See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/257396392 Effects of elevated ammonia concentrations on survival, metabolic rates, and glutamine synthetase activity in the Antarctic pteropod mollusk Clione limacina antarctica Article in Polar Biology · July 2012 DOI: 10.1007/s00300-012-1158-7 CITATIONS READS 4 34 3 authors, including: AE Maas Brad Seibel Bermuda Institute of Ocean Sciences University of Rhode Island 16 PUBLICATIONS 133 CITATIONS 87 PUBLICATIONS 3,474 CITATIONS SEE PROFILE SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, Available from: AE Maas letting you access and read them immediately. Retrieved on: 02 August 2016 1 Short Note 2 3 Effects of Elevated Ammonia Concentrations on Survival, Metabolic Rates and 4 Glutamine Synthetase Activity in the Antarctic Pteropod Mollusc Clione limacina 5 antarctica 6 7 Amy Maas1, 2, Brad A. Seibel1 and Patrick J. Walsh3 8 9 1 Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881 10 2 Current address: Department of Biology, Woods Hole Oceanographic Institute, Woods 11 Hole, MA 02543 12 3Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6N5 13 CANADA 14 15 Corresponding Author Contact Information: 16 P.J. Walsh 17 Dept. of Biology, University of Ottawa 18 30 Marie Curie 19 Ottawa, ON K1N 6N5 Canada 20 Email: [email protected] 21 Phone: 613-562-5800x6328 22 Fax: 613-562-5486 23 2 24 Abstract 25 Information on effects of elevated ammonia on invertebrates in general, and polar 26 Molluscs in particular, is scant. Questions of ammonia sensitivity are interesting for 27 several reasons, particularly since predicted global change scenarios include increases 28 in anthropogenic nitrogen and toxic ammonia. Furthermore, polar zooplankton species 29 are often rich in lipids, and authors have speculated that there is a linkage between 30 elevated levels of lipids/trimethylamine oxide (TMAO) and enhanced ammonia 31 tolerance. In the present study, we sought to examine ammonia tolerance and effects of 32 elevated exogenous ammonia on several key aspects of the physiology and 33 biochemistry of the pteropod mollusc, Clione antarctica limacina. We determined that the 34 96-hour LC50 value for this species is 7.465 mM total ammonia (Upper 95% CL = 8.498 35 mM and Lower 95% CL = 6.557 mM), or 0.51 mg/L as unionized ammonia (NH3) (at a 36 pH of 7.756). While comparative data for molluscs are limited, this value is at the lower 37 end of reported values for other species. When the effects of lower ammonia 38 concentrations (0.07 mM total ammonia) on oxygen consumption and ammonia 39 excretion rates were examined, no effects were noted. However, total ammonia levels as 40 low as 0.1 mM (or 0.007 mg/L NH3) elevated the activity of the ammonia detoxification 41 enzyme, glutamine synthetase, by approximately 1.5 fold. The values for LC50 and 42 observable effects on biochemistry for this one species are very close to permissible 43 marine ammonia concentrations, indicating a need to more broadly determine the 44 sensitivity of zooplankton to potential elevated ammonia levels in polar regions. 3 45 Key Words: global change, nitrogen pollution, Antarctica, pelagic molluscs, O:N ratio, 46 ammonia LC50 values, TMAO 47 4 48 Introduction 49 One of the more toxic forms of nitrogen to animals is ammonia. At least in 50 vertebrates, the mode of action of ammonia as a toxin is primarily through its effects on 51 the central nervous system (CNS). Ammonia CNS toxicity has been relatively well 52 documented in the medical literature (in association with the human disease hepatic 53 encephalopathy), and increases in plasma ammonia levels due to liver dysfunction 54 appear to affect glutamate receptors on neurons, as well as to cause swelling in 55 associated astrocytes (the nutritive and support cells of the vertebrate CNS) (Cooper 56 and Plum 1987; Butterworth 2001). The literature on ammonia toxicity in fish species is 57 smaller but growing, and so far indicates that, although overall mechanisms of toxicity 58 are similar to mammals, there are some important differences, notably: (1) astrocyte 59 swelling seems to be less pronounced in the brains of marine fish; (2) there are wide 60 species differences in the susceptibility of fish to ammonia, with some species showing 61 orders of magnitude greater ability to survive ammonia toxicity than can mammals 62 (Walsh et al. 2007). 63 In contrast, much less is known about mechanisms of ammonia toxicity in 64 marine invertebrates in general and polar invertebrates and molluscs in particular. Data 65 exist on ammonia-induced mortality (e.g., standard Lethal Concentration 50, or LC50 66 values, the concentration that leads to mortality in 50% of a test population after a 67 standard time) for numerous freshwater and some marine invertebrate species, 68 including molluscs (e.g., Boardman et al 2004; USEPA 1989). Although toxic effects 69 leading to mortality are presumed to be primarily neuronal as in vertebrates, very little 5 70 is known. Furthermore, species considered to date have primarily been standard EPA 71 test indicator organisms, or organisms in inland waters that are predicted to be at risk 72 for exposure via close proximity to point sources (examined as part of mandated 73 environmental impact studies). With respect to polar invertebrates, we are not aware of 74 any studies examining ammonia-induced mortality or effects of ammonia on routine 75 physiological processes. In this regard, Seibel and Walsh (2002) previously reported that 76 Clione antarctica has high levels of trimethylamine oxide (TMAO) which is known to 77 counteract ammonia toxicity in some species (Kloiber et al. 1988; Minana et al. 1996). 78 This observation leads to a hypothesis that many polar zooplankton may show 79 enhanced ammonia tolerance because they have high lipid content for over-winter 80 survival and lipid formation is linked to TMAO levels (Seibel and Walsh 2002). It also 81 suggests that ammonia tolerance will depend to some extent on diet. 82 With this scant background in mind, in the present study we examined the 83 effects of ammonia on mortality, routine physiological processes (oxygen consumption 84 and nitrogen excretion), and the activity of an enzyme involved in ammonia 85 detoxification (glutamine synthetase) in the Antarctic pteropod, Clione limacina antarctica 86 in studies complementary to examination of the effects of acidification (Seibel et al. 87 submitted). 6 88 Materials and Methods 89 Collection, Maintenance and Ammonia Exposure of Animals 90 In January 2008, specimens of Clione limacina antarctica (Smith 1902) were collected 91 several meters offshore at Cape Royds (77° 34’ S, 166° 11’ E) on Ross Island near 92 McMurdo Station, Antarctica. Collectors wading in waters of approximately 1m depth 93 dipped animals out of the water using 1L beakers attached to 1 m poles. Organisms 94 were then gently poured into 500mL Nalgene bottles (to a density of 10-12 organisms 95 per bottle), placed in insulated coolers and returned to McMurdo Station by helicopter 96 within 6h of capture. Bottles were then placed in a cold room to maintain temperature 97 at -1.8 oC (also the temperature of all subsequent tests unless noted). Organisms 98 (ranging in body mass from 0.0429 to 0.3616 grams) were held in captivity without food 99 for a period of 24 hours to allow for gut clearance. 100 After initial range finder tests, C. limacina antarctica were exposed to ammonium 101 chloride concentrations of 0, 0.1, 0.5, 1.0, 2.5, 5, 7.5 and 10 mM by adding small volumes 102 of a 1M stock of ammonium chloride to 1L seawater in glass beakers. Seven C. limacina 103 antarctica were placed in each beaker/concentration (only one beaker was used for each 104 concentration) at the start of the experiment, and whether the animals were swimming 105 was monitored every 12h for 96h. Water was changed every 24h. If an organism ceased 106 a normal swimming pattern, it was gently prodded with a jet of seawater from a 107 Pasteur pipette to elicit a response. If no response was noted, revival was attempted in 108 seawater with no ammonium chloride. If no revival was evident, mortality was 109 recorded. At the end of 96h, only surviving animals were removed and briefly blotted 7 110 with a tissue, placed in individual pre-weighed cryovials, reweighed to obtain animal 111 mass, snap frozen in liquid nitrogen, stored at -80oC for several months (including 112 several days on dry ice in transit to Ottawa) prior to analysis of glutamine synthetase 113 activity (see below). Mortality data were subjected to a Trimmed Spearman-Karber 114 analysis, with trim level set at zero, using CETIS software in order to calculate a 96h 115 LC50 value (USEPA 2002). Software and documentation are available for download at 116 http://www.epa.gov/nerleerd/stat2.htm. Because most environmental regulatory 117 agencies set water quality criteria in mg/L of unionized ammonia (NH3), in several 118 places below we transform concentrations of total ammonia (mM) to these values. 119 Conversion of molar values to gram/volume values used the factor of 17.031 120 grams/mole. Calculation of fraction as NH3 used a rearrangement of the Henderson- 121 Hasselbalch equation with a pKa of 10.1483 (USEPA 1998; Bell et al 2007) and the 122 measured pH of seawater in our tests (7.756). 123 124 Measurement of Oxygen Consumption and Ammonia Excretion Rates. 125 Following results of ammonia toxicity testing, we sought to examine the effects of a 126 relatively modest increase in ammonia concentration on two physiological variables.