The calcification process and

measurement techniques

Photo from JC073 Changing Oceans Research Cruise

What is calcification?
• The accumulation of calcium salts into body tissue, such as bones, shells, and
carapaces.

• A biologically-mediated process

• In marine calcifiers, calcification predominantly results in calcium carbonate

structures that are made of either calcite, aragonite or high-Mg calcite.
What is calcification?
Ca2+ + 2HCO3-  CaCO3 + CO2 + H2O

Saturation State – degree to which seawater is saturated (or not) with relevant
ions; provides a measure of the thermodynamic potential for the mineral to form
or to dissolve

Ω = [Ca2+][CO32-] Ω > 1 Supersaturated with respect to CaCO3

Ksp’ Ω < 1 Undersaturated with respect to CaCO3 (dissolution)

Steinacher et al. 2009

Who calcifies, and how?
Major invertebrate calcifying groups:
- Molluscs
- Cnidarians
- Echinoderms
- Crustaceans
Other organism types:
- Formaminifera
- Phytoplankton: Haptophytes (coccolithophores)
- Algae: Rhodophytes (coralline algae)

In most biological systems, the site of mineral deposition is isolated from the
environment, the extent of isolation is variable.

Biologically induced mineralisation – organism uses cellular activities to direct the

nucleation, growth, morphology, and final location of the mineral that is deposited.
Several types, but most CaCO3 forming marine organisms either use an
extracellular biologically-controlled process or an intracellular strategy.
Who calcifies, and how?
Extracellular biologically-controlled process e.g. Molluscs, Corals,

• Basic form of calcification

• Organic matrix important for
defining structure
• Ions can be actively pumped
out of the cell or pumped into a
vesicle within the cell which is
then secreted outside.
Who calcifies, and how?
e.g. Corals
• Model of dissolved inorganic
carbon (DIC) absorption for
coral calcification and
photosynthesis.
• Extracellular space has
controlled pH environment
• Anion exchange pumps are
utilised for control

Furla et al. 2000

Who calcifies, and how?
Intracellular strategy. E.g. Echinoderms (urchins), coccolithophores…
• Can form huge mineralised products within
a vesicle that is the product of many cells
fusing their membranes.
• Mineral is exposed to the environment only
when the membrane is degraded.

Browlee & Taylor 2002

Who calcifies, and how?

• Crustaceans have complex moult cycles

• Able to reabsorb minerals from ‘old’ shell to
incorporate into ‘new’ shell
• High organic component, as well as chitin
• Organic matrix important for structuring
mineral formation
• Different parts of crustaceans (e.g. claws,
carapace, legs) have different mineral
content which determines ‘hardness’ and
Pane & Barry 2007; Photo MBARI (2006)
strength
Why should ocean acidification impact calcification?
1. Direct shifts in acid-base balance (pH, ionic composition) of intracellular fluids
that compromise calcification process
e.g. Corals

Venn et al. 2013

Why should ocean acidification impact calcification?
2. Enhanced dissolution in undersaturated conditions
e.g. dissolution of “dead” structures compared to “live”

Hennige et al. 2015

Findlay et al. 2011 Vad et al. in review

Why should ocean acidification impact calcification?
3. Additional energy requirements needed for maintaining and producing calcium
carbonate material in unfavourable conditions
e.g. trade-offs between physiological process… brittlestars, mussels, many others…

established
40
% calcium/ g of arm ± 95% CI

regrowth
35
30
25
20
15
10
Bibby et al. 2008
5
0
8 7.7 7.3 6.8
pH

Wood et al. 2008

Some definitions
• Gross calcification CaCO3 precipitated by an organism or community

• Net calcification CaCO3 precipitated by an organism or community minus

dissolution of CaCO3 from the organism or community.

• Potential calcification Gross calcification, assuming that the organisms

considered cover 100% of the area

• Net accumulation Amount of CaCO3 precipitated locally plus the amount of

material imported minus dissolution and export
Summary of techniques

• Geological approach
• Sedimentalogical approach
• Alkalinity Anomaly Technique
• pH-O2
• Change in calcium concentration
• Radioisotopes (45Ca, 14C, 3H-tetracycline)
• Changes in particulate calcium content
• X-ray analysis
• Buoyant weight
• “Biological” approach
• Changes in Particulate Inorganic Carbon content
• Molecular tools
Geological
CaCO3 accumulates in sediment over long time periods giving an indication of rates
of calcification.
Net accumulation of CaCO3 is calculated by the thickness of the layer multiplied by
the density, divided by the time increment (measured by radiocarbon dating)
Level: Community
Timescale: 1000-20000 years
Examples: Chave et al. (1972)
Pros: Provides integrated, long-term estimates
Cons: Numerous uncertainties and assumptions. Highly constrained by sea level

Turley et al. 2009

Sedimentalogical
Calcified organisms accumulate within sediments. Net calcification (?) is measured
using the percentage weight contribution in sedimentary skeletal components
Level: Community
Timescale: Months
Examples: Langer et al. (1997), Wienkauf et al. 2013
Pros: Only needs sediment samples.
Cons: It is not clear what this approach measures, it does not account for advection
terms

Weinkof et al. 2013

Alkalinity Anomaly Technique
Alkalinity is lowered by two equivalents for each mole of CaCO3 precipitated.
Net calcification is calculated by measuring the TA before and after an incubation
period, and the ∆TA is scaled to ∆CaCO3 (i.e. calcification = 0.5x∆TA )
Level: Organisms and communities
Timescale: Hours to weeks
Examples: Smith & Key (1975), Gazeau et al. (2007), Martin et al. (2013), Inoue et
al. (2013)
Pros: Very precise (1 SD = 3 µmol/kg or about 0.2%)
Cons: Needs discrete samples (but see Watanabe et al., 2004). A correction for
changes in nutrients may be needed. Need to enclose or know residence time.

Gazeau et al. 2007

pH-O2
Relationships exist between ΔO2 and ΔDICorg, the metabolic quotients.
Net calcification can be measured by estimating net community production and
respiration from changes in the concentration of dissolved O2. ∆DICcalc is then
calculated by subtracting ΔDICorg from the upstream DIC value. ∆DICcalc can be
converted to ∆TA and consequently calcification.
Level: Organisms and communities
Timescale: Hours
Examples: Chisholm & Barnes (1998), Barnes (1983)
Pros: It does not require TA monitor (which is timely)
Cons: Needs DIC (hence TA) upstream. Assumes metabolic quotients

Chrisholm & Barnes 1998

Calcium concentration
Calcium concentration can directly be measured within internal fluids of
organisms. Net calcification can be estimated from calcium removal measured
using chemical titrations or sensors
Level: Organisms and communities
Timescale: Minutes to weeks
Examples: Chisholm & Gattuso (1991), Al-Horani et al. (2003)
Pros: Direct measurement of calcium uptake; no major assumptions
Cons: Low detection limit, high background concentration (10 mmol/l)

Al-Horni et al. 2003

Radio isotopes
Calcium is taken up into the organisms skeletal components, the calcium uptake
can be measured using radiolabelled elements (45Ca, 14C and 3H) to estimate net
calcification
Level: Organisms
Timescale: Minutes to hours
Examples: Fabry et al. (1989), Comeau et al. 2010
Pros: Extremely sensitive, Short-term incubations
Cons: Destructive, Non-biological adsorption, Use of radioisotopes restricted

Comeau et al. 2010

Changes in particulate calcium
Calcium is taken up into the organisms skeletal components, the calcium
concentration can be measured by flame atomic absorption spectroscopy to give
an estimate of net calcification.
Level: Organisms
Timescale: Hours to days
Examples: (Stoll et al., 2002); (Findlay et al. 2011)
Pros: Precision is adequate when growth rates are high (cultures)
Cons: Analytical care Instrumentation

Findlay et al. 2011

X-rays

X-rays (and Computerised tomography (CT) scanning) measure the density and
mass of skeleton, providing a direct measure of net calcification, particularly
through time (using long-lived coral structures).
Level: Organisms
Timescale: days, months, to 100s years
Examples: Lough & Barnes (2000), Crook et al. (2013)
Pros: Enables retrospective analysis, provides an assessment of erosion
Cons: Requires substantial equipment & instrumentation

Crook et al. 2013

Buoyant weight
Increases in mass of an organisms skeleton directly correspond to increases in net
calcification.
Level: Organisms
Timescale: Sub-daily to months/years
Examples: Dodge et al. 1984, Jokiel et al. 2008
Pros: Quite sensitive, Not destructive, No incubation required
Cons: Serious problem of normalization for comparative analysis

Dodge et al. 1984

Biological approaches
Growth measurements or turnover rates (for populations) are associated with an
increase in mass of calcifed structure and can be used to estimate net calcification.
Techniques can include using flurouscent dyes (e.g. calcein staining) to observe
specific growth areas.
Level: Organisms
Timescale: Days, months to years
Examples: Fabry (1990), Smith (1972), Migné et al. (1998), Comeau et al. (2009)
Pros: Simple, individual level
Cons: Short term growth not always significant, lots of variability

Comeau et al. 2009

Changes in PIC
Changes in the content of the particulate carbon content of an organism reflect its
accumlation or loss of carbon and provide an estimate of net calcification.
Total particulate carbon (TPC) and particulate organic carbon (POC) are measured
(CHN analyzer, mass spectrophotometry). PIC = TPC - POC.
Level: Organisms
Timescale: Hours to days
Examples: Riebesell et al. (2000), Sciandra et al. (2003)
Pros: Adequate with cultures and field samples (?)
Cons: Instrumentation, Not amenable to automation

Riebesell et al. 2000

Molecular
Genetics controls the calcification process, by measuring the activity of genes
involved in the calcifcation process (measure mRNA) gives an idea of the gross
calcification (?)
Level: Organisms, perhaps communities?
Timescale: Hours (to days?)
Examples: Lohbeck et al. 2014
Pros: High sampling rate because no incubation required
Cons: Post-translational regulation, Poor precision (semi-quantitative), Reliance on
instrumentation (quantitative real-time PCR), not clearly related to actual
production of calcium carbonate skeleton.

Lohbeck et al. 2014

Generic measuring issues
• Considerably different units across the different techniques

• Measurements tend to need to be normalised

- organism: surface area, skeletal weight, body mass, biomass…
- communities: volumetric, surface area…

• Not trivial to compare!

• Most measure NET calcification – difficult to disentangle the impacts on

the organisms ability to calcify with dissolution.

Chan & Connolly, 2013

Summary
• Calcification ability has a connection to energy budgets

• Feeding rates may overcome some of the costs –will food supply
change?

• Dissolution rates will increase as saturation state decreases – important

for exposed material

• Bio-erosion may also further impact of OA

• Adaptation potential?

• Interactions between organisms

• Complexity of multiple stressors