Across Latitudes: Using Molluscs as Biomonitors to Assess Spatial and Temporal Distribution of Tributyltin (TBT) in Eastern Canada and the British Virgin Islands

by Cassander Patrice Titley-O'Neal

MSc. Applied Marine Science, University of Plymouth, 2001

BSc. Marine Biology, University of North Carolina at Wilmington, 1999

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

Doctor of Philosophy

In the Graduate Academic Unit of Biology

Supervisor: Bruce A. MacDonald, Ph.D, Department of Biology, Saint John

Examining Board: Lucy Wilson, Ph.D, Department of Geology, Saint John (Chair)

Heather L. Hunt, Ph. D, Department of Biology, Saint John

James D. Kieffer, Ph.D, Department of Biology, Saint John

External Examiner: Adrienne J. Bartlett, Ph. D, Environment Canada, Centre for Inland Waters

This dissertation is accepted

Dean of Graduate Studies

THE UNIVERSITY OF NEW BRUNSWICK

January 2011

©Cassander P. Titley-O'Neal, 2011 Library and Archives Bibliotheque et Canada Archives Canada

Published Heritage Direction du Branch Patrimoine de I'edition

395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-89146-9

Our file Notre reference ISBN: 978-0-494-89146-9

NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distrbute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats.

The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission.

In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these.

While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. Canada f Press toward the mark (Phillipians 3:14) DEDICATION

This PhD thesis is dedicated to the two persons that mean the world to me. They

are the ones who gave me life, who believed in me even when I didn't believe in myself, who gave me the strength to push on during the dark days, and who have

instilled in me everything that I am today.

In memory of my Dad (Amos Jerome O'Neal a.k.a Romey):

Your untimely passing on November 27th, 2009 during the last stages of my PhD

thesis rocked me to the core. I miss our late night chats on the telephone until the wee hours of the morning talking about life, fishing or whatever was important at the time.

You always use to tell me, "Sandra... Some birds weren't meant to be in cages and you

are one of them." I reflect on that often. It is because of you I pushed until the end

even when I felt like giving up. I hope that with that bird's eye view from Heaven you

can see that your little girl has made it! You will be in my heart FOREVER. R.I.P. in

Smdm z

ii To my Mom (Barbara Hyacinth Titley):

It is because of you that I am even pursuing my PhD. You continually nagged me until I sent in the application for the scholarship even though I did not feel like applying. Thank you for your foresight. What can I say to the woman who has been and continues to be an unbelievable source of strength emotionally and psychologically? You are my strength, my strong tower, the wind beneath my wings.

During every test and trial you would remind me, "Remember who you are in Christ!"

Hopefully you will be able to rest easier and know that even though my world has turned upside down, and in spite of all the fiery darts, hurdles and speed bumps, both academically and personally, I have made it to the end of this PhD journey!

•Sano

iii ABSTRACT

Three species of molluscs (Nucella lapillus, Mytilus edulis and Strombus gigas) were used as biomonitors to evaluate tributyltin (TBT) pollution spatially and temporally in two Maritime Provinces in Atlantic Canada and the British Virgin Islands

(BVI) during the summers of 2007 to 2009. In addition, a review of the literature on the effects of organotins on female gastropods from 1962 to 2009 was undertaken in an effort to summarize how the terminology, science and theories have evolved over the last 40 plus years.

In Canada, data confirm that TBT was bioavailable based on chemical analysis of samples collected at locations within harbour sites, and TBT levels were high enough to induce imposex, the superimposition of a penis and/or vas deferens, in N. lapillus at harbour sites where they were found. A comparison of the current study with previous surveys in the same geographic areas implies that the 1989 Canadian regulations on

TBT have been effective in reducing TBT levels in sediment and imposex severity in N. lapillus in Saint John Harbour and Halifax Harbour. Conversely, during the same time period, TBT levels in sediment from Sydney Habour increased. Butyltins were also detected in sediment from reference sites where vessels < 25 m in length were docked at marinas and fishing harbours.

In the BVI, data from this study verify that TBT is bioavailable based on the incidence of imposex in female S. gigas collected from areas adjacent to boating activity e.g. shipyards, marinas and harbours, and the presence of TBT and its degradative

iv product, dibutyltin (DBT), in S. gigas, turtle grass (Thalassia testidium) and marine algae from corresponding sites.

To date, female masculinisation (FM) of gastropods has been documented in >

260 species globally. Although most studies attribute FM in gastropods to TBT, there is evidence that "pseudo-imposex" occurred prior to TBT's use as a biocide in antifouling paint in the 1960s, "natural imposex" occurs, and there are other compounds besides

TBT that can cause FM. Even after 40+ years of research, there is no consensus on how the actual mechanism of induction occurs, although five theories have been proposed.

v ACKNOWLEDGEMENTS

I would first of all like to give all honour, praise and thanks to God for without him this PhD would not have been attainable. No man is an island and the achievement of my PhD would not have been possible without financial support, colleagues, and the love and support of family and friends.

At this time I would also like to recognise all the financial stakeholders that have contributed towards my PhD for without them the wheels of research would have ceased before I even got started: the Canadian Bureau for International Education (CBIE), the

Natural Sciences and Engineering Research Council (NSERC) of Canada (Dr. Bruce

MacDonald), Canadian Research Chair in Molecular Ecotoxicology (Dr. Emilien

Pelletier), H. Lavity Stoutt Community College (HLSCC), Hon. Ralph T. O'Neal -

Premier of the Virgin Islands, Ministry of Education and Culture, University of New

Brunswick, Hon. Irene Penn-O'Neal, J. S. Little International Study Fellowship, and

Society of Environmental Toxicology and Chemistry (SETAC).

First and foremost, I must say sincerest appreciation to my supervisor, Dr. Bruce

MacDonald, who allowed me to spread my wings and fly with my project. Thanks for the confidence, encouragement and numerous life lessons taught along the way. As we would say in the Virgin Islands, "Meh son I couldn'ta ask for a better supervisor. Big up!" I would also like to recogonise my supervisory committee, Drs. Karen Kidd, Deb

MacLatchy and Kelly Munkittrick, for their invaluable insight, critiques and suggestions throughout the development and completion of my thesis. I say kudos and a special

vi thank you to Kelly for his foresight, vigilance, and assistance with the development and production of the review article.

To my French collaborators, Drs. Emilien Pelletier and Richard Saint-Louis, at the

Institut des sciences de la mer de Rimouski (ISMER) at the Universite du Quebec a

Rimouski, aka "Remoteski", merci beaucoup for the confidence, support and taking me on as a "stagiaire". Thanks to my field crew in Canada for all your assistance with collecting sediment, mussels and dogwhelks from the various sites throughout New

Brunswick and Nova Scotia: my boat captains, Dr. Bruce Hatcher and Mr. Wayne

Armstrong, and field assistants, Erin Holtz, Adrian Hamer and Matthew Hatcher. I would also like to thank the other arm of my field crew in the British Virgin Islands for their assistance with collecting samples there: Orville Phillip, Lenette Lewis, Geoff

Brooks and my stand behind me captain Reynold "Al" Frazer.

I would like to recognise Adrian Hamer for his invaluable assistance with the production of the review article, and I'm sure that by now you know more than you would ever want to know about imposex, intersex and female penes to last you a life time. Thank you Marie-Josee Abgrall, Roshini Kassie and Sima Usvyatsov for translating the French and Russian articles on imposex as these articles added to my knowledge on the masculinisation of female gastropods. I would also like to acknowledge Ms. Ellen Belyea and Ms. Marie-Eve Engelhart for their assistance with

CHN and organotin analyses.

During the last four years, I have had the opportunity of interacting with several mentors and colleagues that I would like to recognise: (1) Dr. Kate Frego, Director of

Graduate Studies at UNB Saint John, thank you for being the "mother hen" that you always are and giving me those words of wisdom and encouragement when I needed them most; (2) Dr. Jim KiefFer, thanks for the many impromptu conversations on science and life, and the final nudge to complete my thesis when my brain felt like mush; (3) Mrs. Bonnie Sudul, International Students Advisor at UNBSJ for her counsel during my time in Saint John and being a good friend; (4) Professor Jorg Oehlmann at

Goethe Universitat Frankfurt in Germany vielen Dank for taking the time to read and critique my "opus magnus" review article; and (5) my collaborators Dr. Nancy Denslow and Mr. Dan Spade (University of Florida) and Dr. Chris Martyniuk (UNBSJ) for their expertise on mircroarrays which tried and tested my nerves and sanity.

Thank you Lorolie Connor, Dorea Maduro-Corea, Jeri Faulkner, Dr. Almein

O'Neal, David O'Neal, Diana Maduro, Sandra Blaize-McCall, Juel Henley, my NC

FAM, Lindsay Jennings, Heidi Swanson, Leanne Baker and Leah Dalrymple for the long talks, good laughs and unwavering love and support. Special thank you to my extended family in Boston, James "Butchie" & Claudina Eddy for accepting me like one of your kids and making me feel right at home when I was so far from my own. Thank you Yolanda McCoy (Aunty Y.E) for the bungalow when I needed lots of room and peace and quiet during my darkest hours as your gesture was greatly appreciated.

Finally, to my aunts, uncles, cousins, and friends around the globe, who are too numerous to mention individually, your texts, emails, telephone calls, Facebook® messages, and messages via my parents kept me grounded when the days looked bleak.

Thank you ALL for your love and support over the last 4 years. Across Latitudes: Using Molluscs as Biomonitors to Assess Spatial and Temporal Distribution of Tributyltin (TBT) in Eastern Canada and the British Virgin Islands i DEDICATION ii ABSTRACT iv ACKNOWLEDGEMENTS vi List of Tables xii List of Figures xiv List of Symbols, Nomenclature or Abbreviations xvii 1 General Introduction 1 1.1 Organotins and Their Application in Antifouling Paint 1 1.2 Fate of TBT in the Marine Environment 3 1.3 Use of Molluscs as Biomonitors in Toxicology Studies 6 1.4 Thesis Objectives 7 1.5 Thesis Outline 9 1.6 References 11 2 The Relationship between Imposex and Tributyltin (TBT) Concentration in Strombus gigas from the British Virgin Islands 17 2.1 Abstract 18 2.2 Introduction 19 2.3 Materials and Methods 23 2.3.1 Sampling 23 2.3.2 Imposex Analysis 24 2.3.3 Organotin Analysis 25 2.4 Statistical Analysis 27 2.5 Results 28 2.5.1 Imposex Incidence and Severity on a Spatial Scale 28 2.5.2 Comparison of Current Study with Initial 2000 Survey 30 2.6 Discussion 31 2.7 Conclusions 37 2.8 References 39 3 Using Nucella lapillus (L.) as a Bioindicator of Tributyltin (TBT) Pollution in Eastern Canada: A Historical Perspective 49 3.1 Abstract 50 3.2 Introduction 51 3.3 Materials and Methods 53 3.4 Results 59 3.5 Discussion 62 3.6 Conclusion 65 3.7 Literature Cited 67

ix 4 Myth or Truth: Is TBT Still an Issue in Atlantic Canada More Than 20 Years after the 1989 Canadian Regulations Controlling its Use? 80 4.1 Abstract 80 4.2 Introduction 81 4.3 Study Design and Objectives 86 4.4 Materials and Methods 87 4.4.1 Sample Collection 87 4.4.2 Description of Study Sites 88 4.4.3 Butyltin and CHN Analysis 90 4.4.3.1 Butyltin Extraction - Mussel Samples 91

4.4.3.2 Butyltin Extraction - Sediment Samples 92

4.4.3.3 Sample Clean-up and Butyltin Quantification 93

4.4.3.4 Carbon, Hydrogen and Nitrogen Analysis 94 4.5 Statistical Analysis 94 4.6 Results 96 4.6.1 Current study - 2007/2008 Survey 96 4.6.1.1 Distribution of Butyltins in Sediment 96

4.6.1.2 Distribution of Butyltins in Mytilus edulis 98

4.6.2 Temporal Changes in TBT Concentration (1995 to 2007/2008) 99 4.7 Discussion 100 4.8 Conclusion 105 4.9 References 106 5 The Evolution of Masculinised Female Gastropods: What Do We Know After 40+ Years? 119 5.1 Abstract 119 5.2 Effects of Organotins on Female Gastropods 120 5.3 Literature Review from 1962 to 2009 - Global Trends 122 5.3.1 Literature Distribution 122 5.3.2 Types of Studies and Study Parameters 124 5.4 Updated Species Lists (1962 to 2009) 127 5.4.1 Recent Changes in Gastropod Taxonomy 127 5.4.2 Affected Species List (1962 - 2009) 128 5.4.3 "Novel" Unaffected Species List (1962 - 2009) 131 5.5 Global Incidence of Female Masculinisation in Gastropods 134 5.5.1 Europe (including the Mediterranean) 136 5.5.2 Asia and Oceania (including Africa) 137 5.5.3 North America (including Greenland) 138 5.5.4 Latin America 139 5.5.5 Global Incidence - Summary 140 5.6 Inducers of Female Masculinisation in Gastropods 140 5.6.1 Tin Compounds 142 5.6.2 Tin "Cocktails" 144 5.6.3 Steroids 145 5.6.4 Other Contaminants 147

x 5.6.1 Inducers of Female Masculinisation in Gastropods - Summary 151 5.7 Existing Theories on the Masculinisation of Female Gastropods 152 5.7.1 Induction by Disturbance of the Penis Morphogenetic Factor (PMF) 153 5.7.2 Induction by Aromatase Inhibition 154 5.7.3 Induction by Inhibition of Testosterone Excretion 155 5.7.4 Induction by Modulation of Endogenous Testosterone Levels 155 5.7.5 Induction by Retinoid X Receptors (RXR) 156 5.7.6 The Great Induction Debate 157 5.8 Indices Used to Measure Female Masculinisation Incidence and Severity 158 5.8.1 "Classic" Indices 159 5.8.2 Uncommon Indices 166 5.8.3 Recommended Indices 170 5.9 Removing the Veil - Fitting the Remaining Pieces of the Puzzle 171 5.10 Conclusions and Suggestions 174 5.11 References 179 6 General Conclusion 250 6.1 Summary of Thesis 252 6.2 Way Forward 260 6.3 References 264 7 Appendix 270 Curriculum Vitae

xi List of Tables

Table 2.1. Site type, boating activity, sample size (nx0tai), imposex index data [% Imposex (%I), mean female penis length (mFPL) ± SD, mean male penis length (mMPL) ± SD, relative penis length index (RPLI))] and mean imposex grade for Strombus gigas collected in the British Virgin Islands (BVI) during August 2008 and 2009 47

Table 2.2. Site type and mean butyltin tissue concentration (n = 2) for normal female, imposex female and male Strombus gigas collected from the British Virgin Islands during August 2008 and 2009. Tissue concentration reported as ng Sn g"1 dry wt 48

Table 3.1. Imposex surveys conducted on the Pacific and Atlantic coasts of Canada 1987 to 2008 77

Table 3.2. Site type, boating activity, sample size (n), mean shell length (mSL) ± standard deviation (SD), and imposex index data (mMPL ± SD, mFPL ± SD, M:F, % I, RPSI, and VDSI ± SD) for Nucella lapillus at sites in two Maritime Provinces of Canada for data collected during the summers of 2007 and 2008. Boating activity designated as: commercial harbour (CH), cruise ship terminal (CST), fishing harbour (FH), mooring (M) and shipyard (SY) 78

Table 3.3. Imposex index data (% I, mFPL ± SD, RPSI, and VDSI ± SD), boating activity, and tissue concentration in N. lapillus reported as ng Sn g"1 (dry wt.) for surveys conducted at harbour sites in Saint John Harbour (New Brunswick) and Halifax Harbour (Nova Scotia) from 1995 to 2007/2008 79

Table 4.1. Boating activity and butyltin (TBT, DBT and MBT) concentration (n = 1), reported as ng Sn g"1 (dry wt), in sediment and resident blue mussels (Mytilus edulis) at selected harbours and associated reference sites in Atlantic Canada collected in 2007/2008. Boating activity designated as: boat ramp (BR), commercial port (CP), cruise ship terminal (CST), ferry terminal (FT), fishing boats (FB), marina (MA), and shipyard (SY) 114

Table 4.2. Classification of TBT pollution at harbour and reference locations (n = 1) based on TBT concentration in sediment (n = 1) reported as ng Sn g"1 (dry wt) according to Dowson et al. (1992): < 3 = uncontaminated; 3 to 20 = lightly contaminated; 20 to 100 = moderately contaminated; 100 to 500 = highly contaminated; > 500 grossly contaminated 116

Table 4.3. Mean TBT concentration ± standard deviation reported as ng Sn g'1 (dry wt.) comparing the results of a 1995 butyltin survey by Chau et al. 1997 (a, b) with the current survey using Fisher's Exact test 118 Table 5.1. Comparison of known families affected by female masculinisation FM from 1962 to 2009. Families compiled from affected species lists by Jenner (1978), Fioroni et al. (1990; 1991), Matthiessen et al. (1999), Omae (2003), Shi et al. (2005), and new list proposed in this thesis 219

Table 5.2. List of known gastropod species affected by female masculinisation (imposex, intersex and ovo-testis) from 1962 to 2009 220

Table 5.3. List of known gastropods unaffected by female masculinisation (imposex, intersex and ovo-testis) published from 1962 to 2009. Family of sympatric species that displayed female masculinisation in parenthesis where (B) = Buccinidae, (F) = Fasciolariidae, (H) = Hydrobiidae, (M) = Muricidae, (N) = Nassariidae, (S) = Strombidae, (T) = Turbinellidae, and (V) = Volutidae. Region was validated wherever possible based on the available literature 231

Table 5.4. Comparison of orders and families affected and unaffected by female masculinisation (imposex, intersex and ovo-testis) by probable feeding type. Families with < 5 species with all members affected by FM (*); < 5 species with all unaffected by FM (¥), and < 5 species with some affected and unaffected by FM (•) 235

Table 5.5. Comparison by feeding type of species affected and unaffected by female masculinisation (imposex, intersex and ovo-testis) 238

Table 5.6. Summary of gastropod orders and families cited in the literature with signs of female masculinisation (imposex, intersex, and/or ovo-testis) based on laboratory exposure to organotins, organotin "cocktails", steroids, and other contaminants 239

Table 5.7. Types of compounds and mode of exposure that induced female masculinisation (imposex, intersex and ovo-testis) in gastropods 240

Table 5.8. Intensity of penis expression (P) in females adapted from Smith (1981b) to measure imposex intensity in the American mud snail, Ilyanassa obsoleta 244

Table 5.9. Summary of recommended indices to measure imposex and intersex in gastropods used for TBT biomonitoring field studies indicating parameters that can be measured, ecological significance and species applicability 245

Table 5.10. Table of progression on the incidence of imposex, intersex and ovo-testis in the scientific literature from 1962 to 2009 246

Table 5.11. Suggested data to be collected when using gastropods as biomonitors or bioindicators of TBT pollution 249

xiii List of Figures

Figure 2.1. Map of the British Virgin Islands situated in the northern Caribbean depicting sites sampled in the summers of 2008 and 2009 where • delineates boating activity sites and • reference sites. Site A = Nanny Cay, B = Road Harbour, C = Trellis Bay, D = Bluff Bay, E = Guana Island, and F = Anegada 44

Figure 2.2. Linear regression between mean logio TBT and (A) mean female penis length (mFPL) and (B) relative penis size index (RPLI) for female Strombus gigas collected from boating activity and reference sites in the British Virgin Islands during 2008 and 2009. Each point represents the mean logio TBT concentration in relation to mFPL and RPLI for each respective year. Error bars ± female penis length of each Strombus gigas pooled and used for butyltin analysis 45

Figure 2.3. Percent imposex (% I) (A) and mean female penis length (mFPL) ± SD (B) for Strombus gigas collected in the British Virgin Islands in 2000 (Phillip 2000) and 2008-2009 (current study). Significant differences for percent imposex (% I) and mean female penis length (mFPL) denoted with (a) and (b), respectively 46

Figure 3.1 (A-D). (A) Location of imposex sampling sites in harbours (•) in Atlantic Canada - 2007 to 2008 at (B) Saint John Harbour, New Brunswick (NB); (C) Sydney Harbour and (D) Halifax Harbour, Nova Scotia (NS) 73

Figure 3.2. Location of imposex reference sites (•) in (A) New Brunswick and (B) Nova Scotia - 2007 to 2008 74

Figure 3.3. Spatial occurrence of (A) percent imposex (% I), (B) relative penis size index (RPSI), (C) mean female penis length (mFPL) mm ± SD, and (D) vas deferens sequence index (VDSI) ± SD for harbour sites in Saint John Harbour, (New Brunswick) and Halifax Harbour (Nova Scotia) collected during 2007/2008 75

Figure 3.4. Regression analysis between logio TBT levels in female Nucella lapillus and (A) mean female penis length (mFPL) and (B) vas deferens sequence index (VDSI). Each data point represents a site of pooled affected and unaffected females. Error bars ± (A) penis length and (B) vas deferens formation of each Nucella lapillus pooled and used for butyltin analysis 76

Figure 4.1. (A - D). (A) Location of harbour (•) and associated reference (•) sites sampled between 2007 and 2008 in Atlantic Canada. Harbour sites included: (B) Saint John Harbour, New Brunswick (NB); (C) Sydney Harbour, Nova Scotia (NS); and (D) Halifax Harbour, Nova Scotia (NS) 111

xiv Figure 4.2. Logio butyltin concentration reported as ng Sn g'1 (dry wt), in sediment samples collected from harbour and associated reference sites in Atlantic Canada during 2007 and 2008. New Brunswick sites: SJH = Saint John Harbour and BB = Back Bay; Nova Scotia sites: SH = Sydney Harbour, SAB = St. Ann's Bay, HH = Halifax Harbour, and SMB = St. Margaret's Bay. Asterisks (*) and (•) are outliers 112

Figure 4.3. Butyltin concentration reported as ng Sn g"1 (dry wt), in resident blue mussels (Mytilus edulis) collected from harbour and associated reference sites in Atlantic Canada collected during 2007 and 2008. New Brunswick sites: SJH = Saint John Harbour and BB = Back Bay; Nova Scotia sites: SH = Sydney Harbour, SAB = St. Ann's Bay, HH = Halifax Harbour and SMB = St. Margaret's Bay. Asterisks (*) and (•) are outliers 113

Figure 5.1. Distribution of literature on the masculinisation of female gastropods (imposex, intersex, and ovo-testis) by publication type from 1962 to 2009 206

Figure 5.2. Top eleven scientific journals that have published literature associated with the masculinisation of female gastropods from 1962 to 2009 207

Figure 5.3. Publication by year for literature on the masculinisation of female gastropods (imposex, intersex and ovo-testis) from 1962 to 2009 208

Figure 5.4. Distribution of female gastropods affected by female masculinisation (imposex, intersex and ovo-testis) by species and family published from 1978 to 2009.209

Figure 5.5. Geographic regions of the world displaying the proportion of female masculinisation studies with and without chemical analysis on tissue samples between 1962 and 2009 210

Figure 5.6. Distribution of the top six species used as bioindicators of TBT pollution in Europe (1 = Nucella lapillus >2 = Nassarius reticulatus > 3 = Buccinum undatum > 4 = Littorina littorea > 5 = Ocenebra erinacea > 6 = Hexaplex trunculus) and the total number of studies per country that have reported and not reported tissue concentration.211

Figure 5.7. Distribution of the top four species used as bioindicators of TBT pollution in Asia and Oceania (1 = Thais clavigera >2 = 71 distinguenda >3 = T. luteostoma > 4 = T. bitubercularis) and the total number of studies per country that have reported and not reported tissue concentration. * = imposex recorded but not in the top species of that region 212

Figure 5.8. Distribution of the top five species used as biomarkers of TBT pollution in North America and Greenland (1 = Nucella lapillus > 2 = Ilyanassa obsoleta >3 = N. emarginata >4 = N. lamellose > 5 = Searlesia (Lirabuccinum dirum) dira) and the total number of studies per country that have reported and not reported tissue concentration. * = imposex recorded but not in the top species of that region 213

xv Figure 5.9. Distribution of top four species used as bioindicators of TBT pollution in Latin America (1 = Stramonita haemastoma > 2 = Adelomelon briasilina >3=5. rustica > 4 = Strombus gigas) and the total number of studies per country that have reported and not reported their tissue concentration. * = imposex recorded but not in the top species of that region 214

Figure 5.10. Proportion of indices used to measure imposex and/or intersex incidence and severity in field and laboratory studies. Average oviduct convolution (AOS), intersex index (ISI), male:female ratio (M:F), mean female penis length (mFPL), mean male penis length (mMPL), relative penis length index (RPLI), relative penis size index (RPSI) and Uncommon indices. Uncommon indices include: convolution of proximal oviduct, conversion potential females to imposex index (CPFII), coefficient of variance (CV%), female vas deferens length (VDL), imposex index, intersex incidence, mean calibre of the vas deferens (VDC), mean female prostate length (FPrL), mean female vas deferens length (VDLf), penis classification index (PCI), penis expression (P), and percent sterile (female) 215

Figure 5.11. Vas deferens sequence index (VDSI) for Nucella lapillus adapted from Gibbsetal. (1987) 216

Figure 5.12. General scheme of imposex evolution in Nucella lapillus adapted from Oehlmann et al. (1991) and by permission of Oxford University Press 217

Figure 5.13. Penis classification index (PCI) adapted from Mensink et al. (1997), and cited in Strand and Jacobsen (2002) 218

6.1. Comparison of mean female penis length (mFPL) (A and C), relative penis size index (B), and relative penis length index (D) in Nucella lapillus and Strombus gigas collected from Canada and the British Virgin Islands 2006 to 2008. Error bars ± penis length of each female pooled and used for butyltin analysis 268

6.2. Fate of TBT in Canada and the British Virgin Islands showing the three biomonitors used (.Nucella lapillus, Mytilus edulis and Strombus gigas) and the habitats from which they were collected 269

xvi List of Symbols, Nomenclature or Abbreviations

% C - Percent carbon % I - Percent imposex % sterile female - Percent sterile female 3-MC - 3-methylcholanthrene 9-cis RA - 9-cw-retionic acid AOS/OS - Average oviduct convolution APGWamide - Ala-Pro-Gly-Trp-NH2 BPA - Bisphenol A BVI - British Virgin Islands CPA - Cyproterone acetate CPFII - Conversion of potential female to imposex index CRM - Certified reference material Cu - Copper CV% - Coefficient of variance DBT - Dibutyltin DDE - 1,1-di(p-chlorophenyl)-2,2-dichloroethylene DDT - Dichlorodiphenyltrichloroethane DEHA - Adipic acid-di(2-ethylhexyl) DL - Detection limit DOT - Dioctyltin DPT - Diphenyltin DS - Dumpton syndrome E2- 17-(3-estradiol EE2- Ethinyl estradiol EROD - Ethoxyresorufin-O- deethylase FAD - Fadrozole FEN - Fenarimol FM - Female masculinisation Form - Formestane FPL - Female penis length FPrL - mean female prostate length ISI - Intersex index ISMER - Institut des Sciences de la mer de Rimouski K - Sorption coefficient M:F - Male.Female ratio MBT - Monobutyltin mFPL - Mean female penis length MgCh - Magnesium chloride mMPL - Mean male penis length MOT - monooctyltin MPT - Monophenyltin MT - Methyl testosterone Na2S04 - Sodium sulfate

xvii NaBeT4 - Sodium tetraethylborate NP - Nonylphenol OP - Octylphenol OTCs - Organotin Compounds P - Penis expression PAD - Phtalic acid butyl PAHs - Polycyclic aromatic hydrocarbons PCI - Penis classification index PE - Penis expression PI - Penis index PMF - Penis morphogenetic factor PVC - Polyvinyl chloride RPL/RPLI - Relative Penis Length Index RPSI - Relative Penis Size Index RXR - Retinoid X receptors Sn - Tin T - Testosterone TCHT - tricyclohexyltin TBT - Tributyltin TMAH - Tetramethyl ammonium hydroxide TOC - Total organic carbon TOT - Trioctyltin TPT - Triphenyltin TPrT - Tripropyltin TTBT - Tetrabutyltin TTC1 - Tin tetrachloride USVI - United States Virgin Islands VDC - Mean caliber of vas deferens VDL - Mean length of the vas deferens VDLf - Mean vas deferens length VDLI - Female vas deferens length index VDSI - Vas deferens sequence index

xviii 1

1 General Introduction

1.1 Organotins and Their Application in Antifouling Paint

Fouling by marine organisms (e.g. barnacles, sponges and hydroids) on the hull of marine vessels decreases the maximum speed of the vessel and increases fuel consumption due to an increase in turbulent flow and drag (Evans and Smith, 1975;

Evans, 1981). Additional costs related to the fouling of marine vessels include dry docking for hull maintenance and lost ship time while dry docking (Porter and

Miale, 1984). Expenses related to the fouling of organisms on the hulls of marine

vessels can be minimized by applying an effective antifouling system to deter fouling organisms. The oldest antifouling system dates prior to 2,000 BC and consisted of copper (Cu) sheathing on wooden ships (Omae, 2003). Over time, antifouling systems evolved from metal sheathing, to arsenic and sulphur tar-type coatings around 300 BC, and finally to antifouling paints by the 19th century (Omae,

2003). From about 1960 to 1970, the primary biocide used in antifouling paint was

Cu enhanced with boosters such as organomercury, organolead, organoarsenic, and organo halogens e.g. DDT (Stebbing, 1985; Omae, 2003), but by the mid 1960s it

was apparent that Cu-based antifouling paint systems were not very effective in tanker operations (Evans, 1981). Subsequently, Cu-based antifouling paints were

replaced by a more effective and longer-lasting compound, tributyltin (TBT), until

the early 1980s (Morlely et al., 2003).

TBT is a triorganotin compound containing the element tin (Sn) which belongs to Group IV of the periodic table of elements. Organotin compounds (OTCs) are

primarily used as heat stabilizers for polyvinyl chloride (PVC) polymers, industrial catalysts, and biocides (Snoeij et al., 1987). The relative toxicity of OTCs to 2 microorganisms is chemically represented by the following formulae: R^Sn X >

R.2Sn X2 > RSn X3 > R4Sn (Gadd, 2000; Omae, 2003), where R is an alkyl or aryl group and X is an anionic species e.g. halide, oxide or hydroxide. Generally, TBT, tripropyltin (TPrT) and triphenyltin (TPT) compounds are the most toxic organotins

(Gadd, 2000). TBT and TPT were the primary biocides in antifouling paints during the 1970s and early 1980s primarily because:

(1) they were very effective against a wide range of marine fouling

organisms (Evans and Smith, 1975: Omae, 2003);

(2) they did not cause galvanic corrosion problems on aluminum hulls like

paints containing other metal elements e.g. Cu (Omae, 2003);

(3) the amount of organotins required to prevent most fouling organisms is

usually 5 to 10 times less than cuprous oxide (Evans and Smith, 1975;

Omae, 2003);

(4) TBT antifouling systems lasted for 4 to 5 or more years versus 1 year

with Cu-based antifouling systems (Valkris et al., 1985); and

(5) triorganotins are colorless and this allowed for a wide range of paint

colours to be formulated (Omae, 2003).

Biocidal application of organotins was estimated at 10,000 tonnes per year, representing approximately 2,000 to 3,000 tonnes of Sn in 1970 (Evans and Smith,

1975), and by 1983, worldwide annual production of OTCs was estimated at 30,000 3

tonnes (Crompton, 1998). A significant increase in organotins production coupled

with the preferred use of TBT as a biocide in antifouling paint inevitably led to the

introduction of organotins into the marine environment (Seligman et al., 1989).

The first sign that TBT was having adverse effects on non-target organisms

came from France's Atlantic coast in the 1970s. TBT was linked as the causative

agent to shell malformations, "chambering" or "balling", and a decreasing

abundance of the commercially-cultivated Pacific oyster, Crassostrea gigas (Alzieu,

1986). Around the same time that damaging effects were observed in C. gigas, a

"penis-like" structure, dubbed imposex (Smith 1971) or pseudohermaphroditism

(Jenner, 1979), was observed in another group of molluscs, gastropods. Imposex

was first reported in gastropods from the east coast of the US during the early 1960s

(Griffith and Castagna, 1962), and by the 1970s, imposex was also reported in the

UK (Blaber, 1970), France (Poli et al., 1971), and both coasts of the US (Houston,

1971; Smith, 1971). TBT was subsequently linked as the causative agent for

imposex in the 1980s through field transplant and laboratory studies (Smith, 1981;

F6ral and Le Gall, 1982). Because of the adverse effects that TBT imposed on non-

target species, the use of TBT as a biocide in antifouling paints was partially or

totally banned in most developed countries beginning in the 1980s. France was the

first country to regulate the use of TBT in 1982 (Alzieu, 1991) and other developed

countries followed.

1.2 Fate of TBT in the Marine Environment

TBT is an amphiphillic compound that displays both metallic and organic

characteristics (St. Louis et al., 1997; Prasad and Schafran, 2006). Although there 4 are several ways in which organotins may enter the environment, TBT is principally released into marine and estuarine environments via boat repair facilities and via dissolution or erosion from antifouling paint on the hulls of boats (Seligman et al.,

1989). Globally, elevated TBT levels have been reported in water and sediment in temperate and tropical environments at locations with high boating activity in France

(Michel and Averty, 1991; Sarradin et al., 1991), the United Kingdom (Dowson et al., 1992; 1993), Portugal (Coelho et al., 2002), Sweden (Strand et al., 2003), United

States of America (Espourteille et al., 1993; Elgethun et al., 2000), Canada (Maguire et al., 1986; Chau et al., 1997 a, b), Australia (Batley et al., 1989), Fiji (Stewart and de Mora, 1992), Thailand (Kan-Atireklap et al., 1997; Harino et al., 2006), and Hong

Kong (Ko et al., 1995). Cited point sources of TBT into the marine environment include commercial and fishing harbours, marinas, moorings, shipyards or dry dock facilities, and shipping channels. Further, lower TBT levels have also been reported at areas distant from point sources (Maguire, 1987).

TBT that enters the marine environment can remain at source locations, degrade to less toxic components, or move to areas distant from point sources

(Stebbing, 1985). Once TBT enters the marine environment, it tends to adsorb to organic-rich sediments via a hydrophobic interaction, but its degradative products, dibutyltin (DBT) and monobutyltin (MBT), generally adsorb to sandy sediments by way of electrostatic interaction (Ramaswamy et al., 2004). The sorption of TBT in sediments is reversible (Unger et al., 1987; Kram et al., 1989), hence, sediments can act as both a sink and a source by simple desorption and TBT continues to exert toxicity even after TBT inputs into the water column have ceased (Stewart, 1996).

Moreover, TBT can also be re-mobilized from sediments to the water column into the aqueous phase by hydrodynamic perturbations such as storms, tidal currents, 5 dredging operations, and propeller wash (Stewart and de Mora, 1990; Stewart,

1996).

TBT can be removed from an aquatic environment by abiotic (photolysis, hydrolysis, oxidation, solvolysis) and biotic (bioaccumulation and biodegradation) processes (Stebbing, 1985). Removal of TBT by degradation proceeds by first-order kinetics (Stewart and de Mora, 1990) in a step-wise process: TBT DBT 4 MBT

(Cleary and Stebbing, 1987; Dowson et al., 1992) that is slowed by colder temperatures (Stewart and de Mora, 1990). TBT in the water column rapidly diminishes by degradation and adsorption onto particulate matter (Stewart and de

Mora, 1990), and TBT in the water column is more susceptible to biological rather than chemical or photochemical degradation (Maguire, 1987). In sediments, microbes are capable of degrading TBT (Stang et al., 1992), but degradation is much slower than in overlying waters (Stewart, 1996). A slower degradation process in sediments may be attributed to the remarkable stability of triorganotins (including

TBT) in sediment and dark places (Omae, 2003). Any remaining TBT in receiving waters can be diluted and dispersed to the open sea or bioaccumulated by marine and estuarine organisms (Stebbing, 1985); many of which are capable of degrading

TBT into DBT and MBT (Lee, 1986).

TBT exhibits a low to medium persistence in water and moderate persistence in sediment (Maguire, 1987), with an estimated half-life of 15 and 19 days in fresh water (Harino et al., 1997) and sea water (Seligman et al., 1986), respectively, versus

3 months to 2 V2 years (de Mora et al., 1995; Watanabe et al., 1995) in sediments.

The fate of TBT in the marine environment is controlled by its persistence. The persistence of TBT in an aquatic environment is a function of physical (adsorption to particulates and sediment), chemical (chemical and photochemical degradation), and 6 biological (uptake and biodegradation) removal mechanisms in addition to water flow (Maguire, 1987).

1.3 Use of Molluscs as Biomonitors in Toxicology Studies

Although many countries have passed legislation regulating the use of TBT in antifouling paints, environmental levels have shown little or no decline in sea water

(Michel and Averty, 1991) and sediments (Dowson et al., 1993; Waite et al., 1991) after years of regulated use, and numbers of imposex-affected female gastropods remain elevated adjacent to point sources (Barreiro et al., 2004; Sousa et al., 2007;

Rato et al., 2008). Consequently the International Maritime Organization (IMO) announced a worldwide ban on paints containing TBT by 2003 (Christen, 1999).

However, the recovery of environments affected by TBT may be limited or hindered by: (1) the persistence of TBT in sediments; (2) poor water exchange; (3) inconsistencies in control measures (e.g. Caribbean Sea); (4) poor shipyard practices; and (5) continued use of TBT by large marine vessels in deep-sea trade (Stewart,

1996). TBT has proven to be a persistent pollutant in the marine environment even after > 15 years of regulated use in several developed countries, and TBT has been described as quite possibly the most acutely toxic chemical to aquatic organisms that was deliberately introduced to the environment (Maguire, 1987).

Water, sediment, and resident organisms are routinely used to monitor environmental levels of pollutants. Biomonitoring may include the measurement of chemical residues in the tissues of living organisms, quantification of biological endpoints, and traditional ecological measures e.g. species abundance and diversity

(Connell, 1999). As it relates to TBT pollution, molluscs, particularly bivalves and 7 gastropods, have been routinely used as biomonitors in temperate and tropical environments. Chemical analyses of bivalve tissues and sediment have been habitually used in TBT biomonitoring studies to identify "hot spots" and assess temporal changes in TBT over time. On the other hand, for the vast majority of studies using gastropods as biomonitors of TBT, the incidence of imposex and its severity at point sources and distant from point sources are consistently used to assess TBT pollution in the absence of chemical analysis.

1.4 Thesis Objectives

For this study, three species of molluscs; two gastropods, dogwhelks (Nucella lapillus) and queen conch (Strombus gigas), and one bivalve, blue mussels (Mytilus edulis), were used as biomonitors to assess spatial and temporal occurrence of TBT in two Maritime Provinces in Canada and in the British Virgin Islands (BVI).

Biological and ecological aspects of the marine environments used in this study differed in terms of habitat (rocky intertidal, pilings and seagrass beds), feeding type

(carnivore, herbivore and filter feeder), and climate (temperate vs. tropical). Another difference between the two countries relates to TBT legislation. Canada passed legislation regulating the use of TBT in antifouling paint in 1989 (Agriculture

Canada, 1989), but there is no such legislation in the BVI, and antifouling paint containing TBT is currently available for resale (personal observation). Although there are marked differences between the two countries and the species used as biomonitors in this study, previous studies have indicated that TBT was bioavailable at point sources (e.g. harbours, marinas, shipyards), and is a suspected causative agent for imposex in N. lapillus in Canada, and S. gigas in the BVI. The objectives 8 of this study were to:

(1) evaluate the incidence and severity of imposex at several sites with and

without boating activity in the BVI using S. gigas;

(2) assess temporal changes in imposex incidence and severity by comparing

current results with previous studies in the BVI (Phillip, 2000);

(3) use N. lapillus as a biomonitor of TBT pollution in harbours and reference

sites in Atlantic Canada to assess the incidence and severity of imposex on a

spatial scale across New Brunswick and Nova Scotia;

(4) ascertain temporal changes in imposex-affected N. lapillus populations by

comparing the results of this study with previous studies (Prouse and Ellis,

1997; Covert, 1997; Delaney, 2001; Coray, 2007 ) from the same geographic

areas;

(5) determine if there is a difference in TBT concentration in sediment and

resident M. edulis in selected harbours in Atlantic Canada between sites and

site type (harbour vs. reference);

(6) compare current TBT levels in sediment and M. edulis in the Maritimes with

those from Chau et al. (1997 a, b);

(7) use results from the current study to determine the efficacy of the 1989

Canadian regulations on TBT; and 9

(8) review the available literature on the global masculinization of female

gastropods, summarise the progression of the literature from 1962 to 2009,

and outline how the terminology, literature and science have evolved over

time.

1.5 Thesis Outline

Chapter 2 focuses on the use of S. gigas as a biomonitor of TBT pollution in the BVI, a popular tourist destination that hails itself as the sailing capital of the

Caribbean. Previous studies by Phillip (2000) in the BVI and nearby Puerto Rico

(Reed, 1993) reported that S. gigas were affected by imposex, and TBT was present in affected females from point source sites (Phillip, 2000). S. gigas was the species of choice because it is the second most valuable fishery in the Caribbean (Brownell and Stevely, 1981) and is currently on Appendix II of the Convention on

International Trade in Endangered Species (CITES) list (Brautigam, 1992). Hence factors that could possibly affect its reproduction and population ecology were investigated.

In Chapter 3 and Chapter 4, I used sediment, N. lapillus and M. edulis as biomonitors in Saint John Harbour (New Brunswick), Sydney Harbour and Halifax

Harbour (Nova Scotia); these sites have previously been used in other earlier TBT biomonitoring studies. Using well established imposex indices from the literature, percent imposex (% I), mean female penis length (mFPL), relative penis size index

(RPSI), and vas deferens sequence index (VDSI), the incidence and severity of imposex in N. lapillus was assessed on a spatial and temporal scale. Sediment, N. lapillus, and M. edulis were analyzed by gas chromatography and mass spectrometry 10

(GC-MS) for the presence of TBT and its degradative products (DBT and MBT) to determine current environmental levels. Data were compared with those from previous studies at the same geographic areas to evaluate the efficacy of the 1989

Canadian regulations on TBT.

Chapter 5 is a review article that summarizes available literature on the topic of female masculinisation (FM), imposex, intersex, and ovo-testis, in gastropods from 1962 to 2009. This review proposes an updated affected species list of 269 species, presents a "novel" unaffected species list, outlines all the compounds known to induce FM in gastropods including TBT and TPT, and describes how the terminology, science, and literature has evolved over the last 40 plus years. Lastly, in

Chapter 6, I make conclusions about my findings based on data presented in this thesis, and I propose a graphical representation of the environmental fate of TBT for the habitats and organisms used in this study. 11

1.6 References

Agriculture Canada. 1989. Antifouling paints for ship hulls. Canadian Association of Pesticide Control Officials Note 89-02, Ottawa

Alzieu, C. 1986. The detrimental effects on oyster culture in France - evolution since antifouling paint regulation. In Proceedings of the Oceans '86 - Organotin Symposium Proceedings. IEEE: Washington, D. C. USA., pp. 1130 - 1134.

Alzieu, C. 1991. Environmental problems caused by TBT in France: assessment, regulations, prospects. Mar Environ Res. 32: 7-17.

Barreiro, R., Quintela, M., and Ruiz, J. M. 2004. TBT e imposex en Galiciai los sfectos de un diruptor endocrino en problaciones de gasteropodos marinos. Ecosistemas. 13: 13 - 29.

Batley, G. E., Mann, K. J., Brockbank, C. I., and Maltz, A. 1989. Tributyltin in Sydney Harbour and Georges River waters. Aust J Mar Freshwater Res. 40: 39 - 48.

Blaber, S. J. M. 1970. The occurrence of a penis-like outgrowth behind the right tentacle in spent females of Nucella lapillus (L.). Proc Malacol Soc Lon. 39: 231 -233.

Bratitigam, A. 1992. CITES: A Conservation Tool. 3rd Edition. Center for Marine Conservation, Washington, DC.

Brownell, W. N., and Stevely, J. 1981. The biology, fisheries and management of the queen conch, Strombus gigas. U. S. National Marine Fisheries Services, Mar Fish Rev. 43 (7): 1 - 12.

Chau, Y. K., Maguire, R. J., Brown, M., Yang, F., Batchelor, S. P., and Thompson, J. A. J. 1997a. Occurrence of butyltin compounds in mussels in Canada. Appl Organomet Chem. 11: 903 - 912.

Chau, Y. K., Maguire, R. J., Brown, M., Yang, F., and Batchelor, S. P. 1997b. Occurrence of organotin compounds in the Canadian aquatic environment five years after the regulation of antifouling uses of tributyltin. Water Qual Res J Can. 32 (3): 453 - 521.

Christen, K. 1999. IMO will ban the use of a popular biocide. Environ Sci Technol. 33: 11 A.

Cleary, J. J., and Stebbing, A. R. D. 1987. Organotin in the surface microlayer and subsurface waters of Southwest England. Mar Pollut Bull. 18: 238 - 246.

Coelho, M. R., Bebianno, M. J., and Langston, W. J. 2002. Organotin levels in the Ria Formosa lagoon, Portugal. Appl Organomet Chem. 16: 384 - 390. 12

Connell, D., Lam, P., Richardson, B., and Wu, R. 1999. Introduction to Ecotoxicology. Wiley-Blackwell Science, Osney Mead, Oxford, United Kingdom, 184pp.

Coray, C. 2007. Assessing the health of Halifax Harbour's intertidal ecosystem: Impacts of marine pollution on community structure, reproductive health of key predator populations (Nucella lapilius) and immunomodulation in blue mussels (Mytilus edulis & Mytilus trossulus). MSc thesis, Dalhousie University, Nova Scotia, Canada.

Covert, T.J. 1997. Masculinization of Dogwhelks {Nucella lapilius) and periwinkles (Littorina littorea) in Halifax Harbour: Monitoring of Tributyltin. BSc honours thesis, Saint Mary's University, Nova Scotia, Canada.

Crompton, T. R. 1998. Occurrence and analysis of organometallic compounds in the environment. John Wiley and Sons Ltd., Chichester, UK, 248 pp.

Delaney, J. L. 2001. The occurrence of imposex in Nucella lapilius within the Saint John Harbour. BSc honours thesis, University of New Brunswick (Saint John), New Brunswick, Canada. de Mora, S. J., Stewart, C., and Phillips, D. 1995. Sources and rate of degradation of tri(n-butyl) tin in marine sediments near Auckland, New Zealand. Mar Pollut Bull. 30: 50 - 57.

Dowson, P. H., Bubb, J. M., and Lester, J. N. 1992. Organotin distribution in sediments and waters of selected east coast estuaries in the UK. Mar Pollut Bull. 24 (10): 492 - 498.

Dowson, P. H., Bubb, J. M., and Lester, J. N. 1993. Temporal distribution of organotins in the aquatic environment: Five years after the 1987 UK retail ban on TBT based antifouling paint. Mar Pollut Bull. 26 (9): 487 - 494.

Elgethun, K., Neumann, C., and Blake, P. 2000. Butyltins in shellfish, fmfish, water and sediment from the Coos Bay estuary (Oregon, USA). Chemosphere. 41: 953 - 964.

Espourteille, F. A., Greaves, J., and Huggett, R. J. 1993. Measurements of tributyltin contamination of sediments and Crassostrea virginicia in the southern Chesapeake Bay. Environ Toxicol Chem. 12: 305 - 314.

Evans, C. J., and Smith, P. J. 1975. Organo-based antifouling systems. J Oil Col Chem Assoc. 58: 160 - 168.

Evans, L. V. 1981. Marine algae and fouling: A review, with particular reference to ship-fouling. Bot Mar. 24: 167 - 171. 13

Feral, C., and Le Gall, S. 1982. Physiologie des invertebres - Induction experimentale par un pollutant marin (le tributyletain), de I'activite neuroendocrine controlant la morphogenese du penis chez les femelles d'Ocenebra erinacea (Mollusque, Prosobranche gonochorique). C R Hebd Seances Acad Sci. D 295: 627 - 630.

Gadd, G. M. 2000. Microbial interactions with tributyltin compounds: Detoxification, accumulation, and environmental fate. Sci Total Environ. 258: 119-127.

Griffith, G. W., and Castagna, M. 1962. Sexual dimorphism in oyster drills of Chincoteagne Bay, Maryland, Virginia. Chesapeake Sci. 3: 215 - 217.

Harino, H., Fukushima, M., Kurokawa, Y., and Kawai, S. 1997. Susceptibility of bacterial populations to organotin compounds and microbial degradation of organotin compounds in environmental water. Environ Pollut. 98: 157 - 162.

Harino, H., Ohji, M., Wattayakorn, G., Arai, T., Rungsupa, S., and Miyazaki, N. 2006. Occurrence of antifouling biocides in sediment and green mussels from Thailand. Arch Environ Contam Toxicol. 51: 400 - 407.

Houston, R. S. 1971. Reproductive biology of Thais emarginata (Deshayes, 1939) and Thais canaliculata (Duclos, 1832). Veliger. 13: 348 - 357.

Jenner, M. G. 1979. Pseudohermaphroditism!: A newly recognized sexual phenomenon in Ilyanassa obsoleta and other neogastropods. PhD thesis, University of Chapel Hill, North Carolina, USA.

Kan-Atireklap, S., Tanabe, S., and Sanguansin, J. 1997. Contamination by butyltin compounds in sediments of Thailand. Mar Pollut Bull. 34: 894 - 899.

Ko, M. M., Bradley, G. C., Neller, A. H, and Broom, M. J. 1995. Tributyltin contamination of marine sediments of Hong Kong. Mar Pollut Bull. 31: 249 -253.

Kram, M. L., Stang, P. M., and Seligman, P. F. 1989. Adsorption and desorption of tributyltin in sediments of San Diego Bay and Pearl Harbour. Appl Organomet Chem, 3: 523 - 536.

Lee, R. F. 1986. Metabolism of bis(tributyltin) oxide by estuarine animals. In Proceedings of the Oceans '86 - Organotin Symposium Proceedings. IEEE: Piscataway, New York, pp. 1182 - 1188.

Maguire, R. J. 1987. Environmental aspects of tributyltin. Appl Organomet Chem. 1:475 -498.

Maguire, R. J., Tkacz, R. J., Chau, Y. K., Bengert, G. A., and Wong, P. T. S. 1986. Occurrence of organotin compounds in water and sediment in Canada. Chemosphere. 15: 253 - 274. 14

Michel, P., and Averty, B. 1991. Tributyltin analysis in seawater by GC FPD after direct aqueous-phase ethylation using sodium tetraethlyborate. Appl Organomet Chem. 5: 393 - 397.

Morley, N. J., Leung, K. M. Y., Morritt, D., and Crane, M. 2003. Toxicity of anti- fouling biocides to Parorchis acanthus (Digenea: Philophthalmidae) cercarial encystment. Dis Aquat Organ. 54: 55 - 60.

Omae, I. 2003. Organotin antifouling paints and their alternatives. Appl Organomet Chem. 17: 81 -105.

Phillip, O. S. 2000. The relationship between masculinized female conch (Strombus gigas) and tributyltin-based antifouling paints in coastal waters of the British Virgin Islands. MSc thesis - University of Plymouth Thesis, Plymouth, United Kingdom.

Poli, G., Salvat, B., and Streiff, W. 1971. Aspect particulier de la sexualite chez Ocenebra erinacea. Haliotis. 1: 29 - 30.

Porter, R., and Miale, J. B. 1984. Extended control of marine fouling: Formulation of a microencapsulated liquid organometallic biocide and vinyl rosin paint. Appl Biochem Biotechnol. 9 (5 - 6): 439 - 445.

Prasad, R., and Schafiran, G. C. 2006. Characterization of tributyltin in shipyard waters and removal through laboratory and full-scale treatment. Water Res. 40: 453 - 462.

Prouse, N. J., and Ellis, D. V. 1997. A baseline survey of dogwhelk (Nucella lapillus) imposex in Eastern Canada (1995) and interpretation in terms of tributyltin (TBT) contamination. Environ Technol. 18: 1255 - 1264.

Ramaswamy, B. R., Tao, H., and Hojo, M. 2004. Contamination and biomethylation of organotins compounds in pearl/fish culture areas in Japan. Anal Sci. 20: 45 - 53.

Rato, M., Gaspar, M.B., Takahashi, S., Yano, S., Tanabe, S., and Barroso, C. 2008. Inshore/offshore gradients of imposex and organotin contamination in Nassarius reticulatus (L.) along the Portuguese coast. Mar Pollut Bull. 56: 1323 - 1331.

Reed, S. E., 1993. Gonadal comparison of masculinized females and androgynous males to normal males and females in Strombus (Mesogastropoda: Strombidae). J Shellfish Res. 12 (1): 71 - 75.

Sarradin, P. M., Astruc, A., Desauziers, V., Pinel, R., and Astruc, M. 1991. Butyltin pollution in surface sediments of Arcachon Bay after ten years of restricted use of TBT-based paints. Environ Technol. 12: 537 - 543.

Seligman, P. F., Valkirs, A. O., and Lee, R. F. 1986. Degradation of Tributyltin in San Diego Bay, California, Waters. Environ Sci Technol. 20: 1229 - 1235. 15

Seligman, P. F., Grovhough, J. G., Valkirs, A. O., Stang, P. M., Fransham, R., Stallard, M. O., Davidson, B., and Lee, R. F. 1989. Distribution and fate of tributyltin in the United States marine environment. Appl Organomet Chem. 3: 31-47.

Smith, B. S. 1971. Sexuality in the American mud snail, Nassarius obsoletus, Say. Proc Malacol Soc Lon. 39: 377 - 378.

Smith, B. S. 1981. Male characteristics on female mud snails caused by antifouling bottom paints. J Appl Tox. 1 (1): 22-25.

Snoeij, N. J., Penninks, A. H., and Seinen, W. 1987. Biological activity of organotins compounds - An overview. Environ Res. 44:335-353.

Sousa, A., Matsudaira, C., Takahashi, S., Tanabe, S., and Barroso, C. 2007. Integrative assessment of organotin contamination in a southern European estuarine system (Ria de Aveiro, NW Portugal): Tracking temporal trends in order to evaluate the effectiveness of the EU ban. Mar Pollut Bull. 54: 1645 - 1653.

Stang, P. M., Lee, R. F., and Seligman, P. F. 1992. Evidence for rapid, nonbiological degradation of tributyltin compounds in autoclaved and heat- treated fine-grained sediments. Environ Sci Technol. 26: 1382 - 1387.

Stebbing, A. R. D. 1985. Organotins and water quality - Some lessons to be learned. Mar Pollut Bull. 16 (10): 383 - 390.

Stewart, C. 1996. The efficacy of legislation in controlling tributyltin in the marine environment. In de Mora S. J., ed., Tributyltin: Case Study of an Environmental Contaminant. Cambridge University Press, New York, NY, USA, pp 264 - 297.

Stewart, C., and de Mora, S. J. 1990. A review of the degradation of tri(n-butyltin) in the marine environment. Environ Technol. 11: 565 - 570.

Stewart, C., and de Mora, S. J. 1992. Elevated tri(«-butyl)tin concentrations in shellfish and sediments from Suva Harbour, Fiji. Appl Organomet Chem. 6: 507 -512.

St-Louis, R., Pelletier, E., and Marsot, P. 1997. A mechanistic approach to tributyltin (TBT) sorption by marine microflagellated alga {Pavlova lutheri). Appl Organomet Chem. 11: 543 - 550.

Strand, J., Jacobsen, J. A., Pedersen, B., and Granmo, A. 2003. Butyltin compounds in sediment and molluscs from the shipping strait between Denmark and Sweden. Environ Pollut. 124: 7-15. 16

Unger, M. A., Maclntyre, W. G., and Huggett, R. J. 1987. Equilibrium sorption of tributyltin chloride by Chesapeake Bay sediment. In Proceedings of the Organotin Symposium of the Oceans '87 Conference, Halifax, NS, Canada, Sept. 29 - Oct. 1, 1987, pp. 1348 - 1351.

Valkris, A. O., Seligman, P. F., Vafa, G., Stang, P. M., Homer, V., and Lieberman, S. H. 1985. Speciation of butyltins and methyltins in seawater and marine sediments by hydride derivatization and atomic absorption detection. Naval Oceans Systems Center Technical Report 1037, TR 2, 848 pp.

Waite, M. E., Waldock, M. J., Thain, J. E., Smith, D. J., and Milton, S. M. 1991. Reductions in TBT concentrations in UK estuaries following legislation in 1986 and 1987. Mar Environ Res. 32: 89 - 11.

Watanabe, N., Sakai, S., and Takatusuki, H. 1995. Release and degradation half lives of tributyltin in sediment. Chemosphere. 31 (3): 2809 - 2816. 17

2 The Relationship between Imposex and Tributyltin

(TBT) Concentration in Strombus gigas from the

British Virgin Islands

Cassander P. Titley-O'Neal1,3, Bruce A. MacDonald1*, Emilien Pelletier2,

Richard Saint-Louis2, Orville S. Phillip3

'Department of Biology, University of New Brunswick, Saint John, New Brunswick,

Canada, E2L 4L5

2Institut des sciences de la mer de Rimouski (ISMER), University du Quebec a

Rimouski, Quebec, Canada, G5L 3A1

3Natural Sciences Department, H. Lavity Stoutt Community College, Paraquita Bay,

Tortola, British Virgin Islands, VG1110

This manuscript has been submitted to the Bulletin of Marine Science for

publication and is in the format of that journal 18

2.1 Abstract

The queen conch, Strombus gigas (Linnaeus, 1758), was collected from sites with substantial boating activity and adjacent reference sites from the British Virgin

Islands (BVI) in 2008-2009 to determine the incidence and severity of imposex, and to ascertain if there were any changes in imposex over the last decade. Imposex was present at all sites with boating activity, but not reference sites, with tributyltin

(TBT) the suspected cause, a known inducer of imposex in female prosobranch gastropods. TBT and its degradative product, dibutyltin (DBT), were found in conch, turtle grass, Thalassia testidium (Banks ex Konig, 1805), and marine algae from sites with known point sources of TBT, and our data support the assumption of TBT as the causative agent. The highest incidence and severity of imposex along with the highest tissue concentrations were found at Road Harbour > Nanny Cay > Trellis

Bay. S. gigas is the second most important commercial species in the Caribbean so factors that could possibly affect reproduction and population ecology are important in managing a sustainable fishery. We recommend the use of S. gigas as a bioindicator of TBT pollution even though it violates the recommended criterion of non-disperive larvae. 19

2.2 introduction

The coastal environment of the Caribbean is comprised of mangroves, seagrass meadows and coral reefs. Even though each habitat can be viewed as a single entity, they are interconnected and the demise of one will affect the environment as a whole. Environmental managers of these habitats are faced with the daunting task of balancing development and conservation of these precious natural resources which are impacted by both natural and anthropogenic influences. Some notable anthropogenic impacts on the Caribbean coast include eutrophication, coastline development for tourism related activities such as hotels, marinas, villas, cruise ship piers, etc., and the dredging of harbours for marine traffic. Linton and Warner

(2003) suggested the use of biomarkers as a tool to give early warning signs of ecosystems under stress in the Caribbean where natural resources are severely threatened and human resources are limited. Two widely used biomarkers are imposex and intersex which are known to affect female gastropods (Matthiessen et al., 1999). Imposex and intersex have been used globally as bioindicators of organotin pollution, tributyltin (TBT) and triphenyltin (TPT), primarily from their use in antifouling paint (Matthiessen et al., 1999). In females affected by imposex, the entire female genital system is conserved, but superimposed by male organs, i.e. penis and/or vas deferens (Matthiessen et al., 1999), while in intersex females, there is a modification of the female pallial organs that eventually supplants the corresponding male formation (prostate gland) (Matthiessen et al., 1999).

The majority of published literature on imposex and intersex focuses on studies from Europe and other temperate areas (Chapter 5). When it comes to tropical environments, there is a paucity of published studies, especially in the 20

Caribbean. This trend appears to be changing as there has been a surge in imposex studies from Latin America within the last decade. These studies are especially evident from countries bordering the Atlantic Ocean, where the occurrence of imposex has been attributed to boating activity and TBT input from antifouling paint. Imposex has been recently linked to TBT in Argentina (Penchaszadeh et al.,

2001; Goldberg et al., 2004; Biggatti and Penchaszadeh, 2005; Bigatti et al., 2009),

Brazil (Caetano and Absalao, 2002; Camillo et al., 2004; Limaverde et al., 2007),

Chile (Osorio and Huaqurn, 2003; Huaqurn et al., 2004 ), Columbia (Hernandez and

Stotz, 2004), Venezuela (Miloslavich et al., 2007), the British Virgin Islands (Phillip,

2000), and the United States Virgin Islands (Strand et al., 2009).

There is a wide variety of species used in imposex studies, but Strombids are rarely used, even though the occurrence of females growing a male organ is known in members from the Pacific and Atlantic oceans. The first published record that we could find of a Strombid female growing a penis was reported in the 1980s.

Kuwamura et al. (1983) observed that female strawberry conch, Strombus luhuanus

(Linnaeus, 1758), collected from Japan between 1976 and 1977 had a small penis like projection referred to as a "clitoris". Subsequent reports would reveal that other

Strombid females from the Atlantic Ocean were also growing a penis. Reed (1993) collected queen conch, S. gigas (Linnaeus, 1758), fighting conch, S. pugilis

(Linnaeus, 1758), and milk conch S. costatus (Gmelin, 1791) from Puerto Rico and found that some female S. gigas and S. pugulis had an egg groove and verge (penis), coined "Strombus intersex", but this phenomenon did not occur in S. costatus even though 500 specimens were examined. In the neighboring British Virgin Islands,

Philip (2000) observed a similar phenomenon in female S1. gigas, dubbed

"masculinised females", collected in the summer of 2000 from sites with boating 21 activity. Recent studies showed that roughly 37% of female dog conch, S. carnarium (Schumacher, 1817), collected from Malaysia were confirmed females as evident by the presence.of an egg groove, but they also grew a penis, and they were called imposex females (Cob et al., 2008). In this paper, we refer to female

Strombus spp. with an egg groove and verge (penis) as imposex in agreement with

Cob et al. (2008), and as described by Matthiessen et al. (1999) where the female genital system is conserved, but superimposed by male organs, in this case by a pseudo-penis.

In many imposex studies, the incidence of imposex is attributed to TBT from antifouling paint since specimens are often collected adjacent to point sources (i.e. harbours, marinas, shipyards and the like), but the chemical confirmation in tissues is not performed. There are two reasons why a vast majority of imposex studies do not perform chemical analysis of tissue samples: (1) analytical methods for butyltins are expensive and time consuming (Evans et al., 1998; Ramon and Amor, 2001); and (2) even though advances in analytical chemistry have been made in quantifying low concentrations in environmental samples, imposex induction in some species is known to occur at levels close to or below the level of detection (Evans et al., 1998).

Of the four studies that reported imposex in Strombid females, only the study reported by Phillip (2000) performed chemical analysis. Butyltin analysis of imposex specimens from the areas adjacent to boating activity in the BVI recorded elevated TBT levels in affected females, and laboratory induction experiments further confirmed imposex induction in Strombus gigas by TBT (Phillip, 2000).

Further, imposex females from field sites were found to have the highest TBT levels in the gills > gonad > digestive gland > mantle > foot, and TBT levels in the gills ranged from < 3 to 110 ng TBT Sn g"1 dry wt. (Phillip, 2000). 22

Strombus gigas is a tropical gastropod in the Strombidae family whose geographic range encompasses the western Atlantic (Reed, 1993). All Strombus species are gonochoristic (i.e. separate sexes), and exhibit sexual dimorphism where males have a penis (termed "verge") and females have an egg groove (Reed, 1993).

There are five other Strombid gastropods in the Caribbean, S. costatus, the hawkwing conch, S. raninus (Gmelin, 1791), the Florida fighting conch, 5". alatus

(Gmelin, 1791), S. pugilis, and the rooster tail conch, S. gallus (Linnaeus, 1758).

Strombus gigas was the species of choice in this study because: (1) females with an egg groove and penis have been previously reported within its geographic range

(Reed, 1993; Phillip, 2000); (2) S. gigas is a commercially lucrative species in the

Caribbean that was ranked as the second most valuable fishery after the spiny lobster, Panuliris argus (Brownell and Stevely, 1981); (3) S. gigas is an endangered species that is on Appendix II of the Convention on International Trade in

Endangered Species (CITES) list (Bralitigam, 1992), hence factors that may impact its ecology are important; and (4) TBT antifouling paint is still available for sale in the BVI, so the potential for TBT pollution by antifouling paints exists. The objectives of the study are to:

(1) assess imposex incidence and severity on a spatial scale within the BVI in

areas of boating activity and surrounding reference sites by using well

established imposex indices on a spatial scale;

(2) correlate imposex incidence and severity with TBT body concentration;

(3) evaluate temporal changes in imposex occurrence by comparing current

results with a 2000 survey at the same sites; and 23

(4) discuss the implications of using S. gigas as bioindicator of TBT pollution.

2.3 Materials and Methods

2.3.1 Sampling

The British Virgin Islands (BVI) are situated at 18° 30 N and 64° 30 W, roughly 100 km miles east of Puerto Rico (Figure 2.1), and are bordered by the

Atlantic Ocean in the north and the Caribbean Sea in the south. Field collection was completed on August 19 to 21, 2008 and August 19 to 28, 2009, at sites previously surveyed by Phillip (2000) and O'Neal (2001). At each site, depth (m), associated flora and fauna, and type of boating activity were recorded. Boating activity was designated as commercial harbour, cruise ship pier, ferry terminal, marina, mooring, navigational passage, and shipyard and/or drydock facilities. Trellis Bay and

Anegada were the only two sites where data were not collected for both years. Of the six sites studied, Anegada was the single site that had no type of boating activity while the remaining sites all had some type of boating activity. Two sites were used solely as navigational passages (Bluffy Bay and Guana Island), and they were classified as reference sites because they there were no stationary boats in their vicinity for any period of time (e.g. docks, marinas and/or moorings), and there were no shipyards. The other three sites (Nanny Cay, Road Harbour and Trellis Bay) were classified as boating activity sites because they was some type of boating activity where boats would be there for an extended period of time. Qualitatively, boating intensity at boating activity sites was ranked Road Harbour > Nanny Cay >

Trellis Bay > Bluff Bay > Guana Island > Anegada based on the number of activities 24 at each site and local knowledge of the study area. A survey of maritime chandleries operating in the BVI revealed that antifouling paint containing TBT was still available for retail primarily from two manufacturers, SeaHawk® and Pettit®.

Adult Strombus gigas (n = 10 to 30) were collected from all sites using snorkel and/or SCUBA, and kept alive en route to the Marine Centre at the H. Lavity

Stoutt Community College (HLSCC) until imposex analysis the same day they were collected. At each site, five (5) turtle grass, Thalassia testidium (Banks ex K6nig,

1805) or marine algae samples were collected, placed in labelled plastic bags, and stored on ice in ice boxes en route to HLSCC. The species of marine algae collected included: Acanthophora spicifera (Lamouroux, 1813), Avrainvillea longicaulis

(Murray and Boodle, 1889), Dictyota linearis (Lamouroux, 1809) and Ceramium spp. (Roth, 1797). In 2008, T. testidium was initially collected because: (1) seagrasses are sensitive to different types of pollution (Macauley et al., 1990); (2) high tin levels were recorded at the same sites where imposex in S. gigas was found

(O'Neal, 2001); and S. gigas is a herbivore known to feed on T. testidium which could potentially act as a source for TBT (Levine et al., 1990). Butyltin analysis of

T. testidium samples in fall 2008 revealed that butyltin distribution was patchy within sites, so marine algae commonly associated with seagrass beds were collected in 2009.

2.3.2 Imposex Analysis

Imposex analyses were performed at the HLSCC Marine Centre on the same day as sample collection. For each specimen, shell length and penis length (mm) were measured using digital stainless steel calipers. Sexually mature females were 25 differentiated from males by presence of an egg groove (Reed, 1993), and if a determination could not be made on the sex of an animal, it was not used in the analysis. Verges (penises) in males and imposex females were located behind the right ocular tentacle. The degree of imposex in affected females was ranked based on a classification system modified from Andersen (2004). The classification system was comprised of four (4) stages: normal female without a penis (Grade 0); penis length < 0.5 mm, i.e. area is raised, but unable to be measured (Grade 1); penis length 0.5 mm to < 5.00 mm (Grade 2); and penis >5.0 mm (Grade 3). Imposex incidence and severity was assessed at each site using: mean male penis length

(mMPL), mean female penis length (mFPL), percentage of females in each population with imposex (% I), male:female ratio (M:F), and relative penis length index (RPLI), [(mFPL)/(mMPL) x 100] (Oehlmann et al., 1992). RPLI was used to compare imposex intensity and severity between populations (Barroso and Mora,

1998).

2.3.3 Organotin Analysis

The gills and digestive gland of two (2) conchs of each designation (male, female, and imposex female) were pooled and used for organotin analysis since

Phillip (2000) had previously shown that they had the highest butyltin concentration.

All tissues (conch, seagrass and marine algae) were freeze dried for at least 48 to 72 hrs at -80 °C in a LABCONO® Freezezone® freeze drier. Freeze dried samples were kept at -20 'C until they were analyzed for the presence of TBT, dibutyltin (DBT), and monobutyltin (MBT), at the Institut des Sciences de la mer de Rimouski

(ISMER) at the University of Quebec, in Rimouski (Canada). Determination of butyltins in tissue samples was adapted from Chau et al. (1997) with some modifications.

Roughly 0.25 g of freeze dried tissue was digested in 5 ml of tetramethyl ammonium hydroxide (TMAH, 25% H2O) in a 50 ml Teflon® tube. Tissue samples were mixed for approximately 2 mins on a vortex mixer, placed in an ultrasonic bath at 60 °C, and soniccated for 1 hr. Certified reference material (CRM) BCR CRM

462, mussel tissue (European Commission), and a procedural blank were treated in similar fashion to tissue samples. After tissue soniccation, 25 ml of sodium acetate buffered at pH 4.5 (CH3.COONH4, AnalaR®) was added to each Teflon® vial followed by 2 ml saturated NaCl solution, and 2 g NaCl. Organotins were derivatized to an ethylated neutral compound prior to extraction by addition of 600

|il of sodium tetraethylborate (NaBeT4, 2% H20). Extraction of ethylated butyltins was accomplished by two separate, but successive additions of 4 ml 1:1 hexane:toluene mixture. After centrifiigation, the organic layer was recovered and transferred to a glass round-bottom centrifuge tube.

A first clean-up step consisted in the addition of 1 g Florisil® directly to the recovered organic layer (~ 6 ml), in each round-bottom centrifuge tube, placement on a wrist shaker for roughly 2 mins, and centrifugation (3,000 rpm, 10 min, IEC

HN-SII centrifuge). The organic layer was recovered and cleaned on a silica gel micro-column with approximately 0.5 cm of anhydrous sodium sulfate (Na2S04).

The organic fraction was eluted into evaporation vials with 1:1 hexane:toluene and collected in a graduated conical tube to a final volume of 10 ml. Before volume reduction to 200 p.l under a gentle stream of N2 gas at room temperature, 100 jj.1 of tetrapropyltin was added as the internal standard. The concentrated evaporate was transferred to an amber vial with a glass liner. The organotins were analyzed by gas 27 chromatography linked to an ion trap mass spectrometer (GC-MS, Trace GC-Polaris

Q Thermo Finnigan®) equipped with a Valcobond® VB-5 GC column using helium

(He) as the carrier gas.

Butyltin identification was based on the respective mass spectrum and GC retention time. Quantification was performed with reference to the internal standard by Xcalibur®, a software program which manages the Polaris Q® Trace GC.

Recovery of butyltin species was determined with successive extractions (n = 5) of standard CRM 462. Percent recovery for DBT and TBT was 55% (± 2.94) and 72%

(± 1.63), respectively. Due to a low recovery yield, MBT was not analysed in the present study. Butyltin concentrations were not corrected for recovery and are reported as tin (ng Sn g"1 dry wt.). Quality assurance for the analytical component was verified by: (1) calibration of GC, (2) use of CRM (BCR CRM 462 - mussel tissue), every five tissue samples, and (3) use of procedural blanks (TMAH only) for every five samples. The limit of detection expressed as tin (Sn) atom, with one microliter injected, was 2 pg for MBT and 3 pg for DBT and TBT.

2.4 Statistical Analysis

All statistical analyses were performed using SPSS version 17. Where the assumptions of parametric testing were violated, the non-parametric equivalent was used (a = 0.05). Wilcoxon sign ranks test was used to assess temporal and spatial changes in established imposex indices by year and site, respectively. Linear regression was used to determine if there was a relationship between the incidence and severity of imposex (ArcSin % I, mFPL, RPSI & VDSI) with logio butyltin tissue concentration in imposex-affected and non-affected females, while Spearman 28 rho (one-tailed) was used to correlate the incidence of imposex with logio butyltin tissue concentration in all females. For statistical purposes, tissue samples where butyltins were not detected were assigned a value of 0.10, which was the limit of detection.

2.5 Results

2.5.1 Imposex Incidence and Severity on a Spatial Scale

Imposex was observed at all boating activity sites, but not at any of the reference sites (Table 2.1). Percent imposex (% I) at boating sites ranged from 28.6 to 100% for 2008-2009 with the lowest and highest incidence of imposex observed at the same site, Road Harbour. At boating activity sites, mFPL and RPLI ranged from 3.45 ± 1.38 to 20.55 ± 7.71 mm and 15.11 to 129.57, respectively (Table 2.1), and the highest incidence of each occurred at Road Harbour. In several females with imposex, the penis was often bi-lobed and split at its extremity. A comparison of imposex severity between sites for 2009, when data were collected from all 6 sites, showed a significant difference between all sites for % I (z = -3.302, N = 21, p <

0.001), mMPL (2 = -3.931, N = 21, p< 0.001), and RPLI (z = -3.302, N = 21, p <

0.001). No significant among-site differences were found for mFPL (z = -1.310, N =

21, p > 0.05) or M:F (z = -0.611, N = 21, p > 0.05). Mean imposex grade for S. gigas in this study ranged from 0 to 2.5 for all sites surveyed (Table 2.1). All reference sites had a mean score of 0. Of the boating activity sites, the highest mean imposex score (2.5) was observed at Road Harbour while the lowest mean imposex score (0.80) was at Trellis Bay. 29

Tissues of all sexes (i.e. normal females, imposex females and males) of

Strombus gigas were analysed, and butyltin concentration for each sex is reported as the mean concentration (Table 2.2). The highest TBT (22.58 ng TBT Sn g"1 dry wt.) and DBT (163.97 ng DBT Sn g"1 dry wt.) concentrations in any sex were observed in males from Road Harbour. Anegada was the only site of any type (reference or boating activity) where no butyltins were found in any sex analyzed (Table 2.2). On the other hand, butyltins were found in males from the two remaining reference sites,

Bluff Bay and Guana Island, in 2008 and 2009 and in females from Bluff Bay in

2009 although no imposex was observed. TBT tissue concentrations in imposex females from boating activity sites were quantitatively ranked Road Harbour >

Trellis Bay > Nanny Cay > with TBT tissue concentrations of 10.02, 4.34, and 0.57 ng TBT Sn g"1 dry wt., respectively. Regression analysis (Figure 2.2) was used to determine if there was a relationship between logio TBT concentration in females with imposex indices (% I, mFPL and RPLI) while Spearman rho analyses were used to determine if there was a correlation. Best fits for regression analysis were observed for mFPL (R2 = 0.83) and RPLI (R2 = 0.84) while a poorer fit was determined for ArcSin % I (R2 = 0.43), data not shown. Spearman rho statistical analysis confirmed a significant one-tailed correlation between mFPL and RPLI (rs =

0.87, N = 35, p < 0.001).

Turtle grass (n = 20) and marine algae (n = 30) collected in 2008 and 2009, respectively, were used to determine environment levels of butyltins in the BVI. Of the 20 T. testidium samples collected in 2008, TBT was the only butyltin detected in a single sample (5%) collected from Road Harbour (27.79 ng TBT Sn g"1 dry wt.).

The remaining seagrass samples were below the detection limit, suggesting a patchy to non-existent distribution of butyltins for 2008 in T. testidium. Butyltin analysis of 30 marine algae samples in 2009 yielded similar results as T. testidium in 2008. No butyltins were detected at any of the reference sites, but they were detected in four samples (13.3%) collected from boating activity sites. TBT was detected in two samples from Road Harbour (7.44 and 13.97 ng TBT Sn g"1 dry wt.), and one sample from Trellis Bay (5.70 ng TBT Sn g"1 dry wt.). No TBT was detected at Nanny Cay, but DBT, one of TBT's degradative products, was detected in 1 sample (2.36 ng

DBT Sn g"1 dry wt.).

2.5.2 Comparison of Current Study with initial 2000 Survey

The first imposex study reported in the BVI was completed in 2000 by Phillip

(2000) using the same species and sites used in this study, with the exception of

Anegada. The highest incidence of imposex (% I), and the highest recorded mFPL occurred at Road Harbour in the current study (Figure 2.3), as well as in 2000.

Recalculated RPLI values for 2000 were 9.64, 26.67, and 6.06 for Nanny Cay, Road

Harbour and Trellis Bay respectively. In 2008, RPLI values for Nanny Cay (RPLI =

66.79) and Road Harbour (RPLI = 129.57) were calculated, but no values for Trellis

Bay could be determined since no samples were collected during that time (Table

2.1). RPLI values for 2009 for boating activity sites were 81.55, 52.03 and 15.11 for

Nanny Cay, Road Harbour and Trellis Bay, correspondingly.

Between 2000 and 2009, % I at Nanny Cay decreased (Z = -2.231, N = 6, p <

0.05) over time, while at Trellis Bay % I increased (Z = -1.890, N = 5, p > 0.05), although this result was not statistically significant (Figure 2.3). At Road Harbour,

% I was significantly different over time (Z = -2.232, N = 6, p < 0.05) decreasing in

2008 then increasing in 2009 (Figure 2.3). Mean FPL (mFPL) at Nanny Cay and 31

Road Harbour significantly increased in 2008 then decreased in 2009 (Z = -2.232, N

= 6, p < 0.05), while at Trellis Bay there was a marginal, but insignificant, increase

(Z = -1.890, N = 5, p > 0.05). Phillip's (2000) and the current data suggests that sites should be ranked Road Harbour > Nanny Cay > Trellis Bay for % I and mFPL

(Figure 2.3).

2.6 Discussion

To the best of our knowledge this is the third imposex study reported from the

Caribbean. The first study was reported by Phillip (2000) using S. gigas (strombid) collected from field sites in 2000 from the BVI. The second study was published by

Strand et al. (2009) which investigated four harbour sites and seven coastal reference sites in the United States Virgin Islands (USVI) based on data collected in 2007.

The study in the USVI used four species that included: the Muricid widemouth rocksnail, Purpura patula (Linnaeus, 1758), two Deltoids, the rock snail, Thais deltoidea (Lamarck, 1822) and rustic rock snail, T. rustica (Lamarck, 1822), and one

Archaeogastropod, the West Indian topshell, Cittarium pica (Linnaeus, 1758).

Imposex was observed in each species except C. pica even though elevated concentrations of TBT (17 to 119 ng Sn g"1 dry wt.) and DBT (36 to 226 ng Sn g"1 dry wt.) were found in its tissues at sites where imposex was recorded in the other three species (Strand et al., 2009). In comparing the current study from the BVI with that of the neighbouring USVI, the highest incidence of imposex (% I), the highest reported tissue concentrations, and the highest severity of imposex (RPLI or vas deferens sequence index, VDSI) were all recorded at the main ports of entry in the

BVI (Road Harbour) as well as the USVI (Charlotte Amalie). On the other hand, imposex was recorded at six of the seven reference sites in the USVI (Strand et al.,

2009), but not in any of the reference sites in the BVI (n = 3).

In the current study, the mean imposex grade for all reference sites was 0, which is indicative of a site with normal females. However, at boating activity sites, this number ranged from 0.8 to 2.5, illustrating that there were some female S. gigas with some degree of imposex in the BVI. The prevalence of imposex at all boating activity sites, and the absence of imposex at all reference sites suggests that TBT input via antifouling paint is potentially responsible. This is supported by chemical analysis of all females (normal & imposexed) which has a relationship with mFPL and RPLI (Figure 2.2), and there is a correlation between tissue levels and mFPL and

RPLI (Spearman rho test).

In mesocosm studies, the presence of butlytins in turtle grass (Thalassia testidium), lower trophic samples, has been suggested as a potential source of TBT for herbivorous animals (Levine et al., 1990). In this study, TBT and DBT were detected in T. testidium and marine algae, but only rarely at concentrations above the detection limit. Hence, butyltin distribution in the marine flora of this study is described as very "patchy", and they are not likely to be a significant source of TBT for S. gigas. TBT-induced imposex in S. gigas in this field study is most likely due to exposure from TBT in the water column, although not quantified. This hypothesis is based on previous field results that reported that the gills of S. gigas had the highest tissue burden compared with the gonads, digestive gland, mantle, or foot

(Phillip, 2000), and butyltin distribution in marine flora was patchy. In contrast, the presence of TBT and DBT in sessile marine flora suggests that the release of butyltins into the marine environment came from suspected point sources related to boating activity. Imposex is irreversible (Gibbs et al., 1987; Oehlmann, 1998; Foale, 1993; Matthiessen et al., 1999) and the incidence of imposex in S. gigas in this study does not necessarily reflect recent or current TBT exposure.

We observed that some Strombus gigas females affected by imposex had a bi-lobed penis at their extremity and appeared deformed. Females with lobed penises were not unique to this study as bi and tri-lobed penises have been observed in other studies using S. gigas (Reed, 1993, 1994), Hexaplex trunculus (Linnaeus,

1758) (Terlizzi et al., 1999, Lahbib et al., 2008, Abidli et al., 2009), and Bolinus brandaris (Linnaeus, 1758) (Abidli et al., 2009). S. gigas is a lucrative species within its geographic range, so a better understanding of factors that could affect its reproduction and population ecology are important for managing a viable fishery.

Since imposex females have female and male sexual organs (egg groove and penis), it would be important to know if the presence of a penis affects the female's ability to reproduce; however, this was not specifically examined in this study. Reed (1993) concluded that S. gigas females with an egg groove and penis were normal reproductive females because there was no interference in mating behaviour with males, or interference with egg laying since the penis developed from the lip of the egg groove versus the interior. Additionally, all eggs that spawned from egg masses of normal and imposex females were normal in appearance, and showed no differences in hatchability between female type (Reed, 1993). Even though histological and hatchability data from other studies suggests that imposex in S. gigas does not impair its ability to successfully reproduce, it would be interesting to know if TBT affects S. gigas later on in its life cycle or at a molecular level.

The presence of butyltins in male and female 5. gigas from two of the reference sites was unexpected, but considering that those two sites are used as navigational passages, the release of TBT from passing vessels is likely. There are not enough data to conclude why TBT and DBT were present mostly in males from those two sites, but it may be possible that Strombid males are more susceptible to bioaccumulate TBT than their female counterparts as observed in the reticulated nassa, Nassarius reticulatus (Linnaeus, 1758) exposed in the laboratory as well as those collected from field sites (Stroben et al., 1992). When sites are ranked based on the level of TBT tissue concentration of all samples (conch, seagrass and marine algae), % I, mFPL, and RPLI from highest to lowest, rankings for each parameter in the current study always followed the order: Road Harbour > Trellis Bay > Nanny

Cay, which correlates with the level of decreasing boating activity.

When determining if a species should be used as a model bioindicator for TBT pollution, Gibbs and Bryan (1987) suggested the following criteria: (1) wide geographic range; (2) conspicuous and easily identified; (3) limited potential for dispersal; (4) durable egg capsules as a convenient marker of breeding activity; (5) hardy species suitable for experimentation; and (6) highly sensitive to TBT. S. gigas has a wide geographic range encompassing the western Atlantic Ocean (Reed, 1993), and it is easily identifiable from the other five Strombids in its home range. Egg capsules, which are a sign of breeding activity, are mixed with sand in a crescent shape (Davis and Shawl, 2005), and were commonly seen during the time of sample collection. Further, laboratory induction studies by Phillip (2000) have demonstrated 5. gigas' sensitivity to TBT as well as its hardiness for experimentation.

A review of the recommended criteria needed for a model bioindicator indicates that S. gigas appears to comply with all criteria with the exception of a limited potential for dispersal. This is due in part to the reproductive cycle of S. gigas which hatches from an egg mass into planktotrophic veligers drifting in the 35 water column for approximately three weeks (Davis and Shawl, 2005). Hence, S. gigas are capable of colonizing areas far beyond the origin of their egg mass. On the other hand, the dogwhelk, Nucella lapillus (Linnaeus, 1758), which is arguably the best documented species used in imposex studies, meets all the criteria as suggested by Gibbs and Bryan (1987). N. lapillus' dispersal is limited because the veliger equivalent stage as seen in S. gigas is completed within egg capsules laid on rocky shorelines, and young snails develop into miniature versions of the adult form

(Crothers, 1985).

Even though S. gigas does not meet all the recommended criteria for a model indicator of TBT pollution as suggested by Gibbs and Bryan (1987), we recommend its use as a model indicator for three reasons. Firstly, a positive relationship between tissue concentration and RPLI as well as mFPL by regression analysis has already been established in other female gastropods affected by imposex (Oehlmann et al.,

1992; Sole et al., 1998; Smith et al., 2006) and intersex (Bauer et al., 1995), and the same has been established for S. gigas in the current study. Secondly, a model species used in intersex studies already exists that violates the criterion of having a limited dispersal. The periwinkle, Littorina littorea (Linnaeus, 1758) has a pelagic larval veliger stage (Reid, 1996), and has been reported as having intersex as a consequence of TBT pollution in the United Kingdom (Matthiessen et al., 1995);

France (Fioroni et al., 1991); Germany (Oehlmann et al., 1994); and eastern Canada

(Covert, 1997). Lastly, the Oslo/Paris Convention (OSPAR) for the protection of the marine environment in the North-East Atlantic recommends the use of L. littorea as one of their three model species for TBT pollution studies (Davies et al., 1997).

The two imposex studies conducted in the British Virgin Islands used the same species, Strombus gigas, and sites, with the exception of Anegada which was surveyed in 2009 only. In addition, samples were collected in August for both surveys to avoid seasonal differences in imposex indices (Zhengyan, 2005; Chapter

5). When imposex is measured by % I or mFPL from 2000 to present (Figure 2.3), no clear pattern could be drawn for all sites from the available data. However, it appears that a generalization can be drawn on a site by site basis for each site. At

Road Harbour, % I and mFPL varied significantly between survey years, but the highest intensity of each was not observed in the same year. On the other hand, at

Trellis Bay % I and mFPL increased over time, although the mFPL increase at

Trellis Bay was not statistically significant. Additionally, % I declined significantly with each successive survey but mFPL varied significantly at Nanny Cay. RPLI can be used to measure temporal changes in imposex intensity between populations

(Barroso and Mora, 1998), hence, if RPLI values from 2000 and 2009 are compared,

RPLI values increased at all boating activity sites suggesting that imposex severity at these sites also increased over time. A review of a few imposex studies where data were collected between 2007 and 2008, revealed that the incidence of imposex (I %) at Nanny Cay and Trellis Bay was lower than that reported for Nucella lapillus in

Portugal (Galante-Oliveira et al., 2009), Nassarius nitidus (Jeffreys, 1867) in Spain

(Rodriguez et al., 2009), and Hexaplexus trunculus in the Meditterrean (Abidli et al.,

2009) from similar site types (i.e. harbours, marinas, dockyards etc.). On the other hand, maximum % I at Road Harbour was comparable with those reported for N. lapillus in Portugal (Galante-Oliveira et al., 2009) and N. nitidus in Spain (Rodriguez et al., 2009). 37

2.7 Conclusions

Imposex was observed in female queen conch, Strombus gigas, collected in the

British Virgin Islands (BVI) during the summers of 2008-2009 from areas adjacent to boating activity, but imposex was absent from adjacent non-impacted reference sites. Data suggest that TBT input from its use in antifouling paint could be the main cause; this is supported by the presence of TBT and DBT in the tissues of conch, seagrass and marine algae. In addition, a positive linear correlation between mean female penis length (mFPL) and relative penis length index (RPLI) with butyltin concentration in females gives further evidence for TBT being the causative agent of imposex in S. gigas. In the current study, the incidence and severity of imposex was the highest in Road Harbour which also has the most different types of boating activity, and this was also true of the same location 9 years earlier.

While no clear pattern for % I or mFPL exists for all sites, there were patterns for some sites. However, when imposex severity is measured by RPLI, the data suggest an increase over time. S. gigas meets all the requirements suggested for a model bioindicator species of TBT pollution except for one, a limited potential for dispersal. Even though S. gigas does not meet that one criterion, we recommend its use as an indicator of TBT pollution within its geographic range because a positive relationship between tissue concentration and imposex severity (RPLI and mFPL) has been established for S. gigas by regression analysis, and another model species,

Littorina littorea, violates this same criterion and has been recommended and utilised extensively in intersex studies within its geographic range. 38

Acknowledgements. This work was funded by NSERC (Natural Sciences and Engineering

Research Council) of Canada (B. MacDonald), the Canadian Research Chair in Molecular

Ecotoxicology (6. Pelletier), CBIE (Canadian Bureau for International Education), H. Lavity

Stoutt Community College and the Government of the Virgin Islands via the office of the

Premier, Hon. Ralph T. O'Neal. We thank R. Frazer, G. Brooks and L. Lewis for their assistance with field work. 39

2.8 References

Abidli S, Lahbib Y, El Menif NT. 2009. Imposex and genital tract malformation in Hexaplex trunculus and Bolinus brandaris collected in the Gulf of Tunis. Bull Mar Sci. 85 (1): 11 -25.

Andersen LE. 2004. Imposex: A biological effect of TBT contamination in Port Curtis, Queensland. Aus J Ecotox. 10: 105 -113.

Barroso CM, Mora MH. 1998. Reproductive cycle of Nassarius reticulatus in the Ria De Aveiro, Portugal: implications for imposex studies. J Mar Biol Assoc UK. 78: 1233 - 1246.

Bauer B, Fioroni P, Ide I, Liebe S, Oehlmann J, Stroben E, Watermann B. 1995. TBT effects on the female genital system of Littorina littorea: A possible indicator of tributyltin pollution. Hydrobiol. 309:15-27.

Biggatti G, Penchaszadeh PE. 2005. Imposex in Odontyocymbola magellancia (Caenogastropoda: Volutide) in Patagonia. Comunicaciones de la Sociedad Malacologia del Uruguay. 9 (88): 371 - 375.

Bigatti G, Primost MA, Cledon M, Averbuj A, Theobald N, Gerwinski W, Arntz W, Morriconi E, Penchaszadeh PE. 2009. Biomonitoring of TBT contamination and imposex incidence along 4700 km of Argentinean shoreline (SW Atlantic: From 38S to 54S). Mar Pollut Bull. 58: 695 - 701.

BraUtigam A. 1992. CITES; A Conservation Tool. 3rd Edition. Center for Marine Conservation, Washington, DC.

Brownell WN, Stevely J. 1981. The biology, fisheries and management of the queen conch, Strombus gigas. U. S. National Marine Fisheries Services, Marine Fisheries Review. 43 (7): 1-12.

Caetano CHS, Absal&o RS. 2002. Imposex in Olivancillaria vsica vesica (Gmelin) Gastropoda, Olividae from a southeastern Brazilian sandy beach. Rev Bras Zool. 19: 215-218.

Camillo E, Quadros J, de Castro IB, Fernandez M. 2004. Imposex in Thais rustica (Mollusca: Neogastropoda) (Lamark, 1822) as an indicator of organotin compounds pollution at Maceio coast (North-eastern Brazil). Braz J Oceanogr. 52: 101 - 105.

Chau YK, Yang F, Brown M. 1997. Evaluation of derivatization techniques for the analysis of organotin compounds in biological tissue. Anal Chim Acta. 338: 51-55.

Cob ZC, Arshad A, Idris MH, Bujang JS, GhafFar MA. 2008. Sexual polymorphism in a population of Strombus canarium Linnaeus, 1758 (Mollusca: Gastropoda) at Merambong Shoal, Malaysia. Zool Stud. 47: 318 - 325. 40

Covert TJ. 1997. Masculinization of dogwhelks (Nucella lapillus) and periwinkles (Littorina littorea) in Halifax Harbour: Monitoring of Tributyltin. BSc honours thesis, Saint Mary's University, Nova Scotia, Canada.

Crothers J H. 1985. Dog-whelks: An introduction to the biology of Nucella lapillus (L.). Fid Stud. 6: 291 - 360.

Davies IM, Minchin A, Harding A. 1997. OSPAR working group on concentrations, trends and effects of substances in the marine environment (SIME) - Report on the TBT training workshop (September 24 - 26, 1997). Aberdeen Marine Laboratory Report, No. 9/07, 36 pp.

Davis M, Shawl AL. 2005. A guide for culturing queen conch, Strombus gigas. Manual ofFish Culture. Am Fish Soc Symp. 46: 125 - 142.

Evans SM, Nicholson GJ, Browning C, Hardman E, Seligman O, Smith R. 1998. An assessment of tributyltin contamination in the North Atlantic using imposex in the dogwhelk Nucella lapillus (L.) as a biological indicator of TBT pollution. Invertebr Reprod Dev. 34 (2-3): 277 - 287.

Fioroni P, Oehlmann J, Stroben E. 1991. The pseudohermaphroditism of prosobranchs: Morphological aspects. Zool Anz. 226 (1/2): 1 - 26.

Foale S. 1993. An evaluation of the potential of gastropod imposex as a bioindicator of tributyltin pollution in Port Phillip Bay, Victoria. Mar Pollut Bull. 26: 546 - 552.

Galante-Oliveira S, Oliveira I, Jonkers N, Langston WJ, Pacheco M, Barroso CM. 2009. Imposex levels and tributyltin pollution in Ria de Aveiro (NW Portugal) between 1997 and 2007: evaluation of legislation effectiveness. J Environ Monitor. 11: 1405 - 1411.

Gibbs PE, Bryan GW. 1987. TBT paints and the demise of the dog-whelk, Nucella lapillus (Gastropoda). In: Proceedings of Oceans '87: The Ocean - An International Workplace, 28 Sept - 1 October, 1987, Halifax, Nova Scotia. Piscataway, New Jersey: IEEE Service Center, p. 1482 - 1487.

Gibbs P, Bryan G, Pascoe P, Burt G. 1987. The use of the dog-whelk, Nucella lapillus, as an indicator of tributyltin (TBT) contamination. J Mar Biol Assoc UK. 67: 507 - 523.

Goldberg RN, Averbuj A, Cled6 M, Luzzatto D, Sbarbati N. 2004. Search for triorganotins along the Mar del Plata (Argentina) marine coast: finding of tributyltin in egg capsules of a snail Adelomelon brasiliana (Lamarck, 1822) population showing imposex effects. Appl Organomet Chem. 18: 117 -123. 41

Hernandez S, Stotz WB. 2004. Reproductive biology of the "Copey" snail Melongena melongena (Linnaaeus, 1758) in Cispata Bay on the Caribbean coast of Colombia. J Shellfish Res. 23: 849 - 854.

Huaqui'n LG, Osorio C, Verdugo R, Collado G. 2004. Morphological changes in the reproductive system of females Acanthina monodon (Pallas, 1774) (Gastropoda: Muricidae) affected by imposex from the coast of central Chile. Invert Reprod Dev. 46 (2- 3): 111 -117.

Kuwamura T, Fukao R, Nishida M, Wada K, Yanagisawa Y. 1983. Reproductive biology of gastropod Strombus luhuanus (Strombidae). Publ Seto Mar Biol Lab. (5/6): 433 - 443.

Lahbib Y, Abidli S, El Menif NT. 2008. Imposex and penis malformation in Hexaplex trunculus from the Tunisian coast. Am Malacol Bull. 24 (1-2): 79 -89.

Levine SN, Rudnick DT, Kelly JR, Morton RD, Buttle LA. 1990. Pollutant dynamics as influenced by seagrass beds: Experiments with tributyltin in Thalassia microcosms. Mar Environ Res. 30: 297 - 322.

Limaverde AM, Wagener A, Fernandez MA, Scofield ADL, Coutinho R. 2007. Stramonita haemastoma as a bioindicators for organotin contamination in coastal environments. Mar Environ Res. 64: 384 - 398.

Linton DM, Warner GF. 2003. Biological indicators in the Caribbean coastal zone and their role in integrated coastal management. Ocean Coast Manage. 46: 261 -276.

Macauley JM, Clark JR, Pitts AR. 1990. Use of Thalassia and its epiphytes for toxicity assessment: Effects of a drilling fluid and tributyltin. In: Wang W, Gorsuch JW, Lower WR, editors. Plants for toxicity assessment. American Society for Testing and Materials, Philadelphia, pp. 255 - 266.

Matthiessen P, Waldock R, Thain JE, Waite ME, Scrope-Howe S. 1995. Changes in periwinkle (Littorina littorea) populations following the ban on TBT-based antifoulings on small boats in the United Kingdom. Ecotox Environ Safe. 30: 180- 194.

Matthiessen P, Reynoldson T, Billinghurst Z, Brassard DW, Cameron P, Chandler GT, Davies IM, Horiguchi T, Mount DR, Oehlmann J, Pottinger TG, Sibley PK, Thompson HM, Vethaak AD. 1999. In: deFur PL, Crane M, Ingersol CG, Tattersfield LJ, editors. Endocrine disruption in invertebrates: Endocrinology, testing and assessment. Society of Environmental Toxicology and Chemistry (SETAC), pp. 199 - 279.

Miloslavich P, Penchaszadeh PE, Bigatti G. 2007. Imposex in Neogastropods from Venezuela. CiencMar. 33:319-324. 42

Oehlmann J. 1998. Untersuchungen zum Einsatz von Pathomorphosen der ableitenden Geschlechtswege von Vorderkiemerschnecken (Gastropod a: Prosobranchia) ftir ein biologisches TBT-Effektmonitoring. Habilitation thesis, International Graduate School Zittau, Zittau, Germany.

Oehlmann J, Stroben E, Fioroni P. 1992. The rough tingle Ocenebra erinacea (Neogastropoda: Muricidae): an exhibitor of imposex in comparison to Nucella lapillus. Helgoland Mar Res. 46: 311 - 328.

Oehlmann J, Liebe S, Watermann B, Stroben E, Fioroni P, Deutsch U. 1994. New perspectives on sensitivity of littorinids to TBT pollution. Cah Biol Mar. 35: 254-255.

O'Neal CP. 2001. Assessment of environmental levels of organotins in sediments, seawater, and the seagrass, Thalassia testudinum, in coastal waters of the British Virgin Islands. MSc thesis, University of Plymouth, Plymouth, United Kingdom.

Osorio CR, Huaquin LGM. 2003. Alteration de la sexualidad deAcanthina monodon (Pallas, 1774) (Gastropoda: Muricide) en el litoral de Chile Central, inducica pr compuestos organoestafiosos. Cienc Technol Mar. 26 (2): 97 - 107. [In Spanish with English abstract].

Penchaszadeh PE, Averbuj A, Cled6n M. 2001. Imposex in gastropods from Argentina (South-western Atlantic). Mar Pollut Bull. 42 (9): 790 - 791.

Phillip OS. 2000. The relationship between masculinized female conch (Strombus gigas) and tributyltin-based antifouling paints in coastal waters of the British Virgin Islands. MSc thesis - University of Plymouth, Plymouth, United Kingdom.

Ramon M, Amor MJ. 2001. Increasing imposex in populations of Bolinus brandaris (Gastropoda: Muricidae) in the northwestern Mediterranean. Mar Environ Res. 52:463 -475.

Reed SE. 1993. Gonadal comparison of masculinized females and androgynous males to normal males and females in Strombus (Mesogastropoda: Strombidae). J Shellfish Res. 12 (1): 71 - 75.

Reed SE. 1994. Masculinized females in the genus Strombus: Aspects of their biology and possible advantages for mariculture of conch. In Appeldoorn RS, Rodriguez B, editors. Queen Conch Biology, Fisheries and Mariculture. Fundaci6n Cientffica Los Roques, Caracas (Venezuela), pp. 213 - 221.

Reid DG. 1996. Systematics and evolution of Littorina. The Ray Society, London.

Rodriguez JG, Borja A, Franco J, Alonso JIG, Garmendia JM, Muxika I, Sariego C, Valencia V. 2009. Imposex and butyltin body burden in Nassarius nitidus (Jeffreys, 1867), in coastal waters within the Basque country (northern Spain). Sci Total Environ. 407: 4333 - 4339. 43

Smith AJ, Thain JE, Bary J. 2006. Exploring the use of caged Nucella lapillus to monitor changes to TBT hotspot areas: A trial in the River Tyne estuary (UK). Mar Environ Res. 62: 149- 163.

Sole M, Morcillo Y, Porte C. 1998. Imposex in the commercial snail Bolinus brandaris in the northwestern Mediterranean. Environmental Pollution. 99: 241 -246.

Strand J, Jorgensen A, Tairova Z. 2009. TBT pollution in molluscs at US Virgin Islands, Caribbean Sea. Environ Int. 35: 707 -711.

Stroben E, Oehlmann J, Fioroni P. 1992. The morphological expression of imposex in Hinia reticulata (Gastropoda: Buccinidae): a potential indicator of tributyltin pollution. Mar Bio. 113: 625 - 636.

Terlizzi A, Geraci S, Gibbs PE. 1999. Tributyltin (TBT)-induced imposex in the neogastropod Hexaplex trunculus in Italian coastal waters: morphological aspects and ecological implications. Ital J Zool. 66: 141 - 146.

Zhengyan LI. 2005. Seasonal variation in imposex intensity of Thais clavigera. Chin J Oceanol and Limnol. 23 (2): 163 - 168. 44

•j Atlantic Ocean

Caribbean Sea Atlantic Ocean

JostVan Dyke Guana Island tm.

Virgin Goda f island

Sir Francis Drake Channel

Caribbean Sea Peter Is and

Figure 2.1. Map of the British Virgin Islands situated in the northern Caribbean depicting sites sampled in the summers of 2008 and 2009 where • delineates boating activity sites and • reference sites. Site A = Nanny Cay, B = Road Harbour, C =

Trellis Bay, D = Bluff Bay, E = Guana Island, and F = Anegada. 45

(A) 2S.O T V > 8.093x + 9.0923 Ft* * 0.8308

J=

€ + +

O.IO 0.32 l.OO 3.2 10.0 31.6 lOO.O 33.6.2 l.OOO {n togto [TBT3 Strombus gigas as ng Sn gr1 (dry wt.)

y = 53.41x + 58.521 R* * 0.8424

.3

0.10 0.32 l.OO 3.2 lO 31.6 100.0 316.2 1,000

L°g1Q [TBT] in Strombus gigas as ng Sn g~* (dry wt.)

Figure 2.2. Linear regression between mean logio TBT and (A) mean female penis length (mFPL) and (B) relative penis size index (RPLI) for female Strombus gigas collected from boating activity and reference sites in the British Virgin Islands during 2008 and 2009. Each point represents the mean logi0 TBT concentration in relation to mFPL and

RPLI for each respective year. Error bars ± female penis length of each

Strombus gigas pooled and used for butyltin analysis. 46

• % Imposex - 2000 • % Imposex - 2008 * % Imposex - 2009

Nanny Cay Road Harbour Trellis Bay

«r FPL - 2009

Nanny Cay Road Harbour Trellis Bay

Figure 2.3. Percent imposex (% I) (A) and mean female penis length

(mFPL) ± SD (B) for Strombus gigas collected in the British Virgin

Islands in 2000 (Phillip 2000) and 2008-2009 (current study).

Significant differences for percent imposex (% I) and mean female penis length (mFPL) denoted with (a) and (b), respectively. Table 2.1. Site type, boating activity, sample size (niotai), imposex index data [% Imposex (%I), mean female penis length (mFPL) ± SD, mean male penis length (mMPL) ± SD, relative penis length index (RPLI))] and mean imposex grade for Strombus gigas collected in the British

Virgin Islands (BVI) during August 2008 and 2009. 111 2008 n.s n.s n.s n.s n/a n/a n/a n/a n/a Anegada 2009 28 15 13 0.87 0 0 5.42 ±8.26 0 0 2008 10 4 6 1.50 0 0 1.97 ±4.25 0 0 Reference Bluff Bay NP 2009 25 17 8 0.47 0 0 0.73 ±0.35 0 0 2008 24 15 9 0.60 0 0 13.75 ± 15.14 0 0 Guana Island NP 2009 26 16 10 0.63 0 0 5.36 ± 13.99 0 0 2008 13 6 7 1.17 50.0 12.59 ± 13.55 18.85 ±17.55 66.79 1.2 Nanny Cay MA, SH 2009 30 13 17 1.31 46.2 7.07 ± 6.49 8.67 ±5.83 81.55 1.4 CH, CSP, 2008 17 7 10 1.43 28.6 20.55 ± 7.71 15.86 ±7.61 129.57 0.9 Road Harbour FT, MA, Harbour 2009 24 4 20 5.00 100 9.75 ± 8.90 18.74 ± 12.36 52.03 2.5 MO, SH 2008 n.s n.s n.s n.s n/a n/a n/a n/a n/a Trellis Bay MO 2009 18 8 10 1.25 50.0 3.45 ± 1.38 22.82 ± 20.64 15.11 0.8 n.s = no sample; n/a = not applicable Boating activity recorded at sites: commercial harbour (CH); cruise ship pier (CSP); ferry terminal (FT); marina (MA); moorings (MO); navigational passage (NP); and Shipyard (SH). Table 2.2. Site type and mean butyltin tissue concentration (n = 2) for normal female, imposex female and male Strombus gigas collected from the British Virgin Islands during August 2008 and 2009. Tissue concentration reported as ng Sn g"1 dry wt.

Female n/a n/a

.£• oo 49

3 Using Nucella lapillus (L.) as a Bioindicator of

Tributyltin (TBT) Pollution in Eastern Canada: A

Historical Perspective

C. P. Titley-O'Neal1, B. A. MacDonald1*, E. Pelletier2, R. Saint-Louis2

'Department of Biology, University of New Brunswick, Saint John, New Brunswick,

Canada, E2L 4L5

2Institut des sciences de la mer de Rimouski (ISMER), University du Quebec a

Rimouski, Rimouski, Quebec, Canada, G5L 3A1

This manuscript has been accepted by the Water Quality Research Journal of Canada and is in the format of that journal 50

3.1 Abstract

Dogwhelks {Nucella lapillus), a universal bioindicator of tributyltin (TBT) pollution, were used to determine butyltin distribution in three Atlantic Canada

harbours previously surveyed between 1995 and 2006. N. lapillus were analyzed

for the presence of TBT and its degradative products while imposex incidence and severity were compared with previous surveys to assess the efficacy of the Canadian regulations on TBT. Imposex was observed at two harbours that had dogwhelks, but

not at surrounding reference sites. When comparing results with previous surveys in the same geographic area, there appears to be some improvement of affected N. lapillus populations, suggesting that the 1989 Canadian regulations have been effective in decreasing imposex severity for most sites as measured by the vas deferens sequence index (VDSI), but not the occurrence of imposex. The highest

butyltin tissue concentration (63.75 ng Sn g"1, dry wt.) was detected in imposex- affected females from Red Head in Saint John Harbour (New Brunswick), which is adjacent to an area frequented by large oil tankers which under the 1989 regulations are legally allowed to use TBT antifouling paint. This study is the first to illustrate a significant relationship between TBT levels and imposex on a spatial scale in

Atlantic Canada. 51

3.2 Introduction

Between the 1960's and 1970's, the primary biocide in antifouling paint, cuprous oxide (CU2O), was replaced by a more effective one, tributyltin (TBT)

(Hugget et al. 1992). However, it was soon observed that TBT negatively affected non-target organisms such as bivalves and gastropods. The first evidence that TBT was having an adverse effect on non-target organisms came from France's Atlantic coast in the late 1970s where TBT was linked as the causative agent for

"chambering" and decreasing abundance in the commercially cultivated Pacific oyster, Crassostrea gigas (Alzieu 1986). Around the same time, another biological impact was occurring across geographic boundaries in female gastropod molluscs.

Female Dogwhelks (Nucella lapillus) in the United Kingdom (Blaber 1970), sting winkles (Ocenebra erinacea) in France (Poli et al. 1971), emarginate dogwinkles

(Nucella emarginata) and channel dogwinkles (Nucella canalicutata) in California

(Houston, 1971), and the eastern mudsnail (Nassarius obsoletus) in Connecticut

(Smith 1971) were observed growing a "penis-like" structure. This phenomenon where gonochoristic female gastropods were growing a penis has been coined

"imposex" (Smith 1971) or pseudohermaphroditism (Jenner 1979; Fioroni et al.

1990). Laboratory exposure experiments and field transplant studies later confirmed

TBT as the causative agent (Smith 1981; F6ral & Le Gall 1982).

Due to the deleterious effects of TBT on non-target organisms, especially the commercially lucrative C. gigas, several countries regulated the use of organotin in antifouling paint, and have used bivalves and gastropods as biomarkers and bioindicators of TBT pollution. France was the first country to regulate the use of

TBT in 1982 (Alzieu 1991), and other countries followed suit. Canada passed 52 regulations in 1989 to control the use of TBT by banning its use on vessels < 25 m in length (Agriculture Canada 1989). Imposex and intersex, another form of feminine masculinisation, have been used successfully in prosobranch female gastropods as bioindicators of TBT pollution globally, in places such as Europe (Gibbs et al. 1987;

Gibbs & Bryan 1994; Oehlmann et al. 1996), New Zealand (Stewart et al. 1992),

USA (Curtis 1994), Canada (Tester et al. 1996), Thailand (Swennen et al. 1996),

China (Zhu et al. 2005), and Brazil (Camillo et al. 2004).

The use of imposex and intersex as biomonitors of TBT pollution in Canada spans almost 20 years, with the first studies dating back to 1987 on the Pacific coast

(Bright & Ellis 1990) and 1995 on the Atlantic coast (Prouse & Ellis 1997). There has been a total of thirteen gastropod species used in TBT biomonitoring surveys in

Canada, ten on the Pacific coast, and three on the Atlantic coast (Table 3.1). The only species studied in Canada which did not exhibit any signs of imposex was the wrinkled amphissa, Amphissa Columbiana (Bright & Ellis 1990).

This study was designed to assess the incidence and severity of imposex in dogwhelks (Nucella lapillus) in three harbours in the Maritime provinces of Canada and surrounding reference areas. The three harbours were previously surveyed for imposex between 1995 and 2006 at suspected point sources of TBT (e.g. harbours, shipyards, moorings etc.). The objectives of this study were to:

(1) assess the incidence and severity of imposex in N. lapillus in individual

harbours and surrounding reference sites using well established imposex

indices on a spatial scale;

(2) correlate the incidence of imposex in N. lapillus with butyltin body burden; 53

(3) ascertain temporal changes in imposex by comparing these results with those

of previous studies; and

(4) determine if the 1989 Canadian regulations have been effective in reducing

and eliminating imposex at harbour sites.

3.3 Materials and Methods

Dogwhelks, Nucella lapillus, are a common littoral neogastropod found on both shores of the Atlantic Ocean (Hughes 1972), and is gonochoristic (Fretter

1953), having a 1:1 sex ratio in a normal population (Feare 1970). In Europe, N. lapillus has been the preferred bioindicator species used in imposex studies to assess

TBT pollution because: (1) imposex can be induced at very low TBT levels, less than can be detected by chemical means; (2) imposex is specific to TBT in N. lapillus since no other agent has been demonstrated to produce a full response in N. lapillus-, (3) the imposex response is visible and observers can be easily trained to score stages; and (4) no sophisticated equipment is necessary to evaluate the response so surveys are relatively inexpensive (Gibbs 1999). There are three factors that one must consider when interpreting data from imposex studies using Nucella lapillus: (1) a life span of five to six years; (2) the morphological expression of imposex is not reversible; and (3) imposex in adults may reflect effects of exposure of the juveniles (Matthiessen et al. 1998). Even though most of the imposex studies in Canada have been conducted on the Pacific coast, species of Nucella are most often used as bioindicators on either coast (Table 2.2.1). 54

Field collection and study area. Imposex surveys using N. lapillus were carried out in three harbours in Atlantic Canada (Figure 3.1, A - D) during 2007 and

2008. The selected harbours were used as historical data indicated the occurrence of imposex at each harbour initially in 1995. Although three harbours were surveyed, dogwhelks were found only at Saint John Harbour in New Brunswick and Halifax

Harbour in Nova Scotia (Figure 3.1 B and D). No dogwhelks were found at Sydney

Harbour in Nova Scotia (Figure 3.1 C). At each site, egg capsules and juveniles were recorded qualitatively as present or absent since previous studies have noted that severely affected populations are characterized by the absence of egg capsules and juveniles (Bryan et al. 1987a). Dogwhelks (n = 17 to 40) collected from each location were stored in appropriately labelled plastic bags, placed on ice in coolers and transported to the lab until imposex analysis could be done, usually the following day.

Nucella lapillus were collected from four sites within Saint John Harbour and four surrounding reference areas (Figure 3.2 A) from June 7th to 20th, 2007, and June

11th to 23rd, 2008. Musquash Head was selected as a reference site because the site was previously surveyed in 2000 and no incidence of imposex was found (n = 50)

(Delaney, 2001). The remaining reference sites were chosen because N. lapillus were known to occur there, and there were low levels of shipping activity compared to Saint John Harbour.

Three of the four Saint John Harbour sites were situated on the eastern shore of the harbour and one on the western periphery (Figure 3.1 B). A rocky intertidal habitat adjacent to the Princess of Acadia dock was initially surveyed for N. lapillus populations, but this site was abandoned after searching found no live dogwhelks. A preliminary survey in Sydney Harbour was completed on July 16th 2007 at two sites 55

(Figure 2.1 C). A 1995 survey by Prouse & Ellis (1997) indicated imposex in N. lapillus collected from north of South Bar in Sydney Harbour (Figure 3.1 C), but in

2007 no live dogwhelks were found. The other site, North Sydney Slipway, was also surveyed but no dogwhelks were observed there either. As no dogwhelks could be found in Sydney Harbour, imposex analysis for Sydney Harbour was terminated.

Field investigations for Halifax were completed on June 6th and 7th, 2008 at two harbour sites (Figure 3.1 D) and one reference site, Blandford (Figure 3.2 B). St.

Margaret's Bay was another proposed reference site, and previously investigated by

Prouse & Ellis (1997), but no dogwhelks were found during the time of this survey.

Blandford was chosen as an alternative reference site because there was low shipping activity compared with Halifax Harbour, and the site was similar to Back Bay, one of the reference sites in New Brunswick, as both sites were small semi-enclosed fishing harbours.

Imposex analysis. Shell length (mm) and total weight (i.e. shell and wet weight) were measured, and sex was determined in a stepwise fashion using methods adapted from Ellis & Pattisina (1990). An initial determination of sex was made

based on the external body plan of males and females (Gibbs et al. 1987; Gibbs et al.

1990). Final sex was confirmed by the identification of secondary sex organs,

namely the capsule gland, and more specifically, the sperm ingesting gland as it does

not vary with the reproductive state of the female (Gibbs et al. 1987). A final determination of sex was made for each snail as male, female or imposex female.

Incidence and severity of imposex was assessed for each site using the following

imposex parameters: mean male penis length (mMPL), mean female penis length

(mFPL), percent imposex (% I), i.e. the proportion of females in each population

affected with imposex, male:female ratio (M:F), vas deferens sequence index (VDSI; 56 see below), and relative penis size index (RPSI). RPSI is the mean length of the female penis expressed as a percentage of the mean length of the male penis at that site (Gibbs et al. 1987).

The vas deferens sequence index (VDSI) measures vas deferens development from Stage 0 to Stage 6 and was assessed according to Gibbs et al. (1987). Females in Stages 0 to 4 are still capable of reproducing, but those in Stages 5 and 6 are rendered sterile and incapable of reproducing. VDSI has been proposed as the most valid parameter in biomonitoring studies rather than RPSI because: (1) some stages of imposex lack a penis; (2) there is individual variability; and (3) penis lengths in males and females of many species vary throughout the year (Oehlmann et al. 1991,

1992; Stroben et al. 1992; Barroso & Mora, 1998), whereas the VDSI remains fairly constant throughout the year and includes all imposex stages (Stroben et al. 1992), even those without a penis. Even though VDSI by all accounts appears to be the superior measure of imposex intensity, RPSI provides useful supplementary data

(Gibbs 1999), and can be used to compare temporal changes (Smith 1996; Barroso &

Mora, 1998).

Organotin analysis. The soft tissues of about 3 to 4 dogwhelks of each designation (i.e. male, female, and imposex female) were pooled from each site and placed in labelled plastic sampling bags (n = 1). Samples were kept frozen in a dark freezer (-20 °C) to avoid butyltin degradation, and maintain stability until freeze drying (Caricchia et al. 1994; Quevauviller 1996). Tissues were freeze dried for at least 48 to 72 hours at -80 °C in a LABCONO® Freezezone® freeze drier. Samples were then homogenised using a porcelain mortar and pestle that were cleaned between each sample using 10% diluted HC1. Homogenised samples were then placed in appropriately labelled clear glass bottles with screw caps, and stored in a 57 dark freezer (-20 °C) until transported on dry ice for TBT analysis at the Institut des

Sciences de la mer de Rimouski (ISMER) at the University of Quebec in Rimouski.

Determination of butyltins in samples was adapted from Chau et al. (1997) with modifications following a protocol routinely used at ISMER and described below.

Approximately 0.25 g of freeze dried dogwhelk tissue was placed in 5 ml of tetramethyl ammonium hydroxide (TMAH, 25% H2O) in a 50 ml Teflon® tube.

Tissue samples were mixed for roughly 2 min on a vortex mixer, placed in an ultrasonic bath for 60 min at 60 °C, and sonicated for 1 hr to digest tissue. Certified reference material (CRM), BCR CRM 462 (mussel tissue), and a procedural blank were treated in similar fashion to tissue samples for every five samples. Following tissue sonication, 25 ml of sodium acetate (CH3.COONH4 AnalaR®) buffered at pH

4.5 was added to each Teflon® vial followed by 2 ml saturated NaCl solution, and 2 g NaCl. Organotins were derivatized to an ethylated neutral compound prior to butyltin extraction by addition of 600 |xl of sodium tetraethylborate (NaBET*, 2%

H2O). Extraction of ethylated butyltins was accomplished by two separate, but successive additions of 4 ml 1:1 hexane:toluene mixture. The organic layer from each hexane:toluene extraction was recovered from each Teflon® vial using a new glass pipette for each sample to avoid cross contamination, and transferred to correctly labelled glass round-bottom centrifuge tubes with screw caps. A first clean-up step consisted of 1 g Florisil® added directly to the recovered organic layer

(~ 6 ml) in each round-bottom centrifuge tube, placed on a wrist shaker for 2 min, and centrifuged (3,000 rpm, 10 min, IEC HN-SII centrifuge). After centrifiigation, the organic layer was recovered and cleaned on a silica gel micro-column. About 0.5 cm of anhydrous sodium sulfate (Na2S04) was added to the micro-column to remove any water. The organic fraction was eluted with 1:1 hexane:toluene and collected in 58 a graduated conical tube to a final volume of 10 ml. Before volume reduction to 200

Hi under a gentle stream of nitrogen gas at room temperature, 100 \i\ of tetrapropyltin (TepT) at the appropriate concentration was added as the internal standard. The concentrated extract was transferred to amber ICP vials with a glass liner. The organotins were analyzed by gas chromatography linked to an ion trap mass spectrometer (GC-MS, Trace GC-Polaris Q, Thermo Finnigan®) equipped with a Valcobond® VB-5 GC column, with helium as the carrier gas. Butyltin species identification was based on their respective mass spectrum and GC retention time.

Quantification was performed with reference to the internal standard by the

Xcalibur® software program which manages the Polaris Q® Trace GC. Butyltin species recovery was calculated from known butyltin concentration of CRM 462 (n

= 5). Percent recovery for MBT, DBT and TBT was 10%, 55% and 71%, respectively. Butyltin concentrations were not corrected and are reported as tin (ng

Sn g"1 dry wt.) for each type of butyltin examined. MBT levels in N. lapillus are not presented in the results since the percent recovery was too low. Quality assurance for the analytical component was verified by the calibration of the GC, and use of a

CRM (BCR CRM 462) and procedural blank for every five tissue samples. The limit of detection expressed as tin atom, with one microliter injected, was 2 pg for MBT and 3 pg for DBT and TBT.

Statistical analysis. All statistics were performed using SPSS version 17 (a

= 0.05). Data for this study did not satisfy the assumptions of parametric testing even after transformation (logio), so non-parametric equivalents were used. Kruskal-

Wallis was used to assess if there was a difference in shell length between sites and site type. Wilcoxon sign ranks test was used to ascertain if there was a difference in the occurrence and severity of imposex between sites spatially and temporally. 59

Linear regression was used to determine if there was a relationship between the

incidence of imposex (ArcSin % I, mFPL, RPSI & VDSI) with logio butyltin tissue concentration in imposex-affected and non-affected females, while Spearman rho

(one-tailed) was used to correlate the incidence of imposex with logio butyltin tissue

concentration in all females.

3.4 Results

Current study - Spatial assessment. Thirteen sites were surveyed for

incidence of imposex in Nucella lapillus in two Maritime provinces of Atlantic

Canada during the summers of 2007 and 2008. Sample size (n) ranged from 17 to 40

dogwhelks per site except at sites in Sydney Harbour where no dogwhelks were

found at the time of sample collection. Of the 11 sites where dogwhelks were found,

6 were harbour sites and 5 were reference sites. Mean shell length for all sites

ranged from 24.35 ± 2.97 to 35.01 ± 2.29 mm (Table 3.2). There was a significant

difference in shell length between sites (X2 = 212.7, df= 10, p < 0.001), but not site

type (.X2 = 0.40, df= 1, p > 0.05). Differences in mean shell length between sites

were grouped into four distinct groups (Student Newman-Keuls test, a priori).

Imposex was not observed at any of the reference sites, but was found in 83% of

harbour sites. The only harbour site where imposex was not observed was at Fort

Dufferin (Figure 3.1 B). Percent imposex in harbour sites ranged from 0 to 41.2%,

with the highest recorded incidence of imposex at Tuft's Cove. Mean female penis

length (mFPL) at imposex-affected sites varied from 0.42 ± 0.28 to 1.30 ± 0.95, with

Red Head having the highest mFPL and RPSI (Figure 3.3). At harbour sites affected

by imposex, VDSI varied from 0.50 to 1.12 and RPSI from 0.20 to 3.19. Highest

VDSI and RPSI occurred at Tuft's and Red Head, respectively (Table 3.2). M:F 60 ratios at each site ranged from 0.50 to 2.10, but in most cases did not deviate from the expected 1:1 ratio. In only one case did the M:F double from the expected 1:1 ratio, and this was observed at Tin Can Beach (M:F = 2.1) (Table 3.2). Juvenile and egg capsules of N. lapillus were present at all sites during the time of the survey except at Anthony's Cove and Musquash Head where only juveniles were observed.

Spatial occurrence of imposex and severity differed for all imposex parameters for harbour sites: ArcSin % I, mFPL, VDSI, RPSI (z = -2.934, N = 11, p < 0.01); mMPL

(z = -2.134, N = 11,p < 0.05) and M:F (z = -2.701, N = \\,p < 0.01).

Current study - Butyltin accumulation. Measurable levels of butyltins were detected in all imposex-affected female Nucella lapillus from harbour sites, but below the detection limit in all normal females from harbour and reference sites. Red

Head was the only site where both TBT (63.75 ng Sn g*1, dw), and DBT (50.99 ng

Sn g"1, dw), one of TBT's degradative products, were identified in pooled imposex- affected female N. lapillus. TBT was detected in imposex females from Anthony's

Cove (4.75 ng Sn g'1, dw) and Tin Can Beach (8.84 ng Sn g"1, dw), while only DBT was detected in samples from Tuft's Cove (3.84 ng Sn g"1, dw). Regression analysis of mFPL (R2 = 0.75) and VDSI (R2 = 0.82) (Figure 3.4) demonstrated the best relationship with logio TBT levels in female N. lapillus; whereas poorer fits were found between ArcSin % I (R2 = 0.68) and RPSI (R2 = 0.67), data not shown.

Spearman rho test confirmed a significant one-tailed correlation between all parameters tested with logio TBT levels in regression analysis (rs = 0.827 to 0.861, N

= 11, p <0.01).

Temporal assessment (1995 to 2007/2008). The first imposex survey in

Atlantic Canada was performed by Prouse and Ellis (1997) in 1995. After the initial survey, the only subsequent imposex survey conducted in Saint John Harbour prior 61 to this study was completed in the summer of 2000 by Delaney (2001). There were two subsequent surveys in Halifax Harbour in 1996 by Covert (1997) and 2006 by

Coray (2007) after the initial 1995 survey. Imposex index data (% I, mFPL, RPSI and VDSI) and butyltin tissue levels for sites in Saint John Harbour and Nova Scotia where data are available for two or more years are outlined in Table 3.3. Statistical analysis was only performed for harbour sites with consistent previous data.

Red Head is the only site that was consistently surveyed in Saint John

Harbour from 1995 to present. In comparing imposex indices for Red Head, % I

initially increased from the 1995 survey and then declined (Table 3.3), while mFPL

and RPSI continually increased in successive studies. VDSI and butyltin tissue levels in Nucella lapillus were not assessed during all surveys conducted at Red

Head. For the two years where there were data to compare VDSI and tissue concentration, VDSI decreased while tissue concentration increased (Table 3.3).

There was a significant difference by year for % I and RPSI (Z = -2.232, N = 6, p <

0.05), and mFPL (Z = -2.020, N=6,p< 0.05), but no significant difference for M:F or VDSI ip > 0.05). TBT level from 1995 increased significantly from 9.00 to

63.74 ng Sn g1 (dry weight) in 2007/2008, £ = 4.00, df=\,p< 0.05. The only other site within Saint John Harbour with previous data is Anthony's Cove. Percent

imposex, M:F, RPSI and VDSI decreased between 2000 to 2007, while mFPL

increased during the same period. There was a significant difference by year in % I

(Z = -2.070, N= 5, p < 0.05), and mFPL (Z = -2.070, N = 5, p < 0.05) only. Even though there was an apparent increase in mFPL & RPSI, and a decrease in % I at

Point Pleasant, the differences were not significant. 62

3.5 Discussion

Spatial perspective. Statistical analysis verified that there was a difference

in mean shell length between sites, but not site type; which is most likely attributed to differences within populations (Crothers, 1974, 1981). In the current study,

imposex was not observed at any of the reference sites, but was detected at sites

within Saint John Harbour and Halifax Harbour. No determination could be drawn about imposex in Sydney Harbour because no dogwhelks were found during the time of field collection.

In Saint John Harbour, the frequency of imposex decreased from the inner

most harbour site, Tin Can Beach, towards the Bay of Fundy at Anthony's Cove.

The only harbour site in Saint John Harbour where imposex was not observed was at

Fort Dufferin, which is situated on the western periphery of the harbour. The

occurrence of imposex and the measurable levels of butyltins in females affected by

imposex at sites only on the eastern periphery of the harbour suggest that currents

within the harbour flow in an easterly direction. This hypothesis is supported based

on data that verify that the resultant surface currents from the Reversing Falls (in the

Uptown region) flow along the eastern side of the harbour to approximately Mispec

Point before turning westward then continue along the outside of Partridge Island

into the Bay of Fundy (Neu, 1960). The highest degree of imposex in Saint John

Harbour occurred at Red Head, measured by RPSI (Bryan et al.1986) and VDSI

(Evans et al. 2001). In Nova Scotia, imposex was documented in the two harbour sites investigated, but the highest incidence and severity were observed at the inner

most harbour site, Tuft's Cove, and the lowest at the mouth of the harbour, Point

Pleasant. 63

Imposex severity at sites can be assessed by comparing VDSI (Evans et al.

2001), mFPL and RPSI (Bryan et al. 1986) values. A spatial comparison of the severity of imposex between sites in Saint John Harbour and Halifax Harbour revealed that Tuft's Cove in Halifax Harbour is the most severely affected site when severity is measured by %I, mFPL, RPSI and VDSI. However in Saint John

Harbour, Red Head is the most severely affected site when severity is measured by mFPL and RPSI, and Tin Can Beach is the most severely affected when measured by

VDSI (Table 3.2). Even though imposex was identified at harbour sites, the

presence of juveniles and/or egg capsules at the majority of sites indicates recruitment and breeding (Evans & Nicholson 1999), suggesting that although sites may be impacted by imposex, populations were not affected to the point of jeopardising recruitment. The reproductive competency of all populations is further supported by VDSI values which were not > 2 at any site (Gibbs et al. 1987),

indicating that there were no sterile females in any of the investigated populations

(Bryan etal. 1987b).

A significant correlation between TBT levels and imposex parameters is demonstrated for the first time in an imposex study in eastern Canada. Imposex field surveys from other regions have reported positive correlations between Sn/butyltin

levels and the degree of imposex in England (Bryan et al. 1986), France (Oehlmann et al. 1992; Stroben et al. 1992), Japan (Horiguchi et al. 1994), Ireland (Ide et al.

1997), Mediterranean (Sol£ et al. 1998), Scotland (Leung et al. 2001), and Portugal

(Barroso et al. 2005). The discovery of imposex at the majority of sites in the

vicinity of marine activity (harbours, ports, moorings, and shipyards), and the

positive correlation between N. lapillus butyltin tissue levels and imposex indices

(mFPL and RPSI) suggests that the release of TBT into the marine environment of 64

Atlantic Canada is primarily from its usage as a biocide in antifouling paint. This is likely to be fairly recent considering that imposex is irreversible and the lifespan of

N. lapillus is 5 to 6 years (Matthiessen et al. 1998).

In the current study, the only population that somewhat deviated from the expected 1:1 sex ratio was Tin Can Beach. The M:F has been used in imposex biomonitoring studies because ecologically, endocrine disruption in prosobranchs is known to affect population and community levels by skewing the ratios in favour of males (Matthiessen et al. 1998; Oehlmann et al. 2007). This skewing of M:F towards males has been observed in other imposex studies using Nucella lapillus on the Northumbrian coast, M:F = 0.7 to 4.0 (Evans et al. 1991) and the Firth of Forth,

M:F = 0.67 to 9.00 (Miller et al. 1999). Other species recognised as having male- biased populations include Ocinebrina aciculata (Oehlmann et al. 1996) in France and Thais clavigera in Asia (Tan, 1997; Evans et al. 2001; Leung et al. 2006). From the calculated M:F ratios in this study, it is evident populations were generally not skewed towards males as much as has been observed in other studies.

Temporal perspective. In an effort to identify temporal changes in Nucella lapillus populations in the current study, every effort was made to use previously surveyed sites (Davies 2000) from 1995 to present. Temporal changes in populations affected by imposex between years in other studies have been assessed using mFPL (Gibbs & Bryan 1994) and RPSI (Cullon 2001; Barroso & Moreira

2002). Data suggest that even though there has been a decrease in the incidence of imposex at Anthony's Cove, Red Head and Point Pleasant, imposex severity as assessed by mFPL, and to a lesser degree RPSI, did not decline. There was no change in VDSI at Point Pleasant, but a decline was observed at the other two sites, although this was not statistically significant. Even though there was no statistical 65 difference in VDSI at Red Head and Anthony's Cove, OSPAR (2008) considers a change of one unit to be a considerable change in VDSI, and this did occur for both sites. Temporal changes in Nucella lapillus tissue butyltin levels could not be

assessed for Anthony's Cove and Point Pleasant because tissue levels were not

measured for those previous studies. However, at Red Head there was a significant

increase in TBT concentration in dogwhelk tissues between 1995 and 2007; it was

seven times greater in the current study. N. lapillus TBT tissue levels in this study

(63.75 ng Sn g"1, dw) in 2007 were much higher than those observed by Oliveira et

al. (2009) in their 2006 imposex study in the United Kingdom (0.8 to 39 ng Sn g'1,

dw). The fact that no significant change in any of the imposex indices at Point

Pleasant was detected may be because one of the surveys was completed only two

years ago in 2006.

3.6 Conclusion

The incidence and severity of imposex observed in this current study

demonstrate that even though TBT has been regulated in Canada since 1989, TBT is

bioavailable in hot spot areas related to harbours, moorings, and shipyards. Imposex

in adult N. lapillus may reflect effects of exposure as juveniles (Matthiessen et al.

1998), since imposex is not reversible. In addition, since the lifespan of N. lapillus is

five to six years (Matthiessen et al. 1998), data from the current study most likely

represent environmental conditions sometime between 2002 and 2003 and not

2007/2008.

Current data imply that the 1989 Canadian regulations regulating the use of

TBT have been effective in decreasing the incidence of imposex as well as imposex 66 severity if measured by % I and VDSI. On the other hand, if imposex severity is measured by RPSI and/or mFPL, the results are inconsistent across sites, except at

Red Head where there was a consistent increase in RPSI and mFPL from 1995 to

2007/2008. This observation may be due to the fact that Red Head is a mooring site frequented a great deal by large oil tankers, which under Canadian regulations are still allowed to use TBT antifouling paint. This is further substantiated by the high

TBT concentration in Nucella lapillus samples at Red Head when compared with tissue concentration of dogwhelks at other sites affected by imposex in the current study and other comparable studies. This is the first study completed in Atlantic

Canada that demonstrates a positive relationship with the input of TBT into the marine environment, most likely from the use of antifouling paint, with the occurrence of imposex in female N. lapillus. Even though butyltin tissue concentrations, mFPL and RPSI increased during each successive survey at Red

Head, the results of this study suggest that there is some recovery of imposex at two harbours in Atlantic Canada between 1995 to 2007-08.

Acknowledgements. This work was funded by NSERC (Natural Sciences and

Engineering Research Council of Canada (B. MacDonald), the Canadian Research

Chair in Molecular Ecotoxicology (6. Pelletier), and CBIE (Canadian Bureau for

International Education). We thank E. Holtz and A. Hamer for their assistance with field work. 67

3.7 Literature Cited

Agriculture Canada. 1989. Antifouling paints for ship hulls. Canadian Association of Pesticide Control Officials Note 89-02, Ottawa.

Alzieu C. 1986. The detrimental effects on oyster culture in France - evolution since antifouling paint regulation. In Proceedings of the Oceans '86 - Organotin Symposium Proceedings. IEEE: Washington, D. C. USA., pp. 1130 - 1134.

Alzieu C. 1991. Environmental problems caused by TBT in France: assessment, regulations, prospects. Mar Environ Res. 32: 7-17.

Alvarez MS, Ellis DV. 1990. Widespread neogastropod imposex in the northeast Pacific: implications for TBT contamination. Mar Pollut Bull. 21 (5): 244 - 247.

Barroso CM, Mora MH. 1998. Reproductive cycle of Nassarius reticulatus in the Ria De Aveiro, Portugal: implications for imposex studies. J Mar Biol Assoc UK. 78: 1233 - 1246.

Barroso CM, Moreira MH. 2002. Spatial and temporal changes of TBT pollution along the Portuguese coast: inefficacy of the EEC directive 89/677. Mar Pollut Bull. 44: 480 - 486.

Barroso CM, Moreira MH, Richardson CA. 2005. Age and growth of Nassarius reticulatus in the Ria de Aveiro, north-west Portugal. J Mar Biol Assoc UK 85: 151 - 156.

BlaberSJM. 1970. The occurrence of a penis-like outgrowth behind the right tentacle in spent females oiNucella lapillus (L.). Proc Malacol Soc Lon. 39: 231 -233.

Bryan G, Gibbs P, Hummerstone L, Burt G. 1986. The decline of the gastropod Nucella lapillus around south-west England: evidence for the effect of tributyltin from antifouling paints. J Mar Biol Assoc UK. 66: 611 - 640.

Bryan GW, Gibbs PE, Hummerstone, LG, Burt, GR. 1987a. Copper, zinc, and organotin as long-term factors governing the distribution of organisms in the Fal Estuary in southwest England. Estuaries. 10: 208 - 219.

Bryan G, Gibbs P, Burt G, Hummerstone L. 1987b. The effects of tributyltin (TBT) accumulation on adult dogwhelks, Nucella lapillus: long-term field and laboratory experiments. J Mar Biol Assoc UK. 67: 525 - 544.

Bright DA, Ellis DV. 1990. A comparative survey of imposex in northeast Pacific neogastropods (Prosobranchia) related to tributyltin contamination, and choice of a suitable bioindicator. Can J Zoo. 68: 1915 - 1924. 68

Camillo E, Quadros J, deCastro IB, Fernandez M. 2004. Imposex in Thais rustica (Mollusca: Neogastropoda) (Lamark, 1822) as an indicator of organotin compound pollution at Maceio coast (Northeastern Brazil). Braz J Oceanogr. 52: 101 - 105.

Caricchia AM, Chiavarini S, Cremisini C, Morabito R, Scerbo R. 1994. Influence of storage conditions on the determination of organotin in mussels. Anal Chim Acta. 286: 329 - 334.

Chau YK, Yang F, Brown M. 1997. Evaluation of derivatization techniques for the analysis of organotin compounds in biological tissue. Anal Chim Acta. 338: 51 -55.

Coray C. 2007. Assessing the health of Halifax Harbour's intertidal ecosystem: Impacts of marine pollution on community structure, reproductive health of key predator populations (Nucella lapillus) and immunomodulation in blue mussels (Mytilns edulis & Mytilus trossulus). MSc thesis, Dalhousie University, Nova Scotia, Canada.

Covert TJ. 1997. Masculinization of dogwhelks {Nucella lapillus) and periwinkles (Littorina littorea) in Halifax Harbour: Monitoring of Tributyltin. BSc honours thesis, Saint Mary's University, Nova Scotia, Canada.

Crothers JH. 1974. On variation in the shell of the dog-whelk, Nucella lapillus (L.). Fid Stud. 4: 39-60.

Crothers JH. 1981. On variation in Nucella lapillus (L.): Shell shape in populations from Orkney and the north coast of Scotland. J Moll Stud. 47: 182- 189.

Cullon DL. 2001. Evidence of endocrine disruption in neogastropods by organotins: 10 years after the 1989 regulations. MSc thesis, Royal Roads University, British Columbia, Canada.

Curtis LA. 1994. A decade-long perspective on a bioindicator of pollution: Imposex in Ilyanassa obsoleta on Cape Henlopen, Delaware Bay. Mar Environ Res. 38: 291 - 302.

Davies IM. 2000. Kinetics of the development of imposex in transplanted adult dogwhelks, Nucella lapillus. Environ Pollut. 107: 445 - 449.

Delaney JL. 2001. The occurrence of imposex in Nucella lapillus within the Saint John Harbour. BSc honours thesis, University of New Brunswick (Saint John), New Brunswick, Canada.

Ellis DV, Pattisina LA. 1990. Widespread neogastropod imposex: A biological indicator of global TBT contamination? Mar Pollut Bull. 21(5): 248 - 253. 69

Evans SM, Hutton A, Kendall MA, Samosir AM. 1991. Recovery in populations of dogwhelks Nucella lapillus (L.) suffering from imposex. Mar Pollut Bull 22 (7): 331 -333.

Evans SM, Nicholson GJ. 1999. Assessing the impact of antifouling compounds in the marine environment. Lessons to be learned from the use and misuse of biological indicators of TBT contamination. In Proceedings of the Oceans '99 - Organotin Symposium Proceedings. IEEE: Seattle, W. A. USA, pp. 1115-1118.

Evans SM, Bech M, Hawkins ST, Smith R, Stewart A. 2001. Imposex in populations of dogwhelks Thais spp. in relation to shipping intensity and mariculture activity in east Asia. Inverte Reprod Dev. 39: 231 - 237.

FeareCJ. 1970. Reproductive cycle of the dogwhelk {Nucella lapillus). Proc Malacol Soc Lon. 39: 125 - 137.

F6ral C, Le Gall S. 1982. Physiologie des inverttfbres - Induction experimental par un pollutant marin (le tributyl&ain), de P activity neuroendocrine controlant la morphogenese du penis chez les femelles d'Ocenebra erinacea (Mollusque, Prosobranche gonochorique). C R Hebd Seances Acad Sci. D 295: 627 - 630.

Fioroni P, Oehlmann J, Stroben E. 1990. Le pseudohermaphrodisme chez les prosobranches analyse morphologique et histologique. Vie Milieu. 40: 45 - 56.

Fretter V. 1953. The transference of sperm from male to female prosobranchs, with reference, also to the pyramidellids. Proc Linn Soc Lon. 164: 217 - 224.

Gibbs PE, Bryan GW, Pascoe PL, Burt GR. 1987. The use of the dog-whelk, Nucella lapillus, as an indicator of tributyltin (TBT) contamination. J Mar Biol Assoc UK. 67: 507-523.

Gibbs PE, Bryan GW, Pascoe PL, Burt GR. 1990. Reproductive abnormalities in female Ocenebra erinacea (Gastropoda) resulting from tributyltin-induced imposex. J Mar Biol Assoc UK. 70: 639 - 656.

Gibbs PE, Bryan GW. 1994. Biomonitoring of tributyltin (TBT) using the imposex response of neogastropod molluscs, pp 205 - 226. In Kramer KJM (ed) Biomonitoring of coastal waters and estuaries. CRC Press Inc., Boca Raton, FL.

Gibbs, PE. 1999. Biological effects of contaminants: Use of imposex in the dogwhelk (Nucella lapillus) as a bioindicator of tributyltin pollution. ICES Tech in Mar Environ Sci. 24: 1 - 29.

Horiguchi T, Shiraishi H, Shimizu M, Morita M. 1994. Imposex and organotin compounds in Thais clavigera and T. bronni in Japan. J Mar Biol Assoc UK 74: 651 -669. 70

Horiguchi T, Li Z, Uno S, Shimizu M, Shiraishi H, Morita M, Thompson JAJ, Levings CD. 2003. Contamination of organotin compounds and imposex in molluscs from Vancouver, Canada. Mar Environ Res. 57: 75 - 88.

Houston RS. 1971. Reproductive biology of Thais emarginata (Deshayes, 1939) and Thais canaliculata (Duclos, 1832). Veliger. 13: 348 - 357.

Huggett RJ, Unger MA, Seligman PF, Valkris AO. 1992. The marine biocide tributyltin. Assessing and managing the environmental risks. Environ Sci Technol. 26: 232 - 237.

Hughes RN. 1972. Annual production of two Nova Scotian populations of Nucella lapillus (L.). Oecologia. 8: 356 - 370.

Ide I, Witten EP, Fischer J, Kalbfus W, Zellner A, Stroben E, Watermann B. 1997. Accumulation of organotin compounds in the common whelk Buccinum undatum and the red whelk Neptunea antiqua in association with imposex. Mar Ecol Prog Ser. 152: 197 - 203.

Jenner MG. 1979. Pseudohermaphroditisim: A newly recognized sexual phenomenon in llyanassa obsoleta and other neogastropods. PhD thesis, University of Chapel Hill, North Carolina, USA.

Leung KMY, Morgan IJ, Wu RSS, Lau TC, Svavarsson J, Furness RW. 2001. Growth rate as a factor confounding the use of the dogwhelk Nucella lapillus as biomonitor of heavy metal contamination. Mar Ecol Prog Ser. 221: 145 - 159.

Leung KMY, Kwong RPY, Ng WC, Horiguchi T, Qui JW, Yang R, Song M, Jiang G, Zheng GJ, Lam PKS. 2006. Ecological risk assessments of endocrine disrupting organotin compounds using marine neogastropods in Hong Kong. Chemosphere. 65: 922 - 938.

Matthiessen P, Reynoldson T, Billinghurst Z, Brassard DW, Cameron P, Chandler GT, Davies IM, Horiguchi T, Mount DR, Oehlmann J, Pottinger TG, Sibley PK, Thomson HM, Vethaak AD. 1998. Endocrine disruption in invertebrates: Endocrinology, testing, and assessment, p. 199 - 279. In deFur PL, Crane M, Ingersoll CG, Tattersfield LJ (eds). Society of Environmental Toxicology and Chemistry (SETAC), Pensacola, FL: SETAC Press.

Miller KL, Fernandes TF, Read PA. 1999. The recovery of populations of dogwhelks suffering from imposex in the Firth of Forth 1987 -1997/98. Environ Pollut. 106: 183 - 192.

Neu HA. 1960. Hydrographic survey of Saint John Harbour, N. B. Mechanical Engineering Report. National Research Council of Canada, Ottawa. 71

Oehlmann J, Stroben E, Fioroni P. 1991. The morphological expression of imposex in Nucella lapillus (Linnaeus) (Gastropoda: Muricidae). J Molluscan Stud. 57: 375 - 390.

Oehlmann J, Stroben E, Fioroni P. 1992. The rough tingle Ocenebra erinacea (Neogastropoda: Muricidae): an exhibitor of imposex in comparison to Nucella lapillus. Helgoland Mar Res. 46: 311 - 328.

Oehlmann J, Stroben E, Shulte-Oehlmann U, Bauer B, Fioroni P, Markert B. 1996. Tributyltin monitoring using prosobranchs as sentinel organisms. Fresenius J Anal Chem. 354: 540 - 545.

Oehlmann J, Fioroni P, Stroben E, Market B. 1996. Tributyltin (TBT) effects on Ocinebrina aciculata (Gastropoda: Muricidae): imposex development, sterilization, sex change and population decline. Sci Total Environ. 188: 205 -223.

Oehlmann J, Di Benedetto P, Tillmann M, Duft M, Oetken M, Schulte- Oehlmann U. 2007. Endocrine disruption in prosobranch molluscs: evidence and ecological evidence. Ecotoxicol. 16: 29 -43.

Oliveira IB, Richardson CA, Sousa AC, Takahashi S, Tanabe S, Barroso CM. 2009. Spatial and temporal evolution of imposex in dogwhelk Nucella lapillus (L.) populations from North Wales, UK. J Environ Monitor. 11: 1462 - 1468.

OSPAR. 2008. JAMP Guidelines for Contaminant-Specific Biological Effects (OSPAR Agreement 2008-09). In OSPAR Convention for the Protection of the Marine Environment of the North-East Atlantic.

Poli G, Salvat B, Streiff W. 1971. Aspect particulier de la sexualite chez Ocenebra erinacea. Haliotis. 1: 29 - 30.

Prouse NJ, Ellis DV. 1997. A baseline survey of dogwhelk {Nucella lapillus) imposex in Eastern Canada (1995) and interpretation in terms of tributyltin (TBT) contamination. Environ Technol. 18: 1255 - 1264.

Quevauviller Ph. 1996. The analysis of butylated tin compounds in the environment and in biological materials, p. 62 - 93. In Tributyltin: Case study of an environmental contaminant, de Mora SJ (ed). Cambridge University Press, Great Britain.

Reitsema TJ, Thompson JAJ, Scholtens P, Spickett JT. 2002. Further recovery of the northeast Pacific neogastropods from imposex related to tributyltin contamination. Mar Pollut Bull. 44:257-261.

Smith BS. 1971. Sexuality in the American mud snail, Nassarius obsoletus, Say. Proc Malacol Soc Lon. 39: 377 - 378. 72

Smith BS. 1981. Male characteristics on female mud snails caused by antifouling bottom paints. J Appl Tox. 1 (1): 22 - 25.

Smith PJ. 1996. Selective decline in imposex levels in the dogwhelk Lepsiella scobina following a ban on the use of TBT antifoulants in New Zealand. Mar Pollut Bull. 32 (4): 362 - 365.

Sol6 M, Morcillo Y, Porte C. 1998. Imposex in the commercial snail Bolinus brandaris in the northwestern Mediterranean. Environ Pollut. 99: 241 - 246.

Stewart C, de Mora SJ, Jones MRL, Miller MC. 1992. Imposex in New Zealand neogastropods. Mar Pollut Bull. 24 (4): 204 - 209.

Stroben E, Oehlmann J, Fioroni P. 1992. The morphological expression of imposex in Hinia reticulata (Gastropoda: Buccinidae): a potential indicator of tributyltin pollution. Mar Biol. 113: 625 - 636.

Swennen C, Ruttanadakul N, Ardseungnern S, Singh HR, Tan KS, Mensink BP, Ten Hallers-Tjabbes CC. 1996. Sexual aberrations in gastropods in Gulf of Thailand and Strait of Malacca in relation to shipping. Wallaceana 78: 1 -13.

Tan KS. 1997. Imposex in three species of Thais from Singapore, with additional observations on T. clavigera (Kiister) from Japan. Mar Pollut Bull. 34 (7): 577-581.

Tester M, Ellis D. 1995. TBT controls and the recovery of whelks from imposex. Mar Pollut Bull. 30 (1): 90 -91.

Tester M, Ellis DV, Thompson JA. 1996. Neogastropod imposex for monitoring recovery from marine TBT contamination. Environ Toxicol Chem. 15 (4): 560 - 567.

Viglino L, Pelletier E, Lee LEJ. 2006. Butyltin species in benthic and pelagic organisms of the Saguenay Fjord (Canada) and imposex occurrence in common whelk (Buccinum undatum). Arch Environ Contam Toxicol. 50: 45 -59.

Zhu SX, Dong QX, Shen JH, Yu XJ, Du H, Huang CJ. 2005. Imposex in Conus betulinus and Conus Vexillum. Acta Ecol Sin. 25: 289 - 297 [In Chinese with English abstract]. 73

Figure 3.1 (A-D). (A) Location of imposex sampling sites in harbours (•) in

Atlantic Canada - 2007 to 2008 at (B) Saint John Harbour, New Brunswick (NB);

(C) Sydney Harbour and (D) Halifax Harbour, Nova Scotia (NS). 74

Back Bay Bay of Fundy

N

A 5 km *

St. ^Margaret's Bay

Atlantic Ocean N i+ 5 km

Figure 3.2. Location of imposex reference sites (•) in (A) New Brunswick and (B)

Nova Scotia - 2007 to 2008. (A). V> E IS. 00 • •

ZO I" IO 1 O.O Tl^Om ony's" Fort LTuft'l ~BL' lHead Cove Pufferln Cov« pl«M«nt Bfach (B) CD)

1.Tuft's Point Tin Can Red Anthony's Fort Tlift's Point Dufferin Cove Pleasant Head Cove Dufferin Cove Pleasant

Figure 3.3. Spatial occurrence of (A) percent imposex (% I), (B) relative penis size index (RPSI), (C) mean female penis length (mFPL) mm ±

SD, and (D) vas deferens sequence index (VDSI) ± SD for harbour sites in Saint John Harbour, (New Brunswick) and Halifax Harbour (Nova

Scotia) collected during 2007/2008.

UI 76

(A) 3.00 t y = 0.3728x + 0.4518 R1 = 0.7532 -g. 2.SO + E

2.00 • •

a» 1.50 - - o. _a> to E .41 l.OO - -

0.00 -+- -r- 4- -+- -+- 0.10 0.32 1.00 3.2 10.0 31.6 100 316.2 1,000 1 L°g10 CTBT] in Nucella lapillus as ng Sn g (dry wt.)

(B) 3.00 -r y = 0.4294X + 0.5265 R1 = 0.8216 —.t/> 2.SO -• >,a

8 2.00 -•

1.50 - • X s 1.00 -•

3 0.50

0.00 -+• 0.10 0.32 1.00 3.2 10.0 31.6 100 316.2 1,000 1 Log10 [TBT] in Nucella lapillus as ng Sn g (dry wt.)

Figure 3.4. Regression analysis between logio TBT levels in female Nucella lapillus and (A) mean female penis length (mFPL) and (B) vas deferens sequence index

(VDSI). Each data point represents a site of pooled affected and unaffected females.

Error bars ± (A) penis length and (B) vas deferens formation of each Nucella lapillus pooled and used for butyltin analysis. 77

Table 3.1. Imposex surveys conducted on the Pacific and Atlantic coasts of Canada

1987 to 2008.

Survey Year Coast Region(s) Species Reference Amphissa columbiana* Coins halli Neptunea phoenecia Nucella canaliculata 1987/1988 Pacific British Columbia Bright & Ellis (1990) Nucella emarginata Nucella lamellosa Ocenebra lurida Searlesia dira 1989 Ceratostoma foliatium Alvarez & Ellis (1990) 1993 Nucella canaliculata Tester & Ellis (1995) Pacific British Columbia Nucella emarginata 1994 Nucella lamellosa Tester et al. (1996) Searlesia dira New Brunswick Newfoundland 1995 Atlantic Nova Scotia Nucella lapillus Prouse & Ellis (1997) Prince Edward Island Quebec Nucella lapillus 1996 Atlantic Nova Scotia Covert (1997) Littorina littorea Atlantic Quebec Buccinum undatum Viglino et al. (2006) Nucella canaliculata Cullon (2001) Nucella emarginata 1999 Pacific British Columbia Nucella lamellosa Horiguchi et al. (2003) Nucella lima Searlesia dira Atlantic New Brunswick Nucella lapillus Delaney (2001) Nucella canaliculata 2000 Nucella emarginata Pacific British Columbia Reitsema et al. (2002) Nucella lamellosa Searlesia dira Nucella lapillus 2006 Atlantic Nova Scotia Coray (2007) i Littorina littorea New Brunswick 2007/2008 Atlantic Nucella lapillus Current study Nova Scotia * = species that did not exhibit imposex response Table 3.2. Site type, boating activity, sample size (n), mean shell length (mSL) ± standard deviation (SD), and imposex index data

(mMPL ± SD, mFPL ± SD, M:F, % I, RPSI, and VDSI ± SD) for Nucella lapillus at sites in two Maritime Provinces of Canada for data collected during the summers of 2007 and 2008. Boating activity designated as: commercial harbour (CH), cruise ship terminal (CST), fishing harbour (FH), mooring (M) and shipyard (SY).

Tin Can Beach CST 30 35.01 ± 2.29 3.32 ± 0.43 0.42 ± 0.28 2.1 38.5 0.20 1.08 ± 1.04 Red Head M 30 32.78 ± 3.04 4.10 ± 0.70 1.30 ± 0.95 0.5 26.9 3.19 1.04 ± 1.08 Harbour Anthony's Cove M 40 32.09 ± 3.52 2.88 ± 0.90 0.76 ±0.75 1.0 20.0 1.84 0.75 ±0.91 New Fort Dufferin 40 28.85 ± 2.23 3.40 ±0.59 0.00 ±0.00 1.2 0 0 0.00 ± 0.00 Brunswick Musquash Head 30 25.02 ± 3.30 1.87 ±0.19 0.00 ±0.00 1.1 0 0 0.00 ±0.00 Letete Point 40 32.53 ±2.71 3.41 ±0.64 0.00 ±0.00 0.7 0 0 0.00 ± 0.00 Reference Back Bay FH 40 33.63 ±3.68 1.79 ± 0.74 0.00 ±0.00 1.0 0 0 0.00 ±0.00 St. Andrews 17 31.67 ±5.21 3.02 ± 0.52 00.00 ±0.00 0.5 0 0 0.00 ± 0.00 Tuft's Cove SY 30 24.35 ± 2.97 2.77 ±0.50 0.76 ±0.48 0.8 41.2 2.07 1.12 ± 1.22 Point Pleasant CH 30 27.66 ±2.19 2.90 ± 0.58 0.60 ±0.15 1.5 16.6 0.89 0.50 0.67 Nova Harbour North Sydney Slipway SY n/a NF NF NF n/a n/a n/a n/a Scotia South East Bar n/a NF NF NF n/a n/a n/a n/a Reference Blandford FH 30 27.91 ± 1.62 2.47 ± 0.48 0.00 ±0.00 0.5 0 0 0.00 ± 0.00 NF = Not found; n/a = not applicable

00 Table 3.3. Imposex index data (% I, mFPL ± SD, RPSI, and VDSI ± SD), boating activity, and tissue concentration in N. lapillus

reported as ng Sn g"1 (dry wt.) for surveys conducted at harbour sites in Saint John Harbour (New Brunswick) and Halifax Harbour

(Nova Scotia) from 1995 to 2007/2008. mi m 2000 60 0.50 ± 0.38 2.07 3.00 n/a n/a Delaney (2001) Anthony's Cove Mooring 2007/08 20 0.76 ± 0.75 1.84 0.75 ±0.91 4.75 < DL Current Study New 1995 66 1.10 ±0.40 1.40 n/a 9.00 39.00 Prouse & Ellis (1997) Brunswick Red Head 2000 Mooring 71 1.17±0.82 2.07 3.00 n/a n/a Delaney (2001) 2007/08 23 1.30 ±0.95 3.19 1.04 ± 1.08 63.75 50.99 Current Study 1996 87 1.50 14.5 n/a n/a n/a Covert (1997) Nova Commercial Point Pleasant 2006 19 0.10 ± 0.20 0.50 0.50 n/a n/a Coray (2007) Scotia Harbour 2007/08 17 0.60 ±0.15 0.89 0.5010.67 < DL < DL Current Study n/a = not applicable (not calculated/not performed) DL = Detection limit (~ 0.1 ng Sn g"', dry weight)

sO 80

4 Myth or Truth: Is TBT Still an Issue in Atlantic Canada

More Than 20 Years after the 1989 Canadian Regulations

Controlling its Use?

4.1 Abstract

Blue mussel (Mytilus edulis) and sediment samples were collected from Saint

John Harbour, Sydney Harbour and Halifax Harbour in Atlantic Canada in 2007-

2008 to determine if the 1989 Canadian regulations were effective in reducing tributyltin (TBT) concentrations in the marine environment. Even though TBT was not detected at all locations, TBT and its degradative products, dibutyltin (DBT) and monobutyltin (MBT), were detected in sediment and M. edulis at harbours, marinas, and shipyards (known point sources of TBT into the environment via its application as a biocide in antifouling paint). Butyltins were also detected in sediment and mussels from reference sites where vessels < 25 m in length were docked at marinas and fishing harbours, similar to the situation observed in the United Kingdom and

Portugal. Even though Canada passed legislation regulating the use of TBT in antifouling paint, TBT is bioavailable and present in sediment at harbour and reference sites, and TBT levels in sediment have significantly increased over roughly

15 years at Sydney Harbour. A combination of physical (tidal flushing) and anthropogenic (legislation, decrease in boating activity and dredging) factors can be used to explain the differences in TBT levels recorded in sediment and M. edulis at each site. Data suggest that the 1989 Canadian regulations have not been completely effective in reducing TBT levels in the marine environment or its bioavailability to non-target organisms in Atlantic Canada. 81

4.2 Introduction

Biocides are used in antifouling paint on marine vessels to deter fouling organisms and thereby reduce turbulent flow, drag and fuel consumption, and increase speed. Between the 1960s and 1970s, the primary biocide in antifouling paint, cuprous oxide (CU2O), was replaced by a more effective one, tributyltin, TBT

(Huggett et al., 1992). Field studies have confirmed that high TBT concentrations are present in water and sediment from harbours and marinas (Langston et al., 1987;

Michel and Averty, 1991; Dowson et al., 1992; de Mora et al., 1995; Chau et al.,

1997a), shipyards (Dowson et al., 1992; Kan-Atireklap et al., 1997; Elgethun et al.,

2000), and shipping channels (Chau et al., 1997b), verifying the input of TBT into the marine environment via its application as a biocide in antifouling paint. Once

TBT enters the marine environment, it tends to adsorb onto organic-rich sediments via a hydrophobic interaction (Ramaswamy et al., 2004), and data suggest that there is a correlation between organic carbon and TBT (Unger et al., 1987; Kram et al.,

1989; Burton et al., 2004). TBT that has adsorbed to sediments can be re-mobilized into the aqueous phase by hydrodynamic disturbances such as storms, tidal currents, dredging operations, and propeller wash (Stewart, 1996).

The first credible evidence that TBT was having adverse effects on non-target organisms came from Arcachon Bay on France's Atlantic coast in the mid 1980s.

An unusual shell anomaly, known as "chambering" or "balling" was observed in the commercial Pacific oyster, Crassostrea gigas, which was linked to TBT input from boating activity (Alzieu, 1986). Another unusual biological phenomenon, pseudohermaphroditism (Jenner, 1979) or imposex (Smith, 1971), was observed in gonochoristic female gastropods growing a "penis-like" structure in the early 1960s 82

(Griffith and Castagna, 1962) and 1970s (Blaber, 1970; Houston, 1971; Poli et al.

1971; Smith, 1971), however, the causative agent, TBT, was not linked until the

1980s (Smith, 1981).

Because of the adverse effects that TBT had on non-target organisms, its use as a biocide in antifouling paints was eventually regulated by legislation. France was the first country to regulate the use of TBT in 1982 (Alzieu, 1991), but TBT levels in sea water along France's English Channel and Mediterranean coasts showed little or no decrease even after 15 years of legislation (Michel and Averty, 1991). A similar observation was reported in sediments from the UK (Waite et al., 1991; Dowson et al., 1993) although TBT legislation was initially passed in 1986 and revised in 1987

(Waite et al., 1991). Canada passed regulations regulating the use of TBT in 1989

(Agriculture Canada, 1989). The inability of legislation to decrease TBT environmental levels after years of regulation poses a contamination concern, especially for sediments, since data suggest that the sorption of TBT in sediments is reversible (Unger et al., 1987; Kram et al., 1989), i.e. sediments can act as a sink as well as a source.

In Canada, TBT is found primarily in marinas, harbours, and shipping channels

(Maguire, 1986; Maguire et al., 1986; Chau et al., 1997b), which is consistent with the use of TBT as a biocide in antifouling paint. In a 1982/1985 survey across

Canada (n = 265 locations), the highest TBT concentration recorded in water samples (2,340 ng Sn l"1) was discovered at Port Hope, Ontario, while maximum sediment levels (10,078 ng TBT g"1 dry wt) were detected at Vancouver Harbour,

British Columbia (Maguire et al., 1986). A subsequent study in 1993/1994, roughly four to five years after the 1989 Canadian regulations (n = 131 locations), discovered that TBT was still present in both freshwater and marine environments (Chau et al., 1997 a, b). TBT was found in 74% of seawater and 94% of marine sediment samples with the highest recorded concentrations in seawater samples (27.2 ng Sn 1"

') and marine sediments (2,525 ng Sn g'1 dry wt) detected at Burrard Inlet, British

Columbia (Chau et al., 1997b). Moreover, TBT levels from the 1993/1994 survey exceeded the Canadian water quality guidelines of 0.41 ng Sn l"1 (Moore et al., 1992) for the protection of marine life in 100% of the seawater samples in which TBT was detected (Chau et al., 1997b). In comparison to marine samples, TBT was detected in only 13% of freshwater samples and 53% of sediment samples (Chau et al.,

1997b). Maximum TBT concentration in water and sediment samples from freshwater environments was found in samples from Ontario at 17.8 ng Sn l"1 and

975 ng Sn g"1 dry wt, respectively (Chau et al., 1997b).

An in-depth review of the five harbours surveyed in Atlantic Canada by Chau et al. (1997a, b) was completed. The grading of those five harbours based on TBT levels in sediment, reported as ng Sn g"1 (dry wt), was: Saint John Harbour, New

Brunswick (20.9 - 3,212.0) > Halifax Harbour, Nova Scotia (143.0 - 1,198.0) > St.

John's Harbour, Newfoundland (147.3) > Sydney Harbour, Cape Breton Island (4.8

- 8.8) > Charlottetown Harbour, Prince Edward Island (no TBT recorded). Of those five harbours, TBT was recorded in M. edulis at only two harbours, Halifax Harbour

(143 - 1,198 ng Sn g'1 dry wt) and Saint John Harbour (2 - 76 ng Sn g"1 dry wt),

(Chau et al., 1997b).

At the end of the 1993/1994 butyltin survey, Chau et al. (1997b) concluded that:

(1) the 1989 Canadian regulations were only somewhat effective for freshwater environments, but not for marine environments; (2) the regulations had less of an effect on TBT levels in sediments most likely because of TBT's persistence in sediment versus water; and (3) harbours capable of handling vessels that were still 84 allowed to use TBT antifouling paint continued to experience notable levels of TBT contamination. The results and conclusions of the 1993/1994 butyltin survey suggested that TBT would continue to have an effect on marine organisms in Canada since most of the seawater samples collected at the time of that survey had levels which exceeded the Canadian water quality guidelines, and because of the persistence of TBT in sediments which could act as a source as well as a sink.

Subsequent to the 1993/1994 survey by Chau et al. (1997 a, b), Maguire and

Batchelor (2005) carried out another survey (n = 152) at harbours, marinas, and shipping channels across Canada in 1999. In that study, they investigated the occurrence of organotins (butyltins, hexyltins, octyltins and phenyltins) in water and sediment. Results indicated that all butyltins (TBT, DBT and MBT) were found in

50 of 56 marine sediment samples (89.3%), and the maximum concentration of TBT

(5,092 ng Sn g"1 dry wt.), DBT (1,200 ng Sn g"1 dry wt.), and MBT (605 ng Sn g'1 dry wt.) were found at Burrard Inlet, British Columbia (Maguire and Batchelor,

2005). Conversely, in seawater, butyltins were not found in > 20% of samples. TBT was found in 7 of 44 (16%) seawater samples with a maximum concentration of

102.8 ng Sn L*1 at Burrard Inlet, British Columbia. In comparison to TBT, DBT and

MBT were only found in 9% and 5% of seawater samples, respectively. The highest

DBT concentration was found at Burrard Inlet, British Columbia (41.4 ng Sn L"1), and the highest MBT concentration (13.6 ng Sn L-l) at a submarine dock in Halifax

Harbour, Nova Scotia (Maguire and Batchelor, 2005). The main conclusion of the

1999 survey was that the 1989 Canadian regulations on TBT had been effective in reducing TBT contamination in water, but not sediment, small-craft marinas or harbours (Maguire and Batchelor, 2005). 85

More recently published data by Viglino et al. (2006) from the Saguenay Fjord in Quebec identified butyltins in seston, crabs, worms, whelks, fish and sediment collected in 1999 to 2002. In addition, imposex, the super imposition of a penis and/or vas deferens in female gastropods (Smith, 1971), and the best documented example of endocrine disruption by an environmental contaminant (Vos, et al., 2000) was observed in the whelk, Buccinum undatum. Additionally, imposex has also been discovered in the dogwhelk, Nucella lapillus, in Saint John Harbour (New

Brunswick) and Halifax Harbour (Nova Scotia) during a field survey in 2007/2008, and the incidence of imposex has been correlated with TBT levels in N. lapillus tissues (Chapter 3).

It is evident that even though Canada passed legislation to regulate the use of organotins in 1989, these measures did not curtail the input of TBT into freshwater and marine environments, nor did the legislation prevent the effects of imposex in non-target gastropods. The nationwide organotin survey completed in 1993/1994 by

Chau et al. (1997a, b) provides baseline data of TBT levels approximately five years after Canadian regulations; hence, repeated field surveys using the same sites would prove beneficial in determining trends of TBT contamination, and evaluate the effectiveness of Canadian regulations. This study was designed to assess the occurrence of butyltins in sediment and resident M. edulis from three selected harbours in Atlantic Canada (Figure 4.1 A-D) at locations suspected as potential point sources of TBT and previously surveyed by Chau et al. (1997 a, b), and related reference sites. 86

4.3 Study Design and Objectives

Mussels are frequently used in biomonitoring studies because they are widely distributed in sub-Arctic and temperate zones (Cossa, 1989), they have a sedentary

life style and can be easily collected (Smolders et al., 2003), they are exposed to

pollutants in the environment via water, food and suspended matter since they are

filter feeders (Smolders et al., 2003) and they have a very high ability to

bioaccumulate chemicals at magnitudes above those found in the environment

(Salazar and Salazar, 1995; Smolders et al., 2003), but have a low ability to chemically change accumulated chemicals (Smolders et al., 2003). They provide

information about the bioavailability of contaminants in the environment that cannot

be defined by routine chemical analysis (Salazar and Salazar, 1995); and since

mussels are used for human consumption, they are a potential source of contamination (Cossa, 1989). Mytilus edulis was the chosen biomonitor species

because it is commonly used in ecotoxicology studies such as Mussel Watch

(Goldberg, 1975). Specific objectives of this study were to:

(1) ascertain if there is a difference in TBT concentration in sediment and

resident M. edulis between sites (n = 6) and site type (i.e. harbour or

reference site);

(2) establish if TBT concentration in sediment samples correlates with % carbon

(% C) at all sites; and 87

(3) compare TBT concentration at harbour sites from this study with those from

Chau et al. (1997a, b) to determine the efficacy of the 1989 Canadian

regulations on TBT.

4.4 Materials and Methods

4.4.1 Sample Collection

A survey of Saint John Harbour (Figure 4.1 B), Sydney Harbour (Figure 4.1

C), Halifax Harbour (Figure 4.1 D) and reference sites was completed from June 8 to

July 17, 2007, and June 6, 2008. All sediment (n = 5; except at Sydney Harbour where n = 6) and resident M. edulis (n = 30) samples were collected from locations adjacent to suspected point sources including commercial ports, marinas, ship yards, and cruise ship and ferry terminals on a gradient from the inner harbour toward the open Bay of Fundy and Atlantic Ocean (Figure 4.1 B-D). Reference sites were chosen because it was expected that any boating activity at these areas would not be related to TBT since the boats are small enough (generally < 25 m in length) to be encompassed by the 1989 Canadian regulations on TBT, whereas harbor sites have large boats that could still be painted with TBT antifouling paint. Hence, this study was designed to compare sites that should have been affected by the 1989 regulations (i.e. reference sites) to sties that may not have been affected by the regulations (i.e. harbor sites).

Prior to all surveys, low tide time was obtained from local tide tables and at each location, depth and GPS positions recorded (Table 4.1). In this study, location(s) refers to areas where samples were collected within sites. Sediment 88 samples were collected using a stainless steel Ekman® 6x6 grab (30 cm2), and the depth at which the sediment sample was recovered was recorded. The top 2 cm of each sediment grab was retrieved and stored in appropriately labelled amber

Wheaton® Redi-pak wide-mouth glass bottles with TFE-lined closures to avoid butyltin degradation. Resident M. edulis were collected by hand from pilings and other surfaces and stored in labelled plastic bags. Wherever possible, sediment and mussel samples were collected at the same location. Sediment and M. edulis were placed in an ice box en route to the lab, and frozen (-20 °C) until further analysis at the University of New Brunswick - Saint John.

4.4.2 Description of Study Sites

Saint John Harbour (Figure 4.1 B) was surveyed on June 8, 2007. Boating activity at locations in Saint John Harbour included a fishing port (Longwharf

Terminal), a ferry terminal (Princess of Acadia), a commercial port (Rodney

Container Terminal), and cruise ship activity (Pugsley Terminal and Longwharf

Terminal). Partridge Island was the only site with no boating activity in its immediate vicinity, but was selected because it was at the mouth of the harbour and south of all major boating activity within Saint John Harbour (Figure 4.1 B).

Sediment and M. edulis samples were not collected from all locations: (a) no mussels were collected at Partridge Island because there were no pilings or other hard surfaces for mussels to attach to; and (b) no sediment was collected from the cruise ship pier at Pugsley Terminal because the Ekman® grab was dragged by the tide currents on five different attempts to collect sediment from this site. Instead, Tin

Can Beach, an area south of Pugsley Terminal (Figure 4.1 B), was used as an 89 alternate site where mussels and sediment were collected. The associated reference site for Saint John Harbour was Back Bay, a small fishing port with an aquaculture site outside the breakwater. All mussel and sediment samples from Back Bay were collected on a gradient from the boat ramp towards a green navigation marker outside the breakwater from the same sites on June 21, 2007. Sediment and mussels were collected in the vicinity of some of the fishing boats docked at the time of sample collection at Dock 3 (Table 4.1).

Sediment and mussel samples were collected (n = 6) at Sydney Harbour on

July 16, 2007 (Figure 4.1 C) at South Bar, Indian Beach (ferry terminal), North

Sydney Slipway (shipyard site), Barachois Creek (marina), Muggah Creek (harbour), and SYD Port (commercial port). South Bar was initially surveyed to investigate the presence of imposex in Nucella lapillus since imposex was reported in samples collected at this site in 1995 by Prouse and Ellis (1997), and South Bar is also the most northerly site of all boating activity within Sydney Harbour (Figure 4.1 C). St.

Ann's Bay was surveyed on July 17, 2008, and was the reference site for Sydney

Harbour; all sediment and mussel samples were collected from identical spots on a gradient from the mussel aquaculture site towards Fader's Point. St. Ann's Bay was similar to Back Bay, the reference site for Saint John Harbour, in that there was a mussel aquaculture site. However, St. Ann's Bay was substantially larger in geographic size.

Halifax Harbour was the busiest of the harbours studied in terms of boating activity and amount of traffic during the time of sample collection on June 6, 2008.

The five locations in Halifax Harbour comprised: Wright's Cove (marina), Sea View

Park (boat ramp), Tuft's Cove (shipyard site), George's Island (adjacent to Halifax

Sea Port, commercial port), and Imperial Oil Terminal (commercial port) (Figure 4.1 90

D). All sediment and mussel samples were collected from matching locations, except at the Imperial Oil Terminal where no mussels were collected. Halifax Harbour has and continues to play a vital role in the Canadian Navy. Wright's Cove (Figure 4.1

D) serviced warships and submarines during World Wars I & II, but has since been converted into a marina, and at a locality north of Halifax Sea Port, Halifax Harbour is the home base of frigates and submarines of the Canadian Navy. St. Margaret's

Bay was the reference site for Halifax Harbour, and sampling was completed on June

6, 2008. Sediment and mussel samples were collected on a gradient from the mouth of a creek in Hubbard's Cove towards Fox Point. Sediment was collected at all sites, but not mussels since pilings were very rare. Attempts were made to collect mussels from boulders and rocks in areas where sediment samples were collected, however, the rocks and boulders were densely covered with algae including Fucus vesiculosus,

Laminaria saccharina, Chorda spp., and Ascophyllum nodosum. Hubbard's Cove is a marina that had a moderate density of pleasure craft docked at the time of sample collection, and Fox Point had a few (n ~ 8) small fishing vessels < 25 m in length at the time of collection. The remaining areas in St. Margaret's Bay were beaches with no evidence of boating activity except for passing pleasure craft.

4.4.3 Butyltin and CHN Analysis

Sediment and mussel tissues were kept frozen (-20 °C) in a dark freezer to avoid butyltin degradation and maintain stability until and after freeze drying

(Caricchia et al., 1994; Quevauviller, 1996). All mussels were measured for total shell length (mm) and total weight (i.e. shell and soft body) after defrosting at room temperature and epibionts removed from their shells. The soft tissues of roughly two 91 to three mussels were chosen randomly from each location, pooled, and placed in labelled plastic sampling bags (n = 1). Pooled mussel tissues and sediment samples were freeze dried in a LABCONO® Freezezone® freeze drier at -80 °C for approximately 48 to 72 hours. Pooled freeze dried M. edulis tissue was homogenised with mortar and pestle, and sediment samples sieved to 63 (am before

placing them in labelled clear sampling bottles for butyltin (TBT, DBT, and MBT) investigation. Samples were kept in a dark freezer (-20 °C) until transport to the

Institut des Sciences de la mer de Rimouski (ISMER) at the University of Quebec, in

Rimouski (Canada) for chemical analysis.

4.4.3.1 Butyltin Extraction - Mussel Samples

The protocol for determining butyltins in tissue and sediment was adapted

from Chau et al. (1997c) with some modifications, and is routinely used by ISMER.

Roughly 0.25 g of freeze dried tissue was digested in 5 ml of tetramethyl ammonium hydroxide (TMAH, 25% H2O) in a 50 ml Teflon® tube. Tissue samples were mixed for approximately 2 min on a vortex mixer, placed in an ultrasonic bath at 60 °C, and sonicated for 1 hr. Certified reference material (CRM) BCR CRM 462, mussel tissue (European Commission), and a procedural blank were treated in similar fashion for every five samples. After sonication, 25 ml of sodium acetate buffered at pH 4.5 (CH3.COONH4> AnalaR®) was added to each Teflon® vial followed by 2 ml saturated NaCl solution, and 2 g NaCl. Organotins were derivatized to an ethylated neutral compound prior to extraction by addition of 600 ^1 of sodium tetraethylborate (NaBeT4, 2% H20). Extraction of ethylated butyltins was accomplished by two separate, but successive additions of 4 ml 1:1 hexane:toluene 92 mixture. After centrifugation, the organic layer was recovered and transferred to a glass round-bottom centrifuge tube. The recovered organic layer (~ 6 ml) was directly treated with 1 g Florisil®, as a first clean-up step, placed on a wrist shaker for 2 min, centrifuged (3,000 rpm, 10 min, IEC HN-SII centrifuge), and the organic

layer recovered.

4.4.3.2 Butyltin Extraction - Sediment Samples

Approximately 0.25 g of freeze dried sediment was acid-digested in 10 ml of

1 M HC1 in a 50 ml Teflon® tube followed by the addition of 5 ml of a

hexane:tropolone (0.1% tropolone) mixture. CRM for sediment samples (PACS-2,

National Reference Council of Canada), and procedural blanks, for every five samples, were treated in similar fashion to sediment samples. Sediment was mixed

for 2 min on a vortex mixer, agitated on a wrist shaker (Burrell® Wrist-Action

Shaker 75) for 1 hour, and centrifuged (3,000 rpm, 30 min, IEC HN-SII centrifuge).

The first organic layer was extracted from each Teflon® tube and transferred to

labelled round-bottom centrifuge tubes. Following centrifugation, 5 ml of 1:1

hexane:tropolone (0.1% tropolone) mixture was added to each Teflon® tube, mixed

for 2 min on a vortex mixer, followed by simulated wrist shaking (10 min, Burrell®

Wrist-Action Shaker 75), and centrifuged (3,000 rpm, 20 min, IEC HN-SII

centrifuge). The second organic layer was recovered, and added to the

corresponding first extraction (~ 10 ml). The combined organic layers were

derivatized directly in the round-bottom centrifuge tubes to an ethylated neutral

compound by adding 2 ml of sodium acetate buffered at pH 4.5 (CH3.COONH4i

AnalaR®) followed by 500 p.1 of sodium tetraethylborate (NaBeT4, 2% H2O). The organic layer was hand shaken for approximately 1 min, followed by simulated wrist shaking (3 min, Burrell® Wrist-Action Shaker 75) and centrifuged (3,000 rpm, 5 min, IEC HN-SII centrifuge).

4.4.3.3 Sample Clean-up and Butyltin Quantification

The last recovered organic layer of each sample type (mussel and sediment) was cleaned on a silica gel micro-column with approximately 0.5 cm of anhydrous sodium sulfate (Na2S04). The organic fraction was eluted into evaporation vials with 1:1 hexane:toluene and collected in a graduated conical tube to a final volume of 10 ml. Before volume reduction to 200 nl under a gentle stream of N2 gas at room temperature, 100 |al of tetrapropyltin was added as the internal standard. The concentrated evaporate was transferred to an amber vial with a glass liner. The organotins were analyzed by gas chromatography linked to an ion trap mass spectrometer (GC-MS, Trace GC-Polaris Q Thermo Finnigan®) equipped with a

Valcobond® VB-5 GC column using helium (He) as the carrier gas.

Butyltin identification was based on the respective mass spectrum and GC retention time. Quantification was performed with reference to the internal standard by Xcalibur®, a software program which manages the Polaris Q® Trace GC.

Recovery of butyltin species was determined with successive extractions (n = 6) of standard CRM for tissue (BCR 462) and sediment (PACS-2) samples. Percent recovery for TBT, DBT and MBT in M edulis was 54% (±5.1), 69% (± 11.1) and

134% (± 21.7), respectively. Recovery of butyltins for sediment samples was: 84%

(± 2.4) for TBT, 65% (± 4.9) for DBT, and 24% (± 5.6) for MBT. TBT, DBT and

MBT concentrations were not corrected for recovery and are reported as tin (ng Sn g"

1 dry wt). Quality assurance for the analytical component was verified by: (1) calibration of GC, (2) use of CRM for sediment and tissue samples, every five samples, and (3) use of procedural blanks (TMAH only) for every five samples. The limit of detection expressed as tin (Sn) atom with one microliter injected was roughly 0.1 ng Sn g'1, dry wt for all butyltins (TBT, DBT and MBT) in sediment samples. For M. edulis tissues, the detection limit was 1 ng Sn g"1 dry wt for DBT and TBT and 2 ng Sn g'1 dry wt for MBT.

4.4.3.4 Carbon, Hydrogen and Nitrogen Analysis

Freeze dried sediment sieved to 63 urn from each location (n = 3) was weighed (1.5 to 3.0 mg) using a microbalance (Mettler Toledo® MX5, max wt. = 5 g) to determine the percentage of carbon on a CHNS analyzer (Costech® Elemental

Combustion System Instruments). Percent carbon (% C) was analyzed by a combustion reaction at 1,000 °C with helium as the carrier gas. CRM, acetanilide, and procedural blanks were processed every 10 samples to calibrate the CHNS analyzer and validate instrument performance as samples were being processed based on the known percentage of carbon (71.03%), hydrogen (6.70%) and nitrogen

(10.36%) in acetanilide. Percent recovery for carbon, hydrogen, and nitrogen were

100.07% (± 0.40), 97.18% (± 2.91), 100.29% (± 0.57), respectively.

4.5 Statistical Analysis

All statistics were performed using SPSS version 17 (a = 0.05). When the assumptions of parametric testing were violated, data were logio transformed, and if logio transformation still violated the assumptions of parametric testing, the non- parametric equivalent was used. One-way analysis of variance (ANOVA) and

Tukey post hoc tests were used to determine if there was a difference in TBT concentration in sediment between sites and site type (harbour vs. reference).

Alternatively, Kruskal-Wallis and Mann-Whitney U (exact sig. 2-tailed) were used to determine if there was a difference in TBT concentration in mussels between sites and site type, respectively. Mann-Whitney was used for the latter analysis because the data set was small, poorly distributed and had many ties (Hinton et al., 2004).

Regression analysis used to establish if there was a relationship between TBT levels in sediment with % C between sites, while Kendall tau-b test was used to assess if there was a correlation between % C and TBT levels in sediment. Fisher's Exact test was used to compare the results of the current study with those of Chau et al. (1997 a, b) to verify if TBT levels in sediment and mussels have declined since 1993/1994, and conclude if the 1989 Canadian regulations have been effective in reducing butyltin levels in the marine environment. Fisher's Exact test was used instead of

Pearson Chi-Square because it is more conservative, and the analysis involved a 2 x

2 table (Hinton et al., 2004). For statistical purposes, when sediment and tissue samples were below the detection limit for chemical analysis, values were assigned the limit of detection. 96

4.6 Results

4.6.1 Current study - 2007/2008 Survey

4.6.1.1 Distribution of Butyltins in Sediment

During the current survey, butyltins (TBT, DBT and MBT) were detected in sediment at all harbour and reference sites, but not at each location within all sites

(Table 4.1). Chemical analysis (n = 31) identified TBT, DBT and MBT in 55% (n =

17), 45% (n = 14), and 26% of sediment samples, respectively. TBT levels in sediment at each location ranged from below the detection limit (< DL) to 9,424 ng

Sn g"1 dry wt (Table 4.1), and four of the highest TBT concentrations detected in sediment samples were discovered at Sydney Harbour (Table 4.1) locations related to boating activity: SYD Port (commercial harbour) > North Sydney Slipway

(shipyard) > Muggah Creek (harbour and steel mill) > Barachois Creek (marina).

Surprisingly in this survey, butyltins were also detected in sediment from all three reference sites. A closer examination of the occurrence of butyltins at reference locations revealed that for the majority of those areas, the presence of butyltins was also correlated with boating activity such as a boat ramp (Back Bay), a ferry terminal

(near to Black Head, St. Ann's Bay), a marina (Hubbard's Cove in St. Margaret's

Bay), and the presence of fishing boats (Back Bay and St. Margaret's Bay).

There was no significant difference between logio TBT in sediment between site type, i.e. harbour and reference (one-way ANOVA: F (1, 29) = 2.327,/? > 0.05), but there was a significant difference in logio TBT in sediment between sites (one­ way ANOVA: F (5, 25) = 3.279, p < 0.05) (Figure 4.2). Differences between logio

TBT in sediment between sites occurred between Saint John Harbour and Sydney 97

Harbour and Sydney Harbour and St. Ann's Bay (Tukey post hoc test). Sydney

Harbour was the only harbour site that had higher butyltin levels in sediment than its related reference site, St. Ann's Bay (Figure 4.2). Regression analysis (data not shown) and Kendall tau-b Test (Kendall tau-b = 0.070, N = 31, p > 0.05) confirm that there was no relationship or correlation between % C and logio TBT concentration in sediment across all sites.

The degree of contamination at all sites (harbour and reference) appears patchy with localised contamination. The level of TBT contamination at each location (n =

1) was determined (Table 4.2) based on a classification scheme from Downson et al.

(1992), which categorised sites based on TBT concentration in sediment. Of the 31 sediment samples analysed, roughly 70% of all samples were classified as uncontaminated (51.6%; n = 16) or lightly contaminated (19.4%; n = 6), while the remaining samples were labelled as moderately contaminated (6.5%; n = 2); highly contaminated (16%; n = 5); and grossly contaminated (6.5%; n = 2), supporting the notion of localised contamination. Generally, locations classified as moderately-, highly- and grossly contaminated were locations related to boating activity e.g. ferry terminal (Indian Beach), commercial harbour (SYD Port, Sydney Harbour), marina

(Hubbard's Cove Marina), and shipyard activity (North Sydney Slipway). Saint

John Harbour and Halifax Harbour had more uncontaminated and lightly contaminated locations that their associated reference sites, Back Bay and St.

Margaret's Bay, respectively (Table 4.2). Sydney Harbour is the only harbour site that had varying levels of contamination in sediment samples ranging from uncontaminated to grossly contaminated (Table 4.2). 98

4.6.1.2 Distribution of Butyltins in Mytilus edulis

The distribution of butyltins in Mytilus edulis was similar to those of sediment samples (Figure 4.3). TBT, DBT and MBT were detected in 46% (n = 13), 39% (n =

11) and 68% (n = 19), of M. edulis samples (n = 28), respectively, from all sites.

TBT concentration in mussels collected from locations within harbour sites ranged

from < DL to 117 ng Sn g"1 (dry wt), and from < DL to 9 ng Sn g"1 (dry wt) at

reference sites (Table 4.1); however, mean maximum TBT levels for each site did

not exceed 50 ng Sn g"1, dry wt. There was a significant difference in TBT (X2 =

14.89, df= 5,p< 0.05), DBT (X2 = 22.67, df = 5,p < 0.001), and MBT (X2 = 21.88,

df = 5, p < 0.001) concentrations in mussels between all sites and site type (U =

24.00 to 28.00, p < 0.01 to 0.001). Based on the mean butyltin concentration (MBT

+ DBT + TBT) in M. edulis, sites are ranked: Sydney Harbour > Halifax Harbour >

Saint John Harbour > St. Margaret's Bay > Back Bay > St. Ann's Bay.

Further analysis of the presence of butyltins in M. edulis at each site showed that TBT, DBT or MBT were detected at all harbour and reference sites (Figure 4.3,

Table 4.1). MBT was the only butyltin detected at all reference sites, while DBT and

TBT were identified in samples from St. Ann's Bay and St. Margaret's Bay,

respectively (Figure 4.3, Table 4.1). Moreover, maximum butyltin levels in mussels

and sediment at individual locations for the entire study were detected at SYD Port

in Sydney Harbour (Table 4.1). TBT or its degradative products, DBT and MBT,

were detected in all mussel samples from Saint John Harbour and Halifax Harbour,

even though butyltin distribution in sediment was patchy (Table 4.1). The degree of

TBT contamination at each site was categorized based on the mean TBT

concentration (n = 5 to 6) in resident M. edulis on a scale adapted from Page and 99

Widdows (1991). Based on this classification, the degree of contamination at all sites is classified as no to low TBT input (0 to 500 ng Sn g"! dry wt.).

4.6.2 Temporal Changes in TBT Concentration (1995 to

2007/2008)

Statistical analysis confirmed that TBT levels at Saint John Harbour and

Halifax Harbour significantly decreased in M, edulis and sediment from 1995 to

2007/2008 (Table 4.3). Mean TBT levels in sediment significantly decreased from

3,212 to approximately 2 ng Sn g'1, dry wt in Halifax Harbour (x2 = 3,214.00, df= 1,

p < 0.0001), and from 1,348 to 6 ng Sn g"1, dry wt (x2= 1,354.00, df= 1 ,p< 0.0001)

in Saint John Harbour. A similar pattern was observed for Mytilus edulis from the same two harbours. TBT levels in M. edulis from Saint John Harbour noticeably

decreased from roughly 39 to 3 ng Sn g"1, dry wt. (x2 = 42.00, df= 1, p < 0.0001),

and likewise decreased from 429 to 41 ng Sn g"1, dry wt. (x2 = 470.00, df= 1, p <

0.0001) in Halifax Harbour. The obvious increase in mean TBT levels in sediment

at Sydney Harbour was different from the other two harbours since TBT

concentration significantly increased from 9 to 2,204 ng Sn g"1, dry wt (x2 =

2,213.00, df- 1, p < 0.0001) during the same period. No statistical analysis was

performed from M. edulis at Sydney Harbour because no tissue levels were reported

in 1995. 100

4.7 Discussion

TBT or its degradative products, DBT and MBT, were found in sediment and

Mytilus edulis at all harbours and their associated reference sites. Data suggest that

TBT levels have significantly declined in sediment at Saint John Harbour and

Halifax Harbour since the 1993/1994 survey, but have significantly increased in

Sydney Harbour during the same time period. The presence of TBT at the majority of locations within sites is related to boating activity, namely harbours and marinas

(Dowson et al., 1992; Michel and Averty, 1991), shipyards (Dowson et al., 1992;

Elgethun et al., 2000), the presence of fishing vessels (Coelho et al., 2002; Santos et al., 2002), ferry terminals (Harino et al., 1999), and boat ramps verifying the input of

TBT into the marine environment even though TBT has been regulated in Canada since 1989. The degree of TBT contamination at all sites was patchy with localised contamination at several locations (Table 4.2).

TBT concentration in sediment at areas suspected of having high TBT input into the marine environment ranged from < DL to 9,424 ng Sn g'1 (dry wt).

However, mean TBT levels in sediment from Saint John Harbour and Halifax harbour were lower than those in Poland (Radke et al., 2008), Rhode Island (Wade et al., 2004), Tunisia (Mzoughi et al., 2005), and the Antarctic (Negri et al., 2004). On the other hand, mean TBT levels at Sydney Harbour are comparable to those recently recorded in Poland (Radke et al., 2008). Further examination of TBT levels in M. edulis collected from locations within harbour and reference sites indicate that TBT is still bioavailable ranging from < DL to 117 ng Sn g'1 (dry wt), but mean (n = 3 - 6) maximum levels for each site did not exceed 50 ng Sn g"1, dry wt. When mean TBT levels in Mytilus edulis at harbour sites in this study are compared with recent 101 comparable studies that used Mytilus spp. as biomonitors, TBT levels in the current study were lower than those in Rhode Island, 977 ng Sn g"1 (Wade et al., 2004) and the Adriatic Sea, 161 - 597 ng Sn g"1 (Boscolo et al., 2004).

The low ubiquitous TBT levels in sediment from Saint John Harbour and

Halifax Harbour, combined with the incidence of higher TBT levels in their paired reference sites Back Bay and St. Margaret's Bay, respectively, was unexpected.

Plausible explanations for the apparent lower TBT concentration in sediments from harbour sites include well flushed areas (Stang and Seligman, 1986), dredging (Stang and Seligman, 1986), low TBT levels in circulating sea water (Saint-Louis et al.,

1997), and decreased boating activity. As it relates to Saint John Harbour, tidal flushing, dredging, and low TBT levels in circulating seawater (Saint-Louis et al.,

1997) as a result of a decline in boating activity are likely contributing factors to the low TBT levels recorded in this study. Saint John Harbour is situated in the Bay of

Fundy, known for the highest tidal difference in the world, and the inability of the

Ekman® grab to touch the seafloor after numerous attempts during field collection coupled with the large tidal difference in the Bay of Fundy lends credence to the likelihood of tidal flushing. In addition, Saint John Harbour has been dredged every year from 2000 to 2008 for maintenance at sites used in this study (Monahan, 2009; personal communication), which would disturb surface sediments with TBT if present from boating activity in the harbour. Moreover, Saint John was once a thriving shipbuilding hub that built nine frigates for the Canadian navy

(Lewandowski, 2008); however, during field collection the shipyard site was non- operational.

It was surprising that the concentrations of TBT detected in sediments from

Halifax Harbour were so low considering that this site was the busiest harbour 102 surveyed in this study, and is the home base to a Canadian naval fleet. Dredging appears the most likely factor for the decline in sediment levels, although this has not been confirmed, and no recent literature could be found reporting TBT levels from dredge spoils. It is assumed that since the naval base and several commercial harbours (Imperial Oil Terminal and Halifax Sea Port) are located in Halifax

Harbour itself, navigational passages would have to be maintained to ensure proper maneuverability in and out of the harbour. In addition, tidal flushing may have also contributed to the lower detected TBT levels in sediment samples similarly to Saint

John Harbour.

The noticeably higher TBT levels in sediment samples from Sydney Harbour,

Back Bay and St. Margaret's Bay can be attributed to restricted tidal flushing (Stang and Seligman, 1986) and possible current use. Currently, TBT can enter the marine environment of Canada from vessels > 25 m in length, which under the 1989 regulations is legal; and from visiting vessels that have their hulls protected with

TBT antifouling paint. The high TBT levels recorded in Sydney Harbour can be attributed to one or both of these pathways since Sydney Harbour is a commercial harbour. On the other hand, Back Bay and St. Margaret's Bay are not commercial harbours and they do not appear to be capable of accommodating vessels > 25 m in length (personal observation), but an alternative explanation for the high levels observed in this study is the presence of fishing vessels. In a study conducted in

1992/1993, the highest organotin levels recorded in water, sediment and bivalves came from Olhao, a site described as the most important fishing harbour along

Portugal's southern coast (Coelho et al., 2002). In a different study using Nucella lapillus as a bioindicator in 2000/2001 to determine the effectiveness of the EU regulations on TBT, Santos et al. (2002) attributed the increased severity of imposex 103 at fishing harbour sites to the presence of fishing vessels generally < 25 m in length and from pleasure craft, suggesting illegal TBT use. Illegal TBT use on vessels < 25 m in length has been reported in the UK where researchers concluded that the presence of TBT in water samples from a marina site opened in 1988 was the result of illegal use of TBT in antifouling paint because the UK passed legislation restricting TBT's use on vessels < 25 m in length in 1986 and 1987 (Waite et al.,

1991). Moreover, Maguire and Batchelor (2005) concluded that the 1989 Canadian regulations on TBT were not effective in reducing TBT contamination in sediment in small-craft marinas and harbours from a 1999 survey; a similar observation determined for this study. The possibility of illegal TBT use in Canada should be investigated.

The sorption coefficient (K) values for aqueous TBT on sediment and particulate matter suggest that there is a correlation between TBT and total organic carbon (TOC) (Unger et al., 1987), and high TBT levels in sediment were related to fine-grained sediments rich in organic matter % C (Kram et al., 1989; Burton et al.,

2004). Data and statistical analysis from this study did not find a correlation between TBT levels in sediments and % C. The obviously low % C data (0.56 to

10.81 % C) at the majority of sites in this study coupled with the ubiquitous low

TBT levels in sediment may be a confounding factor for the non-correlation between

% C and TBT. In addition, there are other factors reported in the literature responsible for controlling the sorption of TBT to sediments. Some studies have suggested that the sorption of TBT in sediments is controlled by the concentration of butyltins in the overlying water column (Kram et al., 1989), electronic properties of the Sn atom (Sun et al., 1996), nature of the organic matter in sediments (Grathwohl,

1990), pH (Hoch et al., 2002), and salinity (Hoch et al., 2002). Even though TBT 104 concentrations in sediment from harbour and reference sites were not correlated with

% C in this study, TBT is expected to remain in the marine environment of Atlantic

Canada for some time to come based on TBT's behavior in sediment, namely: (1) moderate persistence (Maguire, 1987); (2) an estimated half-life of roughly three months to two and a half years (de Mora et al., 1995; Watanabe et al., 1995), slower degradation in cold environments (Viglino et al., 2004), and reversible sorption of

TBT in sediments (Unger et al., 1987; Kram et al., 1989) enabling sediments to act as both a source and a sink.

A comparison of the data from this study concluded in 2007/2008 approximately 20 years after the 1989 Canadian regulations, with the survey completed in 1993/1994 by Chau et al. (1997a, b) roughly 15 years ago, confirms a decline in TBT concentration in sediment and mussels at Saint John Harbour and

Halifax Harbour, but an increase in sediment only at Sydney Harbour during the same time period. The 1989 Canadian regulations on organotins have been somewhat effective in reducing TBT input into the marine environment of Atlantic

Canada, but not curtailing its bioavailability or biological effects. This conclusion is drawn based on the following evidence: (1) the correlation between TBT levels in N. lapillus and imposex severity, as measured by mean female penis length and vas deferens formation, at Saint John Harbour and Halifax Harbour (Chapter 3) even though the sediment concentrations have declined significantly since 1993/1994; (2) the obvious increase in TBT levels in sediment at Sydney Harbour which increased significantly from 8.8 to 2,204.38 ng Sn g"'(dry wt); (3) moderate contamination of

TBT in sediment at marinas and fishing harbours with vessels < 25 in length which suggests probable illegal TBT use; and (4) the presence of butyltins in 100% of mussel samples collected from locations in Saint John Harbour associated with 105 boating activity although the TBT concentration of associated sediment was < DL at the same locations. Passing of the 1989 Canadian regulations on TBT coupled with dredging, decreased boating activity, and tidal flushing have been effective in reducing TBT levels in Saint John Harbour and Halifax Harbour, but this has not been the same for Sydney Harbour.

4.8 Conclusion

TBT or its degradative products, DBT and MBT, were detected in sediment and resident M. edulis during a survey conducted in 2007/2008 roughly 20 years after the 1989 Canadian regulations. Although TBT levels in sediment and mussels have declined significantly in Saint John Harbour and Halifax Harbour, the 1989

Canadian regulations have not been completely effective in curtailing the input of

TBT into the marine environment in harbours and small-craft areas or the bioavailability of TBT to non-target organisms as is evident by the presence of TBT in resident M. edulis and imposex-affected N. lapillus. In this study, the degree of

TBT contamination at all sites was "patchy" with localised contamination at several locations. Physical and anthropogenic factors that may help explain the differences in TBT levels as seen in harbour and reference sites include an increase or decrease in tidal flushing, dredging, decreased boating activity, low ambient TBT levels in surrounding water, presence of fishing vessels, and probable illegal use. Since TBT has a moderate persistence in sediment, degrades slower in cold environments, and sediments can act as both a sink and a source, it appears that TBT and the effects of

TBT will remain in the marine environment of Atlantic Canada for several more years to come. 106

4.9 References

Alzieu, C. 1986. The detrimental effects on oyster culture in France - evolution since antifouling paint regulation. In Proceedings of the Oceans '86 - Organotin Symposium. IEEE: Washington, DC, USA, Sept. 23 - 25, vol. 4, 1130- 1134.

Alzieu, C. 1991. Environmental problems caused by TBT in France: assessment, regulations, prospects. Mar Environ Res. 32: 7 -17.

Agriculture Canada. 1989. Antifouling paints for ship hulls. Canadian Association of Pesticide Control Officials Note 89-02, Ottawa

Blaber, S. J. M. 1970. The occurrence of a penis-like outgrowth behind the right tentacle in spent females of Nucella lapillus (L.). Proc Malacol Soc Lon. 39: 231 -233.

Boscolo, R., Cacciatore, F., Berto, D., Marin, M. G., and Giani, M. 2004. Contamination of natural and cultured mussels (Mytilus galloprovincialis) from the northern Adriatic Sea by tributyltin and dibutyltin compounds. Appl Organomet Chem. 18: 614 -618.

Burton, E. D., Phillips, I. R., and Hawker, D. W. 2004. Sorption and desorption behaviour of tributyltin with natural sediments. Environ Sci Technol. 38: 6694 - 6700.

Caricchia, A. M., Chiavarini, S., Cremisini, C., Morabito, R. and Scerbo, R. 1994. Influence of storage conditions on the determination of organotin in mussels. Anal Chim Acta. 286: 329 - 334.

Chau, Y. K., Maguire, R. J., Brown, M., Yang, F., Batchelor, S. P., and Thompson, J. A. J. 1997a. Occurrence of butyltin compounds in mussels in Canada. Appl Organomet Chem. 11: 903 - 912.

Chau, Y. K., Maguire, R. J., Brown, M., Yang, F., and Batchelor, S. P. 1997b. Occurrence of organotin compounds in the Canadian aquatic environment five years after the regulation of antifouling uses of tributyltin. Water Qual Res J Can. 32(3): 453-521.

Chau, Y.K., Yang, F., and Brown, M. 1997c. Evaluation of derivatization techniques for the analysis of organotin compounds in biological tissue. Anal Chim Acta. 338: 51 - 55.

Coelho, M. R., Bebianno, M. J., and Langson, W. J. 2002. Organotin levels in the Ria Formosa lagoon, Portugal. Appl Organomet Chem. 16: 384 - 390.

Cossa, D. 1989. A review of the use of Mytilus spp. as quantitative indicators of cadmium and mercury contamination in coastal waters. Oceanol Acta. 12 (4): 417-432. 107 de Mora, S. J., Stewart, C., and Phillips, D. 1995. Sources and rate of degradation of tri(w-butyl) tin in marine sediments near Auckland, New Zealand. Mar Pollut Bull. 30: 50 - 57.

Dowson, P. H., Bubb, J. M., and Lester, J. N. 1992. Organotin distribution in sediments and waters of selected east coast estuaries in the UK. Mar Pollut Bull. 24 (10): 492 - 498.

Dowson, P. H., Bubb, J. M., and Lester, J. N. 1993. Temporal distribution of organotins in the aquatic environment: Five years after the 1987 UK retail ban on TBT based antifouling paint. Mar Pollut Bull. 26 (9): 487 - 494.

Elgethun, K., Neumann, C., and Blake, P. 2000. Butyltins in shellfish, finfish, water and sediment from the Coos Bay estuary (Oregon, USA). Chemosphere. 41: 953 - 964.

Goldberg, E. D. 1975. The Mussel Watch - A first step in global marine monitoring. Mar Pollut Bull. 6: 111.

Grathwohl, P. 1990. Influence of organic matter from soils and sediments from various origins on the sorption of some chlorinated aliphatic hydrocarbons: Implications on Koc correlations. Environ Sci Technol. 24 (11): 1687 - 1693.

Griffith, G. W., and Castagna, M. 1962. Sexual dimorphism in oyster drills of Chincoteagne Bay, Maryland, Virginia. Chesapeake Sci. 3: 215 - 217.

Harino, H., Fukushima, M. and Kawai, S. 1999. Temporal trends of organotin compounds in the aquatic environment of the Port of Osaka, Japan. Environ Pollut. 105: 1 - 7.

Hinton, P. R., Brownlow, C., McMurray, I. and Cozens, B. 2004. SPSS explained. East Sussex, United Kingdom, 377 pp.

Hoch, M., Alonso-Azcarate, J., and Lischick, M. 2002. Adsorption behaviour of toxic tributyltin to clay-rich sediments under various environmental conditions. Environ Toxicol Chem. 21 (7): 1390 - 1397.

Houston, R. S. 1971. Reproductive biology of Thais emarginata (Deshayes, 1939) and Thais canaliculata (Duclos, 1832). Veliger. 13: 348 - 357.

Huggett, R., J., Unger, M. A., Seligman, P. F., and Valkris, A. O. 1992. The marine biocide tributyltin. Assessing and managing the environmental risks. Environ Sci Technol. 26: 232 - 237.

Jenner, M. G. 1979. Pseudohermaphroditism: A newly recognized sexual phenomenon in Ilyanassa obsoleta and other neogastropods. PhD thesis, University of Chapel Hill, North Carolina, USA. 108

Kan-Atireklap, S., Tanabe, S. and J. Sanguansin. 1997. Contamination by butyltin compounds in sediments of Thailand. Mar Pollut Bull. 34: 894 - 899.

Kram, M. L., Stang, P. M., and Seligman, P. F. 1989. Adsorption and desorption of tributyltin in sediments of San Diego Bay and Pearl Harbour. Appl Organomet Chem. 3: 523 - 536.

Langston, W. J., Burt, G. R., and Mingjiang, Z. 1987. Tin and organotin in water, sediments, and benthic organisms of Poole Harbour. Mar Pollut Bull. 18: 634 - 639.

Lewandowski, J. Halifax, B. C. yards win frigate refit contracts. Telegraph Journal. 2008. Apr 22; Sect. B: B1 - B2.

Maguire, R. J. 1986. Review of the occurrence, persistence and degradation of tributyltin in freshwater ecosystems in Canada. In Proceedings of the Oceans '86 - Organotin Symposium. IEEE: Washington, DC, USA, Sept. 23 - 25, vol. 4, 1252- 1255.

Maguire, R. J. 1987. Environmental aspects of tributyltin (Review). Appl Organomet Chem. 1: 475 - 498.

Maguire, R. J., Tkacz, R. J., Chau, Y. K., Bengert, G. A., Wong, P. T. S. 1986. Occurrence of organotin compounds in water and sediment in Canada. Chemosphere. 15: 253 - 274.

Maguire, R. J. and Batchelor, S. P. 2005. Survey for Tributlyin in water and sediment in Canada in 1999. Water Qual Res J Canada. 40 (4): 431 - 447.

Michel, P. and Averty, B. 1991. Tributyltin analysis in seawater by GC FPD after direct aqueous-phase ethylation using sodium tetraethlyborate. Appl Organomet Chem. 5: 393 - 397.

Monahan, M. 2009. Personal communication. Saint John Port Authority.

Moore, D. R. J., Noble, D. G., Walker, S. L., Trotter, D. M., Wong, M. P. and Pierce, R. C. 1992. Canadian water quality guidelines for organotins. Water Quality Branch, Environment Canada, Ottawa.

Mzoughi, N., Lespes, G., Bravo, M., Dachraoui, M., Potin-Gautier, M. 2005. Organotin speciation in Bizerte lagoon (Tunisia). Sci Tot Environ. 349: 211 -222.

Negri, A. P., Hales, L. T., Battershill, C., Wolff, C., and Webster, N. S. 2004. TBT contamination identified in Antarctic marine sediments. Mar Pollut Bull. 48: 1142- 1144.

Page D. S., and Widdows, J. 1991. Temporal and spatial variation in levels of alkyltins in mussel tissues: A toxicological interpretation of field data. Mar Environ Res. 32: 113-129. 109

Poli, G., Salvat, B., and Streiff, W. 1971. Aspect particulier de la sexualite chez Ocenebra erinacea. Haliotis. 1: 29 - 30.

Prouse, N. J., and Ellis, D. V. 1997. A baseline survey of dogwhelk (Nucella lapillus) imposex in Eastern Canada (1995) and interpretation in terms of tributyltin (TBT) contamination. Environ Technol. 18: 1255 - 1264.

Quevauviller, Ph. 1996. The analysis of butylated tin compounds in the environment and in biological materials. In Tributyltin: Case study of an environmental contaminant. Eds. S. J. de Mora. Cambridge University Press, Great Britian. pp 62 - 93.

Radke, B., L^czynski, L., Wasik, A., NamieSnik, J., and Bolalek, J. 2008. The content of butyl- and phenyltin deratives in the sediement from Port of Gdansk. Chemosphere. 73:407 - 414.

Ramaswamy, B. R., Tao, H., Hojo, M. 2004. Contamination and biomethylation of Organotins compounds in pearl/fish culture areas in Japan. Anal Sci. 20: 45 - 53.

Saint-Louis, R., Gobeil, C., and Pelletier, £. 1997. Tributyltin and its degradation Products in the St. Lawrence Estuary (Canada). Environ Pollut. 18: 1209 - 1218.

Santos, M. M., Ten Hallers-Tjabbes, C. C., Santos, A. M., and Vieira, N. 2002. Imposex in Nucella lapillus, as a bioindicator for TBT contamination: re- survey along the Portuguese coast to monitor the effectiveness of EU regulation. J Sea Res. 48: 217 - 223.

Salazar, M. H., and Salazar, S. M. 1995. In Situ bioassays using transplanted mussels: I. Estimating chemical exposure and bioeffects with bioaccumulation and growth. Environmental Toxicology and Risk Assessment - Third Volume, ASTM STP 1218, Jane S. Hughes, Gregory R. Biddinger, and Eugene Mones, Eds., American Society for Testing and Materials, Philadelphia, pp. 216 - 241.

Smith, B. S. 1971. Sexuality in the American mud-snail, Nassarius obsoletus. Proc Malacol Soc Lond. 391: 377 - 378.

Smith, B. S. 1981. Male characteristics on female mud snails caused by antifouling bottom paints. J Appl Tox. 1 (1): 22-25.

Smolders, R., Bervoets, L., Wepener, V., and Blust, R. 2003. A conceptual framework for using mussels as biomonitors in whole effluent toxicity. Hum Ecol Risk Assess. 9 (3): 741 - 760. 110

Stang, P. M., and Seligman, P. F. 1986. Distribution and fate of butyltin compounds in the sediments of San Diego Bay. In Proceedings of the Organotin Symposium of the Oceans '86 Conference, Washington, DC, USA, Sept. 23- 25, 1986, pp. 1256- 1261.

Stewart, C. 1996. The efficacy of legislation in controlling tributyltin in the marine environment. In de Mora SJ, ed, Tributyltin: Case Study of an Environmental Contaminant. Cambridge University Press, New York, NY, USA, pp 264 - 297.

Sun, H., Huang, G., and Dai, S. 1996. Adsorption behaviour and QSPR studies of organotins compounds on estuarine sediment. Chemosphere. 33 (5): 831 - 838.

Unger, M. A., Maclntyre, W. G., and Huggett, R. J. 1987. Equilibrium sorption of tributyltin chloride by Chesapeake Bay sediment. In Proceedings of the Organotin Symposium of the Oceans '87 Conference, Halifax, NS, Canada, Sept. 29 - Oct. 1,1987, pp. 1348 - 1351.

Viglino L., Pelletier E., St Louis, R. 2004. Highly persistent butyltins in northern marine sediments: a long term threat for the Saguenay Fjord (Canada). Environ Toxicol Chem. 23: 2673 - 2681.

Viglino, L., Pelletier £., Lee, L. E. J. 2006. Butyltin species in benthic and pelagic organisms of the Saguenay Fjord (Canada) and imposex occurrence in common whelk (Buccinum undatum). Arch Environ Con Tox. 50: 45 - 59.

Vos, J. G., Dybing, E., Greim, H. A., Ladefoged, O., Lambre, C., Tarazona, J. V., Brandt, I., and Vethaak, A. D. 2000. Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit Rev Toxicol. 30: 71 - 133.

Wade, T. L., Sweet, S. T., Quinn, J. G., Cairns, R. W., and King, J. W. 2004. Tributyltin in environmental samples from the former Derecktor shipyard, Coddington Cove, Newport RI. Environ Pollut. 129: 315 - 320.

Waite, M. E., Waldock, M. J., Thain, J. E., Smith, D. J. and Milton, S. M. 1991. Reductions in TBT concentrations in UK estuaries following legislation in 1986 and 1987. Mar Environ Res. 32: 89 - 11.

Watanabe, N., Sakai, S., and Takatusuki, H. 1995. Release and degradation half lives of tributyltin in sediment. Chemosphere. 31 (3): 2809 - 2816. Ill

Rodney Container ^Terminal Gulf of St. Lawrence

Prince Edwni

Saint John Harbour

WrlghfsCove

Sydney Harbour

Imperial Oil Terminal

Halifax Harbour

Figure 4.1. (A - D). (A) Location of harbour (•) and associated reference (•) sites sampled between 2007 and 2008 in Atlantic Canada. Harbour sites included: (B)

Saint John Harbour, New Brunswick (NB); (C) Sydney Harbour, Nova Scotia (NS); and (D) Halifax Harbour, Nova Scotia (NS). 112

iSedirrtert [MBT]

SJH BB HH SMB SH SAB Site

Figure 4.2. Logio butyltin concentration reported as ng Sn g"1 (dry wt), in sediment samples collected from harbour and associated reference sites in Atlantic Canada during 2007 and 2008. New Brunswick sites: SJH = Saint John Harbour and BB =

Back Bay; Nova Scotia sites: SH = Sydney Harbour, SAB = St. Ann's Bay, HH =

Halifax Harbour, and SMB = St. Margaret's Bay. Asterisks (*) and (•) are outliers. 113

120" •Mussel [MBT] * •Mussel [DBTJ * ^ •Mussel [TBT] s 100- f (0 ? 801 S

60"

£ 40" c

a 20-

0-

"sJH bT "5T SMB SH SAB Site

Figure 4.3. Butyltin concentration reported as ng Sn g"1 (dry wt), in resident blue mussels {Mytilus edulis) collected from harbour and associated reference sites in

Atlantic Canada collected during 2007 and 2008. New Brunswick sites: SJH = Saint

John Harbour and BB = Back Bay; Nova Scotia sites: SH = Sydney Harbour, SAB =

St. Ann's Bay, HH = Halifax Harbour and SMB = St. Margaret's Bay. Asterisks (*) and (•) are outliers. Table 4.1. Boating activity and butyltin (TBT, DBT and MBT) concentration (n = 1), reported as ng Sn g"1 (dry wt), in sediment and resident blue mussels {Mytilus edulis) at selected harbours and associated reference sites in Atlantic Canada collected in 2007/2008.

Boating activity designated as: boat ramp (BR), commercial port (CP), cruise ship terminal (CST), ferry terminal (FT), fishing boats

(FB), marina (MA), and shipyard (SY).

Longwharf Terminal 45" 16.57 N; 66" 04.19 W FB; CST < DL < DL < DL 25 13 6 Rodney Container Terminal 45' 15.01 N; 66' 02.83 W CP < DL < DL < DL 8 < DL 8 Saint John Princess of Acadia 45' 15.19 N; 66' 03.85 W FT 129 < DL < DL 9 < DL < DL Harbour u Harbour Pugsley Terminal 45' 14.02 N; 66" 05.06 W n.s n.s n.s 11 < DL < DL § (/» Tin Can Beach 45' 16.28 N; 66' 03.85 W CST < DL 3.62 7.41 7 < DL < DL 3 k. Partridge Island 45' 15.78 N; 66' 03.25 W CST < DL < DL < DL n.s n.s n.s 00 Dock # 1 45' 15.78 N; 66' 51.25 W BR 215 129 < DL < DL < DL 3 86 <3J Dock # 2 45' 03.04 N; 66' 51.73 W < DL < DL < DL 5 < DL < DL z Reference Back Bay Dock # 3 45'03.37 N; 66'51.82 W FB 77 308 154 < DL < DL < DL Breakwater 45' 03.26 N; 66' 51.70 W 129 < DL < DL < DL < DL < DL Navigation Marker 45' 03.02 N; 66' 51.69 W < DL < DL < DL 12 < DL < DL ' South East Bar 46" 08.38 N; 60' 11.83 W < DL < DL < DL 26 17 14 Indian Beach 46" 12.73 N; 60' 14.41 W FT < DL < DL 79.2 < DL 11 6 Sydney North Sydney Slipway 46' 12.47 N; 60" 15.02 W SY 83 1,733 3,013 21 43 93 Harbour Harbour Barachois Creek 46' 09.66 N; 60' 13.26 W MA < DL 306 267 22 20 47 Muggah Creek 46' 09.84 N; 60" 12.21 W < DL 282 443 11 12 18 SYD Port 46" 09.44 N; 60' 13.17 W CP 201 2,819 9,424 28 44 117 Mussel Farm (Upper) 46' 14.26 N; 60' 34.35 W < DL < DL < DL < DL < DL < DL Mussel Farm (Lower) 46" 15.50 N; 60' 33.53 W < DL < DL < DL 7 < DL < DL St. Ann's m Reference English Town 46' 17.09 N; 60" 32.56 W BR < DL < DL < DL < DL 9 < DL Bay uo Black Head 46" 18.08 N; 60' 31.61 W FT < DL < DL 156 5 < DL < DL Fader's Point 46" 18.53 N; 60" 30.85 W < DL < DL < DL < DL < DL < DL ro > Wright's Cove 44" 42.10 N; 63* 37.06 W MA 0.85 5.56 9.27 26 18 23 o z Seaview Park 44' 40.58 N; 63' 36.90 W BR < DL < DL 7.41 26 23 39 Halifax Harbour Tuft's Cove 44" 40.40 N; 63" 35.65 W SY < DL 1.88 7.69 68 46 60 Harbour George's Island 44' 38.55 N; 63' 33.61 W CP < DL < DL 2.52 71 50 41 Imperial Oil Terminal 44' 38.09 N; 63" 32.03 W CP < DL 1.76 3.99 n/a n/a n/a Hubbard's Cove - Head 44' 63.65 N; 64" 05.28 W < DL 4.96 2.93 33 < DL 9

St. Hubbard's Cove - Marina 44" 63.75 N; 64" 05.88 W MA 19.8 29.6 74.1 < DL < DL < DL Reference Margaret's Outer Cove #1 44' 63.57 N; 64" 06.07 W < DL 3.43 < DL n/a n/a n/a Bay Outer Cove #2 44' 62.86 N; 64' 05.02 W < DL < DL < DL n/a n/a n/a Fox Point 44' 61.36 N; 64' 05.81 W FB < DL 13.4 5.15 < DL < DL < DL n.s = no sample DL for all butyltins (TBT, DBT and MBT) in sediment samples = ~ 0.1 ng Sn g"1, dry wt. DL for MBT in M. edulis samples = 2 ng Sn g"1, dry wt, and 1 ng Sn g"1, dry wt for DBT and TBT 116

Table 4.2. Classification of TBT pollution at harbour and reference locations (n = 1)

based on TBT concentration in sediment (n = 1) reported as ng Sn g"1 (dry wt) according to Dowson et al. (1992): < 3 = uncontaminated; 3 to 20 = lightly contaminated; 20 to 100 = moderately contaminated; 100 to 500 = highly contaminated; > 500 grossly contaminated.

Longwharf Terminal FB; CST < DL Uncontaminated

Rodney Container Terminal CP < DL Uncontaminated

Saint John Harbour Princess of Acadia FT < DL Uncontaminated

Pugsley Terminal (Harbour) Tin Can Beach CST 7.41 Lightly contaminated

Partridge Island CST < DL Uncontaminated

Dock # 1 BR 129 Highly contaminated

Dock # 2 < DL Uncontaminated Back Bay Dock #3 FB 154 Highly contaminated (Reference) Breakwater < DL Uncontaminated

Navigation Marker < DL Uncontaminated

South East Bar < DL Uncontaminated

Indian Beach FT 79.2 Moderately contaminated

Sydney Harbour North Sydney Slipway SY 3,013 Grossly contaminated

Barachois Creek MA 267 Highly contaminated (Harbour) Muggah Creek 443 Highly contaminated

SYD Port CP 9,424 Grossly contaminated

Mussel Farm (Upper) < DL Uncontaminated

Mussel Farm (Lower) < DL Uncontaminated St. Ann's Bay English Town BR < DL Uncontaminated (Reference) Black Head FT 156 Highly contaminated

Fader's Point < DL Uncontaminated

Wright's Cove MA 9.27 Lightly contaminated

Seavlew Park BR 7.41 Lightly contaminated Halifax Harbour Tuft's Cove SY 7.69 Lightly contaminated (Harbour) George's island CP 2.S2 Uncontaminated

Imperial Oil Terminal CP 3.99 Lightly contaminated 117

Hubbard's Cove - Head 2.93 Uncontaminated

Hubbard's Cove - Marina MA 74.1 Moderately contaminated St. Margaret's Bay Outer Cove #1 < DL Uncontaminated (Reference) Outer Cove #2 < DL Uncontaminated

Fox Point FB 5.15 Lightly contaminated DL for all butyltins (TBT, DBT and MBT) in sediment samples = ~ 0.1 ng Sn g", dry wt. Table 4.3. Mean TBT concentration ± standard deviation reported as ng Sn g"1 (dry wt.) comparing the results of a 1995 butyltin survey by Chau et al.

1997 (a, b) with the current survey using Fisher's Exact test.

Mussel 38.75 ± 25.50 3.4013.36 X2 = 42.00, df=l, p<0.001 Saint John Harbour Sediment 3,212.001228.10 1.56 + 3.27 X2= 3,214.00, df=1, p < 0.001 Mussel 429.17 ± 392.74 40.75115.15 X2 = 470.00, df= 1, p < 0.001 Halifax Harbour Sediment 1,347.70 ± 163.80 6.1812.81 X2= 1,354.00, df=l,p<0.001 Mussel Not reported 49.17146.04 n/a Sydney Harbour Sediment 8.8 ± 2.30 2,204.35 1 3,715.05 X2 = 2,213.00, df= 1, p < 0.001 n/a = not applicable 119

5 The Evolution of Masculinised Female

Gastropods: What Do We Know After 40+ Years?

5.1 Abstract

Female masculinisation (FM) in gastropods (imposex, intersex and ovo-testis) has been documented in > 260 species globally. Biomonitoring studies use FM to indirectly assess the presence of tributyltin (TBT), a once popular biocide in antifouling paint, which has since been regulated. Laboratory studies confirm that

FM in gastropods can be induced not only by TBT, but other tin (Sn) compounds, Sn

"cocktails", steroids, and other contaminants such as Aroclor 1260. Even though

FM in gastropods in the field has been attributed to TBT from boating activity, there is evidence that imposex occurred prior to the advent use of TBT in the 1960s, and for some species imposex occurs naturally. There are 42 species that do not elicit a

FM response even if they are exposed to TBT under laboratory conditions, collected sympatrically with other species displaying FM, and/or have TBT in their tissues. A geographic analysis of field studies in four regions indicates that the bulk of studies were conducted in Europe > Asia and Oceania > North America > Latin America.

More data are needed regarding FM in Africa, the Middle East, and Latin America since data regarding the masculinisation of female gastropods in these areas are lacking. The most studied gastropod species used in TBT biomonitoring studies is the dogwhelk, Nucella lapillus. This review summarizes the progression of the literature from 1962 to 2009 outlining how the terminology, science, and theories have evolved over time. 120

5.2 Effects of Organotins on Female Gastropods

Invertebrates are used globally in environmental monitoring programs to measure pollutant levels and the effects of these pollutants on organisms. Some standardized environmental monitoring programs include the global Mussel Watch programme (Goldberg, 1975), the Environmental Effects Monitoring (EEM) programs for metal mining (Environment Canada, 2002) and pulp and paper mills

(Environment Canada, 2005) in Canada, and the Joint Assessment and Monitoring

Programme (JAMP) in Europe (OSPAR, 2008). Molluscs are used globally in ecotoxicology studies because they are common, highly visible, ecologically and commercially globally important, researchers have the ability to work with all life history stages, and the adults have a sedentary or sessile life history (Rittschof and

McClellan-Green, 2005).

Tributyltin (TBT), a once popular biocide in antifouling paint, is a known endocrine disruptor in two classes of molluscs, Bivalvia (bivalves) and Gastropoda

(gastropods). In 1979 the first sign that TBT was having an adverse effect on non- target organisms came from France's Arcachon Bay (Alzieu et al., 1980). TBT was linked as the causative agent to abnormal shell thickening, also known as

"chambering" or "balling" in the commercial Pacific oyster, Crassostrea gigas,

(Alzieu, 1986), a bivalve. TBT also acts as an endocrine disruptor in female prosobranch gonochoristic gastropods (Matthiessen and Gibbs, 1998), and three types of masculinisation have been identified thus far. Two types of female masculinisation in gastropods are noticeable and well-known pseudohermaphroditism (Jenner, 1979; Fioroni et al., 1990) or imposex (Smith,

1971), and intersex (Oehlmann et al., 1994a), but the third, ovo-testis, is 121 inconspicuous and has only been described by histological analysis (Gibbs et al.,

1988; Oehlmann et al., 1996c; Horiguchi et al., 2000, 2004, 2005; Sloan and

Gagnon, 2004). Although TBT was first linked to chambering in C. gigas, the responses of female gastropods to butyltins have been regarded as the most complete example of endocrine disruption caused by an environmental contaminant (Vos et al., 2000). Since there are three different types of masculinisation that are known to occur in female gastropods in response to organotin exposure, I will use the term female masculinisation (FM) when describing all three, and the individual names of each type where appropriate. Quantitatively, the literature on the effects of TBT on female gastropods in field studies (n = 416) has been dominated by imposex (n =

343) > intersex (n = 39) > ovo-testis (n = 34).

In imposex-affected females, the entire female genital system is conserved, but superimposed by male organs, penis and/or vas deferens as seen in dogwhelks,

Nucella lapillus (Matthiessen et al., 1999). On the other hand, in intersex-affected females, such as the periwinkle, Littorina littorea, the pallial organs are modified towards male morphological structures in the early stages that will eventually supplant the corresponding male formation, i.e. prostate gland, in higher stages

(Matthiessen et al., 1999). In the last type of FM, ovo-testis, female gastropods were classified as masculine because oogenesis was suppressed and/or had spermatogenesis and/or seminiferous tube-like structures. This phenomenon was first observed in N. lapillus exposed to TBT under laboratory conditions from hatchlings to maturity for roughly two years (Gibbs et al., 1988). Some of the primary species in which ovo-testis has been detected include: the sharp dwarf winkle, Ocinebrina aciculata (Oehlmann et al., 1996c); the reticulated nassa,

Nassarius reticulatus (Barroso and Moreira, 1998); three species of abalone: Haliotis 122 madaka (Horiguchi et al., 2000, 2004, 2005), H. gigantea (Horiguchi et al., 2001,

2002), and H. roei (Sloan and Gagnon, 2004); and in the ivory shell, Babylonia japonica (Horiguchi et al., 2006b).

The following review constitutes a thorough analysis of the literature (n =

587) (Appendix) on the effects of organotins on female gastropods spanning a 47- year period (1962 to 2009) that extends across four geographic regions consisting of

Europe, Asia and Oceania, North America and Latin America. It includes an updated list of 269 species in 33 families of gonochoristic gastropods affected by

FM; an original unaffected list of 42 species in 20 families; a synopsis of conventional (n = 10) and alternative (n = 11) indices used to measure incidence and severity; a summary of individual compounds and "cocktails" (n = 31) known to induce FM in gastropods; and an outline of the progression of theories as they developed that are used to explain the induction of FM in gastropods. In addition, controversial evidence from museum samples collected prior to the 1960s when there was an increased use of TBT as a biocide in antifouling paint, and the occurrence of "natural" imposex in some species demonstrates that imposex does occur in some species without exposure to tin (Sn) compounds.

5.3 Literature Review from 1962 to 2009 ~ Global Trends

5.3.1 Literature Distribution

A review of the literature related to FM (imposex, intersex, and ovo-testis) in gastropods published from 1962 to 2009 was divided into seven groups: (1) abstracts/meetings (including conference proceedings); (2) articles; (3) editorials

(including letters and notes); (4) reviews/book chapters; (5) reports; (6) risk 123 assessments; and (7) theses. The published literature was dominated by articles (n =

436) > reviews and/or book chapters (n = 67) > abstracts/meetings (n = 46) > theses

(n = 15) > reports (n = 14) > editorials/letters/notes (n = 6) > risk assessment (n = 3)

(Figure 5.1). Further analysis of published articles revealed that the bulk of articles were published in 11 journals (Figure 5.2), and the most popular journals included:

Marine Pollution Bulletin (n = 72) > Marine Environmental Research (n = 43) >

Journal of the Marine Biological Association of the United Kingdom (n = 30) >

Marine Ecology Progress Series (n = 18).

The first publication reporting the incidence of male-like characteristics in female gastropods was completed in the early 1960s (Griffith and Castagna, 1962).

Following this initial publication, there were two intermittent lulls in the 1970s

(Figure 5.3), but by the late 1980s studies focusing on FM in gastropods were becoming well established, reaching a climax in 2006 (Figure 5.3). Literature from the 1960s to mid 1970s focused on the incidence of male characteristics in female gastropods, mainly in Europe and the United States. By the late 1970s and early

1980s studies by Feral (1979; 1980a), Smith (1981b; c) and Feral and Le Gall (1982;

1983) were investigating the causative agent(s) and mechanism of induction, respectively. Subsequent literature advanced knowledge of:

(1) the discovery of masculinisation of other gastropods on a global scale;

(2) use of imposex or pseudohermaphroditism in female gastropods as

bioindicators or biomonitors of organotin pollution adjacent to areas of

boating activity (e.g. harbours, marinas, shipyards, moorings etc.);

(3) establishment of indices to measure FM incidence and severity; 124

(4) the delineation of the different types of masculinisation in female

gastropods (i.e. imposex, intersex and ovo-testis); and

(5) theories to explain the induction mechanism of FM, and potential risk(s)

to humans via consumption of commercial species affected by FM.

5.3.2 Types of Studies and Study Parameters

Female masculinisation studies using gastropods were divided into three types of studies: field (n = 416) > laboratory (n = 112) > risk assessment (n = 3).

Field studies encompassed those that used FM in gastropods as bioindicators or biomonitors of organotin pollution and/or discovered FM in species during field expeditions. On the other hand, laboratory studies included those that exposed gastropods to compound(s) to elicit a masculinised response, bioaccumulation potential, and/or metabolic responses to organotin exposure. Risk assessment studies were geared towards assessing the risk(s) to human health and/or ecology by determining factors that could affect transport, fate, and exposure. For this review, field studies were assessed to determine the sources of organotin as indicated by the author(s), if the study was a recovery study, and if the study used transplants and/or histology. Recovery studies are those that investigated FM in gastropod populations previously documented as being affected by TBT pollution and were re-assessed some time after to determine the severity of FM over time.

Identified sources of organotins attributed to FM by organotin pollution in the literature included: (1) boating activity e.g. commercial harbours (deep sea 125 ports), marinas, ferry terminals, shipyards and/or dry dock facilities, naval facilities, moorings, and fishing harbours (artisanal fishing); (2) shipping activity e.g. shipping lanes; and (3) agriculture. Careful analysis of field studies that verified the source of organotin pollution confirmed sources as follows: boating activity > shipping activity > agriculture. Boating and shipping activity as sources of organotins into the aquatic environment were confirmed in numerous species globally, however, agriculture as a source of organotin into the aquatic environment was cited in only one study using the apple snail, Pomacea canaliculata, in Taiwan (Liu et al., 2006).

The recovery of gastropod populations from organotin pollution can be measured on three levels (Gibbs and Bryan, 1996): (1) a lessening of the severity of imposex; (2) increased breeding capacity for populations where sterilization by imposex has caused a reduction in fecundity; and (3) re-colonization of areas stripped of individuals. Changes in index values measured by the vas deferens sequence index, VDSI (Bailey and Davies, 1991; Harding et al., 1997), relative penis size index, RPSI (Evans et al., 1991; Douglas et al., 1993; Tester and Ellis, 1995;

F0lsvik et al., 1999), male:female ratio, M:F (Oehlmann et al., 1993; Stroben 1996), percent imposex, % I (Stroben 1996; Swennen et al., 2009), and % sterility

(Oehlmann et al., 1993) have been used to evaluate the recovery of gastropod populations from organotin pollution. The re-colonization of gastropod populations with juveniles and/or females to areas where the population size of each was low to non-existent in previous surveys are also indicators that can be used to evaluate populations recovering from TBT pollution (Evans et al., 1991; Stroben, 1996;

Minchin and Davies, 2000).

Transplants in ecotoxicology studies can be used as an in situ bioassay to obtain information on environmental quality that would not be available through 126 routine chemical or biological monitoring (Salazar et al., 1995). In the case of imposex studies, it is known that for some species imposex induction occurs at chemical levels below analytical detection limits (Evans et al., 1998), so transplant studies could provide useful information about organotin environmental levels where chemical monitoring would fall short. Additionally, caged transplant studies using indicator species can be used as a realistic means of obtaining local contamination levels (Giltrap et al., 2009). Female masculinisation studies using gastropods as bioindicators or biomonitors of organotin pollution in transplant studies have used 12 species of gastropods in the families Haliotidae (n = 2), Muricidae (n = 8),

Nassariidae (n = 1), and Buccinidae (n = 1). The first transplant studies using gastropods as biomonitors of imposex dates back to the 1970s using the sting winkle,

Ocenebra erinacea (Feral, 1979) and the American mudsnail, Ilyanassa obsoleta

(Jenner, 1979), but the most widely used species in the literature is the dogwhelk,

Nucella lapillus. In imposex, intersex and ovo-testis studies, histology and scanning electron microsopy were used to identify differences between reproductive structures

(Jenner, 1979), recognise morphological variations in imposex-affected females

(Blaber, 1970; Gibbs and Bryan, 1986; Oehlmann et al., 1996b; Barroso and

Moreira, 1998), and recogonise functional variation(s) of ovo-testis affected females such as ovarian spermatogenesis in affected females (Horiguchi et al., 2000; Sloan and Gagnon, 2004; Horiguchi et al., 2006b).

Laboratory studies were assessed in a similar fashion to field studies, but based on the following parameters: induction, bioaccumulation, imposex suppression and/or reversal, egg production, growth, metabolism, steroid identification, pheromone recognition, reproduction and/or passing on of genetic material, and hormonal changes. Of the laboratory studies analyzed, the majority of studies 127 focused on induction in 28 of the 269 species known to be affected by FM. The dominant species used in laboratory studies included: Nucella lapillus > Ilyanassa obsoleta > Marisa cornuarietis > Thais clavigera. Analysis of the number of studies using T. clavigera may be an underestimate since some studies using this species are published in Chinese. Laboratory experiments have enhanced scientific knowledge especially in the area of FM induction, and it is expected that with further experimentation the actual mechanism that controls FM in gastropods that has eluded scientists will be resolved.

5.4 Updated Species Lists (1962 to 2009)

5.4.1 Recent Changes in Gastropod Taxonomy

One of the challenges of reviewing almost 50 years of literature is consistency in nomenclature. The taxonomy for gastropods was recently changed

(Poppe and Tagaro, 2006) and a "new" classification of gastropod families has been

developed by Bouchet and Rocroi (2005). This "new classification" has spurred

much debate and there does not appear to be a concise agreement on the classification of gastropods. In addition, the names of some species have changed

since their original publication date. For example, when Smith (1971) reported

inconsistencies in the female genital tract of the American mudsnail, the reported species name was Nassarius obsoleta, which has since changed to Ilyanassa

obsoleta. The change in classification and updating of species names and

nomenclature made it challenging to create a "new" list of known prosobranch

species affected by FM (i.e. imposex, intersex and ovo-testis) current to 2009. To 128 this end, the taxonomy of each proposed species for 2009 was gathered from two species databases, Zipcodezoo.com and Appeltans et al. (2009) via the World

Registry of Marine Species® (WoRMS®), and these were cross-checked and compared with the original taxonomy in Matthiessen et al. (1999). The taxonomy from WoRMS® was generally in agreement with Bouchet and Rocroi (2005).

5.4.2 Affected Species List (1962 - 2009)

Lists of known prosobranch gastropods affected by pseudohermaphroditism or imposex were published on at least seven occasions from 1978 to 2005. The first

list of affected gastropods that displayed signs of pseudohermaphroditism was

published by Jenner (1978) with a total of 33 species in 11 families. The number of

gastropod families and species affected by FM progressively increased over time

(Figure 5.4). By 1990, the number of affected species increased from 33 to

51(Fioroni et al., 1990), and the number of families increased from 11 to 17 (Fioroni

et al., 1991). The next four species lists were published by Matthiessen et al. (1999),

Minchin et al. (1999), Omae (2003), and Shi et al. (2005). Between 1999 and 2005

the number of affected families remained fairly constant (Figure 5.4), but the number

of affected species grew dramatically from the lists published by Fioroni et al. (1990;

1991). In 1999, the number of affected species increased from 85 to 140 - 144

species, and by 2005 the number of species had more than doubled to 192 (Figure

5.4; Table 5.1). Not overlooking the affected species lists published in 1999, one

major contribution by Shi et al. (2005) was the addition of newly affected species

from mainland China to the already established lists from Matthiessen et al. (1999).

Building on the existing lists, a "new" composite list has been created for species 129 known to be affected by FM (imposex, intersex, and ovo-testis) from 1962 to 2009

(Table 5.2) by cross referencing all published affected lists from 1978 to 2005 and adding other confirmed species from the literature. For simplicity, any changes in family designation or species name from a previously affected list were corrected to reflect its current status as of 2009.

The new list proposes a total of 269 species in 33 families known to be affected with FM, which is close to the 240+ species suggested in a recent article published by Strand et al. (2009). A comparison of taxonomy by family of all known affected species lists from 1990 to 2005, demonstrated that Muricidae family had the greater number of affected species (Table 5.1 and 5.2), and this trend was consistent throughout all affected species lists published from 1978 to 2009 (Table

5.1). For the current list of affected gastropods (Table 5.2), the top 4 families were ranked (Table 5.1): Muricidae (n = 100) > Buccinidae (n = 33) > Nassariidae (n =

20) > Conidae (n = 16), and the top four genuses were: Thais spp. (n = 25) >

Nassarius spp. (n = 13) > Conus spp. (n = 11) > Nucella spp. (n = 8) (Table 5.2).

In the affected species lists created by Fioroni et al. (1990; 1991), members from the family Littorinidae (n = 6 - 9) were listed, but did not appear in any of the subsequent lists up to 2005 (Table 5.1 and 5.2). This may have occurred because the lists published by Fioroni et al. (1990; 1991) record species as affected by pseudohermaphroditism, also called "imposex", and the definition of imposex and pseudohermaphroditism during the early 1990s was used to describe females growing a "penis-like structure". However, as research advanced into the mid and late 1990s, new data on the effects of TBT on gastropods revealed that two types of

FM could occur (i.e. imposex and intersex), and since the definition of imposex changed from its original conception by Smith (1971), this could justify not 130 including members from the Littorinidae family in subsequent "imposex" lists.

Additionally, in some of the affected species lists, the caption describes the list as

"known imposex-affected" (Matthiessen et al., 1999; Minchin et al., 1999), or

"affected by imposex" (Omae, 2003), and since L. littorea is known to be affected by intersex and not imposex, this could explain why members of the Littorinidae family were not included in lists published after 1991.

On the other hand, a review of available literature reveals conflicting results regarding the occurrence of pseudohermaphroditism in Littorinids, especially for

Littorina saxatilis. The earliest account found investigating pseudohermaphroditism in Littorinids indicated that variations in L. saxatilis and the yellow periwinkle, L. obtustata were similar to those seen in Urosalpiwc cinera (Fioroni et al., 1990).

Additionally, it was observed that in L. obtustata, L. saxatilis, L. saxatilis rudis,

Nucella lapillus, and the sting winkle, Ocenebra erinacea, the vas deferens extended from the bottom of the penis and came into contact with the opening of the female reproductive apparatus (Fioroni et al., 1990). In another study by Fioroni et al.

(1991), it was reported that imposex of Littorinidae (7 species studied, n = 7 to 899) had an "ovipositor" that corresponded with the arrangement of the penis and vas deferens in males. The ovipositor is morphologically and histologically identical to the base of the penis after the shedding of the male copulatory organ (Fioroni et al.,

1991). Later studies investigating pseudohermaphroditism in L. saxatilis from several European coastlines (Germany, Ireland, and France) revealed that no signs of

FM were found in 284 specimens examined (Oehlmann, 1998). Furthermore, analysis of L. saxatilis from seven sites in Greenland (n = 315) by Strand and

Asmund (2003) revealed no signs of FM either although they were collected sympatrically with Buccinum spp. which did show signs of imposex. Even though 131 several Littorinids (n = 6 - 9) were published in two affected species lists in the early

1990s (Fioroni et al., 1990; 1991), their FM status is questionable, with the exceptions of Littorina littorea (Oehlmann et al., 1994a) and L. sitkana (Nohara,

1999). As a result, L. marie, L. neritoides, L. nigrolineata, L. obtusata, L. saxatilis,

L. saxatilis rudis and L. ziczac are not included in the current proposed affected species list for 2009 (Table 5.2).

5.4.3 "Novel" Unaffected Species List (1962 - 2009)

Even though a vast number of female gastropods are "supposedly" masculinised by exposure to TBT, there are 42 species that elicit no response even if they have been exposed to TBT in the laboratory or collected sympatrically from field sites with other species displaying imposex (Table 5.3). What is more, there are two instances where species collected sympatrically from field sites with other species displaying imposex have butyltins in their tissues, yet they display no signs of female masculinisation (Table 5.3). An official list of known species that have been examined and do not elicit an imposex/FM response has never been published prior to this review. On the other hand, Matthiessen et al. (1999) mentions seven species that did not show an imposex response even though they were sampled in highly contaminated areas and/or were exposed to high aqueous TBT concentrations, including: Theodoxus fluviatilis (Neritidae), Bithynia tentaculata (Bithyniidae),

Hydrobia ventrosa and Potamopyrgus antipodarum (Hydrobiidae), Columbella rustica (Columbellidae), Capulus ungaricus (Capulidae), and Turritella communis

(Turritellidae). This review documents 42 species in 20 families (Table 5.3) that were unaffected, including two species that did not show a FM response even though 132 butyltins were detected in their tissues and they were collected adjacent to point sources: Thais tuberosa (Evans et al., 1995), and the West Indian topshell, Cittarium pica (Strand et al., 2009) (Table 5.3).

There are four conceivable reasons for the observed disparity between species that are affected by FM and those that are not. The first likely explanation is interspecies differences in their potential sensitivity to develop imposex. Stroben et al. (1995) collected seven species of gastropods from a highly contaminated area and found differences in species sensitivity in Ocenebra spp. and Nassarius spp. Species with the highest sensitivity to TBT were ranked: Ocenebra aciculata > Nucella lapillus > Ocenebra erinacea > Trivia arctica > T. monacha > Nassarius reticulatus

> Nassarius incrassata (Stroben et al., 1995). The second could be phylogenetic relationships. When a comparison was made of the sensitivity of TBT between

Mesogastropoda and Neogastropoda orders, Mesogastropoda exhibited signs of imposex when exposed to higher concentrations of TBT versus Neogastropods

(Stroben et al., 1992c). Thirdly, the absence of "typical" male anatomical structures in some species and orders of gastropods is normal. For example, when Oehlmann

(1998) investigated several species of gastropods (n ~ 41) of the Archaeogastropoda,

Mesogastropoda, and Neogastropoda from polluted field sites in Europe (Germany,

Ireland, and France) as well as species exposed to butyltins under laboratory conditions, some species did not show an imposex response. It was concluded that

imposex in Capulus ungaricus (Mesogastropoda: Capulidae) and Turritella communis (Mesogastropoda: Turitellidae) was not possible because the males of those species did not possess a vas deferens. Furthermore, the absence of a vas deferens is prevalent in the majority of the males found in the Archaeogastropods and some Mesogastropods. The last plausible reason for the disparity in FM 133 between species is the mode of feeding. Carnivores and/or scavengers have the lowest frequency of negative responses, while detritivores, herbivores, suspension feeders, and omnivores, have the greatest percentages of negative responses (Table

5.4 and 5.5). In the case of C. pica (Trochoidae) this gastropod is a known herbivore that feeds on filamentous algae (Toller, 2003) and is unaffected by imposex (Strand et al., 2009).

Examination of the literature on the FM of gastropods revealed that a total of

41 families has been studied. The bulk of studies that have looked into the use of gastropods as bioindicators of FM in response to butyltin exposure have focused on three families Muricidae > Buccinidae > Nassariidae, and this accounts for < 10% of the total number of the families investigated. On the other hand, there are twenty nine investigated families (~ 71%) that have < 5 species studied in each of them.

This group of twenty nine families was further divided into three groups: (1) < 5 species with all members affected by FM; (2) < 5 species with all members unaffected by FM; and (3) < 5 species with some percentage of members that are unaffected (Table 5.4). An analysis of gastropod families (n = 29) with < 5 species and divided into the above mentioned groups revealed that there are sixteen families

(55%) where all studied species are affected by FM: Haliotidae, Apseudoicdea,

Thiaridae, Lamellariidae, Pterotracheidae, Bursidae, Cypraeidae, Naticidae,

Tonnidae, Personidae, Cancellariidae, Marginellidae, Olividae, Terebridae,

Turbinellidae and Turridae; and eight families (28%) where all species are unaffected by FM: Trochoidae, Neritidae, Capulidae, Ficidae, Turritellidae,

Bithyniidae, Calyptraeidae and Harpidae. The remaining five families (17%):

Hydrobiidae, Aporrhaididae, Cassidae, Strombidae and Columbellidae, had some species that were affected by FM and others that were not. Due to the small number 134 of studies that investigated those twenty nine families that had < 5 gastropod species investigated in them (Table 5.4), it would be ill-advised to make a generalization with regard to the expected FM response. However, within the families

Ampullariidae (n = 6), Conidae (n = 16), Muricidae (n = 100), Nassariidae (n = 20),

Trividae (n = 6), and Volutidae (n = 11), one can assume that any gastropod species collected from a shoreline adjacent to butyltin point sources would be likely to be affected by FM. For those families where there are < 5 species used in FM studies, it is recommended that additional studies be carried out utilising other members within those families that are found adjacent to point sources so as to increase scientific knowledge.

5.5 Global Incidence of Female Masculinisation in

Gastropods

The imposex, intersex and ovo-testis phenomena are not confined to temperate or tropical environments, but are global in their occurrence (Figure 5.5).

However, the documented studies using gastropods as biomarkers or bioindicators of

TBT pollution are dominated by studies conducted in Europe and North America as well as China. A review of the literature that focused on field studies (n = 411) using FM in gastropods as biomonitors and/or bioindicators of TBT pollution, uncovered that there were some studies that reported tissue concentration of TBT and a greater proportion that did not report TBT levels in four geographic regions

(Figure 5.5). The four geographic regions were designated: Europe including the

Meditterrean (Figure 5.6); Asia and Oceania (Australia and New Zealand) including

Africa (Figure 5.7); North America and Greenland (Figure 5.8); and Latin America, comprised of Central and South America and the Caribbean (Figure 5.9). Within 135 each region, the top four to six species cited with signs of FM from field studies were ranked, and the number of studies with and without documented tissue concentrations for the dominant species was determined (Figure 5.6 - 5.9).

Interestingly enough, even though imposex, intersex, and ovo-testis are attributed to female gastropods that have been exposed to organotins, the vast majority of studies do not confirm or perform chemical analyses. Of the field studies reviewed, 32.5% (n = 135) have reported butyltin concentration, while 67.5% (n =

281) have not reported butyltin body levels. The lack of chemical analyses may have occurred because of three reasons. Firstly, as early as the 1980s, female gastropods displaying signs of FM were collected adjacent to "hotspots" (i.e. harbours, marinas, shipyards etc.), while those collected from reference areas did not display any signs of FM (Feral, 1980b; Smith 1981a, b). The incidence of imposex

(Ellis and Pattisina, 1990; Bech, 2002), intersex (Oehlmann, et al., 1994a) and ovo- testis (Horiguchi et al., 2005) occurring at hotspots and absent at reference sites continued throughout the literature. Secondly, chemical analysis for butyltins is expensive and time consuming (Stroben et al., 1995; Evans et al., 1998; Ram6n and

Amor, 2002). Finally, even though advances in analytical chemistry have been made in quantifying low concentrations in environmental samples, imposex induction in some species is known to occur at levels close to/or below the level of detection

(Evans et al., 1998).

Globally, the top four species studied for the incidence of masculinisation in female gastropods in field studies were ranked: Nucella lapillus (n = 171) >

Nassarius reticulatus (n = 46) > Thais clavigera (n = 43) > Littorina littorea (n =

40), but the ranking for T. clavigera may be an underestimation as there were several articles on imposex in T. clavigera published in Chinese that were unattainable via 136 inter-library loan or in English translation. For each region previously characterized above, quantitative analysis of the dominant species studied, and the number of studies with and without tissue concentrations for each dominant species was performed and is described below (Figures 5.6 - 5.9).

5.5.1 Europe (including the Mediterranean)

The top six species studied in Europe include: Nucella lapillus (n = 146) >

Nassarius reticulatus (n = 45) > Buccinum undatum (n = 35) > Littorina littorea (n =

35) > Ocenebra erinacea (n = 27) > Hexaplex trunculus (n = 21) (Figure 5.6). Of those principal species, N. lapillus, B. undatum and L. littorea have been recommended as the preferred bioindicators in the guidelines of the Joint

Assessment and Monitoring Programme (JAMP) in Europe to investigate the intensity of the effects of TBT on female gastropods and the spread of the effects away from point sources (OSPAR, 2008). The bulk of the published literature from

Europe examined species from: France (n = 131) > England (n = 48) > Spain (n =

35) > Scotland (n = 34) > Portugal (n = 31) > Ireland (n = 26). In Europe, 39% of the literature (n = 122) on the top six species also reported chemical analyses (Figure

5.6) and correlated the incidence of imposex and intersex with tissue burdens.

However, the occurrence of FM in the Littorinid, Littorina saxatilis, is one where there are conflicting results (see Section 5.4.2). An extensive field survey across

Europe at point source and reference sites that collects Littorinids along with well established species used in imposex studies such as Nucella lapillus, Nassarius reticulatus, and Ocenebra erinacea would be needed to resolve these inconsistencies. Furthermore, it is advisable that laboratory exposure experiments be 137 completed for those Littorinids that have an ovipositor (L. marie, L. neritoides, L. nigrolineata, L. obtusata, L. saxatilis, L. saxatilis rudis and L. ziczac) which is morphologically and histologically identical with the remaining base of the penis after the shedding of the male copulatory organ (Fioroni et al., 1991) at TBT concentrations similar to those known to induce intersex in L. littorea, as a positive control.

5.5.2 Asia and Oceania (including Africa)

In Asia and Oceania (Australia and New Zealand), including Africa, roughly

30% of the studies using the dominant gastropod species performed chemical analysis. The prevailing gastropod species used as bioindicators of TBT pollution in this region comprised Thais clavigera (n = 43) > T. distinguenda (n = 8) > T. luteostoma (n = 8) > T. bitubercularis (n = 6) (Figure 5.7). When the countries are categorized based on the frequency of documented studies, the bulk of the studies were undertaken in China (n = 63) > Thailand (n = 56) > Japan (n = 46) > Australia

(n = 21) > Taiwan (n = 16) > New Zealand (n = 15). Imposex in T. clavigera is a well established bioindicator of TBT pollution in imposex studies in Asia, and had been confirmed by imposex induction in laboratory experiments with a variety of organotins (Horiguchi et al., 1995; 1997b). The incidence of imposex in Lepsiella albomarginata and L. scobina collected from marina and mooring sites in New

Zealand, and additional confirmation of imposex induction by TBT exposure (Smith and McVeagh, 1991) validates those two Lepsiella species as bioindicators of TBT pollution. Africa and the Middle East were included with Asia and Oceania, since their climate was more similar to this region as opposed to Europe. Unfortunately, 138 in all the literature reviewed, there was only one study that investigated imposex on the entire continent in Africa, and one from the Middle East. The single imposex study in Africa used the Neogastropod Nassarius kraussianus (n = 412) from nine harbours with varying boating activity in South Africa during 2000 (Marshall and

Rajkumar, 2003) where the incidence of imposex at affected sites ranged from 29 to

100%. On the other hand, the sole publication from the Middle East collected

Stramonita haemastoma and Hexaplex truneulus during 1996 - 1997 from four sites close to marinas in Israel (Rilov et al., 2000). Since the number of studies using gastropods as biomonitors of TBT pollution appears to be non-existent for the continent of Africa and the Middle East in comparison to other areas, more species and families should be investigated along those coast lines in areas where there are significant levels of boating activity.

5.5.3 North America (including Greenland)

Within the North America and Greenland region, the most widespread species used for imposex studies included: Nucella lapillus (n = 24) > Ilynassa obsoleta (n = 23) > Nucella emarginata (n = 12) > Nucella lamellosa (n = 11) >

Searlesia dira (Lirabuccinum dirum) (n = 9) (Figure 5.8). The majority of studies were done in Canada (n = 74) with fewer in the USA (n = 56) and Greenland (n =

11). This was the only region with three countries in comparison to the other 3 regions, but further analysis of the dominant species used for imposex and intersex studies in North America revealed a pattern of species use (Figure 5.8), most likely determined by species geographic ranges. Of the east coast of Canada, the foremost gastropod species used as biomonitors of TBT pollution were Nucella lapillus and L. 139 littorea, while on the west coast the main species included N. emarginata and N. lamellosa. The main gastropod species used along the eastern coast of the United

States from Providence to North Carolina was Ilynassa obsoleta, while on the west coast, Nucella emarginata and N. lamellosa stood out (Figure 5.8). Along the southern United States, the principal species was Stramonita haemastoma, but in the north in Alaska, Nucella lima dominated; both species denoted by asterisks. For the

North American region the location of the study will determine which species is to be used as a bioindicator of TBT pollution. Hence, when studying in locations within Canada and the United States, studies should use the dominant species as outlined for the eastern, western, northern and southern coasts in Figure 5.8 when available.

5.5.4 Latin America

Imposex studies have increased substantially in Latin America (Central

America, South America and the islands of the Caribbean) in recent years. The first study to investigate the incidence of imposex at sites adjacent to point sources was in

2000 (Phillip, 2000), although the first record of female gastropods with both a penis and secondary female sex characteristics was published in the early 1990s (Reed,

1993 a, b) using the queen conch, Strombus gigas in both cases. The primary species utilized in imposex studies in Latin America are Stramonita haemastoma (n

= 11) > Adelomelon briasilina (n = 5) > Stramonita rustica (n = 5) > Strombus gigas

(n = 3) (Figure 5.9). An analysis of the number of species in this region (n = 72) by country showed that most studies were conducted in Brazil (n = 22), Argentina (n =

21), and Chile (n = 19). 140

5.5.5 Global Incidence - Summary

In summary, the incidence of imposex, intersex, or ovo-testis (FM) in gonochoristic female gastropods is global, occurring in temperate and tropical environments. The total number of cited species affected by FM up to 2009 is 269 species in 33 families, a dramatic increase from the first published list of Jenner

(1978). Even though there are 269 known species affected by FM, there are 42 known species in 20 families that do not elicit a FM response although they have either been collected from the same sites where other species have displayed signs of

FM, or they have been exposed to TBT under laboratory conditions. The apparent insensitivity of some female gastropods could be related to interspecies and taxonomic differences to TBT sensitivity, and quite possibly their mode of feeding.

For the vast majority of field studies, no contaminant analysis was performed, but since signs of FM were observed at "hot spots" which are suspected point sources, the suspected agent cited in the majority of studies is TBT due to its use as a biocide in antifouling paint. The Latin American region appears to have an increasing number of imposex studies, but data for the continent of Africa and the Middle East are sparse.

5.6 Inducers of Female Masculinisation in Gastropods

Of the 269 gastropod species cited as being affected by imposex, intersex and ovo-testis, laboratory experiments have confirmed induction of masculinisation in only 20 (7.4%) of the species identified thus far. Masculinisation of female gastropods based on laboratory experiments has been verified in three orders and 141 eight families (Table 5.6 and 5.7). From a taxonomic stand point, gastropod FM has been validated in the orders Vestigastropoda (Haliotidae), Mesogastropoda

(Ampullariidae, Hydrobiidae, Littorinidae, Strombidae) and Neogastropoda

(Muricidae, Buccinidae, Nassariidae), with the greatest number of confirmed species and studies occurring in the family Muricidae (Table 5.6).

One of the common erroneous beliefs concerning the masculinisation of female gastropods is that FM is solely the result of exposure to TBT, and to a lesser extent TPT, from antifouling paint, even though no chemical analysis of tissues for the presence of organotins was performed in the majority of studies. This deduction is made based on the fact that in the vast majority of field studies, specimens are collected adjacent to harbours, marinas and high boating areas. Although there is a suite of organotins that can induce FM in gastropods, there are other compounds that can also cause the same response. A review of laboratory studies of gastropods affected by FM (n = 83) revealed that masculinisation can occur in females exposed to four types of compounds: tin (Sn) and/or Sn derivatives, tin "cocktails", steroids, and "other contaminants" (Table 5.7). Further examination of FM induction studies revealed that there are four media by which the tested compounds were exposed to the gastropods: water (n = 57) > injection (n = 17) > other (n = 6) > sediment (n =3)

(Table 5.7). The category of "other" for mode of exposure included painted wooden panels with antifouling paint, painted gastropods with antifouling paint, using a food source pre-exposed to TBT, or bird excreta. 142

5.6.1 Tin Compounds

There are eleven tin (Sn) compounds that have positively induced masculinisation in female gastropods under laboratory conditions (Table 5.7). Not surprisingly, the two most common compounds are TBT and triphenyltin (TPT) which were once popular biocides used in antifouling paint in the 1970s and 1980s

(Evans and Smith, 1975; Snoeij et al., 1987). The remaining Sn compounds and/or derivatives of Sn that positively induced masculinisation in female gastropods include: adipic acid -di (2-ethylhexyl) (DEHA), dioctyltin (DOT), monophenyltin

(MPT), phtalic acid butyl (PAD), tetrabutyltin (TTBT), tin tetrachloride (TTC1), trioctyltin (TOT), tricyclohexyltin (TCHT), and tripropyltin (TPrT).

The Sn compounds known to induce FM in gastropods are used in commercial applications such as poly vinyl chloride (PVC) stabilizers, biocides, pesticides, agrichemicals, and glass coatings. TTC1 is the starting material for organotin compounds (Stannica LLC, 2009), and one of the ingredients used for color stabilization of soaps and perfumes (Gangolli, 1999). TTBT is also a precursor compound, but for the manufacture of TBT (Bryan et al., 1988), an important biocide for wood preservation, marine antifouling, disinfection of circulating industrial cooling waters, and slime control in paper mills (Snoeij et al., 1987).

DEHA, DOT and PAD are plasticizers of PVC compounds that come into contact with food; more specifically, DEHA, PAD and DOT are used in the production of flexible PVC films and sheets (Piver, 1973; IARC, 1982; WHO, 2000), and DOT for

PVC bottles (Piver, 1973). TPT has been used as an agricultural fungicide (Evans and Smith, 1975), particularly in the control of potato blight (Graham, 1964), while

TCHT is applied as a pesticide agent to control arachnids and insects, predominantly 143 ticks and mites (Robinson, 1985). MPT is used in the production of haze-free transparent, non-iridescent, infrared reflecting glass windows (Linder, 1988).

Of the eleven Sn compounds known to induce FM in gastropods, TBT and

TPT are the two most widely used, but only TBT is known to induce all three types

(i.e. imposex, intersex and ovo-testis) of FM. Ovo-testis was induced in Haliotis gigantea (Vestigastropoda: Haliotidae) by TBT and TPT (Horiguchi et al., 2002), whereas, only TBT has been documented to induce intersex in the periwinkle,

Littorina littorea (Mesogastropoda: Littorinidae) (Bauer et al., 1997). Exposure to

TBT and/or TPT under laboratory conditions induced imposex in the remaining species (Table 5.7). Induction by precursor compounds, TTC1 and TTBT, induced imposex in Ilyanassa obsoleta, Nassariidae (McClellan-Green et al., 2006) and

Nucella lapillus, Muricidae (Bryan et al., 1988), respectively. The sharp dwarf winkle, Ocinebrina aciculata, has been cited as the most sensitive species to TBT followed by Nucella lapillus (Oehlmann et al., 1996c); but imposex has been induced by monophenyltin (MPT), a degradation product of TPT in the sting winkle,

Ocenebra erinacea, Muricidae, (Hawkins and Hutchinson, 1990), and by a di- organotin (DOT) in I. obsoleta (McClellan-Green et al., 2006). Imposex induction by the plasticizers PAD and DEHA was documented by Takeda (2001) in the apple snail, Pomacea canaliculata, and by DOT in I. obsoleta (McClellan-Green et al.,

2006).

Even though TBT and TPT were used primarily as biocides in antifouling paints, TPT does not induce FM in all species known to be affected by FM. For example, TPT did not induce imposex in Nucella lapillus (Bryan et al., 1988;

Schulte-Oehlmann et al., 2000), one of the most sensitive and arguably the best documented species affected by imposex. On the other hand, TPT does induce 144 imposex in Thais clavigera (Horiguchi et al., 1994, 1995) an Asian relative of N. lapillus (Oehlmann et al., 2007). The inability of TPT to induce imposex in N. lapillus, the second most sensitive species to imposex induction, under laboratory conditions suggests that: (1) not all butyltins are created equal in their ability to induce imposex in female gastropods; and (2) interspecies differences to the imposex response may be responsible for the observed differences (see Section 5.4.3).

5.6.2 Tin "Cocktails"

There are five tin "cocktails" that have positively induced masculinisation in female gastropods exposed under laboratory conditions (Table 5.7). The five cocktails are: TBT + DDT (dichlorodiphenyltrichloroethane), TBT + estrogen, TBT

+ nonylphenol (NP), TBT + sewage effluent, and TBT + TPT (Table 5.7). To date,

TBT "cocktails" have been used on three (3) species, the rock shell snail, Thais clavigera (Shim et al., 2002), the dogwhelk, Nucella lapillus (Santos et al., 2008), and the purple dye murex, Bolinus brandaris (Santos et al., 2006), all members of the family Muricidae.

DDT and 1,1-di(p-chlorophenyl)-2,2-dichloroethylene (DDE), a metabolite of DDT, are known endocrine disruptors (Colborn et al., 1993; Tyler et al., 1998), and DDE is also a known estrogen agonist (Tyler et al., 1998). The effects of DDT and DDE on the eggshells of some bird species are well documented, as they have been linked to eggshell thinning in a number of birds including the bald eagle,

Haliaeetus leucocephalus fColborn et al., 1993). When Shim et al. (2002) exposed

T. clavigera to TBT + DDT, imposex severity and ethoxyresorufin-O-deethylase 145

(EROD) activity was low. From these results it was concluded that DDT had an antagonistic effect on the induction of imposex by TBT (Shim et al., 2002).

In another mixture containing TBT and sewage effluent from Ponte Moreira,

Portugal, imposex was induced in Nucella lapillus as measured by female penis formation and the development of a vas deferens (Santos et al., 2008). Chemical analysis of the sewage effluent used in the exposure experiments confirmed the presence of three xenoestrogens, bisphenol A (BP A), octylphenol (OP), nonylphenol

(NP) as well as 17-p-estradiol (E2). Of the four estrogens chemically determined in the sewage effluent used by Santos et al. (2008), imposex has been induced solely by

NP in N. lapillus (Evans et al., 2000). The xenoestrogen BPA is an intermediate product for the production of polycarbonate and epoxy resins, and OP is used for a variety of industrial processes including wool washing (Oehlmann et al., 2000).

Exposure to BPA or OP at low concentrations via water induced another phenomenon in female Marisa cornuarietis dubbed "super females" which is the enhancement of oocyte production and enlargement of pallial sex glands (Oehlmann et al., 2000).

5.6.3 Steroids

There are four steroids that have induced masculinisation in six species of female gastropods under laboratory conditions (Table 5.7). Two of the affected species are in the family Ampullariidae (order Mesogastropoda), and the other four species are Neogastropods in the families Nassariidae (n = 2) and Muricidae (n = 2).

Steroidal inducers of FM in gastropods include: ethinyl estradiol (EE2), formestane

(Form), methyl testosterone (MT), and testosterone (T). 146

In an attempt to understand the mechanism(s) of TBT-induced imposex in the dogwhelk, Nucella lapillus, Santos et al. (2005) explored the possible involvement of the P450 aromatase receptors by exposing N. lapillus to formestane. Exposure to

Form (0.3 mg L"1) induced imposex, but was unsuccessful at increasing imposex severity (VDSI and female penis length) when compared to controls (Santos et al.,

2005).

Two groups of steroids that have masculinised female gastropods are androgens and estrogens, which control the development and function of reproductive activity in both sexes (Crain et al., 1998; Wiita et al., 1995). The estrogen group comprises ethinyl estradiol, (EE2), and the androgen group methyl testosterone (MT), and testosterone (T). EE2 is a synthetic derivative of 17-P- estradiol (E2), and an active ingredient in contraceptive pills (Ying et al., 2002), that is often present in raw sewage (Shore et al., 1993). EE2 is resistant to biodegradation but is susceptible to photodegradation (Jurgens et al., 2002). When Marisa comuarietis were exposed to MT and EE2 under laboratory conditions, imposex was induced in a time-dependent manner and imposex severity increased after three and six months, respectively (Tillmann et al., 2001). In another experiment using M. comuarietis, imposex developed after 150 days of exposure to MT (Janer et al.,

2006).

Testosterone as an environmental pollutant can be found in raw sewage

(Shore et al., 1993) and ground water associated with manure (Casey et al., 2004).

Testosterone has exclusively induced imposex in the apple snail, Pomacea canaliculata (Takeda, 2000), N. lapillus (Bettin et al., 1996; Oehlmann et al.,

1996a), the netted dogwhelk, Nassarius reticulatus (Bettin et al., 1996; Oehlmann 147 and Bettin, 1996), and the American mudsnail, Ilyanassa obsoleta (Oberdorster and

McClellan-Green, 2000; 2002) (Table 5.7).

5.6.4 Other Contaminants

The group labeled other contaminants is not distinctly organotins, tin

"cocktails" or steroids. The list of pollutants described herein as "other contaminants" that have induced female masculinisation in gastropods include: 9- cw-retionic acid (9-cis RA), methoprene acid, Ala-Pro-Gly-Trp-Ntb (APGWamide), fenarimol (FEN), magnesium chloride (MgCk), Aroclor 1260, fadrozole (FAD), copper (Cu), nonylphenol (NP), and bird excreta (Table 5.7).

Both 9-cis RA (retinole derivate) and methoprene acid (sesquiterpene) have induced male characteristics in female gastropods through the retinoic acid pathway.

9-cis RA is the natural ligand of RXR (Nishikawa et al., 2004) while methoprene acid is a pesticide (Harmon et al., 1995) and a selective agonist for RXR (Castro et al., 2007). Retinoids are important because they are involved in vitamin A pathways; they can have an effect on cellular differentiation, proliferation and embryonic development (Carlberg et al., 1993) and are required for the development of male and female reproductive tracts.

Studies using Thais clavigera and 9-cis RA have suggested that RXR plays a key role in the differentiation and growth of male genital tracts in female gastropods, i.e. imposex (Nishikawa et al., 2004). 9-cis RA has positively induced imposex in the rock shell, T. clavigera (Horiguchi et al., 2008), but there are conflicting results for two of its European relatives the dogwhelk, Nucella lapillus, and reticulated nassa, Nassarius reticulatus. According to Castro et al. (2007), 9-cis RA and 148 methoprene acid induced imposex in N. lapillus as affected females displayed an increase in female penis length (9-cis RA) and vas deferens formation (methoprene acid). However, imposex induction by methoprene acid was to a lesser degree versus controls (Castro et al., 2007). Contradictory to Castro et al. (2007),

Oehlmann et al. (2007) could not find any imposex-inducing activity of 9-cis RA in

N. lapillus or N. reticulatus.

The American mudsnail, Uyanassa obsoleta, was exposed to four neuropeptides: APGWamide, conopressin, LSSFVRIamide and FMRFamide.

APGWamide is a common neuropeptide found in molluscs related to sex organ growth and reproductive behavior (Oberdorster et al., 2005); conopressin is an antagonist of APGWamide; LSSFVRIamide is an inhibitory or excitatory neuropeptide of various muscles; and FMRFamide is cardio excitatory (Oberd5rster and McClellan-Green, 2000). After fourteen days of exposure, APGWamide significantly induced imposex in I. obsoleta while conopressin, LSSFVRIamide and

FMRFamide did not (Oberdorster and McClelIan-Green, 2000). Subsequent experiments by Oberdorster and McClellan-Green (2002) using I. obsoleta, demonstrated that APGWamide significantly induced imposex by injection over a two-week period, and APGWamide was suggested as the penis morphogenic factor in I. obsoleta.

Fenarimol (FEN) is a synthetic fungicide considered to be non- or weakly genotoxic (de Castro et al., 2005), and inhibits CYP19 aromatase activity in ovarian homogenates from fish (Ankley et al., 2005). In addition, FEN is a pyrimidin carbinole that can act as an estrogen agonist and androgen antagonist (Andersen et al., 2002). Duft et al. (2007) exposed the giant rams-horn, Marisa comuarietis, to

FEN and observed a significant increase in VDSI (i.e. imposex development by way 149 of vas deferens formation) simultaneously with a decrease in egg production. In another study using the same species, Janer et al. (2006) noticed that FEN did not alter esterified steroid levels in males or females, but imposex did develop in exposed females after 150 days. According to Janer et al. (2006), FEN induced imposex in the netted whelk, Nassarius reticulatus, at the lowest test concentration of 300 ng kg"1 dry wt. in sediment as reported by Tillmann (2004).

Garaventa et al. (2006a) exposed the trunculus murex, Hexaplex trunculus, to two different stressors, MgCfe and Aroclor 1260. MgC^ at roughly 7 - 8% is routinely used in some imposex studies to narcotize gastropods (Spooner et al.,

1991; Oehlmann and Bettin, 1996; Oehlmann et al., 1996c; Davies et al., 1999;

Ramasamy and Murugan, 2002), and the other stressor, Aroclor 1260, is a PCB mixture (Fagervold et al., 2007) identified as an endocrine disruptor (Garaventa et al., 2008). Results of this experiment indicated that at least in H. trunculus, MgCk

(Garaventa et al., 2006a, 2008) and Aroclor 1260 (Garaventa et al., 2008) were capable of inducing imposex.

Fadrozole is a non-steroidal inhibitor of aromatase and a known endocrine disruptor (Bonnefoi et al., 1996) used to treat breast cancer (Raats et al., 1992).

Exposure of the rock snail, Thais clavigera, to FAD induced imposex by increasing female penis length, but not imposex severity as measured by vas deferens formation when compared to control specimens (Iguchi et al., 2007).

Nonylphenol (NP) and copper were tested as inducers of imposex by creating paint formulations and exposing gastropods to these paint formulations. In the first experiment, copper (Cu), a popular biocide used in antifouling paint formulations until it was replaced by the more effective TBT (Morley et al., 2003) induced imposex in Lepsiella vinosa, a gastropod from Australia at 100 and 200 ng l"1 (Nias 150 et al., 1993). However, the results of the study by Nias et al. (1993) should be viewed with some caution. Nias et al. (1993) used a Cu-based antifouling paint and they calculated exposure concentrations based on leaching rates without analytical verification. In addition, at the time of the Nias publication (1980 - 1990) most of the "so called" copper antifouling paint contained TBT and organic biocides with endocrine potential e.g. thio-carbamates, s-triazine, thio-urea etc. (Watermann, personal communication). In a similar study by Evans et al. (2000), NP, a known endocrine disruptor (Masuyama et al., 2000) that mimics the natural hormone 170- estradiol (Lee and Lee, 1996), induced imposex in the dogwhelk, Nucella lapillus, measured by the number of females with a penis, female penis growth, and vas deferens formation.

The last inducer of imposex under the grouping of other contaminants is bird excreta. Nucella lapillus collected beneath cliffs that support breeding colonies of sea birds in the United Kingdom, and adjacent to a gull roost in Scotland, had an increased incidence of imposex and imposex severity (Evans et al., 2000).

Moreover, tissue concentration of N. lapillus sampled from beneath cliffs supporting breeding colonies had elevated butyltin levels when compared to reference sites.

From the results of their study, Evans et al. (2000) deduced that it was possible for bird excreta to induce imposex even though no butyltin analysis was performed on the bird excreta in this study. However, butyltins are known to bioaccumulated in the kidneys and liver of seabirds such as the laysan albatross, Phoebastria immutabilis, from the North Pacific Ocean (Guruge et al., 1997), and androgen metabolites e.g. corticosteroid and testosterone have been identified in bird excreta of the European stonechat, Saxicola torquata rubicola (Goymann, 2005). Since butyltins are known to bioaccumulate in the seabirds, and metabolites have been 151 identified in bird excreta, it is reasonable to assume that bird excreta can induce imposex in Nucella lapillus if TBT or some other imposex inducing compound and/or metabolite was present in the bird excreta at those sites.

5.6.1 Inducers of Female Masculinisation in Gastropods - Summary

It is not surprising that several Sn compounds have been used in FM induction studies using gastropods, or that they resulted in a positive female masculinisation response since some Sn compounds have been described as immunotoxic agents e.g. TBT and DOT (Boyer, 1989) and endocrine disruptors e.g.

FAD, PAD (Bonnefoi et al., 1996; Masuyama et al., 2000). As it relates to imposex induction by TBT "cocktails", induction by this group is also obvious since some components of the "cocktails" are known to induce imposex in gastropods solely, e.g. TBT, TPT, estrogen, and NP, or they are well documented endocrine disruptors in the environment, e.g. DDT. Female masculinisation of gastropods by steroidal compounds is realistic since they control or maintain sexual characteristics as androgens, ligands, and/or inhibitors, but the last group of compounds known to induce imposex in female gastropods branded as other contaminants is diverse.

Imposex induction by this last group of compounds is possible since some members of this group are known endocrine disruptors (e.g. Aroclor 1260, NP and FAD); they maintain or control sexual development (e.g. APGWamide); or they may contain compounds or metabolites of compounds known to induce imposex (e.g. bird excreta). 152

5.7 Existing Theories on the Masculinisation of Female

Gastropods

Even though there have been advances in the number of publications on the

topics of imposex, intersex, and ovo-testis in female gastropods, one subject area that

has continued to challenge and elude scientists is the actual mechanism of induction.

Several theories have been proposed and there continues to be much debate on the

different schools of thought. In this section, an outline of the existing theories that

have been proposed to date and the various compounds that have been used in

induction studies are identified.

To date, there are five hypotheses that have been proposed for imposex

induction in female gastropods: (1) disturbance of the penis morphogenetic factor

(PMF) by neuropeptide(s); (2) aromatase inhibition; (3) inhibition of testosterone

excretion; (4) modulation of free versus fatty acid-bound testosterone levels; and (5)

retinoid X receptors (RXR). Even though several hypotheses have been proposed

about the actual mechanism(s) of imposex induction, this topic remains unclear

(Nishikawa et al., 2004; Iguchi et al., 2007), even after 40+ years of research.

Oehlmann et al. (2007) have suggested that the conflicting results on imposex

induction may be a result of intrinsic species differences in imposex induction as

well as the fundamental mechanism itself. 153

5.7.1 Induction by Disturbance of the Penis Morphogenetic

Factor (PMF)

The first hypothesis for the induction of imposex in gastropods was proposed by Feral and Le Gall (1982; 1983) using the sting winkle, Ocenebra erinacea. In vitro studies concluded that TBT induced the penis morphogenetic activity in the pedal ganglia of normal females (F6ral and Le Gall, 1983). Revitalization of the neuropeptide theory was supported by data from Oberdorster and McClellan-Green

(2000, 2002) and Oberdorster et al. (2005) using a different gastropod, Ilyanassa obsoleta. When I. obsoleta was exposed to a suite of neuropeptides (APGWamide, conopressin, LSSFVRIamide and FMRFamide), only APGWamide, which is normally co-localized in the right pedal ganglia, induced imposex (Oberdorster and

McClellan-Green, 2000). From these results, APGWamide was proposed as the penis morphogenetic factor (PMF) which controls the development of secondary male sex characteristics from the right pedal ganglia.

In a subsequent study, I. obsoleta was exposed to TBT, APGWamide, estradiol (E2), 3-methylcholanthrene (3MC), a highly carcinogenic polycyclic aromatic hydrocarbon (Oberdorster and McClellan-Green, 2002), and mixtures of

TBT and 3MC. Exposure to 3MC or E2 alone did not induce imposex, but co- exposure of 3MC+ TBT and E2 + TBT blocked TBT-induced imposex. On the other

hand, when APGWamide was mixed in a cocktail with 3MC or E2, neither cocktail was able to significantly block APGWamide-induced imposex (Oberdorster and

McClellan-Green, 2002). This further supports the hypothesis that APGWamide

induced imposex occurs by neuropeptide action. 154

Further evidence supporting the neuropeptide functioning as the PMF hypothesis was provided by Oberdorster et al. (2005) from two fronts. Firstly, APGWamide administered exogenously induced male penis length confirming that APGWamide can function as the penis morphogenetic factor. Secondly, I. obsoleta females with

TBT-induced imposex had APGWamide levels comparable to control males while testosterone did not alter APGWamide levels. Because testosterone did not alter

APGWamide levels, Oberdorster et al. (2005) concluded that imposex induction by testosterone is different from induction by TBT, and that it is possible that testosterone interferes with a downstream signaling event to induce imposex.

5.7.2 Induction by Aromatase Inhibition

The second hypothesis to evolve was the aromatase inhibition hypothesis.

Data from a study using Nucella lapillus and Nassarius reticulatus, suggested that

TBT induced imposex by inhibiting cytochrome P-450 dependent aromatase.

Aromatase catalyses the aromatization of androgens to estrogens, and the hypothesis suggests that TBT-induced imposex was a result of increased androgen levels and not directly from TBT or organotins themselves (Bettin et al., 1996). Additional studies that have supported the concept that TBT interferes or inhibits aromatization and/or that TBT does not directly induce imposex, but is assisted by androgens, include Oehlmann and Bettin (1996) and Santos et al. (2005). The simultaneous exposure of TBT and an antiandrogen, cyproterone acetate (CPA), suppressed imposex in N. lapillus and N. reticulatus demonstrating that TBT-induced imposex is a result of increasing androgen levels and not by TBT or organotins themselves

(Bettin et al., 1996). Evidence in support of the theory that TBT-induced imposex is 155 the result of aromatase inhibition was verified when formestane (Form), a P-450 aromatase inhibitor, at 0.30 mg l"1 induced imposex in N. lapillus (Santos et al.,

2005). However, even though Form induced imposex in N. lapillus, it failed to increase imposex severity, so Santos et al. (2005) proposed that an additional mechanism(s) besides aromatase inhibition was/were involved in imposex induction.

5.7.3 Induction by Inhibition of Testosterone Excretion

The third hypothesis to emerge from the literature regarding the induction of imposex in female gastropods was the inhibition of testosterone excretion. This theory was first put forth by Ronis and Mason (1996) using the periwinkle, Littorina littorea. Results from an in vivo experiment revealed that TBT inhibited the sulfur conjugation of testosterone and Phase I metabolites, which resulted in an excess of androgens in the tissues of L. littorea. Later studies by Oberdorster (1997) validated that imposex in I. obsoleta occurred as a consequence of altered steroid metabolism and increased retention of unmetabolized testosterone, and not by aromatase

inhibition of P-450.

5.7.4 Induction by Modulation of Endogenous Testosterone

Levels

The fourth theory to come forward concerning the mechanism of imposex

induction in female gastropods was the modulation of testosterone. Early reports by

Gooding (2002) indicated that the conversion rate of free testosterone to fatty acid esters in llyanassa obsoleta was decreasing as a result of TBT which increased free 156 testosterone levels. In another study examining the biotransformation of testosterone in I. obsoleta, > 70% of the metabolized products were identified as fatty acid esters of testosterone which were proposed as a potential target for TBT toxicity (Gooding and LeBlanc, 2001). The idea that TBT was toxic to the esterfication of testosterone in I. obsoleta was confirmed when TBT inhibited I. obsoleta's ability to produce or retain testosterone as a fatty acid ester (Gooding et al., 2003). The ability of TBT to disrupt testosterone esterfication which results in an increase in free testosterone levels may explain why elevated testosterone levels have been reported in a number of species affected by imposex (Gooding and LeBlanc, 2001).

5.7.5 Induction by Retinoid X Receptors (RXR)

The last hypothesis that has been proposed regarding the induction of FM in gastropods is via the activation of retinoid X receptors (RXR). This theory was initially suggested by Nishikawa et al. (2004). In laboratory experiments using

Thais clavigera, 9-cis RA bound to the human retinoid x receptors (hRXRs) in females with high affinity in similar fashion to organotins. Since the ligand binding in T. clavigera with vertebrate RXR, was similar for both organotins and 9-cis RA, it was suggested that RXR plays an important role in imposex induction via growth and differentiation of male genital tracts in female gastropods (Nishikawa et al.,

2004). When Nucella lapillus was exposed to a suite of inducers, APGWamide, 9- cis RA, testosterone, TBT, and methoprene acid, all compounds induced imposex, except APGWamide, the neuropeptide (Castro et al., 2007). When comparing the inducing potential of that suite of contaminants, imposex incidence and severity

(based on female penis length and vas deferens formation) was high in all cases for 157

TBT with similar results for 9-cis RA. Further, even though testosterone and methoprene acid did induce imposex, induction was to a much lesser degree than

TBT (Castro et al., 2007). In other experiments using immunohistochemical staining, male and imposex-affected T. clavigera revealed localization of RXR protein in the epithelial and smooth muscle cells of the vas deferens and epidermis of the penis (Horiguchi et al., 2007) confirming RXR as an integral component of male genital tracts.

5.7.6 The Great Induction Debate

In this review, evidence for the five existing theories on imposex induction has been outlined. Theories regarding imposex induction have evolved from the earliest theory presented by F6ral and Le Gall (1983), induction by a PMF, to the latest, induction via activation of RXR, suggested by Nishikawa et al. (2004). As the schools of thought have developed over time, so has our knowledge on imposex induction. However, the actual mechanism remains unknown, and the hypotheses are hotly debated. Horiguchi et al. (2006a) argue that the evidence supporting all of the theories presented in this review is weak, except for the theory related to RXR.

Their claim is that the proof for the RXR hypothesis has been validated in the literature based on: (1) RXR gene expression; (2) RXR protein content; (3) immunohistochemical staining of RXR after organotin exposure; and (4) time-course

RXR gene expression after organotin exposure to Thais clavigera. On the other hand, Oehlmann et al. (2007) indicated that in their laboratory, the results of Di

Benedetto (2005) contradict those of Nishikawa et al. (2004) using Nucella lapillus and Nassarius reticulatus, two European relatives of T. clavigera. It is not the aim 158 of this review to decide what argument to support, but to present the arguments as stated in the literature. For a detailed review of the arguments associated with the different theories on imposex induction in gastropods, please refer to Horiguchi

(2006), Horiguchi et al. (2006a), Iguchi et al. (2007), and Oehlmann et al. (2007).

5.8 Indices Used to Measure Female Masculinisation Incidence and Severity

A review of the literature (n = 587), revealed a total of twenty one indices used to measure imposex and intersex incidence and severity, a dramatic increase from the six indices first summarized by Gibbs and Bryan (1994). The indices cited in imposex studies include: % imposex (% I); % sterile/% sterile female; average oviduct convolution (AOS/OS); convolution of proximal oviduct; coefficient of variance (CV%); conversion of potential female to imposex index (CPFII); imposex index; intersex incidence; intersex index (ISI); male to female ratio (M:F); mean female penis length (FPL/mFPL); mean male penis length (MPL/mMPL); mean female prostate length (FPrL); penis classification index (PCI); penis expression (P); relative penis length index (RPL/RPLI); relative penis size index (RPS/RPSI); mean calibre of vas deferens (VDC); mean female vas deferens length (VDLf); vas deferens length index (VDLI); and vas deferens sequence index (VDS/VDSI). The indices current to the mid 1990s were reviewed by Oehlmann et al. (1996b) and

Stroben et al. (1996). For the literature reviewed, the "classic" indices used in imposex and intersex studies were quantitatively ranked based on the number of times they were used in the literature: % I > VDSI > RPSI > mFPL > RPLI > mMPL

> % sterile female > M:F > ISI > AOS > uncommon indices (Figure 5.10). The 159 remaining indices referred to as "uncommon indices" were categorized as such because they were used < 5 times in the literature.

5.8.1 "Classic" Indices

Percent imposex (% I) is the proportion of penis bearing females in a population; this was first utilized by Blaber (1970) studying dogwhelks, Nucella lapillus in the United Kingdom. Even though % I is the most widely used measurement in imposex studies, a major drawback is that it cannot differentiate between severely TBT polluted sites (Stroben et al., 1995; 1996). This is especially true for imposex studies where most or all sites have a % I value of 100%. However, within the last decade, % I has been cited as a valid measure of imposex since TBT environmental levels have declined since the 1980s and 1990s (Couceiro et al.,

2009). With the apparent decline in TBT environmental levels, the use of % I in imposex studies is more likely to show a disparity of values which will enable % I to differentiate between sites.

The vas deferens sequence index denoted in the literature as VDS/VDSI is the mean imposex stage of a population. VDSI was first classified into three stages, early, intermediate and late (Gibbs and Bryan, 1986), but there was no classification to describe normal females, i.e. females with no penis and/or vas deferens. The

VDSI was subsequently revised with a more detailed scale (Gibbs et al., 1987) that classified seven stages of imposex development from normal females (stage 0) to females with aborted egg capsules in the lumen (stage 6), which are sterile (Figure

5.11). Populations with a VDSI value > 4 are indicative of a population with sterile females, and in N. lapillus, this affects the reproductive biology of the population 160

(Gibbs et al., 1987). Additional modifications to the revised VDSI were made by

Oehlmann et al. (1991) which had greater applicability to a wider number of species

(Stroben et al., 1996) thus providing an alternate method to describe imposex by vas deferens formation in a wider number of species. The alternative VDSI by

Oehlmann et al. (1991) had the same seven stages characterized by Gibbs et al.

(1987), but the stages were divided into types a-c (Figure 5.12), representing evolutionary lines of imposex development (Oehlmann et al., 1991; Stroben et al.,

1992c).

Although the two established VDSI systems differ somewhat, their calculations are identical (Stroben et al., 1996). No matter what version of the VDSI is used to measure imposex, VDSI has been regarded as the most reliable (Oehlmann et al., 1992), informative (Stroben et al., 1996), and valid (Oehlmann et al., 1991) index for imposex studies versus indices related to penis length (mFPL, mMPL,

RPSI, RPLI). Reasons for recommending VDSI over indices related to penis length are as follows:

(1) VDSI allows for interspecies comparison of TBT sensitivity between

affected prosobranchs (Huet et al., 1995; Stroben et al., 1996);

(2) there are no seasonal changes associated with VDSI (Oehlmann et al.,

1996b; Stroben et al., 1996), so VDSI measurements give a true

indication of vas deferens development due to TBT exposure;

(3) VDSI is the only index that demonstrates ecological significance because

values > 4 confirm that females in that population are sterilized (Stroben 161

et al., 1996), which allows for an accurate determination of reproductive

capability of affected populations (Gibbs et al., 1987);

(4) VDSI exhibits the highest sensitivity because even at low TBT

concentrations VDSI can be detected (Stroben et al., 1996); and

(5) linear correlation between VDSI and TBT levels in the environment and

tissue samples is higher than those for other imposex measurements

(Stroben et al., (1996).

Although the evidence from the literature supports the preferred use of VDSI over other indices, VDSI cannot discriminate between lower concentrations of TBT levels

(Stroben et al., 1996).

The next commonly utilized indices in imposex studies are all related to measures of penis length: RPSI > mFPL > RPLI > mMPL. Mean female penis length and mMPL are required to calculate RPSI and RPLI, but they have been utilized as independent indices to measure imposex severity. Mean female penis length and mMPL are the average penis lengths of females and males, respectively, in a population. Feral (1977) was the first to use mFPL and mMPL to measure imposex severity in sting winkles, Ocenebra erinecia, in France. Relative penis size index (RPSI) was first described by Bryan et al. (1986), for N. lapillus based on the known relationship between the weight and length of the penis. The RPSI is calculated as follows:

RPSI = (mFPV> *ioo (mMPL) 162

The uncubed RPSI, more commonly referred to as the relative penis length index (RPL/RPLI), was developed sometime in the early 1990s using the reticulated nassa, Nassarius reticulatus (Stroben et al., 1992a, b) and the dogwhelk, Nucella lapillus (Stroben et al., 1992b; Schulte-Oehlmann et al., 1992). The RPLI is calculated just as the RPSI, but without cubing the mFPL and mMPL. The RPSI and

RPLI were developed as an index to measure imposex severity (Bryan et al., 1986;

Stroben et al., 1992a). The RPSI appears more applicable to measure imposex severity in Nucella lapillus, the species in which it was originally described, since

RPSI describes the relationship between the shape of the penis to the cube of its

length. However, in species with oddly shaped penises, RPSI is not applicable

(Stewart et al., 1992), and RPLI should be used instead.

There are two drawbacks to using mMPL and measurements related to it

(RPSI and RPLI). Firstly, random seasonal changes in male penis length are known to occur (Stroben et al., 1996) and since RPSI and RPLI use mMPL in their calculations, any random change in mMPL will also affect their values. For imposex studies evaluating changes in severity over time, this issue can be remedied by executing surveys during the same calendar month or biological time frame for the species being studied. Lastly, RPSI and RPLI have an increased number of penis-

less stages, lb, lc, 2b & 3b, (Stroben, 1996; Oehlmann et al., 1996b), and an evaluation of imposex severity based on RPSI/RPLI alone will result in an underestimation of TBT pollution. Even though there are certain drawbacks to using indices related to mMPL (RPSI and RPLI) they can still be useful in providing supplementary data (Gibbs 1999). RPSI and RPLI can be used to compare temporal scale changes (Smith 1996; Barroso and Moreira, 1998) such as a decline in imposex severity at sites (Evans et. al, 1991). 163

Mean FPL can be used as an independent index to measure imposex severity and it is a valid index. Unlike the mMPL, data suggest that female penis length

(FPL) increases directly in response to TBT exposure until attaining a length comparable to that of their male counterparts (Stroben et al., 1996), and FPL does not appear to vary seasonally (Zhengyan, 2005) as seen in the males. Hence, since

FPL is a result of TBT exposure and does not vary seasonally, mFPL can be used as an independent index to measure imposex severity. However, imposex is irreversible (Gibbs et al., 1987; Oehlmann, 1998; Foale, 1993; Matthiessen et al.,

1999) and the occurrence of a penis in females may reflect previous exposure and not current pollution levels.

The next widely used index in imposex studies is % sterile females. The proportion of sterile females was established by Gibbs et al. (1987), and it is evaluated in conjunction with VDSI since the last two stages of the VDSI (stages 5 and 6) are characteristic of females in a population that are sterile. In N. lapillus, sterility results from the over growth of the female genital papilla by vas deferens

(stage 5), or by the presence of aborted egg capsules in the capsule gland (stage 6)

(Bryan et al., 1987). The sterilization of Nucella lapillus has been described based on Sn exposure from 1 to 10 ng Sn l"1. At tin (Sn) levels ranging from 1 to 2 ng l"1 females may be sterilized. If Sn levels were to increase to > 4 ng l"1, reproductive activity would be severely curtailed, and no breeding would occur at 7 to 10 ng Sn l"1

(Gibbs et al., 1987). The proportion of sterile females has been used as an indicator of a population in decline (Gibbs and Bryan, 1987), and is common in areas with boating activity (Gibbs and Bryan, 1987; Bailey and Davies, 1988; Bright and Ellis,

1990; Spence et al., 1990b; Shi et al., 2004a). Percent sterility has also been documented in the periwinkle, Littorina littorea (De Wolf et al., 2004; Sousa et al., 164

2005). In one study using L. littorea, the highest incidence of female sterility was also observed at point source sites (De Wolf et al., 2004) in similar fashion to that of species severely affected by imposex. However, the use of percent sterility in L. littorea in the literature is relatively non-existent. Furthermore, the proportion of sterile females in L. littorea lacks sensitivity, and is only useful in highly contaminated areas because sterile L. littorea are generally not found at TBT levels <

6 ng Sn l"1 (Oehlmann, 2004).

Male to female ratio (M:F) was first established by Feral (1974) to assess if imposex affected populations deviated from the 1:1 expected ratio of male to female prosobranchs. For some reason, use of the M:F as in index to measure imposex declined almost to non-existence in the 1980s, then started reappearing in the literature in the 1990s. One possible reason for the reintroduction of the M:F as an index to measure imposex in the 1990s was the occurrence of male-dominated populations, signaling that sites were severely impacted by imposex (Evans et al.,

1991). M:F values > 1 indicate that gastropod populations are male dominated, while values < 1 indicate that populations are female dominated (Evans et al.,

2001a). Male biased populations affected by imposex in the 1990s were evident in

N. lapillus in the United Kingdom (Evans et al., 1991), France (Oehlmann et al.,

1994b) and Scotland (Miller et al., 1999), the common whelk (Buccinum undatum)

in the North and Clyde Seas (Nicholson and Evans, 1997), the sharp dwarf winkle

(Ocinebrina aciculata) in France (Oehlmann et al., 1996c), and Morula and Thais

spp. in Indonesia (Pandey and Evans, 1996). M:F ratio values have also been used

as an indicator of populations recovering from imposex (Evans et al., 1991; Stroben,

1996). 165

The last common index used in imposex studies is the average oviduct convolution (AOS/OS) first described by Barreiro et al. (2001). The AOS has three stages: female with normal straight oviduct (OS stage 0); female with oviduct slightly sinuous (OS stage 1); and female with clearly convoluted gonadal oviduct

(OS stage 2). Observations by Barreiro et al. (2001) are similar to those noted for

Ilyanassa obsoleta (Smith, 1980), and AOS has been successfully applied in other members of the Nassariidae: Nassarins reticulatus and N. nitidus (Barreiro et al.,

2001; Rodriguez et al., 2007). AOS is declared as a valid index for imposex studies because it can differentiate between highly polluted areas (Barreiro et al., 2001),

AOS data correlated with female tissue levels in N. reticulatus as seen in other established imposex indices such as RPSI and VDSI (Sousa et al., 2007), and it is positively correlated with other indices used to measure imposex, namely VDSI

(Barroso et al., 2002b; Sousa et al., 2007; Rodriguez et al., 2009). There are certain

AOS ranges defined by Barreiro et al. (2001) that allow researchers to interpret how polluted an area is. An AOS value = 0 denotes that there is low pollution (< 400 ng

TBT-Sn g'1 dry wt.), 0.2 to 0.3 signifies moderately polluted (~ 400 to 500 TBT-Sn g"1 dry wt.), 0.4 to 0.5 implies a contaminated site (800 to 1,600 TBT-Sn g"1), and 0.6 represents a severely polluted site (> 2,000 TBT-Sn g"1 dry wt.).

There are four indices in the literature reported to measure intersex in

Littorina littorea: intersex incidence, % sterile (female), intersex index (ISI) and mean female prostate length (FPrL). Of all indices used to measure intersex in L. littorea, ISI is the most widely used and it was first introduced by Bauer et al.

(1995). Intersex incidence and % sterile (female) are the intersex equivalent to % I and % sterile female previously discussed for imposex-affected species. Intersex incidence can be used as a measure to determine the proportion of the population 166 affected by intersex, but not severity because at fairly high TBT levels most, if not all, female L. littorea would develop intersex and you would not be able to rank the severity of TBT pollution between sites since (Oehlmann, 2004). The remaining two indices, ISI and FPrL, will be discussed in greater detail below as they are the indices most commonly used to measure intersex in the literature.

The ISI is the mean intersex stage of a population that characterizes its reproductive capability (Oehlmann, 2004), and is the equivalent to the VDSI for imposex-affected species. There are five distinctive stages described for the ISI

(Bauer et al., 1995; Oehlmann et al., 1994a): normal females (stage 0); females with a ventrally split bursa copulatrix (stage 1); females with a pallial oviduct entirely split; (stage 2); females with a prostate gland instead of the capsule/covering gland

(stage 3); and females with a prostate gland, penis and sperm groove (stage 4). An

ISI value <0.10 describes a population absent of sterilized females that is typically associated with environmental levels at roughly 2 ng Sn l"1 (Oehlmann, 2004). If environmental levels are approximately 6 ng Sn l"1 (ISI = 0.30) TBT can induce sterilization in females, and at levels as high as 23 ng Sn l"1 (ISI = 2.50), females would be rendered incapable of reproduction (Oehlmann, 2004).

5.8.2 Uncommon Indices

The remaining indices are referred to as "uncommon indices" because they were used to measure imposex/intersex < 5 times in the literature. The uncommon indices included: coefficient of variance (CV%), conversion of potential female to imposex index (CPFII), convolution of the proximal oviduct, imposex index, penis classification index (PCI), mean vas deferens length (VDLf), female vas deferens 167 length index (VDLI), penis expression (P), mean female prostate length (FPrL), intersex incidence, and mean caliber of the vas deferens (VDC).

The coefficient of variance (CV %) was used to determine the probability of imposex occurring in two studies, both using Thais orbita as their model species.

The first was conducted by Foale (1993) and the last by Rees et al. (2001). The coefficient of variance was calculated as follows:

CV(x/y)= s(x / y)/(x / y)* 100% where: s(x/y)= 4[{V(x)lx2 +V(y) /(y2)*(x2/y2)] and: V(x) = (s(x))2 ln;V= variance; s= standard deviation; and n = sample size.

The conversion of potential females to imposex index (CPFII) is the potential of females of a given population to convert to imposex, which was cited once in the literature using Ocenebra bombaya, Thais tissoti, T. bufo and T. rudolphi populations from India (Tewari et al., 2002). The CPFII is calculated as:

QPPH_ (Number of imposex animals) ^ ^ (Number of females) + (Number of imposex animals)

The creators of the CPFII claim that this index is more reliable than RPSI and VDSI, two established and widely used indices in imposex studies. Their argument is that RPSI and VDSI measurements are based on individual organelles which change in shape and size depending on environmental conditions, whereas the CPFII gives the impact of the environment on the total female population (Tewari et al., 2002). This claim that CPFII is a more reliable measurement over RPSI and VDSI is not convincing because the components of the CPFII calculation are based on individuals, and CPFII as a measurement of imposex has been used in only one study whereas RPSI and VDSI have been tested and validated in roughly 169 and 182 studies, respectively. The convolution of the proximal oviduct index was developed by Smith (1980) using the American mudsnail, Ilyanassa obsoleta. Subsequent studies that 168 used the convolution of the proximal oviduct to measure imposex severity include Smith (1980; 1981d) and Curtis (1994) using I. obsoleta, and Gibbs (1996) using Ocenebra erinacea. There are three described stages that are used to evaluate imposex severity using the convolution of the proximal oviduct (Smith, 1980): a penis with a duct leading to a vas deferens (stage 1); a vas deferens passed back to the ventral channel of the capsule gland (stage 2); and convolution of the normally straight gonadal oviduct resembling a stunted mimic of the vesicular seminalis in the males (stage 3). Based on the literature that I have read, the convolution of the proximal oviduct seems to be a valid index to measure imposex severity in I. obsoleta and O. erinacea. The imposex index (Ide et al., 1997) and the penis classification index (PCI) (Mensink et al., 1996), are two indices that appear to be very similar. Both indices were developed around the same time as a modified VDSI system for classifying imposex development for the common whelk, Buccinum undatum. The PCI is the proportion of females showing imposex, and describes four stages (Mensink et al., 1996): female with no male characteristics (stage 0), female with a small round bud at the site of the penis (stage 1), enlargement of differently shaped structures (stage 2), and development of a "L"-curved penis similar to adult males (~ 20 - 30 mm in length), but smaller in size, stage 3 (Figure 5.13). Additionally, females with a vas deferens are designated with a OSPAR (2008) has recommended the use of PCI for imposex severity in B. undatum. The imposex index developed by Ide et al. (1997) also has four categories: unaltered females (category 0); female with a penis­ like structure based on the size of the induced "penisanlage" (categories la, 2a, 3a); female with a vas deferens based on the degree of expression (categories lb, 2b, 3b); and female with both penis and vas deferens (category 4). Both the PCI and imposex index have been successfully used to measure imposex severity in members of the family Buccinidae, and both indices are calculated as follows:

, (Number of females in a category)* (Category number) PCI/Imposex Indexr = ° -—- (Total number of female snails)

Poloczanska and Ansell (1999) have suggested that the imposex index allows for comparison of imposex between populations, so since the imposex index and PCI are 169 calculated in the same way, it is assumed that PCI can also be used to compare imposex between populations. The PCI and imposex index have been applied to measure differences in imposex in B. undatum (Ide et al., 1997; Mensink et al.,

1996), and Neptunea antiqua (Poloczanska and Ansell, 1999; Strand and Jacobsen,

2002). Additionally, PCI has also been used to measure changes in imposex in B. finmarkianum in the Arctic (Strand et al., 2006), while the imposex index was used in Colus gracilis (Power and Keegan, 2001), another member of the Buccinidae family.

The mean female vas deferens length (VDLf) was originally developed by

Lahbib et al. (2008) to measure imposex severity in the trunculus murex, Hexaplex trunculus. VDLf is suggested as a valid index to measure imposex severity because it gives more information on areas that may be less impacted, or in areas where females with penises are lacking or less developed (Lahbib et al., 2008). Abidli et al.

(2009a) developed the vas deferens length index (VDLI) for interspecies comparison of imposex sensitivity using H. trunculus and the purple dye murex, Bolinus brandaris. The VDLI is calculated for each female as follows:

VDU = (Femalevasdeferenslength) wtoeyDS4 4 yDS[ (VDS4)

The last index cited in imposex studies that will be discussed in detail here is the penis expression index (P), which is the mean value for penis development. The penis expression index was developed by Smith (1981b) for I. obsoleta on a 10-point scale (Table 5.8) where the minimum penis intensity equals 10 and the maximum penis development is at 40, indicating development of proximal and distal portions of penis (Bryan et al., 1989). Use of the penis expression index did not extend into the 1990s or beyond. An explanation of the stages that describe penis expression 170

(Smith, 1981b) include: no visible protuberance on head mass: cyst, often filled with brown particulate material, in a position normally occupied by base of penis (level

5); proximal portion of penis only that measures the length of the penis on four levels (level 10 to 25); and proximal and distal portions of penis visible that measures the length of the penis on four levels (levels 30 to 40).

FPrL was first established by Bauer et al. (1995) and is the mean prostate length of females in a population. FPrL is applicable only at sites with fairly high

TBT levels (> 15 ng TBT-Sn l'1), and is less sensitive than ISI because females do not develop a prostate gland before they reach stage 3 (Oehlmann, 2004). The mean

FPrL is calculated as follows:

pp £°f prostate lengths of all females sampled number of females sampled

5.8.3 Recommended Indices

There is a wide range of indices that have been used in the literature to measure the incidence and severity of imposex and intersex in prosobranch gastropods. Understanding the benefits and limitations of each index is necessary for a better understanding of the data especially when informed decisions need to be made for the management of commercial species and for legislation purposes. There is no evidence to suggest that imposex and intersex are reversible (OSPAR, 2008), or that any decrease in organotin exposure leads to a remission of a penis (Bryan et al.,

1987; Stroben et al., 1992b). Hence, the values of imposex indices related to penis length (mFPL, RPSI and RPLI) may not reflect pollution levels at the time of sample 171 collection, but could reflect previous pollution levels that may have improved (Gibbs and Bryan, 1994). However, an approximate time-frame of when the affected population was exposed can be deduced that reflects the data given by the imposex indices if the researcher understands the life cycle and ecology of the species used in the study.

To minimize the limitations associated with individual indices used for measuring imposex, it is suggested that a plethora of data for imposex assessment should be gathered (Table 5.9). For species affected by imposex, it is recommended that % I be calculated to measure imposex incidence, and % sterile female, mFPL,

M: F, RPSI, RPLI, and VDSI to determine imposex severity. For members of the family Buccinidae, additional indices that can be used to assess imposex severity include the PCI and the imposex index while for the Nassariidae family severity can also be assessed using AOS/OS. For species affected by intersex, e.g. Littorina littorea, data to evaluate intersex incidence, percent sterile (female), ISI, and FPrL should be gathered.

5.9 Removing the Veil - Fitting the Remaining Pieces of the

Puzzle

Over the last 40 plus years our knowledge regarding the masculinisation of female gastropods has increased. There are three types of FM described to date

(section 5.2); the affected species list is > 260 species spanning tropical and temperate environments globally (section 5.3); inducers have been identified (section

5.6); induction theories have emerged (section 5.7); and indexes developed to measure incidence, severity and recovery of affected gastropod populations (section 172

5.8). Even though our knowledge of FM in gastropods has expanded, some areas remain a mystery, for example the mechanism(s) responsible for FM induction. In spite of this, there is other information that is not as well known, but noteworthy.

A review of the available literature on the masculinisation of female gastropods from 1962 to 2009 is summarized in Table 5.10. Blaber (1970) is cited in the majority of literature as being the original study to describe "a penis-like

appearance" in female dogwhelks, Nucella lapillus. However, this same observation

was published almost a decade earlier for the Atlantic oyster drill, Urosalpinx cinerea, and the thick-lip drill, Eupleura caudata by Griffith and Castagna (1962).

Other female gastropods observed growing a "penis-like" structure during the early to late 1970s that are hardly ever mentioned in the literature are outlined in Table

5.10.

Although FM has been documented in numerous gastropod species, it is often stated or implied that FM is the result of organotin exposure. However, there

is evidence that female gastropods formed a penis and/or another male anatomical structure e.g. vas deferens prior to the time when TBT was initially and

predominantly used as a biocide in antifouling paint during the 1960s (Oehlmann et

al., 1996b). The incidence of imposex in female gastropods prior to the 1960s has

been dubbed pseudo-imposex (Swennen and Horpet, 2008) and this phenomenon has

been recorded in at least four species. Garaventa et al. (2006b) examined 55

museum samples of the trunculus murex, Hexaplexus trunculus, collected between

1845 and 1930, roughly 30 to 115 years prior to TBT's use as a biocide in antifouling paint. Of those 55 H. trunculus specimens examined, four females (7%) displayed signs of imposex (Garaventa personal communication). More recently, females of three species of gastropods (Cymbiola nobilis, C. vespertilio, and Melo 173 amphora) collected between 1908 and prior to 1930 preserved as museum samples

(n = 1) had a penis and a vas deferens (Swennen and Horpet, 2008). Of the three species noted as having pseudo-imposex by Swennen and Hopert (2008), only

Cymbiola nobilis has been documented as having imposex under field conditions

(Swennen et al., 1996, 1997; Swennen and Horpet, 2008).

Another type of imposex, "natural imposex", has been described in the giant ram's horn, Marisa cornuarietis (Schulte-Oehlmann et al., 1994). Using histology and scanning electron micrographs, data suggest that for M. cornuarietis there are four stages of imposex development (Stage 0 to 3). Stage 1 is the primordial of the male penis sheath and penis pouch and this is the natural level of imposex for this species (Schulte-Oehlmann et al., 1994). The occurrence of pseudo-imposex and natural imposex may provide additional evidence for inducers of FM in gastropods other than organotins such as copper (Nias et al., 1993), environmental stress (Nias et al., 1993) and other pollutants e.g. PAHs and PCBs (Garaventa et al., 2006a, 2008;

Maran et al., 2006) which could be working singly or synergistically.

An anomaly worthy of mention is the incidence of the Dumpton Syndrome

(DS), a rare genetic disorder which can result in an underdeveloped or non- developed (aphally) penis, a split vas deferens due to incomplete formation, and/or the inhibition of spermatozoa formation due to limited testis development (Gibbs,

2005). The DS was first described in Nucella lapillus by Gibbs (1993), and laboratory rearing experiments have verified that DS can be passed on to the F2 generation of both sexes when the Fi generation of a DS affected female is crossed with a non-DS-affected male (Gibbs, 2005). To date, DS has not been reported in any other species besides N. lapillus (Gibbs, 2005),and DS has been proposed as a 174 mechanism that lessens the sterilizing effect of TBT in imposex affected populations

(Quintela et al., 2002; Gibbs, 2005; Huet et al., 2008; Ketata et al., 2008).

5.10Conclusions and Suggestions

After 40 plus years of research, three types of female masculinisation (FM) are known to occur in gastropods, namely, imposex, intersex and ovo-testis. The occurrence of FM in gastropods at field sites has been linked to suspected point sources (e.g. marinas, harbours, moorings and shipyard activities), and this phenomenon has been successfully applied in ecotoxicology studies as biomonitors and bioindicators of organotin pollution. Further, imposex has been considered as the most complete example of endocrine disruption caused by an environmental contaminant (Vos et al., 2000). Some of the key findings of this review include:

(1) the first publication describing a "penis-like" structure in female

gonochoristic gastropods occurred in 1962, not in 1970 as is popularly

reported in the literature;

(2) FM studies reached a pinnacle of publication numbers in 2006 with most

of the literature published in 11 journals (Figure 5.2);

(3) even though there has been a recent change in the taxonomy of gastropods,

there has been a dramatic increase in the number of affected gastropods

from 33 species in 11 families in the 1970s to at least 269 species in 33

families by the end of 2009; 175

(4) although some species (n = 42) have been collected at field sites adjacent

to suspected point sources and sympatrically with other species displaying

signs of FM, or have been exposed to organotins under laboratory

conditions (Table 5.3), they remained unaffected possibly due to

interspecies differences, taxonomic evolution, feeding mode, or the fact

that males of some species do not possess a vas deferens.

(5) FM induction by TBT or TPT has been confirmed in only 7.4% (n = 20) of

the species documented in the literature (n = 269);

(6) additional studies investigating FM in gastropods are recommended:

a. to increase scientific knowledge for families used in FM studies

where there are < 5 species examined (Table 5.4) e.g. Haliotidae,

Trochoidae, Aporrhaididae;

b. for members of the Littorinid family with an ovipositor that is

morphologically and histologically identical to the base of the

penis, e.g. Littorina marie, L. saxatilis, L. obtusata etc. (Sections

5.4.2 and 5.5.1), using Littorina littorea as a positive control; and

c. along the African, Middle East, and Latin American coasts; 176

(7) there are four groups of compounds known to induce FM in gastropods, Sn

compounds, Sn "cocktails", steroids, and a group labelled "other

contaminants";

(8) the majority of field studies on FM in gastropods has been linked to the

biocide TBT, and to a lesser extent TPT, found in antifouling paint;

(9) the bulk of biomonitoring studies that used the FM response in gastropods

to monitor TBT pollution have been conducted in Europe > Asia and

Oceania > North America > Latin America;

(10) the establishment of classic and uncommon indices (n = 21) has enabled

researchers to measure FM incidence and severity among affected

populations, and also allowed for a comparison of affected populations

throughout the species geographic range;

(11) "pseudo-imposex" has been observed in museum samples of Hexaplexus

trunculus (Garaventa et al., 2006b), Cymbiola nobilis, C. vespertilio, and

Melo amphora (Swennen and Horpet, 2008) preserved and collected prior

to the 1930s before TBT and TPT were added as biocides in antifouling

paint;

(12) "natural" imposex occurs in the freshwater gastropod Maris cornuarietis

(Schulte-Oehlmann etal., 1994); 177

(13) the occurrence of pseudo-imposex, "natural imposex" and the ability of

steroids (Bettin et al., 1996; Oehlmann et al., 1996a; Oberdorster and

McClellan-Green, 2000, 2002; Santos et al., 2005) and other contaminants

such as Aroclor 1260 (Garaventa et al., 2008) and nonylphenol (Evans et

al., 2000) to induce imposex lends credibility to the inability of researchers

to confirm the actual mechanism of FM in gastropods to date; and

(14) Dumpton syndrome (DS) is a genetic disorder which passes from one

generation to the next, and it has been proposed as a mechanism that

lessens the sterilising effects of TBT in imposex-affected populations

(Quintela et al., 2002; Gibbs, 2005).

As it relates to indices that measure FM incidence and severity, there is no one index used in the literature that can provide all the information about an affected gastropod population. Hence, data for a number of indices should be collected including % I, % sterile female, mFPL, M:F, RPSI, RPLI, and VDSI for all species known to develop imposex, PCI and AOS for the Nassariidae family, and intersex incidence, percent sterile female, ISI and FPrL are suggested for species that develop intersex. Suggested data to be collected for researchers using Nucella lapillus as a bioindicator of TBT pollution studies are outlined in Table 5.11. This information was modified from Ellis and Pattisina (1990) and can be adapted for other gastropod species once the female secondary sex characteristics are known. OSPAR (2008) has recommended a minimum sample size of 40 specimens per station for imposex and intersex studies; however, some authors have affirmed that a sample size of 30 is adequate (Stroben et al., 1995; Schulte-Oehlmann et al., 1998; Bech, 2002; 178

Zhengyan, 2005) for measuring indices such as % I, % sterile females, mFPL and

VDSI.

Even though TBT has been regulated by legislation since the mid 1980s for most developed countries, imposex still occurs, and induction by organotins under laboratory conditions has been confirmed in only 20% of the 269 species known to be affected by FM. One area of science that may have the key to unlocking the mystery of FM induction in gastropods is genomics. The appearance of a penis or vas deferens in masculinised female gastropods is a phenotypic expression of the organism's genes. Is it possible that TBT, other pollutants, and/or environmental stress are affecting masculinised female gastropods on a molecular and/or biochemical level long before the observable response of a penis or vas deferens can be seen? Future studies exploring this area may prove fruitful to finally answering the great induction debate. As TBT is a persistent pollutant in the marine environment, it appears that the effects of it will be found for several years to come. 179

5.11 References

Abidli, S., Lahbib, Y., and El Menif, N. T. 2009a. Imposex and genital tract malformations in Hexaplex trunculus and Bolinus brandaris collected in the Gulf of Tunis. B Mar Sci. 85: 11 - 25.

Abidli, S., Lahbib, Y., and El Menif, N. T. 2009b. Effects of TBT on the imposex development, reproduction and mortality in Hexaplex trunculus (Gastropoda: Muricidae). J Mar Biol Assoc UK. 89: 139 - 146.

Alvarez, M. M. S., and Ellis, D. V. 1990. Widespread neogastropod imposex in the northeast Pacific - Implications for TBT contamination surveys. Mar Pollut Bull. 21: 244-247.

Alyakrinskaya, I. O. 2005. Morphological and biochemical adaptations to feeding in some herbivorous gastropods. Bio Bull. 32 (1): 57-64.

Alzieu, C. 1986. The detrimental effects on oyster culture in France - evolution since antifouling paint regulation. In Proceedings of the Oceans '86 - Organotin Symposium Proceedings. IEEE: Washington, D. C. USA., pp. 1130- 1134.

Alzieu, C., Thibaud, Y., Heral, M., and Boutier, B. 1980. Evaluation des risques dus a l'emploi des peintures anti-salissures dans les zones conchylicoles. Rev. Trav. Inst. Peches. Marit. 44 (4): 306 - 348.

Andersen, H. R., Vinggaard, A. M., Ramussen, T. H., Gjermandsen, I. M., and Bonefeld-Jorgensen, C. B. 2002. Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicol Applied Pharm. 86 (2): 300 - 305.

Ankley, G. T., Jensen, K. M., Durhan, E. J., Makyen, E. A., Butterworth, B. C., Kahl, M. D., Villeneuve, D. L., Linnum, A., Gray, L. E., Cardon, M., and Wilson, V. S. 2005. Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas). Toxicol Sci. 86 (2): 300 - 305.

Appeltans, W., Bouchet, P., Boxshall, G. A., Fauchald, K., Gordon, D. P., Hoeksema, B.W., Poore, G. C. B., van Soest, R. W. M., Stohr, S., Walter, T. C., and Costello, M. J. (eds.). 2009. World Register of Marine Species. Accessed at http://www.marinespecies.org on [2009-11-29].

Arconada, B., and Ramos, M. A. 2002. Spathogyna, a new genus for Valvata (? Tropidina) fezi Altimira, 1960 from eastern Spain: Another case of pseudohermaphroditism in the Hydrobiidae (Gastropoda). J Mollus Stud. 68: 319-327. 180

Axiak, V., Vella, A. J., Micallef, D., Chircop, P., and Mintoff, B. 1995. Imposex in Hexaplex trunculus (Gastropoda, Muricidae) - First results from biomonitoring of tributyltin contamination in the Mediterranean. Mar Biol. 121:685-691.

Bailey, S. K., and Davies, I. M. 1988. Tributyltin contamination in the Firth-of-Forth (1975-87). Sci Total Environ. 76: 185 - 192.

Bailey, S. K., and Davies, I. M. 1991. Continuing impact of TBT, previously used in mariculture, on dogwhelk (Nucella lapillus L.) populations in a Scottish sea loch. Mar Environ Res. 32: 187 - 199.

Barreiro, R., Gonzalez, R., Quintela, M., and Ruiz, J. M. 2001. Imposex, organotin bioaccumulation and sterility of female Nassarius reticulatus in polluted areas of NW Spain. Mar Ecol Prog Ser. 218: 203 - 212.

Barroso, C. M., and Moreira, M. H. 1998. Reproductive cycle of Nassarius reticulatus in the Ria de Aveiro, Portugal: Implications for imposex studies. J Mar Biol Assoc UK. 78: 1233 - 1246.

Barroso, C. M., Reis-Henriques, M. A., Ferreira, M. S,, and Moreira, M. H. 2002a. The effectiveness of some compounds derived from antifouling paints in promoting imposex in Nassarius reticulatus. J Mar Biol Assoc UK. 82: 249 - 255.

Barroso, C. M., Moreira, M. H., and Bebianno, M. J. 2002b. Imposex, female sterility and organotin contamination of the prosobranch Nassarius reticulatus from the Portuguese coast. Mar Ecol Prog Ser. 230: 127 - 135.

Bauer, B., Fioroni, P., Ide, I., Liebe, S., Oehlmann, J., Stroben, E., and Watermann, B. 1995. TBT effects on the female genital system of Littorina littorea - a possible indicator of tributyltin pollution. Hydrobiol. 309: 15-27.

Bauer, B., Fioroni, P., SchulteOehlmann, U., Oehlmann, J., and Kalbfus, W. 1997. The use of Littorina littorea for tributyltin (TBT) effect monitoring results from the German TBT survey 1994/1995 and laboratory experiments. Environ Pollut. 96: 299 - 309.

Bech, M. 1999. Increasing levels of tributyltin-induced imposex in Muricid gastropods at Phuket Island, Thailand. Appl Organomet Chem. 13: 799 - 804.

Bech, M. 2002. A survey of imposex in Muricids from 1996 to 2000 and identification of optimal indicators of tributyltin contamination along the east coast of Phuket Island, Thailand. Mar Pollut Bull. 44: 887 - 896.

Beechey, D. 2010. The seashelf of NewSouth Wales - Based on the collections of the Australian Museum. Accessed at http://www.seashellsofnsw.org.au/index.htm on [2010-06-12]. 181

Bettin, C., Oehlmann, J., and Stroben, E. 1996. TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level. Helgolander Meeresun. 50: 299 - 3 17.

Bigatti, G., and Penchaszadeh, P. 2005. Imposex in Odontocymbiola magellancia (Caenogastropoda: Volutidae) in Patagonia. Communicaciones de la Sociedad Malacologia del Uruguay. 9: 371 - 375.

Bigatti, G., Primost, M. A., Cled6n, M., Averbuj, A., Theobald, N., Gerwinski, W., Arntz, W., Morriconi, E., and Penchaszadeh, P. E. 2009. Biomonitoring of TBT contamination and imposex incidence along 4700 km of Argentinean shoreline (SW Atlantic: From 38S to 54S). Mar Pollut Bull. 58: 695 - 701.

Blaber, S. J. M. 1970. The occurrence of a penis-like outgrowth behind the right tentacle in spent females of Nucella lapillus (L.). Proc Malacol Soc Lond. 39: 231 -233.

Bonnefoi, H. R., Smith, I. E., Dowsett, M., Trunet, P. F., Houston, S. J., daLuz, R. J., Rubens, R. D., Coombes, R. C., and Powles, T. J. 1996. Therapeutic effects of the aromatase inhibitor fadrozole hydrochloride in advanced breast cancer. Brit J Cancer. 73: 539 - 542.

Bouchet, P., and Rocroi, J. P. 2005. Classification and nomenclature of gastropod families. Malacologia, International Journal of Malacology. 47 (1-2): 397pp.

Boyer, I. J. 1989. Toxicity of dibutyltin, tributyltin and other organotins compounds to humans and to experimental animals. Toxicol. 55: 253 - 298.

Bright, D. A., and Ellis, D. V. 1990. A comparative survey of imposex in northeast Pacific neogastropods (Prosobranchia) related to tributyltin contamination, and choice of a suitable bioindicator. Can J Zool. 68: 1915 - 1924.

Bryan, G., Gibbs, P., Hummerstone, L., and Burt, G. 1986. The decline of the gastropod Nucella lapillus around south-west England: evidence for the effect of tributyltin from antifouling paints. J Mar Biol Assoc UK. 66: 611 - 640.

Bryan, G., Gibbs, P., Burt, G., and Hummerstone, L. 1987. The effects of tributyltin (TBT) accumulation on adult dogwhelks, Nucella lapillus: long-term field and laboratory experiments. J Mar Biol Assoc UK. 67: 525 - 544.

Bryan, G., Gibbs, P., and Burt, G. 1988. A comparison of the effectiveness of tri-n- butyltin chloride and five other organotin compounds in promoting the development of imposex in the dog-whelk, Nucella lapillus. J Mar Biol Assoc UK. 68: 733 - 744.

Bryan, G.W., Gibbs, P. E., Huggett, R. J., Curtis, L. A., Bailey, D. S., and Dauer, D. M. 1989. Effects of tributyltin pollution on the mud snail, Ilyanassa obsoleta, from the York River and Sarah Creek, Chesapeake Bay. Mar Pollut Bull. 20: 458 - 462. 182

Caetano, C. H. S., and AbsalSo, R. S. 2002. Imposex in Olivancillaria vesica vesica (Gmelin) (Gastropoda, Olividae) from a southeastern Brazilian sandy beach. Rev Bras Zool. 19: 215 - 218.

Cardoso, R. S., Caetano, C. H. S., and Cabrini, T. M. B. 2009. Biphallia in imposexed females of marine gastropods: new record for Nassarius vibex from Brazil. Braz J Biol. 69: 223 - 224.

Carlberg, C., Saurat, J. H., and Siegenthaler, G. 1993. 9-cw-retinoic acid is a natural antagonist for the retinoic acid receptor response pathway. Biochem J. 295: 343 - 346.

Camillo, E., Quadros, J., de Castro, I.B., and Fernandez, M. A. 2004. Imposex in Thais rustica (Mollusca: Neogastropoda) (Lamark, 1822) as an indicator of organotin compounds pollution at Maceio Coast (northeastern Brazil). Braz J Oceanogr. 52: 101 - 105.

Carpenter, K. E., and Niem, V. H. (eds.). 1998. FAO species identification guide for fishery purposes - The living marine resources of the western Pacific. Volume 1. Seaweeds, corals, bivalves and gastropods. Rome, FAO. 99: 1 - 686.

Casey, F. X. M., Hakk, H., Simunek, J., and Larsen, G. L. 2004. Fate and transport of testosterone in agricultural soils. Environ Sci Technol. 38: 790 - 798.

Castro, L. F. C., Lima, D., Machado, A., Melo, C., Hiromori, Y., Nishikawa, J., Nakanishi, T., Reis-Henriques, M. A., and Santos, M. M. 2007. Imposex induction is mediated through the Retinoid X Receptor signaling pathway in the neogastropod Nucella lapillus. Aquat Toxicol. 85: 57 - 66.

Cledon, M., Theobald, N., Gerwinski, W., and Penchaszadeh, P. E. 2006. Imposex and organotin compounds in marine gastropods and sediments from the Mar del Plata coast, Argentina. J Mar Biol Assoc UK. 86: 751 - 755.

Cob, Z. C., Arshad, A., Idris, M. H., Bujang, J. S., and Ghaffar, M. A. 2008. Sexual polymorphism in a population of Strombus canarium Linnaeus, 1758 (Mollusca: Gastropoda) at Merambong Shoal, Malaysia. Zool Stud. 47: 318 - 325.

Colborn, T., vomSaal, F. S., and Soto, A. M. 1993. Developmental effects of endocrine disrupting chemicals in wildlife and humans. Environ Health Persp. 101 (5): 378 - 384.

Couceiro, L., Diaz, J., Albaina, N., Barreiro, R., Irabien, J. A., and Ruiz, J. M. 2009. Imposex and gender-independent butyltin accumulation in the gastropod Nassarius reticulatus from the Cantabrian coast (N Atlantic Spain). Chemosphere. 76: 424 - 427. 183

Covert, T. 1997. Masculinization of dogwhelks (Nucella lapillus) and periwinkles (Littorina littorea) in Halifax Harbour: Monitoring of tributyltin. BSc. Honours Thesis, St. Mary's University, Nova Scotia.

Crain, D. W., Guillette Jr, L. J., Pickford, D. B., Percival, H. F., and Woodward, A. R. 1998. Sex-steroid and thyroid hormone concentrations in juvenile alligators {Alligator mississippiensis) from contaminated and reference lakes in Florida, USA. Environ Toxicol Chem. 17 (3): 446 - 452.

Curtis, L. A. 1994. A decade-long perspective on a bioindicator of pollution - Imposex in Ilyanassa obseleta on Cape Henlopen, Delaware Bay. Mar Environ Res. 38: 291 - 302. da Costa, M. B., Fernandez, M. A., Barbiero, D. C., de Melo, F. T. V., and Ferreira, B. S. 2008. First record of imposex in Thais deltoidea (Lamarck, 1822) (Mollusca, Gastropoda, Thaididae) in Vitoria, Es, Brazil. Braz J Oceanogr. 56: 145 - 148.

Davies, I. M., and Bailey, S. K. 1991. The impact of tributyltin from large vessels on dogwhelk {Nucella lapillus) populations around Scottish oil ports. Mar Environ Res. 32: 201 -211.

Davies, I. M., Minchin, A., and Harding, M. J. C. 1997. OSPAR working group on concentrations, trends and effects of substances in the marine environment (SIME) - Report on the TBT Training workshop In Marine Laboratory Aberdeen Report. Marine Laboratory, Aberdeen, pp. 36pp.

Davies, I. M., Minchin, A., Bauer, B., Harding, M. J. H., and Wells, D. E. 1999. QUASIMEME laboratory performance study of the biological effects of tributyltin (imposex and intersex) on two marine gastropod molluscs. J Environ Monitor. 1: 233 - 238. de Castro, I. B., Matthews-Cascon, H., and Fernandez, M. A. 2000. Occurrence of imposex in Thais haemastoma (Linnaeus, 1767) (Mollusca: Gastropoda), as an indicator of contamination by organotin on the coastal zone of Fortileza City, Ceara State, Brazil. Arq Cienc Mar. 33: 143 - 148. de Castro, I. B., Ribeiro-Ferreira, V. P., Lima, A. F. A., Meirelles, C. A. O., and Rocha-Barreira, C. A. 2007. Imposex in three prosobranch species from Ilha do Japones, Rio de Janeiro, southeast Brazil. Thalassas. 23: 37 - 42. de Castro, I. B., de Meirelles, C. A. O., Matthews-Cascon, H., Rocha-Barreira, C. D., Penchaszadeh, P., and Bigatti, G. 2008. Imposex in endemic Volutid from northeast Brazil (Mollusca: Gastropoda). Braz Arch Biol Techn. 51: 1065 - 1069. de Castro, V. L. S. S., deMello, M. A., Poli, P., and Zucchi, T. M. A. D. 2005. Prenatal and perinatal fenarimol-induced genotoxicity in leukocytes of in vivo treated rats. Mutat Res. 583: 95 - 104. 184

Deutsch, U., and Fioroni, P. 1996. Effects of tributyltin (TBT) and testosterone on the female genital system in the mesogastropod Littorina littorea (Prosobranchia). Helgolander Meeresun. 50: 105 - 115.

Deutsch, U., Oehlmann, J., and Stroben, E. 1996. Morphological effects of tributyltin (TBT) on the genital system of the Mesogastropod Littorina littorea (Prosobranchia). Malacol Rev Suppl - Molluscan Reproduction. 6: 143- 148.

De Wolf, H., Handa, C., Backeljau, T., and Blust, R. 2004. A baseline survey of intersex in Littorina littorea along the Scheldt estuary, The Netherlands. Mar Pollut Bull. 48: 592 - 596.

Di Benedetto, P. 2005. Investigations in imposex development in marine prosobranchs. Diploma thesis. Johann Wolfgang Goethe University, Faculty Biological Sciences. Frankfurt, Germany. [In German]

Douglas, E. W., Evans, S. M., Frid, C. L. J., Hawkins, S. T., Mercer, T. S., and Scott, C. L. 1993. Assessment of imposex in the dogwhelk Nucella lapillus (L) and tributyltin along the northeast coast of England. Invertebr Reprod Dev. 24: 243 - 248.

Duft, M., Schulte-Oehmann, U., Tillmann, M., Weltje, L., and Oehlmann, J. 2005. Biological impact of organotin compounds on mollusks in marine and freshwater ecosystems. Coast Mar Sci. 29: 95 - 110.

Duft, M., Schmitt, C., Bachmann, J., Brandelik, C., Schulte-Oehlmann, U., and Oehlmann, J. 2007. Prosobranch snails as test organisms for the assessment of endocrine active chemicals - An overview and a guideline proposal for a reproduction test with the freshwater mudsnail Potamopyrgus antipodarum. Ecotoxicol. 16: 169 - 182.

Ellis, D. V., and Pattisina, L. A. 1990. Widespread neogastropod imposex - A biological indicator of global TBT contamination. Mar Pollut Bull. 21: 248 - 253.

Environment Canada. 2002. Metal mining guidance document for aquatic environmental effects monitoring. National EEM Office, Environment Quality Branch, Ottawa, Ontario, www.ec.gc.ca/eem.

Environment Canada. 2005. Revised technical guidance for conducting aquatic environmental effects monitoring for pulp and paper mills. Environment Canada, National EEM Office, National Water Research Institute, Gatineau, QC.

Evans, C. J., and Smith, P. J. 1975. Organotin-based antifouling systems. J Oil Col Chem. Assoc. 58: 160 - 168. 185

Evans, S. M., Hutton, A., Kendall, M. A., and Samosir, A. M. 1991. Recovery in populations of dogwhelks Nucella lapillus (L.) suffering from imposex. Mar Pollut Bull. 22: 331 -333.

Evans, S. M., Dawson, M., Day, J., Frid, C.L.J., Gill, M. E., Pattisina, L. A., and Porter, J. 1995. Domestic waste and TBT pollution in coastal areas of Ambon Island (eastern Indonesian). Mar Pollut Bull. 30: 109 - 115.

Evans, S. M., Nicholson, G. J., Browning, C., Hardman, E., Seligman, O., and Smith, R. 1998. An assessment of tributyltin contamination in the North Atlantic using imposex in the dogwhelk Nucella lapillus (L.) as a biological indicator of TBT pollution. Invertebr Reprod Dev. 34: 277 - 287.

Evans, S. M., Kerrigan, E., and Palmer, N. 2000. Causes of imposex in the dogwhelk Nucella lapillus (L.) and its use as a biological indicator of tributyltin contamination. Mar Pollut Bull. 40: 212 - 219.

Evans, S. M., Barnes, N., Birchenough, A. C., Brancato, M. S., and Hardman, E. 2001a. Tributyltin contamination in two estuaries and adjacent ocean coasts: Puget Sound, Washington, and Narragansett Bay, Rhode Island (USA). Invertebr Reprod Dev. 39: 221 - 229.

Evans, S. M., Bech, M., Hawkins, S. T., Smith, R., and Stewart, A. 2001b. Imposex in populations of dogwhelks Thais spp. in relation to shipping intensity and mariculture activity in East Asia. Invertebr Reprod Dev. 39: 231 - 237.

Fagervold, S. K., May, H. D., and Sowers, K. R. 2007. Microbial reductive dechlorination of Aroclor 1260 in Baltimore Harbor sediment microcosms is catalyzed by three phylotypes within the Phylum Choloroflexi. Appl Environ Microb. 73 (9): 3009 -3018.

Feral, C. 1974. Etude comparee des populations d'Ocenebra erinacea (L.) de Granville et d'Arcachon. Haliotis. 4: 123 - 134.

Feral, C. 1975. Mise en evidence des facteurs determinant 1'apparition et le cycle d'un tractus genital male chez les femelles d'Ocenebra erinacea (L.) du bassin d'Arcachon. B Soc Zool Fr. 100: 250 - 252.

Feral, C. 1977. Analyse experimental de la morphogenese et du cycle du penis les femelles d'Ocenebra erinacea, espece gonochorique. Haliotis. 6: 267 - 271.

Feral, C. 1978. Presence des facterus morphogenetique et dedifferenciateur du penis chez un Mollusque Prosobranche gonochorique Ocenebra erinacea (L.). C R Hebd Stances Acad Sci D. 287: 1235 - 1237.

Feral, C. 1979. Etude des facteurs regissant 1'apparition d'un penis chez les femelles d' Ocenebra erinacea L. (Mollusque Gasteropode gonochorique) de la station d' Arcachon. C R Hebd Seances Acad Sci D. 289: 331 - 334. 186

Feral, C. 1980a. Apparition de femelles atractus genital male externe chez Ocenebra erinacea (L.), Mollusque Gast^ropode gonochorique de la station de Granville: Recherche des facteurs contr61ant de cette anomalie. C R Hebd Stances Acad Sci D. 290: 1003 - 1006.

Feral, C. 1980b. Influence de la qualite de l'eau de mer sur la differenciation d'un tractus genital male externe chez les femelles. C R Hebd Stances Acad Sci D. 291:775- 778.

F6ral, C. L., and Le Gall, S. 1982. Physiologie des invert6br6s - Induction exp^rimentale par un polluant marin (le tributyletain), de 1'activity neuroendocrine controlant la morphogenese du p&iis chez les femelles <¥ Ocenebra erinacea (Mollusque, Prosobranche gonochorique). C R Hebd Stances Acad Sci D. 295: 627 - 630.

Feral, C., and Le Gall, S. 1983. The influence of a pollutant factor (tributyltin) on the neuroendocrine mechanism responsible for the occurence of a penis in the females of Ocenebra erinacea. In Proceedings of the International Minisymposium on Molluscan endocrinology. (Edited by J. Lever and H.H. Boer), Amsterdam, Netherlands, pp. 173 - 175.

Fioroni, P., Oehlmann, J., and Stroben, E. 1990. Le pseudohermaphrodisme chez les prosobranches analyse morphologique et histologique. Vie Milieu. 40: 45 - 56.

Fioroni, P., Oehlmann, J., and Stroben, E. 1991. The pseudohermaphroditism of Prosobranchs - Morphological aspects. Zool Anz. 226: 1 - 26.

Foale, S. 1993. An evaluation of the potential of gastropod imposex as a bioindicator of tributyltin pollution in Port Phillip Bay, Victoria. Mar Pollut Bull. 26: 546 -552.

F0lsvik, N., Berge, J. A., Brevik, E. M., and Walday, M. 1999. Quantification of organotin compounds and determination of imposex in populations of dogwhelks (Nucella lapillus) from Norway. Chemosphere. 38: 681 - 691.

Gangolli, S. D. 1999. The dictionary of substances and their effects (DOSE): A - B. Royal Society of Chemistry; 2nd revised edition, 912 pp.

Garaventa, F. 2006. Personal communication.

Garaventa, F., Faimali, M., Terlizzi, A., Fiorini, S., and Gallo, G. 2006a. Imposex induction in Hexaplex trunculus by tributyltin and other stressors. Mar Environ Res. 62: S230 -S230.

Garaventa, F., Faimali, M., and Terlizzi, A. 2006b. Imposex in pre-pollution times. Is TBT to blame? Mar Pollut Bull. 52: 701 - 702. 187

Garaventa, F., Centanni, E., Fiorini, S., Noventa, S., Terlizzi, A., Faimali, M., and Pavoni, B. 2008. New implications in the use of imposex as a suitable tool for tributyltin contamination: experimental induction in Hexaplex trunculus (Gastropoda, Muricidae) with different stressors. Cell Biol Toxicol. 24: 563 - 571.

Gibbs, P., and Bryan, G. W. 1986. Reproductive failure in populations of the dog- whelk, Nucella lapillus, caused by imposex induced by tributyltin from antifouling paints. J Mar Biol Assoc UK. 66: 767 - 777.

Gibbs, P., Bryan, G., Pascoe, P., and Burt, G. 1987. The use of the dog-whelk, Nucella lapillus, as an indicator of tributyltin (TBT) contamination. J Mar Biol Assoc UK. 67: 507- 523.

Gibbs, P., Pascoe, P., and Burt, G. 1988. Sex change in the female dog-whelk, Nucella lapillus, induced by tributyltin from antifouling paints. J Mar Biol Assoc UK. 68:715-731.

Gibbs, P. E. 1993. A male genital defect in the dogwhelk, Nucella lapillus (Neogastropoda), favoring survival in a TBT-polluted area. J Mar Biol Assoc UK. 73: 667 - 678.

Gibbs, P. E. 1996. Oviduct malformation as a sterilising effect of tributyltin (TBT)- induced imposex in Ocenebra erinacea (Gastropoda: Muricidae). J Mollus Stud. 62:403 -413.

Gibbs, P. E. 1999. Biological effects of contaminants: Use of imposex in the dogwhelk {Nucella lapillus) as a bioindicator of tributyltin pollution. ICES Techniques in Mar Environ Sci. 24: 1 - 29.

Gibbs, P. E. 2005. Male genital defect (Dumpton Syndrome) in the dog-whelk Nucella lapillus (Neogastropoda): Mendelian inheritance inferred, based on laboratory breeding experiments. J Mar Biol Assoc UK. 85: 143 - 150.

Gibbs, P. E., and Bryan, G. W. 1987. TBT Paints and the demise of the dog-whelk, Nucella lapillus (Gastropoda). In Proceedings of Oceans '87- Organotin Symposium Proceedings, pp. IEEE. New York, N.Y., pp. 1482 - 1487.

Gibbs, P. E., and Bryan, G. W. 1994. Biomonitoring of tributyltin (TBT) pollution using the imposex response of neogastropod molluscs. In Biomonitoring of Coastal Waters and Estuaries {Edited by K. J. M. Kramer). CRC Press Incorporated, Boca Raton, Florida., pp 205 - 226.

Gibbs, P. E., and Bryan, G. W. 1996. TBT-induced imposex in neogastropod snails: Masculinization to mass extinction. In Tributyltin: Case Study of an Environmental Contaminant {Edited by S J. de Mora). Cambridge University Press, Cambridge, Great Britain, pp. 212 - 236. 188

Gibbs, P. E., Pascoe, P. L., and Bryan, G. W. 1991a. Tributyltin-induced imposex in Stenoglossan gastropods - Pathological effects on the female reproductive- system. Comp Biochem Phys C. 100: 231 - 235.

Gibbs, P. E., Bryan, G. W., and Pascoe, P. L. 1991b. TBT-induced imposex in the dogwhelk, Nuceila lapillus - Geographical uniformity of the response and effects. Mar Environ Res. 32: 79 - 87.

Gibbs, P. E., Bebianno, M. J., and Coelho, M. R. 1997. Evidence of the differential sensitivity of neogastropods to tributyltin (TBT) pollution, with notes on a species (Columbella rustica) lacking the imposex response. Environ Technol. 18: 1219- 1224.

Giltrap, M., Macken, A., Davoren, M., Minchin, D., McGovern, E., Foley, B., Strand, J., and McHugh, B. 2009. Use of caged Nuceila lapillus and Crassostrea gigas to monitor tributyltin-induced bioeffects in Irish coastal waters. Environ Toxicol Chem. 28: 1671 - 1678.

Godtfredsen, K., Hotchkiss, D., Williston, D., Stern, J., Fox, S., J., G., Andersen, H., and Kohn, A. 2005. Analysis of imposex in neogastropods collected from the lower Duwamish Waterway. In SETAC 26th Annual Meeting (November 13 - 17, 2005), Baltimore, Maryland.

Goldberg, E. D. 1975. The mussel watch - A first step in global marine monitoring. Mar Pollut Bull. 6: 111.

Gomez-Ariza, J. L., Santos, M. M., Morales, E., Giraldez, I., Sanchez-Rodas, D., Vieira, N., Kemp, J. F., Boon, J. P., and Ten-Hallers-Tjabbes, C. C. 2006. Organotin contamination in the Atlantic Ocean off the Iberian Peninsula in relation to shipping. Chemosphere. 64: 1100 - 1108.

Gooding, M., Gallardo, C., and Leblanc, G. 1999. Imposex in three marine gastropod species in Chile and potential impact on muriciculture. Mar Pollut Bull. 38: 1227- 1231.

Gooding, M. P. 2002. The role of steroid metabolism in tributyltin-induced imposex in the eastern mud snail {Ilyanassa obsoleta). PhD. Thesis, North Carolina State University, North Carolina.

Gooding, M. P., and LeBlanc, G. A. 2001. Biotransformation and disposition of testosterone in the eastern mud snail Ilyanassa obsoleta. Gen Comp Endocr. 122: 172 - 180.

Gooding, M. P., Wilson, V. S., Folmar, L. C., Marcovich, D. T., and LeBlanc, G. A. 2003. The biocide tributyltin reduces the accumulation of testosterone as fatty acid esters in the mud snail {Ilyanassa obsoleta). Environ Health Persp. Ill: 426 -430. 189

Goymann, W. 2005. Noninvasive monitoring of hormones in bird droppings physiological validation, sampling, extraction, sex differences, and the influence of diet on hormone metabolite levels. Ann. N.Y. Acad. Sci. 1046: 35 - 53.

Graham, D. C. 1964. The use of organo-tin compounds as potato tuber disinfectants, particularly against Rhizoctonia solani. Potato Res. 7 (1): 33- 44.

Gravel, P., Johanning, K., McLachlan, J., Vargas, J. A., and Oberdorster, E. 2006. Imposex in the intertidal snail Thais brevidentata (Gastropoda: Muricidae) from the Pacific coast of Costa Rica. Rev Biol Trop. 54: 21 - 26.

Griffith, G. W., and Castagna, M. 1962. Sexual dimorphism in oyster drills of Chincoteagne Bay, Maryland, Virginia. Chesapeake Sci. 3: 215 - 217.

Guruge, K. S., Iwata, H., Tanakac, H., and Tanabe, S. 1997. Butyltin accumulation in the liver and kidney of seabirds. Mar Environ Res. 44 (2): 191 - 199.

Hagger, J. A., Depledge, M. H., Oehlmann, J., Jobling, S., and Galloway, T. S. 2006. Is there a causal association between genotoxicity and the imposex effect? Environ Health Persp. 114: 20 - 26.

Harding, M. J. C., Rodger, G. K., Davies, I. M., and Moore, J. J. 1997. Partial recovery of the dogwhelk (Nucella lapillus) in Sullom Voe, Shetland from tributyltin contamination. Mar Environ Res. 44: 285 - 304.

Harmon, M. A., Boehm, M. F., Heyman, R. A., and Mangelsdorf, D. J. 1995. Activation of mammalian retinoid X receptors by the insect growth regulator methoprene. P Natl Acad Sci USA. 92 (13): 6157 - 6160.

Hawkins, L. E., and Hutchinson, S. 1990. Physiological and morphogenetic effects of monophenyltin trichloride on Ocenebra erinacea (L). Funct Ecol. 4: 449 - 454.

Hernandez, S., and Stotz, W. B. 2004. Reproductive biology of the "Copey" snail Melongena melongena (Linnaeus, 1758) in Cispat Bay on the Caribbean coast of Colombia. J Shellfish Res. 23: 849 - 854.

Horiguchi, T. 2006. Endocrine disruption caused by organotin compounds in gastropod mollusks. In 3rd Asia-Pacific Workshop - "Modern approaches to linking exposure to Toxic Compounds and Biological effects", Xiamen, China, pp. 101-109.

Horiguchi, T., Shiraishi, H., Shimizu, M., and Morita, M. 1994. Imposex and organotin compounds in Thais clavigera and T. bronni in Japan. J Mar Biol Assoc UK. 74: 651 -669. 190

Horiguchi, T., Shiraishi, H., Shimizu, M., Yamazaki, S., and Morita, M. 1995. Imposex in Japanese gastropods (Neogastropoda and Mesogastropoda): Effects of tributyltin and triphenyltin from antifouling paints. Mar Pollut Bull. 31: 402-405.

Horiguchi, T., Shiraishi, H., Shimizu, M., and Morita, M. 1997a. Imposex in sea snails, caused by organotin (tributyltin and triphenyltin) pollution in Japan: A survey. Appl Organomet Chem. 11: 451 - 455.

Horiguchi, T., Shiraishi, H., Shimizu, M., and Morita, M. 1997b. Effects of triphenyltin chloride and five other organotin compounds on the development of imposex in the rock shell, Thais clavigera. Environ Pollut. 95: 85 - 91.

Horiguchi, T., Takiguchi, N., Cho, H. S., Kojima, M., Kaya, M., Shiraishi, H., Morita, M., Hirose, H., and Shimizu, M. 2000. Ovo-testis and disturbed reproductive cycle in the giant abalone, Haliotis madaka: possible linkage with organotin contamination in a site of population decline. Mar Environ Res. 50: 223 - 229.

Horiguchi, T., Cho, H.S., Shiraishi, H., Kojima, M., Kaya, M., Morita, M., and Shimizu, M. 2001. Contamination by organotin (tributyltin and triphenyltin) compounds from antifouling paints and endocrine disruption in marine gastropods. Riken Rev. 35: 9 -11.

Horiguchi, T., Kojima, M., Kaya, M., Matsuo, T., Shiraishi, H., Morita, M., and Adachi, Y. 2002. Tributyltin and triphenyltin induce spermatogenesis in ovary of female abalone, Haliotis gigantea. Mar Environ Res. 54: 679 - 684.

Horiguchi, T., Kojima, M., Takiguchi, N., Kaya, M., Shiraishi, H., Morita, M., and Imai, T. 2004. Continuing observation of endocrine disruption in Abalone from an organotin-contaminated site of Japan. In SETAC 25th Annual Meeting (November 14- 18, 2004), Portland, Oregon, USA.

Horiguchi, T., Kojima, M., Takiguchi, N., Kaya, M., Shiraishi, H., and Morita, M. 2005. Continuing observation of disturbed reproductive cycle and ovarian spermatogenesis in the giant abalone, Haliotis madaka from an organotin- contaminated site of Japan. Mar Pollut Bull. 51: 817 - 822.

Horiguchi, T., Shiraishi, H., Nishikawa, T., Shiraishi, F., Nishikawa, J., Ohta, Y., and Morita, M. 2006a. Analysis of induction mechanism of imposex cause by tributyltin and triphenyltin in gastropod molluscs. Biomed Res Trace Ele. 17: 152 - 154.

Horiguchi, T., Kojima, M., Hamada, F., Kajikawa, A., Shiraishi, H., Morita, M., and Shimizu, M. 2006b. Impact of tributyltin and triphenyltin on ivory shell {Babylonia japonica) populations. Environ Health Persp. 114: 13 - 19.

Horiguchi, T., Nishikawa, T., Ohta, Y., Shiraishi, H., and Morita, M. 2007. Retinoid X Receptor gene expression and protein content in tissues of the rock shell Thais clavigera. Aquat Toxicol. 84: 379 - 388. 191

Horiguchi, T., Ohta, Y., Nishikawa, T., Shiraishi, F., Shiraishi, H., and Morita, M. 2008. Exposure to 9-cis retinoic acid induces penis and vas deferens development in the female rock shell, Thais clavigera. Cell Biol Toxicol. 24: 553 - 562.

Houston, R. S. 1971. Reproductive biology of Thais emarginala (Deshayes, 1939) and Thais canaliculata (Duclos, 1832). Veliger. 13: 348 - 357.

Huet, M., Fioroni, P., Oehlmann, J., and Stroben, E. 1995. Comparison of imposex response in three prosobranch species. Hydrobiol. 309: 29 - 35.

Huet, M., Le Goic, N., and Gibbs, P. E. 2008. Appearance of a genetically-based pollution resistance in a marine gastropod, Nucella lapillus, in south-west Brittany: A new case of Dumpton syndrome. J Mar Biol Assoc UK. 88: 1475 - 1479.

IARC. 1982. Some industrial chemicals and dyestufF-IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans, Vol. 29. Lyon, International Agency for Research on Cancer, pp. 257 - 267.

Ide, I., Witten, E. P., Fischer, J., Kalbfus, W., Zellner, A., Stroben, E., and Watermann, B. 1997. Accumulation of organotin compounds in the common whelk Buccinum undatum and the red whelk Neptunea antiqua in association with imposex. Mar Ecol Prog Ser. 152: 197 - 203.

Iguchi, T., Katsu, Y., Horiguchi, T., Watanabe, H., Blumberg, B., and Ohta, Y. 2007. Endocrine disrupting organotin compounds are potent inducers of imposex in gastropods and adipogenesis in vertebrates. Mol Cell Toxicol. 3: 1 -10.

Janer, G., Lyssimachou, A., Bachmann, J., Oehlmann, J., Schulte-Oehlmann, U., and Porte, C. 2006. Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: The effect of xenoandrogenic compounds. Steroids. 71: 435 - 444.

Jenner, M. G. 1978. Pseudohermaphroditism: A newly recognized sexual phenomenon in Ilyanassa obsoleta and other neogastropods. PhD. Thesis, University of Chapel Hill, North Carolina.

Jenner, M. G. 1979. Pseudohermaphroditism in Ilyanassa obsoleta (Mollusca, Neogastropoda). Science. 205: 1407 - 1409.

Jiirgens, M. D., Holthaus, K. I. E., Johnson, A. C., Smith, J. J. L., Hetheridge, M., and Williams, R. J. 2002. The potential for estradiol and ethinylestradiol degradation in English rivers. Environ Tox Chem. 21 (3): 480 - 488.

Kantor, Y. I. 1984. Pseudohermaphroditism in Buccinum undatum (Gastropoda, Prosobranchia). Zool Zh. 63: 1256 - 1258. 192

Ketata, I., Denier, X., Hamza-Chaffai, A., and Minier, C. 2008. Endocrine-related reproductive effects in molluscs. Comp Biochem Phys C. 147: 261 - 270.

Kiran, Y. V., and Anil, A. C. 1999. Record of imposex in Cronia konkanensis (Gastropoda, Muricidae) from Indian waters. Mar Environ Res. 48: 123 - 130.

Kohn, A. J., and Almasi, K. N. 1993. Imposex in Australian Conus. J Mar Biol Assoc UK. 73:241 -244.

Kuwamura, T., Fukao, R., Nishida, M., Wada, K., and Yanagisawa, Y. 1983. Reproductive biology of the gastropod Strombus luhuanus (Strombidae). Publ Seto Mar Biol Lab. 28: 433 - 443.

Lahbib, Y., Labidli, S., Le Pennec, M., Flower, R., and El-Menif, N. T. 2007. Morphological expression and different stages of imposex in Hexaplex trunculus (Neogastropoda: Muricidae) from Tunisian coasts. Cah Biol Mar. 48:315 -326.

Lahbib, Y., Abidli, S., and El Menif, N. T. 2008. Imposex level and penis malformation in Hexaplex trunculus from the Tunisian coast. Am Malacol Bull. 24: 79 - 89.

Lee, P. C., Lee, W. 1996. In vivo estrogenic action of nonylphenol in immature female rats. Bull Environ Contam Toxicol. 57: 341 - 348.

Limaverde, A. M., Wagener, A., Fernandez, M. A., Scofield, A. D. L., and Coutinho, R. 2007. Stramonita haemastoma as a bioindicator for organotin contamination in coastal environments. Mar Environ Res. 64: 384 - 398.

Linder, G. H. 1988. Non-iridescent, haze-free infrared reflecting coated glass structures. US Patent Number: 4,737,388, 5 pp.

Liu, L. L., and Suen, I. J. 1996. Prosobranch gastropod imposex in the west coast of Taiwan. Jap J Malacol (Venus). 55: 207 - 214.

Liu, L. L., Chen, S. J., Peng, W. Y., and Hung, J. J. 1997. Organotin concentrations in three intertidal neogastropods from the coastal waters of Taiwan. Environ Pollut. 98: 113- 118.

Liu, W. H„ Chiu, Y. W., Huang, D. J., Liu, M. Y„ Lee, C. C., and Liu, L. L. 2006. Imposex in the golden apple snail Pomacea canaliculata in Taiwan. Sci Total Environ. 371: 138- 143.

Mann, R., Harding, J.M., and Westcott, E. 2006. Occurrence of imposex and seasonal patterns of gametogenesis in the invading veined rapana whelk Rapana venosa from Chesapeake Bay, USA. Mar Ecol Prog Series. 310: 129 - 138. 193

Maran, C., Centanni, E., Pellizzato, F., and Pavoni, B. 2006. Organochlorine compounds (polychlorinated biphenyls and pesticides) and polycyclic aromatic hydrocarbons in populations of Hexaplex trunculus affected by imposex in the Lagoon of Venice, Italy. Environ Toxicol Chem. 25: 486 - 495.

Marshall, D. J., and Rajkumar, A. 2003. Imposex in the indigenous Nassarius kraussianus (Mollusca: Neogastropoda) from South African harbours. Mar Pollut Bull. 46: 1150-1155.

Martin, P. R. 2002. Evidence for parthenogenesis and natural imposex in the Patagonian freshwater snail Heleobia hatcheri (Gastropoda: Hydrobiidae). J Mollus Stud. 68: 291 - 295.

Masuyama, H., Hiramatsu, Y., Kunitomi, M., Kundo, T., MacDonald, P. N. 2000. Endocrine disrupting chemicals, Phthalic acid and nonylphenol, activate pregnane X receptor-mediated transcription. Mol Endocrinol. 14 (3): 421 - 428.

Matthiessen, P., and Gibbs, P. E. 1998. Critical appraisal of the evidence for tributyltin-mediated endocrine disruption in mollusks. Environ Toxicol Chem. 17: 37-43.

Matthiessen, P., Waldock, R., Thain, J. E., Milton, S., and Scrope-Howe, S. 1991. Changes in periwinkle (Littorina littorea) populations following the ban on TBT-based antifoulings on small boats. ICES C.M. pp. E5 1 -15.

Matthiessen, P., Reynoldson, T., Billinghurst, Z., Brassard, D.W., Cameron, P., Chandler, G. T., Davies, I. M., Horiguchi, T., Mount, D. R., Oehlmann, J., Pottinger, T. G., Sibley, P. K., Thompson, H. M., and Vethaak, A. D. 1999. Field assessment for endocrine disruption in invertebrates (Chapter 4). In Society of Environmental Toxicology and Chemistry, vol. Endocrine Disruption in Invertebrates: Endocrinology, testing, and Assessment (Edited by P. L. deFur, M. Crane, C. G. Ingersoll and L. J. Tattersfield). SETAC Technical Publication Series, pp. 199 - 279.

McClellan-Green, P., Romano, J., and Rittschof, D. 2006. Imposex induction in the mud snail, Ilyanassa obsoleta by three tin compounds. B Environ Contam Tox. 76:581 -588.

Mensink, B. P., Everaarts, J. M., Kralt, H., TenHallers-Tjabbes, C. C., and Boon, J. P. 1996. Tributyltin exposure in early life stages induces the development of male sexual characteristics in the common whelk, Buccinum undatum. Mar Environ Res. 42: 151 - 154.

Mensink, B. P., Van Hattum, B., Ten Hallers-Tjabbes, C. C., Everaarts, J.M., Kralt, H., Vethaak, A. D. and Boon, J. P., 1997. Tributyltin causes imposex in the common whelk Buccinum undatum - mechanism and occurrence. Nederlands Instituut voor Onderzoek der Zee, Rapport 1997-6. 194

Mensink, B. P., Kralt, H., Vethaak, A. D., Ten Hallers-Tjabbes, C. C., Koeman, J. H., van Hattum, B., and Boon, J. P. 2002. Imposex induction in laboratory reared juvenile Buccinum undatum by tributyltin (TBT). Environ Toxicol Phar. 11:49-65.

Millar, R. H. 1971. The biology of ascidians. In Advances in Marine Biology - Volume 9 {Edited by F. S. Russell and M. Yonge). Academic Press Inc., (London) Ltd. pp. 1-100.

Miller, E. R., and Pondick, J. S. 1984. Heavy metal levels in Nucella lapillus (Gastropoda, Prosobranchia) from sites with normal and penis-bearing females from New England. B Environ Contam Tox. 33: 612 - 620.

Miller, K. L., Fernandes, T. F., and Read, P. A. 1999. The recovery of populations of dogwhelks suffering from imposex in the Firth of Forth 1987-1997/98. Environ Pollut. 106: 183 - 192.

Miloslavich, P., Penchaszadeh, P. E., and Bigatti, G. 2007. Imposex in gastropods from Venezuela. Cienc Mar. 33: 319 - 324.

Minchin, A., and Davies, I. M. 1999. Effect of freezing on the length of the penis in Nucella lapillus (L.). J Environ Monitor. 1: 203 - 205.

Minchin, A., and Davies, I. M. 2000. SOTEAG rocky shore monitoring programme. TBT contamination in Sullom Voe, Shetland. 1999 Dogwhelk Survey. Fisheries Research Services, pp. 35pp.

Minchin, A., Davies, I. M., Pymm, H., and Grewar, G. N. 1999. North Sea Imposex Survey: CW0825 August 1998 - July 1999 - Final Report. Fisheries Research Services, pp. 198 pp.

Morley, N. J., Leung, K. M. Y., Morritt, D., and Crane, M. 2003. Toxicity of anti- Fouling biocides to Parorchis acanthus (Digenea: Philophthalmidae) cercarial encystment. Dis Aquat Organ. 54: 55 - 60.

Nias, D. J., McKillup, S. C., and Edyvane, K. S. 1993. Imposex in Lepsiella vinosa from southern Australia. Mar Pollut Bull. 26: 380 - 384.

Nicholson, G. J., and Evans, S. M. 1997. Anthropogenic impacts on the stocks of the common whelk Buccinum undatum (L). Mar Environ Res. 44: 305 - 314.

Nishikawa, J., Mamiya, S., Kanayama, T., Nishikawa, T., Shiraishi, F., and Horiguchi, T. 2004. Involvement of the Retinoid X Receptor in the development of imposex caused by organotins in gastropods. Environ Sci Technol. 38: 6271 -6276.

Nohara, M. 1999. Variation and abnormality of genital system in Littorina sitkana philippi (Mollusca, Gastropoda) in Northern Japan. Zool Sci. 16: 827 - 838. 195

Oberdorster, E. 1997. Sublethal effects of tributyltin on aquatic invertebrate models: Steroid metabolism, protein alterations and physiological responses. PhD. Thesis, Duke University, North Carolina.

Oberdorster, E., and McClellan-Green, P. 2000. The neuropeptide APGWamide induces imposex in the mud snail, Ilyanassa obsoleta. Peptides. 21: 1323 - 1330.

Oberd5rster, E., and McClellan-Green, P. 2002. Mechanisms of imposex induction in the mud snail, Ilyanassa obsoleta: TBT as a neurotoxin and aromatase inhibitor. Mar Environ Res. 54: 715 - 718.

Oberdorster, E., Romano, J., and McClellan-Green, P. 2005. The neuropeptide APGWamide as a penis morphogenic factor (PMF) in gastropod mollusks. Integr Comp Biol. 45: 28 - 32.

Oehlmann, J. 1998. Untersuchungen zum Einsatz von Pathomorphosen der ableitenden Geschlechtswege von Vorderkiemerschnecken (Gastropoda: Prosobranchia) fUr ein biologisches TBT-Effektmonitoring [Habilitation thesis]. Zittau, Germany: International Graduate School Zittau. 162 pp.

Oehlmann, J. 2004. Biological effects of contaminants: Use of intersex in the periwinkle (Littorina littorea) as a biomarker of tributyltin pollution. ICES Techniques in Marine Environmental Sciences. 37: 1 - 22.

Oehlmann, J., and Bettin, C. 1996. Tributyltin-Induced Imposex and the role of Steroids in Marine Snails. Malacol Rev Suppl - Molluscan Reproduction. 6: 157-161.

Oehlmann, J., Stroben, E., and Fioroni, P. 1991. The morphological expression of imposex in Nucella lapillus (L.) (Gastropoda, Muricidae). J Mollus Stud. 57: 375 - 390.

Oehlmann, J., Stroben, E., Bettin, C., and Fioroni, P. 1992. Hormonal disorders and tributyltin-induced imposex in marine snails. In Proceedings of the 27th European Marine Biology Symposium (Edited by J.C. Aldrich), Dublin, Ireland, pp. 301 - 305.

Oehlmann, J., Stroben, E., and Fioroni, P. 1993. Frequence et degree d'expression du pseudohermaphrodisme chez quelques Prosobranches Stenoglosses des c6tes fran?aises (surtout de la baie de Morlaix et de la Manche). 2. Situation jusqu' au printemps de 1992*. Cah Biol Mar. 34: 343 - 362.

Oehlmann, J., Liebe, S., Watermann, B., Stroben, E., Fioroni, P., and Deutsch, U. 1994a. New perspectives of sensitivity of Littorinids to TBT pollution. Cah Biol Mar. 35: 254 - 255.

Oehlmann, J., Schulte-Oehlmann, U., Fioroni, P., and Stroben, E. 1994b. Probable extinction of prosobranch populations as a result of TBT pollution in the Bay of Morlaix. Cah Biol Mar. 35: 255 - 256. 196

Oehlmann, J., Stroben, E., SchulteOehlmann, U., Bauer, B., Fioroni, P., and Markert, B. 1996a. Tributyltin biomonitoring using prosobranchs as sentinel organisms. Fresen J Anal Chem. 354: 540 - 545.

Oehlmann, J., Stroben, E., and Fioroni, P. 1996b. New Facts about Tributyltin- induced imposex in Prosobranchs: General Aspects. Malacol Rev Suppl - Molluscan Reproduction. 6: 149 - 156.

Oehlmann, J., Fioroni, P., Stroben, E., and Markert, B. 1996c. Tributyltin (TBT) effects on Ocinebrina aciculata (Gastropoda: Muricidae): Imposex development, sterilization, sex change and population decline. Sci Total Environ. 188: 205 - 223.

Oehlmann, J., Schulte-Oehlmann, U., Tillmann, M., and Markert, B. 2000. Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part I: Bisphenol A and octylphenol as xeno-estrogens. Ecotoxicol. 9: 383 - 397.

Oehlmann, J., Di Benedetto, P., Tillmann, M., Duft, M., Oetken, M., and Schulte- Oehlmann, U. 2007. Endocrine disruption in prosobranch molluscs: Evidence and ecological relevance. Ecotoxicol. 16: 29 - 43.

Omae, I. 2003. Organotin antifouling paints and their alternatives. Appl Organomet Chem. 17: 81 - 105.

Osorio, C. R., and Huaqui'n, L. G. 2003. Alteraci6n de la sexualidad de Acanthina monodon (Pallas, 1744) (Gastropoda: Muricidae) en el litoral de Chile central, l'nducida por compuestos organoestanosos. Ciencia Technolgia del Mar. 26: 97- 107.

OSPAR. 2008. JAMP Guidelines for Contaminant-Specific Biological Effects (OSPAR Agreement 2008-09). In OSPAR Convention for the Protection of the Marine Environment of the North-East Atlantic.

Pandey, E., and Evans, S. M. 1996. The incidence of imposex in gastropods from Indonesian coastal waters. Asian Mar Biol. 13: 53 - 61.

Pattisina, L. A. 1991. Development of a protocol for imposex measures on tropical neogastropod molluscs. MSc. Thesis, University of Victoria, British Columbia.

Pavoni, B., Centanni, E., Valcanover, S., Fasolato, M., Ceccato, S., and Tagliapietra, D. 2007. Imposex levels and concentrations of organotin compounds (TBT and its metabolites) in Nassarius nitidus from the Lagoon of Venice. Mar Pollut Bull. 55: 505 -511.

Penchaszadeh, P. E., Averbuj, A., and Cledon, M. 2001. Imposex in gastropods from Argentina (south-western Atlantic). Mar Pollut Bull. 42: 790 - 791. 197

Philippart, C. J. M. 1995. Effect of periphyton grazing by Hydrobia ulvae on the growth of Zostrea noltii on a tidal flat in the Dutch Wadden Sea. Mar. Bio. 122(3): 431 -437.

Phillip, O. S. 2000. The relationship between masculinized female conch (Strombus gigas) and tributyltin-based antifouing paint in coastal waters of the British Virgin Islands. MSc. Thesis, University of Plymouth, United Kingdom.

Piver, W. T. 1973. Organotin compounds: Industrial applications and biological investigation. Environ Health Persp. 4: 61 - 79.

Poli, G., Salvat, B., and Streiff, W. 1971. Aspect particulier de la sexualite chez Ocenebra erinacea. Haliotis. 1: 29 - 30.

Poloczanska, E. S., and Ansell, A. D. 1999. Imposex in the whelks Buccinum undatum and Neptunea antiqua from the west coast of Scotland. Mar Environ Res. 47: 203 - 212.

Poppe, G. T., and Tagaro, S. P. 2006. The new classification of gastropods according to Bouchet and Rocroi, 2005. VISAYA - February 2006, 11 pp.

Power, A. J., and Keegan, B. F. 2001. The significance of imposex levels and TBT contamination in the red whelk, Neptunea antiqua (L.) from the offshore Irish Sea. Mar Pollut Bull. 42: 761 - 772.

Quintela, M., Barreiro, R., and Ruiz, J. M. 2002. Dumpton Syndrome reduces the tributyltin (TBT) sterilising effect on Nucella lapillus (L.) by limiting the development of the imposed vas deferens. Mar Environ Res. 54: 657 - 660.

Raats, J. I., Falkson, G., and Falkson, H. C. 1992. A study of fadrozole, a new aromatase inhibitor in postmenopausal women with advanced metastatic breast cancer. J. Clin. Oncol. 10: 111 -116.

RafFaelli, D. 1985. Functional feeding groups of some intertidal molluscs defined by gut content analysis. J Mollusc. Stud. 51: 233 - 239.

Ramasamy, M. S., and Murugan, A. 2002. Imposex in Muricid gastropod Thais biserialis (Mollusca: Neogastropoda: Muricidae) from Tuticorin harbour, southeast coast of India. Indian J Mar Sci. 31: 243 - 245.

Ramon, M., and Amor, M. J. 2002. Reproductive cycle of Bolinus brandaris and penis and genital duct size variations in a population affected by imposex. J Mar Biol Assoc UK. 82: 435 - 442.

Reed, S. E. 1993a. Gonadal comparison of masculinized females and androgynous males to normal males and females in Strombus (Mesogastropoda, Strombidae). J Shellfish Res. 12: 71 - 75. 198

Reed, S. E. 1993b. Size differences between sexes (including masculinized females) in Strombus pugilis (Mesogastropoda: Strombidae). J Shellfish Res. 12: 77 - 79.

Rees, C. M., Brady, B. A., and Fabris, G. J. 2001. Incidence of imposex in Thais orbita from Port Phillip Bay (Victoria, Australia), following 10 years of regulation on use of TBT. Mar Pollut Bull. 42: 873 - 878.

Reid, D. G. 1986. Mainwaringia nevill, 1885, a littorinid genus from Asiatic mangrove forests, and a case protandrous hermaphroditism. J Mollus Stud. 52: 225 - 242.

Reitsema, T. J., and Spickett, J. T. 1999. Imposex in Morula granulata as bioindicator of tributyltin (TBT) contamination in the Dampier Archipelago, western Australia. Mar Pollut Bull. 39: 280 - 284.

Rilov, C., Gasith, A., Evans, S. M., and Benayahu, Y. 2000. Unregulated use of TBT-based antifouling paints in Israel (eastern Mediterranean): High contamination and imposex levels in two species of marine gastropods. Mar Ecol Prog Ser. 192: 229 - 238.

Rilov, G., Benayahu, Y., and Gasith, A. 2001. Low abundance and skewed population structure of the whelk Stramonita haemastoma along the Israeli Mediterranean coast. Mar Ecol Prog Ser. 218: 189 - 202.

Rittschof, D., and McClellan-Green, P. 2005. Molluscs as multidisciplinary models in environment toxicology. Mar Pollut Bull. 50: 369 - 373.

Roberts, R. D., and Forrest, B. M. 1999. Minimal impact from long-term dredge spoil disposal at a dispersive site in Tasman Bay, New Zealand. New Zeal J Mar Fresh. 33:623 -633.

Robinson, A. J. 1985. Pesticidal C6-C25-Mercapto organotin compounds. US Patent Number: 4,560,702, 5 pp.

Rodriguez, J. G., Belzunce, M. J., Borja, A., Franco, J., Garmendia, J. M., Gonzalez Herraiz, I., Muxika, I., Solaun, O., Tueros, I., and Valencia, V. 2007. Organotin bioaccumulation and imposex in female Nassarius reticulatus and N. nitidus in ports and marina areas of the Basque country. In ICES Annual Conference - ICES CM 2007/session I: 12, Helsinki, Finland.

Rodriguez, J. G., Tueros, I., Borja, A., Franco, J., Alonso, J. I. G., Garmendia, J. M., Muxika, I., Sariego, C., and Valencia, V. 2009. Butyltin compounds, sterility and imposex assessment in Nassallus reticulatus (Linnaeus, 1758), prior to the 2008 European ban on TBT antifouling paints, within Basque ports and along coastal. Cont Shelf Res. 29: 1165 - 1173.

Ronis, M. J. J., and Mason, A. Z. 1996. The metabolism of testosterone by the periwinkle (Littorina littorea) in vitro and in vivo: Effects of tributyltin. Mar Environ Res. 42: 161 - 166. 199

Ruppert, E. E., Fox, R. S., Barnes, R. B. 2004. Invertebrate zoology - A functional evolutionary approach, 7 m ed. Brooks Cole Thomson, Belmont, CA. 963 pp. Accessed at http://webs.lander.edu/rsfox/invertebrates/ on [2010-06-12].

Salazar, M. H., Duncan, P. B., Salazar, S. M., and Rose, K. A. 1995. In-situ bioassays using transplanted mussels: II. Assessing contaminated sediment at a superfiind site in Puget Sound. Environmental Toxicology and Risk Assessment -Third Volume, ASTMSTP 1218. Philadelphia, P. A. USA. pp. 242-263.

Santos, M. M., Castro, L. F. C., Vieira, M. N., Micael, J., Morabito, R., Massanisso, P., and Reis-Henriques, M. A. 2005. New insights into the mechanism of imposex induction in the dogwhelk Nucella lapillus. Comp Biochem Phys C. 141: 101 -109.

Santos, M. M., Reis-Henriques, M. A., Vieira, M. N., and Sole, M. 2006. Triphenyltin and tributyltin, single and in combination, promote imposex in the gastropod Bolinus brandaris. Ecotox Environ Safe. 64: 155 - 162.

Santos, M. M., Reis-Henriques, M. A., Guillot, R., Lima, D., Franco-Duarte, R., Mendes, I., Queiros, S., and Castro, L. F. C. 2008. Anti-androgenic effects of sewage treatment plant effluents in the prosobranch gastropod Nucella lapillus. Comp Biochem Phys C. 148: 87 - 93.

Schulte-Oehlmann, U., Oehlmann, J., and Stroben, E. 1992. Tributyltin-induced imposex of Nucella lapillus on Irish Coasts in 1990. In Proceedings of the 27th European Marine Biology Symposium. Dublin, Ireland, pp. 307 - 311.

Schulte-Oehlmann, U., Fioroni, P., Oehlmann, J., and Stroben, E. 1994. The genital system of Marisa cornuarietis (Gastropoda, Ampullariidae) - a morphological and histological analysis. Zool Beitr. 36: 59- 81.

Schulte-Oehlmann, U., Bettin, C., Fioroni, P., Oehlmann, J., and Stroben, E. 1995. Marisa cornuarietis (Gastropoda, Prosobranchia) - A potential TBT bioindicator for fresh-water environments. Ecotoxicol. 4: 372 - 384.

Schulte-Oehlmann, U., Oehlmann, J., Bauer, B., Fioroni, P., and Leffler, U. S. 1998. Toxico-kinetic and dynamic aspects of TBT-induced imposex in Hydrobia ulvae compared with intersex in Littorina littorea (Gastropoda, Prosobranchia). Hydrobiol. 378: 215 -225.

Schulte-Oehlmann, U., Tillmann, M., Markert, B., Oehlmann, J., Watermann, B., and Scherf, S. 2000. Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part II: Triphenyltin as a xeno- androgen. Ecotoxicol. 9: 399 - 412. 200

Seapy, R. R. 2009. Pterotracheoidea Rafinesque, 1814. Heteropoda Lamarck, 1812, heteropods, sea elephants In The Tree of Life Web Project - Version 09 October 2009. Accessed at http://tolweb.org/Pterotracheoidea>'27801/2009.10.09 on [2010-06-12].

Shi, H. H., and Huang, C. J. 2001a. Tributyltin (TBT) pollution and imposex in marine gastropods. Acta Ecol Sin. 21: 1711 - 1717. (Abstract only).

Shi, H. H., and Huang, C. J. 2001b. Morphological characters of imposex in marine gastropod. J Oceanogr Taiwan Strait. 20: 552 - 555.

Shi, H. H., Huang, C. J., Yu, X. J., and Zhu, S. X. 2004a. Imposex and female reproductive failure of Chicoreus brunneus and C. asianus. J Trop Oceanogr. 23: 82 - 87.

Shi, H. H., Huang, C. J., Yu, X. J., Zhu, S. X., and Zhang, Y. B. 2004b. Development of imposex and structural effect on males in three Nassarius spp. Mar Sci (Haiyang Kexue). 28: 36-41. (Abstract only).

Shi, H. H., Huang, C. J., Zhu, S. X., Yu, X. J., and Xie, W. Y. 2005. Generalized system of imposex and reproductive failure in female gastropods of coastal waters of mainland China. Mar Ecol Prog Ser. 304: 179 - 189.

Shim, W. J., Shin, Y. B., Kim, N. S., Oh, J. R., and Jeon, J. K. 2002. Induction of imposex in rock Shell, Thais clavigera, exposed to tributyltin and other antagonistic chemicals. In SETAC 23rd Annual Meeting (November 16-20, 2002), Salt Lake City, Utah.

Shore, L. S., Gurevitz, M., Shemesh, M. 1993. Estrogen as an environmental pollutant. Bull Environ Contam Toxicol. 51: 361 - 366.

Short, J. W., Rice, S. D., Brodersen, C. C., and Stickle, W. B. 1989. Occurrence of tri-n-butyltin-caused imposex in the North-Pacific marine snail Nucella lima in Auke Bay, Alaska. Mar Biol. 102: 291 - 297.

Sloan, S., and Gagnon, M. M. 2004. Intersex in Roe's abalone (Haliotis roei) in western Australia. Mar Pollut Bull. 49: 1122 - 1126.

Smith, B. S. 1971. Sexuality in the American mud-snail, Nassarius obsoletus. Proc Malacol Soc Lond. 39: 377 - 378.

Smith, B. S. 1980. The estuarine mudsnail, Nassarius obsoletus - Abnormalities in the reproductive system. J Mollus Stud. 46: 247 - 256.

Smith, B. S. 1981a. Reproductive anomalies in stenoglossan snails related to pollution from marinas. J Appl Toxicol. 1: 15-21.

Smith, B. S. 1981b. Male Characteristics on female mud snails caused by antifouling bottom paints. J Appl Toxicol. 1: 22 - 25. 201

Smith, B. S. 1981c. Tributyltin compounds induce male characteristics on female mud snails Nassarius obsoletus = Ilyanassa obsoleta. J Appl Toxicol. 1: 141 - 144.

Smith, B. S. 198 Id. Male characteristics in the female Nassarius obsoletus: Variations related to locality. Veliger. 23: 212 - 216.

Smith, P. J. 1996. Selective decline in imposex levels in the dogwhelk Lepsiella scobina following a ban on the use of TBT antifoulants in New Zealand. Mar Pollut Bull. 32: 362 - 365.

Smith, P. J., and McVeagh, M. 1991. Widespread organotin pollution in New Zealand coastal waters as indicated by imposex in dogwhelks. Mar Pollut Bull. 22:409-413.

Snoeij, N. J., Penninks, A. H., Seinen, W. 1987. Biological activity of organotins compounds - An overview. Environ Res. 44: 335 - 353.

Sole, M., Morcillo, Y., and Porte, C. 1998. Imposex in the commercial snail Bolinus brandaris in the northwestern Mediterranean. Environ Pollut. 99: 241 - 246.

Sousa, A., Mendo, S., and Barroso, C. 2005. Imposex and organotin contamination in Nassarius reticulatus (L.) along the Portuguese coast. Appl Organomet Chem. 19:315 -323.

Sousa, A., Matsudaira, C., Takahashi, S., Tanabe, S., and Barroso, C. 2007. Integrative assessment of organotin contamination in a southern European estuarine system (Ria de Aveiro, NW Portugal): Tracking temporal trends in order to evaluate the effectiveness of the EU ban. Mar Pollut Bull. 54: 1645 - 1653.

Spence, S. K., Bryan, G. W., Gibbs, P. E., Masters, D., Morris, L., and Hawkins, S. J. 1990a. Effects of TBT contamination on Nucella populations. Funct Ecol. 4:425 -432.

Spence, S. K., Hawkins, S. J., and Santos, R. S. 1990b. The Mollusk Thais haemastoma - an exhibitor of imposex and potential biological indicator of tributyltin pollution. Mar Ecol P S Z N 1.11: 147 - 156.

Spooner, N., Gibbs, P. E., Bryan, G. W., and Goad, L. J. 1991. The effect of tributyltin upon steroid titers in the female dogwhelk, Nucella lapillus, and the development of imposex. Mar Environ Res. 32: 37 - 49.

Stannica™, LLC. 2009. Anhydrous tin tetrachloride material safety data sheet. Viewed at www.stannica.com/resource/TDS/TTC_Anhydrous_StanicChloride.pdf Seen on July 13, 2010.

Stewart, C., de Mora, S. J., Jones, M. R. L., and Miller, M. C. 1992. Imposex in New Zealand neogastropods. Mar Pollut Bull. 24: 204 - 209. 202

Stickle, W. B., Sharpdahl, J. L., Rice, S. D., and Short, J. W. 1990. Imposex Induction in Nucella lima (Gmelin) Via Mode of Exposure to Tributyltin. J Exp Mar Biol Ecol. 143: 165 - 180.

Strand, J., and Asmund, G. 2003. Tributyltin accumulation and effects in marine molluscs from West Greenland. Environ Pollut. 123: 31 -37.

Strand, J., and Jacobsen, J. A. 2002. Imposex in two sublittoral neogastropods from the Kattegat and Skagerrak: The common whelk Buccinum undatum and the red whelk Neptunea antiqua. Mar Ecol Prog Ser. 244: 171 - 177.

Strand, J., Glahder, C. A., and Asmund, G. 2006. Imposex occurrence in marine whelks at a military facility in the high Arctic. Environ Pollut. 142: 98 - 102.

Strand, J., Jorgensen, A., and Tairova, Z. 2009. TBT pollution and effects in molluscs at US Virgin Islands, Caribbean Sea. Environ Int. 35: 707 - 711.

Stroben, E. 1996. The organotin pollution at the Bay of Morlaix with special reference to biomonitoring with prosobranchs. Malacol Rev Suppl - Molluscan Reproduction. 6: 163 -171.

Stroben, E., Oehlmann, J., and Fioroni, P. 1992a. The morphological expression of imposex in Hinia reticulata (Gastropoda, Buccinidae) - A potential indicator of tributyltin pollution. Mar Biol. 113: 625 - 636.

Stroben, E., Oehlmann, J., and Fioroni, P. 1992b. Hinia reticulata and Nucella lapillus - Comparison of two gastropod tributyltin bioindicators. Mar Biol. 114: 289 - 296.

Stroben, E., Brommel, C., Oehlmann, J., and Fioroni, P. 1992c. The genital systems of Trivia arctica and Trivia monacha (Prosobranchia, Mesogastropoda) and tributyltin induced imposex. Zool Beitr. 34: 349 - 374.

Stroben, E., Schulte-Oehlmann, U., Fioroni, P., and Oehlmann, J. 1995. A comparative method for easy assessment of coastal TBT pollution by the degree of imposex in prosobranch species. Haliotis. 24: 1 - 12.

Stroben, E., Oehlmann, J., Schulte-Oehlmann, U., and Fioroni, P. 1996. Seasonal variations in the genital ducts of normal and imposex-affected prosobranchs and its influence on biomonitoring indices. Malacol Rev Suppl - Molluscan Reproduction. 6: 173 - 184.

Swennen, C., and Horpet, P. 2008. Pseudo-imposex; male features in female volutes not TBT-induced (Gastropoda: Volutidae). Contrib Zool. 77: 17 - 24.

Swennen, C., Ruttanadakul, N., Ardseungnern, S., Singh, H.R., Tan, K. S., Mensink, B. P., and Ten Hallers-Tjabbes, C.C. 1996. Sexual aberrations in gastropods in Gulf of Thailand and Strait of Malacca in relation to shipping. Wallaceana. 78: 1 -13. 203

Swennen, C., Ruttanadakul, N., Ardseungnem, S., Singh, H. R., Mensink, B. P., and Ten Hallers-Tjabbes, C. C. 1997. Imposex in sublittoral and littoral gastropods from the Gulf of Thailand and Strait of Malacca in relation to shipping. Environ Technol. 18: 1245 - 1254.

Swennen, C., Sampantarak, U., and Ruttanadakul, N. 2009. TBT-pollution in the Gulf of Thailand: A re-inspection of imposex incidence after 10 years. Mar Pollut Bull. 58: 526 - 532.

Syasina, I. G., and Shcheblykina, A. V. 2007. Morphofunctional characterization of the reproductive system of the gastropods Littorina brevicula, L. mandshurica, and Nucella heyseana from uncontaminated and contaminated areas of Peter the Great Bay, Sea of Japan. Russ J Mar Biol. 33: 399 - 404.

Takeda, N. 2000. Development of a penis from the vestigial penis in the female apple snail, Pomacea canaliculata. Biol Bull. 199: 316 - 320.

Takeda, N. 2001. Induction of Imposex by plasticizers in the freshwater snail, Pomacea canaliculata. In Abstracts of the 15th ICCE. International Congress of Comparative Endocrinology, Sorrento (Napoli), Italy, pp. P14.19.

Takeda, N. 2006. Induction of imposex by plasticizers in the freshwater snail, Pomacea canaliculata. J Exp Zool. 305A: 183 -183.

Tan, K. S. 1997. Imposex in three species of Thais from Singapore, with additional observations on T. clavigera (Kuster) from Japan. Mar Pollut Bull. 34: 577 - 581.

Ten Hallers-Tjabbes, C. C., Kemp, J. F., and Boon, J. P. 1994. Imposex in whelks (.Buccinum undatum) from the open North Sea - Relation to shipping traffic intensities. Mar Pollut Bull. 28: 311 - 313.

Teso, S. V., and Penchaszadeh, P. E. 2009. Beach filling and imposex in Olivancillaria deshayesiana (Mollusca: Gastropoda: Olividae) from the coast of Mar del Plata, Argentina. J Mar Biol Assoc UK. 89: 557 - 562.

Tester, M., and Ellis, D. 1995. TBT controls and the recovery of whelks from imposex. Mar Pollut Bull. 30: 90 - 91.

Tewari, A., Raghunathan, C., Joshi, H. V., and Khambhaty, Y. 2002. Imposex in rock whelks Thais and Ocenebra species (Mollusca, Neogastropoda, Muricidae) from Gujarat coast. Indian J Mar Sci. 31: 321 - 328.

The Bay Science Foundation. 2009. Zipcodezoo.com. Accessed at http://www.zipcodezoo.com. Accessed on [2009-04-31], 204

Thiriot-Quievereux, C. 1977. Particulaire de l'appareil genital de quelques especes de Rissoidae (Mollusca, Mesogastropod). C R Hebd Seances Acad Sci D. 285: 779-781.

Tillmann, M. 2004. Sediment-toxicological investigations using gastropods and insects with special emphasis on endocrine active substances. Gottingen: Cuvillier-Verlag; ISBN 3-86537-300-3, 196 pp. [in German].

Tillmann, M., Schulte-Oehlmann, U., Duft, M., Markert, B., and Oehlmann, J. 2001. Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part III: Cyproterone acetate and vinclozolin as antiandrogens. Ecotoxicol. 10: 373 - 388.

Toller, W. 2003. U.S.V.I. Animal Fact Sheet #15 - Whelk, Department of Planning and Natural Resources Division of Fish and Wildlife. St. Thomas, Virgin Islands. Accessed at http://www.vifishandwildlife.com on [2010-06-24],

Tyler, C. R., Jobling, S., and Sumpter, J. P. 1998. Endocrine disruption in wildlife: A critical review of the evidence. Crit Rev Toxicol. 28 (4): 319 - 361.

Viglino, L., Pelletier, E., and Lee, L. E. J. 2006. Butyltin species in benthic and pelagic organisms of the Saguenay Fjord (Canada) and imposex occurrence in common whelk (Buccinum undatum). Arch Environ Con Tox. 50: 45 - 59.

Vishwakiran, Y., Anil, A. C., Venkat, K., and Sawant, S. S. 2006. Gyrineum natator: A potential indicator of imposex along the Indian coast. Chemosphere. 62: 1718- 1725.

Vos, J. G., Dybing, E., Greim, H. A., Ladefoged, O., Lambre, C., Tarazona, J.V., Brandt, I., and Vethaak, A. D. 2000. Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit Rev Toxicol. 30: 71 - 133.

Watermann, B. 2010. Personal communication.

WHO. 2000. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 77, 563 pp.

Wilson, S. P., Ahsanullah, M., and Thompson, G. B. 1993. Imposex in negastropods - An indicator of tributyltin contamination in eastern Australia. Mar Pollut Bull. 26: 44 - 48.

Wiita, B., Artis, A., Ackerman, D. M., and Loncope, C. 1995. Binding of 17-a- methyltestosterone in vitro to human sex hormone binding globulin and rat ventral prostate androgen receptors. Ther Drug Monit. 17: 377 - 380.

Ying, G. G., Kookana, R. S., and Ru, Y. J. 2002. Occurrence and fate of hormone steroids in the environment. Environ Int. 28: 545 - 551. 205

Yu, X. J., Huang, C. J., Zhu, S. X., and Chen, X. M. 2003. Histological studies on imposex of three marine prosobranchs. Ecolo Sci. 22: 337 - 340.

Zhengyan, L. 2005. Seasonal variation in imposex intensity of Thais clavigera. Chinese J Oceanol Limnol. 23: 163 - 168. 206

Theses1

Risk Assessments

0 a Reports • ? c 0 V Reviews/Book Chapters a u 3 3 Q. Editorials/Letters/NotesI

Articles Abstracts/Meetings t 0 50 100 150 200 250 300 350 400 450 500

Record Count

Figure 5.1. Distribution of literature on the masculinisation of female gastropods

(imposex, intersex, and ovo-testis) by publication type from 1962 to 2009. 207

Science of theTotal Environment

Marine Polution Bulletin

Marine Environmental Research

a MarineEcology Progress Series E n Z Marine Biology 15 ch • Joumalofthe Marine Biological Association UK 0

Environmental Toxia>k>gv & Chemistry

Environmental Pollution

Ecotoxlcoloiy

Chemosphert

Applied OrganometaDIc Chemistry

0 10203040S0 60 70 80 90 100

Record Count

Figure 5.2. Top eleven scientific journals that have published literature associated with the masculinisation of female gastropods from 1962 to 2009. 208

60 55 50 45 +J C 40 • 0 35 u 30 *0 )m 0 25 u 0 20 cc 15 10 5 0 1960 1970 1980 1990 2000 2010 Publication Year

Figure 5.3. Publication by year for literature on the masculinisation of female gastropods (imposex, intersex and ovo-testis) from 1962 to 2009. 209

• No. of Families No. of Species

300 -]

250 -

£ 200 -

0) jS 150 E | 100

50 J

0 • i I 1978 1990 1991 1999 2003 2005 2009 Publication Year

Figure 5.4. Distribution of female gastropods affected by female masculinisation

(imposex, intersex and ovo-testis) by species and family published from 1978 to

2009. North America Europe (including Greenland) (including the Mediterranean)

Asia and Oceania Latin America (including Africa)

- >/? No. with [Tissue] No. without [Tissue]

Figure 5.5. Geographic regions of the world displaying the proportion of female masculinisation studies with and without chemical analysis on tissue samples between 1962 and 2009. 211

I No. with [Tissue] • No. without [Tissue]

Iceland 1,4

Atlantic Ocean Faroe Islands Sweden Russia 1.2,4 1,4

ingoom 1,2,3,4,5 Germany ekft. 1,2 English Channel

France 1,2,3,4,5

Italy \ CroStia

Spain 1,3,4,6 0 Mediterranean°s?a u C^)

Portugal Malta u 1.2,33.6 6

Figure 5.6. Distribution of the top six species used as bioindicators of TBT pollution in Europe (1 = Nucella lapillus > 2 = Nassarius reticulatus > 3 =

Buccinum undatum > 4 = Littorina littorea > 5 = Ocenebra erinacea > 6 = Hexaplex trunculus) and the total number of studies per country that have reported and not reported tissue concentration. 212

%

a China torea 1,3.* Pacific Ocean

India

Thailand Kong

Indian

liesBj Pacific ll No. with (Tissue) No. without (Tissue] Ocean

Australia New Southern Ocean

Figure 5.7. Distribution of the top four species used as bioindicators of TBT pollution in Asia and Oceania (1 = Thais clavigera >2 = T. distinguenda >3 = 7'. luteostoma > 4 = T. bitubercularis) and the total number of studies per country that have reported and not reported tissue concentration. * = imposex recorded but not in the top species of that region. 213

Arctic Ocean

Greenland

Alaska

Atlantic Ocean Hudson |)Vancouvei7 ewfoundlarvd 3,4," 1 Pacific Ocean

Seattle I NewABrunswick- L4,* 1 Scotia

—Providence!

California ' -Virginal I 3,"

Texas * "N.CarolinaBi

^Florida Louisiana Gulf* 40-1 of 35- I No. with [Tissue] |No. without [Tissue]} o 30. ^Mexico^ Caribbean Sea

Study I'

Figure 5.8. Distribution of the top five species used as biomarkers of TBT pollution in North America and Greenland (1 = Nucella lapillus >2 = Ilyanassa obsoleta > 3 =

N. emarginata > 4 = N. lamellose > 5 = Searlesia (Lirabuccinum dirum) dira) and the total number of studies per country that have reported and not reported tissue concentration. * = imposex recorded but not in the top species of that region. 214

Atlantic Ocean

US. Virgin IslandsI Mexico 3,#

">.« BR.Virgin IslandsI 4 Caribbean Sea I ^ Puerto RicoI

Venezuela

'Colombia

Pacific Ocean

H No. with [Tissue] H No. without [Tissue]

Argentina

Figure 5.9. Distribution of top four species used as bioindicators of TBT pollution in

Latin America (1 = Stramonita haemastoma > 2 = Adelomelon briasilina >3 = 5". rustica > 4 = Strombus gigas) and the total number of studies per country that have reported and not reported their tissue concentration. * = imposex recorded but not in the top species of that region. 215

300 275 ^ 250 C 225 5 200 3 175 _ 150 TJ 125 O ioo

25 --

Figure 5.10. Proportion of indices used to measure imposex and/or intersex

incidence and severity in field and laboratory studies. Average oviduct convolution

(AOS), intersex index (ISI), male:female ratio (M:F), mean female penis length

(mFPL), mean male penis length (mMPL), relative penis length index (RPLI),

relative penis size index (RPSI) and Uncommon indices. Uncommon indices include: convolution of proximal oviduct, conversion potential females to imposex

index (CPFII), coefficient of variance (CV%), female vas deferens length (VDL), imposex index, intersex incidence, mean calibre of the vas deferens (VDC), mean

female prostate length (FPrL), mean female vas deferens length (VDLf), penis classification index (PCI), penis expression (P), and percent sterile (female). 216

Stages in the development of imposex in Mlo lapillus based on vas deferens sequence.

Stage 0 - normal female state with no male character being visibly super imposed; pallial oviduct terminates at a clearly defined opening or vulva situated at the apex of a prominent genital papilla which projects into the mantle cavity.

Stage 1 - development of proximal section of vas deferens commencing by infolding of the mantle cavity epithelium in the region vental Stage to the genital papilla.

Stage 2 • development of the penis initiated with the formation of a ridge a short distance behind the right tentacle.

Stage 3 • small penis formed and development of distal section of vas deferens commencing from base of penis.

Stage 4 • proximal and distal sections of vas deferens now fused and penis enlargment to a size approaching that of the male.

Stage 5 • proliferating vas deferens tissue overgrowing gential papilla causing vulva to be displaced, constricted or no longer visible; blister-like protuberances may appear around site of the papilla (5A) and nodules of hyperplasic tissue often develop (5B).

Stage 6- lumen of capsule gland contains material of aborted capsules; this material may comprise a single capsule or several to many that are compressed together to form a translucent or brown mass(6A&68).

Abbreviatiors: 3 = anus; b="blister"; gp=genital papilla; n=nodule; p = penis; v ~ vulva; vd=vas deferens. Adapted from Gibbs et al. (198?)

Figure 5.11. Vas deferens sequence index (VDSI) for Nucella lapillus adapted from

Gibbs etal. (1987). 217

Schcme of imposcx evolution in Nucellahpillus based on vas cl«.fc-rcns sequence from Stage 0 (normal female! to impose-x stages 1 - 6 divided into types a - c

a /j \ i

Abbreviations: ac = abortive capsules; eg = capsule gland; gp - genital papilla; ocv = occlusion of the vulva; p - penis; pd = penis duct;pr - prostate gland; te = tentacle; vd = vas deferens; vds - vas deferens section. Adapated from Oehlmann et al. (1991) and by permission of Oxford University Press.

Figure 5.12. General scheme of imposex evolution in Nucella lapillus adapted from

Oehlmann et al. (1991) and by permission of Oxford University Press. 218

Penis classification index (PCI) developed for the common whelk, Buccinum undatum.

Imposex stage View from above View from the side

0

Smooth epithelium at the site where males have their perils

/V or

Small knob at perils site

Small penis structure which may wobble and be lifted up from the epithelium of the foot

Penis Is bent and with a shape similar to a normal male penis

Adapted from Mensink et al. (1997) and cited in Strand and Jacobsen (2002).

Figure 5.13. Penis classification index (PCI) adapted from Mensink et al. (1997) and cited in Strand and Jacobsen (2002). 219

Table 5.1. Comparison of known families affected by female masculinisation FM from 1962 to 2009. Families compiled from affected species lists by Jenner (1978),

Fioroni et al. (1990; 1991), Matthiessen et al. (1999), Omae (2003), Shi et al. (2005), and new list proposed in this thesis.

Ampullariidae 0 2 5 6 6 6 6

Aporrhaididae 0 0 1 1 1 1 1

Apseudolcdea 0 1 1 1 1 1 1

Buccinidae 1 2 5 16 16 26 33

Bursidae 0 0 0 0 0 0 1

Cancellariidae 0 0 0 1 1 1 1

Cassidae 0 0 0 1 1 1 3

Columbellidae 2 2 2 2 2 2 2

Conidae 1 3 4 14 14 17 16

Cypraeidae 0 1 0 0 0 1 1

Fasciolariidae 2 2 3 6 6 7 8

Haliotidae 0 0 0 0 0 0 3

Hydrobiidae 0 1 1 1 1 1 3

lamellariidae 0 0 0 1 1 1 1

Littorinidae 0 6 9 0 0 0 4

Marginellidae 1 1 1 1 1 1 2

Melongenidae 3 3 3 4 4 1 8

Muricidae 16 16 31 53 56 78 100

Nassariidae 3 4 5 8 8 13 20

Naticidae 0 0 0 1 1 1 1

Olividae 1 1 1 2 2 2 5

Personidae 0 0 0 0 0 0 1

Pterotracheidae 0 1 1 1 1 1 1

Ranellidae 0 0 0 3 3 5 7

Rissoidae 0 4 6 7 7 7 7

Strom bidae 0 0 0 1 1 1 4

Terebridae 1 1 2 2 2 3 3

Thiaridae 0 0 1 0 0 0 1

Torinidae 0 0 0 1 2 4 4

Triviidae 0 0 3 6 6 6 6

Turbinellidae 0 0 0 0 0 1 1

Turridae 2 0 0 0 0 1 3

Volutidae 0 0 0 0 0 2 11

Total number of species: 31 51 85 140 144 192 269 Table 5.2. List of known gastropod species affected by female masculinisation (imposex, intersex and ovo-testis) from 1962 to 2009.

No. of O Order Family Species Region Source '90 '91 '99 '03 VI '09 Studies

Vestigastropoda Haliotidae Haliotis gigantea' Asia Horiguchi et al. (2002) Y 2

H. madaka" Asia Horiguchi et al. (2000) Y 4

H. roe * Asia Sloan and Gagnon (2004) Y 1

Mesogastropoda Ampullariidae Ampullaria (Pachylabra) cinerea Europe Fioroni et al. (1991) Y Y Y Y Y Y 1

A. gigas Europe Fioroni et al. (1991) Y Y Y Y Y 1

A. polita Europe Fioroni et al. (1991) Y Y Y Y Y Y 1

Marisa cornuarietis* Lab Schulte-Oehlmann et al. (1995) Y Y Y Y 10

Pila globosa Europe Fioroni et al. (1991) Y Y Y Y Y 1

Pomacea canaliculata* Asia Fioroni et al. (1991) Y Y Y Y Y 6

Apseudoicdea Campeloma rufum Unknown n/a Y Y Y Y Y Y n/a

Thiaridae Melania episcopalis Unknown n/a Y Y n/a

littorinidae Uttorina littorea Europe Oehlmann et al. (1994a) Y Y Y 30

N. America Covert (1997) 7

L sitkana' Asia Nohara (1999) Y 1

Mainwaringia nevilf Asia Reid (1986) Y 2

Tanganyikia rufilosa Unknown n/a Y Y n/a

Hydrobiidae Heleobia hatchert S. America Martin (2002) Y 1

Hydrobia ulvae Europe Fioroni et al. (1990) Y Y Y Y Y Y 5

Valvata (Spathogyna)fezi Europe Arconada and Ramos (2002) Y 1

Rissoidae Rissoa auriscalpium Europe Thiriot-Quievreux (1977) Y Y Y Y Y Y 1

R. ventricosa Europe Thiriot-Quievreux (1977) Y Y Y Y Y Y 1

ro K) O R. violacea Europe Thiriot-Quievreux (1977) Y Y Y Y Y 1

Rissoina allanae Unknown n/a Y Y Y Y Y n/a

R. boucheti Unknown n/a Y Y Y Y n/a

R. bruguierei Europe Thiriot-Quievreux (1977) Y Y Y Y Y 1

R. nivea Unknown n/a Y Y Y Y Y n/a

Triviidae Trivia aperta Unknown n/a Y Y Y n/a

T. arctica Europe Fioroni et al. (1990) Y Y Y Y 6

T. costata Unknown n/a Y Y Y n/a

T. millardi Unknown n/a Y Y Y Y n/a

T. monacha Europe Fioroni et al. (1990) Y Y Y Y 6

T. verhoefi Unknown n/a Y Y Y n/a

Lamellariidae Lamellaria perspicua Europe Oehlmann et al. (1993) Y Y Y Y 1

Pterotracheidae Pterotrachea coronata Unknown n/a Y Y Y Y Y n/a

Aporrhaididae Aporrhais pespelecani Europe Fioroni et al. (1991) Y Y Y Y Y 1

Cassidae Cassidaria tyrrhena' Europe Gomez-Ariza et al. (2006) Y 1

Galeodea tyrrhena Unknown n/a Y Y Y Y n/a

Phalium bisulcatum' Asia Swennen et al. (1997) Y 2

Ranellidae Apoilon natator Unknown n/a Y Y n/a

Cabestana spengleri Oceania Foale (1993) Y Y 1

Charonia sauliae saulie Unknown n/a Y Y Y Y n/a

Cymatium parthenopeum' S. America de Castro et al. (2007) Y 1

Fusitriton oregonensis Unknown n/a Y Y Y Y n/a

Gyrineum natator' India Vishwakiran et al. (2006) Y 1

Monoplex echo Unknown n/a Y Y Y Y n/a

Bursidae Bursa rana' Asia Swennen et al. (1997) Y 2

Strombidae Strombus canarium' Asia Cob et al. (2008) Y 1

S. gigas' Caribbean Reed (1993a, b); Phillip (2000) Y 3

INJ to 5. luhuanus Asia Kuwamura et al. (1983) Y Y Y Y 2

S. pugilis* Caribbean Reed (1993a, b) Y 4

Cypraeidae Mauritia arabica Asia Shi et al. (2005) Y Y 3

Naticidae Neverita didyma Asia Shi et al. (2005) Y Y Y Y 1

Tonnidae Galeoocorys leucodoma Unknown n/a Y Y Y n/a

Tonna luteostoma Unknown n/a Y Y Y Y n/a

T. olearium Asia Shi and Huang (2001a); Shi et al. (2005) Y Y 1

T. sulcoso Asia Shi et al. (2005) Y Y 1

Personidae Distorsio reticularis* Asia Swennen et al. (1997) Y 2

Neogastropoda Muricidae Acanthina monodon' S. America Osorio and Huaquin (2003) Y 2

Bolinus brandaris Europe Sole et al. (1998) Y Y 9

Calotrophon ostrearum Unknown n/a Y Y Y Y Y Y n/a

Ceratostoma burnetti Unknown n/a Y Y Y Y n/a

C.foliatum N.America Alvarez and Ellis (1990) Y Y Y Y 1

C. fournieri Asia Shi et al. (2005) Y Y 1

C. roriftuum Asia Shi et al. (2005) Y Y 1

Chicoreus asianus Asia Shi et al. (2005) Y Y 2

C. banksii" Asia Swennen et al. (2009) Y 1

C. brevifrons" S. America Milosiavich et al. (2007) Y 1

C. brunneus Asia Yu et al. (2003) Y Y 3

C. capucinus Asia Swennen et al. (1997) Y Y 6

C. (Phylonotus) margaritensis" S. America Milosiavich et al. (2007) Y 1

Chorus giganteus S. America Gooding et al. (1999) Y Y 2

Conocholepas concholepas' S. America Osorio and Huaquin (2003) Y 1

Coralliophilia abbreviata Unknown n/a Y Y Y Y n/a

C. lamellosa Europe Fioroni et al. (1991) Y Y Y Y 1

Cronia konkanensis India Kiran and Anil (1999) Y Y 1

tsj to to C. margariticola Asia Ellis and Pattisina (1990) Y Y Y Y 2

C. pothuauii Unknown n/a Y Y Y Y n/a

Dicathais orbita Unknown n/a Y Y Y Y Y n/a

Drupelia fragum Unknown n/a Y Y Y Y n/a

D. ochrostoma Unknown n/a Y Y Y Y Y n/a

D. rugosa Asia Ellis and Pattisina (1990) Y Y Y Y 2

Ergalatax contractus Asia Horiguchi et al. (1997a) Y Y Y Y 1

Eupleura caudata N. America Griffith and Castagna (1962) Y Y Y Y Y Y 3

E. caudata etterae Unknown n/a Y Y Y Y n/a

E. caudata sulcidenta Unknown n/a Y Y Y Y n/a

Forreria belcheri Unknown n/a Y Y Y Y Y n/a

Haustrum haustorium Oceania Stewart et al. (1992) Y Y Y Y Y 1

Hexaplex (Murex) trunculus Europe Axiak et al. (1995) Y Y Y Y Y 33

Lataxiena blosvillet Asia Swennen et al. (2009) Y 1

Lepsiella albomarginata Oceania Smith and McVeagh (1991) Y Y Y Y 2

L. scobina Oceania Smith and McVeagh (1991) Y Y Y Y 6

L. vinosa Oceania Foale (1993) Y Y Y Y Y 1

Morula biconica Asia Shi et al. (2005) Y Y 1

M. granulata Asia Pandey and Evans (1996) Y Y Y Y 2

Oceania Reitsema and Spickett (1999) 2

M margaritcola Asia Shi et al. (2005) Y Y 3

M. marginalba Oceania Wilson etal. (1993) Y Y Y Y 2

M. marginatra Unknown n/a Y Y Y Y n/a

M. musiva Asia Ellis and Pattisina (1990) Y Y Y Y 6

Murex altispira' Asia Swennen et al. (2009) Y 1

M. brandaris Europe Fioroni et al. (1991) Y Y Y Y 2

M. florifer dilectus (Chicoreus dilecuts) Unknown n/a Y Y Y Y Y Y n/a

K)to oo M. occa' Asia Swennen et al. (1996,2009)

M. pomum Unknown n/a

M. trapa Asia Swennen et al. (1996); Shi et al. (2005)

M. tribulus' Asia Swennen et al. (1996,1997)

Naquetia capucina Unknown n/a

Nucella canaliculata N. America Houston (1971)

N. crassilabrum S. America Gooding etal. (1999)

N. emarginata N. America Houston (1971)

N. freycineti alabaster Asia Horiguchi et al. (1997a, 2001)

N. freycineti heyseana Asia Horiguchi et al. (1997a, 2001)

N. lamellosa N. America Bright and Ellils (1990)

N. lapillus Europe Blaber (1970)

N. America Miller and Pondick (1984)

Nucella lima Asia Horiguchi et al. (1997a)

N. America Short etal. (1989)

Ocenebra bombayana India Ramasamy and Murugan (2002)

O. erinacea Europe Poli et al. (1971)

O. lumaria Unknown n/a

O. lurida N. America Bright and Ellils (1990)

Ocinebrina aciculata Europe Oehlmann et al. (1993)

O. edwardsi Europe Oehlmann (1998)

Plicopurpura patula Unknown n/a

Purpura patula' Caribbean Strand et al. (2009)

Rapana rapiformis' Asia Swennen et al. (2009)

Rapana venosa Asia Horiguchi et al. (1997a)

N. America Mann et al. (2006)

Semiricinula muricoides' Asia Swennen et al. (2009) Stramonita (Thais) haemastoma Europe Spence et al. (1990b)

Isreal Rilovetal. (2001)

N. America Jenner(1979)

S. America de Castro et al. (2000)

Thais biseriallis India Ramasamy and Murugan (2002)

T. bitubercularis Asia Tan (1997)

T. brevidentata' S. America Gravel et al. (2006)

T. bronni Asia Horiguchi et al. (1997a)

T. bufo India Tewari et al. (2002)

T. clavigera Asia Horiguchi et al. (1994)

T. deltoidea' Caribbean Strand et al. (2009)

S. America da Costa et al. (2008)

T. distinguenda Asia Bech (1999)

T. echinata' Asia Evans et al. (2001b)

T. gradota Asia Swennen et al. (1996)

T. javanica' Asia Pandey and Evans(1996)

T. jubilaeo Asia Tan (1997)

T. kieneri Asia Evans et al. (199S)

T. lacera' Asia Swennen et al. (1997, 2009)

T. luteostoma Asia Ellis and Pattisina (1990)

T. malayensis' Asia Evans et al. (2001b)

T. mancinella Unknown n/a

T. mutabilis Asia Shi and Huang (2001a,b)

T. orbita Oceania Stewart et al. (1992)

T. rudolphi Asia Tewari et al. (2002)

T. rufotincta Asia Evans et al. (2001b)

T. rustica" Caribbean Strand et al. (2009) S. America Camillo et al. (2004)

T. savignyi Asia Evans etal. (1995)

T. tissoti India Tewari et al. (2002)

Trophon geversianus' S. America Bigatti et al. (2009)

Urosalpinx cinerea Europe Fioroni et al. (1990)

N. America Griffith and Castagna (1962)

Urosalpinx perrugata Unknown n/a

Urosalpinx tampaensis Unknown n/a

Xanthochorus cassidiformis S. America Gooding et al. (1999)

Ximenopsis multiformis' S. America Bigatti et al. (2009)

Xymene ambiguus Oceania Stewart et al. (1992)

Buccinidae Austrofusus gians' Oceania Roberts and Forrest (1999)

Babylonia ambulacrum Asia Shi etal. (2005)

B. areolata Asia Shi et al. (2005)

B. formosae formosae Asia Shi et al. (2005)

B. formosae habei Asia Shi et al. (2005)

8. japonica Asia Horiguchi et al. (1997a)

B. luteoma' Asia Shi and Huang (2001a, b)

Buccinum finmarkianum' Greenland Strand et al. (2006)

8. middendorffi Unknown n/a

8. opisthoplectum Unknown n/a

8. undatum Europe Kantor(1984)

N. America Viglino et al. (2006)

Cantharus cecillei Asia Shi et al. (2005)

C. dorbignyi Europe Oehlmann (1998)

Colus gracilis Europe Oehlmann et al. (1993)

C. halli N. America Bright and Ellis (1990) C. sabini" Greenland Strand et al. (2006) Y 1

Cominella adspersa' Oceania Roberts and Forrest (1999) Y

C virgata Oceania Stewart et al. (1992) Y Y Y Y 1

Engina alvedata Asia Shi et al. (2005) Y Y 1

E. armillata Unknown n/a Y Y n/a

Japeuthria ferra Unknown n/a Y Y Y Y n/a

Keltetia lischkei Unknown n/a Y Y Y Y n/a

Nassaria pusUla' Asia Swennen et al. (2009) Y 1

Neptunea antiqua Europe Ide et al. (1997) Y Y 8

N. arthritica arthritica Unknown n/a Y Y Y Y n/a

N. arthritica cumingii Asia Shi et al. (2005) Y Y 1

N. phoenecia N. America Bright and Ellis (1990) Y Y Y Y Y 2

Pareuthria plumbea' S. America Bigatti et al. (2009) Y 1

Pisania tinctus Unknown Jenner (1979) V Y Y Y Y n/a

Pusiostoma mendicaria Unknown Horiguchi et al. (1997a) Y Y Y Y n/a

Searlesia dira (Lirabuccinum dirum) N. America Bright and Ellis (1990) Y Y Y Y Y 8

5. fuscoiabiata Unknown Horiguchi et al. (1997a) Y Y Y Y n/a

Volutharpa ampullacea perryi Unknown Horiguchi et al. (1997a) Y Y Y Y n/a

Melongenidae Buscycon carica Unknown n/a Y Y Y Y Y n/a

B. contarium Unknown n/a Y Y Y Y Y n/a

Hemifusus ternatanus" Asia Swennen et al. (1996) Y 3

H. tuba Unknown n/a Y Y Y Y n/a

Melongena corona Unknown n/a Y Y Y Y Y n/a

M. melongena* S. America Hernandez and Stotz (2004) Y 1

Pugilina cochiidium' Asia Swennen et al. (2009) Y 1

Volema cochilidium' Asia Swennen et al. (1996) Y 2

Nassariidae Buccinanops cochiidium' S. America Bigatti et al. (2009) Y 1

N3 NJ 8. globulosus' S. America Bigatti et al. (2009)

B. monilifer S. America Penchaszadeh et al. (2001)

Bullia rhodostoma Unknown n/a

Cyclope neritea Europe Cledon et al. (2006)

llyanassa obsoleta N. America Smith (1971)

Nassarius hepaticus Asia Shi et al. (2004b, 2005)

N. (Hinia) incrassata Europe Fioroni et al. (1990)

N. jacksonianus* Asia Swennen et al. (2009)

N. kraussianus S. Africa Marshall and Rajkumar (2003)

N. livescens Asia Liu and Suen (1996); Swennen et al. (1996)

N. mendicus' N. America Godtfredsen et al. (2005)

N. (Hinia) nitidus' Europe Pavoni et al. (2007)

N. (Hinia) reticulatus Europe Fioroni et al. (1990)

N. siquijorensis Asia Shi and Huang (2001a, b)

N. stolatus' Asia Swennen et al. (1996,1997)

N. thersites Asia Shi et al. (2004b, 2005)

N. (Hinia) trivittata Unknown n/a

N. (Hinia) vibex S. America Cardoso et al. (2009)

N. America Jenner (1979)

Reticunassa/estiva Unknown n/a

Columbellidae Anachis avara Unknown n/a

Mitrelia lunata Unknown n/a

Fasciolariidae Fasciolaria hunteria Unknown n/a

F. lilium Unknown n/a

Fusinus australis Oceania Foale (1993)

F. perplexus perpiexus Unknown n/a

F. syracusanus Europe Oehlmann (1998) Leucozonia nassa' S. America Miloslavich et al. (2007)

Pleuropoca gigantea Unknown n/a

Taron dubius Oceania Stewart et al. (1992)

Cancellariidae Sydaphera spengleriana Unknown Horiguchi et al. (1997a)

Marginellidae Marginella apicana Unknown n/a

Prunum martin? S. America Bigatti et al. (2009)

Olividae Amalda (Baryspira) australis Oceania Roberts and Forrest (1999)

A. mucronata' Oceania Roberts and Forrest (1999)

Olivella biplicata Unknown n/a

Olivancillaria deshaYiana* S. America Teso and Penchaszadeh (2009)

O. vesica vesica' S. America Caetano and Absalao (2002)

Volutidae Adelomelon ancilla' S. America Swennen et al. (2009)

A. briasilina S. America Penchaszadeh et al. (2001)

A. ferussacif S. America Bigatti et al. (2009)

Cymbiola nobiiis' Asia Swennen et al. (1996,1997)

C. vespertilio1 Asia Swennen and Horpet (2008)

Cymbium melo Asia Shi and Huang (2001b); Shi et al. (2005)

C. olla' Europe G6mez-Ariza et al. (2006)

Melo amphora" Oceania Swennen and Horpet (2008)

M. melo* Asia Swennen et al. (1996)

Odontocymbiola magellancia* S. America Bigatti and Penchaszadeh (2005)

Valuta ebraea' S. America de Castro et al. (2008)

Conidae Conus anemone Oceania Kohn and Almasi (1993)

C. betulinus Asia Shi et al. (2005)

C. coronatus Oceania Kohn and Almasi (1993)

C. dorreensis Oceania Kohn and Almasi (1993)

C. klemae Oceania Kohn and Almasi (1993) C. lischkeanus Oceania Kohn and Almasi (1993) Y Y Y Y 1

C. lividus Oceania Kohn and Almasi (1993) Y Y 1

C. marmoreus bandanus Unknown n/a Y Y Y Y n/a

C. mediterraneus Unknown n/a Y Y Y Y Y Y n/a

C. sponsolis Oceania Kohn and Almasi (1993) Y Y Y Y 1

C. vexillum Asia Shi et al. (2005) Y Y 2

Kurtziella cerina Unknown n/a Y Y Y Y Y Y n/a

Lora (Propebela) tumcula Unknown n/a Y Y Y Y Y Y n/a

Virgiconus lividus Unknown n/a Y Y Y Y n/a

Virroconus ebraeus Unknown n/a Y Y Y Y n/a

V. fulgetrum Unknown n/a Y Y Y Y n/a

Terebridae Duplicaria spectabilis Unknown n/a Y Y n/a

Terebra dislocata N. America Jenner(1979) Y Y Y Y Y 2

T. protexta Unknown n/a Y Y Y Y Y Y n/a

Turbineliidae Vasum turbinellus Asia Evans et al. (1995) Y Y 1

Turridae Lophiotoma leucotroposis Unknown n/a Y Y n/a

Raphitoma reticulata Europe n/a Y Y Y n/a

Turricula javana' Asia Swennen et al. (2009) Y 1

Nota bene'. • Region validated based on the available literature • Source = original publication(s) that identified FM in each species and region * = Freshwater species a = Species found with imposex/intersex/ovo-testis in the literature but never published in an affected species list (n = 67) n/a = not applicable

to oo © Table 5.3. List of known gastropods unaffected by female masculinisation (imposex, intersex and ovo-testis) published from 1962 to 2009.

Family of sympatric species that displayed female masculinisation in parenthesis where (B) = Buccinidae, (F) = Fasciolariidae, (H) =

Hydrobiidae, (M) = Muricidae, (N) = Nassariidae, (S) = Strombidae, (T) = Turbinellidae, and (V) = Volutidae. Region was validated wherever

possible based on the available literature.

Sympatric species Sample [Tissue] Order Family Species Region Showing Source Size ng Sn g"1 Female Masculinisation

Vestigastropoda Trochoidae Cittarium pica NR Caribbean 17 to 119 Purpura patula (M) Strand et al. (2009)

Thais deltoidea (M)

Thais rustica (M)

Neritidae Nerita versicolor 10 S. America NR Chicoreus brevifrons (M) Miloslavich et al. (2007)

Leucozonia nassa (F)

Theodoxus fluviatilis 287 Laboratory NR Oehlmann (1998)

Mesogastropoda Capuiidae Capulus ungaricus NR Europe NR Oehlmann (1998)

Ficidae Ficus ficus 116 Asia NR Babylonia areolata (B) Liu and Suen (1996)

Thais clavigera (M)

Turitellidae Turritella communis NR Laboratory NR Oehlmann (1998)

Littorinidae Littorina brevicula 50 Asia NR Nucella heyseana (M) Syasina and Shcheblykina (2007)

L. mandshurica 10 Asia NR Nucella heyseana (M) Syasina and Shcheblykina (2007)

L. marie 139 Europe NR NR Fioroni et al. (1991)

L. neritoides 7 Europe NR NR Fioroni et al. (1991)

L riigrolineata 587 Europe NR NR Fioroni et al. (1991) L obtusata 899 Eurpoe NR NR Fioroni et al. (1991) L saxatiiis 284 Europe NR Nucella lapillus (M) Oehlmann (1998)

Ocenebra erinacea (M) L saxatiiis rudis 397 Europe NR NR Fioroni etal. (1991) L ziczac NR Europe NR NR Fioroni et al. (1991) Bithyniidae Bithynia tentaculata 745 Laboratory NR Oehlmann (1998) Hydrobiidae Hydrobia ventrosa 120 Europe NR Hydrobia ulvae (H) Oehlmann (1998) Potamopyrgus antipodarum NR NR Matthiessen et al. (1999) Aporrhaidae Aporrhais pespelicani 100 Europe NR Bolinus brandaris (M) Gdmez-Ariza et al. (2006) Buccinum undatum (B)

Hexaplex trunculus (M)

Cassidae Phalium glacum 1; 1 Asia NR 8aby Ionia areolata (B) Swennen et al. (1996,1997)

Thais gradata (M) Ranellidae Charonia lampas 11 Europe NR Bolinus brandaris (M) Gdmez-Ariza et al. (2006)

Buccinum undatum (B)

Hexaplex trunculus (M) Cymatium corrugatum 35 Europe NR Bolinus brandaris (M) Gomez-Ariza et al. (2006) Buccinum undatum (B)

Hexaplex trunculus (M)

Ranella olearia 11 Europe NR Bolinus brandaris (M) Gomez-Ariza et al. (2006) Buccinum undatum (B)

Hexaplex trunculus (M)

Strombidae Stombus costatus 500 Caribbean NR Strombus gigas (S) Reed(1993a)

Strombus pugilis (S) Calyptraeidae Crepidula aculeata 153 S. America NR Adelomelon briasilina (V) Bigatti et al. (2009) Buccinanops cochlidium (N)

Buccinanops globulosus (N)

N3 OJ tsJ Neogastropoda Muricidae Thais hippocastaneum 139 Asia NR Babylonia areolata (B) Swennen et al. (1996,1997)

Thais gradata (M)

Murex trapa (M)

Thais tuberosa ~ 200 Asia NR Thais kieneri (M) Evans et al. (1995)

Thais savignyi (M)

Vasum turbinellus (T) 39 Asia 1.0 to 43.7* Thais clavigera (M) Liu et al. (1997) Trophon fabricii 12 Greenland NR Buccinum undatum (B) Strand and Asmund (2003)

Buccinidae Buccinum humphreysianum 3 Europe NR Bolinus brandaris (M) Gomez-Ariza et al. (2006)

Buccinum undatum (B)

Hexaplex trunculus (M) Colus islandicus 23 Greenland NR Buccinum undatum (B) Strand and Asmund (2003)

Cominella glandiformis ~150 Oceania NR Lepsiella albomarginata (M) Smith and McVeah (1991)

Lepsiella scobina (M)

Neptunea despecta 10 Greenland NR Buccinum undatum (B) Strand and Asmund (2003)

Buccinum finmarkianum (B)

1 Greenland NR Buccinum finmarkianum (B) Strand et al. (2006)

Siphonalia varicosus 15 Asia NR Babylonia areolata (B) Swennen et al. (1996) Thais gradata (M)

Murex trapa (M)

Phos senticosus 2 Asia NR Babylonia areolata (B) Swennen et al. (2009)

Thais bitubercularis (M)

Thais clavigera (M)

Volutopsius norvegicus 4 Greenland NR Buccinum finmarkianum (B) Strand et al. (2006)

Colus sabini (B)

Nassariidae Buccinanops squalidium 12 S. America NR Adelomelon briasilina (V) Bigatti et al. (2009)

Buccinanops cochlidium (N)

K3 OJ Co Buccinanops globulosus (N)

Columbellidae Amphissa columbiana 137 N. America NR Nucella canliculata (M) Bright and Ellis (1990)

Nucella emarginata (M)

Nucella lamellosa (M)

Astyris gausapata 14 N. America NR Nassarius mendicus (N) Godtfredsen et al. (2005)

Cotumbella rustica Europe NR Hexaplex trunculus (M) Gibbs et al. (1997)

Ocenebra erinacea (M)

Nucella lapillus (M)

Fasciolariidae Fasciolaria tulipa S. America N R Chicoreus brevifrons (M) Miloslavich et al. (2007)

Leucozonia nassa (F)

Volutidae Voluta musica 16 S. America N R Chicoreus brevifrons (M) Miloslavich et al. (2007)

Leucozonia nassa (F) Harpidae Harpa articularis 1;1 Asia N R Babylonia areolata (B) Swennen et al. (1996,1997) Thais gradata (M) Nota bene: • Region validated based on the available literature • Source = original publication(s) that identified FM in each species and region * = Tissue concentration reported as tributyltin + triphenyltin NR = Not reported

NJw Table 5.4. Comparison of orders and families affected and unaffected by female masculinisation (imposex, intersex and ovo-testis) by probable feeding type. Families with < 5 species with all members affected by FM (*); < 5 species with all unaffected by FM (¥), and < 5 species with some affected and unaffected by FM (•).

No. of Listed No. No. No. Percent (%) Feeding Order Family Species Examined Affected Unaffected Feeding Type Unaffected Souce Vestigastropoda Haliotidae* 34 3 3 0 Herbivore 0.00 i; 2 Trochoidae* 2** 1 0 1 Detritivore/Herbivore 100.00 1

Neritidae* 533 2 0 2 Herbivore 100.00 3 Mesogastropoda Ampullariidae 120 6 6 0 Omnivore 0.00 2 Apseudoicdea* >35 1 1 0 Herbivore 0.00 4

Thiaridae* 374 1 1 0 Detritovore 0.00 2

Capulidae* 62 1 0 1 Suspension 100.00 10

Ficidae* 37* 1 0 1 Carnivore 100.00 1

TurritellidaeT 442 1 0 1 Suspension 100.00 10

Littorinidae 353 13 4 9 Detritivore/Herbivore 69.23 1

Bithyniidae* 176 1 0 1 Unidentified 100.00

Hydrobiidae* 1001 5 3 2 Herbivore 40.00 5

Rissoidae 789 7 7 0 Herbivore 0.00 6

Triviidae 192 6 6 0 Carnivore 0.00 7

NJ OO cn Lamellariidae* 26 1 1 0 Carnivore 0.00 8

Pterotracheidae* 11 1 1 0 Carnivore 0.00 9

Aporrhaididae* 253 2 1 1 Herbivore 50.00 10

Cassidae* 332 4 3 1 Carnivore 25.00 1

Ranellidae 520 10 7 3 Carnivore 30.00 1; 2

Bursidae* 137 1 1 0 Carnivore 0.00 1;2

Strombidae* 386 5 4 1 Herbivore/Carnivore 20.00 1

Calyptraeidae* 222 1 0 1 Unidentified 100.00

Cypraeidae* 1001 1 1 0 Herbivore/Carnivore 0.00 1

Naticidae* 886 1 1 0 Carnivore 0.00 l; 2

Tonnidae* 78 4 4 0 Carnivore 0.00 1

Personidae* 41 1 1 0 Carnivore 0.00 1 Neogastropoda Muricidae 2201 103 100 3 Carnivore 2.94 i;2 Buccinidae 1399 39 33 6 Carnivore/Scavenger 15.38 l; 2

Melongenidae 119 8 8 0 Carnivore 0.00 l

Nassariidae 457 21 20 1 Carnivore/Scavenger 4.76 i;2

Columbellidae* 828 5 2 3 Omnivore 60.00 i;2

Fasciolariidae 632 9 8 1 Carnivore 11.11 l

Cancellariidae* 361 1 1 0 Unidentified 0.00

Marginellidae* 761 2 2 0 Unidentified 0.00

Olividae* 754 5 5 0 Carnivore/Scavenger 0.00 i; 2

Volutidae 674 12 11 1 Carnivore/Scavenger 8.33 i;2

N3 00 ON Harpidae* 75 2 0 2 Carnivore/Scavenger 100.00 1

Conidae 2108 16 16 0 Carnivore 0.00 1

Terebridae* 482 3 3 0 Carnivore 0.00 1; 2

Turbinellidae* 100 110 Carnivore 0.00 1

Turridae* 1547 3 3 0 Carnivore 0.00 1; 2

Total 41 ~ 19,787 311 269 42 * = does not include species studied; ** = in genus, taxonomy varies. References for Table 5.4: 1 = Carpenter and Niem (1998); 2 = The Bay Science Foundation (2009); 3 = Raffaelli (1985); 4 = Ruppert et al. (2004); 5 = Philippart (1995); 6 = Alyakrinskaya (2005); 7 = Beechey (2010); 8 = Millar (1971); 9 = Seapy (2009); 10 = Appeltans et al. (2010).

tvj 00 -J 238

Table 5.5. Comparison by feeding type of species affected and unaffected by female masculinisation (imposex, intersex and ovo-testis).

Feeding Type Positive (+) Negative (-) Percent (%) Negative Carnivore/Scavenger 236 17 6.72 Detritivore/Herbivore 31 13 29.55 Omnivore 2 3 60.00 Suspension 0 2 100.00 Unidentified 0 7 100.00 Total 269 42 Table 5.6. Summary of gastropod orders and families cited in the literature with signs of female masculinisation (imposex, intersex, and/or ovo- testis) based on laboratory exposure to organotins, organotin "cocktails", steroids, and other contaminants.

Order Family Number of Species Source(s) Vestigastropoda Haliotidae 1 Horiguchi et al. (2002) Mesogastropoda Ampullariidae 2 Schulte-Oehlmann et al. (1994,1995); Takeda (2000, 2006) Hydrobiidae 2 Schulte-Oehlmann et al. (1998); Duft et al. (2007) Littorinidae 1 Deutsch and Fioroni (1996); Deutsch et al. (1996); Bauer et al. (1997) Strombidae 1 Phillip (2000) Neogastropoda Muricidae 9 Feral (1978,1979,1980a) Feral and Le Gall (1982,1983); Stickle et al. (1990); Smith and McVeah (1991); Nias et al. (1993) Horiguchi et al. (1997b); Nishikawa et al. (2004); Garaventa et al. (2006b); Santos et al. (2006); Lahbib et al. (2007); Limaverde et al. (2007); Garaventa et al. (2008) Buccinidae Mensink et al. (1996; 2002) Nassariidae Stroben et al. (1992b); Duft et al. (2005)

Total 20 Table 5.7. Types of compounds and mode of exposure that induced female masculinisation (imposex, intersex and ovo-testis) in gastropods.

Medium Type Compound Family Species Source Injection Sediment Water Other

Tin Adipic acid - di(2-ethylhexyl) (DEHA) Ampullariidae Pomocea canaliculata 3; 4

Dioctyltin (DOT) Nassariidae llyanassa obsoleta 1

Monophenyltin (MPT) Muricidae Ocenebra erinacea 2

Phtalic acid butyl (PAD) Ampullariidae Pomacea canaliculata 3; 4

Tetrabutyltiri (TTBT) Muricidae Nucella lapillus 5

Tin tetrachloride (TTCI) Nassariidae llyanassa obsoleta 1

Tin tetrachloride (TTCI) I. obsoleta 1

Tributyltin (TBT) Haliotidae Haliotis gigantea 6

Ampullariidae Marisa cornuarietis 7

Pomacea canaliculata 8

Hydrobiidae Hydrobia ulvae 9

Littorinidae Littorina littorea 10

Strombidae Strombus gigas 11

Muricidae Bolinus brandaris 12

Hexaplex (Murex) trunculus 13; 14

Lepsiella albomarginata 15

L. scobina 15

L. vinosa 16

Nucella lapillus

to O N. lapillus Y 5

N. lapillus 5; 17

N. lapillus Painted wood panels 18

N. lapillus Y 19; 20

N. lapillus Mussels exposed to TBT 21 Nucella lima Painted specimens 22

N. lima Y 21

Ocenebra erinacea Y 23 Stramonita (Thais) Y 24 haemastoma Thais bronni Y 25

T. clavigera Y 25; 26

T. clavigera Y 27

Buccinidae Buccinum undotum Y 28; 29

Nassariidae llyanassa obsoleta Y 1

I. obsoleta Y 30; 31 Nassarius (Hinia) reticulatus Y 32; 33

N. reticulatus Y 34; 35

Tricyclohexyltin (TCHT) Muricidae Thais clavigera Y 47

Trioctyltin (TOT) Nassariidae llyanassa obsoleta Y 1

I. obsoleta Y 1

Triphenyltin (TPT) Haliotidae Haliotis gigantea Y 6

Ampulla riidae Marisa cornuarietis Y 36 Muricidae Bolinus brandaris Y 12 Stramonita (Thais) Y 24 haemastoma Thais bronni Y 25

T. clavigera Y 25; 26

ro T. clavigera Y 27

Nassariidae Nassarius (Hinia) reticulatus Y 35

Tripropyltin (TPrT) Muricidae Nucella lapillus Y 5

TBT + OOT T. clavigera Y 37 Tin "cocktails" TBT + Estrogen T. clavigera Y 37

TBT + Nonylphenol T. clavigera Y 37

TBT + Sewage Effluent Nucella lapillus Y 38

TBT + TPT Muricidae Bolinus brandaris Y 12

Steroids 4-androsten-4-ol-3,17 dione (FORM) Muricidae Nucella lapillus Y 38

Ethinyl estradiol (EE2) Marisa comuarietis Y 39 Methyl testosterone (MT) Ampullariidae Marisa comuarietis Y 39; 41

Testosterone (T) Ampulla riidae Pomacea canaliculata Y 8 Muricidae Nucella lapillus Y 42

Muricidae Nucella lapillus Y 43

Nassariidae llyanassa obsoleta Y 44; 45

Nassariidae Nassarius (Hinia) reticulatus Y 34; 43

Nassariidae Nassarius (Hinia) reticulatus Y 33

Other 9-c/s-retionic acid (9-c/s RA) Muricidae Nucella lapillus Y 17 Contaminants Thais clavigera Y 27

T. clavigera Y 27

Ala-Pro-Gly-Trp-NH2 (APGWamide) Nassariidae llyanassa obsoleta Y 44; 45

Aroclor 1260 (PCB mix) Muricidae Hexaplex (Murex) trunculus Y 13 Bird excreta Nucella lapillus Bird excreta 18 Copper (Cu) Lepsiella vinosa Y 16 Fadrozole (FAD) T. clavigera Y 40 Fenarimol (FEN) Ampullariidae Marisa comuarietis Y 33 M. comuarietis Y 41; 33

to tsJ Nassariidae Nassarius (Hinia) reticulatus Y 46 via 41

Magnesium chloride (MgCI2) Hexaplex (Murex) trunculus Y 13 Methoprene acid Muricidae Nucella lapillus Y 17

Noriylphenol (NP) Nucella lapillus Y 18

References for Table 5.7: 1 = McClellan-Green et al. (2006); 2 = Hawkins and Hutchinson (1990); 3 = Takeda (2001); 4 = Takeda (2006); 5 = Bryan et al. (1988); 6 = Horiguchi et al. (2002); 7 = Schulte-Oehlmann et al. (1995); 8 = Takeda (2000); 9 = Sculte-Oehlmann et al. (1998); 10 = Bauer et al. (1997); 11 = Phillip (2000); 12 = Santos et al. (2006); 13 = Garaventa et al. (2006a); 14 = Abidli et al. (2009b); 15 = Smith and McVeagh (1991); 16 = Nias et al. (1993); 17 = Castro et al. (2007); 18 = Evans et al. (2000); 19 = Bryan et al. (1987); 20 = Gibbs et al. (1991b); 21 = Stickle et al. (1990); 22 = Short et al. (1989); 23 = Feral and Le Gall (1983); 24 = Limaverde et al. (2007); 25 = Horiguchi et al. (1994); 26 = Horiguchi et al. (1995); 27 = Horiguchi et al. (2008); 28 = Mensink et al. (1996); 29 = Mensink et al. (2002); 30 = Smith (1981b); 31 = Smith (1981c); 32 = Duft et al. (2005); 33 = Duft et al. (2007); 34 = Bettin et al. (1996); 35 = Barroso et al. (2002a); 36 = Schulte-Oehlmann et al. (2000); 37 = Shim et al. (2002); 38 = Santos et al. (2008); 39 = Tillmann et al. (2001); 40 = Iguchi et al. (2007); 41 = Janer et al. (2006); 42 = Spooner et al. (1991); 43 = Oehlmann and Bettin (1996); 44 = Oberdorster and McClellan-Green (2000); 45 = Oberdorster and McClellan- Green (2002); 46 =Tillmann (2004); 47 = Matthiessen et al. (1999) cited Horiguchi et al. (unpublished data).

ro •f* CO 244

Table 5.8. Intensity of penis expression (P) in females adapted from Smith (1981b) to measure imposex intensity in the American mud snail, llyanassa obsoleta.

No visible protuberance on head mass: cyst, often filled with brown b particulate material, in position normally occupied by base of penis. 10 Proximal portion of Denis onlv. measurine (in length): 15 < 0.35 mm(= bump) 20 > 0.35 mm < 0.80 mm 25 > 0.80 mm < 1.70 mm > 1.70 mm 30 Proximal and distal portions of Denis visible, measuring (in lensth): 32 <0.35 mm 35 >0.35 mm <0.70 mm 38 > 0.70 mm £ 1.25 mm 40 > 1.25 mm < 1.70 mm > 1.70 mm Table 5.9. Summary of recommended indices to measure imposex and intersex in gastropods used for TBT biomonitoring field studies indicating parameters that can be measured, ecological significance and species applicability.

Used to Measure Index Ecological Significance Species Applicability Changes over time Incidence Recovery Severity

Average oviduct convolution (AOS/OS) Y Y Y High Nassarius reticulatus

Nassarius nitidus Imposex index Y Y High Buccinum undatum Neptunea antique Intersex incidence Y Y Low Intersex affected species Intersex index (ISI) Y Y Y High Intersex affected species Male to female ratio (M:F) Y Y Y Medium Imposex-affected species Mean female penis length (mFPL) Y Y Y Medium Imposex-affected species

Mean female prostate length (FPrL) Y Y Y Medium Intersex affected species

Mean male penis length (mMPL) Y Low Impoes affected species Penis classification index (PCI) Y Y Y High Buccinidae family Percent imposex (% 1) Y Y Y Low Imposex-affected species Percent sterile female Y Y Y High Imposex-affected species Relative penis length index (RPLI) Y Y Y Medium Imposex-affected species Relative penis size index (RPSI) Y Y Y Medium Nucella lapillus

Vas deferens sequence index (VDSI) Y Y Y High Imposex-affected species

ro ui Table 5.10. Table of progression on the incidence of imposex, intersex and ovo-testis in the scientific literature from 1962 to 2009.

Year Event Source

1962 Verified female Urosalpinx cinerea and Eupleura caudata fromVirginia have a "small vestigal structures resembling a penis" Griffith and Castagna (1962)

1970 Validated female Nucella lapillus have a "penis-like outgrowth" observed in the United Kingdom Blaber (1970)

1971 Known female Ocenebra erinacea detected with a "whole penis at base of right ocular tentacle" along France's Atlantic coast Poli et al. (1971)

1971 "Penis-like structure along the path of vas deferens formation" identified in female llyannasa (Nassarius) obsolete in USA Smith (1971)

1971 "Functional females with a small penis" discovered in female Nucella (Thais) canalicutato & Nucella (Thais) emarginata in USA Houston (1971)

1974 Male:Female ratio (M:F) used as a measure to ascertain if population structure is skewed Feral (1974)

1975 Lab studies suggest that penis formation in females is dominated by the action of a penis morphogenetic factor F^ral (1975)

1977 "Reduced penis" detected in female Rissoa auriscalpium, R. ventricosa, R. violacea & Rissoina bruguierei from France Thiriot-Quievereux (1977)

1977 Lab studies suggest that the penis morphogenetic factor is present in the right ocular tentacle & pedal ganglia of male and limposex females Feral (1977)

1978 "Anomalous sexual attributes of one sort" known to occur in female gastropods in 33 species Jenner(1978)

1979 Pseudohermaproditism introduced to describe male analomous structures Jenner (1979)

1980 Collective male characteristics in female gastropods dubbed "imposex" Smith (1980)

1981 Established imposex indices increased significantly near marinas 8i areas of increased boating activity, but minimal in other areas Smith (1981a)

1983 Induction by penis morphogentic factor (PMF) proposed Feral and Le Gall (1983)

1986 Relative Penis Size Index (RPSI) described as an index to measure imposex intensity Bryan et al. (1986)

1986 Severely imposex population characterized by fewer females, juveniles and scarcity of egg capsules Bryan et al. (1986)

1986 Increased organotin tissue concentration correlated with degree or severity of imposex in Nucella lapillus. Bryan et al. (1986)

1987 Vas Deferens Sequence index (VDSI) described to measure imposex severity (Stage 0 to 6) Gibbs et al. (1987)

1987 Female penis length used as an index to describe imposex severity in females. Bryan et al. (1987)

1988 Histology of laboratory reared Nucella lapillus reveals that oogenesis is suppressed and supplanted by spermatogenesis in imposex females Gibbs et al. (1988)

1988 A suite of organotins {TBT, DBT, TPT, TTBT, and TPrT) used to compare effectiveness of imposex development. Bryan et al. (1988)

NJ % sterile females used as an indicator of imposex severity Spence et al. (1990a)

Laboratory studies demonstrate that monophenyltin (MPT) increases female penis length in O. erinacea Hawkins and Hutchinson (1990)

Nucella lima is capable of bioaccumulating TBT from food (mussels) & water Stickle etal. (1990)

No imposex observed in Amphissa columbiana (n = 130) while other species from the same area exhibited imposex Bright and Ellis (1990)

1991 Mini-review of imposex on gastropods Gibbs et al. (1991a)

1991 Littorina littorea populations increase simultaneously with decreased TBT environmental levels in water and sediment Matthiessen et al. (1991)

1991 Revised VDSI developed expanding on VDSI developed by Gibbs et al. (1987) with evolution of morphological stages (a, b & C) Oehlmann et al. (1991)

1991 Testosterone via injection significantly increases female penis length in Nucella lapillus Spooner et al. (1991)

1991 Field investigation in Scotland documented recovery of Nucella lapillus populations after banning TBT in 1987 Davies and Bailey (1991)

1991 Confirmed effects of TBT released from large ocean going vessels in coastal waters induces imposex in Nucella lapillus Davies and Bailey (1991)

1991 Imposex documented in various tropical species from Indonesia Pattisina (1991)

1991 Updated affected species list (n = 51) Fioroni et al. (1991)

1992 Uncubed Relative Penis Size index (RPL/RPLI) developed as an alternative to RPSI Stroben et al. (1992a)

1992 Even though TBT has been banned previously investigated Nucella lapillus populations (n > 70) still showed signs of imposex Schulte-Oehlmann et al. (1992)

1993 Dumptom Syndrome, a male genital defect described in Nucella lapillus population's increases population survival in TBT-polluted areas Gibbs (1993)

1993 Laboratory exposure in Lepsiella vinosa suggests induction to copper, TBT, and environmental stress are inducers of imposex in gastropods. Nias et al. (1993)

1993 Laboratory experiments demonstrated that imposex induction is suppressed by the antiandrogen cyproterone acetate (CPA) Oehlmann et al. (1993)

1994 Intersex index (ISI) introduced to measure intersex severity in Littorina littorea Oehlmann et al. (1994a)

1994 TPT induces imposex in Thais clavigera and T. bronni Horiguchi et al. (1994)

1994 Increased imposex frequency and occurrence in Buccinum undatum collected from the North Sea linked to shipping intensity Ten Hallers-Tjabbes et al. (1994)

1994 Female Marisa cornuarietis displays some degree of "natural imposex/pseudohermaphroditism" in populations, but is intensified by TBT Schulte-Oehlmann et al. (1994)

1995 Marisa cornuarietis suggested as a bioindicator for TBT in Freshwater environments Schulte-Oehlmann et al. (1995)

1995 Mean female prostate gland used to assess intersex severity in Littorina littorea Bauer et al. (1995) 1996 Female masculinisation induction by aromatase inhibition theory proposed Bettin et al. (1996)

1996 Female masculiniization induced by the inhibition of testosterone excretion is proposed Ronis and Mason (1996)

K) .£• 1996 Comprehensive review of primary species used in imposex studies at the time of this publication Oehlmann et al. (1996b)

1996 Review of primary imposex indices used most commonly at the time for imposex studies (% imposex, FPL, MPL, RPSI, RPL/RPLI & VDSI) Stroben et al. (1996)

1996 Establishment of Penis Classification index (PCI) in Buccinum undatum (Stage 0 to 3) Mensink et al. (1996)

1997 DBT, MBT, DPT and MPT via injection experiments did not increase female penis length Thais clavigera Horiguchi et al. (1997b)

1997 Imposex index developed for Buccinum undatum and Neptunea antiqua Ide et al. (1997)

1997 Freezing has no effect on VDS measurements, but narcotisation increases penis length in Nucella lapillus Davies et al. (1997)

1998 TBT-induced imposex and intersex in gastropods declared as the clearest example of endocrine disruption described in invertebrates to date Matthiessen and Gibbs (1998)

1999 Comprehensive review of endocrine disruption in the field using invertebrates Matthiessen et al. (1999)

1999 Updated imposex-affected species list (n = 140) Matthiessen et al. (1999)

1999 VDSI and RPSI adopted by Oslo and Paris Commission (OSPAR) for their Joint Assessment and Monitoring Programme (JAMP) Minchin and Davies (1999)

2000 Masculinised females of the Order Vestigastropoda nicknamed ovo-testis (ovary with a small amount of testis) Horiguchi et al. (2000)

2000 Exposure to nonylphenol and bird excreta are possible inducers of imposex in Nucella lapillus Evans et al. (2000)

2001 Imposex induction in Pomacea canaliculata not by organotins, but by plasticizers (PAD and DEHA). Takeda(2001)

2001 Average oviduct stage (AOS) introduced as a measure of imposex severity and correlates with tissue concentration Barreiro et al. (2001)

2002 Induction by modulation of endogenous testosterone levels theory proposed Gooding (2002)

2004 Retinoid x receptors (RXR) put forward as possible mode of imposex induction Nishikawa et al. (2004)

2006 New hypothesis proposed for imposex induction - RXR plays an important role in inducing imposex development in female gastropods. Horiguchi et al. (2006a) 2006 Degree of imposex linearly correlated with DNA damage in Nucella lapillus collected from field sites Hagger et al. (2006)

2006 Relationship between tissue concentration of PAHs, PCBs and organotins and female penis length in Hexaplex trunculus Maran et al. (2006) 2008 "Pseudo-imposex" introduced to describe some species of female gastropods found with a penis prior to 1930 Swennen and Horpet (2008)

K) 00 249

Table 5.11. Suggested data to be collected when using gastropods as biomonitors or bioindicators of TBT pollution.

Study Characteristics Population Breeding Capacity Types of Boating Activity

Date specimens collected Are juveniles present? Yes or No Harbour: Yes or No

Date specimens examined Are egg capsules present? Yes or No Naval: Yes or No

Study location Shell and Body Characteristics (Individual Level) Moorings Yes or No

GPS co-ordinates Shell length (mm): Shipyard: Yes or No

Tide status Shell height (mm): Ferry: Yes or No

Time collection started Shell weight [wet in shell) (g): Marina: Yes or No

Time collection ended Fishing port: Yes or No Gender Determination (Individual Level) Initial Gender Determinatipn Additional Observations

Capsule gland: Yes or No Female body arrangement Sperm ingesting gland: Yes or No

Male body arrangement Testis: Yes or No

Initial gender conclusion Male or Female

ImDOsex Parameters

Straight: Yes or No

Penis type Re-curved: Yes or No

Length (mm):

Vas deferens Stage: 12 3 4 5 6

Confirmed gender Female Imposex Female Male Imposex Analysis (Population Level) - Minimum sample size = 30

Total number of females: Percent Imposex =

Total number of imposex females: M:F =

Total number of males: RPSI =

Mean female penis length (mm): RPU =

Mean male penis length (mm): VDSI =

Number of sterile females: Percent Sterile Females = 250

6 General Conclusion

"We have forgotten how to be good guests, how to walk lightly on the earth as its other creatures do."

Barbara Ward (1914 to 1981)

I believe that the above statement caused some heads to turn when Barbara

Ward (1972) wrote it in "Only One Earth: The Care and Maintenance of a Small

Planet." After researching the literature on TBT, this quote struck a chord with me as an aspiring scientist more than 30 years later. From all appearances, it seems that humans in their infinite wisdom have intentionally or unintentionally introduced a

"soup" of pollutants into the environment with an apparent disregard of the effects on the environment and the organisms living in it.

One of the best documented environmental tragedies of an introduced pollutant to control one class of organisms that had adverse effects on non-target species is the effects of the pesticide dichlorodiphenyltrichloroethane (DDT) on bird populations. DDT was introduced as an insecticide during World War II to control malaria and typhus and was very effective against its intended targets and other arthropods as well. After World War II, DDT was used as an agricultural insecticide in the US and due to its undisputed effectiveness, production skyrocketed. In the book "Silent Spring", Rachel Carson (1962) questioned the indiscriminate use of

DDT in the US without full understanding DDT's effects on the environment or 251 human health, but the damage was already done. The use of DDT to control insects resulted in eggshell thinning in birds including the bald eagle, Haliaeetus leucocephalus (Colborn et al., 1993), the American kestrel, Falco sparverius (Bird et al., 1983), and the loggerhead shrike, Lanius ludovicianus (Anderson and Duzan,

1978) by DDT and its metabolite, 1,1 -di(p-chlorophenyl)-2,2-dichloroethylene

(DDE).

Coincidently, around the same time that DDT was impacting birds, TBT was replacing Copper (Cu)-based antifouling paints due to its increased effectiveness and longevity (Evans and Smith, 1975; Omae, 2003). Similar to DDT, TBT's production skyrocketed, and this inevitably resulted in the introduction of TBT into the marine environment. It was not until the 1980s that TBT was linked to chambering in the commercial Pacific oyster, Crassostrea gigas (Alzieu, 1986) and imposex in gonochoristic female gastropods (Smith, 1981; Feral and Le Gall 1982), even though these unusual biological phenomena were first observed in the 1960s and 1970s. Although TBT has been regulated by most developed countries since the mid to late 1980s, environmental levels have shown little or no decline even after many years of regulated use e.g. in France (Michel and Averty, 1991). Stable environmental levels, especially in sediments, are most likely attributed to TBT's persistence in the environment. TBT has been described as quite possibly the most acutely toxic chemical deliberately introduced to the environment (Maguire, 1987).

In addition, the induction of imposex in response to butyltin exposure has been regarded as the most complete example of endocrine disruption caused by an environmental contaminant (Vos et al., 2000). 252

In my thesis, I used Nucella lapillus, Strombus gigas and Mytilus edulis as biomonitors to evaluate TBT pollution in two Maritime Provinces of Canada and in the British Virgin Islands (BVI). In addition, I also undertook the painstaking task of reading and summarising the available literature (n = 587) on the effects of organotins on female gastropods from 1962 to 2009. My aim was to present a review article that illustrated how the terminology, science and theories have evolved over the last 40 or so years, and that further examines the incidence of female masculinization (FM) on a global scale. In the following section I will outline the objectives of my thesis, summarize how I addressed each objective, and conclude with my findings.

6.1 Summary of Thesis

The objectives of this study were to:

(1) evaluate the incidence and severity of imposex at several sites with and

without boating activity in the BVI using S. gigas (Chapter 2);

(2) assess temporal changes in imposex incidence and severity by comparing

current results with previous studies in the BVI (Phillip, 2000) (Chapter 2); 253

(3) use N. lapillus as a biomonitor of TBT pollution in harbours and reference

sites in Atlantic Canada to assess the incidence and severity of imposex on

a spatial scale across New Brunswick and Nova Scotia (Chapter 3);

(4) ascertain temporal changes in imposex-affected N. lapillus populations by

comparing the results of this study with previous studies (Prouse and Ellis,

1997; Covert, 1997; Delaney, 2001; Coray, 2007 ) from the same

geographic areas (Chapter 3);

(5) determine if there is a difference in TBT concentration in sediment and

resident M. edulis in selected harbours in Atlantic Canada between sites

and site type (harbour vs. reference) (Chapter 4);

(6) compare current TBT levels in sediment and M. edulis in the Maritimes

with those from Chau et al. (1997 a, b) (Chapter 4);

(7) use results from the current study to determine the efficacy of the 1989

Canadian regulations on TBT (Chapters 3 and 4); and

(8) review available literature on the global masculinization of female

gastropods, summarise the progression of the literature from 1962 to 2009,

and outline how the terminology, literature and science have evolved over

time (Chapter 5). 254

Objectives 1 and 2 were related to the incidence and severity of imposex in S. gigas collected from the BVI, a popular tourist destination in the Caribbean without any legislation regulating the use of TBT. S. gigas were collected from boating activity areas, e.g. harbours, marinas and moorings and reference sites previously investigated by Phillip (2000). Imposex was present at all boating activity sites, but absent from reference sites. The incidence and severity (using mFPL) of imposex in

S. gigas varied temporally at boating activity sites (Figure 2.3 A and B). Chemical analysis of S. gigas tissues by gas chromatography mass spectrometry (GC-MS) detected TBT in imposex-affected females, but not in normal females from boating activity areas (Table 2.2), supporting my assumption of TBT as the suspected cause.

The patchy distribution of TBT and DBT in turtle grass (n = 1) (Thalassia testidium) and marine algae (n = 4) collected from corresponding boating areas with imposex- affected S. gigas supports the assumption of TBT input into the marine environment from suspected point sources. Even though S. gigas violates the recommended criterion of limited dispersal for a model bioindicator of TBT pollution (Gibbs and

Bryan, 1987), I recommend its use because: (1) there was a positive relationship between S. gigas tissue concentration and RPLI and mFPL (Figure 2.2 A), a relationship that has been established in other well documented female gastropods affected by imposex (Oehlmann et al., 1992; Sole et al., 1998; Smith et al., 2006);

(2) the periwinkle, Littorina littorea, violates the same criterion of limited dispersal, but it is the model species used in intersex studies in Europe (Matthiessen et al.,

1995; Fioroni et al., 1991) and Canada (Covert, 1997); and (3) L. littorea has been 255 recommended by the Oslo/Paris Convention (OSPAR) as one of their model species for TBT pollution studies (Davies et al., 1997). In order to compare the potential of

S. gigas as a model bioindicator to that of N. lapillus, which is quite arguably the best documented gastropod used in TBT imposex studies (Chapter 5), a linear regression between TBT levels and mFPL and RPLI in S. gigas and mFPL and in N. lapillus, were compared. S. gigas had better relationships than N. lapillus between mFPL and RPSI/RPLI (Figure 6.1), and it is evident that S. gigas is indeed a suitable bioindicator of TBT pollution.

In order to address objectives 3 through 6,1 collected sediment, adult N. lapillus and resident M. edulis from reference sites and three harbour sites (Saint John

Harbour, Sydney Harbour and Halifax Harbour) in the Maritime Provinces of New

Brunswick and Nova Scotia. Sediment and M. edulis were collected at suspected point sources on a gradient toward the open sea where vessels > 25 m in length would have still been allowed to use TBT antifouling paint under the 1989 Canadian regulations. Suspected point sources included commercial harbours, shipyards, ferry terminals, and moorings. Sediment and M. edulis samples were analyzed for the presence of butyltins by GC-MS. TBT and its metabolites, DBT and MBT, were detected in sediment at all harbour and "reference" sites, but not all locations within sites (Table 4.1). The presence of butyltins at reference sites was similarly reported in marinas in the UK (Waite et al., 1991) and fishing harbours in Portugal (Coelho et al., 2002) even though these countries had also passed legislation regulating TBT.

The distribution of butyltins in M. edulis and N. lapillus followed similar patterns, with higher concentrations and more frequent detections from harbour sites, 256 and concentrations that were low or below detection limits from reference sites. On the other hand, butyltins were only detected in N. lapillus from harbour sites (Table

3.3). The occurrence of butyltins in M. edulis and N. lapillus at areas associated with boating activity verified that TBT was still bioavailable from suspected sources even though Canadian regulations on TBT were in effect since 1989 (Agriculture

Canada, 1989). In addition, imposex was only observed in N. lapillus from harbour sites, and the severity of imposex correlated with TBT levels in N. lapillus on a spatial scale (Figure 3.4 A and B; linear regression: R2 = 0.67, 0.75 and 0.82 for

RPSI, mFPL and VDSI, respectively). Based on the results of this study, data suggest that the 1989 Canadian regulations have not been completely effective in reducing TBT's bioavailability to non-target organisms in New Brunswick and Nova

Scotia. Conversely, the 1989 Canadian regulations have been effective at decreasing the severity of imposex in N. lapillus at most sites when the data from this study are compared to previous studies (Prouse and Ellis, 1997; Covert, 1997; Delaney, 2001;

Coray, 2007), and at decreasing sediment concentrations in Saint John Harbour and

Halifax Harbour.

The study locations used in this thesis differed in climate, habitat type, sentinel species, mode of feeding, and TBT regulatory status. The Maritime

Provinces of Canada experiences a temperate climate (45° N) and Canada passed regulations limiting TBT's use in 1989 (Agriculture Canada, 1989). The two species investigated in Canada were Nucella lapillus, a carnivore collected from rocky intertidal areas, and Mytilus edulis, a filter feeder sampled from pilings and docks.

In contrast, the BVI experiences a tropical climate (18° N) and there is no legislation 257 regulating TBT's use. As a matter of fact, antifouling paint containing TBT was still available for resale in many of the marine chandleries in the summer of 2009

(personal observation). S. gigas is a herbivore collected from submerged turtle grass

(Thalassia testidium) beds, and was the sole mollusc species collected from the BVI.

Although the two countries and three species used in this study differed in many aspects, all molluscs were effective biomonitors of TBT pollution. The presence of butyltins in all mollusc tissues, sediment and "patchy" distribution of T. testidium and marine algae from commercial and fishing harbours, shipyards, marinas, and moorings (Figure 6.2) support the hypothesis that TBT was released into the marine environment via its application as a biocide in antifouling paint.

A review of the available literature on the masculinization of female gastropods from 1962 to 2009 was completed following more than three years of effort. I feel that this review is unique from previously published reviews on the topic because it outlines how the literature and science of the relationship between organotins and female gastropods has evolved over time. Some of the key findings of the review article include:

(1) masculinised female gastropods were first observed in the early 1960s and

not the 1970s as is cited in the literature;

(2) organotins can cause three different types of female masculinisation (FM) in

gastropods, imposex, intersex and ovo-testis; 258

(3) globally, there are currently > 260 species of gastropods affected by FM

(Table 5.2);

(4) currently, there are > 40 species unaffected by FM (Table 5.3) even though

some have TBT in their tissues, or were collected sympatrically with other

species displaying signs of FM;

(5) currently there are five hypotheses that have been proposed to explain the

induction of FM in gastropods, but no consensus between scientists, and this

topic is hotly debated;

(6) even though FM has been documented in > 260 species, induction by TBT or

TPT has been confirmed in only 20 species (Table 5.6);

(7) there are other compounds besides TBT and TPT that have induced FM in

gastropods (Table 5.7);

(8) analysis of museum samples collected prior to 1960 suggests that imposex

(pseudo-imposex) occurred in some species before TBT's application as a

biocide in antifouling paint; 259

(9) FM induction under field conditions is global in occurrence and the majority

of studies attribute this to TBT although the majority of studies have not

performed chemical analyses;

(10) the bulk of biomonitoring studies that used the FM response in gastropods to

monitor TBT pollution have been conducted in Europe > Asia and Oceania >

North America > Latin America;

(11) additional field studies are recommended for countries including Africa,

India and Latin America;

(12) induction studies are suggested for some Littorinid species (e.g. Littorina

marie) that have an ovipositor that is morphologically and histologically

identical with the remaining base of the penis after the shedding of the male

copulatory organ with L. littorea as a positive control;

(13) there are currently 21 indices used to measure the incidence and severity of

imposex and intersex in gastropods, although some indices are better suited

for certain species and families than others (Table 5.9);

(14) for the freshwater gastropod, Marisa cornuarietis, "natural imposex" has

been described (Schulte-Oehlmann et al., 1994); 260

(15)Dumpton syndrome (DS) is a genetic disorder which can be passed from one

generation to the next, and it has been proposed as a mechanism that lessens

the sterilising effects of TBT in imposex-affected populations (Quintela et

al., 2002; Gibbs, 2005); and

(16)FM studies reached their peak in 2006, and most of the available literature

consists of articles published predominantly in 11 journals (Figure 5.2).

6.2 Way Forward

Even though the results of this study suggest that the 1989 Canadian regulations on organotins have not been effective in preventing TBT's bioavailability to non-target organisms (N. lapillus and M. edulis), data suggest that

TBT levels in sediments in Saint John Harbour and Halifax Harbour have decreased since the 1993/1994 survey by Chau et al. (1997a, b), while increasing during the same time period in Sydney Harbour. I am not aware of any other recently published data on environmental levels in marine sediment or Mytilus spp. on the

Atlantic or Pacific coast, but it would be interesting to compare the results of this study with more recent data from a greater number of sites on both coasts. I feel that such a study would offer a better understanding of current TBT levels in Canada across a greater spatial scale, and this would provide more evidence to conclude if 261 the 1989 Canadian regulations have been effective or ineffective. In addition to collecting sediment and Mytilus spp. from suspected harbour and reference sites,

Nucella spp. and Littorina littorea should also be collected wherever possible to investigate the incidence and severity of imposex and intersex, respectively, at sites that have existing historical data for those two species.

As it relates to the use of gastropods to monitor TBT pollution in the BVI and in the wider Caribbean, I could only find two studies, excluding this thesis, that used gastropods as biomonitors or bioindicators of TBT pollution. The first study reported imposex in S. gigas in the BVI (Phillip, 2000), and the second study reported imposex in the widemouth rocksnail (.Purpura patula), the rock snail (Thais deltoidea), and the rustic rock snail (T. rustica) in the neighbouring United States

Virgin Islands (USVI) (Strand et al., 2009). These two studies are in countries situated in the northern section of the Caribbean and in very close proximity to each other. Linton and Warner (2003) have suggested the use of biomarkers as tools for sustainable management, to give early warning signs of Caribbean ecosystems under stress and that are threatened by humans. In this regard, I would recommend additional field studies throughout the Caribbean using some of the species previously noted for having imposex, e.g. S. gigas, P. patula and Thais spp. to verify if other islands in the Caribbean are also affected by TBT pollution.

The review article highlighted several areas that still need to be addressed as it relates to FM in gastropods. Firstly, there are seven Littorinid species {Littorina marie, L. neritoides, L. nigrolineata, L. obtusata, L. saxatalis, L. saxatilis rudis and

L. ziczac) that have an ovipositor (Fioroni et al., 1991) that is morphologically and 262 histologically identical with the remaining base of the penis after the shedding of the male copulatory organ (Section 5.4.2). I have recommended laboratory exposure experiments using those Littorinid species with an ovipositor along with L. littorea as a positive control at TBT levels known to induce intersex in L. littorea. Secondly, additional studies are also recommended for those families (n = 29) used in FM studies where < 5 species were used where either all were affected e.g. Haliotidae, all were unaffected e.g. Trochidae, or where there was a mixture of affected and unaffected e.g. Strombidae (see Section 5.4.3) to increase scientific knowledge of these families. Thirdly, the incidence of FM is global in occurrence, but there are some countries where data on species affected by FM are lacking. As a result, I have recommended additional field studies from "hot spot" areas in Africa, the

Middle East, and Latin America, even though Latin America appears to be on the rise for studies using imposex in gastropods to monitor TBT pollution. Finally, one intriguing topic that continues to baffle scientists studying the FM response in gastropods is the topic of induction. To date, there are five hypotheses that have been put forth to explain the mechanisms behind FM, but no consensus currently exists among the prevailing schools of thought. In my opinion, the topic of induction is a complex one and is further complicated by three factors:

(1) data from museum samples of some gastropods e.g. H trunculus

(Garaventa et al., 2006b), Cymbiola noblis, C. vespertilio and Melo

amphora (Swennen and Horpert, 2008) verify that there were female

gastropods with signs of FM or pseudo-imposex prior to 1960s when 263

TBT replaced Cu-based antifouling paints (Evans and Smith, 1975;

Omae, 2003);

(2) "natural imposex" has been reported in the giant ram's horn, Marisa

cornuarietis (Schulte-Oehlmann, et al., 1994); and

(3) there are other compounds that can occur in the environment that have

reportedly caused imposex in some species e.g. PAHs and PCBs

(Garaventa et al., 2006a; Maran et al., 2006).

One area of science that may have the key to unlocking the mystery of FM induction in gastropods is genomics. The appearance of a penis or vas deferens in masculinised female gastropods is a phenotypic expression of the organism's genes.

Is it possible that TBT, other pollutants, and/or environmental stress are affecting masculinised female gastropods on a molecular and/or biochemical level long before the observable response of a penis or vas deferens can be seen? Future studies exploring this area may prove fruitful to finally answering the great induction debate.

Man has left his footprint on the Earth forever. Our apparent disregard for the environment, will continue to have unequivocal consequences for generations to come.

C. P. Titley-O'Neal (2010) 264

6.3 References

Agriculture Canada. 1989. Antifouling paints for ship hulls. Canadian Association of Pesticide Control Officials Note 89-02, Ottawa

Alzieu, C. 1986. The detrimental effects on oyster culture in France - evolution since antifouling paint regulation. Proceedings of the Oceans '86 - Organotin Symposium Proceedings. IEEE: Washington, D. C. USA., pp. 1130- 1134.

Anderson, W. L., and Duzan, R. E. 1978. DDE residues and eggshell thinning in Loggerhead shrikes. Wilson Bull. 90 (2): 215 - 220.

Bird, D. M., Peakall, D. B., and Miller, D. S. 1983. Enzymatic changes in the oviduct associated with DDE-induced eggshell thinning in the kestrel Falco sparverius. Bull Environ Contam Toxicol. 31: 22 - 24.

Carson, R. 1962. Silent Spring. The Riverside Press - Cambridge. Houghton Mifflin Company, Boston.

Chau, Y. K., Maguire, R. J., Brown, M., Yang, F., Batchelor, S. P., and Thompson, J. A. J. 1997a. Occurrence of butyltin compounds in mussels in Canada. Appl Organomet Chem. 11: 903- 912.

Chau, Y. K., Maguire, R. J., Brown, M., Yang, F., and Batchelor, S. P. 1997b. Occurrence of organotin compounds in the Canadian aquatic environment five years after the regulation of antifouling uses of tributyltin. Water Qual Res J Can. 32(3): 453 -521.

Coelho, M. R., Bebianno, M. J., and Langston, W. J. 2002. Organotin levels in the Ria Formosa lagoon, Portugal. Appl Organomet Chem. 16: 384 - 390.

Colborn, T., vomSaal, F. S., and Soto, A. M. 1993. Developmental effects of endocrine disrupting chemicals in wildlife and humans. Environ Health Persp. 101 (5): 378 - 384.

Coray, C. 2007. Assessing the health of Halifax Harbour's intertidal ecosystem: Impacts of marine pollution on community structure, reproductive health of key predator populations (Nucella lapillus) and immunomodulation in blue mussels (Mytilus edulis & Mytilus trossulus). MSc thesis, Dalhousie University, Nova Scotia, Canada. 265

Covert, T. J. 1997. Masculinization of Dogwhelks (Nucella lapillus) and periwinkles (Littorina littorea) in Halifax Harbour: Monitoring of Tributyltin. BSc honours thesis, Saint Mary's University, Nova Scotia, Canada.

Davies, I. M., Minchin, A., and Harding, A. 1997. OSPAR working group on concentrations, trends and effects of substances in the marine environment (SIME) - Report on the TBT training workshop (September 24 - 26, 1997). Aberdeen Marine Laboratory Report, No. 9/07, 36 pp.

Delaney, J. L. 2001. The occurrence of imposex in Nucella lapillus within the Saint John Harbour. BSc honours thesis, University of New Brunswick (Saint John), New Brunswick, Canada.

Evans, C. J.,and Smith, P. J. 1975. Organo-based antifouling systems. J Oil Col Chem Assoc. 58: 160 - 168.

Feral, C., and Le Gall, S. 1982. Physiologie des invertebres - Induction experimentale par un pollutant marin (le tributyletain), de l'activite neuroendocrine controlant la morphogenese du penis chez les femelles d'Ocenebra erinacea (Mollusque, Prosobranche gonochorique). C R Hebd Stances Acad Sci. D 295: 627 - 630.

Fioroni, P., Oehlmann, J., and Stroben, E. 1991. The pseudohermaphroditism of prosobranchs: Morphological aspects. Zool Anz. 226 (1/2): 1 - 26.

Garaventa, F., Faimali, M., Terlizzi, A., Fiorini, S., and Gallo, G. 2006a. Imposex induction in Hexaplex trunculus by tributyltin and other stressors. Mar Environ Res, 62: S230-S230.

Garaventa, F., Faimali, M., and Terlizzi, A. 2006b. Imposex in pre-pollution times. Is TBT to blame? Mar Pollut Bull. 52: 701-702.

Gibbs, P. E. 2005. Male genital defect (Dumpton Syndrome) in the dog-whelk Nucella lapillus (Gastropoda). In: Proceedings of Oceans '87: The Ocean - An International Workplace, 28 September - 1 October, 1987, Halifax, Nova Scotia. Piscataway, New Jersey: IEEE Service Center, p. 1482 - 1487.

Gibbs, P. E, and Bryan, G. W. 1987. TBT paints and the demise of the dog-whelk, Nucella lapillus (Gastropoda). In: Proceedings of Oceans '87: The Ocean - An International Workplace, 28 Sept - 1 October, 1987, Halifax, Nova Scotia Piscataway, New Jersey: IEEE Service Center, p. 1482 - 1487.

Linton, D. M., and Warner, G. F., 2003. Biological indicators in the Caribbean coastal zone and their role in integrated coastal management. Ocean and Coast Manage. 46: 261 - 276. 266

Maguire, R. J. 1987. Environmental aspects of tributyltin. Appl Organomet Chem. 1: 475 - 498.

Maran, C., Centanni, E., Pellizzato, F., and Pavoni, B. 2006. Organochlorine compounds (polychlorinated biphenyls and pesticides) and polycyclic aromatic hydrocarbons in populations of Hexaplex trunculus affected by imposex in the Lagoon of Venice, Italy. Environ Toxicol Chem, 25: 486 - 495.

Matthiessen, P., Waldock, R., Thain, J. E., Waite, M. E., and Scrope-Howe, S. 1995. Changes in periwinkle (Littorina littorea) populations following the ban on the TBT-based antifouling on small boats in the United Kingdom. Ecotox Environ Safe. 30: 180 - 194.

Michel, P. and Averty, B. 1991. Tributyltin analysis in seawater by GC FPD after direct aqueous-phase ethylation using sodium tetraethlyborate. Appl Organomet Chem. 5: 393 - 397.

Oehlmann, J., Stroben, E., and Fioroni, P. 1992. The rough tingle Ocenebra erinacea (Neogastropoda: Muricidae): an exhibitor of imposex in comparison to Nucella lapillus. Helgoland Mar Res. 46: 311 - 328.

Omae, I. 2003. Organotin antifouling paints and their alternatives. Appl Organomet Chem. 17: 81 - 105.

Phillip, O. S. 2000. The relationship between masculinized female conch (Strombus gigas) and tributyltin-based antifouling paint in coastal waters of the British Virgin Islands. MSc. Thesis, University of Plymouth, United Kingdom.

Prouse, N. J., and Ellis, D.V. 1997. A baseline survey of dogwhelk (Nucella lapillus) imposex in Eastern Canada (1995) and interpretation in terms of tributyltin (TBT) contamination. Environ Technol. 18: 1255 - 1264.

Quintela, M., Barreiro, R., and Ruiz, J. M. 2002. Dumpton Syndrome reduces the tributyltin (TBT) sterilising effect on Nucella lapillus (L.) by limiting the development of the imposed vas deferens. Mar Environ Res. 54: 657 - 660.

Schulte-Oehlmann, U., Fioroni, P., Oehlmann, J., and Stroben, E. 1994. The genital system of Marisa cornuarietis (Gastropoda, Ampullariidae) - A morphological and histological analysis. Zool Beitr. 36: 59 - 81.

Smith, A. J., Thain, J. E., and Bary, J. 2006. Exploring the use of caged Nucella lapillus to monitor changes to TBT hotspot areas: A trial in the River Tyne Estuary (UK). Mar Environ Res. 62: 149 - 163. 267

Smith, B. S. 1981. Male characteristics on female mud snails caused by antifouling bottom paints. J Appl Tox. 1 (1): 22 - 25.

Sole, M., Morcillo, Y., and Porte, C. 1998. Imposex in the commercial snail Bolinus brandaris in the northwestern Mediterranean. Environ Pollut. 99: 241 -246.

Strand, J., torgensen, A., Tairova, Z., 2009. TBT pollution in molluscs at US Virgin Islands, Caribbean Sea. Environ Int. 35: 707 -711.

Swennen, C., and Horpet, P. 2008. Pseudo-imposex; male features in female volutes not TBT-induced (Gastropoda: Volutidae). Contrib Zool, 77: 17-24.

Vos, J. G., Dybing, E., Greim, H. A., Ladefoged, O., Lambre, C., Tarazona, J. V., Brandt, I., and Vethaak, A. D. 2000. Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit Rev Toxicol. 30: 71-133.

Waite, M. E., Waldock, M. J., Thain, J. E.,Smith, D. J., and Milton, S. M. 1991. Reductions in TBT concentrations in UK estuaries following legislation in 1986 and 1987. Mar Environ Res. 32: 89 - 11.

Ward, B., and Dubos, R. J. 1972. Only one earth: the care and maintenance of a small planet. W. W. Norton and Company, Inc., New York. 268

2.00

LOO

t 0.50

Oil LOO 3,16 U.0 3L6 100.00 11&2 M II Hi III )U it 11 1 Logu [TBT] In Nucellolaplllus as ng Sn g (dry wt) tagto[TBT] In Strombus gigosas ng Sn g 1(dry wt.)

(B) mo

3.50

e 3.00

1 2.50

2.00

LOO

0.00 uo 0.32 LOO 3.16 UJ) 3L« 100.0 316.3 0.H LOO 3.11 1M iLi

in,[TBT] in Nucellolopi/lusss ngSng l(dry wt.) Logu [TBT] in Strombus glgas as ng Sn g1(dry wt.)

6.1. Comparison of mean female penis length (mFPL) (A and C), relative penis size index (B), and relative penis length index (D) in Nucella lapillus and Strombus gigas collected from Canada and the British Virgin Islands 2006 to 2008. Error bars ± penis length of each female pooled and used for butyltin analysis. 269

Canada (Temperate) British Virgin Islands (Tropical)

Chemical Degradation (Hydrolysis, Photolysis, Oxidation,

z /\ /\ TBTIeachatesfrom TBTIeachatesfrom antifouling paint antifouling paint Docks & Pilings Nucella lapillus TBT DBT 4 MBT Strombus Desorption gigas

TBT --> DBT 4 MBT Seagrass Mytilus Rocky edulis Bed Intertidal T8T — > 6BTAM8T

Sediment Sand

6.2. Fate of TBT in Canada and the British Virgin Islands showing the three biomonitors used {Nucella lapillus, Mytilus edulis and Strombus gigas) and the habitats from which they were collected. 270

7 Appendix

Abidli, S., Lahbib, Y., and El Menif, N. T. 2009a. Imposex and genital tract malformations in Hexaplex trunculus and Bolinus brandaris collected in the Gulf of Tunis. B Mar Sci. 85: 11 - 25.

Abidli, S., Lahbib, Y., and El Menif, N. T. 2009b. Effects of TBT on the imposex development, reproduction and mortality in Hexaplex trunculus (Gastropoda: Muricidae). J Mar Biol Assoc UK. 89: 139 - 146.

Alvarez, M. M. S., and Ellis, D. V. 1990. Widespread neogastropod imposex in the northeast Pacific - Implications for TBT contamination surveys. Mar Pollut Bull. 21: 244-247.

Alzieu, C. 2000. Impact of tributyltin on marine invertebrates. Ecotoxicol. 9: 71 - 76.

An, W., and Hu, J. Y. 2006. Effects of endocrine disrupting chemicals on China's rivers and coastal waters. Front Ecol Environ. 4: 378 - 386.

Andersen, L. E. 2004a. Imposex: A biological effect of TBT contamination in Port Curtis, Queensland. Aust J Ecotoxicol. 10: 105 -113.

Andersen, L. 2004b. Imposex in the City - A survey to monitor the effects of TBT contamination in Port Curtis, Queensland. Cooperative Research Centre for Coastal Zone Estuary and Waterway Management, pp. 25 pp.

Arconada, B., and Ramos, M. A. 2002. Spathogyna, a new genus for Valvata (? Tropidina) fezi Altimira, 1960 from eastern Spain: Another case of pseudohermaphroditism in the Hydrobiidae (Gastropoda). J Mollus Stud. 68: 319-327.

Axiak, V., Vella, A. J., Micallef, D., Chircop, P., and Mintoff, B. 1995. Imposex in Hexaplex trunculus (Gastropoda, Muricidae) - First results from biomonitoring of tributyltin contamination in the Mediterranean. Mar Biol. 121:685 -691.

Axiak, V., Vella, A. J., Agius, D., Bonnici, P., Cassar, G., Cassone, R., Chircop, P., Micallef, D., Mintoff, B., and Sammut, M. 2000. Evaluation of environmental levels and biological impact of TBT in Malta (central Mediterranean). Sci Total Environ. 258: 89 - 97.

Axiak, V., Micallef, D., Muscat, J., Vella, A., and Mintoff, B. 2003. Imposex as a biomonitoring tool for marine pollution by tributyltin: some further observations. Environ Int. 28: 743 - 749. 271

Bailey, S. K., and Davies, I. M. 1987. Tributyltin contamination in the Firth of Forth. P Roy Soc Edinb B. 93: 561 - 562.

Bailey, S. K., and Davies, I. M. 1988a. Tributyltin contamination around an oil terminal in Sullom Voe (Shetland). Environ Pollut. 55: 161 - 172.

Bailey, S. K., and Davies, I. M. 1988b. Tributyltin contamination in the Firth-of- Forth (1975-87). Sci Total Environ. 76: 185 - 192.

Bailey, S. K., and Davies, I. M. 1989. The Effects of tributyltin on dogwhelks {Nucella lapillus) from Scottish coastal waters. J Mar Biol Assoc UK. 69: 335 -354.

Bailey, S. K., and Davies, I. M. 1991. Continuing impact of TBT, previously used in mariculture, on dogwhelk {Nucella lapillus L.) populations in a Scottish sea loch. Mar Environ Res. 32: 187 - 199.

Bailey, S. K., Davies, I. M., and Harding, M. J. C. 1995. Tributyltin contamination and its impact on Nucella lapillus populations. P Roy Soc Edinb B. 103: 113 -126.

Bard, S., and Coray, C. 2007. Impacts of Halifax Harbour marine pollution on immunomodulation in blue mussels (Mytilus edulis and Mytilus trossolus), endocrine disruption of key predator populations {Nucella lapillus), and resultant effect on intertidal community structure. In Proceedings of the 34th Annual Toxicity Workshop (September 30th to October 3rd, 2007), Halifax, Nova Scotia, pp 30.

Barreiro, R., Quintela, M., and Ruiz, J. M. 1999. Aphally and imposex in Nucella lapillus from Galicia (NW Spain): incidence, geographical distribution and consequences for the biomonitoring of TBT contamination. Mar Ecol Prog Ser. 185: 229 - 238.

Barreiro, R., Gonzalez, R., Quintela, M., and Ruiz, J. M. 2001. Imposex, organotin bioaccumulation and sterility of female Nassarius reticulatus in polluted areas of NW Spain. Mar Ecol Prog Ser. 218: 203 - 212.

Barreiro, R., Quintela, M., and Ruiz, J. M. 2004. TBT e imposex en Galiciai los sfectos de un diruptor endocrino en problaciones de gasteropodos marinos. Ecosistemas. 13: 13 - 29.

Barroso, C. M., and Moreira, M. H. 1998. Reproductive cycle of Nassarius reticulatus in the Ria de Aveiro, Portugal: Implications for imposex studies. J Mar Biol Assoc UK. 78: 1233 - 1246. 272

Barroso, C. M., and Moreira, M. H. 2002. Spatial and temporal changes of TBT pollution along the Portuguese coast: inefficacy of the EEC directive 89/677. Mar Pollut Bull. 44: 480 - 486.

Barroso, C. M., Moreira, M. H., and Gibbs, P. E. 2000. Comparison of imposex and intersex development in four prosobranch species for TBT monitoring of a southern European estuarine system (Ria de Aveiro, NW Portugal). Mar Ecol Prog Ser. 201:221 -232.

Barroso, C. M., Reis-Henriques, M. A., Ferreira, M. S., and Moreira, M. H. 2002a. The effectiveness of some compounds derived from antifouling paints in promoting imposex in Nassarius reticulatus. J Mar Biol Assoc UK. 82: 249 - 255.

Barroso, C. M., Moreira, M. H., and Bebianno, M. J. 2002b. Imposex, female sterility and organotin contamination of the prosobranch Nassarius reticulatus from the Portuguese coast. Mar Ecol Prog Ser. 230: 127 - 135.

Barroso, C. M., Reis-Henriques, M. A., Ferreira, M., Gibbs, P. E., and Moreira, M. H. 2005a. Organotin contamination, imposex and androgen/oestrogen ratios in natural populations of Nassarius reticulatus along a ship density gradient. Appl Organomet Chem. 19: 1141 -1148.

Barroso, C. M., Moreira, M. H., and Richardson, C. A. 2005b. Age and growth of Nassarius reticulatus in the Ria de Aveiro, north-west Portugal. J Mar Biol Assoc UK. 85: 151 - 156.

Bauer, B., Fioroni, P., Ide, I., Liebe, S., Oehlmann, J., Stroben, E., and Watermann, B. 1995. TBT effects on the female genital system of Littorina littorea - a possible indicator of tributyltin pollution. Hydrobiol. 309: 15 -27.

Bauer, B., Fioroni, P., Schulte-Oehlmann, U., Oehlmann, J., and Kalbfus, W. 1997. The use of Littorina littorea for tributyltin (TBT) effect monitoring results from the German TBT survey 1994/1995 and laboratory experiments. Environ Pollut. 96: 299 - 309.

Bech, M. 1999. Increasing levels of tributyltin-induced imposex in Muricid gastropods at Phuket Island, Thailand. Appl Organomet Chem. 13: 799 - 804.

Bech, M. 2002a. Imposex and tributyltin contamination as a consequence of the establishment of a marina, and increasing yachting activities at Phuket Island, Thailand. Environ Pollut. 117: 421 - 429. 273

Bech, M. 2002b. Imposex and tributyltin contamination as a consequence of the establishment of a marina, and increasing yachting activities at Phuket Island, Thailand. J Plan Lit. 17: 262 - 331.

Bech, M. 2002c. A survey of imposex in Muricids from 1996 to 2000 and identification of optimal indicators of tributyltin contamination along the east coast of Phuket Island, Thailand. Mar Pollut Bull. 44: 887 - 896.

Bech, M., Strand, J., and Jacobsen, J. A. 2002. Development of imposex and accumulation of butyltin in the tropical Muricid Thais distinguenda transplanted to a TBT contaminated site. Environ Pollut. 119: 253 - 260.

Bettin, C., Oehlmann, J., and Stroben, E. 1996. TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level. Helgolander Meeresun. 50: 299 - 3 17.

Bigatti, G., and Penchaszadeh, P. 2005. Imposex in Odontocymbiola magellancia (Caenogastropoda: Volutidae) in Patagonia. Communicaciones de la Sociedad Malacologia del Uruguay. 9:371-375.

Bigatti, G., and Carranza, A. 2007. Phenotypic variability associated with the occurrence of imposex in Odontocymbiola magellanica from Golfo Nuevo, Patagonia. J Mar Biol Assoc UK. 87: 755 - 759.

Bigatti, G., Primost, M. A., Cledon, M., Averbuj, A., Theobald, N., Gerwinski, W., Arntz, W., Morriconi, E., and Penchaszadeh, P. E. 2009. Biomonitoring of TBT contamination and imposex incidence along 4700 km of Argentinean shoreline (SW Atlantic: From 38S to 54S). Mar Pollut Bull. 58: 695 - 701.

Birchenough, A. C., Barnes, N., Evans, S. M., Hinz, H., Kronke, I., and Moss, C. 2002a. A review and assessment of tributyltin contamination in the North Sea, based on surveys of butyltin tissue burdens and imposex/intersex in four species of neogastropods. Mar Pollut Bull. 44: 534 - 543.

Birchenough, A. C., Evans, S. M., Moss, C., and Welch, R. 2002b. Re-colonisation and recovery of populations of dogwhelks Nucella lapillus (L.) on shores formerly subject to severe TBT contamination. Mar Pollut Bull. 44: 652 - 659.

Blaber, S. J. M. 1970. The occurrence of a penis-like outgrowth behind the right tentacle in spent females of Nucella lapillus (L.). Proc Malacol Soc Lond. 39: 231 -233.

Blackmore, G. 2000. Imposex in Thais clavigera (Neogastropoda) as an indicator of TBT (tributyltin) bioavailability in coastal waters of Hong Kong. J Mollus Stud. 66: 1 - 8. 274

Boutilier, K., Rawlings, T. A., and Hatcher, B. G. 2007. Marine gastropod biomonitors: Investigating TBT contamination of Sydney Harbour and surrounding regions. In Atlantic Canada Coastal and Estuarine Science Society. ACCESS 2007, Cape Breton University.

Braga, A. R. C., de Castro, I. B., and Rocha-Barreira, C. A. 2006. Compostos organoestanicos: Um risco potencial para contamina9ao do pescado marinho. Boletim Tecnico - Cientifico do Cepene. 14: 103 - 107.

Bray, S. 2005. The long-term recovery of the bioindicator species Nucella lapillus from tributyltin pollution. PhD. Thesis, University of Southampton, United Kingdom.

Brevik, E. M., F0lsvik, N., Berge, J. A., and Walday, M. 1998. Quantification and effects of organotin compounds in environmental samples from the Norwegian coast. In ACS Symp Ser. 215: 586

Brick, M., and Deutsch, U. 1993. Ultrastructural investigations of the penis epithelia cells of three neogastropods, collected from TBT (tributyltin)-polluted areas. Aquat Toxicol. 27: 113 - 132.

Brick, M., and Bolte, M. 1994. Cytology of the outer penis epithelium of Buccinum undatum (L.) from the Arctic region - An observation of the imposex phenomenon. Helgolander Meeresun. 48: 123 - 131.

Brick, M., Deutsch, U., and Fioroni, P. 1996. Cellular effects of tributyltin (TBT) on the penis epithelium cells of prosobranchs (Hinia reticulata and Ocenibria aciculata). Helgolander Meeresun. 50: 319 - 325.

Bright, D., Cullon, D., and Ikonomou, M. 2001. Evidence of endocrine disruption in neogastropods due to TBT: 10 years after the 1989 regulation. In SETAC - 22nd Meeting (November 11 - 15, 2001), Baltimore, Maryland.

Bright, D. A., and Ellis, D. V. 1988. Imposex in Pacific coast neogastropods related to tributyltin contamination. J Shellfish Res. 8: 318 - 319.

Bright, D. A., and Ellis, D. V. 1990. A comparative survey of imposex in northeast Pacific neogastropods (Prosobranchia) related to tributyltin contamination, and choice of a suitable bioindicator. Can J Zool. 68: 1915 - 1924.

Bryan, G., Gibbs, P., Hummerstone, L., and Burt, G. 1986. The decline of the gastropod Nucella lapillus around south-west England: evidence for the effect of tributyltin from antifouling paints. J Mar Biol Assoc UK. 66: 611 - 640. 275

Bryan, G., Gibbs, P., Burt, G., and Hummerstone, L. 1987. The effects of tributyltin (TBT) accumulation on adult dogwhelks, Nucella lapillus: long-term field and laboratory experiments. J Mar Biol Assoc UK. 67: 525 - 544.

Bryan, G., Gibbs, P., and Burt, G. 1988. A comparison of the effectiveness of tri-n- butyltin chloride and five other organotin compounds in promoting the development of imposex in the dog-whelk, Nucella lapillus. J Mar Biol Assoc UK. 68: 733 - 744.

Bryan, G. W., and Gibbs, P. E. 1991. Impact of low concentrations of tributyltin (TBT) on marine organisms: A Review. In Metal Ecotoxicology: Concepts and Applications (Edited by M.C. Newman and A.W. Mcintosh). Lewis Publishers, Ann Arbour, pp. 323-361.

Bryan, G. W., Gibbs, P. E., Hummerstone, L. G., and Burt, G. R. 1987. Copper, zinc, and organotin as long-term factors governing the distribution of organisms in the Fal Estuary in southwest England. Estuaries. 10: 208 - 219.

Bryan, G.W,, Gibbs, P. E., Hummerstone, L. G., and Burt, G. R. 1989a. Uptake and transformation of C-14-labelled tributyltin chloride by the dog-whelk, Nucella lapillus - Importance of absorption from the diet. Mar Environ Res. 28: 241 - 245.

Bryan, G. W., Gibbs, P. E., Huggett, R. J., Curtis, L. A., Bailey, D. S., and Dauer, D. M. 1989b. Effects of tributyltin pollution on the mud snail, Hyanassa obsoleta, from the York River and Sarah Creek, Chesapeake Bay. Mar Pollut Bull. 20: 458 - 462.

Bryan, G. W., Bright, D. A., Hummerstone, L. G., and Burt, G. R. 1993a. Uptake, tissue distribution and metabolism of C-14 labelled tributyltin (TBT) in the dogwhelk, Nucella lapillus. J Mar Biol Assoc UK. 73: 889 - 912.

Bryan, G. W., Burt, G. R., Gibbs, P. E., and Pascoe, P. L. 1993b. Nassarius reticulatus (Nassariidae, Gastropoda) as an indicator of tributyltin pollution before and after TBT restrictions. J Mar Biol Assoc UK. 73: 913 - 929.

Cadee, G. C., Boon, J. P., Fischer, C. V., Mensink, B. P., and TenHallers-Tjabbes, C. C. 1995. Why the whelk (Buccinum undatum) has become extinct in the Dutch Wadden Sea. Neth J Soc Sci. 34: 337 - 339.

Caetano, C. H. S., and Absalao, R. S. 2002. Imposex in Olivancillaria vesica vesica (Gmelin) (Gastropoda, Olividae) from a southeastern Brazilian sandy beach. Rev Bras Zool. 19: 215 - 218. 276

Cajaraville, M. P., Bebianno, M. J., Blasco, J., Porte, C., Sarasquete, C., and Viarengo, A. 2000. The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: A practical approach. Sci Total Environ. 247: 295 - 311.

Camillo, E., Quadros, J., de Castro, I. B., and Fernandez, M. A. 2004. Imposex in Thais rustica (Mollusca: Neogastropoda) (Lamark, 1822) as an indicator of organotin compounds pollution at Maceio Coast (northeastern Brazil). Braz J Oceanogr. 52: 101 - 105.

Cao, D. D., Jiang, G. B., Zhou, Q. F., and Yang, R. Q. 2009. Organotin pollution in China: An overview of the current state and potential health risk. J Environ Manage. 90: S16 - S24.

Cardoso, R. S., Caetano, C. H. S., and Cabrini, T. M. B. 2009. Biphallia in imposexed females of marine gastropods: new record for Nassarius vibex from Brazil. Braz J Biol. 69: 223 - 224.

Cardwell, R. D., and Tear, L. 1998. Uncertainties in tributyltin-imposex dose- response relationships. Abstr Am Chem Soc. 215: U587 - U587.

Casey, J. D., De Grave, S., and Burnell, G. M. 1998. Intersex and Littorina littorea in Cork Harbour: Results of a medium-term monitoring programme. Hydrobiol. 378: 193 - 197.

Castagna, M., and Kraeuter, J. N. 1994. Age, growth-rate, sexual dimorphism and fecundity of knobbed whelk Busycon carica (Gmelin, 1791) in a western mid-Atlantic lagoon system, Virginia. J Shellfish Res. 13: 581 - 585.

Castro, L. F. C., Lima, D., Machado, A., Melo, C., Hiromori, Y., Nishikawa, J., Nakanishi, T., Reis-Henriques, M.A., and Santos, M.M. 2007. Imposex induction is mediated through the Retinoid X Receptor signaling pathway in the neogastropod Nucella lapillus. Aquat Toxicol. 85: 57 - 66.

Chan, K. M., Cheung, S. G., Cai, Z. W., and Qiu, J. W. 2008a. Sensitivity of different biological responses to accumulation and depuration of butyltins in the neogastropod Thais clavigera-. implications for biomonitoring. Ecotoxicol. 17: 860 - 868.

Chan, K. M., Leung, K. M. Y., Cheung, K. C., Wong, M. H., and Qiu, J. W. 2008b. Seasonal changes in imposex and tissue burden of butyltin compounds in Thais clavigera populations along the coastal area of Mirs Bay, China. Mar Pollut Bull. 57: 645 -651. 277

Chatzinikolaou, E., and Richardson, C. A. 2007. Evaluating growth and age of netted whelk Nassarius reticulatus (Gastropoda: Nassariidae) using statolith growth rings. Mar Ecol Prog Ser. 342: 163 - 176.

Cheng, C. Y., and Liu, L. L. 2004. Gametogenesis in the imposex-affected oyster drills, Thais clavigera and Thais rufotincta. J Fish Soc Taiwan. 31: 55 - 65.

Chiavarini, S., Massanisso, P., Nicolai, P., Nobili, C., and Morabito, R. 2003. Butyltins concentration levels and imposex occurrence in snails from the Sicilian coasts (Italy). Chemosphere. 50: 311 - 319.

Cho, H. S., Seol, S. W., and Horiguchi, T. 2006. Less recovery from imposex and organotin pollution in the rock shell, Thais clavigera in Korea. Mar Environ Res. 62: S224 - S225.

Choi, M., Moon, H. B., Yu, J., Eom, J. Y., and Choi, H. G. 2009. Butyltin contamination in industrialized bays associated with intensive marine activities in Korea. Arch Environ Con Tox. 57: 77 - 85.

Cledon, M., Theobald, N., Gerwinski, W., and Penchaszadeh, P. E. 2006. Imposex and organotin compounds in marine gastropods and sediments from the Mar del Plata coast, Argentina. J Mar Biol Assoc UK. 86: 751 - 755.

Cob, Z. C., Arshad, A., Idris, M. H., Bujang, J. S., and Ghaffar, M. A. 2008. Sexual polymorphism in a population of Strombus canarium Linnaeus, 1758 (Mollusca: Gastropoda) at Merambong Shoal, Malaysia. Zool Stud. 47: 318 - 325.

Colson, I., and Hughes, R. N. 2004. Rapid recovery of genetic diversity of dogwhelk (Nucella lapillus L.) populations after local extinction and recolonization contradicts predictions from life-history characteristics. Mol Ecol. 13: 2223 - 2233.

Coray, C. 2007. Assessing the health of Halifax Harbour's intertidal ecosystem: Impacts of marine pollution on community structure, reproductive health of key predators populations {Nucella lapillus), and immunomodulation in blue mussels {Mytilus edulis and Mytilus trossulus). MSc. Thesis, Dalhousie University, Nova Scotia.

Coray, C., and Bard, C. 2007. Persistence of tributyltin-induced imposex in dogwhelks (Nucella lapillus) and intersex in periwinkles (Littorina littorea) in Atlantic Canada. Water Qual Res. 42: 111 - 122. 278

Coray, C., and Bard, S. M. 2008. Impacts of marine pollution on immunomodulation in blue mussels {Mytilus edulis and Mytilus trossulus), endocrine disruption of key predator populations {Nucella lapillus), and resultant effects on intertidal community structure. Mar Environ Res. 66: 166 - 167.

Couceiro, L., Quintela, M., Ruiz, J. M., and Barreiro, R. 2006. A new case of endocrine disruption: Imposex in the invasive gastropod Cyclope neritea L. Mar Environ Res. 62: S237 - S238.

Couceiro, L., Diaz, J., Albaina, N., Barreiro, R., Irabien, J. A., and Ruiz, J. M. 2009. Imposex and gender-independent butyltin accumulation in the gastropod Nassarius reticulatus from the Cantabrian coast (N Atlantic Spain). Chemosphere. 76: 424 - 427.

Coutinho, R. 2001. Consideration and adoption of the international convention on the control of harmful anti-fouling systems - Annex 1. In International Maritime Organization International Conference on the Control of Harmful Antifouling Systems for Ships AFS/CONF/INF.2.

Covert, T. J. 1997. Masculinization of dogwhelks (Nucella lapillus) and periwinkles (.Littorina littorea) in Halifax Harbour: Monitoring of tributyltin. BSc. Honours Thesis, St. Mary's University, Nova Scotia.

Crothers, J. H. 1989. Has the population decline due to TBT pollution affected shell- shape variation in the dogwhelk, Nucella lapillus (L). J Mollus Stud. 55: 461 -467.

Crothers, J. H. 2003. Further observations on a population of dog-whelks, Nucella lapillus (Gastropoda) recolonizing a site following amelioration of tributyltin (TBT) pollution. J Mar Biol Assoc UK. 83: 1023 - 1027.

Cullon, D. L. 2001. Evidence of endocrine disruption in neogastropods by organotins: 10 years after the 1989 regulations. MSc. Thesis, Royal Roads University, British Columbia.

Curtis, L. A. 1994. A decade-long perspective on a bioindicator of pollution - Imposex in Ilyanassa obseleta on Cape Henlopen, Delaware Bay. Mar Environ Res. 38: 291 - 302.

Curtis, L. A., and Barse, A. M. 1990. Sexual anomalies in the estuarion snail Ilyanassa obsoleta - Imposex in females and associated phenomena in males. Oecologia. 84:371 -375.

Curtis, L. A., and Kinley, J. L. 1998. Imposex in Ilyanassa obsoleta still common in a Delaware estuary. Mar Pollut Bull. 36: 97 - 101. 279 da Costa, M. B., Fernandez, M. A., Barbiero, D. C., de Melo, F. T. V., and Ferreira, B. S. 2008. First record of imposex in Thais deltoidea (Lamarck, 1822) (Mollusca, Gastropoda, Thaididae) in Vitoria, Es, Brazil. Braz J Oceanogr. 56: 145 - 148.

Davies, I. M. 2000. Kinetics of the development of imposex in transplanted adult dogwhelks, Nucella lapillus. Environ Pollut. 107: 445 - 449.

Davies, I. M., and Bailey, S. K. 1991. The impact of tributyltin from large vessels on dogwhelk (Nucella lapillus) populations around Scottish oil ports. Mar Environ Res. 32: 201 -211.

Davies, I. M., and Minchin, A. 2002. Quality assurance of imposex and intersex measurements in marine snails. J Environ Monitor. 4: 788 - 790.

Davies, I. M., Bailey, S. K., and Moore, D. C. 1987a. Tributyltin in Scottish sea lochs, as indicated by degree of imposex in the dogwhelk, Nucella lapillus (L). Mar Pollut Bull. 18: 400 - 404.

Davies, I. M., Drinkwater, J., McKie, J. C., and Balls, P. 1987b. Effects of the use of tributyltin antifoulants in mariculture. In Proceedings of the Oceans '87 - Organotin Symposium Proceedings. IEEE: New York, NY, USA, pp. 1477 - 1481

Davies, I. M., Harding, M. J. C., Bailey, S. K., Shanks, A. M., and Lange, R. 1997a. Sublethal effects of tributyltin oxide on the dogwhelk Nucella lapillus. Mar Ecol Prog Ser. 158: 191 -204.

Davies, I. M., Minchin, A., and Harding, M. J. C. 1997b. OSPAR working group on concentrations, trends and effects of substances in the marine environment (SIME) - Report on the TBT Training workshop In Marine Laboratory Aberdeen Report. Marine Laboratory, Aberdeen, pp. 36pp.

Davies, I. M., Bailey, S. K., and Harding, M. J. C. 1998a. Tributyltin inputs to the North Sea from shipping activities, and potential risk of biological effects. ICES J Mar Sci. 55: 34 - 43.

Davies, I. M., Harding, M. J. C., Bailey, S. K., Shanks, A. M., and Lange, R. 1998b. Reply to comment Tributyltin and imposex: no uncertainty shown' by Ruiz et al. Mar Ecol Prog Ser. 170: 295 - 297.

Davies, I. M., Minchin, A., Bauer, B., Harding, M. J. H., and Wells, D. E. 1999. QUASIMEME laboratory performance study of the biological effects of tributyltin (imposex and intersex) on two marine gastropod molluscs. J Environ Monitor. 1: 233 - 238. 280 de Castro, I. B., Matthews-Cascon, H., and Fernandez, M. A. 2000. Occurrence of imposex in Thais haemastoma (Linnaeus, 1767) (Mollusca: Gastropoda), as an indicator of contamination by organotin on the coastal zone of Fortileza City, Ceara State, Brazil. Arq Cienc Mar. 33: 143 - 148. de Castro, I. B., Meirelles, C. A. O., Matthews-Cascon, H., and Fernandez, M. A. 2004. Thais (Stramonita) rustica (Lamarck, 1822) (Mollusca: Gastropoda: Thaididae), a potential bioindicator of contamination by organotin Northeast Brazil. Braz J Oceanogr. 52: 135 - 139. de Castro, I. B,, de Meirelles, C. A. O., Pinheiro, J. C. L., Matthews-Cascon, H., and Rocha-Barreira, C. A. 2005a. The Increasing incidence of imposex in Stramonita haemastoma (Mollusca: Gastropoda: Muricidae) after the establishment of the Pecem Harbor, Ceara State, northeast Brazil. Thalassas. 21: 71-75. de Castro, I. B., Braga, A. R. C., and Rocha-Barreira, C. A. 2005b. Altos indices de imposex em Stramonita rustica (Mollusca: Gastropoda) em areas port uarias dos estados de Algoas e Sergipe, Brasil. Trop Oceanogr. 33: 121 - 128. de Castro, I. B., Bemvenuti, C. E., and Fillmann, G. 2007a. Preliminary appraisal of imposex in areas under the influence of southern Brazilian harbors. J Braz Soc Ecotoxicol. 2: 73 - 79. de Castro, I. B., Alves de Lima, A. F., Braga, A. R. C., and Rocha-Barreira, C. A. 2007b. Imposex in two Muricid species (Mollusca: Gastropoda) from the northwestern Brazilian Coast. J Braz Soc Ecotoxicol. 2: 81 - 91. de Castro, I. B., Ribeiro-Ferreira, V. P., Lima, A. F. A., Meirelles, C. A. O., and Rocha-Barreira, C. A. 2007c. Imposex in three prosobranch species from Ilha do Japones, Rio de Janeiro, southeast Brazil. Thalassas. 23: 37 - 42. de Castro, I. B., de Meirelles, C. A. O., Matthews-Cascon, H., Rocha-Barreira, C. D., Penchaszadeh, P., and Bigatti, G. 2008. Imposex in endemic Volutid from northeast Brazil (Mollusca: Gastropoda). Braz Arch Biol Techn. 51: 1065 - 1069.

Delaney, J. L. 2001. The occurrence of imposex in Nucella lapillus within the Saint John Harbour. BSc. Honours Thesis - University of New Brunswick, Saint John, New Brunswick. de Lima, A. F. A., de Castro, I. B., and Rocha-Barreira, C. A. 2006. Imposex induction in Stramonita haemastoma floridana (Conrad, 1837) (Mollusca: Gastropoda: Muricidae) submitted to an organotin-contaminated diet. Braz J Oceanogr. 54: 85 - 90. 281

Delmonte, S. 1999. Testosterone and TBT metabolism by Littorina littorea: An investigation of TBT induced imposex in a marine gastropod. MSc. Thesis, California State University, California.

Demaintenon, M. J. 2001. Ontogeny of the pseudohermaphrodite reproductive system in Nassarius vibex (Gastropoda: Buccinidae: Nassariinae). J Mollus Stud. 67: 51 -57.

Depledge, M. H., and Billinghurst, Z. 1999. Ecological significance of endocrine disruption in marine invertebrates. Mar Pollut Bull. 39: 32 - 38.

Deutsch, U., and Brick, M. 1993. Morphological effects of tributyltin (TBT) in vitro on the genital system of the mesogastropod Littorina littorea (Prosobranchia). Helgolander Meeresun. 47: 49 - 60.

Deutsch, U., and Fioroni, P. 1996. Effects of tributyltin (TBT) and testosterone on the female genital system in the mesogastropod Littorina littorea (Prosobranchia). Helgolander Meeresun. 50: 105 - 115.

Deutsch, U., Oehlmann, J., and Stroben, E. 1996. Morphological effects of tributyltin (TBT) on the genital system of the Mesogastropod Littorina littorea (Prosobranchia). Malacol Rev Suppl - Molluscan Reproduction. 6: 143 - 148.

De Wolf, H., De Coen, W., Backeljau, T., and Blust, R. 2001. Intersex and sterility in the periwinkle Littorina littorea (Mollusca: Gastropoda) along the western Scheldt estuary, the Netherlands. Mar Environ Res. 52: 249 - 255.

De Wolf, H., Handa, C., Backeljau, T., and Blust, R. 2004. A baseline survey of intersex in Littorina littorea along the Scheldt estuary, The Netherlands. Mar Pollut Bull. 48: 592 - 596.

Douglas, E. W., Evans, S. M., Frid, C. L. J., Hawkins, S. T., Mercer, T. S., and Scott, C. L. 1993. Assessment of imposex in the dogwhelk Nucella lapillus (L) and tributyltin along the northeast coast of England. Invertebr Reprod Dev. 24: 243 - 248.

Duft, M., Schulte-Oehlmann, U., Tillmann, M., Markert, B., and Oehlmann, J. 2003. Toxicity of triphenyltin and tributyltin to the freshwater mudsnail Potamopyrgus antipodarum in a new sediment bio test. Environ Toxicol Chem. 22: 145 - 152.

Duft, M., Schulte-Oehmann, U., Tillmann, M., Weltje, L., and Oehlmann, J. 2005. Biological impact of organotin compounds on mollusks in marine and freshwater ecosystems. Coast Mar Sci. 29: 95 - 110. 282

Duft, M., Schmitt, C., Bachmann, J., Brandelik, C., Schulte-Oehlmann, U., and Oehlmann, J. 2007. Prosobranch snails as test organisms for the assessment of endocrine active chemicals - An overview and a guideline proposal for a reproduction test with the freshwater mudsnail Potamopyrgus antipodarum. Ecotoxicol. 16: 169 - 182.

Ellis, D. V., and Pattisina, L. A. 1990. Widespread neogastropod imposex - A biological indicator of global TBT contamination. Mar Pollut Bull. 21: 248 - 253.

El Menif, N. T., Lahbib, Y., Le Pennec, M., Flower, R., and Boumaiza, M. 2006. Intensity of the imposex phenomenon - Impact on growth and fecundity in Hexaplex trunculus (Mollusca: Gastropoda) collected in Bizerta lagoon and channel (Tunisia). Cah Biol Mar. 47: 165 - 175.

El-Menif, N., Lahbib, Y., Ramdani, M., Boumaiza, M., and Le Pennec, M. 2007. Imposex in the marine neogastropod Hexaplex trunculus from Tunisian Coasts: Geographical distribution and development intensity. Vie Milieu. 57: 37 - 42.

Evans, S. M. 1998. Biological measures of TBT contamination: Is there sex after imposex? Abstr Am Chem Soc. 215: 586.

Evans, S. M. 1999a. TBT or not TBT?: That is the Question. Biofouling. 14: 117 - 129.

Evans, S. M. 1999b. Tributyltin pollution: The catastrophe that never happened. Mar Pollut Bull. 38: 629 - 636.

Evans, S. M. 2000. Tributyltin pollution: The catastrophe that never happened - Response to the letter from Nichols. Mar Pollut Bull. 40: 713 - 715.

Evans, S. M., and Nicholson, G. J. 1999. Assessing the impact of antifouling compounds in the marine environment. Lessons to be learned from the use and misuse of biological indicators of TBT contamination. In Proceedings of the Oceans '99 - Organotin Symposium Proceedings. IEEE: Seattle, W. A. USA, pp. 1115-1118.

Evans, S. M., and Nicholson, G. J. 2000. The use of imposex to assess tributyltin contamination in coastal waters and open seas. Sci Total Environ. 258: 73 - 80.

Evans, S. M., Hutton, A., Kendall, M. A., and Samosir, A. M. 1991. Recovery in populations of dogwhelks Nucella lapillus (L.) suffering from imposex. Mar Pollut Bull. 22: 331 - 333. 283

Evans, S. M., Hawkins, S. T., Porter, J., and Samosir, A. M. 1994. Recovery of dogwhelk populations on the Isle of Cumbrae, Scotland following legislation limiting the use of TBT as an antifoulant. Mar Pollut Bull. 28: 15 -17.

Evans, S. M., Leksono, T., and McKinnell, P. D. 1995a. Tributyltin pollution - A diminishing problem following legislation limiting the use of TBT-based antifouling paints. Mar Pollut Bull. 30: 14-21.

Evans, S. M., Dawson, M., Day, J., Frid, C. L. J., Gill, M. E., Pattisina, L. A., and Porter, J. 1995b. Domestic waste and TBT pollution in coastal areas of Ambon Island (eastern Indonesian). Mar Pollut Bull. 30: 109 - 115.

Evans, S. M., Evans, P. M., and Leksono, T. 1996. Widespread recovery of dogwhelks, Nucella lapillus (L.), from tributyltin contamination in the North Sea and Clyde Sea. Mar Pollut Bull. 32: 263 - 269.

Evans, S. M., Nicholson, G. J., Browning, C., Hardman, E., Seligman, O., and Smith, R. 1998. An assessment of tributyltin contamination in the North Atlantic using imposex in the dogwhelk Nucella lapillus (L.) as a biological indicator of TBT pollution. Invertebr Reprod Dev. 34: 277 - 287.

Evans, S. M., Kerrigan, E., and Palmer, N. 2000a. Causes of imposex in the dogwhelk Nucella lapillus (L.) and its use as a biological indicator of tributyltin contamination. Mar Pollut Bull. 40: 212 - 219.

Evans, S. M., Birchenough, A. C., and Fletcher, H. 2000b. The value and validity of community-based research: TBT contamination of the North Sea. Mar Pollut Bull. 40: 220 - 225.

Evans, S. M., Barnes, N., Birchenough, A. C., Brancato, M. S., and Hardman, E. 2001a. Tributyltin contamination in two estuaries and adjacent ocean coasts: Puget Sound, Washington, and Narragansett Bay, Rhode Island (USA). Invertebr Reprod Dev. 39: 221 - 229.

Evans, S. M., Bech, M., Hawkins, S. T., Smith, R., and Stewart, A. 2001b. Imposex in populations of dogwhelks Thais spp. in relation to shipping intensity and mariculture activity in East Asia. Invertebr Reprod Dev. 39: 231 - 237.

Fent, K. 1996. Ecotoxicology of organotin compounds. Crit Rev Toxicol. 26: 1 - 117.

Fent, K. 2006. Worldwide occurrence of organotins from antifouling paints and effects in the aquatic environment. The Handbook of Environmental Chemistry. 5: 71 - 100. 284

Feral, C. 1974. Etude comparee des populations Ocenebra erinacea (L.) de Granville et d' Arcachon. Haliotis. 4: 123 - 134.

Feral, C. 1975. Mise en evidence des facteurs determinant l'apparition et le cycle d'un tractus genital male chez les femelles d'Ocenebra erinacea (L.) du bassin d'Arcachon. B Soc Zool Fr. 100: 250 - 252.

Feral, C. 1976a. Etude Statistiques de la presence d'un tractus genital male extern chez les femelles d'un mollusque gasteropode gonochorique: Ocenebra erinacea (L.). Cah Biol Mar. 17: 61 - 76.

Feral, C. 1976b. Repartition geographique des femelles a tractus genital male externe chez Ocenebra erinacea (L.), espece gonochorique. Haliotis. 7: 29 - 30.

Feral, C. 1977. Analyse experimental de la morphogenese et du cycle du penis les femelles d'Ocenebra erinacea, espece gonochorique. Haliotis. 6: 267 - 271.

Feral, C. 1978a. Presence des facterus morphogenetique et dedifferenciateur du penis chez un Mollusque Prosobranche gonochorique Ocenebra erinacea (L.). C R Hebd Seances Acad Sci D. 287: 1235 - 1237.

Feral, C. 1978b. Variations dans revolution du tractus genital male des femelles de Gasteropodes Prosobrances gonochoriques de stations Atlantiques. Haliotis. 9: 90.

Feral, C. 1979. Etude des facteurs regissant l'apparition d'un penis chez les femelles d'Ocenebra erinacea L. (Mollusque Gasteropode gonochorique) de la station d' Arcachon. C R Hebd Seances Acad Sci D. 289: 331 - 334.

Feral, C. 1980a. Apparition de femelles a tractus genital male externe chez Ocenebra erinacea (L.), Mollusque Gasteropode gonochorique de la station de Granville: Recherche des facteurs controlant de cette anomalie. C R Hebd Seances Acad Sci D. 290: 1003 - 1006.

Feral, C. 1980b. Influence de la qualite de l'eau de mer sur la differenciation d'un tractus genital male externe chez les femelles. C R Hebd Seances Acad Sci D. 291: 775 - 778.

Feral, C. 1980c. Variations dans revolution du tractus genital male externe des femelles de trois Gasteropodes Prosobranches gonochoriques de stations Atlantiques. Cah Biol Mar. 21: 479 - 491. 285

Feral, C. L., and Le Gall, S. 1982. Physiologie des invertebres - Induction experimentale par un polluant marin (le tributyletain), de l'activite neuroendocrine controlant la morphogenese du penis chez les femelles d'Ocenebra erinacea (Mollusque, Prosobranche gonochorique). C R Hebd Seances Acad Sci D. 295: 627 - 630.

Feral, C., and Le Gall, S. 1983. The influence of a pollutant factor (tributyltin) on the neuroendocrine mechanism responsible for the occurence of a penis in the females of Ocenebra erinacea. In Proceedings of the International Minisymposium on Molluscan endocrinology. (Edited by J. Lever and H.H. Boer), Amsterdam, Netherlands, pp. 173 - 175.

Fernandez, M. A. 2006. Brazilian experience on TBT pollution: Lessons for future studies. J Coastal Res. 39: 1336 - 1339.

Fernandez, M. A., and Castro, I. B. 2008a. New observations on imposex development in South American marine gastropods. Mar Environ Res. 66: 177.

Fernandez, M. A., and Pinheiro, F. M. 2008b. Will imposex still be useful after the TBT ban? Mar Environ Res. 66: 180.

Fernandez, M. A., Limaverde, A. M., de Castro, I. B., Almeida, A. C. M., and Wagener, A. D. R. 2002. Occurence of imposex in Thais haemoastoma: possible evidence of environmental contamination derived from organotin compounds in Rio de Janeiro and Fortaleza, Brazil. Cad Saude Publica. 18: 463 - 476.

Fernandez, M. A., Wagener, A. D. R., Limaverde, A. M., Scofield, A. L., Pinheiro, F. M., and Rodrigues, E. 2005. Imposex and surface sediment speciation: A combined approach to evaluate organotin contamination in Guanabara Bay, Rio de Janeiro, Brazil. Mar Environ Res. 59: 435 - 452.

Fernandez, M. A., Pinheiro, F. M., de Quadros, J. P., and Camillo, E. 2007. An easy, non-destructive, probabilistic method to evaluate the imposex response of gastropod populations. Mar Environ Res. 63: 41 - 54.

Finnegan, M. C., Pittman, S., and DeLorenzo, M. E. 2009. Lethal and sublethal toxicity of the antifoulant compound Irgarol 1051 to the mud snail Ilyanassa obsoleta. Arch Environ Con Tox. 56: 85 - 95.

Fioroni, P., Oehlmann, J., and Stroben, E. 1990. Le pseudohermaphrodisme chez les prosobranches analyse morphologique et histologique. Vie Milieu. 40: 45 - 56. 286

Fioroni, P., Stroben, E., and Oehlmann, J. 1991a. Frequence et degre d'expression du pseudohermaphrodisme chez quelques Prosobranches Stenoglosses de la Baie Morlaix de la Manche 1. Situation au printemps de 1988. Cah Biol Mar. 32:281 -294.

Fioroni, P., Oehlmann, J., and Stroben, E. 1991b. The pseudohermaphroditism of Prosobranchs - Morphological aspects. Zool Anz. 226: 1 - 26.

Foale, S. 1993. An evaluation of the potential of gastropod imposex as a bioindicator of tributyltin pollution in Port Phillip Bay, Victoria. Mar Pollut Bull. 26: 546 -552.

Folsvik, N., Berge, J. A., Brevik, E. M., and Walday, M. 1999. Quantification of organotin compounds and determination of imposex in populations of dogwhelks (Nucella lapillus) from Norway. Chemosphere. 38: 681 - 691.

Fujinaga, K., llano, A. S., Nomura, H., Miranda, R. T., and Nakao, S. 2006. Present state of imposex in Neptune whelk Neptunea arthritica inhabiting shallow waters around Hokkaido, Japan. Fisheries Sci. 72: 995 - 1003.

Galante-Oliveira, S., Langston, W. J., Burt, G. R., Pereira, M. E., and Barroso, C. M. 2006. Imposex and organotin body burden in the dog-whelk {Nucella lapillus L.) along the Portuguese coast. Appl Organomet Chem. 20: 1 - 4.

Galante-Oliveira, S., Oliveira, I., Jonkers, N., Langston, W. J., Pacheco, M., and Barroso, C. M. 2009. Imposex levels and tributyltin pollution in Ria de Aveiro (NW Portugal) between 1997 and 2007: evaluation of legislation effectiveness. J Environ Monitor. 11: 1405 - 1411.

Galloway, T. S., Bastable, H., and Hagger, J. 2006. Mechanisms of endocrine toxicity: Carbonic anhydrase, calcium metabolism and imposex. Mar Environ Res. 62: S238.

Garaventa, F., Pellizzato, F., Faimali, M., Terlizzi, A., Medakovic, D., Geraci, S., and Pavoni, B. 2006a. Imposex in Hexaplex trunculus at some sites on the North Mediterranean Coast as a base-line for future evaluation of the effectiveness of the total ban on organotin based antifouling paints. Hydrobiol. 555: 281 - 287.

Garaventa, F., Faimali, M., Terlizzi, A., Fiorini, S., and Gallo, G. 2006b. Imposex induction in Hexaplex trunculus by tributyltin and other stressors. Mar Environ Res. 62: S230.

Garaventa, F., Faimali, M., and Terlizzi, A. 2006c. Imposex in pre-pollution times. Is TBT to blame? Mar Pollut Bull. 52: 701 - 702. 287

Garaventa, F., Centanni, E., Pellizzato, F., Faimali, M., Terlizzi, A., and Pavoni, B. 2007. Imposex and accumulation of organotin compounds in populations of Hexaplex trunculus (Gastropoda, Muricidae) from the Lagoon of Venice (Italy) and Istrian Coast (Croatia). Mar Pollut Bull. 54: 615 - 622.

Garaventa, F., Centanni, E., Fiorini, S., Noventa, S., Terlizzi, A., Faimali, M., and Pavoni, B. 2008. New implications in the use of imposex as a suitable tool for tributyltin contamination: experimental induction in Hexaplex trunculus (Gastropoda, Muricidae) with different stressors. Cell Biol Toxicol. 24: 563 - 571.

Gibbs, P., and Bryan, G. W. 1986. Reproductive failure in populations of the dog- whelk, Nucella lapillus, caused by imposex induced by tributyltin from antifouling paints. J Mar Biol Assoc UK. 66: 767 - 777.

Gibbs, P., Bryan, G., Pascoe, P., and Burt, G. 1987. The use of the dog-whelk, Nucella lapillus, as an indicator of tributyltin (TBT) contamination. J Mar Biol Assoc UK. 67: 507 - 523.

Gibbs, P., Pascoe, P., and Burt, G. 1988. Sex change in the female dog-whelk, Nucella lapillus, induced by tributyltin from antifouling paints. J Mar Biol Assoc UK. 68:715 -731.

Gibbs, P., Bryan, G., and Spence, S. 1991. The impact of tributyltin (TBT) pollution on the Nucella lapillus (Gastropoda) populations around the coast of South- East England. Oceanol Acta. 11: 257 - 261.

Gibbs, P. E. 1993. A male genital defect in the dogwhelk, Nucella lapillus (Neogastropoda), favoring survival in a TBT-polluted area. J Mar Biol Assoc UK. 73: 667 - 678.

Gibbs, P. E. 1996. Oviduct malformation as a sterilising effect of tributyltin (TBT)- induced imposex in Ocenebra erinacea (Gastropoda: Muricidae). J Mollus Stud. 62: 403 -413.

Gibbs, P. E. 1999. Biological effects of contaminants: Use of imposex in the dogwhelk (.Nucella lapillus) as a bioindicator of tributyltin pollution. ICES Techniques in Mar Environ Sci. 24: 1 - 29.

Gibbs, P. E. 2005. Male genital defect (Dumpton Syndrome) in the dog-whelk Nucella lapillus (Neogastropoda): Mendelian inheritance inferred, based on laboratory breeding experiments. J Mar Biol Assoc UK. 85: 143 - 150. 288

Gibbs, P. E. 2009. Long-term tributyltin (TBT)-induced sterilization of neogastropods: persistence of effects in Ocenebra erinacea over 20 years in the vicinity of Falmouth (Cornwall, UK). J Mar Biol Assoc UK. 89: 135 - 138.

Gibbs, P. E., and Bryan, G. W. 1987. TBT Paints and the demise of the dog-whelk, Nucella lapillus (Gastropoda). In Proceedings of Oceans '87- Organotin Symposium Proceedings, pp. IEEE. New York, N.Y., pp. 1482 - 1487.

Gibbs, P. E., and Bryan, G. W. 1994. Biomonitoring of tributyltin (TBT) pollution using the imposex response of neogastropod molluscs. CRC Press Incorporated, Boca Raton, Florida., pp 205 - 241.

Gibbs, P. E., and Bryan, G. W. 1996a. Reproductive failure in the gastropod Nucella lapillus associated with imposex caused by tributyltin pollution: A Review. In Organotin {Edited by M. A. Champ and P. F. Seligman). Chapman and Hall, London., pp. 259 - 280.

Gibbs, P. E., and Bryan, G. W. 1996b. TBT-induced imposex in neogastropod snails: Masculinisation to mass extinction. In Tributyltin: Case Study of an Environmental Contaminant {Edited by S.J. de Mora). Cambridge University Press, Cambridge, Great Britain, pp. 212 - 236.

Gibbs, P. E., Bryan, G. W., Pascoe, P.L., and Burt, G. R. 1990. Reproductive abnormalities in female Ocenebra erinacea (Gastropoda) resulting from tributyltin-induced imposex. J Mar Biol Assoc UK. 70: 639 - 656.

Gibbs, P. E., Pascoe, P. L., and Bryan, G. W. 1991a. Tributyltin-induced imposex in Stenoglossan gastropods - Pathological effects on the female reproductive- system. Comp Biochem Phys C. 100: 231 - 235.

Gibbs, P. E., Spencer, B. E., and Pascoe, P. L. 1991b. The American oyster drill, Urosalpinx cinerea (Gastropoda) - Evidence of decline in an imposex- affected population (R-Blackwater, Essex). J Mar Biol Assoc UK. 71: 827 - 838.

Gibbs, P. E., Bryan, G. W., and Pascoe, P. L. 1991c. TBT-induced imposex in the dogwhelk, Nucella lapillus - Geographical uniformity of the response and effects. Mar Environ Res. 32: 79 - 87.

Gibbs, P. E., Bebianno, M. J., and Coelho, M. R. 1997. Evidence of the differential sensitivity of neogastropods to tributyltin (TBT) pollution, with notes on a species {Columbella rustica) lacking the imposex response. Environ Technol. 18: 1219- 1224. 289

Gibbs, P. E., Green, J. C., and Pascoe, P. L. 1999. A massive summer-kill of the dog-whelk, Nucella lapillus, on the north Cornwall coast in 1995: freak or forerunner? J Mar Biol Assoc UK. 79: 103 - 109.

Gibson, C. P., and Wilson, S. P. 2003. Imposex still evident in eastern Australia 10 years after tributyltin restrictions. Mar Environ Res. 55: 101 - 112.

Giltrap, M., Macken, A., Minchin, D., Davoren, M., McGovern, E., Foley, B., Strand, J., and McHugh, B. 2007. An in-situ study using caged Nucella lapillus and Crassostrea gigas to monitor TBT induced bio-effects in Irish coastal waters. In SET AC 28th Annual Meeting (November 11 - 15, 2007), Milwaukee, Wisconsin.

Giltrap, M., Macken, A., Davoren, M., Minchin, D., McGovern, E., Foley, B., Strand, J., and McHugh, B. 2009. Use of caged Nucella lapillus and Crassostrea gigas to monitor tributyltin-induced bioeffects in Irish coastal waters. Environ Toxicol Chem. 28: 1671 - 1678.

Godtfredsen, K., Hotchkiss, D., Williston, D., Stern, J., Fox, S., J., G., Andersen, H., and Kohn, A. 2005. Analysis of imposex in neogastropods collected from the lower Duwamish Waterway. In SETAC 26th Annual Meeting (November 13 - 17, 2005), Baltimore, Maryland.

Goldberg, R. N., Averbuj, A., Cledon, M., Luzzatto, D., and Nudelman, N. S. 2004. Search for triorganotins along the Mar del Plata (Argentina) marine coast: Finding of tributyltin in egg capsules of a snail Adelomelon brasiliana (Lamarck, 1822) population showing imposex effects. Appl Organomet Chem. 18: 117-123.

Gomez-Ariza, J. L., Santos, M. M., Morales, E., Giraldez, I., Sanchez-Rodas, D., Vieira, N., Kemp, J. F., Boon, J. P., and Ten-Hallers-Tjabbes, C. C. 2006. Organotin contamination in the Atlantic Ocean off the Iberian Peninsula in relation to shipping. Chemosphere. 64: 1100 - 1108.

Gooding, M., Gallardo, C., and Leblanc, G. 1999. Imposex in three marine gastropod species in Chile and potential impact on muriciculture. Mar Pollut Bull. 38: 1227- 1231.

Gooding, M. P. 2002. The role of steroid metabolism in tributyltin-induced imposex in the eastern mud snail (Ilyanassa obsoleta). PhD. Thesis, North Carolina State University, North Carolina.

Gooding, M. P., and LeBlanc, G. A. 2001. Biotransformation and disposition of testosterone in the eastern mud snail Ilyanassa obsoleta. Gen Comp Endocr. 122: 172- 180. 290

Gooding, M. P., Wilson, V. S., Folmar, L. C., Marcovich, D. T., and LeBlanc, G. A. 2003. The biocide tributyltin reduces the accumulation of testosterone as fatty acid esters in the mud snail (Uyanassa obsoleta). Environ Health Persp. 111:426-430.

Gooding, M. P., and LeBlanc, G. A. 2004. Seasonal variation in the regulation of testosterone levels in the eastern mud snail {Uyanassa obsoleta). Invertebr Biol. 123: 237 - 243.

Gravel, P., Johanning, K., McLachlan, J., Vargas, J. A., and Oberdorster, E. 2006. Imposex in the intertidal snail Thais brevidentata (Gastropoda: Muricidae) from the Pacific coast of Costa Rica. Rev Biol Trop. 54: 21 - 26.

Griffith, G. W., and Castagna, M. 1962. Sexual dimorphism in oyster drills of Chincoteagne Bay, Maryland, Virginia. Chesapeake Sci. 3: 215 - 217.

Hagger, J. A., Depledge, M. H., Oehlmann, J., Jobling, S., and Galloway, T. S. 2006. Is there a causal association between genotoxicity and the imposex effect? Environ Health Persp. 114: 20 - 26.

Hall, J. G., and Feng, S. Y. 1976. Genital variation among Connecticut populations of the oyster drill Urosalpinx cinera. Veliger. 18: 318 - 321.

Harding, M. J. C., and Davies, I. M. 2000. A field evaluation of international monitoring guidelines for the biological effects of tributyltin. J Environ Monitor. 2: 404 - 409.

Harding, M. J. C., Rodger, G. K., and Davies, I. M. 1994. Effects of tributyltin on Littorina sp. In Fisheries Research Services Report {Edited by S.o.o.A.a.F. Department), pp. 19 pp.

Harding, M. J. C., Rodger, G. K., Davies, I. M., and Moore, J. J. 1997. Partial recovery of the dogwhelk {Nucella lapillus) in Sullom Voe, Shetland from tributyltin contamination. Mar Environ Res. 44: 285 - 304.

Harding, M. J. C., Davies, I. M., Bailey, S. K., and Rodger, G. K. 1999. Survey of imposex in dogwhelks {Nucella lapillus) from North Sea coasts. Appl Organomet Chem. 13: 521 - 538.

Harrison, P. T. C., Holmes, P., and Humfrey, C. D. N. 1997. Reproductive health in humans and wildlife: Are adverse trends associated with environmental chemical exposure? Sci Total Environ. 205: 97 - 106. 291

Hashimoto, S., Watanabe, M., Noda, Y., Hayashi, T., Kurita, Y., Takasu, Y., and Otsuki, A. 1998. Concentration and distribution of butyltin compounds in a heavy tanker route in the Strait of Malacca and in Tokyo Bay. Mar Environ Res. 45: 169- 177.

Hawkins, L. E., and Hutchinson, S. 1990. Physiological and morphogenetic effects of monophenyltin trichloride on Ocenebra erinacea (L). Funct Ecol. 4: 449 - 454.

Hernandez, S., and Stotz, W. B. 2004. Reproductive biology of the "Copey" snail Melongena melongena (Linnaeus, 1758) in Cispat Bay on the Caribbean coast of Colombia. J Shellfish Res. 23: 849 - 854.

Horiguchi, T. 2006a. Masculinization of female gastropod mollusks induced by organotin compounds, focusing on mechanisms of actions of tributyltin and triphenyltin for development of imposex. Environ Sci. 13: 77 - 87.

Horiguchi, T. 2006b. Endocrine disruption caused by organotin compounds in gastropod mollusks. In 3rd Asia-Pacific Workshop - "Modern approaches to linking exposure to Toxic Compounds and Biological effects", Xiamen, China, pp. 101-109.

Horiguchi, T. 2007. Endocrinology and induction mechanism of imposex in gastropod molluscs. Jpn Soc Biomed Res Trace Elem. 18: 231 - 240.

Horiguchi, T., Shiraishi, H., Shimizu, M., and Morita, M. 1994. Imposex and organotin compounds in Thais clavigera and T. bronni in Japan. J Mar Biol Assoc UK. 74: 651 -669.

Horiguchi, T., Shiraishi, H., Shimizu, M., Yamazaki, S., and Morita, M. 1995. Imposex in Japanese gastropods (Neogastropoda and Mesogastropoda): Effects of tributyltin and triphenyltin from antifouling paints. Mar Pollut Bull. 31:402-405.

Horiguchi, T., Shiraishi, H., Shimizu, M., and Morita, M. 1997a. Imposex in sea snails, caused by organotin (tributyltin and triphenyltin) pollution in Japan: A survey. Appl Organomet Chem. 11: 451 - 455.

Horiguchi, T., Shiraishi, H., Shimizu, M., and Morita, M. 1997b. Effects of triphenyltin chloride and five other organotin compounds on the development of imposex in the rock shell, Thais clavigera. Environ Pollut. 95: 85 -91.

Horiguchi, T., Hamada, F., Kajikawa, A., Shiraishi, H., Shimizu, M., and Morita, M. 1998a. Imposex and population decline in the ivory shell, Babylonia japonica. Abstr Am Chem Soc. 215: U586 - U586. 292

Horiguchi, T., Shiraishi, H., Cho, H. S., Shibata, Y., Shimizu, M., and Morita, M. 1998b. Less recovery from imposex in the rock shell, Thais clavigera and organotin contamination after the regulation of organotin usage in antifouling paints in Japan. Abstr Am Chem Soc. 215: U587.

Horiguchi, T., Hyeon-Seo, C., Shiraishi, H., Shibata, Y., Soma, M., Morita, M., and Shimizu, M. 1998c. Field studies on imposex and organotin accumulation in the rock shell, Thais clavigera, from the Seto Inland Sea and the Sanriku region, Japan. Sci Total Environ. 214: 65 - 70.

Horiguchi, T., Takiguchi, N., Cho, H. S., Kojima, M., Kaya, M., Shiraishi, H., Morita, M., Hirose, H., and Shimizu, M. 2000. Ovo-testis and disturbed reproductive cycle in the giant abalone, Haliotis madaka: Possible linkage with organotin contamination in a site of population decline. Mar Environ Res. 50: 223 - 229.

Horiguchi, T., Seiichi, U., Shimizu, M., Shiraishi, H., and Morita, M. 2001a. Contamination of organotin compounds and imposex in molluscs from Vancouver, Canada. In Environmental Assessment of Vancouver Harbour Data Report for the PICES Practical Workshops, vol. 16 (Edited by C.M. Stehr and T. Horiguchi). pp. 53 - 56.

Horiguchi, T., Cho, H. S., Shiraishi, H., Kojima, M., Kaya, M., Morita, M., and Shimizu, M. 2001b. Contamination by organotin (tributyltin and triphenyltin) compounds from antifouling paints and endocrine disruption in marine gastropods. Riken Rev. 35: 9 -11.

Horiguchi, T., Kojima, M., Kaya, M., Matsuo, T., Shiraishi, H., Morita, M., and Adachi, Y. 2002. Tributyltin and triphenyltin induce spermatogenesis in ovary of female abalone, Haliotis gigantea. Mar Environ Res. 54: 679 - 684.

Horiguchi, T., Li, Z., Uno, S., Shimizu, M., Shiraishi, H., Morita, M., Thompson, J. A. J., and Levings, C. D. 2003. Contamination of organotin compounds and imposex in molluscs from Vancouver, Canada. Mar Environ Res. 57: 75 - 88.

Horiguchi, T., Shiraishi, H., Hyeon-Seo, C., Ohta, Y., Iguchi, T., Nishikawa, T., Shiraishi, F., Morita, M., and Nishikawa, J. 2004a. Imposex induced by organotin compounds in gastropods: From molecular mechanism to population-level effects. J Pharm Soc Japan (Yakugaku Zasshi). 124: 48.

Horiguchi, T., Kojima, M., Takiguchi, N., Kaya, M., Shiraishi, H., Morita, M., and Imai, T. 2004b. Continuing observation of endocrine disruption in abalone from an organotin-contaminated site of Japan. In SETAC 25th Annual Meeting (November 14- 18, 2004), Portland, Oregon, USA. 293

Horiguchi, T., Nishikawa, T., Ohta, Y., Mamiya, S., Kanayama, T., Shiraishi, F., Shiraishi, H., Morita, M., and Nishikawa, J. 2004c. Involvement of the Retinoid X Receptor in the imposex development caused by organotins in gastropods. In SETAC 25th Annual Meeting (November 14 - 18, 2004), Portland, Oregon, USA.

Horiguchi, T., Kojima, M., Takiguchi, N., Kaya, M., Shiraishi, H., and Morita, M. 2005. Continuing observation of disturbed reproductive cycle and ovarian spermatogenesis in the giant abalone, Haliotis madaka from an organotin- contaminated site of Japan. Mar Pollut Bull. 51: 817 - 822.

Horiguchi, T., Shiraishi, H., Nishikawa, T., Shiraishi, F., Nishikawa, J., Ohta, Y., and Morita, M. 2006a. Analysis of induction mechanism of imposex caused by tributyltin and triphenyltin in gastropod molluscs. Biomed Res Trace Ele. 17: 152 - 154.

Horiguchi, T., Kojima, M., Hamada, F., Kajikawa, A., Shiraishi, H., Morita, M., and Shimizu, M. 2006b. Impact of tributyltin and triphenyltin on ivory shell {Babylonia japonica) populations. Environ Health Persp. 114: 13 - 19.

Horiguchi, T., Nishikawa, T., Ohta, Y., Shiraishi, H., and Morita, M. 2007. Retinoid X Receptor gene expression and protein content in tissues of the rock shell Thais clavigera. Aquat Toxicol. 84: 379 - 388.

Horiguchi, T., Ohta, Y., Nishikawa, T., Shiraishi, F., Shiraishi, H., and Morita, M. 2008. Exposure to 9-cis retinoic acid induces penis and vas deferens development in the female rock shell, Thais clavigera. Cell Biol Toxicol. 24: 553 - 562.

Houston, R. S. 1971. Reproductive biology of Thais emarginata. Veliger. 13: 348 - 357.

Huang, C. J., Zhu, S. X., Lin, J. D., and Dong, Q. X. 2008. Imposex of Mauritia arabica on the south-eastern coast of China. J Mar Biol Assoc UK. 88: 1451 - 1457.

Huaqui'n, L. G., Osorio, C., Verdugo, R., and Collado, G. 2004. Morphological changes in the reproductive system of females Acanthina monodon (Pallas, 1774) (Gastropoda: Muricidae) affected by imposex from the coast of central Chile. Invertebr Reprod Dev. 46: 111 -117.

Huet, M. 1994. Comparison of imposex response in three prosobranch species. Cah Biol Mar. 35: 247 - 248. 294

Huet, M., Fioroni, P., Oehlmann, J., and Stroben, E. 1995. Comparison of imposex response in three prosobranch species. Hydrobiol. 309: 29 - 35.

Huet, M., Paulet, Y. M., and LePennec, M. 1996a. Survival of Nucella lapillus in a tributyltin polluted area in west Brittany: A further example of a male genital defect (Dumpton Syndrome) favouring survival. Mar Biol. 125: 543 - 549.

Huet, M., Paulet, Y. M., and Glemarec, M. 1996b. Tributyltin (TBT) pollution in the coastal waters of west Brittany as indicated by imposex in Nucella lapillus. Mar Environ Res. 41: 157 - 167.

Huet, M., Paulet, Y. M., and Clavier, J. 2004. Imposex in Nucella lapillus: A ten year survey in NW Brittany. Mar Ecol Prog Ser. 270: 153 - 161.

Huet, M., Le Goic, N., and Gibbs, P. E. 2008. Appearance of a genetically-based pollution resistance in a marine gastropod, Nucella lapillus, in south-west Brittany: A new case of Dumpton syndrome. J Mar Biol Assoc UK. 88: 1475 - 1479.

Hung, T. C., Hsu, W. K., Meng, P. J., and Chuang, A. 2000. Species of organotins in imposex of rock shells and hermaphroditic oysters from the western coast of Taiwan. Bulletin - Institute of Chemistry Academia Sinica. 47: 1 -12.

Hung, T. C., Hsu, W. K., Mang, P. J., and Chuang, A. 2001. Organotins and imposex in the rock shell, Thais clavigera, from oyster mariculture areas in Taiwan. Environ Pollut. 112: 145 - 152.

Hwang, G., Myung, C. S., Park, S. I., Song, S. J., and Maclean, N. 2009. Isolation and characterization of microsatellite loci from rock shell, Thais clavigera. Mol Ecol Resour. 9: 1227 - 1229.

Ide, I., Witten, E. P., Fischer, J., Kalbfus, W., Zellner, A., Stroben, E., and Watermann, B. 1997. Accumulation of organotin compounds in the common whelk Buccinum undatum and the red whelk Neptunea antiqua in association with imposex. Mar Ecol Prog Ser. 152: 197 - 203.

Iguchi, T., Katsu, Y., Horiguchi, T., Watanabe, H., Blumberg, B., and Ohta, Y. 2007. Endocrine disrupting organotin compounds are potent inducers of imposex in gastropods and adipogenesis in vertebrates. Mol Cell Toxicol. 3: 1 -10.

Iguchi, T., Watanabe, H., Ohta, Y., and Blumberg, B. 2008. Developmental effects: Oestrogen-induced vaginal changes and organotin-induced adipogenesis. Int J Androl. 31:263 -268. 295

Janer, G., Bachmann, J., Oehlmann, J., Schulte-Oehlmann, U., and Porte, C. 2006a. The effect of organotin compounds on gender specific androstenedione metabolism in the freshwater ramshorn snail Marisa cornuarietis. J Steroid Biochem. 99: 147 - 156.

Janer, G., Lyssimachou, A., Bachmann, J., Oehlmann, J., Schulte-Oehlmann, U., and Porte, C. 2006b. Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: The effect of xenoandrogenic compounds. Steroids. 71: 435 - 444.

Janer, G., Navarro, J. C., and Porte, C. 2007. Exposure to TBT increases accumulation of lipids and alters fatty acid homeostasis in the ramshorn snail Marisa cornuarietis. Comp Biochem Phys C. 146: 368 - 374.

Jenner, M. G. 1978. Pseudohermaphroditism: A newly recognized sexual phenomenon in Hyanassa obsoleta and other neogastropods. PhD. Thesis, University of Chapel Hill, North Carolina.

Jenner, M. G. 1979. Pseudohermaphroditism in Hyanassa obsoleta (Mollusca, Neogastropoda). Science. 205: 1407 - 1409.

Jorundsdottir, K., Svavarsson, K., and Leung, K. M. Y. 2005. Imposex levels in the dogwhelk Nucella lapillus (L.) - Continuing improvement at high latitudes. Mar Pollut Bull. 51: 744 - 749.

Kantor, Y. I. 1984. Pseudohermaphroditism in Buccinum undatum (Gastropoda, Prosobranchia). Zool Zh. 63: 1256 - 1258.

Kantor, Y. I., and Sysoev, A. V. 1991. Sexual dimorphism in the apertural notch of a new species of gemmula (Gastropoda, Turridae). J Mollus Stud. 57: 205 - 209.

Kawai, S., Kobayashi, M., and Kaneko, H. 2003. Effects of endocrine active substances in wildlife species: Genetic, biochemical, and physiological factors in variable susceptibility to endocrine disruptors. Pure Appl Chem. 75:2335 -2341.

Kendall, C., and Bard, S. 2007. A comparison of the occurence of tributyltin- induced imposex in dogwhelks (Nucella lapillus) in Halifax (1995-2006). In Proceedings of the 34th Annual Toxicity Workshop (September 30th to October 3rd, 2007), Halifax, Nova Scotia, pp 74.

Ketata, I., Denier, X., Hamza-Chaffai, A., and Minier, C. 2008. Endocrine-related reproductive effects in molluscs. Comp Biochem Phys C. 147: 261 - 270. 296

Kiran, Y. V., and Anil, A. C. 1999. Record of imposex in Cronia konkanensis (Gastropoda, Muricidae) from Indian waters. Mar Environ Res. 48: 123 - 130.

Kittridge, A., Reber, J., and McClellan-Green, P. 2004. Recognition and response to sex pheremones in Ilyanassa obseleta following exposure to TBT. In SETAC 25th Annual Meeting (November 14-18, 2004), Portland, Oregon, USA.

Kohn, A. J., and Almasi, K. N. 1993. Imposex in Australian Conus. J Mar Biol Assoc UK. 73: 241 - 244.

Kure, L. K., and Depledge, M. H. 1994. Accumulation of organotin in Littorina littorea and Mya arenaria from Danish coastal waters. Environ Pollut. 84: 149- 157.

Kuwamura, T., Fukao, R., Nishida, M., Wada, K., and Yanagisawa, Y. 1983. Reproductive biology of the gastropod Strombus luhuanus (Strombidae). Publ Seto Mar Biol Lab. 28: 433 - 443.

Lagadic, L. 2002. Biomarkers: useful tools for the monitoring of aquatic environments. Rev Med Vet-Toulouse. 153: 581 - 588.

Lahhib, Y., Trigui El-Menif, N., Le Pennec, M., and Boumaiza, M. 2004. Donnees sur le cycle reproducteur du Mollusque Gasteropode Hexaplex trunculus (Linne, 1758) de la Lagune de Bizerte (Tunisia). B Soc Zool Fr. 129: 407 - 418.

Lahbib, Y., Labidli, S., Le Pennec, M., Flower, R., and El-Menif, N. T. 2007. Morphological expression and different stages of imposex in Hexaplex trunculus (Neogastropoda: Muricidae) from Tunisian coasts. Cah Biol Mar. 48:315 - 326.

Lahbib, Y., Abidli, S., and El Menif, N. T. 2008a. Imposex level and penis malformation in Hexaplex trunculus from the Tunisian coast. Am Malacol Bull. 24: 79 - 89.

Lahbib, Y., Boumaiza, M., and El Menif, N. T. 2008b. Imposex expression in Hexaplex trunculus from the North Tunis Lake transplanted to Bizerta channel (Tunisia). Ecol Indie. 8: 239 - 245.

Lahbib, Y., Abidli, S., Chiffoleau, J. F., Averty, B., and El Menif, N. T. 2009. First record of butyltin body burden and imposex status in Hexaplex trunculus (L.) along the Tunisian coast. J Environ Monitor. 11: 1253 - 1258.

Lamotte, G. 1996. Les messaventures du pourpre Nucella lapillus (Linne, 1758) au cours des siecles. Naturalistes Beiges. 77: 33 - 38. 297

Langston, W. J., Bryan, G. W., Burt, G. R., and Gibbs, P. E. 1990. Assessing the impact of tin and TBT in estuaries and coastal regions. Funct Ecol. 4: 433 - 443.

LeBlanc, G. A., Gooding, M. P., and Sternberg, R. M. 2005. Testosterone-fatty acid esterification: A unique target for the endocrine toxicity of tributyltin to gastropods. Integr Comp Biol. 45: 81 - 87.

Lee, C. C., Jhuang, Y. F., Liu, L. L., Hsieh, C. Y., Chen, C. S., and Tien, C. J. 2009. The major source and impact of phenyltin contamination on freshwater aquaculture clam Corbicula fluminea and wild golden apple snail Pomacea canaliculata. Environ Chem. 6: 341 - 349.

Lemghich, I., and Benajiba, M. H. 2007. Survey of imposex in prosobranchs mollusks along the northern Mediterranean coast of Morocco. Ecol Indie. 7: 209-214.

Leung, K. M. Y., Morgan, I. J., Wu, R. S. S., Lau, T.C., Svavarsson, J., and Furness, R.W. 2001. Growth rate as a factor confounding the use of the dogwhelk Nucella lapillus as biomonitor of heavy metal contamination. Mar Ecol Prog Ser. 221: 145 - 159.

Leung, K. M. Y., Dewhurst, R. E., Halldorsson, H., and Svavarsson, K. 2005. Metallothioneins and trace metals in the dogwhelk Nucella lapillus (L.) collected from Icelandic coasts. Mar Pollut Bull. 51: 729 - 737.

Leung, K. M. Y., Kwong, R. P. Y., Ng, W. C., Horiguchi, T., Qiu, J. W., Yang, R. Q., Song, M. Y., Jiang, G. B., Zheng, G. J., and Lam, P. K. S. 2006. Ecological risk assessments of endocrine disrupting organotin compounds using marine neogastropods in Hong Kong. Chemosphere. 65: 922 - 938.

Limaverde, A. M., Wagener, A., Fernandez, M. A., Scofield, A. D. L., and Coutinho, R. 2007. Stramonita haemastoma as a bioindicator for organotin contamination in coastal environments. Mar Environ Res. 64: 384-398.

Liu, L. L., and Chen, J. L. 1992. Sex ratio changes of the oyster drill, Thais clavigera Kuster in Taiwan. J Fish Soc Taiwan. 19: 85 - 88.

Liu, L. L., and Suen, I. J. 1996a. Organotins promoting the development of imposex in the oyster drill Thais clavigera. J Fish Soc Taiwan. 23: 149 - 154.

Liu, L. L., and Suen, I. J. 1996b. Prosobranch gastropod imposex in the west coast of Taiwan. Jap J Malacol (Venus). 55: 207 - 214. 298

Liu, L. L., Chen, S. J., Peng, W. Y., and Hung, J. J. 1997. Organotin concentrations in three intertidal neogastropods from the coastal waters of Taiwan. Environ Pollut. 98: 113-118.

Liu, W. H., Chiu, Y. W., Huang, D. J., Liu, M. Y., Lee, C. C., and Liu, L. L. 2006. Imposex in the golden apple snail Pomacea canaliculata in Taiwan. Sci Total Environ. 371: 138 -143.

Lu, M., Horiguchi, T., Shiraishi, H., Shibata, Y., Abo, M., Okubo, A., and Yamazaki, S. 2001. Identification and quantification of steroid hormones in marine gastropods by GC/MS. Bunseki Kagaku, 50: 247 - 255. (Abstract only)

Lu, M., Horiguchi, T., Shiraishi, H., Shibata, Y., Abo, M., Okubo, A., and Yamazaki, S. 2002. Determination of testosterone in an individual shell of Thais clavigera by ELISA. Bunseki Kagaku. 51: 21 - 27. (Abstract only)

Lyssimachou, A., Bachmann, J., and Porte, C. 2008. Short-term exposure to the organotin compound triphenyltin modulates esterified steroid levels in females of Marisa cornuarietis. Aquat Toxicol. 89: 129 - 135.

Lyssimachou, A., Ramon, M., and Porte, C. 2009a. Comparative study on the metabolism of the androgen precursor androstenedione in two gastropod species: In vitro alterations by TBT and TPT. Comp Biochem Phys C. 149: 409-413.

Lyssimachou, A., Navarro, J.C., Bachmann, J., and Porte, C. 2009b. Triphenyltin alters lipid homeostasis in females of the ramshorn snail Marisa cornuarietis. Environ Pollut. 157: 1714 - 1720.

Mallon, P., and Manga, N. 2007. The use of imposex in Nucella lapillus to assess tributyltin pollution in Carlingford Lough. J Environ Health Res. 6: 89 - 99.

Mann, R., Harding, J. M., and Westcott, E. 2006. Occurrence of imposex and seasonal patterns of gametogenesis in the invading veined rapana whelk Rapana venosa from Chesapeake Bay, USA. Mar Ecol Prog Series. 310: 129 - 138.

Maran, C., Centanni, E., Pellizzato, F., and Pavoni, B. 2006. Organochlorine compounds (polychlorinated biphenyls and pesticides) and polycyclic aromatic hydrocarbons in populations of Hexaplex trunculus affected by imposex in the Lagoon of Venice, Italy. Environ Toxicol Chem. 25: 486 - 495. 299

Marshall, D. J., and Rajkumar, A. 2003. Imposex in the indigenous Nassarius kraussianus (Mollusca: Neogastropoda) from South African harbours. Mar Pollut Bull. 46: 1150-1155.

Martin, P. R. 2002. Evidence for parthenogenesis and natural imposex in the Patagonian freshwater snail Heleobia hatcheri (Gastropoda: Hydrobiidae). J Mollus Stud. 68: 291 - 295.

Martoja, M., and Bouquegneau, J. M. 1988. Murex trunculus: Un nouveau cas de pseudo-hermaphrodisme chez un gasteropode prosobranche. Bulletin de la Societe de Liege. 57: 45 - 58.

Matthiessen, P., and Gibbs, P. E. 1998. Critical appraisal of the evidence for tributyltin-mediated endocrine disruption in mollusks. Environ Toxicol Chem. 17:37-43.

Matthiessen, P., Waldock, R., Train, J. E., Milton, S., and Scrope-Howe, S. 1991. Changes in periwinkle (Littorina littorea) populations following the ban on TBT-based antifoulings on small boats. ICES C.M. pp. E5 1 -15.

Matthiessen, P., Waldock, R., Thain, J. E., Waite, M. E., and Scropehowe, S. 1995. Changes in periwinkle {Littorina littorea) populations following the ban on TBT-based antifoulings on small boats in the United Kingdom. Ecotox Environ Safe. 30: 180 -194.

Matthiessen, P., Reynoldson, T., Billinghurst, Z., Brassard, D. W., Cameron, P., Chandler, G. T., Davies, I. M., Horiguchi, T., Mount, D. R., Oehlmann, J., Pottinger, T. G., Sibley, P. K., Thompson, H. M., and Vethaak, A. D. 1999. Field assessment for endocrine disruption in invertebrates (Chapter 4). In Society of Environmental Toxicology and Chemistry, vol. Endocrine Disruption in Invertebrates: Endocrinology, testing, and Assessment (Edited by P. L. deFur, M. Crane, C. G. Ingersoll and L. J. Tattersfield). SETAC Technical Publication Series, pp. 199 - 279.

McClellan-Green, P. 2001. Impact of co-exposure to TBT and PAHs on steroid metabolism in mudsnails (Hyanassa obsoleta). In SETAC 22nd Meeting (November 11 - 15, 2001), Baltimore, Maryland.

McClellan-Green, P. 2003. Effects of imposex status and contaminant exposure on steroid metabolism in mud snails {Hyanassa obsoleta). In SETAC 24th Annual Meeting (November 9- 13, 2003), Austin Texas.

McClellan-Green, P., and Robbins, J. 2000. Effects of TBT and 3-MC co-exposure on cytochrome P450 expression and activity in marine organisms. Mar Environ Res. 50: 243 - 243. 300

McClellan-Green, P., Romano, J., and Rittschof, D. 2006. Imposex induction in the mud snail, Ilyanassa obsoleta by three tin compounds. B Environ Contam Tox. 76: 581 -588.

McClellan-Green, P., Romano, J., and Oberdorster, E. 2007. Does gender really matter in contaminant exposure? A case study using invertebrate models. Environ Res. 104: 183 - 191.

McMaster, M. E. 2001. A review of the evidence for endocrine disruption in Canadian aquatic ecosystems. Water Qual Res J Can. 36: 215 - 231.

Meador, J. P., Collier, T. K., and Stein, J. E. 2002. Determination of a tissue and sediment threshold for tributyltin to protect prey species of juvenile salmonids listed under the US Endangered Species Act. Aquat Conserv. 12: 539-551.

Meirelles, C. A. O., Castro, I. B., and Pinheiro, J. C. L. 2007. A first record of biphallia in imposexed female of Leucozonia nassa (Caenogastropoda: Fasciolariidae). J Mar Biol Assoc UK, Biodiversity Records: 1-3.

Meng, P., Han, B. C., Hsu, W. K., Chuang, A., Cheng, J. H., and Hung, T. C. 2003. Bioaccumulation and elimination of tributyltin and triphenyltin in oysters and rock shells in Taiwan. J Food Drug Anal. 11: 96 - 101.

Meng, P. J., Wang, J. T., Liu, L. L., Chen, M. H„ and Hung, T. C. 2005. Toxicity and bioaccumulation of tributyltin and triphenyltin on oysters and rock shells collected from Taiwan mariculture area. Sci Total Environ. 349: 140 - 149.

Meng, P. J., Lin, J., and Liu, L. L. 2009. Aquatic organotin pollution in Taiwan. J Environ Manage. 90: S8 - SI5.

Mensink, B. P., Ten Hallers-Tjabbes, C. C., Kralt, J., Freriks, I. L., and Boon, J. P. 1996a. Assessment of imposex in the common whelk, Buccinum undatum (L.) from the eastern Scheldt, the Netherlands. Mar Environ Res. 41: 315 - 325.

Mensink, B. P., Everaarts, J. M., Kralt, H., TenHallersTjabbes, C. C., and Boon, J. P. 1996b. Tributyltin exposure in early life stages induces the development of male sexual characteristics in the common whelk, Buccinum undatum. Mar Environ Res. 42: 151 -154.

Mensink, B. P., Boon, J. P., Ten Hallers-Tjabbes, C. C., van Hattum, B., and Koeman, J. H. 1997. Bioaccumulation of organotin compounds and imposex occurrence in a marine food chain (eastern Scheldt, the Netherlands). Environ Technol. 18: 1235 - 1244. 301

Mensink, B. P., Fischer, C. V., Cadee, G. C., Fonds, M., Ten Hallers-Tjabbes, C. C., and Boon, J. P. 2000. Shell damage and mortality in the common whelk Buccinum undatum caused by beam trawl fishery. J Sea Res. 43: 53 - 64.

Mensink, B. P., Kralt, H., Vethaak, A. D., Ten Hallers-Tjabbes, C. C., Koeman, J. H., van Hattum, B., and Boon, J. P. 2002. Imposex induction in laboratory reared juvenile Buccinum undatum by tributyltin (TBT). Environ Toxicol Phar. 11:49-65.

Miller, E. R., and Pondick, J. S. 1984. Heavy metal levels in Nucella lapillus (Gastropoda, Prosobranchia) from sites with normal and penis-bearing females from New England. B Environ Contam Tox. 33: 612 - 620.

Miller, K. L., Fernandes, T. F., and Read, P. A. 1999. The recovery of populations of dogwhelks suffering from imposex in the Firth of Forth 1987-1997/98. Environ Pollut. 106: 183 - 192.

Miloslavich, P., Penchaszadeh, P. E., and Bigatti, G. 2007. Imposex in gastropods from Venezuela. Cienc Mar. 33: 319 - 324.

Minchin, A., and Davies, I. M. 1998a. Effect of freezing on penis length in Nucella lapillus (L.). In Marine Laboratory Aberdeen Report. Marine Laboratory, Aberdeen, pp. 12pp.

Minchin, A., and Davies, I. M. 1998b. Effect of sample handling on Imposex measurement in the dogwhelk Nucella lapillus. In Marine Laboratory Aberdeen Report. Marine Laboratory, Aberdeen, pp. 10pp.

Minchin, A., and Davies, I. M. 1999a. Effect of freezing on the length of the penis in Nucella lapillus (L.). J Environ Monitor. 1: 203 - 205.

Minchin, A., and Davies, I. M. 1999b. Imposex measurement in the dogwhelk Nucella lapillus (L.) - Temporal aspects of specimen preparation. J Environ Monitor. 1: 239-241.

Minchin, A., and Minchin, D. 1997. Dispersal of TBT from a fishing port determined using the dogwhelk Nucella lapillus as an indicator. Environ Technol. 18: 1225 - 1234.

Minchin, A., and Davies, I. M. 2000a. BEQUALM and QUASIMEME workshop on imposex and intersex in marine snails, Nucella lapillus, Buccinum undatum and Littorina littorea. In Report on the TBT Training Workshop - Marine Laboratory Aberdeen Report. Marine Laboratory, Aberdeen, pp. 22pp. 302

Minchin, A., and Davies, I. M. 2000b. SOTEAG rocky shore monitoring programme. TBT contamination in Sullom Voe, Shetland. 1999 Dogwhelk Survey. Fisheries Research Services, pp. 35pp.

Minchin, A., Davies, I. M., Pymm, H., and Grewar, G. N. 1998. Survey of imposex in the North Sea CW0825: Interim report for August-December 1998. Fisheries Research Services, pp. 7pp.

Minchin, A., Davies, I. M., Pymm, H., and Grewar, G. N. 1999. North Sea imposex survey: CW0825 August 1998 - July 1999 - Final Report. Fisheries Research Services, pp. 198 pp.

Minchin, D. 2003. Monitoring of tributyltin contamination in six marine inlets using biological indicators. Mar Environ Health Ser. 6: 1 - 15.

Minchin, D., Oehlmann, J., Duggan, C. B., Stroben, E., and Keatinge, M. 1995. Marine TBT antifouling contamination in Ireland, following legislation in 1987. Mar Pollut Bull. 30: 633 - 639.

Minchin, D., Stroben, E., Oehlmann, J., Bauer, B., Duggan, C. B., and Keatinge, M. 1996. Biological indicators used to map organotin contamination in Cork Harbour, Ireland. Mar Pollut Bull. 32: 188 - 195.

Minchin, D., Bauer, B., Oehlmann, J., Schulte-Oehlmann, U., and Duggan, C. B. 1997. Biological indicators used to map organotin contamination from a fishing port, Killybegs, Ireland. Mar Pollut Bull. 34: 235 - 243.

Miranda, R. M., Fujinaga, K., llano, A. S., and Nakao, S. 2007. Incidence of imposex and parasite infection in Neptunea arthritica at Saroma Lagoon and their relationship. Aquat Sci. 55: 9 -15.

Miranda, R. M., Fujinaga, K., and Nakao, S. 2008. Age and growth of Neptunea arthritica estimated from growth marks in the operculum. Mar Biol Res. 4: 224 - 235.

Miranda, R. M., Fujinaga, K., llano, A. S., and Nakao, S. 2009. Effects of imposex and parasite infection on the reproductive features of the Neptune whelk Neptunea arthritica. Mar Biol Res. 5: 268 - 277.

Miyamoto, J., and Klein, W. 1998. Environmental exposure, species differences and risk assessment. Pure Appl Chem. 70: 1829 - 1845.

Morcillo, Y., and Porte, C. 1998. Monitoring of organotin compounds and their effects in marine molluscs. Trac-Trends Anal Chem. 17: 109 - 116. 303

Morcillo, Y., and Porte, C. 1999. Evidence of endocrine disruption in the imposex- affected gastropod Bolinus brandaris. Environ Res. 81: 349 - 354.

Morcillo, Y., Borghi, V., and Porte, C. 1997. Survey of organotin compounds in the western Mediterranean using molluscs and fish as sentinel organisms. Arch Environ Con Tox. 32: 198 - 203.

Morgan, E., Murphy, J., and Lyons, R. 1998. Imposex in Nucella lapillus from TBT contamination in south and south-west Wales: A continuing problem around ports. Mar Pollut Bull. 36: 840 - 843.

Morishita, F., Minakata, H., Takeshige, K., Furukawa, Y., Takata, T., Matsushima, O., Mukai, S. T., Saleuddin, A. S. M., and Horiguchi, T. 2006. Novel excitatory neuropeptides isolated from a prosobranch gastropod, Thais clavigera: The molluscan counterpart of the annelidan GGNG peptides. Peptides. 27: 483 - 492.

Morley, N. J . 2006. Parasitism as a source of potential distortion in studies on endocrine disrupting chemicals in molluscs. Mar Pollut Bull. 52: 1330 - 1332.

Nakano, D., and Nishiwaki, S. 1992. Local Variation of Imposex in Thais clavigera (Prosobranchia: Muricidae). Jap J Malacol (Venus). 51: 79 - 87.

Nakata, H., Kobayashi, S., Hirayama, Y., and Sakai, Y. 2004. Contamination of organochlorines, polycyclic aromatic hydrocarbons and organotin in coastal shellfishes from the Ariake Sea and the effects of TBT on imposex induction in rock shells. Nippon Suisan Gakk. 70: 555 - 566. (Abstract only)

Nehring, S. 2000. Long-term changes in Prosobranchia (Gastropoda) abundances on the German North Sea coast: The role of the anti-fouling biocide tributyltin. J Sea Res. 43: 151 - 165.

Nias, D. J., McKillup, S. C., and Edyvane, K. S. 1993. Imposex in Lepsiella vinosa from southern Australia. Mar Pollut Bull. 26: 380 - 384.

Nichols, C. J. 2000. Tributyltin pollution: The catastrophe that never happened. Mar Pollut Bull. 40:713-713.

Nicholson, G. J., and Evans, S. M. 1997. Anthropogenic impacts on the stocks of the common whelk Buccinum undatum (L). Mar Environ Res. 44: 305 - 314.

Nicholson, G. J., Evans, S. M., Palmer, N., and Smith, R. 1998. The value of imposex in the dogwhelk Nucella lapillus and the common whelk Buccinum undatum as indicators of TBT contamination. Invertebr Reprod Dev. 34: 289 - 300. 304

Nishikawa, J. 2006. Imposex in marine gastropods may be caused by binding of organotins to Retinoid X Receptor. Mar Biol. 149: 117 - 124.

Nishikawa, J., Mamiya, S., Kanayama, T., Nishikawa, T., Shiraishi, F., and Horiguchi, T. 2004. Involvement of the Retinoid X Receptor in the development of imposex caused by organotins in gastropods. Environ Sci Technol. 38: 6271 - 6276.

Nohara, M. 1999. Variation and abnormality of genital system in Littorina sitkana philippi (Mollusca, Gastropoda) in Northern Japan. Zool Sci. 16: 827 - 838.

Oberdorster, E. 1997. Sublethal effects of tributyltin on aquatic invertebrate models: Steroid metabolism, protein alterations and physiological responses. PhD. Thesis, Duke University, North Carolina.

Oberdorster, E. 2001. Peptide hormones versus steroid hormones: Case studies from snail and turtle populations. Ann NY Acad Sci. 948: 75 - 79.

Oberdorster, E., and Cheek, A. O. 2000. Gender benders at the beach: Endocrine disruption in marine and estuarine organisms. Environ Toxicol Chem. 20: 23 -36.

Oberdorster, E., and McClellan-Green, P. 2000. The neuropeptide APGWamide induces imposex in the mud snail, Ilyanassa obsoleta. Peptides. 21: 1323 - 1330.

Oberdorster, E., and McClellan-Green, P. 2002. Mechanisms of imposex induction in the mud snail, Ilyanassa obsoleta: TBT as a neurotoxin and aromatase inhibitor. Mar Environ Res. 54:715-718.

Oberdorster, E., and McClellan-Green, P. 2003. Imposex snails have elevated neuropeptide hormone levels when dosed with TBT or induced in the field. In SETAC 24th Annual Meeting (November 9- 13, 2003), Austin Texas,

Oberdorster, E., and McClellan-Green, P. 2004. Expression of the peptide hormone APGWamide in imposex and normal snails: A neurotoxicity mechanism for imposex induction. In Annual Meeting of the Society for Integrative and Comparative Biology, New Orleans. 43: 893.

Oberdorster, E., Rittschof, D., and McClellan-Green, P. 1998. Testosterone metabolism in imposex and normal Ilyanassa obsoleta: Comparison of field and TBTA Cl-induced imposex. Mar Pollut Bull. 36: 144 - 151. 305

Oberdorster, E., Romano, J., and McClellan-Green, P. 2005. The neuropeptide APGWamide as a penis morphogenic factor (PMF) in gastropod mollusks. Integr Comp Biol. 45: 28 - 32.

Oehlmann, J. 1998. Untersuchungen zum Einsatz von Pathomorphosen der ableitenden Geschlechtswege von Vorderkiemerschnecken (Gastropoda: Prosobranchia) fur ein biologisches TBT-Effektmonitoring [Habilitation thesis]. Zittau, Germany: International Graduate School Zittau. 162 pp.

Oehlmann, J. 2004. Biological effects of contaminants: Use of intersex in the periwinkle (Littorina littorea) as a biomarker of tributyltin pollution. ICES Techniques in Marine Environmental Sciences. 37: 1 - 22.

Oehlmann, J., and Bettin, C. 1996. Tributyltin-induced imposex and the role of steroids in marine snails. Malacol Rev Suppl - Molluscan Reproduction. 6: 157-161.

Oehlmann, J., and Schulte-Oehlmann, U. 2003a. Endocrine disruption in invertebrates. Pure Appl Chem. 75: 2207 - 2218.

Oehlmann, J., and Schulte-Oehlmann, U. 2003b. Molluscs as bioindicators. In Bioindicators and biomonitors: Principles, concepts and applications" (Edited by B. A. Markert, A. M. Breure, H. G. Zechmeister), Elsevier, Amsterdam, pp. 577 - 635.

Oehlmann, J., Stroben, E., and Fioroni, P. 1991. The morphological expression of imposex in Nucella lapillus (L.) (Gastropoda, Muricidae). J Mollus Stud. 57: 375 - 390.

Oehlmann, J., Stroben, E., and Fioroni, P. 1992a. The rough tingle Ocenebra erinacea (Neogastropoda, Muricidae) - An exhibitor of imposex in comparison to Nucella lapillus. Helgolander Meeresun. 46: 311 - 328.

Oehlmann, J., Stroben, E., Bettin, C., and Fioroni, P. 1992b. Hormonal disorders and tributyltin-induced imposex in marine snails. In Proceedings of the 27th European Marine Biology Symposium {Edited by J.C. Aldrich), Dublin, Ireland, pp. 301 - 305.

Oehlmann, J., Stroben, E., and Fioroni, P. 1993. Frequence et degree d'expression du pseudohermaphrodisme chez quelques Prosobranches Stenoglosses des cotes franfaises (surtout de la baie de Morlaix et de la Manche). 2. Situation jusqu' au printemps de 1992*. Cah Biol Mar. 34: 343 - 362.

Oehlmann, J., Liebe, S., Watermann, B., Stroben, E., Fioroni, P., and Deutsch, U. 1994a. New perspectives of sensitivity of Littorinids to TBT pollution. Cah Biol Mar. 35: 254 - 255. 306

Oehlmann, J., Schulte-Oehimann, U., Fioroni, P., and Stroben, E. 1994b. Probable extinction of prosobranch populations as a result of TBT pollution in the Bay of Morlaix. Cah Biol Mar. 35: 255 - 256.

Oehlmann, J., Stroben, E., Schulte-Oehimann, U., Bauer, B., Fioroni, P., and Markert, B. 1996a. Tributyltin biomonitoring using prosobranchs as sentinel organisms. Fresen J Anal Chem. 354: 540 - 545.

Oehlmann, J., Stroben, E., and Fioroni, P. 1996b. New facts about tributyltin- induced imposex in Prosobranchs: General aspects. Malacol Rev Suppl - Molluscan Reproduction. 6: 149 - 156.

Oehlmann, J., Fioroni, P., Stroben, E., and Markert, B. 1996c. Tributyltin (TBT) effects on Ocinebrina aciculata (Gastropoda: Muricidae): Imposex development, sterilization, sex change and population decline. Sci Total Environ. 188: 205 - 223.

Oehlmann, J., Schulte-Oehimann, U., Stroben, E., Bauer, B., Bettin, C., Fioroni, P., and Markert, B. 1996d. Androgenic effects of organotin compounds in Molluscs. Umweltbundesamt, Berlin.

Oehlmann, J., Stroben, E., Schulte-Oehimann, U., and Bauer, B. 1998a. Imposex development in response to TBT pollution in Hinia incrassata (Strom, 1768) (Prosobranchia, Stenoglossa). Aquat Toxicol. 43: 239 - 260.

Oehlmann, J., Bauer, B., Minchin, D., Schulte-Oehimann, U., Fioroni, P., and Markert, B. 1998b. Imposex in Nucella lapillus and intersex in Littorina littorea: interspecific comparison of two TBT-induced effects and their geographical uniformity. Hydrobiol. 378: 199 - 213.

Oehlmann, J., Schulte-Oehimann, U., Tillmann, M., and Markert, B. 2000. Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part I: Bisphenol A and octylphenol as xeno-estrogens. Ecotoxicol. 9: 383 - 397.

Oehlmann, J., Schulte-Oehimann, U., Bachmann, J., Oetken, M., Lutz, I., Kloas, W., and Ternes, T. A. 2006. Bisphenol A induces superfeminization in the ramshorn snail Marisa cornuarietis (Gastropoda: Prosobranchia) at environmentally relevant concentrations. Environ Health Persp. 114: 127 - 133.

Oehlmann, J., Di Benedetto, P., Tillmann, M., Duft, M., Oetken, M., and Schulte- Oehimann, U. 2007. Endocrine disruption in prosobranch molluscs: Evidence and ecological relevance. Ecotoxicol. 16: 29-43. 307

Oetken, M., Bachmann, J., Schulte-Oehlmann, U., and Oehlmann, J. 2004. Evidence for endocrine disruption in invertebrates. In Int Rev Cytol, vol. 236. International Review of Cytology- A Survey of Cell Biology, pp. 1- 44.

Oliveira, I. B., Richardson, C. A., Sousa, A. C., Takahashi, S., Tanabe, S., and Barroso, C. M. 2009. Spatial and temporal evolution of imposex in dogwhelk Nucella lapillus (L.) populations from North Wales, UK. J Environ Monitor. 11: 1462- 1468.

Omae, I. 2003. Organotin antifouling paints and their alternatives. Appl Organomet Chem. 17: 81 -105.

Osorio, C. R., and Huaquin, L. G. 2003. Alteration de la sexualidad de Acanthina monodon (Pallas, 1744) (Gastropoda: Muricidae) en el litoral de Chile central, inducida por compuestos organoestafiosos. Ciencia Technolgia del Mar. 26: 97- 107.

OSPAR 2008. JAMP Guidelines for Contaminant-Specific Biological Effects (OSPAR Agreement 2008-09). In OSPAR Convention for the Protection of the Marine Environment of the North-East Atlantic.

Pandey, E., and Evans, S. M. 1996. The incidence of imposex in gastropods from Indonesian coastal waters. Asian Mar Biol. 13: 53 - 61.

Pattisina, L. A. 1991. Development of a protocol for imposex measures on tropical neogastropod molluscs. MSc. Thesis, University of Victoria, British Columbia.

Pavoni, B., Centanni, E., Valcanover, S., Fasolato, M., Ceccato, S., and Tagliapietra, D. 2007. Imposex levels and concentrations of organotin compounds (TBT and its metabolites) in Nassarius nitidus from the Lagoon of Venice. Mar Pollut Bull. 55: 505 -511.

Pellizzato, F., Centanni, E., Marin, M. G., Moschino, V., and Pavoni, B. 2004. Concentrations of organotin compounds and imposex in the gastropod Hexaplex trunculus from the Lagoon of Venice. Sci Total Environ. 332: 89 - 100.

Pena, J., and Kendall, Y. M. 1989. Imposex en Nucella lapillus como indicador de contaminacion par TBT en el norte de la Penisula Iberica. In Bentos VI: Proceedings of Simposo Iberico de Estudio del Bentos Marino, vol. 6, Palma de Mallorca. pp. 455 - 463.

Penchaszadeh, P. E., Averbuj, A., and Cledon, M. 2001. Imposex in gastropods from Argentina (south-western Atlantic). Mar Pollut Bull. 42: 790 - 791. 308

Penchaszadeh, P. E., Antelo, C. S., Zabala, S., and Bigatti, G. 2009. Reproduction and imposex in the edible snail Adelomelon ancilla from northern Patagonia, Argentina. Mar Biol. 156: 1929 - 1939.

Pessoa, M. F., Fernando, A., and Oliveira, J. S. 2001. Use of imposex (Pseudohermaphroditism) as indicator of the occurrence of organotin compounds in Portuguese coastal waters - Sado and Mira Estuaries. Environ Toxicol. 16: 234-241.

Phillip, O. S. 2000. The relationship between masculinized female conch (Strombus gigas) and tributyltin-based antifouing paint in coastal waters of the British Virgin Islands. MSc. Thesis, University of Plymouth, United Kingdom.

Picado, A., Bebianno, M. J., Costa, M. H., Ferreira, A., and Vale, C. 2007. Biomarkers: A strategic tool in the assessment of environmental quality of coastal waters. Hydrobiol. 587: 79 - 87.

Pinheiro, F. M., Fernandez, M. A., Fragoso, M. R., Quadros, J. P., Camillo, E., and Dos Santos, F. A. 2006. Assessing the impacts of organotin compounds in Ilha Grande Bay, (Rio de Janeiro, Brazil): Imposex and a multiple-source dispersion model. J Coastal Res. 39: 1383 - 1388.

Plejdrup, J. K., Simonsen, V., Pertoldi, C., Schoyen, M., and Bayley, M. 2006. Genetic and morphological diversity in populations of Nucella lapillus (L.; Neogastropoda) in response to tributyltin contamination. Ecotox Environ Safe. 64: 146- 154.

Poli, G., Salvat, B., and Streiff, W. 1971. Aspect particulier de la sexualite chez Ocenebra erinacea. Haliotis. 1: 29 - 30.

Poloczanska, E. S., and Ansell, A. D. 1999. Imposex in the whelks Buccinum undatum and Neptunea antiqua from the west coast of Scotland. Mar Environ Res. 47: 203 - 212.

Pondrick, J. S. 1981. A penis-like structure in female Nucella lapillus from New England, USA. In 4th Annual Meeting of the Malacological Union. B Am Malacol Union Inc, Fort Lauderdale. 50: 39 - 40.

Pondrick, J. S. 1985. Aspects of the biology of the marine gastropod, Nucella lapillus (L.), and its larval Digenea (Parasite, Snail, Trematode, New England). PhD. Thesis, University of Connecticut, Connecticut.

Porte, C., Janer, G., Lorusso, L.C., Ortiz-Zarragoitia, M., Cajaraville, M. P., Fossi, M. C., and Canesi, L. 2006. Endocrine disruptors in marine organisms: Approaches and perspectives. Comp Biochem Phys C. 143: 303 - 315. 309

Power, A. J., and Keegan, B. F. 2001. The significance of imposex levels and TBT contamination in the red whelk, Neptunea antiqua (L.) from the offshore Irish Sea. Mar Pollut Bull. 42: 761 - 772.

Prime, M., Peharda, M., Jelic, K., Mladineo, I., and Richardson, C. A. 2006. The occurrence of imposex in Hexaplex trunculus from the Croatian Adriatic. Mar Pollut Bull. 52:810-812.

Prouse, N. J., and Covert, T. J. 1997. Survey of eastern Canada for imposex, a bioindicator of tributyltin (TBT), in the dogwhelk, Nucella lapillus. Can Tech Rep Fish Aquat Sci, 2163: 44.

Prouse, N. J., and Ellis, D. V. 1997. A baseline survey of dogwhelk (Nucella lapillus) imposex in eastern Canada (1995) and interpretation in terms of tributyltin (TBT) contamination. Environ Technol. 18: 1255 - 1264.

Quadros, J. P., Camillo, E., Pinheiro, F., and Fernandez, M. A. S. 2009. Imposex as an indicator of organotin pollution at Rio de Janeiro South Coast: Sepetiba and Ilha Grande Bays. Thalassas. 25: 19 - 30.

Queiroz, L. R., Castro, I. B., and Rocha-Barreira, C. A. 2007. New imposex development index (IDI) for Stramonita haemastoma (Mollusca: Muricidae): A transplantation experiment in the Brazilian northeast. J Braz Soc Ecotoxicol. 2: 249 - 256.

Quintela, M., Barreiro, R., and Ruiz, J. M. 2000. The use of Nucella lapillus (L.) transplanted in cages to monitor tributyltin (TBT) pollution. Sci Total Environ. 247: 227 - 237.

Quintela, M., Barreiro, R., and Ruiz, J. M. 2002. Dumpton Syndrome reduces the tributyltin (TBT) sterilising effect on Nucella lapillus (L.) by limiting the development of the imposed vas deferens. Mar Environ Res. 54: 657 - 660.

Quintela, M., Couceiro, L., Ruiz, J. M., and Barreiro, R. 2006a. Discovery of imposex in the gastropod Cyclope neritea now invading Galicia (north-west Spain). J Mar Biol Assoc UK. 86: 1171 - 1173.

Quintela, M., Couceiro, L., Ruiz, J. M., and Barreiro, R. 2006b. Imposex in the marine gastropod Nucella lapillus in Galicia (NW Iberian Peninsula): An eight-year re-survey (1996-2003). Mar Environ Res. 62: S236 - S237.

Quintela, M., Couceiro, L., Ruiz, J. M., and Barreiro, R. 2008. Imposex in Nucella lapillus (L.): 10 years of monitoring TBT pollution in Galicia (NW Spain). In SETAC - 18th European Conference, Warsaw. 310

Ramasamy, M. S., and Murugan, A. 2002. Imposex in Muricid gastropod Thais biserialis (Mollusca: Neogastropoda: Muricidae) from Tuticorin harbour, southeast coast of India. Indian J Mar Sci. 31: 243 - 245.

Ramon, M., and Amor, M. J. 2001. Increasing imposex in populations of Bolinus brandaris (Gastropoda: Muricidae) in the north-western Mediterranean. Mar Environ Res. 52: 463 - 475.

Ramon, M., and Amor, M. J. 2002. Reproductive cycle of Bolinus brandaris and penis and genital duct size variations in a population affected by imposex. J Mar Biol Assoc UK. 82: 435 - 442.

Rato, M., Sousa, A., Quinta, R., Langston, W., and Barroso, C. 2006. Assessment of inshore/offshore tributyltin pollution gradients in the northwest Portugal continental shelf using Nassarius reticulatus as a bioindicator. Environ Toxicol Chem. 25: 3213 -3220.

Rato, M., Gaspar, M. B., Takahashi, S., Yano, S., Tanabe, S., and Barroso, C. 2008. Inshore/offshore gradients of imposex and organotin contamination in Nassarius reticulatus (L.) along the Portuguese coast. Mar Pollut Bull. 56: 1323 - 1331.

Rato, M., Ferreira, N., Santos, J., and Barroso, C. 2009a. Temporal evolution of imposex in Nassarius reticulatus (L.) along the Portuguese coast: The efficacy of EC regulation 782/2003. J Environ Monitor. 11: 100 - 107.

Rato, M., Russel-Pinto, F., and Barroso, C. 2009b. Assessment of Digenean parasitism in Nassarius Reticulatus (L.) along the Portuguese coast: Evaluation of possible impacts on reproduction and imposex expression. J Parasitol. 95: 327 - 336.

Reed, S. E. 1992. Histological comparison of masculinized females and androgynous males in the West Indian fighting conch, Strombus pugilis. J Shellfish Res. 11:205.

Reed, S. E. 1993a. Gonadal comparison of masculinized females and androgynous males to normal males and females in Strombus (Mesogastropoda, Strombidae). J Shellfish Res. 12: 71 - 75.

Reed, S. E. 1993b. Size differences between sexes (including masculinized females) in Strombus pugilis (Mesogastropoda: Strombidae). J Shellfish Res. 12: 77 - 79. 311

Reed, S. E. 1994. Masculinized females in the genus Strombus: Aspects of their biology and possible advantages for mariculture of conch. In Queen Conch Biology, Fisheries and Mariculture {Edited by R. S. Appeldoorn and B. Rodriguez), pp. 213 - 221.

Reed, S. E. 1995. Sexual tripmorphism in Strombus luhuanus Linne, 1758 (Mollusca: Gastropoda) at Shirahama, Japan. J Shellfish Res. 14: 159 - 160.

Rees, C. M., Brady, B. A., and Fabris, G. J. 2001. Incidence of imposex in Thais orbita from Port Phillip Bay (Victoria, Australia), following 10 years of regulation on use of TBT. Mar Pollut Bull. 42: 873 - 878.

Reid, D. G. 1986. Mainwaringia nevill, 1885, a Littorinid genus from Asiatic mangrove forests, and a case protandrous hermaphroditism. J Mollus Stud. 52: 225 - 242.

Reijnders, P. J. H., and Brasseur, S. 1997. Xenobiotic induced hormonal and associated developmental disorders in marine organisms and related effects in humans; An overview. J Clean Technol E T. 6: 367 - 380.

Reitsema, T. J., and Spickett, J. T. 1999. Imposex in Morula granulata as bioindicator of tributyltin (TBT) contamination in the Dampier Archipelago, western Australia. Mar Pollut Bull. 39: 280 - 284.

Reitsema, T. J., Thompson, J. A. J., Scholtens, P., and Spickett, J. T. 2002. Further recovery of northeast Pacific neogastropods from imposex related to tributyltin contamination. Mar Pollut Bull. 44: 257 - 261.

Rilov, C., Gasith, A., Evans, S. M., and Benayahu, Y. 2000. Unregulated use of TBT-based antifouling paints in Israel (eastern Mediterranean): High contamination and imposex levels in two species of marine gastropods. Mar Ecol Prog Ser. 192: 229 - 238.

Rilov, G., Benayahu, Y., and Gasith, A. 2001. Low abundance and skewed population structure of the whelk Stramonita haemastoma along the Israeli Mediterranean coast. Mar Ecol Prog Ser. 218: 189 - 202.

Ritsema, R., and Laane, R. 1991. Dissolved butyltins in fresh and marine waters of the Netherlands in 1989. Sci Total Environ. 105: 149 - 156.

Ritsema, R., Laane, R., and Donard, O. F. X. 1991. Butyltins in marine waters of the Netherlands in 1988 and 1989 - Concentrations and effects. Mar Environ Res. 32: 243 - 260.

Rittschof, D., and McClellan-Green, P. 2005. Molluscs as multidisciplinary models in environment toxicology. Mar Pollut Bull. 50: 369 - 373. 312

Roberts, R. D., and Forrest, B. M. 1999. Minimal impact from long-term dredge spoil disposal at a dispersive site in Tasman Bay, New Zealand. New Zeal J Mar Fresh. 33: 623 - 633.

Rodriguez, J. G., Belzunce, M. J., Borja, A., Franco, J., Garmendia, J. M., Gonzalez Herraiz, I., Muxika, I., Solaun, O., Tueros, I., and Valencia, V. 2007. Organotin bioaccumulation and imposex in female Nassarius reticulatus and N. nitidus in ports and marina areas of the Basque country. In ICES Annual Conference - ICES CM 2007/session I: 12, Helsinki, Finland.

Rodriguez, J. G., Tueros, I., Borja, A., Franco, J., Alonso, J. I. G., Garmendia, J. M., Muxika, I., Sariego, C., and Valencia, V. 2009a. Butyltin compounds, sterility and imposex assessment in Nassarius reticulatus (Linnaeus, 1758), prior to the 2008 European ban on TBT antifouling paints, within Basque ports and along coastal. Cont Shelf Res. 29: 1165 - 1173.

Rodriguez, J. G., Borja, A., Franco, J., Alonso, J. I. G., Garmendia, J. M., Muxika, I., Sariego, C., and Valencia, V. 2009b. Imposex and butyltin body burden in Nassarius nitidus (Jeffreys, 1867), in coastal waters within the Basque Country (northern Spain). Sci Total Environ. 407: 4333 - 4339.

Romano, J., and McClellan-Green, P. 2005. Tissue specific localization of APGWamide in TBT exposed mudsnail, Ilyanassa obsoleta. In SET AC 26th Annual Meeting (November 13 - 17, 2005), Baltimore, Maryland.

Ronis, M. J. J., and Mason, A. Z. 1996. The metabolism of testosterone by the periwinkle (Littorina littorea) in vitro and in vivo: Effects of tributyltin. Mar Environ Res. 42: 161 - 166.

Rotchell, J. M., and Ostrander, G. K. 2003. Molecular markers of endocrine disruption in aquatic organisms. J Toxicol Env Heal B. 6: 453 - 495.

Ruiz, J. M., Bachelet, G., Caumette, P., and Donard, O. F. X. 1996. Three decades of tributyltin in the coastal environment with emphasis on Arcachon Bay, France. Environ Pollut. 93: 195 - 203.

Ruiz, J. M., Quintela, M., and Barreiro, R. 1998a. Ubiquitous imposex and organotin bioaccumulation in gastropods Nucella lapillus from Galicia (NW Spain): A possible effect of nearshore shipping. Mar Ecol Prog Ser. 164: 237 - 244.

Ruiz, J. M., Quintela, M., and Barreiro, R. 1998b. Tributyltin and imposex: no uncertainty shown. Mar Ecol Prog Ser. 170: 293 - 294.

Ruiz, J. M., Barreiro, R., and Gonzalez, J. J. 2005. Biomonitoring organotin pollution with gastropods and mussels. Mar Ecol Prog Ser. 287: 169 - 176. 313

Santillo, D., Johnston, P., and Langston, W. J. 2002. Tributyltin (TBT) antifoulants: A tale of ships, snails and imposex. In The Precautionary Principle in the 20th Century: Late Lessons from Early Warnings {Edited by P. Harremoes and D. Gee). Earthscan Publications Limited, pp. 135 - 148.

Santos, M. M. 2006. Comments to "Imposex in Hexaplex (Trunculariopsis) trunculus (Gastropoda: Muricidae) from the Ria Formosa Lagoon (Algarve coast-southern Portugal)". Mar Pollut Bull. 52: 1312 -1313.

Santos, M. M., Vieira, N., and Santos, A. M. 2000. Imposex in the dogwhelk Nucella lapillus (L.) along the Portuguese coast. Mar Pollut Bull. 40: 643 - 646.

Santos, M. M., Ten Hallers-Tjabbes, C. C., Santos, A. M., and Vieira, N. 2002a. Imposex in Nucella lapillus, a bioindicator for TBT contamination: Re- survey along the Portuguese coast to monitor the effectiveness of EU regulation. J Sea Res. 48: 217 -223.

Santos, M. M., Ten Hallers-Tjabbes, C. C., Vieira, N., Boon, J. P., and Porte, C. 2002b. Cytochrome P450 differences in normal and imposex-affected female whelk Buccinum undatum from the open North Sea. Mar Environ Res. 54: 661 - 665.

Santos, M. M., Vieira, N., Reis-Henriques, M. A., Santos, A. M., Gomez-Ariza, J. L., Giraldez, I., and Ten Hallers-Tjabbes, C. C. 2004. Imposex and butyltin contamination off the Oporto Coast (NW Portugal): A possible effect of the discharge of dredged material. Environ Int. 30: 793 - 798.

Santos, M. M., Castro, L. F. C., Vieira, M. N., Micael, J., Morabito, R., Massanisso, P., and Reis-Henriques, M. A. 2005. New insights into the mechanism of imposex induction in the dogwhelk Nucella lapillus. Comp Biochem Phys C. 141: 101 -109.

Santos, M. M., Reis-Henriques, M. A., Vieira, M. N., and Sole, M. 2006. Triphenyltin and tributyltin, single and in combination, promote imposex in the gastropod Bolinus brandaris. Ecotox Environ Safe. 64: 155 - 162.

Santos, M. M., Reis-Henriques, M. A., Guillot, R., Lima, D., Franco-Duarte, R., Mendes, I., Queiros, S., and Castro, L. F. C. 2008. Anti-androgenic effects of sewage treatment plant effluents in the prosobranch gastropod Nucella lapillus. Comp Biochem Phys C. 148: 87-93. 314

Schipper, C. A., Smit, M. G. D., Kaag, N., and Vethaak, A. D. 2008. A weight-of- evidence approach to assessing the ecological impact of organotin pollution in Dutch marine and brackish waters; combining risk prognosis and field monitoring using common periwinkles (.Littorina littored). Mar Environ Res. 66:231 -239.

Schulte-Oehlmann, U., Oehlmann, J., and Stroben, E. 1992. Tributyltin-induced imposex of Nucella lapillus on Irish coasts in 1990. In Proceedings of the 27th European Marine Biology Symposium. Dublin, Ireland, pp. 307 - 311.

Schulte-Oehlmann, U., Fioroni, P., Oehlmann, J., and Stroben, E. 1994. The genital system of Marisa cornuarietis (Gastropoda, Ampullariidae) - A morphological and histological analysis. Zool Beitr. 36: 59 - 81.

Schulte-Oehlmann, U., Bettin, C., Fioroni, P., Oehlmann, J., and Stroben, E. 1995. Marisa cornuarietis (Gastropoda, Prosobranchia) - A potential TBT bioindicator for fresh-water environments. Ecotoxicol. 4: 372 - 384.

Schulte-Oehlmann, U., Oehlmann, J., Fioroni, P., and Bauer, B. 1997. Imposex and reproductive failure in Hydrobia ulvae (Gastropoda: Prosobranchia). Mar Biol. 128: 257 - 266.

Schulte-Oehlmann, U., Oehlmann, J., Bauer, B., Fioroni, P., and Leffler, U. S. 1998. Toxico-kinetic and dynamic aspects of TBT-induced imposex in Hydrobia ulvae compared with intersex in Littorina littorea (Gastropoda, Prosobranchia). Hydrobiol. 378: 215 -225.

Schulte-Oehlmann, U., Tillmann, M., Markert, B., Oehlmann, J., Watermann, B., and Scherf, S. 2000. Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part II: Triphenyltin as a xeno- androgen. Ecotoxicol. 9: 399 - 412.

Schulte-Oehlmann, U., Tillman, M., Casey, D., Duft, M., Markert, B., and Oehlmann, J. 2001. Xeno-estro effects on Prosobranchs (Review). Originalarbeiten. 13: 319 - 333.

Sekizawa, J., Suter, G., and Birnbaum, L. 2003. Integrated human and ecological risk assessment: A case study of tributyltin and triphenyltin compounds. Hum Ecol Risk Assess. 9: 325 - 342.

Shelmerdine, R. L., Adamson, J., Laurenson, C. H., and Leslie, B. 2007. Size variation of the common whelk, Buccinum undatum, over large and small spatial scales: Potential implications for micro-management within the fishery. Fish Res. 86: 201 - 206. 315

Shi, H. H., and Huang, C. J. 2001a. Tributyltin (TBT) pollution and imposex in marine gastropods. Acta Ecol Sin. 21: 1711 -1717. (Abstract only).

Shi, H. H., and Huang, C. J. 2001b. Morphological characters of imposex in marine gastropod. J Oceanogr Taiwan Strait. 20:552-555.

Shi, H. H., Huang, C. J., and Xie, W. Y. 2002. Application of imposex in Thais clavigera to monitoring of marine organotin compound pollution. Mar Environ Sci. 21: 37 - 41.

Shi, H. H., Huang, C. J., Yu, X. J., and Zhu, S. X. 2004a. Imposex and female reproductive failure of Chicoreus brunneus and C. asianus. J Trop Oceanogr. 23: 82 - 87.

Shi, H. H., Huang, C. J., Yu, X. J., Zhu, S. X., and Zhang, Y. B. 2004b. Development of imposex and structural effect on males in three Nassarius spp. Mar Sci (Haiyang Kexue). 28: 36 - 41. (Abstract only).

Shi, H. H., Huang, C. J., Yu, X. J., and Zhu, S. X. 2005a. An updated scheme of imposex for Cantharus cecillei (Gastropoda: Buccinidae) and a new mechanism leading to the sterilization of imposex-affected females. Mar Biol. 146: 717- 723.

Shi, H. H„ Huang, C. J., Zhu, S. X., Yu, X. J., and Xie, W. Y. 2005b. Generalized system of imposex and reproductive failure in female gastropods of coastal waters of mainland China. Mar Ecol Prog Ser. 304: 179 - 189.

Shim, W. J., Kahng, S. H., Hong, S. H., Kim, N. S., Kim, S. K., and Shim, J. H. 2000. Imposex in the rock shell, Thais clavigera, as evidence of organotin contamination in the marine environment of Korea. Mar Environ Res. 49: 435 -451.

Shim, W. J., Shin, Y. B., Kim, N. S., Oh, J. R., and Jeon, J. K. 2002. Induction of imposex in rock Shell, Thais clavigera, exposed to tributyltin and other antagonistic chemicals. In SETAC 23rd Annual Meeting (November 16-20, 2002), Salt Lake City, Utah.

Short, J. W., Rice, S. D., Brodersen, C. C., and Stickle, W. B. 1989. Occurrence of tri-w-butyltin-caused imposex in the North-Pacific marine snail Nucella lima in Auke Bay, Alaska. Mar Biol. 102: 291 - 297.

Siewers, R., and McClelland, P. 2001. Affect of mixed contaminants on reproduction and metabolism in mudsnails (Ilyanassa obsoleta). In SETAC - 22nd Meeting (November 11- 15,2001), Baltimore, Maryland. 316

Skarphedinsdottir, H., Olafsdottir, K., Svavarsson, J., and Johannesson, T. 1996. Seasonal fluctuations of tributyltin (TBT) and dibutyltin (DBT) in the dogwhelk, Nucella lapillus (L.), and the blue mussel, Mytilus edulis L., in Icelandic waters. Mar Pollut Bull. 32: 358-361.

Sloan, S., and Gagnon, M. M. 2004. Intersex in roe's abalone (Haliotis roei) in western Australia. Mar Pollut Bull. 49: 1122 - 1126.

Smith, A. J., Thain, J. E., and Barry, J. 2006. Exploring the use of caged Nucella lapillus to monitor changes to TBT hotspot areas: A trial in the River Tyne Estuary (UK). Mar Environ Res. 62: 149 - 163.

Smith, B. S. 1971. Sexuality in the American mud-snail, Nassarius obsoletus. Proc Malacol Soc Lond. 39: 377 - 378.

Smith, B. S. 1980. The estuarine mudsnail, Nassarius obsoletus - Abnormalities in the reproductive system. J Mollus Stud. 46: 247 - 256.

Smith, B. S. 1981a. Reproductive anomalies in stenoglossan snails related to pollution from marinas. J Appl Toxicol. 1: 15-21.

Smith, B. S. 1981b. Male characteristics on female mud snails caused by antifouling bottom paints. J Appl Toxicol. 1: 22 - 25.

Smith, B. S. 1981c. Tributyltin compounds induce male characteristics on female mud snails Nassarius obsoletus - Ilyanassa obsoleta. J Appl Toxicol. 1: 141 - 144.

Smith, B. S. 198Id. Male characteristics in the female Nassarius obsoletus: Variations related to locality. Veliger. 23: 212 - 216.

Smith, P. J. 1996. Selective decline in imposex levels in the dogwhelk Lepsiella scobina following a ban on the use of TBT antifoulants in New Zealand. Mar Pollut Bull. 32: 362 - 365.

Smith, P. J., and McVeagh, M. 1991. Widespread organotin pollution in New Zealand coastal waters as indicated by imposex in dogwhelks. Mar Pollut Bull. 22:409-413.

Sole, M., Morcillo, Y., and Porte, C. 1998. Imposex in the commercial snail Bolinus brandaris in the northwestern Mediterranean. Environ Pollut. 99: 241 - 246.

Son, M. H., and Hughes, R. N. 2000. Relationship between imposex and morphological variation of the shell in Nucella lapillus (Gastropoda: Thaididae). Estuar Coast Shelf Sci. 50: 599 - 606. 317

Sonak, S., Pangam, P., Giriyan, A., and Hawaldar, K. 2009. Implications of the ban on organotins for protection of global coastal and marine ecology. J Environ Manage. 90: S96 - SI08.

Sousa, A., Mendo, S., and Barroso, C. 2005. Imposex and organotin contamination in Nassarius reticulatus (L.) along the Portuguese coast. Appl Organomet Chem. 19:315 -323.

Sousa, A., Matsudaira, C., Takahashi, S., Tanabe, S., and Barroso, C. 2007. Integrative assessment of organotin contamination in a southern European estuarine system (Ria de Aveiro, NW Portugal): Tracking temporal trends in order to evaluate the effectiveness of the EU ban. Mar Pollut Bull. 54: 1645 - 1653.

Spence, S. K. 1989. Ecotoxicological studies of tributyltin (TBT), using the indicator species Nucella lapillus (L.). PhD. Thesis, University of Liverpool, United Kingdom.

Spence, S. K., Bryan, G. W., Gibbs, P. E., Masters, D., Morris, L., and Hawkins, S. J. 1990a. Effects of TBT contamination on Nucella populations. Funct Ecol. 4: 425 - 432.

Spence, S. K., Hawkins, S. J., and Santos, R. S. 1990b. The Mollusk Thais haemastoma - an exhibitor of imposex and potential biological indicator of tributyltin pollution. Mar Ecol P S Z N 1.11: 147 - 156.

Spooner, N., Gibbs, P. E., Bryan, G. W., and Goad, L. J. 1991. The effect of tributyltin upon steroid titers in the female dogwhelk, Nucella lapillus, and the development of imposex. Mar Environ Res. 32: 37 - 49.

Sternberg, R. M. 2007. The roles of androgen, estrogen, and retinoid signaling in reproductive recrudescence of the eastern mud snail (Ilyanassa obsoleta): Implications for the mechanism of tributyltin-induced imposex. PhD. Thesis, North Carolina State University, North Carolina.

Sternberg, R. M., Gooding, M., and LeBlanc, G. 2004. The mechanism behind the association of tributyltin exposure with high testosterone in prosobranch gastropods: Insights. In 25th Annual SETAC Meeting (November 14 - 18, 2004), Portland, Oregon.

Sternberg, R. M., and LeBlanc, G. A. 2006. Kinetic characterization of the inhibition of acyl coenzyme A: Steroid acyltransferases by tributyltin in the eastern mud snail (.Ilyanassa obsoleta). Aquat Toxicol. 78: 233 - 242. 318

Sternberg, R. M., Hotchkiss, A. K., and Leblanc, G. A. 2008a. Synchronized expression of Retinoid X Receptor mRNA with reproductive tract recrudescence in an imposex-susceptible mollusc. Environ Sci Technol. 42: 1345 - 1351.

Sternberg, R. M., Hotchkiss, A. K., and LeBlanc, G. A. 2008b. The contribution of steroidal androgens and estrogens to reproductive maturation of the eastern mud snail Hyanassa obsoleta. Gen Comp Endocr. 156: 15 -26.

Sternberg, R. M., Gooding, M. P., Hotchkiss, A. K., and LeBlanc, G. A. 2010. Environmental-endocrine control of reproductive maturation in gastropods: Implications for the mechanism of TBT-induced imposex in prosobranchs. Ecotoxicol. 19: 14-23.

Stewart, C., de Mora, S. J., Jones, M. R. L., and Miller, M. C. 1992. Imposex in New Zealand neogastropods. Mar Pollut Bull. 24: 204 - 209.

Stickle, W. B., and Zhang, Z. Y. 2003. Long-term trends in imposex in six populations of Stramonita haemastoma. B Mar Sci. 72: 685 - 694.

Stickle, W., Short, J. W., Rice, S. D., and Brodersen, C. C. 1988. Imposex exists in Alaska, too. Am Zool. 28: A161- A161.

Stickle, W. B., Sharpdahl, J. L., Rice, S. D., and Short, J. W. 1990. Imposex induction in Nucella lima (Gmelin) via mode of exposure to tributyltin. J Exp Mar Biol Ecol. 143: 165 - 180.

Strand, J., and Asmund, G. 2003. Tributyltin accumulation and effects in marine molluscs from West Greenland. Environ Pollut. 123: 31 - 37.

Strand, J., and Jacobsen, J. A. 2002. Imposex in two sublittoral neogastropods from the Kattegat and Skagerrak: The common whelk Buccinum undatum and the red whelk Neptunea antiqua. Mar Ecol Prog Ser. 244: 171 - 177.

Strand, J., Jacobsen, J. A., Pedersen, B., and Granmo, A. 2003. Butyltin compounds in sediment and molluscs from the shipping strait between Denmark and Sweden. Environ Pollut. 124: 7-15.

Strand, J., Glahder, C. A., and Asmund, G. 2006. Imposex occurrence in marine whelks at a military facility in the high Arctic. Environ Pollut. 142: 98 - 102.

Strand, J., Jorgensen, A., and Tairova, Z. 2009. TBT pollution and effects in molluscs at US Virgin Islands, Caribbean Sea. Environ Int. 35: 707 - 711.

Straw, J., and Rittschof, D. 2004. Responses of mud snails from low and high imposex sites to sex pheromones. Mar Pollut Bull. 48: 1048 - 1054. 319

Stroben, E. 1996. The organotin pollution at the Bay of Morlaix with special reference to biomonitoring with prosobranchs. Malacol Rev Suppl - Molluscan Reproduction. 6: 163 - 171.

Stroben, E., Oehlmann, J., and Fioroni, P. 1989. Der pseudohermaphroiditismus bei Nucella lapillus (Prosobranchia, Steneoglossa), eine morphologische und histologische analyse. Zool Beitr. 32: 1-16.

Stroben, E., Oehlmann, J., and Fioroni, P. 1992a. The morphological expression of imposex in Hinia reticulata (Gastropoda, Buccinidae) - A potential indicator of tributyltin pollution. Mar Biol. 113: 625 - 636.

Stroben, E., Oehlmann, J., and Fioroni, P. 1992b. Hinia reticulata and Nucella lapillus - Comparison of two gastropod tributyltin bioindicators. Mar Biol. 114: 289 - 296.

Stroben, E., Brommel, C., Oehlmann, J., and Fioroni, P. 1992c. The genital systems of Trivia arctica and Trivia monacha (Prosobranchia, Mesogastropoda) and tributyltin induced imposex. Zool Beitr. 34: 349 - 374.

Stroben, E., Schulte-Oehlmann, U., Fioroni, P., and Oehlmann, J. 1995. A comparative method for easy assessment of coastal TBT pollution by the degree of imposex in prosobranch species. Haliotis. 24: 1-12.

Stroben, E., Oehlmann, J., Schulte-Oehlmann, U., and Fioroni, P. 1996. Seasonal variations in the genital ducts of normal and imposex-affected prosobranchs and its influence on biomonitoring indices. Malacol Rev Suppl - Molluscan Reproduction. 6: 173 - 184.

Sundermann, G., Bauer, B., and Oehlmann, J. 1998. Ultrastructure of prostate gland tissue in males and females with intersex phenomena of Littorina littorea L. Hydrobiol. 378: 227 - 233.

Svavarsson, J. 2000. Imposex in the dogwhelk (Nucella lapillus) due to TBT contamination: Improvement at high latitudes. Mar Pollut Bull. 40: 893 - 897.

Svavarsson, J., and Skarphedinsdottir, H. 1995. Imposex in the dogwhelk Nucella lapillus (L.) in Icelandic waters. Sarsia. 80: 35 - 40.

Svavarsson, J., Granmo, A., Ekelund, R., and Szpunar, J. 2001. Occurrence and effects of organotins on adult common whelk (Buccinum undatum) (Mollusca, Gastropoda) in harbours and in a simulated dredging situation. Mar Pollut Bull. 42: 370 - 376. 320

Swennen, C., and Horpet, P. 2008. Pseudo-imposex; male features in female volutes not TBT-induced (Gastropoda: Volutidae). Contrib Zool. 77: 17 - 24.

Swennen, C., Ruttanadakul, N., Ardseungnern, S., Singh, H. R., Tan, K. S., Mensink, B. P., and Ten Hallers-Tjabbes, C. C. 1996. Sexual aberrations in gastropods in Gulf of Thailand and Strait of Malacca in relation to shipping. Wallaceana. 78: 1 -13.

Swennen, C., Ruttanadakul, N., Ardseungnern, S., Singh, H. R., Mensink, B. P., and Ten Hallers-Tjabbes, C. C. 1997. Imposex in sublittoral and littoral gastropods from the Gulf of Thailand and Strait of Malacca in relation to shipping. Environ Technol. 18: 1245 - 1254.

Swennen, C., Sampantarak, U., and Ruttanadakul, N. 2009. TBT-pollution in the Gulf of Thailand: A re-inspection of imposex incidence after 10 years. Mar Pollut Bull. 58: 526 - 532.

Syasina, I. G., and Shcheblykina, A. V. 2007. Morphofunctional characterization of the reproductive system of the gastropods Littorina brevicula, L. mandshurica, and Nucella heyseana from uncontaminated and contaminated areas of Peter the Great Bay, Sea of Japan. Russ J Mar Biol. 33: 399 - 404.

Takeda, N. 2000. Development of a penis from the vestigial penis in the female apple snail, Pomacea canaliculata. Biol Bull. 199: 316 - 320.

Takeda, N. 2001. Induction of Imposex by plasticizers in the freshwater snail, Pomacea canaliculata. In Abstracts of the 15th ICCE. International Congress of Comparative Endocrinology, Sorrento (Napoli), Italy, pp. P14.19.

Takeda, N. 2006. Induction of imposex by plasticizers in the freshwater snail, Pomacea canaliculata. J Exp Zool. 305A: 183.

Tallmon, D. A., and Hoferkamp, L. 2009. Long-term changes in imposex frequency in file dogwinkles, Nucella lima G., and tributyltin concentrations in bay mussels, Mytilus trossulus G. B Environ Contam Tox. 83: 235 - 238.

Tan, K. S. 1997. Imposex in three species of Thais from Singapore, with additional observations on T. clavigera (Kuster) from Japan. Mar Pollut Bull. 34: 577 - 581.

Tan, K. S. 1999. Imposex in Thais gradata and Chicoreus capucinus (Mollusca, Neogastropoda, Muricidae) from the Straits of Johor: A case study using penis length, area and weight as measures of imposex severity. Mar Pollut Bull. 39: 295 - 303. 321

Ten Hallers-Tjabbes, C. C. 1997. Tributyltin and policies for antifouling. Environ Technol. 18: 1265 - 1268.

Ten Hallers-Tjabbes, C. C., and Boon, J. P. 1995a. Buccinum undatum L. in the North Sea, state of whelks and imposex phenomena. Mar Environ Res. 39: 354-355.

Ten Hallers-Tjabbes, C. C., and Boon, J. P. 1995b. Whelks (Buccinum undatum L), dogwhelks (Nucella lapillus L) and TBT - A cause for confusion. Mar Pollut Bull. 30: 675 - 676.

Ten Hallers-Tjabbes, C. C., Kemp, J. F., and Boon, J. P. 1994. Imposex in whelks {Buccinum undatum) from the open North Sea - Relation to shipping traffic intensities. Mar Pollut Bull. 28: 311 - 313.

Ten Hallers-Tjabbes, C. C., Everaarts, J. M., Mensink, B. P., and Boon, J. P. 1996. The decline of the North Sea whelk {Buccinum undatum L) between 1970 and 1990: A natural or a human-induced event? Mar Ecol-P S Z N I. 17: 333 - 343.

Ten Hallers-Tjabbes, C. C., Wegener, J. W., Van Hattum, B., Kemp, J. F., Ten Hallers, E., Reitsema, T. J., and Boon, J. P. 2003. Imposex and organotin concentrations in Buccinum undatum and Neptunea antiqua from the North Sea: Relationship to shipping density and hydrographical conditions. Mar Environ Res. 55: 203 - 233.

Terlizzi, A. 2000. Imposex (Pseudoermafroditismo) in Molluschi Gasteropodi Mediterranei: Aspetti morfologici e considerazioni ecologiche. Boll Malacol. 36: 155 - 158.

Terlizzi, A., Geraci, S., and Minganti, V. 1998. Tributyltin (TBT) pollution in the coastal waters of Italy as indicated by imposex in Hexaplex trunculus (Gastropoda, Muricidae). Mar Pollut Bull. 36: 749 - 752.

Terlizzi, A., Geraci, S., and Gibbs, P. E. 1999. Tributyltin (TBT)-induced imposex in the neogastropod Hexaplex trunculus in Italian coastal waters: Morphological aspects and ecological implications. Ital J Zool. 66: 141 - 146.

Terlizzi, A., Fraschetti, S., Gianguzza, P., Faimali, M., and Boero, F. 2001. Environmental impact of antifouling technologies: State of the art and perspectives. Aquat Conserv. 11: 311 - 317.

Terlizzi, A., Delos, A. L., Garaventa, F., Faimali, M., and Geraci, S. 2004. Limited effectiveness of marine protected areas: Imposex in Hexaplex trunculus (Gastropoda, Muricidae) populations from Italian marine reserves. Mar Pollut Bull. 48: 188 - 192. 322

Teso, S. V., and Penchaszadeh, P. E. 2009. Beach filling and imposex in Olivancillaria deshayesiana (Mollusca: Gastropoda: Olividae) from the coast of Mar del Plata, Argentina. J Mar Biol Assoc UK. 89: 557 - 562.

Tester, M., and Ellis, D. 1995. TBT controls and the recovery of whelks from imposex. Mar Pollut Bull. 30: 90 - 91.

Tester, M., Ellis, D. V., and Thompson, J. A. J. 1996. Neogastropod imposex for monitoring recovery from marine TBT contamination. Environ Toxicol Chem. 15: 560 - 567.

Tewari, A., Raghunathan, C., Joshi, H. V., and Khambhaty, Y. 2002. Imposex in rock whelks Thais and Ocenebra species (Mollusca, Neogastropoda, Muricidae) from Gujarat coast. Indian J Mar Sci. 31: 321 - 328.

Thiriot-Quievereux, C. 1977. Particulaire de l'appareil genital de quelques especes de Rissoidae (Mollusca, Mesogastropod). C R Hebd Seances Acad Sci D. 285: 779-781.

Thompson, J. A. J. 1997. Organotin contamination and related imposex studies in southern BC coastal waters. Can Tech Rep Fish Aquat Sci. 2163: 46 - 47.

Tillmann, M. 2004. Sediment-toxicological investigations using gastropods and insects with special emphasis on endocrine active substances. Gottingen: Cuvillier-Verlag; ISBN 3-86537-300-3, 196 pp. [in German].

Tillmann, M., Schulte-Oehlmann, U., Dufit, M., Markert, B., and Oehlmann, J. 2001. Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part III: Cyproterone acetate and vinclozolin as antiandrogens. Ecotoxicol. 10: 373 - 388.

Tyler, C. R., Jobling, S., and Sumpter, J. P. 1998. Endocrine disruption in wildlife: A critical review of the evidence. Crit Rev Toxicol. 28 (4): 319 - 361.

Unger, M. A., Harding, J. M., Mann, R., and Jestel, E. 2008. Tributyltin (TBT) induced imposex development in the veined rapa whelk (Rapana venosa) in Chesapeake Bay, USA. In 29th Annual SETAC Meeting (November 16-20, 2008), Tampa, Florida.

Unknown 1986. More on Imposex. Mar Pollut Bull. 17: 528.

Valentinsson, D., Sjodin, F., Jonsson, P. R., Nilsson, P., and Wheatley, C. 1999. Appraisal of the potential for a future fishery on whelks (Buccinum undatum) in Swedish waters: CPUE and biological aspects. Fish Res. 42: 215 - 227. 323

Van Den Broeck, H., De Wolf, H., Backeljau, T., and Blust, R. 2007. Effects of environmental stress on the condition of Littorina littorea along the Scheldt estuary (The Netherlands). Sci Total Environ. 376: 346 - 358.

Vasconcelos, P. 2006. Response to M. M. Santos comments to: "Imposex in Hexaplex (Trunculariopsis) trunculus (Gastropoda: Muricidae) from the Ria Formosa lagoon (Algarve coast southern Portugal)". Mar Pollut Bull. 52: 1314-1315.

Vasconcelos, P., Gaspar, M. B., and Castro, M. 2006a. Development of indices for nonsacrificial sexing of imposex-affected Hexaplex (Trunculariopsis) Trunculus (Gastropoda: Muricidae). J Mollus Stud. 72: 285 - 294.

Vasconcelos, P., Gaspar, M. B., and Castro, M. 2006b. Imposex in Hexaplex (Trunculariopsis) trunculus (Gastropoda: Muricidae) from the Ria Formosa lagoon (Algarve coast - southern Portugal). Mar Pollut Bull. 52: 337 - 341.

Viglino, L., Pelletier, E., and Lee, L. E. J. 2006. Butyltin species in benthic and pelagic organisms of the Saguenay Fjord (Canada) and imposex occurrence in common whelk (Buccinum undatum). Arch Environ Con Tox. 50: 45 - 59.

Vishwakiran, Y., and Anil, A. C. 1999. Record of imposex in Cronia konkanensis (Gastropoda, Mucricidae) from Indian waters. Mar Environ Res. 48: 123- 130.

Vishwakiran, Y., Anil, A. C., Venkat, K., and Sawant, S. S. 2006. Gyrineum natator: A potential indicator of imposex along the Indian coast. Chemosphere. 62: 1718 - 1725.

Vogt, C., Schmitt, C., Wirzinger, G., Scheider, J., and Oehlmann, J. 2007. Snails as bio-indicators - Imposex of the netted whelk Nassarius reticulars in Brittany along a transect from a point source. In World Congress of Malacology, Antwerp, Belgium.

Vos, J. G., Dybing, E., Greim, H. A., Ladefoged, O., Lambre, C., Tarazona, J. V., Brandt, I., and Vethaak, A. D. 2000. Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit Rev Toxicol. 30: 71 - 133.

Watson, M. 2003. Vancouver workshop: Overview and synthesis. Mar Environ Res. 57: 145 -153.

Wilson, S. P., Ahsanullah, M., and Thompson, G. B. 1993. Imposex in negastropods - An indicator of tributyltin contamination in eastern Australia. Mar Pollut Bull. 26: 44 - 48. 324

Wirzinger, G., Vogt, C., Bachxnann, J., Hasenbank, M., Liers, C., Stark, C., Ziebart, S., and Oehlmann, J. 2007. Imposex of the netted whelk Nassarius reticulatus (Prosobranchia) in Brittany along a transect from a point source. Cah Biol Mar. 48: 85 - 94.

Yu, H., and Zhao, H. 2004. Anatomical and histological studies on imposex of Thais clavigera. Oceanol Limnol Sin. 35: 149 - 155.

Yu, X. J., Huang, C. J., Zhu, S. X., and Chen, X. M. 2003. Histological studies on imposex of three marine prosobranchs. Ecolo Sci. 22: 337 - 340.

Zhengyan, L. 2001. Application of imposex as a biomonitor of tributyltin pollution. Journal of Ocean University of Qingado. 31: 480 - 486.

Zhengyan, L. 2005. Seasonal variation in imposex intensity of Thais clavigera. Chinese J Oceanol Limnol. 23: 163 - 168.

Zhu, S. X., Dong, Q. X., Shen, J. H., Yu, X. J., Du, H., and Huang, C. J. 2005. Imposex of Conus betulinus and Conus vexillum. Acta Ecol Sin. 25: 289 - 297. Curriculum Vitae

Name: Cassander Patrice Titley-O'Neal

Universities attended: University of North Carolina at Wilmington, North Carolina (1996 - 1999) Bachelor of Science (Marine Biology) Minor in Chemistry

University of Plymouth, United Kingdom (2000-2001) Master of Science (Applied Marine Science)

University of New Brunswick, Saint John, New Brunswick (2006 - 2011) Doctor of Philosophy (Biology)

Publications:

Titley-O'Neal, C. P., MacDonald, B. A. Pelletier, £. and Saint-Louis, R. In Press.Using Nucella lapillus (L.) as a Bioindicator of Tributyltin (TBT) Pollution in Eastern Canada: A Historical Perspective, Water Quality Research Journal of Canada.

Titley-O'Neal, C. P., MacDonald, B. A. Pelletier, £., Saint-Louis, R., and Phillip, O. The relationship between imposex and Strombus gigas from the British Virgin Islands. Bulletin of Marine Science. (Accepted pending revisions).

Titley-O'Neal, C. P., Munkittrick, K. R., and MacDonald, B. A. The effects of organotins on female gastropods. Journal of Environmental Monitoring. (Submitted for publication).

British Virgin Islands National Sewerage Programme - Environmental Assessment of Alternatives (2002)

Status of the Red Hind (Epinephelus guttatus) in the British Virgin Islands (2000) Government Report

Preliminary Assessment of Salt Island Sound - September 1999 (2000) - Government Report Conferences:

Titley-O'Neal, C. P., MacDonald, B. A., Pelletier, £. and Saint- Louis, R. 2009. Different latitudes, but same attitude: Using gastropods as bioindicators of TBT pollution in eastern Canada and the British Virgin Islands. SETAC 30th Annual Meeting - New Orleans Hilton Riverside (19-23 November 2009), New Orleans, Louisiana, USA. Oral presentation.