# A new analysis of hypoxia tolerance in fishes using a database of critical oxygen level (Pcrit)

Nicholas Rogers, Mauricio Urbina, Erin Reardon, David J Mckenzie, Rod Wilson

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Nicholas Rogers, Mauricio Urbina, Erin Reardon, David J Mckenzie, Rod Wilson. A new analysis of hypoxia tolerance in fishes using a database of critical oxygen level (Pcrit). Conservation Physiology, 2016, 4 (1), pp.cow012. 10.1093/conphys/cow012. hal-01926467

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Research article

Themed Issue Article: Conservation Physiology of Marine Fishes

# A new analysis of hypoxia tolerance in fishes using a database of critical oxygen level ($P_{crit}$)

Nicholas J. Rogers<fnref n="1" />, Mauricio A. Urbina<fnref n="1" />,†, Erin E. Reardon<fnref n="1" />, David J. McKenzie<fnref n="2" /> and Rod W. Wilson<fnref n="1" />,*

*Corresponding author. Biosciences, College of Life and Environmental Sciences, Geoffrey Pope Building, University of Exeter, Stocker Road, Exeter EX4 4QD, UK. Tel: +44 (0)1392 724652. Email: r.w.wilson@ex.ac.uk

Hypoxia is a common occurrence in aquatic habitats, and it is becoming an increasingly frequent and widespread environmental perturbation, primarily as the result of anthropogenic nutrient enrichment and climate change. An in-depth understanding of the hypoxia tolerance of fishes, and how this varies among individuals and species, is required to make accurate predictions of future ecological impacts and to provide better information for conservation and fisheries management. The critical oxygen level ($P_{crit}$) has been widely used as a quantifiable trait of hypoxia tolerance. It is defined as the oxygen level below which the animal can no longer maintain a stable rate of oxygen uptake (oxyregulate) and uptake becomes dependent on ambient oxygen availability (the animal transitions to oxyconforming). A comprehensive database of $P_{crit}$ values, comprising 331 measurements from 96 published studies, covering 151 fish species from 58 families, provides the most extensive and up-to-date analysis of hypoxia tolerance in teleosts. Methodologies for determining $P_{crit}$ are critically examined to evaluate its usefulness as an indicator of hypoxia tolerance in fishes. Various abiotic and biotic factors that interact with hypoxia are analysed for their effect on $P_{crit}$, including temperature, $CO_2$, acidification, toxic metals and feeding. Salinity, temperature, body mass and routine metabolic rate were strongly correlated with $P_{crit}$; 20% of variation in the $P_{crit}$ data set was explained by these four variables. An important methodological issue not previously considered is the inconsistent increase in partial pressure of $CO_2$ within a closed respirometer during the measurement of $P_{crit}$. Modelling suggests that the final partial pressure of $CO_2$ reached can vary from 650 to 3500 µatm depending on the ambient pH and salinity, with potentially major effects on blood acid-base balance and $P_{crit}$ itself. This database will form part of a widely accessible repository of physiological trait data that will serve as a resource to facilitate future studies of fish ecology, conservation and management.

**Key words:** Carbon dioxide, critical oxygen tension, metabolic rate, oxygen and capacity limitation of thermal tolerance, physiological trait

**Editor:** Steven Cooke

Received 17 December 2015; Revised 17 March 2016; accepted 19 March 2016

**Cite as:** Rogers NJ, Urbina MA, Reardon EE, McKenzie DJ, Wilson RW (2016) A new analysis of hypoxia tolerance in fishes using a database of critical oxygen level ($P_{crit}$). *Conserv Physiol* 4(1): cow012; doi:10.1093/conphys/cow012.

<footnote n="1">¹Biosciences, College of Life and Environmental Sciences, Geoffrey Pope Building, University of Exeter, Stocker Road, Exeter EX4 4QD, UK</footnote>

<footnote n="2">²Centre for Marine Biodiversity Exploitation and Conservation (Marbec), UMR 9190 CNRS-Université Montpellier-Ifremer-IRD, Université Montpellier, Place Eugène Bataillon, Montpellier cedex 5 34095, France</footnote>

<footnote>†Present address: Departamento de Zoología, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.</footnote>
Introduction

In recent decades, there has been growing concern regarding
the increasingly widespread and frequent occurrence of
hypoxia in aquatic environments, associated with the increased
discovery of hypoxic zones globally (Diaz, 2001; Diaz and
Breitburg, 2009; Zhang et al., 2010). Although periods of
hypoxia can develop naturally in many aquatic systems,
anthropogenic influences have been shown to be a major
driver of hypoxic events in both freshwater and marine habi-
tats (Friedrich et al., 2014). In particular, eutrophication asso-
ciated with increased anthropogenic nutrient loading of lakes,
rivers and coastal waters leads to blooms of algae and phyto-
plankton, the death of which subsequently fuels microbial res-
piration and the depletion of dissolved oxygen (Smith, 2003).
Hypoxia has been shown to result in losses of biodiversity and
to trigger widespread mortality events (Vaquer-Sunyer and
Duarte, 2008). In the marine environment, more than 400
coastal systems have been reported as eutrophication-associ-
ated ‘dead zones’ (Diaz and Rosenberg, 2008). Global warm-
ing is likely to exacerbate hypoxia in aquatic systems owing to
increased microbial respiration rates and reduced oxygen solu-
bility with increasing water temperatures (McBryan et al.,
2013). In addition, potential modifications to oceanic circula-
tion linked to future climate change are predicted to result in
greater stratification and ‘deoxygenation’ of the oceans
(Keeling and Garcia, 2002; Keeling et al., 2009). In summary,
in the future, reduced oxygen concentrations are predicted to
occur more extensively, more frequently and for longer periods
of time (IPCC, 2014). Fish are among the more hypoxia sensi-
tive of aquatic taxa and, as such, the sequential loss of fauna
from aquatic ecosystems during hypoxic events is commonly
initiated by the loss or relocation of fish populations (Vaquer-
Sunyer and Duarte, 2008). Understanding the physiological
responses of individual organisms to environmental stressors,
such as hypoxia, provides a mechanistic link between environ-
mental change and population-level effects, which may be key
to predicting future ecological impacts (Chown, 2012;
Seebacher and Franklin, 2012; Cooke et al., 2013).

Fishes can show various behavioural responses to hypoxia,
such as rising to the surface to breathe the uppermost layer of
water in contact with air, increasing activity to escape the
hypoxic area or decreasing activity to reduce oxygen demand
(Chapman and McKenzie, 2009; Urbina et al., 2011;
Domenici et al., 2012). Beyond these behavioural responses,
fishes can engage numerous profound physiological responses,
such as changes in ventilation, cardiac activity and haemoglo-
bin-O₂ binding (Richards, 2009). These physiological
responses work primarily to sustain oxygen extraction from
the environment in order to maintain aerobic ATP produc-
tion. This allows the majority of fishes to maintain stable oxy-
gen uptake rates across a wide range of ambient partial
pressures of oxygen (P₀), a response known as ‘oxyregulation’
(reviewed by Perry et al., 2009). When, however, oxygen
reduces to a threshold below which oxygen uptake rate
cannot be maintained, oxygen uptake declines linearly with a

decrease in ambient P<sub>O</sub>₂, a response known as ‘oxyconform-
ing’ (Pörtner and Grieshaber, 1993; Claireaux and Chabot,
2016). This threshold, when oxygen uptake transitions from
regulation to conforming, is referred to as the critical P<sub>O</sub>₂ (P<sub>crit</sub>;
Beamish, 1964; Ultsch et al., 1978). As a measure of whole-
animal oxygen extraction capacity, which varies extensively
across species and among populations, P<sub>crit</sub> is widely used to
describe the degree of hypoxia tolerance in fishes (Ultsch
et al., 1978; Chapman et al., 2002; Nilsson et al., 2007a,b;
Mandic et al., 2009; reviewed by Chapman and McKenzie,
2009; Speers-Roesch et al., 2012).

Oxygen, the key variable in Pcrit measurements, is used by
aerobic organisms as an electron acceptor in order to drive the
production of ATP. As such, the rate of oxygen uptake is
widely considered as a proxy for the rate of aerobic metabo-
lism, at least when in a steady state (Brown et al., 2004;
Nelson, 2016). Standard metabolic rate (SMR) is the oxygen
uptake rate of an entirely inactive, post-absorptive fish and
reflects its minimal cost of living at a given temperature
(Beamish and Mookherjii, 1964; Chabot et al., 2016). Routine
metabolic rate (RMR) provides a similar estimate of the cost
of living but takes into account energy expended on maintain-
ing posture and making the small movements that are typical
of most fishes even when in a quiescent state (McBryan et al.,
2013). In contrast, maximal metabolic rate (MMR) is the
highest rate of oxygen uptake that can be attained in defined
environmental conditions (Clark et al., 2013; Norin and
Clark, 2016). The difference between SMR and MMR is
referred to as aerobic scope and provides for the oxygen
demands of higher functions, such as locomotion, growth,
behaviour and reproduction (Farrell and Richards, 2009;
Claireaux and Chabot, 2016). In the context of this aerobic
hierarchy, several levels of critical P<sub>O</sub>₂ are represented in
Figure 1. As this conceptual diagram illustrates, MMR is the

<figure><img src="image_2.png" /><figcaption>**Figure 1:** Diagram illustrating the conceptual idea of the effects of hypoxia on the standard metabolic rate (SMR), routine metabolic rate (RMR), maximal metabolic rate (MMR) and aerobic scope (AS) of an oxyregulator. This and may not apply to species with facultative metabolic depression below the critical oxygen level (Pcrit). Pcritmax is defined as the critical exernal oxygen partial pressure at which oxygen supply no longer meets the maximum demand for oxygen (Portner, 2010).</figcaption></figure>
first rate to become limited as ambient oxygen decreases ($P_{cmax}$), from which point a decline in MMR leads to a reduction in aerobic scope. Secondly, the $P_{crit}$ for RMR is reached, whereby oxygen supply cannot sustain even minimal levels of aerobic activity. Finally, the $P_{crit}$ for SMR indicates that oxygen supply cannot meet even basic oxygen demands (Pörtner and Lannig, 2009; Claireaux and Chabot, 2016). Below this threshold, anaerobiosis or suppression of metabolic rate are required to sustain life (Richards, 2009). Each of the three levels of $P_{crit}$ may indicate the difference between mortality and survival. If so, $P_{crit}$ may have major implications for the fitness of fishes living in environments prone to hypoxia and, as such, each of these levels can be considered as functional traits (McGill et al., 2006; Claireaux and Chabot, 2016).

The examination of trait variation across populations and communities, and its ecological implications, are increasingly becoming the basis for predicting and potentially mitigating the effects on biodiversity of environmental change (Chown, 2012). Such trait-based approaches are facilitated by the collection and dissemination of trait data. Large-scale multi-trait databases have been compiled for various taxa, including plants (Kattge et al., 2011), mammals (Jones et al., 2009), marine polychaetes (Faulwetter et al., 2014) and North American freshwater fishes (Frimpong and Angermeier, 2009). As a quantifiable measure of hypoxia tolerance that is measured on individuals and is applicable at population level, $P_{crit}$ is useful for incorporation into trait-based approaches to the conservation physiology of fishes (Frimpong and Angermeier, 2009).

The field of fish physiology has generated a large body of literature on $P_{crit}$, across a wide range of species and in highly variable abiotic and biotic conditions (Perry et al., 2009). Owing to the discrete and nuanced nature of each study, it is challenging to make broad generalizations. The aims of the present work were as follows: (i) to assemble a database of the $P_{crit}$ values reported for fishes, from published literature, in a format suitable for future incorporation into multi-trait-based analyses; (ii) to analyse the data to identify how biotic and abiotic factors (particularly temperature) interact with hypoxia and affect $P_{crit}$; and (iii) to appraise methodologies for measuring $P_{crit}$ critically, and thereby evaluate its usefulness for quantifying hypoxia tolerance in fishes. This new analysis not only provides an opportunity for further quantitative considerations but also serves as a tangible link between the physiology and the conservation of fishes.

# Methods

## Literature search

The citation and abstract indexes, Scopus® and Web of Science®, were used to collect relevant peer-reviewed literature. The literature search was conducted in December 2014 using the following terms: 'critical oxygen', 'critical PO₂', 'oxygen threshold', '$P_{crit}$', 'oxyregulate', 'oxyconform' or 'hypoxia tolerance'. Approximately 400 papers from relevant subject areas were identified. Each of these articles was individually assessed

for relevance based on their title and abstract. Finally, 144 papers were downloaded for a full read of the manuscript. Of these, only 96 papers reported $P_{crit}$ measurements in at least one fish species.

## Database construction

In order to maximize the future usefulness of the database and to ensure that it fully reflects the variation in abiotic/biotic conditions in which $P_{crit}$ has previously been measured in fishes, it was necessary to extract multiple parameters from each study. For each $P_{crit}$ entry, 66 columns summarize information on the species and origin, acclimation parameters, animal characteristics, experimental method, results, statistical analyses, general comments and bibliographic information (Table 1). The database was constructed as a single Microsoft Excel file, with individual columns for each parameter and rows for each $P_{crit}$ determination in a particular species or treatment group. As such, a single study may occupy several rows depending on the number of treatment groups and/or species for which $P_{crit}$ is reported. Values for $P_{crit}$ were reported in a variety of different oxygen units across the literature (millimetres of mercury, torr, percentage air saturation, milligrams of oxygen per litre and micromolar), but were converted here to a partial pressure of oxygen (in kilopascals) based on oxygen solubility values reported by Green and Carrit (1967) and assuming standard atmospheric pressure at sea level (760 mmHg), if not otherwise reported. Likewise, all values of oxygen uptake rate were converted to milligrams of oxygen per kilogram per hour. To enable unbiased inter-species comparison, a subset of the full database was produced, which included only those $P_{crit}$ measurements made in fishes meeting the following conditions: (i) in an unfed or post-absorptive state; (ii) undergoing no additional (to hypoxia) abiotic stressor; and (iii) where temperature acclimation lasted for >2 days.

## Database analysis

The frequency of $P_{crit}$ measurements across families and climate zones was calculated based on the full database. However, comparisons of $P_{crit}$ values were made using the subset 'control' database described above. Based on the latitude of where the studies were conducted, each entry was labelled as tropical, sub-tropical, temperature or polar. Analysis of variance was used to test for an effect of climate zone on $P_{crit}$ using the Sidak *post hoc* test.

Potential influences of varying respirometry methodologies and hypoxia exposure methods on $P_{crit}$ were explored using the subset 'control' database, in which there are 297 data points. Similar to the full database, the majority of studies measured $P_{crit}$ using closed static respirometry on individual fish, where oxygen is reduced via the oxygen consumption of the fish (n = 202). Where there were sufficient data to compare methods between respirometry methods within a species, a Student's unpaired t-test was used to compare between groups. It was not possible to test for differences in hypoxia exposure methods within species because there were insufficient data from at least two methods.
<table><caption>**Table 1:** List of the parameters incorporated into the database alongside each reported critical oxygen level value</caption><thead><tr><th>Species and origin</th><th>Stock acclimation</th><th>Sample characteristics</th><th>Experimental method</th><th>Results</th><th>Statistical analysis</th><th>Comments and reference</th></tr></thead><tbody><tr><td>Family</td><td>Holding time</td><td>Sample size</td><td>Respirometry type</td><td>Oxy regulating or conforming</td><td>Statistical method</td><td>Comments</td></tr><tr><td>Genus</td><td>Acclimation temperature</td><td>Mean mass</td><td>BMR/RMR/SMR/MMR</td><td>M<sub>O₂</sub></td><td>P<sub>crit</sub> calculation method</td><td>Reference</td></tr><tr><td>Species</td><td>Acclimation salinity</td><td>Mass SD</td><td>Determination method</td><td>Critical P<sub>O₂</sub></td><td>SMR determination</td><td>Year</td></tr><tr><td>Origin</td><td>P<sub>O₂</sub> units</td><td>Mass SEM</td><td>Swimming speed</td><td>Critical P<sub>O₂</sub> range</td><td></td><td>Corresponding Author</td></tr><tr><td rowspan="3">Latitude and longitude</td><td>Acclimation P<sub>O₂</sub></td><td>Mass range upper</td><td>Hypoxia method</td><td>Critical P<sub>O₂</sub> SD</td><td></td><td>DOI</td></tr><tr><td>Acclimation pH</td><td>Mass range lower</td><td>Rate of hypoxia onset</td><td>Critical P<sub>O₂</sub> SEM</td><td></td><td>Full citation</td></tr><tr><td>Acclimation time</td><td>Mean length</td><td>P<sub>O₂</sub> set-point time</td><td>Critical P<sub>O₂</sub> units</td><td></td><td></td></tr><tr><td rowspan="2">Diet</td><td rowspan="2">Length SD</td><td rowspan="2">Mass range upper</td><td>Minimal P<sub>O₂</sub></td><td>Air breathing threshold</td><td></td><td></td></tr><tr><td>P<sub>O₂</sub> unit</td><td>Common P<sub>O₂</sub> units</td><td></td><td></td></tr><tr><td>Ration unit</td><td>Length range upper</td><td>Salinity</td><td></td><td></td><td></td><td></td></tr><tr><td>Ration size</td><td>Length range lower</td><td>Temperature</td><td></td><td></td><td></td><td></td></tr><tr><td>Photoperiod (light:dark)</td><td>Life stage</td><td>pH</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Feeding regimen</td><td rowspan="2">Sex</td><td>P<sub>CO₂</sub></td><td></td><td></td><td></td><td></td></tr><tr><td>Photoperiod (light:dark)</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td>Access to air</td><td></td><td></td><td></td><td></td></tr></tbody></table>

Abbreviations: BMR, basal metabolic rate; DOI, digital object identifier; MMR, maximal metabolic rate; M<sub>O₂</sub>, oxygen uptake rate; P<sub>CO₂</sub>, partial pressure of carbon dioxide; P<sub>crit</sub>, critical oxygen level; P<sub>O₂</sub>, partial pressure of oxygen; RMR, routine metabolic rate; SMR, standard metabolic rate.

Stepwise multiple linear regression analysis was used to develop a model for predicting P<sub>crit</sub> based on biotic (body mass, RMR) and abiotic (temperature, salinity) variables. Earlier analysis detected no significant within-species effect of respirometry method (closed or flow through) on P<sub>crit</sub>, and it was therefore not included in the linear regression model. Acclimation variables such as temperature, P<sub>O₂</sub> and salinity were not included in this analysis because they were very highly correlated with the equivalent variables reported during the trials. Minimal P<sub>O₂</sub> was not included in the model because it is driven by P<sub>crit</sub>.

As the multivariate model identified salinity as a relevant factor, the potential effect of salinity on P<sub>crit</sub> was explored further by comparing P<sub>crit</sub> values measured in seawater (150 entries from 82 species) with P<sub>crit</sub> values measured in freshwater (116 entries from 50 species). This approach was taken because most of the studies were conducted either in freshwater [~0.1 practical salinity units (PSU)] or seawater (~30–38 PSU). Values of P<sub>crit</sub> were calculated as the partial pressure of oxygen (in kilopascals) and as the concentration of oxygen (in milligrams per litre), using the solubility coefficient based

on experimental temperature and salinity (Green and Carrit, 1967). Potential differences between groups were then tested by a Mann–Whitney U-test, because normality assumptions were violated.

## Results and discussion

### Database coverage

Of the 96 studies reviewed, 331 measurements of P<sub>crit</sub> across 151 species were incorporated into the database. Across the global database, 58 families are represented, with Cyprinidae (n = 44), Pomacentridae (n = 41), Gobiidae (n = 24), Cichlidae (n = 23), Salmonidae (n = 19), Cottidae (n = 18), Apogonidae (n = 17), Percidae (n = 13) and Sparidae (n = 12) the most frequently represented. Freshwater and marine (including euryhaline) species account for 40 and 60% of P<sub>crit</sub> entries, respectively. Water temperatures at which P<sub>crit</sub> values were determined ranged between –1.5 and 36°C, with a mean (±SD) of 21.7 ± 7.6°C. Values for P<sub>crit</sub> over the entire data set ranged between 1.02 kPa (Pseudocrenilabrus multicolor victoriae; Reardon and Chapman, 2010) and 16.2 kPa (Solea
solea larvae; McKenzie et al., 2008) with a mean (±SD) Pcrit in the ‘control’ data set of 5.15 ± 2.21 kPa. Plots of species and their reported Pcrit values from the subset data set are provided in the Supplementary Data (Supplementary Fig. 1).

The geographical coverage of the database includes at least one entry from every continent, although North America, Europe and Australasia are by far the most heavily represented and, when combined, account for 87% of Pcrit entries. Perhaps unsurprisingly, most studies of Pcrit in fishes have been concentrated around the major fish physiology research groups in Europe, North America and Australia. Arguably, this introduces an element of bias into the database, given the incomplete representation of all habitats and species at a global scale. Based on the full database, tropical studies are the most frequently represented (n = 125 Pcrit measurements, dominated by Lizard Island Research Station, Australia, n = 98), followed by subtropical (n = 104) and temperate regions (n = 100), dominated by Canada and Europe. The polar regions are the most under-represented (n = 2). Within the subset ‘control’ database, there was a significant difference in mean Pcrit across climatic regions (ANOVA, F<sub>2,297</sub> = 4.054, P = 0.018), where tropical fishes had the lowest Pcrit (mean ± SEM: 4.92 ± 0.190 kPa) < sub-tropical fishes (5.0 ± 0.24 kPa) < temperate fishes (5.74 ± 0.24 kPa). However, the Sidak post hoc test suggested that Pcrit values for tropical fishes were significantly lower only than temperate fishes (P = 0.021). There was no difference in mean Pcrit between subtropical and either tropical (P = 0.991) or temperate Pcrit (P = 0.085). Owing to low sample size, the polar Pcrit values were not included in the ANOVA across temperatures but, interestingly, had a higher mean Pcrit than the other three climatic zones (7.9 ± 1.6 kPa).

Additionally, the species studied tend to be those conducive to respirometry trials. In particular, large, active or highly sensitive species, such as those of the Scombridae family (tuna, mackerels and bonitos) are generally under-represented in the literature (Blank et al., 2007). For example, the majority of Pcrit values reported in the database were measured on fish <1 kg body mass.

## Methodology used to determine critical oxygen level

The relationship between ambient PO₂ and oxygen uptake in fishes has been investigated since the study of Keys (1930). Even at that early stage, there was considerable discussion among physiologists regarding the validity of different methodologies. Technological developments, particularly methods for measuring dissolved oxygen content such as galvanic oxygen electrodes and, more recently, fibre-optic sensors, have made the performance of high-resolution measurements of oxygen uptake in fishes increasingly common (Clark et al., 2013; Nelson, 2016). Nevertheless, the literature examined for the purpose of building this database is characterized by considerable variation in terms of methods used to determine Pcrit. For example, the majority of studies (56%) used closed respirometry for Pcrit estimates, 21% used flow-through respirometry, 20% used intermittent respirometry, and 3% used other approaches, such as indirect estimation of gill oxygen uptake (Table 2). Most studies (70%) depleted ambient oxygen through the fish’s own respiration, whereas 30% of studies bubbled nitrogen gas into the water to reduce ambient oxygen levels. The majority of studies (80%) measured RMR for Pcrit estimates; the remaining 20% measured SMR. These methodological differences and their implications are important to consider when interpreting collated Pcrit data.

Closed respirometry, whereby the fish is placed within a sealed chamber from which water is intermittently sampled for measurement of dissolved oxygen content, provides the simplest method of measuring oxygen uptake rate (Steffensen, 1989), as follows:

$$M_{O_2} = [(V_r - V_f) \times \Delta O_2] \div (\Delta t \times bw),$$

where MO₂ represents oxygen uptake rate, Vf is respirometer volume, Vf is fish volume, ΔO₂ is change in ambient oxygen content, t is time, and bw is fish mass (‘body weight’). Importantly, water needs to be recirculated within the chamber to ensure adequate mixing, thus preventing the stratification of dissolved

<table><caption>**Table 2:** The breakdown of the number of data points representing each respirometry type and oxygen removal method in the subset database</caption><thead><tr><th rowspan="2">Respirometry type</th><th colspan="6">Oxygen depletion method</th></tr><tr><th>Fish respiration</th><th>N₂ equilibration</th><th>N₂ and O₂ equilibration</th><th>N₂ and CO₂ equilibration</th><th>N₂, O₂ and air equilibration</th><th>Total</th></tr></thead><tbody><tr><td>Closed static (individual)</td><td>202</td><td>1</td><td>0</td><td>0</td><td>0</td><td>203</td></tr><tr><td>Closed static (grouped)</td><td>13</td><td>0</td><td>0</td><td>0</td><td>0</td><td>13</td></tr><tr><td>Closed flow-through (individual)</td><td>13</td><td>14</td><td>0</td><td>0</td><td>3</td><td>30</td></tr><tr><td>Intermittent flow (individual)</td><td>13</td><td>26</td><td>2</td><td>1</td><td>0</td><td>42</td></tr><tr><td>Mesocosm (grouped, large tuna)</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><td>Open flow-through (grouped)</td><td>7</td><td>0</td><td>0</td><td>0</td><td>0</td><td>7</td></tr><tr><td>Opercular mask</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td></tr></tbody></table>
oxygen within the chamber (Keys, 1930). Whether spontaneous
movements and ventilation are sufficient to provide mixing
depends on the species and achieving the correct fish-to-respi-
rometer volume ratio. For closed determinations of Pcrit, hypoxia
is generated by allowing the fish to deplete available oxygen
through its own respiration, therefore negating the need to strip
dissolved oxygen from the water artificially through equilibra-
tion with nitrogen. For this reason, closed respirometry is par-
ticularly useful for conducting measurements of Pcrit in the field
or at remote locations where facilities such as a supply of N₂ may
not be readily available (Rosenberger and Chapman, 2000;
Nilsson et al., 2007b).

However, there are several important considerations regarding the use of closed respirometry for determination of Pcrit. For instance, the rate of oxygen depletion during closed respirometry is determined by the ratio of fish size (or oxygen uptake rate) to respirometer volume. A lack of control over the development of hypoxia can be problematic in comparative studies that use the same respirometer to measure Pcrit in fish of different size and/or metabolic rate. As an illustrative example, the depletion of oxygen levels from 20 to 1 kPa by Australian barramundi (Lates calcarifer) took between 1.5 and 4 h depending on the temperature (26 or 36°C; Collins et al., 2013). From our database, it is evident that there is very little, if any, standardization in terms of the rate of oxygen depletion between Pcrit studies, irrespective of which respirometry method is employed. This is in contrast to measurements of other physiological threshold traits, such as the determination of critical temperature, which tends to be made at consistent warming or cooling rates among studies (0.2–0.3°C min⁻¹; Beitinger et al., 2000; Mora and Maya, 2006; Murchie et al., 2011). It is unclear whether the rate of decline in ambient oxygen will significantly affect Pcrit, but it is likely that a longer time scale would allow for greater respiratory adjustments, and hence, reveal lower Pcrit values than more acute hypoxic exposures. Indeed, our own anecdotal observations in European flounder (Platichthys flesus) suggest that these fish tend to oxyconform across the entire range of ambient P>O₂ when exposed to a very rapid reduction of oxygen (from 21 to 2 kPa in <2 h).

A further issue associated with closed respirometry is the
build-up of the waste products of metabolism, in particular
CO₂ (Keys, 1930; Steffensen 1989; Urbina et al., 2012). It has
been argued that the level of CO₂ accumulation within a
closed respirometer is unlikely to impact on CO₂ excretion by
fishes significantly, given that they normally exhibit a blood
partial pressure of CO₂ (P<sub>CO₂</sub>) of around 2–4 mmHg, much
higher than normal ambient levels (Ishimatsu et al., 2005;
Nilsson et al., 2007a). However, a precedent has been set,
albeit at more severe levels of hypercarbia (2.25–20 mmHg),
to show that elevated P<sub>CO₂</sub> can increase P<sub>crit</sub> in European eels
(Anguilla anguilla; Cruz-Neto and Steffensen, 1997), although
no effect on P<sub>crit</sub> was observed when eels were given enough
time to acclimate fully in terms of acid–base regulation
(McKenzie et al., 2003), or in spot fish (Leiostomus xan-
thurus) and mummichog (Fundulus heteroclitus; Cochran and

Burnett, 1996). Given the potential influence of hypercarbia,
it would be prudent to report any change in water P<sub>CO₂</sub> along-
side values for P<sub>crit</sub> that have been determined through closed
respirometry, but this has rarely been the case throughout the
existing literature. A single study so far has evaluated this
potential confounding factor in determining P<sub>crit</sub>, but in this
unusual oxyconforming species (inanga, *Galaxias maculatus*)
elevated P<sub>CO₂</sub> had no effect on oxygen uptake rate at any level
of ambient oxygen (Urbina *et al.*, 2012). Furthermore, the
authors pointed out that the effect of CO₂ on M<sub>O₂</sub> in fishes
appears to be species specific (Gilmour, 2001; Ishimatsu *et al.*,
2008).

An important issue that does not appear to have been con-
sidered previously is that the extent to which P<sub>CO₂</sub> increases
within a closed respirometer will be highly dependent on the
starting water chemistry, in particular pH and salinity (Fig. 2).
A higher seawater pH indicates a greater total alkalinity (TA).
In turn, this gives increased capacity for buffering added CO₂
and limiting the increase in P<sub>CO₂</sub> for a given increase in total
CO₂ attributable to net excretion by the fish in a respirometer.
Therefore, the lower the starting water pH, the larger the

<figure><img src="image_3.png" /></figure>

**Figure 2:** Model of the estimated partial pressure of carbon dioxide (P<sub>CO₂</sub>) reached, in water of different salinities and starting pH values, after the addition of 140 µM CO₂. The value of 140 µM approximates the increase in total CO₂ attributable to excretion by a fish at 15°C during a closed respirometry experiment. In this theoretical example, the oxygen level is allowed to decline as a result of respiration from a normoxic partial pressure of >20 kPa (~245 µM) to a common P<sub>crit</sub> value of ~6 kPa (~74 µM), and we have assumed a respiratory quotient (CO₂ excreted ÷ O₂ consumed) of 0.85 for fish (Kieffer et al., 1998). At each starting pH, the total alkalinity (TA) and total CO₂ were calculated from the pH and assuming equilibration with atmospheric P<sub>CO₂</sub> (395 µatm). When excreted CO₂ is dissolved in water, the total CO₂ increases accordingly (in this case, by 140 µM) but TA remains unchanged (Riebesell et al., 2010). For each starting pH, we therefore used the CO₂sys program (for the national bureau of standards pH scale) to calculate the final P<sub>CO₂</sub> that would result from increasing total CO₂ by 140 µM while TA remained constant. This was repeated for salinities of 20, 25, 30, 35 and 40 practical salinity units (PSU) and starting pH values of 7.5–8.5 to cover ranges experienced in many marine laboratories.
overall change in PCO₂ over the course of the Pcrit measurement. From the models shown in Figure 2, it is clear that pH has a massive influence on the ambient PCO₂ reached within such a closed respirometry scenario, with final PCO₂ values ranging by 5-fold, from ~650 µatm (0.49 mmHg) to ~3500 µatm (2.66 mmHg) at the highest (8.5) and lowest (7.5) starting pH values shown, respectively. Note that even the lowest of these final PCO₂ values has been shown (in experiments designed to mimic future ‘ocean acidification’ scenarios) to have significant detrimental effects in fishes (Munday et al., 2009). When the starting pH is low, the highest PCO₂ values of ~3500 µatm occur, which are more than 3.5 times higher than the ‘business as usual’ for end-of-century global CO₂ projections (representative concentration pathway scenario 8.5; Meinshausen et al., 2011). It is also relevant to note that salinity has a major modulating effect, in particular within the middle of the range of starting pH values. For example, at a starting pH of 8.0, the final PCO₂ will vary from slightly <1500 µatm (1.14 mmHg) at the highest salinity (40 PSU) to >2500 µatm (1.90 mmHg) at the lowest salinity (20 PSU).

The larger ambient PCO₂ values indicated above would certainly be expected to cause significant blood acid-base disturbance during the time scale of a typical closed respirometry experiment (minutes to hours) and thus have the potential to influence Pcrit via alterations in the oxygen binding affinity of haemoglobin. It is therefore important to recognize this variability in PCO₂ when conducting closed respirometry experiments to determine hypoxia tolerance, and particularly, when interpreting Pcrit measurements.

Flow-through respirometry is a technique whereby oxygen content of the inflowing (O₂,in) and outflowing (O₂,out) water is continuously measured at a fixed water flow rate through the respirometer (Fw). By application of the Fick principle, oxygen uptake (MO₂) is determined by:

$$MO_2 = F_w(O_2,in - O_2,out) \div bw.$$

Although flow-through respirometry avoids the accumulation of metabolites in the chamber, it suffers from problems primarily related to the ‘wash-out’ effect, whereby a significant lag can develop between changes in the fish’s real MO₂ and changes in observed O₂,out. The degree of wash-out depends on the dilution factor, which is a function of water mixing, volume and flow rate (Steffensen, 1989).

Intermittent flow-through respirometry is generally considered the ideal method of MO₂ determination in fishes because it involves none of the problems associated with closed or flow-through techniques (Steffensen, 1989; Clark et al., 2013). The term ‘intermittent’ or ‘semi-closed’ in this context refers to the transitioning between a closed phase for determination of MO₂ and a flush phase for restoring O₂ to a set level and removing metabolites from the respirometer. As the equipment and software for automating flush-recirculation cycles and simultaneous data acquisition from multiple chambers have become more sophisticated and widely available, intermittent

flow-through respirometry has been increasingly used (Svendsen et al., 2016). However, Pcrit measurements via this preferred technique account for only 20% of values incorporated into the present database.

Flow-through techniques allow for the supply of hypoxic water to the respirometry chamber. This hypoxic water can be produced by bubbling with N₂ via a solenoid valve linked to an O₂ probe (Schurmann and Steffensen, 1997) or by bubbling with set gas mixtures of variable O₂ and N₂ content. Both methods allow for finer control of the hypoxic exposure compared with allowing the fish to deplete ambient oxygen levels dependent on its own MO₂. Progressive hypoxia can be generated in a stepwise fashion such that multiple MO₂ measurements can be made at a specific PO₂, thereby increasing the likelihood of determining an MO₂ that is representative of true SMR or RMR (Rantin et al., 1993).

Using the present database, we were able to explore differences in respirometry methods within three species, Atlantic salmon (Salmo salar), common carp (Cyprinus carpio) and Nile tilapia (Oreochromis niloticus), for which the sample size for at least two methods was greater than n > 2. Between closed static or closed flow-through respirometers, there was no difference in Pcrit of common carp (Student’s unpaired t-test, t = 1.429, d.f. = 6, P = 0.203). Likewise, between closed, static respirometers (individual fish) and open flow respirometry (with grouped fish), there was no difference in Pcrit in Atlantic salmon (Student’s unpaired t-test, t = -0.678, d.f. = 8, P = 0.517). There was no difference in Pcrit between closed, flow-through or intermittent flow-through respirometry within Nile tilapia (Student’s unpaired t-test, t = -0.644, d.f. = 6, P = 0.543). In both Atlantic salmon and common carp, oxygen levels were reduced by the respiration of the fish, whereas in Nile tilapia the oxygen was reduced by nitrogen equilibration. A direct comparison in the shiner perch (Cymatogaster aggregata) found, however, that Pcrit measured by intermittent flow-through respirometry was significantly lower than that measured by closed respirometry (Snyder et al., 2016). Thus, more direct comparisons are needed to investigate whether the two most common methodologies might provide different estimates of Pcrit.

To determine Pcrit, MO₂ is plotted against ambient PO₂ in order to identify the inflection point at which MO₂ transitions from being independent of ambient oxygen to dependent on ambient oxygen. Within this procedure, a great deal of subtle variation exists among studies. Most obvious is the differential use of SMR or RMR, with the majority (84%) of studies reporting a Pcrit for RMR. Arguably, the Pcrit exhibited for RMR is more ecologically relevant, given that this level of MO₂ is likely to be exhibited most of the time in the field (Ultsch et al., 1978; Pörtner, 2010). Indeed, for some highly active species, such as salmonids, Pcrit determined during active swimming may be most useful in considering the ecological implications of hypoxia (Fry, 1957). Activity level may affect Pcrit in unexpected ways, such as in the Adriatic sturgeon (Acipenser naccarii), which exhibits a well-developed ability to
oxyregulate ($P_{crit} = 4.9 \pm 0.5$ kPa) when permitted to swim at a low sustained speed but oxyconforms across the entire range of declining ambient oxygen when its activity is restricted in a static respirometer (McKenzie *et al.*, 2007). Some species exhibit a relatively high $P_{crit}$ for RMR at a $P_{O_2}$ that is well above the $P_{50}$ (half of the hemoglobin oxygen binding sites are saturated with oxygen) of their haemoglobin. In these instances, $P_{crit}$ may indicate a behavioural change and not simply a physical limitation of oxygen supply (McBryan *et al.*, 2013).

Of the studies that determine the $P_{crit}$ for SMR, the methods used for quantifying SMR vary considerably. Some studies use the single lowest $M_{O_2}$ value recorded at normoxia, whereas others take the average of a set number of the lowest $M_{O_2}$ values (Iversen et al., 2010). More sophisticated and robust methods involve extrapolating the average $M_{O_2}$ measured at specified swimming speeds back to zero activity (Wilson *et al.*, 1994; Cook *et al.*, 2014) or the use of percentages and frequency distributions to assess all normoxic $M_{O_2}$ data (Dupont-Prinet *et al.*, 2013). As the critical level for basal metabolism, $P_{crit}$ determinations based on SMR should theoretically reflect a true physiological limitation of oxygen extraction capacity (McBryan *et al.*, 2013), although this may not be true in species for which metabolic depression below $P_{crit}$ has a facultative component. Given that the $P_{crit}$ for RMR is likely to be encountered at higher $P_{O_2}$ than that for SMR (Fig. 1), intra- or inter-species comparisons among studies reporting different levels of $P_{crit}$ may not be entirely valid. Whether SMR or RMR measurements are used to reflect normoxic $M_{O_2}$, it is essential that sufficient time is allowed for the fish to acclimate to the respirometry chamber; otherwise, apparent reductions in $M_{O_2}$ as hypoxia develops may be an artefact of increasing habituation rather than true oxyconforming (Nilsson *et al.*, 2004).

The method used to establish the point of intersection between continuous oxyregulation and oxyconforming $M_{O_2}$ data is also inconsistent among studies. The slope of these lines will determine the $P_{crit}$ and vice versa; therefore, determining which data points should be included within each line is critical to establishing an accurate estimate of $P_{crit}$ (Yeager and Ultsch, 1989). This can be achieved graphically by fitting a least-squares linear regression through data points that show a progressive decline in $M_{O_2}$ such that it intersects with a regression line fitted through normoxic $M_{O_2}$ data (Monteiro *et al.*, 2013). A number of mathematical methods for performing so-called piece-wise or segmented linear regression analyses are available, which provide greater robustness to estimates of $P_{crit}$ and are used in the majority of studies incorporated into the present database (Nickerson *et al.*, 1989; Yeager and Ultsch, 1989; Leiva *et al.*, 2015). These approaches assume that the response of $M_{O_2}$ to declining $P_{O_2}$ is biphasic and consists of two entirely linear elements, with an abrupt transition between the two. Such assumptions are not necessarily met by real-world data, and indeed, concentration-dependent reaction kinetics make truly linear relationships between $M_{O_2}$ and $P_{O_2}$ unlikely (Marshall *et al.*, 2013). Recent developments in non-linear

regression techniques are now being promoted as a more accurate approach to determining biological thresholds such as $P_{crit}$ (Stinchcombe and Kirkpatrick, 2012; Marshall *et al.*, 2013).

## Critical oxygen level as a hypoxia tolerance trait

A low $P_{crit}$ is generally associated with greater hypoxia tolerance because it indicates a higher capacity for oxygen extraction and tissue delivery at low $P_{O_2}$ (Mandic *et al.*, 2009). Maintaining aerobic metabolism during hypoxia is advantageous because it is up to 30-fold more efficient than anaerobic ATP production (per unit substrate consumed) and avoids accumulation of the deleterious by-products (e.g. $H^+$) of anaerobic metabolism (Richards, 2009). Hypoxia-induced physiological modifications that increase oxygen extraction capacity, such as increased gill surface area (Nilsson, 2007) and haemoglobin-O$_2$ binding (Brix *et al.*, 1999), are observed in fishes that frequently encounter hypoxia, suggesting that maintaining aerobic metabolism is a primary hypoxia survival strategy (Mandic *et al.*, 2009). However, when ambient $P_{O_2}$ declines below $P_{crit}$, survival depends on the availability of substrate for O$_2$-independent ATP production (primarily glycolysis) and the ability to reduce metabolic demand (Richards, 2009).

How long a fish can maintain a balance between ATP demand and supply below its $P_{crit}$, and thus delay the onset of cellular dysfunction, necrosis and subsequent death, is a key component of hypoxia tolerance (Nilsson and Östlund-Nilsson, 2008; Urbina and Glover, 2012; Speers-Roesch *et al.*, 2013). Speers-Roesch *et al.* (2013) showed that $P_{crit}$ does not entirely predict hypoxia tolerance at lower oxygen levels. The authors used three species of sculpin (*Blepsias cirrhosis*, *Leptocottus armatus*have and *Oligocottus maculosus*), which exhibit different $P_{crit}$ values (1.76, 1.48 and 1.03 kPa, respectively), and exposed them to hypoxia levels that were 30% below each of their respective $P_{crit}$ values while recording the time to loss of equilibrium. The loss of equilibrium was consistent between only two of the three species (*L. armatushave* and *O. maculosus*). Similar relative hypoxia exposures in the epaulette shark (*Hemiscyllium ocellatum*) and shovelnose ray (*Aptychotrema rostrata*) revealed lower lactate accumulation in epaulette sharks, indicating enhanced metabolic depression in this species (Speers-Roesch *et al.*, 2012). Furthermore, Nilsson and Östlund-Nilsson (2008) showed that $P_{crit}$ did not correlate with body mass in juvenile and adult damselfish (Pomacentridae) ranging between 10 mg and 40 g but that smaller fish were much less tolerant to hypoxia below $P_{crit}$, owing to their limited capacity for meeting ATP demand through anaerobic metabolism. These findings were further supported in *G. maculatus* (Urbina and Glover, 2013). These results illustrate the benefit of considering $P_{crit}$ alongside other methods of determining hypoxia tolerance, such as measurements of tissue-specific lactate accumulation and determinations of the loss of equilibrium of 50% of the fish, in order to assess overall hypoxia tolerance (Urbina and Glover, 2013; Speers-Roesch *et al.*, 2013; Claireaux and Chabot, 2016).
A recent review by Salin *et al.* (2015) argues that whole-animal oxygen consumption measurements may provide only a partial proxy for energy metabolism because of variation, within and between individuals, in the amount of ATP produced per molecule of oxygen consumed by mitochondria (P/O ratio). Environmental factors such as ambient temperature, food intake and diet composition have been shown both to increase and to decrease P/O ratios in the mitochondria of a variety of organisms (Salin *et al.*, 2015). Hence, conclusions based on oxygen consumption rate alone could lead to misleading conclusions regarding respiratory performance during environmental changes. To our knowledge, the effect of hypoxia on P/O ratios in fish has yet to be investigated, and as such, provides an interesting avenue for further research.

As a hypoxia-tolerance trait, low Pcrit can often, but not always, indicate an ability to survive in hypoxic water. It does not consider the use of hypoxia-avoidance strategies, such as adaptations for emersion, aquatic surface respiration and air breathing (Chapman and McKenzie, 2009). The inanga (G. maculatus), which inhabits lowland streams prone to severe hypoxia, is a rare example of a fish species that appears to be an entirely obligate oxyconformer and thus demonstrates no discernible Pcrit (Urbina et al., 2012). Likewise, several species of Gymnotiform electric fishes from South America, which inhabit naturally hypoxic floodplain pools, also appear to be obligate oxyconformers with no Pcrit (Reardon E. E., personal communication), an observation that is also anecdotally supported in Brachyhypopmus brevirostris (Crampton, 1998). In some of these species, such as the inanga, a lack of scales and a large surface area-to-volume ratio indicate a high capacity for cutaneous O2 uptake whilst emersed, and hence, provide a short-term means to escape aquatic hypoxia (Urbina et al., 2011). The oxygen thresholds for aquatic surface respiration, air breathing and emergence were incorporated into the database, but only where they have been reported alongside Pcrit measurements. Such examples demonstrate the limitation of Pcrit as a universal and comparative measure of hypoxia tolerance between species and emphasize the benefit of multi-trait-based approaches.

## Biotic and abiotic interactions

Environmental stressors, such as hypoxia, rarely occur in isolation, and the interaction between stressors is of key concern in the context of predicting the ecological impacts of future environmental change (Crain et al., 2008). As a typical threshold effect, the response of fish to hypoxia is likely to result in ‘ecological surprises’, whereby seemingly resilient populations suddenly collapse once a critical threshold is crossed (McBryan et al., 2013). Additive or synergistic interactions with hypoxia could hasten the arrival of such thresholds, meaning that small environmental shifts could result in large effects on the performance of a population. Theoretically, any abiotic or biotic factor that affects either oxygen supply (cardiorespiratory capacity) or oxygen demand (metabolic rate) of an individual, and the balance therein, will have implications for its hypoxia tolerance. As

an indicator of hypoxia tolerance, the effects of a wide range of abiotic and biotic interactions on Pcrit in fish have been published (Table 3).

The stepwise multiple linear regression found that biotic (body mass, RMR) and abiotic (temperature, salinity) variables were highly correlated with Pcrit (see Table 4). A significant regression (F<sub>4,1154</sub> = 10.565, P < 0.001) predicted 19.5% of the variation in the data, based on an adjusted r<sup>2</sup> (multiple linear regression). Predicted Pcrit is equal to 5.689 + 0.047 (salinity) - 0.083(temperature) + 1.931(body mass) + 0.001 (RMR), where salinity is measured in practical salinity units, temperature in degrees Celsius, body mass in kilograms, and RMR in milligrams of oxygen per litre. All four variables were significant predictors of Pcrit in the full model (Table 4).

Temperature is by far the most widely studied abiotic factor potentially interacting with hypoxia (reported in 30 species) and is particularly relevant, given ongoing global climate change (Ficke et al., 2007; Pörtner, 2010). As ectotherms, oxygen demand in fishes increases in a roughly exponential manner with temperature (inter-species mean Q₁₀ of 1.83; Clarke and Johnston, 1999), and the intrinsic link between temperature and environmental hypoxia has become the basis of an overarching concept termed ‘oxygen and capacity limitation of thermal tolerance’ (Pörtner, 2001, 2010). Essentially, this concept suggests that the thermal tolerance of ectotherms is dictated by their capacity to meet the oxygen demands of aerobic metabolism. Increased temperature both elevates basal oxygen demand (SMR) and reduces oxygen supply (via its effect on oxygen solubility), whereas hypoxia reduces the oxygen supply. Hence, temperature and hypoxia are likely to act synergistically in fishes. Within species, increasing temperature generally results in a higher Pcrit, but among species, the slope of the relationship between temperature and Pcrit is highly variable (Fig. 3). For example, the Atlantic salmon (S. salar) exhibits a steep linear increase of Pcrit in comparison to the shallower slope seen in the common carp (C. carpio) across a similar temperature range (Ott et al., 1980; Remen et al., 2013). A surprising exception to the generally positive intra-species correlation between temperature and Pcrit was observed in four out of six species of darter (Etheostoma), for which Pcrit was lower at 20 than 10°C (Ultsch et al., 1978). Variation in the sensitivity of species to temperature in terms of hypoxia tolerance may arise because of differences in their potential for thermal acclimation. Explanations for this variation may include reducing the metabolic impact of increased temperature or enhancing oxygen extraction capacity (Ott et al., 1980; Pörtner, 2010). Species exhibit highly contrasting capacities for plastic acclimation responses. At opposite ends of this spectrum, crucian carp (Carassius carassius) can dramatically increase respiratory surface area through gill remodelling in response to temperature and hypoxia (Sollid et al., 2005), whereas certain tropical reef fish species (Ostorhinchus doederleini and Pomacentrus moluccensis) demonstrate no thermal acclimation ability even over a relatively modest temperature range (29–32°C; Nilsson et al., 2010).
<table><caption>**Table 3:** Summary of biotic and abiotic factors and their interactions with the intra-species critical oxygen level as reported by studies included in the database</caption><thead><tr><th>Variable</th><th>Species</th><th>Effect on P<sub>crit</sub></th><th>Reference</th></tr></thead><tbody><tr><td rowspan="15">Increasing temperature</td><td>Gadus morhua</td><td>Increase</td><td>Schurmann and Steffensen (1997)</td></tr><tr><td>Lates calcarifer</td><td>Increase</td><td>Collins et al. (2013)</td></tr><tr><td>Scyliorhinus canicula</td><td>Increase</td><td>Butler and Taylor (1975)</td></tr><tr><td>Salmo salar</td><td>Increase</td><td>Barnes et al. (2011)</td></tr><tr><td>S. salar</td><td>Increase</td><td>Remen et al. (2013)</td></tr><tr><td>Dentex dentex</td><td>Increase</td><td>Cerezo Valverde et al. (2006)</td></tr><tr><td>Tautogolabrus adspersus</td><td>Increase</td><td>Corkum and Gamperl (2009)</td></tr><tr><td>Gadus ogac</td><td>Increase</td><td>Corkum and Gamperl (2009)</td></tr><tr><td>Bellapiscis medius</td><td>Increase</td><td>Hilton et al. (2008)</td></tr><tr><td>Bellapiscis lesleyae</td><td>Increase</td><td>Hilton et al. (2008)</td></tr><tr><td>Morone saxatilis</td><td>Increase</td><td>Lapointe et al. (2014)</td></tr><tr><td>Carassius carassius</td><td>Increase</td><td>Sollid et al. (2005)</td></tr><tr><td>Gobiodon histrio</td><td>Increase</td><td>Sørensen et al. (2014)</td></tr><tr><td>Gobiodon erythrospilus</td><td>Increase</td><td>Sørensen et al. (2014)</td></tr><tr><td>Oreochromis niloticus</td><td>Increase</td><td>Fernandes and Rantin (1989)</td></tr><tr><td>Cyprinus carpio</td><td>Increase</td><td>Ott et al. (1980)</td></tr><tr><td>Oncorhynchus mykiss</td><td>Increase</td><td>Ott et al. (1980)</td></tr><tr><td>Pomacentrus moluccensis</td><td>Increase</td><td>Nilsson et al. (2010)</td></tr><tr><td>Ostaghynchus doederleini</td><td>Increase</td><td>Nilsson et al. (2010)</td></tr><tr><td>Carassius auratus grandoculis</td><td>No effect</td><td>Yamanaka et al. (2013)</td></tr><tr><td>Etheostoma boschungi</td><td>Decrease</td><td>Ultsch et al. (1978)</td></tr><tr><td>Etheostoma fusiforme</td><td>Decrease</td><td>Ultsch et al. (1978)</td></tr><tr><td>Etheostoma flabellare</td><td>Decrease</td><td>Ultsch et al. (1978)</td></tr><tr><td>Etheostoma rufulineatum</td><td>Decrease</td><td>Ultsch et al. (1978)</td></tr><tr><td rowspan="4">Increasing salinity</td><td>Cottus asper</td><td>Decrease</td><td>Henriksson et al. (2008)</td></tr><tr><td>Leptocottus armatus</td><td>No effect</td><td>Henriksson et al. (2008)</td></tr><tr><td>Cyprinus carpio</td><td>Increase</td><td>De Boeck et al. (2000)</td></tr><tr><td>Cyprinodon ariegatus</td><td>Increase</td><td>Haney and Nordlie (1997)</td></tr><tr><td rowspan="4">Increased P<sub>CO<sub>2</sub></sub></td><td>Fundulus heteroclitus</td><td>No effect</td><td>Cochran and Burnett (1996)</td></tr><tr><td>Leiostomus xanthurus</td><td>No effect</td><td>Cochran and Burnett (1996)</td></tr><tr><td>Anguilla anguilla</td><td>Increase</td><td>Cruz-Neto and Steffensen (1997)</td></tr><tr><td>Platichthys flesus</td><td>Increase</td><td>Rogers (2015)</td></tr><tr><td rowspan="6">Hypoxic acclimation</td><td>Pagrus auratus</td><td>No effect</td><td>Cook et al. (2013)</td></tr><tr><td>S. salar</td><td>No effect</td><td>Remen et al. (2013)</td></tr><tr><td>Hemiscyllium ocellatum</td><td>Decrease</td><td>Routley et al. (2002)</td></tr><tr><td>Spinibarbus sinensis</td><td>Decrease</td><td>Dan et al. (2014)</td></tr><tr><td>C. auratus</td><td>Decrease</td><td>Fu et al. (2011)</td></tr><tr><td>Poecilia latipinna</td><td>Decrease</td><td>Timmerman and Chapman (2004 a,b)</td></tr></tbody></table>

(Continued)
<table><caption>Table 3: continued</caption><thead><tr><th>Variable</th><th>Species</th><th>Effect on P<sub>crit</sub></th><th>Reference</th></tr></thead><tbody><tr><td>Reared in hypoxic environment</td><td>Pseudocrenilabrus multicolor</td><td>Decrease</td><td>Reardon and Chapman (2010)</td></tr><tr><td>Exercise pre-conditioning</td><td>C. auratus</td><td>Decrease</td><td>Fu et al. (2011)</td></tr><tr><td>Fed</td><td>Astronotus ocellatus</td><td>Increase</td><td>De Boeck et al. (2013)</td></tr><tr><td></td><td>Oreochromis niloticus</td><td>Increase</td><td>Mamun et al. (2013)</td></tr><tr><td></td><td>Perca fluviatilis</td><td>Increase</td><td>Thuy et al. (2010)</td></tr><tr><td>Fatty acid-enriched diet</td><td>Solea solea (larvae)</td><td>Decrease</td><td>McKenzie et al. (2008)</td></tr><tr><td></td><td>S. solea (juveniles)</td><td>Decrease</td><td>McKenzie et al. (2008)</td></tr><tr><td>Increasing body mass</td><td>Hypostomus plecostomus</td><td>Decrease</td><td>Perna and Fernandes (1996)</td></tr><tr><td></td><td>Astronotus ocellatus</td><td>Decrease</td><td>Sloman et al. (2006)</td></tr><tr><td></td><td>Pomacentridae</td><td>No effect</td><td>Nilsson and Östlund-Nilsson (2008)</td></tr><tr><td>Pre- to post-settlement (larvae)</td><td>Chromis atripectoralis</td><td>Decrease</td><td>Nilsson et al. (2007a,b)</td></tr><tr><td></td><td>Pomacentrus amboinensis</td><td>Decrease</td><td>Nilsson et al. (2007a,b)</td></tr><tr><td>Larvae to juveniles</td><td>C. auratus grandoculis</td><td>Decrease</td><td>Yamanaka et al. (2013)</td></tr><tr><td>Juveniles to adults</td><td>Reinhardtius hippoglossoides</td><td>Decrease</td><td>Dupont-Prinet et al. (2013)</td></tr><tr><td>Increasing brood size (mouthbrooders)</td><td>Zoramia fragilis</td><td>Increase</td><td>Östlun-Nilsson and Nilsson (2004)</td></tr><tr><td></td><td>Zoramia leptacantha</td><td>Increase</td><td>Östlun-Nilsson and Nilsson (2004)</td></tr><tr><td>Mycobacteriosis infection</td><td>Morone saxatilis</td><td>Increase</td><td>Lapointe et al. (2014)</td></tr><tr><td>Acidified water</td><td>Salmo gairdneri</td><td>Increase</td><td>Ultsch et al. (1980)</td></tr><tr><td></td><td>Cyprinus carpio</td><td>Increase</td><td>Ultsch et al. (1980)</td></tr><tr><td>Metal exposure</td><td>Brycon amazonicus</td><td>Increase</td><td>Monteiro et al. (2013) (Hg<sup>2+</sup>)</td></tr><tr><td></td><td>C. carassius</td><td>Increase</td><td>Schjolden et al. (2007) (Cu<sup>2+</sup>)</td></tr><tr><td></td><td>Perca fluviatilis</td><td>Increase</td><td>Bilberg et al. (2010) (AgNO<sub>3</sub>)</td></tr><tr><td></td><td>P. fluviatilis</td><td>Increase</td><td>Bilberg et al. (2010) (nano-Ag)</td></tr><tr><td>Organophosphate exposure</td><td>Oreochromis niloticus</td><td>Increase</td><td>Thomaz et al. (2009)</td></tr><tr><td>Anaemia</td><td>Pagrus auratus</td><td>Increase</td><td>Cook et al. (2011)</td></tr></tbody></table>

Abbreviations: P<sub>CO₂</sub>, partial pressure of carbon dioxide; P<sub>crit</sub>, critical oxygen level.
<table><caption>**Table 4:** Results of the stepwise linear regression analysis where salinity, body mass, routine metabolic rate (RMR) and temperature had zero-order *r* correlations with *P*<sub>crit</sub> (*P* < 0.05) and with each other, where values were reported</caption><thead><tr><th rowspan="2">Variable</th><th colspan="6">Zero-order r (n = 159)</th><th rowspan="2">b</th></tr><tr><th>Salinity (psu)</th><th>Temperature (°C)</th><th>Body mass (kg)</th><th>RMR (mg O<sub>2</sub> l<sup>-1</sup>)</th><th>P<sub>crit</sub> (kPa)</th><th>β</th></tr></thead><tbody><tr><td>Salinity</td><td></td><td>0.317</td><td>-0.165</td><td>0.354</td><td>0.279</td><td>0.346</td><td>0.099</td><td>0.047</td></tr><tr><td>Temperature</td><td></td><td></td><td></td><td>0.366</td><td>-0.141</td><td>-0.314</td><td>0.081</td><td>-0.083</td></tr><tr><td>Body mass</td><td></td><td></td><td></td><td>-0.166</td><td>0.166</td><td>0.242</td><td>0.056</td><td>1.931</td></tr><tr><td>RMR</td><td></td><td></td><td></td><td></td><td>0.17</td><td>0.202</td><td>0.032</td><td>0.001</td></tr><tr><td>Mean</td><td>23.54</td><td>23.1</td><td>0.1</td><td>323.84</td><td>5.4</td><td>Intercept = 4.027</td><td></td><td></td></tr><tr><td>SD</td><td>15.36</td><td>7.9</td><td>0.3</td><td>434.04</td><td>2.1</td><td>Adjusted r<sup>2</sup> = 0.195</td><td>P < 0.001</td><td></td></tr></tbody></table>

Abbreviations: *P*<sub>crit</sub>, critical oxygen level; RMR, routine metabolic rate. In the full model, all four variables were significant predictors of *P*<sub>crit</sub>.

<figure><img src="image_4.png" /><figcaption>**Figure 3:** The effect of temperature on inter-species critical oxygen level (*P*<sub>crit</sub>; black dashed line) and intra-species *P*<sub>crit</sub> (continuous lines).</figcaption></figure>

Unlike intra-species *P*<sub>crit</sub>, there is no apparent relationship between temperature and inter-species *P*<sub>crit</sub> (Fig. 3), suggesting that evolution may have nullified the thermal sensitivity of hypoxia tolerance across species. It has been shown that the difference in RMR between a typical cold-water and warm-water fish is less than expected, given the thermal sensitivity of RMR within individual species (intra-species median *Q*<sub>10</sub> = 2.4; Clarke and Johnston, 1999). In addition, gill surface area appears to scale in a linear manner with metabolic rate, implying that natural selection equips fishes with the oxygen extraction capacity required to match demand at higher temperatures (Nilsson and Östlund-Nilsson, 2008). Selective pressures for small gills, such as the osmorespiratory compromise (Nilsson, 1986; Gonzalez and McDonald, 1992; Urbina and Glover, 2015), gill parasites and risks associated with gill

injury, are likely to limit respiratory surface area so that oxygen extraction capacity does not exceed that required by a particular species for survival in its natural range (Nilsson, 2007). Thus, generalizations regarding hypoxia tolerance across temperatures cannot be established firmly at the inter-species level.

Although salinity has long been recognized as a key environmental factor, studies evaluating the effects of salinity on *P*<sub>crit</sub> are scarce. A previous study in the euryhaline sheephead minnow (*Ciprinodon variegatus*), acclimated to salinities from freshwater (0 PSU) to hypersaline waters (100 PSU), showed a marked effect on *P*<sub>crit</sub> (Haney and Nordlie 1997) as environmental salinity rose. Inter-specific comparisons in the database agree with this previous intra-specific finding; that is, salinity had a significant influence on *P*<sub>crit</sub>, whereby freshwater
<figure><img src="image_5.png" /><figcaption>**Figure 4:** The effect of environmental salinity on inter-species critical oxygen level ($P_{crit}$), expressed as partial pressure of oxygen (in kilopascals; **A**) and concentration of oxygen (in milligrams per litre; **B**). Data are shown as means ± SEM, including data from 82 species in seawater and 50 species in freshwater. \*Unpaired t-test, significant when $P < 0.050$.</figcaption></figure>

species (including a few euryhaline species) presented a 23% lower $P_{crit}$ than seawater species (also including a few euryhaline species; Fig. 4A; $P \le 0.001$).

As explained in earlier sections, any factor influencing the oxygen demand (metabolic rate) of an individual will be likely to have implications for its hypoxia tolerance. Given that teleost fishes must maintain a tight regulation of their internal salts and water composition (osmolality), as external salinity changes or becomes extreme, fishes must expend increased efforts to maintain internal homeostasis (Urbina and Glover, 2015). As many of the mechanisms of osmoregulation involve the action of ATP-driven pumps (i.e. Na+,K+-ATPase) in order to pump ions against a concentration gradient, increased costs of osmoregulation may explain, in part, some of these differences in $P_{crit}$, at least for intra-specific comparisons. However,

from our database (inter-specific), where more freshwater vs. seawater species comparison are presented, it is likely that other mechanisms are explaining differences in $P_{crit}$. Given that seawater species separated million years ago from a freshwater ancestor (actinopyergians, 300–180 million years ago; Vega and Wiens, 2012), both fresh- and seawater species have adapted to their respective environments, and therefore, have also optimized their energy allocated to osmoregulation. Thus, the differences in $P_{crit}$ found in the present study, rather than being explained by energy-related/oxygen demand issues, could be associated with intrinsic characteristics of both media (freshwater vs. seawater). Owing to differences in size, organic matter load and stability, hypoxia is much more prevalent and common in freshwater than in seawater environments. As such, the driver for an enhanced hypoxia tolerance (lower $P_{crit}$) could potentially explain the lower $P_{crit}$ found in freshwater species. A future phylogenetic analysis might contribute to test this hypothesis.

It is also worth noting that the difference found in $P_{crit}$ when presented as the partial pressure of oxygen (in kilopascals) was no longer found when $P_{crit}$ was calculated as the concentration (in milligrams per litre; (Fig. 4B; $P > 0.05$). This could potentially highlight the importance of working with partial pressure, because this is what drives diffusion when considering gases. Alternatively, it could indicate that the oxygen concentration is more relevant when considering $P_{crit}$ values, because it determines the total amount of oxygen that is potentially available for diffusion as water flows over the gills, i.e. for the same oxygen uptake, salinity (through its effect on solubility) will have a big effect on the difference between inspired and expired $P_{O_2}$.

The biological processes that consume $O_2$ also produce $CO_2$; therefore, hypoxia and hypercarbia can often co-occur in aquatic environments (Ultsch, 1996; Cruz-Neto and Steffensen, 1997; Gilmour, 2001). Despite this, the interactive effect of environmental hypercarbia on hypoxia tolerance has been relatively understudied. As previously discussed (Table 3), there are conflicting reports within the available literature regarding to the effect of hypercarbia on the $P_{crit}$ of fishes (Cochran and Burnett, 1996; Cruz-Neto and Steffensen, 1997; McKenzie et al., 2003). The most likely mechanism by which hypercarbia could negatively impact hypoxia tolerance is through respiratory acidosis, leading to Bohr/Root effects on haemoglobin and reduced oxygen transport capacity (Jensen et al., 1993; Cruz-Neto and Steffensen, 1997). In this respect, hypercarbia is partly akin to the far more extreme acidosis that can occur in poorly buffered freshwater environments subjected to acid precipitation or drainage. Acidification of the surrounding water by addition of sulphuric acid (water pH range 7.4–4.0, at constant atmospheric $P_{CO_2}$) increases $P_{crit}$ in both rainbow trout (*Oncorhynchus mykiss*) and common carp (*Cyprinus carpio*; Ultsch et al., 1980). The time required to compensate for acid-base disturbance is highly variable among species (10–24 h during moderate hypercarbia; Melzner et al., 2009), and as such, the effect of hypercarbia and acidification on hypoxia tolerance is likely to be dependent
largely on the species in question as well as the severity and
duration of the hypercarbic or acid exposure (Jensen et al.,
1993).

Exposure to toxicants, such as trace metal contamination,
appears to reduce hypoxia tolerance in fishes. Specifically,
exposure to elevated concentrations of copper (300 µg l⁻¹),
mercury (150 µg l⁻¹) and silver (63 µg l⁻¹) have been demon-
strated to increase Pcrit in various species (Table 3). The accu-
mulation of toxic metals on the gills can stimulate the
hypersecretion of mucus, which acts as a barrier to diffusion
of external toxicants into the blood (McDonald and Wood,
1993; Wilson et al., 1994). In addition, some trace metals
cause hyperplasia and hypertrophy of gill epithelia cells that
results in the fusing and thickening of gill lamellae (Schjolden
et al., 2007; Bilberg et al., 2010). As a consequence, respira-
tory function is compromised as a result of reduced diffusion
area and increased diffusion distance (McDonald and Wood,
1993). The organophosphate insecticide trichlorfon has been
shown to increase Pcrit by inducing similar changes in gill mor-
phology as well as by promoting vasoconstriction that reduces
lamellar blood flow in Nile tilapia (Oreochromis niloticus;
Thomaz et al., 2009). These potential interactions between
toxic contaminants and hypoxia in fishes are clearly of con-
cern, particularly given that both stressors predominantly
threaten freshwater and coastal marine systems and are there-
fore likely to coincide (McDonald and Wood, 1993; Diaz and
Rosenberg, 2008).

Determinations of Pcrit in fishes have almost universally
been made in unfed, post-absorptive individuals which,
although providing a useful basis for comparing absolute
hypoxia tolerance among species and individuals, does not
fully account for the digestive state typical of fishes in their
natural setting. An increase in oxygen uptake following inges-
tion of food, termed specific dynamic action (SDA), is required
in order to meet the energetic costs associated with mechani-
cal and biochemical digestion and assimilation (Jobling,
1993). Shortly after a meal, oxygen uptake in fish typically
rises rapidly, reaching a peak two to three times higher than
pre-fed levels within a few hours. The shape and duration of
the SDA is highly dependent on the species in question as well
as the meal size and composition (Secor, 2009). Measurements
of Pcrit in fishes undergoing SDA have revealed significant
increases in Pcrit compared with unfed control fishes, showing
that increased aerobic demand during digestion has negative
consequences for hypoxia tolerance (Table 3). In common
perch (Perca fluviatilis) force-fed a 5% body mass ration, Pcrit
at 20 h post-feeding was increased by 1.44-fold compared
with sham-fed individuals (Thuy et al., 2010). Likewise,
oscars (Astronotus ocellatus) fasted for 14 days showed a 1.6-
fold lower Pcrit than individuals fed a daily 1% body mass
ration up to 24 h prior to Pcrit determination (De Boeck et al.,
2013). In such experiments, the requirement for a stable MO₂
on which to base a determination of Pcrit means that measure-
ments at peak SDA are not feasible, and thus, are likely to
underestimate the effect of digestion on hypoxia tolerance
(Thuy et al., 2010).

Several studies have investigated the effect of hypoxia acclimation on Pcrit (Table3). Broadly, short-term physiological acclimation to hypoxia appears to be achieved through either enhanced O₂ extraction capacity or metabolic depression. In goldfish (*Carassius auratus*), 48 h of severe (0.63 kPa) hypoxia induced dramatic increases in both lamellar surface area and blood haemoglobin content, leading to a 49% reduction in Pcrit compared with individuals held at normoxia (Fu *et al.*, 2011). Likewise, sailfin molly (*Poecilia latipinna*) demonstrated increased haemoglobin and red blood cell concentrations and a reduced Pcrit following a 6 week exposure to severe hypoxia (Timmerman and Chapman, 2004a). Depression of RMR at normoxia and a subsequent reduction in Pcrit following chronic hypoxic exposure has been observed in the epaulette shark (*H. ocellatum*; Routley *et al.*, 2002) andqingbo (*Spinibarbus sinensis*; Dan *et al.*, 2014). However, some less hypoxia-tolerant species appear to demonstrate no physiological acclimation potential through hypoxic pre-conditioning. Daily exposure to 6 h of moderate hypoxia (10.5 kPa) for 33 days had no effect on Pcrit in post-smolt Atlantic salmon (*S. salar*; Remen *et al.*, 2013). Additionally, chronic (6 week) moderate hypoxia produced no change in the Pcrit of juvenile snapper (*Pagrus auratus*; Cook *et al.*, 2013).

As hypoxia is likely to become an increasingly predomi-
nant aquatic perturbation in the future (Vaquer-Sunyer and
Duarte, 2008; Keeling et al., 2009), the degree of physiologi-
cal plasticity for hypoxia tolerance will be a key determinant
of species performance. The potential for long-term and trans-
generational hypoxia acclimation with respect to Pcrit has been
largely unstudied. A transgenerational transfer of hypoxia
tolerance has been demonstrated in zebrafish (Danio rerio)
larvae after 2–4 weeks of parental hypoxia exposure, but this
was based on determinations of time to loss of equilibrium
(4 kPa O₂) rather than through measurement of Pcrit (Ho and
Burggren, 2012). Reardon and Chapman (2010) demon-
strated a strong element of developmental plasticity in the Pcrit
of the Egyptian mouthbrooder (Pseudocrenilabrus multi-
color) when reared in hypoxic conditions. In addition, intra-
species population effects on Pcrit across habitats of differing
O₂ regimens have been observed in several species, indicating
that a high degree of phenotypic plasticity for Pcrit exists
within these populations (Timmerman and Chapman, 2004b;
Reardon and Chapman 2010; Fu et al., 2011).

Future applications

The comprehensive Pcrit database presented here provides the
opportunity for a variety of further analyses with potential to
offer fundamental physiological, as well as wider ecological,
insights. For example, further analyses could involve compar-
ing species Pcrit values within a phylogenetic context as a
means to investigate the evolutionary relationships of hypoxia
tolerance among species (Mandic et al., 2009). Likewise, com-
bining species Pcrit data with information on the spatial distri-
bution of populations would help to refine our understanding
of the ecological relevance of Pcrit as a physiological trait. Such
an analysis would be particularly relevant to predicting the
impacts on fish populations likely to arise from the increas-
ingly widespread occurrence of hypoxic zones in aquatic envi-
ronments around the globe (Friedrich *et al.*, 2014). Given the
variability found in the reported Pcrit for different fish species,
it is likely that hypoxic events will have consequences that are
very dependent on individual species. This highlights the com-
plexity of predicting the effects that hypoxia will have at
community and ecosystem levels, and the potential for
hypoxia to have differential effects on predator-prey interac-
tions, migrations, and ultimately, global fisheries.

The integration of the present database with similar data-
bases of other widely measured physiological parameters in
fishes should offer useful insights into interactions among
traits. Such physiological data are of great value for improv-
ing the predictive capacity of models as an aid to the manage-
ment and conservation of aquatic systems (Jørgensen et al.,
2012; Cooke *et al.*, 2013). Traits for which databases are cur-
rently under construction include the metabolic response to
feeding (SDA), aerobic scope, growth rate and critical tem-
perature. On completion, the combined data set will be made
widely accessible via an online data repository facility, such as
that provided by Dryad (http://datadryad.org/). Thus, it is
envisaged that these data will prove to be a tangible link
between the field of fish physiology and future studies of ecol-
ogy, conservation and management.

Supplementary material

Supplementary material is available at *Conservation Physiology* online.

Acknowledgements

The authors wish to thank Silvana Birchenough and Julian
Metcalfe (Cefas, Lowestoft, UK) for their mentoring and
encouragement in the creation of the Pcrit database.

Funding

This work was supported by a Natural Environment Research Council (NERC, UK) PhD studentship awarded to N.J.R./R.W.W. and NERC and Biotechnology and Biological Sciences Research Council (BBSRC) research grants (NE/ H010041/1, BB/D005108/1 and BB/J00913X/1) awarded to R.W.W. The physiological database is a contribution of the European Union Cooperation in Science and Technology (COST) Action (FA1004) on the ‘Conservation Physiology of Marine Fishes’. The same EU COST Action supported this work as a Short Term Scientific Mission (STSM). For more information, see: http://fish-conservation.nu/.

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