Mass extinctions and ocean acidification: biological constraints on geological dilemmas

The five mass extinction events that the earth has so far experienced have impacted coral reefs as much or more than any other major ecosystem. Each has left the Earth without living reefs for at least four million years, intervals so great that they are commonly referred to as ‘reef gaps’ (geological intervals where there are no remnants of what might have been living reefs). The causes attributed to each mass extinction are reviewed and summarised. When these causes and the reef gaps that follow them are examined in the light of the biology of extant corals and their Pleistocene history, most can be discarded. Causes are divided into (1) those which are independent of the carbon cycle: direct physical destruction from bolides, ‘nuclear winters’ induced by dust clouds, sea-level changes, loss of area during sea-level regressions, loss of biodiversity, low and high temperatures, salinity, diseases and toxins and extraterrestrial events and (2) those linked to the carbon cycle: acid rain, hydrogen sulphide, oxygen and anoxia, methane, carbon dioxide, changes in ocean chemistry and pH. By process of elimination, primary causes of mass extinctions are linked in various ways to the carbon cycle in general and ocean chemistry in particular with clear association with atmospheric carbon dioxide levels. The prospect of ocean acidification is potentially the most serious of all predicted outcomes of anthropogenic carbon dioxide increase. This study concludes that acidification has the potential to trigger a sixth mass extinction event and to do so independently of anthropogenic extinctions that are currently taking place.     

Genetic variation in mortality of chinook salmon during a bloom of the marine alga Heterosigma akashiwo

Mortality in a netpen-reared population of 3-year-old chinook salmon Oncorhynchus tshawytscha during an extensive 1997 bloom of the alga Heterosigma akashiwo in Puget Sound, Washington, was low (7·2%), and corresponded to a reduction in variance effective population size of 9·4%. Under a liability threshold model, the heritability of mortality (± S.E.), based on paternal half-sibs, was estimated at 0·15 ± 0·04. No significant genetic variation was detected for date of death. Despite the low overall mortality, the consequences for variation in family size underscore the importance of maximizing genetic variation in cultured fish populations later released to the wild as a precaution against mortality and losses of genetic variation over the life cycle. The pattern of family variation in response to this algal bloom provides evidence for potentially selective mortality of anadromous salmonids in the marine environment during natural perturbations.     

Phylogeographic characteristics of hypervariable regions in the mitochondrial genome of a cosmopolitan,bloom‐forming raphidophyte,Heterosigma akashiwo

The global distribution of phytoplankton is defined by many events, including long‐term evolutionary processes and shorter time span processes (e.g., global climate change). Furthermore, human‐assisted, unintentional dispersion, including the transport of live fish and spat for aquaculture, and transfer of aquatic microorganisms contained in ship ballast water, may aid the spread of phytoplankton. To understand the phylogeographic history of a species, the development of useful molecular markers is crucial. We previously reported a hypervariable mitochondrial gene in the cosmopolitan bloom‐forming alga, Heterosigma akashiwo. In this study, we identified two additional hypervariable segments in the H. akashiwo mitochondrial genome, one a protein coding sequence, and the other an intergenic region, by comparing the whole mitogenomes of strains obtained from various geographic origins. Interestingly, the newly identified hypervariable protein coding sequence was a paralogue of the previously identified gene, and both sequences showed tendencies to correlate with latitude of geographic origin. However, the hypervariable intergenic sequence did not show a clear correlation with origin. Our results demonstrated that the protein coding sequences may serve as useful tools for understanding the phylogeographic history of H. akashiwo, and they may crucially function in adaptation of the species to the environment.     

Impacts of ocean acidification and burrowing urchins on within-sediment pH profiles and subtidal nematode communities

Rising carbon dioxide levels in the atmosphere will affect the ocean carbonate system, resulting in a predicted future decrease in the pH of seawater by 0.3-0.5 units. To investigate whether the presence of a burrowing urchin, Echinocardium cordatum, might influence the impact of ocean acidification on subtidal sediment pH profiles and nematode community structure an experiment was conducted using subtidal sediment, with urchins present or absent and seawater at either pH 8.0 (ambient) or 7.5. The presence of urchins, and a reduction in pH, both had significant effects on within-sediment pH profiles. Where urchins were present sediment profiles were more consistent and sediment pH was lower than that of the overlying seawater. There were significant differences in nematode abundance between treatments. The primary effect was a higher abundance of nematodes in replicates with urchins in natural seawater. All treatments had similar nematode community structure and diversity. Ocean acidification could therefore lead to changes in nematode communities in subtidal sediments affected by burrowing urchins.     

Coralline algae elevate pH at the site of calcification under ocean acidification

Coralline algae provide important ecosystem services but are susceptible to the impacts of ocean acidification. However, the mechanisms are uncertain, and the magnitude is species specific. Here, we assess whether species‐specific responses to ocean acidification of coralline algae are related to differences in pH at the site of calcification within the calcifying fluid/medium (pH_cf) using δ¹¹B as a proxy. Declines in δ¹¹B for all three species are consistent with shifts in δ¹¹B expected if B(OH)₄^? was incorporated during precipitation. In particular, the δ¹¹B ratio in Amphiroa anceps was too low to allow for reasonable pH_cf values if B(OH)₃ rather than B(OH)₄^? was directly incorporated from the calcifying fluid. This points towards δ¹¹B being a reliable proxy for pH_cf for coralline algal calcite and that if B(OH)₃ is present in detectable proportions, it can be attributed to secondary postincorporation transformation of B(OH)₄^?. We thus show that pH_cf is elevated during calcification and that the extent is species specific. The net calcification of two species of coralline algae (Sporolithon durum, and Amphiroa anceps) declined under elevated CO₂, as did their pH_cf. Neogoniolithon sp. had the highest pH_cf, and most constant calcification rates, with the decrease in pH_cf being ¼ that of seawater pH in the treatments, demonstrating a control of coralline algae on carbonate chemistry at their site of calcification. The discovery that coralline algae upregulate pH_cf under ocean acidification is physiologically important and should be included in future models involving calcification.     

Effects of increasing temperature and acidification on the growth and competitive success of Alexandrium catenella from the Gulf of Maine

Climate driven increases in ocean temperature and pCO₂ have the potential to alter the growth and prevalence of future Harmful Algal Blooms (HABs), but systematic studies on how climate drivers influence toxic algal species relative to non-toxic phytoplankton are lacking. In particular, little is known about how future climate scenarios will affect the growth of the toxic dinoflagellate Alexandrium catenella, which is responsible for the paralytic shellfish poisoning (PSP) events that threaten the health and economy of coastal communities in the Gulf of Maine and elsewhere. The growth responses of A. catenella and two other naturally co-occurring dinoflagellates in the Gulf of Maine—Scrippsiella sp., and Amphidinium carterae—were studied in mono and mixed species cultures. Experimental treatments tested the effects of elevated temperature (20 °C), lower pH (7.8), and the combination of elevated temperature and lower pH on growth rates relative to those in near-current conditions (15 °C; pH 8.1). Growth rates of A. catenella decreased under elevated temperature and lower pH conditions, a response that was largely attributable to the effect of temperature. In contrast, growth rates of Scrippsiella sp. and A. carterae increased under elevated temperature and lower pH conditions, with temperature also being the primary driver of the response. These trends did not change substantially when these species were grown in mixed cultures (A. catenella + Scrippsiella sp., and A. catenella + A. carterae), indicating that allelopathic or competitive interactions did not affect the experimental outcome under the conditions tested. These findings suggest that A. catenella blooms may become less prevalent in the southern regions of the Gulf of Maine, but potentially more prevalent in the northeastern regions of the Gulf of Maine with continued climate change.     

New ocean,new needs: Application of pteropod shell dissolution as a biological indicator for marine resource management

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Diurnal fluctuations in seawater pH influence the response of a calcifying macroalga to ocean acidification

Coastal ecosystems that are characterized by kelp forests encounter daily pH fluctuations, driven by photosynthesis and respiration, which are larger than pH changes owing to ocean acidification (OA) projected for surface ocean waters by 2100. We investigated whether mimicry of biologically mediated diurnal shifts in pH—based for the first time on pH time-series measurements within a kelp forest—would offset or amplify the negative effects of OA on calcifiers. In a 40-day laboratory experiment, the calcifying coralline macroalga, Arthrocardia corymbosa, was exposed to two mean pH treatments (8.05 or 7.65). For each mean, two experimental pH manipulations were applied. In one treatment, pH was held constant. In the second treatment, pH was manipulated around the mean (as a step-function), 0.4 pH units higher during daylight and 0.4 units lower during darkness to approximate diurnal fluctuations in a kelp forest. In all cases, growth rates were lower at a reduced mean pH, and fluctuations in pH acted additively to further reduce growth. Photosynthesis, recruitment and elemental composition did not change with pH, but δ¹³C increased at lower mean pH. Including environmental heterogeneity in experimental design will assist with a more accurate assessment of the responses of calcifiers to OA.     

Natural acidification changes the timing and rate of succession,alters community structure,and increases homogeneity in marine biofouling communities

Ocean acidification may have far‐reaching consequences for marine community and ecosystem dynamics, but its full impacts remain poorly understood due to the difficulty of manipulating pCO₂ at the ecosystem level to mimic realistic fluctuations that occur on a number of different timescales. It is especially unclear how quickly communities at various stages of development respond to intermediate‐scale pCO₂ change and, if high pCO₂ is relieved mid‐succession, whether past acidification effects persist, are reversed by alleviation of pCO₂ stress, or are worsened by departures from prior high pCO₂ conditions to which organisms had acclimatized. Here, we used reciprocal transplant experiments along a shallow water volcanic pCO₂ gradient to assess the importance of the timing and duration of high pCO₂ exposure (i.e., discrete events at different stages of successional development vs. continuous exposure) on patterns of colonization and succession in a benthic fouling community. We show that succession at the acidified site was initially delayed (less community change by 8 weeks) but then caught up over the next 4 weeks. These changes in succession led to homogenization of communities maintained in or transplanted to acidified conditions, and altered community structure in ways that reflected both short‐ and longer‐term acidification history. These community shifts are likely a result of interspecific variability in response to increased pCO₂ and changes in species interactions. High pCO₂ altered biofilm development, allowing serpulids to do best at the acidified site by the end of the experiment, although early (pretransplant) negative effects of pCO₂ on recruitment of these worms were still detectable. The ascidians Diplosoma sp. and Botryllus sp. settled later and were more tolerant to acidification. Overall, transient and persistent acidification‐driven changes in the biofouling community, via both past and more recent exposure, could have important implications for ecosystem function and food web dynamics.     

Methods of pH calibration of sedimentary diatom remains for reconstructing history of pH in lakes

The pH history of lakes can be inferred from diatom remains in dated sediment cores. To derive transfer functions for pH inference in acidic lakes, we counted diatoms in surface-sediment from 31 soft-water lakes in n. New England (NE) and 36 in Norway (N), covering pH 4.4–7.1. Cluster analysis of each data set indicates that pH 6 is an upper limit for a group of similar diatom assemblages. For each set, we developed multiple linear regressions to relate three versions of the diatom data to pH of surface-waters: (1) relative frequencies of selected diatom taxa, (2) the first principal component (1 PC) of these frequencies, and (3) the frequencies of Hustedt pH groups. Also, simple linear regressions were developed for two versions: (1) Index B and (2) Index Alpha, both based on pH groups. Regressions were run separately for lakes with pH 6; these are most relevant for pH inference in acidic lakes. The best regressions (N: taxa & 1 PC taxa) have r² 0.69–0.91 and S_e 0.24–0.31 pH units, the worst (NE: log alpha) have r² 0.27–0.57 and S_e 0.51. In all cases, errors for NE are greater than N, partly due to greater diversity of NE lakes. Regressions based on pH groups (directly & by indices) have smaller r² and larger S_e than those based on taxa and 1 taxa. The Index Alpha is least useful because its requirement for alkaline diatom units is unsatisfied at many acidic lakes. Regressions based on taxa may give erratic pH inferences due to sensitivity to unusual frequencies of individual taxa; this effect is reduced by using 1 PC taxa. Four regressions based on pH 6 lakes were used for inferring pH in a ²¹⁰Pb dated core from Nedre Målmesvatn, N (now pH 4.6). There is good agreement among three of the four (not for the regression based directly on taxa) that there has been a decrease of ca. 0.6 pH units starting in the late 1800's.     

Ecological differences influence the thermal sensitivity of swimming performance in two co-occurring mysid shrimp species with climate change implications

Temperature strongly affects performance in ectotherms. As ocean warming continues, performance of marine species will be impacted. Many studies have focused on how warming will impact physiology, life history, and behavior, but few studies have investigated how ecological and behavioral traits of organisms will affect their response to changing thermal environments. Here, we assessed the thermal tolerances and thermal sensitivity of swimming performance of two sympatric mysid shrimp species of the Northwest Atlantic. Neomysis americana and Heteromysis formosa overlap in habitat and many aspects of their ecological niche, but only N. americana exhibits vertical migration. In temperate coastal ecosystems, temperature stratification of the water column exposes vertical migrators to a wider range of temperatures on a daily basis. We found that N. americana had a significantly lower critical thermal minimum (CT_min) and critical thermal maximum (CT_max). However, both mysid species had a buffer of at least 4 °C between their CT_max and the 100-year projection for mean summer water temperatures of 28 °C. Swimming performance of the vertically migrating species was more sensitive to temperature variation, and this species exhibited faster burst swimming speeds. The generalist performance curve of H. formosa and specialist curve of N. americana are consistent with predictions based on the exposure of each species to temperature variation such that higher within-generation variability promotes specialization. However, these species violate the assumption of the specialist-generalist tradeoff in that the area under their performance curves is not constant. Our results highlight the importance of incorporating species-specific responses to temperature based on the ecology and behavior of organisms into climate change prediction models.     

An overview of cyanobacterial bloom occurrences and research in Africa over the last decade

Cyanobacterial blooms are a current cause for concern globally, with vital water sources experiencing frequent and increasingly toxic blooms in the past decade. These increases are resultant of both anthropogenic and natural factors, with climate change being the central concern. Of the more affected parts of the world, Africa has been considered particularly vulnerable due to its historical predisposition and lag in social economic development. This review collectively assesses the available information on cyanobacterial blooms in Africa as well as any visible trends associated with reported occurrences over the last decade. Of the 54 countries in Africa, only 21 have notable research information in the area of cyanobacterial blooms within the last decade, although there is substantial reason to attribute these blooms as some of the major water quality threats in Africa collectively. The collected information suggests that civil wars, disease outbreaks and inadequate infrastructure are at the core of Africa’s delayed advancement. This is even more so in the area of cyanobacteria related research, with 11 out of 21 countries having recorded toxicity and physicochemical parameters related to cyanobacterial blooms. Compared to the rest of the continent, peripheral countries are at the forefront of research related to cyanobacteria, with countries such as Angola having sufficient rainfall, but poor water quality with limited information on bloom occurrences. An assessment of the reported blooms found nitrogen concentrations to be higher in the water column of more toxic blooms, validating recent global studies and indicating that phosphorous is not the only factor to be monitored in bloom mitigation. Blooms occurred at low TN: TP ratios and at temperatures above 12 °C. Nitrogen was linked to toxicity and temperature also had a positive effect on bloom occurrence and toxicity. Microcystis was the most ubiquitous of the cyanobacterial strains reported in Africa and the one most frequently toxic. Cylindrospermopsis was reported more in the dry, north and western parts of the continent countries as opposed to the rest of the continent, whilst Anabaena was more frequent on the south eastern regions. In light of the entire continent, the inadequacy in reported blooms and advances in this area of research require critical intervention and action.     

Dynamic energy budget modeling reveals the potential of future growth and calcification for the coccolithophore Emiliania huxleyi in an acidified ocean

Ocean acidification is likely to impact the calcification potential of marine organisms. In part due to the covarying nature of the ocean carbonate system components, including pH and CO₂ and CO₃^2? levels, it remains largely unclear how each of these components may affect calcification rates quantitatively. We develop a process‐based bioenergetic model that explains how several components of the ocean carbonate system collectively affect growth and calcification rates in Emiliania huxleyi, which plays a major role in marine primary production and biogeochemical carbon cycling. The model predicts that under the IPCC A2 emission scenario, its growth and calcification potential will have decreased by the end of the century, although those reductions are relatively modest. We anticipate that our model will be relevant for many other marine calcifying organisms, and that it can be used to improve our understanding of the impact of climate change on marine systems.     

Meta‐analysis reveals complex marine biological responses to the interactive effects of ocean acidification and warming

Ocean acidification and warming are considered two of the greatest threats to marine biodiversity, yet the combined effect of these stressors on marine organisms remains largely unclear. Using a meta‐analytical approach, we assessed the biological responses of marine organisms to the effects of ocean acidification and warming in isolation and combination. As expected biological responses varied across taxonomic groups, life‐history stages, and trophic levels, but importantly, combining stressors generally exhibited a stronger biological (either positive or negative) effect. Using a subset of orthogonal studies, we show that four of five of the biological responses measured (calcification, photosynthesis, reproduction, and survival, but not growth) interacted synergistically when warming and acidification were combined. The observed synergisms between interacting stressors suggest that care must be made in making inferences from single‐stressor studies. Our findings clearly have implications for the development of adaptive management strategies particularly given that the frequency of stressors interacting in marine systems will be likely to intensify in the future. There is now an urgent need to move toward more robust, holistic, and ecologically realistic climate change experiments that incorporate interactions. Without them accurate predictions about the likely deleterious impacts to marine biodiversity and ecosystem functioning over the next century will not be possible.     

Effects of ocean acidification on Antarctic marine organisms: A meta‐analysis

Southern Ocean waters are among the most vulnerable to ocean acidification. The projected increase in the CO₂ level will cause changes in carbonate chemistry that are likely to be damaging to organisms inhabiting these waters. A meta‐analysis was undertaken to examine the vulnerability of Antarctic marine biota occupying waters south of 60°S to ocean acidification. This meta‐analysis showed that ocean acidification negatively affects autotrophic organisms, mainly phytoplankton, at CO₂ levels above 1,000 μatm and invertebrates above 1,500 μatm, but positively affects bacterial abundance. The sensitivity of phytoplankton to ocean acidification was influenced by the experimental procedure used. Natural, mixed communities were more sensitive than single species in culture and showed a decline in chlorophyll a concentration, productivity, and photosynthetic health, as well as a shift in community composition at CO₂ levels above 1,000 μatm. Invertebrates showed reduced fertilization rates and increased occurrence of larval abnormalities, as well as decreased calcification rates and increased shell dissolution with any increase in CO₂ level above 1,500 μatm. Assessment of the vulnerability of fish and macroalgae to ocean acidification was limited by the number of studies available. Overall, this analysis indicates that many marine organisms in the Southern Ocean are likely to be susceptible to ocean acidification and thereby likely to change their contribution to ecosystem services in the future. Further studies are required to address the poor spatial coverage, lack of community or ecosystem‐level studies, and the largely unknown potential for organisms to acclimate and/or adapt to the changing conditions.     

Role of vacuolar adenosine triphosphatase in the regulation of cytosolic pH in hepatocytes

The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2,7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of ¹⁴C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25–37.5 mm in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO ₃ ^– , both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A₁ caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO₂) was sensitive to both 25 mm NO ₃ ^– and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5–1.0 mm ouabain resulted in an initial alkalinization (0.5–2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mm NO ₃ ^– , but not by 25 mm gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H⁺ uptake by V-ATPase activity. We conclude that V-ATPases make an important contribution to the regulation of the cytosolic pH of hepatocytes.This work was supported in part by National Institutes of Health B.R.S. Grant 507 RR05417 to Temple University.     

Origins of root-mediated pH changes in the rhizosphere and their responses to environmental constraints: A review

The aim of the present review is to define the various origins of root-mediated changes of pH in the rhizosphere, i.e., the volume of soil around roots that is influenced by root activities. Root-mediated pH changes are of major relevance in an ecological perspective as soil pH is a critical parameter that influences the bioavailability of many nutrients and toxic elements and the physiology of the roots and rhizosphere microorganisms. A major process that contributes root-induced pH changes in the rhizosphere is the release of charges carried by H⁺ or OH^– to compensate for an unbalanced cation–anion uptake at the soil–root interface. In addition to the ions taken up by the plant, all the ions crossing the plasma membrane of root cells (e.g., organic anions exuded by plant roots) should be taken into account, since they all need to be balanced by an exchange of charges, i.e., by a release of either H⁺ or OH^–. Although poorly documented, root exudation and respiration can contribute some proportion of rhizosphere pH decrease as a result of a build-up of the CO₂ concentration. This will form carbonic acid in the rhizosphere that may dissociate in neutral to alkaline soils, and result in some pH decrease. Ultimately, plant roots and associated microorganisms can also alter rhizosphere pH via redox-coupled reactions. These various processes involved in root-mediated pH changes in the rhizosphere also depend on environmental constraints, especially nutritional constraints to which plants can respond. This is briefly addressed, with a special emphasis on the response of plant roots to deficiencies of P and Fe and to Al toxicity. Finally, soil pH itself and pH buffering capacity also have a dramatic influence on root-mediated pH changes.     

On the potential for ocean acidification to be a general cause of ancient reef crises

Anthropogenic rise in the carbon dioxide concentration in the atmosphere leads to global warming and acidification of the oceans. Ocean acidification (OA) is harmful to many organisms but especially to those that build massive skeletons of calcium carbonate, such as reef corals. Here, we test the recent suggestion that OA leads not only to declining calcification of reef corals and reduced growth rates of reefs but may also have been a trigger of ancient reef crises and mass extinctions in the sea. We analyse the fossil record of biogenic reefs and marine organisms to (1) assess the timing and intensity of ancient reef crises, (2) check which reef crises were concurrent with inferred pulses of carbon dioxide concentrations and (3) evaluate the correlation between reef crises and mass extinctions and their selectivity in terms of inferred physiological buffering. We conclude that four of five global metazoan reef crises in the last 500 Myr were probably at least partially governed by OA and rapid global warming. However, only two of the big five mass extinctions show geological evidence of OA.     

Acidic extracellular pH neutralizes the autophagy-inhibiting activity of chloroquine: Implications for cancer therapies

Acidic pH is an important feature of tumor microenvironment and a major determinant of tumor progression. We reported that cancer cells upregulate autophagy as a survival mechanism to acidic stress. Inhibition of autophagy by administration of chloroquine (CQ) in combination anticancer therapies is currently evaluated in clinical trials. We observed in 3 different human cancer cell lines cultured at acidic pH that autophagic flux is not blocked by CQ. This was consistent with a complete resistance to CQ toxicity in cells cultured in acidic conditions. Conversely, the autophagy-inhibiting activity of Lys-01, a novel CQ derivative, was still detectable at low pH. The lack of CQ activity was likely dependent on a dramatically reduced cellular uptake at acidic pH. Using cell lines stably adapted to chronic acidosis we could confirm that CQ lack of activity was merely caused by acidic pH. Moreover, unlike CQ, Lys-01 was able to kill low pH-adapted cell lines, although higher concentrations were required as compared with cells cultured at normal pH conditions. Notably, buffering medium pH in low pH-adapted cell lines reverted CQ resistance. In vivo analysis of tumors treated with CQ showed that accumulation of strong LC3 signals was observed only in normoxic areas but not in hypoxic/acidic regions. Our observations suggest that targeting autophagy in the tumor environment by CQ may be limited to well-perfused regions but not achieved in acidic regions, predicting possible limitations in efficacy of CQ in antitumor therapies.