Ocean acidification may increase calcification
rates, but at a cost
Hannah L. Wood1,*, John I. Spicer2and Stephen Widdicombe1
1Plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth PL1 3DH, UK
2Marine Biology and Ecology Research Centre, School of Biological Sciences, University of Plymouth,
Plymouth PL4 8AA, UK
Ocean acidification is the lowering of pH in the oceans as a result of increasing uptake of atmospheric
carbon dioxide. Carbon dioxide is entering the oceans at a greater rate than ever before, reducing the
ocean’s natural buffering capacity and lowering pH. Previous work on the biological consequences of
ocean acidification has suggested that calcification and metabolic processes are compromised in acidified
seawater. By contrast, here we show, using the ophiuroid brittlestar Amphiura filiformis as a model
calcifying organism, that some organisms can increase the rates of many of their biological processes
(in this case, metabolism and the ability to calcify to compensate for increased seawater acidity).
However, this upregulation of metabolism and calcification, potentially ameliorating some of the effects
of increased acidity comes at a substantial cost (muscle wastage) and is therefore unlikely to be
sustainable in the long term.
Keywords: ocean acidification; echinoderm; regeneration; muscle; hypercapnia; Amphiura filiformis
Since the start of the industrial revolution, atmospheric
levels of carbon dioxide (CO2) have been rising at a far
greater rate than previously experienced in the Earth’s
history. This is primarily a result of burning fossil fuels.
The oceans are a natural carbon sink and have so far
absorbed approximately half of all anthropogenically
produced CO2(Siegenthaler & Sarmiento 1993). When
CO2enters the ocean it reacts with seawater and alters
the chemical properties of the sea itself (Zeebe &
Wolf-Gladrow 2001). Among other things this process
produces hydrogen ions thus increasing the acidity
reflected in a lowering of the value of pH. Seawater pH
currently ranges between 7.8 and 8.2 and is already on
average 0.1 pH unit lower than it was prior to the
industrial revolution (Caldeira & Wickett 2003). Predic-
tions, based on realistic scenarios for future CO2
emissions, suggest that ocean pH will decrease by a
further 0.3–0.4 by 2100 (Caldeira & Wickett 2003),
a phenomenon termed ocean acidification. While the
magnitude of impact will vary with depth (Caldeira &
Wickett 2003), latitude (Orr et al. 2005) and habitat, the
effects of ocean acidification on seawater chemistry will
affect all marine organisms. An organism can be affected
by ocean acidification in two ways; firstly through reduced
pH and secondly through increased CO2(hypercapnia).
Here we use the term ocean acidification to include both.
Different species and groups of marine animals vary
in their ability to cope with, and compensate for,
hypercapnia and lowered pH (e.g. Po ¨rtner et al. 2004,
2005) with implications for marine trophic interactions.
Species with calcium carbonate skeletons, such as
molluscs, crustaceans and echinoderms, are particularly
susceptible to ocean acidification. As pH decreases, so
too does carbonate availability that has led some authors
to conclude that ocean acidification will result in
reduced rates of calcification (e.g. Gattuso et al. 1999)
and shell dissolution (e.g. Feely et al. 2004) for all
calcified organisms, as well as metabolic depression
resulting in reduced growth (e.g. Michaelidis et al.
2005). Echinoderm skeletons are composed of magne-
sium calcite that is particularly susceptible to dissolution
as ocean pH decreases (Shirayama & Thornton 2005).
Work on the effect of acidification on echinoderms is
currently restricted to investigations of survival, growth
and extracellular acid–base balance in a limited
number of groups, mainly echinoids (e.g. Shirayama &
Thornton 2005; Miles et al. 2007). One of the key
characteristics of many echinoderm species is their
ability to regenerate, which involves alterations in
calcification rates (Bowmer & Keegan 1983; Bannister
et al. 2005). However, we know nothing of the effect
of CO2-induced acidification on such regeneration.
Consequently, we have investigated the effect of CO2-
induced acidification on the ability of a calcifying
organism (the ophiuroid brittlestar Amphiura filiformis)
to regenerate calcium carbonate structures (arms). In
addition, we have examined the potential energetic costs
associated with regeneration in terms of metabolism and
reproduction. Amphiura filiformis is a key species in
many seafloor communities. Using sediment-filled cores
supplied with filtered seawater, brittlestars were exposed
for 40 days to varying degrees of acidification; nominally
a control of pH 8.0, the worst case scenario for the end
of the century of pH 7.7, a 2300 scenario of pH 7.3 and
finally pH 6.8.
Proc. R. Soc. B
Research was conceived by H.L.W., S.W. and J.I.S., performed and
analysed by H.L.W. The paper was written by H.L.W. with
contributions and advice from J.I.S. and S.W.
Electronic supplementary material is available at http://dx.doi.org/10.
1098/rspb.2008.0343 or via http://journals.royalsociety.org.
*Author for correspondence (firstname.lastname@example.org).
Received 12 March 2008
Accepted 11 April 2008
This journal is q 2008 The Royal Society
2. MATERIAL AND METHODS
(a) Experimental set-up
The experiment was carried out in a mesocosm facility
(Widdicombe & Needham 2007). Amphiura filiformis
collected from Plymouth Sound, UK, were maintained
in sediment cores (five individuals per core) supplied with
filtered seawater of the allocated pH (pH modified using
CO2). Each pH treatment (8.0, 7.7, 7.3 and 6.8) had four
cores (20 individuals per pH). Half the individuals in each
treatment had one arm removed and the remaining had
two arms removed. Total carbon dioxide content (TCO2),
pHNIST, salinity and temperature of the seawater were
measured for each treatment three times a week (figure 4).
(b) Oxygen uptake
Closed-bottle respirometry technique adapted from
Pomory & Lawrence (1999). Dissolved O2 measured
using an automated titration system with photometric
(c) Arm regeneration
Regenerated arm discernable from original arm by lighter
colour. Measured to 0.05 mm with vernier calipers.
(d) Measurement of calcium content
Arms were digested in nitric acid and the total calcium
content was determined using atomic absorption spectro-
photometer (Varian SpectrAA 50) as in Spicer & Eriksson
(e) Arm structure and measurement of egg size
Central discs were embedded in methacrylate (Lewis &
Bowen 1985), sectioned with a glass knife and stained
(Lee’s methylene blue/basic fuchin). Egg feret diameter
was measured by image analysis software (IMAGE-PRO
PLUS v. 4.5 Media Cybernetics) using a digital image
(!40 mag. Reichert Polyvar microscope).
(f ) Statistical analyses
All statistical analyses were run using MINITAB v. 14. The
two-way analysis of variance (ANOVA) was used to test for
effects of pH treatment or number of arms regenerating on
O2uptake, calcium content, arm regeneration rate, egg
size and arm structure. A Kolmogorov–Smirnov test was
used to test for normality.
3. EFFECT OF OCEAN ACIDIFICATION ON
CALCIFICATION IN A. FILIFORMIS
One of the most surprising results is that there was no
decrease in the total amount of calcium carbonate in
individuals exposed to acidified water. Indeed, individuals
from lowered pH treatments had a greater percentage of
calcium in their regenerated arms than individuals from
control treatments, indicating a greater amount of calcium
carbonate (two-way ANOVA using log transformed data,
table 1c). Established arms had a significantly lower
percentage of calcium carbonate content than regenerated
arms (figure 1c); however, number of arms removed had
no effect. The interaction between pH and arm type
(established or regenerated) was significant due to the
Table 1. Results from two-way ANOVA comparing the impact of different levels seawater pH (8.0, 7.7, 7.3 and 6.8) and number
of arms regenerating (1 or 2) on (a) oxygen uptake (mmol dK1gK1animal); nZ80, data were of normal distribution after square
root transformation (nZ80). (b) Length of arm regeneration (mm); nZ40 and normally distributed. (c) Calcium content of arm
(%). Comparing pH treatments (8.0, 7.7, 7.3 and 6.8) and number of arms regenerating (1 or 2) and established arms (denoted
as arm regeneration); nZ60 and data were normally distributed after log transformation. The significance level was 0.05. (d)
Mean egg feret (mm); nZ40 and normally distributed.
2 H. L. Wood et al. Biological cost of ocean acidification
Proc. R. Soc. B
different amount of calcium found in each of the arm
types; the regenerated arms having significantly greater
calcium levels than established ones (figure 1c). This was
due to the more developed skeletal structure seen in the
established arms. Throughout the exposure, and therefore
during the period of regeneration, the brittlestars
were maintained in sediment cores (also collected from
Plymouth Sound) in order to simulate natural conditions
for the species. The inorganic carbon levels (%) for this
sediment are 2.207 (G0.176; S. Widdicombe 2005, 2007,
unpublished data). However, it is unlikely that the
sediment is being used as a carbon source for the
calcification process; a previous study found no change
in the carbon (TIC) content of the same type of sediment
(fine muddy) containing species including A. filiformis
after a 20-week exposure to pHs of 7.3, 6.5 and 5.6
(Widdicombe et al. submitted).
The sediment pH profiles from another acidification
study using sediment cores (S. Widdicombe 2005, 2007,
unpublished data) show that the pH of the sediment is
lower than that of the overlying water even under
normocapnic conditions; the pH is 7.64 at a depth of
5 cm, the depth at which A. filiformis is typically found.
However, after a four-week exposure to mild hypercapnic
conditions (overlying water pH 7.7) the sediment pH at
5 cm deep was still 7.64, while more severe hypercapnia
(pH 7.3 and 6.5) only reduced sediment pH at 5 cm depth
by 0.16 and 0.22 pH units, respectively. In a study by
Widdicombe et al. (in preparation), cores of both muddy
and sandy sediment were exposed to acidified seawater
(pH 7.8, 7.4 and 6.8) for 60 days. After this time oxygen
profiles were measured through the sediment. It was
demonstrated that seawater acidification had no signi-
ficant impact on the sediment oxygen profiles in either the
sand or the mud, indicating no increase in sediment
anoxia. pH imaging of Nereis succinea burrows showed that
the porewater pH was dependent on the burrow profile,
animal size and rate of irrigation, with high porewater pH
associated with periods of irrigation (Zhu et al. 2006).
Amphiura filiformis continually ventilate their burrows by
arm undulation; therefore, the pH of their burrow
porewater is expected to be related to surface water pH
rather than the surrounding sediment. As such, the
burrowing lifestyle of this study species is not counter-
acting or altering the experimental pH conditions created
for the purposesof this study and the results shown areas a
result of altering seawater pH.
To disentangle the direct chemical effect of pH on the
calcium carbonate within A. filiformis arms from the active
biological processes used by the species to maintain
calcium carbonate structures, a separate 7-day exposure
at all four pH treatments was carried out on ‘dead’ arms.
The arms were removed from the animal, frozen for a
period in excess of 7 days to K808C to kill, and then
brought back to seawater temperature. In this experiment,
the dead arms were placed in small pots with no sediment
and supplied continuously with seawater of appropriate
pH. Under these conditions calcium levels decreased with
Figure 2. Calcium content (%) of arms which had been
exposed to lowered pH after being removed from animal. All
values are means G95% CI.
calcium content of
arm per gram
arm regrowth (mm)
1 arm regenerating
2 arms regenerating
( mol d–1 per gram animal)
egg diameter ( m)
Figure 1. Impact of seawater pH on (a) oxygen uptake (mmol per day per gram animal), (b) length of arm regeneration (mm),
(c) calcium content of established and regenerated arms (%; hatched bars, established; dotted bars, regenerated) and (d) egg
feret diameter (mm) following a 40-day exposure. All values are means G95% CI.
Biological cost of ocean acidification
H. L. Wood et al.
Proc. R. Soc. B
pH (figure 2). As these arms were detached from the
individual and therefore could not replenish the calcium
carbonate skeleton, the decrease in calcium indicates that
this structure is susceptible to dissolution at lowered pH.
Therefore, in live (attached) arms, an increased rate of
calcification is required merely to maintain calcium
carbonate structures in their original condition. In
regenerated arms, calcium levels were greater in those
organisms exposed to acidified seawater than in those held
in untreated seawater (figure 1c). This was true for all
three levels of acidified seawater. The data from the
detached (dead) arms (figure 2) showed that lowered pH
caused dissolution of arm calcium carbonate. Therefore,
where these three lowered pH treatments appear to have
had a similar response, there was actually an increasing
rate of calcification with lowered pH. Calcium carbonate
in established arms was also affected by lowered pH. At
pH 6.8, calcium levels increased and at pH 7.7 and pH
7.3, calcium levels were equal to the control indicating
that A. filiformis actively replaced calcium carbonate lost
4. EFFECT OF OCEAN ACIDIFICATION ON
A. FILIFORMIS METABOLISM
Rates of oxygen (O2) uptake (as a measure of metabolic
rate), or MO2, were significantly greater at reduced pHs
(7.7, 7.3 and 6.8) than in controls (pH 8; figure 1a).
However, MO2was not significantly different between the
three lowered pH treatments (figure 1a). Increased rates
of physiological processes that require energy are paral-
leled by an increase in metabolism; this relationship is seen
with growth and metabolism here in our results.
Figure 3. Longitudinal cross sections of (a) established and (b) regenerated arms (!10 mag) mounted in methacrylate resin and
stained with Lee’s basic blue fuchin. ((i) pH 8, (ii) pH 7.7, (iii) pH 7.3 and (iv) pH 6.8).
4 H. L. Wood et al. Biological cost of ocean acidification
Proc. R. Soc. B
5. EFFECT OF OCEAN ACIDIFICATION ON
A. FILIFORMIS GROWTH AND REGROWTH
Seawater acidification stimulated arm regeneration.
After the 40-day exposure, the length of the regenerated
arm was greater in acidified treatments than in the
controls (two-way ANOVA, table 1b; figure 1b). This
increased rate of growth coincided with increased
metabolism. Regeneration was not affected by the
number of arms removed, nor was there a significant
difference in any of the physiological parameters
measured as a result of having two arms regenerating
instead of one. The ability to regenerate lost arms faster
meant a reduction in the length of time animal function
(e.g. burrow ventilation and feeding) was compromised
by reduced arm length.
6. THE BIOLOGICAL COST OF OCEAN
The internal structure of A. filiformis arms was affected by
pH (figure 3); muscle wastage occurred at lowered pH.
Longitudinal sections of the arm showed distinct loss of
the control individuals, these sections were filled with
muscle. As pH decreased, large empty spaces were clearly
visible (figure 3). Candia Carnevali et al. (2001) found that
muscle de-differentiation occurred in regenerating arms as a
result of PCBs, which appeared visually similar to muscle
in structure not seen in our study; the arm muscle from
lowered pH samples has the same visual structure as the
controls, with just less present. In addition, our results
showed muscle loss in established arms as well as
regenerating, whereas Candia Carnevali et al. (2001) found
de-differentiation in the regeneration process. The absence
of muscle as a result of lowered pH is not de-differentiation
but rather muscle loss. In conclusion, arms can be
regenerated under hypercapnic conditions but they are
unlikely to function as well as arms regenerated under
food particles and irrigateits burrow. Therefore, muscle loss
turn will affect both feeding and respiration and ultimately
survival. This species is also predated on by the commercial
flatfish dab, Limanda limanda (Bowmer & Keegan 1983),
which crop the arms extended into the water column. If the
muscle mass in these arms is significantly reduced, so too is
the nutritional value, indicating the effects of ocean
acidification could be transferred between trophic levels.
7. EFFECT OF OCEAN ACIDIFICATION ON
A. FILIFORMIS REPRODUCTION AND MORTALITY
Egg size (feret diameter) and structure were not affected
by seawater acidification (two-way ANOVA, table 1d;
figure 1d ). However, the timing of this study (December–
January) falls in a latent period of egg growth; develop-
ment of eggs laid down the previous autumn typically
begins in March (Bowmer 1982). Therefore, while no
degeneration of eggs was found in this study, egg
development may still be affected by hypercapnia.
A further experiment encompassing the egg growth
phase is required to assess the impact of ocean acidifi-
cation on egg development. A study by Lowe et al.
(in preparation) has found that the process of vitellogen-
esis in the surface dwelling ophiuroid Ophiura ophiura was
disrupted by lowered pH, highlighting the potential for
disruption in the growth phase. Spermatogonia were not
investigated in the current study as all individuals sampled
were female. While the sex ratio of A. filiformis is thought
to be 1 : 1, patchiness in the distribution of sexes has been
documented (Bowmer 1982), which may explain the
reason for the absence of males from the samples fixed for
While some ophiuroids reallocate energy from gonadal
to somatic growth, and a decrease of egg size is seen when
arm regeneration is undertaken, this was not seen in
A. filiformis (figure 1d). Ocean acidification has the
potential to also affect reproductive success indirectly; as
a broadcast spawner, A. filiformis must come to the
sediment surface to spawn. This behaviour requires
the arms to move the individual through the sediment
and should arm muscle wastage reduce motility then
individuals may release gametes within their burrows and
far fewer gametes would enter the water column;
significantly reducing reproductive success.
The duration of this experiment (40 days) was chosen
to investigate long-term physiological responses to
hypercapnia. Shirayama & Thornton (2005) have
elegantly demonstrated with echinoids that mortality as
TCO2 (mmol l–1)
Figure 4. Water conditions during experiment on (a) pH(NIST)(diamonds, 8.0; squares, pH 7.7; filled triangles, pH 7.3; and
open triangles, pH 6.8) and (b) TCO2(mmol lK1). All values are means G95% CI.
Biological cost of ocean acidification
H. L. Wood et al.
Proc. R. Soc. B
a result of a 0.05 pH decrease (560 ppm) may only
occur after several months. Interestingly, even at high
levels of hypercapnia (the 6.8 pH treatment crosses the
threshold into acidic water, i.e. pH!7.0) investigated
here, no mortality was observed. In light of the results
regarding the trade-off between calcification and muscle
mass it is probable that mortality at low pH will occur as
an indirect result of lowered pH, and this may take
longer than the experimental duration. Any loss, or
impairment, of an important ecosystem engineer (Jones
et al. 1994) will profoundly affect the biotic and abiotic
environments where they occur; therefore, the potential
for loss of this species would alter ecosystems on a large
All previous ocean acidification studies on benthic marine
invertebrates have reported reduced calcification rates
(Gazeau et al. 2007) and hypometabolism (Michaelidis
et al. 2007) as common outcomes. Here we have shown
the opposite; that in some species at least, ocean
acidification can increase both the rate of calcification
and metabolism. These results change the face of
predictions for future marine assemblages with respect to
ocean acidification. Whereas it was previously assumed
that all calcifiers would be unable to construct shells or
skeletons, and inevitably succumb to dissolution as
carbonate became undersaturated, we now know that
this is not the case for every species. However, by
investigating the functional consequences of hypercapnia
and lowered pH at an organism level rather than focusing
on a single process, we have also detected a cost to these
increased activities. Arm muscle mass decreased with pH,
i.e. as calcification and metabolism increase. There was a
trade-off between maintaining skeletal integrity and arm
function. pH decreased the arm muscle mass by causing
the brittlestar to use the muscle as an energy source. As
muscle loss was seen in established as well as regenerated
arms, it is clearly not just a failure to synthesize muscle
tissue under hypercapnic conditions. For this particular
ophiuroid species, the loss of muscle mass experienced at
low pH has implications for survival and ecosystem
function; arm movement is necessary for feeding (Loo
et al. 1996), burrow aeration (Woodley 1975) and
predator avoidance (O’Reilly et al. 2006). In areas where
this animal is present, burrow creation and irrigation by
A. filiformis is responsible for up to 80% of all bioturbation
(Vopel et al. 2003); therefore, the effects of ocean
acidification will also alter the surrounding environment.
Results of a previous study indicate that this trade-off of
increased calcification against reduced muscle mass is
occurring in other species; Shirayama & Thornton (2005)
found that the decrease in test thickness did not account
for total mass loss of the echinoderms Hemicentrotus
pulcherrimus and Echinometra mathaei exposed to hyper-
capnic conditions. These species may also be decreasing
muscle mass as a cost of increasing calcification and
metabolism. Here we show that A. filiformis, and almost
certainly other species, will attempt to cope with changes
in seawater acid–base balance. Unfortunately, it appears
that the physiological responses to combat the effects of
ocean acidification may themselves reduce survival and
fitness as much as acidification itself.
The Intergovernmental Panel on Climate Change
predicts that under their worst case scenario of carbon
dioxide emissions, seawater pH will reach our experi-
mental level of 7.7 by 2100. Here we show that some
species at least can modulate their biological processes in
response to ocean acidification and while calcified
structures are affected by ocean acidification, so too is
the rest of the animal. Such trade-offs are likely to be
present in other species but to identify these, future studies
need to work at the organism rather than the process level.
To place the importance of calcification above other
factors without empirical evidence leads to false assump-
tions and therein the capacity of some species to respond
effectively may be overlooked.
This study was funded by a NERC Blue Skies PhD
studentship awarded to H.L.W. The study used the mini-
mum number of animals necessary to ensure scientific
robustness. Under the Animals (Scientific Procedures) Act
1986, work with echinoderms is not a licensable activity.
We thank H. Findlay for critical discussions and assistance in
the laboratory, D Lowe & C. Pascoe for advice on using
methacrylate and sectioning technique and A. Beesley for
assistance with collecting water data.
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