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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
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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.
127 We chose 70 µM total ammonia as an exposure concentration that would clearly be well
128 below lethal limits (some 1/100th the LC50, see ‘Results’ and only 3.5 to 15 fold above
129 current background levels of 5-20 µM in seawater), but one which has shown biological
130 effects in fish species in simulated global change studies (Linton et al 1998). For these
131 tests we randomly selected C. limacina antarctica that had been held in captivity between
8 132 24 and 36 hours and placed them in air-tight glass syringes with a known volume of 0.2
133 micron-filtered and well aerated seawater. Ammonium chloride was added to half of
134 the trials to achieve a 70 µM total ammonia concentration. A blank syringe containing
135 no organism was set up for every 1-2 experimental syringes and allowed to incubate
136 simultaneously to monitor background (presumably microbial/bacterial) respiration.
137 After a 20-28 hour period we measured the O2 concentration in the glass syringes by
138 drawing a water sample using a Hamilton gas tight syringe (500 μL) and then injecting
139 the sample through a water-jacketed Clarke-type microcathode oxygen electrode
140 (Strathkelvin Instruments, North Lanarkshire, United Kingdom; Marsh and Manahan
141 1999). We then removed the animals from their syringe, gently blotted them dry and
142 weighed them on an analytical balance. This method has been used successfully to
143 determine effects of body mass, feeding, temperature and carbon dioxide on pteropod
144 metabolism (Seibel and Dierssen 2003; Seibel et al 2007; Maas et al 2011; Seibel et al
145 submitted).
146 At the termination of the respiration measurements, a water sample was
147 analyzed for ammonia concentration by the phenol-hypochlorite method (Ivancic and
148 Degobbis 1984). Notably, in preliminary experiments, no urea excretion was detected
149 using a standard colorimetric method (Rahmatullah and Boyd 1980).
150
151 Measurement of Glutamine Synthetase Activity.
152 Glutamine Synthetase (GS; L-glutamate:ammomnia ligase (ADP forming), E.C. 6.3.1.2)
153 activity was measured using the glutamyl transferase assay as previously applied to
9 154 fish tissues (e.g., Walsh 1996). Individual pteropods were homogenized on ice in 5
155 volumes per weight in 50 mM Hepes, pH 7.5 using a Fisher Powergen 125 with a 5 mm
156 tip, and then centrifuged at 16,100 x g for 5 min at 4oC in an Eppendorf 5415D
157 microcentrifuge. An aliquot of 50 µl of the supernatant was added to a 1.5 ml
158 microcentrifuge tube with 1 ml of a reaction cocktail containing (in mM): glutamine
159 (60), hydroxylamine (15), ADP (0.4), KH2AsO4 (20), MnCl2 (3), Hepes (50) (pH 6.7), and
160 the reaction proceeded for 20 min at 20oC. The reaction was terminated and color
161 developed by addition of 0.3 ml Ferric Chloride reagent (containing equal parts 50%
162 HCl : 24% Trichloroacetic acid : 10% FeCl3 in 0.2 N HCl). The reaction mixture was
163 then centrifuged and 200 µL of the supernatant was read for absorbance at 540 nm in a
164 Molecular Devices Spertra Max Plus microtitre plate spectrophotometer. A time zero
165 blank absorbance (Ferric Chloride reagent added before addition of supernatant) was
166 subtracted from the measured sample absorbance and then the concentration of product
167 was calculated from a standard curve (absorbance vs. concentration) of gamma
168 glutamyl monohydroxamate reacted with the Ferric Chloride reagent. Using
169 micromoles of product, time of reaction, body mass, and homogenization dilution
170 factors, enzyme activities were calculated in µmols Substrate Product min-1 g wet
171 mass-1.
172
173 Results
174 The 96h LC50 value for C. limacina antarctica exposed to ammonium chloride was 7.465
175 mM (Upper 95% CL = 8.498 mM and Lower 95% CL = 6.557 mM). Since most
10 176 comparisons of ammonia toxicity data are compared in the literature as mg/L
177 unionized ammonia available (NH3), the above 96h LC50 value converts to 0.51 mg/L
178 NH3. Notably the mortality curve yielding this value was rather steep with no deaths
179 occurring up to 5 mM, 3 out of 7 animals dying at 7.5 mM, and all animals dying at 10
180 mM.
181 Oxygen consumption and nitrogen excretion rates fit well to standard mass-
182 scaling equations (Table 1), and there were no significant effects of ammonia on either
183 rate or on the O:N ratio (Figure 1).
184 The sub-lethal exposure concentrations used in the mortality experiment had
185 significant effects on the activity of GS (Figure 2) with a pronounced 1.5-fold increase in
186 GS activity at even the lowest concentration employed (0.1 mM) and then declining
187 activities at higher concentrations until there was no significant difference from controls
188 (nominal 0 mM).
189
190
11 191 Discussion
192 Studies of ammonia toxicity effects on marine organisms are rather scant, certainly in
193 comparison to the body of information for freshwater organisms (Boardman et al. 2004;
194 USEPA 1989; 1998), and this is understandable since most significant ammonia
195 pollution point sources are freshwater or estuarine. From the data on marine
196 invertebrates available, marine molluscs can be among the more ammonia tolerant
197 invertebrate species, showing for example 96h LC50 values in quahog clams (Mercenaria
198 mercenaria) of up to 36.3 mg/L NH3 (Boardman et al 2004). Thus the 96h LC50 value we
199 obtained for C. limacina antarctica at 0.51 mg/L NH3 is considerably lower and indicates
200 a high sensitivity to ammonia (see below). Noting this low LC50 value, it does not
201 appear that high levels of lipids and TMAO confer ammonia tolerance to at least this
202 species of polar zooplankton as initially hypothesized.
203 The measured O:N ratios and underlying rates were in line with previously
204 reported data for this species (Maas et al 2011), and more generally indicate that
205 metabolism in C. limacina antarctica is being fueled exclusively by proteins/amino acids;
206 Mayzaud and Conover (1988) point out that O:N ratios of 3 to 16 are indicative of pure
207 protein catabolism in zooplankton. At more realistic concentrations of ammonia, we
208 observed no effects on the processes of oxygen consumption and nitrogen excretion.
209 The lack of effect on ammonia excretion rates is somewhat surprising in light of what is
210 known about mechanisms of ammonia excretion in aquatic organisms in general. At
211 least in fish, ammonia excretion is now known to take place largely through ammonia
212 channels in gill/respiratory surfaces, the so-called Rhesus (or Rh) glycoproteins
12 213 (Weihrauch et al 2009; Wright and Wood 2009) and is largely a facilitated diffusion
214 process determined by the numbers/density of transporters and the partial pressure
215 gradients of dissolved ammonia gas from the internal to seawater compartments.
216 Raising external ammonia concentrations even slightly often causes fish to show a net
217 uptake of ammonia from the environment, albeit usually briefly (e.g., for up to 24h),
218 until an outwardly directed gradient is re-established and ammonia excretion can
219 resume (see reviews by Weihrauch et al 2009; Wright and Wood 2009). Excretion
220 pathways in invertebrates, while appearing also to rely on Rh glycoproteins, may be
221 more complicated, with possible mechanisms in crustaceans involving initial
222 sequestration of ammonia in gill vesicles (Weihrauch et al 2009). The fact that ammonia
223 excretion could continue without change at the elevated test concentrations used in the
224 present study (Figure 1) perhaps reflects that these potential specialized mechanisms
225 exist in molluscs, or that the 24h measurement period was sufficient for gradients and
226 total excretion rates to be reestablished.
227 In this study, we also wished to examine a biochemical process or endpoint that
228 might show greater sensitivity to low concentrations of environmental ammonia,
229 namely activity of the ammonia detoxification/metabolism enzyme glutamine
230 synthetase. Even the lowest test concentration used, 0.1 mM total ammonia or 0.007
231 mg/L NH3 led to a significant increase in the activity of this enzyme in C. limacina
232 antarctica (Fig. 2). Several environmental regulatory agencies have set water quality
233 levels very close to both this concentration and the LC50 value we measured. For
234 example, the US EPA has set Criteria Continuous Concentration water quality levels for
13 235 ammonia in seawater at 0.019-0.030 mg/L NH3 (at 0oC, 30 ppt and representative
236 seawater pHs of 7.8 to 8.0) (USEPA 1989). Similarly, the UK Environment Agency has
237 proposed a short-term (96h) PNEC (predicted no effect concentration) for ammonia in
238 seawater under similar conditions at 0.0057 mg/L NH3 (UK Environment Agency 2007).
239 Again, data on GS activation by elevated ammonia in marine molluscs have not been
240 previously reported, so it is difficult to compare our data to other species, and to know
241 whether the elevation of GS activity by low ammonia concentrations is sufficient to
242 protect the organism from neuronal/behavioral impairment at sub-lethal
243 concentrations. Clione limacina (the northern congener) has been used extensively as a
244 model for the neural basis of behavior and these studies indicate that elements of the
245 feeding system of this species are activated by the neurotransmitter gamma amino
246 butyric acid (GABA) (Arshavsky et al 1993). In mammalian models, some of the
247 symptoms of hepatic encephalopathy are believed to be the result of imbalances
248 between GABA- vs. glutamate-mediated neuronal pathways (Cooper and Plum 1987;
249 Butterworth 2001). In this regard, it would be instructive to examine effects of modest
250 ammonia concentrations on feeding behavior in Clione sp.
251 Interestingly, while anthropogenic ammonia point sources in Antarctic waters
252 are certainly rare, potential naturally occurring sources of ammonia might exist in
253 runoff from the substantial guano mounds associated with penguin rookeries. It would
254 be informative to obtain information on nearshore ammonia concentrations adjacent to
255 these rookeries. Furthermore, in examining the potential effects of global change
256 scenarios on polar marine organisms, investigators have largely focused on increased
14 257 temperature and carbon dioxide (and resulting acidification) as important variables
258 (Orr et al 2005; Trathan et al 2007; McNeil and Matear 2008; Moline et al 2008).
259 However, nitrogen loading (notably as potentially toxic ammonia and nitrite) in the
260 marine environment is also expected to increase, primarily due to anthropogenic
261 sources such as fertilizers and sewerage (Vitousek et al 2009), all with potential
262 disruptions to the natural nitrogen cycle (Canfield et al 2010). While most polar regions
263 are currently relatively shielded from direct anthropogenic point sources of nitrogenous
264 pollution, eventually, any increase in background oceanic levels could potentially reach
265 polar oceans and species, and therefore it would be prudent to obtain additional
266 information on the effects of toxic nitrogenous molecules on polar organisms. One
267 study has shown that GS mRNA transcript levels in Crassostrea virginica are elevated by
268 pesticides, hydrocarbons and hypoxia (Tanguy et al 2005). Our enzymatic data indicate
269 that GS could potentially be used as one important bioindicator of environmental
270 degradation/exposure in polar mollusc species. Certainly, the high sensitivity of this
271 one species to ammonia toxicity warrants additional study of the effects of elevated
272 nitrogen on the physiology of polar zooplankton.
273
274 Acknowledgements
275 This research was supported by a US National Science Foundation grant (OPP#
276 0538479) to BAS and VJ Fabry, and by a Discovery Grant from the Natural Sciences and
277 Engineering Council of Canada to PJW, who is also supported by the Canada Research
278 Chair Program. The authors wish to thank Drs. Martin Grosell and Andrew Esbaugh of
15 279 the University of Miami Rosenstiel School for advice on calculation of LC50 values.
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364
20 365 Fig 1: Effect of ammonia on oxygen consumption and nitrogen excretion rates of Clione
366 limacina antarctica. There is no statistical difference between the oxygen consumption
367 rate (A, p = 0.77), ammonia excretion rate (B, p = 0.25) and O:N ratio (C, p = 0.50) for
368 organisms exposed to nominal 0 µM ammonia (white circles) and 70 µM ammonia
369 (black circles).
370
371
21 372 Fig. 2: Effect of 96h exposure to variable levels of ammonium chloride on Glutamine
373 Synthetase Activity (µmols S P min-1 g-1) for Clione limacina antarctica. Values are
374 means + 1 S.E.M. and N = 7 for all treatments except 0 mM where N = 14. Total
375 ammonia concentration has a significant effect on the Glutamine Synthetase Activity
376 (ANOVA, F6,49 = 3.1, p = 0.011) and bars with common letters are not significantly
377 different.
378 379 380 381
22 382 Table 1: Mean oxygen consumption and ammonia excretion rates and O:N ratio of Clione
383 limacina antarctica exposed to 0 µM vs. 70 µM ammonia. P values calculated using a
384 Welch’s t-test. Regression constants are in the form of Y=aXb where oxygen consumption or
385 ammonia excretion rate = Y and organismal mass = X.
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
23