At the end of the Cretaceous era, about 66 million years ago (Mya), there was a

mass extinction of many animal species around the world, including the large and
diverse family of dinosaurs. The challenge of explaining this huge loss of biological
diversity has been the province of geologists and palaeontologists. Their expertise
in dating rocks and in reconstructing the characteristics of these long-gone life
forms, from fossil bone and tooth fragments, is a remarkable achievement of
scientific observation and analysis. Nevertheless, there has not been unanimous
agreement in interpretations amongst these expert investigators. Because, from
the vantage point of the present, the extinction of such a vast range of species
seemed to have suddenly occurred, the concept has been proposed that some
catastrophic environmental change is a unifying hypothesis to explain their
disappearance. Some investigators, on the other hand, suggest that the time
frame of the process of extinction from a biological viewpoint was very long and
that the various species had declined in number over many generations. The
conflict between catastrophism and gradualism to describe the process of
extinction has been debated extensively during the second half of the 20th
century.

The case for a catastrophic cause of mass extinction was strengthened by the
discovery of a worldwide enrichment of the rare element iridium in a narrow
band of geological strata formed 66 Mya(,1). The source of that iridium was
proposed to be from a huge asteroid or comet that collided with the Earth,
coinciding with the time of the mass extinctions(,2–4). The discovery of the
Chicxulub impact crater in the Yucatan Peninsula on the Gulf of Mexico
confirmed, from its size and structure, that a large celestial object had collided
with the Earth at the time that the mass extinctions had occurred(,5). The
consequences of that impact would indeed have been catastrophic, not just in the
geographic region around the impact site, but worldwide because of
environmental changes from an atmospheric dust and aerosol cloud that would
have remained for years in the stratosphere(,2).

Those who favour a gradual decline towards extinction of all species of the
dinosaur family, except the ancestors of modern birds, have some plausible
arguments. The dating of fossils of all known dinosaur species is incomplete and is
only well established for a range of dinosaurs in North America. It has been
argued that long-term environmental changes had diminished the number of
species over millions of years before the asteroid or comet impact, particularly
the large-bodied Saurischian and Ornithischian dinosaurs(,6). Careful review of
fossil records of both classes of dinosaurs has indicated that the number of
species was in decline over millions of years. Thus the family Dinosauria would
have been susceptible to extinction by a range of environmental changes to which
they could not adapt(,7). Nevertheless, others have concluded that there is little
evidence for a gradual decline worldwide in the diversity of dinosaur species, in
contrast to that in North America, over the long term before the fossil record
indicated that all non-avian dinosaurs had become extinct(,8).

Although the debate between the gradualists and the catastrophists appears to
be still unresolved, both groups of palaeontologists do agree that the dating of
dinosaur fossils ended in the era of the Chicxulub impact event. Even if the
diversity of dinosaur species had been in decline, the gradualists do acknowledge
that the worldwide environmental changes caused by that impact could have led
to their final extinction.

Clearly the effect of the asteroid or comet collision would have been devastating
to all life forms in a wide geographical region around the impact site. The
proposed environmental changes that would have occurred worldwide include a
period of dim sunlight because of solar radiation absorption by ejected particles
and aerosols in the atmosphere and stratosphere. Consequently, there would
have been a short-term temperature drop(,9), which, together with diminished
sunlight, would have inhibited or killed photosynthetic plants. However, perhaps
a more significant effect, as far as the dinosaurs were concerned, was a
consequence of the geological site of impact. The Chicxulub site contains vast
amounts of limestone (CaCO3), anhydrite (CaSO4) and gypsum (CaSO4.2H2O)(,10)
as well as hydrocarbons(,11). A result of the asteroid impact would have been the
ejection into the stratosphere of particulate carbon soot, CO2 and sulfate
aerosols(,12). This would have had a marked cooling effect on the climate from
the absorption of much solar radiation. Because such geological sites that are rich
in sulfur and carbon deposits are not plentiful, it has been suggested that if the
asteroid impact had occurred in most other regions of the world, the probability
of mass extinctions would have been much less(,11).

Conclusions about the biological effects of the Chicxulub impact have focused on
the undoubted environmental changes of temperature, light intensity, sea
temperatures, sea levels and climate disruption in the months and probably years
following the impact. The implications of these changes are that a consequent
restricted food supply for many life forms that disappeared would have
contributed to their extinction.

One result of the asteroid collision with Earth, that has not been considered from
a biological perspective, is a particular effect from the ejection into the
stratosphere of the sulfur aerosols. There are various calculations of the amount
of sulfur, as sulfate or SO2, based on estimates of the size of the impacting
asteroid and the density of sulfur deposits at the impact site. One estimate is that
326–527 gigatonnes of SO2 were distributed globally in the stratosphere(,13).
Another, in broad agreement, put the quantity of stratospheric SO2 in the range
of 200–400 gigatonnes(,10). The effect of such quantities of SO2 on global climate
can be deduced from observations on the changes from sulfur ejected into the
upper atmosphere from volcanic eruptions(,14). As an example, the Tambora
eruption in Indonesia in 1815, the largest volcanic event in the past 200 years,
produced only about 0·15 gigatonnes of stratospheric SO2. Yet in the following 12
months, this relatively small amount, compared with that from the Chicxulub
impact, had a noticeable cooling effect on the world climate(,10).

An alternative environmental catastrophe postulated as the cause of dinosaur

extinctions is a series of vast volcanic eruptions in India which created the Deccan
basalt larval floods, known as the Deccan Traps. It has been argued that the
timing of this volcanism is more likely to have been associated with the demise of
the dinosaurs than the Chicxulub impact event(,15). Nevertheless, geological
evidence puts the timing of the Deccan volcanism and the asteroid collision so
close together that it has been suggested that the Chicxulub impact actually
triggered the largest of the volcanic eruptions which gave rise to the Deccan
Traps(,16). Although volcanic activity in general has not been considered to
produce stratospheric sulfate aerosols in the quantities anywhere near that of the
Chicxulub impact, the Deccan volcanism may have been an exception.
Calculations from paleomagnetic measurements in a Deccan basalt escarpment
identified single eruptive events, the largest of which could have released
quantities of SO2 over years or decades, comparable with that released by the
impact at Chicxulub(,17). Together, from volcanism and the asteroid collision, the
SO2 emitted into the stratosphere could have had a prolonged climatic effect that
would have lasted for years.

One biologically significant property of stratospheric SO2 is that it strongly

absorbs solar UV light in the UVB wavelength range of 290 to 320 nm. In the
steady atmospheric state of the present-day era, the intensity of UV light reaching
the Earth's surface is attenuated by ozone(,18). However, the UVB-absorbing
power of SO2 is 2·5 times greater than that of ozone(,19). The traces of SO2 from
human activity such as that emitted by cities has a limited effect on UVB at
ground level(,20). However, the mass of stratospheric sulfur from the Chicxulub
impact, perhaps combined with that from the Deccan volcanism, would have
been sufficient to completely block UVB reaching the Earth's surface for a decade
or longer(,13).

One of the effects of UVB radiation is that it acts on 7-dehydrocholesterol in cells

of the superficial integument of terrestrial animals to make, by a photochemical
reaction, the pre-hormone, cholecalciferol. A characteristic of animals that
depend on solar UVB radiation to supply cholecalciferol is that its precursor, 7-
dehydrocholesterol, the penultimate metabolite in the synthesis of cholesterol,
accumulates in superficial skin cells. In other cells, its concentration is very low
because of rapid conversion to cholesterol. Cholecalciferol is more popularly
known as vitamin D3 but under natural conditions it is not a nutrient. The food of
most animals contains, at most, only traces of cholecalciferol. An adequate supply
of cholecalciferol can therefore only be obtained by exposure of skin to UVB
radiation from the sun. A deficiency of cholecalciferol causes a range of
pathological changes, the most prominent of which is the bone disease of rickets
or osteomalacia.

Much of the knowledge about the metabolism and function of cholecalciferol was
obtained in the mid-20th century from studies of the domestic chicken (Gallus
domesticus), a member of the avian descendants of the dinosaur family. Like the
extinct dinosaurs, modern birds reproduce by depositing eggs encased in a hard
shell of crystalline calcium carbonate. The egg shell not only protects the
developing embryo inside, but it also is the source of Ca to mineralise the bony
skeleton towards the end of the incubation period. Thinning of the egg shell from
dissolution of calcium carbonate also weakens it, so that pecking by the beak of
the chicken at full term fractures the shell, allowing the chicken to emerge.

Early in the development of knowledge about the biology of cholecalciferol, it was

observed that hatchability of domestic hen eggs produced during winter was low,
particularly if the laying hens had been kept indoors. Hatchability improved when
hens were directly exposed to sunlight in spring(,21). It was therefore postulated
that cholecalciferol, incorporated into yolk during egg production, was an
essential requirement for avian embryonic development. When hens, reared
away from sunlight, were fed a diet containing no cholecalciferol, the embryos in
the eggs they produced died at 18–19 d of embryonic life, just before the 21-d
scheduled time of hatching(,22). Likewise, the embryos in eggs from
cholecalciferol-deficient turkey hens died at day 26–27 of their somewhat longer
incubation period of 28 d(,23). Embryos in eggs from hens deprived of
cholecalciferol developed hypocalcaemia and low bone mineralisation compared
with embryos from hens fed a diet containing cholecalciferol(,24). From this
pathology it was suggested that the embryos were unable to mobilise Ca from the
shell and that the consequent hypocalcaemia caused muscle weakness which
prevented the chicken from breaking the egg shell(,24,25).
The yolk of bird eggs, in contrast to other biological material, is quite rich in
cholecalciferol. This is surprising because the yolk substances are derived from
blood circulating through the ovary of the laying hen and the majority of
cholecalciferol molecules in blood are in the form of its metabolite, 25-
hydroxycholecalciferol, the precursor of the hormone, 1,25-
dihydroxycholecalciferol. 25-Hydroxycholecalciferol has a long residence time in
blood in comparison with its parent cholecalciferol which, after diffusing from its
site of formation in the skin, is rapidly removed from blood by the liver. A
specialised form of a cholecalciferol-binding protein transfers the trace
cholecalciferol from blood into the developing yolk follicles in the ovary(,26).

In recent years there has been much interest in the many thousands of whole
fossilised dinosaur eggs and egg shell fragments found around the world. Most
attention has been directed at identifying the dinosaur species of each egg type
and in drawing conclusions about the reproductive strategies from the
arrangement of groups of eggs and of fossilised dinosaur embryos or juvenile
dinosaurs that had died after hatching. In comparison with fossil dinosaur bones
and teeth, little attention has been devoted to determining the age of the
fossilised eggs and shell fragments. In his comprehensive book on dinosaur eggs,
Carpenter(,27) catalogued the era of sites around the world where fossilised eggs
had been discovered. There were twenty sites where eggs of the Jurassic era
(130–80 Mya) were located and twenty-eight sites with eggs from the early
Cretaceous era (100–130 Mya). However, most fossilised eggs have been dated
from 187 sites of the late Cretaceous era (66–100 Mya). Despite considerable
palaeontological study of the eggs, some containing fossilised embryos, the
question does not seem to have been asked as to why so many eggs of this extinct
family have survived for analysis over 66 million years. Why did so many whole
eggs become fossilised? Were the eggs infertile or did the embryos die before
hatching?

A study of Hypselosaurus dinosaur eggs from southern France and the Spanish
Pyrenees concluded that shell thickness was abnormally thin in up to 90 % of the
samples(,28). By comparison with egg biology of modern birds, the thinning of the
shells was attributed to abnormal hormonal control of the calcification process, as
a result of environmental stress. Strangely, as a possible explanation, there was
no mention of cholecalciferol deficiency which is a well-established cause of thin
shells in eggs of the domestic hen(,29).

Apart from shell thickness, another feature of dinosaur eggs that has been
explored is the length of incubation. By remarkable deductive analysis it has been
concluded that the incubation times of eggs of various dinosaur species are longer
than those of their descendant modern-day birds. Lee(,30) estimated the
metabolic rate of embryonic dinosaurs in comparison with that of birds and
crocodiles and concluded that dinosaur egg incubation times were up to 76 d.
However, Erickson et al.(,31), by measuring the growth lines in fossilised dinosaur
embryo teeth estimated, more directly, the incubation times of dinosaur eggs. On
the presumption that dinosaurs, like modern birds, were endothermic, they
deduced that dinosaur egg incubation times were at least twice as long as those
of birds, being up to 171 d, which is comparable with those of eggs of
poikilothermic modern reptiles. Using this tooth growth-line technique, Varricchio
et al.(,32) calculated that the incubation time of a theropod dinosaur (Troodon
formosus) was 74 d compared with an average avian incubation time of 44 d.

Even if female dinosaurs had sufficient 25-hydroxycholecalciferol in their bodies

to produce the hormone 1,25-dihydroxycholecalciferol to allow egg shell
formation in the oviduct, unless they had a daily input of cholecalciferol to
incorporate into the yolk being laid down in ovarian follicles, the eggs would have
contained insufficient cholecalciferol to meet the needs of embryos during their
prolonged period of incubation. The cholecalciferol-deficient dinosaur embryos,
like those of the modern birds would either die during development, or else just
before hatching. Although environmental changes leading to failure of food
supply would certainly threaten the survival, over years or decades, of the wide
variety of dinosaur species, the most effective mechanism of extinguishing the
dinosaur family altogether would be one that prevented reproduction. One could
speculate that if the modern bird descendants of theropod dinosaurs were today
unable to obtain cholecalciferol from their environment, then over a short period
of time they too would become extinct from reproductive failure.

Of course, if reproductive failure because of cholecalciferol deficiency was the

cause of all dinosaur species becoming extinct, why then did other terrestrial
vertebrates survive and evolve into the animals we know today? It is apparent
that modern birds do not have a storage mechanism for cholecalciferol. Could
other animals, such as mammals, have developed a means of conserving
cholecalciferol to allow its many functions to continue when the environmental
supply ceased? Present-day nocturnal mammals which seldom are exposed to
solar UVB radiation seem to be able to maintain the functions of cholecalciferol
without any apparent input from its formation in skin. There is evidence that the
ancestors of modern mammals at the time of the dinosaurs had a nocturnal
lifestyle(,33). By being both viviparous and nocturnal they may have avoided the
reproductive failure of egg-laying dinosaurs. It is also possible that some dinosaur
species survived the catastrophic events that were associated with most dinosaur
extinctions. A few species that lived within the Antarctic Circle had anatomical
features that suggested they were able to see in the dim light of winter and may
have died out long after other Dinosauria had become extinct(,34). Apart from
being able to see in the darkness of the Antarctic winter and accommodate to a
cooling climate from the asteroid impact and volcanic eruptions, these polar
dinosaurs may have also been able to conserve sufficient cholecalciferol,
produced during the Antarctic summer, to maintain their capability for oviparous
reproduction.

Would it be possible to test a cholecalciferol-deficiency hypothesis by further

study of fossilised dinosaur eggs and embryos? Evidence could come from
determining whether fossilised dinosaur embryos had bone pathology
comparable with that, for example, of cholecalciferol-deficient turkey embryos
that had died before hatching. Likewise, this theoretical cause of complete
elimination of the dinosaur family would be supported if fossilised dinosaur egg
shells, dated more exactly to the era of extinction, had the defective structure of
egg shells produced by cholecalciferol-deficient birds.