Received: 15 December 2020 | Revised: 26 March 2021 | Accepted: 31 March 2021 DOI: 10.1002/jez.2468 REVIEW The physiology of fish in acidic waters rich in dissolved organic carbon, with specific reference to the Amazon basin: Ionoregulation, acid–base regulation, ammonia excretion, and metal toxicity Carolyn Morris1,2 | Adalberto L. Val3 | Colin J. Brauner1 | Chris M. Wood1,2,3 1Department of Zoology, University of British Columbia, Vancouver, British Columbia, Abstract Canada Although blackwaters, named for their rich content of dissolved organic carbon 2Department of Biology, McMaster (DOC), are often very poor in ions and very acidic, they support great fish biodi- University, Hamilton, Ontario, Canada 3Laboratory of Ecophysiology and Molecular versity. Indeed, about 8% of all freshwater fish species live in the blackwaters of the Evolution, Brazilian National Institute for Rio Negro watershed in the Amazon basin. We review how native fish survive these Research of the Amazon, Manaus, Brazil harsh conditions that would kill most freshwater fish, with a particular focus on the Correspondence role of DOC, a water quality parameter that has been relatively understudied. DOC, Carolyn Morris, Department of Zoology, which is functionally defined by its ability to pass through a 0.45‐µm filter, com- University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. prises a diverse range of compounds formed by the breakdown of organic matter Email: [email protected] and is quantified by its carbon component that is approximately 50% by mass. Funding information Adaptations of fish to acidic blackwaters include minimal acid–base disturbances Natural Sciences and Engineering Research associated with a unique, largely unknown, high‐affinity Na+ uptake system that is Council of Canada, Grant/Award Numbers: resistant to inhibition by low pH in members of the Characiformes, and very tight RGPIN 2018‐04172, RGPIN‐2017‐03843; Coordenaçaõ de Aperfeicoamento de Pessoal regulation of Na+ efflux at low pH in the Cichliformes. Allochthonous (terrigenous) de Nıveĺ Superior: CAPES; finance code 001; DOC, which predominates in blackwaters, consists of larger, more highly colored, Fundaçaõ de Amparo a Pesquisa do Estado do Amazona, Grant/Award Number: FAPEAM; reactive molecules than autochthonous DOC. The dissociation of protons from 062.1187/2017; INCT ADAPTA allochthonous components such as humic and fulvic acids is largely responsible for (INCTADAPTA–CNPq), Grant/Award Number: 465540/2014‐7 the acidity of these blackwaters, yet at the same time, these components may help protect organisms against the damaging effects of low water pH. DOC lowers the transepithelial potential (TEP), mitigates the inhibition of Na+ uptake and ammonia excretion, and protects against the elevation of diffusive Na+ loss in fish exposed to acidic waters. It also reduces the gill binding and toxicity of metals. At least in part, these actions reflect direct biological effects of DOC on the gills that are beneficial to ionoregulation. After chronic exposure to DOC, some of these protective effects persist even in the absence of DOC. Two characteristics of allochthonous DOC, the specific absorbance coefficient at 340 nm (determined optically) and the PBI (de- termined by titration), are indicative of both the biological effectiveness of DOC and the ability to protect against metal toxicity. Future research needs are highlighted, including a greater mechanistic understanding of the actions of DOCs on gill J Exp Zool A Ecol Integr Physiol. 2021;1–21. wileyonlinelibrary.com/journal/jez © 2021 Wiley Periodicals LLC | 1 2 | MORRIS ET AL. ionoregulatory function, morphology, TEP, and metal toxicity. These should be in- vestigated in a wider range of native fish Orders that inhabit one of the world's greatest biodiversity hotspots for freshwater fishes. KEYWORDS acid–base regulation, Amazon basin, ammonia excretion, dissolved organic carbon, ionoregulation, low pH, metal toxicity, transepithelial potential 1 | INTRODUCTION ammonia, there are data that support this (Duarte et al., 2016, 2018; Wood et al., 2003). Here, our major focus is on ionoregulatory Dissolved organic matter, often loosely referred to as dissolved or- physiology and associated functions (acid–base balance, ammonia ganic carbon (DOC), is central to the function, structure, and di- excretion), where there remain significant gaps in our understanding versity of aquatic ecosystems (Findlay & Parr, 2017; Lindeman & of the effect(s) of DOC on ion transport in freshwater fish. DOC Lindeman, 2007). It is defined by passage through a 0.45‐µm filter source, physicochemical properties, and the species of interest all (therefore “dissolved”), and quantified by its carbon content (there- influence the effect of DOC on freshwater organisms under acidic fore “DOC”), which generally makes up about 50% of the molecules conditions (Wood et al., 2011). We will first review the nature, ori- by mass (Wood et al., 2011). When molar units are used, these are gins, ecological roles, and chemistry of DOC, and then provide an based only on the moles of carbon content (molecular weight overview of previous investigations on the effects of DOC in acidic [MW] = 12.01 Da), because the exact structures of most DOC mo- water on ion transport including active ion uptake and passive ion lecules are unknown. It should be recognized that there are many loss, acid–base balance, electrical properties of the gill (transe- carbon atoms in each DOC molecule. These heterogeneous com- pithelial potential [TEP]), ammonia excretion, and the amelioration of pounds are naturally occurring transformed products of the break- metal toxicity. Blackwater systems are found globally from tropical down of organic matter by microbes, light, heat, and enzymes in soil peatlands in Southeast Asia, European peat bogs, swamps along the and water (Zara et al., 2006). Allochthonous DOCs (also known as Atlantic coast of North America to South American tropical rain- terrigenous) are formed largely on land from the breakdown of plant forests (Gandois et al., 2020; Robroek et al., 2017; Sabater products such as lignins, whereas autochthonous DOCs are formed et al., 1993). Blackwater systems have a characteristically high largely in lakes and rivers by the metabolism of very small organisms concentration of DOC, low pH, and often low‐ion concentration. Our (invertebrates, plankton, and bacteria). DOC is often the most focus will be on the best studied of these habitats, the Amazon basin, abundant dissolved component in freshwater. It provides essential particularly the fish native to Rio Negro blackwaters. nutrients to organisms and influences the productivity of trophic interactions within an ecosystem (Findlay & Parr, 2017; Lindeman & Lindeman, 2007). The effects of fluctuations in other water quality 2 | PHYSICOCHEMICAL PROPERTIES parameters (e.g., pH, alkalinity, Ca, Na, salinity) on gill function have OF DOC been extensively studied by physiologists but the effects of DOC on the ion transporting properties of the gill remain relatively un- Humification is a poorly understood biological process where DOC is explored. DOC is a critically important water quality parameter to formed by the microbial processing of lignin‐rich plant materials and consider for ecological understanding and risk assessment as it may the decay of organic remains of animals (Ertel et al., 1984; Hatcher & have comparable or greater effects on freshwater fish physiology Spiker, 1988). Humification yields complex chemical structures that (e.g., ionoregulation, acid–base balance, and acute metal toxicity) are more stable than the original compounds from which they are than the previously mentioned and well‐studied water quality para- derived (Boggs et al., 1985). DOC is often the largest component by meters (Duarte et al., 2016; Gonzalez et al., 2002; Sadauskas‐ mass of the dissolved components of natural waters. However, there Henrique et al., 2019; Wood et al., 2011; Wood et al., 2003, 1998). is a wide variation in concentrations (5–35 mg/L in Rio Negro; The dissociation of protons from allochthonous DOC is the Table 1) found within freshwater systems that may fluctuate sea- major cause of acidity in peat bogs and many other natural black- sonally and differ among regions (Boggs et al., 1985; Holland waters. However, there is now extensive evidence in freshwater fish et al., 2018, 2017; Thurman, 1985; Tipping, 2002). DOC is a com- that natural DOCs may actually mitigate the ionoregulatory dis- bination of humic substances which are generally larger molecules turbances of the ambient low pH which they help to create (Duarte than nonhumic substances such as amino acids, fatty acids, phenols, et al., 2016, 2018; Gonzalez et al., 2002; Wood et al., 2003, 1998). It sterols, natural sugars, hydrocarbons, urea, and porphyrins (Boggs has even been suggested that DOCs may affect ventilation (Holland et al., 1985; Gaffney et al., 1996). Humic substances are primarily et al., 2014; Perry et al., 2021) and respiratory gas exchange (O2, composed of carbon, nitrogen, oxygen, and sulfur that make CO2, and ammonia) at the gills (Matsuo et al., 2004), and at least for up organic acids that have highly oxygenated functional groups MORRIS ET AL. | 3 TABLE 1 General water chemistry ranges for acidic, DOC‐rich (Boggs et al., 1985; Zara et al., 2006). Operationally, humic sub- black waters of the Rio Negro watershed, based on the experience stances are fractionated into “humic acids” which precipitate