Carbon emission associated with respiration and calcification of nine gastropod species from the intertidal rocky shore of Western Europe

Intertidal rocky shores are characterized by vertical zonation that results from the interplay between environmental conditions, organism physiology, and species interactions. Metabolism of intertidal organisms is highly variable between species and it changes with vertical position along the intertidal gradient. The present study aimed to quantify the carbon metabolism of nine intertidal rocky shore gastropods, in order to clarify their respective roles in carbon production during emersion and immersion. The influences of monthly temperature variation and tidal level were tested for each species. Analyses were performed in the laboratory using the infrared gas analyzer method for measuring aerial respiration rates, and the dissolved inorganic carbon and total alkalinity technique for measuring aquatic respiration rate and calcification. Hourly carbon fluxes were calculated for the mean annual temperature of 13 °C measured in both air and underwater in the study area. Respiration rates were similar for emersion (8–25 μmol CO₂ g AFDW^?1 h^?1) and immersion (10–23 μmol DIC g AFDW^?1 h^?1). For all species, underwater respiration fluxes were more influenced by monthly temperature variation than by air fluxes, probably as an adaptation to the rapid changes occurring during emersion. Calcification was an important factor influencing annual carbon fluxes for all studied species; every species showed different calcification rates according to its size and position on the intertidal zone. Annual carbon emissions were calculated using the mean immersion/emersion time of each species. Intertidal gastropod carbon emission was primarily influenced by body biomass and their vertical position within the intertidal zone.     

Respiratory metabolism of crabs from marine and estuarine habitats: an interspecific comparison

The gill-surface area and R-T (metabolic rate-temperature) response during aquatic and aerial respiration of three different species of crabs, the subtidal Scylla serrata, the intertidal Sesarma quadratum, and the supratidal Ocypoda platytarsis, were compared. Scylla serrata has the highest, Sesarma quadratum the second highest, and O.platytarsis the lowest gill-surface area. Oxygen-consumption values in water under different ambient partial pressures of oxygen indicate that all three species display equally efficient aquatic respiration despite variation in gill area and number. There is no coherent R-T response either inter- or intra-specifically. However, the ability to extract oxygen from the surrounding medium (respiratory efficiency) conditions the R-T response in the different species. The R-T trends in aquatic respiration of Scylla serrata and aerial respiration of O.platytarsis reveal a normal response to temperature. The proportion of aquaticoxygen uptake to total respiration in all sizes in both Scylla serrata and Sesarma quadratum remains more or less constant (between 88 and 92%), whereas in the supratidal O.platytarsis, this proportion decreases with increasing weight (from 88 to 64 %), indicating a progressive loss of gill respiration to total respiration. All three species are metabolically equally efficient; what one lacks in respiratory surface it compensates by respiratory efficiency.     

Adaptive phenotypic differentiation across the intertidal gradient in the alga Silvetia compressa

Populations of intertidal species span a steep environmental gradient driven by differences in emersion time. In spite of strong differential selection on traits related to this gradient, the small spatial scale over which differences occur may prevent local adaptation, and instead may favor a single intermediate phenotype, or nongenetic mechanisms of differentiation. Here I examine whether a common macroalga, Silvetia compressa, exhibits phenotypic differentiation across the intertidal gradient and evaluate how local adaptation, developmental plasticity, and maternal effects may interact to shape individual phenotypes. Reciprocal transplants of both adults and embryos showed a "home-height advantage" in two of the three populations tested. In laboratory trials, the progeny of upper-limit individuals survived exposure to air significantly better than lower-limit progeny from the same population. I compared the emersion tolerance of full-sib families generated from gametes produced in the field to those produced under common garden conditions. The relative advantage of upper-limit lineages was robust to maternal environment during gametogenesis; this pattern is consistent with genetic differentiation. The possible role of local adaptation has historically been ignored in studies of intertidal zonation. In S. compressa, phenotypic differentiation may have important consequences for vertical range, both within and among sites.     

Nutritional status affects the capacity of the snail Concholepas concholepas to synthesize Hsp70 when exposed to stressors associated with tidal regimes in the intertidal zone

Synthesis of the heat shock protein Hsp70 is one of the most important physiological mechanisms that intertidal organisms possess to counteract damage to macromolecules caused by stressors associated with the tidal cycle. However, the synthesis and activity of Hsp70 involves an elevated energetic cost. We evaluated the effect of the nutritional status (fed vs. starved for 2 weeks) of juvenile Concholepas concholepas mollusc on their capacity to synthesize Hsp70 during emersion (i.e. low tide) and immersion (i.e. high tide) at high temperatures (24 °C, e.g. summer conditions) and at low temperatures (7 °C, e.g. winter conditions). In addition, we evaluated whether Hsp70 is induced directly upon exposure to stress (emersion) or during recovery (re-immersion). Starvation decreased the content of stored energy substrates of juveniles as well as their ability to synthesize Hsp70 during emersion under thermal stress, especially at high temperatures. Additionally, analysis of environmental factors associated with laboratory simulation of tidal regimes indicated that juveniles in starvation, in contrast to fed juveniles, did not significantly increase their levels of Hsp70 during cold emersion (7 °C) or warm emersion (24 °C) or upon re-immersion. Induction of Hsp70 occurred during exposure to stress (low-tide conditions) and not when juveniles returned to “normal” conditions (high-tide conditions). Thus, the synthesis of Hsp70 for the juveniles of this intertidal snail species was coordinated and adapted to the tidal cycle, and the species responds in a similar way to hot and cold emersion conditions. The observed levels of Hsp70 reflect the ability of the individual to synthesize these proteins, which is dependent on the nutritional status of the individual.     

Huddling up in a dry environment: the physiological benefits of aggregation in an intertidal gastropod

In many intertidal gastropods, the formation of aggregations and closing of the opercular opening are behaviors commonly assumed to be associated with water conservation and maintenance of body temperature during tidal emersion periods. In the laboratory, we quantified the relationship between these two behaviors in a littorinid snail common to the north-central shores of Chile, Echinolittorina peruviana, and evaluated any benefit of these behaviors during desiccating conditions. We predicted that solitary individuals would maintain their opercula open for less time than aggregated snails when exposed to drier conditions due, at least in part, to differences in evaporative water loss. In laboratory trials, where relative humidity was manipulated, we observed that aggregated snails maintained their opercula open for longer periods of time than solitary snails under increasingly drier conditions. These results, together with observations of body temperature, suggest that aggregated animals may able to maintain gaseous exchange with their environment for longer periods of time than solitary individuals in response to desiccation stress. Our results suggest an interactive effect of two behaviors that confer physiological benefits when confronted with extreme physical conditions experienced during periods of emersion.     

Effects of emersion and temperature upon growth of the tropical brown alga Padina japonica,using a tide-simulation apparatus

A simple tide-simulation apparatus was used to investigate the influence of emersion and temperature on the intertidal growth of Padina japonica Yamada in both the juvenile and adult growth phases. The upper zonation limits are not determined by the sensitivity of any particular growth phase, since all phases show the same emersion tolerance limits. The species grows best when continuously submerged, and growth rates decrease with increasing emersion up to a clear upper zonation boundary. High temperature reduces the emersion tolerance and is therefore an important factor in tropical intertidal zonation.     

滨海不同生境下盐地碱蓬生物量分配特征研究

盐地碱蓬是黄河三角洲重要的先锋植物,抗盐能力强,分布范围广,对维持该地区生态系统稳定和演替发挥着重要作用。通过野外取样测定,研究了潮间带和潮上带盐地碱蓬种群的形态及生物量分配特征。在个体大小上,潮间带盐地碱蓬在株高和地径上显著低于潮上带盐地碱蓬;潮上带盐地碱蓬密集分布的株高高于散生分布,而生物量低于散生分布。在生物量分配特征上,潮间带盐地碱蓬Ⅰ以发展花、叶为主,潮上带盐地碱蓬Ⅱ的密集分布以优先发展茎为主,潮上带盐地碱蓬Ⅲ的散生分布以发展枝为主,属于种群发展的稳定阶段。因此,盐地碱蓬通过形态特征及生物量分配特征的调节,以适应潮间带和潮上带的不同生境特征,从而达到种群维持和土壤改良的目的。     

Corallina and Ellisolandia (Corallinales,Rhodophyta) photophysiology over daylight tidal emersion: interactions with irradiance,temperature and carbonate chemistry

The photophysiology of three geniculate coralline algal species (Corallina officinalis, C. caespitosa and Ellisolandia elongata) was determined in intertidal rock pools in the south-west UK at Combe Martin (51°12′31N 4°2′19W) and Heybrook Bay (50°31′66N 4°11′41W), at the start, middle and end of summer (September 1 and 2) and winter (February 9 and 10) daylight tidal emersion periods, in relation to prevailing irradiance, temperature and carbonate chemistry conditions. Algal photophysiology was assessed from rapid light curves performed using pulse amplitude modulation fluorometry. Corallina and Ellisolandia experienced significant fluctuations in irradiance, temperature and carbonate chemistry over seasonal and tidal cycles. Rock pool carbonate chemistry was predictable (R ² = 0.82, P < 0.0001) by photodose (summed irradiance) plus water temperature, but not significantly related to photophysiology. In contrast, Corallina and Ellisolandia relative maximum electron transfer rate showed a significant negative relationship (R ² = 0.65, P < 0.0001) with irradiance plus water temperature. At a seasonal resolution, photoacclimation to maximize both light harvesting during winter months and photoprotection during summer months was observed for all species. Dynamic photoinhibition was apparent over both summer and winter tidal emersion, in relation to irradiance fluctuations. More effective photoinhibition was apparent during summer months, with greater sensitivity to irradiance and slower recovery in F _v/F _m, observed during winter. With sustained high irradiance over tidal emersion, the establishment of high pH/low inorganic carbon conditions may impact photochemistry. This study represents the first assessment of C. officinalis, C. caespitosa and E. elongata photophysiology underpinned by clear species concepts and highlights their ability to adapt to the dramatically fluctuating conditions experienced in intertidal rock pools.     

滨海不同生境下盐地碱蓬生物量分配特征研究

盐地碱蓬是黄河三角洲重要的先锋植物,抗盐能力强,分布范围广,对维持该地区生态系统稳定和演替发挥着重要作用。通过野外取样测定,研究了潮间带和潮上带盐地碱蓬种群的形态及生物量分配特征。在个体大小上,潮间带盐地碱蓬在株高和地径上显著低于潮上带盐地碱蓬;潮上带盐地碱蓬密集分布的株高高于散生分布,而生物量低于散生分布。在生物量分配特征上,潮间带盐地碱蓬Ⅰ以发展花、叶为主,潮上带盐地碱蓬Ⅱ的密集分布以优先发展茎为主,潮上带盐地碱蓬Ⅲ的散生分布以发展枝为主,属于种群发展的稳定阶段。因此,盐地碱蓬通过形态特征及生物量分配特征的调节,以适应潮间带和潮上带的不同生境特征,从而达到种群维持和土壤改良的目的。     

Mechanisms of crustacean egg hatching: Evidence for enzyme release by crab embryos

During the summer of 1989, we examined mechanisms of egg hatching in three species of brachyurans that occupy different habitats as adults near Beaufort, North Carolina, USA:Neopanope sayi (Smith) (subtidal),Uca pugilator (Bosc) (intertidal) andSesarma cinereum (Bosc) (supratidal). Results of casein assays indicated that embryos of all species release proteolytic enzymes near the time of egg hatching. Species differences in specific enzyme activity were suggested, with increased activity in the more terrestrial crabs. Embryos ofN. sayi released enzymes several hours before larval release by the female, whileU. pugilator andS. cinereum released enzymes closer to the time of larval release; enzyme release coincided roughly with time of egg-membrane breakage in all species. Direct observations of hatching showed a sequence of outer-membrane breakage apparently followed by inner-membrane breakage and emergence of the larva. Egg volumes increased most during early and/or mid-stages of development, with a marked slowing of the increase during the several days before hatching. Thus, a gradual osmotically-driven increase in water content may also be involved in egg hatching.     

Desiccation as a factor in the intertidal zonation of barnacles

Four species of balanomorph barnacles, Balanus crenatus Brugière, B. balanoides (L.), Elminius modestus Darwin and Chthamalus stellatus (Poli), were studied to assess the susceptibility of intertidal barnacle species to desiccation. Known sized samples of barnacles were exposed to controlled desiccating conditions and subsequent survival and water loss were determined. It is clear that the ability to live high on the shore is dependent on a reduction of the overall permeability to water loss. Because of greater surface area to volume ratios, small stages are particularly prone to desiccation. In normal intertidal emersion periods, small stages of B. crenatus particularly, and also of B. balanoides and E. modestus which are similar in their desiccation resistance, would be susceptible to desiccation at normal temperatures and low humidities. Large barnacles would be more prone to death from high temperatures when the tide is out. The spat of C. stellatus, although surviving much longer than spat of larger dimensions of the other species, must also be prone to prolonged emersion conditions at high shore levels.     

Comparisons of genetic population structures in four intertidal brachyuran species of contrasting habitat characteristics

Genetic population structures along the Japanese coast, analyzed by sequence data from the mitochondrial DNA COI region, were determined for four intertidal brachyuran species in the superfamily Thoracotremata (Ocypode ceratophthalma, Gaetice depressus, Chiromantes dehaani and Deiratonotus japonicus), which were characterized by different habitat requirements. O. ceratophthalma (seashore; supratidal sand) and C. dehaani (estuarine; supratidal marsh) showed no significant genetic differentiation among Japanese populations. The Japanese populations of O. ceratophthalma, however, were found to genetically differentiated from the Philippine population. G. depressus (seashore; intertidal cobbles) exhibited significant genetic differentiation between the Amami-Ohshima population and other local populations. D. japonicus (estuarine; intertidal cobbles) showed significant genetic differentiation among many local populations separated by about 30–1,200 km. The different patterns of genetic population structure recorded for the four species, thus, do not simply correspond to habitat type.     

Einfluß von 2,4-dinitrophenol auf die atmung und die Konzentration einiger metabolite bei embryonen des herings Clupea harengus

In rearing experiments with herring eggs (temperature=14.0°±0.1°C; salinity=15‰), oxygen consumption under normal conditions and after addition of 2,4-DNP (concentration=0.1 mM/l; pH=8.1) was measured over the period of embryonic development by means of the Wabburg-technique. Additionally, the concentration of low molecular sugars, polysaccharides, free amino-acids, and adenosintriphosphate (ATP) was determined. The oxygen consumption increases during embryonic development; this increase is not linear. Periods of high intensity of oxygen consumption are followed by others with only slight increase. Immediately before hatching, the respiration curve distincly declines (Fig. 1). Under the influence of 2,4-DNP (dinitrophenol), the embryos increase their respiration intensity after a short period of incubation. The maximum rise in percentage over the normal values reaches up to 400% at the beginning of gastrulation, falls to 50% even before the locking of the blastopore, and decreases slightly to about 30% until hatching. The immense decline in the percentage increase in respiration following the addition of 2,4-DNP at the end of the first day of development is caused by the rapid increase in normal respiration. After poisoning with DNP at different stages of development, the uncoupled respiration curves are normally almost equal. This holds both for the temporal position of the respiration maxima (about 12 to 24 h after poisoning), and for the absolute amounts of the increased respiration over the normal values (5 to 7 μl/h/100 embryos). Excluded from these regularly repeated findings are two stages of development: (1) the stage of epiboly after exceeding the yolk equator until shortly before locking of the blastopore (26 to 32 h after fertilization at 14°C); (2) the period at the end of the 4th day of development when the eyes become pigmented (100 to 120 h after fertilization). These two stages are characterized by the fact that, at the moment of poisoning, the normal respiration shows retarded activity. On the other hand, these two stages are well able to undergo periods of development in which long-living embryonic deformations can occur after uncoupling of respiration with 2,4-DNP. The content in low molecular sugars and polysaccharides decreases slightly in the course of embryonic development and, following the addition of 2,4-DNP, decreases considerably during the first 24 h. After 48 h, accelerated decomposition of carbohydrates continues. Under the influence of 2,4-DNP, the embryos metabolize more carbohydrates in 1 day than during the whole normal development period. The changeover of the metabolism to increased decomposition of carbohydrates can be explained as a dislocation of the energetic sources from the respiration chain to glycolytic phosphorylation. In accordance with these facts, the concentration of free amino-acids, almost equal during normal embryonic development, remains unchanged under the influence of 2,4-DNP.     

Comparisons of genetic population structures in four intertidal brachyuran species of contrasting habitat characteristics

Genetic population structures along the Japanese coast, analyzed by sequence data from the mitochondrial DNA COI region, were determined for four intertidal brachyuran species in the superfamily Thoracotremata (Ocypode ceratophthalma, Gaetice depressus, Chiromantes dehaani and Deiratonotus japonicus), which were characterized by different habitat requirements. O. ceratophthalma (seashore; supratidal sand) and C. dehaani (estuarine; supratidal marsh) showed no significant genetic differentiation among Japanese populations. The Japanese populations of O. ceratophthalma, however, were found to genetically differentiated from the Philippine population. G. depressus (seashore; intertidal cobbles) exhibited significant genetic differentiation between the Amami-Ohshima population and other local populations. D. japonicus (estuarine; intertidal cobbles) showed significant genetic differentiation among many local populations separated by about 30–1,200 km. The different patterns of genetic population structure recorded for the four species, thus, do not simply correspond to habitat type. An erratum to this article can be found at     

Hypoxic life of intertidal acorn barnacles

Oxygen levels were monitored within the mantle cavities of three barnacle species ( Chthamalus stellatus, Semibalanus balanoides, Elminius modestus), using optode microsensors. Conditions were always hypoxic, even when barnacles were actively using the prosoma and cirri to pump aerated seawater into the mantle cavity. Mantle fluid oxygen concentrations were extremely variable; behaviour and oxygen concentrations were not closely coupled. Ventilation of the mantle cavity depended partially on external water flow, with higher and more stable mantle fluid oxygen concentrations being sustained when the water around barnacles was agitated. During emersion, barnacles initially pumped seawater between the mantle cavity and the cone above the opercular plates to achieve ventilation. As water was lost it was replaced by air bubbles, eventually resulting in an air-filled mantle cavity. In S. balanoides and E. modestus, once the mantle cavity was filled with air, the barnacle usually used up the oxygen within the bubble within 2–3 h and did not regain oxic conditions until the barnacle was reimmersed in seawater. In C. stellatus, the air bubble was repeatedly refreshed for many hours by pneumostome formation. In response to low environmental salinity, all three species closed the opercular plates firmly and rapidly used up oxygen within the mantle fluid.     

Photosynthetic performance,oxidative damage and antioxidants in <Emphasis Type="Italic">Cylindrotheca closterium</Emphasis> in response to high irradiance,UVB radiation and salinity

Cylindrotheca closterium is a common marine diatom living in intertidal environments where it can be present both in the water column and on sediments, depending on the tidal regime. In the present work this diatom was employed to investigate the responses to desiccation and to increase in PAR and UVB intensity, as occurs during emersion. Under these circumstances, the production of active oxygen species (AOS) may be enhanced resulting in an oxidative stress. Stress responses in this species were measured by exposing it to normal (30) and double salinity (60), supplying light of low or high intensity for 12 h, in the latter case either without or with moderate dose rates of UVB. Pulse amplitude modulated fluorometry was used to measure Chl a autofluorescence (F ₀), an index of photosynthetic efficiency of PSII (F _v/F _m) and the relative electron transfer rate (rETR). The oxidative stress was evaluated by analysing GSH pools and SOD activity. It was observed that at double salinity and under low light, intracellular pools of reduced glutathione (GSH) were higher than under the two conditions of high light without and with UVB at both salinities. The antioxidative defence activity of superoxide dismutase (SOD) was far higher under hypersaline conditions. The oxidative damage was evaluated as protein and lipid damage. The results showed that it expressed itself mainly through protein peroxidation: at normal salinity relative protein carbonyl content was (a) twice as high as in cells grown at double salinity, and (b) three times as high under UVB. Total unsaturated lipid contents doubled under hypersalinity conditions. The lipid peroxidation marker malondialdehyde showed the strongest response to low light and UVB at salinity value of 60. Lipid peroxide content was significantly higher at salinity of 60 compared to normal salinity and was the highest under low light and high light with UVB. The simulated emersion condition of the diatom seems to lead to the establishment of a balance between damage and repair, expressed mainly as (a) oxidative protein damage at normal salinity, in particular due to UV radiation, (b) sufficient protection by SOD activity mainly under hypersaline conditions.     

Freezing rate and duration determine the physiological response of intertidal fucoids to freezing

Differential thermal analysis (DTA) was used to measure the freezing temperature of nine species of red brown intertidal macroalgae from the coast of Maine, USA in 1991. Using slow and rapid cooling rates approximating those found in the field for Ascophyllum nodosum (L.) Le Jol. we found that, for a given rate, the freezing points of all species were similar: -7.06 to -8.02°C for slow cooling (ca. 0.25°C min^-1) and -3.42 to -4.56°C for rapid cooling (ca. 5.0°C min^-1). In the low shore species, Fucus evanescens C. Ag., photosynthesis was inhibited to a greater extent when plants were frozen or thawed rapidly than after slow freezing or thawing. However, in the upper shore species, F. spiralis (L.), photosynthesis recovered rapidly and completely regardless of freezing rate. Rapidly frozen F. evanescens also experienced greater loss of plasmalemmal integrity, evidenced by a greater loss of cellular contents on re-immersion, than those frozen slowly. Light-limited photosynthesis following freezing was more severely inhibited than light-saturated photosynthesis. Respiration was generally enhanced immediately after freezing, but then declined to rates below those of unfrozen controls within 2 h following re-immersion, with control rates of respiration being achieved after a 24 h recovery period. Our data suggest that the physiological consequences of winter emersion at sub-zero temperatures may vary widely between individual plants of freezing-susceptible species, due to the wide variations in freezing rate associated with microhabitat effects.     

The oxygen microenvironment adjacent to the tissue of the scleractinian Dichocoenia stokesii and its effects on symbiont metabolism

The biology of symbiotic scleractinians is profoundly influenced by their intracellular zooxanthellae, and many studies have focused on the mechanistic basis of this influence. This has usually been accomplished by examining the metabolism of zooxanthellae under physical conditions measured in the open reef and assumed to be similar to conditions in hospite. Recent advances in the measurement of conditions near and within coral tissue suggests that this assumption may result in substantial errors. To address this possibility, the role of water flow in determining oxygen saturation adjacent to the tissue of Dichocoenia stokesii was investigated, and the effect of these measured oxygen saturations on the respiration and photosynthesis of zooxanthellae isolated from the same species was quantified. Using a microelectrode (700 μm diam), we measured oxygen saturations above (≤4 mm) the tissue in two flow speeds over 24 h periods in a flume receiving sunlight at in situ levels. The results were used as a proxy for ecologically relevant intracellular oxygen saturations, which were applied to zooxanthellae in vitro to assess their effect on symbiont metabolism. Microenvironment oxygen saturations (% air saturation) ranged from 74–159% in slow flow (2.7 cm s⁻¹) to 88–110% in faster flow (7.5 cm s⁻¹) over day–night cycles. Therefore, the metabolic rates of zooxanthellae were measured at 50 to 54% (hypoxia), 98 to 102% (normoxia) and 146 to 150% (hyperoxia) oxygen saturation. Oxygen saturation significantly affected the metabolism of zooxanthellae, with gross photosynthesis increasing 1.2-fold and dark respiration increasing 2-fold under hyperoxia compared to hypoxia. These results suggest that the metabolism of zooxanthellae in hospite is affected markedly by their microenvironment which, in turn, is influenced by flow-mediated mass transfer. Received: 13 July 1998 / Accepted: 30 April 1999     

Combined effects of dissolved oxygen concentration and water temperature on embryonic development and larval shell secretion in the marine snail <Emphasis Type="Italic">Chorus</Emphasis><Emphasis Type="Italic">giganteus</Emphasis> (Gastropoda: Muricidae)

The present study was undertaken to determine the effects of both extracapsular oxygen concentration and temperature on embryonic development in Chorus giganteus. In normoxia increasing water temperature from 12°C to 18°C reduced by 15 days the median time required for the capsules to hatch. Hypoxia (oxygen content at 50% of air saturation) generated a low development rate and totally prevented both shell secretion and larval hatching from the egg capsule. Experimental transfer at weekly intervals, from normoxia to hypoxia and vice versa, induced a decrease and increase in the embryonic ash content, respectively, but did not affect the number of hatched larvae. Such an effect was more pronounced at 12°C than at 15°C or 18°C. The embryonic inability to produce a shell under hypoxia is likely to be a result of the low intracapsular oxygen concentration (IPO₂) generated as the combined effect of a low extracapsular oxygen concentration (environmental) added to the intracapsular embryonic oxygen demands, which lowers the IPO₂ still further. Under such conditions, a decrease in intracapsular pH is likely to take place, and, if so, embryos might divert carbonates away from shell calcification to balance such changes in pH.