STABLE ISOTOPE SmYOF RWERINE BENTHIC FOOD WEBS INFLLJ'ENCED BY ANTHROPOGENIC DEVELOPMENTS

A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph

by ANDREA J.C. FARWELL

In partial fulfilment of requirements for the degree of Doctor of Philosophy January, 2000

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STABLE ISOTOPE STUDY OF RIVERINE BENTHIC FOOD WEBS iNFLüENCED BY ANTHROPOGEMC DEVELOPMENTS

Andrea Joan Catherine Farwell Advisors: University of Guelph, 2000 KR.Solomon and KR.Munkittrick

Stable carbon and nitrogen isotopes were used to assess the potential impacts of hydroelectric development and pulp miIl activity on benthic food web interactions in the tributaries of the Drainage Basin in Northeastem . Secondly, the appiicability of stable carbon isotopes as a tool to indicate exposure to pulp mil1 effluent in wild white sucker (Catostomus commerso~~i)located downstrearn of hydroelectric facilities and pulp miIl emuent discharges was assessed. Stable isotope analysis (SIA) of the benthivorous white sucker From an undeveloped tnbutary (Missinaibi River) showed no significant spatial or temporal variability in the 6'3~(mean 2 SE (n) = -29.3 960 f 0.2 (47)) and 6"~values (mean It SE

(n) = 8.3 %O f O. 1 (47)) of muscle tissue. The range of 613c (-3 1.4 to -26.5 960) and 6lb (6.3 to 9.4) were used as an indicator of normal fluctuation in the isotopic composition of white sucker in an undeveloped river in the Moose River Basin. Stable isotope analysis of white sucker muscle fiom a regulated river () were found to be "C and 1% depleted upstream of hydroelectric development. Downstrearn, there were indications (fiom pre-impoundment and post-impoundment liver comparisons) of 13c depletion and 15N e~chmentof white sucker tissues associated with this recent hydroeiectric development (commissioned in fall of 199 1 ). Isotopic trends of white sucker established for the Missinaibi and Groundhog Rivers were used as a reference to detennine changes in benthic food web interactions associated with histoncal log drives and pulp mill effluent discharges on regulated nvers (Mattagarni and Kapuskasing Rivers). Bark and wood accumulations from historical log drives did not appear to infiuence benthic food web dynamics. ')c depleted particulate organic material fiom upstream of the dam combined with "C enriched particulate organic material from pulp mil1 eWuent discharges resulted in the cornbineci 13c values detected in white sucker. The ability to dicerentiate the 613c of white sucker exposed to pulp mill effluent versus the 6"~of white sucker fiom non-impacted sites may be dependent on several factors, including the sire of the reservoir, water discharge, efiluent composition and discharge, and fish mobility. S"N of white sucker did not appear to be infiuenced by pulp mil1 activities. The implications of this study of benthic invertebrates and benthivorous fish fiom the tributaries of the Moose River were discussed with regard to the use of stable isotopes to define fish exposure to pulp mil1 effluent, and to detine suitable reference sites for assessing the impacts of anthropogenic developments on wild fish populations. The thesis is organized as a series of papers (Chapter 2-5) that will be subtnitted for publication. Chapter 1 provides the general introduction and Chapter 6 provides the thesis conclusions. References to Chapters 1-6 are presented in Chapter 7 (References). To the memory of

my father, John Francis Fanvell

and

a fnend, Arthur Woodgate

Love always, Andrea Joan iii Acknowleâgmeats

The best of life begins with fnends and family! My family and friends mean the worid to me and it is with their support and encouragement that 1 am able to accomplish this and al1 tiiture endeavors. You may never completely understand, but 1 know what rnakes each and every one of you special to me and for this, 1 thank you dl. 1 also know there is no way in heaven that 1 can deny you the 'glory' of seeing your name in print. Special thanks to Michele Burley, Linda Bailey, Susan Gloguaer, Rhonda Wadel, Shona McIntyre and Ingrid Burgetz for putting up with me and making me la@. Thanks to my family, al1 27 of you! I could not image what my life would be like without you! Special thanks to my mother, Jean Nice Farwell, for always being there for al1 of us. Your strength and devotion to your family are tmly inspirational. I thank God for you! It is an honor to dedicate this thesis to the memory of my father, John Francis Fmell and have it serve as a reminder of the value of even the smallest gestures of kindness. An act as simple as changing a bumt out headlight in my car, not because 1 couldn't, but because Dad could, and he could do it for me! Dad, I love you, and miss you, always! 1 am extremely fortunate to have had the opportunity to interact with several research groups including Kelly Munkittick's and Mark Servos's crews at the Canada Centre for Inland Waters (CCiW), Burlington, Keith Solomon's group at the Centre of Toxicology (Environmental Biology), University of Guelph and the Environmental Isotope Lab in Earth Sciences, University of Waterloo. There were times when I believe I was lost in this mysterious Burlington - Guelph - Waterloo triangle. Loa perhaps but never for long, thanks to a group of reliable, helpfùl and Wendly individuals. Special thanks to Elayne Starr, Centre of Toxicology for keeping me up to date on University of Guelph events and for your helpful assistance when I was on campus. Elayne and the many friendly faces, both staff and graduate students, have made the Centre of Toxicology a most enjoyable place, and their comedic stories will always be remembered. Also, 1 have had the privilege to meet various fiin-loving, hard working individuals at CCIW. Special thanks to Michele Burley, Bev Blunt, Janet Jardine, Lynne Luxon, and Lynda McCarthy for always knowing where to find or how to get exactly what 1 needed and for bemg such Rice people! 1 am forever indebted to Bob Drimmie for allowing me to work out of the Environmental Isotope Lab, Earth Sciences, University of Waterloo. Special thanks to Bob Drimmie and Bill Mark, for the srniling faces that always made me fa1 wekome in iv the isotope lab. Also, the Biology Department at the University of Waterloo has a very positive and energetic group of isotope people and 1 have enjoyed the formal and not so fomal isotope debates over the years. In particular, 1 would Iike to acknowledge Karin Guiguet, a fiiend and fellow PhD thesis writer, for her emotional and scientific suppon during the final stages of thesis writing. This thesis would not be possible without field collections and the tireless effort of so many individuals. Special thanks to Cam Pom and his associates, for the collection of Bsh samples for this project. Over the years there have been numerous individu& fishg the tributanes of the Moose River including Mark McMaster, Wade Gibbons, Mark Hewitt, Michael van den Heuvel, Dana Boyter. Janet Jardine, John Nickle, Lisa Ruemper, Ken Oakes and more! 1 gratefidly acknowledge the efforts of J.S. Griffiths, R.W. Sheehan and E.A. McLeod from Natural Sciences Department, Technical Resources, Ontario Hydro Technologies for the collection of samples and data for various sites on the Missinaibi River in the fall of 1996. Individuals fiom Ontario Hydro aiso collected samples downstream on the at the Kipling dam. Thanks for al1 the effort! Special thanks to Michelle Burley (CCIW) for the collection of water wnples for chernical analysis and field assistance. 1 thank Bruce Gray, Ken W. John Kraft and Ross Neureuther from Technical Operations (CCIW), for their hvaluable assistance in the survey and colleaion

Water chemistry parameters were analyzed at the Enviromnent Canada National Lab for Environmental Testing (N'LET) at CCIW, Burlington. Isotope values for DIC were analyzed thanks to Ray Hesslein. Freshwater Institute, Winnipeg. Stable isotope analysis at the Environmentai Isotope Laboratory (EL), Earth Sciences Department, University of Waterloo, was made possible thanks to Bill Mark Bob Drimmie and vaiious members of the support staff. The question remains, where would 1 be without my PhD advisory cornmittee? 1 am truly fortunate to have had a comminee assembled of such dynamic individuals including my CO-supervisors, Dr. Kelly Munkittrick, and Dr. Keith Solomon, and cornmittee members Dr. Mark Servos and Dr. Narinder Kaushik. 1 greatly appreciate al1 the suppon and advise throughout the course of this degree. It has been a privilege to interact with you on professional and persona1 levels, and now 1 have a better undetstanding of the whole business of science. Special thanks to Kelly Munkittrick for your financial assistance, and your endless encouragement and enthusiasm. Kelly, it has v been an honor to work with you! 1 think it would be extremely difficult to find a more supportive supervisor, anywhere! Research funding for this project was provided by the Electricity Association, Ontario Hydro, Department of Fishenes and Oceans (PERD) and Environment Canada. Additional funding was provided by personal student scholarships from the Natural Sciences and Engineering Research Council (NSERC) and the Mary Edmunds Williams Scholarship, University of Guelph.

... Vlll 5.2.3 Stable Isotope Andysis ...... 132 5.2.4 Statistics ...... 132 5.3 Results ...... 132 5.4 Discussion ...... 148 5.4.1 Trophic levd comp~~ns...... 159 5.5 conclusions ...... 162 CHAPTER 6 General Discussion ...... 163 6.1 Simmmy of research conclusions...... 163 6.1.1 Isotopic variability and trends for reference sites ...... 163

6.1.2 lsotopic trends associated with hydroeiectrk development ...... S.. 169 6.1.3 Isotopic trends associated with hydroelectric development and pulp mil1 aaifity...... 173 6.1.4Spatial and temporal isotopic trends for burrowing mayflies ...... 181 6.2 Future research areas ...... 182 CHAPTER 7 References ...... 184 ix List of Tables

Table 1.1 Taxa richness, EPT richness, and percentage of EPT richness for riven in the Moose River drainage basin surveyed by the Muiistry of Naturd Resources fr0m 198 1 to 1986 (Fiset 1995)...... 9

Table 2.1 Selected water chemistry data for sites dong the Missinaibi River in the spring (9and fd (0of 1994, 1995 1996 ...... 24

Table 2.2 Length, weight, age, condition factor (K), liversomatic (LSI) and gonadosornatic (GSI)indices of mature male and female white sucker at sites on the Missinaibi River for the fa11 of 1994. 1996, and spring of 1995. Mdlers represent mean * SE (n)...... 26

Table 2.3 Summary of spatial and temporal variation in 8l3cand ô'% mean * SE(n), maximum. minimum, and range for dorsal muscle of adult white sucker c~ktedin the Missinahi fiver...... 27

Table 2.4 Summary of 613c and 6% mean * SE@) for dorsal muscle of male and female adult white sucker collected in the Missinaibi River...... 28

Table 2.5 Summary of 613c and 6% mean i SE(n), maximum, minimum, and range for whole and lipid extracted muscle. liver, and sonad tissue of white muscle collected at Mattice on the Missinaibi River in the fdl of 1996 @=IO, 2 femdes, 8 males)+...... 34

Table 3.1 Selected water chemistry parameters collected at Fauquier on the Groundhog fiver in the fa11 of 1994 1995...... 53

Table 3.2 Sumrnary of 8I3c and 6''~ mean ISE(n), ststistical significance. maximum, minimum and range for lipid extracted and non-lipid extracted muscle. liver and gonad tissue of white muscle collected downstrearn at Fauquier (1995) and upstream of Carmichael Dam (1996) on the Gmundhog River...... 59

Table 3.3 Length, weight, age. condition factor (K), liversornatic (LSI) and gonadosomatic (GSI) indices of mature male and fde white sucker X collected by gillnet during Fall sampling at sites dong the Groundhog River.

Table 3.4 Surnmary of 6')~(lipid extracted) and (whole) mean I SE (n) for muscle. liver and gonad tissue of female white sucker collected downstrearn at Fauquier (1995) and upstream of Cannichael Dam (1996) on the Groundhog River...... 66

Table 3.5 Surnrnary of 6''~and 8% mean * SE (n), statistical sigruficance, maximum, minimum, and range for whole muscle of white sucker collected upstrearn (Carmichael) and downstream (Fauquier, Whist Falls) of the Carrnichael dam on the Gmundhog River in the fdof 1997...... 66

Table 4.1 Location of generating station, in-seMce year, installeci capacity, drainage area. nomal storage and mean flow rate for sites on the Mattagarni and Kapuskasing Rkrs...... 90

Table 4.2 Sediment characteristics upstream and downstream of the hydroelectric facilities and pulp rnills on the Kapuskasing and Mattagarni Rivers (1994 and 1995)...... 92

Table 4.3 Selected nutrient data presented as mean k SE(n=3) for mil1 effluent and water samples fiom upstream and downstream of the mills on the Kapuskasing and Mattagarni Rivers...... 94

Table 4.4 Water chemistry data presented as mean t SE for mil1 effluent and water samples from upstream and downstream of the rnills on the Kapuskasing and Mattagarni Rivers...... 95

Table 4.5 8 '.' C and 6 l5 N values of dissolved inorganic carbon, sources of terrestrial and aquatic primary production and aquatic secondary production collecteci fiom sites dong the Kapuskasing River. Numben represent pooled samples.98

Taôle 4.6 Length, weight, age, condition factor O(), liversomatic (LSI) and gonadosomatic (GSI) indices of mature male and fedewhite sucker for sites xi along the Kapuskasing River. Fish were collected in the fall by gillnet and

Table 4.7 Length, weight, condition factor (K),liversomatic (LSI) and gonadosomatic (GSI) indices of mature male and femde white sucker for sites along the Kapuskasing River. Fish were collected in the spnng and numbers represent means * SE (n)...... 1 O 1

Table 4.8 Sumrnary of 6 '' C and 6 lS N values of mature white sucker for sites along the Kapuskasing River...... 102

Table 4.9 6 l3 C and 6 l5 N values of dissolved inorganic carbon, sources of terrestrial and aquatic primary production and aquatic secondary production collected fiom sites dong the Mattagami River. Numbers represent pooled samples ...... 104

Table 4.10 Length, weight, age, condition factor (K), liversomatic (LSI) and gonadosornatic (GSI) inclices of mature male and female white sucker for sites along the Mattagami River collected in the spring (S) andor fall (F) of 1994 to 1996. hhnbers represent means * SE (n)...... 106

Table 4.1 1 Summary of 6 l3 C and 6 '' N values of mature white sucker for sites dong the Mattagarni River...... 107

Table 5.1 Profiles of temperature, pH, conductivity, and dissolved oxygen summarized for subsurfhce water, and maximum depths for sites located at distances ups~am and downstrearn of the Carmicheal dam on the Groundhog River and the Smooth Rock Falls on the Matta& River (hg~st,1997)...... 131

Table 5.2 d3cand 6% values of young Hemgetia (0-4 mg dry weight) during summer and fd growth periods. Isotope valws are presented as mean (4* SE (n)...... 140

Table 5.3 Summary of mean, minimum, mar

Figure 2.1 Site locations for collections made in 1994, 1995, and 1996 on the Missinaibi River situated in the western region of the Moose River drainage basin (Nofiheastem Ont am...... 18

Figure 2.2 Relationship between age of white sucker (C. commerso~~i)and 613c (960) (a) and b''~(960) (b) values of dorsal muscle tissue from the Missinaibi River at Mattice (fall of 1994), Skunk Island (fdl of 1996) and Thunderhouse Falls (fall of 1996). Data for male white sucker are labeled (M), the remainder are fernale white sucker...... 29

Figure 2.3 Relationship between weight of white sucker (C. commerso~,ri)and 6I3c(%O)

(a) and 6'% (%O) (b) values of dorsal muscle tissue collected along the Missinaibi River at Mattice, Skunk Island and Thunderhouse Falls fiom 1994 to 1996...... 32

Figure 2.4 Male (n=8) and female (n=2) differences in S"C (%O) and 6% (%O) mean f SE for lipid-extracteci (6"~only) tissues of muscle, liver, and gonad tissues of white sucker (C. commerso~~i)collecteci in the Missinaibi River at Mattice in

Figure 2.5 Range of 6"~(%O) values of primary production fiom terrestrial and aquatic sources, benthic invertebrates, and small foraging fish (modified fiom

Munkittrick et al., 1999) related to the 6I3c (%O) and 6'% (%O) composition of benthic fish species, including white sucker (C. commersoni) (dashed box = range). and longnose sucker (C. catmtoms)(LS), and predatory fish species, such as waileye (S. virruum) (W), northem pike (E. lricit~)(NP), srnallmouth bass (Microptenis do!omietti) (SB), and fdffish (Semotiftts corporah) (FF) from Mattice on the Missinaibi River in 1994 and 1995...... 38

Figure 3.1 Site lOcBfions f8r samples coUected on the Groundhog River, a triiof the Moose River (Northeastern -0) in 199 1, and 1994 to 1997...... 50 xiii Figure 3.2 613c (%) values of terresaial and aquatic prirnary producers and benthic invertebrates including gastropoda (G), decapoda @), ephemeroptera (E) and

613c(%O) and 6'k (960) values of muscle tissue of white sucker (C. commerxmi) (open cucles, dashed rectangle = range), Iongnose sucker (C. cat~omi~s)(LS), and northem pike (E. Iucius) (NP) wllected downstrm of the Carmichael dam at FauWer in the fiiU of 1994- ...... 57

Figure 3.3 6"~(760) mean t SE(n=5) for iipid-artracted musde, liver and gonad tissue and

6'% (%O) mean f SE(n=S exqtfor liver, n=4) for whole muscle, liver and gonad tissue of female and male white sucker (C. comm~rsoni)fiom upstream of the Carmicheal hmon the Groundhog (fdof 19%)...... 6 1

Figure 3.4 613c(%O) (a) and 6'% (960) (b) mm?: SE for whole muscle of male and female white sucker (C.commerstmi) and tipid-extracteci liver (613c only) of fernale white sucker ttom upstream of the Carmichael Dam (CM), downstream at Fauquier (FQ), and downstrearn at Whist Falls (WF) on the Groundhog River for the years of 1991 (pre-irnpoundment) and 1994 to 1997 (post-impoundment). Male and fdewhite sucker whole muscle data fiom Mattice (MT) on the Missinaibi River (1996F) represents the reference site of an unaltered tniutaiy of the Moose River basin @me11 et al. 1999a). Numbers above the syrnbols represent sample

Figure 3.5 Age and 6'" (960) for lipid-extracted muscle, liver and gonad tissue (a) and 6'%(%) for whole muscle. liver and gonad tissue @) of fdeand male white sucker (C. comnttirswti) from uppstrea of the Carmicheal Dam on the Growdhog fivn ( 19%)...... 71

Figure 3.6 White sucker age and. 6 I3c(960) (a) ami 6' k(%)(b) values of whole muscle white sucker (C. commerwt~)60m upstream of the Carmicheal Dam on the GroundhOg River (1996) and Mattjce on the Missinaiii River (1994)...... 73

Figure 3.7 Measured 613c(960) values for individual annuîi of white sucka operuda coiiected upstrearn of the Carmichsel Dam in the W of 1996. Each point represents one year's growth (annulus) of the opercular bone and iines represent the operculwn xiv of an individual white sucker. 613c (%) values of whole muscle for each white aicker are presented with the pph...... 76

Figure 4.1 Sampüng site locations dong the Mattagami and Kapuskashg Rivers of the Moose River Drainage Basin (Northeastm Ontario) (shaded rectangles) and other study site locations on the Missinaibi (Farwell et ai. 1999a) and Groundhog (Fawell et ai. 199%) Rivns (open rectangles)...... 87

Figure 4.2 6'3~(-4and 8% (L~~)values of dorsal muscle for fish species collected at sites upstream (GR-UP 1-3 km) (open symbols) and downstream (GRFQ-DN - 22 km; GRWF-DN - 50 km) (closed syrnbols) of the hydroelecvic dam on the Groundhog River (a) (FanveU et al. 199%). and upstream (open symbols) and downstrem (closed symbols) of the hydroeIectric dam and miil effluent discharge on the Kapuskasing (b) and Mattagarni (c) Rivers. Open square syrnbols represent the reservoir sites for all rivers and closed square symbols represent downstream sites nearest the dam, including areas aposed to pdp miil duent discharge on the Kapuskasing and Mattagami Rivers. Symbds were labeled for fish species including waüeye (W),northem pike (NP), longnose sucker (LS), and troutperch (TP). Symbols with no labels represent the isotope values of white sucker for the

diffèrent sites. The range of 613~(%0)and 6'% (%.) values for white sucker fiom three sites on the Missinaibi River are presented in the dashed box (Fanvell et al. 199%)...... 1 09

Figure 4.3 Phosphorus (a) and nitrogen @)data collected fiom sites on the Missinaibi (Mattice and Thunderhouse Falls), Groundhog (Fauquier), Mwtagami (upstream and downstream of the dam), ond Kapuskasing (upstream and downstream of the dam) Rksin the fall of 1994 ad *ring of 1995-...... 112

Figure 5.1 Site locations for the benthic invertebrate swvey on the Mattagarni River (fall, 1996; wmmer, 1997). and the Growdhog River (summmer, 1997), miesof the Moose River in Northeasteni Ontario...... 1 29

Figure 5.2 6l3c(-) and 8'%J(qb0) values for Hexclgeniu sp. wUected at distances (km) umtream of the hvdroelecüic dam at Smooth Rock Falls in the mainstream and in XV srnaller tributaries of the Mattagami River in the fd of 1996. Open reaches hduded mainstream sites, upstreaml-% (4.0 km) and upstream 2-96 (7.0 km) and sites 0.5 km upstream in Bradbum creek, upstream 3-% (8.0 km) and un- named creek, upstream 6-96 (10 km). Shaded reaches in Bradbum creek (7.5 km fiom dam) included upstream 4-96 (1.8 km 60m mouth) and upstream 5-96 (2.7

FigureY 5.3 613~(~3r..2)and 6'%(*c) values for iYexage11ia sp. collected at various depths at sites upstream of the Smooth Rock Fds dam on the Mattagami River in the fall of 1996. Water depth (m) of the sarnple is presented by the symbol...... 136

Figure 5.4 Hexagenicl sp. weight (mg) versus (a) 613cvalues and (b) 6% values for samples collected at depths of < 3m and 2 3m in the mainstreem, upstrearn of the dam on the Mattagami River at Smooth Rock Falls in the fd of 19% and the summer of 1997...... 138

Figure 5.5 Linear regrwsion of Hex~gti~liasp. (a) 6"~values and (b) 6% values venus water depth for samples collectai in the rnainstream, upstream of the Smooth Rock Falls dam on the Mattagami River in the fd of 1996 and the summer of 1997...... i41

Figure 5.6 6'3~(ac)and 6"~(~4values for Hyxage~Nasp. collected at distances upstream and downstream of the Carmicheal dam on the Groundhog River in the sumer (August) of 1997. The numbers next to the symbols are the water depths (m) at which the sarnples were ~011ected. Symbols with no comsponding number repmt samples wllected at dephof 1 ln or less-...... 144

Figure 5.7 s"c(.,) and s'%(.

Groundhog and Mattagarni Rivers in 1997. Mean 6%(3ba) values are presented as symbols with maximum and minimum 6"~(%~)values as vertical lines. The mmhr&ove the s~mhlis the sample size (n)...... 149

Figure 5.9 Comparison of 613~(a~c)values for Hexage~,Msp. (-) and white sucker (x) ~Uected€rom sections of river upstrearn and downstream of the hydroeiectnc dams on the Groundhog and Managami Rivers in 1997. Mean 613~(q-..)values are presenteâ as symbols with maximum and minimum 6'3~(.:,sp)values as vertical bries. The Unber un ove the ~ymbolis the sample (n)...... 152

Figure 6.1 Frequency distribution of 5"~(,i.) values of white sucker coliected fiom 1994 to 1997 from referenw sites on the undevetoped Missinaibi River (n=47) (Mattice. Skunk Island, and Thunderhouse Falls) (a). reference sites on the developed rivers (n=34) (GRWF-DN(Whist Falls), KAPWF-DN (Woman's Falls). and MATCY-DN (Cypus Falls)) (b), hydroelectnc sites (n=68) on the Gmundhog (GR-UP and GRFQ-DN) and Mattagarni (MATSM-DN and MATKP-DN) Rivas (c). and des duend by the combiion of hydroelectric development and pilp mill activity (n=76) on the Mattagarni (MAT-UPand MAT-DN) and -*8 ~~~ (KAP-UP, KAP-DN. and KAPFF-DN) (dl...... 164

Figure 6.2 Frequency distribution of 8% (4.) values of white sucker collected 6orn 1994 to 1997 ffom refèrence sites on the undevdopsd Missinaibi River (n=47) (Mattice, Skunk Island, and Thunderhouse Falls) (a). reference sites on the developed rivers (n=34) (GRWF-DN(Whst Falls), KAPWF-DN (Woman's Falls). and MATCY-DN (Cypus Falls)) (b), hydroelectric sites (n=68) on the Gmmdhog (GR-UP and GWQ-DN) and Maîtagami (MATSM-DN and MATKP-DIU) Rivers (c). and sites uifluenced by the cornbition of hydroelectric deveiopmmt and pulp rdi activity (n=76) on the (MAT-UP and MAT-DN) and Kapiskariiig (KAP-up, W-DN, md WFF-DN) (a...... 166 rn Figure 6.3 615~(%O) values of white sucker from sites upstream and downstream on the Missinaibi, Groundhog, Kapuskasing, and Mattagami Rivers ( 1994- 1997) (a) and Htrxagertiu (ô) fiom sites upstream and downstream on the Groundhog

and MawJami avers (summer of 1997)- .a .. . . . a ...... , ...... 175

Figure 6.4 Condition factor (K) (a) and body weight (b) of white sucker analyzed for stable isotopes. White sucker were collected from sites upstream and downstream on the Missinaibi, Groundhog, Kapuskasing, and Mattagami

fivers ( 1994- 19971...... *...... 178 1 CHAPTER 1 General Introduction

1.1 Overview Stable isotope analysis (SIA) was used as a tool to study benthic food web dynamics of white sucker in rivers influenced by hydroelectric development and pulp mil1 activity within the Moose River Drainage Basin in Northeastem Ontario, Canada. In order to assess possible alterations in the benthic food web associated with the combined impacts of hydroelectric development and pulp mil1 activity, using stable carbon and nitrogen isotopes as indicaton of change, the following questions must be answered: 1. What is the isotopic composition and variability of selected benthivorous fish, and does it differ with differences in habitat within sections of an unregulated tnbutary? 2. What effect does water regulation imposed by a hydroelectric development have on the isotopic composition and vanability of selected benthivorous fish upstream and downstream of a dam? Knowledge of the isotopic variability at sites upstream and downstream of an unregulated and regulated tributary is used as a reference to assess the impacts of pulp mill activity on food web dynamics in regulated rivers. Food web dynarnics may be altered due to pulp mill practices including historical log drives which resulted in the accumulation of bark and wood upstream of the dam, and pulp mil1 effluent discharges downstream of the dam. Stable isotope analysis of ubiquitous species in the tributaries of the Moose River system were used as short-term (benthic invertebrate) and long-term (benthivorous fish) indicators of change to determine if pulp mil1 activities alter benthic food web interactions. The questions to be addressed were: 3. Does the accumulation of bark and wood upstream of the dam alter the isotopic composition of dietary items (benthic invertebrates), the abundance and diversity of dietary items ador the accessibility of dietary items for benthivorous fish as indicated by changes in the 613c and 6'% values of benthivorous fish? 4. 1s pulp MU effluent assimilated and incorporateci into the tissues of aquatic biota located downstream of effluent discharge as indicated by changes in 613cvalues? 5. 1s there an indirect effi, where pulp mil1 effluent exposure alters the abundance and diversity of dietary items for benthivorous 6sh, thus changing the 6'% values of 2 benthivorous fish downstream of discharge? Information acquired from this study is useful in determining the applicability of SIA for characterizing fish exposure to pulp mil1 effluent, in addition to characterizing the feeding habits of fish species and detennining the suitability of reference sites used in assessments such as the Environmental Effects Monitoring programs for pulp and paper mills.

1.2 Stable isotopes in ecologicai studies Stable isotopes (12c, 13c, I JN, are atoms that retain their composition of protons and neutrons over time. The average nature abundance of 13cand "N in carbon and nitrogen is 1.1100 % and 0.3663 %, respectively (Fogel and Cifbentes 1993). An isotope ratio is defined as R = XhB(, where Xh represents the heavier isotopes (l3c,'?U) and Xi represents the lighter isotopes (12~,"~)of a sample or reference standard. 6I3cand 6% values are denved from the difference between the isotope ratio (R = 13~1'*cor 15 N/%) of a sample and a reference standard such that 6X(s.) = (R,,.) - (Itundad)/

(Rwndd) x lo3. The delta (6) value is expressed as a paris per thousand, or per mil (1. ..J. The reference standard used to calculate 613c values is PDB (Pee Dee Belemnite) which is the fossil skeleton of an extinct cuttlefish (Bdemnitella americanu) found in the Pee Dee formation (South Carolina)(Craig 1957). Atmosphenc nitrogen (N2) is the reference standard used to calculate 6% values. When comparing the 6'" or 6"~values of different samples, a more negative value represents a decrease in the quantity of the heavier isotopes (l3c,'5relative to the lighter isotopes ('2~,'4N)and is referred to as a more depleted 13cor 'h value relative to the primary standard. A more positive 6')~or 6'h value represents an increase 'in the heavier isotopes (')C,'Nrelative to the lighter isotopes ( 12C, 14N) and is referred to as a more enriched "C or "N value. Naturally occurring stable carbon and nitrogen isotopes have been utilized to study various aspects of biogeochemical cycling in aquatic ecosystems. The application of stable isotopes to aquatic food web studies is based on the principk that changes in the isotope ratios of consecutively higher consumer levels are predictable and thai differences in the isotope ratios have ecological ador biologicd relevance (Peterson and Fry 1987; Rounick and Winterboum 1986). The 613c and 6% composition of a consumer tissue 3 may be affected by isotopic fkactionation, whereby discrimination of the lighter ('*c.13c) or heavier ('"N,'%) isotope occurs during the process of assimilation and incorporation into tissue. Minimal carbon isotopic fi-actionation occurs dunng assimilation and incorporation. therefore the 6')~values of primary producers and secondary consumers are relatively conservative (DeNiro and Epstein 1978; Fry and Arnold 1982; Macko et al. 1982). The ecologicd relevance of consumer 613c values is based on the ability to detemiine the contribution of different food sources, assuming the 613c values of the food sources are isotopically distinct. In the present study. the 6"~values of pulp mil1 effluents were used to test the nu11 hypothesis that no carbon sources from pulp miIl effluent were assimilated and incorporated into benthic invertebrate and fish tissues. Significant nitrogen isotopic fkactionation occurs whereby the 6"~values of 15 consumers become increasingly more N e~ched(- 3 - 5 -:O per trophic level) at consecutively higher trophic levels, due to the preferential excretion of '"N during rnetabolism (DeNiro and Epstein 1981; Macko et al. 1982; Minigawa and Wada 1984). The 6'% composition of aquatic biota could be used to identiQ probable dietary items of consurners and quantitatively define trophic positions. In the present study, 6% values of bent hivorous fish were used to test the nul1 hypo t hesis t hat ant hropogenic develo pmen t does not alter the quantity and quality of dietary items conwmed by benthivorous fish, therefore there is no change in the 6% composition of benthivorous fish. The usefùlness of stable isotopes in an assessrnent of nutrient cycling alterations in river systems is lirnited by the need for sufficient badine data to detemiine the natural isotopic variability of the riverine benthic food chain. However, there is limited information on the ecological applications of stable isotopes with benthivorous fish species (France 1995b; Rounick and Hicks 1985) and few studies conducted in river systems with low productivity such as are found in Northem Canada (BUM et al. 1989; Hesslein and Ramla1 1993). Longnose sucker (Cutostomw catostomt~s)were examined as part of the food web in a biomagnification study using nitrogen isotopes in Lake Laberge, Yukon Temtory (Kidd et al. 1994), and there are limited data avdable for white sucker (Ca~ostomnrscommersoni) 6om three temperate lakes (Northwestem Ontario) (Hecky and Hesslein 1995). Assimilation and incorporation of carbon and nitrogen hto various biochemical 4 constituents within the different tissues of fish is complicated by the physiologicd condition of the fish. Factors including seasonal fluctuations in the isotopic composition, abundance and diversity of dietary items, and dietary changes associated with diflerences in the life stage of fish are eliminated in controlled laboratory studies. Lab studies of broad whitefish (Coregoms ?lasus)examined the replacement of carbon, nitrogen and sulfir in tissues following a change in the isotopic composition of the diet (Hesslein et al. 1993). Field studies of the isotopic variability of individual species related to growth parameters is critical when attempting to hterpret whether the changes in the isotopic composition of a species is due to dietary changes or physiologicd changes associated with growth. The majonty (60-83%) of the 6'" variability ((6"~ range = 94of crayfish (Orco~iectrsvirilis) from the littoral zones of four oligotrophic headwater lakes (Northwestem Ontario) was attributed to physiological changes associated with growth based on carapace length (France 1996). Few field studies have assessed the isotopic variability in different fish tissues associated with growth parameters. Sholto-Douglas et al. (1991) examined the relationship between fish length and the isotopic composition of muscle and vertebral bone (decalcified) for the pelagic coastal fish species, cape anchovy (E~i~attiiscqttsis) and roundhemng (Etn~meliswhiteheudi). Abiotic and biotic factors intluencing the 613c variability of pnmary producers and consumen in Stream ecosystems have been reviewed by France (1995b). However, the compilation of data sets in this review included a wide range of strearn sizes, predominately headwater streams. Fewer studies have characterized the isotopic variability of food web components in medium and large riven. Variability in 613c values of benthic invertebrates frorn 3" and 4* order tributaries was attributed to hydrological differences whereas less 6"~variability was observed in the mainstram, River Usk, draining southem regions of England and Wales (Winterboum et al. 1986). ldormation on the 613c variability of producers and consumen in different habitats and the ability to isotopically distinguish between habitats within non-impacted larger rivers in northem Canadian regions is of particular interest in the present study. The tundra river study by BUM et al. (1989) included highly diverse habitats ranging from tributaries draining small lakes to rapids and a deep basin (maximum depth - 75 m) in the main channe1 of the Koroc River (4' order) which discharges hoUngava Bay in northem Quebec. Unfomuuitely, the ability to isotopically distinguish between the benthic invertebrates and fish inhabithg lotic 5 versus Ientic regions within the river system was complicated by sources of '3~enriched marine matter, introduced into the rivenne food web via the excrement and decay of migrating arctic char (Saivelims a/pimds). In addition to the 6I3cvalue of aquatic biota, the 6I3c vwiability within a systern may be an important parameter for distinguishing between anthropogenic enrichment and natural variation within river systems assuming that habitat differences are not a factor. In theory, if the anthropogenic source contributed significantly to the carbon flow in the food web than variability in isotopic composition of consumers rnay be reduced.

1.3 Stable isotopes in ecotoricologicd studies Stable isotopes have been utilized in ecotoxicological studies to estimate biomagnification of persistent contaminants (PCBs and organochlorine pesticides) in aquatic biota (Broman et al. 1992; Kidd et al. 1994; Kiriluk et al. 1995; Schindler et al. 1995) and waterfowl (Mazak et al. 1997). The research is based on evidence that aquatic organisms assimilate persistent contaminants by consumption of contaminated dietary items and storage of contaminants in the lipid Fractions of the tissues such that contaminant concentration increases fiom lower to higher levels of the food chah (biomagnification). The accumulation of contaminants by biomagnitication may be correlated to the trophic level of the organism which is defined using stable nitrogen isotope values. Stable isotopes may also be applied to ecotoxicology studies to trace contaminants based on the principle that the isotope values From an anthropogenic source are isotopically distinct fiom the isotopic values of organic constituents naturally found in the aquatic system. Stable isotopes have been used to examine changes associated with industrial developments, including the influence of pulp miIl effluent discharge (Wassenaar and Culp 1994) and agricuitural practices (MacLeod 1998) in hic systems.

1.4 Anthropogenic deveiopment in the Moosc River drainage barin The Moose River is a large drainage basin (109,000 km2) located in , Canada. The abundance of natural resources in the Moose River drainage basin have been recognized since the early 1900's (Brousseau and Goodchild 1989). Development within the basin was initiated when resource exploration lead to the 6 discovery of gold in the region of Timmins in 1905. Shonly thereafler, stands of black spruce were harvested for pulp mills at Kapuskasing (1909) on the Kapuskasing River, at Smooth Rock Falls (1918) on the Mattagarni River and at Iroquois Falls (1923) on the Abitibi River. Power fiom the first hydroelectnc dam on the Mattagarni River was supplied by 1910 to meet the energy requirements of the prosperous mining and pulp industries. Generating stations s~pplyingpower to the mills at Smooth Rock Falls and Kapuskasing have been established since 19 17 and 1923, respectively. The most recent hydroelectric development, the Camiichael dam on the Groundhog River, began operations in the fail of 1991. There are 14 existing hydroelectnc facilities within the basin, and more than 200 additional sites with hydroelectric potential (Greig et al. 1992).

1.5 Environmental impact studies in the Moose River drainage basin Present activity and the potential for further development on the Moose River tributaries is a concem for fisheries management. In addition to the potential impacts of hydroelectric facilities on fish within the rivers (Brousseau and Goodchild 1989), there are potential impacts on fish associated with pulp mills (Munkittfick et al. 1994, 1998a; Nickle et al. 1998) and mining (Munkittrick and Dixon 1988) within the basin. Conservation of existing fish habitat is of particular importance with regard to proposais for fiiture hydroelectric development, and there have been several large studies within the basin concemed with developing methodology for assessing the potential cumulative effects of development (Fiset 1995; EIP 1998; Munkinrick et al. 1999). Alterations to fish habitat may adversely affect fish productivity in this system which has a relatively low fish production as a consequence of the harsh clirnatic conditions and subsequent short growing seasons (Brousseau and Goodchild 1989; Roy 1989). A series of studies have been conducted to examine the potential impacts of pulp mil1 activity and hydroelectric facilities on white sucker populations in tributaries of the Moose River system (Munkittrick et al. 1994, 1999; Nickle et al. 1998). [Ntial studies within the Moose River Basin found that white sucker downstream of developed areas were larger than those at reference sites (Nickle et al. 1998; Rwmper 1998; Munkittrick et al. 1999). There were several possible explanations for the change in fish size, including potential impacts of the hydroelectric facilities and pulp mil1 etnients on productivity and food web structure in the rivers. Stable isotope analysis may be useful as a tool to determine changes in food weô interactions associated with hydroelectric development and 7 pulp mil1 effluent discharges. Stable isotope analysis of white sucker was used to study changes in benthic food web interactions in the Moose River system. White sucker are a widely distributed benthivorous fish species that are sensitive to pulp mil1 effluent exposure and has been used to investigate alterations in fish growth and reproductive parameters within nvers and Iakes receiving pulp mil1 effluent (Munkittrick et al. 1991, 1998b; McMaster et al. 1992; Gagnon et al. 1994, 1995). White sucker feeding habits have been studied in terms of the dietary composition of juveniles (Mgen 19904b) and the foraging patterns of adults (Logan et al. 1991). Previous impact assessments have shown white sucker to be generalist feeders (Munkittrick et al. 1991). In the Moose River Basin, white sucker, yellow perch (Percaflmescns)and nonhem pike (Esox Iircius) are the most predominant species, followed by walleye (Slizostedkm vineont) (Brousseau and Goodchild 1989). In addition to detemiinhg the changes in benthic food web interactions, stable isotope analysis could be used to define fish exposure to pulp mil1 effluent. One of the difficulties in assessing the impacts of pulp mil1 effluent on fish populations is determining the relationship between the observed effects and the level of exposure of wild fish populations. The ability to define the level of effluent exposure to mobile aquatic orgmisms is findamental to the assessrnent of the impact on wild fish populations. Undefined exposure was recognized as one of the dominant problems with fieshwater monitoring audies at Canadian pulp and paper milk, based on the Expert Working Group (EWP) review of Cycle One reports for the Environmental Effects Monitoring (EEM) program (Munkittrick et al. 1998b). The Environmental Effkcts Monitoring program was implemented in 1992 as a result of arnendments to the Fishenes Act.

1.6 Impacts OC pulp mU efïiuent on food web dynimier Environmental impacts of pulp mil1 effluent in fieshwater systerns have ken reviewed by Owens (199 1). Adverse population and community effects summarized from field studies were caused by three poilutant factors, including fiber and suspended solids, color and turbidity, and organic and nutrient enrichment loads. Fiber and suspendecl dids may eliMnate sedentary ôenthic organisms, or hthic organisms wit h limited mobility, and reduce feeding and reproductive habitats of mobile organisms. Colour and turbidity increases light absorption thereby reducing primary productivity. A study of riverine algal 8 production noted light adaptation of periphyton in the region of an effluent plume whereas the productivity of phytoplankton entering the plume was affected by color (Davis et al. 1988). Also, organic and nutnent ennchment may accelerate growth of primary and secondary producers and consumers. As a result of these pollutant factors, food web interactions in the regions exposed to pulp mil1 effluent may differ fiom other regions.

1.7 Impacts of hydroelectric development on food wcb dynamics Hydroeiectric deveiopment aiters tlow rate, sedientation (turbidity), temperature and oxygen profiles, water depth, and the cycling of nuhients which in tum may alter the abundance, distribution and composition of prYnary and secondary producers, includhg fish species (Brousseau and Goodctiild 1989; Roy 1989; Petts et al. 1995). Hydroelectnc development impacts on aquatic invertebrates in the Moose River drainage basin have been reviewed by Fiset ( 1995). Qualitative surveys of aquatic inveriebrates from 198 1 to 1986 by the Ministry of Natural Resources (MNR) showed differences in taxa richness, EPT richness (EPT = Ephemeroptera, Plecoptera, Trichoptera) and percentage of EPT ricimess between undeveloped and developed rivers (Table 1.1). Improvements in taxa nchness and EPT nchness were found downstream of the hydroelectric complex of four dams located on the Mattagarni River (Fiset 1995). In theory, changes in the aquatic invertebrate community associated with hydroelectric development has the potential to alter food web interactions which may be detected by changes in the isotopic composition of consumers. The composition of the aquatic invertebrate community is dependent on habitat characteristics. Different lotic macrohabitats are created by changes in the flow of water which occun naturally as a function of basin morphology and lithology or artificially due to man-made dams. Changes in habitat was proposed to explain the differences observed in the invertebrate assessrnents on the Groundhog River. Aquatic invertebrate surveys conducted by the MNR in 1985 pnor to construction of the hydroeleçtric dam on the Groundhog River (completed fdl of 1991) generally showed higher taxa richness, EPT richness, and percentage of EPT richness at sites upstream and downstream of the fhre development compared to sites fùrther downstream of La Duke Rapids (Fiset 1995). Thus, determining the food web interactions within diffkrent natural habitats is critical to the assessrnent of impact from anthropogenic development. The review of Moose River studies by Fiset (1995) emphasized the importance of cornparisons with rivers of similar 9 Table 1.1 Taxa richness. EPT richness. and percentage of EPT richness for nvers in the Moose River drainage basin surveyed by the Ministry of Natural Resources hm1981 to 19% (Fiset 1995). Rivers Taxa Richness EPT

klopcd Upper Mattagamin 24.0 Lowr Mattagami 2 1.7 Kapuskasing 21.5

Headwaters Ivanhoe 20.5 6.8 32.9 Chapleau 15.6 2.6 16.5 - - 'upareamsSmooihRod<~allr:bdo\n>sirramollea~-. '' 10 stream order for impact assessrnents since taxa richness was found to be related to stream order, with the highest taxa richness observed in 4' and 5' order streams relative to higher and lower order streams.

1.8 Thesis objectives and organization The overall objective of this work was to use stable isotope analysis as a tool to determine if hydroelectric development and pulp mil1 activity influenced the riverine benthic food webs in the Moose River Drainage Basin. The following is the list of objectives addressed within various chapters of the thesis. The main objectives were to: 1. Detennine if there was spatial ancilor temporal changes in benthic food web interactions linked to the benthivorous fish species, white sucker, in an undeveloped tributary of the Moose River system. 2. Examine different white sucker tissues on spatial and/or temporal scales to define the changes in benthic food web interactions related to hydroelectric development, including: a) present day site isotopic differences in white sucker muscle; b) temporal isotopic differences in white sucker muscle; C) pre-impoundment and post-impoundment isotopic differences in white sucker liver; d) isotopic differences in white sucker annuli from back-calculated opercular bone. 3. Define the combined impact of hydroelectric development and pulp miIl activities using white sucker as a long-term integrated indicator of food web dynamics in two rivers (Mattagarni and Kapuskasing Rivers). 4. Determine the isotopic trends of a seâentary benthic invertebrate group, burrowing magies (Hexagenia sp.) within micmhabitats and the isotopic relationship between sedentary invertebrates and mobile fish species (white suc ker) foraging in rnacrohabitats.

In Chapter 2, "Stable carbon and nitrogen isotope characterization of a riverine benthic food web. 1. Isotopic variability in an undeveloped tributary of the Moose River (Northeastem Ontario)", spatial isotopic variabiüty was determined for muscle of the 1 I benthivorous fish species, white sucker (C. cornmersot~i)to test the nul1 hypothesis that there is no difference in the isotopic composition of white sucker from different habitats within an unregulated river (Missinaibi River). Temporal isotopic vanability was evaluated using yearly isotope data sets for muscle tissue, in addition to age-muscle isotope comparisons to test the nul1 hypothesis that is no temporal variation in the isotopic composition of white sucker as a result of physiological changes in adult white sucker or changes in the type of dietary items consumed. Muscle-liver-gonad isotope compansons were also evaluated to test the nul1 hypothesis that there is no seasonal changes in the isotopic composition of dietary items as indicated by the similarity in the isotope values of muscle and gonad. In Chapter 3. "Stable carbon and nitrogen isotope characterization of a riverine benthic food web. II. Isotopic change in a tributary of the Moose River (Northeastem Ontario) with recent hydroelectric development", spatial and temporal differences in the isotopic composition of white sucker tissues from sites influenced by hydroelectric development were determined and used as an indicator of change in nutrient dynamics and food web interactions associated with the alteration in habitat. The nul1 hypothesis is that there is no difference in the isotopic composition of white sucker tissues upstream and downstream of the hydroelectric development compared to reference sites on the same river and sites on an unregulated river (Chapter 2). Since the development is recent (1 99 1), isotope values of muscle were compared for several years post-impoundment (1994-1997), to determine if there was a gradua1 shifl in the isotopic composition of muscle as adult white sucker becarne acclimated to the new environment and the isotopic composition of dietary items in this environment. Muscle, liver, and gonad were also examined as a method to determine if the isotopic steady state of muscle had been achieved several years post-impoundment. The nul1 hypothesis states that there is no difference between the isotopic composition of muscle and gonad tissues which would suggest that both the short term (gonad) and long terni (muscle) turnover rate tissues are in isotopic steady state with the diaary items consumed in this new habitat. Pre- impoundment and pst-impoundment liver samples were compared to detede if the differences observed between upstream and downstream of the dam are the result of irnpoundment or due to natural conditions. The nuil hypothesis is that there is no difference between pre-impoundment and pst-impoundment liver, thus, the hydroelectric dam has no effect on nutrient dynamics and food web interactions, as indicated by isotopic 12 change. Variability associated with lipid content and possible differences between sexes were considered. Age and weight of white sucker were regressed against muscle isotope values to determine if there was a gradient in exposure to differences in isotopes associated with development as a function of time. In Chapter 4, "Stable carbon and nitrogen isotopes as nutnent tracers in northeastern Ontario rivers with hydroelectric development and pulp mil1 activity. 1. Study of fish populations at distances upstream and downstream of development", stable isotopes of white sucker were used as an indicator to determine the impact of hydroelectric development and pulp mil1 effluent on nutrient dynarnics and food web interactions. Isotope data for white sucker muscle collected at sites upstream and downstream of pulp miIl activity on two different rivers were used to determine if historical log drives resulting in accumulations of bark and wood provided carbon sources or influenced food web dynarnics by altenng the species composition and abundance of potential dietary items andlor the availability of dietary items at upstream sites. Downstream, stable isotopes were used to determine if pulp mil1 effluent directly influences the isotopic composition of white sucker due to enrichment in the system (indicated by changes in 613c)or whether the toxic impacts of effluent influence the species abundance and composition of potential dietary items (indicated by changes in 6%). The study considered two pulp mills which differ in terms of pulping process and treatment, thus the effluents discharged have sligbtly different physical and chernical charactenstics. The nul1 hypothesis states that there is no difference in the impact of pulp miIl effluent on the food webs at the two different sites. Isotope data from an unregulated (Chapter 2) and a regulated river (Chapter 3) were used to determine the impact of pulp mil1 efnuent exposure on nutrient dynamics and food web interactions in a regulated system. If stable carbon isotopes can be used to trace nutrient sources fiom the effluent then 6I3cvalues could be used to trace etnuent exposure in mobile species such as white sucker. The nul1 hypothesis states that there is no different between sites located immediately downstream of effluent discharges and non-impacted reference sites. in Chapter 5, "Stable carbon and nitrogen isotopes as nutrient tracers in northeastern Ontario rivers with hydroelectric development and pulp mil1 activity. 1. Study of benthic invertebrates at distances upstrearn and downstream of development", sedentaiy coliector-gatherer invertebrates (Hexagenia)were examined to determine if there 13 were dfierences in the isotopic composition of invertebrates related to changes in season and habitat. Characterization of the isotopic variability of potentid dietary items was required to explain why there was high 613c variability in fish species from impounded areas and why there was 6'% differences in fish @es between sites infiuenced by impoundment, and the combineci effkcts of impoundment and pulp miii inputs (Chapters 3 and 4). 6I3c and ~'k values of fht level consumers represents the cycling of carbon and Ritrogen within microhabitats, therefore providing a baseline to distinguish feeding habitats of benthivorous fish species within the reservoirs utilizing 613c values and to differentiate between microhabitat cycling of nivogen and changes in the diet of benthivorous fish species at dflerent sites utilking 6% values. The general discussion is presented in Chapter 6. This chapter summarizes the signifiant findings of this study and the scientific ment of utilizing stable isotopes in this particular application. Further areas of research are proposed to enhance the understanding of food web dynamics. CHAPTER 2

Stable carbon and nitrogcn isotope chancterization in a riverine benthic food web. 1. lsotopic variability in an undeveloped tributary of the Moose River (Northeastern Oa tario) 15 2.1 Introduction The objective of this study was to use stable isotopes of carbon and nitrogen as a tool to evaluate benthic food web interactions in an undeveloped tributary in the Moose River Drainage Basin in Northeastem Ontario, Canada. Differences in aquatic invertebrate abundance and diversity within various habitats, including pools and swifls, in the medium sized tributaries of the Moose River system (Fiset 1999, constitute potential dietary items for fish species. In theory, changes in primary and secondary production arnong different habitats, and changes in the isotopic composition of primary and secondary production associated with physical changes in habitat (MacLeod 1998) may be detected at higher consumer levels using stable isotopes. In this study, the benthivorous fish species, white sucker (C. commerso~ii),which represents one of the dominant species in the Moose River system, is used io detect possible changes in the isotopic composition of consumers in different habitats and to define the natural isotopic variability within an undeveloped, relatively pristine environment. White sucker are a widely distributed benthivorous fish species that have been used to investigate alterations in fish growth and reproductive parameters within rivers and lakes receiving pulp mil1 effluent (Munkittrick et al. 199 1, 1998b; McMaster et al. 1992; Gagnon et al. 1994, 1995). Previous impact assessments have shown white sucker to be generalist feeden (Munkittrick et ai. 1991). White sucker feeding habits have been studied in terms of the dietary composition of juveniles (Ahlgren 1990a,b) and the foraging patterns of adults (Logan et al. 1991). However, little information is available on the isotopic variability of benthivorous fish species (Rounick and Hicks 1985) and few studies conducted in river systems with low productivity such as are found in Northem Canada (BUMet al. 1989; Hesslein and Mal1 993). Limited isotope data is available for longnose sucker (Catostom~~scatostomcs) and white sucker (C. commersorii) from investigations of lake food webs (Kidd et al. 1994; Hecky and Hesslein 1995). Isotope data fiom this study, characteriring the diet and feeding habitats of white sucker in an undeveloped river, will be used as a badine data set in funher studies to assess alterations to fish habitat and feod web dynarnics in developed rivers. Alterations to fish habitat may adversely affect fish produdivity in this system which has a relatively low fish production as a wnseqwnce of the harsh cümatic conditions and subsequent short growing seasons (Brousseau and Goodchild 1989; Roy 1989). Present development, 16 including pulp and paper, rnining and hydroelectric facilities and the potential for îùrther hydroelectric development on the Moose River tributaries is a concem for fishenes management, in particular, with regard to the conservation of existing fish habitat. Studies have investigated the potential impacts on fish associated with hydroelectric facilities (Brousseau and Goodchild 1989), pulp mills (Munkittrick et al. 1994, 1998a; Nickle et al. 1998) and mining (Munkittrick and Dixon 1988) within the basin. In addition, several large studies have focused on developing methodology for assessing the potential cumulative erects of development within the basin (Fiset 1995; EIP 19998; Munkinrick et ai. 1999). Although, information on the potential impacts on invertebrate and fish populations in the Moose River system is available, indirect effects on fish populations via aiterations in food web dynamics due to alterations in habitat is not well understood. Stable isotopes were utilized as a tool to assess changes in food web interactions due to habitat alterations associated with anthropogenic development within the Moose River drainage basin. The present study focuses on the Missinaibi River, which represents one of the few undeveloped tributaries in the Moose River Basin, and wiil be used as a reference in further studies that assess the changes in the food web interactions associated with development on other rivers within the system. Three sites were studied to test the nul1 hypothesis that there is no significant difference in the 6"~and 6% values of white sucker dorsal muscle collected fiom sections of river with different habitat characteristics (specifically aquatic macrophyte development, substrate characteristics and water depth). The increase in strearn width fiom the uparearn site to sites some distances downstream was also considered in tems of strearn ecosystem dynamics (Vannote et al. 1980; Minshall et al. 1983; Minshall et al. 1985). Temporal 8l3cand 6% variability of the resident white sucker population was assessed by analyzing dorsal muscle of white sucker from the uparearn site in the faIl of 1994 and 1996 and spring of 1995 (collected during spring migration to spawning grounds in smaller tributaries). Defining temporal variability in low turnover rate tissue of muscle is important in funher studies that assess recent alterations in habitat associated with impoundment. Temporal isotopic variation was investigated by comparing tissues of muscle, liver and gonad, npresenting tissues of varying turnover rates. Lipid extracted tissue and whole tissue (muscle, liver, and gonad) were compared to detemine the degree to which lipid composition inQuenceci 6'" and 6'% values since 17 lipid has been found to be more 13 C depleted compareci to other biochemicai fractions (DeNiro and Epstein 1977). Age, weight and sex of fish were considered to detennine if these factors are a potential source of physiological isotopic variability based on the assumption that the diet of white sucker is relatively consistent throughout the life cycle of the fish. Components of the nverine food web were analyzed to determine trophic position and potential primary sources.

2.2 Materials and methods 2.2.1 Smpling site description The Missinaibi River is one of the major tributaries of the Moose River flowing north-northeast to in northeastem Ontario, Canada. The Missinaibi River is 430 km in length with a drainage basin encompassing an area of 22 530 km2 (Brousseau and Goodchild 1989). This river is classified as a medium sized river with a Stream order of 6 (Brousseau and Goodchild 1989). High fiow rates of 1740 m3/s have been reponed with an average of 105 mS/s(Brousseau and Goodchild 1989). Samples were collected at three locations on the Missinaibi River: Mattice (2-4 km upstream of Highway II); Skunk Island (-25 km downstream of Mattice, upstream of the confluence with the Mattawishkwia River from Hearst); and Thunderhouse Falls (-67 km downstream of Mattice at Bell's Bay, downstream of the naturai barrier at Thunderhouse Fdls) (Figure 2.1). The river runs through the crystalline granites of the Precambrian Shield at Mattice and Skunk Island. The site ai Thunderhouse Falls, located immediately downstream of the Precambrian Shield - Hudson's Bay Lowlands boundary is compnsed of glacial deposits. Mixed deciduous (white birch, and trembling aspen) and coniferous (white spruce, black spruce, and jack pine) stands fom the treeline dong the Missinaibi River. Bands of woody ~h-~bs(dogwood, alder, and willow), grasses and sedges (Cmex), and emergent plants (rushes (Jt~nct~s),horsetails (Equisetum), and arrow heads (Sagit~arja)colonize the shoreline at Mattice. Species of Potamogetotr and Ceratophytlt~mare cornmon aquatic macrophytes. Shoreline vegetation and aquatic macrophytes become less abundant fùrther downstream as the dominant substrate shifts fiom a siit, sand and gravel composition (Mattice) to a gravel, cobble and boulder composition (Skunk Island and Thunderhouse Falls). Sediment composition at Mattice consists of 0.14 % gravel, 97.73 % sand and 2.13 Figure 2.1 Site locations for collections of water samples. benthos and fish along the Missinaibi River situated in the western region of the Moose River drainage basin (Northeastem Ontario).

20 % silt-clay. Habitat surveys (5 transects per site) were conducted in the fa11 of 1996 for the sampling sites along the Missinaibi River. Natural barriers including rapids and falls alter channel morphology fiequently and are evident at distances of approximately 1-2 km upstream of al1 sarnpling sites. Mean Stream width (i SE) increased downstream of Mattice (1 50.2 rn f 4.8) at Skunk Island (182.6 m f 26.2) and Thunderhouse Falls (182.8 m t 30.5). Maximum depths of 6.3 m at Mattice (mean k SE = 3.8 m + 0.7) and 7.0 m at Thunderhouse Falls (mean k SE = 4.0 rn f 0.9) were recorded in the faIl of 19%. The site at Skunk Island was shallower with a maximum depth of 3.7 m (mean k SE = 2.7 m f 0.3). Low water levels in the fa11 increase the occurrence of shallow rocky swifts in the region of Skunk Island and Thunderhouse Falls. Evidence of recent ice scounng at Skunk Island and Thunderhourse Falls indicate natural seasonal water level fluctuations of approximately 3 - 4 m.

2.2.2 Physical and chernical panmeten Physical parameters were obtained for 5 transects per site, inciuding depth (meten) profiles recorded using an echosounder, Stream width (m), substrate composition from ponar grabs, presencdabsence of submersed, and emergen t macro p hyt es, and riparian vegetation. Temperature (OC), pH, dissolved oxygen (mg/'), conductivity (mS/cm), and turbidity (NTU)were measured 0.5 m below the water surface at the left bank, center and right bank. Water samples were collected at the left bank, center and right bank at each site to detennine various water chemistry parameters. Water samples were collected for the analysis of dissolved inorganic carbon (DIC),dissolved organic carbon (DOC) and total phospotus-P (TP-P) in 1251111 boston round glass bottles and stored in the dark at 4OC. DIC for isotope analysis were collected in evacuated 250 ml glas bottles containing potassium chloride preservative. Water samples for aikalinity and total suspended solids were collected in 500d and 1L plastic bottles, respectively, and stored in the dark at 4OC. Particulate organic carbon (POC)and nitrogen (PON) were obtained by filtering a known volume of water through precombusted GFIC filters, followed by an acid se with 3û% HzSOJ and stored in separate petri dishes at 4°C in the dark. AU samples were analyzed at the Canada Center for Inland Waters (CCIW) according to the procedures established by the Environment Canada National Lab for 2 1 Environmental Testing (MET) (Environment Canada 1995).

2.2.3 Sam pk collection Fish were collected at Mattice on the Missinaibi River using hoop nets in srnall tributhes in the spnng of 1995 (May 12 - 25) and gill nets with 3.5 and 4 inch mesh size in the mainstream in the fdl of 1994 (Sept. 29-30) and 1996 (Sept. 11 - Oct. 1). In the fall of 1996, additional collections were made at two mainstream sites located downstream of Mattice at Skunk lsland (Sept. 18 - Oct.3) and downstream of Thunderhouse Falls (Sept. 24 - 27). Sex, fork length (cm), and total weight, liver weight, and gonad weight (g) were determined for each fish. Measurements were used to calculate condition factor, K, (K= 100 * total weight (g) / fork length3 (cm)), iiversomatic index, LSI, (percent liver weight / total weight) and gonadosomatic index, GSI, (percent gonad weight / total weight) for male and female white sucker. The lefi operculum of each fish was removed, and fiozen. In the lab, operculi were cleaned by immersion in 90°C water for 5 seconds to remove the skin covering, and annuli were counted to determine age. Approximately 5-10 g of dorsal white muscle, liver and gonad of white sucker were collected, wrapped in precombusted foi1 (400°C), contained in whirlpac bags and stored on ice in the field. Dorsal muscle was obtained for other fish species and stored in a sirnilar rnanner. Small foraging fish obtained from D-hesamples in shallow water along the shore were fiozen whole. Al1 samples were kept in -20°C freezers prior to preparation for isotope analysis. Terrestrial (primarily leaves) and aquatic plant matenal were colleneci along the shore and in shallow water, cleaned of debris. wrapped in precombusted foil. and stored in plastic whirl pac bags. Fine particulate organic matter (FPOM)was obtained by filtering river water collected in 20 L nalgene containers ont0 precombusted GFIC filters under vaccum. Al1 filters used in this study were precombusted at 400°C for 4 h. Biofilm was collecd fiom small rocks and placed into large ziploc bags underwater to minimize material loss. Rocks were scrubbed with a plastic brush to remove biofilm the biofdm was nnsed, and the nnse water filtered through precombusted GFIC filters. Periphyton on submerged macrophytes was coliected by placing the macrophyte into a large ziploc bag with minimal disturbance. Later, the macrophyte was washed to remove periphyton, and nnse water filtered ont0 precombusted GF/C füters. AM fdters were placed in precombusted foi1 and whirl pac bags or in 7 or 20 ml glass scintillation Mals. Both 22 allochthonous and autochthonous material was stored at -20 OC pior to preparation for stable isotope anaiysis. The majority of benthic invertebrates were obtained using a D-heaquatic net in shallow water to a depth of approximately 1 m. Ponar grabs were used to collect invertebrate samples in deeper water, although success was minimal due to the compressed nature of the substrate. The samples were placed in 500 pm nitex mesh bags and rinsed in river water to remove fine sediments. Each sarnple was placed in a large àploc bag with adequate water, and kept on ice. Later, live invertebrates were picked, cleaned of detritus, sorted and held for 18-24 h in filtered river water to allow the gut to clear. Invertebrates were placed in precombusted foi1 and whirl pac bags or in 7 - 20 ml glass scintillation vials, and frozen at -20 OCuntil being prepared for isotope analysis.

2.2.4 Lipid estraction of tissues Subsarnples (- 0.2 - 0.4 g) of previously freeze-dried, ground muscle, liver and gonad tissues of white sucker were weighed and placed in glas test tubes with 4 ml of methylene ch1oride:hexane (60:40). Samples were vonexeâ at high speed for 1 minute and centrifugai for 5 minutes. The supematant was decanted and the remaining pellet was resuspended in solvent. The procedure was repeated in triplicate. The final pellet was dried, ground to ensure sample homogeneity and prepared for isotopic analysis foilowing the protocol outlined previously. The remairhg supematant was pooled, evaporated, and weighed to estimate lipid content.

23.5 Prepiration and analysis of stable isotopa Material including aquatic macrophytes, FPOM, and biofilm removed fiom rocks and macrophytes were acidified with 1.0 N HCI to remove inorganic carbon fonned as calcium carbonate. AU material was freeze-drieci, removed from filten (where applicable) and ground to a powder using a bal1 mil1 grinder. Benthic invertebrates were categorized by ftnctional feeding groups and family (or genus). The intestinal tract of most invertebrates was removed with the exception of small amphipods, gastropods, and bivalves. Whole (minus intestine) invertebrates were tieeze-dned and ground except for crayfish, where only the muscle tissue tiom the caudai region was used. Dorsal muscle fiom large and sdfi& species as weil as liver and gonad fiom white sucker were also freeze-dried and ground. Dry, ground sarnples were anaiyzed for carbon ("CI 12c)and nitrogen ('%/'"N) isotope ratios using a VG Optima continous flow isotope ratio mass spectrometer at the Environmentai Isotope Laboratory (EL), Department of Earth Sciences, University of Waterloo (Waterloo. Ontario). IAEA standards were used to monitor analytical precision of t 0.20 %O and f 0.30 %O for 6'" and 615~,respectively, within the range of linearity. 613c of DIC and pCOz (partial pressure of dissolved COz) collected in evacuated bottles were analyzed using VG 602 rnicromass duai-inlet mass spectrometer (Hesslein et al. 1997) at the Freshwater Institute Science Laboratory, Winnipeg, Manitoba.

2.2.6 Statistics Analysis of variance (ANOVA) was applied to test for significant isotopic differences between sampling years and sites. Two factor ANOVA and Tukey's HSD test (pairwise comparisons) was applied to isotope data of whole and lipid extracted muscle, liver and gonad tissues of white sucker. 6'" and 6% trends for dorsal muscle of white sucker related to age and weight were evaluated using linear regression analysis.

2.3 Rtsults

The 6I3cof dissolved inorganic carbon PIC) at Mattice was -1 1.4 %O in the fa11 of

1994 (October 1). A sirnilar DIC 6I3c of - 1 1.2 %O was obtained in the spring of 1995 (May 22). In May of 1995, at a pH of 6.45, total DIC (983 pmoüL) consisted of aqueous COt (566 pmoVL) and HCOi (417 pol/L). Alkalinity in the range of 54 to 96 mg/i (Table 2.1) indicated soft to medium hard water. Seasonal fluctuations included reduced levels of alkalinity, dissolved inorganic carbon and pH in the spring (1995) relative to the fàll (1994, and 1995) at Mattice. In contrast, levels of total suspendeâ solids, particdate organic carbon and nitrogen were slightly elevated in the spring (1995) compared with the fa11 (1994, and 1995). Chernical parameters including pH, dissolved oxygen, conductivity and turbidity vked little between sites (Mattice, Skunk Island, and Thunderhouse) in the faIl of 1996. Habitat differences, specifically, decreased abundance of shoreline vegetation and macrophytes, increased proportion of couser sediment (gravel, cobble, and boulder) and increased stream width were evident at both sites downstrem of Mattice. The site at

25 Skunk Island was slightly shallower than the other two sites. Female white sucker were consistently longer and heavier than males for sites on the Missinaibi River (Table 2.2). Condition factor (K)and liversomatic index (LSI) were slightly elevated in females compared to males. in contrast, the gonadosomatic index (GSI) for males was often higher than for females with the exception of the spnng sampling period (1995) at Mattice where the GSI for females was 4-fold higher than males. Cornpansons of carbon and nitrogen isotope data for white sucker dorsal muscle collected in 1994, 1995 and 1996 at Mattice showed no statistically significant difference between years (p > 0.05). 613c and 6% means varied by < 0.50 %O within the three year sampling period (Table 2.3). The S'~Crange was higher in the spnng collection (1995) than Ml collections ( 1994 and 1996). Similarly, no statistically significant difference was observed between sites for 6'" and 6''~values of white sucker dorsal muscle collected at Mattice, Skunk Island and Thunderhouse Falls in 1996 (p>O.OS). S"C and 6% means varied by < 1 %O among the different sites. The range in 613c and 6i5~tended to increase at sites fùrther downstream with the highest range of values reported at Thunderhouse Falls. Isotopic variability was consistently higher for 6"~values compared to 6'% values for al1 sites and sampling periods. White sucker sex. age, and weight were used to detemine if these factors contributed to 6I3c and variability in dorsal muscle. No significant 613c or 6'% difference was observed between male and female white sucker dorsal muscle collected from the Missinaibi River (p>0.05) (Table 2.4). The mean sex difference (n = 23 females and 24 males) for the combined spatial and temporal data for 6I3cand 6'- values was < 0.50 960. The available age data were pooled for male and female white sucker at al1 sampling sites. Adult white sucker varied in age fiom 4 to 13 years. 613c (Figure 2.2a) and 6% (Figure 2.2b) values for muscle showed a similar trend of slight isotopic e~chrnent with an increase in age (s'~c 8 = 0.37, p= 0.009; 6'% 8 = 0.44, p=0.004). White sucker body weight ranged from 401 g to 1,45 1 g (n=47) for data pooled fiom al1 sites and sampling periods. 13C enrichment of dorsal muscle was observed with an increase in

27 Table 1.3 Summary of spatial and temporal variation in 813c and 6% mean k SE(n). maximum, minimum, and range for dorsal musde of adult white sucker coiiected in the Missinaiii River. - Isotope Site Year Mean (%) * SE(n) Maximum Minimum Range (Fall,Spring) (960) ...***.,.*.* ^.-*----.----.~-.-.~~~~...... ~..--*..-.*..-....".....~--....---*...-....-...... *..~~--.*-~-.-----...... -...-.--...... ~..-~...~.- 8I3c Mattice 199JF -28.9 10.3 (10) -27.8 -30.7 2.9 1996F -29.3 f 0.3 ( 10) -28.1 -31.1 3 .O 1995s -29.1 k O.4 (IO) -27.6 -31.4 3.8 Skunk Island 1996F -29.9 i 0.3 (10) -28.2 -3 1.3 3.1 Thunderhouse 1996F -29.5 I0.5(7) -26.5 -30.6 4.1 Total -29.3 I 0.2 (47) -26.5 -3 1.4 4.9

6% Matticc 1994F 8.2 1 O. 1 (10) 8.8 7.6 1.2 1996F 8.5 S O. 1 (10) 9.0 7.8 1.2 1995s 8.6 f 0.1 (10) 9.4 8.1 1.3 Skunk Island 19%F 8.0 I0.2(10) 8.7 6. Y 1.8 Thunderhouse 19%F 7.9 f 0.3 (7) 8.7 6.3 2.4 Total 8.3 f O. 1 (47) 9.4 6.3 3.1 28 Table 2.4 Summary of 8"~and 6% mean * SE(n) for dorsal muscle of male and female adult white sucker collected in the Missinaibi River

Isotope Site Ycar Female Male

(Fal1,Spring) Mean (%O) I SE(n) Mean (%O) k SE(n) 8I3c Manice 1994F -28.9 10.4 (6) -28.9 f 0.4 (4) 19%F -28.3 (2) -29.5 i 0.3 (8) 1995s -28.8 f 0.6 (5) -29.4 f 0.7 (5) Skunk Island 19%F -30.0 f 0.2 (5) -29.8 f 0.5 (5) Thunderhoux 19%F -29.3 f 0.7 (5) -30.1 (2) T~tai -29.1 1: 0.2 (23) -29.5 f 0.2 (24)

8% Maitice 199JF lW6F 1995s Skunk Island 19%F Thunderhouse 19%F Total Figure 2.2 Relationship between age of white sucker (C cornrnersoui) and 6"~(%O)

(a) and S"N (%O) (b) values of dorsal muscle tissue fiom the Missinaibi River at Mattice (fall of 1994), Skunk Island (fa11 of 1996) and Thunderhouse Falls (fall of 1996). Data for male white sucker are labeled (M), the remainder are female white sucker. a O -30 -- a Skunk Island

1 -3 1 1 1

iMattice Skunk Island A Thunderhouse Falls 3 1 weight (8 = 0.38, p < 0.001) (Figure 2.3a). 6% values were poorly correlated to white sucker body weight (8 = 0.17, p = 0.004) (Figure 2.3b). 6''~variability was highest at low body weight. Muscle, liver and gonad tissues of individual white sucker collected at Mattice in the fail of 1996 were analyzed to determine if gonadal biomass accumulation throughout the summer and fall of 1996 was comparable to lower turnover rate tissues of liver and muscle. Lipids were extracted fiom each tissue to eliminate 613c variability related to differences in lipid content. Approximate estimates of mean percent lipid were highea in the liver (1 4.9 %), followed by gonad (4.9 %) and muscle (1.4 %). Whole tissues of muscle, liver, and gonad were I3c depleted by 0.6 %O, 1.3 960. and 0.6 %O, respectively, compared to the lipid-extracted tissues (Table 2.5). Tukey HSD pairwise cornpansons showed significant "C depletion of whole liver compared with whole muscle (p=0.029) and gonad (p=0.009).6')~ values of lipid-extracted muscle, liver, and gonad tissues were not significantly different (mean difference = 0.2 %O, p > 0.05). There was no significant difference in the 6"~values of lipid-extracted tissue and whole tissue (p > 0.05). The mean 6% values were progressively more 15~enriched (gonad, 6.8 %O < liver, 7.6 960 < muscle. 8.6 !%O) for lipid extracted tissues of lower tumover rates relative to gonad tissue. Tukey HSD pairwise comparisons indicated significant differences between the 6% values of al1 the tissues (pc0.00 1). The range of isotopic values increased for tissues with higher turnover rates relative to muscle for both 6l'~(muscle, 1.0 %O < liver, 1.3 %O < gonad, 3.2 %O) and 6')~(muscle, 3.1 %O < liver. 4.2 %a < gonad, 1.3 !%O) values. Isotopic variability in different tissues was also assessed for male and female white sucker. The mean 613cvalues for white sucker males were found to be slightly 13cdepleted for ail lipid extracted tissues in cornparison to females (Figure 2.4). Two factor ANOVA indicated a significant difference between the 613c values of male vs. female tissues (p=0.004). The 613c values of the tissues within each sex were not significantly different. Mean 6% values showed a trend of '% depletion ftom muscle to gonad for male white sucker. Tukey HSD pairwise cornparisons indicated significant differences between the 6% values of al1 three tissues for male white sucker (pc0.00 1). No significant difference was Figure 2.3 Relationship between weight of white sucker (C cummersw~i)and 6I3c (%O) (a) and S"N (%O) (b) values of dorsal muscle tissue collected dong the Missinaibi River at Mattice, Skunk Island and Thunderhouse Falls from 1994 to 1996. o female 1 male 1 weight (g)

1 rn male 1 34 Table 2.5 Summar). of 6"~and 8''~mean f SE(n). muimum. minimum. and range for whole and lipid estractcd niusclc. livcr. and gonad tissue of white muscle collectecl ai Mattiœ on the Missinaibi River in the fall of 19% (n=10. 2 fendes, 8 males).

Isotopc Tissue Lipid Mcan (%) I SE(n) Ma..um Minimum

(%O) (%O)

-.--7-.- ...-....---...-...-...-..------.--*---*.--~-.".-.~--*---...-~...... ~....*...*.*.-~-~***.**..~*--.~...... -.-,...*...... -.- d'k muscle whole -29.3 f 0.3 (10) -28.1 -3 1.1 e?rtra~ted -28.7 +0.3(10) -27.5 -10.6 livcr whole -30.2 1 0.4 (10) -29.0 -33.5 emcted -28.910.4(10) -27.8 -3 1.9 gonad whole -29.2 1: 0.3 (10) -28.1 -32.0 cstracted -28.7 i: 0.4 ( 10) -27.3 -3 1.6

s'% muscle wholc 8.5 10.1 (10) 9.0 7.8 1.2 cvtracted 8.6 I O. I ( 10) 9.0 8.0 1 .O liver w hole 7.6 10.1 (10) 8.0 6.7 1.3 extracted 7.6 f O. 1 ( 10) 8.0 6.7 1.3 gonad whole 6.8 1: 0.3 (10) 9.0 5.9 3.1 cxtractcd 6.8f0.3(10) 9.1 5.8 3.3 Figure 2.4 Male (n=8)and female (n=2) differences in 6I3c(%O) and 6% (%O) mean k SE for lipid-extracted (613conly) tissues of muscle. liver. and gonad of white sucker (C. commersooni) collected in the Missinaibi River at Mattice in fa11 of 1996. imuscle (m) o muscle (f) A liver (m) A liver (f) a gonad (m) O gonad (f) 37 observed between the 6"~values of fedewhite sucker tissues since fernale gonad were similarly enriched compared to muscle and liver (pXl.05). 613c values of white sucker were related to the 6I3crange of FPOM and biofilm, aquatic rnacrophytes, terrestrial leaf matter, benthic invertebrates and small foraging fish species. Individual 6"~values of primary producen, different fùnctional feeding groups for benthic invertebrates and species of small foraging fish (Figure 2.5) are modified fiom Munkittnck et al. (1999). Trophic levels were identified using 6'?U values of benthic and predatory fish species for sampfes collected at Mattice in 1994 and 1995. 6"~values of primary sources included terrestrial material (-29.0 %O), FPOM and macrophyte biofilm (-

24.5 %O to -28.6 %O) and aquatic macrophytes (-22.2 %O). The range of benthic invertebrate S"C values (-28.5 960 to -33.9 %O) fiom shallow water dong the shoreline was more depleted than the primary sources identified. 813c values of small foraging fish were similar to the benthic invertebrates. Adult benthic and predatory fish species were increasingly more I3c eenriched with an increase in 15~enrichment. Predators, including northem pike, smallmouth bass and fallfish were slightly more "N enriched than benthic feeders, white sucker and longnose sucker. Walleye was the most '% emiched fish species analyzed. '% enrichment of walleye relative to white sucker was also observed at

Thunderhouse Falls (mean + SE(n) = 10.9 %O f 0.3(4)). 613c values of walleye (mean + SE(n) = -28.8 %O k 0.4 (4)) at Thunderhouse Falls were slightly 13cenriched compared to white sucker.

2.4 Discussion The 613c values of periphyton and aquatic macrophytes are dependent on the 6')~ of DIC, DIC concentration (Raven et al. 1993), and isotopic kinetic fiactionation associated with the photosynthetic process. The quantity and composition of total DIC (HCOi , CO*) are influenced by photosynthesis, respiration, atmospheric gas exchange, and carbonate rock weathering (Paerson and Fry 1987; Hesslein et al. 1997). Isotopic variation in fiactionation is influenceci by the diffusion of inorganic carbon species (CO2 and HCO33 into the ce11 (Raven et al. 1993) at difkent flow rates of water (France 1995a; Hecky and Hesslein 1995). ûther parameters, including water temperature, hadiance, and nutrients, affect isotopic hctionation (Raven et al. 1993; Rau et al. 1997; MacLeod and Barton 1998). Figure 2.5 Range of 613c (%O) values of primary production fiom terrestrial and aquatic sources, benthic invertebrates, and small foraging fish (modified

from Munkittrick et al. 1999) related to the 6"~(%O) and 6% (%O) composition of benthivorous fish species, including white sucker (C. cornmerson/) (dashed box = range), and longnose sucker (C. catostomi~s) (LS), and predatory fish species, such as walleye (S. vifretim) (W), northem pike (E. hci11.s) (NP), srndlmouth bass (Microptms Jolomieui) (SB), and fallfish (Semotilics corpordis) (FF) from Mattice on the Missinaibi River in 1994 and 1 995. WHITE SUCKER ***..Cl.l*.* @ .& A FF eI QQi LS

FORAGING FISH 1

O TERRESTRIAL LEM MACRWHYE 40 The 6')~sirnilarity of DIC in the spring and fall in the Missinaibi River suggests that the processes that detemiine the sources of DIC, the concentration of DIC and species composition do not fluctuate significantly to influence the 613cvalues of total DIC (- -1 1 %) in the Miuinaibi River. A study of periphyton cultivated on glas slides in a small Ontario Stream with DIC concentrations and DIC 613c values similar to those in the Missinaibi River showed seasonal 6I3c trends in periphyton ranging from approximately -

27 %O to -33 %O in the summer and -3 1 %O to -36.5 %O in the fall (MacLeod and Barton 1998). DIC with a "C depleted value indicates the influence of respiration (Rounick and Winterboum 1986) in the Missinaibi River. 613c values were more ennched for aquatic macrophytes (-22 %), and represent the oniy pure sample of aquatic primary production collected fiom the Missinaibi River. Both FPOM and biofilm 6'" values (-24 %O to -28 %) fall within the range of aquatic macrophytes and terrestrial leaf matter (-29 %), and are more closely linked to the terrestrial 613c value, suggesting the possible influence of terrestrial sources in these samples. Fractionation of 6')~varies 6orn primary sources to first and second level consumers. The range of 613c values of benthic inveriebrates are more depleted than the primary sources identified. This 13C depletion of the benthic invertebrates relative to dietary sources has been observed in other studies (Hecky and Hesslein 1995). Assuming that al1 the 613c values of different primary dietary sources have been characterized in the samples analyzed, the ')cdepletion in benthic invertebrates may be the result of 6I3c fiactionation due to differences in the biochemical composition of invertebrates. Percent lipid variability in aquatic invertebrates is associated with species differences, life stage of the species, and seasonal changes (Guiguer 1999; Legget 1998) and may alter the 6I3c value of the invertebrate. The 613c range of white sucker is slightly more enriched than the benthic invertebrate 6'" range. "C enrichment corn one trophic level to the next in the Missinaibi

River is similar to the 1 %O I3cenrichment obsewed in the laboratory studies (DeNiro and Epstein 1978; Fry and Arnold 1982). The 6"~values for aduit white sucker collected at Mattice over a period of years is suggestive of the importance of terrestrial materid in this aquatic ecosystem, however, it is also possible that the isotopic composition of benthic algae could be in the same range as terrestrial matenal (France 199Sb). The tissues of white sucker were lipid-extracted to eliminate the isotopic variability associated with diaerences in percent lipid content of the tissues. Lipids have been found 41 to be more 13c depleted in comparison to other biochemical constituents such as protein and carbohydrate, due to carbon isotope fiadonation dunng the process of lipid synthesis (DeNiro and Epstein 1977). As a result, whole tissues of muscle, liver, and gonad were more I3c depleted than the lipid-extracted tissues. Liver, with the highest lipid content. showed a greater than 2-fold "C depletion (-1.3 %O) in whole versus extracted tissue, in cornparison to the lower percent lipid content of muscle and gonad (-0.6 %O). There was no difference in the 615~values of lipid-extracted tissue and whole tissue. Hesslein et al. (1993) also observed no difference in 6% values for whole and lipid-extractecl liver of broad whitefish (Coregomis nasus). Lipid extraction eliminated the 613c variability associated with differences in percent content of biochernical constituents For different tissues. Lipid extraction of laboratory reared juvenile rainbow trout (OI~CO~~YIIC~IIS mykiss) tissues (white muscle, red muscle, liver and heari) resulted in no statistical 6I3c difference between tissues and increased 613c variance (Pinnegar and Polunin 1999). In conclusion, any 6'" differences in the lower turnover rate tissues of lipid-extracted muscle and liver compared to the seasonal biomass of lipid-extracted gonad tissue may be attributed to seasonal changes in the dietary items consumeci dunng the gonadal deposition (sumrner months from post spawning in spnng to collection).

The mean 6% values were progressively more '% enriched (- 1 .O %O) fiom gonad, liver, to muscle. Laboratory reared broad whitefish (C. nastîs) were found to differ by 0.5 f 0.5 %O for 6% values of muscle and liver (Hesslein et al. 1993). White muscle of juvenile rainbow trout (0.ntykss) was found to be more "N e~chedthan red muscle, liver and heart tissues (Pinnegar and Polunin 1999). Variation in the 6% composition of different rainbow trout tissues may be a function of differences in the relative abundance of essential and non-essential amino acids within the tissues (Pinnegar and Polunin 1999).

The greater 6% variability between tissues (mean difference, 1.8 %O) in cornparison to the combined 6% variability between sites and sampling penods for muscle (mean difference,

0.8 %O) suggests that isotopic variation between tissues may be intluenced by the ratio of essential to non-essential amino acids. There is preferential excretion of 'k during metabolism resulting in '% e~chment(- 3.4 %O) firom one trophic level to the next (Muiigawa and Wada 1984). However, wit hin a t rophic level (benthivorous fish species), the degree of preferentiaî excretion of 'k associated with physiological changes with age and sexual maturity of 42 males and females is not well understood. Isotopic fiactionation due to differences in assimilation efficiencies resuiting fiom ph y siological changes rnay ex plain the high 6 l 'N variability in dorsal muscle of smaller white sucker and the slight ''N depletion in younger, white sucker for 6lk of muscle versus age and weight regressions, based on the assumption that the diet of white sucker is independent of age. The '% depletion of male gonad relative to muscle differs from the relationship of female gonad and muscle which suggests possible sex differences in the composition of amino acids in gonad tissue. DifTerences in the S"N values of male and femaie gonad may also be a function of sex differences in energy reallocation, however the sample size for females is too small to determine if the differences are valid. Cornparisons of lipid-extracted muscle, liver, and gonad tissues for white sucker showed no significant difference between tissues for 613c mean values. Rounick and Hicks (1985) also reported similarities in the 613cvalues for muscle and liver of foraging Bsh in several New Zealand rivers. The low 6"~variability between tissues (mean difference, 0.2 1%0) in cornparison to the combined 613c variability between sites and sampling periods for muscle (mean difference, 0.99 960) suggests the importance of dserences in the 613c composition of dietary items dong the Missinaibi River versus 6')~fiactionation during the process of biosynthesis of different tissues. Low 13c fiactionation between tissues at Mattice indicates the relative stability of the 613c values of dietary items consumed by white sucker in the short and long term, thus suggesting there is no signifiant seasonal changes in the isotopic composition of dietary items. However, there was slight depletion for male relative to female white sucker when each tissue type was compared. This "C fiactionation may be attributed to physiological differences between sexes during the process of tissue biosynthesis. Although not strongly correlated. age and weight regressed against 6I3cof muscle also indicated slight 13cdepletion in Young, smaller white sucker. The range of isotopic values increased for tissues with higher turnover rates

relative to muscle for both 6'% (muscle, 1.0 960 < liver, 1.3 %O < gonad, 3.2 %) and 6"~

(muscle, 3.1 %O < liver, 4.2 %O < gonad, 4.3 %O) values. White sucker gonad tissue is produced during a feeding period of approximately 5 months fiom the the of spring spawning to the faIl collection. The isotopic variability of the gonad integrates possible seasonal changes in the isotopic composition of dietaiy sources and the relative proportion of dietary items. Throughout the year, the abundance of ûenthic invettebrates varies and. 43 subsequently, the diet of white sucker rnay shift to increased consumption of detrital material as the densities of benthic invertebrates decreased (Ahlgren 1990a). Although the protein content of detritus is too low to support somatic growth, the combination of detritus and invertebrates provides sufficient energy to promote growth or reduce the rate of weight loss when invertebrate abundance is low (Ahlgren l99Ob). The advantage of utilizing muscle of white sucker to assess long-term change in developed nver systems is that muscle incorporates seasonal isotopic variability of the detrital material and benthic invertebrates and the relative contributions of these sources to the diet of white sucker. The inherent reduction of isotopic variability at the level of white sucker muscle tissue must therefore be recognized when assessing different populations of white sucker. The dynamics of stream ecosystems have been defined by the gradual change in the structure of the cornmunity and the fiinctional processes fiom headwaters to midsized rivers (Vannote et al. 1980; Minshall et al. 1983). Variation in species composition and the contribution of heterotrophic and autotrophic processes within a nver are influenced by local differences in watershed climate and geology, riparian vegetation, tributaries and location-specific lithology and geomorphology (Minshall et al. 1983). Along the sites of the Missinaibi River, noticeable differences included changes in the abundance of shoreline vegetation, lithology (substrate composition). and geornorphology of the sites and. to a lesser extent, geological change at Thunderhouse Falls. All these parameters have the potential to alter the isotopic values of stream flora and fauna. 613c and S"N values of prirnary producers andor benthic invertebrates may vary spatially, in terms of macro- environmental and micro-environmental direrences, and temporal (Winterboum et al. 1986; MacLeod and Barton 1998). 613c and 6% values of muscle tissue of white sucker represent the long-term feeding habits of this benthivorous fish species, the integration of spatial and temporal isotopic variability of dietary items, the relative abundance of different dietary items, and the biological isotopic variability of white sucker. As a result, the range of isotopic values of white sucker muscle were similar for 6''~values (1994, 1995 and 1996) and for 6"~values (1994 and 1996). However, a greater range of G"C values (range=3.8 %, SE4.443)was obxrved for white sucker collected at Mattice in spring of 1995. The higher 613c range in spnng may represent fish mignting fiom a wider range in territory to spawning grounds in contrast to faIl collections in 1994 and 1996, although migration is ümited by naturd barriers. The slight decrease in 6"~and 8% means downstream and the süght increase in the isotopic range rnay be the result of habitat 44 differences. The site at Mattice represents a more uniform habitat whereas changes in the lithology downstream introduces a combination of shallow rocky swifts and deeper pools. The increased diversity of habitat may explain the wider range in isotope values at the downstream sites. The ultimate goal of the project was to use the isotopic values of white sucker muscle tissue as an integrated long term indicator to determine the impacts on food web interactions associated with anthropogenic environmental changes in different tributaries of the Moose River system. Data from the Missinaibi River was used to establish a baseline of the natural variability in isotope values for the benthivorous fish species, white sucker, in an unaltered nver. Analyses of white sucker muscle tissue fiom Mattice showed minimal differences between 6I3c and 6% means (< 0.5 %O) for 1994, 1995, and 1996. Also, 6I3cand 6% means for white sucker muscle tissue collected at three different sites in the fall of 1996 showed differences of < I %O. Hecky and Hesslein (1995) also observed similarity between the 6 "N means (n= 1 O; mean = 7.34 %O, 6.36 %O, 6.85 %O) for white sucker collected from three temperate lakes on the Canadian Shield in Northwestem

Ontario. However, the 6')~means (n=lO;mean = -26.16 %O, -22.63 %O, -22.07 %O) differed by - 4.0 %O for the three lakes.

2.5 Conclusions Muscle tissue is a good indicator of the integrated long term isotopic composition of dietary sources for male and female white sucker compareâ to the higher 6I3cand 6% variability of liver and gonad tissues. No significant isotopic differences were found between sampling periods (years) and sites for muscle tissue of white sucker in the undeveloped nver. The 6I3cvalues of white sucker were comparable to the 613cvalues of the benthic invertebrates analyzed at Mattice. The isotopic composition of white sucker in the Missinaibi River can be defined at -29.3 %O f 0.2(47) and 8.3 % f O. 1 (47) (mean f

SE(n)) for 613cand 6% values, respectively. The range of 6'" (-3 1.4 to -26.5 %O) and 6% (6.3 to 9.4) cm be used as an indicator of nonnal fluctuation in the isotopic composition of white sucker in an undeveloped river in the Moose River Basin. Isotope data fiom the reference sites on the Missinaibi River will be used in combination with isotope data fiom reference sites on developed riven in the Moose River Basin. Carbon and nitrogen isotope values of white sucker âom developed sites outside of the range for reference site 6'" andor 6'% values may be considered as evidence of signifiant isotopic change. Stable carbon and nitmgen isotope characterizition of a riverine bcnthic food web. II. Isotopic change in a tributary of the Moose River (Northeastcrn Ontario) with ment hydroektric developmmt. 47 3.1 introduction In Northemtern Ontario (Canada), the water rewurces of the tnbutaries of the Moose River have been used by industry to support hydroelectric generation, pulp and paper, and mining activities since the early 1900's (Brousseau and Goodchild 1989). Studies within these riven have identified potential impacts on fish associated with hydroelectric facilities (Brousseau and Goodchild 1989), pulp mills (Munkinrick et al. 1994, 1998a; Nickle et al. 1998) and mining (Munkittrick and Dkon 1988). There are 14 existing hydroelectric facilities nithin the Moose River basin (Greig et al. 1992). Proposais for tùture development of more than 200 additional sites (Greig et al. 1992) with hydroelectric potential has prompted several large studies focusing on the development of methods for assessrnent of the potential cumulative effects of development within the basin (Fiset 1995; EiP 1998; Munkittrick et ai. 1999). Hydroetectric development alters flow rate, sdmentation (turbidity), temperature and oxygen profiles, water depth, and the cycling of nutrimts (Brousseau and Goodchild 1989; Roy 1989; Petts et al. 1995). Physical and chemical changes associated with dams alter the abundance, distriibution and composition of primary and secondary producers, including fish species (Brousseau and Goodchild 1989; Pms et al. 1995). A senes of studies have been conducted to examine the potential impacts of pulp miIl activity and hydroelectric facilities on the performance of white sucker populations in tributaries of the Moose River system (Munkittrick et al. 1994, 1998b; Nickle et al. 1998). However, information on the potential impact of dams on nutrient cycling and food web structure of midsized rivers in northem regions is limited. Preliminary stable carbon and nitrogen isotope studies used the benthivorous fish species, white sucker (Chtostomr~scommersonii) as a long term integrated indicator to trace the natural cycling of carbon and nitrogen in an undeveloped tributary (Missinaibi River) in the Moose River Drainage Basin (FarweU et al. 1999a). Low spatial and temporal variabiity in the 613c and d"~values of benthivorous fish are indicative of the biogeochernid and hyûrological stabilty of this unaltmed systern. White sucker (C. commersoni) has also bcen examined in the study of lake fdwebs in Northwestem Ontano where stable isotopes were useû to detemine the contribution of benthic algae to different trophic levels (Hecky and Hesslein 1995). Longnose sucker (Cutostomus cutos~is)have been investigateâ in a biomagnification study using nitrogen isotopes to assess the food web of a Yukon Territory lake (Kidd et al. 1994). 48 The primary objective of this study is to determine if nutrient cycling, upstream and downstream, is altered by hydroelectric development utilizing stable isotopes as an indicator of change in nutrient dynarnics and food web interactions in the system. Numerous stable isotope studies have documented spatiai and tempoml isotopic variability in primaiy and secondas, producers related to the physical and chernical characteristics of the aquatic environment (Hecky and Hesslein 1995; France 1995a; Leggett et al. 1997% b; MacLeod and Barton 1998; Servos et al. 1998; Guigua 1999). in this study, isotope analysis of white uicker muscle rqresents the iineption of the isotopic variability in prunaiy and secondary producers and allows for species-specific cornparisons of river sections in undevelopeâ (FanveU et al. 1999a) and developed riven. The regulated river investigated in this study is the Groundhog River which is one of several tributaries in the Moose River Basin supporting hydroelectric facilities. The Grst hydroelectric development (Carmichael dam) on the Groundhog River was cornpleted with initial operation in October of 1991. Since the development is recent, the 6"~and 6% variability of resident white sucker populations upstream and downstream of the dam were assessed by analyzing dorsal muscle tissue of white sucker collected fiom 1994 to 1997. The ability to utilize adult white sucker muscle isotope vaiues to establish isotopic trends in recently altered systems is dependent on the isotopic steady state of muscle to the isotopic composition of dietary items in the existing habitat. Thus, 613cand 6% values of gonad were compared to muscle to detemiine if muscle was at an isotopic steady state relative to the recent isotopic composition of gonad tissue. Tissues of different turnover rates (muscle, liver, and gonad) were analyzed to determine if there are any differences in the isotope values and to determine if the use of muscle tissue is a good indicator of isotopic changes associated with regulation. Lipid extracted tissues and whole tissues of the muscle, liver and gonad were compared to determine the degree to which lipid content infiuenced the 6"~and 6% values. Male and fernale white sucker tissues were compared to determine if there are sex differences since there is a possibility that the isotopic composition of the different tissues, particularly the 6% vaiues of gonadai tissue, may Vary with sex (Fawell et al. 1999a). Since the âam was consmicted in 1991, age and weight of white sucker males and females were also evaluated to test the hypothesis that recently mature fish (age 6 and 7) have more depleted isotope values for muscle than older fish wllected upstream of the dam (fall 1996). In 1991, white sucker hers wne coliected hmthe Groundhog River at 49 Fauquier for use as a reference in a survey of the environmental impacts associated with pulp miil discharges (Munkittnck et al. 1994). Isotope values of tiver tissue were compared for white sucker coiiected at pre-impoundment ( 199 1) and pst-irnpoundment intervals for upaream (1996) and downstream sites (1994 and 1995) to detennine if hydroelectric development altered the isotopic composition of white sucker. Isotope values of liver were compared to muscle for downstream (1995) and upstrearn (19%) sites to determine if' liver provides an accurate measure relative to muscle since the majority of isotopic cornparisons in diis study involves muscle tissue. Yearly growth rings (annuii) of the operailar bone white sucker were isolated and individuaily anaiyzed to detemine the potential use of operculi as a hiaoric record of carbon isotopic change in an aquatic system. Components of the riverine food web were analyzed to determine trophic position and the importance of primary sources. Information from this study was correlatai with the isotopic trends of the unregulated Missinaibi River (Fanvell et al. 1999a) located in the western region of the Moose River Basin.

3.2 Materials and Methods 3.2.1 Sampling Site Description The Groundhog River is a tnbutary of the Moose River (length 106 km) flowing noith- northeast to James Bay (Northeastem Ontario) (Figure 3.1). The Groundhog River is 363 km in length, supplieci with drainage water fiom a catchmmt area of 12 5 18 bn' (Brousseau and Goodchild 1989). The geology of the basin was crystalline granites characteristic of the Precambrian Shield. The sumxindiig landscape consisteci of mixed deciduous and coniferous forests with agriniltural land established in most regions This medium sized river is clded as a 5' order Stream (Brousseau and Goodchild 1989). A mean flow rate of 145 m3 s" was recurded prior to hydroelectric deveiopment on the Groundhog River (Brousseau and Goodchüd 1989). Construction of the hyâroetectric Mtyat Carmicheal Falls was initiatecl in 1989 and completed with turbine operation in October of 1991. Mean flow rates of approximately 102, 173, and 140 m3 i' were observed at the Fauquier flow station in 1995. 1996, and 1997 following irnpoundrnent @oug Lawler, Water Management SeMces, Water Resoucces Division, pers. mm.) There were three sampling sites on the Groundhog River. Samples were collected in a 1 to 3 km section of the reservou upstrepm of the Carmichad drun This te~ervoirwas a considerable distance downsirram of the swrce waters of the ûroundhog River âom Figure 3.1 Site locations for samples coiiected on the Groundhog River, a tribuary of the Moose River (Northeastern Ontario) in 199 1, and 1 994 to 1 997.

52 Horwood Lake and fiom the possible influences of the Ivanhoe River. However, there are a number of permanent and temporary tributaries within the region of the reservou. The second site was located where Highway 1 I crosses over the Gromdhog River at Fauquier, approxhately 22 km downstrearn of the hydroelectric dam. The third sampling site was located at Whist Falls, approxhately 28 km downstrearn of Fauquier and 10 km upstream of the confluence of the Groundhog and Mattagarni Rivers. Habitat was characterized in terms of water depth, substrate composition, presencelabsence of aquatic macrophytes and riparian vegetation for 5 transects per site, upstream and downstrearn of the Carmich1 dam on the Groundhog River. Upstream of the hydroelectric dam, high water levels limited shoreline development to fdlen logs, shnibs and trees with no aquatic mamphytes. Maximum depth ranged fkom 11.3 to 21.2 m (mean maximum depth; 15.5 m) and substrate was composed of silt, clay, and sand. Downstream of the dam at Fauquier, Iow water levels exposed sporadic roc@ zones between clay-sand beaches with grasses and emergent macrophytes approximately 1 m 6om the water line. M&um depth ranged 6om 3.0 to 5.8 m (mean maximum depth; 4.0 m) with submerseû macrophytes in shallow water where the substrate was adequate to support growth. Analysis of water chemistry at Fauquier in the fiIl of 1994 and 1995 (Table 3.1 ) showed minimai variation and values were comparable to the water chemistry of the Missinaibi River (FanveU et al. 199%). Details of the collection protocol are outüned in Fmell et al. (1999a). Water sarnples were analyzed according to the procedures established by the Environment Canada National Lab for Environmental Testing (NLET) (Environment Canada 1995) at the Canada Center for inland Waters (CCIW) (Burlington, Ontario).

3.2.2 Sample Collection Fish were obtained from gill nets (8.8 and 10.2 cm mesh size) placed in the mainstream at Fauquier in the fall of 199 1 (Aug.20-Sept. 1S), 1994 (Sept. 19-25), 1995 (Sept.25-26) and 1997 (Sept.2 1-22). Mainstream collections were made in the fall of 1996 (Sept.26) and 1997 (Sept. 19-20) upstream of the Carmichael dam and in 1997 (Sept. 9- 24) at Whist Falls. Sex, fork length (cm), and total weight, liver weight, and gonad weight (g) were determined for each fish. Measurements were used to calculate condition factor, K, (K=lOO*total weight(g)/fork ~ength(cm)~),üversomatic index, LSI, (percent liver weightltotal weight) and gonadosomatic index, GSI, (percent gonad weighthotal weight) for male and female white sucker. Both operculi of each fish were collecteci, Frozen and 53 Table 3.1 Selected water chemistry parameters collecied at Fauquier on the Groundhog River in the fdl of 1994 and 1995.

PH dissolved o.ygen (mgiî) conductivity (mS/cm) colour (hazeniin) auralinity (mgil) dissolved inorganic carbon (mg) dissolved organic cartion (mg/') particulale organic carbon (md) dissolved organic nitrogen [mgfl) particulatc organic nitrogen (mgll) kjedahl-N (mgfl) dissolved phosphorus (mg/l) particulaie phosphorus (mg) toial suspendcd solids (mg/!)

------.- -- - Note: 'panmeters reprcsent the avcrage of IcA bank center and right bank measurements 54 later, irnmersed in 90°C water for 5 sec. to remove the skin covering. The lefi operculum was used to detemine age by counting annuli and the nght operculum was used for isotope analysis. Dorsal white muscle, liver and gonad of white sucker (-S-log)were collected, wrapped in precombusted foil (400°C), contained in whirlpac bags and stored on ice in the field. Dorsal muscle was also obtained for other fish species and stored in a similar rnanner. All sarnples were kept in -20°C freezers pnor to preparation for isotope analysis. Terrestrial (prirnarily leaves) and aquatic plant material were collected dong the shore and in shallow water, cleaned of debris, wrapped in precombusted foi1 and stored in plastic whirl pac bags. Fine particulate organic matter (FPOM)was obtained by filtering river water collected in 20L nalgene containers ont0 precombusted GF/C filters under vaccum. Ai! filten used in this study were precombusted at 400°C for 4 h. Separate biofilm sarnples fiom rocks and macrophytes were collected by gently placing the substrate in large ziploc bags underwater to minimize loss of biofilm. Within the bag, the biofilrn was removed from its substrate, the substrate rinsed and discarded, and the remaining rinse water filtered ont0 precombusted GFIC filters. MI filters were placed in precornbusted foi1 and whirl pac bags or in 7 or 20ml glass scintillation vials. All filters and plant material were stored at -20°C phor to preparation for stable isotope analysis. The majority of benthic invertebrates were obtained using a Dframe aquatic net in shallow water to a depth of approximately 1 m. Ponar grabs were used to collect invertebrate sarnples in deeper water, althwgh success was minimal due to compacted sediments downstream. The samples were placed in 500pm nitex mesh bags and rinsed in river water to remove fine sediments. Each sample was placed in a large ziploc bag with adequate water, and kept on ice. Later, live invertebrates were picked, cleaned of detntus, soried and held for 18-24 h in ûltered river water to ailow the gut to clear. lnvertebrates were placed in precombusted foil and whirl pac bags or in 7 or 20ml glass scintillation vials, and fkozen at -20°C until being prepared for isotope anaiysis.

3.2.3 Lipid Extraction of Tissues Subsamples (- 0.2 - 0.4g) of previously fieeze-dried, ground muscle, liver and gonad tissues of white sucker were weighed and placed in giass test tubes with 4 ml of methylene ch1onde:hexane (60:40). Samples were vortexed at high spdfor 1 min. and centrifiiged for 5 min. The supernatant was decanted and the remaining pellet was 55 resuspended in solvent. The procedure was repeated in triplicate. The final pellet was dned, ground to ensure sample homogeneity and prepared for isotopic analysis. The remairing supernatant was pooled, evaporated, and weighed to estimate lipid content.

3.2.4 Preparation and Aoalysis or Stable Isotopes Material including aquatic macrophytes, FPOM, and biofilm removed from rocks and macrophytes were acidified with 1.0 N HCI to remove inorga~ccarbon fonned as caicium carbonate. Mi material was freeze-dried, rernoved hmfilters (where applicable) and ground to a powder using a bal1 mil1 grinder. Benthic invertebrates were categorized by tùnctional feeding groups and family (or genus). The intestinal tract of most invertebrates were removed with the exception of smdl arnphipods, gastropods, and bivalves. Whole (minus intestine) invertebrates were freeze dried and ground except for crayfish where only the muscle tissue from the caudal region was used. Dorsal muscle fiom large and small species as well as liver and gonad fiom white sucker were also fieeze-dned and ground. Ody the welldefined central reghn of the opernilum was used for anaîysis. Beginning at the outer edge of the operailurn (representative of the most recent growth), individual rings (annuli) were cut dong the ring Iuie using a sharp-eâged cutîing pliers and placed in separate Mals. The individual Migs were ground to a homogenous powder and prepared for isotopic analysis. Dry, ground samples were analyzed for carbon ("c/'~c)and nitrogen ("N/"N) isotope ratios using a VG Optima continous flow isotope ratio mass spectrometer at the Environmental Isotope Laboratory (EL),Department of Earth Sciences, University of Waterloo (Waterloo, Ontario). IAEA and NlST standards were used to monitor analytical precision of k 0.20 %O and t 0.30 %O for 6"~and 6'%, respectively, within the range of linearity. 6'" of DIC and pC02 (partial pressure of dissolved CO2) collezted in evacuated bottles were analyzed using VG 602 micromass dual-inlet mass spectrometer (Hessiein et al. 1997) at the Freshwater Institute Science Laboratory (Winnipeg, Manitoba).

3.2.5 Statistics Analysis of variance (ANOVA) and Tukey pairwise cornparisons were applied to test for significant isotopic differences between whole and lipid extracted tissues; muscle, liver, and gonad; male and fende; and spatial and temporal effects for white sucker tiom sites upstream and downstream of the Carmichael dam on the Groundhog River. Linear 56 regressions were applied to determine the correlation between age and 6"~or 6'% values of white sucker muscle.

3.3 ksulb Werent sources of wbon in the fomi of fine particle organic matter (FPOM), biofiim, aquatic mecrophyte and leaf litter constitute the organic carbon base of the benthic food web in the Groundhog River. Preliminaiy stable carbon isotope analysis of sarnples collected downstream of the dam at Fauquier in the fdOF 1994 showed "C depietion of FPOM relative to bioîdm, leaf litter and rnacrophytes (Figure 3.2). The depleted "C value of FPOM suggests the presence of a combination of aüochthonous and autochthonous material. The 613cvalue of fine partidates from a terrestrial source would probaMy by simüar to the 6% values of leaf material (-29 %O). The &n isotopic composition of benîhic invertebrates and benthivorous fish suggested the importance of carbn sources which were more depleted than terrestrial sources. The 613c and 6%l values of longnose sucker were comparable to those for white sucker, whereas the top predator was slightly more 13cand '% enriched. Four different approaches were used in this study to determine if hpoundment altered the isotope vdues of the components of the food web. The methods involved differerrt tissue analyses of white aicker which represented an integrated indicator of isotopic change. The standard approadi involved comparing present &y conditions upstream and downstrearn of the Camiichael dam. Muscle, liver and gonad tissues of male and fdewhite sucker were compared to determine if muscle is a good indicator of isotopic chge and to estabiish a relationship bnween the tissues since there are archived white sucker fdeliver samples fiom downstrearn of the dam, prior to impmdment. Cornparisons of white sucker musde, liver and gonad fiom downsaearn (Fauquier, 1995) and upstream of the Carmichel Dam (1996) required lipid donof tissues to eliminate 613c variability associated with diffices in iipid content (Table 3.2). -factor ANOVA indicated sigruûcant "C depleiion of üpid extfacted tissue versus wMe tissue (p4.002). Whole muscle was considered a good indicator of 6I3cchange ôased on evidence that the S'~Cvafues of whole and lipid extracted muscle were Marend that aii lipid extracted tissues wae similar. SpeQfic testing (MGLH) of each tissue showed signifiant 6I3c ~éraieesbetween whde and extra34 tissue for the downstrram (fl.028) and upstream (p=û.002) sites. Spccific (MGLH) compPnsons lipid aaracted musele, liver, Figure 3.2 613c (%O) values of terrestrial and aquatic primary producers and benthic invertehates including gastropoda (G),decapoda O),ephemeroptera (E) and

613c (%O) and 6'% (%O) values of musde tissue of white sucker (C cornmerso~~i)(open circles, dashed rectangle = range), longnose sucker (C. calostom~~s)(LS), and northem pike (E lucirs) @IP) deaed downstrearn of the Carmichael dam at Fauquier in the tiill of 1994. FPOM O BlOFlLM O II MACROPHYTE a TERRESTRIAL MATERIAL Table 3.2 Summar). of ô"~and 8"~mcan f SE(n), s~ltisîicalsignificance, maximum. minimum and rangc for lipid cxtriicted and non-lipid extracîed muscle. -of -of white ~au~uieqichael lW0pe Site Lipid Mean (%O) f SEtn) Sfatistical Maximum Minimum Range -...... (P values) 8' 3~ ~auquiefl mus& (m) whok -3 1.5 I 0.5 (10) -29.6 -34.1 4.5 ewacted whole extraçtd whole estractcd whole cxtracted whole ehqracted whole cdracted

w hole extractcd wholc estraclûd whole estracîed whole exlractcd whole eamed wholc extracted . Noie: ' n=IO, 8 femlcs, 2 males: n=IO, 5 females, 5 males escept for liver, 5 females. 4 males: siaîisiical diffcrences betwcn wholc vs. lipid estradcd tissues. *tissue t)p.' sites. 60 and gonad 613c values were not statistically diierent at the downstream or upstream sites. Three-factor ANOVA also indicated a signiticant 8"~difference between sites (p<0.001). Specific (MGLH)cornparisons for üpid-extracted tissues between sites showed rigiuficant 13c depletion upstream of the dam for muscle (p=0.005) and liver (p=0.014). Whole muscle showed a sirnilar trend with significant I3c depletion upmearn (p=0.007). The signifiant ditference between sites for lipid extracted iiver is important for Uiterpreting cornparisons of archived liver 6om downstream, prior to the hpowidment. 8% values of whole and lipid extracted muscle, üver and gonad were compared for the dowostream (Fauquier-1995) and upstream (Carnichael-19%) sites to determine if' the process of lipid extraction altered the 6I%J values of the tissues (Table 3.2). Three-faor ANOVA otX1?U values Uidicated no signifiant clifference bnween lipid extracted and whole tissues for the two sites (Table 3.2). Although no signifiant 6% differences were observed, the following comparisons used the 6'%J values of whole tissue. Whole muscle was considered an adequate indicator of 6% changes due to Unpoundment based on the consistent relationship to the other tissues, speafically, whole muscle was emiched compareci to liver and similar to gonad, for both sites. The 6% values of whole muscle were also fodto be statistically different between sites. Effects of impoundment upstrearn and downstream of the dam and the influence of the Walnisirni River (Figure 3.1) downstream of the dam must be considered when imerpreting the isotopic differences obsewed for whole muscle between upstream and downstrm sites. Spedc (MGLH)comparisons baween whole muscle, liver, and gonad 6% values showed significant '%J e~chmentof whole muscle versus liver, downstream (p4.013) and upstrram (p4.002)of the dam. Whole gonad was also found to be significantly ')N enriched compared to tiver (w.006)at the downstream site. Three-factor ANOVA also Udicated a sisnifiant 6% difference béhveen sites (p

Zn-A h 2 s C'eZ- -F m 222$1444 Cheg Q0 9 65 between sites will be re-evaluated for female white sucker only, since the ratio of male to fdewas different for the upstrearn (5 male5 fernale) and downstream (2 male: 8 female) sites (Table 3.4). Two-factor ANOVA showed no significant 6I3c diffefence between lipid extracteci muscle, liver, and gonad, however, there was a sisraficant ciifference between sites (p<0.001). Specific comparisons between sites for each tissue type showed signifiant differences for muscle (p<0.001), liver (p=û.040), and gonad (~4.035).The cornparison of female gonad with siBnificant 13cdepletion upstrearn relative to downstrem, diièrs from the cornparison of cornbined deand fende gonaà data (Table 3.2). 6'hcomparisons of fede tissues showed signifiant site ciifferences for all three tissue types (Table 3.4) which is the sarne result obtained for site comparisons of combine. male and fdetissues (Table 3.2). Therefore, gonad uui be used as an indicator of site diffèrences for 613c and s'% values, however, the ratio of male to fernale within each site must be taken into consideration. Present day conditions refleaed in the isotopic composition of white sucker, upstream versus downstream of the Carmichael Dam on the Groundhog River can be successfùlly detennined with a single par data set of whole dorsal muscle. 6I3cand 6*kvalues of whole muscle fiom upstrearn and downstrearn were found to be statistidy ciiffirent (ANOVA, ~~0.001) (Table 3 S). Tukey's HSD painvise cornparisons revded significant 13c depletion upstream cumpared to downstream sites at Fauquier (pC0.00 1) and Whist Falls (p<0.001). A ndar trend was observecl for 6'% values with sigmiicanf depletion upstrearn relative to downstrearn sites at Fauquier (@.O0 1) and Whist Falls (p

Isotope Site Tissue Mean (%O) k SE(n) Statist ical DiBtérenccs

...... *...... *.**-~~~.~-...... **.~.~.*.*~*~~..~.~.-"--...-.--.*-~--..------..--~.~~...*...*...... (p...... *- values) " b"~ Fauquier muscle -30.8 f 0.6 (8) b<~.oO1 lsver -32.2 f 0.5 (8) 0.040 gonad -32.3 f 0.5 (8) 0.035 Cannic hacl musclc -34.4 f 0.8 (5) liver -33.8 f 0.3 (5) gonad -34.0 f 0.4 (5)

ri' 'N Fauquier muscle 8.7 f 0.2 (8) 0.010 Live r 8.1 * 0.2 (8) 0.001 (1.g): ~0.001 gonad 9.1 * 0.2 (8) 0.001 Carmic hael muscle 7.8 f 0.3 (5) a

Tac 3.5 Sununa- of 613cand 6% mm k SE (n). statisticai tignilicance. m-hum. minimum, and range for whole muscle of whitc sucker allccied upstrcam (Carmichael) and downsscam (Fauquier. Whist Falls) of rhc Carmichael dam on the GmRiver in the fa11 of 1W7.

lsoro~e Site Mean (%) f SE(nJa Statisrical Maximum Minimum Rang

haerenœJ (%) (%O) (%O)

(p

.-l....--*.f--..--ff1.-.--.-~~...C --W.-.-.-..-y-. "-----**-...... -.-."~~.*...... "...... --..*~-.*.*--..~..---.-....*.~... 6"~ Carmicheai -35.6 I0.8 (9f -32.6 -39.7 7.1 Fauquier -30.4 f 0.5 (10) <0.001 -28.5 -33.3 4.8 Whist Falk -32.6i 1.4 (10) -29.1 4.1 15.0 -3 1.3 f 0.7 (9)' 4.001 39.1 -35.0 5.9

FauqWer 8.6 fO. 1 (10) 0.00 1 9.4 7.9 1.5 Whisi Falis 8.7 k 0.2 (10) dl.001 9.2 7.5 1.7

Note: a sample Pze amsis& of5 fkmak and 5 niales eyoepI: 4 te5 niala :Whist Falls 1997 without outiier (44.07). 5 fernales. 4 males; 'p valws fa amparhm dupsnepni aid dmmûmm sites. Figure 3.4 (%) (a) and PN(%O) (b) mean f SE for whole muscle of male 'and fede white sucker (C. commermi) and tipid-extracted liver (6"~ody) of female white sucker fiom upstream of the Cadchael Dam (CM), downstream at Fauquier (FQ),and downstream at Whia Falls (WF) on the Groundhog River for the years of 1991 (pre-impoundment) and 1994 to 1997 (pst- impoundment). Male and female white sucker whole musde data 60m Manice (MT) on the Missinaibi River (19960 reprrsents the reference site of an unahered tnhtary of the Mwse River basin (Farweii et al. 1999a). Numbers above the symbols represent sample sii(n). sitelyear 69 sampled. Also, the similarity between the reference site (Mattice- 19%) and sites downstream of the hydroelectric dam suggested that changes within the resewoir did not influence the isotopic composition of white sucker muscle at do~ll~frearnsites. Additionai information is provided with the 613cvalues of fdeliver (lipid extracted) for pre-impoundment (1 99 1) and pst-irnpoundment (1994 and 1995) years, downstream at Fauquier. Tukey pMsecornparisons hdicated no signifiant 6'3~difference between 1991 and 1994 liver, however, 1995 liver was signincantly more "C depleted than 1991 liver (~4.001).613c values of 1996 tiver from upmeam of the dam wae significamly more "C depleted than 1991 (p

O a 7 *- O' A 0 t A a F-muscle 6 -- a# A F-liver O F-gonad -- A M-muscle 5 A M-liver a Mgonaâ

1 1 1 1 1 1 1 1 1 4 1 1 1 I 5 T 1 1 1 I 567 8 9 10 11 12 13 14 15 aga (vil Figure 3.6 White sucker age and 6I3c(%O) (a) and ~"N(%o)(b) vaiues of whole muscle white nicker (C commerso~ti)from upstrearn of the Carmicheal Dam on the Groundhog River ( 19%) and Mattice on the Missinaibi River ( 1994). 1 o Mattice 1 / 75 impoundment samples and is therefore a usefiil methoci for deteminhg isotopic changes within the impoundment. individual anndi of the operaila of white sucker were analyzed to detemwie if 6"~ changes as.sociated with Unpoundrnent were isotopically archived in the yearly rings of the opercula. The average of the 6I3cvalues of the annuli for each operculum were generaüy 3 to 6 %o more I3cenriched relative to the corresponding muscle tissue (Figure 3.7). Young white sucker with very I3c depleted muscle (< - 34.0 %O) showed a trend of 13c depletion of the annuli for the 1993 and /or 1994 grovhg season compared to the 1991 and 1992 groiving seasons. This pattern was not evident for white sucker with more "C enriched muscle (> -34.0 %). The results aiggest that the annulus technique for establishing 613c changes in a recently altered system is liMted to situations where there are extreme fluctuations in the 613cvalues of the corresponding muscle tissue of young white sucker. Also, in terms of the technique of idating individual growth rings, working with older fish is more ciifficult and the probability of contamination is higher due to the narrow Nig of bone that is deposited yearly by older, slower growing fish.

3.4 Discussion The preliminaiy 1994 stable isotope analysis of cornponents of the food web downstream of the hydroelectric dam at Fauquin on the Groundhog River suggested that 13c depleted autochthonous materiai, in addition to allochthonous material, was an important carbon source. This resulted in the 13c depletion of white sucker muscle tissue (-3 1.2 f 0.6(10)). 613c data from an undeveloped tributary (Missinaibi River) of similar siie to the Groundhog River, located within the same latitudinal zone of the Mwse River Basin showed 13 C enrichment for benthic feeders includhg white sucker (-28.9 k 0.3(10)) (Fanvell et al. 1999a). The isotopic difference observed between the two sites for white sucker wggested that there rnay be changes in the benthic food web 6om hydroelectric deveiopment on the Grounâhog River. White sucker muscle and liver were good indicators of 6'3~and 6'k diffefeflce~ between upstream (Camiichaet-1996) and downstream (Fauquier-1995) sites based on whole and lipid extracted comparisons of muscle, liver, and gonad for each site. Lipid extraction of tissws eLiminated the 613câitfidinerences betwem tissues for the downstrerim and upstream sites. The 6I3cvalues of lipid extracted muscle, liver, ad goMd were also fodto be similar for the refiaence site on the Missinai'bi River (Farwell et al. 1999a). This is indicative of isotopic Figure 3.7 Measured 6')~(%) values for individual annuli of white sucker opercula coiiected upstream of the Carmichael Dam in the fd of 1996. Each point represents one year's growth (annulus) of the opercular bone and lines represent the opercuium of an individual white sucker. 6"~(960) values of whole muscle for each white sucker are presented within the graph. lrnpoundment Muscle G"C%

annuli 78 equiiibnum between the white sucker tissues of dflerent turnover rates and the dietary sources consumeci by white sucker. Tissues of different turnover rates would be expeded to be similar in an undeveloped river, however, the lack of si3cdifferentiation between tissues in a recently developed system suggests that an isotopic steady state has been estabiished within the tissues ofwhite sucker at sites upstream and downstream ofthe dam. White sucker gonad samples were poor indiators and showed no 6'3~ciifferences between upstream (Cadchael-1996) and downstream (Fauquier-1995) sites. The lack of digerentiiation between sites may be aççociated with diffaences in the ratio of fdesto des for the two sites since female gonads were found to be more 13cdepleted than male gonads. Thus, gonads of the same sex, or same ratio of male to ferde. should be used to compare site differences. in the reference river (Missinaibi River), fede white sucker tissues were consktently 13ce~ched relative to rnale white sucker tissues (Fanvell et al. 1999a). However, tissue cornparisons of rnale and €dewhite sucker fiom upstrearn of the dam (19%) on the Groundhog River showed that fdetissues were consistently 13cdepleted compareci to male tissues. 6"~values of pre-impoundment white sucker female livers indicate that pst- impoundment white sucker from upstream and downsûwm sites on the Groundhog River were recently feeding on more I3c depleted dietary sources cornpared to historic dietary sources. Merences in the 6I3ctrends for muscle, liver and gonad for upstream (1996) and downstream (1995) sites are therefore a fùnction of the period of exposure and the rate of growth of white sucker. Female white sucker coiiected upstream of the Cannichael Dam with an average age of 7.2 years, weighuig 847 g, have been feeding in the impounded ara for the majonty of their lives (5 years). The 6''~difi'et~ce of 0.59 %O for the three tissues suggests that there is isotopic steady state between tissues and dietary sources. The higher residence the and higher growth rate of fernales with higher energetic requirements suppliecl by new I3c depleted dietary sources may also explain the 13cdepletion of fdesrelative to males (mean weight = 791 .8 g, mean age = 9 yrs) at the upstrm site in 19%. The isotopic ~~mposiionof white sucker coiiected in dewloped (Groundhog) and undeveloped (Missinaibi) rivm is a fùnction of the isotopic composition of dietaiy items and the abundarice, âistn'bution and diversity of dietary items in microhabitats and macrohabitats within the system. Microhabitat is detined by the anall area of habitat encompashg the home range of the organism wtiereas the macfohabitat rekto the habitat conditions in a region of the river that influences the longitudllial distribution of the organisms (Fiset 1995). W~nthe macrohabitat of rivers in the Moose Riwr Bssin, thae are two distinct types of habitat, fest 79 moving water and slow moving water, that influence the diversity of aquatic invenebrates (Fiset 1995). Aquatic invertebrate midies conduaed in the Moose River Basin indicate that the diversity of invertehates is generaily greater in habitats of fist moving water than in slow moving water (Fiset 1995). Substrate composition, water velocity and depth were identifid as factors iduencing invertebrate taxa richness and density in the Moose River (Mârea et al. 1984). Lower benthic invertebrate densities were found at sites with a high percentage of clay. Also, low water depths (< 1 m) and low velocities (s 0.5 mis) were characteristic of habitats apporthg higher nuinbers of taxa (taxa richness) (McCrea et al. 1984). Within an unaltered river, the macrohabitat characteristics Uitluencing benthic invertebrate abundance and diversity are a fiinction of time (seasonai changes in hydrology) and space (basin morphology, lithology). Prior to hyâroeleetnc development on the Groundhog River, benthic hvertebrate su~eys conductecl by the Ministry of Natural Resources (MNR) in 1985 showed diffmces in the numbers of taxa, numbers of taxa in EPT and percentage of EPT at sites dong the river due to differences in habitat types (Fiset 1995). The similarity of 6'hvalues of white sucker liven âom the Missnaibi River (FarweU et al. 1999a) and fiom Fauquier on the Groundhog River prior to hydroelectnc operations (1991) indicates similarities in the factors determinhg the isotope values of dietary items and the composition of dietary items consumed by white sucker. Fiset (1995) reviewed surveys of benthic invertebrates in the region of the construction site, prior to and &er dam construction, to evaluate changes in or@c enrichment associated with impoundment. Rior to dam construction ( 1989). evaluation of water resource quality indicated that there was no organic enrichment. Similar benthic invertebrate densities and taxa richness were observeci in 1990 upon completion of construction. A 1992 surwy, upstrearn of the headpond, showed lower diversity and higher mean density than in 1989. Speafidy, the diversity and density of Chironomidae increesed in contrast to decreases in both parameters for stoneflies and odonates, while mayfly den* was similar and diversity decreased in 1992. However, naturai environmental fàctors, specificaily temperature complicateâ the ability to interpret divenity and density data for aquatic invertebrates for pre- and post-hpoundment piods. Observations of lower diversity also evident at the refiisite in 1992 were proûably due to eady emergence of aquatic invaiebrates, since water temperatutes were 4O or 5' C higher in 1992 than in 1989 (Fiset 1995). The available ùenthic invertebrate data rnakes it difticult to determine if the changes in the 6'hcomposition of white sucka associateci with impoundment were poss~iilya funaion of changes in the diversity and density of hthic invaiebrates available for 80 consumption by white sucker or changes in the cycling of inorganic and organic nitrogen. Regulation of the Groundhog River alters the fiequency and distribution of habitat types (fast and slow moving water) particularly upstream of the dam where increased water depth and reduced flow rate are constant, reducing the naturai heterogeneity of the river habitat. 13c and "N depletion of white sucker liver upstream of the Cdchael Dam relative to pre-impoundment and reference site (Farwell et al. 1999a) data suggests the influence of vertical cycling associated with plankton production in the rese~or.Studies of the isotopic composition of prirnary producers in lakes have observed depletion in the isotope values of planktonic algae relative to benthic algae (France 1995a; Hecky and Hesslein 1995). In a river system with diverse habitats, BUMet al. (1989) reported s'ilar or more ')c enriched benthic invertebrates and fish coUected in Aiik Lake (a deep basin within the main river channel) in cornparison to srnall hibutaries of a tundra river system in northem Quebec. However, the explanation of the I3c enrichent in this deep basin was possibly due to sources of manne 613c values (fish excretion, fish decay) from fish migration. Downstream at the Fauquier site, I3c values were depleted while "N values were e~chedfor post-impoundment sainples (1 994, 1995) relative to pre-impoundment samples (1991). The sirnilarity between the flow rates before and after hpoundment downstream at Fauquier implies that rtutrients are cycled in a spiral pattern consistent with water flow. Discharge from the Carmichael Dam rdting in the release of water saturated with "C depleted respired Caand "C depleted POM would possibly explain the I3c depletion of white sucker liver downstream at Fauquier following impoundment compared to pre- impoundment values. of -9 for DIC at Fauquier (1994) is indicative of respired carbon. The 613c values of FPOM at Fauquier (1994,1995) represent the combined influences of terrestrial carbon and the ')c depleted sources fiom upstream. FPOM collected at Fauquier (1994) was > 3 %O more ')c depleted than FPOM fiom the Missinaibi River (Farwell et al. 1999a). if the cycling of nitrogen in the reservoir influenced the isotopic values of dietary items downstream, then one would expect to see sirnilar 6"~values for white sucker livers at upstrem and downstream sites foUowing impowdrnem. Howwer, this is not the case, so perhaps the 6"~Merence between sites is a hnction of the isotopic composition, diversity and abundance of dietary Uivertebrates. Unfiortwiately, environmentai variables such as temperature inmase the difllicuity of hterpteting pre- and pst impoundmait benthic invertebrate brotey chta (Fiset 1995). Further study on comparing the 6% trends of sederrtary benthic invertebrates compareû to white 8 1 sucker is in progress to determine if the pre- and post-impoundment til% trends upstream and downstream are a fiinction of nutrient cyciing or changes in the consumption of dietary invert ebrates. Tissue analysis of fernale white sucker collected downstream at Fauquier in the fd of 1995 indicated a lack of isotopic steady state with new dietary sources. 613c values for liver and gonad were similar. indicating isotopic steady state with dietary sources. However, 8% values of muscle (- 1.5 %O) were more I3cenriched for the heavier (mean 1O76 g), older (mean 8.5 yean) fernale white nicker feeding in regulated waters for a perd of 4 ym. This indicates that the low turnover rate muscle tissue has not reached a new isotopic steady state. Laboratory experhents monitoring the replacement of carbon in juvenile (2.5 year) broad whitefish (Coregoni~s~llclst~s) following changes in the isotopic composition of the diet showed similar rates of replacement for liver and muscle (Hesslein et al. 1993). The rate of 6I3cchange in fad-growing fish is a &naion of the growth rate (Hesslein et al. 1993). in this dtered system, the low tissue turnover rate of muscle in adult white sucker represents a cumulative measure of the historic and recent stahis of the isotopic composition of dietary items influenced by residence time and growth rate. As a result mean 6'" vdues of muscle sarnpled over a period of years at sites upstream and downstrearn of the dam showed more variability (1.1 960 to 1.5 %O) than mean 6"~variability (0.4 %O) of white sucker muscle collected over a three year period in an unaltered reference river (Fanveli et al. 1999a).

6'% valws of iipid-extractecl muscle. liver and gonad varied by approxirnately 2 %O for white sucker collected from an undeveloped river (Missinaibi River) in the Mwse River Basin (Fanvell et al. 1999a). No difference was observed between the 6"~values of lipidextracted tissues and whole tissues. Hesslein et al. (1993) also found no ditlerace (O. 1%0) between whole and lipid-extracted liver of broad whitefish (C. mws). However, slight '% e~~hmnt (- 0.3 560) of iipidcxtracted liver compared to whole liver was observed for white sucker cdected at sites upstream and down~earnof the dam on the Groundhog River. Sex differences in the 6% vaiues of gonad wae observed at the derence river (FarweU et al.

1999a). The 6% values of muscle were approtamately 1 %O mire '%J enriched than liver for both fede and male white sucker. Male gonad was approximsteiy 1 960 more '%J depleted than male liver while fdegonad was more isotopically smilar to fdemuscle (FarweU et al. 1999a). This 6'"N aend for white sucker male and fdetissues was also evident upstream (1996) of the Gnwdhog River- The variation in 6% vdws may k a fundion of differences in the structural proteins comprishg the tissues of the muscle, her and gonad. The protein 82 content of female gonad (eggs) is generally expected to be higher than the protein content of male gonad. Hesslein et al. ( 1993) found a 6'% difference of O. 5 %O between muscle and liver of broad whitefish (C. ms).Cornparisons of fernale white sucker tissues for sites upstream (1996) and downstrearn (1995) of the Carmichael dam show that liver is consistentiy more 'h depleted in cornparison to muscle and gonad. There is m, 6'% trend for tissues of different turnover rates associateci with residence time or growth rate (age or weight). ûpercula backcalculation and isotopic detennination of individual yearly growth rings (annufi) was examùied as a possible method br assessing recent isotopic changes due to impoundment. The trend of "C enrichment of opercular bone relative to muscle have been observed in other studies of bone collagen and musde comparisons. Adysis of anchovy and roundhening veitebrae, decalcifiecl in 1.5% hydrochloric acid to obtain bone collagen, showed that it was more 13c enricheci than muscle tissue (Sholto-Douglas et al. 1991). Wiro and Epstein (1978) studied the 613c values of biochmiical components wch as collagen, chitin and insoluble organic hction of shells and the relationship of these components to the 6I3c value of the diet of the animal. The niccess of the use of the operadar bone to document isotopic change is a fùnction of the magnitude of the isotopic change in the dietary items conwned by white sucker influencecl by residence tirne, growth rate and the range of feeding habitats. The significant I3c depletion of white sucker liver collecteci upstrearn of the Camiichael dam relative to pre-impoundrnent 613c values for white sucker liver, indicated that the upstrearn site had isotopicaily changeci. Only white sucker with a highly I3c depleted value for muscle were exposed to dcient quamites of "C depleied dietary sources to allow detection of isotopic diffkrences within the opercular bone. 13 C depletion of annuli approximately 2 years following impoundment was estabüshed for white sucker muscle with "C depletion of values to -35 %O. No trends were established for white sucker with 6I3c values in the range of -3 1 to -34 960 for muscle. The technique is limited to the analysis of younger white sucker with high grow rates which are easier to work with since the annuü are larger. Therdore, there is sufticient uncontaminated material available for isotopic analysis. However, in older white sucker, with very narrow growth rings, the degree of comamination was probably high.

3.5 Conclusions Merences in the isotopic composition of white sucker muscle tissue wae observed betweai upstrearn and do- sites. The upstream site was fwnd to be si@cantly more 13C depleted than both downstrearn sites on the Gnnindhog River and reference sites on 83 the Missinaibi River. The 6')~values of FPOM and benthic invertebrates from downstream at Fauquier were comparable to the 6')~values of white sucker muscle. Muscle is a good indicator of isotopic composition of white sucker for both 6"~and 6'h values since minid preparation was required (no bpid extraction), the isotopic composition of muscle was independent of sex, and musde was sensitive to recent changes resulting in isotopic depletion upstream of development. The 6')~and 6'w values of white sucker muscle downstream of the dam were not different from the reference river suggesting that there is no impact on food web interadions (based on isotopic anaiysis) downstrearn of the hydroelectnc development . The relationship ôetween 6 "C and 6 '%J vahies of muscle with age were usefùi for interpreting graduai changes in the isotopic composition pst-impoundment. No lipid extraction of gonad was required pnor to analysis, however there were sex differences in the isotopic composition (6"~)of gonad which must be considered when using gonad tissue as an indicator of change. The high lipid content of white sucker liver significantly 13c depleted whole liver compared to lipid-extracted liver. The process of lipid extraction did not alter the 6% values of tissues. The cornparison of archived liver sarnples (pre-impoundment) and post-irnpoundment liver samples was the only method which showed a change (13c depletion) in the isotopic composition of white sucker downstream of the recent hydroelectnc development. White sucker liver were found to be 'k depleted upstream and enriched downstrearn compared to pre-impoundment white sucker liver samples. The use of opercular bone has iimited application to Uiterprethg change due to development. Stabie carbon and nitmgen isotopes rs nutrient trmn in Nortbtastern Ontario rivm with hydroclectiir dcvdopment and pulp mil activity. 1. Shidy orfsh popuhtions at distances rpsmim and downstream of devdopmcnt. 85 4.1 Introduction Stable isotope analyses have been applied to ecotoxicology studies to trace persistent organoctilorines at diaérent trophic levels in fieshwater and marine environments (Broman et al. 1992; Kidd et al. 1994; Kiriluk et ai. 1995; Schindler et al. 1995). sewage organics in estuarine and marine food webs (Gearing et al. 1991; Van Dover et al. 1992). and pulp mil1 effluent in riverine biota (Wassenaar and Culp 1994). Isotopic variabüity in the benthivorous fish species white sucker (Cutostom~scommerso~ii), has been quantifid for an undeveloped tributary (FanveU et ai. i 999a) and isotopic changes associated with hyâroelectric development have been defineci for a recently developed tributary (Farwell et al. 1999b) within the Moose River drainage basin in northeastem Ontario. This study examines the combined impacts of hydroelectric development and pulp mil1 effluent discharge dong miutaries of the Moose River using difKerences in the 613cand 6'3composition of benthivorous 6sh spies as an indicator of environmental change. Amendments to the Fisheries Act in 1992 required the irnplementation of an Environmental Effects Monitoring (EEM) program at Canadian pulp and paper mills. Reports corn Cycle One of EEM were reviewed by an Expert Working Group (EWP) which outlined the dominant problems associated with monitoring studies at pulp and paper mills (Munkittrick et ai. 1998b). Undefined levels of exposure and concems about the witability of reference sites were two of the several problems outlined for fieshwater studies of fish populations. Several pulp mill studies have used white sucker to investigate the potential impacts of pulp mill effluent on fish growth and reproduction (Munkittrick et al. 199 1, 1998a ; McMaster et al. 1992; Gagnon et al. 1994, 1995). The ability to use 6"~ values of white sucker as an indicator of exposure to pulp niiU effluent may be a usefiil tao1 in monitoring of impacts on wild fi& populations. The applicabiiity of 6'" anaiysis as an scponw indicator is dependent on the distinction behveen the 6I3c composition of effluent and the autochthonous and ailochthonous material fùehg the benthic food web. Wassenaar and Culp (1994) trad pulp mill effluent exposure using stable isotopes in biota Grom an unregulated river in westem Canada. However, in the reguîated tnîtaries of the Moose River the isotopic composition of biota downstream of pulp d efauent discharge and hydroeleçtric development is influenced by the combination of organic inputs fiom up- of the dam and tiom efEuent discharges. Isotope vahies of 6sh fkom two rivers inûuenced by the combineû effêcts of hydroelectric developmait and pilp dlduent disctiarge were useû to test the null hypotheses that there is no dinerrna in food web interactions (as inâicated by chmges in 86 stable isotopes) associated with pulp mil activity in regulated rivers. There are puip milis locateâ on the Kapuskasing, and Mattagami Rivers within the Moose River Basin. White sucker populations upstmm and downstream of both pulp dl discharges are separated by hydroelectric dams. The miUs have diffèrent pulping processes with a 1ûû%chlorine dioxide bleached Iwft mil1 at Smooth Rock Falls (Mattagami River) and a thennomechanical mil1 at Kapuskasing (Kapuskashg River). ûver the past sevd year~,both miüs have undergone a number of internai process changes as well as an upgrade f?om primary to secondary eauent treatment. Upstream of the dam, inputs of bark and wood Eom log drives continueû until 1986 and 1993 at Smooth Rock Falls and Kapuskasimg, respectively. isotope studies of wiid white sucker populations in a developed tributary of the Moose River showed depletion of 13c and 'h upstream of the dam compareâ to downstream and reference sites (Fanvell et al. 1999b). It was hypothesized that the isotopic shifl in this resewoir was due to increased phytoplankton production, which is more ')c depleted than temestrial particulate organic matter (France 1995a; Leggett et al. 1997a). However, on the Kapuskasing and Mattagami rivers. the impded regions are potentiaily inauenced by bark and wood accumulations, while downstrearn sites are influenced by pulp effluent discharges Stable &n and nitrogen isotopes of food web components were analyzeâ for sites upstream and downstrearn of mills to detemine if biota inhabiting regions where pulp miil activiiy is prevalent were isotopically distinct ftom other regions on the Khpuskasing and Mattagami rivers. Fish coiiected pnor to and &er secondary treatment were e;uamined to determine if the addition of seçondary treatment alterd the isotopic composition of benthivorous 6sh. Isotope values of white sucker muscle tissue from this study were comped with isotope data ftom an undeveloped river (Missinaibi River) (Fanweli et al. 1999a) and a recentiy developed river (Farwell et al. 1999b) to distinguish nahiral isotopic variabiility and hydroelectric impacts fiom effluent impacts.

4.2 Materiais and Methods 4.2.1 Suapihg Site Daeription The Moose River watded drains 90,643 km2 flowing north to James Bay in northeastern Ontario (Figure 4.1). The Mattagarni River is one of the major tributaries of the Moose River with a drainage area of 36,257 km2,augmented by the Grcudhog and Kapaskasing Rivers. The Mattagami River is clasyfied as a medium Yzed riva (morder, 6) with a length of491 km (Brousseou and Goodchild 1989). The Kapuskasing River is also a Figure 4.1 Sampling site locations dong the Mattagarni and Kapuskasing Rivers of the Moose River Drauiage Basin (Northeastern Ontario) (shaded rectangles) and other study site Iodons on the Missinaibi (Farwell et al. 1999a) and Groundhog (Farwell et al. 1999b) Rivers (open rectangles). t 1 Moose River L ,' _C( /' /' 6 - 2 Mlssi"lssinaiM River ' *- - -wKPMJ '- - , , 3 Magarni MVW Harmcn ( 4 Kapuskasing River 1 - -" 2'. - 5 ~roundhogRiver - *- ,Fw - - -' MaK3Y-DN- ' ' -- \ -- C rus Falls - A fUp and Pope* MIH - Dam I ! 89 medium sid river (Stream order, 5) stretching 324 km with a drainage ara of 8,633 km2 (Brousseau and Goodchild 1989). Hydroelectric developments are present at the town of Kapuskasing on the Kapuskasimg River and at Smooth Rock Falls on the Mattagarni River adjacent to the pulp mil facilities. There is a series of four dams incluâing Little Long, Smoky Falls, Hannon, and Kipling dams located downstrearn of the confluence of the Kapuskasing and Mattagami Rivers. Monnation regardhg the year of operation, installeci capacity, drauiage area, normal storage and mean flow rates is provided in Table 4.1. The thamornechanical pulp (TMP) mil1 at Kapuskasing began operations in 1909 utiiizing spnice (98%) as the major wood fiiniish in addition to srnall arnounts of balsam. The mil1 produces 1050 t/d of newsprint and 225 t/d of recycled pulp. The dl has undergone a number of process and treatment changes over the years. In ûctober of 199 1, paper machine # 1 was shutdown and, in 1992, the process of de-inking recycled paper (5%) was introduced. The shutdown of the magnefite process in May of 1993 rdted in decrduse of water and reductions of BOD.Thennomechanical processing increased with the addition of TMP line #3 in July of 1993. In the fd of 1993, groundwood processing was shut down (September, 1993), dry debarking was initiated (October, 1993) and the practice of log drives on the Kapuskasing River was discontinued (October, 1993). The conversion of the mill io 100 % thennomechanical processing was complete by July of 1994. A sawmill was openeci and de- inking capacity was increased in Apd of 1995, followed by the addition of TMP line #4 in April of 1996. improvements to duent discharge included the retouting of digester condensates (1993) fiom the sewer and rerouting ash sewer to the primary clarifier (Aprii, 19%). Secondary treatment of e8iuent via activated sludge began in Apd of 1995. The treatment system consisted of 34 submerged mahanical aerators with a holding capacity of 79,700m3. Mill ettluent was discharged approximately O. 1 km downstream of the dam. Fish were sampled in the fàil of 1994 (Sept. 19-27), 1995 (Sept. 22-30), and 1996 (Sept. 1 1- 19) at two mainstream sites located upstream of the hydroelectric dam and pulp mil1 on the Kapuskasllig River (KAPWF-UP; -40 Lm upstream, KAP-UP; 1-2 km upstrearn) (Figure 4.1). DownsVeam sites includeâ KAPTB-DN ( 1-2 km downstream) and KAPFF-DN (-1 5 bn downstream). In the sp~gof 1995 (May 13-22), fisi were colîecteû using hoop nets in tributaries a fw kilometers upshwm (KAPUNK-UP and KAPBB-UP) and downstream (KAPGC-DN, KAPû'B-DN, and KAPUNK-DN). The pulp mill at Smooth Rock Falls was buüt in 191 8 and Uiitially operateci as an unbleached kral'k mill und 1927 when it was converted to a bleached kraft d.Production 90 Table 4.1 Location of generating station, in-service year, installeci capacity. drainage area. normai storage and mean flow rate for sites on the Mattagami and Kapuskasing Rivers.

------Site River In- Installed Drainage Normal Mmflow service Capacity Ara Storage k SE Year (MW)' (sq-km)' (m3/day)& (m3/s)'

Srnooth Rock Fdls Matcgami 1917 6,250 - - -

Little Long Mattagami 1%3 1 15,200 36.72 1 1874.0 313 i73

Smoky Falls Maitagami 1928 52.800 36.736 77.4 280 f 63

Hannon Mattagarni 1965 122.40 36.750 67.8 281 i60

Kipling Mattagarni 1%6 1 18.600 36,781 36.5 284 I 61

' Ontario Hydm RefemCentre b nord storagc is the storage volume aSSQciakd with the band of water aiii;rincd within the storage mcnroir's normal qmating range. average flav rate dculakd hm mean monthly flow rates for 1997 (monthly flow mtc &ta hm Doug Lawlcr. Water Management Senices Watcr Raources mision) 91 increased to 295 t/d by 1966. The miil utilized 65% black spruce and 35% jackpine in the fonn of wood chips. Logs were transported to the rniU headpond from upstream locations on the Mattagami River until 1986. The kraft prunaiy process of 16% CIO2 substitution was converted to 1000/o Cl02 substitution in 1992. The production capacity of the mil1 was 170,000 airdned metric tonnes of ECF Iwft pulp per year (465W) (Robinson et ai. 1994) using continuous flow digestion. The bleaching sequence changed from CdEopDED to DEopDEpd in 1992, with the addition of oxygen delignitication in 1993. The treatment of efiiuent, initially established with a mechanical clarifier in 1976 was augmentai with secondq treatment in ûctober of 1994, with an aeration stabilization basin with a retention period of 7.5 days. In addition, increased recovery of black liquor resulted in BOD redudon. Effluent was discharged into the Mattagarni River approximately 0. t 5 km downstream of the dam by a submerged diser at a rate of approximately 0.8 m3s" (Acres 1994). Fish sarnples were collected in the fd of 1994 (Sept. 15- 17), 1995 (Sept. 19-2 1), and 1996 (Sept. 19-24) and the spring of 1995 (May 9- 14) at sites upstream and downstream of the hydroelectric dam and mill effluent discharge on the Mattagami River (Figure 4.1). Sites MAT-üP (upstream) and MAT-DN (downsiream) are located approximately 5-7 km above and 1-3 km below the dam. Further downstrearn at MATCY-DN (- 60 km downstream) was also sampled in the fiill of 1996. In 1997 (Sept. 9-16), additional sites were mpled downstrearn of the Smoky Falls (MATSM-DN)and Kipüng (MATKP-DN) hydroeltxtric facüties located approxirnately 100 and 124 km respectively, frorn the mil1 at Smooth Rock Falls. Habitat suweys consisting of 5 transects per site were conducted in fall of 1994 (October) for sites upstream and downstream of the dams on the Kapuskashg and Mattagami Rivers. Submerge. macrophytes, predorninately, Potamogrtm~species, covered 10 to 20 % of the shorehe at KAP-UP and MAT-W.ûther maaophytes, including MjmphyiItmr and Failis~n'uwere present at KAP-UP. Macrophyte divefsity was higher tùrther upstrem on the Kapuskashg River (KAPWF-UP). Vegetation at KAPWF-üP Uiduded Scirpüs, VaIIis7neria, MyriopliyIIt~m,Nynrphriea, EIaka, Najas and Putamugetan (dominant germa). The hmct of years of log drives on these rivers was evident by the high nwnber of subrnerged logs and ûee stumps covering the bottom seâiments upstrram (KAP-UP, MAT-UP)of the dams. Sedimnit composition and percent carbon and nitmgen are consistent with river hydrology with high Sedilnentation upstream in the fesetvoirs compared to downstrwm sites (TaMe 4.2). Nutrient data and water chemistry parameters for pulp mis effluent anci water samples 92 Table 4.2 Sediment characteristics upstream and downNeam of the hydrwkcvic facilities and pilp Mlls on the Kapuskasing and Mattagarni Rivers ( 1994 and 1995). Site Sediment composition Carbon (Y&) Nitmgen (%) sand (%) day (%) silt (%) Kapuskasing River KAPW-Uf' 6.1 23.5 70.4 3 .O0 O. 17 KAP- W - - - 14.60 0.22 K AP-ON 06.5 3.5 - 1.19 O. 12 (silt and chy)

Mattagarni River MAT-UP 16.3 16.8 66.9 1.W-5.20 0.054.29 MAT-DN 93.8 2.8 2.8 0.18 0.0 1 (0.6 gravel) - no data available 93 collected upstream and downstream of the miils on the Kapuskasing and Mattagami Rivers are represented in Tables 4.3 and 4.4, respectively. Water samples were collected and analyzed according to the procedures established by the Environment Canada National Lab for Environmental Testing NET) (Environment Canada 1995) at the Canada Center for Inland Waters (CCIW), Burlington, Ontario.

4-22 Sample Collection Fish were obtained by gill nets (8.8 and 10.2 cm mesh size) placed in the mainstream upstream and downstream of the dams in the fall of 1994 (Sept. 15-27), 1995 (Sept. 19-30), 1996 (Sept. 1 1-24), and 1997 (Sept. 12-26) and hoop net in the spring of 1995 (May 9-22). Fish parameters such as sex, fork length (cm), total weight (g), liver weight (g), and gonad weight (g) were detennined for each fish. Measurements were used to calculate condition factor, K = 100 * total weight (g) 1 fork length (cm)), liversomatic index, LSI = percent liver weight 1 total weight, and gonadosomatic index, GSI = percent gonad weight 1 total weight for male and female white sucker. The lefl operculum of each fish was collected, fiozen and later imrnersed in 90°C water for 3 seconds to remove the skin covering. The age of the fish was determined by counting annuli. Dorsal white muscle, liver and gonad of white sucker (-5-10 g) were collected, wrapped in precombusted foi1 (400°C), contained in whirlpac bags and stored on ice in the field. Dorsal muscle was obtained for other fish species and stored in a similar manner. All samples were kept in -20°C freezers prior to preparation for isotope analysis. Terrestrial (primarily leaves) and aquatic plant material were collected dong the shore and in shallow water, cleaned of debns, wrapped in precombusted foi1 and stored in plastic whirl pac bags. Fine particulate organic matter (FPOM)was obtained by filtering river water collected in 20L nalgene containers ont0 prewmbusted GFIC filters under vaccum. Al1 filters used in this study were precornbusted at 400°C for 4 h. Separate biotilrn samples from rocks and mamphytes were collected by gently placing the substrate in large ziploc bags underwater to minimize loss of biofilm. Within the bag, the biofilm was removed fiom its substrate, the substrate rinsed and discarded, and the remaining rinse water filterd ont0 prccombusted GF/C filters. Al1 mers were placed in precombusted foü and whirl pac bags or in 7 - 20ml glas scintillation vials. Al1 î3ters and plant material were stored at -20 OCprior to preparation for stable isotope adysis. 94 Table 4.3 Sclected nutrient data presented as mean f SE(n=3) for mil1 effluent and water samples from upstream and downsircam of the mills on the Kapuskasing and Mattagarni Rivers.

Kapuskasing River emuent I9YJF 1995F 1996F kap-dn 199 1P 1994F 1995s 1995F kapffdn 1994F bp-up 1991F' 1994F 1995s 199 5F kapwf-up 1994F Mattagarni River effluent 1994F 1995F 19%F mat-dn 199lF 1994F 1995s 1995F mt-up 1991F IrnF 1995s 1995F

96 The majonty of benthic invertebrates were obtained using a D-Me aquatic net in shallow water to a depth of approximately 1 m. Ponar grabs were used to collect invertebrate samples in deeper water, although success was minimal due to compacted sediments downstrea.cn. The samples were placed in 500 prn Ntex mesh bags and rinsed in river water to remove fine sediments. Each sample was placed in a large ziploc bag with adequate water, and kept on ice. Later, live invertebrates were picked, cleaned of detritus, sorted and held for 18-24 h in filtered river water to allow the gut to clear. lnvertebrates were placed in precombusted foi1 and whirl pac bags or in 7 - 2Oml glass scintillation vials, and fiozen at -20 OC until being prepared for isotope analysis.

4.2.3 Sampk preparation and analysis of stable isotopes Autochthonous and allochthonous material, including aquatic macrophytes, FPOM,and biofilm removed fiom rocks and macrophytes were acidified with 1 .O N HCI to remove inorganic carbon fonned as calcium carbonate. Al1 material was freeze-dried, removed fiom filters (where applicable) and ground to a powder using a bail mil1 grinder. Benthic invertebrates were categorized by fiinctional feeding groups and family (or genus). The intestinal tract of most invertebrates were removed with the exception of small amphipods, gastropods, and bivalves. Whole (minus intestine) invertebrates were freeze- dried and ground except for craytish where only the muscle tissue fiom the caudal region was used. Dorsal muscle fiom large and small fish species white sucker were fieeze-dried and ground. Dry, ground samples were analyzed for carbon (13~/'2~)and Ntrogen ('%JI'% isotope ratios using a VG Optima continous flow isotope ratio mass spectrometer at the Environmental Isotope Laboratory (EU), Department of Earth Sciences, University of Waterloo (Waterloo, Ontario). IAEA and NIST standards were used to monitor analytical precision of t 0.2 % and 0.3 960 for 613c and 6'%, respectively, within the range of linearity. 6% of DIC and pC9 (partial pressure of dissolved COr)collected in evacuated bottles were analyzed using VG 602 micromss dual-inlet mass spectrometer following the methods by (Hesslein et ai. 1997) at the Freshwater Institute Science Laboratory (Winnipeg, Manitoba). 97 4.2.4 Statistics Analysis of variance (ANOVA) and Tukey pairwise cornparisons were applied to test for significant isotopic dicerences for white sucker collected upstream and downstream of the hydroelectric dams and pulp mil1 effluent discharge on the Kapuskasing and Mattagarni Rivers.

4.3 &dtS 4.3.1 Kapuskasing River lmplementation of secondary treatment at the Kapuskasing miIl in April of 1995 resulted in a decrease of DIC and DOC concentrations in mil1 effluent fiom the fa11 of 1994 to 1995 and 1996 (Table 4.3). Secondary treatment of mil1 effluent also resulted in an increase in alkalinity, and a decrease in colour and turbidity (Table 4.4). Concentrations of various parameten (DIC, DOC, TSS) and other measurements including pH, conductivity, alkalinity, colour and turbidity at sites upstrearn (KAP-UP) and downstream (KM-DN) of the miIl were similar in years following secondary treatment. Seasonal trends were noted with consistently iower DIC. and higher DOC in the spring relative to fall. POC and PON concentrations measured downstream of the miIl (KM-DN) were reduced by greater than 50% compared to the sampling period prior to secondary treatrnent in the faIl of 199 1 (Robinson et al. 1994). Preliminary isotope analyses of dissolved inorganic carbon and primary and secondary components of the benthic food web for sites dong the Kapuskasing River indicated isotopic differences of bent hic invertebrates between sites (Table 4.5). The S')C values of DIC for upstream and downstrearn sites (fdl of 1994) were similar which suggests that levels of DIC in miU effluent do not innuence the isotopic composition of DIC downstream of effluent discharge. Seasonal DIC trends w here DIC concentrations were decreased (Table 4.5) and "C of DIC were depleted in the spring (1995) compared to the fall (1994) rnay result in seasonal fluctuations in the 6"~values of primary producers at upstream and downstrearn sites. Aquatic macrophytes (Potumogeton spp.) were 13c and '% dched upstream relative to downstream. Biofilm, which was slightly 13 C enricheâ downstream relative to upstream, was comparable to the S"C of mil1 effluent. Chironomids and otigochaetes were consistently "C and '% depleteâ upstream relative te ciodownsaam The distinct clifference beniveen sites for benthic invertebrates suggests 98 Table 4.5 6 l3 C and 6 l5 N values of dissolved inorganic Farbon sources of terrestrial and aquatic primary production and aquatic secondary production coUected from sites dong the Kapuskasing River. Numbers represent pooled samples.

Material Site SeamnYcat ô"~(%) s '%(s)

DIC KAP-UP KAP-UP KAPWF-UP KAP-DN KAP-DN KAPFF-DN Pot;irnogeton ric-nii KAPWF-UP Pamogetoncphyirus KAP-UP KAP-DN(S)' epiphytic matenal KAP-UP epilithic maienal KAP-DN

Chir~n~miQe KAP-UP S 1995 -12.3 4.6 KAP-DN SlW5 35.8 10.2 Tuôificidae KAPIüP SIN5 -32.7 4.5 KAP-DN SI995 -2 4.6 14.7

"down~rrramof sewage treatment disEharge; na = not able 10 &ed 99 that there are measurable isotopic impacts upstream and downstream of the hydroelectric dam and miil effluent. The dorsal muscle of white sucker was analysed to determine if the isotopic differences observed in benthic invertebrates upstream and downstream of the hydroelectric dam and pulp mil1 on the Kapuskasing River were reflected in the isotopic trends in fish at the top of the benthic food web. Characteristics of wild white sucker populations inhabiting upstream and downstream reaches on the Kapuskasing River were summarized in Tables 4.6 and 4.7. -4 subsample of white sucker donal muscle hm upstream and downstream sites in 1994, 1995, andor 1996 were analysed and the 6% and 6% values presented in Table 4.8.In 1994, the site immediately upstream of the dam (KM-UP) was significantly more 13c depleted than the site fùnher upstream (KAPWF- LJP) (p=0.005)which suggested the impact of reservoir effects on 6"~composition. Both upstrearn sites were significantly more 13c depleted than the downstream site (KAPTB- DN) (p<0.001).In 1996, the site immediately downstream of mil1 effluent discharge (KAPTB-DN)was significantly more 13cenriched (p=0.0 18) compared to the site further downstream (KAPFF-DN) which suggested the impact of effluent on 6"~composition. The significant 13c enrichment (p<0.001) irnmediately downstream (KAPTB-DN) compared with upstrearn (KAPWP-0) in the fall of 1996 is consistent with 1994 observations. The significant difference (p=0.005) between sites which were a greater distance fiom the impacts of the reservoir (KAPWF-UP, 1996) and effluent discharge (KAPFF-DN, 1996) indicated that possibly one or both of these sites may be infiuenced by development, or natural variability. Yearly 13cvariability upstream at KAPWF-UP (mean difference = 1.9 %O) was greater than the 13c variability at ICAP-DN (mean difference =

0.8 %O) and at the reference site on the Missinaibi River (Farwell et al. 1999a). Kapuskasing sites were compared with reference sites on the Missinaibi River (Farwell e!t al. 1999a) and Groundhog River (Farwell et al. 1999b) to detennine the level of impact fiom pulp miIl development (see Section 4.3.3). The 6'% values of dorsal muscle of white sucker upstream and do~ll~freamof the hydroelectric dam and deffluent discharge on the KepuslcsPng River were not as distinctly different as the S'~Cvalues of white sucker or the s'% dues of benthic bertebrates (Table 4.8). Data fiom 1994 sbowed signifiant '% enrichment at the dowiismam site (KAPTB-DN) compared to the up~aeamsites at KAP-üP (pO.OS) in 19%. Yearly 6'% variabdity for upstream and downstrearn sites was consistent with the yearly variability fiom the reference site on the Missinaibi River (Farwell et al. 1999a). The lack of statistical clifferences within upstrearn or downstrearn sites and between upstream and downstream sites in 1996 rnay be a bction of the miall sample six ador the subtle impact of development on the 6" cumposkbn of white sucker. in summary, Born the overall sarnpling effort, extrema in 'b depletion and '% e~chmentwere evident at the sites imediately upstream of the dam (KAP-UP) and downstream of discharge (KM-DN), respectively.

4.3.2 Mattagami River Secondaiy treatment at the Smooth Rock Falls miIl (October, 1994) resulted in increased concentrations of DIC and DOC for the year (fall of 1995) following treatment compared to pre- secondary treatment concentrations (fall of 1994) (Table 4.3). In the fdl of 1996, DIC concentrations were lower than pre-secondary concentrations. Other effluent parameters such as aikalinity, AOX, colour and turbidity increased in the years following the implementation of secondary treatment (Table 4.4). Nutrient data and water chemistry parameters were similar upstream and downstrearn of pulp miIl effluent following secondary treatment. Trends, specifically, decreased DIC concentrations and alkaîinity and increased colour were observed in spring relative to fall mple periods for sites upstream and downstream of effluent discharge. Isotope analyses of efffuent, dissolved inorganic carbon, and components of the benthic food web upstream and downstream of the hydroelectric dam and pulp miIl effluent discharge on the Mattagarni River are presented in Table 4.9. Wood chips collected pior to processing at the miIl were slightly I3c enriched compared to effluent discharge. Percent composition of wood and black spruce bark were similar with > 50 % C and c 0.5 % N content. Both wood and effluent were I3cenriched relative to leaf litter. 613c values of DIC were sirnilar at upstream (MAT-UP) and downstream (MAT-DN) sites. Trends of "C depletion and decreased DIC concentrations in the spring relative to the fall were similar to trends observed on the Kapuskasing River. Biofilm and FPOM were I3cenriched upstream (MAT-UP)relative to downstream (MAT-DN) while effluent 1O4 Table 4.9 8 l3 C and 8 l5 N values of dinolveâ inorga~ccarbon. sources of tenatrial and aquatic primaty production and aquatic sccondary production collecteci hmsites dong the Mattagami River. Numbers represent pooled samples.

Ma teriai Site Season. Year 8') c (%) PN(G) .....1...~...... ~_...... *.-.--..-*...... *.~.....*...... **.*...... --~~..*-...~...... *---.-.--.*...... -...-~...... *.....------*------.-..*--.~..-.....*...... *. efnuent miil S1995 -26.2 nii effluent mil1 waxi chips mil1

DIC MAT-UP MAT-UP MAT-DN MAT-DN MAT-UP

MAT-DN MAT-UP MAT-DN MAT-UP NAT-DN MAT-DN

MAT-UP MAT-DN MATCY-DN MAT-UP MAT-DN MATCY-DN MAT-UP MAT-DN MAT-UP MAT-DN 1O5 6'" values were median to the enriched and depleted "C values of upstream and downstream sites. No consistent trends were observed for the benthic invertebrate groups sarnpled at sites upstream (MAT-üP) and downstream (MAT-DN and MATCY-DN)of the dam. Chironomidae, Ephemeridae, and Physidae were 13c enriched upstream (MAT- UP) relative to downstream which was consistent with the trends observed for biofilm and FPOM.6% values were depleted for Chironomidae, Tubificidae, and Physiâae from upstream (MAT-DN) compared to downstream (MAT-DN) whiie invertebrates from tiirther downstrearn ai Cypnis Falls (MATCY-DN) were more '% depleted than bodi of the other sites. Isotope values for white sucker muscle fiom sites upstream and downstream of development on the Mattagarni River were used as a long ten integrated indicator to determine if the trends observed for particulate organic matter and benthic invertebrates were similar to trends at the top of the benthic food web. Measurements of wild white sucker collected upstream and downstream on the Mattagami River were sununarized in Table 4.10. Subsamples of white sucker from upstream and downstream were used in isotope analyses over a period of years from 1994 to 1997 (Table 4.1 1 ). Comparisons between years showed no significant 6"~ciifferences for the upstrearn (MAT-UP)(pM.05) or downstrm (MAT-DN and MATCY-DN)(H.05) sites. Site cornparisons for 1997 data reveded no significant difference in the 613c values upstream of the dam (MAT-UP) and downstream at Cypnis Falls (MATCY-DN)(p>O.OS). Tukey painvise comparisons showed both the MAT-UP and MATCY-DN sites were significantly "C depleted relative to sites immediately downstream of the dams at Smooth Rock Falls (MAT-DN),Smoky Falls (MATSM-DN), and Kipling (MATKP-DN). No significant statistical 6I3c differences were observed between sites located immediately downstream of the dams (MAT-DN, MATSM-DN,and MATKP-DN) (p>O.OS). In conclusion, the 13c depletion at MAT-UP and MATCY-DN suggests the influence of wata retention, whereby the site at MAT-UP is controiied by the dam and the site at MATCY-DN is contr01led by the natural morphology upstream of Cyprus Falls. Ah, there is no indication that efRuent influenas the 6')~ composition of white sucker based on the similarity of the 6"~values of white sucker inhabiting sites downstrearn of the dams. The complexity of the natural and anthropogenic factors potenbaUy inûuencing the 6I3ccomposition of white rarcker in the Msnagann River will be ad- tiiruier by cornparhg data from otha rivers (see Won4.3.3). Comparisons between yeers showed no signincant 6'% difference for the upstnam

Sl:\T-LI' i997F -34.9 i0.9(10) bcO.OO1 MAT-DN (97). -3 1.9 -10.1 8.2 *-0.001 LLTSXI-DN (97). c0.00 1 Lf ATW-DN (97)

11AT-DN 1996F -27.6 f 0.6 (4) -26.6 -29.3 2+7

ICI AT-DN 19941: 10.6 f O.3(4) ' 0.018 Sf=\T-DN (97) 11.3 9.7 1.6 hl AT-DN 1995s 1 5 k O 1 (4) 4.001 MAT-DN (97) 11.8 11.2 0.6 I O8 site (MAT-üP) (p>0.05) or downstream site at Cypnis Falls (MATCY-DN) (pM.05) (Table 4.1 1). However, there was significant '% depletion irnrneâiately downstream (MAT-DN)of mil1 effluent discharge in the fa11 of 1997 compared to earlier sarnpling periods in the fa11 of 1994 (p=O.Ol8) and the spring of 1995 (p<0.001). Site cornparisons for 1997 data showed signifiant '% depletion downstream at Cypnis Falls (MATCY-DN,p=0.031) and '% enrichment downstream at Smoky Falls (MATSM-DN, p4.001 ) relative to upstrearn of the Smooth Rock Falls dam (MAT-UP).No sigruficant ciifferences were observeci between MAT- W.MAT-DN, and MATKP-DN (pû.05). Cornparisons of downstream sites indicated that sites at MAT-DN and MATCY-DN were significantly '% depleted relative to sites tùrther downstream of the rdat MATSM-DN and MATKP-DN. In conclusion, the SMin the 6% composition of white sucker €?om 1994 and 1995 to 1997, a minimum period of two years der secondas, treatment was implemented, suggests that primary treated etmient may have Uidirectly resulted in "N e~chment.Comparisons with other reference sites on the Missinaibi and Groundhog Rivers may explain the '% enrichment further downstrearn at the series of four dams (Section 4.3.3).

4.3.3 Comparisoas of fih spceks upstream and dowastmm of devclopment on the Groundhog, Kapuskuing, and Mattagarni Riven Isotope data for fish species fiom the Missinaibi (Fanireil et al. 1999a) and Groundhog (Fanvell et al. 199%) rivers were used to diferentiate between the impacts of hydroektric development and the combined impacts of hydroelectric dams and pulp mil1 activity on the Kapuskashg and Matîagami Rivers. Carbon and nitrogen isotope values of white sucker and other fish species collected fiom 1994 to 1997 fiom the Mwse River trilutaries are presented in Figure 4.2 a, b, and c. Revious isotope investigations showed ct'ierences bmeen yean for correlations between up- of the dam on the Grounâhog River and the reference site (Mamce) on the Missinaibi Riva (FarweU et al. 1999b), therefore, the most recent data were useâ to idemi@ present day Merences between rivers. Isotope data for white sucker musde from Mattice (1996) on the Missiw'bi River, and upstrearn sites on the Growidhog (GR-UP, 1997), Kapiskasng (KAP-üP, 1994), and Mattagami (MAT-LJP, 1997) Rivers were compared to Wefentiate between the impact of impoundment and the combhed impact of unpoundmait and pJp rdactivity. Analysis of vaiiance (ANOVA) showed no sigmtiant Merence benVeen the 6I3cdues of white wcker for the hpoded sites 0.05). Tukey pairwise cornparisons indicated si@cant 13c Figure 4.2 6'3~(-cc)and 6'% (L~~..)values of dorsal muscle for fish species coUected at sites upstream (GR-UP1-3 km) (open symbols) and downstrearn (GRFQ-DN - 22 km; GRWF-DN - 50 km) (closed symbols) of the hydroeI&c dam on the Groundhog River (a) (FanveU et al. 1999b), and upstrearn (open symbols) and downstream (closed symbols) of the hydroelectric dam and mill effluent discharge on the Kapuskasing (b) and Mattagami (c) Rivers. Open square symbols represent the reservoir sites for dl rivas and closed square symbols represent downstrearn sites nearest the dam, including areas exposed to pulp mill effluent discharge on the Kapuskasing and Mattagami Rivers. Symbols were labeled for fish species uicluding walleye (W), northem pike (NP), longnose sucker (LS), and troutperch (TP). Symbols with no labels reptesent the isotope values of white sucker for the dEerent sites. The range of 6'3~(i0) and 6% (+values for white sucker fiom three sites on the Missinaibi River are presented in the dashed box (FanveU et al. 1999a). a 8 -rrr*rrrrr+-œ MlSSlNAlBl RIVER

13

iKAP-ON wo

13 -

A MATCY-DN O

-- 9 :

8 8 -- af: 48 8 :rn -- 8 D 7 O O 8 8 Lrrrr-rrr-rrrœ 6 -- MlSSlNAlBl RIVER 111 enrichment at Mattice relative to the irnpoundment sites on the other three rivers (p

macrophyte, Polmnogetm sp (8%= 3.7 qia) from upstream of the Kapuskasimg River (KAP- UP) was '% depleted compared to the same genus coîlected hmthe Mattagami River (til%J

= 5.8 -au). Referetlce sites downstrtsm on the Grounâhog Riwr (GRFQ-DN, 1997) and at Figure 4.3 Phosphorus (a) and Ntrogen data (b) coUected f?om sites on the Missinaibi (Manice and Thunderhouse Falls), Groundhog (Fauquier), Mattagarni (upaream and downsiream of the dam), and Kapuskasing (upstrearn and downstream of the dam) Rivers in the fall of 1994 and spring of 1995. Mattagarni River II site location, year and season of collection

site location, year uid season of collection 114 Mattice on the Missinaibi River (1996) were compared with sites downstream of the dams on the Mattagami River (MATSM-DN, 1997; MATKP-DN, 1997) and downstream of d emuent discharge on the Mattagami (MAT-DN,1997) and Kapuskasing (KAP-DN, 1996) Rivers to distinguish the Muence of pulp d effluent on the isotopic composition of resident white sucker populations. 6I3c values of white aicker downstream of the mil on the Kapuskashg River were significantly 13c enricheci relative to the reference sites on the Groundhog (p<0.001) and Missinaibi (p4.002) Rivers as weli as sites fùrther downstream on the Mattagarni River (MATSM-DN, p=O.ûû 1; MKXP-DN, ~34.002).White sucker 6"C values downstream of the mill on the Mattagami River were similar to al1 downstream sites with the exception of downstream on the Groundhog River (GRFQ-DN, p=0.016). in conclusion, there is evidence to suggest that the pulp miii discharge influences the 613c composition of white sucker downstream on the Kapuskasing River. Howewr, the discharges at Kapuskasing that contributes to sigruficant 613c ditlierences relative to other downstrearn sites rnay be a combination of pulp miil eWuent and sewage discharge. On the Mattagarni River, the combination of I3c depleted inputs fiom upstream and I3c e~chedinputs from effluent discharge results in the 6I3c composition of white sucker which is indistinguishable from the undeveloped reference river. However, there was sufficient "C enrichment downstream on the Mattagarni River to distinguish the combined influence of the hydroelectric dam and effluent discharge firom the deiduence of the hydroelectric dam downstream of the Groundhog River. Presentday conditions showed no sigrUficant 6% differences between downstream sites on the Missinaibi (Mattice, l996), Groundhog (GRFQ-DN, 1997), Kapuskasing (KAP- DN, 1996), and Ma- (MAT-DN,1997) Rivers. Thmefore, duent discharge was not a factor intluencing the 6% composition of white sucker for recmt yean (19% and 1997). However, there was a shifi in the 6% composition of white sucker downstrearn of discharge on the Mattagarni River (Section 4.3.2) foUowing seconchy treatment in October of 1994. Cornparisons of 1994 data indiateci significant '% enrichment down~beamon the Mattagami River (MAT-DN,1994) compared to the ret'erence site on the Missinsibi River (Mattice, 1994, p<0.001)ad the downstream site on the Grwndhog River (GRFQ-DN, 1994, @.(Ml). No statisticaî Merence was observed between the down~aeamsites on the Kapuskasing (KAP. DN, 1994) and Mattagami (MAT-DN, 1994) Rivas (@.OS). This data suggests that, prior to secondary treatment. efEuent discharge contriied to the 6% composition of white sucker downstreem on the River. It shouid be noted tbat 1994 conditions showed the same 115 statistical trends in 6'" as the more ment conditions outhed in the above paragraph for downstream sites. The isotopic composition of white sucker fiom sites tiirther downstream on the undeveloped river (MiSanaibi Riwr) were compared to non-Unpacted sites on developeci rivers to detemillie the isotopic variability associated with changes in the naturai morphology of the river. Consider the ditferences in the range O€G"C values of white sucker fiom Skunk Island (- 28.2 to -3 1.3':,) and Thunderhouse Falls (-26.5to -30.6.cG)on the Missinaiii River compared to Whia Falls (GRWF-DN;-29.1 to -3S.Ov,,, outlier 4.hC) on the Groundhog River and Cypms Falls (MATCY-DN; -31.6 to -37.6(,*)on the Mattagarni River. Both sites on the Missinaibi River are refatively homogenous in terms of basin morphology and water flow (Fanveli et al. 1999a) and have similar 6"~ranges (mean range = 3.6%). However, sites at Whist Falls and Cyprus Falls have a wider range of si3cvalues (mean range = 5.9~-.,)due to the presence of "C depletion associated with pooled water upstrem of the falls. Although these sites are not statistically Werent fiom one another (W.05).the 13cdepletion associated with a natural resetvoir must be recognized, otherwise, the use of these sites as refmence sites rnay resuit in erroneous interpretations when compared with sites potentially impacted with sources of ''c enricheâ matnial. For example, these sites rnight be used as downstrearn reference sites to detemine if the 13cenrichment observed at KAP-DN was also evident - 15 downstrearn of the point source (KAPFF-DN).Statistical difF'érences between KAPFF-DN compared with GRWF-DN(p=0.014) and MATCY-DN @=0.004) wodd suggest that there was 13cairichment fiom pulp miJi efauent at this site. However, the KAPFF-DN site was not statisticaiiy different from sites (Skunk Island and Thunderhouse Falls) on the Missinaibi River. In temis of the natural morphology and hydrology, the downsir~amsites on the Missnaibi River provide a more reaiistic frame of derence. In another example, consider the usef'ulness of MATCY-DNas a refèrence site to Merentiaie impacts associates with the series of dams on the Mattagarni River (MATSM-DN,MATKP-DN). The significant difference benVeen MATCY-DN and MATKP-DN (w.016)may suggest that impounbmit resulted in 13c enrichment. However, cornparisons between the MATKP-DN site and other downstream sites (MATSM-DN, GRFQ-DN, GRWF-DN, KAPFF-DN,Skunk lslud and Thunderhouse Falls) showed no statistical difference. In conclusion, due to the nature of rivers, carrfiJ consideration must be given with regard to the choice of reférence sites adthe Runkr of &kence sites in order to adequately assess ddopmental impacts on the 613ccomposition offi& species. 116 NaW isotopic variability of the 6% composition of white sucker from the various riven was also considerd for non-impacted sites to determine the influence of habitat dflerences. The range of 6% valws for white sucker Grom Skunk Island (6.9 to 8.7~~~). Thunderhouse Falls (6.3 to 8.7~-,),Whist Falls (GRWF-DN; 7.5 to 9.2-i), Cyprus Falls (MATCY-DN;7.1 to 1O.@,) and Freddie Hats WFF;8.3 to 9.0-00.n=4) were les variable than the range of 6I3c values. There was no indication that the '% depletion observed in the man-rnade impoundments on the Groundhog and Kapuskasing rivers was evident in the pools located upstream of Whist Fails (GRWF-DN)and Cypms Falls (MATCY-DN).Therefore, natural variations in habitat do not appear to strongly inûuence the 6%i composition of white suc ker. The lack of 6% variability associated with habitat differences suggests that it is reasonable to use the downstream sites located outside the region of pdp mil1 activity to determine the influence of the series of dams on the 6% composition of white sucka. Both MATSM-DN and MATKP-DN were significantly more enriched (p<0.001) than downstrearn sites on the Missinaiibi (SMIsland and Thunderhouse Falls), Groundhog (GRWF-DN),Kapuskasing O(APFF-DN) and Mattagami (MATCY-DN) Rivers. The '% enrichment within the series of dams was consistent with the 'h e~chmentobsetved downstream of the Carmichael dam (GRFQ-DN)relative to upstream, however, both sites (MATSM-DN and MATKP-DN) were statistically '% e~chedcompared to GRFQ-DN. '% enrichment on the Mattagami River rnay be infiuenced by P:N ratios.

4.4 Discussion The isotopic composition of white sucker is a function of the type and quantity of dietary items consumed and the isotopic composition of the dietary items. These variables are dependent on the habitat characteristics that exist within the tributaries of the Moose River Basin. The review of benthic invertebrate studies in the Moose River Basin by (Fiset 1995) stressed the importance of habitat in assessments of benthic invertebrate diversity and density. Numerous studies within the Moose River Basin have documented differences in the diversity (numôer of taxa) of benthic invertebrates in areas of slow moving and fast moving water, which represent the two natural types of rnacrohabitat found in rivers (Fiset 1995). McCrea et al. (1984) found shdlow water (4 m) and low velocities (1 0.5 ms") promoted higher benthic invertebrate diversity whereas low benthic inveriebrate 117 abundance was evident in areas with a high clay content. Slight increases in benthic invertebrate taxa richness and density were evident dong the margins of Groundhog River (1989 and 1990) compared to the middle channel (Fiset 1995). Seasonal fluctuations in water depth, water velocity, water temperature (insect emergence) will also influence the diversity and density of benthic invertebrates (Fiset 1995). In addition, spatial and temporal factors will influence the isotopic composition of primary and secondary consumers (Winterboum et al. 1986; France 199%; MacLeod and Barton 1 998). Spatial and temporal variability associated with benthic invertebrate diversity, density, and isotopic composition, and the mobility of white sucker required the exarnination of isotopic variability at the trophic level of white sucker within reaches of a natural flowing river (Missinaibi River)(FarweIl et al. 1999a). Based on the similarity of white sucker 6I3c and 6% values for dfierent sites on the Missinaibi River, the isotopic variability of white sucker in an undeveloped river was defined (6I3cmean ISE(n) = -29.3 %O t 0.2 (47) and

6'% mean k SE(n) = 8.3 %O f 0.1 (47)) for use in distinguishing natural isotopic variability fiom variability associated with anthropogenic developments within the Moose River Basin (Farwell et al. 1999a). The influence of hydroelectric development on the isotopic composition of white sucker was evident in the irnpounded areas of the Groundhog (Fanvell et al. 1999b), Kapuskasing and Mattagarni rivers where depleted I3cvalues were obwed. The depleted 13 C values of white sucker observed in the reservoirs were comparable to the 6"~composition of phytoplanlaon in lake systems (France 1995~Hecky and Hessiein 1995; Rau 1980). 613c values of lotic attached algae range nom to -2@, (mean i SD = -29 t 454 based on the compilation of published stream data, the mpjoriîy of which were headwater streams (France i99Sb). The range of lotic attached algae encompasses the range of terrestrial leaf litter (rnean +- SD = -28 f 144(France 199Sb). The distinct '.'c depldon of phytoplankton compared to attached algae is baseâ on the relative proportion of the bounâary layer munding the cefi whae turbulence in open water reduces the boundary laya of the planktonic ceU. Constant exposure to quantities of '*c and I3c dtsin the preferential utiliEation of '*c by the planktonic ce4 thereby depleting the I3cvalues of phytoplsnkton (France 199%; Leggett et al. 1997a). Therrfore, the I3c depletion of white sucker residing in the resewoirs is probably a Wonof the quentity and quality of planktonic primary production. The 6I3c similarity beenthe reservoir sites incücates that 6I3c vaîues of temsaial ongin îiom historical inputs 118 of bark and wood do not duence the 613c composition of white aicker at the miii sites on the Mattagami and Kapuskasing Rivers. Noticeable ')c depletion at the site upstream of Cypnis Falls (MATCY-DN) on the Mattagami River is sidar to the I3cdepletion observeci in the Smooth Rock Falls resewoir and statistically diffèrent fiom other do~ll~freamsites. Although the MATCY-DN is not statistically diffkrent fiom downstrearn sites on the Missinaibi River, the shift toward "C depletion is indicative of pools and therefore must be considered when using this site as a reference to interpret the influence of 13ce~chment from pulp mill discharge on downstream sites. A s'iarscenario involving '% depletion must also be taken hto consideration. The lack of 13cdepletion in the Hannon head pond located downstream of the Smoky Falls dam (MATSM-DN)is probably linked to the siorage volume and flow rates associated with the series of four dams downstream on the Mattm River. The majority of the water fiom upstrearn is retained by the fht dam (Little Long) which has a mnnal storage volwne of 1874 m3/days whereas the storage volume for each of the remaining three dams is greatly reduced ((18 m3/days)(Table 4.1). In addition, the comparable flow rates of the four dams mggests that the retention time of water in the head ponds of the Smoky Fds, Harmon, and Kipting dams is not sufficient to produce the 13 C depletion obsmed at the Carmichael, Kapuskasing, and Smooth Rock Falls dams. The similarity of the 613c values of white sucker from MATSM-DNand MATKP-DN compared to other downstrearn reference sites (Ath the exception of MATCY-DN)inâicates that the proxhity of the dams, in co~ectionwith the retention volwne and the, redts in a 6"~composition SimiIar to that of an wvegulated river (Farwe11 et al. 1999a). One exception would be the impowdment area upstream of the Little Long dam where pater retention volume and the would kely promote increased phytoplankton biomass rdting in the addition of I3c depleted primary production to the system, iunilar to the "C depletion observeci upstream of the Camiichaei, Kapuskasing, and Smooth Rock Falls dams. The fact that the samphg sites within the region of the secies of dams provided no evidence of the of hydroelectric dams on the isotopic composition of white sucker, litsthe usetiilness of these sites to dinerentiate the downstream impact of dams eom the combined impacts of hydrodecüic developrnent and pdp mill efnient discharges. The 613ccomposition of white sucker fiom sites downstrem of the dam and efüuent discharge on the Mattapni and Kapuskasng RiMs (MAT-ON, KAP-DN) were sigiiicanfly I3cenriched relative to downstream on the Groundhog River (GRFQDN), however, only the Kapuskasuig site Mèreû hmthe rekence site on the hiissinaiii River. The asses~manof the 119 impact of pulp mill effluent on the 6I3c composition of white sucker is complicated by the potential impact of impoundment on sites located immediately do~ll~frearnat both mills and the combined impact of mil1 effluent and sewage discharge on the Kapuskasing River. There was no indication that the 13c depletion in the reservoir Uifluenced the 613c composition of white sucker muscle downstrearn of the dam at Fauquier on the Groundhog River (GRFQ- DN) bdon the fact that no statistical Merence was found benVeen this site and the reference site on the Missinaibi River (FarweU et al. 1999b). However, Farwell et al. (1999b) observed "C depietion in pst-irnpoundment white sucker liver wmpared to historical pre- impoundment white sucker üver fiom the Fauquier site and concluded there was a SMto ')c depletion downstream of the dam. Therefore, at sites located downstrearn of the hydroelectric dams and pulp mil1 eflluent discharges, there is a potential influence 6om inputs of I3cdepleted POM (particulate organic matter) from upstrearn and inputs of "C enriched POM firom miIl effluent. The 6I3ccomposition of mil1 effluent was I3cenricheci by appronmately 2 %O relative to terrestrial matter. As a result, the influence of mil1 effluent discharge at the Smooth Rock Falls site, may be obscured by impoundment effkcts whereby 13cdepleted POM from upstrearn of the dam mk with 13cenriched POM fiom mil1 discharge to produce a 6I3ccomposition of white sucker which is consistent with the reférence sites on the MissUiaibi River. However, the combination of effluent and sewage discharges rnay have provided a s~fficientquantity of I3c e~chedPOM to enrich the I3c value of white sucker inhabited the site downstream at Kapuskasing thereby distinguishing it fiom the reference site on the Missinaiii River. Another possible explanation for the I3cciifferences in white sucker downstream of pulp dldischarges on the Mattagami and Kapuskasing Rivers rnay be related to differences in the biomass of 13c depleted plankton within the resewoir. Merences in plankton productivity within the reservoirs, the size of the reservoir adthe tlow rates would inauence the quantity of I3c depleted POM downstream, therefore, altering the proportion of 13c depleted POM (fkom plankton) and I3c e~chedPOM (hm efhent) available for consumption by benthic invertebrates adwhite sucker. Greater productivity and size of the Mat- Rim reservoir rnay explain the inabüity to Werentiate 13ce~chcnent fi0111 pilp miN duent in white sucker downstream of the Smooth Rock Falls miU relative to the reference sites on the Missuiaibi River (Farwell et al. 1999a). Sites fùrther dowllstfeam of discharge were sampleû to determine if I3cenricheci POM UiBuehced the 6"~composition of white sucker et these sites. Downstmm on the Kapuskashg River (KAPFF-DN), the 13cvalues of white sucker wae more depkted than the 120 site immediately downstrearn of discharge (KAP-DN) and sdar to reference sites at Skunk Island and Thunderhouse on the Missinaibi River. Therefore, 13ce~ched POM tiom etDuent and sewage discharge do not influence the 613c composition of white sucker inhabithg waters a distance of approximately 15 km fiom the point source. The '% depletion of white aicker upstream of the &uns compared with downstream sites on the same river was consistent for ail the developed rivers, however, there were diffaences in the 6% trends among the upstream sites of the regulated rivers compared to the unr@ated river. The depletion of '% in the reservoin was consistent ~iththe '% depletion of phytoplankton in l&es (Leggett et al. 199%; Servos et al. 1998). Both the Groundhog and Kapuskashg reservoirs were more '% depleted compared with the Missinaibi reference site (Fawell et al. 1999a), however, the Groundhog reservoir was not as '% depleted as the Kapuskasing reservoir. The Kapuskasing resenoir represents a well-established system which has been in existence for ddeswhereas the Groundhog reseivoir is a recent developrnent (6 years). Comparisons of pre-impoundment white sucker liver samples (Fauquier, 199 1) to post- irnpoundment sarnples âom upstream of the dm (GR4.JP, 19%) on the Groundhog River indicated a SMto '% depletion in the resenoir (FarweU et al. 1999b). Therefore, the 6% difference observed between the Groundhog and Kapuskasing Rivers is probably a fûnction of the low turnover rate of muscle where white sucker 6om the Groundhog River has not reached nitrogen isotopic steady state with dietary items in this recently developed system. Resident white sucker in the Mattagarni River were '% depleted upstream of the Srnooth Rock Falls dam (MAT-UP) relative to downstrearn sites (with the exception of MATCY-DN), which is consistent with the other regulated rivers. However, the reservou site at Srnooth Rock Fdls (MAT-0) was significantly more '%ienricheci comparai to reservou sites on the Grwndhog and Kapuskasing rivers. In generai, the 6% composition of white sucker 6om the Msttagami River was more eruiched ththe other three rivers investigated in this study. A sumrnary of values of aquatic coiilwner$ tern$rid plants, iitter and mils from the Long TmEcologid Research (LTER) sites in the United States and Puerto Rico provided a guiddim for the potential6'W variabiiity in mlatively undishutKd lakes and streams (Fry 199 1). Aquatic and terreaial plants ffom various des in lakes and streams were generally isotopicslly similar with a wide 6% range of 4 %O to 3 960. Frequency distributions of N2 hgand non Ni 6xing plants, litter, organic soi1 and nillwnl soü were provided for a variety of climatic zones inciuding desats and arid grassfanâs which wem oflm more 'k enricheci.

Generally, minerai soi1 (O 960 to 8 960) was more '?U enricheci rdative to plants (-9 960 to 5 %O), 121 litter (-5 L to 5 960) and organic soi1 (-3 960 to 2%0), badon the exclusion of plant material from desens and arid grasslands. Fiy (199 1) also noted that plants and soils fiom other LTER studies were generatly 'h depleted compared to agricultural plants and soüs in North America. Theoraicaily, practices within the Mwse River Basin including tree harvesting, minllig, rd and dam construction and agriculture could contribute to the mobility of '% e~chedmineral soils, transported in dissolved form @Dl) in rainwater, and dischargecl to tributaries of the Moose River. '% enrichment of the bemhic food web in the Mattagarni River. Ma uptake and metabolism of nitrogen fiom mineral soii sources by bacteria and pRmaty producers, would be differentiated firom the 6'k composition of the benthic food webs in the other triiutaries based on the type and duration of activities with the drauiage ara of each of the fivers. In ternis of water regulation, the Managami River is fiir more developed, with a greatn number of dams, compared to development on the Groundhog and Kapuskashg Rivers Another possible expianation for the trend of 'k enrichment of white sucker hm the Mattagarni River relative to the other rivers in this study could be related to differences in nutrient status. In lake ecosystem, the 6% values of phytoplankton may Vary depending on nutrient limitation of P versus N (Peterson and Fry 1987). ff ~trogenis limited, all available dissolved inorganic Ntrogen (N2, W',NO< Nw is utilized by prVnary producers, therefore no isotope fiadonation OCCUIS. The resulting 6% value of primary producers is a t'unction of the type of Dm available, the quantity of DiN and the isotope values of the nitrogm species (fonn) utilized and the flux of nitrogen in and out of the cell (Handley and Raven 1992). If phosphorus is Sied, and nitrogen is abundant, SI% dismmination occurs during nitrogen uptake (Peterson and Fy 1987). In the Mariagami River, higher phosphonis levels promoting increased prirnary production ddresult in the utilization of a4 available Ntrogen (no isotope fhctionation), thuq the '% enrichment of primary producers and consumers (white sucker). In contrast, the Iowa levels of phosphonis in the 0th- rivers suggests that nitmgen limitation is not a factor, thérefore, uptake by primary proâum mlts in 6'% Wonation which would be reflected in the ')N depletion of primary producers and consumers. Investigations of several Roda lakes showed depleted '% values for PûM (partiailete o@c rnatter) in oligotrophic lakes relative to eutrophic lakes (Gu et al. 1996).

4.5 Conclusions The 6'3~snalanty between the resewou sites on the Gromdhog, Mattagarni and 122 Kapuskasing Rivers indicated that 613c values of terrestrial origin tiom historical inputs of bark and wood did not influence the 613c composition of white sucker at the miü sites on the Mattagarni and Kapuskasimg Rivers. "C depleted particulate organic matenal fiom upstream of the dam and 13cetuiched particulate organic material from pulp mil1 effluent discharges resulted in the combined 613c values detecteci in white sucker. The ability to differentiate the 613c values of white sucker exposed to pulp mil1 effluent venus the 613c values of white sucker fiom non-impacted sites was dependent on several factors, including the size of the resetvoir, water discharge, effluent composition and discharge, and fish mobility. 6% values of white sucker did not appear to be influenced by pulp mil1 activities. StaMe carbon and nitmgen isotopes as triem in noitheastem Ontario riven with hyddectric dcvebpmmt and pulp mül activity. IL Study of benthif inveriebntes at distances upstream and downstmm of dcvekpment. Stable isotopes have been utilized in ecologid studies to evaluate various aspects of carbon and/or nitrogen cycling in lotic systems iduenced by different types of land use and development, including, defining the contribution of allochthonous and autochthonous sources of carbon in grassland and forest catchments (Rounkk et ai. 1982), describing trophic relationships in agriculturai and forest catchments (Macleod 1998; Roumkk and Hicks 1985) and assessing nutrient flow dynamics associated with river impoundments (Fanvell et al. 1999qb). Stable isotopes have also been used to quant@ expsure of riveMe biota to pulp miIl effluents (Wassenaar and Culp 1994) and to edne the cornbined impacts of hydroelectric development and pulp mil1 efnuent discharges on riverine food webs (Farwell et al. 1999b,c). Stable isotope values of cdledor-gatherer invertebrates are used in this study to characterize the isotopic variability of sedentary dietary items cunsumed by benthivorous 6sh species in order to explain observations of high 613c variability in 6sh species tiom impounded areas and 6% differences in fish species between sites influenced by impoundment, and the combined affects of irnpoundment and pulp mill inputs (Fanveil et al. 1999b.c). 613c and 6'?U values of htlevel conmers represents the cycling of carbon and Mtrogen within microhabitats, therefore providing a baseiine to distinguish fesding habitats of benthivorous fish species within the reservoirs utiiizing values and to differmtiate between microhabitat cycting of nitrogen and changes in the diet of benthivorous fish spesies at dierent sites utilizing 6'kvalues. Populations of white wcker (Ca!ostm~sccarnrnerso~t~) have be!en used to tmx nutrient flow between trophic levels in ciiffirent miutaries of the Moose River to assess impacts of hydroeletric development (FanueIl et al. 199%) and the combined impacts of hydroelecûic development and pulp mül activity (FarweU et al. 1999~).Reference river baseline studies hdicate sniilarities in the isotopic composition (6"~and 6%) of white der coiiected from the same site on the Missinaibi river in 1994, 1995, and 19% as weU as at Werent sites downsmain in 1996 (FarweU et al. 1999a). Minimai isotopic variability in fish for the reference river was observed basai on the long term isotope integration of muscle in adult 6sh and the relative homogeneity of the river dons. In contrast, the 613c and 6% values of white sucker were highly variable with signifiant 6I3c and/or 6% sshat bbenupstream addo~ll~frem sites of the hydroelecbic dewloprnent on the Groundhog Riwr and the pulp rnills and hydroelectric developments on the Mamgmi and Kapiskasing Rivers (Fmd et al. Efforts to assess the impact of development on changes in the isotopic composition of the riverine food web for white sucker 6om the Moose River tributaries has been difficult due to 13c depletion and high 613c variabiIity of white sucker in cornparison to the benthic invertebrate population at the sarne site (Fanvell et al. 199%). In theory, carbon isotope values of prirnary producers, consumed by secondary producers and fish, undergo minimal isotopic fiactionation during rnetaholic processing between trophic levels @eNiro and Epstein 1978). Therefore, 6'" vaiues of white sucker and benthic invertebrates should be within the wune range for sites on the developed rivers. Evidence of the discrepancy benveen 6I3c values of white sucker and benthic invertebrates upsfream of the hydroelectric dam on the Mattagami River prompted a closer investigation of the benthic invertebrates inhabiting these donsof the river. Previous benhic invertebrate samphg efforts were generally restricted to shallow water (< lm) dong the shoreline collected in the fd. However, studies have shown depletion in the 13cvalues of planktonic algae compared to littoral benthic dgae in various lakes (Fmce 1995a; Hecky and Hesslein 1995) as weU as seasonal changes in strearn periphyton (MacLeod and Barton 1998). The present study considers the rnovement and feeding of white sucker at various depths within the reservou upstream of the dams and in srnaller tributaries as well as cornparisons of upstream and downstream sites of developed areas. The primary objective of this survey is to dhbethe fiIl range of 6I3c and 6% values of benthic invertebrates available for consumption by white sucker within reaches of the river. The benthic invertebrate survey was conducted upstream and downsiream of the hydroeiectric dam and pulp miIl on the Mattagami River as well as upstream and downstream of the dam on the Groundhog River at sites where white sucker have been collected. Invertebrates collected fiorn these two rivers are used to différentiate between the isotopic idlueme of hydroelectric development on the Groundhog River, in tem of aiterations in water flow, temperature, sed'imentation and bain morphology versus the combined inauence of impou-nt and incnased organic inputs Eom bark, wood and mil1 duent associated with pulp miIl activity on the Mattagami River. Isotopic analyses of all benthic invertebrate groups is impracfical. Therefore 6I3cand 6% values were obtained for HexqpNa sp., a genus of burrowing myfly (Ephemeroptera) commoniy fowid in the tnitaries on the Moose River Basin (Fiset 1995). Hkxqge~~iain the nymph stage inhabits the mu4 muddy sand or dcbris (Pennak 1978) and d-süt depositional 126 zones of lentic and lotic habitats (Edmunds 1978). In ternis of fùnctional groups, Edmunds (1978) classiS> Hexagenia as collectors and gathners. Collectors represent the dominant fùnctional feeding group in regulated and unregulated streams (Short and Ward 1980). Species of Hruge~iiaare classined as facultative taxa based on the wide range of tolerance to organic contamination (Fiset 1995). Freshwater biomonitoring studies usiig benthic invertebrates found Hcxagettia to be intolerent to conditions of organic pollution (Johnson et al. 1993). However, in the Moose River Basin, Heugtwia has been collected in areas of organic enrichment (Fiset 1995). The widespread distribution and abundance of sedentary Hrxng~ia in the Moose River tributaries as weil as the siiof individual mayflies providecl sufficient invertebrate biomass for the isotope examination of spatial variation. The use of a sedentary benthic invertebrate as an indicator of isotopic variability within the river system was based on observations of Haugenia in white sucker gut contents in addition to inhabithg areas where benthic white sucker forage. Merences in the isotopic composition of Hexaget~iawithin different microhaôitats will enhance the understanding of 6I3cvariability in white sucker and dehe the importance of feeâing habitats of white sucker in resewou areas. DifKerences in the 6% trends of Hexagetria and white sucker are used to determine whether changes in 6% composition of white sucker at different sites are the result of inorganic and organic nutrient cycling or changes in the invertebrate composition of white sucker diet. In theory, the 6'% values of Hexageeia, fèeduig on decornposing particdate organic matter, will reflect the cycling of nitrogen in the system, whereas white sucker are influenced by the cycling of nitrogen and the species composition and abundance of invertebrates in the diet, wtiich dehes the trophic position of white sucker. Theortically, dif5erences in benthic invertebrate density and diversity due to hydtoelectric development and pulp mil activity (Fiset 1995) rnay be reflected in the 6'k values of white sucker at diierent sites. The nul hypothesis states that there is no Metence in the 6'% trends of Hexuge~~iaand white sucker upstream and downstream of development. in this study, Hmgenia were utilized as an benthic invertebrate indicator of isotopic trends, however, it is recognvad that Hmgenia does not constitute a signiscant dietaiy item of the white sucker ôaseû on gut content examination. This siudy was conducted to examine 613c and 6'k variabii between years for Htmgenia coileaed at similar locations upstream on the Matta@ Riva in 1996 and 1997. 127 Isotopic variability associated with Hexrgenia size as a measure of age was used to interpret possible shifis in 6I3c and 6'% values associated with seasonal changes. Hexage~~iafrom mainstrearn and sder tributaries were compared with regard to water depth and distance ffom the dam for upstream sites on the Mattagami River in 1996. In 1997, Hemge~riatiom upstream and downstream of the dams on the Groundhog end Mattagami were compared to determine dif5erences between sites on the sarne river and ciifferences between rivers iduenced solely by hydroelectric development (Groundhog River) versus hydrodectric development and pulp dl activity (Mattagami River). The isotopic data for H'uge~riu is compared with white sucker data (Fanveil a al. 1999b,c) cuilected in 1997 to determine if the sources of organic carbon consumed by white sucker and Hmgettia have umilar 6"~composition. Comparisons of 6'% vahies for Hrxagnia and white sucker were used to deterrnine changes in 6% values of white sucker at sites upstream and downstream are a fùnction of nimgen cycling in the system or related to changes in conswnption of benthic invertebrates due to differences in species composition and abundance influenceci by developrnent.

5.2 Materials and Methods 5.2.1 Sampling Site Description The Groundhog (5h order) and Mattagami (6' ordo) Rivers are medium sized developed tributaries of the Moose River Basin draining northward into James Bay in northeastem Ontano, Canada. Glaciai, glaciofluvial and glacolacushine deposits cover the crystaîline granites of the Precambrian Shield. The region of shidy was located in the middle reaches of these rivers within the latitudinal zone of 48" 60' to 49O 50' and extended north to the Kipling dam (50' 08'). Detailal chemistry &ta for water and effluent samples were coUected in 1994 and 1995 for the Groundhog and Managami Rivers (Fmell et al. 199%,c). Development on the Groundhog River consists of the recent hydrdectric fàcility (Carmichitel Dam) with initial construction in 1989 and completion with tuhii operation in ûctober of 1991. The hydroelearic development on the Mattagarni River was instaned in 1917 with a capacity of 6250 MW. The bleachd lrraft miil at Smooth Rock Falls (Managami River), in operation since 19 18, has recentiy undergone a number of proceta changes (Fanivell and others 199%) in addition to the implementation of secondary duent memmi (aeration stabiition basin, 1994). 128 52.2 Sampk Coiiection and Preparation The sampling sites foi benthic invertebrates were located in the same regions where white wcker have been captured (Farwell et al., 1999b,c). Benthic sampks were mllected in the fall of 19% (Sept. 28 io Oct.5) dong latitudinal transects in the mainstrearn and in srnaller tributaries in the region upstream of the Smooth Rock Falls dam on the Mattagarni River (Figure 5.1). Site # upstream 1-96 was located 4.0 km (width:-220 m) upstream of the Smooth Rock Falls dam. This site was approhately 1.O km dowmtream of the confluence of the North Muskego River. The size of the North Muskego River represents a si@cant source of inputs to the study region. Further upstream, site # upstrearn 2-96 was located 7.0 h frorn the dam (width: - 180 m) in a more narrow stretch of river. Sarnples were also collecteci from Bradbum and un-med creeks locateû 7.5 la and 9.5 km fiom the dm, respectively. In Bradbum creek, samples were collected at distances upstream of the mouth of the creek, at sites upstream 3-96 (distance: 0.5 km, width: -30 m), upstream 4-% (distance: 1.8 km, width: -16 m), and upstrearn 5-96 (distance: 2.7 km, width: 7 m, ma>

132 precombusted foi1 (4 hrs @ 400°C) and frozen at -20°C. Preparation for isotope analysis included the removal of the intestinai tract, and dehydration in a fieeze-drier. Dry, whole invertebrates were weighed and ground using a mortar and pestle.

5.2.3 Stable Isotope Andysis Dry, ground sarnples were analyzed for carbon (13~/12~)and Ntrogen ('?V/'''N) isotope ratios using a VG Optima continous flow isotope ratio mass spectrometer at the Environmental Isotope Laboratory (EIL), Department of Earth Sciences, University of Waterloo (Waterloo, Ontario). lAEA and NIST standards were used to monitor analytical precision of + 0.2 %O and f 0.3 960 for 6I3c and 6'%, respectively, within the range of linearity .

5.2.4 Statistics Analysis of variance (ANOVA) was used to differentiate isotope values upstream and downstream of development. Regression analysis was used to determine the correlation between 613cand 6'% values of Hexugen~aand the water depth at which the mayfiies were collected.

5.3 Results Spatial variation in 6I3c and 6'% values of Hexagda sp. was investigated in the mainstrearn and in smaller tributaries in the region upstream of the dam at Smooth Rock Falls on the Mattagarni River in the fdl of 1996. The 6% values of Hexageniu sp. showed differing trends in the shadeâ, nmwupstream reaches of small tri'butaries versus the open regches of the mainsiream (Figure 5.2). Hexage~nia6'%J values ranged Erom 2.8a0 to 4.oâ at the more shaded upstream sites of Bradburn creek (upstream 4-96 & 59%). More e~ched'% values variecl from S.ho to 8.6~~at the open reacties downstream on Bradbum (upstream 3-%) and "un-named" creeks (upstrm 6-96) and in the mainstream (upstream 1-% & 20%). The 6'% valws for Hexuget~iacoilected in the shaded upstream reaches of Bradbum creek were similar to the open sections of the river. However, at open sites, the range of G"C vaiues was 8.h and included more "C depleted values (-36.2 to -27.3%) in comast to a range of 3.6% for shaded sites (-32.lym to -28.5~~). Figure 5.2 6'3~(-.-)and 6'%((4 values for Hexagefra sp. coileaed at distances (km) upaream of the hydroelectrk dam at Smooth Rock Falls in the maitlstceam and in smaller tributaries of the Mattagarni River in the fdl of 1996. Open reaches included rnainstream sites, upstream 1-96 (4.0 km) and upstrearn 2-96 (7.0 km) d sites 0.5 km upstream in Bladbum creek, upstream 3-96 (8.0 km) and un- narned creek, upstream 6-96 (10 km). Shaded reaches in Bradburn aeek (7.5 km Rom dam) included upsiream 4-96 (1.8 km fiom mouth) and upstream 5- % (2.7 km from mouth). O upstream 1-96 O upstream 2-96 A upstream 3-96 Shaded reaches upstream 4-96 A upstream 5-96 O upstream 6-96 135 6')~and 6% values of HexagetnM coUected at various depths dong two transects in the mainstream (upstream 1-96 & 2-96) and one iransea located 0.5 km from the mouth of Bradbum Creek (up~fream3-96) on the Mattagarni River were plotted in Figure 5.3. At dl three sites, HexagenM coilected from shallow water (< 3 m) were generally more I3cenricheci

(-33.1 -,:. to -27.3~~~)relative to Hruigettia firom deeper water (-35.!h to -32.3-bc).6I3c values of Haagettia 6om shallow (< 3 m) and deep (23 m) water overlapped by les than I %:.:. No depth trends weobsewed for the 6% cdues of Hxageniu. Seasonal isotopic variation of Heuge~tiawas evaluated by comparing 6I3c and 6'3N values to the size of individual Hexagetlia f?om Mattagami mainstream sites at depths < 3rn and 2 3m collecteci in early fdl (early October) 1996 and in late sumrner (mid-August) 1997 (Figure 5.4a & b). Hexagenia ranged in size fiom 0.15 to 25.2 mg dry weight (n=66). No relationship was found between depth and Hexagenia Ne (8 = 0.02). The limited sampling pends precludes an understanding of the iife histories of Hcxagenia, dthough the low weights (0.1-0.3 g) of Hexagenia tom the fdl of 1996 indicated ment hatching. The 6'3~values of newly hatched Hexagwia were similar to the mature mayûies collected within the sarne depth zone in the fd of 1996 (Figure 5.4a). However, lower weight rnayaies were generally more 13C e~chedcompared to heavier madies colieeted in the deep water zone late in the mer of 1997. No relationship was observeû between 6'% values of Hrxagettio and ske (Figure 5.4b). Isotope values of low weight Hemge~~ia(O. 15-4 mg dry weight) were used to compare Sumer (1997) vernis fd (1996) growth of Hc.wgeer>iacoUected at depths (3m and Sm (Table 5.2). Theoretically, low weight Hexaget~iacollected in early October ( 19%) have been accumulating biomass in sumer and eady fa11 whereas Hexagettia collected in rnid-August have been accwnuiating biomass in spring and sumrner. The mean 613c of Hexugenia inhaùiting deeper water (Sm) was 2.8 wm more "C depleted in the fa11 of 1996 relative to the summer of 1997. Simüarly, the mean 6'% of Hexagenia inhabithg deeper water (Sm) was slightiy (0.7 .:+ho) more 'h depleted in the fidi of 1996 relative to the su~nerof 1997. Linear regression showed a stronger correiation bnween the 613c of Hmgmia and water depth in the tàll of 1996 (84.46. p<0.001) compad with the sumtner of 1997 (h.31, @.007) (Figure 5.5a). 6% of Henalgenia versus water depth in the fidl of 19% showed a minimal Figure 5.3 6I3c(-<) and 6'%( -) dues for Hexageni~sp. collected at various depths at sites upstream of the Smooth Rock Falls dam on the Mattagarni River in the fall of 19%. Water depth (m) of the sample is presented by the symbol. depth < 3 m

,--...-..--...-.depth z 3 m D 4 b 0 9.1 I

O upstream 1-96 iupstream 2-96 Figure 5.4 Hexqet~iasp. weight (mg) venus (a) 613c values and (b) 6'% values for sarnples coliected at depths of c 3m and 2 3m in the mainstream, upstream of the dam on the Mattagarni River at Smooth Rock Falls in the faIl of 1996 and the wmmer of 1997. weight (mg) 140 Table 5.2 6"~and 5'k values of young Hexagenia (O. 1S-i mg dry weight) during surnrncr and fail growth

lsorOpe Year SmpihgDate Giwvth Mean (-) k SE (n) - stalm 6' Tc c 3m 1996 Oct- s~mmer-w -30.0 i 0.5 (1 1)

spring - siimmer -3 1 .O * l .O (8) summ~r-fall 7.2 10.4 (11)

1997 August spring - summer 6.9 f 0.4 (8) Figure 5.5 Linear regression of Hexagenia sp. (a) S'~Cvalues and (b) ~'kvalues versus water depth for samples collected in the rnainstream, upstrearn of the Smmth Rock Falls dam on the Mattagarni River in the fa1 of 1996 and the sumer of 1997. isummer 97 O

R~ = 0.31

R2 = 0.46

4 6 8 10 12 14 watet depth (m)

4 6 8 10 12 14 water âepîh (m) 143 comlation (&=4.16, p4.008) (Figure 5.5b). No significant correlation was found between 6% of Hexngeeia versus water depth in the merof 1997 (h.1 1, pX.05) (Figure 5.Sb). Differences in basin rnoiphology were documented with depth profiles dong transect lines (minimum of 5 aansects) upstream and dowll~treamof the dams on the Groundhog and Mattagami Rivers in August of 1997. Upstream of the Carmicheal dam, maximum depths ranged âom 11.3 m to 21.2 m (mean depth, 15.5 m) in cornparison to the shallower waters upstream of the Smooth Rock Falls dam with maximum depths of 9.8 m to 11.9 m (mean depth, 10.7 m). Water levels downstrearn of the Carnichael (2.7 m - 5.8 m) and Smooth Rock Falls (2.5 m - 6.2 m) dams were sùnilar with mean dept hs of 4.0 m and 4.6 m. respectively . Vertical profles of temperature, pH, conductivity, and dissolved oxygen are surnmarized for sub-surface, and dmumdepths at sites upstrm and downstreem of the dams (Table 5.1). Slight declines fiom surface to sediment were obsewed for pH, temperature

(-2 - 3 OC) and dissolved oxygen (-1 - 3 mg/L) at upstream sites. The downstream sites are generally homogenous in tmof the parameters measured at various depths and distances. One exception is the increase in conductivity of surface waters (+57 mS/w distance 1.1 km) immediately downstream of pulp mill effluent plume (Mattagarni River) and at distances fwther downstrearn (+40 rnskm) relative to upstrearn sites. Secchi depth transparency was sirnilar at upstrm and downstream sites on the Groundhog and Matta@ Rivers. The 6I3ctrend on the Groundhog River showed 13cdepletion for Hemge~Naupstream of the dam relative to enrichment at sites downstrearn (Figure 5.6). A siniilar trend was observeci upstrearn and downstrearn on the Mattagarni River (Figure 5.7). The diffaence between the two rivers is a distinct separation of 6I3cvalues betweeri upstfesm (-38. to - 30.6%:G)and downstream (-28.8~~to -24.4~~) sites on the Groundhog River, whereas on the Mattagarni River, 613c values upstream (-34.7~~to -27.b.) and downstream (-3 1.8n0 to - 26. O.,) ovedapped by 4.8~~. The range in 6% values of Hc?mgniu at sites upstream (1.7.r3. to 6.04 and downstream (3.1a, to 5 -7.4 of the Groundhog River were similar (Figure 5-6). Upstream of the Mattagami River (Figure 5.n Heragnia were wnsiderabiy more enrichcd (5.hto 9.h)relative to upstream of the Groundhog River. 6% of Hexage~ifacoUected immediately Figure 5.6 6'3~'c) and 6'%(-,:-)values for Hexagenia sp. collected at distances upstream and downstrearn of the Cannicheal dam on the Groundhog River in the summer (August) of 1997. The numbers next to the symbols are the water depths (m) at which the samples were collecteci. Symbols with no comesponding number represent sarnples «>Uected at depths of 1 m or les. upstream GR3-97 + upstiearn GR2-97 A upstream GRl-97 o downstream GR1 -97 O downstream GW-97 A dowstream GR3-97 Figure 5.7 6l3c(&. ) and 6'%(-..) values for Hexnge~~iirsp. coilected at distances upstream and downstrearn of the hydroelectric dam at Smooth Rock Falls on the Mattagarni River in the mer(August) of 1997. The numbers presented by the symbol are the water depths (m) at which the sarnples were dected. Symbols with no comesponding number represent sarnples wllected at depths of I m or les. iupstream MR3-97 + upstream MW-97 A upstream MRl -97 a downstream MR1-97 O downstream MR2-97 A downstream MR3-97 O downstream MR4-97 x downstream MR5-97 + downstream MR6-97 x Cypus - Kipling 148 downstream (downstrearn MR 1-97) of the dam and upstream of effluent discharge were in the same range as upstrearn sites. Hexageniu from sites downstream of mil1 effluent discharge (downstream MW-97 to Cypus Falls) were more '% depleted with a range of 3.1%; to 6.31:. Downstream of the Kipling dam, most of the Hexage~iufd into the same range as downstrearn of the Smooth Rock Falls dam with the exception of one invertebrate. This invertebrate was 'SN enricheci (8.k)and 13c depleted (-31.8%0)which is consistent with isotope values hmupstream sites and suggests the possible transport f?om an upstream ongui. The 6% trends for all sites on both the Groundhog and Mattagamj Rivers showed '%J enrichment of white sucker relative to Hexagtaia (Figure 5.8). Trophic enrichment (mean 6% difference) between these two groups varied fiom 2.3r, to 5.11.: with an average of 3.6~!?., between trophic levels (Table 5.3) for al1 sites on both rivers. Upstream of the Mattagami River, '% enrichment between trophic levels was lower (2.3%")than the average (3.6+, with elevated rnean 6'v of Hcixtc~ge~tia(7.62!,.-) and white sucker (9.9-4 in cornparison to Groundhog upstrearn (mean 6%: Hrragenia, 4.5--, white sucker, 7.6$,). The '% enrichment of white sucker downstrearn of the Kipling dam on the Mattagami River is greater than white sucker fiom the other dowllstrearn sites. The siiarity ôetween 6% values of white sucker from upstrearn of the Smooth Rock Falls dam and downstream of the Kipling dam suggests the importance of transporteci material fiom upstream ofthe Kipling dam. The general 6I3ctrend for the invertebrate-fish cornparisons for both the Groundhog and Mattagarni Rivers was 13c depletion of white sucker relative to Hexaget~iu(Figure 5.9). Trophic SMof 613c mean values between whte sucker and Hexagmia ranged from 1.Su, to 5.3- I3C. depletion (Table 5.3). The ody exception is the site located immediately downstrearn (MAT-DN) of mil1 effluent discharge on the Mattagami Rivet where trophic 13ce~chment of 0.8%=was evident.

5.4 DWcussion Movments of white sucker in the large tributaries of the Moose River are restricted by hydroelecaic dams and natural bedrock formations aesting nJls and rapids in areas of low water at down~aeamdes. nie longitudinaf restriction in movement is puticuiariy usetir1 in the Figure 5.8 Cornparison of 6'%(~,~)values for Hexagenia sp. (0) and white sucker (x) collecteci Eiom sections of river upstream and downstrearn of the hydroelectric dams on the Groundhog and MattagaM Rivers in 1997. Mean 6'%(~(+-.~)values are preented as symbols with maximum and minimum 6'%~~4values as vertical lines. The number above the symbol is the sample size (n).

,..,...IiaoPc Si& spcCies Mm(4 Maximun(s) Minimum(s~) Sunple Mn) 8% MAT-UP W@ -3 1.O -27.0 -34.7 7.7 22 M AT-DN MATCY-DN

MATKPIDN GRCF-UP GRFQ-DN GRW-DN

MAT-UP

MAT-DN MATCY-DN MATKP-DN

GRCF-UP GRFQ-DN GRWF-DN Figure 5.9 Cornparison of 613c(s.) values for Hexagenia sp. (-) and white sucker (x) collecteci from sections of river upstream and downstream of the hydroekctric dams on the Groundhog and Mattagarni Rivers in 1997. Mean 6I3c(-.) values are presented as syrnbols with maximum and minimum 6"~(+-~)values as vertical lines. The number above the symbol is the sample size (n).

154 assessment of the influence of pulp d activity on the isotopic composition of fish species. However, tnîutaries that difi in Cie (width, depth) rnay also inhience the isotopic composition of benthic invertebrates and fish species fdgin these various habitats (Winterboum et al. 1986). Isotope analyses of burrowing rnayûies were used as an indicator of the isotopic composition of seûentary invertebrates, potential dietary items of white sucker, in headwater and do~eamsites. Burrowing rnayflies coliected tom shaded shallow headwaters @rd 1.8 km and Brd 2.7 km) were more '%Idepleted than maytiies fiom downstream sites on Bradbum and unnamed creeks. The I3c and '% depletion of mafies collecteci at heaclwater sites rnay be a fùnction of light intensity and season. MacLeod and Barton ( 1998) examineci strearn periphyton grown on glass slides under âiierent conditions of light intensity and water velocity in the sunimer and fd. "C and '% values of periphyton were found to be more depleted under low-tight conditions with seasonal shifis to 13c and '% depletion in the fdl. The similarity of 6% values from downstream sites (Bradbum and unnamed creeks) compared to the mainstream indiates that the transport of inorganic and organic sources €rom headwaters do not appear to infiuence the isotopic composition of invertebrates at downstream sites. Burrowing mayflies (kagmia) inhabithg the irnpded region upstream of the

Srnwth Rock Falls dam ( 19%) vary in their 6I3ccomposition by - 9.0 ;:.>. The eruiched 13c values of Hexugenia coilected fiom shallow water (< 3 meters) were comparable to other benthic invertebrates fiom the refmence sites on the Misgnaibi River (Fanveil et al. 1999a) and downstream at Fauquier on the Groundhog River (Farweil et al. 199%). The 813c distinction between shallow water coUections (< 3 meters) and deep water collections (2 3 meters) was related to water transparency estimated by secchi dephs of approimately 2.5 m. Secchi disk transparency deph relates to the depth reœiving, on avenige, approximately 10 % of surfacc irradiame (Wetzel 1983). Light transmission dierentiates primary proâuctivity dominated by aquatic macrophytes end periphyton in the littoral zone hmprimsry productivity dominated by phytoplankton in the pelagic zone. Isotope stuclies have documented the 13c depletion of phytoplankton relative to peciphyton in lakes (Fr- 1995a; Guiguer 1999; Hecky and Hesslein 1995). The aôiüty to isotopically differentiate between benthic invertebrates fesdng 155 on periphyton fiom the littoral zone and phytoplankton 6om the pelagic zone in the Mwse River study rnay provide information on the feediiig habitats of benthivorous fish species in these reservoirs. The life span of most mayfly nyrnphs (Order Ephemeroptera) range from 3 to 6 months, however Hexagertia limhta nymphs may siMve as long as 2 years in some northem Canadian lakes (Memt and Cunimins 1978). fixagenia Iimbata was the most widely âistributed species found in the tributaries of the Moose River system (Fi* 1995). The size (O. 153 to 25.214 mg dry weight) of Hexage~~iiacoUected upstrem of the Smooth Rock Falls dam in the fdl of 1996 indicates a wide range of ages Eiom low weight nymphs that have recently hatched (swnmer of 1996) to higher weight nymphs that probably hatched early in the 1996 growing season or late in 1995. The growth of mayfües involves a senes of molts whete a portion of the existing aiticle is digested and absohî, in conjundon with the deposition of a new cuticle beneath, followeâ by the shedding of the old exoskeleton (Merrit and Cummins

1978). In ternis of stable isotope turnover, the rnajority of the biochernical constituents required to produce a new exoskeleton probably originate 60m dietary items recently consumed. The process of molting is important when interpreting data to support the hypothesis of seasonal trends in isotope values. Comparisons of all size ranges of Hexagenia and their 6I3cand nd'% values showed that low weight mayflies collected in the summer of 1997 Eom deeper water (Sm)were more I3c enricheci than heavier mayflies suggesting the growth of larger mayûies over a number of seasons. Low weight mayflies (5 4 mg dry weight) were compared to Mer understand differences in the isotopic values of rnayflies growing in the su~nerand faIl of 19% (dezted in ûctok, 19%) and the spring ad sumof 1997 (cdlected in mid-August, 1997). The 613cand 6'% values of littoral mayflies were similar for both growth periods. The similarity in mayfiy isotope values indicates that there was no seasonai variabtiity in the isotope values of the primiuy sources cod.This suggests that temsaiel sources which do not Vary isotopidy with season rnay ôe important carbon sources for ôenthic hvenebrates in the linoral zone. Mayûies inhabithg deeper water showed similar isotope values to littoral rnayflies

in the spring ancl summer of 1997 suggesting the inbaice of tanstrial socicces hmspring 156 runoff waters. As the growing season progreses, fine particdate matter including U~ and '?V depleted phytoplankton settles to the bottom and becornes available for consumption by youiig ma* producing more isotopically depleted mafies coUected in the fd of the year. This succession may explain the "C depletion of young, recently hatched rnaytiies growing in the faIl of 19% in cornparison to the spring of 1997 in deeper, offshore habitats. The differences in the 6% values of mayfhes were not si@cantly diffèrent to estabüsh any seasonal trends. The availability of ''c enriched benthic inveriebrates in spring and summer in impounded areas may also explain the general trend of 13c enrichment of white sucker gonad relative to muscle fiom the reservoir upstream of the Carnichael dam on the Groundhog River (Farwell et al. 1999b). White sucker spawn in the spring, followed by the accumuiation of new gonadal biomass as the seasons progress. Therefore, white suckn capturd in mid-September wiU have accumulated the majority of the godbiornass during the period when dietary invertebrate sources were I3cenricheci. The prirnary objective of the Hexage~r,iasurvey and the determination of 6% for these Uivertebrates was to establish a 6% baseîine of tirst level consumers for cornparison with second level consumers (white sucker) at different sites on both nvers. The 6'%ldata for Hcxagmia revded ciifKerences in the 6% trends between upstream and downstream sites on the Groundhog and Mattagarni Rivers. The mean 6% of Hexupia varied < 1 -= (0.8 b.-) between upsiream and downstream Groundhog sites in contrast to the > 2 -.Q (2.8 -ha,) difference between upstream and downstream Mattagarni sites. The greater 6% difference on the Mattagami River relative to the Groundhog River was due to '% hlctunent of Hexagenia upstream of the Smooth Rock Falls dam on the Mattagami River. Physicai and chemid parameters myexplain the '% e~chmentof Hyxagenia upstream of the Smooth Rock Falls dam (mean 6*% 7.6 qo,) relative to the downstrearn site

(mean 6% 4.8 J&)and upstream of the Cannichael dm(mean 6% 4.5 %,). Hrugmia are categorued as coUector-gatherers féeding on decomposing fine partidate organic matter (particle size < id pm)(Mefnt and Cummins 1978). The source of the decomposecl material originates from a combination of pht and anid matter. 6% dues of Hexagwiia are dependent on the availabii, the the and the 6% of the PON (partiCulate organic 157 nitrogen). 6% values of different types of aiiochthonous (leaves, bark and wood) and autochthonous (rnacrophytes, algae, bacteria and t'ungus) sources in streams and rivers are highly variable (Fry 199 1; UacLeod and Barton 1998). As a result, it is difficult to determine the 6'% sources that constitute the 6')N values of Hemgenia. However, analysis of the quantity of particdate and dissolved organic nitrogen in the water and sediments may be usefiil to identie dEerences between the sites on the Groundhog and Mattagami Rivers. Paticulate and dissolved or@c nitropen were mmedin the fall of 1994, 1995 and the spring of 1995 for sites on the Mattagami and Groundhog Rivers (Fanveil et al. 1999b.c). Particulate organic nitrogen IpoN) vaned fiom 0.053 m& to 0.082 m& upstream and 0.068 mg/L to 0.233 mg& downstream on the Mattagami River. The increase in the quantity of PON downstrearn on the Mattagami River is probably the result of higher levels of PûN (9.410 mg/L) in mil1 effluent discharged downstream. Downsiream on the Groundhog River, PON varied fiom 0.032 mg/L to 0.042 mg/L. Levels of dissolved organic nitrogen were found to be similar at sites upstream (0.406-0.475 m@), and downstream (0.4 17-0.439 mg,L) on the Mattagami River and downstream (0.4 12 mg.)on the Groundhog River. nie similady of 6% values for Hexagmia coUected fiom downstream of the Smooth Rock Falls (mean 4.8 a,) and

Carmicheal (mean 5.2 ,+a) dams wggests that the higher levels of PûN 60m an effluent source are not isotopicaily distinct fiom natural sources and therefore do not alter the isotopic composition of t he benthos. Particdate organic nitrogen (PON) wnsists of allochthonous (lems, bark and wood) and autochthonous (maaophytes, algie, bacteria and fingus) sources transportai fiom upstream. The depth of the photic zone iimits macrophyte abundance in the reservoirs, and therefore limits the quantity of nitrogen, âom decayhg aquatic plant material, available to Hruigenia especidy in deep water zones. Medium sized rivers such as the Groundhog (stream order 5) and M- (Stream order 6) Rivers have a stmwidth of signifiant size to minünize the importance of coprse partidate organic material (0hm ripaian vegetation based on a ratio of laigth of bank to area of the river bottom (Anderson and SdeU 1979). However, historical log dmie aaivities on the hUagami River introduceû large quantities of terrestrial mataial in the fom of bark and wood to the site upstream of Smooth 158 Rock Falls dam. The mill at Smooth Rock Falls uses approxixnately 65% black spnice and 35% jackpine as wood fùrnish (Acres 1994). A study on wood decomposition predicted a period of 75 years for 95% of black spmwd to disappear 6om a ninth order Stream in eastem Quebec (MeMo et al. 1983). The slow rate of decay of black spruce was related to the high initiai lignin concentration. High initiai lignin content (24.6 %) and low nitrogen content (0.038 %) of black spruce determine the quantity of nitrogen mniobilized per gram of initial material (MelUo et al. 1983). The use of bark and wood as substrates for microbial colonization upstream ofthe Smooth Rock Falls dam may explain the higher percentage oforganic nitrogen in the sediments upstrearn. The percent composition of organic Ntrogen in the sediments was 0.05% to 0.29% upstrearn and 0.01% to 0.03% downstream of the Smooth Rock Falls dam (sprhg 1995 and fd 1994 & 1995, unpublished data). Downstrearn at Fauquier on the Groundhog River, organic nitrogen was 0.05% to 0.1% of the total SediRlent ms(fa11 1994 & 1995, unpublished data). Particdate organic Ntrogen in the form of microbes donizing bark and wood is possibly the source of nitrogen intluencing the '% e~chmentof Hexagtwia upstream of the Smooth Rock Falls dam. The presence of large quantities of decomposing bark and wood upstrearn of the dam at Smooth Rock Falls rnay also explain the differences in the 6'3~trends between upstream and downstream sites on the Groundhog and Mattagami Rivers. Log drives on the Mattagami River were discontinued in 1986, therefore bark and wood accwnulations from log drives have been decomposing for a minimum pend of 10 years. Hexagenta from the Groundhog Riva showed a distinct separation between the I3cdepleted mayflies collecteci iipstrm of the dam and the 13c enriched mayflies collected downstream. Mafies hmdeeper water upstream on the Groundhog River were -3 more I3c depleted than maytlies fiom deeper water upstrem on the Mattagami River. Microbial populations colonizing the large quantities of deçomposîng bark and wood with emiched 13cvalues in the range of -22.7 to -24. (FmeU et al. 199%) may contribute to the dietary carbcjn sources consumed by mayflies. Hence, the combii âietaiy sources of bacteria colonking terrestrial mptter and phytopllanldon €rom the pelagic zone rnay diin Htmqpnia that are more ')c enriched at sites upsoe~mof the Mattagami River compared to upstream of the Grounâhog River. 159 5.4.1 Trophic levd corn parisons The 6% cornparison of first (Hexageniu) and ndnd (white sucker) level consumers was used to investigate possible dietary changes of white sucker in sections of the river influencecl by anthropogenic development. Theorehlly, trophic enrichment fiom fht to second level mnsumers should be sirnilar if the quantity and quality ofdietary items consumed by white sucker are comparable for sites on the Groundhog and Maîtagarni Rivers. The 6% trends for al1 sites on both the Groundhog and Mattapi River showed '% enrichment of white sucker relative to Hexagettia. 6'hvalues of consumer groups were used to define trophic levels for temperate lakes in Northwestem Ontario considing of second (6'w < 6-4, third (6'h 6-91>*9e)and fourth (6% >94trophic levels (Hecky and Hesslein 1995). White sucker 60m these lakes were class'ied as a third trophic level group. H'xageniu and white sucker fiom the Moose River tributaries are categorized in the second and third trophic levels, respectively, based on the 6% defined trophic levels. Trophic enrichment (mean 6% ditference between levels) variai from 2.3icto 5.1 with an average of 3.61. between trophic levels. The dflerence in 6% is consistent with trophic level enrichment reported by @eNuo and Epstein 1981). Upstrearn on the Mattagarni River, '% enrichment between trophic levels was lower (2.3~~~)than expected with elevated mean 6% of Hrxcgwiu (7.6~4and white sucker (9.9-4beyond the range of trophic levels observed by Hecky and Hdein (1995). The basic theory of trophic '% enrichment is related to the ciifferences in dietary items consumed by different species. Changes Ui '%lenrichment of the same species may reflect differences in the dietaiy items available for consumption. hpoundment dten the composition of benthic invertebrates in terrns of the dominant species and diversity due to changes in curtent, water level fluctuations and ~rbiditydowll~tream of the dam (McCrea et al. 1984). Howwer, similu '3enrichment from the second to third trophic levels was observed for sites upstream (GRCF-üP, 3.&4 and downstream (GRFQ-DN, 3.494 of the Carmicheal dam. in cornparison, e~chmentdittéred considerably fiom upstream (MAT-UP,2.306~) of the Smooth Rock Falls dam relative to dowmüeam (MAT-DN,4.2%). The low level of 'k e~chmentbetweerr trophic ievels suggests that bacteria colonkhg bark and wood substrates may be an important dirra dietaty source for white sucker in the reservoir upstmm of the 160 Smooth Rock Falls dam. The presence of large quantities of bark and wood chips rnay provide an element of protection 60m predation for benthic invertebrates. if white sucker are not successfùl at capturing large quantities of benthic invertebrates, the diet rnay be supplemented by bactena colonizing detrital material. Therefore, if the burrowing mafies and white sucker are feeding on simiiar die@ the 6% vdues d be more sdk, and the trophic e~chment between levels wiii be less. nie benthic invertehate meywas conducted as a result of anodes in the isotope data for trophic level cornparisons of benthic invertebrates and white sucker upstream of the Mattagami River (Farwel et al. 199%). Specifically, when the 613c values of white sucker were compared with a variety of benthic invertebrates (potentid dietary items), the 613cvalues of white sucker were more I3c depleted than the benthic invertebrates. There were conms that the previous invenebrate sampling efforts restricted to the iittoral zones did not provide an adequate representation of the potentid feeding habitats of white sucker. The isotope data collecteci on burtowing mayflies for the present study were used to establish a baseline of the range of 613c and 6'k values of invertebrate detritivores inhabithg pools and backwaters, and vegetative zones where retention and sedirnentation of FPOM is high. Sections of the river with high levels of deüitus and consequently high abundance of invertebrates provide ideal habitats for benthic feeders such as white sucker. The general 6I3c trend for the invertebrate-fish cornparisons for both the Mattagimi and Groundhog Rivers was 13c depletion of white sucker relative to Hexugwiu. The 613c mean values for white sucker were 1.8~~~- 5.3.- more depleted than Hexage~~iawith the exception of the site Unmediately downstream of the mil1 on the Mattapi River (0.8.a I3c e~chment).Theoreticdy, the preferentid utiiization of 12~in the process of biological tissue synthesis wou~remît in a siight enriclmient (-1 .O *-,) of 613c from one trophic ~eve~to the next @eN'io and Epstein 1978). 13cenrichment 60m the second to the third trophic level has been descn'bed for a temperate lake (ELA 373) on the Precambh Shield in Northwestern Ontho (Hecky and Hessiein 1995). In dSs study, trophic levds were defbd for dsbased on th& G'%vaiues, where second (61,thûd (6-9xm) and fm (%) trophic levels were represented in the system. Hecky dHessleiti (1995) also observed a reduction in the range of 161 613c values firom planktonic and benthic hvertebrates (second trophic level) to foraging fish including white sucker (third trophic level) in two temperate lakes. Reduction Ui the range of values for fish is related to a SMfiom a herbivorous diet of aquatic and terrestrial plant matter with a wide 6I3c range to an omnivorous diet at higher consumer levels with a more narrow 6"~range. In the Moose River tniutaries, the "C depletion and the similar or increased 613c range of white sucker relative to Hexagenia rnay be indicative of the comibution of primaiy production to the diet of white sucker versus contributions fiom secondary production. The contribution of detriial material in white sucker diet may fiinction to compliment or maintain energetic requirements in waters of iow productivity. Nutrient e~chmentof the waters downstrearn of the miu effluent discharge represent the most enricheci and productive site on the Moose River s'stem. Here, the abundance of the benthic invertebrates is sufticiently high to support the maintenance and growth of white sucker. The hi@ commption of fint level consumers by white sucker downstrearn of the mil1 in wmparison to the combined consurnption of the primary producers and first level consumers at other sites results in the 13c e~chmentof white sucker (second trophic Ievel) relative to Hexagettia (first trophic level). 13 C e~chmemtof white sucker relative to Hmgeniu was only observed at the downstream site intluenced by pulp rnill effluent. Higher consurnption of benthic invertebrates downstrearn of the rniil may also be reflected in the grrater eruichrnent between trophic levels in contrast to upstream on the Mattagami River. The I3c depletion of Hruge11ia in deeper water, upstream of the dams on the Mattagami and Groundhog Rivers, confhns the presence of depleted 13csources available for white sucker consumption. The 13cdepletion of white wcker indicates white wcker feeding in deeper offkhore water in the reservoir. In a large temperate lake (ara 346 h2:maximum depth 47 m) in Northwestem Ontario, white sucker 6"~ranged from -24.%,, to -19.8~~ suggestng a littoral feeâing habitat in cornparison to pelagic and protiuidal fding of coregonid species (613c-29.2% to -22.1 (Hecky and Hesslein 1995). Logan et al. (1 99 1) studied the foraging pattm of white sucker in two small thermal stratifieci oligotrophic laka (maicimum depths 38 m and 32 m) and fano signiscant evidence to support the theory of 162 epilirnnetic féeding foilowed by migration to the hypolimnion based on gut content analysis of white sucker captureci in the epilimnion, metalimnion and hypolion.

5.5 Condusions The isotopic trends established for burrowing mayaies upstream and downstream of developments on the Groundhog and Mattagami Rivers provided valuable idormation used to increase the understanding of isotopic trends of white sucker 6om these regions. The isotopic composition of white sucker gonds were found to be related to the seasonal isotopic composition of potential dietaiy items. Spatial trends in the isotopic composition of benthic invertebrates were used to define fdnghabitats of white sucker upstream of the dams and the contribution of pulp mil1 efnuent as a nutritive source for white sucker downstream. Similarities in '% enrichment from first level (benthic invertebrate) to second level (benthivorous fish) consumers for sites upstream and downstream on the Groundhog and Mattagarni Rivers vecified that differences in nitrogen cychg at the prirnary productivity level resulted in the diifferences in the 6% composition of white sucker between the two rivers. Thus, the greater '% e~chmentof white sucker from the Mattagami River cm not be due to changes in the abundance and diversity of dietary items. 163 CHAPTER 6

Cenerai Discussion

6.1 Summary OF rrserrch conclusions The 613c and 6'% composition of white sucker were examined at 15 sites within the Moose River Basin with the overall objective of deteminhg the carbon and nitropen cycling of the benthic fdweb in unregulated and regulated nvers. The objective was to differentiate the influence of hydroelectric development from the combined influence of hydroelectric development and pulp mil1 activity. The ability to detect isotopic enrichment associated with efüuent discharges at sites dowmtream of hydroelectric facilities will detennine the app1icabTt.y of using 6"~values as a tracer for pulp miIl effluent exposure. Secondly, the ability to distinguish specific habitat diferences using stable isotopes in addition to establishing a relationship between site-specific isotope values and alterations in ecosystem structure or function may be useful in the selection of adequate reference sites for ecotoxicological studies. Frequency distribution plots were used to provide an ove~ewof the isotopic composition of white sucker from reference sites in unregulated (Missinaihi River) and regulated (Groundhog, Mattagarni, and Kapuskashg nvers) tribut& and impacted sites upstream and downstream OP hydroelectric development as weli as upstream and downstream sites iduenced by the combination of hydroelectric developrnent and pulp mil activity (Figure 6.1 and 6.2).

6.1.1 htopic variability and -di for dhncesites Temporal and spatial variability in the 6"~and 6% velues of white wcker were determined to define the normal fluctuation of the isotopic composition of a bemhivorous fish species in an undeveloped tributary (Chapter 2). Isotopic variability of white sucker between sites and years was minimal. The range of 613c (-3 1 -4 to -26.5 !%O) and 6% (6.3 to 9.4 960) values defined the isotopic composition of white sucker muscle from the Figure 6.1 Frequency distribution of 6"~(.) values of white sucker COU& fiom 1994 to 1997 from reference sites on the undeveloped Missinaibi River (n47) (Mattice, Skunk Island, and Thunderhouse Falls) (a), reference sites on the developed nvers (n=34) (GRWF-DN (Whist Falls), KAPWF-DN (Woman 's Falls), and MATCY-DN (Cypus Falls)) (ô), hydroelectric sites (n=68) on the Groundhog (GR-UP and GRFQ-DN) and Mattagami (MATSM-DN and MATKP-DN) Rivers (c), and sites inQuend by the combination of hydroelectric development and pulp mill activity (n=76) on the Mattagarni (MAT-UP and MAT-DN)and Kapuskasing Rivers (KAP-UP, KAP-DN, and KAPFF-DN) (d).

Figure 6.2 Frequency distribution of 6% (--) values of white sucker collected fiom 1994 to 1997 6om reference sites on the undeveloped Missinaibi River (n=47) (Mattice, Skunk Island, and Thunderhouse Falls) (a), reference sites on the developed rivers (n=34) (GRWF-DN (Whist Falls), KAPWF-DN (Woman's Falls), and MATCY-DN (Cypus Falls)) (b), hydroelectric sites (n=68) on the Groundhog (GR-üP and GRFQ-DN) and Mattagami (MATSM-DN and MATKP-DN) Rivers (c), and sites innuend by the combination of hydroelectric development and pulp mil activity (n=76) on the Mattagami (MAT-üP and MAT-DN) and Kapuskasing Rivers (KAP-W, KAP-DN, and KAPFF-DN) (d). frequency frequency frequency frequency NO a Cu O a h) O Cu W P O000 O O O O O O O O O O O O O 168 Missinaibi River. This data provided a baseline for cornparisons within and between the other sites collected during these studies. The variability of 5 %O 613c and 3 960 S"N were used as a baseline to interpret changes that were outside the range of normal variability. There was also an interest in cornparhg the stability and responsiveness of isotopes within different tissues. This was necessary for a variety of reasons, but especially because of the availability of archived liver sarnples that had been collected pnor to the construction of the hydroelectric facility on the Groundhog River. Within the Missinaibi River, the 6I3c composition of lipid-extracted white sucker muscle, liver, and gonad ivere similar.

However, the 6% composition of tissues differed by 2 %O with progressive 6''~ enrichment (-1 %O) from low, medium and high turnover rate tissues (gonad c liver < muscle). Relationships between isotopic composition of white sucker and fish measurements (age and weight) were also examined to provide a badine of isotopic trends associateci with fish growth in a stable systern. 13cenrichment of white sucker was positively correlated to increases in age and weight. 6''~values versus age showed a sirnilar correlation, but 6% values showed more variability at lower body weights. Since the Missinaibi River is relatively pristine, the isotopic composition and the density and diversity of dietary items should be relatively stable from year to year. The diet of benthic feeders does not change significantly from juvenile to adult. Therefore, the isotope trends related to age and weight were probably linked to isotopic fiactionation resulting from metabolic changes as the fish aged and grew. Other reference sites, distances (> 40 km) fiom anthropogenic development on the Groundhog (GRWF-DN), Kapuskasing (KAPWF-UP), and Mattagarni (MATCY-DN) Rivers showed a sirnilar range of 6l'~variability as the sites sampled on the Missinaibi

River. The range of 6% composition was 7.1 to 10.0 %O, reflecting again a 3 %O difference between years and sites. However, these sites were found to be more S'~C variable, showing a range of values that differed by 15 %O (range = -44.1 to -29.1 %O). The upper limit of "C enrichment of white sucker (-26 960) defined for the Missinaibi River was not exceeded at the reference sites on the regulated rivers which is particularly important in ternis of tracing I3c e~chedeffluent sources. However, the magnitude of depletion of white sucker fiom reference sites on the developed rivers exceeâed the lower 6"~limit (-3 1 960) def'ined for the Missinaibi River. The increased.fraquency of 13cdepkted white sucker (x31 96) fiom teference 169 sites on the developed riven may be a fùnction of reduced water level fluctuations and regulation of water flow irnposed by the hydroelectric facilities in addition to the natural hydrology of the river intluenced by basin morphology. The lentic habitat of the reference sites, upstream of naturai barriers including Whist Falls (Groundhog River), Woman's Falls (Kapuskasing River), and Cypnis Falls (Mattagami River) may have infîuenced the isotopic composition of white sucker by enhancing the productivity of planktonic algae, thus introducing greater quantities of "C depleted sources to the benthic food web via sedimentation. The natural barriers restrict fish movement to feeding habitats upstream, resulting in a shifl to more 6''~depleted values for white sucker at these reference sites in comparison to sites on the Missinaibi River where fish movement is less restncted. The variety of reference sites (Mattice, Skunk Island, Thunderhouse Falls, GRWF-DN (Whist Falls), KAPWF-DN (Woman's Falls), and MATCY-DN (Cypus Falls)) sampled in this study provided a reasonable representation of the range of isotope values of white sucker fiom different habitats in the mainstream. The isotopic variabiüty of white sucker, especially 6"~variability associated with lotic and lentic habitats in these medium sized rivers emphasizes the importance of the selection of reference sites and the number of reference sites necessary to adequately assess alterations in the cycling of carbon and nitrogen in developed reaches. The species specific SIA of sexually mature white sucker eliminates the isotopic variability associated with differences in life stage and species. The ability to isotopically differentiate between habitats using benthivorous fish is indicative of the sensitivity of 613c values to detect structural and functiond changes in primary productivity in larger rivers.

6.1.2 Isotopic trends associated witb hydroelectric development The impact of hydroelectric development on nutrient cycling was assessed by examining the isotopic composition of white sucker upstream and downstream of a recent hydroelectric facility on the Groundhog River and at sites located within a series of established hydroelectric facilities on the Mattagami River. The pnmq objective was to isotopically detine the idluence of hydroelectric development in order to dserentiate the impacts of hydroelectric development fiom the combined impacts of hydroelectric development and pub mil1 activity. The isotopic composition of white sucker associated with hydroelectric impacts were summarized for sites on the Groundhog (GR-UP and GRFQ-DN) and Mattagarni Rivers (MATSM-DNand MATKP-DN) (Figure 6.lc and 170 6.2~).Frequency distributions showed that the range of 6"~values was consistent with the range normally found for reference sites in the undeveloped and developed rivers. However, the frequency of occurrence of depleted 613c values (S -33~~~)of white sucker fiom the impoundment upstream of the Camiichael dam was greater. The 6% distribution showed a sirnilar trend comparable to the reference sites in the range of 6 to 9 %O.

However, the 6equency of occurrence of enriched 6'w values (MO -Q) was strongly influenced by the elevated 6'w values of white sucker at MATSM-DN and MATKP-DN which was consistent and unique to sites on the Mattagarni River. The selection of hydroelectric sites as a reference to isotopically differentiate the impacts of hydroelectric development and pulp mil1 activity was complicated by temporal and spatial factors that influence the isotopic composition of white sucker. The hydroelectric facilities upstream of pulp mil1 effluent discharges on the Kapuskasing and Mattagami Rivers were well established, therefore fish species with a life span of 15+ years were acclimated to the environmental conditions which influence the isotopic composition of fish at sites upstream and downstream of development. However, due to the recent construction of the hydroelectric facility on the Groundhog River ( 199 1 ), the isotope values of muscle in older white sucker may not be representative of the present isotopic composition of dietary items due to the lower turnover rate of muscle tissue. Thus, further understanding of the magnitude of isotopic change in the low turnover rate tissue of adult white sucker muscle due to changes in the isotopic composition of dietary items would be beneficial for differentiating between isotopic trends associated with hydroelectric development and the combined impacts of hydroelectnc development and pulp mil1 effluent discharge at downstream sites. Also, the distance of the downstream site at Fauquier on the Groundhog River is significantly greater than the sampling sites downstream of hydroelectric developments and pulp mil1 effluent discharges on the Mattagarni and Kapuskasing Rivers, and therefore must be taken into consideration when interpreting the impacts of hydroelectric development for downstream sites. White sucker were collected at Fauquier and Whist Falls sites downstream of the

Carmichael Falls facility six times during the studies. The variability was less than 5 %O for

6I3c and 2 %O for 6'%, N, to the basdine data coilected on the dérence river (hissinaibi). The values seen for 6'" in fish at Carmichael Falls were far outside of the "normal" range for these rivers. In present day conditions, 6 years &et commissioning of the Carmichad Dam in the faIl of 1991, white sucker muscle were found to be significantly 171 13 C and '%Idepleted upstrearn of the dam (GR-W) compared to downstream sites (GRFQ-DN and GRWF-DN) (Chapter 3). SIA of historical liver amples from the downstream site at Fauquier (GRFQ-DN, 1991) indicated that the recent impoundment resulted in a shift to 13cand '% depletion of white sucker upstream of the dam, based on the assumption that, pnor to impoundment, the isotopic composition of white sucker at Carnichael Falls were comparable to other sites on the Groundhog River. Since the shifi to 13c depletion of white sucker in the irnpoundment was more pronounced than the shift to '% depletion, the change in 13 C composition was dso examined in fish of different ages and younger fish were more "C depleted than older fish. Younger fish with strongly depleted "C (< -34.4 values for musde also showed a progressive depletion in the isotopic composition of annuli for pst-impoundrnent years. The applicability of the annular technique to interpret 'y change is Iimited by fish age and the level of 13cdepletion of muscle. Cornparisons of Iimited temporal data for the low turnover rate tissue of white sucker muscle from 1996 and 1997 showed no progression of I3c and 15~depletion, which is a function of the years sarnpled, the slow turnover rate of muscle, and the age structure of the sample population. In terms of higher turnover rate tissues, gonad deposition throughout the sumrner showed 613c and s'% compositions comparable to muscle (no statistical difference); at other sites the gonad showed a trend of isotopic depletion of gonad relative to muscle. The similar composition is related to the slow rate of isotopic depletion in muscle as a fùnction of age and residence time in the impoundment and the seasonal isotopic enrichment of dietary sources incorporated into gonadal biomass. Seasonal trends were observed for burrowing mayfly populations upstream of Mattagami dam where 'y and enrichment in the spring - summer shifled to depletion in the fall (Chapter 5). Since gonad was strongly influenced by seasonal isotopic fluctuations in the reservoir, the interpretative power of gonad-muscle comparisons to predict isotopic equilibrium or isotopic shifts to 13c and '% depletion due to recent impoundment based on low vernis high turnover rate tissues is minimal. Therefore, the most powerful method to dete* changes in the isotopic composition of white sucker upstream of development for dual SIA was comparisons of the pre-impoundment and post-impoundment white sucker liver samples. Upstream of the recent hydroelectnc developmt there is documentation of the 172 shifi to I3c and '%J depletion in white sucker. These changes are comparable to the relative ')cand '% depletion in white sucker observed in well established impoundments upstream of the Mattagarni (MAT-UP) and Kapuskasing (KAP-üP) hydroelectric facilities (Chapter 4). It is necessary to understand the transport of 13cand '% depleted dissolved and particulate organic matter from upstream sources and the incorporation of this depleted organic matter into components of the benthic food web. These data are critical to the understanding of multiple inputs of organic material fiom "C depleted upstream sources and 13c eniiched effluent sources dowmstream of the hydroelectric facilities and pulp miIl effluent discharges on the Mattagarni and Kapuskasing Rivers. Isotope data (fall 1994) indicated that fine particulate organic matter (FPOM) fiom Fauquier on the Groundhog River (downstrearn of Carmichael Dam) was more "C depleted than FPOM fiom Mattice on the Missinaibi River (fa11 1994). Yearly 6')~and 6''~values for white sucker muscle (1994. 1995, and 1997) downstream at Fauquier (GWQ-DN) were similar and there was no significant difference between the Fauquier site and the other downstream site at Whist Falls (GRWF-DN)or the reference sites on the Missinaibi River. This suggested that the muscle of white sucker collectecl at Fauquier located a distance of -20 km downstream of the dam was not influenced isotopically by the impoundment. Cornparisons of 6'j~values of pre-impoundment white sucker liver fiom downstream at Fauquier (GWQ-DN,1991) were similar to white sucker liver sampled 3 years &er impoundment (GRFQ-DN,1994), however 4 years post-impoundment (GRFQ-DN,1995) significant 13cdepletion was evident (Chapter 3). While this trend was statistically significant, the values were still within the range of 5 +IQ 613c seen between years at the Missinaibi sites. Gonadal tissue deposited in the summer was 13c depleted (mean difference -1a2h) relative to muscle and similar to liver while the 6'% composition of gonadal tissue was sirnilar to muscle and statistically '%J enriched compared to liver. It may be that it will take more time for changes evident in the reservoir to be reflected in the downstream fish. It should be noted that the changes in 6'" downstream of the pulp miIl and dam on the Kapuskasing River were much stronger 2 km downstream relative to sites located 10 or 12 km downstream. The S'~Cdifferences in FPOM samples suggest that changes should be eventually detectable at this site. Isotope values of white sucker from downstream of the Smoky Falls (Harmond head pond) and Kipling dams on the Mattagarni River were examiBed to provide 173 information on the isotopic composition of benthivorous fish in an established system where hydroelectric facilities have been operating for decades (Chapter 4). The isotopic composition of white sucker muscle from the Hannon head pond (MATSM-DN) was significantly more "C enriched than predicted based on the I3c depletion of white sucker from impounded sites upstream of the Carmichael (ûroundhog River), Smooth Rock Falls (Mattagami River), and Kapuskasing dams. The proximity of the four hydroelectnc dams within the Mattagami cornplex, and the low retention volume and retention time upstream of the Smoky Falls and Kipling dams may explain the lack of 13c depletion observed for white sucker at MATSM-DN and MATKP-DN and the sirnilarity of the 613c values to reference sites on the Missinaibi River. The influence of hydroelectric facilities on the isotopic composition of fish will be a function of the design of the system, the age of the facility, the retention times, the depth of the impoundment , and the distance downstream that amples are collected. The Cannichael Falls site demonstrated the potential for depletion of heavier isotopes upstream of some facilities.

6.1.3 Isotopic trends assoeiatd with hydroelectric devdqment and pulp miU activity Stable isotopes of resident white sucker were examined upstream and downstream of the hydroelectric deveiopments and pulp mills on the Mattagami and Kapuskasing Rivers to assess the impact of pulp mil1 inputs on the cycling of carbon and nitrogen in benthic food webs (Chapter 4). The 6"~composition of white sucker upstrearn of the Smooth Rock Falls (Mattagami River) and Kapuskasing impoundments were similar to the values found upstream of the Cdchael dam on the Groundhog River. Both the Mattagami and Kapuskasing Rivers were used to transport logs to the pulp mills until quite recently (1986 on the Mattagami and 1993 on the Kapuskasing). The similarity of results suggested that terrestrial sources of excess carbon (bark and wood) from histoncal log drives were not significant sources of carbon incorporated into the benthic food web. The 8% composition of white sucker upstrm of the dams (Carmichael, Smooth Rock Falls, and Kapuskasing) were significantly different from one another and from reference sites on the Missinaibi River. The hydrodectnc sites showed consistent trends of "N depletion in white sucker upstream compared to downstream. The low turnover rate of musde in the recently developed Groundhog River would explain why 174 samples collected upstream of the Carmichael dam were not as depleted as upstream of the Kapuskasing dam. However, other factors, partieulady increased phosphorus levels may be responsible for the significant "N e~chmentof white sucker upstream of the Smooth Rock Falls Dam relative to upstream of the Carmichael and Kapuskasing Dams and reference sites on the Missinaibi River. The 6% composition of white sucker from upstream and downstream sites on the Mattagami River was noticeably more enriched compared to the other riven in this study. A plot of the frequency distribution of 6% values for white sucker collected from ail sampling sites on the different rivers indicated distinct '% enrichment of white sucker on the Mattagami River independent of the presence or absence of pulp mil1 activity andor hydroelectnc development (Figure 6.3a) (Chapter 4). Similar enrichment in the '%J composition of the burrowing mayfly, Hexagenia, fiom upstream and downstream on the

Mattagarni River relative to the Groundhog River (Figure 6.3b) indicated that the 'IN enrichment of aquatic biota from the Mattagami River was a function of '% enrichment at the base of the benthic food chah (primary production) (Chapter 5). Since the '% enrichment (average 3.6 %O) from collecter-gatherer benthic invertebrates to benthivorous fish from both rivers was consistent with reporteci trophic level e~chment.the '% enrichment of white sucker fiom the Mattagami River was not related to possible site differences in the diversity and density of benthic invertebrates comprising the diet of white sucker. In theory, increased primary productivity as a function of elevated total phosphorus in the Mattagami River would result in nitrogen limitation. Therefore both isotopes of N ('%J and "N) would be readily incorporated into prirnary biomass to meet the productivity requirement for N. Lower phosphoms levels in the other rivers (phosphorus limitation) would result in lower rates of primary productivity, reducing the requirement for N. In situations where N is not limiting productivity, the lighter isotope ('+IV) would be preferentially incorporated into biomass. Therefore, pnrnary production in the Mattagarni River would be '% eruiched compared to the other rivers. '% enrichment or '% depletion of primary production within the different rivers would therefore be reflected in the 6% composition of benthic invertebrates and fish, regardless of the trophic position of the aquatic biota. The ''N e~chmentof white sucker was also evident, to a lesser degree, downstream of the Kapuskasing dam (KAP-DN) where probable sources of phosphoms Figure 6.3 6''~(%O) values of white sucker from sites upstream and downstream on the Missinaibi, Groundhog, Kapuskasing, and Mattagami Rivers ( 1 994- 1997) (a) and Hexagerria (b) from sites upstream and downstream on the Groundhog and Mattagarni Rivers (summer of 1997). au , 0 Missinaibi River 30 -- Groundhog River .II Kapuskasing River 25 -- O Mattagarni River % e 20 -- Q) 3 177 fiom treated sewage water discharge increased primaxy productivity and caused nitrogen limitation. Observations of increased periphyton and green filamentous algal growth at the end of the discharge pipe indicated that sewage water was nutrient rich and there was documentation of elevated total phosphorus at this site (Chapter 4). Northern rivers in Canada are typically classified as low productivity systems. The reduced growing season in northern regions infiuence fish performance resclting in lower growth rates, advanced age to matunty and increased longevity. If phosphonis concentrations are the limiting factor controlling primary productivity in the tributaries of the Moose River basin then increased phosphorus levels in the Mattagami River would increase pnmary and secondary productivity. Higher abundance of preferred dietary items (benthic invertebrates) with high nutritional value, in theory would promote increased growth in white sucker in the Mattagami River. In contrast, lower pnmary and secondary productivity and higher consurnption of detritus by white sucker to maintain energetic requirements would affect the growth of fish in the Missinaibi, Groundhog, and Kapuskasing rivers (exception KAP-DN). Hypotheticdly, if '?U enrichment of white sucker is indicative of increased primary productivity in the Mattagami River, then changes in the growth of white sucker should correspond to changes in the 6"~ composition of white sucker in the Mattagami River relative to the other riven. Frequency distribution plots of the 6% values of white sucker (Figure 6.3a) and the corresponding condition factor (Figure 6.4a) and body weight (Figure 6.4b) showed similar trends of increased "N enrichment, condition factor and body weight for white sucker fiom the Mattagami River relative to the Missinaibi, Groundhog, and Kapuskasing Rivers. The increase in white sucker condition in the Mattagami River is indicative of increased weight independent of length. Further research on pnmary productivity would be required to verify this hypothesis. However, if the theory is valid, there are implications regarding the selection of reference sites in other rivers for use in assessing the impacts of hydroelectnc development and pulp mil1 effluent discharges on fish growth. In conclusion, the application of SIA for an overall assessrnent of benthic food web dynamics using a benthivorous fish and invertebrate as indiators may be usehl as an ecological tool to assess the suitability of reference sites in toxicological field studies. The duence of hydroelectric developments and pulp dleffluent discharges on the isotopic composition of white sucker fiom upstream and dowwtream sites is depicted in the birn0dal6'~~distribution (Figure 6.1). "C depletion of white sucker was indicative Figure 6.4 Condition factor (K) (a) and body weight (b) of white sucker analyzed for stable isotopes. White sucker were collected fkom sites upstrearn and downstrearn on the Missinaibi, Groundhog, Kapuskasing, and Mattagarni Rivers ( 1994- 1997). E Missinaibi River Groundhog River .B Kapuskasing River

1.5 1.6 1 -7 condition factor (K)

Groundhog River M Kapuskasing River 180 of impoundment eRects (upstream) while 13cenrichment exceeding the 613c lirnit (-26 %O) observed for rivers where pulp mil1 activity was absent, suggested the incorporation of ')c enriched pulp miIl effluent in the benthic food chah However, white sucker collected downstream of effluent discharges were not consistently more 13c eenrihed relative to other sites, instead there was a range of overlap (-30 to -26 %O) for the site imediately downstream of eflluent discharge on the Mattagami River (-30.1 to -25.8 960) compared to reference sites on the undeveloped and developed rivers and sites influenced by hydroelectric development (Figure 6.1). In contrast, white sucker imediately downstream of the Kapuskasing River were consistently more 13ce~ched (6°C range of

-25.7 to -23.6 %O) compared to reference and hydroelectric sites. The distinct 13c e~chmentobserved downstream on the Kapuskasing River relative to the Mattagami River may be related to a combination of factors including multiple anthropogenic discharges (sewage and pulp mil1 effluents), mil1 effluent composition and concentration, characteristics of the hydroelectric facilities (type and size of dam, dixharge) and site-specific basin morphology related to fish mobility. The "C enrichment of white sucker downstream of the Kapuskasing mil1 relative to the 6"~ composition of effluent suggested that a) the portion of effluent readily assimilated and incorporated into tissue was 13C enriched compared to the 6I3c composition of whole effluent or b) 13c e~chedsources (presently undeterrnined) from sewage discharge influenced the 6')~composition of white sucker in addition to effluent sources. The magnitude of the difference in effluent POC (fall of 1995) between the two mills (Smooth Rock Falls, 528 mg/l; Kapuskasing, 4 mg/l) and the similarity in the 6"~composition of effluent suggested that the incorporation of effluent organic carbon into the benthic food chain, resulting in 13c e~chmentof white sucker would be more pronounced at the Smooth Rock Falls site, however this was not the case. There was evidence to suggest that 13c depleted sources from upstream of the dam were transporteci downstream and incorporated into components of the benthic food chain, specifically in white sucker (Groundhog River, Chapter 3) and Hexagenîa (Mattagami River, Chapter 5). Downstream of mil1 sites, the influence of I3cdepleted POM fiom upstrearn is a function of the concentration of POM and discharge relative to the concentration of POM and discharge of ')c enriched effluent. Hexagenia 6I3c values fiom downstream of the dam and upstrem of effluent discharge (Maitagarni River) were 13c depleted compared to Hexagenia imediately downstream of effluent discharge (1-2 km). Hexagenia 181 downstream of effluent discharge (1-2 km) were not as 13cenriched as effluent indicated the possible mixing of ')c enriched (effluent) and I3c depleted (upstream) sources. Also. in terms of fish mobility, white sucker From the Mattagami River had unrestricted movement downstream of effluent discharge whereas white sucker on the Kapuskasing River were more restricted to the area immediately downstrearn of discharge due to a higher proportion of shallow rocky swifts. Therefore, downstream of the Mattagami River there was evidence to suggest that "C depleted sources fiom upstream and ')cenriched sources hmeffluent as well as fish mobility influenced the isotopic composition of white sucker resulting in increased 613c variability compared to downstream on the Kapuskasing River. In conclusion, 6')~values of white sucker have the potential to be used to trace exposure of white sucker to pulp mil1 effluent in systems impacted by the combination of hydroelectnc development and pulp mill activity. Theoretically, endpoints used to assess the impact of pulp mill effluent on fish populations including growth (sue-at-age) and condition factor could be correlated with the degree of exposure to effluent based on the 613c composition of white sucker. Further research in this area would be beneficial to the assessrnent of pulp miIl effluent impacts of wild fish populations. Also. research focused on the relationship between muscle, liver and gonad and confounding factors such as age, weight and sex would provide a better understanding of exposure-response relationships for parameten such as GSI and LSI in systems that have recently undergone changes in effluent treatment.

6.1.4 Spatial and temporal isotopic trends Cor burrowiag mayflies Evaluation of the spatial and temporal isotopic trends of the burrowing mayfly, Hexagmia providexi valuable information in tenns of seasonal isotopic changes in the syaem which influence white sucker gonad - muscle relationships and spatial isotopic variability in micro and macrohabitats which would be isotopically integrated in mobile fish species (C hapter 5). The '% enrichment of Hexugenia in the mainstream (upstream of the Smooth Rock Falls) compared to the smaller tributaries may be a function of the factors limiting photosynthetic production. In theory, for the more shaded tributaries, light was probably the limiting factor controllhg the rate of photosynthesis. Therefore, the dissolved inorganic nitrogen pool was able to support the low rate of photosynthesis and 6% discrimination occdwhere the '% isotope was preferentially incorporated hto 182 algal biomass that resulted in the depletion of 6'% values of primary and secondary (Hexugeiria) biomass. However, with the increased light intensity in the mainstream, the factor limiting photosynthetic growth would be the concentration of dissolved inorganic nitrogen, thus no "N discrimination occurred and both 'IN and 15~were incorporated in biomass. hence the "N enrichment of primary and secondary production. Further research on the 6% values, and the concentration and composition of dissolved inorganic and organic nitrogen pools and the relationship to planktonic and benthic algae would be required to ver@ this theory. The slight '%I depletion of Hexugertia fiom the profùnda! zone relative to the littoral zone upstrearn of the dam (Mattagami River) and the ratio of profÙndaf:littoral area may explain why white sucker were '% depleted upstrearn relative to downstream. The S"C composition of Hexagenia from littoral and prolùndal zones upstrearn of the hydroelectric facilities explaineci the 13cdepletion and high 6I3cvariability of white sucker. The trend of I3c e~chmentof first level consumen (Hexage~iia) compared to second level consumen (white sucker) was consistent for al1 sites on the Groundhog and Mattagami Riven with the exception of downstream of pulp mil1 effluent discharge which demonarates the incorporation of I3c eenriched effluent sources into the benthic food web. Similarities in the trophic enrichment of "N fiom Hexagenia to white sucker for sites on the Groundhog and Mattagami Rivers indicated that factors infiuencing the 6"~enrichment of white sucker from the Mattagami River (particulady upstrearn of the dam) were related to differences in the cycling of nitrogen at the base of the food chah (primary production).

6.2 Future research areas The use of benthivorous fish tissues of different turnover rates has the potential to be a powd ecologicai tool for assessing isotopic changes related to seasonal changes and alterations associatecl with anthropogenic devdopment (changes in duent treatment) within benthic food webs. However, knowledge of stable Ntrogm isotope framonationation associated with lower tumover rate tissues of liver and gonad is limiteci. Laboratory studies of a smsll fi& spacies raid to seiaiel matunty on an isotopicaily consistent dKt foilowed by biochemical analysis to detemine the composition and isotope values of the major biochernid constituents of muscle, liver and gonad, and the relationship between the biochemical composition and whole tissue SIA wddbe bemdcial. Derenriinasions of cîi&mce ôetween sexes would also be required, particulariy in tams of malefernale dinaauxs in the isotopic composition of 183 gonadal tissues based on differences observed in the Moose River tributaries. The taboratory based biochemical constitutent composition - whole tissue isotope correlations couid be used to assess changes in wild fish where the isotopic composition of whole tissues were determined in addition to the biochemical composition and the isotope values of biochemid constituents. Selection of wiid fish within the same age and weight class would eiiminate the isotopic variability @ated witb these factors based on information of age and weight trends related to the isotopic composition of the white sucker muscle from the Missinaibi River. The benefit of SM of Ssh tissues is the &&y to assess change at a temporal and spatial scale that is relevant to ecosystern structure and bction ushg an mdpoint (fish species) that has ecologicai, social and economic value. References Acres IL. 1994. Predesign study and study plan for the first EEM cycle. Prepared for Malette Kraft Pulp and Power Company. Smooth Rock Falls. Toronto, Ontario: Aces International Ltd. Ahlgren MO. 1990a. Diet selection and the contribution of detritus to the diet of the juvenile white suc ker (Catos1om11.scommersoiii). Canadian Journal of Fisheries and Aquatic Sciences 47:4 1-48.

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