Pure water has both a freezing point and melting point of 0˚C; however water also has colligative properties meaning that the freezing point can vary depending on the number of molecules dissolved in it. As the concentration of molecules increases, the freezing point decreases (and the boiling point increases). For example, pure water will have a higher freezing point than salt water. This is because the presence of dissolved molecules in the water makes the formation of hydrogen bonds less energetically favourable, such as those formed by ice crystals. The change in freezing point is known as freezing point depression.

Freezing Point Depression

The decrease in the temperature at which a fluid freezes (or freeze point depression) can be represented by ΔFp. To work out how ΔFp varies the equation below is used:

ΔFp= -1.86˚C x Osm

Where:

• ΔFp = The change in freezing point
• -1.86˚C = Constant
• Osm = The osmolarity of the fluid

A 1M solution of glucose has an osmolarity of 1 Osm, a 0.5M solution of NaCl also has an osmolarity of 1 Osm (0.5M of Na^– and 0.5M of Cl^–). If we consider that seawater has contains 0.5M of NaCl, we can determine the ΔFp of seawater. This would give:

ΔFp= -1.86˚C x 1

ΔFp= -1.86˚C

Therefore the freezing point of seawater is -1.86˚C, which is lower than pure water due to the dissolved NaCl molecules.

Ice Nucleation

The formation of ice crystals usually requires a form of ‘catalyst’ known as an ice nucleating agent (INA). The presence of an INA allows water molecules to begin forming an ice crystal around it. Such agents include; dust, bacteria, proteins or already present ice crystals. Without the presence of an INA, much lower temperatures can be reached before freezing occurs, this is known as supercooling.

Supercooling

Supercooling is a process where liquids can reach very low temperatures before rapid freezing occurs, this is because there are no ice nucleating agents (INAs) present. Without an INA present, water molecules struggle to begin forming ice crystal and hence the fluid stays in liquid form.

For example, a beaker of non-distilled water is cooled slowly and freezes at around 0˚C. Another beaker of pure water is cooled and care is taken to remove all INAs. This requires using pure H₂0 and a clean, sterilised glass free from dust and impurities etc. The experiment must also be carried out in a clean atmosphere or the beaker must be covered to prevent INAs such as dust entering. If no INAs are present, then the beaker of water can be cooled past 0˚C, it is possible to reach -40˚C before spontaneous rapid freezing occurs. The point at which a liquid will freeze in the absence of INAs is known as its super cooling point.

Even though the 2^nd beaker of water froze at -40˚C, its freezing point is still 0˚C, this means that spontaneous freezing can occur anywhere between 0 to -40˚C. Freezing post-freeze point can often be induced by disturbing the liquid i.e. tapping the glass.

Freeze Injuries

Freezing of internal fluids is dangerous and can cause damage to living organisms by a number of means:

• Physical damage, for example, the formation of ice crystals which could pierce and burst the lining of small capillaries.
• Freezing can lower the rate of blood flow (ischemia), which would reduce the rate at which tissues receive oxygen and nutrients. This in turn could lead to anoxia.
• The thawing process can lead to the release of reactive oxygen species which can damage DNA
• Freezing may also cause osmotic shock, this can lead to collapse of the cell membrane, swelling or bursting of the cell.

Osmotic Shock

Organisms without adaptations to cope in freezing conditions may experience osmotic shock, a cell damaging process caused by the formation of ice crystals in tissues. Typically concentration gradients between intracellular and extracellular fluids are at equilibrium; however at freezing temperatures the formation of ice crystals can alter this:

• At temperatures below zero, ice crystals can form from the water in the extracellular fluids
• The formation of the ice crystal reduces dissolved H₂0 and thus increases the concentration of the extracellular fluid
• H₂0 within the intracellular fluids (i.e. the fluid within cells) leaves the cell along the concentration gradient to form equilibrium again, as a result the cell shrinks
• As the ice crystal continues to grow, the concentration of the extracellular fluid continues to rise as does the intracellular fluid as it attempts to maintain equilibrium
• This process continues until the cell shrinks to a volume known as the critical minimal volume
• Normally the cell membrane is fluid-like but at the critical minimal volume, it becomes rigid and transport across the membrane ceases
• At this volume, the cell can no longer maintain its cell membrane and thus the cell membrane collapses, this is irreversible
• This leads to the cell rupturing, widespread occurrence can cause severe tissue damage

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Surviving At Sub-zero Temperatures

To survive at freezing temperatures, organisms must combat the freezing process. Mammals and birds are endothermic, thermoregulators and thus produce their heat to stay warm. Thermoconformers, i.e. ectothermic organisms such as invertebrates and lower vertebrates, do not produce their own heat and must avoid freezing by other means. The body temperature of thermoconformers (T_b) is equal to the environment temperature (T_a) i.e. T_b=T_a. Problems generally occur when T_a < 0^oC.

For example, freshwater fish have a body fluid freezing point of around -0.6^oC. The freezing point of freshwater is around 0^oC. Because the environment freezing point is higher than that of the body fluids it is unlikely the fish will be harmed by freezing.

However marine fish (still with a freezing point of around -0.6^oC) face a bigger threat. This is due to the freezing point of seawater being lower, at around -1.38^oC (due to a higher osmolarity as discussed earlier). This means the marine fish can freeze before their environment; therefore they must adapt different survival strategies.

To survive freezing conditions there are two options; freeze tolerance and freeze avoidance. Tolerance requires that the organism’s body learns to cope with the freezing process, whereas avoidance employs certain strategies to avoid freezing altogether.

These strategies include, super cooling and freeze point depression. Supercooling is widespread amongst arthropods; it involves the voidance of all ice nucleating agents from the body, thus allowing body tissue to supercool. An example of how this is achieved in insects is the emptying of the gastrointestinal tract. This prevents ice nucleating agents from entering the body. By entering hibernation at the same time it is possible to prevent the disturbance of bodily fluid and thus reduce the risk of freezing.

Freeze Point Depression as a Means of Survival

Utilising freeze point depression as a means of survival requires the regulation of body fluid freezing points. One method of achieving this is to increase the osmolarity of the body fluids which will decrease their freezing point. This technique is known as colligative defence. There are a few candidate molecules which could be used for colligative defence:

• Electrolytes, such as salts and ions, however these can be damaging to the body at high concentrations as they can disrupt membrane potentials and proteins.
• The other candidates are low mass organic solutes such as short chain sugars and alcohols; these have the benefit of not being harmful to the body at the concentrations required for freeze protection.

Colligative Defence

The typical molecule used for colligative defence is glycerol; such use has been demonstrated in Alaskozetes antarcticus, a species of mite which is able to survive in freezing temperatures. During the winter A. antarcticus cellular concentrations of glycerol are around 7 times higher than in summer. This rise in concentration of glycerol causes ΔFp (the change in freeze point depression) to fall drastically.

A 5M concentration of glycerol means a ΔFp of -9.3^oC allowing survival at much lower temperatures. A fall in freezing point also means a fall in super cooling point too; a 5M concentration of glycerol will lead to a super cooling point of around -55^oC.

Non-Colligative Freeze Point Depression

An alternative to the typical colligative defence is the use of non-colligative proteins which work in a different manner to prevent the formation of ice crystals. The proteins responsible for this are known as antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs).

AF(G)Ps work by lowering the temperature at which ice crystals can grow, thus reducing the freeze point of body fluids. However they do not alter the melting point, a concept known as thermal hysteresis. This means that the inclusion of AFPs in the blood will lower the freezing point to around -2.0^oC, but the melting point of the blood will not change, staying at around -0.9^oC.

Under normal circumstances, the freeze point (Fp) is equal to the melting point (Mp). Colligative defence mechanisms decrease both Fp and Mp, but non-colligative defences, via AFPs, reduce the Fp leaving the Mp constant.

AFPs also differ from colligative defences in another manner. With colligative defence, the freeze point changes in a linear relationship to the osmolarity of the colligative molecule. However AFPs depress the freeze point greater than expected. They reduce the freeze point to the maximum possible at concentrations which would barely see a reduction in freeze point by typical colligative molecules.

AFPs inhibit recrystalisation, that is, they prevent smaller ice crystals joining together to form large ice crystal lattices. They do this by attaching to the, typically flat, growing planes of the ice crystal. By doing this, they cause highly curved fronts to form in the ice crystal. This disrupts the structure and prevents further water molecules adding to the lattice as doing so would require much larger amounts of energy than normal. This is known as the adsorption-inhibition model.

The production of AFPs may exhibit seasonality, for example, polar fish of the Antarctic (where there is a continuous ice sheet all year round) continuously produce AFPs. However polar fish of the Arctic (where seasonality is observed) only produce AFPs in the winter when they are at most risk of internal freezing. By doing this, they are able to conserve energy for the winter period (as the production of AFPs is energetically expensive).

The seasonal production of AFPs is controlled by the photoperiod (i.e. the day length). A long daylength stimulates the hypothalamus to produce GHRH (growth hormone regulation hormone) which in turn stimulates the pituitary gland to produce growth hormone (GH). Receptors in the liver for GH are activated by GH and induce a signal within liver cells. This signal inhibits the transcription of the AFP gene and thus no AFP mRNA is transcribed and no AFPs produced.

During shorter photoperiods (i.e. winter) instead of GHRH, the hypothalamus produces somatostatin (also known as growth hormone inhibiting hormone GHIH). This prevents the secretion of GH by the pituitary gland and thus liver cell GH receptors produce no signal. The AFP gene is not inhibited, produces mRNA and mature AFPs are produced and exported in to the blood.

Freeze Tolerance

Some organisms choose to cope with freezing by a different means, instead of trying to prevent freezing they actually induce freezing at ‘high’ temperatures so they are able to cope. This is widespread amongst insects as well as some intertidal invertebrates (e.g. barnacles), amphibia (e.g. green tree frog) and reptilia (e.g. garter snake).

Their method of coping with freezing is to control the formation of ice crystal (and thus physical damage). This allows them to freeze slowly and avoid super cooling (which would cause rapid freezing). They initiate freezing at higher than usual temperatures by not allowing body fluid temperatures to fall below freezing; they do this by ‘innoculative freezing’, a process where ice nucleating agents (INAs) are actively taken up from the environment. This prevents super cooling from occurring. However, freezing must only occur extracellularly, even freeze tolerant organisms cannot survive intracellular freezing.

An example of innoculative freezing is observed in the wood frog, this species of frog actively internalises ice crystals from its environment to act as INAs. It does this by pressing its body against ice which allows the thin membrane of the under belly to be penetrated by ice crystals. Another example is the crane fly, which instead of taking INAs from the environment, has its own protein ice nucleators which allow initiation of freezing at higher temperatures.

As discussed earlier, with osmotic shock, the formation of extracellular ice crystals causes the extracellular osmolarity to increase. The intracellular concentration equilibrates and, as a result of increased osmolarity, freeze point depression occurs. Normally this cycle of increased extracellular osmolarity and equilibration of the intracellular osmolarity would cause the cell to shrink to its critical minimal volume and rupture; however freeze tolerant organisms have developed ways to deal with these major changes in cell volume and osmolarity.

Cryoprotectants

Cryoprotectants are molecules found in freeze tolerant organisms which protect them against the cold. Cryoprotectants can be colligative or non-colligative:

• Colligative – Colligative cryoprotectants require higher concentrations than non-colligative to function well. They work by accumulating in the intra- and extracellular fluids and limiting the amount of water transfer between them.
• Non-colligative – Required in lower concentrations, non-colligative cryoprotectants work by binding to the cell membrane. Typically, as the cell osmolarity increases due to freezing, the cell membrane becomes more rigid as the phospholipids of the move closer together. Non-colligative cryoprotectants bind to the phospholipids of the cell membrane and prevent them from moving close enough to form a rigid structure, thus keeping the cell membrane as fluid as possible and preventing cell collapse.

The most common cryoprotectants are sugars and polyols (alcohols), they have the benefits of being non-toxic, highly soluble and metabolically inert. Often, multiple cryoprotectants are used, as opposed to relying on just one form. Examples of typical cryoprotectants are; glycerol, trehalose and sorbitol.

Cryoprotectants are synthesised from glycogen which means that to survive long, cold winters, the organisms must have large glycogen stores in the liver and fatty tissues. Two other considerations for survival are how to deal with the anoxic conditions endured during freezing and coping with the reactive oxygen species which occur during the thawing process.

Freeze Tolerance of the Wood Frog (Rana sylvatica)

The way in which the wood frog copes with freezing is to control ice formation in a manner that is slow and occurs from the periphery to the core. Freezing of the peripheral extremities causes signals to be sent to the liver to begin producing cryoprotectants. The glycogen stores begin to be broken down into glucose which acts as a cryoprotectant for the wood frog.

An advantage the wood frog has for surviving freezing are the large fluid filled spaces within the abdomen. The process of freezing in the wood frog is as follows:

• Periphery
• Large fluid filled spaces
• Organs
• Heart and liver

This is where the large fluid filled spaces are advantageous – when freezing begins to occur in the organs, ice is actively transported from the organs to the large fluid filled spaces limiting the amount of freezing which occurs in the organs. When the organs begin to freeze, there is minimal water content as the majority now resides in the large fluid filled spaces. These spaces are less likely to suffer from ice damage when compared to important organs, which is why the organs shift the water there.

The cryoprotectant, glucose, is transported around the body by the blood, but as the periphery begins to freeze, no further glucose can diffuse. However, as glucose is still being produced, the organs still receive glucose i.e. their concentration of glucose rises. As the body begins to freeze totally, the last places to receive glucose are the liver and heart, which means they have the highest concentration of glucose – this is important for the thawing process.

When completely frozen, breathing ceases as does the heartbeat however the frog continues its metabolism at a highly reduced rate. This keeps the frog alive and allows it to deal with the anoxic conditions.

When the wood frog eventually begins to thaw, it melts from the core to the periphery (the opposite direction to which it froze). This is because the highest concentration of glucose is found in the heart, liver and other organs. As glucose is a colligative defence molecule, at higher concentrations it reduces the freezing point, but it also reduces the melting point. Therefore, the organs with the highest concentration of glucose (liver and heart) have the lowest melting point and thus thaw first.

As the frog thaws from inside to out, it must cope with the readjusting volumes of cells and fluid filled spaces due to the ice melting. It must also readjust its metabolism from the reduced anoxic levels to increased normoxic levels. This involves the resumption of breathing and the heartbeat. One final concern is the presence of reactive oxygen species which occur during the thawing process, this requires the use of antioxidant molecules to inhibit oxidation of other molecules by the reactive oxygen species.

References: Voelkel, S (2010), “Living Below Zero”, BIOL 445, University of Liverpool, Unpublished.