The Chronic Toxicity of Ion Mixtures to Freshwater Organisms: Ceriodaphnia dubia and Pimephales promelas (fathead minnow)

Transcription

1 Clemson University TigerPrints All Disserttions Disserttions The Chronic Toxicity of Ion Mixtures to Freshwter Orgnisms: Ceriodphni dubi nd Pimephles promels (fthed minnow) Ktherine Anne Johnson-Couch Clemson University, Follow this nd dditionl works t: Recommended Cittion Johnson-Couch, Ktherine Anne, "The Chronic Toxicity of Ion Mixtures to Freshwter Orgnisms: Ceriodphni dubi nd Pimephles promels (fthed minnow)" (2018). All Disserttions This Disserttion is brought to you for free nd open ccess by the Disserttions t TigerPrints. It hs been ccepted for inclusion in All Disserttions by n uthorized dministrtor of TigerPrints. For more informtion, plese contct kokeefe@clemson.edu.

2 The chronic toxicity of ion mixtures to freshwter orgnisms: Ceriodphni dubi nd Pimephles promels (fthed minnow) A Disserttion Presented to the Grdute School of Clemson University In Prtil Fulfillment of the Requirements for the Degree Doctor of Philosophy Environmentl Toxicology by Ktherine Anne Johnson-Couch My 2018 Accepted by: Dr. Peter vn den Hurk, Committee Chir Dr. Stephen J. Kline Dr. Juli L. Brumghim Dr. Willim C. Bridges, Jr Dr. Thoms Schwedler

3 ABSTRACT Dissolved ions re nturl components of qutic systems, the concentrtion nd composition of which re gretly controlled by the surrounding geologicl mteril present. Although nturlly occurring, mny nthropogenic ctivities, including mountintop removl mining, rod deicing prctices, griculturl irrigtion nd col-fired power plnt effluents, cn gretly increse dissolved ion concentrtions, s well s lter the ionic composition of freshwter systems. An increse in dissolved ions is positively correlted with n increse in slinity nd conductivity. Dissolved ions hve mny importnt physiologicl functions within qutic orgnisms, one of which is to crete electrochemicl grdients within cells. These grdients re necessry for controlling wter nd ion movement within the orgnism. To estblish these electrochemicl grdients, freshwter orgnisms must blnce osmotic gin nd pssive ion loss by utilizing ctive trnsport through series of pumps nd trnsporters locted t the gill. If the externl environment exceeds threshold tolerble for freshwter orgnisms, they my rellocte more energy for ionoregultion, ultimtely reducing the energy vilble for growth, reproduction nd even survivl. The overll gol of this reserch, therefore, ws to chrcterize the chronic toxicity of single ions nd ion mixtures to two freshwter orgnisms, nd to investigte the key mechnisms by which they exert these chronic effects. Reproductive effects of elevted dissolved ions were initilly evluted in Ceriodphni dubi, smll cldocern freshwter invertebrte. Following eight-dy sttic renewl biossys, divlent ions (clcium, mgnesium, sulfte) were determined to ii

4 hve the gretest effect on C. dubi reproduction, while monovlent ions (sodium, chloride, bicrbonte) produced the lest. Additionlly, binry ion mixtures resulted in dditive, less-thn-dditive, nd greter-thn-dditive responses, depending on the specific ion mixtures. Seven-dy sttic renewl biossys were lso performed utilizing Pimephles promels (fthed minnow), smll vertebrte species. Similr to reproductive effects described for C. dubi, divlent ions resulted in the lrgest reduction in growth compred to monovlent ions. Furthermore, P. promels demonstrted mostly dditive nd less-thn-dditive effects following binry ion mixture exposures, with sulfte reducing chloride toxicity, nd clcium reducing mgnesium toxicity. A positive correltion between chloride, clcium, mgnesium, nd sodium EC 50 vlues between C. dubi reproduction nd P. promels growth my indicte similr dissolved ion potencies between n invertebrte nd vertebrte species. Although some mixture interction differences were identified, similrities in concentrtion-response slopes between the two orgnisms my indicte similr toxicologicl modes-of-ction. These similrities suggest tht C. dubi reproduction my be useful predictor of P. promels growth. These results will be useful in the development of future predictive models, which cn id in estblishing site-specific wter qulity criteri. The inclusion of physiologiclly-bsed prmeter; however, would gretly improve the ccurcy nd predictbility of such models. For this purpose, it is impertive to identify the underlying modes-of-ction for these dissolved ions. In the present study, the toxicity of ion interctions were identified by compring the slopes of concentrtion-response curves. Concentrtion-response curves exhibiting iii

5 similr slopes possibly indicte similr modes-of-ction between contminnts. It hs been suggested tht effects of elevted dissolved ions on freshwter orgnisms my be ttributed to the relloction of energy for ionoregultory purposes. If the ion concentrtion in the externl environment exceeds tolerble threshold, freshwter fish my utilize more energy in the synthesis nd use of ionoregultory essentil enzymes, including ATPses nd crbonic nhydrse. As such, it would be expected tht n increse in enzymtic ctivity would occur, which would reduce the energy vilble for growth nd reproduction. Totl ATPse nd crbonic nhydrse ctivity were mesured in dult P. promels gill tissue following 3- nd 7-dy single ion nd multi-ion exposures, s well s 7-dy post-exposure recovery period. Sodium bicrbonte significntly reduced crbonic nhydrse ctivity, while sodium nd chloride significntly incresed totl ATPse ctivity. Significnt effects on totl ATPse nd crbonic nhydrse ctivity in the gills of P. promels did not result from sodium sulfte exposures. This ws somewht expected, s the fish gill is impermeble to divlent nions, such s sulfte. Insted, ltertions in enzymtic ctivity long the intestinl trct my occur, where sulfte is known to increse the permebility of sodium nd chloride. This possibility presents need for identifying chnges in enzymtic ctivity for other ionoregultory importnt tissues, such s the intestines nd even kidney. The results of this reserch not only dd to the limited dtset regrding the chronic toxicity of elevted dissolved ions to freshwter orgnisms, but lso further demonstrte the complex nture of ion toxicity. Differences between previously described cute ion toxicity, nd the chronic toxicity estblished by the present study, iv

6 indicte tht sub-lethl effects cnnot be simply explined by their effects on survivl. Although cute events, such s fish kills, re very brupt nd esily identified, chronic events should not be discounted. Reproduction nd growth re importnt spects tht contribute to orgnisml fitness, nd ultimtely ffect the success of n ecosystem. Chnges in these spects cn disrupt ecosystem processes over time, nd re oftentimes too subtle to tke erly corrective ctions. For this reson, estblishing wter qulity guidelines for elevted dissolved ions bsed on sub-lethl effects is criticl. Overll, these results provide essentil informtion tht cn help mnge wter qulity by serving s the foundtion for the development of future predictive models. v

7 DEDICATION I would like to dedicte this work to Dr. Stephen J. Kline ( ) who, through his unending guidnce, support nd pssion, helped shpe me into the scientist I m tody. vi

8 ACKNOWLEDGMENTS First nd foremost, I would like to cknowledge my dviser, Dr. Stephen J. Kline, who llowed me the privilege of studying under his guidnce for five yers. His eternl optimism nd contgious pssion for ll things science will follow me in my future endevors. And lthough he is not here tody to see this work completed, his cretive vision will live on in these pges. With tht being sid, I cnnot thnk Dr. Peter vn den Hurk enough for ccepting four very high-strung ldies into his lb, myself included. Not only did he step up to the plte nd ccept us without hesittion, he lso mde the trnsition into his lb s esy s possible. He ws n incredible support system when times were hrd, when experiments filed, nd when procedures refused to work. He believed in me, even when it ws hrd to believe in myself, nd for tht I will be forever thnkful. I would lso like to cknowledge my committee members, Dr. Juli Brumghim, Dr. Thoms Schwedler, nd Dr. Willim Bridges, for ll of their hrd work nd dediction towrds this project. To Dr. Juli Brumghim who reminded me to step bck nd exmine the big picture, nd to tke credit where credit is due. To Dr. Thoms Schwedler for providing me with the tools necessry to estblish successful fish colony, nd for ssisting in their cre. To Dr. Willim Bridges, who spent mny hours helping me nlyze dtset fter dtset, then only hving to help me re-nlyze the sme dtsets. Our coffee shop meetings with Steve will lwys hold specil plce in hert. Specil thnk to John Smink for helping me become very skilled fthed minnow colony mnger nd for being friend throughout my time t Clemson. Thnk vii

9 you to Norm Ellis for being such gret friend over the yers, for going bove nd beyond wht ws required to help mke this project success. Thnk you so much, Ron Gossett, for lwys being vilble to help with fish culture, nd for keeping ENTOX flot these pst few months. I cn t thnk Dr. Holly Zhner enough for her lb notebooks mde of gold nd Dr. Mrtin Grosell of University of Mimi enough for nswering ll of my questions regrding the enzyme ctivity procedures. Thnk you to both Ron Dumis nd Rob Silv with Hnn Instruments for being so quick to help me with my ph probe issues, nd keeping my smple nlysis schedule on time. A specil thnk you to Dr. Joe Bisesi for lending me his homogenizer for much longer thn plnned. It ws life sver. I would lso like to cknowledge nd thnk the Electric Power Reserch Institute nd Mr. John Goodrich-Mhoney for providing me with the support nd funding necessry to complete this project. I m incredibly grteful for their understnding nd id following Steve s pssing. This project would hve been impossible without the unending support of my fellow Klinics. A specil thnks to Dr. Austin Wry for showing me the ins nd outs of ENTOX, to Dr. Eric Linrd for providing shoulder to len on, nd to Drs. Srh Au nd Json Corl for mking light from even the drkest of hours. I would lso like to cknowledge Luren Grci-Chnce for opening up her home to me, nd for letting me become prt of her fmily this pst yer. I cnnot put into words how thnkful I m for my prents, Slly nd Glenn Johnson. For being my biggest support system nd cheerleders during my pst seven viii

10 yers t Clemson, for teching me not to ccept mediocre life, to fulfill my drems nd to follow my hert, to grow confidently, but to not forget where I cme from. I m undeserving of them. And finlly, thnk you to my husbnd Lucs Couch, for lifting my spirits during hrd times nd for celebrting with me during the good. Thnk you for helping me to understnd my own self worth, nd for never giving up on me. ix

11 TABLE OF CONTENTS Pge TITLE PAGE... i ABSTRACT... ii DEDICATION... vi ACKNOWLEDGMENTS... vii LIST OF TABLES... xiv LIST OF FIGURES... xvii CHAPTER I. LITERATURE REVIEW... 1 Dissolved Ions... 1 Sources of Dissolved Ion Contmintion... 2 Conductivity versus Ionic Composition... 4 Acute Ion Toxicity... 7 Chronic Ion Toxicity... 9 Ionoregultion in Freshwter Orgnisms Overll Reserch Gol References II. THE CHRONIC TOXICITY OF MAJOR IONS AND ION BINARY MIXTURES TO Ceriodphni dubi Introduction x

12 Tble of Contents (Continued) Pge Mterils nd Methods Ceriodphni dubi Culture Methods Test Solutions Biossy Procedure nd Experimentl Design Chemicl Anlysis Sttisticl Anlysis Results Ion-Only Toxicity Anion Binry Mixture Toxicity Ction Binry Mixture Toxicity Discussion Conclusions Tbles nd Figures References III. THE CHRONIC TOXICITY OF MAJOR IONS AND ION BINARY MIXTURES TO Pimephles promels (fthed minnow) Introduction Mterils nd Methods Pimephles promels Mintennce nd Culture Test Solutions Biossy Procedure nd Experimentl Design Chemicl Anlysis Sttisticl Anlysis Results Ion-Only Toxicity Anion Binry Mixture Toxicity Ction Binry Mixture Toxicity Discussion xi

13 Tble of Contents (Continued) Pge Conclusions Tbles nd Figures References IV. THE CHRONIC TOXICITY OF SINGLE IONS AND BINARY ION MIXTURES OF AN INVERTEBRATE SPECIES (C. dubi) AND A VERTEBRATE SPECIES (P. promels): A COMPARISON Species Comprison Future Predictive Modeling Tbles nd Figures References V. THE EFFECT OF MULTI-ION EXPOSURES ON NA + /K + -ATPASE AND CARBONIC ANHYDRASE ACTIVITY AND RECOVERY IN Pimephles promels (fthed minnow) Introduction Mterils nd Methods Pimephles promels Culture Methods Test Solutions Biossy Procedure nd Experimentl Design N + /K + -ATPse Activity Protocol Crbonic Anhydrse Activity Protocol Sttisticl Anlysis Results Crbonic Anhydrse Activity Preliminry Results: N + /K + -ATPse Activity Totl ATPse Activity Discussion xii

14 Tble of Contents (Continued) Pge Conclusions Tbles nd Figures References VI. CONCLUSIONS xiii

15 LIST OF TABLES Tble Pge 1.1 The chnge in conductivity nd specific ion concentrtion from n un-mined to mined site Wter qulity chrcteristics nd specific ion concentrtion of three sites impcted by mining opertions EC 50 vlues for C. dubi reproduction estimted from conductivity nd specific ion concentrtion Sttisticl nlysis of C. dubi nion binry mixture toxicity Sttisticl nlysis of C. dubi ction binry mixture toxicity EC 50 vlues for growth nd LC 50 vlues for survivl in P. promels The effect of sulfte nd chloride on bicrbonte toxicity to P. promels The effect of bicrbonte nd sulfte on chloride toxicity to P. promels The effect of chloride nd bicrbonte on sulfte toxicity to P. promels The effect of mgnesium nd sodium on clcium toxicity to P. promels The effect of sodium nd clcium on mgnesium toxicity to P. promels The effect of clcium nd mgnesium on sodium toxicity to P. promels Ion-only slope comprison for growth nd survivl of P. promels A summry of the chronic toxicity of ion-only exposures to C. dubi nd P. promels A summry of the chronic toxicity of binry nion mixtures to C. dubi nd P. promels A summry of the chronic toxicity of binry ction mixtures to C. dubi nd P. promels xiv

16 List of Tbles (Continued) Tble Pge 5.1 p vlues for chnges in crbonic nhydrse ctivity following chloride-only exposures p vlues for chnges in crbonic nhydrse ctivity following sulfte-only exposures p vlues for chnges in crbonic nhydrse ctivity following bicrbonteonly exposures p vlues for chnges in crbonic nhydrse ctivity following sulfte:bicrbonte mixture exposures p vlues for chnges in crbonic nhydrse ctivity following chloride:bicrbonte mixture exposures p vlues for chnges in crbonic nhydrse ctivity following chloride:sulfte mixture exposures p vlues for crbonic nhydrse response between tretments following chloride-only exposures p vlues for crbonic nhydrse response between tretments following sulfte-only exposures p vlues for crbonic nhydrse response between tretments following bicrbonte-only exposures p vlues for chnges in crbonic nhydrse ctivity following sulfte:bicrbonte mixture exposures p vlues for chnges in crbonic nhydrse ctivity following chloride:bicrbonte mixture exposures p vlues for chnges in crbonic nhydrse ctivity following chloride:sulfte mixture exposures p vlues for chnges in totl ATPse ctivity following chloride-only exposures xv

17 List of Tbles (Continued) Tble Pge 5.14 p vlues for chnges in totl ATPse ctivity following sulfte-only exposures p vlues for chnges in totl ATPse ctivity following bicrbonte-only exposures p vlues for chnges in totl ATPse ctivity following sulfte:bicrbonte mixture exposures p vlues for chnges in totl ATPse ctivity following chloride:bicrbonte mixture exposures p vlues for chnges in totl ATPse ctivity following chloride:sulfte mixture exposures p vlues for totl ATPse response between tretments following chlorideonly exposures p vlues for totl ATPse response between tretments following sulfte-only exposures p vlues for chnges in totl ATPse ctivity following bicrbonte-only exposures p vlues for chnges in totl ATPse ctivity following sulfte:bicrbonte mixture exposures p vlues for chnges in totl ATPse ctivity following chloride:bicrbonte mixture exposures p vlues for chnges in totl ATPse ctivity following chloride:sulfte mixture exposures xvi

18 LIST OF FIGURES Figure Pge 1.1 A schemtic of freshwter teleost gill A grphicl representtion of the titrtion experimentl design Slope nlysis pproch to determine contminnt mixture interctions Averge percent reproduction for C. dubi exposed to incresing concentrtions of bicrbonte, sulfte, nd chloride (s sodium slts) Averge percent reproduction for C. dubi exposed to incresing concentrtions of clcium, mgnesium, nd sodium (s chloride slts) The effect of chloride with fixed sulfte (7.84 mm) nd fixed bicrbonte (11.2 mm) on C. dubi reproduction The effect of sulfte with fixed chloride (9.81 mm) nd fixed bicrbonte (11.2 mm) on C. dubi reproduction The effect of bicrbonte with fixed sulfte (7.84 mm) nd fixed chloride (9.81 mm) on C. dubi reproduction The effect of mgnesium with fixed clcium (3.45 mm) nd fixed sodium (30.2 mm) on C. dubi reproduction The effect of clcium with fixed mgnesium (4.67 mm) nd fixed sodium (13.2 mm) on C. dubi reproduction The effect of sodium with fixed clcium (3.26 mm) nd fixed mgnesium (4.27 mm) on C. dubi reproduction A grphicl representtion of the titrtion experimentl design Slope nlysis pproch to determine contminnt mixture interctions The effect of bicrbonte, chloride, nd sulfte on P. promels growth The effect of bicrbonte, chloride, nd sulfte on P. promels survivl The effect of clcium, mgnesium, nd sodium on P. promels growth xvii

19 List of Figures (Continued) Figure Pge 3.6 The effect of clcium, mgnesium, nd sodium on P. promels survivl The effect of chloride nd sulfte on bicrbonte toxicity on P. promels growth The effect of bicrbonte nd sulfte on chloride toxicity on P. promels growth The effect of chloride nd bicrbonte on sulfte toxicity on P. promels growth The effect of chloride nd sulfte on bicrbonte toxicity on P. promels survivl The effect of bicrbonte nd sulfte on chloride toxicity on P. promels survivl The effect of chloride nd bicrbonte on sulfte toxicity on P. promels survivl The effect of mgnesium nd sodium on clcium toxicity on P. promels growth The effect of sodium nd clcium on mgnesium toxicity on P. promels growth The effect of clcium nd mgnesium on sodium toxicity on P. promels growth The effect of mgnesium nd sodium on clcium toxicity on P. promels survivl The effect of sodium nd clcium on mgnesium toxicity on P. promels growth The effect of clcium nd mgnesium on sodium toxicity on P. promels growth The correltion of EC 50 vlues estimted for C. dubi reproduction nd P. promels growth xviii

20 List of Figures (Continued) Figure Pge 4.2 The correltion of EC 50 vlues estimted for C. dubi reproduction nd P. promels growth seprted by exposure type The correltion of EC 50 vlues estimted for C. dubi reproduction nd P. promels growth seprted by binry mixture type The correltion of the concentrtion-response slopes for C. dubi reproduction nd P. promels growth The correltion of the concentrtion-response slopes for C. dubi reproduction nd P. promels growth seprted by exposure type The correltion of the concentrtion-response slopes for C. dubi reproduction nd P. promels growth seprted by binry mixture type The correltion of EC 50 vlues estimted for C. dubi reproduction nd P. promels growth Crbonic nhydrse ctivity in P. promels gill tissue following chloride-only exposures Chnges in crbonic nhydrse ctivity in P. promels gill tissue following chloride-only exposures Crbonic nhydrse ctivity in P. promels gill tissue following sulfte-only exposures Chnges in crbonic nhydrse ctivity in P. promels gill tissue following sulfte-only exposures Crbonic nhydrse ctivity in P. promels gill tissue following bicrbonteonly exposures Chnges in crbonic nhydrse ctivity in P. promels gill tissue following bicrbonte-only exposures xix

21 List of Figures (Continued) Figure Pge 5.7 Crbonic nhydrse ctivity in P. promels gill tissue following sulfte:bicrbonte mixture exposures Chnges in crbonic nhydrse ctivity in P. promels gill tissue following sulfte:bicrbonte mixture exposures Crbonic nhydrse ctivity in P. promels gill tissue following chloride:bicrbonte mixture exposures Chnges in crbonic nhydrse ctivity in P. promels gill tissue following chloride:bicrbonte mixture exposures Crbonic nhydrse ctivity in P. promels gill tissue following chloride:sulfte mixture exposures Chnges in crbonic nhydrse ctivity in P. promels gill tissue following chloride:sulfte mixture exposures Preliminry Results: The lck of inhibition of N + /K + -ATPse by 1.5 mm oubin in P. promels gill tissue Preliminry Results: The lck of inhibition of N + /K + -ATPse by 13.0 mm oubin in P. promels gill tissue Preliminry Results: The inhibition of N + /K + -ATPse by 157 mm copper in P. promels gill tissue Preliminry Results: The inhibition of N + /K + -ATPse by 1.5 mm oubin in P. heteroclitus gill tissue Preliminry Results: The inhibition of N + /K + -ATPse by 10.0 mm orthovndte in P. promels gill tissue Preliminry Results: The lck of inhibition of N + /K + -ATPse by 6.5 mm digoxin in P. promels gill tissue Totl ATPse ctivity in P. promels gill tissue following chloride-only exposures xx

22 List of Figures (Continued) Figure Pge 5.20 Chnges in totl ATPse ctivity in P. promels gill tissue following chlorideonly exposures Totl ATPse ctivity in P. promels gill tissue following sulfte-only exposures Chnges in totl ATPse ctivity in P. promels gill tissue following sulfteonly exposures Totl ATPse ctivity in P. promels gill tissue following bicrbonte-only exposures Chnges in totl ATPse ctivity in P. promels gill tissue following bicrbonte-only exposures Totl ATPse ctivity in P. promels gill tissue following sulfte:bicrbonte mixture exposures Chnges in totl ATPse ctivity in P. promels gill tissue following sulfte:bicrbonte mixture exposures Totl ATPse ctivity in P. promels gill tissue following chloride:bicrbonte mixture exposures Chnges in totl ATPse ctivity in P. promels gill tissue following chloride:bicrbonte mixture exposures Totl ATPse ctivity in P. promels gill tissue following chloride:sulfte mixture exposures Chnges in totl ATPse ctivity in P. promels gill tissue following chloride:sulfte mixture exposures xxi

23 CHAPTER ONE LITERATURE REVIEW Dissolved Ions Totl Solids, term encompssing dissolved, suspended, nd settleble solids, re further defined by their bility to pss through 2 µm filter. Suspended nd settleble solids re lrge substnces, including silt, lge, nd plnkton, nd s such cnnot pss through this defined filter. Dissolved solids, which cn pss through 2 µm filter, re lso commonly referred to s dissolved ions. These ions cn either be defined s ctions, those ions possessing positive chrge including sodium (N + ), clcium (C 2+ ), mgnesium (Mg 2+ ), nd potssium (K + ), or s nions, those possessing negtive chrge such s chloride (Cl - ), sulfte (SO 2 4 ), crbontes (HCO - 3, CO 2-3 ), nd bromide (Br - ) (US EPA, 1997). The surrounding terrestril system gretly controls the dissolved ion concentrtions nd ionic composition of freshwter rivers nd strems due to the mkeup of the geologicl mteril present (Sigee, 2005). The chemicl mkeup of rocks nd soils is the gretest contributor to freshwter dissolved ions. Other indirect methods controlling dissolved ion concentrtions nd ionic composition include rinfll nd nthropogenic ctivities (Sigee, 2005). In North Americ, cid volcnic rock hs the highest percent totl re (53%), while the rest is comprised of crbonte rocks (22.4%), shles (18.4%), sndstones (7.3%), shield rocks (19.7%) nd bslt (8.4%)(Suchet et l., 2003). Acidic volcnic rocks, or cid igneous rocks, re comprised mostly of silic (i.e. qurtz) nd do little to 1

24 contribute to ionic mkeup of freshwter systems. In fct, most soft wters, those comprised of little clcium or mgnesium, re found in res with high cidic igneous rock. Sndstones nd crbonte rocks hve greter contributions to overll ion concentrtion within surrounding freshwter systems (Thorp nd Covich, 2010). There re mny methods for mesuring dissolved ion concentrtions within qutic systems. Some mesurements include slinity (ppt), Totl Dissolved Solids (mg/l) nd conductivity (µs/cm). Slinity is simply the concentrtion of dissolved slts in wter, nd is typiclly <0.5ppt for freshwter systems, compred to 30ppt of sltwter (US EPA, 2006). Totl Dissolved Solids (TDS) is the weight in milligrms of ll ions tht cn ps through 2 µm filter in known volume of wter. For freshwter systems, TDS is typiclly 0-1,000 mg/l for freshwter, but cn be s high s 35,000 mg/l in sltwter (Brlow, 2004). Due to the electricl chrges produced by ech ion, wter is lso ble to pss n electric current tht is mesured s conductivity. For freshwter systems, conductivity is typiclly between 0 nd 1,300 µs/cm, while sltwter cn be s high s 29,000 µs/cm (Li nd Migliccio, 2011). Simply put, s the ion concentrtion of wter source increses, so does the slinity, TDS nd conductivity. One issue with utilizing these forms of mesurements is tht they re ll non-specific. So, lthough these mesurements re good representtion of the overll ion concentrtion, they do not indicte which ions re present nd t specific concentrtions. Sources of Dissolved Ion Contmintion Low ion concentrtions re primrily chrcteristic of freshwter systems. Mny nthropogenic ctivities, however, cn contribute to elevted dissolved ions ultimtely 2

25 incresing the slinity of these freshwter systems. For exmple, to lower the freezing point of ice during snowstorms, mny sttes will pply thin lyer of brine or highly sline wters to rod surfces. Consequently, the residul wter nd slt remining once the snow melts will eventully runoff from rodwys nd into nerby qutic systems. In the United Sttes, it hs been estimted tht nerly 18.5 million tons of rod slt used for deicing purposes ws pplied to rod surfces in 2003 lone (Kelly nd Mtos, 2013). Corsi et l. (2010) smpled 13 freshwter strems in Milwukee, Wisconsin nd found conductivity vlues s high s 30,800 µs/cm, nd chloride concentrtions of 11,200 mg/l. At 55% of the sites tested, chloride concentrtions exceeded the U.S. EPA cute wter qulity criteri (860 mg/l), while 100% of sites surpssed chronic criteri (230 mg/l)(corsi et l., 2010). Mining opertions hve lso been reported to increse dissolved ion concentrtions of freshwter strems, s well s modify the ionic composition. One prctice in prticulr involves mountintop removl mining. This form of mining utilizes explosives to remove surfce soils, nd expose col sems. The excess soil nd rock, lso referred to s overburden, is then plced djcent to the mountin in vlley fills. The mine dringe tht results from these vlley fills cn ultimtely impct nerby smll freshwter strems (Griffith et l., 2012). Pond et l. (2008) reported significnt increses in conductivity nd dissolved ion concentrtions between sites impcted by mountintop removl effluents (n=27) nd unmined strems (n=10). More specificlly, reference sites hd men conductivity of 62 µs/cm, which ws significntly higher thn the conductivity recorded for impcted sites (1023 µs/cm). Contributing to the 3

26 significnt increse in conductivity ws significnt increse bicrbonte, clcium, chloride, sodium, mgnesium, nd sulfte concentrtions (Pond et l., 2008)(Tble 1.1). Tble 1.1. The chnge in conductivity nd specific ion concentrtion from n un-mined to mined site (Pond et l., 2008). Vlues indicte men mesurements, while prentheticls indicte the rnge. p vlues indicte significnt differences between the un-mined nd the mined sites. Un-Mined Mined p Conductivity (μs/cm) 62 (34 133) 1023 ( ) HCO (6.1-35) 183 ( ) C (2.7 12) (38 269) Cl (< 2.5 4) 4.6 (< ) Mg (2.3 7) (28 284) N ( ) 12.6 (2.6 39) SO 4 16 ( ) ( ) *All vlues represent dt collected nd sttisticl nlysis performed by Pond et l., Studies hve demonstrted not only chnges in wter qulity conditions, but lso severe biologicl impirment ssocited with mountintop removl mining (Pond et l., 2008; Timpno et l., 2010; Pond et l., 2014). Further studies hve documented more specific effects of individul orgnisms, such s reproduction nd growth, due to elevted dissolved ions nd conductivity of impcted freshwter systems (Dwyer et l., 1992; Dickerson et l., 1996; Kennedy et l., 2005; Corsi et l., 2010; Kunz et l., 2013). These chnges cn ultimtely led to chnge in community structure. Conductivity versus Ionic Composition The U.S. EPA hs mde gret efforts in developing regultory criteri for dissolved ions bsed on conductivity due to the strong positive correltion between conductivity nd ionic concentrtion, s well s its ese of mesurement in environmentl settings. Becuse ech ion produces its own current, depending on the chrge nd 4

27 electron mobility, it is necessry to understnd the specific ions tht re present in wter body (U.S. EPA, 2011). As result, wter qulity criteri bsed on conductivity must be mde on site-specific bsis. Currently, Benchmrk for Conductivity hs been estblished for the Applchin Region, site tht is hevily impcted by mountintop removl mining prctices. The dominnt ions tht mke up this region include clcium, mgnesium, sulfte nd bicrbonte, nd these ions were tken into ccount in the development of this benchmrk. Typiclly, toxicity is reported in either Lethl (LC) or Effective (EC) Concentrtions. These vlues indicte toxicity bsed on the mortlity of orgnisms exposed to contminnt, or the sub-lethl effects such s reproduction nd growth. However, in the development of the conductivity benchmrk, Extirption Concentrtions (XCs) were utilized. The Extirption Concentrtion ws defined s the conductivity vlue below which 95% of the observtion of the genus occur. This correltes with 5% mortlity rte (LC 5 ), or disppernce of prticulr genus (U.S. EPA, 2011). Although this is very conservtive pproch in generting wter qulity criteri, previous reserch hs suggested tht s vlue devites from the medin lethl or effect concentrtion (LC 50, or EC 50 ), the confidence intervls surrounding those predictions increse significntly, ultimtely effecting the bility to derive ccurte vlues (Vn Gestle nd Hensbergen, 1997; Shw et l., 2006). Additionlly, conductivity is n effective estimte for totl dissolved ion concentrtion; however, it does not specify the individul ions present within solution. As such, conductivity-bsed toxicity mesures would ssume tht ll ions produce the 5

28 sme toxic response. Mny studies, however, hve suggested tht the chemicl mkeup of solution is extremely importnt in identifying the overll solution toxicity. For exmple, Kunz et l. (2013) smpled three freshwter strems tht were impcted by mining effluents in West Virgini. Two of these strems, identified s Winding Shols nd Bordtree, were representtive of sites impcted by n lkline mine dringe, while the ionic composition of the third strem (Upper Dempsey) ws chrcteristic of neutrlized, or chemiclly treted, mine dringe. Although the ionic compositions for the three sites were different, the conductivities reported were similr (1,800 2,100 µs/cm). Winding Shols nd Bordtree were comprised mostly of mgnesium, clcium, nd sulfte, while sodium nd bicrbonte were dominte in the Upper Dempsey site. Acute toxicity biossys were performed to determine the mortlity ssocited with ech site, using reconstituted wters simulting ech individul site. Of the three sites, the Upper Dempsey ws the only one to result in significnt reduction in Ceriodphni dubi (C. dubi) survivl. These results suggest tht toxicity is strongly dependent on the ctul ions present, nd not necessrily how mny (Tble 1.2). These results corroborte previous reserch tht indictes mesurements such s TDS or conductivity re not idel surrogtes for specific ion concentrtions in evluting ion toxicity (Kennedy et l., 2005; Mount et l., 1997; Soucek et l., 2011; Kunz et l., 2013). 6

29 Tble 1.2 Wter qulity chrcteristics nd specific ion concentrtion of three sites impcted by mining opertions (Kunz et l., 2013). All ion concentrtions re represented in mg/l. Winding Shols Bordtree Upper Dempsey Conductivity (µs/cm) Mjor Ion Concentrtion (mg/l) N Mg C K SO HCO3- (s mg/l CCO3) Acute Ion Toxicity Mny sources of elevted dissolved ions in freshwter systems occur quickly, nd t very high concentrtions. For exmple, griculturl irrigtion drin wters in Nevd hve been reported to exhibit conductivities s high s 30,000 µs/cm, conductivity chrcteristic of sewter. Exposure to such high ion concentrtions, even for short durtions, typiclly results in mortlity of freshwter species. As such, gret efforts hve been mde to scertin the cute toxicity of these dissolved ions. One of the lrgest studies ws performed by Mount et l. (1997), which investigted the cute toxicity of over 2,900 ions nd ion combintions to three freshwter species. The purpose of this reserch ws to not only to gin better understnding of mjor ion toxicity, but to lso use these results to generte predictive models for survivl. Following 48-hour Ceriodphni dubi nd Dphni mgn nd 7

30 96-hour Pimephles promels toxicity biossys, the reltive order of toxicity for dissolved ions ws determined to be K + > HCO - 3 Mg 2+ > Cl - > SO 2-4, with the toxicity of N + nd C 2+ being insignificnt. These results suggested tht nions produced the lrgest toxic response, wheres ction toxicity ws mostly ttributed to the ssocited nion. However, subsequent studies indicte the potentil for C 2+ to influence N + nd Mg 2+ toxicity, s well s N + to control K + toxicity (Mount et l., 2016). The role of ech ion to contribute to the overll toxicity of ion mixture solutions is still complex; however, significnt efforts hve been mde to fully understnd the significnt effects of ech ion. Doing so is impertive in not only building predictive models, but lso setting pproprite wter qulity criteri. Additionl studies hve been conducted to help differentite the interctions between ions nd ion mixtures for this purpose. Soucek nd Kennedy (2005) demonstrted tht chloride decresed sulfte toxicity to Hyllel ztec, smll freshwter invertebrte. More specificlly, they noted tht the protective effect of chloride did not occur in lower concentrtions (5 mg/l nd 13 mg/l Cl - ), but insted t the higher concentrtions (18 mg/l, nd 36 mg/l Cl - ). Interestingly, sulfte did not seem to hve the sme meliortive effect on chloride in studies utilizing C. dubi (Soucek et l., 2011). This suggests tht different freshwter species do not respond similrly to elevted dissolved ions. Subsequent studies hve exmined this phenomenon further: Wng et l. (2016) demonstrted tht increses in chloride concentrtions (10 mg/l to 25 mg/l Cl - ) did not ffect sulfte toxicity to lrvl P. promels, vertebrte fish species; however, smll ltertions in potssium concentrtion (1 mg/l to 3 mg/l K + ) significntly reduced sulfte toxicity. 8

31 Another importnt fctor to note is tht the hrdness (due to mg/l CCO 3 ) of n exposure medium hs been demonstrted to ply n importnt role in reducing the toxicity of elevted dissolved ions (Dwyer et l., 1992; Kennedy et l., 2003; Soucek nd Kennedy, 2005; Soucek et l., 2011). Mount et l. (1997) ws ble to tke this ide step further, nd suggested tht it my not necessrily be hrdness, but insted the number of ctions in solution tht provide this protective effect. For exmple, when NCl nd CCl 2 were tested independently, the EC50 vlues estimted for Cl - ws nerly identicl. Although these solutions hd similr toxicities the hrdness vried gretly, where the hrdness of the CCl 2 solution ws much higher thn NCl. Furthermore, when NCl nd CCl 2 were tested in combintion, the resulting Cl - toxicity ws significntly reduced from tht of ech slt independently. The resons behind this meliortive effect re still lrgely unknown. It hs been hypothesized tht perhps clcium reduces gill permebility nd tightens junctions, reducing the pssive diffusion of ions nd wter into the orgnism (Potts nd Fleming, 1970; Soucek nd Kennedy, 2005). Chronic Ion Toxicity Due to heightened federl regultions, mny dischrgers re implementing new strtegies to reduce the high ion content of effluents. Although there hve been significnt reductions in conductivity, nd s result dissolved ion concentrtions, reported conductivities re still bove wht is typicl of freshwter systems. Conductivities nd TDS mesurements, ssocited with lower dissolved ion concentrtions, hve been reported to decrese reproduction nd growth in freshwter species (Jop nd Askew, 1994; Kennedy et l., 2005; Chpmn et l., 2010; Lsier nd 9

32 Hrdin, 2010; Armsted et l., 2016). Decreses in reproduction nd growth re typicl sub-lethl responses of chronic, or long-term, exposures to contminnts. Although the effects of chronic exposures re not immeditely pprent, unlike mss mortlity events ssocited with cute exposures, their consequences within ecosystems should not be discounted. Chnges in orgnisml helth nd performnce cn reduce popultion growth nd fitness, fctors tht gretly impct the success of ecosystems. Due to the problems ssocited with chronic exposures, it is impertive to understnd the sub-lethl toxicity ssocited with elevted dissolved ions, not only the cute toxicity. The mjority of chronic toxicity studies hve focused on the effects of nions specificlly, due to cute toxicity results tht suggest nions re mjor contributors to toxic effects. Initilly, Lsier nd Hrdin (2010) focused on the reproductive effects of these three ions s single components nd in mixtures on C. dubi. Effective concentrtions for 50% decrese in reproduction (EC 50 vlues) for chloride (653 mg/l), sulfte (1,252 mg/l) nd bicrbonte (725 mg/l) were reported. Similr vlues for C. dubi reproduction hve been reported for chloride (697 mg/l), nd sulfte (1,267 mg/l)(elphick et l., 2011; Elphick et l., 2011b). Frg nd Hrper (2014) reported n EC 20 (effective concentrtion tht reduced reproduction by 20%) for bicrbonte s 274 mg/l, but did not report n EC 50 vlue. A few other studies hve reported effective concentrtions for other species, including fthed minnows (P. promels), white suckers (Ctostomus commersoni), mussels (Lmpsilis siliquoide), rinbow trout (Oncorhynchus mykiss), nd midge (Chironomus dilutus)(chpmn et l., 2010; Frg nd Hrper, 2014; Wng et l., 2016). 10

33 Ameliortive effects of wter hrdness hve lso been demonstrted in chronic toxicity studies. Elphick et l. (2011) reported decrese in the EC 50 vlues derived for chloride from 161 mg/l Cl - to 700 mg/l Cl - when hrdness incresed from 10 mg/l CCO 3 to 320 mg/l CCO 3. Similr results were lso reported for sulfte (EC 50 : 465 mg/l SO 2-4 t hrdness of 40 mg/l CCO 3 ; EC 50 : 1257 mg/l SO 2-4 t hrdness of 160 mg/l CCO 3 )(Elphick et l., 2011b). The effects of hrdness on chronic ion toxicity hve not been extensively studied, nd dt hve not been extrpolted to the degree t which cute toxicity hs, including differences in sources of hrdness (Mg 2+ or C 2+ ) or multiple ction effects. Although elucidting the toxicity of single ion is the initil step in understnding differences between ion effects, it is not necessrily environmentlly relevnt. Ions do not occur s single entities within environmentl settings. Insted, ions occur in mixtures, nd the ionic composition of those mixtures cn shift gretly due to nthropogenic ctivities. As such, it would lso be necessry to identify multi-ion toxicities, nd better yet, exmine how the toxicity of one ion cn influence the toxicity of nother. Very few chronic ion mixture studies hve been performed to dte. One such study investigted reproductive effects to C. dubi chloride, sulfte, nd bicrbonte mixtures in three wter types (low hrdness/low lklinity, low hrdness/moderte lklinity, nd moderte hrdness/moderte lklinity)(lsier nd Hrdin, 2010). Concentrtions utilized for ech ion included their respective EC 6, EC 12, nd EC 25 vlues to crete full fctoril experimentl design. The results of this study indicted tht these 11

34 ions rect in n dditive mnner, mening the presence of one ion does not impct the toxicity of the second (Lsier nd Hrdin, 2010). Due to the limited dt regrding the chronic toxicity of dissolved ions, it is impertive tht dditionl work be completed to understnd how these ions cn impct orgnisms on sub-lethl bsis, especilly with regrds to ion mixtures. These results would mke significnt dditions to the development of future wter qulity criteri. Ionoregultion in Freshwter Orgnisms The toxicity of these dissolved ions my be best explined by the ionoregultory cpcity of freshwter orgnisms. The osmolrity, or the mount of solute prticles per liter of solution, in freshwter systems is typiclly <25 mosm, compred to sltwter which roughly 1,000 mosm (Hunter nd Rudy Jr, 1975; Kefford et l., 2016). Aqutic orgnisms, both freshwter nd sltwter, mintin n osmolrity of bout 300 mosm (Pelster, 2008). Becuse freshwter orgnisms mintin n osmolrity much higher thn their surrounding medi, they re considered hypertonic to their externl environment. This mens they must hve strtegic mechnisms to combt the pssive loss of ions nd gin of wter. For this purpose, freshwter orgnisms must expend energy in the form of ctive trnsport systems. It is believed tht this energy is derived from mitochondrionrich (MR) cells, which re equipped to hndle the energy lod needed for ionoregultion (Perry nd Fryer, 1997). In fct, it hs been estimted tht rinbow trout (Oncorhynchus mykiss) utilize 1.5% of their resting metbolic rte for ionoregultory purposes in freshwter, nd this rte decreses to 0.5% when in sewter (Eddy, 1982). Krogh (1938) ws the first to suggest ionic uptke s the mechnism controlling this homeosttic 12

35 blnce. Since then, extensive reserch hs been conducted to investigte the specific mechnisms occurring within the gills of fish, nd how they interct. Following extensive studies utilizing goldfish (Crssius urtus), Krogh (1938) discovered tht sodium nd chloride were tken up independently from one nother. This work lso suggested tht, due to lws of electroneutrlity, these fish must utilize nother set of ions to initite the uptke of sodium nd chloride. It ws suggested tht these ions might be byproducts of CO 2, nmely H + nd HCO - 3, nd lso serve s mens for wste excretion. To this dy, the hypothesis suggested by Krogh (1938) is still believed to be the min mechnisms for ion uptke. Additionl studies hve suggested tht pssive uptke of sodium in exchnge for H + could not hppen due to the lws of thermodynmics, further evidence of the need for energy during ion exchnge nd uptke (Avell nd Bornncin, 1989; Kirschner, 2004). Current freshwter fish gill models denote two distinct MR cell subtypes locted within the gills of fish, often referred to s penut lectin-insensitive (PNA) cells. The first, identified s the cid-secreting cell (PNA-), is believed to be the site for N + /H + exchnge, wheres Cl - /HCO - 3 exchnge occurs t the bse-secreting cell (PNA+)(Figure 1.1)(Mrshll nd Grosell, 2005). These models further demonstrte the presence of V- type H + -ATPse enzyme within the picl membrne of cid-secreting cells, which provides source of energy for N + uptke through picl chnnels. More specificlly, energy (ATP) is initilly required to excrete H + outside the MR cytoplsm. The excretion of H + cretes n electrochemicl grdient, which encourges N + diffusion in the cytoplsm through sodium chnnels (Avell nd Bornncin, 1989). The possibility for 13

36 such n intricte system ws further evidenced by studies tht reveled decrese in sodium uptke during inhibition of the V-type ATPse inhibitor, bfilomycin A (Bury nd Wood, 1999; Fenwick et l., 1999; Perry et l., 2003). Movement of sodium through the bsolterl membrne of MR cells into the plsm of freshwter fish is believed to be primrily by N + /K + -ATPse enzymes. These enzymes function by hydrolyzing one ATP molecule for every 3 N + ions trnsferred externlly nd 2 K + ions moved internlly, reltive to the cell. This provides net movement of 1+ chrge, creting n internl negtive chrge nd n externl positive chrge. These enzymes re locted throughout the body, occurring more prominently in the gills, kidney, intestines, Red Blood Cells (RBCs), nd muscle nd nerve cells (Duhl nd Hokin, 1974). This chrge difference drives the movement of mny mcromolecules, such s glucose nd mino cids, s well other biologiclly importnt molecules, such s oxygen (Thoms nd Egee, 1998). Although extensive reserch suggests tht chloride is most likely exchnged for bicrbonte on the picl membrne of freshwter fish, it is still lrgely unknown how this mechnism properly functions due to electrochemicl grdients (Perry et l., 2003). The presence of V-type ATPse on the bsolterl membrne hs been suggested to provide the electrochemicl grdient needed for this exchnge (Piermrini nd Evns, 2001). Crbonic nhydrse, member of the zinc metlloenzyme fmily, is responsible for providing the bicrbonte ion needed for its 1:1 exchnge with chloride. It does so by ctlyzing the reversible hydrtion nd dehydrtion rections of crbon dioxide, byproduct of respirtion. During the hydrtion process, one ATP molecule is consumed 14

37 to trnsform crbon dioxide into bicrbonte nd hydrogen (H + ), the H + ion being further excreted through the piclly locted V-type H + -ATPse pump. The kidney, s well s the intestine plys supporting role to the gills in ionoregultion nd ion uptke. The kidney in prticulr is importnt in this process for freshwter fish. In fct, pproximtely 95% of sodium chloride is rebsorbed through the glomerulus (Perry et l., 2003). Additionlly, bicrbonte is lso highly regulted t the kidney to help mintin proper blood ph. The exct mechnisms for the rebsorption of N + nd Cl -, s well s the regultion of HCO - 3 within the kidney re still mostly unknown. However, similrities between the gill nd kidney hve been mde in terms of N + /H + interction, N + /K + -ATPse, nd Cl - /HCO - 3 exchnge. Furthermore, evidence of V-type ATPse inititing rebsorption of HCO 3-, similr to the gill, hs been demonstrted in the kidney (Perry nd Fryer, 1997). Due to the rebsorption of nerly 100% of sodium nd chloride, s well s the pssive gin of wter, freshwter orgnisms typiclly produce lrge volumes of extremely dilute urine (Perry et l., 2003). The intestine, lthough not s significnt s the kidney, is lso responsible for the uptke of ions. Clcium uptke is prticulrly importnt long the intestinl tissue of freshwter fish. Euryhline fish re those cpble of moving between freshwter nd sltwter. A 20% increse in the osmolrity of euryhline fish plsm hs been recorded during this trnsition (Bone nd Moore, 2008). Furthermore, the mummichog (Fundulus heteroclitus, nother euryhline species, hs been reported to rpidly increse N + /K + - ATPse ctivity within gill tissue following exposure to sewter. Becuse these fish 15

38 nturlly move between the two environments, they re better dpted to shifts in slinity or increses in dissolved ion concentrtions thn freshwter fish tht re limited by their ionoregultory cpcity. Figure 1.1. A schemtic of freshwter teleost gill. Drk circles represent ATPse pumps, gry cylinders indicte ion chnnels, nd open circles indicte ion exchngers (Mrshll nd Grosell, 2005). 16

39 Overll Reserch Gol The overll gol of this reserch ws to gin better understnding of the chronic toxicity of ions nd multi-ion mixture exposures to freshwter orgnisms. To chieve this gol, I completed four objectives: 1. Chrcterize the chronic toxicity of elevted dissolved ions, both s single ions nd in binry mixtures, to n invertebrte species (Ceriodphni dubi) using reproduction s the endpoint. 2. Chrcterize the chronic toxicity of elevted dissolved ions, both s single ions nd in binry mixtures, to vertebrte species (Pimephles promels) using growth s the endpoint. 3. Compre the effects from C. dubi reproduction to P. promels growth to determine if the toxicity of one species could be useful predictor for nother species. 4. Investigte the mechnism-of-ction for single ions nd ion mixtures by mesuring chnges in enzyme ctivity essentil to ionoregultion. My first nd second objectives were conducted to contribute to the limited dtset describing the chronic toxicity of ions nd ion mixtures to freshwter orgnisms. Next, I conducted comprison of the results obtined from objectives one nd two to compre between the two species tested. More specificlly, I wnted to determine if the toxicity demonstrted by one species could be used to predict the toxicity of the second species, nd to describe the potentil for the development of predictive models bsed on these findings. Lstly, I wnted to gin some understnding of the mechnisms tht contribute 17

40 to ion toxicity within the gill of freshwter fish, nd potentilly provide physiologiclly bsed prmeter tht would be importnt for future predictive model development. 18

41 References Armsted MY, Bitzer-Crethers L, Wilson M The effects of elevted specific conductivity on the chronic toxicity of mining influenced strems using Ceriodphni dubi. PLoS ONE. 11 (11): e Avell M, Bornncin M A new nlysis of mmoni nd sodium trnsport through the gills of the freshwter rinbow trout (Slmo girdneri). Journl of Experimentl Biology. 142: Brlow PM Occurrence nd Flow of Freshwter nd Sltwter in Costl Aquifers in Ground Wter in Freshwter-Sltwter Environments of the Atlntic Cost. U.S. Geologicl Survey, Reston, Virgini, USA. Bone Q, Moore RH Osmoregultion nd Ion blnce in The Biology of Fishes (3 rd edition) pp Tylor nd Frncis Group, New York, NY, USA. Bury NR, Wood CM Mechnism of brnchil picl silver uptke by rinbow trout is vi the proton-coupled N+ chnnel. Americn Journl of Physiology. 277: R1385-R1391. Chpmn PM, Biley H, Cnri E Toxicity of totl dissolved solids ssocited with two mine effluents to chironomid lrve nd erly life stges of rinbow trout. Environmentl Toxicology nd Chemistry. 19: Corsi SR, Grczyk DJ, Geis SW, Booth NL, Richrds KD A fresh look t rod slt: Aqutic toxicity nd wter-qulity impcts on locl, regionl, nd ntionl scles. Environmentl Science nd Technology. 44: Duhl JL, Hokin LE The sodium-potssium denosinetriphosphtse. Annul Review of Biochemistry. 43: Dwyer FJ, Burch SA, Ingersoll CG, Hunn JB Toxicity of trce element nd slinity mixtures to striped bss (Morone sxtilis) nd Dphni mgn. Environmentl Toxicology nd Chemistry. 11: Eddy FB Osmotic nd ionic regultion in cptive fish with prticulr reference to slmonids. Comprtive Biochemistry nd Physiology. 73B: Elphick JRF, Bergh KD, Biley HC Chronic toxicity of chloride to freshwter species: Effects of hrdness nd implictions for wter qulity guidelines. Environmentl Toxicology nd Chemistry. 30:

42 Elphick JR, Dvies M, Gilron G, Cnri EC, Lo B, Biley HC. 2011b. An qutic toxicologicl evlution of sulfte: The cse for considering hrdness s modifying fctor in setting wter qulity guidelines. Environmentl Toxicology nd Chemistry. 30: Frg AM, Hrper DD The chronic toxicity of sodium bicrbonte, mjor component of col bed nturl gs produced wters. Environmentl Toxicology nd Chemistry. 33: Fenwick JC, Wendelr Bong SE, Flik G In vivo bfilomycin-sensitive N+ uptke in young freshwter fish. Journl of Experimentl Biology. 202: Griffith MB, Norton SB, Alexnder LC, Pollrd AI, LeDuc SD The effects of mountintop mines nd vlley fills on the physiochemicl qulity of stem ecosystems in the centrl Applchins: A review. Science of the Totl Environment : Hunter KC, Rudy Jr RP Osmotic nd ionic regultion in the Dungeness crb, Cncer mgister dn. Comprtive Biochemistry nd Physiology Prt A: Physiology. 51: Jop KM, Askew AM Toxicity identifiction evlution using short-term chronic test with Ceriodphni dubi. Bulletin of Environmentl Contmintion nd Toxicology. 53: Kefford BJ, Buchwlter D, Cnedo-Arguelles M, Dvis J, Duncn RP, Hoffmnn A, Thompson R Slinized rivers: degrded systems or new hbitts for slt-tolernt funs? Biology Letters. 12: Kelly TD, Mtos GR Historicl sttistics for minerl nd mteril commodities in the United Sttes. Report No.: 140. U.S. Geologicl Survey, Reston, Virgini, USA. Kennedy AJ, Cherry DS, Currie RJ Field nd lbortory ssessment of colprocessing effluent in the Leding Creek Wtershed, Meigs Co., Ohio. Archives of Environmentl Contmintion nd Toxicology. 44: Kirschner LB The mechnism of sodium chloride uptke in hyperregulting qutic nimls. Journl of Experimentl Biology. 207: Kunz JL, Conley JM, Buchwlter DB, Norberg-King TJ, Kemble NE, Wng N, Ingersoll CG Use of reconstituted wters to evlute effects of elevted mjor ions ssocited with mountintop col mining on freshwter invertebrtes. Environmentl Toxicology nd Chemistry. 32:

43 Lsier PJ, Hrdin IR Observed nd predicted reproduction of Ceriodphni dubi exposed to chloride, sulfte, nd bicrbonte. Environmentl Toxicology nd Chemistry. 29: Li Y, Migliccio K Wter Qulity Stndrds: Designted Uses nd Numeric Criteri Development in Wter Qulity Concepts, Smpling, nd Anlyses. Tylor nd Frncis Group, LLC, Boc Rton, FL, USA. Mrshll W, Grosell M Ion trnsport, osmoregultion, nd cid-bse blnce. In The Physiology nd Fishes. pg Pelster, B Bouyncy in Evns DH(ed) The Physiology of Fishes Second Edition pp CRC Press LLC, Boc Rton, Florid, USA. Perry SF, Shhsvrni A, Georglis T, By M, Furimsky M, Thoms SLY Chnnels, pumps, nd exchngers in the gill nd kidney of freshwter fishes: Their role in ionic nd cid-bse regultion. Journl of Experimentl Zoology. 300A: Piermrini PM, Evns DH Immunochemicl nlysis of the vcuolr proton- ATPse B-subunit in the gills of the euryhline stingry (Dsytis Sbin): effects of slinity nd reltion to N+/K+-ATPse. Journl of Experimentl Biology. 204: Pond GJ, Pssmore ME, Borsuk FA, Reynold L, Rose CJ Downstrem effects of mountintop col mining compring biologicl conditions using fmily- nd genus-level mcroinvertebrte biossessment tools. Journl of North Americn Benthologicl Society. 27: Pond GJ, Pssmore ME, Pointon ND, Felbinger JK, Wlker CA, Krock KJG, Fulton JB, Nsh WL Long-term impcts on mcroinvertebrtes downstrem of reclimed mountintop mining vlley fills in centrl Applchi. Environmentl Mngement. 54: Potts WTW, Fleming WR The effects of prolctin nd divlent ions on the permebility to wter of Fundulus kinse. Journl of Experimentl Biology. 53: Shw JR, Dempsey TD, Chen CY, Hmilton JW, Folt CL Comprtive toxicity of cdmium, zinc, nd mixtures of cdmium nd zinc to Dphnids. Environmentl Toxicology nd Chemistry. 25: Sigee DC Inorgnic nutrients: Uptke nd cycling in freshwter systems in Freshwter Microbiology: Biodiversity nd Dynmic Interctions of Microorgnisms in the Aqutic Environment. John Wiley & Sons Ltd, West Sussex, London. 21

44 Soucek DJ, Kennedy AJ Effects of hrdness, chloride, nd cclimtion on the cute toxicity of sulfte to freshwter invertebrtes. Environmentl Toxicology nd Chemistry. 24: Soucek DJ, Linton TK, Trr CD, Dickinson A, Wickrmnyke N, Delos CG, Cruz LA Influence of wter hrdness nd sulfte on the cute toxicity of chloride to sensitive freshwter invertebrtes. Environmentl Toxicology nd Chemistry. 30: Suchet PA, Probst JL, Ludwig W Worldwide distribution of continentl rock lithology: Implictions for the tomospheric/soil CO2 uptke by continentl wethering nd lklinity river trnsport to the ocens. Globl Biogeochemicl Cycles. 17: Thoms S, Egee S Fish red blood cells: Chrcteristic nd biologicl role of the membrne ion trnsporters. Comprtive Biochemistry nd Physiology. 19: Thorp JH, Covich AP An overview of inlnd qutic hbitts in Ecology nd Clssifiction of North Americn Freshwter Invertebrtes Third Edition. Elsevier, Sn Diego, CA, USA. Timpno AJ, Schoenholtz SH, Zipper CE, Soucek DJ Isolting effects of totl dissolved solids on qutic life in centrl Applchin colfield strems. Proc Am Soc Mining Reclm. 27: U.S. Environmentl Protection Agency Volunteer Strem Monitoring: A Methods Mnul. EPA-841-B Wshington, DC, USA. U.S. Environmentl Protection Agency Voluntry Estury Monitoring Mnul Chpter 14: Slinity. EPA-842-B Wshington, DC, USA. U.S. Environmentl Protection Agency A field-bsed qutic life benchmrk for conductivity in Centrl Applchin strems. EPA-600-R F. Office of Reserch nd Development, Ntionl Center for Environmentl Assessment, Wshington, DC, US. Vn Gestel CAM, Hensbergen PJ Interction of Cd nd Zn toxicity for Folsomi cndid Willem (Collembol: Isotomide) in reltion to biovilbility in soil. Environmentl Toxicology nd Chemistry. 16: Wng N, Dormn RA, Ingersoll CG, Hrdesty DK, Brumbugh WG, Hmmer EJ, Buer CR, Mount DR Acute nd chronic toxicity of sodium sulfte to four freshwter orgnisms in wter-only exposures. Environmentl Toxicology nd Chemistry. 35:

45 CHAPTER TWO THE CHRONIC TOXICITY OF MAJOR IONS AND ION BINARY MIXTURES TO Ceriodphni dubi Introduction Dissolved ions re nturlly occurring compounds in qutic systems tht influence slinity nd re frequently mesured s either Totl Dissolved Solids (TDS) or conductivity. Totl Dissolved Solids is defined s the weight of ll ions in given solution, both orgnic nd inorgnic, tht cn pss through 2 µm filter (U.S. EPA, 1997). Conversely, conductivity mesures the electricl current tht results from ctionic (e.g. C 2+, N +, Mg 2+, K + ) nd nionic (e.g. PO 3-4, Cl -, HCO - 3, SO 2-4 ) ion interctions. Typiclly, both conductivity nd TDS mesurements for freshwter systems re extremely low, but cn esily be influenced by nthropogenic ctivities nd disruption of surrounding geologicl mterils. Anthropogenic ctivities, such s mountintopremovl mining, griculturl irrigtion, rod slt ppliction nd col-fired power plnt effluents hve ll been reported to increse dissolved ions of freshwter systems. For exmple, Pond et l., (2008) reported significnt increse (p vlue < 0.05) in conductivity between reference sites (verge 63 µs/cm; n=10) nd those impcted by surfce col mining opertions (verge 1023 µs/cm; n=27). Previous reserch lso suggests tht n increse in dissolved ion concentrtion nd chnge in ionic composition cn led to impired orgnisml helth nd even chnges in community structure (Dickerson et l., 1996; Tietge et l., 1997; Chpmn et l., 2000; Kennedy et l., 2003; Pond et l., 2008; Kennedy et l., 2005). Furthermore, previous studies lso suggest tht 23

46 utilizing mesurements such s conductivity nd TDS my not be the most ccurte pproch to explin dissolved ion toxicity (Mount et l., 1997; Goodfellow et l., 2000; Soucek et l., 2005; Kunz et l., 2013; Mount et l., 2016) The mjority of ion toxicity dt published hs focused on the cute toxicity of individul ions or multi-ion mixtures (Mount et l., 1997; Soucek nd Kennedy, 2005; Soucek et l., 2010; Mount et l., 2016; Erickson et l., 2017; Erickson et l., 2018). However, few studies hve reported the implictions for chronic exposures (Lsier nd Hrdin, 2010; Elphick et l., 2011; Elphick et l., 2011b; Frg nd Hrper, 2014). Understnding both the cute nd chronic toxicity of dissolved ions is impertive for setting dischrge regultions, developing federl wter qulity criteri, which re essentil for mnging the relese of ions of both point (e.g. col fired power plnt effluents) nd nonpoint (e.g. runoff from rodwy slt ppliction) sources. The chronic toxicity of mjor nions (HCO - 3, SO 2-4, Cl - ) to Ceriodphni dubi (C. dubi) s single ions, nd in binry mixtures hs previously been reported (Johnson, 2014). To compliment this previous reserch, nd to gin full understnding of the chronic toxicity of mjor ions to freshwter invertebrte species, the gol of this reserch ws to chrcterize the effect of mjor ctions (N +, C 2+, Mg 2+ ) on Ceriodphni dubi reproduction. Additionlly, results described by Johnson, 2014 were evluted further. 24

47 Mterils nd Methods Ceriodphni dubi Culture Methods Rering nd testing of C. dubi followed the U.S. EPA guidelines for short-term methods for estimting chronic toxicity (U.S. EPA, 2002), s well s the frmework previously described in Johnson, Briefly, orgnisms were cultured in-house t the Clemson University Institute of Environmentl Toxicology (CU-ENTOX). Reconstituted modertely hrd wter ws prepred ccording to U.S. EPA guidelines (U.S. EPA, 2002) with regent grde chemicls (96 mg/l NHCO 3, 60 mg/l CSO 4 2H 2 0, 60 mg/l MgSO 4, nd 4.0 mg/l KCl). To id in reproduction, the reconstituted modertely hrd wter ws spiked with 2 μg/l selenium (s N 2 SeO 4 ). Individul cultures were initited in order to ssess orgnisml helth by seprting neontes into individul 30 ml culture chmbers contining 15 ml of culture medi (reconstituted modertely hrd wter nd 2 µg/l selenium), nd kept in n incubtor set t 25 C (±1 C) with 16:8 light/drk cycle. Dily wter renewls were performed nd reproduction/mortlity were recorded. Orgnisms were fed diet of 1:1 Pseudokirchneriell subcpitt (~3.0 x 10 7 cells/ml) nd YCT (yest, trout chow, nd CEROPHYLL ). Both lge nd YCT were prepred t CU-ENTOX using methods described by the U.S. EPA (2002). Test orgnisms were selected from successful dults producing 15 neontes cross 3-broods within 7-dy period. Test Solutions Test solutions were prepred using ACS grde chloride-slts of sodium, mgnesium, nd clcium. Sodium chloride (CAS ; Fisher Scientific, Atlnt, 25

48 GA), clcium chloride dihydrte (CAS ; Fisher Scientific, Atlnt, GA), nd mgnesium chloride hexhydrte (CAS ; Fisher Scientific, Atlnt, GA) stock solutions were creted using the sme reconstituted modertely hrd wter recipe s the culture medium. Test solutions were creted 24-hours prior to use by dding the pproprite volume of ech slt stock solution to culture medi for ll biossys. All test solutions were spiked with 2 µg/l selenium. Once prepred, test solutions were llowed to come to equilibrium in the sme temperture controlled test chmber s the test orgnisms (25 ±1 C) to ensure no temperture chnges between wter chnges. A smple of ech test solution nd controls were collected for ion nlysis vi ICP-MS. Biossy Procedure nd Experimentl Design Biossys procedures nd chronic toxicity were ssessed by following the bsic frmework of the short-term methods for chronic toxicity estimtion guidelines described by the U.S. EPA (2002), with modifictions. Similr to the individul cultures, one C. dubi neonte produced by successful culture dults ws plced in 30 ml plstic test chmber contining 15 ml of test solution t the strt of ech biossy. Wter renewls occurred every 24-hours over the eight-dy exposure, t which time reproduction nd mortlity were recorded. Orgnisms were fed 250 µl 1:1 mix of Pseudokirchneriell subcpitt nd YCT immeditely following wter renewls. Wter qulity prmeters, including temperture ( C), DO (mg/l), conductivity (µs/cm), ph, lklinity (s mg/l CCO 3 ) nd hrdness (s mg/l CCO 3 ) were monitored for the initil test solution, nd fter 24-hours of exposure. Results for ech biossy were only ccepted if control 26

49 tretments 60% of individuls produced 15 neontes cross 3 broods nd mintined 80% survivl over the eight-dy exposure. Briefly, biossys consisted of six tretments with ten replictes per tretment. Tretments were either single-ction exposures, or ction binry mixtures. Initilly, single-ction exposures were conducted to estimte Effective Concentrtions (EC) vlues for decresed C. dubi reproduction for use in binry mixture biossys (EC 10, EC 30, EC 50 nd EC 70 ). The experimentl design utilized in this pproch for ction binry mixtures followed the titrtion method described by Johnson (2014) nd Meyer et l., (2015) with modifictions. This type of pproch quntittively evlutes the toxic response of one titrted ion (Ion B), when combined with fixed concentrtion of nother ion (Ion A) (Figure 2.1). A negtive control tretment, only contining reconstituted modertely hrd wter, ws used to ssess the success of ech binry mixture, s well s to stndrdize reproduction in ll exposure tretments. A positive control tretment ws lso estblished nd only contined the fixed concentrtion of Ion A. One ction-only biossy ws performed in conjunction with two binry mixture biossys. The ctiononly biossy contined the sme ction s the titrted ction present in the binry mixtures. For exmple, sodium-only biossy ws performed with titrted sodium with fixed mgnesium nd titrted sodium with fixed clcium. This mens tht t ny given time, three biossys were being performed concurrently. The two binry mixture biossys were then compred bck to the ction-only biossy tht ws simultneously performed. Ction concentrtions utilized were the sme for the ction-only nd binry mixture tretments. 27

50 Chemicl Anlysis Smples were obtined for ech test solution prior to introduction of orgnism (initil) nd 24-hour fter exposure (finl) everydy of the eight-dy biossy. Wter smples were filtered using 0.45 µm filter to remove ny food prticles or debris then trnsferred to 15 ml plstic tube. Proper dilutions were performed using 3% HNO 3 solution with nnopure wter using Trce-Grde HNO 3. Sodium, clcium nd mgnesium concentrtions were identified using n ICP-MS t the CU-ENTOX fcility. Chloride concentrtion, the ssocited nion to the three ctions tested, ws derived from the mesured ction concentrtions. Sttisticl Anlysis JMP ws utilized to perform ll sttisticl nlyses. For ll biossys, percent reproduction ws clculted by dividing the totl number of neontes produced per replicte by the verge neontes produced in the negtive control tretment (the tretment only contining modertely hrd wter) for their corresponding biossy. Both liner regressions nd Hill equtions (Logistic 4P) were pplied to ech ction-only nd ction binry mixture dtset to determine the best-fit curve. The best-fit curve ws chrcterized s the curve hving the lrgest R 2 vlue. Polynomil fits were only used for grphicl representtion in figures, nd not for EC vlue estimtions or slope. Inverse interpoltion ws then performed on ech ction-only best-fit curve to estimte the Effective Concentrtion t which 50% decrese in reproduction occurred (EC 50 ) with 95% confidence intervls (α=0.05). For ech ction-only dtset, both conductivity (µs/cm) nd specific ion concentrtion (mm) were used to estimte EC 50 28

51 vlues. Significnt differences between ech estimted EC 50 vlue ws determined bsed on overlpping 95% confidence intervls. Using n Anlysis of Covrince (ANCOVA), significnt differences between the slope of the best-fit curve for the ction-only concentrtion-response line, nd the best-fit curve for the ction binry mixture concentrtion-response line were verified. Less-thndditive interctions were concluded if the slope of the mixture concentrtion-response line ws sttisticlly shllower thn the corresponding ction-only concentrtion-response line. Alterntively, if the mixture slope ws sttisticlly steeper thn the corresponding ction-only slope, greter-thn-dditive interction ws concluded. No significnt differences in slope resulted in dditive toxicity (Figure 2.2). This sme nlysis ws lso employed to dt collected by Johnson (2014) in order to better chrcterize the chronic toxicity of nions nd nion binry mixtures to C. dubi. Results re reported below. Results Ion-Only Toxicity Estimtes for 50% decrese in reproduction for C. dubi were clculted from both overll conductivity mesurements (µs/cm) nd specific ion concentrtions (mm) (Tble 2.1). The order of toxicity, s described by EC 50 vlues estimted from totl conductivity, ws HCO - 3 Mg 2+ > C 2+ > N + Cl - > SO 2-4. This series indictes tht some EC 50 vlues bsed on conductivity were significntly different from one nother. For exmple, bicrbonte ws similr to mgnesium, but not clcium b, while sodium c nd chloride c were similr, nd sulfte d ws sttisticlly different. Alterntively, the 29

52 order of toxicity, s described by EC 50 vlues estimted from specific ion concentrtions, ws C 2+ Mg 2+ SO 2-4 > HCO - 3 > Cl - > N +. Although no prticulr ctegoriztion ws indicted by conductivity, the order of toxicity bsed on ion concentrtion indicted tht divlent ions, such s clcium, mgnesium, nd sulfte, were the most toxic of the six ions tested, while monovlent ions, including chloride, bicrbonte, nd sodium were less toxic. For nion-only exposures, the bicrbonte concentrtion-response curve exhibited the shrpest slope 14.9 (R 2 : 0.746), followed by sulfte (slope: 4.94; R 2 : 0.862). Chloride hd the shllowest slope of the three concentrtion-response curves t 3.08 (R 2 : 0.739). The three nion-only concentrtion-response curves were sttisticlly different (p vlue < ) (Figure 2.3). The concentrtion-response curve for clcium (slope: 5.34; R 2 : 0.855) ws sttisticlly similr when compred to both mgnesium (slope: 7.07; R 2 : 0.859) (p vlue: ), s well s sodium (slope: 4.89; R 2 : 0.766) (p vlue: ). Additionlly, mgnesium nd sodium lso possessed similr concentrtion-response slopes (p vlue: ) (Figure 2.4). Anion Binry Mixture Toxicity Anion binry mixtures contining titrted chloride with fixed concentrtion of bicrbonte resulted in steeper slope thn tht of the chloride-only concentrtionresponse curve, suggesting greter-thn-dditive response between these two ions (p vlue: ; Figure 2.5). Conversely, when titrted chloride ws in the presence of 30

53 fixed concentrtion of sulfte, significntly shllower slope resulted, indicting lessthn-dditive interction (p vlue: ; Figure 2.5) (Tble 2.2). Concentrtion-response curves produced for mixtures contining titrted sulfte were not significntly different from the corresponding sulfte-only concentrtionresponse curve, indicting dditive interctions when combined with fixed concentrtion of both chloride (p vlue: ; Figure 2.6) nd bicrbonte (p vlue: 0.701; Figure 2.6) (Tble 2.2). Furthermore, the concentrtion-response slope of sulfte with fixed concentrtion of chloride ws not significntly different from the concentrtion-response slope of sulfte in the presence of bicrbonte (p vlue: 0.714). It should lso be noted tht the p vlue clculted for the sulfte concentrtion-response curve with fixed concentrtion of chloride ws close to being sttisticlly significnt, suggesting the potentil for greter-thn-dditive interction between these two ions. Both binry mixture concentrtion-response curves contining titrted bicrbonte resulted in less-thn-dditive interctions when combined with chloride (p vlue: ; Figure 2.7) nd sulfte (p vlue: ; Figure 2.7), s compred to the bicrbonte-only slope (Tble 2.2). There ws no significnt difference between the slope of the concentrtion-response curve for titrted bicrbonte combined with fixed chloride or sulfte (p vlue: ). Ction Binry Mixture Toxicity The mjority of ction binry mixture concentrtion-response curves resulted in dditive interctions, when compred bck to their ction-only counterprt. More specificlly, concentrtion-response curves produced for mixtures contining titrted 31

54 clcium were not significntly different from the corresponding clcium-only concentrtion-response curve, indicting dditive interctions with both sodium (p vlue: 0.292; Figure 2.9) nd mgnesium (p vlue: 0.125; Figure 2.9) (Tble 2.3). Similr dditive results occurred between the sodium-only concentrtion-response curve nd tht produced for sodium in the presence of 4.27 mm mgnesium (p vlue: 0.820; Figure 2.10) nd in the presence of 3.26 mm clcium (p vlue: 0.660; Figure 2.10). Titrted mgnesium, combined with fixed concentrtion of sodium, lso resulted in n dditive interction (p vlue: 0.501; Figure 2.8). Alterntively, when titrted mgnesium ws in the presence of 3.45 mm clcium, less-thn-dditive interction occurred (p vlue: ; Figure 2.8). Discussion Dissolved ions in freshwter systems hve been mesured t elevted concentrtions due to mny nthropogenic ctivities (Kunz et l., 2013; Pond et l., 2008; Timpno et l., 2010). Due to the positive correltion between conductivity nd ion concentrtion, s well s its ese of mesurement, it would be idel to build wter qulity criteri bsed on toxicity resulting from overll conductivity. Becuse conductivity combines ll ions into one prmeter, it would ssume tht ech ion hs the sme effect on biologicl systems. As result, the sme toxic effect would be expected regrdless of ion or ionic composition. Reproductive effects, described by EC 50 vlues derived from conductivity in the present study, resulted in significnt differences between individul ions. These significnt differences suggest tht ech ion produces the sme effect, 50% decrese in reproduction, but t vrying conductivities. Bicrbonte ws the most toxic 32

55 with n EC 50 vlue of 1322 µs/cm, while sulfte, with n EC 50 of 2514 µs/cm, ws the lest toxic. These results further support previous findings tht mesurements, such s conductivity, re limited in their ppliction (Mount et l., 1997; Kennedy et l., 2005; Kunz et l., 2013; Mount et l., 2016). Anion-only biossys utilized sodium-slts of chloride, bicrbonte nd sulfte to chrcterize the toxicity of the ssocited nion (Johnson, 2014). Sodium ws used s the stndrd ction due to its minor contribution to toxicity, unlike clcium, mgnesium, or potssium (Mount et l., 1997; Lsier nd Hrdin, 2010; Elphick et l., 2011; Elphick et l., 2011b). Point estimtes (EC 50 ) derived from specific ion concentrtions for chlorideonly, bicrbonte-only, nd sulfte-only biossys were similr to previously published vlues (Lsier nd Hrdin, 2010; Elphick et l., 2011; Elphick et l., 2011b). However, comprisons for clcium nd mgnesium point estimtes could not be completed becuse previous EC 50 vlues for these individul ions were not found in the published literture. Additionlly, point estimtes clculted in the present study suggest tht divlent ions (clcium, mgnesium, sulfte) hd the lrgest effect on C. dubi reproduction, more so thn monovlent ions (chloride, bicrbonte, sodium). In this regrd, it seems tht the ssocited ction (sodium) or nion (chloride) did not ffect the reltive order of toxicity, further suggesting tht both sodium nd chloride contribute less, thn their counterprts, to overll toxicity. Previous studies hve demonstrted tht elevted clcium concentrtions cn reduce gill tissue permebility to both ions nd wter, prticulrly in sltwter cclimted orgnisms (Potts nd Fleming, 1970; Eddy FB, 1975; Lucu nd Flik, 1999; Pic nd Metz, 1981). More specificlly, clcium reduces pssive sodium loss by 33

56 blocking prcellulr tight junctions (Grosell et l., 2002). Soucek et l. (2011) suggested tht the sme mechnism by clcium, nd to lesser extent mgnesium, might occur in freshwter orgnisms. However, these sme divlent ions re known to increse intestinl uptke of chloride nd sodium, which could result in divlent ion dependent toxicity. Bicrbonte-only exposures, unlike sulfte nd chloride-only exposures, experienced high mortlity rte. Previous studies hve generted n estimted 50% - decrese in C. dubi survivl (48-hour LC 50 ) between nd mg/l HCO 3 (Mount et l., 1997; Hrper et l., 2014). The present study estimted n EC 50 vlue similr to the published LC 50 vlues, indicting tht the mechnism by which bicrbonte exerts its toxic effect might be the sme s tht for reducing survivl nd reproduction in C. dubi. Results of the nion binry mixture biossys indicte tht the only mixture resulting in greter-thn-dditive effect ws fixed bicrbonte with titrted chloride. This suggests tht the ddition of bicrbonte to chloride-dominted wters increses its effect on C. dubi reproduction. However, the reverse combintion (fixed chloride with titrted bicrbonte) resulted in less-thn-dditive effect, indicting tht the ddition of chloride to bicrbonte-dominted wters reduces the overll toxicity. Becuse sodiumslts were utilized in ll nion-only biossys, the ddition of sodium bicrbonte nd sodium chloride to these mixtures nerly doubled the concentrtion of sodium. The increse in sodium concentrtion, however, is most likely not mjor component of the mixture toxicity. This is further explined by the decrese in reproduction for ech 34

57 concentrtion-response curve reltive to the sodium concentrtion. Fixed bicrbonte with titrted chloride, t 24 mm sodium, the percent reproduction ws 13%; however, for fixed chloride with titrted bicrbonte, percent reproduction ws 8% t 28 mm sodium. It would be expected tht if sodium ws the overll cuse of toxicity for these mixtures, tht the higher sodium concentrtion would produce greter effect on reproduction. Currently, the exct mechnism by which chloride nd bicrbonte gin entrnce into freshwter invertebrte species is unknown; however, it is thought tht n increse in externl osmollity leds to firly proportionl increse in internl osmollity. Some studies hve suggested tht mino cid metbolism in freshwter invertebrtes cn ffect their bility to ionoregulte properly, prticulrly with regrds to chloride. If the extrcellulr concentrtion of chloride shifts beyond wht is physiologiclly cpble for the orgnism, decreses in reproduction nd/or deth will occur (Greenwy P, 1979). The ddition of sulfte t fixed concentrtion of 7.84 mm, to chloride nd bicrbonte, resulted in less-thn-dditive effect. This effect hs been previously noted with regrds to cute ion toxicity. Although in Hylell ztec, Soucek nd Kennedy (2005) demonstrted tht the toxicity of sulfte, fixed t 2,846 mg SO 2 4 /L cross tretments, decresed with n incresing concentrtion of chloride. These results indicte tht for both sub-lethl nd cute ion mixtures, the ddition of n incresing concentrtion of chloride results in decresed sulfte toxicity. Interestingly, mixtures contining fixed chloride with titrted sulfte resulted in dditive interctions, indicting tht sulfte does not hve the sme meliortive effect on chloride toxicity. The sme dditive response ws further demonstrted in cute toxicity studies (Soucek et l. 2011). 35

58 Dt regrding the meliortion in toxicity of fixed sulfte with titrted bicrbonte is firly limited. Although not explicitly stted, it ppers tht bsed on published LC 50 vlues, Mount et l. (1997) did not see ny toxicity meliortion in N 2 SO 4 (LC 50 = 3080 mg/l) nd NHCO 3 (LC 50 =1080 mg/l) mixtures (LC 50 = 2630 mg/l). Although extensively investigted with regrds to invertebrtes nd cute ion toxicity, the meliortive effect of clcium on sodium-dominted wters ws not illustrted in the present study (fixed clcium with titrted sodium) (Dwyer et l., 1992;). More specificlly, Kennedy et l. (2005) reported tht C. dubi mortlity ws significntly reduced up to 1:20 rtio of C 2+ :N +. Further studies hve lso indicted tht clcium hs the sme meliortive effect for the cute toxicity of chloride in invertebrtes (Mount et l., 1997; Soucek et l., 2011), lthough lso not indicted by fixed clcium with titrted sodium. To lesser extent, mgnesium hs lso been described s producing n meliortive effect on cute chloride toxicity. However, in the present study, the only ction binry mixture producing less-thn-dditive effect ws fixed clcium with incresing mgnesium. These differences between cute toxicity nd the chronic toxicity demonstrted in the present study my be result of different mechnisms of ction of these ions t vrying combintions. These discrepncies between cute nd chronic toxicity hve been well documented for mny contminnts. For exmple, copper is known to impir proper ionoregultory cpbilities t the gill following cute exposures; however, in chronic exposures, copper ws shown to 36

59 ccumulte within intestinl nd liver tissues, creting lesions nd nodules (Luren nd McDonld, 1985; McGeer et l., 2000; Bielmyer et l., 2005). The lck of interpretbility between cute nd chronic studies suggests tht chronic toxicity cnnot be simply predicted or described by the cute toxicity of mjor ions. On the bsis of environmentlly relevnt exposures, it is importnt to not discredit the effects tht sub-lethl responses cn hve on popultions of orgnisms. Furthermore, the mjority of published literture chrcterizing the chronic toxicity of these mjor ions to invertebrte species focus on either single ion exposures (Lsier nd Hrdin, 2010; Elphick et l., 2011; Elphick et l., 2011b). In qutic systems, mjor ions do not occur s single constituents, but rther in mixtures. To better understnd the effects tht these mjor ions cn hve on the qutic environment, the underlying mechnisms of the chronic effects of ion mixtures should be further exmined. Conclusions The order of chronic toxicity for mjor ions (chloride, sulfte, bicrbonte, clcium, mgnesium, nd sodium) ws C 2+ Mg SO 4 > HCO 3- > Cl - > N +, s described by their effects on C. dubi reproduction (EC 50 ). This order cn further be extrpolted to indicte tht divlent ions tended to be the most toxic on n ion-only bsis, thn nions. Results of binry mixture exposures indicted vriety of toxicity interctions including dditive, greter-thn-dditive, nd less-thn-dditive. Furthermore, these results differ from previously published literture describing the cute toxicity of these mjor ions, prticulrly for clcium, which is generlly regrded s hving n meliortive effect on toxicity. These differences further emphsize the need 37

60 for dditionl informtion regrding chronic toxicity, nd sub-lethl responses to mjor ions. Especilly becuse mny nthropogenic ctivities cn potentilly increse ion concentrtions in freshwter systems bove wht is physiologiclly tolerble for these orgnisms. Furthermore, developing wter qulity criteri derived from chronic endpoints re essentil for mnging the relese of ions both point (e.g. col fired power plnt effluents) nd nonpoint (e.g. mountin top removl mining) sources. 38

61 Figure 2.1. A grphicl representtion of the titrtion experimentl design. One ion (Ion A) is held t constnt bckground concentrtion, second ion (Ion B) increses in concentrtion cross tretments. 39

62 Ion-Only Additive Greter-thn-Additive Less-thn-Additive (Percent of Control) Ion Concentrtion (mm) Figure 2.2. Slope nlysis pproch to determine contminnt mixture interctions. Results indicte dditive, greterthn-dditive, nd less-thn-dditive. 40

63 Bicrbonte Sulfte Chloride Reproduction (Percent of Control) Anion Concentrtion (mm) Figure 2.3. Averge percent reproduction for C. dubi exposed to incresing concentrtions of bicrbonte, sulfte, nd chloride (s sodium slts). Individul dt points represent the verge reproduction for ech tretment with error brs indicting 95% confidence intervls (α = 0.05). 41

64 Clcium Mgnesium Sodium Reproduction (Percent of Control) Ction Concentrtion (mm) Figure 2.4. Averge percent reproduction for C. dubi exposed to incresing concentrtions of clcium, mgnesium, nd sodium (s chloride slts). Individul dt points represent the verge reproduction for ech tretment with error brs indicting 95% confidence intervls (α = 0.05). 42

65 Figure 2.5. The effect of chloride with fixed sulfte (7.84 mm) nd fixed bicrbonte (11.2 mm) on C. dubi reproduction. Dt points represent verge reproduction for ech tretment, stndrdized to the negtive control, with error brs indicting 95% confidence intervls (α = 0.05). 43

66 Figure 2.6. The effect of sulfte with fixed chlorde (9.81 mm) nd fixed bicrbonte (11.2 mm) on C. dubi reproduction. Dt points represent verge reproduction for ech tretment, stndrdized to the negtive control, with error brs indicting 95% confidence intervls (α = 0.05). 44

67 120 Bicrbonte-Only Fixed Sulfte; Titrted Bicrbonte Fixed Chloride; Titrted Bicrbonte Reproduction (Percent of Negtive Control) Bicrbonte Concentrtion (mm) Figure 2.7. The effect of bicrbonte with fixed sulfte (7.84 mm) nd fixed chloride (9.81 mm) on C. dubi reproduction. Dt points represent verge reproduction for ech tretment, stndrdized to the negtive control, with error brs indicting 95% confidence intervls (α = 0.05). 45

68 Reproduction (Percent of Negtive Control) Mgnesium-Only Fixed Clcium; Titrted Mgnesium Fixed Sodium; Titrted Mgnesium Mgnesium Concentrtion (mm) Figure 2.8. The effect of mgnesium with fixed clcium (3.45 mm) nd fixed sodium (30.8 mm) on C. dubi reproduction. Dt points represent verge reproduction for ech tretment, stndrdized to the negtive control, with error brs indicting 95% confidence intervls (α = 0.05). 46

69 Reproduction (Percent of Negtive Control) Clcium-Only Fixed Mgnesium; Titrted Clcium Fixed Sodium; Titrted Clcium Clcium Concentrtion (mm) Figure 2.9. The effect of clcium with fixed mgnesium (4.67 mm) nd fixed sodium (13.2 mm) on C. dubi reproduction. Dt points represent verge reproduction for ech tretment, stndrdized to the negtive control, with error brs indicting 95% confidence intervls (α = 0.05). 47

70 Reproduction (Percent of Negtive Control) Sodium-Only Fixed Clcium; Titrted Sodium Fixed Mgnesium; Titrted Sodium Sodium Concentrtion (mm) Figure The effect of sodium with fixed clcium (3.26 mm) nd fixed mgnesium (4.27 mm) on C. dubi reproduction. Dt points represent verge reproduction for ech tretment, stndrdized to the negtive control, with error brs indicting 95% confidence intervls (α = 0.05). 48

71 Tble 2.1. EC 50 vlues for C. dubi reproduction estimted from conductivity (µs/cm) nd specific ion concentrtion (mm *. Ion Conductivity (µs/cm) EC 50 Ion Concentrtion (mm) Chloride Sulfte Bicrbonte Clcium Mgnesium Sodium 2122 c (2028, 2217) 2514 d (2402, 2625) 1322 (1287, 1357) 1649 b (1500, 1799) 1411 (1324, 1498) 2061 c (1916, 2206) 16.2 c (15.4, 17.1) 12.0 (11.5, 12.6) 13.7 b (13.3, 14.1) 9.74 (8.54, 10.9) 10.4 (9.50, 11.5) 23.6 d (21.2, 26.1) Vlues in prenthesis indicte 95% confidence intervls for ech EC 50 vlue (α = 0.05). * Letters next to EC 50 vlues for conductivity nd ion concentrtion indicte sttisticl differences between single ions derived from comprison between 95% confidence intervls. 49

72 Tble 2.2. Sttisticl nlysis of C. dubi nion binry mixture toxicity. Results include slope (mm), estimted EC 50 vlue, R 2 for the best fitting line, p vlue clculted from nion-only nd binry mixture comprisons, s well s the overll effect. Anion Binry Mixtures Slope (mm) EC 50 R 2 P-Vlue Effect Chloride-Only (-3.41, -2.75) Fixed Sulfte; Titrted Chloride (-2.87, -2.25) Fixed Bicrbonte; Titrted Chloride (-4.05, -1.45) Sulfte-Only (-5.30, -4.57) Fixed Chloride; Titrted Sulfte (-9.08, -4.41) Fixed Bicrbonte; Titrted Sulfte (-5.83, -3.55) Bicrbonte-Only (-17.8, -11.9) Fixed Chloride; Titrted Bicrbonte (-13.5, -7.14) Fixed Sulfte; Titrted Bicrbonte (-12.2, -3.91) 16.2 (15.4, 17.1) (10.4, 12.4) 1.59 (0.145, 3.03) 12.0 (11.5, 12.6) 8.0 (7.23, 8.78) 3.04 (1.40, 4.67) 13.7 (13.3, 14.1) 8.76 (7.95, 9.87) 7.32 (6.00, 8.64) * Less-thn-Addi<ve * Greter-thn-Addi<ve Addi<ve Addi<ve * Less-thn-Addi<ve * Less-thn-Addi<ve 50

73 Tble 2.3. Sttisticl nlysis of C. dubi ction binry mixture toxicity. Results include slope (mm), estimted EC 50 vlue, R 2 for the best fitting line, p vlue clculted from nion-only nd binry mixture comprisons, s well s the overll effect. Ction Binry Mixtures Slope (mm) EC 50 R 2 P-Vlue Effect Mgnesium-Only (-12.3, -8.05) Fixed Clcium; Titrted Mgnesium (-7.69, -0.28) Fixed Sodium; Titrted Mgnesium (-19.2, -9.25) Clcium-Only (-10.0, -2.68) Fixed Mgnesium; Titrted Clcium (-13.7, -7.78) Fixed Sodium; Titrted Clcium (-14.2, -6.32) Sodium-Only (-9.96, -3.85) Fixed Clcium; Titrted Sodium (-9.03, -2.82) Fixed Mgnesium; Titrted Sodium (-10.9, -3.93) 9.92 (9.33, 10.5) 7.52 (6.57, 8.47) 5.59 (4.18, 7.00) 6.5 (6.31, 6.69) 7.58 (5.54, 9.62) 18.3 (13.3, 15.3) 14.9 (10.3, 19.7) * Less-thn-Addi<ve Addi<ve Addi<ve Addi<ve Addi<ve Addi<ve The EC 50 vlue for fixed clcium; titrted sodium nd fixed clcium; titrted mgnesium could not be estimted due to the lck of 50% decrese in reproduction for both mixtures. 51

74 References Bielmyer GK, Gtlin D, Isley JJ, Tomsso J, Kline SJ Responses of hybrid striped bss to wterborne nd dietry copper in freshwter nd sltwter. Comprtive Biology nd Physiology Prt C. 140: Chpmn PM, Biley H, Cnri E Toxicity of totl dissolved solids ssocited with two mine effluents to Chironomid lrve nd erly life stges of rinbow trout. Environmentl Toxicology nd Chemistry. 19: Dickerson KK, Hubert WA, Bermn HL Toxicity ssessment of wter from lke nd wetlnds receiving irrigtion drin wter. Environmentl Toxicology nd Chemistry 15: s Dwyer FJ, Burch SA, Ingersoll CG, Hunn JB Toxicity of trce elements nd slinity mixtures to striped bss (Morone sxtilis) nd Dphni mgn. Environmentl Toxicology nd Chemsitry. 11: Eddy FB The effect of clcium on gill potentils nd on sodium nd chloride fluxes in the goldsfish, Crssius urtus. Journl of Comprtive Physiology. 96: Elphick JRF, Bergh KD, Biley HC Chronic toxicity of chloride to freshwter species: Effects of hrdness nd implictions for wter qulity guidelines. Environmentl Toxicology nd Chemistry. 30: Elphick JRF, Dvies M, Gilron G, Cnri EC, Lo B, Biley HC. 2011b. An qutic toxicologicl evlution of sulfte: the cse for considering hrdness s modifying fctor in setting wter qulity guidelines. Environmentl Toxicology nd Chemistry. 30: Erickson RJ, Mount DR, Highlnd TL, Hockett JR, Hoff DJ, Jenson CT, Norberg-King TJ, Peterson KN The cute toxicity of mjor ion slts to Ceriodphni dubi. II. Empiricl reltionships in binry slt mixtures. Environmentl Toxicology nd Chemistry. 36: Erickson RJ, Mount DR, Highlnd TL, Hockett JR, Hoff DJ, Jenson CT, Norberg-King TJ, Peterson KN The cute toxicity of mjor ion slts to Ceriodphni dubi. III. Mthemticl models for mixture toxicity. Environmentl Toxicology nd Chemistry. 37: Frg AM, Hrper DD The chronic toxicity of sodium bicrbonte, mjor component of col bed nturl gs produced wters. Environmentl Toxicology nd Chemistry. 33:

75 Goodfellow WL, Ausley LL, Burton DT, Denton DL, Dorn PB, Grothe DR, Heber MA, Norberg-King TJ, Rodgers Jr JH Mjor ion toxicity in effluents: review with permitting recommendtions. Environmentl Toxicology nd Chemistry. 19: Greenwy, P Freshwter invertebrtes: In G.M.O. Mloiy ed. Comprtive Physiology of Osmoregultion in Animls. Acdemic, London, UK. Pg Grosell M, Nielson C, Binchini A Sodium turnover rte determines sensitivity to cute copper nd silver exposure in freshwter nimls. Comprtive Biochemistry nd Physiology Prt C. 133: Johnson, K Chrcterizing the chronic toxicity of ion mixtures to Ceriodphni dubi using two experimentl designs. All Theses in TigerPrints. Pper Kennedy AJ, Chrry DS, Currie RJ Field nd lbortory ssessment of colprocessing effluent in the Leding Creek Wtershed, Meigs Co., Ohio. Archives of Environmentl Contmintion nd Toxicology. 44: Kennedy AJ, Cherry DS, Zipper CE Evlution of ionic contribution to the toxicity of col-mine effluent using Ceriodphni dubi. Archives of Environmentl Contmintion nd Toxicology. 49: Kunz JL, Conley JM, Buchwlter DB, Norberg-King TJ, Kemble NE, Wng N, Ingersoll CG Use of reconstituted wters to evlute effects of elevted mjor ions ssocited with mountintop col mining on freshwter invertebrtes. Environmentl Toxicology nd Chemistry. 32: Lsier PJ, Hrdin IR Observed nd predicted reproduction of Ceriodphni dubi exposed to chloride, sulfte, nd bicrbonte. Environmentl Toxicoogy nd Chemistry. 29: Luren DJ, McDonld DG Effects of copper on brnchil ionoregultion in the rinbow trout, Slmo girdneri Richrdson. Journl of Comprtive Physiology B. 155: Lucu C, Flik G N+-K+-ATPse nd N+/C2+ exchnge ctivities in gills of hyperregulting Crcinus mens. Americn Journl of Physiology. 276: R490-R499. McGeer JC, Szebedinszky C, McDonld DG, Wood CM Effects of chronic sublethl exposure to wterborne Cu, Cd, or Zn in rinbow trout 1: Iono-regultory disturbnce nd metbolic costs. Aqutic Toxicology. 50:

76 Meyer JS, Rnville JF, Pontsch M, Gorusch JW, Adms WJ Acute toxicity of binry mixtures nd ternry mixtures of Cd, Cu, nd Zn to Dphni mgn. Environmentl Toxicology nd Chemistry. 34: Mount DR, Gulley DD, Hockett JR, Grrison TD, Evns JM Sttisticl models to predict the toxicity of mjor ions to Ceriodphni dubi, Dphni mgn, nd Pimephles promels (fthed minnows). Environmentl Toxicology nd Chemistry. 16: Mount DR, Erickson RJ, Highlnd TL, Hockett R, Hoff DJ, Jenson CT, Norberg-King TJ, Peterson KN, Polske ZM, Wisniewski S The cute toxicity of mjor ion slts to Ceriodphni dubi: I. Influence of bckground wter chemistry. Environmentl Toxicology nd Chemistry. 35: Perry SF The chloride cell: Structure nd function in the gills of freshwter fishes. Annul Review of Physiology. 59: Pic P, Metz J Role of externl clcium in sodium nd chloride trnsport in the gills of sewter-dpted Mugil cpito. Journl of Comprtive Physiology B Pond GJ, Pssmore ME, Borsuk FA, Reynolds L, Rose CJ Downstrem effects of mountintop col mining: compring biologicl conditions using fmily- nd genus-level mcroinvertebrte biossessment tools. Journl of the North Americn Benthologicl Society. 27: Potts WTW, Fleming WR The effects of prolctin nd divlent ions on the permebility to wter of Fundulus kinse. Journl of Experimentl Biology. 53: Soucek DJ, Kennedy AJ Effects of hrdness, chloride, nd cclimtion on the cute toxicity of sulfte to freshwter invertebrtes. Environmentl Toxicology nd Chemistry. 24: Soucek DJ, Linton TK, Trr CD, Dickinson A, Wickrmnyke N, Delos CG, Cruz LA Influence of wter hrdness nd sulfte on the cute toxicity of chloride to sensitive freshwter invertebrtes. Environmentl Toxicology nd Chemistry. 30: Tietge JE, Hockett JR, Evns JM Mjor ion toxicity of six produced wters to three freshwter species: Appliction of ion toxicity models nd TIE procedures. Environmentl Toxicology nd Chemistry. 16:

77 Timpno AJ, Schoenholtz SH, Zipper CE, Soucek DJ Isolting effects of totl dissolved solids on qutic life in centrl Applchin colfield strems. Proceedings, Ntionl Meeting of the Americn Society of Mining nd Reclmtion. P U.S. Environmentl Protection Agency Volunteer Strem Monitoring: A Methods Mnul. EPA-841-B Wshington, DC, USA. U.S. Environmentl Protection Agency Short-term methods for estimting the chronic toxicity of effluents nd receiving wters to freshwter orgnisms, fourth edition. EPA/824/R/02/013. Wshington, DC. 55

78 CHAPTER THREE THE CHRONIC TOXICITY OF MAJOR IONS AND ION BINARY MIXTURES TO Pimephles promels (fthed minnow) Introduction Dissolved ions, commonly mesured s conductivity (µs/cm) nd Totl Dissolved Solids (TDS), re nturl constituents mong qutic systems. Freshwter systems typiclly exhibit very low conductivities nd possess ionic compositions tht re dependent on the surrounding geologic mteril of the wter body. For exmple, typicl freshwter strem in the Centrl Applchin Mountins is dominted by sulfte nd bicrbonte with conductivity of 62 μs/cm nd TDS concentrtion of 21 mg/l (Timpno et l., 2010). However, these chrcteristics re subject to chnge in res impcted by nthropogenic ctivities such s griculturl irrigtion, the ppliction of slt to rods, mining opertions, nd col-fired power plnt effluents. Timpno et l. (2010) reported n increse in sulfte (SO 2-4 ), clcium (C 2+ ) nd potssium (K + ) concentrtions of twenty-two mining sites in Virgini with men TDS concentrtion of 406 mg/l. Further studies describe similr trends, lthough to higher degree with men TDS concentrtion of 1,165 mg/l nd substntil increse in sulfte concentrtion (Pond et l., 2008). Although conductivity nd TDS re utilized in environmentl settings in order to chrcterize chnges from norml conditions, the use of these mesurements to predict or describe potentil toxic responses to elevted dissolved ions hs been demonstrted to be poor predictor of toxicity (Mount et l., 1997; Goodfellow et l., 2000; Kennedy et l., 2005; Mount et l., 2016). Inconsistencies between the toxicity of environmentl 56

79 freshwter systems nd conductivity hve been observed. For exmple, the LC 50 for Ceriodphni dubi (C. dubi) hve been reported for col mining effluent t 7,000 µs/cm (Kennedy et l., 2005), but only 3,000 µs/cm for irrigtion drin wters (Dickerson et l., 1996). It is ssumed tht specific ion concentrtions nd ionic composition re better predictors of ion toxicity. Current federl wter qulity criteri in the United Sttes focus on chloride s single ion (230 mg/l chronic; 860 mg/l cute); however, criteri for mny other mjor ions including N + 2-, HCO 3-, SO 4 or even TDS currently do not exist. To develop wter qulity guidelines, it is importnt to consider not just cute toxicity, but more importntly chronic toxicity. The cute toxicity of ions nd ions mixtures hs been extensively investigted, with new reports being published frequently (Mount et l., 1997; Soucek nd Kennedy, 2005; Soucek et l., 2011; Hrper et l., 2014; Mount et l., 2016; Wng et l., 2016; Erickson et l., 2017; Erickson et l., 2018). Few studies hve demonstrted the potentil for chronic toxicity of ions, nd more specificlly, ion mixtures (Lsier nd Hrdin, 2010; Elphick et l., 2011; Elphick et l., 2011b; Frg nd Hrper, 2014). Sublethl effects, however, should not be discounted. Unlike cute toxicity, such s mortlity tht cn ffect popultions nd ecosystems bruptly, chronic toxicity, including growth nd reproductive effects, cn disrupt ecosystem processes over time nd my eventully led to ecosystem collpse (Pond et l., 2008). The effects of chronic toxicity re often subtle, nd cn initilly go undetected. The reltive order of cute toxicity for mjor ions hs previously been reported s K + > HCO 3 Mg 2+ > Cl > SO 2-4 (Mount et l., 1997). It ws further noted tht N + nd 57

80 C 2+ did not contribute to overll cute toxicity; however, current reports indicte tht these ctions my ctully hve significnt influence on ion mixture toxicity (Mount et l., 2016). Alterntively, the chronic toxicity of individul ions to C. dubi indicte n order of C 2+ Mg 2+ SO 2-4 > HCO 3 > Cl > N + (see Chpter Two). Differences exist between cute nd chronic toxicity of ions nd ion mixtures, nd becuse most of the published literture emphsizes cute effects, further dt is required in order to elucidte how these ions ffect orgnisms on sub-lethl bsis. Additionlly, little dt hs been generted regrding vertebrte effects (Frg nd Hrper, 2014; Wng et l., 2016). Therefore, the gol of this reserch ws to chrcterize the chronic toxicity of mjor ions nd ion binry mixtures to Pimephles promels (fthed minnow), vertebrte fish species. Results from this study will expnd on the limited dtset regrding mjor ion effect on the growth of higher-order species. Mterils nd Methods Pimephles promels Mintennce nd Culture Lrvl Pimephles promels (P. promels) were obtined from n in-house culture of dult P. promels t the Clemson Institute of Environmentl Toxicology (CU- ENTOX, Pendleton, SC, USA). Adult fish were rered in ~350-gllon freshwter recirculting system consisting of four troughs (retention time between 5-6 hours) with wter qulity prmeters mintined s follows: Temperture: 25 ± 1 C; Alklinity: 56 mg/l s CCO 3 ; Hrdness: 100 mg/l s CCO 3 ; ph: 7.5. Reproduction ws monitored dily by the presence of eggs on polyvinyl chloride tiles, which were plced in ech trough to enhnce reproduction. Tiles with eggs were immeditely removed from the 58

81 recirculting system nd plced in seprte holding tnk contining U.S. EPA reconstituted modertely hrd wter (18 MΩcm ultrpure wter, 96 mg/l NHCO 3, 60 mg/l CSO 4 2H 2 O, 60 mg/l MgSO 4, nd 4.0 mg/l KCl) nd n ir stone to redistribute ny sttic wter (U.S. EPA, 2002). Eggs were gently rinsed twice dily with deionized wter, ny non-vible eggs were discrded, nd the number of htched fry ws recorded. Test Solutions Stock solutions of ech ion were mde dily with ACS certified slts nd reconstituted modertely hrd wter. Prepred stock solutions included sodium chloride (CAS ), clcium chloride (CAS ), mgnesium chloride (CAS ), sodium bicrbonte (CAS ), nd sodium sulfte (CAS ) (purchsed from Fisher Scientific, Atlnt, GA, USA). The pproprite mount of ech stock solution ws dded to 2-L of reconstituted modertely hrd wter to crete test solutions. After preprtion, solutions were llowed to come to equilibrium for 24-hours prior to use nd mintined t temperture of 25 ± 1 C. Test solutions were creted dily. A smll smple ws collected for ech test solution to quntify ccurte ion concentrtions using ICP-MS. Biossy Procedure nd Experimentl Design Procedures for conducting 7-dy P. promels biossys followed the U.S. EPA short-term chronic toxicity estimtion guidelines frmework (U.S. EPA, 2002). Specificlly, ech replicte contined ten < 24-hour old lrvl P. promels, with five replictes per tretment. During test initition, the ten fish were trnsferred t rndom to one 600-mL polyethylene beker contining 300 ml test solution. Test chmbers were 59

82 kept in temperture (25 ± 1 C) nd light (16:8 light/drk cycle) controlled room with wter renewls occurring every 24-hours. During renewls, wter qulity mesurements were recorded for both the initil test solution (prior to orgnism introduction) nd finl test solution (24-hours fter exposure). These mesurements included temperture ( C), dissolved oxygen (mg/l), ph, lklinity (mg/l s CCO 3 ), hrdness (mg/l s CCO 3 ), totl mmoni (mg/l), nd conductivity (µs/cm). Any decesed test orgnisms were promptly removed from the test chmber. Test orgnisms were fed twice dily, t 4-hour intervls, concentrted solution of <24-hour old Artemi nuplii cultured s described by the U.S. EPA (2002). On dy seven of the biossy, fish were euthnized using 1.0 g/l MS-222 solution buffered with NHCO 3 to ph of 7.5. Ech replicte ws filtered using 2-inch borosilicte cylinder ffixed with 120 µm mesh nd rinsed with pproximtely 50 ml deionized wter. Orgnisms were then trnsferred to seprte pre-weighed luminum tins for ech replicte nd plced in drying oven t 60 C for 24-hours. Averge fish dry weights were clculted by subtrcting the initil tin-only weight from the finl dry weight of the tin + fish, nd then dividing by the number of fish plced in ech tin (the number of fish tht survived per replicte). Single-ion biossys were performed initilly to determine the pproprite concentrtion of ech ion needed for binry mixtures. Following single-ion biossys, binry mixture biossys were conducted. For these mixture exposures, titrtion experimentl design ws employed. This type of design consists of one ion being held t constnt bckground concentrtion (Ion A), while the second ion (Ion B) increses in 60

83 concentrtion cross tretments 3.1). Ech biossy included negtive control tretment tht only contined reconstituted modertely hrd wter. Results of biossys were ccepted if survivl ws 80% in the negtive control. A second tretment, identified s the positive control, ws composed of reconstituted modertely hrd wter nd the fixed concentrtion of Ion A. Subsequent tretments still contined the reconstituted modertely hrd wter, fixed concentrtion of Ion A, nd incresing concentrtions of Ion B. One single-ion biossy ws performed in conjunction with one binry mixture biossy. This mens tht t ny given time, two to four biossys were being conducted simultneously. The concentrtion of the titrted ion (Ion B) ws the sme for both single-ion nd binry mixture biossys. Chemicl Anlysis Smll liquots, filtered through 0.45 µm filter, were collected dily for ech control nd tretment in order to confirm ion concentrtions. Smples were plced in 15 ml plstic tube nd then diluted within the detection limit to ph <3.0 using HNO 3 nd ultrpure wter (18 MΩ resistivity). Ction concentrtions were then confirmed using ICP-MS t the Clemson University Institute of Environmentl Toxicology. Mesured ction concentrtions were used to clculte nion concentrtions. For exmple, chloride concentrtions were clculted bsed on the confirmed concentrtion of sodium, but corrected for the chloride present in the reconstituted modertely hrd wter. 61

84 Sttisticl Anlysis For ll sttisticl nlyses, JMP ws utilized. All results, s weight (mg), were stndrdized to the verge weight (mg) of the negtive control tretment (the tretment only contining modertely hrd wter) for ech biossy. In doing so, orgnisml rndomiztion nd helth were ccounted for. Ech concentrtion-response curve ws nlyzed by both liner regressions nd Hill equtions (Logistic 4P). The bestfit curve, determined by the fit with the highest R 2 vlues, ws used in ll subsequent dt nlysis. Estimtions of Effective Concentrtions for 50% decrese in reproduction (EC 50 ), nd the ssocited 95% confidence intervls (α = 0.05), were clculted by inverse interpoltion. Conductivity (μs/cm) nd specific ion concentrtion (mm) for iononly biossys were used in estimtions of EC 50 vlues. Non-overlpping 95% confidence intervls indicted significnt differences between ion-only EC 50 vlues. Depending on the best-fit curve, either overll liner slopes or pproximte Hill slopes were compred using Anlysis of Covrince (ANCOVA) to determine significnt differences between ion-only nd ion binry mixture concentrtion-response curves. For grphing purposes, polynomil fits re represented for Hill equtions. However, slopes nd EC 50 vlues were estimted from Hill equtions. Anlyses indicting no sttisticl differences resulted in dditive interctions for the binry mixture. In contrst, sttisticl differences could either indicte less-thn-dditive interctions where the binry mixture produces shllower slope thn the ion-only slope, or greter-thn-dditive where the binry mixture produces steeper slope thn the ion-only slope (Figure 3.2). 62

85 Results Ion-Only Toxicity Conductivity (µs/cm) nd specific ion concentrtion (mm) were used to estimte Effective Concentrtions (EC 50 ) tht reduced P. promels growth by 50% compred to control growth. Effective concentrtions estimted from conductivity indicted reltive order of toxicity s HCO - 3 > SO 2-4 Mg 2+ > N + Cl - > C 2+. Significnt differences occurred between conductivity EC 50 vlues, s determined by overlpping 95% confidence intervls (Tble 3.1). For exmple, clcium ws significntly less toxic thn ny other ion. Conversely, estimtions derived from specific ion concentrtions indicted n order of SO 2-4 Mg 2+ - > HCO 3 C 2+ > Cl - > N +. Significnt differences due to non-overlpping confidence intervls were lso noted for these vlues (Tble 3.1). Due to lrge degree of mortlity in most exposures, 7-dy Lethl Concentrtions, resulting in 50% decrese in survivl (LC 50 ), were lso clculted (Tble 3.1). Sulfte (Figure 3.3; slope: 9.08; R 2 : 0.859) hd the lrgest concentrtionresponse slope suggesting tht it hs the gretest effect on P. promels growth per millimolr bsis compred to both bicrbonte (slope: 1.54; R 2 : 0.939; p vlue: *) nd chloride (slope: 3.33; R 2 : 0.856; p vlue: *). Furthermore, the chloride concentrtion-response slope ws significntly different from the slope of bocrbonte (p vlue: 0.002*). Bicrbonte hd the shllowest slope of the three nions (Figure 3.3). Similr to growth effects, sulfte lso resulted in the lrgest decrese in survivl (slope: 4.76; R 2 : 0.799) nd ws significntly different from bicrbonte (slope: 1.26; R 2 : 0.936; p vlue: *) nd chloride (slope: 1.28; 0.725; R 2 : 0.725; 63

86 p vlue: *). Chloride nd bicrbonte hd similr concentrtion-response slopes (p vlue: 0.965); however, LC 50 vlues were significntly different (Figure 3.4). Slopes clculted for ction-only concentrtion-response curves indicted tht sodium (slope: 7.12; R 2 : 0.728) hd the lest overll effect on P. promels growth compred to mgnesium (Figure 3.5; slope: 10.2; R 2 : 0.861; p vlue: *). The concentrtion-response slope mesured for sodium ws not significntly different from clcium (slope: 9.01; R 2 : 0.653; p vlue: 0.364), nor ws clcium significntly different from mgnesium (p vlue: ) (Figure 3.5). Although the effect on P. promels growth were similr of sodium, clcium nd mgnesium on P. promels growth were similr on milli-molr bsis, EC50 vlues were significntly different. Effects on survivl of P. promels, for sodium, clcium, nd mgnesium were similr to those on growth (Figure 3.6). Overll slopes for both ctions nd nions suggest tht divlent ions, including clcium, sulfte, nd mgnesium, resulted in the lrgest effect on both P. promels survivl nd growth. However, these results were not similr to the reltive order of toxicity developed from EC 50 or LC 50 vlues. This indictes tht the potency of these ions does not correspond with the effective or lethl concentrtions. Anion Binry Mixture Toxicity The concentrtion-response curve for bicrbonte in the presence of 14.7mM sulfte resulted in n dditive interction for effects in both growth (slope: 1.81; R 2 : 0.903; p vlue: 0.099) nd survivl (slope: 1.78; R 2 : 0.918; p vlue: 0.613) of P. promels. Similrly, the ddition of 35mM chloride to titrted bicrbonte lso resulted 64

87 in n dditive interction (growth - slope: 1.53; R 2 : 0.977; p vlue: 0.123; survivl - slope: 1.44; R 2 : 0.926; p vlue: 0.772) (Figure 3.7; Figure 3.10; Tble 3.2). Regrding growth (p vlue: 0.104) nd survivl (p vlue: 0.177), there ws no significnt difference between fixed chloride with titrted bicrbonte, nd fixed sulfte with titrted bicrbonte, suggesting tht the ddition of either nion to bicrbonte results in similr response. Similr growth effects resulted from exposures with titrted sulfte in the presence of both 25.2mM chloride (slope: 3.12; R 2 : 0.876; p vlue: 0.927), nd 22.7mM bicrbonte (slope: 3.51; R 2 : 0.873; p vlue: 0.511) (Figure 3.9). Moreover, survivl dt indicte dditive interctions for these mixtures s well (Figure 3.12; Tble 3.4). Although the two sulfte-only survivl concentrtion-response curves produced resulted in sttisticlly different slopes (p vlue: *; * indicting significnt difference), the results were not different between the two mixtures. Additive growth effects were lso result of titrted chloride in the presence of 22.7mM bicrbonte (slope: 1.98; R 2 : 0.924; p vlue: 0.751). This dditive interction did not extend to the survivl effects of the sme mixture. Insted, survivl ws significntly more for the mixture of fixed bicrbonte with titrted chloride (slope: 0.988; R 2 : 0.935; p vlue: *), thn for chloride-only (slope: 2.10; R 2 : 0.861). Less-thn-dditive interctions were lso concluded for both growth (slope: 2.23; R 2 : 0.959; p vlue: *) nd survivl (slope: 1.05; R 2 : 0.946; p vlue: *) effects for chloride in the presence of 13.9mM sulfte (Figure 3.8; Figure 3.11; Tble 3.3). 65

88 Ction Binry Mixture Toxicity Mixtures contining titrted clcium in the presence of 18.4mM mgnesium resulted in sttisticlly similr slopes (slope: 2.49; R 2 : 0.564; p vlue: 0.921) for growth effects when compred to clcium-only (slope: 2.49; R 2 : 0.926), indicting dditivity (Figure 3.13). Although not s strong, comprison of the survivl concentrtionresponse curves for the sme mixture lso indicted dditive effects (p vlue: 0.086). This slight similrity for survivl effects could indicte tht shift towrds greter-thndditive, or n increse in toxicity due to this ction mixture (Tble 3.5). Survivl dt revel tht s clcium exceeds 28.9mM, in the presence of 18.4mM mgnesium, considerble reduction from 86% to 12% in survivl occurs (Figure 3.16), lthough not significnt. Further dditive interctions resulted from titrted chloride in the presence of 43.7mM sodium for both growth (p vlue: 0.690) nd survivl (0.521) effects (Tble 3.5). The concentrtion-response curve for incresing sodium concentrtions in the presence of 28.7mM clcium (slope: 1.16; R 2 : 0.745), indicted dditive interctions for both growth (p vlue: 0.211) nd survivl (p vlue: 0.529) effects (Tble 3.7; Figure 3.15; Figure 3.18). Combintions of sodium in the presence of 16.4mM mgnesium further demonstrted dditive responses (Tble 3.7). A less-thn-dditive interction between titrted mgnesium in the presence of 29.2mM clcium ws demonstrted for both growth (slope: 0.619; R 2 : 0.113; p vlue: 0.009*) nd survivl (slope: 3.78; R 2 : 0.876; p vlue: *) effects. Both responses suggest tht clcium decreses mgnesium toxicity, nd hs this sme effect for survivl nd growth. Although the ddition of 40.0mM sodium to titrted mgnesium produced 66

89 the sme protective effect s clcium for survivl, nd resulted in less-thn-dditive interctions (Figure: 3.17; slope: 3.74; R 2 : 0.877; p vlue: *), this effect did not extend to growth effects (slope: 3.10; R 2 : 0.149; p vlue: 0.315) (Tble 3.6; Figure 3.14). The sttisticlly similr slopes indicte dditive effects on growth for mgnesium in the presence of sodium. Discussion Conductivity, mesurement tht combines ll ions into one prmeter, hs been previously used s surrogte to explin mjor ion toxicity (Kennedy et l., 2005; Pond et l., 2008; U.S. EPA, 2011). By describing ion toxicity on the bsis of conductivity lone ssumes tht ll ions hve similr chemicl ctivity nd effect on biologicl systems. As such, it would be expected tht there would be no significnt differences between growth effects, described by their EC 50 vlues estimted from conductivity. Conversely, utilizing specific ion concentrtion s mens to explin toxicity would ssume tht ech ion hs the potentil to exert its own specific effect t the site of ction, most prominently the gill. In this cse, there should be cler toxicity grdient in which some ions produce more considerble response thn others. Results from the present study indicte significnt difference between ions, nd their corresponding EC 50 vlues tht were estimted from conductivity. Although some ions demonstrted similr responses, it is importnt to note tht these re vlues estimted from single-ion exposures. In cses where multiple ions exist t elevted concentrtions, their electrochemicl behviors nd interctions cn chnge, ultimtely influencing the solution conductivity. These results re comprble to previous studies tht hve 67

90 suggested conductivity is not the best pproch for estimting ion toxicity (Mount et l., 1997; Kennedy et l., 2003; Timpno et l., 2010; Soucek et l., 2011; Kunz et l., 2013; Mount et l., 2016). Initil ion-only biossys identified sulfte, mgnesium nd bicrbonte s being the most toxic of the six ions tested, s described by their EC 50 vlues. Clcium demonstrted similr toxicity s bicrbonte, indicting tht 50% decrese in growth occurred t comprble ion concentrtions. Both sodium nd chloride were identified s the lest toxic for both survivl nd growth of fthed minnows. Direct comprisons between the estimted EC 50 vlues in the present study, nd those described in the published dt could not be mde. Frg nd Hrper (2014) estimted P. promels IC 20 for bicrbonte s 8.17mM (95% confidence intervls, mM bicrbonte). This estimte is roughly 42% lower n EC 20 vlue estimted for the present study: 14.1mM HCO3- (95% confidence intervls, mM bicrbonte). A seven-dy LC 50 of 22.1mM bicrbonte ( mM bicrbonte) ws lso reported, nd ws pproximtely 47% lower thn the present study (Tble 3.1). Furthermore, other studies hve reported lower seven-dy LC 50 s for P. promels s mM sulfte (Wng et l., 2016). Typicl 96-hour cute toxicity exposures of NCl, N2SO4, nd NHCO3 s single slts to P. promels resulted in 109mM chloride ( mM chloride), 56mM sulfte ( mM sulfte) nd 10.5mM bicrbonte ( mM bicrbonte) s LC 50 vlues respectively (Mount et l., 1997). These differences between nion-only exposures could be explined by differences in testing procedures nd the ge of the fish t test initition. For exmple, Mount et l. (1997) initited testing with lrvl 68

91 fish between one nd seven dys post-htch, while Frg nd Hrper (2014) initited testing t < 48-hours post-htch. Additionlly, Mount et l. (1997) utilized fish tht hd been cultured in-house, while Frg nd Hrper (2014) hd to trnsport fish to the testing loction. The ge of fish utilized in exposures my be of greter importnce when determining effects due to incresed slinity. Previous studies hve indicted tht some fish, prticulrly tilpi nd killifish, do not possess functionl gills until pproximtely 96-hours post-htch (Ayson et l., 1994; Ktoh et l., 2000). The bility for the yolk sc in P. promels to regulte ion intke t the sme rte nd cpcity s fully functioning gill is unknown. However, this could be reson for differences between nion results in these studies. Ction-only seven-dy LC 50 vlues estimted in the current study were firly comprble to the five-dy LC 50 vlues reported by Mount et l. (1997), suggesting tht P. promels my regulte ctions to greter extent thn nions. It is lso importnt to note tht the LC 50 vlues were not significntly different from EC 50 vlues estimted for sulfte, bicrbonte, mgnesium, nd clcium. This my suggest tht for these ions in prticulr, the mechnism of ction resulting in cute toxicity my be the sme for chronic, specificlly growth. These ions ply mny importnt physiologicl roles in freshwter orgnisms, one of which is the formtion of electrochemicl grdients within cells. By generting these grdients, they ultimtely produce delicte blnce tht controls wter nd ion movement within the orgnism. Becuse the internl ion concentrtion of freshwter orgnisms is typiclly greter thn their externl environment, it is necessry for them to utilize ctive trnsport through system of pumps nd trnsporters within the gills to trnslocte ions (Ahern et l., 1998; 69

92 Perry et l., 2003). It is possible tht these orgnisms increse the energy expenditure used to properly ionoregulte during periods of elevted slinity, while decresing the mount of energy used for reproduction nd growth. Once the ionoregultory cpbilities of the orgnism hve been exhusted, the overll function of the gill my cese (Greenwy P, 1979). Currently, the precise mechnism behind the sub-lethl effects of these ions is unknown. Determining tht mechnism my help identify the wy in which these ions interct nd contribute to the understnding nd prediction of their toxicity in environmentl situtions. Binry mixture toxicity for mjor nions nd ctions ws identified through titrtion experimentl design. The titrtion experimentl design is useful in identifying how the toxicity of one ion cn be influenced by the presence of second ion. In the present study, most ion binry mixtures resulted in n dditive response, mening tht the ddition of second ion did not lter the toxicity of the first. Previous studies hve suggested tht contminnts with similr concentrtion-response curves, resulting in dditive interctions, my hve similr modes of ction (vn der Geest et l., 2000). This could further exemplify tht these ions crete high-stress environment for freshwter orgnisms, where more energy is utilized in order to ionoregulte properly regrdless of the ions present. However, growth effects showed shift from dditivity when sulfte ws introduced with chloride (fixed sulfte with titrted chloride). Similr results hve been demonstrted in invertebrte species. More specificlly, decrese in toxicity occurred in Hyllel ztec (H. ztec) fter exposure to chloride (5 25 mg/l chloride) with the 70

93 ddition of fixed sulfte (Soucek, 2007). However, this sme llevition in toxicity did not occur in embryonic P. promels when exposed to fixed sulfte with incresing concentrtions of chloride (10 25 mg/l chloride) over fourteen-dys (Wng et l., 2016). In the fourteen-dy exposure, the chloride concentrtions utilized were much lower thn the current study. In fct, chloride concentrtions were only incresed from 0.14 to 0.71mM, wheres, in the present study, chloride ws between 0.05 nd 3.10mM. For this prticulr mixture, sodium is dded t 1:2 molr rtio due to sodium chloride nd sodium sulfte. An increse in the externl concentrtions of both chloride nd sodium hs been shown to increse the pssive loss of both ions (Bourguet et l., 1964). A pssive loss mens tht the energy required to remove excess sodium nd chloride from the plsm of freshwter fishes decreses, possibly mening tht more energy is vilble for other functions. So quite possibly, the llevition in toxicity of sulfte nd chloride is not relted to them directly, but more so the high concentrtion of sodium. However, this sme reduced toxicity did not occur in mixtures contining sodium bicrbonte nd sodium sulfte, which hd similr sodium concentrtions. So nother explntion my lie in sulfte directly. Although the exct mechnism of sulfte toxicity is mostly unknown, it my block the bility of chloride to gin ccess to the gill due to its lrge moleculr size, leding to n overll decrese in chloride toxicity. This my explin why the sme result did not occur for sulfte mixed with bicrbonte. Bicrbonte is excreted through chloride/bicrbonte exchnger locted on the picl membrne of the gill fter CO 2 wste is converted into H + nd HCO - 3 by the crbonic nhydrse enzyme to control cid-bse blnce within the orgnism. However, in this prticulr mixture, 71

94 bicrbonte increses while chloride remins low. The bility for the chloride/bicrbonte exchnger to remove bicrbonte from the chloride cell my be reduced from lck of chloride, s well s the high bicrbonte concentrtion. Clcium lso seemed to provide protective effect when introduced to mgnesium (fixed clcium with titrted mgnesium). The protective effect of clcium hs been previously described in lge, qutic plnts, snil species (Amerinn cumingi), the Northern trout gudgeon (Morurnd mogurnd) nd C. dubi (Mount et l., 1997; vn Dm et l., 2010, Mount et l., 2016; Wng et l., 2016). Mgnesium, lthough n importnt ion in mny cellulr processes such s ctivtion of enzymes nd protein synthesis, is lso reltively toxic t low concentrtions (Jhnen-Dechent nd Ketteler, 2012). As such, the control of mgnesium within the orgnism is especilly controlled (Cowey et l., 1997). In fct, it hs been reported in sticklebck (Gsterosteus culetus), Mozmbique Tilpi (Oreochromis mossmbicus) nd goldfish (Crssius urtus) tht s externl mgnesium concentrtions increse, plsm mgnesium remins reltively constnt (Wendelr Bong, 1978; Wendelr Bong et l., 1983; Olivereu et l., 1987). One of the key wys mgnesium exerts its toxicity is by blocking the uptke of clcium, eventully producing clcium deficiency (Hrdwick et l., 1991). However, s the concentrtion of clcium increses, it my compete with mgnesium t binding sites locted on the chloride cells of fish, ultimtely reducing mgnesium uptke nd toxicity (vn Dm et l., 2010). The fct tht sodium does not compete for the sme binding sites s clcium nd mgnesium my explin why clcium did not hve the sme meliortive effect on sodium s it did for mgnesium. 72

95 Attention hs been directed towrds the toxicity of mjor ions primrily due to n increse in these constituents from col-mining, col-fired power genertion, griculturl irrigtion, s well s mny other nthropogenic ctivities. Due to the numerous nthropogenic sources tht contribute to incresing ions, it hs become criticl to crete wtershed mngement prctices in order to mintin helthy qutic ecosystem. While the mechnisms behind their toxicity is, for the most prt, unknown, considerble efforts hve given rise to significnt dt illustrting their effects on survivl. Limited work hs been conducted to better understnd sub-lethl effects, such s growth nd reproduction. However, inconsistencies between previously reported dt, nd those presented in the present study indicte the complexities tht re ssocited with these ion mixtures. Vritions between concentrtion-response curves my demonstrte difference in the mechnisms tht produce sub-lethl effects. The development of predictive model for environmentl monitoring purposes, similr to the BLM model for metls toxicity, would help reduce the need for niml testing, s well s site-specific wter qulity criteri. Understnding some of the bsic mechnisms behind these toxic effects is fundmentl in creting ccurte predictive models. Conclusions Significnt differences between the toxicity of mjor ions, described by conductivity, further emphsize tht conductivity my not be the most ccurte predictor of ion toxicity. As such, the remining nlysis focused on specific ion concentrtions (mm). The chronic toxicity of mjor ions to Pimephles promels indicted n order of 2- toxicity s SO 4 Mg 2+ - > HCO 3 C 2+ > Cl - > N +. The most toxic of the six ions tested 73

96 included divlent ions, with bicrbonte not being significntly different from clcium. Binry mixture exposures concluded in mostly dditive interctions between the six ions; however, fixed sulfte with incresing concentrtions of chloride, s well s fixed clcium with incresing concentrtions of mgnesium resulted in less-thn-dditive interctions. These results demonstrte the complex nture of ion toxicity, especilly sub-lethl effects, nd the potentil difficulty with developing predictive models. Incorporting the bsic mechnisms of sub-lethl responses to ions nd ion mixtures will significntly increse the ccurcy nd vlidity of these models, s well s decrese the need for site-specific wter qulity criteri. 74

97 Figure 3.1. A grphicl representtion of the titrtion experimentl design. Where one ion (Ion A) is held t constnt bckground concentrtion, second ion (Ion B) increses in concentrtion cross tretments.

98 Ion-Only Additive Greter-thn-Additive Less-thn-Additive (Percent of Control) Ion Concentrtion (mm) Figure 3.2. Slope nlysis pproch to determine contminnt mixture interctions. Results indicte dditive, greterthn-dditive, nd less-thn-dditive. 76

99 Bicrbonte-Only Chloride-Only Sulfte-Only Growth (Percent of Control) Anion Concentrtion (mm) Figure 3.3. The effect of bicrbonte, chloride, nd sulfte on P. promels growth. Dt points represent the verge percent growth for ech tretment (± 95% confidence intervls (α = 0.05)). 77

100 Bicrbonte-Only Chloride-Only Sulfte-Only Survivl (Percent of Control) Anion Concentrtion (mm) Figure 3.4. The effect of bicrbonte, chloride, nd sulfte on P. promels survivl. Dt points represent the verge percent survivl for ech tretment (± 95% confidence intervls (α = 0.05)). 78

101 Clcium-Only Mgnesium-Only Sodium-Only Growth (Percent of Control) Ction Concentrtion (mm) Figure 3.5. The effect of clcium, mgnesium, nd sodium on P. promels growth. Dt points represent the verge percent growth for ech tretment (± 95% confidence intervls (α = 0.05)). 79

102 Clcium-Only Mgnesium-Only Sodium-Only Survivl (Percent of Control) Ction Concentrtion (mm) Figure 3.6. The effect of clcium, mgnesium, nd sodium on P. promels survivl. Dt points represent the verge percent survivl for ech tretment (± 95% confidence intervls (α = 0.05)). 80

103 Bicrbonte-Only Fixed Chloride; Titrted Bicrbonte Bicrbonte-Only Fixed Sulfte; Titrted Bicrbonte 140 Growth (Percent of Control) Bicrbonte Concentrtion (mm) Figure 3.7. The effect of chloride nd sulfte on bicrbonte toxicity on P. promels growth. Individul dt points denote verge percent growth for ech tretment (± 95% confidence intervls (α = 0.05)). 81

104 Chloride-Only Fixed Bicrbonte; Titrted Chloride Chloride-Only Fixed Sulfte; Titrted Chloride Growth (Percent of Control) Chloride Concentrtion (mm) Figure 3.8. The effect of bicrbonte nd sulfte on chloride toxicity on P. promels growth. Individul dt points denote verge percent growth for ech tretment (± 95% confidence intervls (α = 0.05)). 82

105 Sulfte-Only Fixed Chloride; Titrted Sulfte Sulfte-Only Fixed Bicrbonte; Titrted Sulfte Growth (Percent of Control) Sulfte Concentrtion (mm) Figure 3.9. The effect of chloride nd bicrbonte on sulfte toxicity on P. promels growth. Individul dt points denote verge percent growth for ech tretment (± 95% confidence intervls (α = 0.05)). 83

106 Bicrbonte-Only Fixed Chloride; Titrted Bicrbonte Bicrbonte-Only Fixed Sulfte; Titrted Bicrbonte Survivl (Percent of Control) Bicrbonte Concentrtion (mm) Figure The effect of chloride nd sulfte on bicrbonte toxicity on P. promels survivl. Individul dt points denote verge percent survivl for ech tretment (± 95% confidence intervls (α = 0.05)). 84

107 Chloride-Only Fixed Bicrbonte; Titrted Chloride Chloride-Only Fixed Sulfte; Titrted Chloride Survivl (Percent of Control) Chloride Concentrtion (mm) Figure The effect of bicrbonte nd sulfte on chloride toxicity on P. promels survivl. Individul dt points denote verge percent survivl for ech tretment (± 95% confidence intervls (α = 0.05)). 85

108 Sulfte-Only Fixed Chloride; Titrted Sulfte Sulfte-Only Fixed Bicrbonte; Titrted Sulfte Survivl (Percent of Control) Sulfte Concentrtion (mm) Figure The effect of chloride nd bicrbonte on sulfte toxicity on P. promels survivl. Individul dt points denote verge percent survivl for ech tretment (± 95% confidence intervls (α = 0.05)). 86

109 Clcium-Only Fixed Mgnesium; Titrted Clcium Clcium-Only Fixed Sodium; Titrted Clcium Growth (Percent of Control) Clcium Concentrtion (mm) Figure The effect of mgnesium nd sodium on clcium toxicity on P. promels growth. Individul dt points denote verge percent growth for ech tretment (± 95% confidence intervls (α = 0.05)). 87

110 Mgnesium-Only Fixed Sodium; Titrted Mgnesium Mgnesium-Only Fixed Clcium; Titrted Mgnesium Growth (Percent of Control) Mgnesium Concentrtion (mm) Figure The effect of sodium nd clcium on mgnesium toxicity on P. promels growth. Individul dt points denote verge percent growth for ech tretment (± 95% confidence intervls (α = 0.05)). 88

111 Sodium-Only Fixed Clcium; Titrted Sodium Sodium-Only Fixed Mgnesium; Titrted Sodium Growth (Percent of Control) Sodium Concentrtion (mm) Figure The effect of clcium nd mgnesium on sodium toxicity on P. promels growth. Individul dt points denote verge percent growth for ech tretment (± 95% confidence intervls (α = 0.05)). 89

112 Clcium-Only Fixed Mgnesium; Titrted Clcium Clcium-Only Fixed Sodium; Titrted Clcium Survivl (Percent of Control) Clcium Concentrtion (mm) Figure The effect of mgnesium nd sodium on clcium toxicity on P. promels survivl. Individul dt points denote verge percent survivl for ech tretment (± 95% confidence intervls (α = 0.05)). 90

113 Mgnesium-Only Fixed Sodium; Titrted Mgnesium Mgnesium-Only Fixed Clcium; Titrted Mgnesium Survivl (Percent of Control) Mgnesium Concentrtion (mm) Figure The effect of sodium nd clcium on mgnesium toxicity on P. promels survivl. Individul dt points denote verge percent survivl for ech tretment (± 95% confidence intervls (α = 0.05)). 91

114 Sodium-Only Fixed Clcium; Titrted Sodium Sodium-Only Fixed Mgnesium; Titrted Sodium Survivl (Percent of Control) Sodium Concentrtion (mm) Figure The effect of clcium nd mgnesium on sodium toxicity on P. promels survivl. Individul dt points denote verge percent survivl for ech tretment (± 95% confidence intervls (α = 0.05)). 92

115 Tble 3.1. EC 50 vlues for growth nd LC 50 vlues for survivl in P. promels. Estimtions derived for both conductivity nd specific ion concentrtion. Conductivity (µs/cm) Ion Concentrtion (mm) Ion EC 50 LC 50 EC 50 LC 50 Chloride 5354 c (4981, 5728) (9538, 10777) 50.7 d (47.1, 54.4) (90.9, 109.8) Sulfte 3841 b (3622, 4059) 4258 (3799, 4718) 19.2 (17.9, 20.5) 21.6 (19.0, 24.2) Bicrbonte 3029 (2663, (2858, 3925) 33.6 c (30.9, 36.2) 41.8 (34.5, 49.2) Clcium Mgnesium 8224 d (7554, 8893) 4313 b (3916, 4709) 6660 (5739, 7581) 4072 (3790, 4353) 38.7 c (35.5, 41.8) 24.5 b (23.7, Sodium c d (4981, 5728) (9265, 10890) (72.2, 75.6) Prentheses indicte 95% confidence intervls for ech EC 50 nd LC 50 (α = 0.05) (26.5, 35.7) 23.3 (21.4, 25.3) 95.6 (91.0, 100.2) Letters next to EC 50 vlues denote significnt differences, determined by non-overlpping confidence intervls. Differences were determined for the two mesurements independently. 93

116 Tble 3.2. The effect of sulfte nd chloride on bicrbonte toxicity to P. promels,b,c,d. Bicrbonte Binry Mixtures Slope (mm) EC 50 R 2 P-Vlue Effect Bicrbonte-Only Fixed Sulfte; Titrted Bicrbonte Bicrbonte-Only Fixed Chloride; Titrted Bicrbonte (51.9, 62.7) 22.3 (19.9, 24.8) 48.6 (43.5, 53.8) 18.8 (15.8, 21.9) Additive Additive Slope (mm) LC 50 R 2 P-Vlue Effect Bicrbonte-Only Fixed Sulfte; Titrted Bicrbonte Bicrbonte-Only Fixed Chloride; Titrted Bicrbonte Prenthesis indicte 95% confidence intervls (α = 0.05) 26.0 (19.5, 32.7) 28.2 (24.1, 32.2) 33.7 (25.2, 42.2) 20.6 (15.9, 25.3) Additive Additive b R 2 vlues were derived from the best-fit curve for ech concentrtion-response line. c p vlues indicte significnt differences between bicrbonte-only slope, nd the bicrbonte mixture slope. d ANCOVA ws performed for effect on growth (EC 50 ) nd survivl (LC 50 ). 94

117 Tble 3.3. The effect of bicrbonte nd sulfte on chloride toxicity to P. promels,b,c,d. Chloride Binry Mixtures Slope (mm) EC 50 R 2 P-Vlue Effect Chloride-Only Fixed Bicrbonte; Titrted Chloride Chloride-Only Fixed Sulfte; Titrted Chloride (44.8, 51.8) 26.4 (22.4, 30.3) 48.4 (44.8, 51.8) 83.1 (81.3, 84.8) Additive * Less-thn-Additive Slope (mm) LC 50 R 2 P-Vlue Effect Chloride-Only Fixed Bicrbonte; Titrted Chloride Chloride-Only Fixed Sulfte; Titrted Chloride Prenthesis indicte 95% confidence intervls (α = 0.05) 58.6 (53.2, 63.9) 52.4 (49.0, 55.8) 59.7 (54.3, 65.1) 91.8 (86.6, 96.9) * Less-thn-Additive * Less-thn-Additive b R 2 vlues were derived from the best-fit curve for ech concentrtion-response line. c p vlues indicte significnt differences between bicrbonte-only slope, nd the bicrbonte mixture slope. d ANCOVA ws performed for effect on growth (EC 50 ) nd survivl (LC 50 ). 95

118 Tble 3.4. The effect of chloride nd bicrbonte on sulfte toxicity to P. promels,b,c,d. Sulfte Binry Mixtures Slope (mm) EC 50 R 2 P-Vlue Effect Sulfte-Only Fixed Chloride; Titrted Sulfte Sulfte-Only Fixed Bicrbonte; Titrted Sulfte (24.6, 30.9) 13.0 (10.5, 15.5) 20.1 (17.8, 22.5) 15.0 (13.0, 17.0) Additive Additive Slope (mm) LC 50 R 2 P-Vlue Effect Sulfte-Only Fixed Chloride; Titrted Sulfte Sulfte-Only Fixed Bicrbonte; Titrted Sulfte Prenthesis indicte 95% confidence intervls (α = 0.05) 28.1 (21.0, 35.3) 14.8 (12.8, 16.9) 20.1 (19.4, 20.9) 8.05 (5.79, 10.3) Additive Additive b R 2 vlues were derived from the best-fit curve for ech concentrtion-response line. c p vlues indicte significnt differences between bicrbonte-only slope, nd the bicrbonte mixture slope. d ANCOVA ws performed for effect on growth (EC 50 ) nd survivl (LC 50 ). 96

119 Tble 3.5. The effect of mgnesium nd sodium on clcium toxicity to P. promels,b,c,d. Clcium Binry Mixtures Slope (mm) EC 50 R 2 P-Vlue Effect Clcium-Only Fixed Mgnesium; Titrted Clcium Clcium-Only Fixed Sodium; Titrted Clcium (26.0, 35.9) 21.4 (24.6, 32.7) 45.4 (39.9, 50.9) 13.3 (6.99, 19.6) Additive Additive Slope (mm) LC 50 R 2 P-Vlue Effect Clcium-Only Fixed Mgnesium; Titrted Clcium Clcium-Only Fixed Sodium; Titrted Clcium Prenthesis indicte 95% confidence intervls (α = 0.05) 28.7 (24.6, 32.7) 37.2 (29.3, 45.1) 38.7 (33.8, 43.7) 24.9 (20.9, 28.9) Additive Additive b R 2 vlues were derived from the best-fit curve for ech concentrtion-response line. c p vlues indicte significnt differences between bicrbonte-only slope, nd the bicrbonte mixture slope. d ANCOVA ws performed for effect on growth (EC 50 ) nd survivl (LC 50 ). 97

120 Tble 3.6. The effect of sodium nd clcium on mgnesium toxicity to P. promels,b,c,d. Mgnesium Binry Mixtures Slope (mm) EC 50 R 2 P-Vlue Effect Mgnesium-Only Fixed Sodium; Titrted Mgnesium Mgnesium-Only Fixed Clcium; Titrted Mgnesium (13.0, 17.4) 15.4 (8.42, 22.5) 16.9 (15.2, 18.7) 52.1 (40.1, 64.2) Additive * Less-thn-Additive Slope (mm) LC 50 R 2 P-Vlue Effect Mgnesium-Only Fixed Sodium; Titrted Mgnesium Mgnesium-Only Fixed Clcium; Titrted Mgnesium Prenthesis indicte 95% confidence intervls (α = 0.05) 14.9 (14.3, 15.5) 12.8 (8.54, 17.1) 14.8 (13.9, 15.6) 17.7 (15.4, 20.1) * Less-thn-Additive * Less-thn-Additive b R 2 vlues were derived from the best-fit curve for ech concentrtion-response line. c p vlues indicte significnt differences between bicrbonte-only slope, nd the bicrbonte mixture slope. d ANCOVA ws performed for effect on growth (EC 50 ) nd survivl (LC 50 ). 98

121 Tble 3.7. The effect of clcium nd mgnesium on sodium toxicity to P. promels,b,c,d. Sodium Binry Mixtures Slope (mm) EC 50 R 2 P-Vlue Effect Sodium-Only Fixed Clcium; Titrted Sodium Sodium-Only Fixed Mgnesium; Titrted Sodium (47.1, 55.2) 20.2 (8.6, 31.8) 68.3 (54.8, 81.8) 67.8 (51.7, 83.8) Additive Additive Slope (mm) LC 50 R 2 P-Vlue Effect Sodium-Only Fixed Clcium; Titrted Sodium Sodium-Only Fixed Mgnesium; Titrted Sodium Prenthesis indicte 95% confidence intervls (α = 0.05) 62.9 (55.5, 70.4) 64.8 (59.6, 69.8) 74.0 (66.4, 81.8) 61.1 (53.6, 68.7) Additive Additive b R 2 vlues were derived from the best-fit curve for ech concentrtion-response line. c p vlues indicte significnt differences between bicrbonte-only slope, nd the bicrbonte mixture slope. d ANCOVA ws performed for effect on growth (EC 50 ) nd survivl (LC 50 ). 99

122 Tble 3.8. Ion-only slope comprisons for growth nd survivl of P. promels. Ion-only concentrtion-response lines produced for comprison during binry mixture exposures. Growth P-Vlue Survivl Bicrbonte Chloride Sulfte * Clcium Mgnesium Sodium * An sterisk (*) indictes significnt differences between slopes. 100

123 References Ahern GA, Duerr JM, Zhung Z, Brown RJ, Aslmkhn A, Killebrew DA Ion trnsport processes of crustcen epithelil cells. Physiologicl nd Biochemicl Zoology. 72: Ayson FG, Kneko T, Hsegw S, Hirno T Development of mitochondrion-ric cells in the yolk-sc membrne of embryos nd lrve of tilpi, Oreochromis mossmbicus, in freshwter nd sltwter. Journl of Experimentl Zoology. 272: h Bourguet J, Lhlou B, Metz J Modifictions experimentles de I équilibre hydrominérl et osmoregultion chez Crssius urtus. Generl nd Comprtive Endocrinology. 4: Cowey CB, Knox D, Adron JW, George S, Pirie B The production of renl clcinosis by mgnesium deficiency in rinbow trout (Slmo girdneri). British Journl of Nutrition. 38: Dickerson KK, Hubert WA, Bergmn HL Toxicity ssessment of wter from lkes nd wetlnds receiving irrigtion drinwter. Environmentl Toxicology nd Chemistry. 15: Elphick JRF, Bergh KD, Biley HC Chronic toxicity of chloride to freshwter species: effects of hrdness nd implictions for wter qulity guidelines. Environ. Toxicol. Chem. 30: Elphick JR, Dvies M, Gilron G, Cnri EC, Lo B, Biley HC. 2011b. An qutic toxicologicl evlution of sulfte: the cse for considering hrdness s modifying fctor in setting wter qulity guidelines. Environ. Toxicol. Chem. 30: Erickson AJ, Mount DR, Highlnd TL, Hockett JR, Hoff DJ, Jenson CT, Norberg-King TJ, Peterson KN The cute toxicity of mjor ion slts to Ceriodphni dubi. II. Empiricl reltionships in binry slt mixtures. Environmentl Toxicology nd Chemistry. 36: Erickson AJ, Mount DR, Highlnd TL, Hockett JR, Hoff DJ, Jenson CT, Norberg-King TJ, Peterson KN The cute toxicity of mjor ion slts to Ceriodphni dubi. III. Mthemticl models for mixture toxicity. Environmentl Toxicology nd Chemistry. 37: Frg AM, Hrper DD The chronic toxicity of sodium bicrbonte, mjor component of col bed nturl gs produced wters. Environ. Toxicol. Chem. 33:

124 Greenwy, P Freshwter invertebrtes: In G.M.O. Mloiy ed. Comprtive Physiology of Osmoregultion in Animls. Acdemic, London, UK. Pg Hrper DD, Frg AM, Skr D Acute toxicity of sodium bicrbonte, mjor component of col bed nturl gs produced wters, to 13 qutic species s defined in the lbortory. Environ Toxicol Chem. 33: Jhnen-Dechent W, Ketteler M Mgnesium Bsics. Clinicl Kidney Journl. 5: i3-i14. Ktoh F, Shimizu A, Uchid K, Kneko T Shift of chloride cell distribution during erly life stges in sewter-dpted killifish, Fundulus heteroclitus. Zoologicl Sciences. 17: Kennedy AJ, Cheery DS, Currie RJ Evlution of Ionic Contribution to the Toxicity of col-mine effluent using Ceriodphni dubi. Archives for Environmentl Contmintion nd Toxicology. 49: Kennedy AJ, Cherry DS, Zipper CE Evlution of ionic contribution to the toxicity of col-mine effluent using Ceriodphni dubi. Archives of Environmentl Contmintion nd Toxicology. 49: Hrdwick LL, Jones MR, Brutbr N, Lee DBN Mgnesium bsorption: Mechnisms nd the influence of Vitmin D, clcium nd phosphte. Journl of Nutrition. 121: Kunz JL, Conley JM, Buchwlter DB, Norberg-King TJ, Kemble NE, Wng N, Ingersoll CG Use of reconstituted wters to evlute effects of elevted mjor ions ssocited with mountintop col mining on freshwter invertebrtes. Environmentl Toxicology nd Chemistry. 32: Lsier PJ, Hrdin IR Observed nd predicted reproduction of Ceriodphni dubi exposed to chloride, sulfte, nd bicrbonte. Environ. Toxicol. Chem. 29: Mount DR, Gulley DD, Hockett JR, Grrison TD, Evns JM Sttisticl models to predict the toxicity of mjor ions to Ceriodphni dubi, Dphni mgn, nd Pimephles promels (fthed minnows). Environ Toxicol Chem. 16: Mount DR, Erickson RJ, Highlnd TL, Hockett JR, Hoff DJ, Jenson CT, Norberg-King TJ, Peterson KN, Polske ZM The cute toxicity of mjor ion slts to Ceriodphni dubi. I. Influence of bckground wter chemistry. Environmentl Toxicology nd Chemistry. 35:

125 Olivereu M, Oliveru JM, Lmbert JF Differentil effect of sodium nd mgnesium on the pituitry of goldfish dpted to clcium-free environments. Act Zoologicl. 68: Perry SF, Shhsvrni A, Georglis T, By M Chnnels, pumps nd exchngers in the gill nd kidney of freshwter fishes: Their role in ionic nd cid-bse regultion. Journl of Experimentl Zoology. 300: Pond GJ, Pssmore ME, Borsuk FA, Reynolds L, Rose CJ Downstrem effects of mountintop col mining: compring biologicl conditions using fmily- nd genus-level mcroinvertebrte biossessment tools. Journl of North Americn Benthologicl Society. 27: Soucek DJ, Kennedy AJ Effects of hrdness, chloride, nd cclimtion on the cute toxicity of sulfte to freshwter invertebrtes. Environ Toxicol Chem. 24: Soucek DJ Comprison of hrdness- nd chloride-regulted cute effects of sodium sulfte on two freshwter crustcens. Environmentl Toxicology nd Chemistry. 26: Soucek DJ, Linton TK, Trr CD, Dickinson A, Wickrmnyke N, Delos CG, Cruz LA Influence of wter hrdness nd sulfte on the cute toxicity of chloride to sensitive freshwter invertebrtes. Environ Toxicol Chem. 30: Timpno AJ, Schoenholtz SH, Zipper CE, Soucek DJ Isolting effects of totl dissolved solids on qutic life in centrl Applchin colfield strems. Proceedings, Ntionl Meeting of the Americn Society of Mining nd Reclmtion. P U.S. EPA Short-term methods for estimting the chronic toxicity of effluents nd receiving wters to freshwter orgnisms, fourth edition. EPA 824-R Wshington, DC, US. U.S. Environmentl Protection Agency Ntionl recommended wter qulity criteri. EPA 800-R Wshington, DC, USA. U.S. EPA A field-bsed qutic life benchmrk for conductivity in Centrl Applchin Strems. Office of Reserch nd Development, Ntionl Center for Environmentl Assessment, Wshington, DC. EPA/600/R-10/023F. vn Dm RA, Hogn AC, McCullough CD, Houston MA, Humphrey CL, Hrford AJ Aqutic toxicity of mgnesium sulfte, nd the influence of clcium, in very low ionic concentrtion wter. Environmentl Toxicology nd Chemistry. 29:

126 Vn der Geest HG, Greve GD, Boivin ME, Krk MHS, vn Gestel CAM Mixture toxicity of copper nd dizinon to lrve of the myfly (Ephoron virgo) judging dditivity t different effect levels. Environmentl Toxicology nd Chemistry. 19: Wng N, Dormn RA, Ingersoll CG, Hrdesty DK, Brumbugh WG, Hmmer EJ, Buer CR, Mount DR Acute nd chronic toxicity of sodium sulfte to four freshwter orgnisms in wter-only exposures. Environmentl Toxicology nd Chemistry. 35: Wendelr Bong SE The effects of chnges in externl sodium clcium nd mgnesium concentrtions on prolctin cells, skin nd plsm electrolytes of Gsterosteus culetus. Generl Comprtive Endocrinology. 34: Wendelr Bong, SE, Lowik CJM, Vn der Meij JCA Effects of externl Mg2+ nd C2+ on brnchil osmotic wter permebility nd prolctin secretion in the teleost fish Srotherodon mossmbicus. Generl Comprtive Endocrinology. 52:

127 CHAPTER FOUR THE CHRONIC TOXICITY OF SINGLE IONS AND BINARY ION MIXTURES OF AN INVERTEBRATE SPECIES (C. dubi) AND A VERTEBRATE SPECIES (P. promels): A COMPARISON Species Comprison An initil comprison ws mde by plotting the reltionship between effective concentrtions tht resulted in either 50% decrese in growth for P. promels or 50% decrese in reproduction for C. dubi. The initil plot reveled low correltion nd R- squre vlue of only (Figure 4.1). Although the R-squre vlue ws low, positive trend ws observed; s the EC 50 vlue incresed for P. promels, it lso tended to increse for C. dubi. The estimtes were then seprted into EC 50 vlues derived from single-ion exposures, nd those derived from binry mixture exposures (Figure 4.2). A liner regression for single-ion exposures produced much higher R-squre vlue of 0.789, indicting strong reltionship between the ion toxicity in the two orgnisms. In ll cses, the EC 50 vlues estimted for P. promels were much lrger, suggesting tht the vertebrte species is less sensitive to nd my be better t controlling the influx of elevted ions thn the invertebrte species. These results corroborte similrly with those previously published for cute ion toxicity (Mount et l., 1997; Kennedy et l., 2003; Wng et l., 2016). In the present study, the smllest difference mong EC 50 estimtes between the two orgnisms occurred with sulfte, wheres the lrgest difference included sodium nd chloride. Although the EC 50 estimtes were very different, the slopes for most ions tended to be similr, prticulrly for chloride, clcium, mgnesium, nd sodium 105

128 (Tble 4.1). This similrity my indicte tht even though P. promels my tolerte lrger ion concentrtions, they respond similrly to certin threshold s C. dubi with chnges in ion concentrtion on milli-molr bsis. Initil ion toxicity for C. dubi resulted in n overll toxicity grdient of C 2+ Mg 2+ SO 2-4 > HCO - 3 > Cl - > N +. Although this ws slightly different thn tht described for P. promels (SO 2-4 > Mg 2+ > HCO - 3 C 2+ > Cl - > N + ), there re some similrities. For exmple, C. dubi tended to be more sensitive to divlent ions (clcium, mgnesium, sulfte), while less sensitive to monovlent ions (bicrbonte, chloride, sodium). Generlly, this sme trend holds true to P. promels in tht sulfte nd mgnesium re mong the most toxic, followed by bicrbonte, which is not significntly different from clcium. Furthermore, both orgnisms were less ffected by chloride nd sodium, possibly indicting tht these orgnisms re well dpted to mnging fluctutions in the concentrtion of these ions. Binry mixture effects showed the lrgest difference in nion responses between the two species. Similrities between P. promels nd C. dubi resulted from both titrted sulfte mixtures (dditive) nd fixed sulfte with titrted chloride (less-thndditive). Wheres other nion binry mixtures demonstrted dditivity for P. promels, results from C. dubi exposures reveled tht titrted bicrbonte mixtures result in dditionl meliortive effects, while fixed bicrbonte with titrted chloride demonstrted n enhnced toxic response (greter-thn-dditive) (Tble 4.2). Unlike nion results, ction binry mixtures results were similr for both species (Tble 4.3). All ction binry mixtures resulted in dditivity, with the exception of fixed 106

129 clcium with titrted mgnesium, which resulted in less-thn-dditive response. These similrities indicte tht sub-lethl mixtures of clcium, mgnesium, nd sodium hve the sme effects on C. dubi reproduction s they do for P. promels growth. Although ion-only EC 50 vlues showed strong correltion between the two species, plot of binry mixture EC 50 vlues did not (R 2 : 0.448)(Figure 4.2). Further seprting these EC 50 vlues into ction nd nion binry mixture results still did not give rise to ny definite trends (Figure 4.3). Becuse C. dubi nd P. promels presented similr trends in toxicity with regrds to ction binry mixtures, elucidting trends through effective concentrtions my not be the most suitble. For this reson, determining species response trends bsed on concentrtion-response slopes for ech ion nd ion mixture my hve better correltions. Plots of concentrtion-response slopes for ech species were plotted initilly in one plot (Figure 4.4). A distinct trend, however, ws not immeditely pprent. Further seprtion into single-ion nd binry mixture slopes, s well s ction nd nion binry mixture slopes did not revel ny dditionl informtion (Figure 4.5; Figure 4.6). The internl environment of freshwter orgnisms is hyperosmotic to the surrounding medi, mening tht the externl environment contins much lower ion concentrtion thn the orgnism itself. Nturlly, ions wnt to move from n re of bundnce to n re of insufficiency, which cretes problem for these hyperosmotic orgnisms. Freshwter orgnisms must utilize ctive trnsport of ions cross the gill, intestinl trct, nd kidney in order to mintin the proper ion rtios needed for cellulr functions. The gills of freshwter vertebrte species, fish in prticulr, re comprised of 107

130 mitrochondri-rich (MR) cells tht possess series of ion chnnels, ion exchngers, nd ntiporters tht id in ion uptke (Perry et l., 2003). The gill structure of fish hs been studied extensively (Perry, 1997; Perry nd Fryer, 1997; Goss et l., 1998; Perry et l., 2003; Evns et l., 2005; Eddy, 2006). However, less is understood bout the prticulr structure nd function of the Dphni gill. Epithelil cells within the gills of Dphni species re comprised of drk cells, which contin bundnt mitochondri, nd light cells, which contin fewer mitochondri. For the most prt, the drk cell types re believed to be primrily responsible for ionoregultion within the Cldocern order (Kikuchi, 1983). The drk cell types function by ctively bsorbing N + nd Cl - from their surrounding environment, but the presence of more intricte chnnels nd ion exchngers is less understood (El-Deeb Ghzy et l., 2009). One distinctly chrcteristic pprtus of Dphni is their shell, or crpce. The crpce hs been linked to offering protection from ion chnges in the outside environment by providing both n impermeble surfce round the body, s well s ctively prticipting in ionoregultion (Ebert, 2005; El-Deeb Ghzy et l., 2009). Furthermore, C. dubi hve open circultory systems, where hemolymph is llowed to flow freely throughout the body cvity (Ebert, 2005). This is different from their vertebrte counterprts, where blood is comprtmentlized. Most ion combintions resulted in n dditive interction for both species. Additivity ws concluded if the concentrtion-response lines produced sttisticlly similr slopes. Comprble concentrtion-response curves hve been suggested to indicte similr modes-of-ction or mechnisms of toxicity between compounds (vn der Geest et l., 2000). 108

131 Orgnisms utilize energy to mintin the biologicl functions necessry to ensure ecologicl success, through functions such s growth nd reproduction. Freshwter orgnisms prticulrly rely on ionoregultion to control ion movement into nd out of the body, which lso requires energy in the form of denosine triphosphte (ATP). If the ion concentrtion in the externl medi exceeds wht is physiologiclly mngeble for the orgnism, they my need to llocte more energy towrds ionoregultion nd reduce the energy for other functions needed in order to grow nd reproduce. For exmple, it hs been demonstrted in C. dubi tht reduction in energy linked to decresed filter feeding rtes occurs following exposure to sodium sulfte (Soucek, 2007). Most ion chnnels nd pumps require energy to trnsport ions ginst their concentrtion grdient, from n re of low concentrtion to high concentrtion (Perry et l., 2003). If the concentrtion of the externl medi increses to concentrtion resulting in sub-lethl effects, it my indicte tht the freshwter orgnisms re llocting more energy to id in proper ionoregultion thn for other energy dependent functions (Frg nd Hrper, 2014). This would indicte tht ll ions hve the sme mode-of-ction, nd tht energy deficiency is the min route of toxicity. The fct tht the toxicity dt for both orgnisms resulted in mostly dditive responses could indicte tht the route of toxicity, or energy deficiency, is similr between the two freshwter species. Binry nion mixture toxicity resulted in mostly less-thn-dditive responses for C. dubi. This less-thn-dditive response is unusul becuse invertebrte species tend to hve greter toxic responses compred to tht of their more physiologiclly complex counterprt, P. promels. Unfortuntely, more is 109

132 known ionoregultion in freshwter fish nd little is known regrding the ionoregultory cpbilities of Cldocern species. In freshwter fish, crbonic nhydrse is the enzyme importnt in breking down CO 2 wste into H + nd HCO - 3 ions, which re ultimtely excreted by the orgnisms (refer to Figure 1.1). It is then ssumed tht bicrbonte is exchnged for chloride on the picl membrne vi Cl - /HCO - 3 exchnger, the ction of which is controlled by the ctivity of V-type ATPse locted on the bsolterl membrne of mitochondrion-rich cells (Piermrini nd Evns, 2001). When the bicrbonte concentrtion in the externl environment increses, it hs been demonstrted tht the N + /K + -ATPse specific ctivity decreses in P. promels (Frg nd Hrper, 2014b). A decrese in N + /K + -ATPse ctivity could result in decrese in Cl - - /HCO 3 exchnge rte. If this were to hppen, crbonic nhydrse my become inhibited due to buildup of bicrbonte ions, ultimtely stopping the brekdown of CO 2 wste. Higher concentrtions of CO 2 wste in the plsm of these freshwter fish my contribute to decrese in blood ph nd eventully deth possibly due to hypercpni nd cidosis. Future Predictive Modeling Extensive work hs been performed to develop predictive models of cute ion toxicity for field ppliction purposes. After ssessing the cute toxicity of over 2,900 ion combintions to Ceriodphni dubi, Dphni mgn, nd Pimephles promels, Mount et l. (1997) developed multivrite logistic regression models for ech individul species. Initilly, regression equtions were developed for single ions pirs (i.e., C2+ nd Cl- s CCl2). These models dvnced to incorporte either two ctions nd one nion (i.e., N +, Mg 2+, Cl - s NCl nd MgCl 2 ) or one ction with two nions (i.e., N +, 110

133 Cl -, SO 2-4 s N 2 SO 4 nd NCl). These more dvnced models exhibited poor predictbility, in prticulr for C. dubi. It ws determined tht the dvnced models were underestimting the toxicity of solutions contining more thn one ction, specificlly for solutions with two sources of chloride. For exmple, similr toxicities occurred for single slt solutions contining NCl nd CCl 2 ; however, when in combintion, the toxicity of chloride decresed. These results implied tht multiple ctions in solution hd n importnt effect on the overll toxicity of tht solution, which ws not incorported into the model prediction eqution. This discovery prompted the inclusion of NumCt vrible, which ws simply the number of ctions present. Finl models produced R-squre vlues of for 24-hour LC 50 vlues. Conversely, P. promels cute toxicity estimtes showed tht the NumCt vrible ws not significnt fctor in the prediction equtions. R-squred vlues for P. promels were between nd Field-bsed testing reveled tht models developed by Mount et l. (1997) performed well when observed toxicity of irrigtion drin wter sites ws similr to predicted toxicity for C. dubi (Dickerson et l., 1996). This ws dissimilr to reports indicting tht P. promels toxicity ws over-predicted (Tietge et l., 1997). Since then, more dvnced models hve been expnded for C. dubi to include greter understnding of the roles in which C 2+ nd Mg 2+ ply in the meliortive effect on ion combintions, with little work being done to strengthen the predictbility for P. promels (Erickson et l., 2018). In the present study, similrities between the chronic toxicity of P. promels nd C. dubi my suggest tht prediction models my be built from combined species dt, 111

134 rther thn one model for ech orgnism, incresing model pplicbility. These similrities further suggest tht P. promels growth my be useful predictor of decresed C. dubi reproduction. Currently, mthemticl models predicting cute ion toxicity hve been developed mostly incorporting LC 50 vlues nd impcts of multiple ctions. However, chronic toxicity dt presented in this study demonstrte tht nion toxicity my hve further significnce in model development. The resons for these differences my lie in the mechnisms behind which these ions exert their sub-lethl vs. lethl effects on freshwter orgnisms. For tht reson, incorporting physiologicl vribles such s chnges in enzymtic ctivity, osmotic pressure, blood ph nd/or whole orgnism ion composition my increse the ccurcy of dditionl models. The incorportion of these chrcteristics would help develop model similr to Physiologiclly Bsed Phrmcokinetic (PBPK) model. Chrcterizing the effect of elevted mjor ions on these spects of biologicl functions would be difficult to complete on smller orgnisms, such s C. dubi. However, becuse of the similrities described in the present study between C. dubi nd P. promels, the mechnistic response of P. promels my be comprble to effects seen for C. dubi. These types of PBPK models seprte the orgnism into multiple comprtments nd help understnd the flow of ions through the ionoregultory importnt orgns within fish, such s the gill, intestinl trct, integument, nd kidney. The toxicity of mjor ions, s described bove, is very complex system. It hs been demonstrted repetedly tht ech ion individully produces its own toxic response for multiple endpoints, nd multiple orgnisms. Ion mixtures hve lso been shown to 112

135 not necessrily ct in simply dditive mnner (Mount et l., 1997; Wng et l., 2016; Soucek et l., 2011; Soucek nd Kennedy, 2005; Mount et l., 2016; Erickson et l., 2017). This implies tht utilizing comprehensive prmeters, such s conductivity or TDS, to predict or estimte toxicity my not be the most suitble. Additionlly, strong ccurte models could lso reduce the need for site-specific regultions, nd ultimtely reduce the time nd expense required for such significnt endevor. 113

136 25 Ceriodphni dubi EC50 (mm) y = 0.161x R² = Pimephles promels EC50 (mm) Figure 4.1. The correltion of EC 50 vlues estimted for C. dubi reproduction nd P. promels growth. Dt points represent EC 50 vlues estimted for ll ion-only nd binry mixture exposures (R 2 : 0.485). 114

137 C. dubi EC50 Vlues (mm) y = x R² = y = x R² = P. promels EC50 Vlues (mm) Ion-Only Binry Mixtures Figure 4.2. The correltion of EC 50 vlues estimted for C. dubi reproduction nd P. promels growth seprted by exposure type. Dt points represent EC 50 vlues estimted for nd seprted by ll single-ion nd binry mixture exposures. The liner regression for ion-only exposures indictes n R 2 of 0.789, wheres the binry mixture R 2 is

138 16 C. dubi EC50 Vlues (mm) Anion Binry Mixtures Ction Binry Mixtures P. promels EC50 Vlues (mm) Figure 4.3. The correltion of EC 50 vlues estimted for C. dubi reproduction nd P. promels growth seprted by binry mixture type. Dt points represent EC 50 vlues estimted for nd seprted by ction nd nion binry mixture exposures. No distinct trends between the two types of binry mixtures were noted. 116

139 Pimephles promels Slopes Ceriodphni dubi Slopes Figure 4.4. The correltion of the concentrtion-response slopes for C. dubi reproduction nd P. promels growth. Dt points represent slopes estimted for ll single-ion nd binry mixture exposures. 117

140 P. promels Slope C. dubi Slope -12 Ion-Only Binry Mixtures Figure 4.5. The correltion of the concentrtion-response slopes for C. dubi reproduction nd P. promels growth seprted by exposure type. Dt points represent slopes estimted for nd seprted by ll single-ion nd binry mixture exposures. 118

141 y = x R² = y = x R² = P. promels Slope C. dubi Slope -12 Anion Binry Mixtures Ction Binry Mixtures Figure 4.6. The correltion of the concentrtion-response slopes for C. dubi reproduction nd P. promels growth seprted by binry mixture type. Dt points represent slopes estimted for nd seprted by ction nd nion binry mixture exposures. No distinct trends between the two types of binry mixtures were noted. 119

142 Tble 4.1. A summry of the chronic toxicity of ion-only exposures to C. dubi nd P. promels. Results for C. dubi indicte effects on reproduction, while P. promels results indicte effects on growth. Dt re previously described in Chpter Two nd Chpter Three. C. dubi P. promels Ion Slope EC 50 (mm) R 2 Slope EC 50 (mm) R 2 Chloride (-3.41, -2.75) 16.2 (15.4, 17.1) (-4.51, -2.15) 50.7 (47.1, 54.4) Sulfte (-5.30, -4.57) 12.0 (11.5, 12.6) (-13.8, -4.39) 19.2 (17.9, 20.5) Bicrbonte (-17.8, -11.9) 13.7 (13.3, 14.1) (-1.70, -1.38) 33.6 (30.9, 36.2) Clcium (-6.39, -4.27) 9.74 (8.54, 10.9) (-11.8, -6.19) 38.7 (35.5, 41.8) Mgnesium (-8.66, -5.48) 10.4 (9.50, 11.5) (-18.2, -2.19) 24.5 (23.7, 25.7) Sodium (-6.66, -3.13) 23.6 (21.2, 26.1) (-9.65, -4.59) 73.9 (72.2, 75.6)

143 Tble 4.2. A summry of the chronic toxicity of binry nion mixtures to C. dubi nd P. promels. Results for C. dubi indicte effects on reproduction, while P. promels results indicte effects on growth. Dt re previously described in Chpter Two nd Chpter Three. C. dubi P. promels Fixed Ion Titrted Ion Slope P-Vlue Effect Slope P-Vlue Effect Bicrbonte Chloride (-4.05, -1.45) 0.004* Greter-thn-Additive (-2.38, -1.58) Additive Sulfte Chloride (-2.87, -2.25) 0.033* Less-thn-Additive (-2.44, -2.03) 0.018* Less-thn-Additive Chloride Sulfte (-9.08, -4.41) Additive (-4.23, -2.02) Additive Bicrbonte Sulfte (-5.83, -3.55) Additive (-6.89, -3.39) Additive Chloride Bicrbonte (-13.5, -7.14) 0.007* Less-thn-Additive (-1.76, -1.30) Additive Sulfte Bicrbonte (-12.2, -3.91) 0.010* Less-thn-Additive (-2.02, -1.59) Additive 121

144 Tble 4.3. A summry of the chronic toxicity of binry ction mixtures to C. dubi nd P. promels. Results for C. dubi indicte effects on reproduction, while P. promels results indicte effects on growth. Dt re previously described in Chpter Two nd Chpter Three. C. dubi P. promels Fixed Ion Titrted Ion Slope P-Vlue Effect Slope P-Vlue Effect Mgnesium Clcium (-13.7, -7.78) Additive (-4.20, ) Additive Sodium Clcium (-14.2, -6.32) Additive (-3.63, ) Additive Sodium Mgnesium (-19.2, -9.25) Additive (-7.95, 1.74) Additive Clcium Mgnesium (-7.69, -0.28) 0.004* Less-thn-Additive (-3.52, 0.739) 0.009* Less-thn-Additive Clcium Sodium (-9.03, -2.82) Additive (-2.05, ) Additive Mgnesium Sodium (-10.9, -3.93) Additive (-1.56, ) Additive 122

145 References Ebert D Chpter Two: Introduction to Dphni Biology in Ecology, Epidemiology, nd Evolution of Prsitism in Dphni. Bethesd, MD. Ntionl Cente for Biotechnology nd Informtion (US). r Piermrini PM, Evns DH Immunochemicl nlysis of the vcuolr proton- ATPse B-subunit in the gills of the euryhline stingry (Dsytis Sbin): effects of slinity nd reltion to N+/K+-ATPse. Journl of Experimentl Biology. 204: Kikuchi S The fine structure of the gill epithelium of freshwter fle, Dphni mgn (Crustce: Phyllopod) nd chnges ssocited with cclimtion to vrious slinities. Cell nd Tissue Reserch. 229: El-Deeb Ghzy MM, Hbshy MM, Koss FI, Mohmmdy EY Effects of slinity on survivl, growth nd reproduction of the wter fle, Dphni mgn. Nture nd Science. 7: Perry SF, Fryer JN Proton pumps in the fish gill nd kidney. Fish Physiology nd Biocehmistry. 17: Perry SF The chloride cell: Structure nd function in the gills of freshwter fishes. Annul Review of Physiology. 59: Goss GG, Perry SF, Fryer JN, Lurent P Gill morphology nd cid-bse regultion in freshwter fishes. Comprtive Biochemistry nd Physiology. 119A: Erickson RJ, Mount DR, Highlnd TL, Hockett JR, Hoff DJ, Jenson CT, Norberg-King TJ, Peterson KN The cute toxicity of mjor ion slts to Ceriodphni dubi. III. Mthemticl models for mixture toxicity. Environmentl Toxicology nd Chemistry. 37: Evns DH, Piermrini PM, Choe KP The multifunctionl fish gill: Dominnt sit of gs exchnge, osmoregultion, cid-bse regultion, nd excretion of nitrogenous wste. Physiology Review. 85: e vn der Geest HG, Greve GD, Boivin ME, Krk MHS, vn Gestel CAM Mixture toxicity of copper nd dizinon to lrve of the myfly (Ephoron virgo) judging dditivity t different effect levels. Environmentl Toxicology nd Chemistry. 19: Perry SF, Shhsvrni A, Georglis T, By M, Furimsky M, Thoms SLY Chnnels, pumps nd exchngers in the gill nd kidney of freshwter fishes: their role in ionic nd cid-bse regultion. Journl of Experimentl Zoology. 300A:

146 Frg AM, Hrper DD A review of environmentl impcts of slts from produced wters on qutic resources. Interntionl Journl of Col Geology. 126: Metz J Trnsport of ions nd wter cross the epithelium of fish gills In Lung Liquids. Americn Elsevier, New York, NY. pg Frg AM, Hrper DD. 2014b. The chronic toxicity of sodium bicrbonte, mjor component of col bed nturl gs produced wters. Environmentl Toxicology nd Chemistry. 33: Mount DR, Gulley DD, Hockett JR, Grrison TD, Evns JM Sttisticl models to predict the toxicity of mjor ions to Ceriodphni dubi, Dphni mgn, nd Pimephles promels (fthed minnows). Environmentl Toxicology nd Chemistry. 16: Kennedy AJ, Chrry DS, Currie RJ Field nd lbortory ssessment of colprocessing effluent in the Leding Creek Wtershed, Meigs Co., Ohio. Archives of Environmentl Contmintion nd Toxicology. 44: Wng N, Dormn RA, Ingersoll CG, Hrdesty DK, Brumbugh WG, Hmmer EJ, Buer CR, Mount DR Acute nd chronic toxicity of sodium sulfte to four freshwter orgnisms in wter-only exposures. Environmentl Toxicology nd Chemistry. 35: Eddy FB Drinking in juvenile Atlntic slmon (Slmo slr L.) in response to feeding nd ctivtion of the endogenous renin-ngiotensin system. Comprtive Biochemistry nd Physiology, Prt A. 148: Tietge JE, Hockett JR, Evns JM Mjor ion toxicity of six produced wters to three freshwter species: Appliction of ion toxicity models nd tie procedures. Environmentl Toxicology nd Chemistry. 16: Soucek DJ Bioenergetic effects of sodium sulfte on the freshwter crustcen, Ceriodphni dubi. Ecotoxicology. 16: Dickerson KK, Hubert WA, Bergmn HL Toxicity ssessment of wter from lkes nd wetlnds receiving irrigtion drinwter. Environmentl Toxicology nd Chemistry. 15: Soucek DJ, Linton TK, Trr CD, Dickinson A, Wickrmnyke N, Delos CG, Cruz LA Influence of wter hrdness nd sulfte on the cute toxicity of chloride to sensitive freshwter invertebrtes. Environmentl Toxicology nd Chemistry. 30:

147 Soucek DJ, Kennedy AJ Effects of hrdness, chloride, nd cclimtion on the cute toxicity of sulfte to freshwter invertebrtes. Environmentl Toxicology nd Chemistry. 24:

148 CHAPTER FIVE THE EFFECT OF MULTI-ION EXPOSURES ON N + /K + -ATPASE AND CARBONIC ANHYDRASE ACTIVITY AND RECOVERY IN Pimephles promels (fthed minnow) Introduction Dissolved ions hve severl importnt physiologicl functions within qutic orgnisms. Primrily, ions crete electrochemicl grdients tht ultimtely control the movement of wter nd other electrolytes between the internl nd externl environment of these orgnisms (Thoms nd Egee, 1998). In freshwter species, the internl ion concentrtion (~274 mosm) is typiclly greter thn tht of their externl environment (<50 mosm) (Evns et l., 2005). As result, freshwter species must utilize ctive trnsport to mintin homeosttic ion blnce. The primry site of ctive trnsport for the purpose of ionoregultion nd cid-bse regultion in freshwter fish is the gill (Gilmour nd Perry, 2009). Although there re mny pumps, trnsporters nd enzymes locted in the gill tht re responsible for this osmotic control, crbonic nhydrse nd N + /K + -ATPse re essentil contributors to these processes (Perry et l., 2003). Not only is crbonic nhydrse importnt in cid-bse regultion through the conversion of CO 2 wste into H + nd HCO - 3 ions, it is lso indirectly responsible for the exchnge of Cl - nd N + (Gilmour nd Perry, 2009). Briefly, HCO - 3 ions re exchnged for Cl - ions t the picl surfce of the gill llowing HCO - 3 to exit the body while llowing Cl - inside. In similr fshion, H + ions re exchnged for N + ions on the picl membrne. As result, crbonic nhydrse is importnt in mintining the delicte 126

149 blnce between blood ph nd ionic concentrtion (refer to Figure 1.1; Perry nd Fryer, 1997). Little work hs been done to describe the response of crbonic nhydrse following exposure to elevted ions, prticulrly freshwter fish, nd in fct, mny studies re contrdictory. Zhnyszek nd Smith (1984) suggested tht crbonic nhydrse ctivity in the gills of coho slmon (Oncorhynchus kisutch), n ndromous species, ws higher when dpted to sltwter conditions versus freshwter. Similrly, crbonic nhydrse ctivity incresed lso in tilpi (Oreochromis mossmbicus) gills, euryhline species, when exposed to incresing slinity concentrtions (Kultz et l., 1992). Alterntively, the euryline species Pltichthys flesus (flounder) did not show ny significnt chnges in gill crbonic nhydrse ctivity between sewter nd freshwter dpted fishes (Mshiter KE, MRJ Morgn, 1975). N + /K + -ATPse, being bsolterlly locted on mitochondrion-rich cells (MR Cell) of fish gill tissue, is responsible for hydrolyzing one ATP molecule (energy) to exchnge three sodium ions for every two potssium ions (Evns et l., 1999). In doing so, n electrochemicl grdient is produced tht llows for the entrnce of mny other ions through picl chnnels, including clcium (Evns et l., 2005). As result, N + /K + - ATPse is especilly importnt in controlling internl ionic blnce. The N + /K + -ATPse ctivity in euryhline species (Anguill Anguill) ws shown to increse by fctor of 2.5 when moved from freshwter conditions to sltwter (Thomson nd Srgent, 1977). Freshwter rinbow trout (Oncorhynchus mykiss) lso showed n increse in N + /K + - ATPse ctivity when exposed to incresing concentrtions of sewter (Hwkings et l., 2004). Unlike crbonic nhydrse, these studies suggest tht N + /K + -ATPse ctivity increses in sltwter nd freshwter species like when exposed to increses in overll 127

150 slinity. When this ctivity is monitored during exposures to n increse in single slt, specificlly sodium bicrbonte, decrese in ctivity results (Frg nd Hrper, 2014). This demonstrtes tht different dissolved ions my hve vrying roles in ionoregultion. These results suggest tht freshwter fish exposed to elevted dissolved ions my experience incresed energy expenditure, either direct or indirect, with regrds to crbonic nhydrse nd N + /K + -ATPse ctivity to combt osmoregultory stress. This my leve less energy vilble for other importnt biologicl functions, such s growth nd reproduction, ultimtely leding to decreses in exposed fish popultions. Although extensive reserch hs been done with regrds to single slt exposures, prticulrly for sodium chloride nd sodium bicrbonte, s well s chnges in overll slinity, limited work hs focused on the effects of sodium sulfte, or multi-ion exposures. Furthermore, most of the published literture hs focused on either N + /K + - ATPse or crbonic nhydrse enzymtic response to chnges in slinity, but not both. Additionlly, little work hs been done to describe recovery of the ctivity of these enzymes following elevted dissolved ion exposures. This reserch, therefore, exmined the response nd recovery of both crbonic nhydrse nd N + /K + -ATPse in the gills of the fthed minnow during chronic exposures to sodium chloride, sodium sulfte, nd sodium bicrbonte described s single nions, nd in binry mixtures. Mterils nd Methods Pimephles promels Culture Methods Adult Pimephles promels (fthed minnows) were rered in n in-house culture mintined t the Clemson Institute of Environmentl Toxicology (CU-ENTOX, Pendleton, SC, USA). All fish were cultured nd held in ccordnce with the pproprite 128

151 Institutionl Animl Cre nd Use Committee protocols (Clemson University IACUC, AUP nd AUP ). All fthed minnows were housed in 3m by 1m wter trough ffixed to ~ 350-gllon recirculting wter system with biologicl filter. Culture wter within the recirculting system ws prepred using regent grde chemicls (96 mg/l NHCO3, 60 mg/l CSO4 H2O, 60 mg/l MgSO4, nd 4.0 mg/l KCl) nd Ultrpure (18 MΩ cm resistivity) wter (U.S. EPA, 2002). Temperture ws recorded dily ( C), while lklinity (mg/l CCO 3 ), hrdness (mg/l CCO 3 ), ph, mmoni (ppt NH 3 ) nd nitrte (ppt NO 2 ) were recorded weekly. Fish were fed Tetrmin flke food purchsed from Dr. Foster nd Smith Aqutics, Inc., (Rhinelnder, WI, USA). Test Solutions Four 300L crboys were used in order to prepre solutions 72-hours prior to test initition to ensure ll solutions were thoroughly nd evenly dissolved. One crboy ws used for ech tretment: control, low, medium, nd high. All solutions were creted using the sme reconstituted modertely hrd wter in which the fish were cultured, nd the sodium-slts of chloride, bicrbonte, nd sulfte. Sodium chloride (CAS ; Fisher Scientific, Atlnt, GA), sodium bicrbonte (CAS ; Fisher Scientific, Atlnt, GA), nd sodium sulfte (CAS ; Fisher Scientific, Atlnt, GA) were utilized in the preprtion of test solutions. Once sodium-slts hd been dded to their respective crboys, n ir stone ws ffixed to ech to increse slt dissolution, s well s prevent the solution from becoming stgnnt. Solutions were kept in the sme temperture controlled room s test orgnisms to mintin the sme temperture throughout the exposure. Smples were collected for every solution during wter renewls nd sent to the Clemson Agriculturl Services Lbortory for ion quntifiction. 129

152 Biossy Procedure nd Experimentl Design Exctly 144 fthed minnows (between 0.75 nd 2.5 g wet weight) were trnsferred from the recirculting culture system into temperture controlled (22 ± 1 C) room, including 16:8 light/drk cycle for ech biossy. The fthed minnows were then rndomly divided into twelve 5-gllon quri contining 15L of reconstituted modertely hrd wter (U.S. EPA, 2002). Fish were then llowed to cclimte to the sttic nture of the biossy 24-hours prior to test initition once twelve fish were plced into ech qurium. At the strt of ech biossy, quriums contining twelve fish were divided into four tretment ctegories for both single-ion nd binry mixture exposures. Single-ion exposures included control, low, medium, nd high concentrtions for ech ion. Binry mixtures included control, low:high, medium:medium: nd high:low concentrtions, with the ion concentrtions being the sme s from the single-ion exposures. Ech tretment ws mde up of three quriums (n=3). Once initited, biossys were continued for fourteen dys. These fourteen dys were split into seven-dy exposure period (dys one through seven), nd seven-dy post-exposure recovery period (dys eight through fourteen). The first seven dys, fish were exposed to either single ions or binry ion mixture solutions. Two smpling events, where one tnk from ech tretment ws collected, occurred during this exposure period to understnd the potentil chnge in response over multiple dys of exposure. The first smpling event occurred on dy three of the exposure, while the finl smpling event occurred on dy seven for the exposure period. At the conclusion of the exposure period, fish from the finl qurium for ech tretment were removed from the single ion 130

153 or binry ion mixture test solutions, nd trnsferred into reconstituted modertely hrd wter. The finl smpling event for the biossy occurred on dy fourteen to nlyze post-exposure recovery. During ech smpling event, fish were removed from their quri nd immeditely euthnized in 1L modertely hrd wter mixed with 1.5g MS-222 buffered to ph = 7.5 with NHCO3 (Clemson University Institute for Environmentl Toxicology SOP ). Fish were considered decesed 10-minutes fter lst operculum movement. The wet weight of ech fish ws recorded prior to dissection of gills, kidney nd intestines. Ech of the three tissues ws plced on seprte piece of luminum foil, folded nd seled, then flsh frozen in liquid nitrogen nd stored in 80 C freezer for no longer thn 3-months. Test solution renewls occurred every 24-hours to ensure mmoni concentrtions remin well below lethl limits (0.02 mg/l NH 3 ) for both exposures nd post-exposure recovery periods. Wter qulity criteri including conductivity (µs/cm), ph, wter hrdness (mg/l CCO 3 ), lklinity (mg/l CCO 3 ), temperture ( C), dissolved oxygen (mg/l), nd mmoni (ppt NH 3 ) were performed. Alklinity nd hrdness were only performed on the test solution prior to every 24-hour wter renewl, nd mmoni ws only recorded for test solutions fter the 24-hour exposure period. Fish were fed Tetrmin flke food once dily. Tetrmin ws weighed prior to feedings t 200mg/qurium to reduce ny influence on enzymtic ctivity ttributed to ions present in the feed. The lst feeding occurred 24-hours prior to dissection to ensure the intestinl trct ws mostly cler of ll food substnces. 131

154 N + /K + -ATPse Activity Protocol Methods used to quntify N + /K + -ATPse ctivity followed those described by McCormick (1993) nd Zhner (2009). These methods were developed to cquire smples through nonlethl gill smpling, nd re dequte for use on smll smple sizes. Becuse the tissues extrcted from the dult fthed minnows were very smll, the use of this method ws idel. Furthermore, it is one of the most common methods in use for this type of nlysis, nd is used throughout the published literture due to its reproducibility nd high sensitivity (McCormick, 1993). Gill tissues for ech fish were removed from the 80 C freezer one tretment t time (n=5), immeditely removed from the luminum foil pouch nd plced into individul 2.0 ml micro-centrifuge tubes on ice. To ech tube, 100 µl of SEI buffer nd 25 µl of SEID buffer t pproximtely 4 C ws dded. To prepre the SEI buffer, 150 mm sucrose, 10 mm EDTA, nd 5 0mM imidzole were dded to volumetric flsk with Ultrpure (18 MΩ cm resistivity) wter until fully dissolved. Once dissolved, this mixture ws trnsferred to wide-mouthed flsk. SEID buffer ws prepred by dding 0.25 g sodium deoxycholte to 50 ml SEI buffer. Both solutions were plced in 4 C refrigertor for 24-hours prior to use. Prior to use, the ph of both buffer solutions ws djusted to 7.5 with 0.1 M HCl. Next, the smples were thoroughly homogenized on ice using hnd-held IKA T-10 Bsic Ultr-Turrx. After homogeniztion, smples were centrifuged in n Eppendorf 5810R centrifuge t 8000g for 2-minutes t 4 C. Following centrifugtion, 5 µl of superntnt were plced into four wells on 96-well plte. To two wells, 150 µl of ctivtion solution ws dded. The ctivtion solution ws 132

155 prepred by mixing 4-enzymtic units/ml of lctte dehydrogense, 5-enzymtic units/ml of pyruvte kinse, 2.8 mm phosphoenolpyruvte, 3.5 mm denosine triphosphtse (ATP), 0.22 mm nicotinmide denine dinucleotide phosphte (NADH), nd 50 mm imidzole. To the remining two wells, 150 µl of dectivtion solution ws dded. The dectivtion solution ws prepred by dding known N + /K + -ATPse specific inhibitor to n liquot of the ctivtion solution. Five inhibitors were tested in the present study to determine which, if ny, were best suited for use in fthed minnows. The first inhibitor tested ws oubin since it iss the most studied nd well-known N + /K + -ATPse specific inhibitor. Both 1.5 mm nd 13.0 mm oubin were utilized, s well s 157 mm copper, 89.0 mm cdmium, 10 mm vndte, nd 6.5 mm digoxin. Both the ctivtion solution nd dectivtion solutions were prepred less-thn 24-hours before use, nd ph djusted to 7.5 with 0.1 M HCl. Just prior to nlysis, 50 µl of slt solution (189mM NCl, 10.5mM MgCl2, 42mM KCl, nd 50mM imidzole; ph djusted with 0.1M HCl to ph = 7.5) ws dded to ech well to initite the rection. Smples were then nlyzed on temperture controlled Moleculr Devices Gemini microplte reder with UV detection t 340 nm every 45-seconds over 10-minutes. The resulting N + /K + -ATPse ctivity ws then clculted using the following eqution: (1) Results were stndrdized to protein content, mesured using Protein Assy kit (Pierce, Rockford, IL, USA) nd reported in mmol ADP/mg protein/hour. Crbonic Anhydrse Activity Protocol The Delt ph method is one of the most common methods used for mesuring crbonic nhydrse ctivity tody. Ese of use, utilizing reltively inexpensive 133

156 equipment, nd providing ccurte nd relible results re just some of the dvntges of this technique (Henry, 1991). The Delt ph method employed for these studies follows the frmework of Henry (1991); however, it includes modifictions by Gervis nd Tufts (1998) nd Tufts et l. (1999). To strt, gill smples were removed from 80 C storge nd immeditely trnsferred from their luminum foil pouch to 2 ml microcentrifuge tube contining n ice-cold TRIS buffer solution t ph of 7.4 (10 mm TRIS bse, 225 mm mnnitol-d, nd 75 mm sucrose; ph djusted using 10% H 3 PO 4 ). After homogeniztion with hnd-held IKA T-10 Bsic Ultr-Turrx, smples were centrifuged using n Eppendorf 5810R centrifuge t 8000g for 2-minutes t 4 C. Once centrifuged, either 2 μl or 5 μl (depending on the whole wet weight of ech fish) of superntnt nd 2 ml of TRIS buffers solution ws dded to custom-mde 2 ml glss well ffixed to recirculting wter bth held t 4 C. The enzyme rection ws initited with the ddition of 40 µl of CO 2 sturted UltrPure (18 MΩ cm resistivity) wter, during which time the ph nd mv chnge ws recorded using double, open junction micro-ph probe (Hlo HI13302, Hnn Instruments, Woonsocket, RI, USA) connected to the Hnn Lb App on n Apple ipad vi Bluetooth Smrt Technology. The ctlyzed rection for ech smple ws performed three times (n = 3). Another rection to determine the unctlyzed rte ws lso performed in duplicte (n = 2). This rection ws initited in the sme custom-mde glss well with 2 ml TRIS buffer solution; however, the fish smple ws omitted. The ph nd mv chnge ws recorded fter the ddition of 40 µl CO 2 sturted UltrPure (18 MΩ cm resistivity) wter. The unctlyzed rte ws indictive of the ph shift creted solely by the CO 2 sturted UltrPure (18 MΩ cm resistivity) wter. Both the ctlyzed nd unctlyzed rection 134

157 rtes for 0.15 unit ph chnge were determined by plotting time (seconds) nd mv. The verge of both rtes ws divided by the buffer cpcity to ccount for the rte of H + produced during the rection. Buffer cpcity ws the chnge in mv, mesured fter the ddition of 40 µl of 0.1N HCl to 2 ml of TRIS buffer solution in the sme custom-mde glss well, over time (seconds) divided by 4 µmoles. Crbonic nhydrse ctivity ws clculted by subtrcting the djusted unctlyzed rection rte from the ctlyzed rection rte. Totl crbonic nhydrse ctivity ws then stndrdized to protein content, mesured by BCA Protein Assy kit (Pierce, Rockford, IL, USA) nd reported in µmol H + /mg protein/hour. Sttisticl Anlysis All sttisticl nlyses were performed utilizing JMP Pro For both N + /K + -ATPse nd crbonic nhydrse ctivities, five smples were nlyzed. The verge ctivities (n = 5) for ech enzyme ws determined nd then plotted ginst tretment. Anlysis of Vrince (ANOVA) nd Fisher s LSD ws used to determine sttisticl differences between tretments nd exposure dys. Results Crbonic Anhydrse Activity Chloride-only exposures to P. promels resulted in no sttisticlly significnt chnges in crbonic nhydrse ctivity on dy 3 (p vlue: ) nd dy 7 (p vlue: ). A significnt difference, however, occurred on Dy 14, following the postexposure recovery period. A significnt increse in crbonic nhydrse ctivity occurred between the tretment contining low (8.56 mm) chloride nd high (85.2 mm) chloride (p vlue: *). The medium chloride tretment (34.2 mm chloride) did not result in 135

158 significnt differences from the control (Figure 5.1; Figure 5.2; Tble 5.1). No significnt chnges in crbonic nhydrse ctivity occurred between dys or within tretments for sulfte-only exposures (Figure 5.3; Figure 5.4; Tble 5.2; Tble 5.8). Exposures to bicrbonte-only further resulted in significnt chnges in crbonic nhydrse ctivity. By dy 3, ll tretments contining elevted bicrbonte resulted in significnt reduction in crbonic nhydrse ctivity from the control. However, by dy 7, only the high tretment contining 23.8 mm bicrbonte remined significntly different (p vlue: *). Following the 7-dy post-exposure recovery period, ll three tretments contining low (5.95 mm), medium (11.9 mm) nd high (23.8 mm) bicrbonte resulted in sttisticlly decresed crbonic nhydrse ctivity from the control. It ws noted tht decrese in crbonic nhydrse ctivity occurred on dy 7 for the control group, potentilly resulting in less significnt difference between tretments (Figure 5.5; Tble 5.9). Significnt chnges in crbonic nhydrse did not result between dy 3, dy 7 nd dy 14 for 5.95 mm nd 11.9 mm bicrbonte, wheres there ws significnt reduction in ctivity on dy 7 for the control group, nd dy 7 for the 23.8 mm bicrbonte tretment (Figure 5.6; Tble 5.3). Crbonic nhydrse ctivity significntly decresed from the control group on Dy 3 of exposure to 8.35mM sulfte nd 23.8mM bicrbonte (p vlue: <0.0001*), s well s 14.1 mm sulfte nd 11.9mM bicrbonte (0.0172*). However, fish exposed to 35.2 mm sulfte nd 5.95 mm bicrbonte did not result in ny sttisticl difference from the control (p vlue: ) (Figure 5.8; Tble 5.4). By dy 7 nd dy 14, ll tretments hd similr crbonic nhydrse ctivities. Unlike sulfte with bicrbonte mixtures, chloride with bicrbonte mixtures did not induce significnt response until dy 7, 136

159 when the tretment consisting of 34.2 mm chloride nd 11.9 mm bicrbonte demonstrted significnt increse in crbonic nhydrse ctivity compred to the control (p vlue: *). By dy 14, both tretments contining 34.2 mm chloride:11.9 mm bicrbonte nd 85.2 mm chloride: 5.95 mm bicrbonte demonstrted sttisticlly significnt increse in crbonic nhydrse ctivity from the control (Figure 5.10; Tble 5.5). Chnges in crbonic nhydrse ctivity were only sttisticlly significnt between dys in the tretment contining 85.2 mm chloride nd 5.95 mm bicrbonte (Figure 5.9; Tble 5.11). Mixtures contining 8.56 mm chloride with 35.2 mm sulfte, nd 34.2 mm chloride with 14.1 mm sulfte were slightly decresed by exposure dy 3; however, the 85.2 mm chloride with 8.35 mm sulfte ws significntly reduced compred to the control (p vlue: *). Furthermore, by exposure Dy 7, 34.2 mm chloride with 14.1 mm sulfte ws lso reduced significntly compred to the control. Interestingly, dy 14 indicted significntly reduced crbonic nhydrse ctivity in the tretment contining 8.56 mm chloride nd 35.2 mm sulfte, which ws not reduced t ny other dy during the exposure. The other two tretments (34.2 mm chloride with 14.1 mm sulfte nd 85.2 mm chloride with 8.35 mm sulfte), which hd previously been significntly reduced on dys 3 nd 7, were only slightly different from the control by dy 14 (Figure 5.12; Tble 5.6). The only tretment to produce significnt reduction in crbonic nhydrse ctivity over the 7-dy exposure nd 7-dy post-exposure recovery period consisted of 8.56 mm chloride nd 35.2mM sulfte. Although there ws slight increse in ctivity of the control group on dy 7 of the exposure, it returned to similr ctivity by Dy 14 (Figure 5.11; Tble 5.12). 137

160 Preliminry Results: N + /K + -ATPse Activity Initilly, methods for quntifying N + /K + -ATPse ctivity were employed to demonstrte the enzymtic effects of sodium chloride, sodium sulfte, nd sodium bicrbonte to P. promels. Methods published by McCormick (1993) proposed the use of 0.5 mm oubin, known N + /K + -ATPse specific inhibitor. However, it ws noted by Zhner (2009) tht P. promels ppered to be firly insensitive to oubin exposure, nd so 1.5 mm oubin ws utilized. Since the ctivtion solution demonstrted ll ATPse ctivity, nd is not specific to ny one of the ATPse enzymes, quntifying the difference between the totl ATPse ctivity nd residul ATPse ctivity fter the ddition of oubin, the specific N + /K + -ATPse ctivity cn be clculted. However, there ws no inhibition prompted by 1.5 mm oubin, with the Vmx clculted s 46.0 µmol H+/minute for the ctivtion solution, representing the totl ATPse ctivity, nd Vmx of 40.1 μmol H+/minute for the dectivtion solution contining 1.5 mm oubin (Figure 5.13). Due to the lck of inhibition, further preliminry studies utilizing higher concentrtions of oubin were lso performed. Even t 13.0 mm oubin, the highest concentrtion tested due to the wter solubility limit of oubin, inhibition of N + /K + - ATPse did not occur (ctivtion Vmx: 27.1 μmol H+/minute; 13 mm oubin Vmx: 26.4 μmol H+/minute) (Figure 5.14). To vlidte the ccurcy of the procedure, copper, known nonspecific ATPse inhibitor for P. promels, ws utilized. Unlike oubin, copper did show slight ATPse inhibition by decresing the overll ATPse Vmx from 50 μmol H+/minute to 33.3 μmol H+/minute (Figure 5.15). Although copper is not 138

161 N + /K + -ATPse specific, this inhibition experiment did show tht inhibition ws chievble nd thus, the procedurl methods were ccurte. The mjority of the published literture utilizing oubin demonstrtes ttinble N + /K + -ATPse inhibition in mummichogs (Fundulus heteroclitus), Atlntic slmon (Slmo slr) nd rinbow trout (Oncorhynchus mykiss) (Morgn et l., 1997; Mncer nd McCormick, 2000). To further demonstrte procedurl ccurcy, the sme methods were employed using 1.5 mm oubin on F. heteroclitus gill tissue. A significnt reduction in N + /K + -ATPse ctivity occurred between the ctivtion solution (Vmx: 48.1 µmol H+/minute) nd the dectivtion solution (Vmx: 30.3 µmol H+/minute) (Figure 5.16). Becuse the methods were successful in inhibiting ATPse ctivity using copper in P. promels, s well s oubin-inhibition occurrence in well-documented species, F. heteroclitus, the sme procedure ws used in ll subsequent studies. Oubin, member of the digitlis fmily, effectively inhibits potssium binding on the N + /K + -ATPse enzyme. Although the effectiveness of oubin hs been demonstrted in mny euryhline species, including slmon nd mummichogs, freshwter P. promels were insensitive. Differences in these species must indicte tht P. promels hs slightly different structurl conformity which limits the binding of oubin. This structurl difference my either result from environmentl differences between species (freshwter versus euryhline) or some evolutionry benefit. Due to the insensitive nture of P. promels to oubin, other inhibitors were utilized to try nd inhibit N + /K + -ATPse in P. promels, the first being orthovndte. This prticulr inhibitor is believed to mimic phosphte ions, nd, s result, inhibits phosphte binding on the enzyme during ctivtion (Lindquist et l., 1973; Boyd nd 139

162 Kustin, 1984). Although extreme inhibition occurred in the present study, it ws not specific to N + /K + -ATPse. As result, ll ATPse ctivity ws impeded (Figure 5.17). Subsequently it ws noted tht vndte is not specific to N + /K + -ATPse lone, but insted, inhibits the ctivity of ll ATPse enzymes (Boyd nd Kustin, 1984). The finl inhibitor tested ws digoxin. Digoxin is similr to oubin in tht they re both members of the digitlis fmily, nd specific for inhibiting N + /K + -ATPse enzymes. However, little to no inhibition occurred (ctivtion Vmx: 54.9 µmol H+/minute; digoxin Vmx: 48 µmol H+/minute) (Figure 5.18). A lck of inhibition for both oubin nd digoxin my indicte P. promels is insensitive to ll members of the digitlis group of inhibitors. Due to the difficulty in obtining N + /K + -ATPse specific inhibitor for P. promels, finl results were reported s totl ATPse ctivity, mesured by the ctivtion solution. Although these results do not indicte chnges in specific enzyme ctivities, they my indicte the lloction of more energy, in the form of ATP, for proper ionoregultory processes. Totl ATPse Activity Chnges in totl ATPse ctivity for P. promels exposed to chloride-only did not show ny significnt chnges on dy 3 (p vlue: ) of exposure; however, by dy 7 significnt difference between the four tretments occurred (p vlue: *). Specificlly, fish exposed to 34.2 mm nd 85.2 mm chloride resulted in significnt increse in totl ATPse ctivity from the control. By dy 14, following 7-dy postexposure recovery period, ll tretments hd similr ctivities (p vlue: ) (Figure 5.19; Tble 5.13). The low tretment, contining 8.56 mm chloride, hd significnt increse in totl ATPse ctivity from dy 3 to dy 14 (p vlue: *) nd dy 7 to 140

163 dy 14 (p vlue: *) indicting elevted ctivity post-exposure. However, it ws lso noted tht n increse in totl ATPse ctivity lso occurred between dy 3 nd dy 14 (p vlue: *) nd dy 7 to dy 14 (p vlue: 0.001*) for the control tretment (Tble 5.19). Fish exposed to sulfte-only did not exhibit significnt chnge in totl ATPse ctivity between dys (Figure 5.22; Tble 5.14) or within tretments (Figure 5.21; Tble 5.20). Furthermore, results produced by bicrbonte-only exposures lso reveled no significnt differences in totl ATPse ctivity between dys (Figure 5.23; Tble 5.15) or within tretments (Figure 5.24; Tble 5.21). Both mixtures contining bicrbonte did not produce ny significnt differences in totl ATPse ctivity for either sulfte (Figure 5.25; Figure 5.26; Tble 5.16; Tble 5.22) or chloride (Figure 5.27; Figure 5.28; Tble 5.17; Tble 5.23). Mixtures contining chloride nd sulfte resulted in sttisticlly significnt decrese in totl ATPse ctivity cross tretments for dy 3, dy 7 of exposure, nd dy 14, the post-exposure recovery period. By dy 3, significnt reduction in overll ATPse ctivity occurred between the control group nd mixtures contining 34.2 mm chloride nd 14.1 mm sulfte, s well s 85.2 mm chloride with 8.35mM sulfte. However, the tretment contining 8.56 mm chloride nd 35.2 mm sulfte ws not significntly different from the control group by dy 3. By dy 7 nd dy 14, both tretments continued to show significnt reduction in totl ATPse ctivity (Figure 5.30; Tble 5.18). There were no significnt chnges in totl ATPse ctivity between exposure dys for ech tretment (Figure 5.29; Tble 5.24), indicting tht once reduced ATPse ctivity occurred, it did not improve following post-exposure recovery period. 141

164 Discussion Aqutic systems re nturlly comprised of mny different dissolved ions, which re reflective of their surrounding environment. The concentrtion of dissolved ions in these qutic systems ultimtely controls the slinity of ech system. A low slinity environment, such s freshwter system, consists of very low dissolved ion concentrtions. Becuse these dissolved ions re criticl for vrious functions within living orgnisms, freshwter fish hve dpted to the osmotic pressures chrcteristic of their surrounding environment. One such dpttion includes the use of ctive trnsport in order to move ions ginst their concentrtion grdient, from the low ion concentrtion of their externl environment, to the high ion concentrtion typicl of their internl environment (refer to Figure 1.1; Evns, 1980; Evns et l., 1999; Perry et l., 2003; Evns et l., 2005; Gilmour nd Perry, 2009). Becuse freshwter orgnisms re dpted to mnging low dissolved ion concentrtions, it is uncler exctly how n increse in these ions would impct ctive trnsport. Elevted dissolved ions re of concern for freshwter orgnisms minly due to the mny nthropogenic ctivities tht led to incresed dissolved ions, s well s chnges in ionic composition, of their surrounding environment (Timpno et l., 2010; Pond et l., 2004; Frg nd Hrper, 2014; Brittinghm et l., 2014). Previous studies hve reported dverse effects rnging from chnges in ecosystem structure, reproductive impirment, decresed growth nd even mortlity for freshwter orgnisms exposed to incresed ion concentrtions (Dickerson et l., 1997; Mount et l., 1997; Tietge et l., 1997; Blsius nd Merritt, 2002; Kennedy et l., 2003; Soucek nd Kennedy, 2005; Vosyliene et l., 2006; Pond et l., 2008; Soucek et l, 2011; Frg nd Hrper, 2014b; Mount et l., 2016; 142

165 Wng et l., 2016; Johnson-Couch (Chpter 2); Johnson-Couch (Chpter 3)). It hs been previously suggested tht n increse in energy used for ionoregultion nd ctive trnsport my decrese the energy vilble for other criticl functions, hence decrese in growth nd reproduction (Frg nd Hrper, 2014). However, cler toxicity grdient is exhibited, where some ions produce more toxic response thn others. In iononly studies, the chronic toxicity of divlent ions (SO 2-4, Mg 2+, C 2+ ) elicited more pronounced response in reproduction of Ceriodphni dubi nd growth of Pimephles promels thn their monovlent counterprts (N +, Cl -, HCO - 3 ) (Johnson-Couch (Chpter 2); Johnson-Couch (Chpter 3)). Although this finding is interesting, increses in dissolved ions in the environment do not occur s one constituent, but in mixtures. How these multi-ion mixtures interct t the site of ction, predominntly the gill could gretly influence the energy necessry for mnging excess dissolved ions. Mitochrondrion-Rich (MR) cells nd pvement cells, locted on the gill of freshwter orgnisms, re responsible for regulting the movement of ions between their externl nd internl environment (refer to Figure 1.1). It is well known tht sodium chnnels locted on the picl membrne, s well s N + /K + -ATPse enzymes on the bsolterl membrne of these MR cells re responsible for combting the diffusive loss of sodium through prcellulr junctions on the picl membrne (Evns et l., 2005). Although the sodium chnnel is responsible for the trnsport of sodium into the MR Cell cytoplsm, N + /K + -ATPse is principlly responsible for sodium gining ccess to the plsm, nd ultimtely the internl environment of the orgnism (Evns et l., Furthermore, H + exits the MR cell through n H + -ATPse locted on the picl membrne, which drives the movement of sodium into the MR cell cytoplsm (Evns et 143

166 l., 1999). Not only re these series of trnsporters, chnnels, nd exchngers importnt for mintining ionic regultion, but lso cid-bse blnce (Perry et l., 2003; Evns et l., 2005; Mrshll nd Grosell, 2005; Gilmour nd Perry, 2009). The cid-bse blnce cpbilities of fish is predominntly controlled by crbonic nhydrse, which is locted within the cytoplsm of MR cells. Endogenous CO 2 wste diffuses into the MR cell cytoplsm, where crbonic nhydrse rpidly converts it with wter into HCO - 3 nd H + ions. The H + ion is then excreted through the H + -ATPse, while llowing the entrnce of one N + ion through the sodium chnnel. Additionlly, one HCO - 3 is excreted while one Cl - ion enters through the Cl - /HCO 3- exchnger. Chnges in externl ion concentrtions, which hve been shown to negtively impct freshwter orgnisms, could be driven by n effect on ionoregultory systems. Due to the lrge interply between these ionoregultory enzymes, n effect on one, could led to n effect on nother or ll, resulting in the negtive impcts on growth nd reproduction. Becuse N + /K + -ATPse nd crbonic nhydrse prompt most ionoregultory enzymtic ctions, s well s utilize energy either directly or indirectly to perform their functions, the gol of the present study ws to understnd the chnges in their ctivity cused by elevted dissolved ions, both s single ions nd multi-ion mixtures. Any chnges could reflect on ionoregultory impirment, nd/or energy lloction. Unfortuntely, due to difficulties in identifying N + /K + -ATPse specific inhibitor in P. promels, only totl ATPse ctivity could be reported. Although this limits the interprettion of the results, chnges in totl ATPse ctivity could still provide insights into ny ionoregultory disturbnces nd chnges in energy lloction. 144

167 Three-dy exposures to sodium chloride did not produce ny significnt chnges in both crbonic nhydrse nd totl ATPse ctivity, indicting tht freshwter fish cn withstnd concentrtions s high s 85 mm chloride without ny obvious initil consequence. By dy 7, however, fish exposed to 34.2 mm nd 85.2 mm chloride showed significntly elevted ATPse ctivity, incresing by roughly 42%, while crbonic nhydrse ctivity remined stble. The estimted Effective Concentrtion (EC) reducing lrvl P. promels growth by 50% compred to the control ws 50.7 mm chloride (Johnson-Couch (Chpter 3). This corresponds pproximtely with the chloride concentrtions producing elevted ATPse ctivity, which could indicte reduction in growth due to the lloction of more energy for ionoregultory purposes. Although it is quite well recognized tht n picl Cl - /HCO 3- exchnge protien is responsible for Cl - uptke by MR cells (Metz nd Grci-Romeu, 1964; Metz, 1976; Perry, 1997), it is still uncler wht truly drives its function. The possibility of V-type proton pump locted on the bsolterl membrne of the MR cell my provide the electrochemicl grdient necessry for Cl - /HCO 3- exchnge to occur (Piermrini nd Evns, 2001). During this exchnge, one chloride ion enters into the cytoplsm of the MR cells, while one bicrbonte ion exits. For chloride in the externl medi to enter through the gill tissue, more bicrbonte must be present inside the cyptoplsm of the MR cell for n exchnge to occur. In fct, n increse in CO 2 efflux hs been demonstrted in goldfish (Crssius urtus) during exposure to high externl chloride concentrtions (Dejours, 1969). This my indicte tht upon exposures to high externl chloride concentrtions, freshwter fish increse chloride influx nd bicrbonte efflux. 145

168 If more bicrbonte is present within the fish, mesured s CO 2 excretion, it would be expected tht n upregultion of crbonic nhydrse would result. However, when sodium chloride incresed in concentrtion over the 7-dy exposure period, no significnt chnges in crbonic nhydrse occurred. It hs been suggested tht sodium nd chloride co-trnsporter on the picl membrne of the MR Cell is present, nd could potentilly led to n overll increse in totl ATPse ctivity (Evns, 2011). The presence of sodium nd chloride co-trnsporter would ultimtely bypss the Cl - - /HCO 3 exchnge system nd thus, not ffect crbonic nhydrse ctivity. Although it is believed tht chloride nd bicrbonte re dependent on one nother for trnsloction, in the present study, bicrbonte-only exposures did not significntly ffect totl ATPse ctivity similr to chloride-only exposures. Insted, bicrbonte-only exposures resulted in significnt decreses in crbonic nhydrse ctivity, by pproximtely 64%, s erly s dy 3 of the exposure. By dy 7, 5.95 mm nd 11.9 mm bicrbonte were not significntly different from the control; however, 23.8 mm bicrbonte remined significntly lower. It is importnt to note tht on dy 7, the control group hd significnt reduction in crbonic nhydrse ctivity itself, nd my be the reson why some differences were not significnt. However, becuse the highest tretment (23.8 mm bicrbonte) ws still significntly lower, it speks volumes to the hindrnce of crbonic nhydrse ctivity by bicrbonte, or more specificlly, sodium bicrbonte. The reson for decrese in crbonic nhydrse ctivity in the control fish on dy 7 is unknown. Although it my be due to stress in the orgnism, it is more likely vrition within ech tretment. Lrge 95% confidence intervls surrounding the control group, especilly on dys 3 nd dy 7 of the post-exposure recovery period (dy 14) 146

169 could indicte lrge typicl differences tht some fish disply in ionoregultion. Chnges in sodium plsm concentrtions re known to fluctute within freshwter fish, depending on where the fish is in its osmotic cycle (Prior et l., 1995). Rndom smpling my contribute to the selection of fish t different points, whether low or high, in their osmotic cycle. Nevertheless, decrese in control crbonic nhydrse ctivity ws shortlived, s the control tretment crbonic nhydrse rebounded following the 7-dy postexposure recovery period, nd ws not significntly different from dy 3 ctivity. Additionlly, ll tretments following the 7-dy post-exposure recovery period gin showed significnt decreses in crbonic nhydrse ctivity, further demonstrting tht n nomly within the control fish on dy 7 ws most plusible. The movement of bicrbonte from the plsm of freshwter fish, in exchnge for chloride, is most likely relted to the need for mintining criticl blood ph. It my lso be relted to the movement of bicrbonte from n re of high concentrtion within the fish, to n re of low concentrtion in the externl environment. However, when the bicrbonte concentrtion in the externl environment exceeds wht is physiologiclly tolerble for freshwter fish, they my no longer be ble to excrete bicrbonte. A buildup of bicrbonte within MR cells my lter the internl ph, ultimtely reducing further bicrbonte production nd crbonic nhydrse ctivity. An ccumultion of bicrbonte within the plsm of fish hs been demonstrted in rinbow trout (Oncorhynchus mykiss) following exposure to lkline wters (ph = 10.5) (McGeer nd Eddy, 1998). Without proper function of crbonic nhydrse enzymes, CO 2 my ccumulte within the fish, leding to cidosis. Previous studies hve shown tht freshwter fish quickly respond to cidotic conditions by decresing Cl - /HCO 3- exchnge, 147

170 leding to decrese in both chloride uptke nd bicrbonte efflux (Goss et l., 1992). In ddition, the influx rte of sodium hs been shown to increse drsticlly during times of cidosis, which should correspond to increses in ATPse ctivity (Cmeron, 1976). However, chnges in totl ATPse ctivity did not occur during bicrbonte-only exposures. This is unlike previous studies tht demonstrte decrese in N + /K + -ATPse enzyme mount nd ctivity in P. promels following chronic sodium bicrbonte exposures (Frg nd Hrper, 2014b). However, this decrese does seem to be time dependent. Frg nd Hrper (2014b) found significnt decreses in N + /K + -ATPse t 300 mg/l NHCO 3 t dy 60, but not dy 37. This could indicte tht seven-dy exposures re not long enough to elicit response in ATPse ctivity. Additionlly, chloride is ctively pumped out of the gill by the indirect energetic coupling with bsolterl V-type ATPse ctivity in sltwter fish (Metz, 1972). Although this occurrence hsn t been explicitly documented in freshwter fish, it hs been suggested tht chloride uptke does require n ctive trnsport system (Kerstetter nd Kirschner, 1972). If this is true, freshwter fish my utilize some form of ctive trnsport to trnslocte chloride nd bicrbonte. Chnges in bicrbonte efflux, s well s crbonic nhydrse ctivity, my be responsible for overll decreses in N + /K + -ATPse ctivity over time if ctivity of this V-type ATPse is in fct coupled to Cl - /HCO - 3 exchnge. In fct, during inhibition of crbonic nhydrse, complete inhibition of chloride uptke nd 75% reduction in sodium uptke hs been exhibited in goldfish (Metz nd Grci Romeu, 1964). Becuse of this limited uptke in physiologiclly importnt ions, toxicity of bicrbonte my be linked to chloride or sodium deficiency. 148

171 Precipittion of clcium crbonte, white solid, on the test chmbers during elevted bicrbonte exposures hs lso been linked to increses in bicrbonte concentrtion within fish (Frg nd Hrper, 2014b). As the bicrbonte concentrtion rises, so does the rte of precipittion of clcium crbonte, ultimtely reducing the concentrtion of wter-soluble clcium in the wter column. Among its mny functions, clcium is known to reduce cell permebility in freshwter fish gills, prticulrly with regrds to sodium (Hunn, 1985). If the wter-soluble clcium concentrtion is reduced due to elevted bicrbonte concentrtions, it could subject the gill to higher influx of sodium through unprotected prcellulr junctions. One issue with evluting totl ATPse ctivity, nd not the chnges in specific ATPses, is the fct tht certin ATPses my be upregulted while others re downregulted so tht the overll totl ATPse level remins unchnged. For exmple, while it hs been reported tht N + /K + -ATPse ctivity decresed during elevted bicrbonte exposures, it hs been suggested tht C 2+ -ATPse upregultes in order to combt low clcium concentrtions in externl medi (Frg nd Hrper, 2014b; Hunn, 1985; Mrshll, 2002). Nevertheless, regrdless of the differences in specific ATPse ctivity, incresed energy lloction in the gill through ATPses nd crbonic nhydrse for ionoregultory purposes does not seem to be the primry route of bicrbonte toxicity with respect to decresed growth in P. promels. Since the gill is impermeble to divlent nions, such s SO 2-4, it is not surprising tht n ssocition between sulfte-only exposures nd chnges in gill totl ATPse nd crbonic nhydrse ctivity ws not found (Grci-Romeu nd Metz, 1964; Metz, 1971). Chnges in sodium influx rtes, however, hve been reported in goldfish 149

172 (Crssius urtus) following sodium sulfte exposures (Grci-Romeu nd Metz, 1964). It would be expected tht tht n increse in sodium influx would result in higher ATPse ctivity, primrily ttributble to N + /K + -ATPse. Furthermore, sodium concentrtions utilized in the present study were similr to the previously estimted sodium EC 50 for growth effects in P. promels (73.9 mm) (Johnson-Couch (Chpter 3)). If decreses in growth occurred due to the relloction of energy for ionoregultory purposes, n increse in ctive trnsport systems would hve been expected, however, chnges in totl ATPse nd crbonic nhydrse ctivity within the gill of P. promels did not occur in the present study. Tking these spects into considertion, perhps the min contributor to toxicity is sulfte controlled. This hs lso been suggested by previous studies following cute nd chronic sodium sulfte exposures to multiple species (Mount et l., 1997; Soucek et l., 2011; Johnson-Couch (Chpter 3)). Due to the impermebility of the gill to sulfte, this prticulr ion must hve mechnism of ction seprte from the gill. The most likely route of toxicity is the intestinl trct. Although sltwter fish drink copious mounts of wter to combt osmotic wter loss, freshwter fish typiclly drink much less due to pssive wter gin (Metz, 1971). However, it would be expected tht freshwter fish would ingest lrger quntities of wter if the ion concentrtion in the externl environment exceeded tht present inside the fish. In situtions where sodium nd sulfte re present t elevted concentrtions, long with incresed wter uptke, the fish would lso ingest high concentrtions of sodium nd sulfte. Sulfte, nd other divlent ions such s mgnesium nd clcium re known to increse monovlent ion (sodium nd chloride) uptke with the gstrointestinl trct of mmmls, nd my be the cse in fish s well (Ingrhm nd 150

173 Visscher, 1936; Ingrhm nd Visscher, 1938; Shehdeh nd Gordon, 1969). Additionlly, euryhline fish excrete more sulfte when exposed to elevted mgnesium sulfte, suggesting they do not utilize much energy in the ctive uptke of sulfte long the gstrointestinl trct (Oikri nd Rnkin, 1985). If this is the cse, perhps incresed energy lloction for ionoregultion is occurring within the gstrointestinl trct nd not necessrily the gills. This would suggest tht in fct, sodium is hving toxic effect on freshwter fish, most likely due to chnges in electrochemicl grdients, but is enhnced through elevted sulfte concentrtions. Chnges in gill totl ATPse ctivity did not occur in mixtures contining sulfte nd bicrbonte, which ws to be expected since both ions independently resulted in no significnt chnges in totl ATPse ctivity. However, significnt reduction in crbonic nhydrse ws pprent on dy 3 in ll tretments, with fish returning bck to norml by dy 7. This suggests tht bicrbonte elicited n initil response within the gill of P. promels, but due to the presence of sulfte, the fish were ble to recover. The initil toxicity from higher bicrbonte concentrtions my be the result of decresed chloride uptke long the gills. Recovery by exposure dy 7 my be due to the incresed bsorption of chloride long the gstrointestinl trct by elevted sulfte concentrtions. Alterntively, chloride nd bicrbonte mixtures resulted in significntly elevted crbonic nhydrse ctivities by tretments consisting of higher chloride nd lower bicrbonte concentrtions by exposure dy 7, while decrese in totl ATPse ctivity occurred on dy 3 in tretments consisting of lower chloride nd higher bicrbonte concentrtions. These results re unlike bicrbonte-only nd chloride-only results, where bicrbonte-only produced decrese in crbonic nhydrse ctivity with no effect 151

174 on totl ATPse, nd chloride-only resulted in n increse in totl ATPse ctivity, with no effect on crbonic nhydrse. These results re further evidence of the complex nture of dissolved ion toxicity. Growth effects exhibited by P. promels following exposure to mixtures contining chloride nd bicrbonte resulted in n dditive interction, mening tht the ddition of one ion does not ffect the toxicity of the second. Although it does not pper enzymtic ctivity necessrily cts in n dditive mnner, it does seem s though the ddition of bicrbonte prevents chloride effects on ATPse ctivity. Also, the ddition of chloride prevents bicrbonte effects on crbonic nhydrse ctivity. Interestingly, combintions of chloride nd sulfte significntly reduced totl ATPse ctivity in tretments contining 34.2 mm chloride nd 14.1mM sulfte, s well s 85.2 mm chloride nd 8.25 mm sulfte. There ws no response of totl ATPse ctivity when chloride ws low (8.96 mm) nd sulfte ws high (35.2 mm). This mkes sense considering ll sulfte-only tretments nd the low (8.96 mm) chloride tretment during chloride-only exposures did not hve ny significnt effects on totl ATPse ctivity. However, it is interesting tht combintions with higher chloride concentrtions resulted in such drstic decrese on gill totl ATPse ctivity. Perhps the decrese in totl ATPse ctivity is not necessrily due to cellulr destruction or improper enzyme function. If freshwter fish drink lrge volumes of wter to combt osmotic wter loss during periods of elevted dissolved ions, they would lso ingest high quntities of sodium, sulfte nd chloride. The elevted sulfte would led to incresed bsorption of sodium nd chloride long the gstrointestinl trct, thus reducing the ctivity needed to increse uptke long the gill. Furthermore, if freshwter fish rech mximum tolerble 152

175 sodium nd chloride concentrtion, they my reduce ctivity uptke long the kidney nd produce highly concentrted urine. This could lso explin why less-thn-dditive effects exhibited by growth in P. promels occurred. If the orgnism requires less energy for ionoregultion long the gill nd kidney, they my be ble to utilize more energy for feeding, escping predtion, reproduction, nd other importnt biologicl functions. Due to the possibility for chnges within the other ionoregultory importnt tissues, such s intestines nd kidney, in P. promels following certin exposures, it my be beneficil to mesure totl ATPse nd crbonic nhydrse ctivity within these tissues s well. This could id in the understnding of toxicity for these specific ions. Moreover, specific ion concentrtions within the plsm of fish could further explin if ion trnsloction is ctully occurring vi pssive diffusion, specificlly for ions tht did not seem to result in ny chnge in enzymtic ctivity. It my lso be importnt to note tht lthough most fish species utilize Cl - - /HCO 3 exchngers, tht my require energy through bsolterl ATPses, others pssively trnsport chloride without the use of ATP (Mrshll nd Grosell, 2005). For exmple, mummichogs (Fundulus heteroclitus) utilize ctive trnsport for sodium uptke, but not chloride (Wood nd Mrshll, 1994; Ptrick et l., 1997; Tomsso nd Grosell, 2005; Bucking et l., 2013). F. heteroclitus is lso unique in tht it lcks stomch (Bbkin nd Bowie, 1928; Bucking et l., 2013). This could be of importnce considering P. promels is member of the Cyprinide fmily, which lso consists of crps, group of fish tht lso lck true stomchs (Krtz, 1924). Specificlly, P. promels lck gstric glnds, structure which ids in cid secretion. This mens tht P. promels does not utilize cid to brek down food prticles, nd insted mintins ph similr to their 153

176 externl environment in the digestive trct (Dy et l., 2011). This similrity between P. promels nd F. heteroclitus could indicte tht they lso osmoregulte similrly, prticulrly with regrds to intestinl uptke. It would be interesting to ssess the ctul processes employed during ionoregultion in P. promels to ensure utilizing crbonic nhydrse ctivity s mens to ssocite energy usge nd chloride uptke is dequte. Similrities between concentrtion-response curves of different contminnts hve been suggested to hve similr modes-of-ction (vn der Geest et l., 2000). Chronic exposures estimting growth effects due to elevted ion exposures resulted in similr concentrtion-response curves for sodium, bicrbonte, nd chloride, while sulfte ws different. This could men tht sulfte, unlike sodium, bicrbonte, nd chloride, does not ctully ffect ion uptke long the gill, but insted increses uptke of sodium nd chloride within the gstrointestinl trct of freshwter fish contributing to its toxic effect. Differences in enzymtic ctivities nd toxicities occurred between chloride, bicrbonte nd sulfte ions nd ion combintion exposures, indicting tht these prticulr ions elicit different responses in the gill of P. promels. However, the belief tht freshwter fish utilize more energy in order to ionoregulte nd mintin proper internl ion concentrtion during times of elevted ion exposure my still hold true, lthough the energetic cost for ech ion, nd ion combintion my be different. Conclusions Freshwter orgnisms hve been suggested to lter energy lloction for proper ionoregultory function nd mintining proper ion blnce when exposed to elevted dissolved ions t concentrtions exhibiting sub-lethl effects. By doing so, freshwter orgnisms would decrese the energy vilble for survivl nd biologicl success. In the 154

177 present study, totl ATPse nd crbonic nhydrse ctivity were mesured in order to elucidte mechnism of ction for elevted dissolved ions. The totl ATPse nd crbonic nhydrse ctivity of dult P. promels gill tissue ws different between orgnisms exposed to elevted sulfte, chloride, bicrbonte nd sodium, s single ions nd in multi-ion mixtures. Most effects on crbonic nhydrse ctivity were in response to elevted bicrbonte, while totl ATPse ws mostly ffected by sodium nd chloride. Sulfte did not produce response in either totl ATPse or crbonic nhydrse ctivity of gill tissue. Becuse the gill is impermeble to divlent nions, such s sulfte, it my be tht sulfte incresed the uptke rte of sodium long the gstrointestinl trct. As result, further studies should be completed on other ionoregultory importnt tissues, including kidney nd intestines, in order to understnd the toxicity of elevted ions throughout the entirety of the orgnism. Mesuring ion concentrtions within the plsm of ech orgnism would further our understnding of ctive uptke versus pssive diffusion of freshwter orgnisms exposed to higher slinity solutions. Differences between enzymtic ctivities in the gill tissue of P. promels exposed to elevted dissolved ions indicte tht ech ion nd ion combintion exerts its own energetic cost by either incresing or decresing enzymtic ctivity. 155

178 Crbonic Anhydrse Activity umol H+/mg protein/hour b b Dy 3 Dy 7 Dy 14 0 Control Low Medium High (8.56mM) (34.2mM) (85.2mM) Chloride Tretment Figure 5.1. Crbonic nhydrse ctivity in P. promels gill tissue following chloride-only exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within tretments re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 156

179 Crbonic Anhydrse Activity (umol H+/mg protein/hour b b b Control Low (8.56mM) Medium (34.2mM) High (85.2mM) Dy Figure 5.2. Chnges in crbonic nhydrse ctivity in P. promels gill tissue following chloride-only exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within smpling dys re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 157

180 Crbonic Anhydrse Activity umol H+/mg protein/hour Control Low Medium High (3.50mM) (14.1mM) (35.2mM) Sulfte Tretment Dy 3 Dy 7 Dy 14 Figure 5.3. Crbonic nhydrse ctivity in P. promels gill tissue following sulfte-only exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within tretments re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. b 158

181 Crbonic Anhydrse Activity (umol H+/mg protein/hour Control Low (83.50mM) Medium (14.1mM) High (35.2mM) Dy Figure 5.4. Chnges in crbonic nhydrse ctivity in P. promels gill tissue following sulfte-only exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within smpling dys re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 159

182 Crbonic Anhydrse Activity umol H+/mg protein/hour b Control Low Medium High (5.95mM) (11.9mM) (23.8mM) Bicrbonte Tretment Dy 3 Dy 7 Dy 14 Figure 5.5. Crbonic nhydrse ctivity in P. promels gill tissue following bicrbonte-only exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within tretments re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. b 160

183 Crbonic Anhydrse Activity (umol H+/mg protein/hour b b b b b b b Control Low (5.95mM) Medium (11.9mM) High (23.8mM) Dy Figure 5.6. Chnges in crbonic nhydrse ctivity in P. promels gill tissue following bicrbonte-only exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within smpling dys re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 161

184 Crbonic Anhydrse Activity umol H+/mg protein/hour b b b b b Dy 3 Dy 7 Dy 14 0 Control Low Medium High High Medium Low Sulfte Bicrbonte Figure 5.7. Crbonic nhydrse ctivity in P. promels gill tissue following sulfte:bicrbonte mixture exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within tretments re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 162

185 14 Crbonic Anhydrse Activity (umol H+/mg protein/hour c b b Control Low:High Medium:Medium High:Low Dy Figure 5.8. Chnges in crbonic nhydrse ctivity in P. promels gill tissue following sulfte:bicrbonte mixture exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within smpling dys re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 163

186 Crbonic Anhydrse Activity umol H+/mg protein/hour * b b Dy 3 Dy 7 Dy 14 0 Control Low Medium High High Medium Low Chloride Bicrbonte Figure 5.9. Crbonic nhydrse ctivity in P. promels gill tissue following chloride:bicrbonte exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within tretments re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 164

187 Crbonic Anhydrse Activity (umol H+/mg protein/hour * b b b b b Control Low:High Medium:Medium High:Low Dy Figure Chnges in crbonic nhydrse ctivity in P. promels gill tissue following chloride:bicrbonte mixture exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within smpling dys re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 165

188 Crbonic Anhydrse Activity umol H+/mg protein/hour b * b Dy 3 Dy 7 Dy 14 0 Control Low Medium High High Medium Low Chloride Sulfte Figure Crbonic nhydrse ctivity in P. promels gill tissue following chloride:sulfte mixture exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within tretments re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 166

189 12 Crbonic Anhydrse Activity (umol H+/mg protein/hour b b b b b b b b b Control Low:High Medium:Medium High:Low Dy Figure Chnges in crbonic nhydrse ctivity in P. promels gill tissue chloride:sulfte mixture exposures. Brs represent verge crbonic nhydrse ctivity mesured s µmol H + /mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within smpling dys re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 167

190 Activtion Dectivtion (1.5mM Oubin) 0.4 OD Units :00:00 0:02:36 0:05:11 0:07:47 0:10:22 Time (Minutes) Figure Preliminry Results: The lck of inhibition of N + /K + -ATPse by 1.5 mm oubin in P. promels gill tissue. 168

191 Activtion Dectivtion (13mM Oubin) 0.4 OD Units :00:00 0:02:36 0:05:11 0:07:47 0:10:22 Time (Minutes) Figure Preliminry Results: The lck of inhibition of N + /K + -ATPse by 13.0 mm oubin in P. promels gill tissue. 169

192 Activtion Dectivtion (Copper) OD Units :00:00 0:02:44 0:05:28 0:08:12 0:10:57 Time (Minutes) Figure Preliminry Results: The inhibition of N + /K + -ATPse by 157 mm copper in P. promels gill tissue. 170

193 Activtion Dectivtion (1.5mM Oubin) 0.4 OD Units :00:00 0:02:36 0:05:11 0:07:47 0:10:22 Time (MInutes) Figure Preliminry Results: The inhibition of N + /K + -ATPse by 1.5 mm oubin in F. heteroclitus gill tissue. 171

194 OD Units :00:00 0:02:36 0:05:11 0:07:47 0:10:22 Time (Minutes) Activtion Dectivtion (Vndte) Figure Preliminry Results: The inhibition of N + /K + -ATPse by 10.0 mm orthovndte in P. promels gill tissue. 172

195 Activtion Dectivtion (Digoxin) OD Units :00:00 0:02:36 0:05:11 0:07:47 0:10:22 Time (Minutes) Figure Preliminry Results: The lck of inhibition of N + /K + -ATPse by 6.5 mm digoxin in P. promels gill tissue. 173

196 ATPse Activity (mmol ADP/mg protein/hour) b b b b Control Low Medium High (8.56mM) (34.2mM) (85.2mM) Dy 3 Dy 7 Dy 14 Chloride Tretment Figure Totl ATPse ctivity in P. promels gill tissue chloride-only exposures. Brs represent verge ATPse ctivity mesured s mmol ADP/mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within tretments re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 174

197 ATPse Activity (mmol ADP/mg protein/hour) c bc b Control Low (8.56mM) Medium (34.2mM) High (85.2mM) Dy Figure Chnges in totl ATPse ctivity in P. promels gill tissue chloride-only exposures. Brs represent verge ATPse ctivity mesured s mmol ADP/mg protein/hour ± 95% confidence intervls (α = 0.05). Significnt differences within smpling dys re indicted by different letters derived from Fisher s LSD (p vlue 0.05). Error brs with the sme letter indicte no significnt difference. 175