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An organism's oxygen supply capacity, measured as a ratio of a metabolic rate to its critical oxygen partial pressure, describes the efficacy of oxygen uptake and transport. This metric is sensitive to errors in oxygen measurement, especially near anoxia where the magnitude of instrument error as a proportion of total signal is magnified. Here, we present a conceptual and mathematical method that uses this sensitivity to identify, quantify, and therefore correct oxygen measurements collected using inaccurately calibrated sensors. When appropriate, adding a small correction value to each oxygen measurement counteracts the effects of this error and provides results that are comparable to data from accurately calibrated oxygen probes. We demonstrate, using simulated, laboratory, and literature datasets, how this method can be used post hoc to diagnose error in, correct the magnitude of, and reduce the variability in repeat measures of traits relevant to oxygen tolerance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Air-breathing fish risk losing aerially sourced oxygen to ambient hypoxic water since oxygenated blood from the air-breathing organ returns through the heart to the branchial basket before distribution. This loss is thought to help drive the evolutionary reduction in gill size with the advent of air-breathing. In many teleost fish, gill size is known to be highly plastic by modulation of their anatomic diffusion factor (ADF) with inter-lamellar cell mass (ILCM). In the anoxia-tolerant crucian carp, ILCM recedes with hypoxia but regrows in anoxia. The air-breathing teleost Chitala ornata has been shown to increase gill ADF from normoxic to mildly hypoxic water by reducing ILCM. Here, we test the hypothesis that ADF is modulated to minimize oxygen loss in severe aquatic hypoxia by measuring ADF, gas-exchange, and by using computed tomography scans to reveal possible trans-branchial shunt vessels. Contrary to our hypothesis, ADF does not modulate to prevent oxygen loss and despite no evident trans-branchial shunting, C. ornata loses only 3% of its aerially sourced O 2 while still excreting 79% of its CO 2 production to the severely hypoxic water. We propose this is achieved by ventilatory control and by compensating the minor oxygen loss by extra aerial O 2 uptake.

Levels of dissolved oxygen in open ocean and coastal waters are decreasing (ocean deoxygenation), with poorly understood effects on marine megafauna. All of the more than 1000 species of elasmobranchs (sharks, skates, and rays) are obligate water breathers, with a variety of life-history strategies and oxygen requirements. This review demonstrates that although many elasmobranchs typically avoid hypoxic water, they also appear capable of withstanding mild to moderate hypoxia with changes in activity, ventilatory responses, alterations to circulatory and hematological parameters, and morphological alterations to gill structures. However, such strategies may be insufficient to withstand severe, progressive, or prolonged hypoxia or anoxia where anaerobic metabolic pathways may be used for limited periods. As water temperatures increase with climate warming, ectothermic elasmobranchs will exhibit elevated metabolic rates and are likely to be less able to tolerate the effects of even mild hypoxia associated with deoxygenation. As a result, sustained hypoxic conditions in warmer coastal or surface-pelagic waters are likely to lead to shifts in elasmobranch distributions. Mass mortalities of elasmobranchs linked directly to deoxygenation have only rarely been observed but are likely underreported. One key concern is how reductions in habitat volume as a result of expanding hypoxia resulting from deoxygenation will influence interactions between elasmobranchs and industrial fisheries. Catch per unit of effort of threatened pelagic sharks by longline fisheries, for instance, has been shown to be higher above oxygen minimum zones compared to adjacent, normoxic regions, and attributed to vertical habitat compression of sharks overlapping with increased fishing effort. How a compound stressor such as marine heatwaves alters vulnerability to deoxygenation remains an open question. With over a third of elasmobranch species listed as endangered, a priority for conservation and management now lies in understanding and mitigating ocean deoxygenation effects in addition to population declines already occurring from overfishing., (© 2024 The Author(s). Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.)

Sinking or floating is the natural state of planktonic organisms and particles in the ocean. Simulating these conditions is critical when making measurements, such as respirometry, because they allow the natural exchange of substrates and products between sinking particles and water flowing around them and prevent organisms that are accustomed to motion from changing their metabolism. We developed a rotating incubator, the RotoBOD (named after its capability to rotate and determine biological oxygen demand, BOD), that uniquely enables automated oxygen measurements in small volumes while keeping the samples in their natural state of suspension. This allows highly sensitive rate measurements of oxygen utilization and subsequent characterization of single particles or small planktonic organisms, such as copepods, jellyfish, or protists. As this approach is nondestructive, it can be combined with several further measurements during and after the incubation, such as stable isotope additions and molecular analyses. This makes the instrument useful for ecologists, biogeochemists, and potentially other user groups such as aquaculture facilities. Here, we present the technical background of our newly developed apparatus and provide examples of how it can be utilized to determine oxygen production and consumption in small organisms and particles.

In hypoxia, air-breathing fish obtain O 2 from the air but continue to excrete CO 2 into the water. Consequently, it is believed that some O 2 obtained by air-breathing is lost at the gills in hypoxic water. Pangasionodon hypophthalmus is an air-breathing catfish with very large gills from the Mekong River basin where it is cultured in hypoxic ponds. To understand how P. hypophthalmus can maintain high growth in hypoxia with the presumed O 2 loss, we quantified respiratory gas exchange in air and water. In severe hypoxia (PO 2 : ≈ 1.5 mmHg), it lost a mere 4.9% of its aerial O 2 uptake, while maintaining aquatic CO 2 excretion at 91% of the total. Further, even small elevations in water PO 2 rapidly reduced this minor loss. Charting the cardiovascular bauplan across the branchial basket showed four ventral aortas leaving the bulbus arteriosus, with the first and second gill arches draining into the dorsal aorta while the third and fourth gill arches drain into the coeliacomesenteric artery supplying the gut and the highly trabeculated respiratory swim-bladder. Substantial flow changes across these two arterial systems from normoxic to hypoxic water were not found. We conclude that the proposed branchial oxygen loss in air-breathing fish is likely only a minor inefficiency.

Oxygen is essential for most eukaryotic lifeforms, as it supports mitochondrial oxidative phosphorylation to supply ∼90% of cellular adenosine triphosphate (ATP). Fluctuations in O2 present a major stressor, with hypoxia leading to a cascade of detrimental physiological changes that alter cell operations and ultimately induce death. Nonetheless, some species episodically tolerate near-anoxic environments, and have evolved mechanisms to sustain function even during extended hypoxic periods. While mitochondria are pivotal in central metabolism, their role in hypoxia tolerance remains ill defined. Given the vulnerability of the brain to hypoxia, mitochondrial function was tested in brain homogenates of three closely related triplefin species with varying degrees of hypoxia tolerance (Bellapiscis medius, Forsterygion lapillum and Forsterygion varium). High-resolution respirometry coupled with fluorometric measurements of mitochondrial membrane potential (mtMP) permitted assessment of differences in mitochondrial function and integrity in response to intermittent hypoxia and anoxia. Traditional steady-state measures of respiratory flux and mtMP showed no differences among species. However, in the transition into anoxia, the tolerant species B. medius and F. lapillum maintained mtMP at O2 pressures 7- and 4.4-fold lower, respectively, than that of the hypoxia-sensitive F. varium and exhibited slower rates of membrane depolarisation. The results indicate that dynamic oxic-hypoxic mitochondria transitions underlie hypoxia tolerance in these intertidal fish., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The speckled peacock bass Cichla temensis is a popular sport and food fish that generates substantial angling tourism and utilitarian harvest within its range. Its popularity and value make this species important for management and a potential aquaculture candidate for both fisheries enhancement and food fish production. However, little is known of optimal physiochemical conditions in natural habitats, which also are important for the development of hatchery protocols for handling, spawning and grow-out. Speckled peacock bass have been documented to have high sensitivity to extreme temperatures, but the metabolic underpinnings have not been evaluated. In this study, the effects of temperature (25, 30 and 35°C) on the standard metabolic rate (SMR) and lower dissolved oxygen tolerance (LDOT) of juvenile speckled peacock bass (mean ± standard error total length 153 ± 2 mm and wet weight 39.09 ± 1.37 g) were evaluated using intermittent respirometers after an acclimation period of 2 weeks. Speckled peacock bass had the highest SMR at 35°C (345.56 ± 19.89 mgO 2  kg -1  h -1 ), followed by 30°C (208.16 ± 12.45 mgO 2  kg -1  h -1 ) and 25°C (144.09 ± 10.43 mgO 2  kg -1  h -1 ). Correspondingly, the Q 10 , or rate of increase in aerobic metabolic rate (MO 2 ) relative to 10°C, for 30-35°C was also greater (2.76) than from 25 to 30°C (2.08). Similarly, speckled peacock bass were the most sensitive to hypoxia at the warmest temperature, with an LDOT at pO 2 of 90 mmHg (4.13 mg l -1 ) at 35°C compared to pO 2 values of 45 mmHg (2.22 mg l -1 ) and 30 mmHg (1.61 mg l -1 ) at 30 and 25°C, respectively. These results indicate that speckled peacock bass are sensitive to temperatures near 35°C, therefore we recommend managing and rearing this species at 25-30°C., (© 2023 Fisheries Society of the British Isles.)

Ocean deoxygenation is a pressing concern in the face of climate change. In response to prolonged hypoxia, fishes have demonstrated the ability to dynamically regulate hemoglobin (Hb) expression to enhance oxygen (O 2 ) uptake. Here, we examined hypoxia-inducible Hb expression in red drum (Sciaenops ocellatus) and the subsequent implications on Hb-O 2 binding affinity and aerobic scope. Fish were acclimated to 30 % air saturation for 1 d, 4 d, 8 d, 2 w, or 6 w, and red blood cells were collected for gene expression and biochemical profiling. Hypoxia acclimation induced significant up-regulation of one Hb subunit isoform (hbα 2) relative to control by 4 d with consistent upregulation thereafter. Hematocrit increased in hypoxia, with no changes in the allosteric modulator [NTP] at any time point. Changes in Hb expression co-occurred with a reduced Root effect (~26 % in normoxia, ~14 % in hypoxia) at a physiologically relevant pH while increasing O 2 binding affinity (i.e., lower P 50 ). These changes correlated with increased maximum metabolic rate and aerobic scope relative to controls when fish were tested in hypoxia. These results demonstrate an important role for Hb multiplicity in improving O 2 affinity and maximizing respiratory performance in hypoxia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Like all taxa, populations of aquatic insects may respond to climate change by evolving new physiologies or behaviors, shifting their range, exhibiting physiological and behavioral plasticity, or going extinct. We evaluated the importance of plasticity by measuring changes in growth, survival and respiratory phenotypes of salmonfly nymphs (the stonefly Pteronarcys californica) in response to experimental combinations of dissolved oxygen and temperature. Overall, smaller individuals grew more rapidly during the 6-week experimental period, and oxygen and temperature interacted to affect growth in complex ways. Survival was lower for the warm treatment, although only four mortalities occurred (91.6% versus 100%). Nymphs acclimated to warmer temperatures did not have higher critical thermal maxima (CTmax), but those acclimated to hypoxia had CTmax values (in normoxia) that were higher by approximately 1°C. These results suggest possible adaptive plasticity of systems for taking up or delivering oxygen. We examined these possibilities by measuring the oxygen sensitivity of metabolic rates and the morphologies of tracheal gill tufts located ventrally on thoracic segments. Mass-specific metabolic rates of individuals acclimated to warmer temperatures were higher in acute hypoxia but lower in normoxia, regardless of their recent history of oxygen exposure during acclimation. The morphology of gill filaments, however, changed in ways that appeared to depress rates of oxygen delivery in functional hypoxia. Our combined results from multiple performance metrics indicate that rising temperatures and hypoxia may interact to magnify the risks to aquatic insects, but that physiological plasticity in respiratory phenotypes may offset some of these risks., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The hypoxic constraint on peak oxygen uptake (ṀO2,peak) was characterized in rainbow trout over a range of ambient oxygen tensions with different testing protocols and statistical models. The best-fit model was selected using both statistical criteria (R2 and AIC) and the model's prediction of three anchor points for hypoxic performance: critical PO2 (Pcrit), maximum ṀO2 and a new metric, the minimum PO2 that supports 50% of absolute aerobic scope (PAAS-50). The best-fitting model was curvilinear using five strategically selected PO2 values. This model predicted PAAS-50 as 70 mmHg (coefficient of variation, CV=9%) for rainbow trout. Thus, while a five-point hypoxic performance curve can characterize the limiting effects of hypoxia in fish, as envisaged by Fry over 75 years ago, PAAS-50 is a promising metric to compare hypoxic constraints on performance in a standardized manner both within and across fish species., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

One of the most air-reliant obligate air-breathing fish is the South American Arapaima gigas, with substantially reduced gills impeding gas diffusion, thought to be a result of recurring aquatic hypoxia in its habitat. In normoxic water, A. gigas is reported to satisfy 70-80% of its O2 requirement from the air while excreting 60-90% of its CO2 to the water. If this pattern of gas exchange were to continue in severely hypoxic water, O2 loss at the gills would be expected. We hypothesized therefore that partitioning of CO2 would shift to the air phase in severe aquatic hypoxia, eliminating the risk of branchial O2 loss. By adapting a respirometer designed to measure aquatic ṀO2/ṀCO2, we were able to run intermittent closed respirometry on both water and air phase for both of these gasses as well as sample water for N-waste measurements (ammonia-N, urea-N) so as to calculate metabolic fuel utilization. In contrast to our prediction, we found that partitioning of CO2 excretion changed little between normoxia and severe hypoxia (83% versus 77% aquatic excretion, respectively) and at the same time there was no evidence of branchial O2 loss in hypoxia. This indicates that A. gigas can utilize distinct transfer pathways for O2 and CO2. Routine and standard ṀO2, N-waste excretion and metabolic fuel utilization did not change with water oxygenation. Metabolism was fuelled mostly by protein oxidation (53%), while carbohydrates and lipids accounted for 27% and 20%, respectively., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The identification of novel physiological regulators that stimulate energy expenditure through brown adipose tissue (BAT) activity in substrate catalysis is of utmost importance to understand and treat metabolic diseases. Myoglobin (MB), known to store or transport oxygen in heart and skeletal muscles, has recently been found to bind fatty acids with physiological constants in its oxygenated form (i.e., MBO 2 ). Here, we investigated the in vivo effect of MB expression on BAT activity. In particular, we studied mitochondrial function and lipid metabolism as essential determinants of energy expenditure in this tissue. We show in a MB-null (MBko) mouse model that MB expression in BAT impacts on the activity of brown adipocytes in a twofold manner: i) by elevating mitochondrial density plus maximal respiration capacity, and through that, by stimulating BAT oxidative metabolism along with the organelles` uncoupled respiration; and ii) by influencing the free fatty acids pool towards a palmitate-enriched composition and shifting the lipid droplet (LD) equilibrium towards higher counts of smaller droplets. These metabolic changes were accompanied by the up-regulated expression of thermogenesis markers UCP1, CIDEA, CIDEC, PGC1-α and PPAR-α in the BAT of MB wildtype (MBwt) mice. Along with the emergence of the "browning" BAT morphology, MBwt mice exhibited a leaner phenotype when compared to MBko littermates at 20 weeks of age. Our data shed novel insights into MB's role in linking oxygen and lipid-based thermogenic metabolism. The findings suggest potential new strategies of targeting the MB pathway to treat metabolic disorders related to diminishing energy expenditure., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The critical oxygen partial pressure (Pcrit), typically defined as the PO2 below which an animal's metabolic rate (MR) is unsustainable, is widely interpreted as a measure of hypoxia tolerance. Here, Pcrit is defined as the PO2 at which physiological oxygen supply (α0) reaches its maximum capacity (α; µmol O2 g-1 h-1 kPa-1). α is a species- and temperature-specific constant describing the oxygen dependency of the maximum metabolic rate (MMR=PO2×α) or, equivalently, the MR dependence of Pcrit (Pcrit=MR/α). We describe the α-method, in which the MR is monitored as oxygen declines and, for each measurement period, is divided by the corresponding PO2 to provide the concurrent oxygen supply (α0=MR/PO2). The highest α0 value (or, more conservatively, the mean of the three highest values) is designated as α. The same value of α is reached at Pcrit for any MR regardless of previous or subsequent metabolic activity. The MR need not be constant (regulated), standardized or exhibit a clear breakpoint at Pcrit for accurate determination of α. The α-method has several advantages over Pcrit determination and non-linear analyses, including: (1) less ambiguity and greater accuracy, (2) fewer constraints in respirometry methodology and analysis, and (3) greater predictive power and ecological and physiological insight. Across the species evaluated here, α values are correlated with MR, but not Pcrit. Rather than an index of hypoxia tolerance, Pcrit is a reflection of α, which evolves to support maximum energy demands and aerobic scope at the prevailing temperature and oxygen level., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Oxygen uptake, heart rate and contraction frequencies of slow oxidative (SO) and fast glycolytic (FG) muscle were measured simultaneously in gilthead seabream Sparus aurata submitted to stepwise increases in current speed in a swimming respirometer. Variation in oxygen uptake was closely related to variation in heart rate, over initial steps these rose in concert with an increase in contraction frequency of SO muscle. There was an asymptote in oxygen uptake and heart rate at high speeds that reflected a transition from exclusive use of aerobic SO muscle to a combination of SO and anaerobic FG muscle, and which preceded fatigue., (© 2020 The Fisheries Society of the British Isles.)

The critical O 2 tension ( P crit ) is the lowest P O 2  at which an animal can maintain some benchmark rate of O 2 uptake ( Ṁ O 2 ). This P O 2  has long served as a comparator of hypoxia tolerance in fishes and aquatic invertebrates, but its usefulness in this role, particularly when applied to fishes, has recently been questioned. We believe that P crit remains a useful comparator of hypoxia tolerance provided it is determined using the proper methods and hypoxia tolerance is clearly defined. Here, we review the available methods for each of the three steps of P crit determination: (1) measuring the most appropriate benchmark Ṁ O 2  state for P crit determination ( Ṁ O 2 ,std , the Ṁ O 2  required to support standard metabolic rate); (2) reducing water P O 2 ; and (3) calculating P crit from the Ṁ O 2  versus P O 2  curve. We make suggestions on best practices for each step and for how to report P crit results to maximize their comparative value. We also discuss the concept of hypoxia tolerance and how P crit relates to a fish's overall hypoxia tolerance. When appropriate methods are used, P crit provides useful comparative physiological and ecological information about the aerobic contributions to a fish's hypoxic survival. When paired with other hypoxia-related physiological measurements (e.g. lactate accumulation, calorimetry-based measurements of metabolic depression, loss-of-equilibrium experiments), P crit contributes to a comprehensive understanding of how a fish combines aerobic metabolism, anaerobic metabolism and metabolic depression in an overall strategy for hypoxia tolerance., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Oxygen- and capacity-limited thermal tolerance (OCLTT) is a controversial hypothesis claiming to explain variation in, and mechanistically determine, animal thermal limits. The lack of support from Insecta is typically argued to be a consequence of their high-performance respiratory systems. However, no studies have reported internal body oxygen levels during thermal ramping so it is unclear if changes in ambient gas are partially or fully offset by a compensatory respiratory system. Here we provide such an assessment by simultaneously recording haemolymph oxygen (pO 2 ) levels-as an approximation of tissue oxygenation-while experimentally manipulating ambient oxygen and subjecting organisms to thermal extremes in a series of thermolimit respirometry experiments using pupae of the butterfly Pieris napi. The main results are that while P. napi undergo large changes in haemolymph pO 2 that are positively correlated with experimental oxygen levels, haemolymph pO 2 is similar pre- and post-death during thermal assays. OCLTT predicts that reduction in body oxygen level should lead to a reduction in CTmax. Despite finding the former, there was no change in CTmax across a wide range of body oxygen levels. Thus, we argue that oxygen availability is not a functional determinant of the upper thermal limits in pupae of P. napi.

Microalgae-bacteria consortia application to wastewater treatment is considered as a potential and cheap strategy towards a self-sustaining oxygen-carbon dioxide gas exchange. However, microalgae can also carry out mixotrophy, thus reducing the net oxygen production, due to consumption of organic substrates. In this work, respirometric tests were used to quantify the oxygen reduction in the presence of biodegradable COD (chemical oxygen demand), which resulted up to 70%, depending on the biodegradability of the carbon substrate. The implication of mixotrophic metabolism on nutrient removal in urban wastewater was also measured by co-cultivating C. protothecoides with bacteria from activated sludge. To better understand the contribution of different populations, ad hoc experiments under controlled conditions were designed to quantify the nutrient consumption of bacteria and microalgae. Microalgae and bacteria were cultivated together and separately, with and without external bubbling, so to better ascertain the specific role of gas production and nutrient removal. Results showed that microalgae can remove up to 100 and 85% of P and N respectively, but the contribution on COD consumption may affect the net O 2 supply to heterotrophic bacteria. However, a mutual COD consumption by microalgae and bacteria was proved by both experimental growth curves and mass balance application, based on stoichiometry experimentally adjusted.

Platelet activation plays a key role in normal haemostasis and pathological thrombosis. Platelet activation is rapid; within minutes of stimulation, platelets generate bioactive phospholipids, secrete their granule contents, activate integrins and aggregate together to form a haemostatic plug. These events are dependent on ATP synthesis. Mitochondrial function in platelets from healthy volunteers and patients with a range of diseases indicate an important role for oxygen consumption in oxidative phosphorylation in normal and pathological function. Platelets also consume oxygen during oxidation reactions, such as cyclooxygenase-dependent thromboxane A 2 synthesis. In this study, we used high-resolution respirometry to investigate rapid changes in oxygen consumption during platelet activation. We demonstrated a rapid, transient increase in oxygen consumption rate within minutes of platelet stimulation by the physiological activator, thrombin. This was partly inhibited by aspirin and by oligomycin. This shows that high resolution respirometry can provide information regarding rapid and dynamic changes in oxygen consumption during platelet activation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, the monitoring of reactive oxygen species and the regeneration of the ferrous ions catalyst were performed during electro-Fenton (EF) process to highlight the influence of operating parameters. The removal of metronidazole (MTZ) was implemented in an electrochemical mono-compartment batch reactor under various ranges of current densities, initial MTZ and ferrous ions concentrations, and pH values. It was found that under 0.07 mA cm -2 , 0.1 mM of ferrous ions and pH = 3, the efficiency of 100 mg L -1 MTZ degradation and mineralization were 100% within 20 min and 40% within 135 min of electrolysis, respectively. The highest hydrogen peroxide and hydroxyl radical concentrations, 1.4 mM and 2.28 mM respectively, were obtained at 60 min electrolysis at 0.07 mA cm -2 . Improvement of the biodegradability was reached from 60 min of electrolysis with a BOD 5 /COD ratio above 0.4, which was reinforced by a respirometric study, that supports the feasibility of coupling electro-Fenton and biological treatment for the metronidazole removal., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Animals are bombarded with information about their environment and must select and interpret the relevant cues to make behavioral adjustments critical to survival. How animals integrate and balance the many signals they receive about their environment is rarely assessed. We investigated how signals from the social and physical environment interact to influence environmental preferences in the endemic Tanganyikan cichlid Neolamprologus pulcher. Specifically, we explored how fish respond to the physiological challenge of declining O 2 levels in light of embedded social preferences using a modified shuttle box apparatus to test O 2 preferences. In the presence of a conspecific, the average (preferred) partial pressure of oxygen (Po 2 ) and minimum Po 2 experienced were significantly lower ([Formula: see text] and [Formula: see text] kPa, respectively) than in trials without a conspecific ([Formula: see text] and [Formula: see text] kPa, respectively). Fish with conspecifics also spent more time in the low Po 2 zone of the shuttle box and moved between the high and low Po 2 zones less frequently. Hence, O 2 preferences were modified, and fish willingly remained in an area of continuously declining O 2 availability to associate with a conspecific. The O 2 preferences of an individual during social trials correlated with its excess postexercise O 2 consumption following an exhaustive chase but not with its aerobic scope, routine O 2 consumption rate, or body mass. These results suggest that some aspects of respiratory and metabolic physiology (such as the propensity to use anaerobic metabolism) but not others (such as O 2 transport capacity) underpin some variation in social behavior under environmental stress.

The NDHA model comprehensively describes nitrous oxide (N 2 O) producing pathways by both autotrophic ammonium oxidizing and heterotrophic bacteria. The model was calibrated via a set of targeted extant respirometric assays using enriched nitrifying biomass from a lab-scale reactor. Biomass response to ammonium, hydroxylamine, nitrite and N 2 O additions under aerobic and anaerobic conditions were tracked with continuous measurement of dissolved oxygen (DO) and N 2 O. The sequential addition of substrate pulses allowed the isolation of oxygen-consuming processes. The parameters to be estimated were determined by the information content of the datasets using identifiability analysis. Dynamic DO profiles were used to calibrate five parameters corresponding to endogenous, nitrite oxidation and ammonium oxidation processes. The subsequent N 2 O calibration was not significantly affected by the uncertainty propagated from the DO calibration because of the high accuracy of the estimates. Five parameters describing the individual contribution of three biological N 2 O pathways were estimated accurately (variance/mean < 10% for all estimated parameters). The NDHA model response was evaluated with statistical metrics (F-test, autocorrelation function). The 95% confidence intervals of DO and N 2 O predictions based on the uncertainty obtained during calibration are studied for the first time. The measured data fall within the 95% confidence interval of the predictions, indicating a good model description. Overall, accurate parameter estimation and identifiability analysis of ammonium removal significantly decreases the uncertainty propagated to N 2 O production, which is expected to benefit N 2 O model discrimination studies and reliable full scale applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hypoxia resulting from aquatic eutrophication threatens the population health of the New Zealand freshwater crayfish (koura), Paranephrops zealandicus. An integrated study, combining behavioural, physiological and biochemical approaches, was therefore conducted to characterise the tolerance of this species to hypoxia. When provided with a choice between water flows of high or low dissolved oxygen in short-term laboratory assays, crayfish did not preferentially inhabit waters of higher PO 2 . However, when an aerial refuge was provided and dissolved oxygen was progressively decreased, crayfish emersed at a PO 2 of 0.56±0.03kPa, suggesting a relatively high tolerance to hypoxia. Closed-box respirometry delineated a P crit , the point at which crayfish transition from oxyregulating to oxyconforming, of 6.0kPa. Simultaneous measurement of heart rate showed no changes across the PO 2 range. In response to 6-h exposures to fixed dissolved oxygen levels (normoxia, 19.3kPa; moderate hypoxia, 3.5kPa; and severe hypoxia, 1.7kPa), P. zealandicus showed a haemolymph PO 2 that declined with the magnitude of hypoxia, and while plasma pH declined in severe hypoxia, there were no changes in plasma PCO 2 . Plasma glucose concentrations fell, and plasma lactate increased in both hypoxic groups. There were no changes in tissue glucose or lactate concentrations. These data indicate that P. zealandicus is relatively tolerant of hypoxia, and possesses biochemical and physiological mechanisms that facilitate survival during short-term exposures to acute hypoxia. If hypoxia is severe and/or prolonged, then this species is capable of escaping to aerial refugia., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Flying insects have the highest known mass-specific demand for oxygen, which makes it likely that reduced availability of oxygen might limit sustained flight, either instead of or in addition to the limitation due to metabolite resources. The Glanville fritillary butterfly (Melitaea cinxia) occurs as a large metapopulation in which adult butterflies frequently disperse between small local populations. Here, we examine how the interaction between oxygen availability and fuel use affects flight performance in the Glanville fritillary. Individuals were flown under either normoxic (21 kPa O2) or hypoxic (10 kPa O2) conditions and their flight metabolism was measured. To determine resource use, levels of circulating glucose, trehalose and whole-body triglyceride were recorded after flight. Flight performance was significantly reduced in hypoxic conditions. When flown under normoxic conditions, we observed a positive correlation among individuals between post-flight circulating trehalose levels and flight metabolic rate, suggesting that low levels of circulating trehalose constrains flight metabolism. To test this hypothesis experimentally, we measured the flight metabolic rate of individuals injected with a trehalase inhibitor. In support of the hypothesis, experimental butterflies showed significantly reduced flight metabolic rate, but not resting metabolic rate, in comparison to control individuals. By contrast, under hypoxia there was no relationship between trehalose and flight metabolic rate. Additionally, in this case, flight metabolic rate was reduced in spite of circulating trehalose levels that were high enough to support high flight metabolic rate under normoxic conditions. These results demonstrate a significant interaction between oxygen and energy availability for the control of flight performance., (© 2016. Published by The Company of Biologists Ltd.)

Naked mole rats are among the most hypoxia-tolerant mammals identified and live in chronic hypoxia throughout their lives. The physiological mechanisms underlying this tolerance, however, are poorly understood. Most vertebrates hyperventilate in acute hypoxia and exhibit an enhanced hyperventilation following acclimatization to chronic sustained hypoxia (CSH). Conversely, naked mole rats do not hyperventilate in acute hypoxia and their response to CSH has not been examined. In this study, we explored mechanisms of plasticity in the control of the hypoxic ventilatory response (HVR) and hypoxic metabolic response (HMR) of freely behaving naked mole rats following 8-10 days of chronic sustained normoxia (CSN) or CSH. Specifically, we investigated the role of the major inhibitory neurotransmitter γ-amino butyric acid (GABA) in mediating these responses. Our study yielded three important findings. First, naked mole rats did not exhibit ventilatory plasticity following CSH, which is unique among adult animals studied to date. Second, GABA receptor (GABAR) antagonism altered breathing patterns in CSN and CSH animals and modulated the acute HVR in CSN animals. Third, naked mole rats exhibited GABAR-dependent metabolic plasticity following long-term hypoxia, such that the basal metabolic rate was approximately 25% higher in normoxic CSH animals than CSN animals, and GABAR antagonists modulated this increase., (© 2016 The Author(s).)

Respirometry is a robust method for measurement of oxygen uptake as a proxy for metabolic rate in fishes, and how species with bimodal respiration might meet their demands from water v. air has interested researchers for over a century. The challenges of measuring oxygen uptake from both water and air, preferably simultaneously, have been addressed in a variety of ways, which are briefly reviewed. These methods are not well-suited for the long-term measurements necessary to be certain of obtaining undisturbed patterns of respiratory partitioning, for example, to estimate traits such as standard metabolic rate. Such measurements require automated intermittent-closed respirometry that, for bimodal fishes, has only recently been developed. This paper describes two approaches in enough detail to be replicated by the interested researcher. These methods are for static respirometry. Measuring oxygen uptake by bimodal fishes during exercise poses specific challenges, which are described to aid the reader in designing experiments. The respiratory physiology and behaviour of air-breathing fishes is very complex and can easily be influenced by experimental conditions, and some general considerations are listed to facilitate the design of experiments. Air breathing is believed to have evolved in response to aquatic hypoxia and, probably, associated hypercapnia. The review ends by considering what realistic hypercapnia is, how hypercapnic tropical waters can become and how this might influence bimodal animals' gas exchange., (© 2015 The Fisheries Society of the British Isles.)

The objective of the study was to investigate the achievable limits of aerobic sludge stabilization applied on waste-activated sludge generated in domestic, tannery, and pharmaceutical wastewater treatment plants. Stabilization study involved monitoring of conventional parameters and model evaluation of oxygen uptake rate and particulate components of waste sludge. Multi-component biomass approach was adopted based on death-regeneration mechanism. The results showed that sludge stabilization efficiency ranged between 25% and 30%, which was closely related to the fate of different particulate fractions of biomass, that is, viable biomass, hydrolysable particulates, and microbial metabolic products. Model calibration exercises yield in rate coefficient ranges of 0.18-0.32/day for biomass decay and 0.60-0.65/day for hydrolysis of non-biomass components. Degradation rates of particulate metabolic products were estimated as 0.035, 0.04, and 0.01/day for domestic, tannery, and pharmaceutical sludge, respectively. Relatively low degradation rates compared to conventional biological treatment processes confirmed reduced microbial activity in the course of aerobic stabilization.

One hypothesis for the small size of insects relative to vertebrates, and the existence of giant fossil insects, is that atmospheric oxygen levels constrain insect body sizes because oxygen delivery is more challenging in larger insects. This study tested this hypothesis in dragonflies by measuring the oxygen sensitivity of flight metabolic rates and behavior during hovering for 11 species of dragonflies that ranged in mass by an order of magnitude. We measured flight times and flight metabolic rates in seven oxygen concentrations ranging from 30% to 2.5% to assess the sensitivity of their flight to atmospheric oxygen. We also assessed the oxygen sensitivity of flight in low-density air (nitrogen replaced with helium) in order to increase the metabolic demands of hovering flight. Lowered atmospheric densities did induce higher flight metabolic rates. Flight behavior was more sensitive to decreasing oxygen levels than flight metabolic rate. The oxygen sensitivity of flight metabolic rates and behaviors were not correlated with body size, indicating that larger insects are able to maintain an oxygen supply-to-demand balance even during flight., (© 2014. Published by The Company of Biologists Ltd.)

Unsteady water flows are common in nature, yet the swimming performance of fishes is typically evaluated at constant, steady speeds in the laboratory. We examined how cyclic changes in water flow velocity affect the swimming performance and energetics of a labriform swimmer, the shiner surfperch, Cymatogaster aggregata, during station holding. Using intermittent-flow respirometry, we measured critical swimming speed (Ucrit), oxygen consumption rates (O2) and pectoral fin use in steady flow versus unsteady flows with either low- [0.5 body lengths (BL) s(-1)] or high-amplitude (1.0 BL s(-1)) velocity fluctuations, with a 5 s period. Individuals in low-amplitude unsteady flow performed as well as fish in steady flow. However, swimming costs in high-amplitude unsteady flow were on average 25.3% higher than in steady flow and 14.2% higher than estimated values obtained from simulations based on the non-linear relationship between swimming speed and oxygen consumption rate in steady flow. Time-averaged pectoral fin use (fin-beat frequency measured over 300 s) was similar among treatments. However, measures of instantaneous fin use (fin-beat period) and body movement in high-amplitude unsteady flow indicate that individuals with greater variation in the duration of their fin beats were better at holding station and consumed less oxygen than fish with low variation in fin-beat period. These results suggest that the costs of swimming in unsteady flows are context dependent in labriform swimmers, and may be influenced by individual differences in the ability of fishes to adjust their fin beats to the flow environment.

Dragonfly nymphs breathe water using tidal ventilation, a highly unusual strategy in water-breathing animals owing to the high viscosity, density and low oxygen (O2) concentration of water. This study examines how well these insects extract O2 from the surrounding water during progressive hypoxia. Nymphs were attached to a custom-designed respiro-spirometer to simultaneously measure tidal volume, ventilation frequency and metabolic rate. Oxygen extraction efficiencies (OEE) were calculated across four partial pressure of oxygen (pO2) treatments, from normoxia to severe hypoxia. While there was no significant change in tidal volume, ventilation frequency increased significantly from 9.4 ± 1.2 breaths per minute (BPM) at 21.3 kPa to 35.6 ± 2.9 BPM at 5.3 kPa. Metabolic rate increased significantly from 1.4 ± 0.3 µl O2 min−1 at 21.3 kPa to 2.1 ± 0.4 µl O2 min−1 at 16.0 kPa, but then returned to normoxic levels as O2 levels declined further. OEE of nymphs was 40.1 ± 6.1% at 21.3 kPa, and did not change significantly during hypoxia. Comparison to literature shows that nymphs maintain their OEE during hypoxia unlike other aquatic tidal-breathers and some unidirectional breathers. This result, and numerical models simulating experimental conditions, indicate that nymphs maintain these extraction efficiencies by increasing gill conductance and/or lowering internal pO2 to maintain a sufficient diffusion gradient across their respiratory surface. [ABSTRACT FROM AUTHOR]

Given the vulnerability of early life stages to predation, many species elicit antipredator responses to predator-associated cues (e.g. alarm cues, necromones). However, the response used may vary between predatory stimuli. Antipredator responses, while ostensibly enhancing survival, are costly, either resulting in missed opportunities (e.g. foraging) through decreased activity and crypsis strategies (i.e. predator avoidance) or incurring high energetic costs through the use of generalized stress responses and increased activity (i.e. predator evasion). Juvenile Port Jackson sharks, Heterodontus portusjacksoni , were exposed to the scent of dead conspecifics (conspecific necromones) coupled with a simulated predator chase to determine the energetic effects of a predator 'attack'. When exposed to necromone cues alone, juvenile sharks depressed their oxygen uptake rates for up to 3 h following exposure, indicating that they used crypsis to avoid detection by predators. However, when forced to swim and evade a predator, sharks increased their oxygen uptake rates, regardless of whether or not they were exposed to predatory cues. Juvenile sharks exposed to conspecific necromones may primarily rely on strategies that depress activity to avoid detection by predators; however, if forced to evade capture they use energetically costly escape strategies similar to typical 'fight or flight' responses. • Necromones elicit context-dependent antipredator responses. • Exercise, such as that from a predator chase, negates crypsis responses. • Changes in oxygen uptake rates persisted for at least 3 h. • Concomitant stressors will decrease effectiveness of these strategies. [ABSTRACT FROM AUTHOR]

Background: Industrial wastewaters with a high content of nitrogen are a relevant environmental problem. Currently, treatments to remove nitrogen are not efficient, so is necessary to develop alternative methods. The objective of this study is to investigate a consortium of microalgae — nitrifying, that due to the symbiosis between them could be an interesting alternative. Results: In this study, it was possible to obtain a consortium of nitrifying bacteria (NB) and microalgae (MA) capable of operating with low requirements of dissolved oxygen, using aerobic sludge from wastewater treatment plants. During the operation, this consortium presents removal percentages above 98% of ammonia, even at concentrations of DO of 0.5 mg O2 L-1. It is estimated that the removal was caused both by the action of nitrifying bacteria and microalgae. It was determined that approximately 60% of the ammonia feed was oxidized to nitrate by nitrifying bacteria, while the algae assimilated 40% of the nitrogen feed at steady state. A methodology for measuring the specific activities of nitrifying bacteria and microalgae by comparing the rates in the variation inorganic nitrogen compounds was established with satisfactory results. An average specific activity of 0.05 and 0.02 g NH4+ gVSS-1 d-1 for nitrifying bacteria and microalgae was determined, respectively. Conclusions: The consortium it can be obtained in a single continuous operation, and has a high capacity for nitrogen removal with low oxygen content. The consortium could prove to be a more economical method compared to traditional.

Background: Energy expenditure (EE) calculated from respirometric indirect calorimetry is most accurate when based on oxygen consumption (VO2), carbon dioxide production (VCO2) and estimated protein metabolism (PM). EE has a substantial dependence of ~7% on the respiratory quotient (RQ, VCO2/VO2) and a lesser dependence on PM, yet many studies have instead estimated EE from VO2 only while PM has often been ignored, thus reducing accuracy. In 1949 Weir proposed a method to accurately calculate EE without using RQ, which also adjusts for estimated PM based on dietary composition. This RQ- method utilizes the calorimeter airflow rate (FR), the change in fractional O2 concentration (ΔFO2) and the dietary protein fraction. The RQ- method has not previously been empirically validated against the standard RQ+ method using both VO2 and RQ. Our aim was to do that. Methods: VO2 and VCO2 were measured repeatedly in 8 mice fed a high protein diet (HPD) during exposure to different temperatures (n = 168 measurements of 24h gas exchange). The HPD-adjusted RQ+ equation was: EE [kcal/time] = VO2 [L/time]×(3.853+1.081RQ) while the corresponding RQ- equation was: EE = 4.934×FR×ΔFO2. Agreement was analyzed using the ratios of the RQ- to RQ+ methods along with regression and Bland-Altman agreement analyses. We also evaluated the standard equation using the dietary food quotient (FQ) of 0.91 as a proxy for RQ (FQ+ method). Results: Ratio analysis revealed that the mean error of the RQ- method was only 0.11 ± 0.042% while the maximum error was only 0.21%. Error using the FQ+ method was 4 -and 10-fold greater, respectively. Bland-Altman analysis demonstrated that the RQ- method very slightly overestimates EE as RQ decreases. Theoretically, this error can be eliminated completely by imposing an incurrent fractional oxygen concentration at a value only slightly greater than the atmospheric level. Conclusions: The Weir ‘RQ-free’ method for calculating EE is a highly valid alternative to the ‘gold standard’ method that requires RQ. The RQ- approach permits reduced cost and complexity in studies focused on EE and provides a way to rescue EE measurement in studies compromised by faulty CO2 measurements. Practitioners of respirometry should consider adjusting EE calculations for estimated protein metabolism based on dietary composition. [ABSTRACT FROM AUTHOR]

Abstract Oviparous reptile embryos must tolerate fluctuations in oxygen availability and incubation temperature during development. In this study, regional hypoxia was simulated by painting eggs of Eublepharis macularius with melted paraffin wax to decrease the available surface area for gas exchange by approximately 80%. Experimental and control eggs were incubated at either 28 or 34 °C and embryo mass, stage, heart mass, relative heart mass, and oxygen consumption (V̇O 2) were measured at 15 and 30 days of incubation. Embryo mass from the regional hypoxia treatment was reduced by about 50% at day 15 and by about 30% at day 30 of incubation, independent of incubation temperature compared to controls. Embryo stage from the regional hypoxia treatment was reduced by about 2 stages at day 15 independent of incubation temperature but there was no effect of hypoxia treatment at day 30. Absolute heart mass was reduced by about 60% in regional hypoxia embryos sampled at day 15 while relative heart mass was increased by about 30% in regional hypoxic embryos at day 30 compared to controls, suggesting that heart mass is conserved at the expense of somatic growth. Embryo V̇O 2 was affected by incubation temperature at both 15 and 30 days of incubation but not by regional hypoxia treatment. These results indicate that embryos of E. macularius possess plasticity in their capacity to respond to reduction in oxygen availability during incubation, and are able to survive and continue developing when gas exchange surface area is severely limited. Highlights • Gecko embryos tolerate wide fluctuations in O2 and temperature • Embryos continue development despite 80% reduction in respiratory membrane • Regional hypoxia results in retarded development and reduced embryo mass • There is no interactive effect of temperature and hypoxia on embryo development • During late development heart mass is maintained at the expense of somatic growth [ABSTRACT FROM AUTHOR]

Inactin is a long lasting anesthetic agent commonly used in rat studies, but is also shown to exert physiological effects such as reducing renal blood flow, glomerular filtration rate and depressing tubular transport capacity. The effect of inactin on isolated kidney mitochondria is unknown and may be important when studying related topics in anaesthetized animals. The aim of this study was to determine whether inactin exerts effects on mitochondrial function and production of reactive oxygen species. Kidney mitochondrial function and production of reactive oxygen after acutely (5 min) or longer (1.5 hour) anesthetizing rats with inactin was evaluated using high-resolution respirometry. The results demonstrate that inactin significantly improves respiratory control ratio, inhibits complex I in the mitochondrial respiratory chain, reduce both unregulated proton leak and time dependently reduce the regulated proton leak via uncoupling protein-2 and adenine nucleotide translocase. Inactin also contributes to increased mitochondrial hydrogen peroxide production. In conclusion, inactin exerts persistent effects on mitochondrial function and these profound effects on mitochondrial function should to be considered when studying mitochondria isolated from animals anesthesized with inactin. [ABSTRACT FROM AUTHOR]

The aim of this study was to analyze the mechanisms, stoichiometry, and stability of 4-chlorophenol (4CP) biodegradation kinetics by phenol-acclimated activated sludge using open respirometry. While the removal of 4CP was higher than 98%, the removal of chemical oxygen demand (COD) ranged between 69 and 79% due to the accumulation of an intermediate metabolite. The value obtained from respirometric profiles for the stoichiometric ratio of O2 to 4CP (YO2/4CP) was 1.95 ± 0.04 mol of oxygen consumed per mol of 4CP removed. This YO2/4CP value reflected the action of the oxygenases responsible for the first steps of the aerobic oxidation of 4CP. The 4CP degradation activity decreased noticeably when successive pulses of 4CP were added to the respirometer. A mathematical model was developed to represent the aerobic biodegradation of 4CP. The fitted model adequately predicted the oxygen consumption rate, total phenols, and soluble COD concentrations as a function of time. The results presented could help to predict the dynamic of biodegradation of chlorophenols in a biological wastewater treatment system. [ABSTRACT FROM AUTHOR]

Outline: In chronic lung infections by Pseudomonas aeruginosa (PA) the bacteria thrive in biofilm structures protected from the immune system of the host and from antibiotic treatment. Increasing evidence suggests that the susceptibility of the bacteria to antibiotic treatment can be significantly enhanced by hyperbaric oxygen treatment. The aim of this study is to simulate the effect of ciprofloxacin treatment in a PAO1 biofilm model with aggregates in agarose when combined with hyperbaric oxygen treatment. This is achieved in a reaction-diffusion model that describes the combined effect of ciprofloxacin diffusion, oxygen diffusion and depletion, bacterial growth and killing, and adaptation of the bacteria to ciprofloxacin. In the model, the oxygen diffusion and depletion use a set of parameters derived from experimental results presented in this work. The part of the model describing ciprofloxacin killing uses parameter values from the literature in combination with our estimates (Jacobs, et al., 2016; Grillon, et al., 2016). Micro-respirometry experiments were conducted to determine the oxygen consumption in the P. aeruginosa strain PAO1. The parameters were validated against existing data from an HBOT experiment by Kolpen et al. (2017). The complete oxygen model comprises a reaction-diffusion equation describing the oxygen consumption by using a Michaelis-Menten reaction term. The oxygen model performed well in predicting oxygen concentrations in both time and depth into the biofilm. At 2.8 bar pure oxygen pressure, HBOT increases the penetration depth of oxygen into the biofilm almost by a of factor 4 in agreement with the scaling that follows from the stationary balance between the consumption term and diffusion term. Conclusion: In the full reaction-diffusion model we see that hyperbaric oxygen treatment significantly increases the killing by ciprofloxacin in a PAO1 biofilm in alignment with the experimental results from Kolpen et al. (Kolpen, et al., 2017; Kolpen, et al. 2016). The enhanced killing, in turn, lowers the oxygen consumption in the outer layers of the biofilm, and leads to even deeper penetration of oxygen into the biofilm. [ABSTRACT FROM AUTHOR]

One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Background: The protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT) is proposed as a potential clinical non-invasive tool to monitor mitochondrial function. This technique has been evaluated in several animal studies. Mitochondrial respirometry allows measurement in vivo of mitochondrial oxygen tension (mitoPO2) and mitochondrial oxygen consumption (mitoVO2) in skin. This study describes the first use of a clinical prototype in skin of humans. Methods: The clinical prototype was tested in 30 healthy volunteers. A self-adhesive patch containing 2 mg 5-aminolevulinic acid (ALA) was applied on the skin of the anterior chest wall (sternal) for induction of mitochondrial protoporphyrin IX and was protected from light for 5 h. MitoPO2 was measured by means of oxygen-dependent delayed fluorescence of protoporphyrin IX. MitoVO2 was determined by dynamic mitoPO2 measurements on the primed skin, while locally blocking oxygen supply by applying local pressure with the measurement probe. MitoPO2 was recorded before and during a 60-s period of compression of the microcirculation, at an interval of 1 Hz. Oxygen consumption (i.e. the local oxygen disappearance rate) was calculated from the decay of the mitoPO2 slope. Results: Oxygen-dependent delayed fluorescence measurements were successfully performed in the skin of 27 volunteers. The average value (± SD) of mitoPO2 was 44 ± 17 mmHg and mean mitoVO2 values were 5.8 ± 2.3 and 6.1 ± 1.6 mmHg s-1 at a skin temperature of 34°C and 40°C, respectively. No major discomfort during measurement and no long-term dermatological abnormalities were reported in a survey performed 1 month after measurements. Conclusion: These results show that the clinical prototype allows measurement of mitochondrial oxygenation and oxygen consumption in humans. The development of this clinically applicable device offers opportunities for further evaluation of the technique in humans and the start of first clinical studies. [ABSTRACT FROM AUTHOR]