Biogeosciences Coupled CO 2 and O 2-driven compromises to marine life in summer along the Chilean sector of the Humboldt Current System

Carbon dioxide and coupled CO 2 and O2-driven compromises to marine life were examined along the Chilean sector of the Humboldt Current System, a particularly vulnerable hypoxic and upwelling area, applying the Respiration index (RI = log10 pO2 pCO2 ) and the pH-dependent aragonite saturation ( ) to delineate the water masses where aerobic and calcifying organisms are stressed. As expected, there was a strong negative relationship between oxygen concentration and pH orpCO2 in the studied area, with the subsurface hypoxic Equatorial Subsurface Waters extending from 100 m to about 300 m depth and supporting elevated pCO2 values. The lowest RI values, associated to aerobic stress, were found at about 200 m depth and decreased towards the Equator. Increased pCO2 in the hypoxic water layer reduced the RI values by as much as 0.59 RI units, with the thickness of the upper water layer that presents conditions suitable for aerobic life (RI >0.7) declining by half between 42 ◦ S and 28 S. The intermediate waters hardly reached those stations closer to the equator so that the increased pCO2 lowered pH and the saturation of aragonite. A significant fraction of the water column along the Chilean sector of the Humboldt Current System suffers from CO 2–driven compromises to biota, including waters corrosive to calcifying organisms, stress to aerobic organisms or both. The habitat free of CO 2-driven stresses was restricted to the upper mixed layer and to small water parcels at about 1000 m depth. Overall pCO2 acts as a hinge connecting respiratory and calcification challenges expected to increase in the future, resulting in a spread of the challenges to aerobic organisms.


Introduction
The evolution of the concentrations of atmospheric CO 2 and O 2 over the history of the Earth has played a crucial role in the evolution of life (Dudley, 1998;Berner, 2002).After 800 000 years of relative stability, anthropogenic emissions have driven atmospheric CO 2 to reach 385 ppmv, well above the range of 172-300 ppmv observed over the 800 000 years preceding industrial development (Lüthi et al., 2008), with a dramatic impact in the Earth's climate (Meehl et al., 2007).
Oceans have absorbed almost 50 % of the 7 Gt C yr −1 released by anthropogenic activities (Sabine et al., 2004), and its surface waters now hold approximately 45 µmol kg −1 of CO 2 in excess compared to preindustrial concentrations (Broecker et al., 1985).Increased CO 2 in ocean waters has already lead to a decline of 0.1 units in ocean pH, and may decrease by an additional 0.3 pH units by the end of the century, with a large impact on marine calcifying organisms (Orr et al., 2005;Doney et al., 2009).The thresholds of ocean acidification to marine calcifying organisms are given by the aragonite and calcite saturation values, (Feely et al., 2004; Published by Copernicus Publications on behalf of the European Geosciences Union.E. Mayol et al.: Coupled CO 2 and O 2 -driven compromises Orr et al., 2005), with aragonite saturation being more sensitive to ocean acidification than that for calcite.Indeed, calcification processes are already affected at aragonite values <2 (Hauri et al., 2009;Hendriks et al., 2010), although these thresholds are species-specific.Ocean acidification has received considerable attention as the main direct impact of increased ocean CO 2 , but other potential impacts of increased CO 2 have been overlooked.Indeed, increased CO 2 and lowered pH also affect respiratory processes by driving reduced binding affinity for oxygen in blood (Pörtner et al., 2004) and a direct ventilatory sensitivity to CO 2 (Burleson and Smatresk, 2000;McKendry et al., 2001).Hence, increased CO 2 also poses challenges to aerobic respiration, threatening marine life, an impact observed long ago on marine fishes in controlled laboratory conditions (Powers, 1922), and recently addressed by Brewer and Peltzer (2009) in a study of the changing ocean conditions.
Indeed, the efficiency of aerobic respiratory processes depends on the partial pressures of both CO 2 and O 2 , which are tightly coupled through the metabolic activity of marine organisms.Brewer and Peltzer (2009) indicated that the efficiency of aerobic respiratory processes is dependent on the ratio of the partial pressures of O 2 and CO 2 , which defines the range of conditions compatible with aerobic marine life.Hence, present concerns on the threat posed by on-going declines of marine oxygen in the ocean (Díaz and Rosenberg, 2008;Vaquer-Sunyer and Duarte, 2008;Gilbert et al., 2010) are further aggravated by the parallel increase in CO 2 (Brewer and Peltzer, 2009).Yet, the impacts of hypoxia on marine biota have been traditionally studied in isolation from the effects of increased CO 2 .Brewer and Peltzer (2009) highlight the importance of studying the coupled effects of changes in both CO 2 and O 2 on aerobic marine life, based on the notion that elevated dissolved CO 2 concentrations may impose physiological strain and less available energy on marine animals.This results from the fact that hemoglobin has an optimum pH to carry oxygen (Powers, 1922).Hence, these authors use the basic oxic respiration equation (C org + O 2 → CO 2 ) associated with the free-energy relation ) to derive a Respiration Index (RI), which is used to parametrise the combined effect of O 2 and CO 2 on the efficiency of aerobic respiration.The RI is a simple numerical constraint that is linearly related to available energy, given by the expression: where RI ≤ 0 corresponds to the thermodynamic aerobic limit, a formal dead zone; at RI = 0 to 0.4 aerobic respiration does not occur; the range RI = 0.4 to 0.7 represents the practical limit for aerobic respiration, and the range RI = 0.7 to 1.0 delimits the aerobic stress zone.Thus, increased CO 2 aggravates the impacts of hypoxia (Brewer and Peltzer, 2009).Elevated CO 2 acts as a hinge, connecting two otherwise independent threats to marine life, acidification and hypoxia.This connection has been poorly studied to date.The areas of the world ocean most sensitive to both these threats are upwelling regions, as they are typically low in oxygen (Grantham et al., 2004) and corrosive to carbonate structures due to high CO 2 levels (Feely et al., 2008).
A particularly vulnerable area is the Humboldt Current System along the Chilean coast, the largest naturally hypoxic area and an important upwelling center (Thiel et al., 2007;Ulloa and Pantoja, 2009), where CO 2 , O 2 and pH levels across the water column result from natural variation.In this study we examine the Chilean sector of the Humboldt Current System using data from one single summer cruise, when upwelling is typically strongest and habitat stresses greatest.Whereas the pCO2 and carbon chemistry in this region has been extensively studied in the past (Torres et al., 2002;Torres et al., 2011), the link between increased CO 2 and hitherto considered independent threats to marine life, ocean acidification and hypoxia has not yet been addressed.Here we provide a first perspective of the latitudinal changes in CO 2 -driven compromises to marine life over a distance of 1700 km along a very complex system.We demonstrate how RI and can be used to delineate the water masses where aerobic and calcifying organisms are stressed.Our main objective is to examine, in a predominantly along-shore transect, the connection, through increased CO 2 , between challenges to respiration arising from the combination of reduced oxygen values and high pCO 2 levels and challenges to calcification derived from reduced pH levels with high pCO 2 levels.Increased awareness of the connection between compromises to marine biota derived from these two effects of increased CO 2 , will hopefully lead to further studies on the seasonal, interannual and long-term trends of the CO 2 , O 2 and pH levels.

Study site
The study was conducted along the Humboldt 2009 cruise on board the R/V Hespérides from 5 to 16 March 2009.The cruise track followed the Chilean coast, starting in the Patagonia channels (54.9 • S) proceeding North along the Humboldt Current System until Antofagasta (Chile, 23.6 • S, Fig. 1).The Humboldt Current System is one of the largest naturally hypoxic areas of the world's oceans (Levin, 2002;Thiel et al., 2007;Ulloa and Pantoja, 2009), characterized by upwelling of cold, oxygen-poor waters supersaturated in CO 2 (Torres et al., 2002).The Humboldt Current System is a quite complex dynamic region, characterized by the presence of a system of along-slope currents that brings waters of both tropical and subpolar origin.The dominant current is the far-offshore equatorward Humboldt Current but near shore a system of poleward and equatorward currents is found, the former formed by the Peru-Chile Counter Current and the near-slope Poleward Undercurrent and the latter by the Peru-Chile Coastal Current (Strub et al., 1998;Silva et al., 2009).The poleward currents are responsible for bringing Subtropical Waters (STW) and Equatorial Subsurface Waters (ESSW) while the equatorward flow brings Subantarctic Waters (SAAW) and Antarctic Intermediate Waters (AAIW).Each of these water masses is characterized by distinct properties, including the levels of CO 2 and O 2 .
The Humboldt Current System, and therefore the associated CO 2 and O 2 levels, has substantial interannual and seasonal cycles.Much of the variability in the biogeochemical cycles north of 35 • S is directly driven by the interannual ENSO cycle (Halpin et al., 2004).As a result of warm ENSO events, the Oxygen Minimum Layer (OML) deepens and higher oxygen concentrations in the top 100 m layer are found (Morales et al., 1999).The Humboldt Current System is controlled, to a large extent, by the coastal equatorward winds linked to the Pacific Subtropical Anticyclone.These winds drive all year long coastal upwelling along northern and central Chile, extending to southern Chile in summer.The maximum upwelling-favorable winds occur in summer, the winter-summer difference increasing with latitude.In southern Chile, beyond about 35 • S, upwelling only occurs in summer while in winter the polar front provides downwelling-favorable winds (Thiel et al., 2007).
As a result of changes in upwelling most variability occurs in the upper 150 m, therefore, affecting STW and SAAW and, to a lesser degree, ESSW (Blanco et al., 2001;Antezana, 1978); deeper waters, such as AAIW, experience much more moderate seasonal changes, in any case, not directly linked to coastal upwelling.During summer, therefore, we expect there will major near-surface coastal changes associated to the onshore and upward transport of the oxygendeficient (the Oxygen Minimum Layer, OML) and strongly CO 2 supersaturated tropical and equatorial waters (in surface to reach 100 % near 23 • S and 200 % near 30 • S) (Torres et al., 2002).The high primary production (PP) values in the upwelling region, particularly intense near several geomorphological coastal features (Strub et al., 1998), strengthens the oxygen-minimum zone and results in CO 2 supersaturated reaching near-surface waters.

Sampling
A series of 15 stations spaced along the meridional cruise track were sampled.Hydrographic properties were profiled down to 1400 m depth using a Seabird 9 CTD probe.Water samples were collected at different depths (5,15,30,50,100,200,300,600,1000, 1400 m) using 12 L Niskin bottles fitted on a Rosette sampler system.Water samples were analyzed for pCO 2 , O 2 , and pH immediately after sampling.

CO 2 measurements
The partial pressure of CO 2 in the water (pCO 2 ) was measured using a non dispersive infrared gas analyzer (EGM-4, PP systems) that measures pCO 2 with a precision of ±1 ppm.For pCO 2 , near surface water (about 1 m depth) was collected and passed through a gas exchange column (Mini-Module Membrane Contactor) and pCO 2 measured, details of this methodology have been described elsewhere (Calleja et al., 2005;Silva et al., 2008).

pH, RI and aragonite saturation ( ) measurements
All seawater samples for pH, collected immediately after sampling the Niskin bottles for oxygen determinations, were siphoned into 500 mL glass bottles, allowed to overflow and immediately stopped.After temperature stabilization on a water bath at 25 • C, the pH samples were carefully transferred to 10 cm path-length optical glass cells (fitted with a jacket that circulates water at 25 • C) to carry out the doublewavelength spectrophotometric measurements (Clayton and Byrne, 1993).Oxygen concentrations were converted into pO 2 and RI were calculated following Brewer and Peltzer (2009).
values for aragonite saturation were calculated from pH, pressure, temperature, salinity and alkalinity using CO 2 SYS (Pierrot et al., 2006).Because the pH-pCO 2 paired couple is not a good predictor of alkalinity, total alkalinity was obtained from the CDIAC data base (Lamb et al., 2001).

Description of water masses and its associated pCO 2 , O 2 and pH levels
The ship's meridional transect encompassed waters of equatorial and Antarctic origin, displaying substantial changes in pCO 2 and O 2 .The surface waters, down to 100-150 m, correspond to STW and SAAW, characterized by pCO 2 and O 2 concentrations close to atmospheric equilibrium and by the highest pH values found in the water column (Fig. 2).Immediately below were the hypoxic ESSW, their thickness increasing towards the Equator, where they extend from 100 m to about 300 m depth (Fig. 2).Below this layer and down to about 1000 m we found AAIW, characterized by much higher oxygen concentrations (Fig. 2).The hypoxic ESSW were also characterized by elevated pCO 2 and pH values (> 1000 µatm pCO 2 and <7.8 pH units, Fig. 2), while the AAIW were characterized by comparatively low pCO 2 and intermediate pH values (Fig. 2).Further below we find the moderately oxygen-depleted Pacific Deep Waters (PDW).
We also present three temperature-salinity diagrams with colour-coded values of the oxygen concentration (Fig. 3a), pCO 2 (Fig. 3b), and pH (Fig. 3c).The oxygen-coded diagram shows interleaving between oxygen-rich AAIW and ESSW, with the latter overlaying the former (Fig. 3a).The STW and SAAW surface waters have relatively large oxygen concentrations, with maximum values corresponding to the high-latitude relatively cold SAAW (Fig. 3a).The oxygendepleted equatorial subsurface waters were also characterized by elevated pCO 2 (>1000 µatm pCO 2 ; Fig. 3b) and acidic (pH <8.0) waters (Fig. 3c).Indeed, our study area was characterized by a strong negative relationship betweenpCO 2 and oxygen levels, and between pCO 2 and pH levels, while an opposite relationship was found between oxygen concentrations and pH values (Fig. 4).

Respiration index and threatened aerobic life
The respiration index, which describes the adequacy of the gaseous composition of the water to maintain aerobic life, reached a minimum at about 200 m depth, with the minimum RI values generally decreasing towards the Equator (Fig. 5).These minimum RI values were below the 0.7 threshold value across most of the region (Fig. 5).The 0.4 and 0.7 RI thresholds were associated with higher CO 2 and lower O 2 values (O 2 < 70 µmol kg −1 ; Fig. 4a).We examined the contribution of changes in pCO 2 to the observed variability in RI by holding pCO 2 constant at atmospheric equilibrium (Fig. 6a), and calculating the difference between the observed RI and that calculated if pCO 2 was constant (Fig. 6b).This exercise showed that the increased pCO 2 levels in the hypoxic water layer (observed in Fig. 5b) enhance the thickness of the water column that has RI values below the 0.7 threshold, as can be observed in Fig. 6b, and reduces the RI values of the water column below 100 m by as much as 0.59 RI units at the oxygen minimum zone.Indeed, the thickness of the water column with RI values below the 0.7 threshold increases greatly towards the Equator, encompassing 1/3 of the studied water column at 28 • S (Fig. 7a).It is important to emphasize that the pattern described also involves a reduction toward the Equator in the thickness of the upper water layer that presents conditions suitable for aerobic life (RI>0.7),declining by half between 42 • S and 28 • S (Fig. 7b).

pH and saturation of aragonite levels as a threat to calcification processes
In addition to reducing the RI values, the increased pCO 2 in intermediate waters also lowers pH and, therefore, the saturation limit for aragonite (Fig. 8a).The aragonite saturation levels may compromise calcification processes ( < 2) everywhere in the top 1400 m of the water column except in the uppermost 75 to 125 m, with the thickness of this surface layer increasing from 42 • S to 28 • S, respectively (Fig. 8b).This pattern is opposite to that observed in RI due to the increase in pCO 2 in the oxygen minimum zone toward the Equator, and the parallel warming of the waters that result in increased saturation levels, by as much as 50 % across the 3 • C meridional gradient encompassed by surface waters.

Combined threats
The aragonite saturation levels ( < 2) were also observed in most of the water column, except in the upper waters and in small scattered water parcels below 600 m.In the northern end of the cruise track the water depths between about 200 and 400 m, corresponding to ESSW, were also associated with RI values (RI < 0.7) that may compromise aerobic respiration (Fig. 9).The thickness of this layer reduced towards the south and almost disappeared in the southern limit of the cruise track.In these waters the aragonite saturation levels were always such that they may also compromise calcification processes ( < 2) (Fig. 9).

Discussion
The bulk of the water column (0-1400 m) along the Chilean sector of the Humboldt Current System, where summer winds are usually favorable to upwelling events, is strongly acidic or has low RI values which compromise habitat.These waters are associated with the subsurface hypoxic Equatorial waters that flow South through both the Peru-Chile Counter Current and the Poleward Undercurrent.Compromises are particularly acute for aerobic organisms, as hypoxia rises close to the sea surface (O 2 concentrations <8 µmol kg −1 at 100 m depth in 30.51 • S) towards the north of the study area.pCO 2 levels were also very high (up to 1460 ppm) in association with the hypoxic layer.The negative relationship between CO 2 and O 2 levels is enhanced due to upwelling in this area.The pH values decline in this area, as a typical chemical response to rising CO 2 levels.Therefore, the positive relationship between O 2 and pH is a consequence of the inverse relation with CO 2 (Fig. 4), which is linked to O 2 through metabolic processes and to pH through its role in the seawater carbon buffer system.Indeed, the close relationship between O 2 and pH allows the scaling of two threats, thus far mostly treated as independent, to rising CO 2 levels.Llanillo et al. (2011) have recently examined the distribution of water masses along the cruise track, in particular the relation between oxygen concentration and the presence of ESSW.These authors found that ESSW are characterized by relatively high salinity and nutrient concentrations, and very low oxygen values, leading to an extensive subsurface oxygen minimum zone as reported by previous studies (Fuenzalida et al., 2009;Silva et al., 2009).Llanillo et al. (2011) found that the ESSW, centered at 200-250 m depths, flow poleward progressively loosing their identity through mixing with the overlying SAAW and the underlying AAIW until they are no longer recognizable at 41.6 • S.
The additional stress to biota in the hypoxic water mass of the Humboldt Current System arising from the high pCO 2 levels has not been discussed earlier.Our results showed that a significant fraction of the water column along the Chilean sector of the Humboldt Current System suffers from CO 2driven compromises to biota, including corrosive waters to calcifying organisms, stress to aerobic organisms or both.Only those waters shallower than 100 m (STW and SAAW) present conditions free of stress to aerobic organisms (Fig. 9).The threshold imposing challenges to aerobic organisms, as indicated by the RI values and particularly by RI < 0.7, was associated with O 2 values below 70 µmol kg −1 .This value is slightly higher than the typical threshold for hypoxia (Gray et al., 2002), but is consistent with experimental evidence that yields a median lethal oxygen concentration among studied taxa start at 60 µmol L −1 , while the hypoxic threshold for the most resistant organisms is 25 µmol L −1 (Vaquer-Sunyer and Duarte, 2008;Keeling et al., 2010).This challenge is highly increased by consideration of the increased pCO 2 levels, which lowered the RI value by up to 0.59 RI units and increased the thickness of the water column with RI < 0.7.Hence, our results concur with those of Brewer and Peltzer (2009) to suggest that increased pCO 2 levels aggravate the challenges to aerobic organisms in oxygen deficient waters, such as those in the Humboldt Current System.
Whereas our study represents a quasi-synoptic assessment of the extent of challenges derived from pCO 2 , and associated pH levels, and O 2 in the water column of the Humboldt Current System, these were expected to be highly dynamic.The oxygen minimum of the Humboldt Current System shows seasonal and interannual variability, driven by upwelling events and large-scale perturbations in regional circulation, such as those accompanying El Niño events.The oxygen content in the top 100 m layer is higher in the region during El Niño events (Morales et al., 1999;Ulloa et al., 2001).In addition to seasonal and interannual oscillations, the CO 2 -driven challenges to biota reported here are expected to increase in the future.AtmosphericpCO 2 levels are expected to reach 700 to 1000 ppm by the end of the 21st Century (Meehl et al., 2007), with an increase inpCO 2 at depth more than 1000 µatm in the Pacific (Brewer and Peltzer, 2009), resulting in a spread of the respiratory challenges to aerobic organisms.The corresponding pH levels are expected to continue to decline, being reduced by 0.3 units below present values by the end of the 21st Century and by up to 0.7 units by 2300 (Caldeira and Wickett, 2003;Doney et al., 2009).In addition, oxygen concentrations are declining in many areas of the ocean (Stramma et al., 2008;Keeling et al., 2010), further affecting the RI ratio.
The area where biocalcification processes may be close to being compromised can be delineated from the water column with saturation levels, for aragonite <2 (cf.Orr et al., 2005;Yates and Halley, 2006;Guinotte and Fabry, 2008;Hauri et al., 2009, Hendriks et al., 2010), which encompasses most of the water column except for the upper layer (above 70 m).The thickness of the water column where biocalcification processes may be impacted is largest at mid-latitudes (between about 30 and 37 • S) and decreases slowly towards high latitudes and rapidly towards the Equator.This swift change in the equatorial region is opposite to what happens to the layer where aerobic respiration is compromised.The preindustrial conditions (CO 2 level of around 260-270 ppmv (Wigley, 1983), and ocean temperatures approximately 1 • C lower than at present (Hughes, 2000)) suggest that for aragonite in the surface of the ocean could have been 0.2 units higher than present (calculations not shown).Reduced aragonite saturation levels and RI, are driven by pCO 2 , each includes a second, independent, driver: temperature in the case of biocalcification and oxygen concentration in the case of aerobic respiration.pCO 2 acts, therefore, as a hinge connecting respiratory and calcification challenges.
In summary, ocean acidification affects most waters below 150 m, while respiratory compromises are located within the 200 to 400 m layer (Fig. 9).These two challenges show similar trends at mid and high latitudes but have opposite trends within equatorial waters.The whole cruise track carried out in summer was influenced by upwelling, but yet the temperature displayed a significant latitudinal gradient at all depths.This clearly must have affected the chemical processes in the water column, as for example the O 2 solubility, which increases with lower temperatures (Keeling et al., 2010).In general, the subsurface depth layer is affected by biocalcification and hypoxia, its thickness being maximum at low latitudes (200 to 400 m) and decreasing to nearly zero at high latitudes (Fig. 9), which is consistent with the increase of the ESSW thickness towards the Equator and in summer (Blanco et al., 2001).The habitat free of CO 2 -driven stresses was restricted to the upper mixed layer and to small water parcels at about 1000 m depth (Fig. 9).Increased pCO 2 in the future may increase the thickness of the water column with both RI and aragonite saturation reaching values under the threshold that compromises marine life, therefore, compressing the vertical extent of the stress-free habitat.
Probably, both the aragonite saturation threshold for biocalcification and the threshold RI affecting respiration vary across taxa (cf.Hendriks et al., 2010 andVaquer-Sunyer andDuarte, 2008, respectively), depending on their metabolic capacities.Indeed, whereas most metazoans are excluded from the oxygen minimum zone of the Humboldt Current System, specialized crustacean communities, including copepods and euphasids, have been reported to enter this hypoxic layer (Escribano et al., 2009).The temperature also is a factor that may lead to a differentiated biological resistance to the RI threshold.Higher temperatures are mostly associated with higher metabolic rates and respiration (Doney et al., 2012).Therefore, organisms affected by the same RI value could be more stressed in the North of Chile.Use of the RI value as a predictive tool to evaluate and project the impact of increased pCO 2 on aerobic organisms in this region requires, therefore, experimental evidence of the RI thresholds for aerobic respiration of the main taxa in the ecosystem.
The most intense upwelling in Chile takes place near small capes and bays, where subsurface waters are exported offshore by ∼125 km (Fonseca and Farias, 1987).These upwelling centers are the site for major fisheries, with catches that represent 40 % of the annual landings of the Humboldt Current System (Thiel et al., 2007).Along the Humboldt Current System about 40 important commercial species of fishes, crustaceans, molluscs, echinoderms and seaweeds are found (Montecino and Lange, 2009).Our study showed that the extent of the threats toward the surface layer is presumably larger towards the coast when deep upwelling events, reaching ESSW layers, are most intense.These events could lead to RI and values closer to the threshold values that compromise marine life, therefore possibly affecting the large fisheries supported at these sites.Additionally, events associated with changes in temperature and O 2 levels, such as El Niño, have been extremely adverse on anchovy and positive on sardine populations (Alheit and Bernal, 1993).It is likely that these events are also associated with concurrent changes in CO 2 levels, leading to a compression of the habitat suitable for aerobic organisms.
Pörtner and Langenbuch ( 2005) have described mechanisms of short and long-term sensitivity to CO 2 in fish and have shown how elevated CO 2 levels, particularly when combined with other factors, may become a life risk for different organisms.The trends towards increased pCO 2 and reduced O 2 concentrations in the future may compress the water column available for aerobic organisms and expand the minimum oxygen zone until zones where fisheries species, such as the Humboldt squid and fish species, are located.

Conclusions
In this study we presented the risk for aerobic and calcifying marine organisms associated to high pCO 2 and low O 2 levels.The study was centered in an area naturally low in oxygen and with high pCO 2 levels, potentially corrosive to carbonate structures.The main point of this manuscript is to  examine the co-variation between pCO 2 and O 2 to explore how ocean acidification and hypoxia trends are not independent threats, but are connected to one another through the effect of CO 2 on both respiratory activity and calcification rates.With this purpose we extended the thermodynamic model of Brewer and Peltzer (2009) on joint effects of pCO 2 and O 2 on respiration to also address the effects of pCO 2 , pH and calcification.With the definition of the RI index (respiration index) and saturation states of aragonite, we have attempted to delineate water masses in the Humboldt Current System where respiration and calcification may be compromised.
This study can be used as a predictive model of the future situation that oceans are likely to exhibit, when considering the expected trends in the evolution of both O 2 and pCO 2 levels.Relating pCO of this index indeed shows that high pCO 2 contributes to exacerbate the challenges to respiration in Humboldt Current System.As well as this respiratory threat, it is also necessary to take into account the stress inflicted upon calcifying processes, associated with increased pCO 2 levels, resulting in decreased pH levels and low saturation levels for aragonite, where calcification may be compromised.The RI and the saturation state of aragonite have been used in this work as predictive tools to evaluate and project the impact of increased pCO 2 on aerobic and calcifying organisms, showing that along the Chilean sector of the Humboldt Current System the habitat free of CO 2 -driven stresses was restricted to the upper mixed layer and to small water parcels at about 1000 m depth.< 2; aerobic respiration compromised, RI < 0.7) to marine life along the studied transect.+/+ = both biocalcification and respiration compromised; +/-= only biocalcification compromised; -/-= no compromises.The missing combination (-/+ = only respiration compromised) was not observed.Note that the 28 • station had data only at 200 and 600 m depths, therefore, the region with compromises to both biocalcification and respiration was likely missed by the data; this region is hatched with vertical lines.

Figure 2 610Fig. 2 .
Figure 2 610 Contour plots showing the variation in pCO 2 , O 2 and pH levels along the studied transect.611 The black points represent the location of the samples within the water column.612 613 614 615 616 617 618

Fig. 3 .
Figure 3 620 Temperature-salinity diagram colour-coded for oxygen (a), pCO 2 (b), and pH (c) with 621 potential-density isolines superposed.The dashed lines in (a) illustrate the location of 622 intermediate waters (points denser than defined by line A-A') and their partitioning 623 between waters of Equatorial origin (points above line B-B' and lighter than 27.0) and 624 those of Antarctic origin (points to the left of line B-B').Deep-water points are found to the 625 right of line B-B' and with densities higher than 27.0.Oxygen values in (a) were derived 626 from the CTD-mounted oxygen sensor calibrated with Winkler analyses from bottle casts, 627 while pCO 2 (b) and pH (c) correspond to the values measured from the bottle casts.628 629 630 631 632 633 634

Figure 7 677Fig. 7 .
Figure 7 677 Relationships between the thickness of the water column and latitude: (a) RI < 0.7 in the 678 top 1,400 m; (b) RI > 0.7 in the top 200 m.679 Fig. 7. Relationships between the thickness of the water column and latitude: (a) RI <0.7 in the top 1400 m; (b) RI > 0.7 in the top 200 m.
Figure 8 681 (a) Contour plot showing the distribution of aragonite saturation index (Ω), where the 682 indicated by gray lines correspond to the thresholds of aragonite saturation (Ω = 1 an 683 (b) Thickness of the water column with Ω < 2 along the studied transect.684 685 686

Fig. 9 .
Fig. 9. Distribution of compromises (biocalcification compromised,< 2; aerobic respiration compromised, RI < 0.7) to marine life along the studied transect.+/+ = both biocalcification and respiration compromised; +/-= only biocalcification compromised; -/-= no compromises.The missing combination (-/+ = only respiration compromised) was not observed.Note that the 28 • station had data only at 200 and 600 m depths, therefore, the region with compromises to both biocalcification and respiration was likely missed by the data; this region is hatched with vertical lines.

Table 1 .
Depth, position and distance to the coast for each station.