Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/257396392

Effectsofelevatedammoniaconcentrationsonsurvival,metabolicrates,andglutaminesynthetaseactivityintheAntarcticpteropodmolluskClionelimacinaantarctica

ArticleinPolarBiology·July2012

DOI:10.1007/s00300-012-1158-7

CITATIONS

4

READS

34

3authors,including:

AEMaas

BermudaInstituteofOceanSciences

16PUBLICATIONS133CITATIONS

SEEPROFILE

BradSeibel

UniversityofRhodeIsland

87PUBLICATIONS3,474CITATIONS

SEEPROFILE

Allin-textreferencesunderlinedinbluearelinkedtopublicationsonResearchGate,

lettingyouaccessandreadthemimmediately.

Availablefrom:AEMaas

Retrievedon:02August2016

Short Note 1

2

Effects of Elevated Ammonia Concentrations on Survival, Metabolic Rates and 3

Glutamine Synthetase Activity in the Antarctic Pteropod Mollusc Clione limacina 4

antarctica 5

6

Amy Maas1, 2, Brad A. Seibel1 and Patrick J. Walsh3 7

8

1 Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881 9

2 Current address: Department of Biology, Woods Hole Oceanographic Institute, Woods 10

Hole, MA 02543 11

3Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6N5 12

CANADA 13

14

Corresponding Author Contact Information: 15

P.J. Walsh 16

Dept. of Biology, University of Ottawa 17

30 Marie Curie 18

Ottawa, ON K1N 6N5 Canada 19

Email: pwalsh@uottawa.ca 20

Phone: 613-562-5800x6328 21

Fax: 613-562-5486 22

2

23

3

Abstract 24

Information on effects of elevated ammonia on invertebrates in general, and polar 25

Molluscs in particular, is scant. Questions of ammonia sensitivity are interesting for 26

several reasons, particularly since predicted global change scenarios include increases 27

in anthropogenic nitrogen and toxic ammonia. Furthermore, polar zooplankton species 28

are often rich in lipids, and authors have speculated that there is a linkage between 29

elevated levels of lipids/trimethylamine oxide (TMAO) and enhanced ammonia 30

tolerance. In the present study, we sought to examine ammonia tolerance and effects of 31

elevated exogenous ammonia on several key aspects of the physiology and 32

biochemistry of the pteropod mollusc, Clione antarctica limacina. We determined that the 33

96-hour LC50 value for this species is 7.465 mM total ammonia (Upper 95% CL = 8.498 34

mM and Lower 95% CL = 6.557 mM), or 0.51 mg/L as unionized ammonia (NH3) (at a 35

pH of 7.756). While comparative data for molluscs are limited, this value is at the lower 36

end of reported values for other species. When the effects of lower ammonia 37

concentrations (0.07 mM total ammonia) on oxygen consumption and ammonia 38

excretion rates were examined, no effects were noted. However, total ammonia levels as 39

low as 0.1 mM (or 0.007 mg/L NH3) elevated the activity of the ammonia detoxification 40

enzyme, glutamine synthetase, by approximately 1.5 fold. The values for LC50 and 41

observable effects on biochemistry for this one species are very close to permissible 42

marine ammonia concentrations, indicating a need to more broadly determine the 43

sensitivity of zooplankton to potential elevated ammonia levels in polar regions. 44

4

Key Words: global change, nitrogen pollution, Antarctica, pelagic molluscs, O:N ratio, 45

ammonia LC50 values, TMAO 46

47

5

Introduction 48

One of the more toxic forms of nitrogen to animals is ammonia. At least in 49

vertebrates, the mode of action of ammonia as a toxin is primarily through its effects on 50

the central nervous system (CNS). Ammonia CNS toxicity has been relatively well 51

documented in the medical literature (in association with the human disease hepatic 52

encephalopathy), and increases in plasma ammonia levels due to liver dysfunction 53

appear to affect glutamate receptors on neurons, as well as to cause swelling in 54

associated astrocytes (the nutritive and support cells of the vertebrate CNS) (Cooper 55

and Plum 1987; Butterworth 2001). The literature on ammonia toxicity in fish species is 56

smaller but growing, and so far indicates that, although overall mechanisms of toxicity 57

are similar to mammals, there are some important differences, notably: (1) astrocyte 58

swelling seems to be less pronounced in the brains of marine fish; (2) there are wide 59

species differences in the susceptibility of fish to ammonia, with some species showing 60

orders of magnitude greater ability to survive ammonia toxicity than can mammals 61

(Walsh et al. 2007). 62

In contrast, much less is known about mechanisms of ammonia toxicity in 63

marine invertebrates in general and polar invertebrates and molluscs in particular. Data 64

exist on ammonia-induced mortality (e.g., standard Lethal Concentration 50, or LC50 65

values, the concentration that leads to mortality in 50% of a test population after a 66

standard time) for numerous freshwater and some marine invertebrate species, 67

including molluscs (e.g., Boardman et al 2004; USEPA 1989). Although toxic effects 68

leading to mortality are presumed to be primarily neuronal as in vertebrates, very little 69

6

is known. Furthermore, species considered to date have primarily been standard EPA 70

test indicator organisms, or organisms in inland waters that are predicted to be at risk 71

for exposure via close proximity to point sources (examined as part of mandated 72

environmental impact studies). With respect to polar invertebrates, we are not aware of 73

any studies examining ammonia-induced mortality or effects of ammonia on routine 74

physiological processes. In this regard, Seibel and Walsh (2002) previously reported that 75

Clione antarctica has high levels of trimethylamine oxide (TMAO) which is known to 76

counteract ammonia toxicity in some species (Kloiber et al. 1988; Minana et al. 1996). 77

This observation leads to a hypothesis that many polar zooplankton may show 78

enhanced ammonia tolerance because they have high lipid content for over-winter 79

survival and lipid formation is linked to TMAO levels (Seibel and Walsh 2002). It also 80

suggests that ammonia tolerance will depend to some extent on diet. 81

With this scant background in mind, in the present study we examined the 82

effects of ammonia on mortality, routine physiological processes (oxygen consumption 83

and nitrogen excretion), and the activity of an enzyme involved in ammonia 84

detoxification (glutamine synthetase) in the Antarctic pteropod, Clione limacina antarctica 85

in studies complementary to examination of the effects of acidification (Seibel et al. 86

submitted).87

7

Materials and Methods 88

Collection, Maintenance and Ammonia Exposure of Animals 89

In January 2008, specimens of Clione limacina antarctica (Smith 1902) were collected 90

several meters offshore at Cape Royds (77° 34’ S, 166° 11’ E) on Ross Island near 91

McMurdo Station, Antarctica. Collectors wading in waters of approximately 1m depth 92

dipped animals out of the water using 1L beakers attached to 1 m poles. Organisms 93

were then gently poured into 500mL Nalgene bottles (to a density of 10-12 organisms 94

per bottle), placed in insulated coolers and returned to McMurdo Station by helicopter 95

within 6h of capture. Bottles were then placed in a cold room to maintain temperature 96

at -1.8 oC (also the temperature of all subsequent tests unless noted). Organisms 97

(ranging in body mass from 0.0429 to 0.3616 grams) were held in captivity without food 98

for a period of 24 hours to allow for gut clearance. 99

After initial range finder tests, C. limacina antarctica were exposed to ammonium 100

chloride concentrations of 0, 0.1, 0.5, 1.0, 2.5, 5, 7.5 and 10 mM by adding small volumes 101

of a 1M stock of ammonium chloride to 1L seawater in glass beakers. Seven C. limacina 102

antarctica were placed in each beaker/concentration (only one beaker was used for each 103

concentration) at the start of the experiment, and whether the animals were swimming 104

was monitored every 12h for 96h. Water was changed every 24h. If an organism ceased 105

a normal swimming pattern, it was gently prodded with a jet of seawater from a 106

Pasteur pipette to elicit a response. If no response was noted, revival was attempted in 107

seawater with no ammonium chloride. If no revival was evident, mortality was 108

recorded. At the end of 96h, only surviving animals were removed and briefly blotted 109

8

with a tissue, placed in individual pre-weighed cryovials, reweighed to obtain animal 110

mass, snap frozen in liquid nitrogen, stored at -80oC for several months (including 111

several days on dry ice in transit to Ottawa) prior to analysis of glutamine synthetase 112

activity (see below). Mortality data were subjected to a Trimmed Spearman-Karber 113

analysis, with trim level set at zero, using CETIS software in order to calculate a 96h 114

LC50 value (USEPA 2002). Software and documentation are available for download at 115

http://www.epa.gov/nerleerd/stat2.htm. Because most environmental regulatory 116

agencies set water quality criteria in mg/L of unionized ammonia (NH3), in several 117

places below we transform concentrations of total ammonia (mM) to these values. 118

Conversion of molar values to gram/volume values used the factor of 17.031 119

grams/mole. Calculation of fraction as NH3 used a rearrangement of the Henderson-120

Hasselbalch equation with a pKa of 10.1483 (USEPA 1998; Bell et al 2007) and the 121

measured pH of seawater in our tests (7.756). 122

123

Measurement of Oxygen Consumption and Ammonia Excretion Rates. 124

Following results of ammonia toxicity testing, we sought to examine the effects of a 125

relatively modest increase in ammonia concentration on two physiological variables. 126

We chose 70 µM total ammonia as an exposure concentration that would clearly be well 127

below lethal limits (some 1/100th the LC50, see ‘Results’ and only 3.5 to 15 fold above 128

current background levels of 5-20 µM in seawater), but one which has shown biological 129

effects in fish species in simulated global change studies (Linton et al 1998). For these 130

tests we randomly selected C. limacina antarctica that had been held in captivity between 131

9

24 and 36 hours and placed them in air-tight glass syringes with a known volume of 0.2 132

micron-filtered and well aerated seawater. Ammonium chloride was added to half of 133

the trials to achieve a 70 µM total ammonia concentration. A blank syringe containing 134

no organism was set up for every 1-2 experimental syringes and allowed to incubate 135

simultaneously to monitor background (presumably microbial/bacterial) respiration. 136

After a 20-28 hour period we measured the O2 concentration in the glass syringes by 137

drawing a water sample using a Hamilton gas tight syringe (500 μL) and then injecting 138

the sample through a water-jacketed Clarke-type microcathode oxygen electrode 139

(Strathkelvin Instruments, North Lanarkshire, United Kingdom; Marsh and Manahan 140

1999). We then removed the animals from their syringe, gently blotted them dry and 141

weighed them on an analytical balance. This method has been used successfully to 142

determine effects of body mass, feeding, temperature and carbon dioxide on pteropod 143

metabolism (Seibel and Dierssen 2003; Seibel et al 2007; Maas et al 2011; Seibel et al 144

submitted). 145

At the termination of the respiration measurements, a water sample was 146

analyzed for ammonia concentration by the phenol-hypochlorite method (Ivancic and 147

Degobbis 1984). Notably, in preliminary experiments, no urea excretion was detected 148

using a standard colorimetric method (Rahmatullah and Boyd 1980). 149

150

Measurement of Glutamine Synthetase Activity. 151

Glutamine Synthetase (GS; L-glutamate:ammomnia ligase (ADP forming), E.C. 6.3.1.2) 152

activity was measured using the glutamyl transferase assay as previously applied to 153

10

fish tissues (e.g., Walsh 1996). Individual pteropods were homogenized on ice in 5 154

volumes per weight in 50 mM Hepes, pH 7.5 using a Fisher Powergen 125 with a 5 mm 155

tip, and then centrifuged at 16,100 x g for 5 min at 4oC in an Eppendorf 5415D 156

microcentrifuge. An aliquot of 50 µl of the supernatant was added to a 1.5 ml 157

microcentrifuge tube with 1 ml of a reaction cocktail containing (in mM): glutamine 158

(60), hydroxylamine (15), ADP (0.4), KH2AsO4 (20), MnCl2 (3), Hepes (50) (pH 6.7), and 159

the reaction proceeded for 20 min at 20oC. The reaction was terminated and color 160

developed by addition of 0.3 ml Ferric Chloride reagent (containing equal parts 50% 161

HCl : 24% Trichloroacetic acid : 10% FeCl3 in 0.2 N HCl). The reaction mixture was 162

then centrifuged and 200 µL of the supernatant was read for absorbance at 540 nm in a 163

Molecular Devices Spertra Max Plus microtitre plate spectrophotometer. A time zero 164

blank absorbance (Ferric Chloride reagent added before addition of supernatant) was 165

subtracted from the measured sample absorbance and then the concentration of product 166

was calculated from a standard curve (absorbance vs. concentration) of gamma 167

glutamyl monohydroxamate reacted with the Ferric Chloride reagent. Using 168

micromoles of product, time of reaction, body mass, and homogenization dilution 169

factors, enzyme activities were calculated in µmols Substrate Product min-1 g wet 170

mass-1. 171

172

Results 173

The 96h LC50 value for C. limacina antarctica exposed to ammonium chloride was 7.465 174

mM (Upper 95% CL = 8.498 mM and Lower 95% CL = 6.557 mM). Since most 175

11

comparisons of ammonia toxicity data are compared in the literature as mg/L 176

unionized ammonia available (NH3), the above 96h LC50 value converts to 0.51 mg/L 177

NH3. Notably the mortality curve yielding this value was rather steep with no deaths 178

occurring up to 5 mM, 3 out of 7 animals dying at 7.5 mM, and all animals dying at 10 179

mM. 180

Oxygen consumption and nitrogen excretion rates fit well to standard mass-181

scaling equations (Table 1), and there were no significant effects of ammonia on either 182

rate or on the O:N ratio (Figure 1). 183

The sub-lethal exposure concentrations used in the mortality experiment had 184

significant effects on the activity of GS (Figure 2) with a pronounced 1.5-fold increase in 185

GS activity at even the lowest concentration employed (0.1 mM) and then declining 186

activities at higher concentrations until there was no significant difference from controls 187

(nominal 0 mM). 188

189

190

12

Discussion 191

Studies of ammonia toxicity effects on marine organisms are rather scant, certainly in 192

comparison to the body of information for freshwater organisms (Boardman et al. 2004; 193

USEPA 1989; 1998), and this is understandable since most significant ammonia 194

pollution point sources are freshwater or estuarine. From the data on marine 195

invertebrates available, marine molluscs can be among the more ammonia tolerant 196

invertebrate species, showing for example 96h LC50 values in quahog clams (Mercenaria 197

mercenaria) of up to 36.3 mg/L NH3 (Boardman et al 2004). Thus the 96h LC50 value we 198

obtained for C. limacina antarctica at 0.51 mg/L NH3 is considerably lower and indicates 199

a high sensitivity to ammonia (see below). Noting this low LC50 value, it does not 200

appear that high levels of lipids and TMAO confer ammonia tolerance to at least this 201

species of polar zooplankton as initially hypothesized. 202

The measured O:N ratios and underlying rates were in line with previously 203

reported data for this species (Maas et al 2011), and more generally indicate that 204

metabolism in C. limacina antarctica is being fueled exclusively by proteins/amino acids; 205

Mayzaud and Conover (1988) point out that O:N ratios of 3 to 16 are indicative of pure 206

protein catabolism in zooplankton. At more realistic concentrations of ammonia, we 207

observed no effects on the processes of oxygen consumption and nitrogen excretion. 208

The lack of effect on ammonia excretion rates is somewhat surprising in light of what is 209

known about mechanisms of ammonia excretion in aquatic organisms in general. At 210

least in fish, ammonia excretion is now known to take place largely through ammonia 211

channels in gill/respiratory surfaces, the so-called Rhesus (or Rh) glycoproteins 212

13

(Weihrauch et al 2009; Wright and Wood 2009) and is largely a facilitated diffusion 213

process determined by the numbers/density of transporters and the partial pressure 214

gradients of dissolved ammonia gas from the internal to seawater compartments. 215

Raising external ammonia concentrations even slightly often causes fish to show a net 216

uptake of ammonia from the environment, albeit usually briefly (e.g., for up to 24h), 217

until an outwardly directed gradient is re-established and ammonia excretion can 218

resume (see reviews by Weihrauch et al 2009; Wright and Wood 2009). Excretion 219

pathways in invertebrates, while appearing also to rely on Rh glycoproteins, may be 220

more complicated, with possible mechanisms in crustaceans involving initial 221

sequestration of ammonia in gill vesicles (Weihrauch et al 2009). The fact that ammonia 222

excretion could continue without change at the elevated test concentrations used in the 223

present study (Figure 1) perhaps reflects that these potential specialized mechanisms 224

exist in molluscs, or that the 24h measurement period was sufficient for gradients and 225

total excretion rates to be reestablished. 226

In this study, we also wished to examine a biochemical process or endpoint that 227

might show greater sensitivity to low concentrations of environmental ammonia, 228

namely activity of the ammonia detoxification/metabolism enzyme glutamine 229

synthetase. Even the lowest test concentration used, 0.1 mM total ammonia or 0.007 230

mg/L NH3 led to a significant increase in the activity of this enzyme in C. limacina 231

antarctica (Fig. 2). Several environmental regulatory agencies have set water quality 232

levels very close to both this concentration and the LC50 value we measured. For 233

example, the US EPA has set Criteria Continuous Concentration water quality levels for 234

14

ammonia in seawater at 0.019-0.030 mg/L NH3 (at 0oC, 30 ppt and representative 235

seawater pHs of 7.8 to 8.0) (USEPA 1989). Similarly, the UK Environment Agency has 236

proposed a short-term (96h) PNEC (predicted no effect concentration) for ammonia in 237

seawater under similar conditions at 0.0057 mg/L NH3 (UK Environment Agency 2007). 238

Again, data on GS activation by elevated ammonia in marine molluscs have not been 239

previously reported, so it is difficult to compare our data to other species, and to know 240

whether the elevation of GS activity by low ammonia concentrations is sufficient to 241

protect the organism from neuronal/behavioral impairment at sub-lethal 242

concentrations. Clione limacina (the northern congener) has been used extensively as a 243

model for the neural basis of behavior and these studies indicate that elements of the 244

feeding system of this species are activated by the neurotransmitter gamma amino 245

butyric acid (GABA) (Arshavsky et al 1993). In mammalian models, some of the 246

symptoms of hepatic encephalopathy are believed to be the result of imbalances 247

between GABA- vs. glutamate-mediated neuronal pathways (Cooper and Plum 1987; 248

Butterworth 2001). In this regard, it would be instructive to examine effects of modest 249

ammonia concentrations on feeding behavior in Clione sp. 250

Interestingly, while anthropogenic ammonia point sources in Antarctic waters 251

are certainly rare, potential naturally occurring sources of ammonia might exist in 252

runoff from the substantial guano mounds associated with penguin rookeries. It would 253

be informative to obtain information on nearshore ammonia concentrations adjacent to 254

these rookeries. Furthermore, in examining the potential effects of global change 255

scenarios on polar marine organisms, investigators have largely focused on increased 256

15

temperature and carbon dioxide (and resulting acidification) as important variables 257

(Orr et al 2005; Trathan et al 2007; McNeil and Matear 2008; Moline et al 2008). 258

However, nitrogen loading (notably as potentially toxic ammonia and nitrite) in the 259

marine environment is also expected to increase, primarily due to anthropogenic 260

sources such as fertilizers and sewerage (Vitousek et al 2009), all with potential 261

disruptions to the natural nitrogen cycle (Canfield et al 2010). While most polar regions 262

are currently relatively shielded from direct anthropogenic point sources of nitrogenous 263

pollution, eventually, any increase in background oceanic levels could potentially reach 264

polar oceans and species, and therefore it would be prudent to obtain additional 265

information on the effects of toxic nitrogenous molecules on polar organisms. One 266

study has shown that GS mRNA transcript levels in Crassostrea virginica are elevated by 267

pesticides, hydrocarbons and hypoxia (Tanguy et al 2005). Our enzymatic data indicate 268

that GS could potentially be used as one important bioindicator of environmental 269

degradation/exposure in polar mollusc species. Certainly, the high sensitivity of this 270

one species to ammonia toxicity warrants additional study of the effects of elevated 271

nitrogen on the physiology of polar zooplankton. 272

273

Acknowledgements 274

This research was supported by a US National Science Foundation grant (OPP# 275

0538479) to BAS and VJ Fabry, and by a Discovery Grant from the Natural Sciences and 276

Engineering Council of Canada to PJW, who is also supported by the Canada Research 277

Chair Program. The authors wish to thank Drs. Martin Grosell and Andrew Esbaugh of 278

16

the University of Miami Rosenstiel School for advice on calculation of LC50 values.279

17

References 280

Arshavsky YI, Deliagina TG, Gamkrelidze GN, Orlovsky GN, Panchin YV, Popova LB, 281

Shupliakov OV (1993) Pharmacologically induced elements of the hunting and 282

feeding behavior in the pteropod mollusc Clione limacina. I. Effects of GABA. 283

Journal of Neurophysiology 69:512-521. 284

Bell TG, Johnson MT, Jickells TD, Liss PS (2007) Ammonia/ammonium dissociation 285

coefficient in seawater: a significant numerical correction. Environ Chem 4:183-286

186 287

Boardman GD, Starbuck SM, Hudgins DB, Li X, Kuhn DD (2004) Toxicity of ammonia 288

to three marine fish and three marine invertebrates. Environ Toxicol 19:134-142 289

Butterworth RF (2001) Glutamate transporter and receptor function in disorders of 290

ammonia metabolism. Ment Retard Dev Disabil Res Rev 7:276-279. 291

Canfield DE, Glazer AN, Falkowski PG (2010) The evolution and future of earth’s 292

nitrogen cycle. Science 330:192-196 293

Cooper AJL, Plum F (1987) Biochemistry and physiology of brain ammonia. Physiol Rev 294

67:440-519 295

Ivancic I, Degobbis D (1984) An optimal manual procedure for ammonia analysis in 296

natural waters by the indophenol blue method. Water Res 18:1143-1147 297

Kloiber O, Banjac B, Drewes LR (1988) Protection against acute hyperammonemia: the 298

role of quaternary amines. Toxicology 49:83-90 299

Linton TK, Morgan IJ, Walsh PJ, Wood CM (1998) Chronic exposure of rainbow trout to 300

18

simulated warming and sublethal ammonia: a year-long study of their appetite, 301

growth, and energetics. Can J Fish Aq Sci 55:576-586 302

Maas AE, Elder LE, Dierssen H, Seibel BA (2011) The metabolic response of Antarctic 303

pteropods (Mollusca: Gastropoda) to food deprivation and regional productivity. 304

Mar Ecol Prog Ser 441:129-139 305

Marsh AG, Manahan DT (1999) A method for accurate measurements of the respiration 306

rates of marine invertebrate embryos and larvae. Mar Ecol Prog Ser 184:1-10 307

Mayzaud P, Conover RJ (1988) O:N atomic ratio as a tool to describe zooplankton 308

metabolism. Mar Ecol Prog Ser 45:289-302 309

McNeil BI, Matear RJ (2008) Southern ocean acidification: A tipping point at 450-ppm 310

atmospheric CO2. Proc Nat Acad Sci 105:18860-18864 311

Minana M, Hermenegildo C, Llansola M, Montoliu C, Grisola S, Felip V (1996) 312

Carnitine and choline derivatives containg a trimethylamine group prevent 313

ammonia toxicity in mice and glutamate toxicity in primary cultures of neurons. 314

J Pharmacol Exp Ther 279:194-199. 315

Moline MA, Karnovsky NJ, Brown Z, Divoky GJ et al (2008) High latitude changes in ice 316

dynamics and their impact on polar marine ecosystems. Ann NY Acad Sci 317

1134:267-319 318

Orr JC, Fabry VJ, Aumont O, Bopp L et al (2005) Anthropogenic ocean acidification over 319

the twenty-first century and its impact on calcifying organisms. Nature 437:681-320

686 321

19

Rahmatullah M, Boyde TRC (1980) Improvements in the determination of urea using 322

diacetyl monoxime; methods with and without deproteinisation. Clin Chim Acta 323

107:3-9 324

Seibel BA, Walsh PJ (2002) Trimethylamine oxide accumulation in marine animals: 325

relationship to acylglycerol storage. J Exp Biol 205:297-306. 326

Seibel BA, Dierssen HM (2003) Cascading Trophic Impacts of Reduced Biomass in the 327

Ross Sea, Antarctica: Just the Tip of the Iceberg? Bio Bull 205:93-97. 328

Seibel BA, Dymowska A, Rosenthal J (2007) Metabolic temperature compensation and 329

co-evolution of locomotory performance in pteropod molluscs. Integrative and 330

Comparative Biology 47: 880-891. 331

Seibel BA, Maas AE, Dierssen HM Energetic plasticity underlies a variable response to 332

ocean acidification in the pteropod, Limacina helicina antarctica., PLoS One, 333

submitted. 334

Tanguy A, Boutet I, Moraga D (2005) Molecular characterization of the glutamine 335

synthetase gene in the Pacific oyser Crassostrea gigas: expression study in 336

response to xenobiotic exposure and developmental stage. Biochim Biophys Acta 337

1681:116-125 338

Trathan PN, Forcada J, Murphy EJ (2007) Environmental forcing and southern ocean 339

marine predator populations: Effects of climate change and variability. Phil 340

Trans B 362:2351-2365 341

United Kingdom Environment Agency (2007) Proposed EQS for Water Framework 342

20

Directive Annex VIII substances: ammonia (un-ionised) Bristol, England (Report: 343

SC040038/SR2) 344

United States Environmental Protection Agency (1989) Ambient water quality criteria 345

for ammonia (saltwater). Washington, DC (EPA 440/5-88-004) 346

United States Environmental Protection Agency (1998) Update of ambient water quality 347

criteria for ammonia. Washington, DC (EPA 822-R-98-008) 348

United States Environmental Protection Agency (2002) Method for measuring the acute 349

toxicity of effluents and receiving waters to freshwater and marine organisms, 5th 350

edition. (EPA-821-R-02-012) USEPA Office of Water (4303T), Washington, DC 351

Vitousek PM, Naylor R, Crews T et al (2009) Agriculture. Nutrient imbalances in 352

agricultural development. Science 324:1519-1520. 353

Walsh PJ (1996) Purification and properties of hepatic glutamine synthetases from the 354

ureotelic gulf toadfish, Opsanus beta. Comp Biochem Physiol 115B:523-532 355

Walsh PJ, Veauvy, CM, McDonald, MD, Buck, LT, Wilkie MP (2007) Piscine insights 356

into comparisons of anoxia tolerance, ammonia toxicity, stroke and hepatic 357

encephalopathy. Comp Biochem Physiol 147A:332-343 358

Weihrauch D, Wilkie MP, Walsh PJ (2009) Ammonia and urea transporters in gills of 359

fish and aquatic crustaceans. J Exp Biol 212:1716-1730 360

Wright PA, Wood CM (2009) A new paradigm for ammonia excretion in aquatic 361

animals: roles of Rhesus (Rh) glycoproteins. J. Exp Biol 212:2303-2312 362

363

364

21

Fig 1: Effect of ammonia on oxygen consumption and nitrogen excretion rates of Clione 365

limacina antarctica. There is no statistical difference between the oxygen consumption 366

rate (A, p = 0.77), ammonia excretion rate (B, p = 0.25) and O:N ratio (C, p = 0.50) for 367

organisms exposed to nominal 0 µM ammonia (white circles) and 70 µM ammonia 368

(black circles). 369

370

371

22

Fig. 2: Effect of 96h exposure to variable levels of ammonium chloride on Glutamine 372

Synthetase Activity (µmols S P min-1 g-1) for Clione limacina antarctica. Values are 373

means + 1 S.E.M. and N = 7 for all treatments except 0 mM where N = 14. Total 374

ammonia concentration has a significant effect on the Glutamine Synthetase Activity 375

(ANOVA, F6,49 = 3.1, p = 0.011) and bars with common letters are not significantly 376

different. 377

378

379 380

381

23

Table 1: Mean oxygen consumption and ammonia excretion rates and O:N ratio of Clione 382

limacina antarctica exposed to 0 µM vs. 70 µM ammonia. P values calculated using a 383

Welch’s t-test. Regression constants are in the form of Y=aXb where oxygen consumption or 384

ammonia excretion rate = Y and organismal mass = X. 385

386

O2 NH3 O:N

0 uM 70 uM 0 uM 70 uM 0 uM 70 uM

Mean 1.31 1.28 0.42 0.33 8.18 9.25

Std Error 0.09 0.08 0.06 0.04 1.17 1.07

p 0.77 0.25 0.5

a 0.54 -0.50 0.22 0.22

b 0.71 -0.30 -0.28 -0.14

R 0.83 0.64 0.31 0.11

387