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, c
). Established arms had a significantly lower percentage of calcium carbonate content than regenerated arms (c
); however, number of arms removed had no effect. The interaction between pH and arm type (established or regenerated) was significant due to the different amount of calcium found in each of the arm types; the regenerated arms having significantly greater calcium levels than established ones (c
). 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 The inorganic carbon levels (%) for this sediment are 2.207 (±0.176; S. Widdicombe 2005, 2007, unpublished data).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
Figure 1 Impact of seawater pH on (a) oxygen uptake (μmol 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 (more ...)
The sediment pH profiles from another acidification study using sediment cores (S. Widdicombe 2005, 2007, unpublished data) show that the pH of the sediment is lower than that of the overlying water even under normocapnic conditions; the pH is 7.64 at a depth of 5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 significant 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 counteracting or altering the experimental pH conditions created for the purposes of this study and the results shown are as 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 −80°C 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 pH (). 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 (c). This was true for all three levels of acidified seawater. The data from the detached (dead) arms () 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 by dissolution.
Calcium content (%) of arms which had been exposed to lowered pH after being removed from animal. All values are means ±95% CI.