dissolution of the apatite in bone as well as by the microbial decay of soft tissue. The mineral ion concentration and the pH level determine where the precipitation of a mineral might occur, as well as which particular mineral might preserve the soft tissue. The microbial decay of organic matter provides the ions needed for mineralization and the pH level needed to keep the ions in solution until they are deposited in soft tissue.

Preservation in Silica

Wood can be infiltrated and permeated by water carrying silica in solution. Volcanic ash is often the source of the silica. The vacant spaces in the wood fill up with silica in a process called permineralization. This produces petrified wood, some of which still contains the original carbon and organic compounds.7 Sometimes the original organic matter degrades and is also replaced with silica. Silicious fluids from hot springs have preserved biota that still contain much of the original organic matter.8

This process does not take millions of years. Wood has silicified quickly in the natural environment.9 Pieces of fresh wood 5–8 cm in diameter and 10 cm long were placed in the Tateyama Hot Spring. The spring water, with a pH of 3, an average water temperature of 70ºC, and a high silica content, precipitates amorphous opal spheres that range in size up to 10 µm in diameter. The water permeates the wood and the opal likely silicifies the wood by depositing spheres on the surfaces. After just seven years almost 40% of the wood’s weight was silica. Since the texture of the experimentally silicified wood was similar to petrified wood found in rocks in the region, Akahane concluded that “some silicified wood near volcanic or pyroclastic rocks could have formed due to the flow of hot ground water with high silica content during a fairly short period of time, in the order of several tens to hundreds of years.”10 When the right chemical conditions are present, permineralization happens quickly.

Preservation in Carbonates

Calcite as well as silica is often involved in preserving the form of soft tissues. When a creature is buried in rapidly cementing sediment the soft tissues can subsequently decay leaving a void in the stiff matrix. If the void is filled by precipitating minerals, the outward shape of the soft tissues will be preserved. An arthropod fossil was preserved in this way.11 The animal was trapped in stiffened volcanic ash. After the arthropod’s tissues decayed, the void left behind was filled with calcite. The matrix had protected it from compaction before the minerals preserved its form. If the conditions are right, this process of preservation will proceed quickly.

Shells, bones, and teeth of creatures are most of what is found in the fossil record. These decay-resistant parts were biomineralized by processes in the animal while it was still alive. The original hard parts are often permineralized when the spaces in the bone fabric filled with minerals precipitating from water solution. The original bone material often remains in place, but it may be replaced to a greater or lesser extent. Reports even tell of the original organic soft tissue remaining in place.12^, 13 What appeared to be blood vessels containing little round cells and flexible elastic tissue remained after a fossil dinosaur bone matrix was demineralized in an acid solution. How these tissues were preserved is a mystery; however, the mineralization process had to be quick, proceeding before the soft tissues could deteriorate.

Permineralization of plant material has intricately preserved plant cell contents—starch grains, nuclei, spores, gametophytes, and pollen drops and tubes—in coal balls.14 These may have been formed when decay increased the HCO₃- ion concentration trapped below a layer of sediment. If the sediment were suddenly washed away, carbon dioxide would quickly escape. As a result, the water would suddenly become less acidic causing calcium or magnesium carbonates in solution to quickly precipitate and infiltrate the buried plant material. If the carbonates continued to precipitate, the permineralized region would grow outward like a snowball. The extent of the details preserved in the plants depended on their state of deterioration before permineralization started. Quick permineralization occurring before deterioration set in would produce the exquisite detail in the plant cell contents.

Many fossils are found in concretions, which may grow much like coal balls. After the soft tissue fossilizes in an authigenic mineral in a fairly acidic environment, the pH returns to a higher level, precipitating calcite around the mineralized tissue. These can continue to grow outward, like the coal balls mentioned before. The concretion protects the fossil from compaction and keeps the three-dimensional preservation of the authigenic minerals intact. These concretions grow in response to changes that occur quickly.

Preservation in Clay Minerals

Some fine examples of fossilization of soft tissue are found in the Soom Shale in South Africa and the Burgess Shale in Canada. In these shales the fossilizing agent is the clay mineral illite. There is evidence that microbes are involved in the process,15 but so far no bacteria have been found fossilized in illite.16 They may set up the conditions that either directly precipitate the illite into the soft tissues, or aid the adsorption of the pre-existing clay mineral particles onto the organic matter. This may have taken place in a highly acidic environment, possibly because minerals that release Ca⁺² ions, like limestone, were not present in order to buffer the solution. There are fewer examples of illite soft tissue preservation, perhaps because the conditions for preservation are so specific. Experiments have shown that in the presence of bacteria, pre-existing silica and clay minerals can be attached to the surface of lobster eggs.17 After incubating 31 days at 15ºC, vials with non-sterile sediment in seawater showed (1) a color change to black from the formation of iron sulfide by sulfate reducing bacteria, (2) a drop in the pH of the seawater from 7.2 to 6.9, and (3) attachment of clay particles onto the lobster eggs. Soft tissues preserved in clay may have been fossilized either in pre-existing minerals or by direct precipitation of the mineral. The means of preservation and the role of microbes in the replication of the form of soft tissue by clay minerals are still being debated. Either way, the process must be fairly quick in order to preserve the soft tissues before they decay. The mud rock at the Burgess Shale deposit, where soft tissues have been preserved in illite, contains suspended grains of sand and shells, leading some researchers to conclude that the creatures died quickly, from being buried in continuous pulses of dense muddy slurry.18

Preservation in Pyrite.

Delicate plant tissues and some animals are sometimes found preserved in pyrite. For example, an arthropod, showing its feathery appendages and body segments is exquisitely preserved by pyrite in the Hunsrück Slate in Germany.19 Experiments have been done to try to replicate the preservation of plants in pyrite, both using a purely chemical approach and by allowing the presence of bacteria.20 A source of sulfate ions, SO₄^2-, such as is found in ocean water, and a source of Fe³⁺ ions are necessary to form pyrite. As the bacteria metabolize the organic matter, they reduce the sulfur and the iron, eventually forming pyrite. During the experiment that reproduced the precipitation of amorphous pyrite, the pH started at 7.5, went down to 6.5, rebounded to 8.5, then remained constant. The temperature was 15ºC, and the glass jar was open to the air. The researchers concluded that “The microbiological experiments demonstrated how rapid plant pyritization can occur under natural conditions (within 80 days) with intense anaerobic bacterial activity.”21

Preservation in Apatite

Apatite is the mineral which has preserved the soft tissue of animals in the finest detail. For example, the wing membrane of a pterodactyl is preserved, not as just a skin impression, but showing distinct layers of mineralized tissues including muscle.22 This style of fossilization has happened so quickly that in the Santana Formation in Brazil it has preserved in apatite the gills of fish, showing the arteries, veins, and secondary lamellae.23 Fish gills will begin to deteriorate in four to five hours after the fish dies. Knowing more about this particular preservation process helps us understand the unusual conditions that so quickly brought about the fossilization of tissues as delicate as fish gills.

Like preservation in pyrite, attempts have been made to replicate this apatite mineralization process in the laboratory.24 In decay experiments using modern shrimp, amorphous calcium phosphate preserved cellular details of muscle as well as bacteria. The source of phosphate was the decaying carcass of the shrimp itself. The experimental fossilization proceeded in an environment closed to additional oxygen. The pH began at 8, dropped to between 6 and 7 after three days, then recovered to near 8 within four weeks. Mineralization of the soft tissue began less than two weeks after the start of the experiment and continued to progress throughout the experiment. In the Santana Formation the apatite, precipitating in anoxic and acidic conditions, must have begun to preserve the fish gills within five hours of the fish’s death.

Evidently bacteria play a major role in the precipitation of apatite. The microbes’ metabolic activity increases the phosphate ion concentration and decreases the pH in the immediate vicinity. If there are also calcium ions in solution, apatite can precipitate if the pH is not too acidic. This fossilization process is referred to as phosphatization. It is often restricted to one area of a carcass. The first tissues that decay supply the phosphate ions which later precipitate as apatite in another area of the carcass. This seems to have happened in some clams that died with their shells tightly closed from being buried alive in sediment.25 The microbe-infested belly of the clam metabolizes first. The phosphate ion concentrations build up and subsequently mineralize the muscle of the clam before the muscle totally decays.

A buff-colored mineral encrusts the inside of a clam shell from the Desoto Shell Pit, Florida. The shell was found tightly closed. Perhaps the buff-colored mineral is apatite fossilizing muscle tissue. The mineral appears where the clam tissues would have collapsed on one side of the shell.

Autolithification of Microbes

The microbes that set up the conditions that precipitate apatite can be overcome by the very conditions their metabolic activity produce. The microbes autolithify as calcium phosphate precipitates on their own cell walls. These fossilized bacteria form mats leaving a mould about the soft tissue. The intricacy of the detail of this kind of preservation depends on the size and shape of the microbe. Smaller ones make a more precise mould. This is the style of soft tissue fossilization found in the bivalves mentioned above. (See photo.)

Direct Mineralization

Apatite can nucleate directly onto the collagenous protein around muscle fibers. This is microbially induced mineralization, even though the microbes themselves may not be fossilized. The detail of the preservation depends on the size of the precipitated microspheres. Since they are generally smaller than the microbes, this method usually preserves more detail than the mould method. Direct mineralization can rapidly preserve intricate features such as the white collagen fibers around muscle, muscle fibers, myoseptal membranes, muscle cells, and cell nuclei in fish muscle and gills. These features were observed in the Brazilian phosphatized fishes found in the Santana formation.26 After the apatite precipitated directly, and as the pH was rebounding to a less acidic level, a calcite concretion precipitated around specimens protecting and preserving the fossils in three dimensions. Soft tissues phosphatized in the laboratory closely resemble the phosphatized tissue in the Santana Formation, implying that similar processes were involved.27

A Mass Mortality Event Preserved in Apatite

In the Jurassic Oxford Clay formation in England are world-famous cephalopod fossils. During the 1800s at Christian Malford, Wiltshire, over 100 of these coleoids—squid-like creatures—were collected for their soft tissues exceptionally preserved in apatite.28 Belemnotheutis antiquus was among three species of coleoids found with well-preserved mantles, ink sacs, and arms with suckers and hooks.29

The style of preservation at the Christian Malford site had been cited as a unique occurrence within the Oxford Clay30; however, a new exposure of a laterally equivalent bed at the Rixon Gate Quarry near Ashton Keynes, Wiltshire, has revealed similarly preserved cephalopods and has allowed further study of the taphonomy of these fossils.31 Wilby reports that Belemnotheutis antiquus specimens found there had soft tissues phosphatized both as moulds and by direct mineralization. Their ink sacs were also preserved and generally maintained their shape. The fossils were usually encased in a calcite concretion which protected them from compaction. The shaly mudstone in which the concretions were found was made up of 14.6 to 16.1 per cent organic carbon. Other isolated fossils were also preserved, generally within the concretions. Wilby writes,

Included in this category are the phosphatized coleoids, a crocodile skull, a fully articulated fish, and abundant driftwood. One ‘log’ observed was c. 2 m long and 0.5 m in diameter, and extended between two concretions. In the mudstone it was black, as is usual for the Oxford Clay, but within the concretions it was yellow-brown and fibrous.32

From observations and study of the specimens and the site, Wilby and his associates concluded that the coleoids were “killed en masse, together with other elements of the associated fauna, in one or more catastrophic mass mortality events that affected a significant area of the Peterborough Member Sea.”33 While the major source of the phosphate came from the creatures themselves, the extensive phosphatization of the soft tissues of the cephalopods came about because they were in a zone “where levels of dissolved phosphorus were greatly augmented by the large number of associated decaying carcasses.”34

A Belemnotheutis antiquus ink sack has recently been found at Towbridge, Wiltshire, at another laterally equivalent site.35^, 36 Wilby said about it, “It is difficult to imagine how you can have something as soft and sloppy as an ink sac fossilized in three dimensions, still black, and inside a rock that is 150 million years old.”37 “We call it the Medusa effect—specimens turn to stone within a matter of days, before the soft parts can be eaten away.”38 How the ink sac was separated from its owner is also a mystery. Wilby’s research team has removed several thousand fossils from the site, hoping that studying them will give clues as to how so many creatures perished over such a large area and why some of them are so well preserved. This continuing research is an example of a taphonomic study using fossil forensics.

Embryonic Cells Preserved in Apatite

Another study concerns the authigenic phosphatization of the embryos of a worm-like creature.39 These microscopic fossils were retrieved from 12 metric tons of rock found in China and Siberia. “We picked through every single grain to determine whether it was sand or embryos,” said Donoghue.40 A new microscopy technique that is non-invasive showed that the embryo preservation was three dimensional, much more extensive that just a mould of the surface structure. These delicate embryos “are just gelatinous balls of cells that rot away within hours,” according to Donoghue. What else was in that mudstone along with the suspended sand and fossil grains? If investigated, would there be evidence of organic carbon present, as there was in the Oxford Clay? How was that mudrock deposited and cemented without destroying the embryos, and yet preserving their form in apatite? Do the sand grains present suggest that the embryos were suddenly trapped in a hyper-concentrated mud flow, as has been suggested for the exquisitely preserved fossils of the Burgess Shale? A source of calcium and phosphate ions, and pH levels in the sediments that kept the phosphate ions in solution before precipitating apatite into the embryonic cells would have been necessary for their rapid fossilization. Where did theses ions come from? From decaying organic matter?

Conclusions

Millions of years are not needed for the fossilization process. Most fossils were originally hard parts such as bones and shells that were biomineralized by the creature while it was still was alive. The subsequent permineralization of the material does not require extensive time as much as the right conditions. Furthermore, the rarer fossilization of soft parts does require haste or the tissues will decay. Fossilization in apatite is promoted by the presence of anaerobic microbes metabolizing many associated dead and decaying creatures. Again, the right conditions, not time, are what is necessary for fossilization, and the process fits into the biblical time scale.

The surprise at the recovery of soft tissue from the thighbone of a Tyrannosaurus rex shows how little we understand about the fossilization process, or in this case the preservation process. Whatever the mysterious process is that preserved the organic tissue, it had to be quick, or the tissue would have deteriorated. A report of preserved organic bone marrow in frogs and salamanders attributes the exceptional preservation to “cryptic preservation: the bones of the amphibians formed protective microenvironments, and inhibited microbial infiltration.”41 Unusual conditions are necessary to have preserved these organic tissues. Once preserved, if the conditions do not change, the preservation continues. This is what would be expected if the preserved organic tissue was left undisturbed for thousands of years.

In the past many have assumed that much time is necessary for fossilization, and few have attempted to reproduce the fossilization processes in the laboratory. We have just begun to understand the fossilization process. Many questions remain. For instance, recall the log in the Oxford Clay Formation stretching between two concretions. Why is it black in the mudstone, yet yellow-brown and fibrous in the concretions? Both ends of the log are obviously the same age. How did it get suspended in the mud and become part of the two concretions? Is this Oxford Clay the deposit of a hyper-concentrated mud flow that trapped the coleoids as well as the log? We still know very little about the mysterious process of soft tissue fossilization. We do know that the soft tissues are fossilized in minerals that are released by micro-organisms as they metabolize flesh and bone. Soft tissue fossilization points to unusual conditions in the fossil record: rapid successive events of catastrophic burial, sudden death, and extensive decay. Where soft tissue fossilization has occurred, more taphonomic research must be done to uncover the history of the fossil deposit. The results of this biogeochemical research will not conflict with the biblical record that “every living substance was destroyed which was upon the face of the ground.” The evidence points to catastrophic watery burial of living creatures in a region that was already high in phosphates from other decaying carcasses. These conditions are consistent with the extensive destruction of life as recorded in the Genesis account of the worldwide Flood.

Appendix A

Appendix B

Experiment: Effect of pH on the Precipitation of Apatite

Objective:

Observe the effect of pH on the precipitation of apatite.

Description:

A solution of the soluble salt calcium chloride mixed with a solution of the soluble salt sodium phosphate forms the relatively insoluble salt, hydroxyapatite. By mixing the same amount of the salt solutions in solutions of regularly varying concentration of HCl, the effect of the pH on the precipitation of the apatite can be observed.

Safety:

Sodium phosphate dibasic is a skin and eye irritant. Hydrochloric acid is also corrosive. Wear goggles and an apron. Do not ingest the chemicals. Wash spills with abundant water. Wash hands well after the experiment.

Student Materials:

• 35 mL of 0.10 M CaCl₂
• 21 mL of 0.10 M Na₂ HPO₄
• 2 mL of 0.10 HCl solution
• Distilled water
• 5 small beakers for the acid dilutions
• 7 25 mL test tubes for precipitating the hydroxyapatite in various pH solutions
• test tube rack
• 1 10 mL graduated cylinder
• 1 clean stirring rod; (rinse it with distilled water between stirring different solutions)
• pH paper indicating a range of pH from 0-14
• Test tube cleaners
• Colored index card

Procedure:

Make the acid dilutions:

• Mix 1 mL of 0.10 M HCl with 9 mL distilled water in a small beaker. Label 0.01 M HCl.
• Mix 1 mL of 0.01 M HCl with 9 mL distilled water in a small beaker. Label 0.001 M HCl.
• Repeat pattern for making 0.0001 M, 0.00001 M, and 0.000001 M HCl. Label each.
• Check the pH of the 0.10 HCl and each dilution and record.
• Check the pH of the distilled water and record.
• Check the pH of the salt solutions and record.

Prepare the test tubes:

• Label test tubes # 1 to #7.
• Put 5.0 mL of distilled water in test tube #7
• Put 5.0 mL of 0.000001 M HCl in test tube #6.
• Put 5.0 mL of 0.00001 M HCl in test tube #5.
• Repeat the pattern for test tubes #4, #3, #2, #1.

Do the experiment:

• Add 5.0 mL of the 0.10 M CaCl₂ solution to each test tube. Stir, check and record the pH.
• Add 3.0 mL of the 0.10 M Na₂HPO₄ solution to each test tube. Observe carefully each time the solution is added. Record observations. Stir each test tube, check and record the pH.
• Hold a colored index card behind the test tubes. Do you notice any difference in the cloudiness? Is there more or less precipitate in the more acidic solutions?
• Let the precipitate settle in the test tubes. Record your observations.

Clean up:

• Wash the beakers and test tubes out at the sink. Run lots of water down the sink. Wash hands.
• Put away equipment.

Data:

pH of CaCl₂ solution:           pH of Na₂ HPO₄ solution:          

Questions:

1. If the HCl dissociates totally, what is the calculated pH of the acid dilutions in each test tube? Were the measured pH values similar? Did the pH change after the addition of CaCl₂? Did it change after the addition of Na₂HPO₄?
2. The formula for hydroxyl apatite is Ca₅(PO₄)₃(OH). Assuming this is the precipitate, write a balanced chemical equation for the precipitation reaction. Remember that water can ionize to OH^- and H⁺. What else is formed?
3. Using LeChatelier’s Principle, would you expect more or less precipitate in acidic conditions? Explain.
4. What happened when the salt solutions were mixed at the various acidities? Was the amount of precipitate similar in each of the test tubes? Describe what you observed and give a possible explanation for any differences.
5. What are the major sources of error in this experiment?

Effect of pH on the precipitation of apatite: Teacher Guide

Curriculum:

This experiment should be done near the end of a high school chemistry course, as it requires an understanding of chemical equations, solutions, pH, and LeChatelier’s Principle. It would make a good lab for a review of these topics. It could be used to teach about fossilization; the precipitation of apatite into soft tissue is one form of soft tissue mineralization. It can also be used to demonstrate the insolubility of most phosphate salts, a problem for an evolutionary formation of DNA and RNA which have an alternating phosphate/sugar backbone.

Time:

Teacher preparation: 20 minutes; Class time: 40 minutes

Materials for teacher preparation:

• 100 mL of 0.10 M CaCl₂ – Dissolve 1.47 g calcium chloride dihydrate (CaCl₂ •2H₂O) in enough distilled water to make 100 mL solution. This chemical is put on roads to reduce dust. It is very hygroscopic. Because it readily absorbs water it has to be kept in a tightly sealed container. It is relatively safe to handle, but avoid breathing it as calcium chloride reacts exothermically with water and can burn the mouth and esophagus. Wear goggles to protect eyes and avoid contact.
• 100 mL of 0.10 M Na₂ HPO₄ – Dissolve 2.68g sodium phosphate dibasic heptahydrate (Na₂ HPO₄ •7H₂O) in enough water to make 100 mL solution. Avoid contact with the skin and eyes. Do not ingest or breath the dust. If ingested, seek medical advice immediately. (The solute will dissolve at room temperature in this amount of solution, but it takes longer to dissolve than the CaCl₂ solution.)
• 100 mL of a 0.10 HCl solution.
• Scale for weighing chemicals
• 100mL graduated cylinder
• 3 large beakers for the salt and acid solutions

Disposal:

The materials used in this experiment may be discarded safely at the sink using large volumes of water.

Data:

pH of CaCl₂ solution:     6     pH of Na₂ HPO₄ solution:     9    

Answers:

1. Test tube #1 has pH =1, etc.; pH = -log[H⁺];
   
   Since HCl is a strong acid, it will dissociate totally at concentrations less than 0.1 M. The measured pH should be approximately the same as the calculated pH.
   
   The addition of CaCl₂ did not significantly affect the pH.
   
   The addition of Na2HPO4 raised the pH in test tubes 1, 2, 3, and possibly 4. Test tube 2 showed the most change.
2. 5CaCl₂ (aq)+ 3Na₂ HPO_4(aq) + HOH → Ca₅(PO₄)₃(OH) + 6NaCl + 4HCl
   
   HCl and salt are by products of the precipitation of apatite.
3. Precipitation of apatite is less likely to occur in acidic conditions. Since H⁺ is a product, increasing acidity (greater concentration of H⁺) will drive the reaction toward the reactants.
4. Test tube #1: When the salt solutions were mixed in, a swirling cloud of white precipitate appeared momentarily, but then cleared as the solutions mixed thoroughly.
   
   Test tube #2: The white precipitate remained visible, but the solution behaved differently, somewhat like a gel. Perhaps it is a colloidal solution.
   
   Test tubes #3 through #7: The cloudy white precipitate remained visible. It soon started settling, leaving a clear upper layer.
   
   No difference in cloudiness was apparent when the colored index card was held behind the test tubes, with the possible exception of test tube #2. Letting the test tubes settle overnight might show up a difference in the precipitation of apatite, but it does not appear to be significant.
   
   The presence of a high concentration of H⁺ ions seems to have prevented the apatite from precipitating in test tube #1. The presence of H⁺ ions in test tube #2 seems to be keeping the apatite from clumping in larger spheres. The precipitation and dissolution process may be continuing at that pH. Letting the test tubes settle overnight might show a difference in the settling time of the mixtures.
5. Different results may be obtained if care is not taken to accurately make the dilutions or to prevent contamination by not rinsing the stirring rod between solutions.

Elaboration and Extension

Try letting the test tubes settle overnight and observe the results.

Can fossilization in apatite be done in the lab? How might you get the apatite to precipitate in bone or muscle? Try putting bone or meat in a phosphate ion solution. Let it soak, remove it from the phosphate solution and put it in the calcium ion solution. After leaving it for a time, check the cells or bone structure under a microscope. Look for a precipitate.

The precipitation of a rapid setting calcium phosphate mineral has medical uses. It can be used to produce bone-like materials in broken or deteriorating bones.

Cavities in teeth may be the result of acidic demineralization of tooth apatite. Check out the following journal article: