EFFECTS OF AND OTHER ON FISH CYPlA MONOOXYGENASES, EMBRYONIC AND REPRODUCTIVE DEVELOPMENT

A Thesis Submitted to the Committee of Graduate Studies in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Faculty of Arts and Science

Trent University

Peterborough. Ontario. Canada

0 Y iannis Kiparissis 200 1

Watershed Ecosystems Ph.D. Program June 2001 National Library Bibliothbque nationale du Canada Acquisitions and Acquisitions et Bibliographic Sewices services bibliographiques 395 Wellington Street 395, rue WeUington Ottawa ON KIA ON4 Ottawa ON KIAON4 Canada Canada

The author has granted a non- L'auteur a accorde une Licence non exclusive licence allowing the exclusive pernettant a la National Library of Canada to Bibliotheque nationale du Canada de reproduce, loan, distr!iiute or sell reproduire, preter, distniuer ou copies of this thesis in microform, vendre des copies de cette these sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format electronique.

The author retains ownership of the L'auteur conserve la propriete du copyright in this thesis. Neither the droit d'auteur qui protege cette these. thesis nor substantial extracts &om it Ni la these ni des extraits substantiels may be printed or othenvise de celfe-ci ne doivent &re imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT

Effects of Flavonoids and Other Phytochemicals on Fish CYPIA Monooxygenases, Embryonic and Reproductive Development

The purpose of this study was to demonstrate that biological responses in fish such as induction of CYP1 A-dependent monooxygenases. embryonic defects. and reproductive failure that are observed in feral fish exposed to bleached kraft mill effluents (BKME) may be attributed to exposure to phytochemicals. Aqueous leachates from pulp and selected phytochemicals induced etboxyresorufin-0-deethylase (EROD)in rainbow trout

(Oncorhynchzu mykiss). The same phytochemicals caused a multitude of developmental abnormalities in exposed embryos of Japanese medaka (Oryzias Zutipes). Further work with flavonoids. which are a specific class of phytochemicals. indicated that molecuiar structure and position of hydroxy-substitution are variables that affect EROD induction potency and embryotoxicity. The compounds. flavone. flavonol. . . . . galangin, , . kaernpferol and all altered gonadal development in Japanese medaka. Intersex gonads. delayed maturation. reduced numbers of germ cells. ovarian atresia and alterations to secondary sex characteristics were evident in medaka from most treatments. Results from in vitro studies showed that flavonoids may affect reproductive development in fish by binding to either the estrogen or androgen receptor. or by binding to the sex steroid carrier proteins.

Analysis of BKME by liquid chromatography mass spectrometry indicated that the , genistein was present in wood pulp and in mill effluent at ppb concentrations. Thus. flavonoids may be responsible for the biological effects observed in fish downstream of pulp mills. To my wife Barb. daughter Lucy and parents ABav&aroand AopoBCa ACKNOWLEDGEMENTS

First of all, I thank my supervisor Dr. Chris Metcalfe for his continued support throughout this study, for his editing skills and for his patience and faith in me. For their advice and useful comments on this dissertation. I thank the members of my committee.

Dr. Art Niimi. Dr. Alicia Zobel and Dr. Brendan Hickie. I would like to acknowledge my good Friend Dr. Dave Janz who encouraged me (under the influence of plentiful ouzo and retsina) to convert this project From a Master's to a Ph.D. thesis. Special thanks to Dr.

Richard Hughes for analyzing the effluent samples with the LC-MS and LC-MS-MS techniques. I was fortunate to work with some special people during this study: thanks to my labrnates. Tracy Metcalfe. Dr. Brenda Koenig. Dr. Gord Balch and Erin Bennett for helping me with various aspects of this study. I also thank Jason Allen and Mike Sanbom for taking good care of the medaka in the genistein experiment and Colin Khan for rnicrotoming ail the samples in the same experiment.

This research was financially supported. in part. by an operating grant from the

Natural Sciences and Engineering Research Council (NSERC), a grant from the Canadian

Network of Toxicology Centres (CNTC). a grant from the international ofice of the

Ministry of Education and Research (BMBF) in Germany. contracts from the Department of Fisheries and Oceans (DFO) and NSERC Graduate Scholarships. TABLE OF CONTENTS

Abstract ...... i . Acknowledgments ...... 11

List of Figures ...... ix

Chapter 1 Genera1 Introduction ...... 1 1.1 Identification of the ProbIem ...... 1 1.2 Adverse Effects on Feral Fish ...... 3 1.3 Phytochemicals ...... 6 1.4 Objectives ...... 9

Chapter 2 induction of Hepatic EROD in Fish Exposed to Leachates from Wood Pulp ...... 12 2.1 Abstract ...... 12 2.2 Introduction ...... 13 2.3 Material and Methods ...... 15 2.3.1WoodPulp ...... 15 2.3.2 Fish Exposure and Preparation of Hepatic Microsomes ...... 15 2.3.3 EROD Analysis ...... 17 2.3.4 Statistical Analyses ...... 18 2.4 Results ...... 19 2.5 Discussion ...... --73

Chapter 3 Induction of Hepatic CYP 1A-dependent Monooxygenases in Rainbow Trout (Oncorhynchw mykiss) Dosed to Phytochemicals ...... 26 3.1 Abstract ...... 26 3.2Introduction ...... 27 3.3 Material and Methods ...... 30 3.3.1 Chemicals ...... 30 3.3.2 CYPl A Induction Assays ...... 30 33.3 Determination of OctanoVwater Partition Coefficients for Phytochemicds ...... 32 Chapter 4 Embryonic Potential of Phytochemicals to Japanese Medaka (Oryzias latipes) .... 42 4.1Abstract ...... 42 4.2 Introduction ...... 43 4.3 Material and Methods ...... 46 4.3.1 Chemicals and Solutions ...... 46 4.3.2 Japanese Medaka Embryotoxicity Assay ...... 47 4.4Results ...... 48 4.5 Discussion ...... 54

Chapter 5 Effects of Various Flavonoids on Fish: EROD induction. Embryotoxicity and Reproductive Development ...... 62 5.1 Abstract ...... 62 5.2 Introduction ...... 64 5.3 Material and Methods ...... 70 5.3.1 Chemicals ...... 70 5.3.2 EROD Induction ...... 71 5.3.3 Japanese Medaka Embryotoxicity Assay ...... 71 5.3.4 Japanese Medaka Reproductive Development Studies ...... 73 5.3.4.a Reproductive Development Experiment # 1 ...... 73 5.3.4. b Reproductive Development Experiment #2 ...... 75 5.3.4.c Reproductive Development Experiment #3 ...... 76 5.3.5 in vitro assays ...... 76 5 -35.a Yeast Estrogenicity Assay (YES Assay) ...... 76 5.3.5.b in vitro Binding Assays ...... 77 5.4Results ...... 78 5.4. l EROD Bioassay ...... 78 5 A.2 Japanese Medaka Embryotoxicity Assay ...... 80 5.4.3 Japanese Medaka Reproductive Development Studies ...... 90 5.4.3.a Reproductive Development Experiment # 1 ...... 90 5.4.3.b Reproductive Development Experiment #2 ...... 96 5 .4.3 .c Reproductive Development Experiment #3 ...... 117 5.4.4 in vim Studies ...... 124 5.4.4.a YES Estrogenicity Assay (YES Assay) ...... 124 5.4.J.b in vitro Binding Studies ...... 124 5.5Discussion ...... 132

Chapter 6 Identification of the Isoflavonoid. Genistein in Bleached Kraft Mill Effluent ...... 158 6.1Abstract ...... I58 6.2Introduction ...... 159 6.3 Material and Methods ...... 160 6.3.1 Chemicals ...... 160 6.3.2 Sample Collection ...... 161 6.3.3 Preparation of Extracts ...... 161 6.3.4 Analysis ...... 163 6.4 Results and Discussion ...... 165

Chapter 7 Conchsions ...... 175

References ...... 180

Appendices Appendix1 ...... 105 Appendix 2 ...... 208 Appendix 3 ...... 211 Appendix4 ...... 216 Appendix5 ...... 219 Appendix 6 ...... 220 Appendix 7 ...... 231 Appendix 8 ...... 224 Appendix 9 ...... 229 Appendix 10 ...... 266 LIST OF TABLES

Page Table 2.1. Relative ethoxyresorufin-0-deethylase induction (x-fold) in rainbow trout during 3 weeks of exposure and 2 weeks of post-exposure to leac hates from softwood and hardwood pulp taken From two different pulping processes (i.e. prior and after the oxygen delignification stage) ...... 2 1 Table 3.1 Hepatic ethoxyresorufin-0-deethylase (EROD) activity in immature rainbow trout at 72 h after a single intraperitoneal injection with anthrone, juglone. trans-stilbene. sitosterollcampesterol and l3-naphthoflavone at a dose of 10 mgkg ...... 34 Table 3.2 OctanoYwater partition coefficients (log KO,,)of the tested phytochemicals estimated by the reverse-phase high- performance liquid chromatography technique of McDufie (1981) ...... 37 Table 4.1 Summary of results from the Japanese medaka embryotoxicity assay. Dead. unhatched. partially hatched and hatched embryos from all treatments are presented as percent (%) of all viable embryos at day 1 of' exposure. Dead. abnormal and viable larvae are also expressed as percent (%) of the viable embryos at day 1 ...... 49 Table 4.2 Median lethal concentrations (LCSOs) and median effective concentrations (EC50s). expressed in ppm (pg/mL). for Japanese medaka embryos exposed to several phytochemicals ...... 50 Table 4.3 Embryonic responses of Japanese medaka embryos exposed to the CYP 1A-inducing phytochemicals. flavone. . 7-methoxy-coumarin and ...... 58 Table 5.1 Flavonoid class and hydroxyl substitution of individual flavonoid compounds tested for their endocrine-disrupting potential with the in vitro assays ...... 70 Table 5.2 Range of concentrations (pM or mg/L) or doses ( mgkg) of flavonoids tested in vivo in the: a) rainbow trout hepatic ethoxyresorufin-0-deethylase(EROD) assay; b) Japanese medaka embryotoxicity assay; and c) Japanese medaka reproductive development assay ...... 72 Table 5.3. Median lethal concentrations (LCSOs) and median effective concentrations (ECSOs). expressed in pM. for Japanese medaka embryos exposed to several flavonoids ...... 8 1 Table 5.4. Reproductive development experiment #I. Numbers of phenotypic male and female Japanese medaka identified histologically after exposure to the flavonoids. c hrysin and quercetin as well as to the androgen, testosterone. The number and percent (in parenthesis) of the intersex condition, testis-ova observed in medaka From all treatments are also presented ...... 92 Table 5.5. Reproductive development experiment #2. Numbers of phenotypic male and female Japanese medaka identified histologically after exposure to various flavonoids and to synthetic estrogen. 1 7a-ethinyl-estradiol. The number and percent (in parenthesis) of the intersex condition. testis-ova observed in males from all treatments is also presented ...... 98 Table 5.6. Reproductive experiment #2. Histological observations in female medaka with total length 2 17 mm. Stage of oocyte development such as. pre-vitellogenic (pre-VtG). vitellogenic (VtG) or post-ovulatory(post-VtG). Gonadal effects including, incidence of atresia increase of ovarian lumen. presence of primordial germ cells (PGCs). development of ovarian stroma and incidence of intersex (testis-ova)are also presented. Extragonadal effect includes the development of ectopic oocytes ...... 99 Table 5.7. Reproductive experiment #2. Histological observations in male medaka with total length 2 17 mm. Number of medaka entering different stages of spermatogenesis such as. immature (absence of spermatozoa). all stages and advanced (increased number of spermatozoa) is present. in addition. gonadal effects including. an increase in fibrotic tissue, abnormal architecture and testis-ova incidence are also presented ...... 100 Table 5.8. Reproductive experiment #2. Comparisons between the phenotypic gonadal sex and secondary sex characteristics of Japanese medaka exposed to individual flavonoids and to 1 7a-ethinylestradiol. The terms ...... 10 1 Table 5.9. Reproductive development experiment #3. Number of Japanese medaka prepared for histology. number of medaka examined as well as number of phenotypic male and female. Incidence of testis-ova is also presented ...... 1 19 Table 5.10. Reproductive experiment #3. Histological observations in female medaka with total length 2 17 mrn. Stage of oocyte development such as. pre-vitellogenic (pre-VtG), vitellogenic (VtG) or post-ovulatory(post-VtG). Gonadal efikcts including, incidence of atresia increase of ovarian lumen. presence of primordial germ cells (PGCs) are also presented ...... 120 Table 5.1 1. Reproductive development experiment #3. Histological observations in male medaka with total length r 17 mm.

vii Number of medaka entering different stages of spermatogenesis such as, immature (absence or presence of few spermatozoa), all stages and advanced (increased number of spermatozoa) is present. In addition, gonadal effects including. an increase in fibrotic tissue or interstitial connective tissue (CT) abnormal testicular architecture and reduction in density of mature spermatozoa ( 1 spz)are also presented ...... 121 Table 5.12. Estrogenic potential of flavonoids used in this study as assessed with a yeast recombinant (YES) assay with the human estrogen receptor (hER). and the Bgalactosidase reportergene ...... 129 Table 5.1 3. Relative potencies of flavonoids for binding to the fish sex steroid binding proteins (SSBP). estrogen receptor (ER) and androgen receptor (AR). Values represent the mean of 2-4 different experiments. The binding of t 7p-estradiol in the SSBP and ER assays and testosterone in the AR assay were setatl ...... 131 Table 5.14 External secondary sex characteristics in Japanese medaka. For each sex characteristic its persistence status (permanent or temporary). its dependence to hormones (male- or female-positive) and a brief description are provided ...... 145 Table 5.15 Summary of positive (+) or negative (-) responses by flavonoids in the in vitro assays. Intensity of the response (+ to +++) was arbitrarily set fiom Table 5.13. These 10 flavonoids were tested in the Japanese medaka reproductive development studies ...... 150 Table 6.1 Concentrations of genistein by LC-MS in extracts of wood pulp and in untreated and treated mill effluent collected fiom a bleached krafi mill in Ontario. Canada in August. 1998 Extracts were subfractionated into Fractions 1.2 and 3 by LH-20 gel filtration chromatography and the fractions analyzed separately ...... 169

viii LIST OF FIGURES

Page Figure 2.1 Continuous flow-through apparatus for dosing of rainbow trout with leachates from wood pulp ...... 16 Figure 2.2 Relative EROD activities in rainbow trout during continuous-flow exposure to leachates from softwood and hardwood pulp at 7, 14 and 2 1 days during exposure. and at 3.7 and 14 days during post-exposure ...... 20 Figure 3.1 Molecular structure of phytochemicals used in this study for i.p. exposures to immature rainbow trout ...... 29 Figure 3.2 Time-dependent hepatic EROD activity in experiment with immature rainbow trout following a single intraperitoneal (i.p.) injection of DMSOPBS (control). harmane, 7- methoxycournarin and tropolone at a dose of 10 mg/kg ...... 35 Figure 3.3 Time-dependent hepatic EROD and AHH activities. expressed as x-fold induction. in experiments with immature rainbow trout following a single intraperitoneal (i.p.) injection of 7-methoxy-coumarin at a dose of 10 mgkg ...... 36 Figure 4.1 Molecular structure of phytochemicals used in the Japanese medaka embryotoxicity assay ...... 46 Figure 4.2 Cumulative mortality (%) of Japanese medaka embryos/larvae exposed to various concentrations (0.1. 1.5 and 10 pg/rnL) of the phytochemicals. flavone. harmane. juglone and tropolone ...... 5 1 Figure 4.3 Histograms depicting daily hatching frequencies (%) of Japanese medaka embryos exposed to various phytochemicals. Arrow shows the median hatching time. and asterisks ("* * *") indicate significant differences between treatments and Controls (P c 0.05) ...... 52- - Figure 4.4 Viable Japanese medaka embryo from the Control treatment ...... 33 Figure 4.5 Non-viable Japanese medaka embryo exposed to 1 pg/rnL of flavone showing pericardial edema and the development * C oftube-heart ...... 33 Figure 4.6 Viable newly hatched Japanese medaka showing a successfd swim bladder inflation ...... 56 Figure 4.7 Abnormal newly hatched Japanese medaka exposed to 10 pg/mL of 7-methoxycoumarin showing scoliosis. tail curvature. Note also the absence of a successfbl swim bladder inflation ...... 56 Figure 5.1 Molecular structure of flavonoid classes used in this study. Flavonoids are structurally similar C- 15 compounds comprised of two aromatic rings (A and B) and a heterocyclic C ring, except for chalcones. Note the presence of a double bond (C2-C3) in . and its absence fiom . Presence of a C-3 hydroxyl group in the flavone nucleus is the structural characteristic of flavanols. whereas a C-3 bridging of C and B nucleus is the characteristic of . -3-01s lack the ketone group. giving the molecule a positive charge at C-4 ...... 68 Figure 5.2 Molecular structure of hydroxylated flavonoids used in the Japanese medaka reproductive assay ...... 69 Figure 5.3 Concentration-dependent hepatic EROD activity in experiment with immature rainbow trout following a single intraperitoneal (i-p.) injection of several non-substituted flavonoids (A). mono-hydroxyflavonoids (B). poly- hydroxyflavonoids (C). and the positive control a- naphthoflavone (D) ...... 79 Figure 5.4 Hatching success (%) throughout the 17 day assay period in Japanese medaka exposed to: (A) non-substituted flavonoids. D-NF. flavone. trans-chalcone. flavanone; (B) mono-hydroxy flavonoids. flavonol. 6-hydroxyflavone. 7- hydroxflavone: (C) di- and tri-hydroxy-substi tuted flavonoids. chrysin. galangin. apigenin. naringenin: and (D) poly-hydroxy-substituted flavonoids. . catechin. quercetin and taxifohn ...... 82 Figure 5.5 Cumulative mortality (%) at Day 1 7 in Japanese medaka embryos exposed to:@) non-substituted flavonoids. t3-NF. flavone. trans-chalcone. flavanone; (B) mono-hydroxy flavonoids. flavonol. 6-hydroxyflavone. 7-hydroxyflavone: (C) di- and tri-hydroxy-substituted flavonoids. chrysin. galangin. apigenin. naringenin: and (D) poly-hydroxy- substituted flavonoids. kaempferol. catechin. quercetin and tifolin ...... 83 Figure 5.6 Viability or successful "swim-up" (%) at Day 17 day of Japanese medaka exposed to: (A) non-substituted flavonoids. Q-NF. flavone. trans-chalcone. flavanone; ( I3 ) mono-hydroxy flavonoids. tlavonol. 6-hydroxyflavone. 7- hydroxyflavone; (C) di- and ui-hydroxy-substituted flavonoids. chrysin. galangin. apigenin, naringenin; and (D) poly-hydroxy-substituted flavonoids. kaempferol, catechin. quercetin and ...... 84 Figure 5.7. Abnormal newly hatched Japanese medaka exposed to 0.5 pM of LI-NF. Note the presence of the yolk sac and the absence of a successfbl swim bladder inflation ...... 87 Figure 5.8 Newly hatched Japanese medaka exposed to 25 pM of flavanone showing the symptoms of *blue-sac" disease. Note the presence of yolk edema and haemorrhaging. The yolk sac was not absorbed and the swim bladder is absent ...... 87 Figure 5.9 Japanese medaka exposed to 25 pM of flavonol showing caudal haemorrhaging, anisophthalmia microcephaly and yolk edema. The development of this embryo is retarded ...... 9 1 Figure 5.10 Ovary of a Control Japanese medaka showing stage lI pre- vitellogenic oocytes in the periphery and stage IY pre- vitellogenic oocytes in the middle ...... 9 1 Figure 5.1 1 Testis of a Control male Japanese medaka showing all stages of spermatogenesis. Immature sperm cells. sperrnatogonia and spermatocytes are located in the periphery. whereas mature spermatozoa are found in lobules adjacent to efferenct duct ...... 95 Figure 5.12 Ovary of a chrysin-treated phenotypic female Japanese medaka showing advanced oogenesis. Note the large vitellogenic oocytes at the VII stage of development. having incorporated vitellogenin in their oolemma ...... 93 Figure 5.13 Reproductive development experiment # 1. Sex ratio and stage of oogenesis in female Japanese medaka exposed to 3 nominal concentrations of chrysin and quercetin for 3 months ...... 95 Figure 5-14 Testis of a phenotypic male Japanese medaka exposed to flavone for 4 months. Note the presence of oocytes within a testicularlobule ...... 102 Figure 5.15 Testis of a phenotypic male Japanese rnedaka exposed to flavone for 4 months. The architecture of this testis resembles the structure of testes in intersex conditions. Note the increase in interstitial tissue and intestitial space around the testicular lobules ...... 102 Figure 5.1 6 Ovary of a phenotypic female Japanese medaka exposed to chrysin for 5 months. Note an increased development in ovarian lumen and a simultaneous reduction in oocytes. This female has entered the vitellogenic stage of oogenesis as indicated by the presence of stage VI oocytes ...... 106 Figure 5.17 Ovary of a phenotypic female Japanese medaka exposed to quercetin for 5 months. Note the presence of somatic tissue (stroma) and the presence of many atretic oocytes as indicated by the separation of oolemma from the nucleus. This female is in the late vitellogenic stage of oogenesis ...... 106 Figure 5.18 Japanese medaka exposed to the binary mixture of chrysin and quercetin. Note the absence of the dorsal fin and deformed anal fin ...... I08 Figure 5.19 Ovary of a Japanese medaka exposed to 0.05 mg/L of kaempferol for 5 months. The fish is an adult female experiencing a delayed sexual maturity: Note the reduced number of oocytes and the increased development of ovarian lument ...... 108 Figure 5.20 Ovary of a Japanese medaka exposed to 0.05 mg.L of kaempferol for 5 months. This adult female medaka is not sexually mature since the majority of oocytes are in the pre- vitellogenic stage of oogenesis ...... 109 Figure 5 .2 1 Development of testis-ova in a Japanese medaka exposed to 0.5 mg/L of naringenin for 3 months. Immature pre- vitellogenic oocytes are mainly located in the posterior end of the testicular tissue. Sperm cells at different stages of spermatogenesis are found in the anterior end of the testis ...... 109 Figure 5.22 Female Japanese medaka exposed to 0.1 mg/L of naringenin for 5 months. There is a development of ectopic germ-cells in the hepatic region ...... 1 12 Figure 5.23 (A) Development of testis-ova in a phenotypic male Japanese medaka exposed to 0.5 mg/L of apigenin. as indicated by the presence of a single oocyte within a testicular lobule. Note the increase of fibrotic tissue around the lobules. (B) A testis from a male medaka exposed also to 0.5 meof apigenic showing similar architectural pattern of testicular development ...... 1 12 Figure 5.24 A case of testis-ova with the ovarian component predominating in the gonad. This Japanese medaka was exposed to 0.5 mg/L of apigenin for 4 months ...... 1 13 Figure 5.25 A phenotypic female Japanese medaka exposed to 0.5 mg/L of galangin for 5 months. showing most of the VIII oocytes being atretic and the consequent development of the somatic tissue. stroma ...... 1 13 Figure 5.26 Development of testis-ova in a male Japanese medaka exposed to 10 ng/L of 17~bethinylestradiol for 4 months. Oocytes are confined in the posterior end. whereas testicular lobules with sperm cells are mainly in the anterior testis ...... 1 16 Figure 5.27 A nearly sex-reversed male Japanese medaka exposed to 10 ng/L of 17a-ethinylestradiol for 5 months. Remnants of the testicular component are located in the anterior gonad. whereas pre-vitellogenic and early vitellogenic oocytes are distributed throughout the gonad ...... 1 16 Figure 5.28 Graph depicting the agreement between the phenotypic gonadal sex and secondary sex characteristics in Japanese medaka exposed to genistein for 3 months ...... 122 Figure 5.29 A testis of a Japanese medaka exposed to 100 pg/L of genistein for 3 months. Note the increase of connective tissue. and the decrease in density of spermatozoa in the

xii efferent duct ...... 123 Figure 5.30 A Japanese medaka exposed to pg/L of genistein showing arrhenoid expression of the dorsal and anal fins. Note the notches in both fins and papillary processes in the anal fin. The anal fin is deformed in the middle ...... 123 Figure 5.3 1 YES Assay. The 96-well plate showing the expression of D- galactosidase in the presence of the flavonoids. (+)- catechin. chrysin. naringenin. galangin. flavanone and tram-chalcone (rows A-F), the procedural blank (G) and the positive control. 1 7bestradiol (H). Positive estrogenic response is indicated with magenta whereas no response is indicated with orange. Cytotoxic effects are found in wells with yellow colouration ...... 125 Figure 5.32 Response of the recombinant yeast estrogen screen. expressed as D-galactosidase activity (%). to the flavonoids naringenin. kaempferol and apigenin at various concentrations (M). The response of 17D-estradiol was used as the positive controi ...... 126 Figure 5.33 Response of the recombinant yeast estrogen screen. expressed as B-galactosidase activity (%). to the flavonoids 4'-hydroxyflavone. 4'-hydroxyflavanone and 4.6- dihydroxyflavone at various concentrations (M). The response of 17D-estradiol was used as the positive control ...... 127 Figure 5.34 Response of the recombinant yeast estrogen screen. expressed as a-galactosidase activity (%), to the . genistein and equol at various concentrations (M). The response of 17D-estradiol was used as the positive control ...... 128 Figure 5.35 Male medaka. In the posterior end of the dorsal fin there is a notch, a maie characteristic. Also note a notch in the anal fin. In addition. in the posterior end there are papillary processes used during mating The urogenital pore is visible ...... 142 Figure 5.36 Female medaka. Both dorsal and anal fins lack the notches. Mature Female have a well developed papilla which cover both genital and urinary pores ...... 142 Figure 5.37 (A) A phenotypic male Japanese medaka exposed to 1 mg/L of naringenin for 5 months. This specimen shows a female- like urogenigal papilla and male-like dorsal and anal fins with papillary processes. (B)The testis of the same fish showing the development of testis-ova as indicated by the presence of four oocytes ...... 146 Figure 6.1 Total ion chromatographs (m/z = 253.269.271.285.389. 30 1) generated by LC-ESEMSanalysis(Hew1ett Packard Model 1 100) in negative ion mode of extracts prepared £?om LH-20 Fraction 2 of extracts prepared from: A) wood pulp, B) mill effluent after treatment and C) a procedural blank. The compound identified as genistein was subsequently confirmed by LC-MS-MS.The retention time of the unknown compound did not correspond to any of the flavonoid compounds in the analytical standard ...... 167 Figure 6.2 CID mass spectra generated by LC-MS-MS analysis (i.e. Micromass Quattro) in negative ion mode of the peak monitored at m/z 269 that was tentatively identified as genistein in: A) extract prepared From mill effluent after treatment (Fraction 2). B) extract prepared from mill effluent before treatment (Fraction 2). and C) a standard of genistein ...... 168 Figure 6.3 Total ion chromatographs (m.z = 23 7) generated by LC- ESI-MS (Micromass Quattro) in negative ion mode of extracts prepared from: A) a standard of 4'-hydroxyflavone. B) an extract of mill effluent before treatment (Fraction 2). C) an extract of mill effluent after treatment (Fraction 2) ...... 172 Figure 6.4 CID mass spectra generated by LC-MS-MS analysis (Micromass Quattro) in negative ion mode of the peak monitored at rn/z 237 in: A) an extract from mill effluent before treatment (Fraction 2). B) an extract from mill emuent after treatment (Fraction 2) ...... 173

xiv CHAPTER 1

General Introduction

I. 1. Identification of the Problem

The pulp and paper industry is an important contributor to Canada's national and local economies. However. this industry also impacts the environment. including health effects on feral fish. In 1990. across Canada there were approximately 150 operational pulp mills: consistent with the conclusion that pulp and paper is a major industrial sector that drives economic growth in this country (Sinclair. 1990). These mills typically each discharge 50.000 to 170.000 metric tons of effluent into receiving environments

(Robinson er ul. 1994). To minimize and remedy associated environmental problems. the pulp and paper industry is investing approximately 3% of its sales revenues to new technologies for preventing and controlling pollution (Turoski. 1 998). However. despite the investments. fish populations near pulp mill discharges still show signs of sub-lethal biological effects. including induction of mixed hction oxidases (MFOs). alteration of hormonal status. reproductive dysfunctions. alterations to secondary sex characteristics. vertebrae and gill deformities. imbalances on ionoregulation. and immunosuppression

(Kovacs. 1986: hdersson et ul. 1988: Sodergren. 1989: Owens. 199 1 : Mukittrick ei cd.

199%. 1992b. 1992~).These effects probably have a chemical etiology since installations of primary and secondary wastewater treatment facilities have reduced emuent parameten such as chemical and biological oxygen demand (COD and BOD). nutrient loadings. total suspended solids (TSS), and acute toxicity to fish (Berry et al. 199 1).

The main purpose of pulping is to separate the wood cellulose from the wood lignin and other extractives and this process is facilitated either by mechanical processes in thennomechanical pulp mills or by chemical treatment in sulfite and kraft mills

(Environment Canada 1983). The kraft process utilizes a mixture of sodium hydroxide and sodium sulfide to '-cook" the pulp and delignify the wood fibres (Sjostrom. 1981 ).The kraft process currently is the dominant pulping practice in Canada and in other countries because of chemical recovery of the cooking agents. versatility with most wood species and cornpatability with the bleaching technology (Environment Canada. 1983).

Attention has been focussed on environmental impacts of pulp mills using

bleaching kraft processes because persistent chlorinated compounds such as chlorinated dioxins and dibenzohrans. formed during the bleaching stages of the pulping process.

have been detected in bleached krafi mill effluents (BKME) and in biota near these mills

(Kovacs. 1986: Owens. 199 1 ). Long-term ecological studies in Scandinavia (Gulf of

Bothnia) demonstrated a strong association between exposure to BKME and toxic

impacts on lish. but these studies failed to positively identify the chemicals responsible

for the toxic effects (Sodergren. 1989). Since these chemicals are believed to be

constituents of the total burden of chlorinated compounds in the effluent. which is usually

measured as Adsorbable Organic Halogens (AOX). it has been proposed that reduction or

elimination of AOX from the effluents may eliminate toxic impacts on the aquatic

environment (Sodergren. 1989). Based mainly on this ecological study. as well as

supporting evidence from studies conducted in North America (Hodson rr a!. 1992: Servos et al. 1992). the provincial governments of British Columbia and Ontario imposed guidelines for reductions in AOX discharges (Williamson, 1992; Mettelstaedt and

Mahood. 1993). Subsequently. most pulp mills attempted to reduce AOX discharges by using chlorine dioxide (ClO,) instead of the widely used chlorine as an alternative bleaching method. However. a number of recent in viva and in vitro studies have shown that similar toxic responses are observed in fish exposed to primary and/or secondary treated BKME. and also to effluents from mills that use kraft or sulfite pulping technology. This suggests that the bioactive chemicals may not be chlorinated compounds

( Lindstrom-Seppa et ai. 1992: Pesonen and Andersson. 1992). Furthermore. the failure of

secondary treatment systems to remove toxic impacts to fish. evidence of rapid declines in

biochemical effects in fish after mill shutdowns. and elevation of ethoxyresorufin-0-

deethylase (EROD)' activities in fish exposed to pulp leachate indicate that bioactive

compounds are relatively hydrophilic. and therefore are not chlorinated compounds

(Payne and Fancey. 198 1: Servos ct uf. 1992: Martel rt ui. 1994). In light of the this

evidence. regulation of AOX discharges may not be effective in reducing toxic effects to

fish near mill discharges. Consequently. Environment Canada has made it a priority to

identify and remove the toxic compounds from BKME effluents. rather than regulating

levels of AOX in effluents (Berry et (11. 199 1).

1.2. Adverse effects on feral fish

'Description of this enzyme system is given in the next section (Section 1.3) The most consistent response of fish to pulp mill effluent is induction of the hepatic MFOs. especially the P-4501 A-dependent monooxygenases' such as ethoxyresorufin-0-deethyiase(EROD) and aryl hydrocarbon hydroxylase (AHH).

Induction of the teleost P-4501A (CYP1 A) oxygenases is a highly sensitive cellular response to exposure to several classes of aromatic chemicals (i.e. polynyclear aromatic hydrocarbons. chlorinated dibemodioxins. chlorinated dibenzohrans and coplanar polychlorinated biphenyls). CYPl A cytochromes are involved in Phase I metabolic pathways. and consequently they are capable of metabolizing lipophilic xenobiotics to more hydrophilic metabolites. resulting in their elimination from the organism or their bioactivation (Nebert and Gonzalez. 1987). Structure-activity studies indicate that the toxicity of the CYP 1 A-inducing xenobiotics is mediated through binding to a cytosolic

Ah receptor (Poland and Knutson. 1982). Hepatic EROD activities in caged and feral fish exposed to pulp mill effluents are correlated with the distance from mill discharges

(Andenson et al. 1988). which demonstrates the usefulness of this biochemical marker as

a monitoring tool near pulp effluents . Field data are supported by induction of EROD in

fish exposed to diluted and undiluted pulp mill effluents under laboratory conditions

(Andersson et rrl. 1987).

Field studies have shown that induction of P-4501A proteins in fish exposed to

pulp effluents often occurs simultaneously with alterations in the endocrine and

reproductive systems. These effects include reduction of plasma steroid levels and delay

of sexual maturation. reduced gonadosomatic index. lower fecundity with age. reduced

'~ccordingto the new nomenclature this enzyme system is also denoted as CYP 1 A

4 numbers of viable eggs and alterations to secondary sex characteristics. Most of these responses have been observed in white sucker (Ca~ostomzrscomrnersoni) populations downstream of Canadian pulp mills (McMaster et ut. 199 1 : Munkittrick ei ul. 1992a:

Munkittrick el ul. 1992b ). In addition. development of gonopodium (an arrhenoid secondary sex characteristic) and a male-like courtship behaviour were observed in female mosquitofish (Gambrrsh uffinis) exposed to pulp effluent (Howell et ul. 1980: Schroder and Peters. 1988). Thus. it is plausible that the active constituents of pulp eRluents affect normal reproductive Function by interfering with the development and differentiation of gonadal tissues and secondary sex characteristics.

Despite the circumstantial evidence for a relationship between EROD induction and reproductive dysfunction in fish. a direct cause-and-effect relationship has not be established. However. it has been proposed that a chronically-induced EROD system might contribute to hormonal dysfunction and consequently to reproductive failure in fish exposed to xenobiotics (Nebert and Gonzalez. 1987: Lehtinen. 1990). For instance. in rats exposed to 2.3.7.8-tetrachloro-dibenzodioxin (TCDD).a potent P-4501 A inducer. there were reduced levels of androgens in serum (Moore et ui. 1985). Moreover. induction of the P-450 1A protein in rainbow trout pituitary resulted in alterations of the endocrine system as indicated by increased plasma concentrations of gonadotropin (GTH II) and testosterone ( Andersson er al. 1993). It is possible that P-450I A inducing compounds in pulp effluents may elicit endocrine responses in fish by binding to steroid hormone receptors. However. chemicals that are not CYP1 A-inducers may also elicit similar estrogenic or androgenic effects. Munki ttrick er a&.( 1994) suggested that C YP 1A induction and reproductive effects are not associated with the same chemicals in pulp effluents. Other studies supported these findings. including evidence that the natural . O-sitosterol. a major constituent of the pulp plume (Fox. 1977), was linked to the masculinization of the female mosquitofish (Howell and Denton. 1989). but was not a

CYPIA inducer (Hewitt er nl. 1996).

Although CYPI A induction. reproductive dyshnction and alterations to sex characteristics are commonly observed responses in fish exposed to pulp mill effluent. other adverse effects have been documented such as alterations in carbohydrate . increases in red blood cells and haematocrit counts. depression of the immune system. decreases in the liversomatic index. and higher incidences of skeletal and fin deformities (Larsson ct ul. 1988). In addition. in the vicinity of a mill located on the Gulf of Bothnia (Sweden). recruitment of perch (Perccr.flwicitilisL. ) was impaired

(Sandstrom. 1994) and the density and diversity of fish communities were negatively impacted (Neuman and KMs. 1988).

13. P hytochemicals

Scientific efforts have been directed towards identifying the toxic cornpound(s) present in the effluent of the pulp mill indusw. Pulp effluents are complex mixtures comprised of a myriad of chemicals. Effluent composition varies greatly. depending on:

(a) the tree species. (b) the age of the wood chips used during mill operations. (c) the pulping processes and (d) the wastewater treatment facilities used in each pulp mill (O'Connor et al. 1992). Therefore. it is unlikely that we will identify and isolate all chemicals that induce biological effects in tish.

Up until recently. most of the attention regarding identification of active chemicals has been focussed on chlorinated byproducts formed during removal of lignin at the bleaching stages (Munkittrick et ul. l992b). or on chemical constituents of the black liquor formed by the cooking process (Martell et ui. 1997). Recently. there has been interest in the degradation products of resin acids (Fragoso et ul. 1998) . However. wood extractives may contribute to the toxic impacts of pulp effluent. For instance. there is evidence that natural phytochemicals such as juvabiones can induce EROD activity and are toxic to fish (O'Connor et d 1993: Martel er ul, 1997).

Plants synthesize many secondary metabolites (i.e. phytochemicals) to combat attacks by pathogenic organisms and herbivory by insects and animals. High concentrations of these biologically active "estractives" in the tree sapwood and heartwood make "disease-resistant" (Sjostrom. 198 1 ). The toxic potential of

various classes (eg. . lignans. terpenoids. etc.) and effects have

been documented in other disciplines. especially in pharmaceutical and ontological fields.

The phytochemical families present in wood chips include C, acids (ferrulic. sinapic.

cinnamic and cournaric acids). coumarins. flavonoids. chromones. xanthones. quinones.

anthra- and naphtha-quinones. stilbenes and ( Bate-Smith. 1962: Gardner.

1962). It is not known. however. whether these chemicals are persistent enough to

--survive" the pulping and effluent treatment processes and occur in final effluent at

concentrations suficient to induce biochemical and physiological responses in fish. Of the phytochemicals described above. the flavonoid family is one of the most widespread groups of secondary substances in the plant kingdom; consisting of a large group of structurally-related compounds comprising at least 10 classes (Harborne.

1986). Flavonoids may be of toxicological importance since they affect many physiological and biological processes in animals (Harbome and Grayer. 1994) and may be important in pulp mill effluents since they are present in the heartwood of tree species at relatively high concentrations (Sjostrom. 198 1 : Fang et ul. 1 987). Dietary administration of several unsubstituted flavonoids caused induction of CYP 1A and

CYP2B activities in mammals (Brouard et al. 1988: Siess et ui. 1993,). In addition. individual flavonoids affect reproduction in animals. probably by binding either to the estrogen receptor (Miksicek. 1993) or by inhibiting the function of aromatases: enzymes that catalyze the conversion of androgens to estrogens (Kellis and Vickery. 1984). The troublesome corrosive action of flavonoids (i.e. tlavonols) in mill digesters and other pulp mill equipment provides indirect evidence that they are present in high concentrations in wood pulp (Hillis. 1962). Also. it has been proposed that colour changes in wood chips or wood pulp could be partially attributed to the enzymatic oxidation of the colourless to their corresponding yellow such as kaempferol and quercetin

(Hillis and Swain. 1962). Currently. flavonoids have not been isolated or identified analytically in pulp effluent.

Although the primary emphasis of this thesis is on the potential impacts of

flavonoids in pulp mill effluents. this class of compounds may also be of environmental

importance because recent evidence suggests that fish are exposed to them via dietary or waterborne routes. For instance. Pelissero et a1 (1 989) isolated equol. an isoflavonoid derivative. from the plasma of sturgeon (Acipenrer baerr) which had been fed on a diet consisting of 30% soya. Later work showed that the vegetable component of fish food for fmed fish is very rich in isoflavonoid content (Pelissero and Sumpter. 1992). Also. there is evidence that the same isoflavonoid (equol) is present in the effluent of municipal wastewater treatment plants and in agricultural runoffs (Bumison er al. 1000).

1.4. Objectives

The main objective of this study was to investigate and evaluate the toxic potential of several phytochemicals to fish. with an emphasis on flavonoids. Induction of CY P I A- dependent monooxy genases. embryonic development. and reproductive effects were the end-points used to assess toxicity. The selection of the test phytochemicals was based on evidence of their presence in the wood of the tree species used in pulping and/or in the pulp effluent. The objective of this study is complementary to ongoing research which focuses on the isolation of the toxic/bioactive fraction(s) or chemical(s) from pulp mill effluents. For instance. Dr. Glen Van Der Kraak and colleagues at the Univentity of

Guelph directed their efforts to understanding the role and mode of action of . especially l3-sitosterol. on the reproductive endocrine processes in fish (MacLatchy and

Van Der Kraak. 1995: MacLatchy er cd 1997: Tremblay and Van Der Kraak. 1999). In addition, there have been several attempts by other research groups to characterize the toxic potential of several natural plant constituents such as juvabiones (Martel el (II. 1997). terpenoids. fatty acids (Hewitt er al. 1996). resin acids (Ferguson er cd. 1992). as well as some degradation products of resin acids under anoxic conditions. such as retene

(Fragoso et al. 1998).

To meet the main objective. this study was divided into seven sections. with each unit having a well-defined goal. In Chapter 2. we attempted to veriFy findings reported in other studies that relatively hydrophilic chemicals which are constituents of wood pulp are hepatic EROD inducers. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to leachates from wood pulp samples from a mill prior to chlorination stage. In Chapter 3. we screened various phytochemicals for their potency to induce hepatic EROD activity in rainbow trout either after 3 days post injection. or alternatively in time-dependent experiments at 3.7. 14 and 21 days post injection. In Chapter 4. the same phytochemicals were tested for their toxicity to early life stages of Japanese medaka (Oryzkis laripes) exposed to compounds from just after fertilization to the swim-up stage (i.e. 18 days).

Hatching success. mortality. and developmental anomalies were the end-points used to assess the toxicity of the phytochemicals. Following preliminary screening experiments with several phytochemical classes. the focus of this study was shifted to tlavonoids

(Chapters 5-6). Several unsubstituted and hydroxylated flavonoids were tested in Chapter

5 for their potential to induce EROD and for embryotoxicity. as well as to affect reproduction of Japanese rnedaka. In the reproductive experiments. newly hatched medaka embryos were subjected to waterborne exposure to flavonoid for two to five months. Expression of the secondary sex characteristics. gonadal development and induction of intersex (i.e. testis-ova) were the end-points used to evaluate the effects of flavonoids on sexual development. In addition to in vivo studies, we used a battery of in vitro techniques for binding to androgen and estrogen receptors and serum sex steroid binding proteins to evaluate the potential of flavonoids to disrupt endocrine responses through binding to hormonal receptorsJ. In Chapter 6. we isolated a flavonoid Fraction

From wood pulp and final pulp mill effluent, and analyzed these samples for flavonoid

compounds by Liquid Chromatography-Mass Spectrometry (LC-MS)".Finally. study

conclusions were presented in Chapter 7.

'Most of the in vitro assays were performed at the facilities of Dr. Glen Van der Kraak at the University of Guelph.

'Dr. Richard Hughes analyzed the samples for flavonoids using the LC-MS technique at Trent University. Verification of the results were carried out in Germany. CHAPTER 2

Induction of Hepatic EROD in Fish Exposed to Leachates from Wood Pulp

Yiannis Kiparissis. Chris D. Metcalfe and Arthur J. Niimi

Published in Chemosphere. 32(9): 1833-1 841

2.1. ABSTRACT

Aqueous leachates from softwood and hardwood pulp collected from two different pulping stages in a kraft mill (Le. pre- and post-oxygen delignification) were assessed for their ability to induce hepatic ethoxyresorufin-0-deethylase(EROD) in rainbow trout

(Oncorhynchrcs mykiss). Immature rainbow trout were exposed to aqueous leachates From wood pulp for 3-1 days in a flow-through system. The leachates. from softwood pulp collected at both pulping stages and leachate from pre-oxidation hardwood pulp elevated

EROD activities 2.5- to 6-fold above reference fish. beginning at 7 days from the stm of exposure. The EROD activity remained elevated in these treatments throughout exposure. but declined rapidly during a I4 day post-exposure period. Leachate from the softwood pulp appeared to be a more potent EROD inducer than leachate from hardwood pulp. The results of this study indicate that relatively hydrophilic chemicals capable of inducing

MFOs in fish are present in wood pulp even before extensive mill processing. 2.2. INTRODUCTION

The impact of the pulp and paper industry on the aquatic environment has been well documented (Kovacs. 1 986: Owens. 199 1 ). Induction of mixed function oxygenases

(MFOs) such as ethoxyresorufin-0-deerhylase (EROD) is the most consistent biological response observed in fish populations exposed to pulp mill emuents (Andersson el ui.

1987: Oikari ei ul. 1988: Munkittrick er a/. 199 1. 1992a: Gagnon er ul. 1995). Despite the

lack of evidence to directly link EROD induction with other toxic responses noted in fish

exposed to pulp mill effluents. elevated EROD activities do indicate that fish have been

exposed to biologically active compounds that are discharged in mill wastewater

(Klopper-Sams and Benton. 1 994: Klopper-Sams et uf. 1994).

Induction of MFOs associated with the P-450I A subfamily (i-e. EROD and AHH)

is a sensitive cellular response in fish and higher vertebrates to exposure to halogenated

aromatic hydrocarbons (Janz and Metcalfe. 199 1 :Stegeman and Hahn. 1994). It was

initially believed that the halogenated compounds present in the Adsorbable Organic

Halogen (AOX) component of bleached kraft mill effluent (BKME) were the major

contributors to the EROD induction response in fish (Sodergren. 1989). However. the

association between EROD induction and AOX in mill effluents has been called into

question. since the same biochemical response has been documented in fish exposed to

the effiuents from mills that do not bleach with chlorine (Lindstrom-Seppa rt ul- 1992:

Pesonen and Andenson, 1992; Huskonen and Lindstrom-Seppii. 1995). Pulp effluents are

complex mixtures comprised of thousands of known and unknown chemicals (Suntio et al. 1988). Martel et al. ( 1994) suggested that bioactive chemicals are formed during the kraft "cooking" process. since fish exposed to "black liquor" showed elevated MFO activities. It is also possible that naturally occurring constituents of wood. such as planar terpenoids may contribute to the overall toxic potential of the pulp mill effiuents

(Klopper-Sams and Benton. 1994).

Although it is apparent that some EROD-inducing compounds are present in effluents from mills that do not use chlorine bleaching. it is not clear if these bioactive chemicals are natural wood constituents or are formed during the pulping process

( Lindstrom-Seppa el al. 1992). Since installation of secondary treatment facilities at some

mills has been documented to have little effect on the EROD inducing potency of the final

effluent (Munkittrick et al. 1992b). we may postulate that some of the active compounds

are relatively hydrophilic. Furthermore. a rapid decline in EROD activity observed in fish

during a temporary pulp mill shutdown (Munkittrick ct ul. 1992c) provided evidence that

the active compounds in the effluents were not persistent in the environment.

The purpose of this study was to investigate whether chemicals that are leached by

water from relatively unprocessed wood pulp have the potential to induce EROD in

rainbow trout (Oncorhynchus mykiss). These tests were also designed to determine

whether aqueous leachates of pulp prepared from different tree tam (i.e. softwood vs

hardwood) and from different pulping stages (i.e. pre- and post-oxygen delipification)

differed in their €ROD-induction potency. 2.3. MATERIALS AND METHODS

2.3.1. Wood Pulp

Wood pulp was obtained from a krafi mill located in Espanola. Ontario. Canada in

November of 1994. and was stored at 4°C for 2 months prior to leachate tests. This pulp mill processes softwood (85% jackpine. 15% spruce) and hardwood (33% birch. 33% maple. 33% poplar) in two separate pulping and bleaching lines. Wood chips are digested either in a single continuous hydraulic digester (softwood line) or in a batch of five digesters (hardwood line). and the wood pulp produced (brownstock) is subjected to extensive washing For the recovery of the black liquor. Afierwards. the washed wood pulp is subjected to partial delignitication in oxygen reactors before bleaching. The softwood and hardwood pulp used in this research were collected from storage tanks just prior to and after the oxygen delignification stage, and this material is referred to in this manuscript as "pre- and post-oxidation stage" wood pulp. respectively.

2.3.2. Fish Exposure and Preparation of Hepatic Microsomes

Immature rainbow trout of the Kamloops strain ( 100-230 g) were purchased from

Lynwood Acres Trout Farm. CampellcroFt. Ontario. The fish were acclimated for 3 weeks at 121°C in holding tanks receiving sand-filtered water from the Otonabee River.

Peterborough- Ontario (pH 8.1-8.3: hardness = 80-1 00 mg CaCOJL: alkalinity = - 80 mg I ater Inflow f Header Tank ! I (e-16 Urnin1 I (400 L) I I I

Water Flaw I6-8 l/mtnl Leaching A00aratu8 I I

Water Outflow A -- 1 I Pice -- - [ Trout (n-31) (8-0 L/rnm) Holding Tank ------. 1 z. (650 Li .____-Ci-___j I - /

Figure 2. i : Continuous flow-through apparatus for dosing of rainbow trout with Ieachates from wood pulp.

CaCOJL; Cr = 8.0-9.0 mgL; DOC = 5.0-5.5 mg/mL). Fish were given a maintenance ration ( - 2% weight/day) of commercial trout food (Martin Feed Mills, Elmira, Ontario).

In February 1995, fish were simultaneously exposed to aqueous leachates kom both softwood and hardwood pulp m two separate flow-through systems (Fig.2.1). Each experimental system was comprised of a header tank (400 L) receiving river water at a rate of 12 to 16 Uminute, and two holding tanks (650 L), each holding 31 trout. Above each holding tank, there was a leaching apparatus (22.5 x 15 x 17 cm) which was packed with wood pulp (Fig.2.1.1). This apparatus was packed with pre- and post-oxidation softwood or hardwood (2 kg) every day during the 21 day exposure period. Before packing, pulp was washed with river water to remove excess black liquor. Water tiom the header tank was directed into the bottom of the leaching apparatus at a rate of 6 to 8

Urnin (Fig. 2.1 ). In both systems. the rate of water replacement (>99%) in the holding tanks was empirically estimated to be approximately 7 hours. Rainbow trout (n=4-5) were removed from each tank at days 0 (reference). 7. 14.21 (exposure). and 2428.35

(post-exposure). Feeding was stopped J8h prior to each sampling day. Fish were sacrificed and their livers (minus the gall bladder) excised and rinsed with 0.154 M KC1 solution. Hepatic microsomes were prepared as described by Hodson et (11. ( 1 99 1 ).

Briefly. livers were homogenized using a Potter-Elvehjem homogenizer in KCI-HEPES buffer (pH 7.5). The liver homogenates were centrifuged at 10.000xg at 4OC for 25 min to prepare an S-9 fraction. and the S-9 was centrifuged at 105.000xg for 1 h to prepare a microsomal fraction. The precipitated microsomal pellets were resuspended and homogenized in 2 mL storage buffer solution (pH 7.4). and were stored at -80°C for a maximum of 2 months prior to analysis of EROD activity.

7.33. EROD .Analysis

All chemicals used for the EROD assay were purchased from Sigma Chemical

Co.. St Louis. Mo. USA. EROD activity was determined by the fluorometric method of

Pohl and Fouts (1980). as modified by Muir rr (11. ( 1990). Each sample was analyzed in triplicate. The reaction mixtures were prepared in 16 x 100 mm screw-top glass tubes as follows: 1300 pL of KEPES buffer (0. l M HEPES); 20 pL MgSO, ( 154 mg/mL): 40 pL BSA (40 mg/mL); 30 pL NADPH (20 mg/mL). The mixtures were incubated at 25°C for at least 5 min prior the addition of 50 pL of microsomal suspension. The reaction was stopped after 7 min by adding 3.2 mL methanol. In the blank mixtures. methanol was added prior to the addition of microsomal preparation. The samples were centrifbged at

6.800xg for 15 min to precipitate the protein and the clear solutions (3.5 mL)were transferred into glass tubes ( 13 x 100 mrn). The fluorescence of resorufin was measured with a Turner 1 10 fluorometer with excitation h of 460 - 580 nm (#58 and #65 excitation filters) and emission h of ~570nm (#23A emission filter). The fluorescence units were converted to units of EROD activity (pmoles resorufin/min/mg protein) using a standard curve (F.U. = 0.0629 x resorufin). The microsomal protein was analyzed as described by

Bradford (23)using bovine serum albumin (BSA) as the standard. Hepatic EROD activity data in exposed fish were standardized relative to the reference fish (day OI in each treatment by calculating "x-fold EROD induction" relative to the mean EROD activity in reference fish (note: standardized value for reference fish = 1 ).

2.3 -4. Statistical Analyses

Differences in relative EROD activities for fish sampled on different experimental days within each treatment and for the reference fish from the two experimental systems were tested for statistical significance by the non-parametric Kruskall-Wallis one-way

ANOVA and the Mann- Whitney test. respectively (P<0.05). Differences in the mean

relative EROD activities in fish from treatments with different wood pulp (softwood vs hardwood) and different pulping stages (pre- and post-oxidation) were tested for significance with a two-way ANOVA (P<0.05). The statistical program. SAS for

VAXNMS (Version 5.5-2). was used for the statistical analyses.

2.3. RESULTS

The EROD activities in reference fish from the softwood leachate experiment

(n=4) and the hardwood leachate experiment (n=4) varied between 4.1-8.4 pmol/min/mg

and 7.0-1 8.6 prnol/min/mg. respectively. EROD activities in reference fish from the two

experiments were not statistically different (Mann-Whitney. P=O. 13). To facilitate

statistical comparisons among the mean EROD activities in fish from the various

treatments. the EROD activities of the experimental fish were standardized relative to the

mean EROD activity for the reference fish from each treatment (i.e."x-fold" EROD

induction). It was assumed that the EROD activity of the reference fish would remain the

same to the end of the experiment, as has been demonstrated in our laboratory in previous

time-dependent studies.

Fish exposed to leachate From both pre- and post-oxidation softwood pulp and the

pre-oxidation hardwood pulp showed elevated mean EROD activities by day 7. and

EROD remained elevated (3.5 to 6-fold) in these treatments throughout the 21 day

exposure periods (Figure 2.2a-c). However. during the 14 day post-exposure period. mean

EROD activities in trout returned to near reference values in these treatments. An

unexplained deviation from this pattern was observed at 3 days post-exposure for the a. Softwood Before Oxidation b. Softwood After Oxidation

Day Day c. Hardwood Before Oxidation d. Hardwood After Oxidation

Figure 2.2 : Mean (k I standard error) for ethoxyresorufin-0-deethylase (EROD) activity in rainbow trout (n = 4-5)relative to reference fish during continuous-flow exposure to leachates from sofbood pulp (2.2a. 2.3)and hardwood pulp (22. 2.2d) at 7. I4 and 2 1 days during exposure. and at 3.7 and 14 days during a post-exposure period (PE3. PE7. PElJ). Pulp tested was taken From the mill prior to and after the oxygen delignification stage in pulp processing. EROD activity is expressed as an "x-fold" change in relation to the mean EROD activity in the reference (Day 0) fish. Those treatments in which mean EROD activities were not significantly different (Kruskall-Wallis: Pc0.05) are notated with the same lower case letters above the standard error bars.

"softwood pre-oxidation" treatment where the EROD activity declined to reference levels

(Fig. 2.2a).

In the present study. there was no statistically significant difference in the magnitude of EROD induction in rainbow trout exposed for 21 days to leachate from the pre- and post-oxidation sohood pulp (Table 2.1 ). These data indicate that the leachable compounds in softwood that induce EROD are not removed during the oxidative delignification process. However. there was a difference in the response to leachates From pre- and post-oxidation hardwood pulp (Table 2.1). Mean EROD activity in fish from the post-oxidation treatment was only slightiy elevated (I .8 fold) above reference fish after 2 1 days of exposure, and the mean EROD activity fell below reference fish by the end of the post-exposure period (Figure Xd).

Analysis by two-way ANOVA of the EROD data for all rainbow trout in the softwood treatments and hardwood treatments (Table 2. 1) showed that EROD induction was significantly greater in magnitude among fish exposed to leachate from the sofiwood pulp (P=0.002). ANOVA analysis (Table 2.1 ) also confirmed that there were significant differences in the responses of trout to leachates from the pre- and post-oxidation hardwood pulp (P=0.03).

Table 2. I :Relative ethoxyresorufin-O-deethylase induction (x-fold) in rainbow trout during 3 weeks of exposure and 2 weeks of post-exposure to leachates from softwood and hardwood pulp taken from two different pulping processes (i.e. prior and after the oxygen defignification stage). P-values, as calculated by a two-way ANOVA. are also presented.

Pulp Type EROD (-fold) Pulping Processing Stage EROD (-fold)

Softwood (n=62) 2.7k0.2 Before Oxidation (n=3 1) 2.5k0.3

After Oxidation (n=3 1 ) 2.8i0.3

Hardwood (n=62) 1.7k0.2 Before Oxidation (n=3 1) 2.1k0.2

After Oxidation (n=3 1) 1-2kO.1 I 1 P,,, = 0.002 Ppmesss = 0.169 Ppulps ptocess = 0.030 2.5. DISCUSSION

Trout exposed to all leachate treatments (excluding the post-oxidation hardwood pulp treatment) showed elevated mean EROD activities by days 7 to 2 1. Afterwards. during the 14 day post-exposure period mean EROD activities declined to reference levels. MFO activity does not rapidly decline to baseline levels in fish exposed to persistent halogenated aromatic compounds (Janz and Metcal fe. 199 1 : Stegeman and

Hahn. 1994). These data may indicate that the EROD inducing constituents in softwood pulp (pre- and post-oxidation) and hardwood (pre-oxidation)are relatively hydrophilic chemicals that are cleared rapidly from the fish post-exposure.

The rapid post-exposure decline in EROD activity is consistent with a field study showing that EROD activity in white suckers ( Crtrusromzis commersoni) declined during a

1 week pulp mill shutdown (Munkittrick el tri. 1992~).The authors of this study suggested that the compounds in mill effluent responsible for EROD induction were: a) not effectively retained by the secondary treatment facilities at the mill. b) did not originate from contaminated sediments near the mill. and c) were easily cleared by the fish; which is consistent with exposure to relatively hydrophilic. non-persistent compounds. In addition. EROD induction has been observed in rainbow trout caged at sites 2 and 3 km (but not at 28 km) downstream from the pulp mill in Espanola (Williams er al. 199 1 ). which may indicate that the bioactive chemicals are relatively water-soluble compounds that can be transported long distances in the effluent plume.

It is apparent that EROD induction was significantly greater in trout exposed to leachates fiom the softwood pulp. Our findings are consistent with the results from other studies. For example, Andenson et al. (1987) observed differences in the responses of fourhorn sculpin (Myoxocephalus quadricornis) to exposure to BKME fiom a softwood

(pine) line and hardwood (birch) line. In this study. EROD induction from the pine line or a combination of pinehirch effluents was generally several orders of magnitude higher than that of the birch line effluents. OIConnoret al. (1 992) found that simulated effluents prepared from different tree species showed different degrees of toxicity to aquatic biota. but in this study. effluents prepared from two softwood species (i.e. white pine and balsam fir) appeared to be the most toxic.

Other studies have shown that EROD activity is elevated in fish exposed to the hydrophilic compounds in black liquor (Martel et ul. 1994: Hodson et al. 1994). Most of the black liquor was removed from the pulp used in this study. but it is possible that residual material normally removed as black liquor was responsible For EROD induction.

Williams ( 1993) found that EROD induction in fish exposed to black liquor from softwood and hardwood was not different when normalized for DOC content.

Nevertheless, the data from the present study could indicate that there are more potent

EROD inducers in the material leached from softwood pulp in comparison to the leachates fiom hardwood pulp. or alternatively. the data could indicate that hardwood contains higher levels of compounds that inhibit this biochemical response. Future studies of this type should include analysis of the induction of P-JjOl A-specific mRNA. the rates of hepatic DNA synthesis. and other biochemical parameters that may provide some insight into the mechanisms governing the variable responses of fish exposed to these complex mixtures.

EROD is a catalytic probe for the P-4501A sub-family of MFOs. which is the primary metabolic system for Phase I biotransformation of aromatic xenobiotics

(Kloepper-Sams et al. 1994). Model chemicals for induction of PA50 1 A-associated

MFOs generally include halogenated aromatic compounds with planar molecular configurations that bind readily to the Ah-receptor (Williams el d..1 99 1 ). However. there is evidence that EROD activity in organisms can be induced by compounds from other chemical classes. For example. treatment of rats with acetone elevated hepatic

EROD activity by almost 2-fold (Barnett et ui. 1992). Treatment of rat hepatocytes with dimethyl sulfoxide induced EROD in a dose-dependent manner (Jeong et d.1992).

Payne and Fancey ( 198 1 ) found that leachates from bark and spruce needles slightly increased gill and liver MFO activities in trout.

Since the oxygen delignification reactors in the pulp mill are installed before the chemical bleaching stage in both the hardwood and sofiwood lines. the results of this study confirm that non-halogenated chemicals have the potential to induce EROD in fish.

We may also infer that some of the bioactive compounds. whether they are natural constituents of wood pulp or pulping by-products. are relatively water-soluble.

Overall. these data indicate that more research is needed to determine the identity of bioactive consituents of wood pulp and pulp mill effluents. No attempt was made in this study to relate the volumes of pulp and water used in exposure experiments to actual effluent discharge volumes in pulp mills. These types of studies are needed to develop pulping techniques and effluent treatment systems that remove bioactive compounds from final mill effluent. Further studies could also identify which tree species contain the highest concentrations of these potentially toxic chemicals. CHAPTER 3

Induction of Hepatic CYP1 A-dependent Monooxygenares in Rainbow Trout

(Oncorhyncltus mykiss) Dosed to Phytochemicals

Yiannis Kiparissis. Arthur J. Niirni and Chris D.Metcalfe

Submitted to Aquatic Toxicology

3.1. ABSTRACT

Natural chemical constituents of wood pulp may contribute to the induction of hepatic cytochrome P-450 monooxygenases (CYPI A) that have been observed in tish populations exposed to pulp mill effluents. In this study. eight phytochemicals. tlavone. anthrone. juglone. [runs-stilbene. tropolone, harmane. 7-methoxycoumarin and a mixture of sitosteroVcampestero1 were tested For their ability to induce ethoxyresorutin-0-

deethylase (EROD)in 72 hours following a single i.p. dose ( 10 mg/kg) to immature

rainbow trout (Oncorhynchus mykiss). The chemicals harmane. flavone and tropolone

caused a 2.4.2.8- and 3.5-fold increase in hepatic EROD activity. respectively. A time-

dependent experiment indicated that EROD activity declined rapidly post-exposure. The

test compound 7-methoxycoumarin was not an EROD inducer although in a preliminary

study. it was found to cause a 4.7-fold increase in the activity of aryl hydrocarbon

hydroxylase (Am.another catalytic marker of the CYP l A family. The CYPl A inducing

compounds in our study were relatively hydrophilic (log K, 0.53 to 2.9 1): indicating that they are unlikely to induce EROD or AHH in fish once aqueous exposures cease. These results indicate that relatively hydrophilic natural phytochemicals derived from wood pulp may contribute to the elevated CYP 1A activities observed in feral fish exposed to pulp mill efnuents.

3.2. INTRODUCTION

The pulp and paper industry discharges numerous organic substances into the aquatic environment (Suntio er al. 1988). Feral fish populations downstream of pulp mills experience a variety of adverse effects. including skeletal and gill deformities. alterations in serum sex steroids. impairment of reproduction and increased activity of hepatic cytochrome P-450-dependent monooxygenases (Andenson el ul. 1988: Bengtsson cf id.

1988: Sodergren. 1989: McMaster el cii. 199 1 : Munkittrick er d. 1992a 199%).

Induction of the hepatic ethoxyresorufin-0-deethylase(EROD) has been used extensively as a biomarker of exposure to pulp mill efnuents since it is the most sensitive and commonly observed response in fish (Andersson et af. 1988: Bengtsson er ul. 1988:

Martel et ul. 1994; McMaster er ul. 199 1 ; Munkittrick er ul. 1999). Although it has been

suggested that EROD is induced by exposure to some chlorinated compounds formed as

by-products of pulp bleaching (Sodergren. 1989). or chemical constituents of "black

liquor" (Martel el cd 1994). most of the bioactive chemicals in the pulp plume have not

been identified.

In a previous study (Chapter 2). we demonstrated that Ieachates from wood pulp induce hepatic EROD activity in immature rainbow trout (0ncorhynchu.smykiss). and postulated that at least some of the inducing chemicals are narurally-occurring plant metabolites or "phytochemicals" (Kiparissis et al. 1996). Plants synthesize many biologically-active secondary chemicals for protection against herbivory and parasitism

(Bell. 198 1 ). In the heartwood of tree species used in pulping operations there are numerous classes of phytochemicals. collectively referred to as wood extractives

(Sjostrijrn. 198 1). The biocidal properties of phytochemicals such as terpenoids. alkaloids. lignans arid (po1y)phenolic compounds such as flavonoids and coumarins have been extensively documented (MacRae and Towers. 1984: Towers. 1984; Zobel and Brown.

1995). Herbivores may have evolved biochemical processes. including cytochrome P-JSO enzymes. to detoxify and excrete these phytochemicals (Nebert and Gonzalez. 1 987).

During pulping operations. the effluent flows of typical pulp mills range between 50 to

170 thousand m5/day (Robinson et a!. 1994): hence. feral fish may be exposed to large amounts of phytochemicals present in pulp effluent.

The primary objective of this study was to screen several phytochemicals representative of different chemical families for their potential to induce hepatic EROD in immature rainbow trout. The selected phytochemicals were 7-methoxycoumarin. tropolone. harmane. flavone, anthrone. rruns-stilbene. juglone and a mixture of D-

sitos~erol/campesterol(Fig.3.1) and B-naphthoflavone was used as a positive control

since it is known to be a potent inducer of CYP 1A-dependent monooxygenase activities.

Chemicals were selected on the basis of evidence that they or their metabolites are present

in the heartwood of trees or in pulp mill effluents. that they have a steric molecular Anthrone

0 Jug lone

- -

CH HC '

trans-stii bene

Tropolone

rahbow trout (hcorhynch~mykiss). structure appropriate for binding to the Ah receptor, and that they are commercially available. Juvenile rainbow trout were exposed in vivo to each chemical with a single i.p. dose and liver microsomes were prepared for analysis of hepatic EROD activity.

.9 9 MATERIAL AND METHODS

3 -3 1 . Chemicals

Anthrone. flavone (2-phenyl-4H-l -benzopyran-4-one). juglone ( 5-hydroxy- 1.4- naphthoquinone). harmane ( I -methyl-9H-pyrido[U-blindole;P-carboiine ). trans-stilbene

(trans-1 .2-diphenylethylene). tropolone (2-hydroxy-2.4.6-cycloheptatrienone). 0- naphthoflavone (5.6-benzoflavone) and all reagent chemicals used for the EROD bioassay were purchased from Sigma Chemical Co. (St. Louis. MO). 7-methoxycoumarin was purchased from ICN Biochemicals Inc. (Aurora. OH). The purity of these compounds was

>97%. A mixture of p-sitosterol(60%) and carnpesterol(40%) was purchased from

Aldrich Chemical Co. (Milwaukee. WI). For the determination of microsomal protein concentration. a concentrated dye reagent was purchased from Bio-Rad Laboratories

(Hercules, CA). and bovine serum albumin was purchased From Sigma Chemical Co. (St.

Louis. MO).

3.3.2 CYP l A induction assays The in vivo EROD induction assay was conducted with immature rainbow trout

(100 - 250 g) as described previously (Sections 2.3.2 and 2.3.3) with some modifications.

Briefly, rainbow trout were purchased from Linwood Acres Trout Farm and placed in 650

L holding tanks (n=30-40) receiving sand-filtered Otonabee River water. The fish were acclimated to IM"C for at least 3 weeks prior to chemical exposure. During that period. the fish were fed ad libitrcm with commercial trout pellets and feeding was stopped 3 days prior to exposure in short term tests. or 3 days prior to sacrifice in the time-response experiment.

The phytochemicals were dissolved in dimethyl sulfoxide (DMSO) and phosphate buffer saline (PBS)( 1O/9O. v/v). with the exception of the non-polar chemicals sitosterol/carnpesterol. trans-stil bene and t3-nap htho flavone (O-NF) which were dissolved only in DMSO.Fish (n = 4-5) were anaesthetized with MS-212 and i.p. injected with a single dose of 10 mgkg (dose determined with previous preliminary tests) of each test chemical at a constant volume of 2 mVkg. Control fish (n=3) were i.p. injected with the camer solvent DMSO or DMSOPBS. D-NF ( 10 mg/kg) was used as the positive control.

Trout i.p. injected with flavone. trans-stilbene. juglone. anthrone. and sitosteroll campesterol were sacrificed at 72 h post-injection. The time-dependent experiments with trout exposed to harmane. tropolone and 7-methoxycoumarin were terminated at intervals of 3.7. 14 and 2 1 days post-injection while control fish were sacrificed at days 3 and 2 1.

The hepatic microsomal hctions were prepared with differedal centrifugation as

described previously (Janz and Metcalfe. 1991 : Kiparissis er al. 1 996).

EROD activity. expressed as pmoUmin1mg. was determined fluorometrically as described previously (Kiparissis et a/. 1996). AHH activity, expressed as pmol3-hydroxy-

BaP/min/mg, was also determined fluorimetrically according to Janz and Metcalfe

(199 1). The microsomal protein content was determined as described by Bradford ( 1976)

using bovine serum albumin (BSA) as the standard. Each sample was analyzed in triplicate. EROD activities were log transformed and differences in mean EROD activity

between control and exposed fish were analysed with either a t-test in short-term tests. or

ANOVA (Dunnett's test) for the time-dependent experiment. Hepatic EROD and AHH

activity data in exposed fish were standardized relative to the Controls in each treatment

by calculating "x-fold CYP1 A induction".

3.3 2. Determination of octanol/water partition coeficients for phytochemicals

A reverse-phase high-performance liquid chromatography (HPLC) approach. as

described by McDuffie ( 198 1 ). was used for estimation of the octanollwater partition

coefficients (KO,,)of the tested phytochemicals. All phytochemicals were dissolved in

methanol at a concentration of SO pghl and 20 p1 samples were injected and

isocratically eluted From a reverse phase C,,column with a mobile phase of 7525:

MeOH:H20 (vlv) at a flow rate of 1.0 ml/min using a Waters model 600E HPLC

equipped with a stainless steel 5 pm reverse phase C,, column (Supelco). 0.46 x 25 cm

(ID). and a Waters C,,Novapak (4 pm) guard column. Analytes were detected with a

Waters 484 variable absorbance detector set at 254 nm. The injection port was equipped

with a Waters U6K injector unit (2 ml sample loop). The software used was the Baseline 8 10 (version 3) software by Dynamic Solutions (Millipore).

This HPLC approach assumes a linear relationship between log Kowsand the capacity factor. k'(i.e. t/to; retention time of solutes/ time for the solvent chemical uracil to reach the detector). Thus, retention times for the test compounds were compared to retention times for the reference compounds. acetophenone. benzene. toluene. naphthalene and phenanthrene, with known Ko,s. Log Ko,s of the test compounds were calculated by the linear relationship of log = 2.56 +2.65 log k' (McDuffie. 198 1).

3.4. RESULTS

The mean EROD activities in control fish at 72 h post-injection were 7.6k0.8

(DMSO) and 6.5*0.4 (DMSOPBS) pmol/min/rng. Exposure to flavone at a dose of 10 mg/kg caused a 2.8-fold increase in hepatic EROD activity (i.e. 1 7.Ok2.l prnol/min/mg) at

72 h post-injection; significantly higher than EROD activity in control trout (P = 0.006).

On the other hand. exposure to the same dose of the phytochemicals anthrone. juglone. trans-stilbene and the mixture of R-sitosterol/campesterol failed to significantly induce

EROD activity after 72 h of exposure. The positive control compound R-NF at a dose of

I0 mg/kg significantly increased EROD activity by 32-fold (Pc0.00 1 ) (Table 3.1 ).

In the time-dependent experiment. the mean EROD activities of the control fish at

3 and 2 1 days post-injection were 6. I* 1.0 and 5.4*0. 1 pmol/min/mg. respectively. Since these activities were not significantly different. the data were combined and then statistically compared to the EROD activities of the tish exposed to phytochemicals. Table 3.1 : Hepatic ethoxyresorufin-0-deethy lase (EROD) activity in immature rainbow trout (Oncorhynchus mykiss) at 72 h after a single intraperitoneal (i.p.) injection with anthrone, juglone, oms-stilbene, sitosterol/campesteroI and O-naphthoflavone at a dose of 10 mgfkg. EROD activities are expressed as pmol/min/mg protein and as x-fold (mean * standard error) in relation to their corresponding controls (DMSOor DMSOIPBS exposed fish).

Chemical N EROD (prnol/minlmg) Relative Induction (x-fold)

SitosteroI/campestero1 5 rrm-stilbene 4 Control (DMSO) 3

Flavone 5 1 7.0 i 2.1 (P

There were similar patterns of EROD induction in Ash i.p. injected with tropolone and

harmane. Monooxygenase activities were significantly elevated by 3.5- and 2.4-fold (day

3). 3.3- and 1.9-fold (day 7) and 1.9- and 1 &fold (day 14). respectively (P < 0.005).

EROD activities declined to basal levels by day 2 1 post-injection. The phenolic

compound. 7-methoxycoumarin (7-MC) failed to induce EROD activity at any point post-

injection (Fig. 3.2). However. the same chemical in a preliminary study induced AHH

induction by 2- and 4.7-fold at day 3 and day 7 post-injection. respectively (P<0.0005)

(Fig. 3.3).

The retention times from the reverse-phase HPLC indicated that the Ko,,s of the

CYP 1A inducing chemicals tropolone. 7-MC and flavone are 0.53. 1.14 and 2.9 1.

respectively (Table 3.2). Log K,,s calculated by this method for tropolone and tram-

stilbene were identical to those reported by Hansch and Leo ( 1979) . We were unable to ': ': tlamanr.

.. --.-.- Tropolone

------7-MC

Control

Figure 3.2: Timedependent hepatic EROD activity in esperiment with immature rainbow trout (Oncorhynchzismykrss) following a single intraperitoneal (i.p.) injection of DMSO/PBS (control). harmane. 7-methosycoumarin and tropolone at a dose of 10 mg/kg. Each bar and vertical line represent the mean * standard error: asterisks represent significantly elevated EROD activities relative to controls at cx s 0.05. -- AHH

-0- EROD

...... +...... *-.".* ...... t...... *."-a Figure 3 -3: Timedependent hepatic EROD and AHH amvities. eztpressed as x-fold induction. in esperiments with immature rainbow tmut (0ncurhynchli.v myhss) following a single intraperitond (i.p.) injection of %metho?c~coumarinat a dose of I0 mgkg. Each bar and vertical line represent the mean * standard error: asterisks represent significantly elevated CYP IA activities at I 0.05. Table 3.2: Octanolfwater partition coefficients (log Y,) of the tested phytochemicals estimated by the reverse-phase high-performance liquid chromatography technique of McDufie ( 198 1 ).

Chernical tm t, t, k' log k' Ioe; K, Reference Compounds uracil 1.41 1 .41 0.00 - - - acetophenone 1.89 0.48 0.34 -0.46 1.58 benzene 2.58 1-17 0.83 -0.08 2.13 toluene 3.39 1.98 1.40 0.15 2.65 naphthalene 4.19 2.78 1.97 0.29 3.4 1 phenanthrene 8.26 6.85 4.86 OA9 4.57

Phvtochemicals anthrone 4.59 3.1 8 2.26 0.3 5 3.49 tlavone 3.33 1.93, 1.36 0.13 2.9 1 juglone 2.26 0.85 0.60 -0.22 1-98 7-methoxycoumarin I .82 0.41 0 .29 -0.54 1.14 trans-st i l bene 9.18 7.77 5.51 0.74 3.52 tropolone 1.65 0.24 0.1 7 -0.76 0.53

determine the KO,for harmane due to retention of the compound on the column: however

by using the software program. KOWWM v 1.65 (43 W. Meylan. 1993-1995) the log KO,,

for hannane was estimated to be 2.75.

3.5. DISCUSSION

Flavone induced hepatic EROD in immature rainbow trout. which is consistent

with induction of CYP 1A enzymes by flavone in in vitro and in vivo studies with

mammalian models (Siess and Vemevaut. 1982: Siess et a!. 1995: Canivenc-Lavier et ui.

1996). No responses were observed in trout exposed to anthrone and juglone. No previous

studies in fish or mammals have demonstrated induction of CYP 1 A activities by these compounds. Tests with the mixture of O-sitosteroVcampesterol in this study are in agreement with the findings from a similar teleost study which showed that l3-sitosterol does not induce EROD activity in immature rainbow trout (Hewitt et ul. 1996). Although trans-stilbene did not induce EROD in this study. trans-stilbene oxide was previously shown to be an inducer of the CYP2B enzyme subfamily in mammals (Meijer el al.

1982). but not for CYPIA (Kuo el al. 1984).

The significance of the time-response data is threefold. First. for the first time it was shown that harmane and tropolone have the potential to induce hepatic CYP 1A enzymes in fish. Although in the present study 7-MC did not elevate EROD. in a preliminary study it increased significantly the activity of ary 1 hydrocarbon hydroxy lase

( AHH), another CYP 1A marker. in immature rainbow trout. Second. the decline in EROD activity in fish exposed to these phytochemicals is consistent with the pattern of field observations where EROD activity declined rapidly to basal levels in white suckers

(Cutostomus commersoni) during a two week pulp mill shutdown. indicating that the Ah agonists affecting the fish were relatively hydrophilic (i.e. non-persistent) and not efficiently eliminated by the primary and secondary treatment facilities (Munkinrick et (11.

1992~).Third. the findings from this study justified our previous hypothesis that EROD inducing compounds present in wood pulp leachates may have been of natural origin

(Kiparissis et al. 1996).

The primary objective of this study was to investigate the contribution of secondary plant constituents to the EROD induction observed in feral fish populations exposed to pulp and mill effluents. The findings show that four of eight phytochemicals tested at an i.p. dose of 10 mgkg significantly elevated the CYPl A1 (ERODand AHH) activity in immature rainbow trout. Considering the facts that pulp effluent is a mixture of numerous chemicals and that pulp mills typically process thousands of tomes of wood chips daily. fish downstream of the pulp mills may be exposed to large amounts of several phytochemical classes. It has been theorized that the function of some sub-families of cytochrome P-450-dependent monooxygenases to catalyze exogenous substrates in vertebrates are products of selective pressures attributed to dynamic plant-animal interactions (Nebert and Gonzalez. 1987). Thus. considering the evolutionary importance of the cytochrome P4Os in animals for metabolizing toxic chemicals of plant origin. our results were not unexpected and are consistent with other studies. For instance. the phytochemical eugenol at a relatively large dose of 1000 mg/kg caused an approximate 1- fold increase in EROD in male Wistar rats exposed by gavage (Rompelberg el d.1993).

As shown in our experiments with sitosterol/campesterol. [runs-stilbene. j uglone and anthrone. at doses of 10 mgkg. not all phytochemicals induce CYPI A enzymes in fish. It has also been demonstrated that resin acids (abietic. dehydroabietic and a resin mixture) are not responsible for EROD induction (Ferguson el ui. 1992). In another study. resin acids. fatty acids. and several terpenoids. as well as R-sitosterol were ruled out as

EROD inducers (Hewitt el al. 1996). On the other hand. juvabione and dehydro- juvabione. phytochemicals present only in balsam fir (Abies bulsumea) significantly

increased hepatic EROD activity in immature rainbow (Martel el ul. 1997). However. the

authors ruled out the possibility that these chemicals affect fish populations near pulp

mills since they are removed by secondary wastewater treatment (Martel el ul. 1997). Among the phytochemicals tested in our study, only sitosterol has been documented as a constituent of pulp mill effluents (Fox. 1976). Although the parent compounds flavone. tropolone and stilbene are not expected to be found in pulp mill effluents, many of their

substituted derivatives are likely to be present. Several flavonoids (quercetin. kaempferol. taxifolin. chrysin). and stilbenes (rhapontin and pinosylvin) are heartwood constituents in tree species commonly used by the pulp mill industry (Bate-Smith. 1962: Sjostrom.

198 1). In addition. the methylated tropoiones, a-. 6- and y- are found in the

heartwood of the western red cedar (Thujuplicata) and also in tree species of the

Cupressaceae family (Gardner. 1962). Furthermore. the presence of juglone and anthrone.

representatives of naphthaquinones and respectively. has been detected in

the heartwood of trees (Bate-Smith. 1 962).

Another significant finding of our study is associated with the low octanol/water

partition coefficients (log KO,)of the EROD inducing chemicals. Burnison er ul ( 1996)

used Fractionation techniques for bleached-haft mill effluent (BKME) to indicate that

most of the EROD inducing compounds are moderately hydrophobic (log KO,between

4.6 to 5.1). The K,,, of retene. an alkyl-substituted phenanthrene that is a degradation

product of resin acids and induces EROD activity in fish. fits the above specification

(Fragoso el d 1998). Our results showed that more hydrophilic phytochernicals (KO,<

3.0) may significantly elevate the CYPlA enzyme activities in tish exposed by i.p.

injection.

The boclassical"process of CYP 1A induction requires binding of non-polar

compounds with an appropriate steric conformation (eg. PAHs. TCDD. etc) to the dh receptor, followed by the translocation of the activated receptor to the nucleus. causing transcriptional, translational and posttranslational events that result in the synthesis of

CYP 1A protein (Stegeman and Woodin, 1984). However. Celander and Forlin ( 199 1) showed that the EROD inductive responses to rainbow trout exposed to isosafiole were different from these of O-NF exposed trout. indicating alternative unknown mechanism(s) for the activation of CYP 1 A enzymes. It is possible that the observed elevated EROD activities in trout exposed to the test phytochemicals. especially the most hydrophilic (i.e. tropolone and 7-methoxycoumarin) occurred by mechanisms that do not involve binding to the Ah receptor. Increases in total P-450 content and induction of other

CYP isoenzymes may be an alternative mechanism for the elevated EROD activity.

Future studies using DNA probes or imrnunochemical techniques to measure the levels of the CYP 1A-rnRNAs and the CYP I A proteins may be needed to investigate such alternative mechanisms of induction.

This study demonstrates the importance of investigating several classes of phytochemicals for their potential to induce CYP l A enzymes and cause other biological responses in fish exposed to pulp mill effluents. Natural constituents of the heartwood of pulpwood species may induce responses in fish which are similar to these observed in fish exposed to by-products of chlorine bleaching. However. this study shows that large amounts of these phytochemicals are required to cause modest EROD induction in fish relative to the potency of CYP 1A induction by chlorinated dioxins. dibenzofurans. and coplanar PCBs (Janz and Metcalfe. 199 1). CHAPTER 4

Embryotoxic Potential of Phytochemicals to Japanese Medah (Oryzius latipes)

4.1. ABSTRACT

The phytochemicals anthrone. flavone. harmane. juglone. 7-methoxycoumarin and tropolone were evaluated for their toxic potential to fish by exposing Japanese medaka (Orpius latipes) embryos to various nominal concentrations (0.1 to 10 pg/mL) from fertilization to swim-up stage (day 18). Hatching time and success. mortality. and gross pathomorphological defects were the embryonic endpoints used for assessing the toxicity of the test phytochemicals. All tested phytochemicals but anthrone were embryotoxic to medaka with median effective nominal concentrations (ECSOs) estimated between 0.2 and 0.6 pg/rnL. Juglone was lethal at 1 - 10 pg/mL. Flavone. harmane. tropolone and 7-methoxycoumarin at the higher nominal concentrations elicited a multitude of embryonic responses. such as developmental retardation. circulatory defects. craniofacial aberrations. yolk and pericardial edemas. failure of successful swim bladder inflation. failure of yolk sac absorption and skeletal abnormalities. Delay in median hatching time was observed at the lower tested nominal concentrations in treatments with juglone. flavone and 7-methoxycomarin. Some of the aforementioned embryotoxic responses were chemical dependent. whereas others were common to more than one treatment. In addition. the findings of the present study indicate indirectly that exposure of fish during the early life history stages could affect population recruitment. Since fish may be exposed to phytochemicals through a variety of pathways. including exposure to pulp and paper mill effluent, the potential for effects on fish at the biochemical. individual and population level should not be underestimated or overestimated.

4.2. INTRODUCTION

Fish exposed to pulp mill effluents experience a variety of adverse biochemical and physiological responses. including reproductive dyshction depression of serum sex steroids. higher incidence of skeletal abnormalities. altered carbohydrate metabolism and induction of the cytochrome P.JjO 1A (CYPI A) monooxygenases ( Andersson ei al. 1988:

Bengtsson et a/. 1 988; SMergren. 1989: Munkittrick er a&.1992). Large capital investment by the pulp and paper industry to modernize pulp and paper mills by installing secondary treatment facilities and changing bleaching technologies has resulted in minimal reduction in the aforementioned biological responses (Lindstrom-Seppa cr ul.

1992; Pesonen and Andenson. 1992: Munkittrick er d 1 994). indicating that by-products of chlorine bleaching may not contribute to the overall toxicity of pulp mill emuents.

Consequently. a research priority is characterization of the effluent and identification of the toxic chemicals or fraction of the pulp plume that may have environmental impacts on /. biota in areas receiving discharges (Ahtianet (11. 1996: Hall et ul. 1996: LaFleur. 1996:

Verta et al. 1996).

CYP 1A induction. especially EROD. has been the most commonly used biomarker to characterize the bioactive potential of pulp and paper mill effluents

(Lindstrom-Seppa and Oikari, 1990; Servos. et al. 1992; Ahokas el al. 1994; Martel el ui.

1994; Martel and Kovacs, 1997). It has been demonstrated that CYP I A inducing compounds are present in most effluents. regardless of the differences in pulping processes. wood species. bieaching sequences and treatment of effluents (Martel et ul.

1994: Martel and Kovacs. 1997: Munkittrick et al. 1999). However. there are questions regarding the use of the EROD bioassay as the sole biomarker for assessing environmental impacts to feral fish since EROD induction is a biochemical response to stressors rather than a necessarily negative toxicological effect to fish ( Lehtinen. 1 996).

Therefore. it seems appropriate that EROD induction should be coupled with other meaningfid toxic endpoints. especially at the organismal level. to characterize the toxicity of pulp effluents (Lehtinen. 1996: Sandstrom. 1996).

In Canada. currently there is legislation requiring that all pulp mill effluents be tested and their toxic potential assessed by short term experiments using only early life stages of fathead minnows ( Pimephules promelm) and the cladoceran. C'erioduphniu ditbia (Environment Canada. 1W2a: 1 W2b). The tests with fathead minnows or other

short-term tests are believed to be sufficient for screening chemicals or mixtures of chemicals for their toxicity since the experimental species are subjected to testing at the

very early sensitive stages of their life histories (McKim. 1977). However. by using this

approach. the embryonic stage of fish is not utilized for testing when important ontogenic

kctions such as cell differentiation. cell migration and organogenesis occur (McKim.

1977). Furthermore, embryo-lama1 tests have been shown to be more sensitive tests than the post swim-up early life stages of fish in assessing the toxicity of individual chemicals or chemical mixtures (Kovacs and Megraw, 1996).

The Japanese medaka (Oryzias latipes) embryolarval assay may be a usehl short- term model to measure and assess developmental toxicities by exposure to environmental chemicals (Metcalfe et al. 1999). There are certain advantages to using the medaka model since: (a) ovulation is induced by manipulation of temperature and photoperiod: (b) the egg chorion is clear and all developmental processes and defects are easily visible: (c) the hatching time of embryos and the swim-up stages of larva are short: and (d) acute toxicity and teratogenic effects are consistent with effects in other fish models (Kirchen and West.

1976: Shi and Faustman. 1989). in addition. the Japanese medaka life history. especially the timing of the developmental stages has been thoroughly described (Iwamatsu. 1994)

It was previously reported (Chapter 3) that several phytochemicals. representative of different chemical families. were tested for their ability to induce EROD activity in rainbow trout. It was the purpose of this study to screen the same phytochemicals (Fig.

4.1) for their potential to cause toxicity or to induce developmental abnormalities in

Japanese medaka embryos exposed over a period from just after fertilization to L 7 days after fertilization. The phytochemicals. l3-sitosterol and trans-stilbene were not tested in the present study since in preliminary studies the first compound was not embryotoxic at the highest nominal concentration used (i.e. 10 pg/rnL). whereas the second compound was not soluble in water at concentrations between I and 10 pg/rnL. 0 Anthrone Tropolone Flavone

'Ll u' \./\N---.2N U' CH iw/\d'% CHI 0 Harmane Juglone 7-methoxycoumarin

Figure 4. I. Molecular structure of phytochemicals used in the Japanese medaka (0ryriu.s luripes) embryotoxicity assay.

4.3. MATERIAL AND METHODS

4.3.1. Chemicals and Solutions

The suppliers of the phytochemicals anthrone. flavone. hamane. juglone. 7- methoxycoumarin. and tropolone were listed previously in Chapter 3 (Section 3.1). The embryo rearing solution (1 rnL 10% NaCI. 1 mL 0.3% KCI. 1 mL 0.4% CaC12.2H20.1 mL 1.63% MgS0,.7H,O. 1 mL 0.01% and 95 mL glass distilled H,O) was purchased fiom Carolina Biological Supply Company, Burlington NC, USA.

4.3.2. Japanese medaka embryotoxicity assay

The embryotoxic potential of all phytochemicals was tested with embryos of

Japanese medaka using a static non-renewal assay slightly modified from an assay described previously (Wisk and Cooper. 1990: Harris et d.1994). All phytochemicals were dissolved in acetone (distilled in glass). and 50 or 100 pL aliquots of these solutions were placed in glass autosampler vials prior to testing. In the control vials. only acetone was added. The acetone was allowed to evaporate in the fume hood. and 1 mL of embryo rearing solution was added. Nominal concentrations of all phytochemicals were 0.1. 1. 5 and 10 pg/mL.

Japanese medaka eggs from several female fish were collected after fertilization

(0.5 to I h). separated fiom each other and pooled in a petri dish with embryo rearing solution. All eggs were thoroughly mixed and each individual egg was placed in one exposure vial (n = 25 per treatment) which was immediately capped with a Teflonk cap.

All embryos were examined under a dissecting microscope to ensure that no damage occurred during handling, and were placed in an incubator at 25 "C.For the next 18 days. approximately at the same time daily. the embryos were examined to determine the stage and pattern of development. occurrence of lesions. hatching success and mortality.

The median lethal concentrations (LCSOs) and median effective concentrations (ECSOs) for abnormals embryos were estimated by Probit analysis. All results were expressed as percentages. and the overall toxicity of the individual phytochemicals was tested on the basis of the proportion of abnormal embryos in relation to the corresponding

Controls using the f-test. The non-parametric Kruskal-Wallis test was used to compare the time of hatching for embryos exposed to phytochemicals relative to Controls.

4.4. RESULTS

Hatching success in the two Control treatments ranged from 85% to 100Y0. with

12% of the embryos in the second treatment failing to inflate the swim bladder. No mortalities throughout the duration of the experiment were reported. For all phytochemicals. at the lowest nominal concentration (0.1 pg/mL) used for all phytochemicals. the proportion of abnormal medaka embryos did not differ significantly

From the corresponding Controls. The most toxic phytochemicals were: 7-MC. tlavone. harmane. juglone and tropolone (P < 0.01 ) (Table 4.1 ). Anthrone was not embryotoxic at a11 concentrations tested (Table 4.1 ). The calculated ECSOs for the abnormal embryos

were 0.2.0.3.0.3.0.5 and 0.6 pg/mL for flavone. juglone. tropolone. harmane and 7-MC.

respectively (Table 4.2). All phytochemicals except 7-MC. were lethal (> 50%) to rnedaka

embryos at the higher nominal concentration(s). The estimated LCSOs. based on the

nominal concentrations were 0.2.0.3.2.3 and 6.7 @rnL for juglone. flavone. harrnane

5 Term that excludes only the embryos that were viable at the end of the 1Fday experiment. Table 4.1. Summary of results from the Japanese medaka (0.lafipes) embryotoxicity assay. Dead. unhatched, partially hatched (P.Hatched) and hatched embryos from all treatments are presented as percent (%) of all viable embryos at day 1 of exposure. Dead. abnormal and viable larvae are also expressed as percent (%) of the viable embryos at day I.

-. - 0bservat ions Embrvos (Oh) Larvae (%) Chemical Conc'n (~e/rnLI Dead Unhatched P. Hatched Hatched Dead Abnormal Viable Flavonea 0 0 0 89

Juglone" 0 0 0 88

Hannanea 0 34 50 88

Tropolone' 0 96 85 78

7-MC' 92 72 90 82

Anthrone 92 84 77 92

Control 100 85

a significant decrease of viable medaka at P < 0.05 Table 42. Median Iethal concentrations (LCSOs) and median effective concentrations (ECSOs). expressed in pg/mL. for Japanese medaka (0.lutipes) embryos exposed to several phytochernicals.

flavone 0.3 0.2 harmane 2.3 0.5 juglone 0.2 0.2 7-methoxycoumarin > 10.0 0.6 tropo lone 6.7 0.3 anthrone > 10.0 > 10.0

and tropolone. respectively: an indication that these natural secondary plant compounds

are acutely toxic to fish embryos (Table 4.2).

No embryos exposed to the higher concentrations (1 - 10 pg/mL) of juglone

survived beyond 48 h (Fig. 4.2). The median hatching time at day 13 of rnedaka embryos

at the lowest concentration of juglone (0.1 pg/mL) was significantly greater than the

median hatching time at day 12 of the Controls (P<0.05)(Fig. 4.3).

Tmpoione was acutely toxic to rnedaka embryos at the highest concentration.

since all embryos died by day 8 (Fig. 4.2). Although this phytochemical was not lethal at

lower concentrations. most of the hatched embryos did not successfully inflate their

swim-bladder (64% inflation at 5 @nL. 30% inflation at I pg/mL). The median time of

hatching at day 12 at the lowest concentration was similar to the median hatching time of

the Controls.

In embryos exposed to flavone. the development of lesions and cumulative

mortalities were concentration-dependent. For the highest concentration. all mortalities .-3 40- 2 to-

-+-I ppm * e-

Day Trow lane

and tropolone. Control

- -- j uglone 7-MC 0. I ppm

Day Day

Figure 4.3. Histograms depicting daily hatching frequencies (%) of Japanese medaka (0- latipes) embryos esposed to various phytochemicds. Arrow shows the median hatching time. and asterisks ('-***") indicate significant differences between treatments and Controls (P < 0.05). occurred before 48 h. with embryos failing to reach the gastrulation stage. At 5 pg/mL. the development of embryos was retarded ( 100%). there were circulatory anomalies

(hemorrhages and blood stasis) appearing by days 3 to 5 (100%). and mortalities occurring between days 6 to 12 ( 100%). At 1 pg/mL. most of the embryos (88%) developed pericardial edema by day 9 (Fig. 4.5). and mortalities occurred by days 1 I to 16

( 100%) (Fig. 4.2). Some of the embryos exposed to tlavone at 1 @mL (28%) were only partially hatched (Table 4.1 ). Although medaka exposed to 0.1 pg/mL medaka appeared normal. the median time of hatching was at day 13. significantly greater than the median hatching time of the Controls (P

Mortalities among embryos exposed to harmane at 1 - I0 pg/mL ranged from 50 to 70%. with death occurring at days 10 to 17 (Fig. 4.2). [n the treatment of the highest nominal concentration ( 10 pg/mL). 70% of the embryos died. 10% were partially hatched. and the rest completely failed to hatch. All of the embryos exhibited multiple lesions.

Arrest or retardation of development was especially pronounced during the neurulation stage. There were subsequent appearances of yolk edemas. eye deformities

(anisophthalmia. microphthalmia. cyclopia. absence of lenses). cephalic aberrations

(absence of head. undifferentiated brain. microcephaly). and absence of the tail in mrdaka embryos exposed to 10 pg/mL of harmane. In the 5 pg/mL treatment. only 15% of the embryos were viable. 2 1% did not hatch and 2 1% were partially hatched. Circulatory malfunctions (i.e. blood stasis and haemonhage) and spasmodic movements indicative of neurotoxicity were the most common pathological effects observed. In the 1 pg/mL treatment. 14% of the embryos failed to hatch. 23% were partially hatched. and only 32% were viable (Table 4.1). The median time of hatching in medaka exposed to 0.1 pg/mL was the same as in Controls.

The 7-methoxycoumarin was embryotoxic but not acutely lethal at nominal concentrations between 1 to 10 pg/mL. At the highest concentration. embryos examined prior to hatching showed a high incidence of pericardial edema (36%). and the hatched embryos failed to inflate their swim bladder (100%). or to absorb their yolk sac (65%). In

addition. 57% of embryos developed either vertebral or tail deformities (Fig. 4.7). The

latter response was only observed in larva exposed to 7-MC. At 5 pg/mL. similar lesions

were observed but at lower incidences (26%. 60%. 27% and 20%. respectively). Median

time of hatching at day 13 in the I pg/rnL treatment was different than the hatching time

of Controls (PC 0.05). but there was no significant difference in time to hatch at the 0.1

pg/mL treatment (Fig. 4.3).

4.5. DISCUSSION

This study showed that the tested phytochemicals with the exception of anthrone.

have the potential to adversely affect the swival and viability of fish embryos. Survival

of medaka was very poor in embryos exposed to all nominal concentrations except the

lowest one for treatments with juglone. flavone, harrnane. and tropolone. with LCjOs

calculated as 0.2.0.3.2.3 and 6.7 pg/mL. respectively. Viability of the embryos was

impaired at lower nominal concentrations, as shown by the EC5Os of 0.2 pg/mL (juglone).

0.2 pg/mL (flavone). 0.3 pg/rnL (tropolone). 0.5 pg/mL (harmane) and 0.6 pg/mL (7-MC) Figure 4.4. Normal Japanese medaka (0.latipes) embryo from the Control treatment.

Figure 4.5. Non-viable Japanese medaka (0.latipes) embryo exposed to 1 pg/mL of flavone showing pericardial edema (PE) and the development of tube-heart (TH). Figure 4.6. Viable newly hatched Japanese medaka (0.latipes) showing a successful swim bladder inflation (sB).Oil droplet (OD) is also visible (Courtesy of Glenn Harris).

Figure 4.7. Non-viable newly hatched Japanese medaka (0.lutipes) exposed to 10 pg/mL of 7 methoxycoumarin showing scoliosis (Sc), tail curvature (TC). Note also the absence of a successful swim bladder inflation (ASB) and the presence of the oil droplet (OD). (Table 4.2). However, by taking into consideration the relatively hydrophilic nature of the test phytochemicals (Ch.3), and the fact that lipid solubility and trans-chorionic permeability of toxicants are linearly correlated (Helrnstetter and Alden 111, 1995). it is possible that the actual LCSOs and EC5Os may be even lower than that estimated from the nominal concentrations. This is the first study that has attempted to assess the embryotoxicity of phytochemicals or other structurally related compounds; therefore. there are no prior references for comparisons of their embryotoxic potential. However. the embryotoxicity of these phytochemicals is comparable to that of the potato alkaloids. a- chaconine and a-solanine to rnedaka embryos (Crawford and Kocan. 1993). In addition. the embryotoxic potential of the tested phytochemicals is similar to that of various polychlorinated diphenyl ethers (PCDE)(Metcalfe et ul. 1997), and polychlorinated biphenyl (PCB)congeners (i.e. PCB 77 or PCB 8 l), but lower than that of PCB 126 and

2.3.7.8-tetrachlorodibenzo-p-dioxin (TCDD)(Hams et a/. 1994).

The results of the present study showed that juglone caused total mortality at the nominal concentrations tested at the very early stages of development. whereas the phytochemicals that induce EROD or AHH (Chapter 3), flavone. 7-MC, tropolone and harmane elicited a multitude of responses to Japanese medaka at later stages of embryonic development. Developmental retardation, vascular hemorrhaging, pericardial and yolk edema. inability of embryos to penetrate the chorion during hatching time (i.e. partial hatching). unsuccessfbl swim bladder inflation and yolk sac absorption, as well as skeletal defects were some of the developmental lesions and abnormalities observed in medaka exposed to the latter phytochemicals. Some of these effects were chemical-specific. Table 4.3. Embryonic responses of Japanese medaka (0.lutipes) embryos exposed to the CYP I A-inducing phytochemicals, flavone. harmane. 7-methoxycoumarin (7-MC)and tropolone.

Embrvonic Response Phvtoc hem ica1 Developmental Retardation flavone, harmane, tropoIone Vascular hemorrhage flavone, harmane Pericardial edema flavone, harmane, 7-MC Tube heart flavone Yolk sac edema harrnane. 7-MC Abnormal yolk sac tropolone Cran iofacial defects harmane Neurotoxicity harmane Partial hatching flavone Swim bladder inflation failure 7-MC. tropolone Yolk sac absorption failure 7-MC Skeletal and tail deformities 7-MC whereas others were common to fish exposed to most chemicals (Table 4.3). With this study it is difficult to assess mechanisms of toxicity since most of the aforementioned teratogenic aberrations may be linked to phytochemicals acting upon early stages of the developing embryos. such as cellular differentiation. apoptosis. migration. communication and metabolism (Weis and Wris. 1987). For instance it has been suggested that skeletal and craniofacial defects are associated with early tissue damage or inhibition of enzymatic action (Von Westernhagen, 1988). Pericardial and yolk edema are probably associated with imbalanced ionoregulation resulted from retarded circulation and vascular damage

(Laale and Lerner. 198 1 ).

Xenobiotics that are inducers of CYP I A-dependent monooxygenases, induction initiated by binding to the aryl hydrocarbon (AhR) receptor. have been shown in medaka studies to cause early vascular damage. pericardial edema tube-heart formation.

inhibition of swim bladder inflation. and failure of yolk sac absorption (Wisk and Cooper.

1990; Harris et al. 1994). Of the tested phytochemicals that were found to induce the CYP l A monoxygenases (Chapter 3), flavone appeared to cause similar effects. For instance. at the 1 pg/mL treatment with flavone. circulatory defects were observed early in development. followed by pericardial edema and the formation of tube-hearts. Mortalities occurred either during hatching when embryos died during unsuccessful hatching. probably due to the inhibition of the hatching enzyme. or alternatively at day 16 for medaka that failed to hatch. Exposure to 5 and 10 pg/mL of 7-MCappeared to cause some of the same responses previously observed in medaka exposed to dioxins such as. occurrence of pericardial edema. absence of inflated swim bladders and inhibition of yolk sac absorption. In addition. skeletal and tail abnormalities observed in embryos exposed to 7-MCcould be attributed to competition for ascorbic acid between cytochrome P-450 enzymes and collagen metabolism in the bones as previously described by Mayer et ai.

( 1978). As for the other CYP 1A-inducing phytochemicals. tropolone inhibited the inflation of the swim bladder, whereas harmane treatment did not induce the same effect. although this elicited a variety of other adverse effects. These responses may be linked to the binding of the phytochemicals with the aryl hydrocarbon receptor (AhR). although the same embryotoxic responses have been found to be AhR independent

(Villalobos et al. 1996).

There is evidence that the embryotoxic effects observed in our study were also observed in feral fish exposed to pulp mill effluents. For instance. in our study. exposure of medaka embryos to 7-MCresulted in the development of skeletal and tail deformities.

At the highest nominal concentration, 57% of the hatched medaka had either scoliosis or bent tail. whereas at the 5 pg/mL treatment the incidence of these responses declined to 20%. In the Gulf of Bothnia (Scandinavia), fourhom sculpin (Myoxocephdus quadricornis)exposed to bleached kraft mill effluent (BKME), at distances less than 20 km from the discharge area, showed significantly elevated frequencies of vertebral deformities (Bengtsson ef al. 1985). Consequent laboratory work with juvenile fourhorn sculpin exposed to various types of pulp mill effluent confirmed these field observations

(Bengtsson er al. 1988; Hiirdig ef al. 1988). Bengtsson et al. ( 1988) exposed juvenile fourhorn sculpin for 4 1/2 months to both bleached and unbleached pulp mill effluents. and concluded that the development of skeletal deformities was not correlated with the chlorinated Fraction of the effluents but rather with less bio-accumulative chemicals. Our study supports their conclusions showing that phenolic phytochemicals have the potential to affect the vertebral structure of exposed fish.

In other field studies, skeletal and craniofacial defects were observed in the marine fish. Tasmanian blemy (Parab[enniustasmanianus) when exposed to pulp mill effluents from eucalypt trees. at dilutions ranging from 2.5 to 10%. Also. in the same study. hatching success was impaired and high mortality around hatching was observed at >7% effluent dilutions (Deavin, 1994). In experimental stream studies, exposure of trout to

1.3% of BKME resulted in greater egg mortality and a decline in hatching success

(NCASI, 1989). Egg mortality in pike (Esox lucius) was also increased &er exposure to dilutions of 3.5% of partially treated BKME effluents (Tana and Nikunen. 1986). Somatic growth and mortality of the blenny (Zoarces viviparus L.) was also drastically affected at

0.5% untreated effluent dilutions (Jacobsson er al. 1986). In our study. medaka embryos exposed to harmane and flavone caused high mortality just around hatching time. It has been reported that reproduction in fish exposed to pulp mill effluents is seriously affected at individual, population and community level. Sijdergren (1989) reported unsuccessfbl recruitment in fish species such as perch, sand goby and herring as a consequence of reproductive failure or avoidance behavior in response to exposure to pulp mill effluents. Other studies were in accordance with the previous study since decreased rate of recruitment and lower densities of larvae were reported for the Baltic perch (P. fluviatilis), as a result of a higher incidence of malformed embryos (10%). and an increased rate of mortality around hatching time (Kariis et al. 199 1 ). Follow-up studies. after the local kraft mill was modernized. indicated that the perch population did not fully recover since larval survival was still low (Sandsuom. 1996). The structure of fish communities in Scandinavian inland waters receiving pulp mill effluents was altered with the disappearance of the most sensitive salmonids and their replacement by other

indigenous species (Lindstrom-Seppa and Oikari, 1989). Donaldson ( 1990) argues that

reproduction is a continuous process throughout ontogeny. and thus. toxicants could affect

reproduction by acting during the early fish life history stages, as well as during

maturation and spawning. Correspondingly. developmental retardation. teratogenicity and

hatching failure or delay may ultimately affect the successful recruitment of the species.

and thus the propagation of the population (Donaldson, 1990). Clearly, then. the findings

of the present study indicate that the phytochemicals flavone. tropolone. harmane. 7-MC

and juglone could affect reproduction at the early stages and consequently impair

successful recruitment in fish. CHAPTER 5

Effects of Various Flavonoids on Fish: EROD induction, Embryotoxicity and

Reproductive Development

5.1. ABSTRACT

The potential of several flavonoid compounds to affect normal biochemical. physiological and reproductive processes in teleosts was evaluated with a battery of in

vivo and in vitro studies. Several of the flavonoids tested in this study are present in the heartwood of tree species used in pulping. Overall. the data of this study suggest that

flavonoid compounds may contribute to the toxicity of the pulp mill effluents in feral fish.

Immature rainbow trout (Oncorhynchzis mykiss) were intraperitoneally ( i .p. ) injected with

flavonoids at a single dose ranging from 0.1 to 100 rngkg. At Day 3 post-injection. only

flavone. chalcone and 7-hydroxyflavone caused significant induction of the hepatic

etho?cyresorufin-0-deethylase ( EROD) by Sfold, I 0- fold and 3-fold. respectively.

indicating that the planar molecular structure of the unsubstituted flavonoids. as well as

pattern and degree of hydroxy substitution are important factors in the expression of

hepatic CYP I A activity. Exposure of early life stages of Japanese rnedaka ( Ovzias

laf@es)to flavonoids at a range of 0.5 to 75 pM with a 17d non-renewal assay showed

that many flavonoids induced embryonic responses that affected the hatching success.

survival and the overall health of the embryos. The synthetic flavonoid. I3-naphthotlavone

was the most toxic compound (ECSO = 0.08 pM), followed by flavone (I -6 pM). quercetin (2.7 pM), chdcone (3.2 pM), flavonol(4.2 pM), 6-hydroxyflavone (4.7 pM). galangin (5.1 pM ) and flavanone (5.5 pM). Apigenin (23.8 pM)and taxifolin (5 1 pM) were moderately toxic, whereas Fhydroxflavone, chrysin, naringenin. kaempferol and catechin were not toxic at the range of concentrations tested. These data indicate that the unsubstituted parent flavonoids are the most toxic. and that toxicity decreases with the addition of hydroxyl groups to the flavonoid nucleus. except for the pentahydmxyflavone. quercetin. In addition. planarity and pattern of hydroxy 1 substitution significantly affect the embryotoxic potential of flavonoids. In the Japanese medaka reproductive development assay. all flavonoids tested (i.e. flavone. flavonol. flavanone, chrysin. galangin. apigenin. kaempferol. quercetin. genistein. naringenin. catechin and a mixture of chrysidquercetin) had an impact on gonadal development and differentiation. as evident

with: (a) a low to moderate incidence ( 3 - 15%) of inter-sex (i.e. testis-ova): (b) inhibition or delay of gonadal maturation: (c) a decrease in number of mature germ cells: (d) an

increased incidence of ovarian atresia: and (e)a high proportion of phenotypic female

medaka with reduced or altered secondary sex characteristics. Results from in vimassays

for binding to fish estrogen and androgen receptors indicated that developmental effects

may be attributed to the ability of flavonoids to bind to either the estrogen receptor

(genistein. naringenhapigenin. kaempferol. quercetin) or androgen receptor ( flavonol.

flavone. kaempferol, flavanone and naringenin) and act either as steroid hormone agonists

or antagonists. The potential of some flavonoids to bind to the sex steroid binding

globulins (flavone. apigenin. flavonol. galangin. kaempferol. flavanone and naringenin).

may be another mode of endocrine disruption. 5.2. INTRODUCTION

The pulp and paper industry has been recognized as a major point-source for pollutants discharged into the aquatic environment and its impacts on fish populations have been well documented (Kovacs. 1986: Sodergren. 1989; Owens. 1 99 1).

Reproductive failure. imbalance of sex hormones. carbohydrate metabolism alterations. immunosuppression and induction of the hepatic cytochrome CYP 1A-dependent monooxygenases (such as EROD)are some of the observed sub-lethal effects attributed to pulp mill effluent (Andersson et ul. 1988: Munkittrick et ul. 1992). It has been suggested that organochlorines. formed as by-products during the bleaching stages. are the chemicals that cause the aforementioned adverse effects (Kovacs. 1986: Sodergren. 1989:

Owens. 199 1 ). However, as other in vivo and in vitro studies show. similar biological and toxic responses are also observed in Ash exposed to unbleached andlor biologically treated pulp mill emuent. indicating that other non-chlorinated chemicals may contribute to overall effluent toxicity (Lindstrom-Seppa cr uI. 1992: Pesonen and Andersson. 1997:

Servos et a/. 1992: Martel er al. 1994; Kiparissis el 01. 1996). The isolation and identification of the responsible bioactive chemical(s) of the pulp mill emuent. which eludes researchers so far. have been made priorities by Environment Canada (Munkittrick et al. 1998).

The task of isolating and identifjring the bioactive fraction or chemicals From the pulp plume is difficult since pulp mill effluent is a complex mixture of numerous chemicals which vary in composition. depending on factors such as type of pulping processes. wood type, bleaching technique and treatment technology (O'Comor et al.

1992). Studies have shown that the chemicals that cause an elevation in CYP 1A activities could be constituents of the black liquor (Martel et al. 1994), could be also degradation products of resin acids (Fragoso et al. 1998). or alternatively could be natural plant products such as juvabiones (Martel et al. 1997). In addition wood-derived phytosterols

(i.e. a-sitosterol) could be partially responsible for the documented reproductive dysfunctions observed in fish exposed to pulp mill effluents (Denton et ui. 1985:

Mellanen er al. 1996: MacLatc hy el d. 1997).

Our earlier screening tests (Chapter 3 and 4) showed that individual phyto- chemicals of the coumarin, harmane. tropolone and flavone families were able to induce hepatic CYP 1A activity in rainbow trout (0.mykiss) and were also embryotoxic to

Japanese medaka (0.hipes). Of these phytochemicals. the flavonoid family may be of great ecotoxicological importance in pulp mill effluent. First. flavonoids. a large family of structurally related compounds (Fig. 5.1 ). are widespread in the piant kingdom. and their presence in the heartwood of tree species used in the pulping process has been documented (Sjostrom. 198 1; Fang et d 1987). Second. flavonoids affect many biochemical and physiological functions in animals such as the expression of the phase I and 11 detoxifying enzymes. and reproduction (Harborne and Grayer. 1994). The relative toxic potencies of flavonoids depend on their molecular structure. For example. the structural class (flavone. flavanone. . etc.), the type and degree of substitution

(hydroxylation. methoxylation. isoprenylation. glycosylation. etc.) and the degree of polymerization of each individual flavonoid are important for its pharmacological or toxic properties (Cody et ul. 1985). Thus, a study focussed on the possible toxic potential of flavonoids to fish is warranted.

The induction of the hepatic ethoxyresorufin-0-deethylase(EROD) and other cytochrome P450 1A monooxygenases (CYP1 A) have been extensively used as a biomonitoring tool for assessing the impact of pulp mill effluent on fish (Andersson et d

1988: Lindstrom-Seppa et al. 1992: Pesonen and Andersson. 1992: Ahokas et al. 1 994:

Huuskonen and Lindstrom-SeppH 1995). However. EROD induction is an adaptive response to xenobiotics. and thus in many field studies other biological endpoints. including hormonal imbalance. expression of secondary sex characteristics and reproductive failure were used to assess the impact of pulp mill effluents (Andersson et al. 1988: McMaster et al. 199 1 : McMaster et ul. 1992: Munkittrick et ul. 1992b: Gagnon et al. 1993: Gagnon el al. 1995). Conclusions regarding a link between EROD induction and other adverse sub-lethal responses are inconclusive. For instance. Lehtinen ( 1990) addressed the possibility that a chronically induced CYP I A system could indirectly interfere with normal steroidogenesis and adversely affect reproduction in fish. On the other hand. it has been suggested that chemicals in the pulp plume that elevate the hepatic

EROD activities are not necessarily the same chemicals that interfere with the metabolism of steroids (Munkittrick ct al. 1992b). or other adverse biological effects (Swanson et rd.

1992). Therefore. it has been recommended that other meaningful biologic endpoints should be utilized in addition to CYPI A assays to assess toxicity of pulp mill effluents

(Lehtinen, 1996; Sandstr6m. 2 996).

Chemical stresson have the potential to impact reproduction in fish exposed during ontogeny at the early embryolarval stages or during sexual maturation and spawning (Donaldson, 1990). Thus, multigenerational studies may be the appropriate experimental approach to assess the reproductive impact of chemical or mixture.

However. for screening many chemicals. these chronic studies are logistically impractical.

McKim (1977) suggested that short-term tests during sensitive early life stages of fish could be used as a surrogate for chronic studies. Bresch (1 982) argues that early embyolarval tests alone may not be sufficient to predict reproductive dysfunction and therefore. other reproductive tests of short duration could be included to replace full life- cycle studies. For instance. the phytosterol. l3-sitosterol was not embryotoxic to Japanese medaka at all concentrations tested (Chapter 4). even though it has been shown to be an endocrine-modulating substance (Denton er ul. 1985: Mellanen et ccl. 1996).

The primary objective of this study was to test several flavonoids representative of different flavonoid classes (Fig. 5.1 ) with a battery of in vivo and in vitm assays for their potential to: (a) induce hepatic CYP 1 A-monooxypnases in immature rainbow trout: (b) be embryotoxic in Japanese medaka: and (c)affect sexual differentiation. gonadal development and expression of secondary sex characteristics in Japanese medaka. The selection of the flavonoid compounds. chrysin. kaempferol. apigenin. galangin. naringenin. taxifolin. catechin. quercetin and genistein (Fig. 5.2) was based on evidence that they are present in the heartwood of tree species and that they are readily available fiom commercial sources. The unsubstituted parent flavonoids. flavone. flavonol. flavanone and chalcone (Fig. 5.1). as well as other hydroxylated flavonoids were selected for establishing structure-activity relationships in the aforementioned in vivo assays. The I Flavone ~sorravone

. > Q- , .- 0 Chalcone

I Figure 5.1. Molecular structure of flavonoid classes used in this study. Flavonoids are structurally similar C-15 compounds comprised of two aromatic rings (A and B) and a heterocyclic C ring, except for chalcones. Note the presence of a double bond (CZ-C3) in flavones. and its absence from flavanones. The three rings in flavonotds witha CX3double bond are in the same plane (planar molecular configuration). Presence of a C-3 hydrosyl group in the flavone nucleus is the structural characteristic of flavanols. whereas a C-3 bridglng of C and B nucleus is the characteristic of isoflavones. Flavan-hls lack the ketone group. giving the molecule a positive charge at C4. gaiangin , ,,OH

.\. \, -. -4 V OH 0 OH 0 apigenin naringenin OH

>. >. / - _ ,OH - .-OH

.. . .- \- 'OH - 'OH OH 0 OH 0 kaempferol quercetin OH

1 (+)atechi n genistein I Figure 5.2. Molecular structure of hydrosylated flavonoids used in the Japanese medaka (0. laripes) reproductive development assay. Table 5. I. Flavonoid class and hydroxyl substitution pattern of individual flavonoid compounds tested for their endocrine-disruptingmodulating potential with the in vimassays used in this study.

Flavonoid Class Hydroxy I substitution pattern A ring C ring B ring Flavone Flavones Chalcone Chalcones Flavanone Flavanones Flavonol Flavonols 5-hydroxyflavone Flavones 6-hydroxyflavone Fiavones 7-hydroxyflavone Flavones 4'-hydroxyflavone Flavones 4-hydroxy flavanone Flavanones 4',6-dihydroxy flavone Flavones Chrysin Flavones Equol lsoflavone Galangin Flavonols Apigenin Flavones Naringenin Flavanones Genistein [so flavones Kaempferol Flavonols Luteolin Flavones Fisetin Flavonols Quercet in FIavonols

synthetic benzoflavone. O-naphthoflavone was used as positive control in the CYP l A and

embryotoxicity assays. whereas 17~-ethinylestradioland testosterone were used as the

positive controls in the reproductive studies. Finally. a battery of in virro assays were

used to understand the mode of action of flavonoids as endocrine-disrupting modulating

substances. For these assays. additional hydroxylated flavonoids were used (Table 5.1 ) to

develop structure-activity relationships.

5.3. MATEWANDMETHODS

5.3.1. Chemicals All flavonoids were purchased from Sigma- Aldrich (Toronto. ON. Canada). unless otherwise specified.

5.3 2. EROD induction assay

The in vivo hepatic EROD assay was conducted with immature rainbow trour

(100-200 g) as previously described (Chapter 2 & 3) with few modifications. All flavonoids were dissolved in DMSO and intraperitoneally (i.p.) injected into rainbow trout with a single dose of 0.1. 1. 10 or 100 mg/kg (Table 5.2) at a constant volume of 2 ml/kg. For P-naphthoflavone. kaempferol and taxifolin. the doses used were 0.1. 1 and 10 mglkg. Control fish (n=34) were i.p. injected with the carrier solvent. DMSO.After 72 h post-injection. the fish were sacrificed with an overdose of MS-222. the livers were immediately excised (minus the gall bladder). and were washed through the portal vein with ice-cold 0.1 5 M KCI. Next. the livers were homogenized in a KCI-HEPES buffer

(pH 7.5). centrifbged at 10.000 x g for 25 min (4 " C) to obtain the supernatant fraction (S-

9) and stored at -80 "C until further analysis. EROD activity was determined fluorometrica1Iy as described previously (Chapter 2 & 3). Each sample was analyzed as a duplicate (plus the blank). and the non-parametric Knrskal-Wallis test was used for statistical analyses.

5.3.3. Japanese rnedaka embryotoxicity assay

The embryotoxic potential of all tested flavonoid compounds was determined with embryos of Japanese medaka using a static 17 day non-renewal exposure. as described Table 5.2. Range of concentrations (pM or mg/L) or doses ( mglkg) of flavonoids tested in vivo in the: a) rainbow trout (0.mykiss) hepatic ethoxyresoru fin-0-deethylase (EROD)assay: b) Japanese medaka (0.latipes) embryotoxicity assay; and c) Japanese medaka reproductive development assay. Assavs Rainbow trout Ja~anesemedaka Flavonoid EROD(me/kgl Embwotoxicitv (uMl Re~roductiveDevelooment (mdL 1 Flavones flavone 0.1, 1, 10, 100 0.5, 5,25.75 0.0 1 6-hydroxyflavone 0.1. I. 10, 100 0.5-5.25,75 - 7-hydroxyflavone 0.1. 1. 10. 100 0.5.5.25.75 - chrysin 0.1. 1, 10. 100 0.5. 5, 25. 75 0.1.0.5. 1 apigenin - 0.5, 5, 25, 75 0.05.0.5 F lavonols flavonol galangin kaemp ferol quercetin Flavanones flavanone naringen in taxi fohn C halcones chalcone

Isoflavonoids genistein - Benzoflavones 13-naphthoflavone 0.1. 1. 10 0.5.5.25.75 -

previously (Chapter 4). The nominal concentrations of all flavonoids tested were 0.5. 5.

25 and 75 pM (Table 5.2). The primary endpoints used to evaluate the toxic potential of

flavonoids were the mortality and the incidence of medaka reaching successfblly the

swim-up stage of development (i.e. viable medaka) at the end of the experiment. Hatching

success and gross morphological defects were also noted. The significance of test results

was tested with f-analysis. The median lethal concentration (LCSOs) and the median

72 effective concentration (ECSOs) for the aon-viable medaka for all flavonoids were estimated with Probit analysis (softToxa, Los Angeles, CA. USA).

5 3 -4. Japanese medaka reproductive development studies

Three reproductive development studies were conducted in the present study. The purpose of the first study was to determine whether flavonoids have the potential to affect gonadal development and differentiation in Japanese medaka afker 3 months of exposure.

In the second study. a more detailed approach was used to test more flavonoids with various patterns of hydroxy substitutions to determine structure activity relationships

(SARs). In addition. the development of adverse responses as a function of exposure time

(i.e. 2.3.4 and 5 months) was also tested. In the third experiment. Japanese medaka were exposed to the isoflavonoid. genistein since its presence in the final effluent from a pulp mill has been determined (see Chapter 6). The various developmental endpoints monitored in the present reproductive developmental assays. were: sex differentiation

(normal. inter-sex. sex-reversal). gonadal development. gametogenesis and development of secondary sex characteristics.

5.3.La. Reproductive development experiment $1

In the fim preliminary experiment. the flavonoids tested were chrysin and quercetin. and the duration of the experiment was 3 months. A treatment with testosterone (1 8 pg/L) was used as a positive control substance for androgens. Newly hatched medaka embryos (n = 50-70) were collected daily and placed in aquaria containing 5 L of filtered water from the Otonabee River. Both flavonoids and testosterone were dissolved in acetone (distilled in glass) and administered in 1 mL aliquots into the static exposure systems. The aqueous exposure media in aquaria

(approximately 95%) were replaced every 48 hrs. The nominal concentrations of chrysin were 0.1.0.5 and 1 in@. and for quercetin were 0.0 1.0.05 and 0. I mg/L (Table 5.2).

These nominal concentrations were selected as below the LOEL in embryotoxicity tests.

The water in the Control treatment was spiked with 1 mL of acetone.

At the termination of the experiment (i.e. 3 mo.). medaka (n=25-30) were randomly taken from the aquaria. anaesthetized with MS-222 and placed in a watch dish for morphological observations. including total length (mm) and weight (g). Under a dissecting microscope. the shape of the urogenital pore (a female positive characteristic). as well as papillary processes in the anal fin (a male positive characteristic) were observed. Medaka were then placed in tissue cassettes and fixed in Bouin's decalcitjring solution for 48 hrs. and preserved in 70% . From all treatments. some medaka

(n= 10- 15) were retained for another month in "clean" water for other developmental observations.

All medaka histological samples were dehydrated in a graded alcohol series and embedded in paraffin. Sagittal sections (5-7 pm) were taken from each fish in a step-

section manner (6 to10 sections per fish), mounted in microscope slides and stained with

haematoxy lin and eosin ( H&E). using standard staining techniques. and a few randomly

chosen slides were stained with "Periodic-acid Schiffs" reagent (PAS) using 1% aqueous

orange G and 1% aqueous light green as the counterstains. The Schiff s reagent gives a positive response (magenta) with glycoproteins, such as vitellogenin; therefore, it was used in this experiment to differentiate between oocytes that are immature (pre- vitellogenic stages) or oocytes that are at the vitellogenic stage of development. The type of the gonadal tissue. various stages and expression of gametogenesis of all individual fish were evaluated under a compound microscope.

5.3.4.b. Reproductive development experiment $2

In the second experiment. newly hatched medaka were exposed to only one nominal concentration of: (a) the parent flavonoids. flavone. flavanone and flavonoi: (b) the previously tested chrysin and quercetin; and (c) the binary mixture of chrysirdquercetin. Two nominal concentrations were used for the (poly)-hydroxy- substituted flavonoids. apigenin. galangin. naringenin. kaempferol and catechin (Table

5.2). Also. two concentrations (I and 10 ng/L) of the synthetic estrogen. 17~ethinyl- estradiol were used as a positive control for an estrogen.

The experimental protocol was similar to that of the first reproductive development experiment (5.4.3.a). with a few modifications. First. for the first month of exposure. 70 to 80 medaka were placed in 2 L glass containers (Pyrex). and afterwards were transferred into glass aquaria containing 7.5 L of river water. Second. sub-samples of fish were taken at 2.3.4 and 5 months of exposure. Using a dissecting microscope. the phenotypic sex for medaka was assessed by the shape of the secondary sex characteristics. urogenital papilla (or pore). dorsal and anal fins. Fixation and preparation of the histological samples were carried out as described previously (5.4.3.a). 5.3.4c. Reproductive development experiment #3

The third experiment with the isoflavonoid, genistein was initiated after it was identified in the fmal effluent from a pulp mill (see Chapter 6). Once again. the experimental protocol was carried out in a similar way. with few modifications. Medaka were exposed to four nominal concentrations (0.00 1.0.0 1.0.1 and 1 mg/L) of genistein dissolved in distilled in glass (DIG) acetone administered in a volume of 25 pL. Glass containers (2 L) were used throughout the 3 month exposure period. Dechlorinated tap water was used. rather than river water. All samples were stained only with H&E.

5.3 5. In vitro assays

5.3.5.u. Yeast Estrogenicity Assuy (YES assay)

A recombinant yeast strain for screening for estrogenicity was provided by J.

Surnpter of Brunel University. U.K. The assay was conducted as described by Routledge

and Sumpter ( 1996). The assay system utilizes a recombinant strain of yeast in which

DNA sequences of the human estrogen receptor (hER) are integrated into the yeast

genome and human estrogen responsive elements (hEREs) are integrated into plasmids.

Binding of chemicals to the hER expressed in yeast is followed by binding of the ligand-

receptor to ERE. which signals transcription of the reporter gene for B-galactosidase

which is detected by a colorimetric response in the presence of the chromogenic marker.

chlorophenol red-a-galactopyranoside (CPRG). All medium components were prepared as

described by Routledge and Sumpter ( 1996). Assay procedures were also carried out in a

similar manner but with some slight modifications. All flavonoids were dissolved in methanol (distilled in glass). The highest concentrations of the flavonoids in the assays applied to each test well varied between 10 and 100 pg/mL; the choice of highest concentration depending upon the toxicity observed in the test wells with preliminary

studies (data not shown). Blanks consisted of methanol only in the test wells. The highest concentration of the positive control. 170-estradiol was 2.5 ng/mL ( 10.' M).

In a laminar flow hood. the chemicals were serially diluted ( 1 2)and 10 pL

aliquots of each dilution were transferred to a 96-well microtitre plate. The methanol

solvent was allowed to evaporate to dryness in the plate and then 200 pL aliquots of assay

medium were dispensed into each well. The plates were incubated at 32 "C. and after

approximately 3 days of incubation, a plate reader (Biorad) was used to measure color

development of the medium at 540 nm. with corrections for turbidity measured at 630 tun.

The absorbance readings From each well were corrected against their corresponding blank

readings. and then converted to percent (%) a-galactosidase activity using the maximal

absorbance reading of the dose-response relationship for 1 7bestradiol as the reference

point. All flavonoids were tested in replicates (n=3) and data were presented as the mean

response * the standard error. In order to quantify the relative estrogenic potency of the

flavonoids that tested positive in the YES assay. data from the linear part of their dose-

response curves were used to estimate the concentrations that caused 25% maximal

response (EC25) induced by the positive control. 1713-estradiol (El).

5.3-5. b. in virro binding assrrys

Flavonoids were sent to the University of Guelph in the laboratory of Prof. Glen Van Der Kraak for testing of binding affinity with the rainbow trout estrogen receptor

(ER), goldfish androgen receptor (AR)and sex steroid binding protein (SSBP)assay.

Tests were conducted as described by Tremblay and Van Der Kraak ( l998), by Wells and

Van Der Kraak (in press) and by Van Der Kraak and Biddiscombe (1999). respectively. In all of these three assays. the percentage of displacement of the natural labelled ligands. 2.5

nM of ['HI- l 7R-estradiol (SSBP. ER) and 1 25nM of [3H]-testosterone(AR). in the

presence of the individual flavonoids (0.16 to 90.000 nM). was used to determine their

binding affinity. The estrogenic or androgenic potency was estimated relative to the

endogenous steroids. estradiol and testosterone. In the SSBP assay. the potency of

flavonoids to displace the natural ligand. [)H]-I 70-estradiol was also estimated in relation

to this endogenous estrogen.

5.4. RESULTS

5.4.1. EROD bioassay

The EROD activities in control Ash for all treatments ranged from 0.23*0.03

(flavonol) to 2.7k0.7 (quercetin) pmol/min/mg protein. EROD measurements were

obtained at various dates. Therefore. in order to compare EROD activities at different

times. EROD activities were converted into x-fold induction in relation to the

corresponding controls. The dose-response curves for EROD induction by four flavones

(flavone. 6-OH-flavone. FOH-flavone and chrysin). three flavonols (flavonol. kaempferol

and quercetin). two flavanones (flavanone and taxifoiin). one chalcone (truns-chalcone)

and P-NF (positive control) are shown in Figure 5.3. Statistical analyses showed that 3A ?Or 'Z -5 IS-

IFigure 5 -3. Concentration-dependent hepatic EROD activity in esperiment with immature rainbow trout (0.mykius) following a single intraperitond (i.p.)injection of several non- substituted flavonoids (A). mono-hydrox~avonoids(B). poly-hy&os).tlavonoids (C). and the oositive control D-naphthoflavone (D).The nominal concentrations of the i.p. injected flavonoids ranged from 0.1 to 100 mgkg. EROD is expressed as s-fold induction in relation to Controls. Each bar and vertical line represent the mean * standard error. and asterisks represent elevated EROD activities relative to controls at a I0.05. flavone, trans-chalcone, 7-hydroxyflavone and P-NF significantly increased the hepatic

EROD activities (P4.05). Injections of flavone and P-NF caused gradual dose- dependent increases in EROD activities, ranging from 3.2- (1 mgkg) to 4.4- (100 mgkg). and 14.1 - (0.1 mgkg) to 60.1 - (1 0 mgkg) fold induction. respectively. On the other hand. the EROD induction in fish exposed to chalcone was highest at the dose of 1 mgkg (15.2- fold) and declined in a dose-dependent manner at higher concentrations (Fig. 5.3). EROD induction caused by exposure to 7-hydroxyflavone was significantly elevated in the 1 mgkg (2.6-fold) and 10 mglkg (2.4-fold) treatments. All other flavonoids did not induce

hepatic EROD in immature rainbow trout (Fig. 5.3).

5.4.2. Japanese medaka embryotoxicity assay.

Hatching success for medaka in Control treatments ranged from 80% to 97%.

Overall. the proportion of Control medaka that successfully reached the "swim-up" stage

at the end of the exposure period (i.e. viable or normal medaka) was 82 * 4%. Using the

incidence of normal embryos by Day 17 as the endpoint of evaluation. the most toxic

flavonoids were: P-NF. flavone. chalcone. flavanone. flavonol. 6-hydroxyflavone.

galangin and quercetin (f > 9.49: P < 0.05). Apigenin. taxifolin and 7-hydroxyflavone

were toxic only at the highest nominal concentration (75 pM). whereas chrysin.

naringenin. kaempferol and catechin were not toxic at all concentrations tested. Based on

the median effective concentration (ECSOs) of the aforementioned endpoint. the ranking

of flavonoids for their relative embryotoxic potential was: O-NF (0.08 pM) > flavone ( 1.6

pM) > quercetin (2.7 pM) > chalcone (3.2 pM) > flavonol ( 4.2 pM) t 6-hydroxyflavone (4.7 pM) 2 galangin (5.1 pM))t flavanone (5.5 pM) > apigenin (23.8 pM) > taxifolin

(50.9 pM). ECSOs for chrysin, 7-hydroxyflavone, naringenin, kaempferol and catechin were greater than 75 pM, as estimated with Probit analysis (Table 5.3). Using the median lethal concentration (LCSOs). the toxic potential of the flavonoids to Japanese medaka embryos during the 17 d exposure were ranked as: BNF (0.5 pM) > flavone ( 1.6 pM) > quercetin (3.9 pM) > chalcone (7.3 pM) > 6-hydroxyflavone (1 1.5 pM) > flavanone

(19.1 pM), flavonol(30.2 pM) t galangin (32.0 pM). The rest of the tested flavonoids.

7-hydroxyflavone. chrysin. apigenin. naringenin. kaernpferol. catechin and taxi fohn were

not lethal within the range of the nominal test concentrations (Table 5.3). Hatching

success. mortalities and incidence of viable medaka exposed to flavonoids are presented

Table 5.3. Median lethal concentrations (LCSOs) and median effective concentrations (ECSOs)". expressed in pM. for Japanese medaka (0.kutipes) embryos exposed to several flavonoids. 95% confidence limits (C.L.)are given in parenthesis. FIavonoid LCjOs (pM) ECSOs (uM) a-naphthoflavone 0.5 (0.2 - 1 .O) 0.08 (0.03 - 0.2) flavone 1.6 (0.9 - 2.6) 1.6 (0.9 - 2.6) chalcone 7.3 (4.2 - 13.4) 3.2 (1.5 - 7.4) flavanone 19.1 (10.4 - 41.7) 5.5 (3.1 - 10.3) flavonol 30.2 (13.7- 109.1) 4.2 (2.2 - 8.5) 6-hydroxyflavone 11.5 (6.5 -21.4) 4.7 (2.6 - 8.9) 7-hydroxyflavone >75 .O >75.0 chrysin >75 .O >75.0 apigenin >75 .O 23.8 (9.4- 1 19.4) galangin 32.0 (15.8- 100.1) 5.1 (2.1 - 9.7) naringenin >75 .O ~75.0 kaempferol >75 .O >75.0 catechin >75.0 B75.0 quercetin 3.9 (0.9 - 1930) 2.7 (0.6 - 537) taxi fohn >75.0 50.9 ( 1 3.5 - 4505) 'ECSOs refers to viable (or normal) medaka that success€blly hatched and had no morphological defects at Day 17.

in Figures 5.4 - 5.6. Detailed descriptions of embryotoxicity data are provided below for the toxic flavonoid compounds.

Flavone treatment

Flavone was lethal to Japanese medaka embryos at the three highest nominal concentrations (5 - 75 pM). At the 75 pM treatment, all embryos died prior to the gastrulation stage (c 48 h), whereas in the 25 pM treatment, mortality occurred between days 3 and 9. In the 5 pM treatment, embryos showed circulatory abnormalities (blood stasis and haemorrhage), development of pericardial edema accompanied by the formation of tube hearts (Fig. 4.5) and mortality occumng from Day 9. Of the embryos that survived to hatch, three embryos failed to exit from the eyg chorion and died (partial hatching), and only one embryo hatched but died the next day. At the lowest concentration tested (i-e. 0.5 pM), most of the embryos were normal.

ha~hthoflavonetreatment

B-NF was lethal to Japanese medaka embryos at concentrations ranging from 5 -

75 pM. The mortality pattern at this range of nominal concentrations was similar to that of the 5 pM flavone treatment. At the 0.5 pM treatment, 79% of the embryos hatched, with only one embryo being normal at the end of the experiment. Few (29%) hatched medaka died, whereas 43% filed to absorb the yolk sac (Fig. 5.7), or failed to inflate their swim bladder (Fig. 4.5). At the lowest concentration, 73% of the exposed medaka successfully reached the swim-up stage (7% died and 20% failed to inflate the swim bladder). Chalcone treatment

Chalcone was acutely toxic to exposed medaka embryos at the 25 and 75 pM treatments, with mortality occurring at the first 3 days of exposure. At 5 pM, 78% of embryos hatched, with 2 1% failing to inflate the swim bladder and 57% of embryos being normal. At the lowest nominal concentration, most of the medaka were normal.

Flavanone treatment

Flavanone was acutely lethal at the highest test concentrations with most embryos

(75%) dying at Day 2 or 3, and the rest (25%) dying at Day 8 of exposure. At the 25 pM treatment, 16% of embryos died after 14 days of exposure, 5% died during hatch and 16% failed to hatch. All of the hatched embryos (63%) developed symptoms resembling blue- sac disease; failure to absorb the yolk sac, failure to inflate the swim bladder, tail or vertebrae curvatures and inability to swim normally (Fig. 5.8). At the 5 pM treatment,

85% of the embryos hatched, with 20% failing to inflate the swim bladder and 65% being normal at the end of the experiment. At the lowest nominal concentration, development of medaka embryos was similar to Controls.

Flavonol treatment

Exposure to flavonol(3-hydroxyflavone) caused mortality to medaka embryos in a dose-dependent manner (Fig. 5.4), with more than 50% of the exposed medaka dying at the two highest concentrations. At the same concentrations ( 25 and 75 pM), as well as at the 5 pM treatment, flavonol caused a multitude of responses such as, haemorrhaging

(caudal cephalic and ophthalmic regions), blood stasis, anisophthalmia (i.e. unequal eye sizes), rnicrocephaly, and yolk edemas Fig. 5.9). Embryos that hatched showed Figure 5.7. Abnormal newly hatched Japanese medaka (0.lafipes) exposed to 0.5 pM of B-NF. Note the presence of the yolk sac and the absence of a successfhlly inflated swim bladder

Figure 5.8. Newly hatched Japanese medaka (0.latipes) exposed to 25 pM of flavanone showing the symptoms of "blue-sac" disease. Note the presence of yolk edema (YE), haemorrhaging (Hm). The yolk sac was not absorbed and the swim bladder is absent, symptoms similar to those observed at the 25 pM flavanone treatment. By the end of the experiment, there were no normal embryos at 25 and 75 pM treatments, whereas the proportion of normal embryos at 5 and 0.5 pM concentrations were 65 and 85%, respectively.

6-h~droxyflavonetreatment

6-hydroxyflavone was lethal at the 75 pM (100%) and 25 pM (70%) treatments. with death occurring at days 9 to 1 7. In both treatments, the development of the embryos was retarded and multiple circulatory aberrations were commonplace. Pericardial and yolk edemas were observed in the 25 pM treatment. At the two lowest concentrations. 6- hydroxflavone was not toxic with 70 to 90% of the exposed embryos successfully reaching the swim-up stage of development.

Galangin treatment

The prevalence of morphological defects among embryos exposed to galangin at 5

- 75 pM ranged From 70 to 90%. with generalized edema and circulatory stasis developing at Day 4 and thereafter as the most Frequently observed abnormalities. Mortalities in medaka were 35,40 and 70% at the 5.25 and 75 pM galangin treatments. Hatching

success was also impaired at these nominal concentrations. with ~50%(25 pM) of

embryos hatching.

Ouercetin treatment

Quercetin was not dissolved in the embryo rearing medium at the two highest

nominal concentrations as was evident with the presence of a yellow precipitate. Thus. the

LC50 and EC50 parameters were calculated by using results from the two lowest treatments, plus the Controls. Quercetin at the 5 pM treatment caused retardation in embryo development (65%). Circulatory abnormalities or development of yolk edemas were also evident in this treatment. Of the 85% of embryos that hatched, 45% of them died, 5% failed to inflate the swim bladder and 35% were viable at the end of the experiment. At the lowest quercetin concentration. 12% of the medaka died. 11% failed to inflate the swim bladder and 76% were normal.

A~i~enintreatment

Apigenin was embryotoxic to medaka embryos at the highest nominal concentration since only 10% of hatched larvae were normal at the last day of the experiment. During the early developmental stages. some embryos (55%) showed signs of caudal hemorrhaging and appearance of yolk edemas. There were two incidences of anisophthalmia. Of other affected embryos. 25% died and 30% failed to hatch. Most of the hatched rnedaka (35%) showed symptoms similar to those described in the flavanone treatment (25 1M). in addition to spinal deformities. Viability of medaka increased to 60.

85 and 75% at the 25, 5 and 0.5 pM treatments. respectively.

Taxifolin treatment

Taxifolin was toxic only at the highest concentration (75 pM). with only 39% of the medaka being normal at the end of the exposure. Of the exposed embryos in this treatment. 22% died, 6% failed to hatch and 5% died during hatching. Of the hatched larvae (67%), 17% died and 1I% failed to inflate their swim bladders. Besides a low incidence of haemorrhaging or development of yolk edemas, there were few other gross morphological defects observed during the experiment. 7-h~droxvflavonetreatment

Probit analysis indicated that the EC50 and LC50 for Fhydroxyflavone in the

Japanese medaka embryotoxicity assay were greater than 75 pM (the highest nominal concentration tested). However, observations at the 75 pM treatment showed that from the 80% of the embryos that hatched, only 30% were normal and 15% died and 35% showed adverse developmental responses such as failure to inflate the swim bladder and failure to absorb the yolk sac. At all other nominal concentrations of 7-hydroxyflavone, the proportion of normal medaka were 70 to 78%; similar to Controls (84%).

5 -4.3. Japanese medaka reproductive development studies

5-43.a. Reproductive development study #I

Control Treatment

The gonads of all control fish were differentiated by the end of the exposure period at 3 mo. There were 15 (58%) female and 1 1 (42%) male in the control group. as determined histologically (Table 5.4). The majority of the female medaka (93%) reached the perinucleus pre-vitellogenic stage of ovarian development since the more mature oocytes in these ovaries were only at stage N. as classified using the criteria developed by Iwamatsu et al. (1988) for medaka. Younger oocytes (stage I - II) were predominately located in the periphery of the ovarian lamellae, whereas the more mature oocytes were more centrally located (Fig. 5.10). The testes of male medaka appeared normal in all

Controls. All stages of spermatogenesis were apparent, with spermatogonia present in lobules next to the testicular membrane and the more mature spermatocytes and Figure 5.9. Japanese medaka (0.latipes) exposed to 25 pM of flavonol (3-hydroxyflavone) showing caudal haemonhaging (Hm), anisophthalmia (AnO), microcephaly and yok edema (YE). The development of this embryo was retarded.

Figure 5.10. Ovary of a Control Japanese medaka (0.latipes) showing stage II pre-vitellogenic oocytes in the periphery and stage N pre- vitellogenic oocytes in the middle (H&E, X 100). Table 5.4. Reproductive development experiment #I. Numben of phenotypic male and female Japanese medaka (0.Zafipes) identified histologically after exposure to the flavonoids. chrysin and quercetin as well as to the androgen. testosterone. The number and percent (in parenthesis) of the intersex condition, testis-ova observed in medaka from all treatments are also presented.

Treatment Concentration (ma) N Female Male Testis-ova (%)

Control 0 25 I5 I I 0 Chrysin 0. I 28 12 IS 1 (4) 0.5 27 13 14 0 1 26 11 IS 1 (4) Quercetin 0.0 1 29 15 14 0 0.05 26 13 15 0 0.1 28 8 20a 0 Testosterone 0.0 18 57 29 28 6 (11) " significant results at P < 0.05

spermatids present in lobules next to vas efferent. Spermatozoa were present in the efferent duct of all male rnedaka (Fig. 5.1 1). The urogenital papilla was not very pronounced in all females, and papillary processes in the anal fin of all male medaka did not develop.

Chrvsin Treatment

The proportions of females in the 0.1,0.5 and 1.0 mg/L chrysin treatments were

44%, 48%, and 42% respectively, which are not significantly different from the control medaka (Table 5.4). The ovaries of most female fish in all chrysin treatments were in advanced stages of oogenesis. as indicated by the incorporation of the PAS-positive

vitellogenin vesicles into the oocyte yolk mass (Fig 5.12). In the treatment at the lowest concentration (0.1 mg/L), 16.7% of the fish were in the pre-vitellogenic stage (oocyte stage I-rv), 66.7% in the viteilogenic stage (oocyte stage I-VII) and 16.7% in the Figure 5.1 I. Testis of a Control male Japanese medaka (0. latipes) showing all stages of spermatogenesis. Immature germ cells, spermatogonia (spg) and spennatocytes (spc) are located in the periphery,whereas mature spermatozoa (spz) are found in lobules adjacent to efferent duct (ed) (ME, 60X).

Figure 5.1 2. Ovary of a chrycin-treated phenotypic female Japanese medaka (0. latipes) showing advanged oogenesis. Note the large vitellogenic oocytes at the YII stage of development, having incorporated vitellogenin in their oolernrna (PAS- positive). Also note the pre-vitellogenic oocytes in the periphery of the ovary (PAS- negative) (PAS, 60X). post-ovulatory phase (oocyte stage VIII-[X). In the test at the intermediate concentration, the proportion of gonadally immature medaka was 23.1 %. Finally, in the highest chrysin concentration (1 mgL), the oocytes in 6.7% of female tish had not developed past the IV stage. 80% were vitellogenic, and 13.3% were post-vitellogenic. In ali treatments. oogenesis in female medaka was more advanced than in that of the Controls. In addition. two fish fiom this treatment were egg-bearers. which is quite unusual for 3 months old fish (Fig. 5.13). Two large females (25 mm) in the highest concentration had pronounced urogenital papillae.

In the testicular tissue of two male fish from the 0.1 and 1.0 mgL treatments. there was development of oocytes. an intersex condition known as "testis-ova". The oocytes (stage I to IV) in the one fish From the 0. I mg/L chrysin treatment were centrally located. whereas in the other case of testis-ova the oocytes were at stage III of development and were scattered mostly at the periphery of the testicular tissue. Testes

From the chrysin-treated male fish. in comparison to the male in Controls. were more fibrotic (40-85%) and had enlarged primary sperrnatogonia. Papillary processes in the anal fins were observed in two male medaka. and in one fish with testis-ova.

Ouercetin treatment

The proportions of female in the 0.01.0.05 and 0.1 m@ quercetin treatments were 52%. 46% and 29% for. respectively (Table 5.4). The latter was significantly different from Controls and all other treatments. Quercetin had an effect on the ovarian development at all concentrations tested. For example, the ovaries of female fish did not reach sexual maturity. as is evident fiom higher proportions (60% to 70%) of pre- Chrysin (0.1 mgR) Quercetin (0.01 ma)

Quercetin (0.05 m&)

Quercetin (0.1 mg/L)

Control

Figure 5.13. Reproductive development experiment # 1. Ses ratio (pie) and stage of oogenesis (co1umn) in Japanese medaka (0.Iaripes) e~~osedto 3 nominal concentrations of chqsin and quercetin for 3 months (pre-VtG = pre-vitellogenic stage oocytes: VtG = vitellogenic oocytes). vitellogenic oocytes (Fig. 5.13). The remaining (30%-40%) of ovaries had few oocytes in stage V. as was apparent from the presence of PAS-positive yolk vesicles in the ooplasm.

In most ovaries, the majority of the vitellogenic oocytes were atretic, as indicated by the separation of ooplasm From: (a) the nucleoplasm; or (b) the oocyte membrane. Testes were in advanced stages of spermatogenesis. since in most of the fish the proportion of mature spermatozoa exceeded by far the proportion of immature sperm cells. The urogenital papillae were not fully developed in all female medaka. However. four phenotypic female fish developed papillary processes in the anal fin. a secondary male sex characteristic. In addition. nine male medaka also developed the same processes in their anal fins.

Testosterone treatment

The proportion of females (50%) and males (50%) were similar to those of

Controls (Table 5.4). There were six cases of testis-ova development. four of which were observed in gonads with the ovarian component being the most predominant tissue. Very

few female medaka (n4)reached the vitellogenic stages of oocyte development. whereas

most were at the pre-vitellogenic or early vitellogenic stages (V) of oogenesis.

Spermatogenesis was advanced in only a few (~5)male medaka. as indicated by an

increased proportion of spermatozoa observed in the efferent duct and in the lobules next to it. All medaka, both male and female. developed papillary processes in the 7 posterior

rays of the anal fin (1 00%). which is a male positive characteristic.

5.4.3.b. Reproductive development study #2 Control Treatment

The proportions of female and male medaka in the control treatment were 42% and 58%. respectively (Table 5.5). Gonadal development in the Control treatment was normal over the 5 months of exposure. Female medaka entered the ovarian vitellogenic stage at total lengths of 17 rnrn or greater (Table 5.6). In general. all stages of spermatogenesis in the testes were observed in male medaka with lengths >l5 mm and advanced stages were observed in male medaka with lengths of 18 rnm or greater (Table

5.7). In males > 17 mm (n=17) there was total agreement between external secondary sex characteristics and gonadal sex. whereas in all females with lengths greater than 17mm

(n43). the urogenital papilla dorsal and anal fins from three females (23%) were scored as phenotypically male (Table 5.8).

Flavone Treatment

The proportion of female (41%) and male medaka (59%) did not differ from

Controls. Development of testis-ova were observed in males at 4 months (n=2) and 5 months (n= I ) of exposure. In all testis-ova cases. spermatogenesis was in advanced stages and oogonia were observed within peripheral testicular lobules (Fig. 5-14). In two other exposed males. the structural organization of the testes was altered in a manner similar to that observed in the intenex testes (although oogonia were not present): that is. there was an increase in the interstitial spaces and in connective tissue around the testicular lobules

(Fig. 5.15). In all other male medaka. testicular development and spermatogenesis appeared to be normal (Table 5.7). Ovarian development and oogenesis appeared to be affected by flavone (Table 5.6). In all females but one. regardless of the stage of somatic Table 5.5. Reproductive development experiment #2. Numbers of phenotypic male and female Japanese medaka (0.latips) identified histologically after exposure to various flavonoids and to synthetic estrogen, 17a-ethinyl-estradiol (EEZ). The number and percent (in parenthesis) of the intersex condition, testis-ova observed in all treatments is also presented.

Treatment Concentration (rng/L) N Female Male Testis-ova (%)

Control Flavone Flavonol Flavanone Chrysin Quercetin ChrysidQuercetin Galangin

Apigenin

Naringenin

Kaempferol

Catechin

" significant results at P < 0.05

growth. primordial germ cells (PGCs) were present. and in the ovaries of five females

there were many atretic follicles. as indicated with the separation of oolemma from the

oocyte membrane. Vitellogenic oocytes (stage VI) were observed in four females (>19.5

mm TL,).There was a total agreement (100%) between gonadal sex and secondary sex

characteristics in all males (n=12). To the contrary. the dorsal and anal fins of 3 females

were identified as arrhenoid (i.e. male-like). whereas the fins in the other 3 females were

reduced. making it impossible to definitely identify the sex from external secondary sex

Table 5.8. Reproductive development experiment #?. Conlparisons between the phenotypic gonadal sex and secondary sex characteristics of Japanese medaka (0,lutipes) exposed to individual flavonoids and to 17~-ethi~lylestradiol.The terms "Total" refers to the disagreement of both urogenital area and fins (dorsal and anal), whereas "reduced" refers to secondary sex characteristics that were not positively identified.

Phenotypic Male Phenotypic Female Flavonoid (mglL) N Agreement (%) Disayement (%) Reduced(%) N Agreement (%) Disagreement (%) Reduced (%) Total UGP Fins Total UGP Fins Control 17 Flavone 12 17 - Flavonol 6 Flavano~ie 13 - 23 Chrysin 20 5 10 Quercetin 20 10 15 Chrysin & Quercetin 24 - 8 Galangin (0.05) 29 3 3 c1 a (0.5) 25 - C Catechin (0.1 ) 2 1 10 9 (1) 24 - 21 Kaempferol(0.05) 22 5 4 (0,s) 15 - 13 Naringenin (0.I ) 15 7 26 (1) 23 - 17 Apigenin (0.05) 19 - II (0.5) 23 4 9 Estradiol ( l n@L) 7 14 -

( I0 11glL) 9 2 2' 44 - 33 12 42" 16 - - "significant resul~sar P *: 0.05 ^significantresults at P -. 0.01 "significant results at P 0.005 Figure 5.14. Testis of a phenotypic male Japanese medaka (0.latipes) exposed to flavone for 4 months. Note the presence of wcytes (TO) within a testicuiar lobule (H&E, 1 SOX).

Figure 5.15. Testis of a phenotypic male Japanese medaka (0. latipes) exposed to flavone for 4 months. The architecture of this testis resembles the structure of testes in intersex conditions. Note the increase in intdal tissue (IT) and interstitial space (IC)around the testicular lobules (K&E, LOOX). characteristics. However, in all 6 cases, the urogenital papilla of the female phenotype corresponded :a the gonadal phenotype (Table 5 .a).

Flavonol treatment

The proportion of females (65%) and males (35%) did not significantly differ from

Controls (Table 5.5). Two male fish (20%) developed testis-ova, as indicated by either the presence of a single or multiple oogonia confined within testicular lobules of the anterior part of the testis. Testicular growth and spermatogenesis appeared to be normal in the males without testis-ova (Table 5.7). The ovaries of the majority of females exposed to flavonol showed an increased incidence of atresia (57%). and an increase in the amount of ovarian lumen (36%). indirectly indicating a reduction in number of oocytes. Primordial germ cells were noted in the ovaries of even large size female fish (>19 mm).Secondary sex characteristics in males were in agreement (100%) with the phenotypic gonadal sex.

In females. the agreement was only 29% (4/14). with 6% of females having reduced secondary sex characteristics and 65% having arrhenoid urogenital pore and fins (Table

5.8).

Flavanone treatment

The proportions of females (55%) and males (45%) were not statistically different ftom Controls (Table 5.5). In three exposed male medaka ( 16%). the development of testis-ova was evident. A case of testis-ova was observed in a fish after 2 months of exposure. where gonadal cells were equally distributed between male spermatogonia or spermatocytes and pre-vitellogenic oogonia. The other two cases of testis-ova were observed in male medaka exposed to flavanone for 5 months. with oogonia present throughout the testis. The oocytes in the majority of ovaries in females were pre- vitellogenic (78%), with only two reaching the early vitellogenic stage of oogenesis

(22%). Six ovaries had increased rates of atresia (66%), and PGCs were present in gonads of three large females (>1 7 mm).

In males. development of testis and spermatogenesis appeared to be normal. with

69% of males being at the advanced stages. Agreement of secondary sex characteristics and gonadal sex in males was 77% (10113): with three urogenital pores in males identified as female-like. Only in 33% of the females (319) was agreement between gonadal sex and external appearance. The secondary sex characteristics in 67% of female fish were male- like.

Chrvsin treatment

The proportions of female (47%) and male medaka (53%) were not statistically different from Controls. There was only one testis-ova (4%) in a male medaka exposed to chrysin for four months. The single oogonium was present within a testicular lobule in a testis showing all the stages of spermatogenesis. The testicular structure of 8 male medaka

(42%) was affected with an increase in interstitial space separating individual lobules and with a subtle thickening of the lobular membrane (Fig, 5.1 5). In addition. male medaka reached advanced stages of spermatogenesis at larger sizes (> 2 1 mm). Few females

(20%)were at the pre-vitellogenic stage of development. The ovaries of the remaining females were at the vitellogenic stage (60%) and few were at the late stages of oogenesis

(20%). There were a few incidences of ovaries with increased atresia (20%). increased development of the ovarian lumen (20%) (Fig. 5.16) and increased number of PGCs. Within the posterior liver of one female fish there was development of an ectopic oocyte

(III stage). Agreement between the gonadal phenotypic sex and secondary sex characteristics in males was only 65% (P < 0.0 1). whereas in females agreement was 40%

(P < 0.05).

Ouercetin treatment

The proportions of females and males (48 and 52%. respectivelyo) were not different from the Control treatment (Table 5.5). There were two cases (6%) of testis-ova in medaka after 4 and 5 months of exposure. respectively. Gonadal development in the rest of the male medaka appeared to be normal (Table 5.7). On the other hand. quercetin had an effect on ovarian development in females. In ovaries of 20 female medaka (>I7 mm). there were 16 cases (80%) of atresia. 5 cases (25%) of stroma development (Fig.

5.17). 6 cases (30%) of an increase in inter-ovarian space (ovarian lumen). and 10 cases

(50%) of ovaries having PGCs (Table 5.6). In the liver of one female fish. there was development of an oocyte (i.e. ectopic oocyte). Agreement between gonadal sex and secondary sex characteristics in males was 75%: significantly different From Control males (P < 0.05). In females. agreement was only 33% (P < 0.05).

Chrysin and Ouercetin treatment

The proportion of females (46%) and males (54%) did not differ from the Controls

(Table 5.5). There were 10 cases of testis-ova ( 14%). a response which was observed in

medaka sampled throughout the duration of the experiment. The testicular development of

four male specimens was affected (Table 5.7). as indicated by an increase in interstitial

space and thickening of the lobular membrane without any inter-sex (Fig. 5.15). Ovarian Figure 5.16. Ovary of a phenotypic female Japanese medaka (0.latipes)exposed to chrysin For 5 months. Note an increased development in ovarian lumen (OL) and a simultaneous reduction in oocytes. This female has entered the vitellogenic stage of oogenesis as indicated by the presence of stage YI oocytes (HbE,1 OOX).

Figure 5.17. Ovary of a phenotypic female Japanese medaka (0. latipes) exposed to quercetin for 5 months. Note the presence ofsomatic tissue (stroma) and the presence of many atretic oocytes as indicated by the separation of oolemma from the nucleus. This female is in the late viteUogenic stage of wgenesis (PAS-positive VUI oocytes) (PAS, LOOX). development was greatly affected in females from this treatment (Table 5.6). Atresia, from moderate to severe. was observed in 86% of the phenotypic female medaka. In addition, 52% of the ovaries had a reduced number of oocytes as indicated by an increase in ovarian lumen. Furthermore, PGCs were present in 48% of the female fish as large as

25 mm in length. In the periphery of one liver. there was development of an ectopic oocyte. The most dramatic effects of the binary flavonoid mixture. chrysin and quercetin. was seen in the development of the secondary sex characteristics. In 75% of all examined medaka (s67)there were gross deformities in the dorsal. anal, pectoral or caudal fin. and in many occasions. fins were completely absent (Fig. 5.18). In addition. agreement between gonadal sex and secondary sex characteristics was poor in both males (67%: P <

0.01) and females (1 0%. P < 0.005). as presented in Table 5.8.

Kaempferol treatments

The proportions of females in the two kaempferol treatments were 43% (0.05 mg/L) and 54% (0.5 mg/L). which were not different from the control treatment (Table

5.5). In both kaempferol treatments. there were a total of 2 cases of testis ova formation.

Spermatogenesis in the fish exposed to the lower nominal concentration of kaempferol was affected more than in the higher concentration (Table 5.7). Specifically. in the 0.05 mg/L. treatment. spermatogenesis was advanced in only 7 male medaka (32%) in comparison to 67% in the 0.5 mg/L treatment.

In Female medaka exposed to the lower kaernpferol concentration. the ratio of pre- vitellogenic to vitellogenic oocytes was 67 to 33%. whereas in the higher concentration the ratio was 45 to 55%. Ovarian development was also affected in both kaempferol Figure 5.18. Japanese medaka (0.latipes) exposed to the binary mixture of chrysin and quercetin. Note the absence of the dorsal fin and the deformed anal fin.

Figure 5.19. Ovary of a Japanese medaka (0. latipes) exposed to 0.05 mgL of kaempferol for 5 months. The fish is an adult fanale experiencing a delayed sexual maturity (pre-vitellogenic oocytes). Note the reduced number of oocytes and the increased development of ovarian lumen (OL) (H&E, 100X). Figure 5.20. Ovary of a Japanese medaka (0.latipes) exposed to 0.05 mg/L of kaempferol for 5 months. This adult female medaka is not sexually mature since the majority of oocytes are in the pre-vitellogenic stage of oogenesis (H&E SOX).

Figure 5.2 1. Development of testis-ova in a Japanese medaka (0.latipes) exposed to 0.5 mg/L of naringeoin for 3 months. Immature prc vitellogenic oocytes (0)are mainly located in the posterior end ofthe testicular tissue. Sperm cells at dierent stages of spermatogenesis are found in the anteriorend of the testis WE,100X). treatments (Fig. 5.19 and 5.20). The incidence of atresia, the presence of ovarian lumen and PGCs were 60%. 53% and 33%. respectively. in the 0.05 mgL treatment. and 65%.

75% and 596, respectively, in the 0.5 mg/L treatment. Agreement between gonadal sex and secondary sex characteristic in males exposed to 0.05 and 0.5 mgL treatments was

41% (P < 0.005) and 80%. respectively. In females. the agreement was 26% (P < 0.01) and 21% (P < 0.005). respectively.

Naringenin treatments

The proportions of females both naringenin treatments were 6 1% and 58%. respectively. both statistical different than the Controls (PcO.05) (Table 5.5). Japanese medaka exposed to the lower nominal concentration (0.1 mg/L) of naringenin showed a

3% incidence of testis-ova. whereas in the higher concentration (1 mgL)the incidence of testis-ova increased to I 1% (Table 5.7). It appean that the development of testis-ova in medaka exposed to this test compound follows a gradient. fiom posterior to anterior. and it can be observed in medaka after 2 months of exposure (Fig. 5.21).

Oogenesis advanced to vitellogenic stages in 57% and 39% of female medaka exposed to the lower and higher naringenin nominal concentrations. respectively. The rest of the females had only pre-vitellogenic oocytes (Table 5.6). Atresia was observed in 48%

(0.1 mg/L) and 65% (1 mgL) of females. increased incidence of PGCs in 5% (0.1 mgL) and 30% (1 m@) of females. and increase in ovarian lumen in 62% (0.1 rn@) and 39%

(I ma)of females fiom the two treatments. Ectopic oocytes (II - Wstages) were observed in the hepatic region of one female medaka exposed to the lowest concentration of naringenin [Fig. 5.22). Agreement between external and gonadal sex was 65% (1 mg/L) and 67% (0.1 mg/L) in males (P < 0.0 1), and was 9% and 10% in females (P <

0.005).

Apieenin treatments

The proportion of females at the 0.05 mglL and 0.5 mgR kaempferol treatments were 54 and 53%. respectively. statistically not different from Controls (Table 5.5).

Testis-ova formation was observed in 8% and 1 1% of medaka exposed to 0.05 and 0.5 mg/L of apigenin. respectively (Table 5.5). The development of this intersex condition varied amongst individual medaka as was shown by the appearance of a single (Fig 5.23) or a few oocytes within testicular lobules or alternatively by the presence of many pre- vitellogenic oocytes in nearly the entire gonad (Fig. 5.24). The testicular development in four male medaka (one in the highest and three in the lowest exposure concentration) was affected. as was evident by the proliferation of the connective tissue that surrounds the testicular lobules (Fig. 5.23). Similar developmental responses were observed also in medaka with testis-ova (Fig. 5.23). All other male medaka appeared to be normal.

On the other hand. ovarian development was greatly affected in both apigenin exposure concentrations (Table 5.6). as indicated by a high incidence of atresia (72 and

53%: 0.05 and 0.5 mg/L. respectively). presence of stroma (28 and 13%). presence of ovarian lumen (60% both). and presence of PGCs (28 and 40%). Agreement between

secondary sex characteristics and phenotypic male medaka was 79% (P < 0.05) at the

lowest concentration. with the urogenital pore identified as female in two occasions or

both fins being reduced (difficult to identify) in two other cases (Table 5.8). At the hi&est

concentration, also 78% (P < 0.05) the secondary sex characteristics were identified as Figure 5.22. Female Japanese medaka (0. latipa) exposed to 0.1 mg/L of naringenin for 5 months. There is a development ofectopic genn-cells in the hepatic region (H&E, 100X).

Figure 5.23. (A) Development of testis-ova m a phenotypic male Japanese me& (0. laripes) exposed to 0.5 mg/L of apigenh, as indicated by the presence of a single oocyte (0) within a testicular lobule. Note the increase in fibroric tissue (Fb) around the lobules. (B) A testis Corn a male medaka exposed also to 0.5 m& of apigenin showing similar architectural pattern of testicular development (H&E, 1 SOX) Figure 5.24. A case of testissva with the ovarian component predominating in the gonad. This Japanese medaka was exposed to 0.5 m@ of apigenin for 4 months (HLE,100X).

Figure 5.25. A phenotypic female Japawse medaka (0.Iutipes) exposed to 0.5 mg/L of galangin for 5 months, showing most of the YIII oocytes being atretic and the consequent deveIopment of the somatic tissue, stroma (PAS, 40X). female. The expression of urogenital papilla or dorsal and anal fins were more drastically affected in phenotypic females by exposure to apigenin, since agreement with gonadal sex was only 12% at the lower concentration. whereas at the higher concentrations all exposed phenotypic female medaka (1 00%) had either male-like (87%) or unidentified (1 3%) secondary sex characteristics (Table 5.8).

Galangin treatments

The proportion of females in the galangin treatments were 41% (0.05 mg/L) and

46% (0.5 mglL). which were not different from Controls (Table 5.5). There were three cases of testis-ova (5%) at the lower concentration and two cases (3%) at the higher concentrations (Table 5.5). Testicular development and spermatogenesis were normal in all male medaka. with the exception of one specimen that showed a reduced density of spermatozoa in both the testicular lobules and the efferent duct (Table 5.7).

Ovarian development was affected by exposure to galangin (Table 5.6). More than

50% of ovaries in both treatments had an increased number of atretic oocytes (Fig 5.25).

In addition, in both treatments, 6 1 - 65% of ovaries showed a decrease in the number of oocytes and an increase in ovarian lumen. At the lower concentration. in 40% of the examined females (>I 7 rnrn), there were PGCs present. On the other hand.at the 0.5 mg/L treatment. there were fewer female medaka with increased number of PGCs ( 17%) (Table

5.6). There was good agreement between secondary sex characteristics and gonads in male medaka exposed to galangin (84 and 100%); however. this agreement was very poor

(25%) in females (Table 5.8). Catechin treatments

The proportions of females in the catechin treatments were 55% and 54% (0.1 and

1 mgL, respectively) which did not differ from Controls (Table 5.5). Testis-ova were observed in 4 fish (5%) at the lower concentration. and in 3 fish (4%) at the higher concentration (Table 5.5). Overall catechin did not affect spermatogenesis and testicular development in male medaka exposed to both concentrations. with the exception of one medaka showing an increase in intestitial connective tissue (Table 5.7).

Once again. the female fish were the most affected by catechin. At the 0.1 and 1 mg/L treatments. the development of testis-ova was 6 and 8%. incidence of atresia reached 59 and 65%. increase in ovarian lumen was observed in 73 and 56%. and PGCs were present in 32 and 65%. respectively (Table 5.6). Agreement regarding the appearance of secondary sex characteristics and gonadal sex was at 76 - 79% in males. with affected males showing a feminized urogenital pore. In females. this agreement was very low at 4 - 9%. with either the urogenital papilla andfor the dorsal and anal fins being masculinized (57%). or the fins being reduced (24 - 29%) (Table 5.8).

17a-ethinylestradiol(EE2) treatements

The proportion of females in the EE2 treatments were 63% ( 1 n&) and 58% ( 10

ng/L). not different from Controls (Table 5.5). At the lower concentration. 9% of the

exposed medaka developed testis-ova in comparison to a 2 1% incidence at the higher

concentration (Table 5.5). Oocytes were developing from the posterior to the anterior part

of the testes (Fig. 5.26). or they were scattered throughout the gonad. In some cases. there

were remnants of testicular tissue (Fig. 5.27) showing a near complete sex-reversal. Figure 5.26. Development of testis-ova in a phenotypic male Japanese medaka (0.latipes) exposed to 1 0 ng/L of 17a-ethiny l-estradiol (EE2) for 4 months. Oocytes are contined in the posterior end, whereas testicular lobules with sperm cells are mainly in the anterior testis (H&E, 1 SOX).

Figure 5.27. A nearly sex-reversed male Japanese medaka (0.lutipe.s) exposed to 10 a& of I fa-ethinyl-estradiol (EE2) for 5 months. Remnants of the testicular component (Scs) are located in the anterior gonad, whereas previtefI~genic(N)and early vitellogenic (V)oocytes are distn'buted throughout the gonad W&E, 2 SOX). Exposure to the higher concentration of EE2 retarded spermatogenesis (P < 0.05) with only one male medaka (9%) reaching the advanced stages (Table 5.7).

Ovarian development was also affected by EE2 at 10 ng/L. as indicated by the high incidence of atresia and presence of PGCs in 46 and 77% of females. respectively.

At the lower concentration. the incidences of the same ovarian response were 1 1 and

56%. respectively (Table 5.6). The agreement between gonadal sex and secondary sex characteristics was affected in male medaka (Table 5.8). For instance. at the lower concentration 72% of the fish identified from secondary characteristics as males had feminized urogenital pores. At the higher concentration. 22% of the males were identified correctly from secondary sex characteristics. but among the other males in 33% the urogenital pore was feminized and in 55% both the urogenital pore and fins were female- like. The agreement in &males was 67 and 42% for the I and 10 ng/L treatments. respectively (Table 5.8). Most of the incorrect characterizations were related to the shape of the fins: especially the dorsal fin (22 and 42%).

5.43~.Reproductive development study $3

Controi treatment

There were 34 (56%) females and 27 (44%) males histologically examined from the Control treatment (Table 5.9). In females > 17 mm. the ovarian development appeared to be normal (91%). except for two females: one having an increased number of PGCs and the other showing an increase in amount of ovarian lumen (Table 5.10). Two of the female medaka were at the pre-vitellogenic stage of oogenesis. whereas the rest were at the early vitellogenic stages as indicated by the presence of stage V and VI oocytes. In 17 male medaka, the testicular development was normal: three males (18 %) were sexually immature. five (29%) had all stages of sperm cells present in the testes and the rest (53%) were at the advanced stages of spermatogenesis (Table 5.1 1). The expression of secondary sex characteristics was in agreement with the gonadal sex at 96 and 100% for the female and male phenotypes. respectively (Fig. 5.28).

Genistein treatments

The numbers of female and male medaka examined in the different genistein treatments are presented in Table 5.9. Testis-ova were observed in only two fish of the highest nominal concentration (i.e. 1000 pg/L). The stages of oogenesis were different

(XI-analysis; P < 0.05) From Controls at the 10 pg/L treatment. where 47% of phenotypic female medaka were at the pre-vitellogenic and 53% at the vitellogenic stages of oogenesis ( V and Vl). with only one female being at the late ( VIII) stage (Table 5.10). One female from each of the 1 and 100 pg/L genistein treatments was in spawning condition.

However. other characteristics of ovarian development were affected in all treatments (f- analysis: P < 0.05). The incidence of oocyte atresia increased in a dose-dependent manner

From 9 to 38%. whereas reduction in oocytes and a consequent increase in ovarian lumen area were present in 10 to 27% of the female fish examined. Finally. the incidence of ovaries having PGCs increased from 5% in the I pg/L treatment to 19% at the highest exposure concentration (Table 5.1 0).

Spermatogenesis was not different between Controls and genistein-treated male

Japanese rnedaka (Table 5.1 1). However. the testicular development was altered in all genistein treatments. There was a significant increase in connective tissue inside the testis and fibrosis around the testicular lobules (28 - SO%), as well as a decrease in the density of spermatozoa (1 1 - 50%) in both lobules and efferent duct (Fig. 5.29; Table 5.1 1).

Also, the architecture of some testes resembled that of gonads with testis-ova, as evident by an increase in fibrosis and interstitial separation in testicular lobules. In phenotypic female medaka the agreement between gonadal sex and secondary sex characteristics ranged from 56 to 61% (Fig. 5.28). which was statistically different from Controls ( P <

0.05). In general. the dorsal and/or anal fins of the affected fish were arrhenoid. In phenotypic male rnedaka, there was poor agreement in the highest genistein concentration

(62%) between external characteristics and gonadal sex (Fig. 5.28). The shape of urogenital pore was altered to the female phenotype (i.e. feminized) in the affected male medaka. However. there was an over-expression of the male phenotype in the shape of dona1 and anal fins (Fig. 5.30).

Table 5.9. Reproductive development experiment #3. Number of Japanese medaka (0.kuiipes) prepared for histology. number of medaka examined as well as number of phenotypic male and female. Incidence of testis-ova is also presented.

Genistein r 1.~9n-1 N(al11 N(examined) Female Male Testis-ova control 6 1 6 1 34 27 - 1 66 64 34 30 - 10 87 65 43 22 100 67 57 35 22 - 1000 84 48 3 1 17 -3

- -

- * - male

-- Control (tiemule) = 96% -.- - -m. B== Control (mule) = 100a/o - -

concentration

Figure 5.28. Graph depicting the agreement between the phenotypic gonadal sex and secondary sex characteristics in Japanese mPdaka (0.lattpes) exposed to genistein for three months Asterisks (**) represent significant results at P < 0.05. Figure 5.29. A testis of a Japanese medaka (0.lutipes) exposed to 100 ofgenistein for 3 months. Note the increase of connective tissue (cT), and the decrease in density of spermatozoa (spz) in the efferent duct (ed) (H&E, IOOX).

Figure 5.30. A Japanese medaka (0.lutipe.s)exposed to 100 p& of genistein showing arrhenoid expression of the dorsal and anal tin. Note the notches in both fins and papillary processes (ppl) in the anal fin. The anal fin is defonned in the 5.4.4. in vitro studies

5.4.5.a. Yeast Estrogenici~Assay (YES assay)

The flavonoids, apigenin, naringenin, kaempferol, 4'-hydroxyflavanone. 4'- hydroxyflavone. 4.6-dixydroxyflavone and the isoflavonoids. genistein. equol. and daidzein exhibited estrogenic activity in the recombinant yeast assay (Fig 5.32 - 5.34). At the concentrations tested. only the isoflavonoids reached the maximal response relative to

E2 (Fig. 5.34). whereas the flavone. apigenin and the flavanone. naringenin reached 75% and 80%. respectively of this maximal response (Fig. 5.32). The flavanol. kaempferol reached 38% of the maximal response, and 4'-hydroxyflavanone. 4'-hydroxyflavone and

4'.ddihydroxyflavone induced only 29%. 30% and 50% of the maximal response. respectively (Fig. 5.33). The rest of the flavonoid compounds failed to induce a response in this assay. The concentrations of flavonoids required to induce 25% maximal response

(i.e. EC25) are presented in Table 5.12. Based on these values. the relative estrogenic potential of all flavonoids used in this study was calculated (Table 5.12). and the ranking of the estrogenic flavonoids was determined as: equal> genistein > apigenin > daidzein >

4'.6-dihydroxyflavone > naringenin = 4'-hydroxyflavanone > kaempferol > 4'- hydroxyflavone.

5.44 b. in vitro binding studies6

6 Work in these in vitro assays were completed at Dr. Glen Van Der Kraak's lab at University of Guelph. Figure 5.3 1. YES Assay. The 96-well plate showing the expression of 8-galactosidase in the presence of the flavonoids, (+)-catechin, chrysin, naringenin, galangin, flavanone and mans-chalcone (rows A-F), the procedural blank (G) and the positive control. 170- estradiol (H). Positive estrogenic response is indicated with magenta whereas no response is indicated with orange. Cytotoxic effects are found in wells with yellow colouration, Concentration (M)

Figure 5.32. Response of the recombinant yeast estrogen screen. expressed as D-gdactosidase activity (%), to the flavonoids naringenin (nu). kaempferol (kae) and apigenin (api) at various concentrations (M). The response of 17D-estradiol (E2)was used as the positive control. Figure 5.33. Response of the recombinant yeast estrogen screen. expressed as D-galactosidase activity (%). to the flavonotds. 4'-hydrosyflavanone (4'-fnn). 4'-hydrosyflavone (4'-fla)and J.6- dilyimxyflavone (4.6-fla) at various concentrations (M). The response of 17D-estndiol (E2) was used as the positive control. - " dai - equol

Concentration (M)

Figure 5.34. Response of the recombinant yeast estrogen screen. expressed as B-galactosidase activity (%), to the isoflavonoids d;udzein (dai). genistein (gen) and equol at various concentrations (M). The response of 17D-estradiol (E2)was used as the positive control. oao ooaC41 0000m 00000 00000 99999 00000 Binding to sex steroid binding b rote in (SSBP)

Competitive binding of [3H]17Bestradiol to the goldfish plasma SSBP was reduced by many flavonoids (Table 5.13). The parent flavonoids, flavone. flavonol and

flavanone were highly effective in binding to the SSBP,with &nities relative to estradiol ranging from l OOx I Od to 158x 104. FIavonoids with hydroxy-substituted flavonoids in the

A ring only (i-e. 5--6-, 7-hydroxyflavone and chrysin) bound to the SSBP with lower affitnities (8 to 19x 104) In addition. the 4'-hydroxy-substituted flavonoids including.

apigenin. kaempferol. fisetin. naringenin. and 4'-hydroxy-flavanone were also competitors

for SSBP at ranges of relative affinity of 30 to JOOx lod. The binding affinity of luteolin

and quercetin. flavonoids with an extra hydroxy group in the 3' position. lowered the

binding affinity (3.7 to 4.5~10~).Finally. chalcone and the synthetic benzoflavones. a-

and B-naphthoflavone were poor competitors for SSBP (Table 5.13).

Trout estrogen receDtor (ER)assav

Flavonoids having 3' and/or C-hydroxy substitution in the B ring bound effectively

to the rainbow trout hepatic ER. For instance. the flavones. apigenin and luteolin. the

flavonols. kaempferol. fisetin and quercetin. and the flavanones. 4'-hydroxyflavanone and

naringenin bind to ER with an affinity relative to estradiol of 1 14 to 642x 104. Although

the flavan-3-01, catechin has a similar hydroxy-substitution pattern with quercetin, this

compound bound very poorly to the ER. All other flavonoids. which were either parent or

only A-ring hydroxy-substituted flavonoids bound poorly to the trout ER. with potencies

ranging from < 1 to 3.5~1O4 (Table 5.13). Table 5.13. Relative potencies of flavonoids for binding to the fish sex steroid binding proteins (SSBP), estrogen receptor (ER) and androgen receptor (AR). Values represent the rilean of 2-4 different experiments. The binding of I7p-estradiol in the SSBP and ER assays and testosterone in the AR assay were set at 1.

Flavonoid Class Hydroxyl substitution pattern --_--_-----_------A!!I~~~~_C_I~~%-~C~~IJ: ------SSBP ER AR Flavone Flavones - - 0.000 1 583

The flavonols. flavonoI(3'-hydroxyflavone) and kaempferol had the highest

binding affinity for the goldfish testis androgen receptor (AR). with potencies of 189x10'

and 120x 10". respectively. The parent compound, flavone also bound to AR. but with a

lower finity of 25x 1Ob. followed by the 4'-OH-flavanone ( 1 1x 1 04). and flavanone

(8x10"). Apigenin. fisetin and naringenin weakly bound to AR (2 to 6x 1Oh). whereas all

other flavonoids bound poorly to AR (Table 5.13).

5.5 DISCUSSION

EROD induction

The present study reveals that the naturally occurring phytochemicals flavone and

trans-chalcone are moderate hepatic EROD inducers in immature rainbow trout. since

they caused 4.5-fold ( 100 mglkg) and 1 5-fold ( 1 mg/kg) increases in EROD activities.

respectively. The 7-hydroxyflavone is a weak EROD inducer (2.6-fold). On the other

hand. all other flavonoids had no effect on EROD activity at the concentrations tested.

Based on these findings. we may infer that: (a) planar unsubstituted flavonoids induce the

teleost hepatic EROD system: (b) mono-hydroxy substituted flavonoids may induce

EROD. depending on the pattern of substitution: (c) compounds with di- to poly-hydroxy

substitution of the flavone molecule do not induce ER0D:and (d) compounds with

saturation of the 23 double bond in the pyrone ring also do not induce EROD. Other in

vivo mammalian studies are in agreement with our results. For example. flavone given to rats at a concentration of 0.25% in the diet for 14 days increased hepatic EROD. PROD

(pentoxyresorufin-0-deethylase)and ECOD (ethoxycoumarin-0-deethylase) activities by

20-, 30- and 2.5-fold respectively. whereas quercetin at a concentration of 1% failed to activate the same drug metabolizing enzyme systems (Brouad et a[. 1988). In another study. when rats were fed with diets containing either flavone. flavanone and tangeretin (a pentoxymethoxylated flavone) at various concentrations (20 - 2000 ppm). flavone induced the monooxygenases EROD. AHH and PROD. whereas tangeretin induced the EROD system only at the highest level (Siess et crl. 1992). In addition. oral administration of several flavones. flavanones and chalcone derivatives at a dose of 0.1 nM in rats revealed that only flavone caused a Cfold increase in CYP 1A activity (Wattenberg et al. 1968). In the same study. it was suggested that methoxylation of the flavone molecule slightly increases the AHH activity. hydroxylation has no effect. and halogenation at the 4'- position of the B ring significantly enhances the induction potency of the flavonoids. The authors of these studies (Wattenberg et uI. 1968: Brouad et ul. 1988: Siess cr (11. 1992) all suggested that flavone is both a phenobarbital and a 3-methylcholanthrene-type inducer.

Dose-dependent induction of EROD activity by flavone and 8-NF suggests that these flavonoids activate the CYP 1A-monooxygenase system via the Ah receptor. Chalcone may also elicit a response via the same mechanism. although there was some decline in

EROD activity at higher doses. The weak EROD induction caused by 7-hydroxyflavone may be attributed to other mechanisms (as discussed in Chapter 1). However. unless other techniques (Western blot. DNA probes. etc) are used. the mechanistic aspect of EROD

induction by flavonoids is speculative. Once again. the class of the flavonoids or substitution pattern influences their CYP l A inducing properties as seen in the present study and discussed el(Wood et al. 1986).

The specific biologic activities of flavonoids appear to depend on the experimental protocol used. the species used. and the administration route. For example. flavanone in our study had no effect on EROD activity in teleost hepatic microsomes: however. in in virro studies with human and rodent liver microsomal homogenates. the same chemical exhibited inhibitory properties (Siess cr ul. 1995). In our experiment. we preferred to expose the rainbow trout to flavonoids with a single i.p. injection. instead of a waterborne-exposure. since we were able to screen a larger number of flavonoid compounds with relatively small amounts of the test compounds.

Embrvoto~icitvof flavonoids

This study is the first to assess the embryotoxic potential of a number of unsubstituted and hydroxy-substituted flavonoids in fish. Survival and health of exposed

Japanese medaka embryos were greatly afFected in most of the flavonoid treatments.

Some generalizations can be made regarding the structural requirements for flavonoid compounds that elicit embryotoxic responses to Japanese medaka. The synthetic benzoflavone. R-NF was the most toxic compound (EC=0.08 pM). followed by the unsubstituted parent compounds. flavone (1.6 pM) and trans-chalcone (3.2 pM). The

EC50 for the other parent flavonoid. flavonone was at 5.5 pM. Addition of one hydroxyl

group in the flavone nucleus reduces the toxicity. as indicated by the EC5Os of 4.2 and 4.7

pM. respectively for the 3-hydroxjlavone (flavonol) and 6-hydroxyflavone treatments. However. the ECjO for 7-hydroxyflavone was above the range of nominal concentrations tested(>75 pM). indicating that the placement of hydroxyl group in the flavone molecule is a very important factor governing its embryotoxicity. Chrysin with hydroxy substitutions in the 5 and 7 positions had no effect on the development of rnedaka embryos. whereas addition of another hydroxy group in the 3 position (galangin) or 4' position (apigenin) caused toxicity. with calculated ECSOs of 5.1 and 23.8 pM. respectively. However. when there a simultaneous dihydroxy substitution at the same sites (ie. 3 and 4') to form kaempferol (3.4'5.7- tetrahydroxy-flavonol). there was no embryotoxicity. Quercetin (3.3'.4.5.7-pentahydroxyflavone) showed a high embryotoxic potential (2.7 pM). indicating that the additional hydroxy substitution in 3' position is an important structural requirement for flavonoid toxicity. Between flavonoid classes. it appears that the planar flavones (i.e. flavones. flavonols). which share the same hydroxy- substitution pattern with flavanones and flavan-3-01s. are more toxic. For example. flavone is more toxic than flavanone. as are apigenin (vs naringenin) and quercetin (vs taxifolin and catechin).

The patterns of mortality differ among flavonoids. suggesting a different mode of action. To elaborate. flavone and trrmss-chalcone at the highest treatments were acutely lethal within 1 to 2 days post-fertilization to exposed medaka embryos. probably by interfering with early mitotic processes during cell differentiation. Addition of a benzene ring on the flavone nucleus (ie. R-naphthoflavone) increased the embryotoxicity by a

factor of 10-fold. However. mortality in the case of O-NF occured at later days. resulting

fi-om *gdioxin-likeg'symptoms: namely circulatory aberrations. pericardia edemas and blue-sac disease. The same mortality pattern was observed in the 25 pM flavone treatment. indicating a common mechanism of embryotoxicity amongst CYP L A-inducing compounds. However. the other EROD-inducing flavonoid. chalcone did not cause similar responses. mainly because it was acutely toxic at the 25 and 75 pM treatments.

and toxicity was not observed at 5 pM. Additional testing involving exposure of

Japanese medaka embryos to nominal concentrations between 5 and 25 pM of chalcone

may provide evidence that this EROD-inducing flavonoid also causes dioxin-like

responses. Lethality in all other treatments with the most toxic flavonoids was a sequela to

embryonic abnormalities such as non-specific circulatorory malfomations. generalized

edemas and optic. cardiac. cephalic or skeletal deformities.

Reproductive DeveIovment Effects

The in vivo Japanese medaka reproductive assay used in the present study showed

evidence of the potential of all flavonoid compounds. including flavone. flavonol.

flavanone. chrysin. quercetin. apigenin. galangin. catechin. naringenin and kaempferol to

affect normal reproductive development and differentiation at nominal concentrations

ranging from 50 to 1000 pg/L. One of the most well-defined developmental responses

observed in all flavonoid testosterone and 17~~ethinylestradioltreatments was the

induction of testis-ova. Exposure to known xenoestrogens. endogenous steroids or

synthetic estrogens has been shown to induce testis-ova. in male medaka. as indicated by

the presence of pre-vitellogenic oocytes within the testis (Wester and Canton. 1986: Gray

and Metcdfe. 1997: Gray et al. 1999. Metcalfe er al. 2000b: Metcalfe el 01- 2000a). This is the first study to show that exposure of fish to natural phytochemicals such as flavonoids can induce this intersex condition at comparable pg/L concentrations to those of other xenoestrogens. All of the aforementioned flavonoids induced a low to moderate incidence of testis-ova (2 - 14%) after a period of 2 to 5 months of exposure. Statistics to determine which flavonoid treatments significantly increased the incidence of testis-ova were not used since Japanese medaka is a true differentiated gonochoristic species and thus. spontaneous sex-reversal or hermaphrodism never occurs (Yarnarnoto, 1958).

The progression or intensity of testis-ova development varied amongst treatments.

For instance. in some treatments (e.g. tlavone. tlavonol. etc) this intenex condition was evident by the presence of one or few pre-vitellogenic oocytes confined within testicular lobules. In other treatments (eg. apigenin. galangin. quercetin and 17ðinylestradiol) oocytes were distributed throughout the gonad with sperm cells located only in the anterior end. Similar results were documented in medaka exposed to intermediate concentrations of the potent estrogens. 17R-estradiol. 17~~ethinylestradiol.rstrone and estriol (Yamarnoto. 1959. 1965: Metcalfe er ul. 2000a). It was suggested in these studies

that testis-ova were induced by feminization of genotypic males due to the binding of

these estrogens to the estrogen receptor (ER): however. in the present study the same

could not be stated since flavonoids appear to affect multiple sites of the endocrine teleost

system besides the classical binding to the estrogen receptor. For instance. in the

testosterone treatment testis-ova was induced probably by the aromatization of

testosterone to estrogens.

In the majority of the treatments. the sex ratios of flavonoid treated medaka were not significantly different From the sex ratios of the Controls. In the first reproductive development experiment. at the highest concentration (0.1 mg/L) of quercetin there were

20 phenotypic male medaka and 8 females identified. whereas in Controls the numbers were 11 and 14. respectively (P < 0.05). It has been suggested that the masculinization of genotypically female medaka to phenotypic males is possible if fish are exposed to the

"ideal" concentration of androgens and the exposure is continuous during a critical time of their life history (Yamamoto. 1975). In this experiment. exposure started immediately after hatch when the germ cells are differentiated (Satoh and Egami. 1972). indicating that the increased proportion of male medaka may be attributed to the androgenic effect of quercetin. However. histological evaluation of the .gsurvivors**of this treatment. which were placed in clean water for a month. showed that 9 fish were female and 4 male. changing the female to male ratios to 2417: statistically no different than the Controls.

Thus. either the male-skewed ratio in the 0.1 mg/L quercetin treatment at 3 mo. was a statistical artifact due to the small sample size. or there was reversion of males to females over the month post-exposure. In the second reproductive development experiment with naringenin. the sex ratio was skewed toward the female phenotype (P

(Metcalfe el ul. in press). YES and rtER assays confirmed that naringenin is a weak phytoestrogen. Thus. it is possible that there was sex-reversal in some male Japanese medaka exposed to naringenin through similar mechanisms to those of the potent endogenous and synthetic steroids. A statistical error is quite unlikely to happen since the sample size ( n=78-79)was relatively large for both treatments.

Flavonoids had pronounced effects on ovarian development and oogenesis. In general. the ovary of the Japanese medaka is an unpaired sac-like organ attached to the wall of the coelomic cavity. It is lined with epithelium cells and within the epithelium there is a layer of mesenchymal connective tissues. From the ovarian wall, there are numerous folds projected towards the middle of the ovary that are comprised of oocytes at various stages of development. attached to a loosely connective tissue or stroma. The ovarian lumen is located in the dorsal part of the ovary and is connected to the oviduct

(Robinson and Rugh. 1972). The development of oocytes is asynchronous. with oocytes developing continuously from one stage to the next older stage. During post-ovulation. many oocytes become atretic as evident by the deformed shapes and hypertrophied follicular cells of the oocytes. The absorbed atretic oocytes are the precursors of the stromal tissue in the ovary (Yamaxnoto and Yoshioka 1964). All flavonoids. except chrysin and flavone. caused a high incidence of oocyte atresia in exposed female medaka.

Atretic oocytes are easily recognized in the stained tissues since there is a separation of the oocyte membrane from the ooplasm.or alternatively. the separation of ooplasm from the nucleus. Atresia is a natural process of normal ovarian development at the post- ovulatory stages: however. it is uncommon in pre-vitellogenic ovaries (Guraya. 1986). It

has been suggested that atresia is enhanced by exposure to chemicals with androgenic activity and it is inhibited by estrogens (Billig et al. 1993). It appears that this adverse

response in very consistent. since in both experiments (1 and 2) quercetin caused ovarian atresia in more than 80% of females. Increased rates of ovarian apoptosis. which is the molecular mechanism of atresia. in the presence of quercetin supports our histological findings (Janz et a/. 2000). The flavonoids. apigenin and quercetin caused a significant proliferation of intraovarian stromal tissue. even in ovaries with oocytes at the VI

(vitellogenic) of development. A reduction in the number of oocytes and a simultaneous increase in '~oid"intraovarian space (ovarian lumen) were observed in most of the flavonoid treatments. The incidence of affected female rnedaka ranged from 36%

(flavonol) to 75% (kaempferol. 0.5 mg/L). Finally. the presence of primordial germ cells

(PGCs) was evident in the ovaries of fish exposed to most of the flavonoids. Their presence in the ovaries of mature medaka ( TL r 17 mm) is quite uncommon and it may be attributed to the inhibitory action of flavonoids on oocyte growth.

Histological observations in a few Female medaka exposed to flavanone. naringenin. quercetin and the mixture of chrysin/quercetin treatments ( n =I for each treatment) revealed the development of ectopic oocytes in the liver or in the viscera outside the liver (Fig. 5.22). The ectopic oocytes appeared to be well-developed at the pre-

VtG stage (III or In. In other studies. one female Japanese medaka exposed to 100 p@L of octylphenol (OP) developed an ectopic oocyte (Gray rt ul. 1999). During embryonic developmental events. primordial germ cells found mainly in the mesentoderm

(gastrulation stage) migrate first in the mesoderm. where an increase in their numbers take

place. and they then migrate in the peripheral endoderm (somite Formation stage).

followed by translocation to the dorsal mesentery to form the gonad primordia (tail

elongation stage). where they became "definitive germ cells" (Gamo. 1 96 1 ). However. some primordial cells are incorporated to other tissues such as the neural keel or myotomes as dormant, non definitive germ cells (Gamo. 196 1). Our findings indicate that some flavonoids have the ability to act upon these dormant primordial cells by triggering cellular events that lead to their expression as "viable" oocytes. Although the exact mode of action is not known. it may be possible that ectopic oocyte development is the end result of the endocrine-disrupting potential of individual flavonoids.

Apart from the testis-ova induction. only a few flavonoids elicited some adverse effects on the testis of the exposed male medaka. Chrysin. the binary mixture of chrysinlquercetin. naringenin ( l mg/L) and apigenin (0.05 rng/L) caused some architectural changes in the testicular structure. For instance. there was an increase in interstitial space between the testicular lobules rendering a resemblance to ovarian architecture. In some cases there was also a visible increase in fibrotic tissue around the lobules: however. this response it is not easily quantifiable. It is interesting to note that the same abnormal structures were observed in testes developing testis-ova. Genistein at all concentrations tested (1 to 1000 pg/L) caused a significant increase in fibrotic and connective tissue in the testis with a simultaneous decrease in the density of mature sperm cells. conditions that are the prerequisites for the development of oocytes (Egami. 1955:

Komo and Egami. 1966). Similar effects were observed in medaka exposed to 10 ng/L of

170-estradiol in this study. or documented in other experiments with 17B-estradiol. 17a- ethinylestradiol and estriol at a concentration range of 10- 1000 ng/L (MetcalFe rr rd

2000a). Testicular fibrosis was also observed in sheepshead minnows (Cyprinodon variegarrcs) exposed to 200 - 800 ng/L of 17~~ethinylestradioI(Zilliowc. et a[. 2000. 5.35. Male rnedaka. In the posterior end of the dorsal fin there is a notch, a male- characteristic, Also note the notch in the anal fin. In addition, in the posterior end there are papillary processes (ppl) used during mating. Urogenital pore (Ugp) is visible

Figure 5.36. Female rnedaka. Both dorsal and anal fin lack the notches. Mature female have a well developed papilla (UGP) which cover both the genital and Ltrinary pores, suggesting that this adverse testicular effect is common to a variety of fish species. as a consequence of exposure to estrogenic chemicals.

Gametogenesis was the developmental characteristic least affected by exposure to flavonoids. Only flavanone and kaempferol at the lower concentration (0.05 mg/L.) seem to have an inhibitory effect on the stages of oogenesis. since the majority of the exposed females were at the pre-VtG stages. Interestingly. at the same treatment of kaempferol. spermatogenesis was also impacted in male medaka. with only 36% of exposed

individuals reaching spermatocyte maturation. Data from in vimstudies suggest that

flavanone may bind to plasma sex steroid binding proteins (SSBP) and thus. interfere with the transport of endogenous hormones to target sites. However. it is not possible to

speculate on the exact mechanism by which kaempferol (only in the lower concentration)

affects gametogenesis since this flavonoid binds to SSBP. androgen receptor (AR) and

both human (hER) and rainbow trout estrogen receptors (rtER).

Secondary sex characteristics

The medaka is a sexually dimorphic species. and mature males are recognizable

from the shape of the dorsal fin where a characteristic shallow notch exists in the

posterior end (Egami. 1975: Kirsten and West. 1979). Another male-like feature during

maturation is the presence of papillary processes at the posterior rays of the anal fin which

are used during mating behaviour (Fig. 5.35). Females are recognizable from a distinct

round-shaped urogenital papilla which covers both the genital and urinary pores (Fig.

5.36) (Metcalfe et a/. 1999). The rest of the dimorphic external morphological features are presented in Table 5.14. Flavonoids had a dramatic impact on the expression of these secondary sex characteristics. To elaborate, in most of the female medaka (60 - 100%) from all flavonoid treatments. the appearance of the dorsal andfor anal fins was arrhenoid or reduced. In the quercetin (0.1 mgL) and quercetin/chrysin treatments there were some phenotypic females with papillary processes in their dorsal fin. Papillary processes were also present in all medaka. regardless of the sex. that were exposed to testosterone at a nominal concentration of 18 pg/L for 3 months of exposure. On the other hand. in male medaka exposed to flavanone. chrysin. quercetin. chrysinlquercetin. catechin. kaempferol

(0.05 mg/L). naringenin. apigenin. genistein and ethinylestradiol the expression of the urogenital pore (2 1- 86%) was female-like or male with estrogenized appearance. In female fish. there was uncertainty in recognizing the phenotype sex externally since the identification of the secondary sex characteristics in three female Control fish (n = 13). with length sizes ranging from 17 and 18.5 mm. was not correct.

It is postulated that the expression of secondary sex characteristics is under steroidal hormone control (Jost 1972). Therefore. their alteration in the presence of flavonoids could be attributed to the endocrine-modulating potential of this class of phytochemicals. In many cases. gonadal effects were correlated with the altered expression of secondary sex characteristics (Fig, 5.37). and in other cases. there was no correlation.

Endocrine-disru~tinp- Dotentid- of flavonoids

We used a battery of in vim assays for the identification of the endocrine- Table 5.14. External secondary sex characteristics in Japanese medaka. For each sex characteristic its persistence status (permanent or temporary). its dependence to hormones (male- or female-positive) and a brief description are provided.

2" Sex Characteristic Description Dependence (Persistence) body size and shape males have larger body depth 6 - positive (permanent)

nuptial coloration increasing number of leucophores in the caudal fin 8 - positive (temporary) of males during the breeding period teeth size males have a greater number of large distal teeth 8 - positive (permanent) in both jaws

-- -- - pectoral fins males develop papillary processes on the second a" - positive (temporay) ray during the breeding period ventral fins shorter in males. almost reaching the base of the 6 - positive (permanent) anal fin dorsal fin -larger in males with a distinguishable notch 6 - positive (permanent) present in the posterior end of the saw-toothed distal edge -rays are longer and thicker in males anal fin -shape and size are different between sexes 6 - positive (permanent) -males have a longer parallelogram-like shaped fin with a characteristic notch in the saw-toothed distal edge -females have a smaller right-angled triangle4 ike shape -in mature males there are papillary processes on the posterior rays Urogenital pore (UGP) -more pronounced in mature females with the pair 9 - positive (permanent) of protuberances extending from the anus to the oviduct opening giving a distinguishable spherical shape -in males it is a unilobed prominence between anus and urogenital pore (not advanced) Figure 5.37. (A) A phenotypic male Japanese medaka (0.laripes) exposed to 1 m@ of naringenin for 5 months. This specimen shows a femalelike urogenital papilla and malelike dorsal and anal fins with papillary processes. (B) The testis of the same specimen showing the development of testis-ova as indicated by the presence of four oocytes (H&E, ISOX) disrupting potential of various flavonoid compounds. The structure of flavonoid compounds appeared to play a pivotal role in their binding to both the human and rainbow trout estrogen receptors. In the YES assay. maximal estrogenic activity was displayed by the isoflavonoids. equol, genistein and daidzein. whereas substantial estrogenic activity was evident in wells treated with the flavonoids. apigenin. naringenin. kaempferol. 4.6'- hydroxyflavone. 4'-hydroxyflavone and 4'-hydroxyflavanone. These data indicate that a hydroxy substitution in the 4' position of the flavonoid nucleus is essential for binding to the estrogen receptor. Absence of a hydroxy group in this position (i.e. parent flavonoids. chrysin. galangin) or additional hydroxy substitution at the 3' position (i.e. quercetin.

luteolin. fisetin. catechin) inhibits or diminishes the binding potential of the flavonoid

molecule. The estrogenic potency of flavonoids in relation to the positive control. 1713-

estmdiol is 4 to 5 orders of magnitude lower for the isoflavonoids and 5 to 7 orders of

magnitude lower for the other flavonoids (Table 5.12). In other words. a C3-bridging of C

and B rings enhances the estrogenic potential of flavonoids. Also. in isoflavonoids the

optimal conditions for maximizing the binding affinity for binding to estrogen receptor is

a 4' and 7 hydroxy substitution pattern with the absence of the ketone group in the C ring

(i.e. equol). Presence of the ketone group in the C ring and presence of a double bond in

C2-C3 positions reduces the binding ability of the isoflavonoids, daidzein and genistein

for the hER by 1 order of magnitude. In the other flavonoids, a hydroxy substitution in the

4'5.7 positions (i.e apigenin and naringenin) or 4' and 6 positions (4.6-dihydroxyflavone)

favours the binding to hER whereas absence of hydroxy group from the A ring. or a

hydroxy substitution in the 3 position (i.e. kaempferol) diminishes the binding affinity for hER. These conclusions are in relatively good agreement with results from other studies.

For instance. Zava and Duwe (1997) also suggested the paramount importance of a 4'-

hydroxy and 7-hydroxy substitution for the estrogenicity of flavonoids. Miksisec (1 995)

reported that the flavonoids. genistein. daidzein. apigenin. naringenin. kaempferol. and 4'-

hydroxyflavone have a stimulatory effect on the ER. However. in this study it was found

that the 6'-hydroxyflavone and luteolin bind to the ER and also that kaempferol binds with

higher affinity to ER than apigenin or daidzein. In Miksisec's study the flavonoids were

tested by using a transient transfection assay in HeLa cells and having as the reporter

element the plasmid pERE-TK-CAT. Thus. some differences in the results between these

studies could be attributed to the different in virro studies used.

With the rainbow trout hepatic estrogen receptor (rtER) competition in vifroassay.

it was revealed that the 4'-hydroxy substituted tlavonoids also gave a positive response.

This was expected since it has been shown that rtER is an analogous system to hER:

however. rtER binds to its ligand with10 times less sensitivity (Le Drean rt d 1995).

Other flavonoids such as quercetin. luteolin and fisetin that were effective in binding to

rtER contained hydroxy substitutions in both 3' and 4' positions (Table 5.13). This

discrepancy could be attributed to the Fact that in the estradiol- sensitive target tissues (eg.

liver. ovary) there are other estradiol specific binding sites, such as type 11 binding sites.

that can bind not only endogenous estrogens but also non-steroidal xenoestrogens such as

flavonoids (Die1 and Michna 1998). For instance. it has been demonstrated that quercetin

and luteolin compete for ['Hlestradiol binding to both cytosol and nuclear type I1 sites in

rat uterine tissues (Markaverich a al. 1988). It is possible that in the rainbow trout liver homogenates there are both type I and U estradiol binding sites, whereas in the the YES assay it is known that there are only type I receptors. Another possible explanation could be that transactivation of rtER is induced in the presence of high androgen concentrations

(Le DrCan et 01. 1995). Nevertheless. it is worth noting that the YES assay has been used for screening chemicals of natural. municipal or industrial origin for their estrogenic potential on aquatic biota (Sumpter and Routledge. 1996: Metcalfe et ul. 2000a).

Although the YES assay is very sensitive in vitro technique for assessing estrogenicity. caution is recommended since in the present study the YES approach failed to recognize positive results (i.e. false negatives) for the flavonoids. quercetin. luteolin and fisetin.

To our knowledge. this study is the first to show that binding of ['HI 1 7bestradioI to teleost plasma SSBP was reduced by many flavonoids. SARs indicate that a 4'-hydroxy substitution in the B ring (4'-hydroxflavanone. apigenin. naringenin. kaempferol. fisetin).

a 3-hydroxy substitution (flavonol. galangin) or no hydroxy substitution (flavone.

flavanone) are some of the structural prerequisites for the displacement of the natural

endogenous ligand from the SSBP binding sites. To the contrary. flavones with other

hydroxy substitutions in the A ring (besides in the C3 site) bind to SSBP with much lower

affinity. Additional substitution of the B ring in the 3' position (quercetin and luteolin)

resulted in a very diminished binding affinity.

Also. for the first time it was demonstrated that flavonoids have the potential to

bind to the androgen receptor. Flavonol and kaempferol. both having hydroxy groups in

the C3 position. were the most active flavonoids in displacing [3~]testosteronefrom the

androgen receptor. On the other hand. the other flavonoids with 3'-hydroxy substitution (i.e. galangin, fisetin and quercetin) bound poorly to the AR. The unsubstituted parent compound flavone bound also with relatively high affinity, whereas flavanone and 4'- hydroxyflavanone showed a moderate affinity for AR.

Positive results of flavonoids in the different in vitro assays are summarized in the

Table 5.15. These flavonoids were the ones tested in the Japanese medaka reproductive development studies. Apigenin (4.5.7-trihydroxyflavone), naringenin (4.5.7- trihydroxyflavanone) and kaempferol(3.4'.5.7-tetrahydroxyflavone) bound with different affinities to the receptors in all the in vitro systems. Thus. theoretically they can interfere with the endocrine system by either binding to the estrogen or androgen receptors and

inhibiting (antagonists) or amplifying (agonists) the biological responses. or by binding to the SSBP with a consequent reduction in the availability of the endogenous hormones to

the target cells (Van der Kraak et al. 1998). In the medaka system. these

Table 5. i 5. Summary of the positive (+) or negative (-) responses by the flavonoids in the in vitro assays. Intensity of the response (+ to +++) was arbitrarily set from the Table 5.13. These 10 flavonoids were tested in the Japanese medaka reproductive development studies.

in vimassays Flavonoid hER rtER AR SSBP flavone - - ++ +++ fiavonol - . +++ +++ flavanone . - + +++ chrysin - - - + galangin - - - +++ apigenin +++ +++ + ++ naringenin +++ +++ + + kaempfero I ++ ++ +++ ++ quercetin - ++ - - catechin - - - - genistein +++ NT NT NT

NT = not tested three flavonoids greatly affected the ovarian development with a >48% incidence of atresia and >39% increase in development of the ovarian lumen. In addition, exposure of male medaka to 0.05 mgkg of kaempferol caused an increase in the rate of spermatogenesis and a decrease in the rate of oogenesis. All three flavonoids also significantly affected the expression of the secondary sex characteristics in both phenotypic male and female medaka. More specific. the urogenital pore in phenotypic males was feminized. whereas the dorsal and anal fins in phenotypic females were masculinized. In other studies, naringenin was found to be a weak estrogen that also exhibited an antiestrogenic activity (Ruh er al 1995). The parent flavonoids. flavone and tlavonol evoked reproductive impacts on only female medaka. probably by either displacing the endogenous estrogens from the SSBP or by binding to the AR and acting as agonists. The other parent flavonoid. flavanone may also affect the reproductive development in female medaka through binding to AR and SSBP. although there was a small incidence of male medaka (23%) with feminized urogenital pore. Galangin acted in the medaka assay as an antiestrogen. possibly by competing with estradiol and displacing it from the SSBP sites. Quercetin. as mentioned earlier. gave a positive response in the rtER system by probably binding to the type I1 estrogen receptor (Markaverich et d.1988:

Scambia et al. 1990). and acting as an antiestrogen as evident by the high incidence of

ovarian atresia (80%) or high incidence (67%) of male-like secondary sex characteristics

in the gonadaily phenotypic female fish. The effects of chrysin on the ovarian

development or on the altered expression of the fins could be explained by its weak

binding activity to SSBP (Table 5.15) or alternatively by its inhibitory action on aromatases, enzymes that convert estrogens to androgens (Kellis and Vickery, 1984;

Pellisero et al. 199 1). On the other hand, the effects of the same flavonoid on testicular development in 42% of exposed males or on the expression of the secondary sex characteristics in 35% males could be explained by the results of the in vitro studies.

Catechin also affected reproductive development in medaka but did not give any positive endocrine-disrupting response in all four in vifro systems. Future studies with other in vitro studies (eg. arornatase inhibition) may elucidate the mode of action of this flavonoid. Finally. the isoflavonoid. genistein bound efficiently to hER. Unfortunately. genistein was not tested with the other in vifro assays with teleost models. Adverse impact on testicular development of male medaka exposed to waterborne genistein at concentrations ranging between 1 and 1000 pg/L is an indication of its estrogenic potential. However. ovarian development and expression of secondary sex characteristics was also impacted in all genistein treatments. suggesting an additional mode of endocrine- disrupting potential. Sex-related differences regarding the uptake and metabolism of genistein exist in mammals (Coldham and Sauer. 2000) and may also apply to teleosts.

The parodoxicd action of genistein (both estrogenic and antiestrogenic effects) was also mentioned in other studies when low concentrations of genistein ( 1 nM to I pM) cause estrogenic biological effects whereas concentrations > I0 pM caused antiestrogenic effects (Zava and Duwe. 1997). Zava and Duwe ( 1997) were puzzled with the growth- stimulating effects of genistein on MCF-7 cells in vitro and the tumor growth-inhibitory potential of genistein in vivo. They concluded that although genistein is an estrogen agonist it also --cross-talks" with other mechanisms that are ER-independent. a notion that has been recognized elsewhere (Cassidy. 1998; Die1 and Michna. 1998; Klein-Hitpab et a!. 1998).

Ecotoxicological Relevancv

Findings from this study illustrate the ability of unsubstituted and hydroxy-substituted flavonoids to induce the hepatic CYP l A enzyme system. to cause embryo developmental abnormalities and impair reproductive development in fish. responses that are also observed in feral fish exposed to bleached krafi mill effluent (Sodergren. 1989: Andersson

et ul. 1988: Owens. 199 1 ;Munkittrick cr d. 1Wa. 1992b. 1992~).There is evidence that

quercetin. kaempferol. chrysin, taxifolin. apigenin. galangin. catechin. naringenin and

genistein are present in the heartwood of tree species used in pulping operations (Bate-

Smith. 1962: Sjostrom. 198 1 : Fang er ul. 1987). Also. the isoflavonoid. genistein and

unidentified putative monohydroxyflavone(s)were isolated in the final bleached krafi mill

effluent (see Chapter 6).

The unsubstituted flavonoids. flavone and chalcone were the most potent EROD-

inducing phytochemicals in the present study. However. to our knowledge. neither is

found in the heartwood or in pulp mill plume. The other hydroxyflavonoids whose

presence was documented in the heartwood of trees were not EROD inducers. The

potential of some methoxylated flavonoids such as pinocembrin and tangeretin to induce

hepatic EROD system has not been tested in our study since these were not commercially

available. Therefore. the role or the contribution of flavonoids in activating the CYP 1A

enzyme system of the feral fish downstream the pulp mill effluent should not be underestimated and fbrther research is needed.

Exposure of Japanese medaka embryos to the test flavonoids caused a multitude of developmental defects which can reduce survival of individual fish and ultimately the successfbl recruitment of fish populations (Donaldson. 1990). As discussed in more detail previously (Chapter 4). feral fish exposed to BKME experienced skeletal and craniofacial deformities. a decline in hatching success and a high mortality incidence around hatching

(Bengtsson er a/. 198% 1988: Tana and Nikunen. 1986: Hiirdig ef ul. 1988: NCASI. 1989:

Deavin. 1996). responses that were also observed in the flavonoid treatments in the present study. The aforementioned developmental defects. especially the skeletal defects. were not correlated with AOX levels in the effluent but rather their occurrence was attributed to contamination by phenolic organic compounds. a notion that was supported with our previous study (Chapter 4) and is further supported with the present study.

Therefore. flavonoid compounds if present in pulp mill effluents in critical concentrations could affect the viability of fish population at the early life history stages and may also affect successfbl recruitment.

Field studies have shown that fish exposed to pulp mill effluents experience endocrine modulation and reproductive dyshnction. Reduction of plasma steroid levels and delay of sexual maturity. lower gonadosomatic indices. reduced numbers of viable eggs and alteration of secondary sex characteristics were observed in white sucker populations exposed to BKME ( McMaster er d..1 99 1 : Munkittrick er rd.. 1992a: I 992b).

The same effects were observed in the present Japanese medaka reproductive assays either directly or indirectly. Plasma steroid levels were not measured in the present study because of the small size of medaka; however. binding of most tested flavonoids to SSBP indicate indirectly a possible reduction of circulated endogenous hormones (Van der

Kraak et al.. 1998). Once again. due to the small size of fish and to the small size of the gonadal tissue, gonadosomatic index data were not available in our study. However, in the ovaries of female medaka exposed to most flavonoids there was a significant increase in the development of the ovarian lumen (i.e. reduction in number of oocytes) which indirectly suggests a reduction in ovarian weight and consequently a lower gonadosomatic index. The other reproductive effects were histologically observed in our study. For instance. in some flavonoid treatments (i.e. kaempferol. quercetin). the proportion of pre- vitellogenic oocytes exceeded by far the proportion of vitellogenic oocytes in mature females (i.e. >20 rnm). suggesting a delay in sexual maturity. The most pronounced ovarian developmental effect observed in most of the flavonoid treatments was the reduction of viable eggs in the ovarian cavity which is possibly linked to an increased rate of ovarian atresia. Ovarian atresia was also observed in feral fish exposed to mill effluents (Janz ct d. 1997: 2000). The most pronounced impact of flavonoids in the

chronic study with Japanese rnedaka was the reduction or alteration of the phenotypic

expression of secondary sex characteristics. namely the appearance of the urogenital

region as well as the shape of the dorsal and anal fins. Female mosquitofish populations in

the vicinity of pulp mills developed male gonopodia and showed male-like courtship

activity and reproductive behaviour (Howell et al. 1 980: Bortone et ul. 1989: Cody and

Bortone. 1997: Bortone and Cody. 1999). Installation of secondary treatment and changes

in bleaching technology at Canadian pulp mills resulted in the regression of secondary sex characteristics in males to reference levels. However, 50% of the BKME-exposed female fish still have male-like secondary sex characteristics (Munkittrick et al. 1998). To summarize. medaka exposed to flavonoids during the earlier life stages show reproductive developmental defects similar to those observed in feral fish exposed to BKME. indicating that flavonoids may play a significant role in the overall toxicity of the pulp mill effluent.

Reproductive development results from the present study could be of interest in other studies dealing with municipal wastewater discharges and agricultural runoff. For instance. the isoflavonoid equol was found in hog manure at concentrations approximately 6 mg/L and application of manure to fields resulted in the runoff of this phytoestrogen to adjacent waterbodies (Bumison el al. 2000). The authors of this study admitted that the biological significance of these compounds in the aquatic biota is unknown. In addition. dietary routes of exposure to flavonoids in fish may be of concern in aquaculture. The isoflavonoid. genistein and its glucoside genistin are found at high concentrations in soya and alfalfa. plants that are used as the basis of the vegetable component in commercially available fish food (Pelissero ei ui. 1989: Pelissero and

Sumpter. L 992).

Most of the hydroxyflavonoids that are present in the heartwood of trees failed to induce the hepatic CYP 1A system: however. all these flavonoids affected embryonic and reproductive development. These results are in accordance with the speculations of other researchers that the bioactive chemicals of the pulp plume that induce EROD or impact reproductive processes in feral fish are not necessarily the same (Munkittrick et ui- 1994). However, the alternative notion that CYP 1A-inducing chemicals affect reproduction should not be rejected since in our study flavone, an EROD inducer, affected the gonadal development of medaka.

Conclusion

To summarize. the planar unsubstituted flavone and chalcone were EROD inducers in rainbow trout. whereas all the other non-planar (flavanones)and hydroxylated flavonoids (but 7-hydroxyflavone) were not inducing chemicals. Unsubstituted flavonoids were the most toxic to Japanese medaka embryos. whereas the least toxic were the polyhydroxy-substituted compounds: however. the pattern of hydroxy substitution also affects embryotoxicity. Ten flavonoids were tested for their potential to affect reproductive development in Japanese medaka. All flavonoids caused developmental effects at various intensity levels: however. most of the test flavonoids caused more pronounced effects on female ovarian development and expression of secondary sex characteristics, results that are in accordance with field studies. All flavonoids but one

(catechin) exhibited endocrine-disrupting potential since they were found to bind to ER.

AR. and SSBPs. and reproduction development was affected via these endocrine pathways. Results of the present study suggest that flavonoids. if present in the final effluent. may also play an important role in the overall toxicity observed in feral fish exposed to pulp mill effluents. CHAPTER 6

Identification of the Isoflavonoid, Genistein in Bleached Kraft Mill Effluent

Yiannis Kiparissis, Richard Hughes. Chris Metcalfe, Thomas Temes

Accepted in Environmental Science and Technology

6.1 ABSTRACT

Plants synthesize many phytochemicals. including flavonoids. which may be present in the heartwood of trees used in the pulp and paper industry. Extracts were prepared from wood pulp and mill effluent collected from a bleached kraft mill in

Ontario. Canada. and these extracts were subfractionated by LH-20 gel filtration chromatography and analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) and LC-ESI-tandem mass spectrometry (LC-ESI-MS-MS).

Initial LC-MS analysis in negative ion mode was conducted by monitoring ions corresponding to the deprotonated molecular ions of a range of flavonoid compounds.

Total ion chromatograms generated by LC-MSindicated that putative flavonoid compounds were present in both mill effluent and wood pulp. Of these compounds. the isoflavonoid. genistein was positively identified by LC-MS-MS and was quantified at a concentration of 30.0 pgkg in wood pulp. and concentrations of 13.1 pgL and 10.5 pg/L in untreated and treated (find) effluent. respectively. Genistein is a known endocrine disruptor substance: and therefore. could contribute to the alterations in sex steroid levels and reduced reproductive capacity observed in fish captured near the discharges of pulp mills. Work continues on identifying other putative flavonoids in the extracts; in particular, compounds that may be monohydroxy-flavonoids.

Plants synthesize many secondary metabolites such as terpenoids. alkaloids. lignans. tannins. coumarins. and tlavonoids For protection against pathogens and herbivores. In the heartwood and sapwo~dof trees. the presence of these phytochemicals makes the wood disease-resistant (Sjostr6m. 198 1 ). Compounds from the flavonoid group of phytochemicals have been identified in significant quantities in the heartwood of tree species that are used for manufacturing wood pulp (Bate-Smith. 1962: Sjostrom.

1981). The presence of flavonoid compounds in wood pulp may have environmental significance since several flavonoid compounds. especially the ones lac king sugar moities

(i.e. aglycones). are known to be biologically active (Harbome and Grayer. 1994). For instance. flavonoids affect reproduction in mammals by acting upon the pituitary-gonadal axis. either as competitors for steroid receptor sites (Miksisek. 1993) or by inhibiting aromatase (Kellis and Vickery. 1984).

Many sub-lethal biochemical and physiological responses have been observed in populations of fish collected near pulp mills. including induction of hepatic cytochrome

P-450-dependent monooxygenases. changes to serum steroid levels. alterations in secondary sex characteristics. and reproductive dysfunction (Andenson et ul. 1988:

SiSdergren, 1989; McMaster er a&.199 1; Munkitnick el al. 1992: Cody and Bortone. 1997). Several studies have shown that these responses may be induced in fish through exposure to phytochemicals originating from the wood pulp. including chemicals in black liquor (Martel et al. 1994), alkyl-substituted polynuclear aromatic hydrocarbons (Fragoso et a/. 1998), phytosterols (MacLatchy et al. 1997) and other hydrophilic compounds of plant origin (Kiparissis et al. 1996). It is possible that flavonoids may also contribute to the biological responses observed in fish near pulp mill discharges. Surprisingly. there

have been no previous studies directed at identifying flavonoid compounds in the effluents of pulp mills. even though these compounds have been detected in tree

heartwood (Bate-Smith. 1962: Sjostrom. 198 1 ).

Final pulp mill effluents are complex mixtures of numerous chemicals and their

composition depends on various parameters. including the pulping process. wood species.

bleaching technology and wastewater treatment (O'Connor et ul. 1992). Thus. research

directed at isolating and identifying the toxic or bioactive chemicals in these effluents is a

challenging task. The objective of this study was to analyze effluents and wood pulp from

a bleached kraft mill to determine whether flavonoid compounds are present in these

complex mixtures. Samples of wood pulp after oxygen delignification. as well as mill

effluents collected prior to treatment and afier treatment were analyzed by LC-MS and

LC-MS-MS techniques to identify and quantify flavonoid compounds.

6.3. MATEEUAL AND METHODS

6.3. I. Chemicals Flavone (2-pheny l-4H- 1- benzopyran-4-one), chrysin (5,7-dix ydroxyflavone), apigenin (3',5,7-trihydroxflavone). kaempferol(3,3',5,7-tetrahydroxyflavone), quercetin

(3,3'T4',5T7-pentahydroxyflavone),naringenin (4'5.7-trihydroxflavanone). (+)-catechin,

flavanone, flavonol(3-hydroxyflavone). 6-hydroxflavone. 7-hydroxyflavone. trans- chalcone. and galangin (33.7-trihydroxyflavone) were purchased from Sigma- Aldric h.

Inc..Toronto. ON. Canada. Genistein (43.7-trihydroxyiso flavone. 4'-hydroxyflavone and daidzein (4.7- dihydroxyisoflavone) were purchased from Apin Chemicals Ltd.

Oxfordshire. England.

6.3 2. Sample Collection.

Wood pulp and pulp mill ef'fluent were collected in August. 1998 from a pulp mill

located in Espanola in northern Ontario. Canada. Wood pulp was obtained from the

softwood pulping line immediately after oxygen delignification. The pulp mill effluent.

which originated from both the softwood and hardwood pulping lines. was collected in 4L

glass bottles from points in the mill: either: (a) prior to its biological treatment in the

secondary wastewater treatment facility (i.e. before treatment). or (b) after treatment just

prior to its discharge into receiving waters (i.e. after treatment). Upon arrival at Trent

University. the samples were immediately stored in the dark at C C. All samples were

extracted within 4-7 days of sample collection.

6.3 -3. Preparation of Extracts. Effluent samples and procedural blanks of distilled water were extracted on a column of Arnberlite XAD-7 resin (BDH.Toronto, ON. Canada). using the methods described by Metcalfe et al. (1995). The effluent sample was filtered with 0.45 pm glass fibre filters (Whatman. 934-4H).which were previously extracted in a Soxhlet apparatus with hexane. A ZL aliquot of the filtrate was acidified to pH 2 with 16 mom HC1. and then passed through a glass chromatography column (35 cm x 2 cm I.D.) packed with

XAD-7 resin. Prior to extraction. XAD resin was sequentially extracted in a Soxhlet apparatus with hexane (1.5 h). acetone (1.5 h) and methanol (2h). and stored in excess methanol. Glass wool (hexane washed) was added to the bottom of the column and resin was added to within 3 cm of the top of the column. Another glass wool plug was placed on the top of the column prior to extraction of samples.

The column was initially eluted with 100 mL of methanol. followed with 100 mL

HPLC-grade water. and then the effluent sample was passed through the column at a rate of 25 mllrninute. The column was allowed to run dry and any residual effluent was forced out by air pressure. After removal of the top plug. 100 mL of methanol was added to the column and mixed thoroughly with the resin. .After 10 minutes. the methanol was eluted from the column and passed through sodium sulfate (hexane washed) into a 500 mL boiling flask. Finally. the volume of the extract was reduced on a rotary evaporator to approximately 20 mL.

A sample of air-dried softwood pulp (100 g) was placed in 5 thimbles (solvent washed) and each subsample was extracted with 100 mL of methanol in a Soxhlet apparatus for 3 h. The methanol extracts From all flasks were pooled in a 1000 mL boiling flask and the volume was reduced in a rotary evaporator to a volume of 20 mL.

The extracts of wood pulp. effluents and procedural blanks were subfractionated by gel filtration chromatography with Sephadex LH-30 (BDH.Toronto. ON. Canada) using methods based on those described by Johnston et a1 ( 1967). LH-20 resin (40 g dry weight) was soaked overnight in excess methanol and then packed into a glass chromatography column (2.5 x 33 cm ID). The extracts were applied to the column and eluted with methanol at a rate of 4-5 Wmin. Three eluent fractions of 0-1 50 mL

(Fraction 1 ). 1 5 1-260 mL (Fraction 2) and 26 1-400 mL (Fraction 3) were collected. the volumes were reduced in a rotary evaporator to 15 mL. and stored at 4 "C until further analysis. The dihydroxy- and pentahydroxy-flavonoids. chrysin and quercetin. respectively. were used as the model standards to determine the elution patterns in the above fractionation process. Chrysin and quercetin were eluted in the 2nd and 3rd

fractions. respectively.

6.3.4. Analysis

Extracts were initially analyzed by liquid chromatography-electrospray ionization-

mass spectrometry in selected ion mode (LC-ESI-MS-SIM) using a Hewlett Packard 1 100

Model LC-MSD.The HPLC was equipped with a 3 prn Phenomenex Luna C 18 column.

150 x 4.6 mrn. There was gradient elution of analytes with a binary mobile phase

consisting of solvent A of 0.5% acetic acid in HPLC-grade water. and solvent B of HPLC-grade methanol. The gradient solvent program at a constant flow rate of 670 pUmin consisted of 100% solvent A at t=O. followed by immediate linear addition of solvent B to a concentration of 70% solvent B by t-3 min. followed by a hold time of 10 min. Solvent A was then added so that by t=l 1 min. there was 0% solvent B. The column was flushed with 100% solvent A until t=15 min. The electrospray ionization source was operated at 350°C in negative ion mode with nitrogen drying and nebulizer gas. The compounds eluting from the column were analyzed for putative flavonoids by monitoring at mass to charge ratios corresponding to the deprotonated molecular ions for

7 flavonoid compounds in an analytical standard: specifically. chrysin ( m/z 2% ). genistein. apigenin and galangin (mlz 269). kaempferol (dz285). catechin (m/z289). and quercetin (m/z 301). The Limits of Detection (LOD). determined from analysis of procedural blanks according to the method of Keith rt d.( 1983). were between 100-200

ngIrnL for the flavonoids in the standard.

To confirm identification of specific tlavonoids. extracts were Further analyzed by

liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS-

MS) using two instruments: a Perkin Elmer (PE) HPLC and PE-Sciex API 365 Tandem

Mass Spectrometer and a Micromass -'Quanro" LC-Tandem Mass Spectrometer. The PE-

Sciex system consisted of a PE 200 Series quaternary HPLC pump and autosampler and

separation was performed on a Lichrosphere 100 RP-18 endcapped (5pm) stationary

phase (Merck. Darmstadt. Germany) packed in a Merk EcoCart column ( 125 r 3 mm).

The binary gradient solvent program at a constant flow rate of 300 pUmin consisted of a

5545 solution (v/v) of 0.1% acetic acid in HPLC-grade acetonitrile. and the content of acetonitrile was increased linearly to a 20:80 solution (vh) of 0.1 % acetic acid in acetonitrile. The electrospray ionization source was operated in positive ion mode at a temperature of 47j°C and nitrogen was used as a desolvation gas. nebulizer gas and collision gas. MS-MS analysis was conducted using product ion scans and multiple reaction monitoring. The Micromass LC-MS-MS system consisted of a Waters 600 quaternary HPLC pump and Waters 71 7 autosampler and separation was performed on a

Phenornenex Luna C 18 column (5pm. 150 x 4.6 mm). Analytes were eluted with an

isocratic mobile phase consisting of 65% methanol:35% formic acid (20% solution) at a

flow rate of 0.3 mL/min. The 2-spray electrospray ionization source was operated in

negative ion mode at a temperature of 300°C and nitrogen was used as a desolvation gas

and nebulizer gas. In MS-MS experiments. UHP argon was used as the collision gas.

MS-MS analysis was conducted using product ion scans.

6.4. RESULTS AND DISCUSSION

When extracts from wood pulp and treated and untreated mill effluent were analyzed by

LC-MS (negative ion mode). several peaks were detected by monitoring ions

corresponding to the deprotonated molecular ions of the 7 flavonoid compounds in the

analytical standard (i.e. chrysin. genistein. apigenin. galangin. kaempferol. catechin. and

quercetin). Total ion chromatograms of the different subfractions of the extracts were

compared to identify any peaks that consistently appeared in all extracts. Of the major

peaks detected in all extracts. only one eluted at a retention time corresponding to a flavonoid compound in the analytical standard; genistein (Figure 6.1). Subsequent analysis of the extracts by LC-MS-MS using both the PE-Sciex and Micromass systems confirmed identification of genistein by analysis of product ion spectra (Figure 6.2).

Quantitation of genistein by LC-MSanalysis of the various subfiactions of the extracts indicated that this isoflavone compound was present in wood pulp at a concentration of 30.0 pgkg and was present in the mill effluent before treatment at 13.1 pg/L and after treatment at 10.5 pg/L (Table 6.1 ). The majority of the genistein in the extract from wood pulp was present in Fraction 2 and Fraction 3 (Table 6.1 ). However. in extracts from the effluent samples. genistein appeared in significant quantities in all 3 subfractions (Table 6.1 ). Although Sephadex LH-20 gel filtration chromatography efficiently fractionated quercetin and chrysin in preliminary trials of the method. the more complex matrix of the extracts prepared from effluent appeared to cause channeling in the

LH-20 column that interfered with the efficiency of the subfractionation process.

However. gel filtration appeared to remove some other components of the effluent that interfere with analysis of raw extracts. and so was considered a useful step for sample clean-up.

These findings indicate that the isoflavone. genistein is present in the wood pulp generated from mixed hardwood and softwood. In addition. it appears that genistein persists through the vigorous pulping and oxidation process and the secondary wastewater treatment to the point of discharge into receiving waters. Genistein has been identified in a range of plant species. including clover (Nicolier and Thompson. 1982). alfalfa Wood Pulp (Fraction 2) I 1 -I I ~“OO~OI unknown --& , +-genistein

lmmi0 a] ,,. If' .?. I .., ,.... 5 10 1s zo 2s Final Effluent (Fraction 2) I ! unknown + 4- genistein

'-I 0 . s 1 1' 5 10 15 20 : Blank (Fraction 2)

0 1 I I 5 10 15 20 25 m Figure 6. I. Total ion chromatographs (mfz = 253,269,27 1,285,289,30 I) generated by LC-ESI-MS analysis(Hewlett Packard Model 1 100) in negative ion mode of extracts prepared from LIi-20 Fraction 2 of extracts prepared from: A) wood pulp, B) mill effluent after treatment and C) a procedural blank. The compound identified as genistein was subsequently confirmed by LC-MS-MS. The retention time of the unknown compound did not correspond to any of the flavonoid compounds in the analytical standard.

Table 6.1. Concentrations of genistein by LC-MS in extracts of wood pulp and in untreated and treated mill effluent collected from a bleached krafi mill in Ontario, Canada in August. 1998. Extracts were subfractionated into Fractions 1.2 and 3 by LH-20 gel filtration chromatography and the fractions analyzed separately.

SAMPLE UNITS GENISTEM CONCENTRATION

Effluent before treatment I-WL 2.5 6 -3 4 -3 13.1 Effluent after treatment p#L 4.0 2.1 4.0 10.5

(Pettenon and Kiessling. 1984) and soybeans (Setchell et ul. 1998). This is the first study to identify genistein or any other flavonoid compound in a pulp mill effluent. This finding is of environmental significance because genistein is an endrocrine-disrupting substance that binds to the estrogen receptor and mimics the action of endogenous sex steroids (Price and Fenwick. 1985: Cassidy. 1998). Thus. genistein may be one of the phytochemicals in pulp mill effluents that is responsible for the biological effects observed in fish near pulp mills. such as alterations in serum steroid levels. effects on secondary sex characteristics and reproductive dysfunction (Andersson et al. 1988:

Siidergren. 1989: McMaster et al. 199 1 : Munkittrick et al. 1992: Cody and Bortone.

1997).

Genistein persisted through the "OD,E,D,D" bleaching process (i.e. elemental oxygen. molecular chlorine with chlorine dioxide. caustic extraction with oxygen. chlorine dioxide with neutralization) utilized by the pulp mill (Robinson et al. 1994). [n addition. this compound persisted through the wastewater treatment process. which consisted of a settling basin (primary treatment) and a 7-d aerated stabilization basin (secondary treatment). Considering that the pulp mill sampled in this study is one of the most modernized in the province of Ontario, Canada, it is possible that flavonoids are present in the receiving-water of other pulp mills.

As mentioned previously. LC-MS analysis of wood pulp and effluent samples generated total ion chromatograms in which several putative flavonoid compounds were identified. Unfortunately. none of these compounds eluted at retention times corresponding to flavonoid compounds in the analytical standard. including quercetin. kaempferol. naringenin. chrysin. apigenin. galangin and catechin. All of these flavonoids have been reported in the heartwood and bark of trees that are commonly used in the pulp and paper indusuy. For instance. quercetin and kaempferol are the most abundant aglycone flavonoids in a number of trees. such as. Douglas fir and Lark species (Bate-

Smith. 1962). The change in color of wood chips during storage at pulp mills is attributed to the formation of flavonoids from their precursors by aerial oxidation (Hillis and Swain. 1962). Catechin is the monomer flavonoid that comprises the condensed tannins in chestnut wood and in Eucalyptus species (Sjostrom. 198 1 ). Fang et (11. ( 1987) extracted 9.4 g of flavonoids from I. 1 kg of the heartwood of a pine species (Pinzrs morrisonicolu) native to Taiwan. including relatively large quantities of chrysin (7.1 g). apigenin ( 14.5 mg) and galangin ( 17.4 mg). That these compounds were not identified in this study may be explained either by their absence from the tree species used in the pulp mill (i.e. jackpine. spruce. birch. maple and poplar). or by their inability to persist through the pulping. bleaching and effluent treatment processes at the mill. In any event. further studies of effluents from other pulp mills should not be confined to analysis of only genistein, as other flavonoid compounds may occur in effluents. Methyl-substituted flavonoids have been identified as important classes of flavonoid glycosides in some plant species (Chaves et al. 1998).

Work continues to identify some of the putative flavonoid compounds in the effluent samples. One promising avenue of research involves identification of a group of' compounds originally identified by LC-MSanalysis by monitoring in negative ion mode at the mass to charge ratio that corresponds to the deprotonated molecular ion For 4'- hydroxyflavone (i.e. m/z = 237). Several poorly resolved peaks were identified in the pulp and effluent samples (Figure 6.3) and on this basis. these compounds were putatively identified as monohydroxy-flavonoids. LC-MS-MS analysis of the peak eluting at a retention time of 5.96 min. as indicated in Figure 6.3. generated the CtD mass spectra illustrated in Figure 6.4 in which there are two major product ions at m/z 1 67 and 1 69.

This spectrum did not correspond to any of the C ID spectra generated for several monohydroxy- flavonoid standards. including 4'-hydroxy. 3-hydroxy. 6-hydroxy and 7- hydroxy flavonoids. Since individual monohydroxy-flavonoid compounds appear to generate unique CID spectra (Hughes el a!. 2000). further analysis of standards may result in identification of this compound.

In conclusion. LC-MS analysis of wood pulp and effluents (before and after treatment) of a bleached kraft pulp mill in Ontario. Canada indicated that putative flavonoid compounds were present in both mill effluent and wood pulp. Of these compounds. the isoflavonoid. genistein was positively identified by LC-MS-MS and was

CID mass spectra of m/z 237

0 3 Dauahters- or 237ES 9 822 B) Treated effluent

Figure 6.4 CID mass spectra generated by LC-MS-MSanalysis (Le. Micromass Quattro) in negative ion mode of the peak monitored at mlz 237 in: A) an extract from mill effluent before treatment (Fraction 2), B) an extract from mill emuent after treatment (Fraction 2) quantified at pgkg concentrations in wood pulp, and pg/L concentrations in effluent samples. Similar concentrations of genistein in untreated and treated (final) effluent are consistent with persistence of genistein through the effluent treatment process. Genistein is a known endocrine disruptor substance; and therefore. could contribute to the alterations in sex steroid levels and reduced reproductive capacity observed in fish captured near the discharges of pulp mills. More work is required to identify whether flavonoids are present in the effluents from other pulp mills and whether these compounds are present in receiving waters near mill discharges.

Acknowledgments: This work was financially supported by grants to Metcalfe from the

Natural Sciences and Engineering Research Council (NSERC)of Canada and From the

Canadian Network of Toxicology Centres (CNTC).and a grant to Temes from the

international ofice of the Ministry of Education and Research (BMBF).Germany within

the bilateral GermanyKanada cooperation program. CHAPTER 7

Conclusions

The environmental impact of bleached haft mill emuents on fish populations attracted the attention of many researchers worldwide in the late 1980s. The adverse effects on fish were initially attributed to chlorinated organic compounds which are formed as by-products during the bleaching processes (Sodergren. 1 989). Documentation of similar adverse responses (i.e. EROD induction. reduction in plasma sex steroids. reproductive failure) on fish exposed to effluents from thennomechanical mills or from mills that have reduced or eliminated organochlorines indicated otherwise. In the heartwood of trees there are many secondary plant substances (i.e. phyto-chemicals) for protection against pathogens. parasites and herbivores. Thus. a study focusing on the potential of phytochemicals to affect the same biochemical and physiological processes in fish and their contribution to the overall toxicity of the pulp mill effluent is warranted.

The first goal of this thesis (addressed in Chapter 2) was to verify the findings of previous studies that chemicals other than organochiorines can cause induction of hepatic

EROD in fish. a sensitive biochemical response indicative of exposure to certain bioactive chemicals. To test our hypothesis. we exposed immature rainbow trout for 2 1 days to aqueous leachates From unbleached wood pulp. We found that wood puip leachates elevated EROD activities by 2.5 to 6-fold after 7 days of exposure and this activity was sustained throughout exposure. EROD activities declined rapidly during a 2 week post- exposure period. suggesting that the EROD-inducing chemicals are relatively IabiIe. This conclusion is in agreement with the results of a field study showing that EROD activity in feral fish declined during a 2 week pulp mill shutdown (Munkittrick et al. 1992~).Other results of the present study demonstrated that: a) EROD activities were higher in trout exposed to softwood leachates. and b) EROD activities declined in fish exposed to leachates from hardwood pulp that were oxidized. Thus. we inferred that tree species used in pulping and pulping processes play a role in the overall bioactive potential of the effluent.

In the first experiment we postulated that the EROD-inducing chemicals were phytochemicals, since they are present in relatively high concentrations in the heartwood and sapwood of trees. Thus. the next objective was to demonstrate that phytochemicals can induce EROD in fish (Chapter 3). One approach to testing our hypothesis was to isolate and identify the bioactive phytochemicals (an approach taken by many laboratories) from pulp mill effluents. However. this approach would have been site specific. since the composition of BKME. a complex mixture of chemicals. depends on many parameters such as wood species. pulping. bleaching and wastewater treatment processes and the receiving environment. The alternative was to select a number of phytochemicals. representative of larger families of compounds. assuming that if some of those can induce EROD in fish. then it could be quite possible that related phytochemicals present it the effluents will also induce EROD in feral fish. Flavone. uopolone. harmane.

7-methoxycournarin. anthrone. trans-stilbene. [I-sitosterol and juglone were injected intraperitoneally (i-p.) to rainbow trout. Results of this experiment showed that CYP l A I activities were elevated (2.4 to 4.7-fold) 72 h post-injection in the treatments of harmane. tropolone, flavone and 7-methoxy-coumarin (50% of the tested phytochemicals). The time-dependent experiment indicated that EROD activity declined rapidly post-exposure. a trend observed in the previous experiment (Chapter 2). and thus. justified our assumption that EROD inducing compounds present in wood pulp leachates may have been of natural origin. The test phytochemicals that caused EROD induction are relatively hydrophilic (log KO,= 0.53 to 2.9 1 ). These results are complementary to those of other studies (Bumison et al. 1996: Fragoso er at. 1998) that have shown that the EROD inducing compounds present in BKME are less hydrophillic (log KO,= 4.6 to 5.1 ).

EROD induction is a biochemical response to specific pollutants rather than a toxicological response of fish. Thus. the next question that needed to be addressed was whether the same phytochemicals can be toxic to fish. especially during the early ernbryo- larval stages of development (Chapter 4). All EROD-inducing phytochemicals (flwone. harmane. tropolone and 7-methoxycoumarin) plus juglone were acutely toxic to Japanese medaka embryos at the higher nominal concentrations tested (i.e. 1 to 10 pg/mL) when exposure started immediately after fertilization. Hatching time and success. mortality. and a multitude of developmental defects were apparent at the pg/L level (ECjO is 200 to 600 pa).Embryotoxic effects. such as skeletal and tail deformities. declines in hatching success. and a high incidence of mortality around hatching were also observed in feral fish exposed to pulp mill effluents. Finally. we inferred that phytochemicals. by affecting early embryo-larval development. could ultimately impair success~lrecruitment in fish.

Next. we concentrated our efforts on assessing the toxic potential of flavonoids. a group of structurally related phytochemicals. since a) they interfere with numerous biological processes. and b) are present in the heartwood of tree species used in pulping

(Chapter 5). In general, the unsubstituted flavonoids are the most toxic, and toxicity decreased with the addition of hydroxyl groups. as shown with the Japanese medaka embryotoxicity assay. However. all test flavonoids (i.e. flavone. flavonol. flavanone. chrysin. galangin. apigenin, naringenin. genistein. kaempferol. quercetin and catechin) affected gonadal development in exposed Japanese medaka as evident by: a) testis-ova development. b) a delay of gonadal maturation. c) a high incidence of ovarian atresia d) a decreased number of germ cells. and e) a high proportion of phenotypic females (gonadal sex) with reduced or altered secondary sex characteristics. A batter): of in vitro assays showed that these reproductive development effects could possibly be mediated via the binding of tlavonoids to ER and AR receptors. or alternatively via the binding to SSBP.

Similar reproductive development effects were observed in published field studies. stressing the importance of flavonoids and other phytochemicals in the overall toxicity shown by feral fish exposed to pulp mill effluents.

Prior to this study. flavonoid compounds had not been found in BKME. Thus. the last objective of this study was to analyze effluents from a bleached krafi mil1 to determine whether tlavonoids are present in these complex mixtures (Chapter 6). LC-MS analysis indicated that putative flavonoid compounds are present in the effluent. LC-MS-

MS analysis identified the isoflavonoid. genistein as one of these flavonoid compounds which was quantified at a concentration of 13.1 pg/L and 10.5 pg/L in untreated and treated (final) effluent. respectively. It is worth noting. that genistein at similar concentrations affected medaka gonadal development and the expression of the secondary sex characteristics. Whether flavonoid compounds are present in effluents From other pulp mills, has to be determined in future studies.

S~dergrensaid '-In 1991. I think we were a little bit too optimistic and assumed that we were at last going to find Pandora's box around the corner and finally identify the culprit. or culprits. behind the observed environmental disturbances. Today it is safe to say that there was no box -- instead we found several new comers!" ( 1996. pg. xv). We believe that the present study found another "comer" in this research area since for the first time flavonoids were identified in the final effluent.

Beside the specific objectives of the present study. our results showed the importance of Japanese medaka reproductive studies in conjunction with in virro studies as sensitive bioassay for endocrine modulation in fish species. Also. the presence of

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Treatment Exposure TL Wt Liver LSI CF EROD time (d) (mm) (g) (9 (pmole!rng/min) SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO SBO Sf30 SBO SBO SBO SBO

'LSI = [(Liver) I (Wt)] * 100

?CF= [(wt)/ (n);j* 1 ooooo Treatment Exposure Liver CF EROD time (d) (9) (prnole/mg/min)

SBO SBO

SAO 0 SAO 0 SAO 0 SAO 0 SAO 7 SAO 7 SA0 7 SAO 7 SAO 7 SAO I4 SAO 14 SAO 14 SAO 14 SAO 14 SAO 2 1 SAO tt SA0 21 SA0 2 1 SA0 2 1 SAO 3PE SAO 3PE SAO 3PE SA0 3PE SAO 7PE SAO 7PE SAO 7PE SAO 7PE SAO 14PE SAO I SPE SA0 13PE SAO i4PE

HBO HBO HBO HBO HBO HBO HBO HBO HBO HBO HBO HBO HBO HBO HBO Treatment Exposure TL Wt Liver LSI CF EROD time (d) (m) (9) (9) (prnole/mp/min)

HBO 2 1 HBO 2 1 HBO 21 HBO 21 HBO 3PE HBO 3 PE HBO 3PE HBO 3PE HBO 7PE HBO 7PE HBO 7PE HBO 7PE HBO 14PE HBO I4PE HBO I4PE HBO I4PE

HA0 0 HA0 0 HA0 0 HA0 0 HA0 7 HA0 7 HA0 7 HA0 7 HA0 7 HA0 14 HA0 14 HA0 I4 HA0 14 HA0 I4 HA0 2 1 HA0 11 HA0 2 1 HA0 2t HA0 11 HA0 3PE HA0 3PE HA0 3PE HA0 3PE HA0 7PE HA0 7PE HA0 7PE HA0 7PE HA0 ISPE HA0 ISPE HA0 14PE HA0 IJPE Appendix 2. Hepatic EROD activity (expressed as pmole activityhg microsomal proteidmin) in rainbow trout intraperiteoneally (i.p.) injected with various phytochemicals with a single dose of 10 mgkg (data used in Chapter 3). Total length (TL). body and liver weights and the calculated LSI and CF are also presented.

Phytochemical Exposure TL Wt Liver LS 1 CF EROD time (d) (mm) (g) (g) (pmo Vmghin) l3-naphthoflavone 3

Control (DMSO)

Harmane

Tropolone

Control (DMSO/PBS)

sitosteroU campestero 1

stilbene

j uglone

flavone Phytochemical Exposure Liver LSI CF EROD time (d) (g) (pmol/mg/min)

0.9 t .O 1.3 1.1 1. 1 1 -0 1.2 1.1

anthrone 3 1.1 1.1 3 1.3 1.3 3 0.7 1.1 3 1 .2 I .2 3 1.1 1 .o

hannane 3 I .O 0.9 3 1 .Z 0.9 3 0.9 0.9 3 0 -9 1.1 7 1 .o 0.9 7 0.3 0.9 7 1.6 I .3 7 0.9 0.9 14 0.9 1.2 I4 1.1 1 .o 14 1 .o 0.9 1 J 1 .o 1.1 2 1 0.9 0.9 2 1 0.9 0.8 2 1 I .8 I .4

tropolone 3 0.5 1.3 3 0.6 I .O 3 0.5 1.1 3 0.5 1.3 7 0.5 l .Z 7 0.7 1.5 7 0.5 1.3 7 0.8 1.4 14 0.4 0.8 14 0.5 1 .o 14 0.6 1.1 I4 0.6 1.1 21 0.5 1.0 2 1 0.5 0.8 21 0 -4 0.8

0.5 1.3 0.7 I-4 0.7 1.4 1 .Z 2.5 1.3 2.1 I -2 1.7 1.0 1.8 Phytochemical Exposure TL Liver LSI CF EROD tirne(d) (mm) (g) (pmoVmp/min)

Control 3 3 3 2 1 2 1 2 1 Appendix 3. Dose-dependent hepatic ethoxyresorufin-0-deethylaseactivity (expressed as pmoielmg proteidmin) in rainbow trout exposed to various flavonoids (data used in Chapter 5). Total length (cm), body and liver weights (g) and the calculated liver somatic index (LSI) and condition factor (CF) From all individual fish are also presented.

Flavonoid Dose Liver EROD (mp/kg) (9) (pmollrng/min) Flavone 0 -.-7 7 1.7 0 1.6 1.0 0.1 1.4 1.7 0.1 2.4 3.5 0. I 1.8 4.3 0. I 2.6 4.3 0.1 2.0 3.8 1 1.7 2.5 i 2.0 4.2 I 1.2 6.3 1 1.9 3.5 10 2.3 3. I 10 1.8 3.3 10 1.9 7.2 10 LO 4.2 t 0 t .7 4 -4 100 1.7 4.0 100 1.8 4-6 100 1.9 8.5 1 00 I .4 6.4 100 1.7 7.4

Flavonol 0 1 -2 1.5 0 1.7 I .3 0 I. 1 2.0 0 1.6 1 -2 0.1 1.7 1.1 0.1 I .6 0.9 0.1 1 .-I 1 .Z 0.1 1.6 1.4 0.1 1.4 3 -6 1 1.3 4.7 I 1.9 2.9 1 1.7 3.5 1 2.0 1.5 10 1.3 1.1 7 7 10 1.3 -*- 10 1.8 2.3 i 0 1.7 I *-I 100 1.3 3 -4 too 23 1.7 100 1 -6 2.0 1 00 1*8 3 2 FIavonoid Dose Liver EROD (mp/kg) (g) (prnoVmg/min)

Flavanone 0 2.1 0 I .9 0 2.1 0 1.9 0.1 1.6 0.1 LO 0.1 2.1 0.1 1.9 0.1 1.4 1 --7 7 I I .9 I 1.6 1 1.9 1 1.6 10 2.0 I0 1.6 10 3.1 10 1 .5 10 1.9 1 00 1.8 100 1.9 100 2.0 100 I .8 100 I -6

Chalcone 0 1.3 0 1.7 0 2.1 0.1 1.5 0. i 1.3 0.1 1.7 0.1 2.0 I 1.5 1 1.5 I 1.o I I. I I 1.4 I0 1.6 10 2.1 10 l .5 10 1.7 10 1.6 100 1.8 100 ---7 7 100 -7 .-3 100 1.8

2.5 2.0 Flavonoid Dose Liver LSI EROD (mflg) (g) (pmol/mg/min)

1.5 0.9 3 -0 1.6 1 -0 1 -9 2.1 1.0 0.8 2.1 I .O 2.8 1.9 1.1 2.9 2.3 1 -0 I .O 2.5 I -4 I .J 1.9 1 -0 1.9 1.1 0.8 2.9 2.0 1 .o 0.8 22 I .O 0.7 1.8 1 -2 1.6 0.9 I .7 1.3 0.8 1.7 2.0 0.9 5 .o 2.0 0 -9 -.--7 7 2.0 I .O 2.6 2.1 0.9 3 -0 2.1 1.1 2.4 1.9 I .O 3.8 2.2 I -0 3.9 2.8 1.1 0.8

7-OH-fl avone 0 2.5 1.2 1.2 0 2.0 1.1 0.5 0 I .s 0.9 3 .o 0 1.6 1 .o 1.9 0. I 2.5 1.1 2.9 0.1 1.3 0.8 2.7 0.1 1.8 1 .o 7 7 0.1 2.4 I .o -*- 0.1 2.4 1.1 3.1 I [ .8 0.9 5.2 1 2.1 0.9 1.1 I -.-3 7 1.3 3 3 I 1.6 I. 1 I -.-7 7 1 .o 6.7 10 1.6 0.9 3 -7 I0 1.7 0.9 3.6 10 2.0 1 .o 3.6 10 1.8 1.1 3.8 10 1.8 1.1 4.4 too I .8 1.0 3.7 100 1.7 1.1 3.5 1 00 2.5 I .3 2.0 100 1.7 1 .o 1 -6

Chrysin 0 1.8 0 -9 3 -3 0 1.4 I -0 1.3 0 1.8 0.9 2.0 Flavonoid Dose Liver LSI EROD (mflg) (9) (pmol/mg/min)

I .3 0.8 1.4 t -9 0.9 3 -4 1.4 0.9 1 -9 1.5 0.9 0.6 1 -7 I -0 3.8 1.5 1 -0 2.2 -.-7 7 1 .o 1.8 2.0 1 .o 0.9 I .4 0.8 2.4 3.1 1 -2 2.3 1.7 0.9 2.9 -7 .-7 1.2 3.3 1.6 0.9 2.4 2.4 1.1 3 .O i .9 1 .O 5.3 1.6 0.9 8 .o I .3 0.8 5 -6 2.5 1.2 2.4 1.7 0.9 0.7 kaempferol 0 1.2 0.7 2.3 0 1.2 0.9 0.9 0 1.8 1.1 1.1 0.1 1 .Z 0.9 I .O 0.1 l .5 1 .o 1.3 0.1 1.7 1.1 1.5 0.1 2.1 i .Z 3 .s 0.1 2.1 1 .o 2.8 I 1.6 0.9 1.5 I 2.0 1.0 1 -2 1 1.8 l .2 0.3 1 1.7 1 -0 I .4 I 1.2 0.9 0.7 10 1.5 0.8 1 - 1 10 2.0 0.8 t .3 10 1 -6 1 .o 3.5 10 2.0 1.2 3 .o 10 1.6 1.0 3.6

quercetin 0 1 -2 0.8 2.3 0 1.2 1 .o 3.3 0 1.7 1.2 0.9 0 1.8 1 .o 4.3 0.1 1.6 1 .o 8.6 0.1 1.7 1.2 2.9 0.1 1 -9 1 -2 3 -3 0.1 1.8 1.1 1 3 0.1 2.1 1.1 4-0 I 2.2 f -0 4.0 1 I -6 1.0 2-6 Flavonoid Dose CF EROD (wk) (prnoUmp/min)

O-NF 0 0 0 0 0.1 0.1 0.1 0. I I 1 I 1 I0 10 10 10 Appendix 4. Summary of results from the Japanese medaka embryotoxicity assay with flavonoids (data used in Chapter 5). Dead, unhatched, partially hatched (P-Hatched) and hatched embryos fiom all treatments are presented as percent (%) of all viable embryos at day 1 of exposure. Dead, non-viable and viable larvae are expressed as percent of the successfblly hatched embryos.

Observations Embryos (%) Larvae (96) Chemical Conc'n (nM) Dead Unhatched P. Hatched Hatched Dead Non-viable Viable MF 75 25 5 0.5 0.05 Cntl

Flavone 75 25 5 0.5 Cntl

Chalcone 75 25 5 0.5 Cntl

Flavanone 75 25 5 0.5 Cntl

Flavonol 75 25 5 0.5 Cntl

75 25 5 0.5 Cntl Observations Ernbrvos (%) Larvae (%) Chemical ConcTn(nM) Dead Unhatched P. Hatched Hatched Dead Non-viable Viable

7-0H 75 25 5 0.5 Cntl

Chrysin 75 25 5 0.5 CntI

Galangin 75 25 5 0.5 Cnt l

Apigenin 75 25 5 0.5 Cntl

Kaempfetol 75 25 5 0.5 Cntl

Naringenin 75 25 5 0.5 Cntl

Catechin 75 25 5 0.5 Cnti Observations Embwos (94) Larvae (%) Chemical Conc'n (nM) Dead Unhatched P. Hatched Hatched Dead Non-viabIe Viable

Quercetin 75 16 16 - 68 10 11 47 25 5 - - 95 10 - 85 5 15 - - 85 45 5 35 0.5 6 - - 94 6 12 76 Cntl - 13 - 87 - 3 84

Taxifolin 75 22 6 5 67 17 I I 39 25 15 - - 85 10 5 70 5 5 - - 95 5 5 85 0.5 5 - - 95 10 5 80 Cntl - 3 - 97 3 8 86 Appendix 5. HBE and PAS staining histological techniques (used in Chapter 5)

H&E staining PAS staininp;

- xylene 10 min xy lene 10 min - xylene 10 min xy lene 10 min - 100% ethanol 10 rnin 100% ethanol 10 min - 100% ethanol 10 min 100% ethanol 10 rnin - 90% ethanol 5 min 90% ethanol 3 min - 70% ethanol 5 min 70% ethanol 5 min - distilled water 3 rnin distilled water 3 rnin - hematoxylin 4 min periodic acid (5%) 5 min - running tap water 20 rnin distilled water rinsing - eosin 1 min Schiff s reagent 15 min - 70% ethanol I0 sec wingtap water 10 min - 95% ethanol 30 sec orange G ( 1%) 20 sec -100% ethanol 2 rnin phosphotungstic acid (5%) 20 sec - 100% ethanol 2 rnin running tap water 20 sec - xylene 3 min light green ( 1 %) 20 sec - xylene 3 min acetic acid ( 1%) ZOsec 70% ethanol 30 sec 95% ethanol 1 rnin 100% ethanol 2 min 100% ethanol 2 rnin xy lene 3 rnin xy lene 3 rnin Appendix 6. Morphometric measurements of Japanese medaka (Oryzias Iutipes) exposed to various concentrations of chrysin and quercetin. Total lenght (TL) and body weight (Wt) are expressed as mm and gpresented as mean k st. dev. The condition factor (C.F.)has been calculated with the formula: (Wflp)*100.000. Measurements with different small letters (a and b) are statistically different at P<0.05)

Chrysin 0.1 28 23 .B1 .9b 0.10Ok0.024~ 0.7%0.08b 0.5 27 22.3* I .7b 0.085*0.02 I 0.75i0.07" 1 .O 26 2331.3b 0.096k0.0 1 8b 0.76i0.07b

Quercetin 0.0 1 29 22,lrtl.T 0.077i0.018" 0.7 1 k0.04" 0.05 26 2 1.% 1.5" 0.079=tO.O1 8" 0.74i0.05" 0.1 28 2 1.W2.8" 0.08 1 k0.028" 0.74iO. 1 0" Appendix 7: Morphometric measurements of Japanese medaka (Oryzias latipes) exposed to various flavonoids for a period of 5 months (data used in Chapter 5). Total length (mm). body weight (g) and condition factor in all treatments are expressed as me-standard error (Abbreviation in headings: TL = total lenght; Wt =weight :CF=condition factor).

Treatment Concentration Time TL Wt CF (mEm (mo) (mm) (g)

Flavone 0.0 1

Flavonol 0.1

Flavanone 0.1

Quercetin 0.1

Chrysin & 0.5 Quercetin 0.1

Kaempferol 0.05 Treatment Concentration Time (ma-) (mo)

Kaempfero 1 0.5

Apigenin 0.05

0.5

Galangin 0.05

Catechin 0.1

1 Treatment Concentration Time (mgn) (mo) Appendix 8. Determination of the gonadal phenotypic sex in Japanese medaka (0.latipes) exposed to chrysin, quercetin (flavonoids), testosterone and procedural control from hatching to 3 months post-hatching (data used in Chapter 5). Medaka with feminized urogenital pore or with papillary processes in the anal fin are presented. Total length (TL). body weight (Wt) and condition factor (CF)are also noted.

Sample TL (rnrn) C.F. Gonadal Sex UGP Anal fin

Control treatment

CCQ-1 0.74 CCQ-2 0.76 CCQ-3 0.72 CCQ4 0.72 CCQd 0.67 CCQ-6 0.70 CCQ-7 0.72 CCQ-8 0.75 CCQ-9 o.n CCQ-I 0 0.75 feminized CCQ-11 0.74 CCQ-12 0.72 CCQ-13 0.76 feminized CCQ-14 0.75 CCQ- 1 5 0.74 CCQ-16 0.64 CCQ-17 0.64 CCQ-18 0.69 CCQ-19 0.76 CCQ-20 0.72 CCQ-21 0.76 fern wed CCQ-22 0.73 feminized CCQ-23 0.77 feminized CCQ-24 0.74 CCQ-25 0.72

Chrysin treatment (0.1 mg/L)

CHR-L-1 0.80 C H R-L-2 0.88 CHR-L-3 0.82 CHR-L-4 0.72 CHR-L-5 0.84 CHR-L-6 0.7 1 CHR-L-7 0.8 1 CHR-L-8 0.82 C H R-L-9 0.83 CHR-L- I0 Sample C.F. Gonadal Sex UGP Anal fin

CHR-L-I I 0.73, F CHR-L- 12 0.9 1 M CHR-L- 13 0.77 M feminized CHR-L- 14 0.55 M papilla CHR-L- 15 0.84 F CHR-L- 16 0.83 M CHR-Ll7 0.78 F feminized CHR-L- I8 0.7 I CHR-L- I9 0.78 testis-ova CHR-L-20 0.83 M CHR-L-2 I 0.77 M C HR-L-22 0.89 F feminized CHR-L-23 0.73, F feminized CHR-L-24 0.78 M CHR-L-25 0.93 M CHR-L-26 0.74 M CHR-L-27 0.83 F feminized CHR-L-28 0.78 F feminized

Chrysin treatment (0.5 m@)

CHR-M- I 0.85 M CWR-M-2 0.83 F feminized C HR-M-3 0.86 F CHR-M-4 0.65 M CHR-M-5 0.76 F feminized C HR-M-6 0.65 F feminized CHR-M-7 0.74 M CHR-M-8 0.7 1 F CHR-M-9 0.76 F feminized CHR-M- I0 0.76 M feminized CHR-M-I I 0.68 M CHR-M- I2 0.77 M CHR-M- l3 0.73 M CHR-M- I$ 0.74 M CHR-M- I 5 0.64 F CHR-M- 16 0.78 M CHR-M- 17 0.79 F CHR-M- I8 0.78 M CHR-M- I9 0.80 M CHR-M-20 0.82 M CHR-M-2 I 0.6 1 F CHR-M-22 0.76 F feminized CHR-M-23 0.79 M CH R-M-24 0.73 F feminized CHR-M-25 0.74 M papilla Sample TL (mm) Wt (g) Gonadal Sex UGP Anal fin

F feminized F feminized

Chrysin treatment ( l mg/L)

CHR-H- I F feminized CHR-H-2 F feminized CHR-H-3 F CHR-H-4 F CHR-H-5 ? CHR-H-6 F feminized CHR-H-7 M CHR-H-8 F CHR-H-9 M CHR-H- 10 M CHR-H-11 ? CHR-H-I2 F fern inized CHR-H- I3 M CHR-H-I 4 F CHR-H- I5 F CHR-H- I6 F CHR-H-17 M CHR-H- 1 8 M CHR-H- 19 F CHR-H-20 M CHR-H-2 I F CHR-H-22 F fern inized CHR-H-23 testis-ova papilla CHR-H-24 F CHR-H-25 F C H R-H -26 M

Quercetin treatment (0.01 mg/L.)

feminized- -- feminized feminized feminized feminued

papilla Sample TL (mm) C.F. Gonadal Sex UGP Anal fin

Q-L-14 22.0 M Q-L-15 20.0 F feminized Q-L-16 24.0 M Q-L-17 22.5 M Q-L-18 22.5 M Q-L-19 21 .o M Q-L-20 24.0 M Q-1-21 23.5 F a-L-22 23.0 F feminized Q-L-23 26.0 F feminized Q-L-24 24.0 F feminized Q-L-25 17.0 F Q-L-26 20.5 M Q-L-27 21.5 M Q-L-28 23.0 F Q-L-29 20.5 F feminued

Quercetin treatment (0.05 rng/L)

femmized feminized

feminized

feminized fernin~ed femtnued

papiila papilla Sample TL (mm) Wt (g) C.F. Gonadal Sex UGP Anal fin

Quercetin treatment (0.1 mgL)

papilla

papilla feminlted

femin~zed feminized papilla

fern mued

feminized feminued

Testosterone treatment ( 18 pg/L)

papilla feminized papilla feminized papilla papilla papilla papilla feminized papilla feminized papilla feminized papilla fem~nlzed papilla papilla Appendix 9: Determination of the gonadal phenotypic sex and expression of secondary sex characteristics in Japanese medaka (Oryzias latipes) exposed to various treatments of flavonoids from hatching to 2.3.4 and 5 months post-hatching (data used in Chapter 5). Total lenght (mm), body weight (g) and condition factor From all individual medaka are also presented (Abbreviations in headin~s:TL = total length; Wt = wet weight; C.F. = condition factor: UGP = urogenital pore or urogenital papillu; D F = dorsal fin; d F = unal fin Abbreviations under "Gonadal Sex": M = male: F =-female.Abbreviations under "20 Sex Characters": U = unidentified: ki = male; F =/emale: ib1M = definite male; FF = definite female; ppl = papillary processes; hi?or F? = identification with uncertainly: A = ubsent: D = deformed: F-M =female probably changing lo male; M-F = male probably changing to female; M(E) = estrogenized male; F(A) = androgenizedfemale. Abbreviution and Terms under "Observations": spg = only sperrnatogonia are present, immatzire: spc = spermatocytes present. immature; spd = spermutids, present, immture; ull stages = spermatozoa present. immature: all stages, udianced = active spermutogenesis; advunced = advanced sperma f ogenesis; TO = testis-ovu;jib = fibrosis; pre Vg = oocytes ut the previtellogenic stage: early Vg = onset of vitellogenesis. oocyles in IV andjiw in Vstuges of development: C'g = viteflogenic stage, oocytes at V und M stuges of developmenr; lute Vg = oocytes at VII and lip stage of development; PGCs = increased number of primordial germ cells: atresia = increased number oj'atretic oocytes; OL = increase in ovarian lumen). Samples with strike outs were not processed.

Gonadal 2" Sex Characters Sample TL(mm)Wt(g) C.F. Sex UGP DF AF Observations

Control - Month 2 Gonadal 2" Sex Characters Sample TL (mm)Wt (g) C.F. Sex UGP DF AF Observations

Control - Month 3

spd

all stages

prevg

spd

preVg: PGCs

preVg

preVg: PGCs preVg; PGCs

Control - Month 4

M all stages. advanced F early Vg M all stages M preVg 6 atresia M all stages, advanced M at1 stages MM,ppl advanced M all stages, advanced M all stages. advanced Gonadal 2" Sex Characters SampIe TL (mrn) Wt (g) C.F. Sex UGP DF AF Observations

all stages all stages early Vg: PGCs Vg; some atresia all stages. advanced all stages all stages early Vg

Control - Month 5

CFIS- I MM advanced CFIS-2 MM.ppl advanced CFIS-3 FF late Vg CFIS4 F Vg €m+ M CF15-6 MM advanced CF15-7 MM.ppl advanced CFIS-8 F? Vg CF15-9 M advanced CF15-I0 M advanced CFI5-I I M Vg CFIS- I2 MM,ppl advanced CFIS- 13 MM advanced CFIS- 14 M? advanced CFIS- 15 F late Vg CFIS- 1 6 F Vg CF15- 1 7 F? Vg CFIS- 18 M advanced CFIS- 19 F late Vg Gonadal 2" Sex Characters Sample TL (mrn) Wt (g) C.F. Sex UGP DF AF Observations

Flavone - Month 2 - Concentration 0.01 mg/L

=PC preVg: PGCs

preVg; PGCs

Flavone - Month 3 - Concentration 0.0 1 mg/L

M preVg; PGCs: atresia M prrVg: PGCs: atresia. fib M all stages u SPg u SPS F all stages

Flavone - Month 4 - Concentration 0.0 I mg/L

FLAJ- I MM MM preVg; PGCs: OL FLAJ-2 MM MM all stages FLA4-3 MM MM TO FLA4-4 M M preVg: PGCs: atresia FL AII-5 MM MM TO FLA4-6 F-M? F-M? all stages FLA4-7 M M Vg; some atresia FM4-8 M M FLA4-9 M M preVg; PGCs; atresia

Flavone - Month 5 -Concentration 0.0 I mg/L

FLAS-I Z 1.0 0.082 0.89 F F M? Vg: PGCs; atresia FLAS-2 21.0 0.072 0.78 F F M? Vg FLAS-3 22.0 0,077 0.72 M MM MM advanced FLA5-4 23.0 0.09 1 0.75 M M MM advanced - ..- Gonadal 2" Sex Characters Sample TL (mm)Wt (a) C.F. Sex UGP DF AF Observations

MM MM advanced M M all stages M M all stages M? F? Vg: PGCs, atresia MM MM,ppl advanced MM TO: advanced

Flavonol- Month 2 - Concentration 0.1 mg/L

preVg; PGCs: "spc-like" cells

preVg; PGCs: "spc-like" cells

Flavonol- Month 3 - Concentration 0.1 mg/L

U U U preVg: atresia U preVg: atresia M prevg U preVg; PGCs; fib

Flavonol- Month 4 - Concentration 0.1 mg/L

preVg: atresia early Vg; OL early Vg; atresia earIy Vg; PGCs: atresia early Vg: atresia all stages preVg: atresia all stages. advanced preVg; PGCs Gonadal 2" Sex Characters Sample TL (mm)Wt (g) C.F. Sex UGP DF AF Observations

Flavonol - Month 5 - Concentration 0.1 mg/L

FN LS- I F M M Vg: PGCs: "spc-like" cells FNL5-2 MM M MM allstages, FNLS-3 F( A)? F M Vg; atresia: OL FNLS-4 FF F? F Vg: stroma; OL FNL5-5 MM M? M all stages: a lone oocyte; TO FN L5-6 M( E)? M F Vg; PGCs: atresia: OL FN LS-7 M MM MM advanced FNL5-8 F M M Vg: atresia: OL FNL5-9 M MM MM TO: a11 stages FNLS- I0 MM MM MM advanced

Flavanone - Month 2 - Concentration 0.1 mg/L

preVg PGCs: "spc-like" cells preVg: PGCs preVg: PGCs: "spc-like" cells preVg: PGCs: "spc-like" cells

preVg; PGCs: "spc-like" cells SPg TO: preVg; PGCs: spc-like cells preVg: PGCs: atresia preVg: PGCs

Flavanone - Month 3 - Concentration 0.1 mg/L

FVN3- I 13.5 0.017 0.69 M F? M? M? spg FW3-2 13.0 0.013 0.59 M F F F SPg FVN3-3 16.0 0.025 0.61 F F? F F preVg FVN3-4 17.0 0.028 0.57 M U M M SPg FVN3-5 18.0 0.041 0.70 M M M M all stages FVN3-6 13.0 0.013 0.64 M U U u SPg Flavanone - Month 4 - Concentration 0.1 mg/L Gonadal 2" Sex Characters Sample TL (mrn) Wt (g) C.F. Sex UGP DF AF Observations

preVg: PGCs preVg; PGCs; atresia spd preVg; PGCs: atresia P~W prevg preVg preVg: PGCs; "spc-like" cells prevg

Flavanone - Month 5 - Concentration 0.1 mg/L

M preVg; PGCs; atresia; OL M advanced M all stages: UGP with Z pores (F) M all stages M Vg; atresia M all stages. advanced U early Vg: PGCs MM advanced M all stages. advanced M all stages. advanced M? preVg; atresia MM all stages. advanced F TO: all stages F preVg MM TO: all stages. advanced M ppl all stages. advanced MM advanced

Chrysin - Month 2 - Concentration 0.1 mg/L

preVg; PGCs Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

Chrysin - Month 3 - Concentration 0.1 mg/L

preVg; PGCs

all stages. advanced

preVg: PGCs: "spc-like" cells

preVg; PGCs: atresia

preVg

Chrysin - Month 4 - Concentration 0.1 mg/L

F preVg; PGCs: OL M-F TO: one oocyte present M all stages, abnormal M preVg: PGCs: "spc-like" celb M all stages U spd M-F preVg; PGCs: atresia M all stages: lobular IT separation M early Vg M all stages, abnormal U preVg; PGCs Gonadal 2" Sex Characters Sample TL (mm)Wt (g) C.F. Sex UGP DF AF Observations

CHR4-12 all stages: 3 pores in UGP CHRJ- 13 U u SPg CHRrt-14 M M Vg; PGCs CHRLC- 15 U U preVg: PGCs; "spc-like" cells CHR4- I6 all stages CHRJ- I7 M M all stages. abnormal CHR4-I 8 M M all stages, advanced CHRJ-19 M M all stages

Chrysin - Month 5 - Concentration 0.1 mg/L

CHRS-I MM MM.ppl advanced CHRS-2 M? M Vg: PGCs; sperrn-like cells: OL CHRS-3 FF FF Vg; atresia: OL CHRS-4 MM MM advanced CHRS-5 F F Vg CHRS-6 miss. miss. all stages, advanced CHRS-7 F? D Vg CHR3-8 F FF Vg CHRS-9 F F Vg; atresia: OL CHRS-I0 MM MM.ppl advanced CHRS-I I M? M? Vg; some atresia CHR5- 12 MM F advanced CHR5- 13 FF FF all stages. abnormaI CHRS-I4 M-F? M all stages. advanced CHRS-I S F? M? Vg CHRS-16 F F? preVg CHRS-17 MM M all stages. advanced CHRS- I8 M-F? M? Vg; some atresia CHRS- I9 M-F? M al t stages. advanced

Quercetin - Month 2 - Concentration 0.1 mg/L

spd preVg; PGCs: atresia

preVg: PGCs; "spc-like" cells Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

Quercetin - Month 3 - Concentration 0.1 mg/L

F MU? M? F u U U U M U M U M U M U U U U M

Quercetin - Month 4 - Concentration 0.1 mg/L

QUES- I 18.0 0.034 0.58 M M M all stages QUES-2 18.0 0.036 0.62 F M M preVg: PGCs; atresia QUES-3 19.0 0.057 0.83 F F M preVg; PGCs: atresia QUE4-4 17.0 0.039 0.79 M M M TO: all stages Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

QUE4-5 U preVg: PGCs; atresia; OL QU E4-6 U ail stages QU E3-7 F SPg QUE4-8 F pteVg; PGCs: auesia QUEJ-9 U sPg QUEJ- I0 MM preVg; PGCs; increased atresia QUEJ-I I M all stages QUEJ-I2 M all stages QUEJ- I3 M all stages QUEJ-14 M ail stages QUEJ- I5 F preVg; PGCs: atresia QUE4- I6 U preVg; atresia QUE4- I7 F preVg; atresia: OL QUE4- I8 M all stages QUEJ- I9 M preVg: PGCs: atresia: OL QUEJ-20 F preVg

Quercetin - Month 5 - Concentration 0.1 mg/L

QUES-I M M advanced QUES-2 M M all stages QUES-3 F-M M Vg stroma QUES-4 MM MM advanced QUES-5 M M advanced QUES-6 M M spd QUES-7 F F spd QU ES-8 F F Vg QUE5-9 M M? all stages,advanced QUE5-I0 F-M F preVg; atresia QUES-I I F-M M advanced QUES- 12 F F? allstages QUES-13 M M Vg; PGCs; some atresia QUES-I 3 F-M M V_r: stroma: atresia QUE5-I 5 F F Vg; stroma: atresia QUES- 16 U M eariy Vg: PGCs; OL: atresia QUES-17 F F vg QUES- I8 F F all stages. advanced QUES- 19 M M preVg; atresia QUES-20 F-M F-M all stages Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

M M M all stages, TO M M M? all stages, advanced F MM M Vg, atresia; OL MM MM MM advanced FF F-M M Vg: stroma: atresia M MM MM advanced FF F F early Vg; PGCs M M U all stages M M M early Vg: atresia FF M M Vg; OL: atresia; ectopic oocytes MM MM MM.ppl advanced FF FF FF Vg: stroma; atresia

Chrysin and Quercetin - Month 2 - Concentration 0.1 and 0.05 mg/L

CHRQUE2-1 TO: one ectopic oocyte CHRQUEZ-2 SPg. CHRQUEZ-3 sPg CHRQUE2-4 preVg: PGCs: atresia: "spc-like" CHRQUE2-5 SPg CHRQUEZ-6 TO CHRQUE2-7 SPg CHRQUEZ-8 preVg: PGCs CHRQUEZ-9 spd CHRQUEZ- 10 preVg; "spc-like" cells CHRQUE2- I 1 spd CHRQUE2- I2 preVg: "spc-like" cells CHRQUEZ- I 3 preVg; "spc-like" cells CHRQUE2- I4 TO CHRQUE2- I5 preVg: PGCs

Chrysin and Quercetin - Month 3 - Concentration 0. l and 0.05 mgL

mEz+- 12.5 0.012 0.61 U D D 13.5 0.015 0.61 U D D CHRQUE3-3 16.5 0.030 0.67 F M D D preVg; PGCs; atresia 17.0 0.035 0.71 M? M? D €E+RQCE 5 14.5 0,023 0.75 M? F? F? Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

M preVg D D D preVg; PGCs; atresia U preVg: PGCs: atresia U spd D preVg U D spd U all stages

Chrysin and Quercetin - Month 4 - Concentration 0.1 and 0.05 mg/L

CHRQUE4-I preVg; PGCs; atresia: Ot CHRQUE4-2 spd CHRQUE4-3 preVg: PGCs: atresia; OL CHRQUE4-4 all stages. advanced CHRQUE4-5 Vg: PGCmtresia CHRQUE4-6 TO CHRQUE4-7 early Vg; atresia: PGCs CHRQUE4-8 preVg; atresia; "spc-like" cells CHRQUE4-9 preVg; atresia: PGCs:"spc-like" CHRQUEJ- I0 sPg CHRQUES-I I all stages: abnormal CHRQUE4- I2 all stages CHRQUEJ- 13 preVg: atresis CHRQUEJ- t 4 sll stages; abnormal CHRQUE4- I5 CHRQUEJ-I 6 all stages. abnormal CHRQUEJ-I7 preVg: PGCs: atresia CHRQUE4-18 TO CHRQUEJ- 19 all stages. advanced CHRQUES-20 all stages. abnormal

Chrysin and Quercetin - Month 4 - Concentration 0. l and 0.05 mgL

CHRQUES-I 22.0 0.086 0.81 F F FD F Vg CHRQUES-2 22.0 0.082 0.77 M M? MD UD advanced CHRQUES-3 21.5 0.078 0.78 M MM D MM advanced Gonadal 2" Sex Characters Sarnp le TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

CHRQUES-4 20.5 F-M F earl Vg: atresia; OL CHRQUES-5 18.0 miss. MD all stages. advanced CHRQUESd 20.0 D D preVg: atresia: OL CHRQUES-7 20.0 MM MM advanced CHRQUES-8 22.0 F M Vg: PGCs: atresia; OL CHRQUES-9 20.5 MD MM all stages, advanced CHRQUES-10 22.0 MM MM advanced CHRQUES-11 22.0 M M advanced CHRQUES-I2 23.0 MM MM.pp1 advanced CHRQUES-I 3 19.0 MD M advanced CHRQUES-13 2 1.0 MM MM all stages, advanced CHRQUES- IS 25.0 MM MM.ppl Vg, PGCs. atresia; OL CHRQUES-16 2 1.0 D D Vg: atresia: OL; abnormal CHRQUES-17 18.0 UD M all stages. advanced CHRQUES-18 17.0 M MM all stages. advanced CHRQUES-19 19.0 MD MD Vg: atresia CHRQUES-20 20.0 D M Vg; atresia: OL CHRQUES-21 19.0 M M advanced CHRQUES-22 17.0 MM MM TO CHRQUES-23 2 1.0 M FD advanced CHRQUES-24 16.0 M MD all stages CHRQUES-25 24.0 F? MM.ppI advanced. abnormal CHRQUES-26 19.5 miss. UD Vg: atresia; OL CHRQUES-27 23.0 miss. MM Vg; PGCs: atresia: OL CHRQUES-28 23.0 MD UD early Vg: PGCs: atresia CHRQUES-29 23.5 M D all stages, advanced CHRQUES-30 19.5 miss. D all stages, advanced CHRQUES-3 1 20.0 MD F all stages. advanced CHRQUES-52 20.5 MD MD Vg: atresia: OL

Kaempferol - Month Z - Concentration 0.05 mp/L

KAEZL-I 13.0 0.017 0.77 M *Pg KAEZL-2 13.0 0.017 0.77 M SPS KAEZL-3 10.0 0.006 0.60 F preVg: PGCs: atresia; "spc-like" KAE2L-4 f 1.5 0.010 0.66 M KAEL-5 12.0 0.0 13 0.75 M KAE2L-6 1 1.5 0.01 1 0.72 M Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

KAE2L-9 KAEZL- I0 KAEZL-11 KAE2L- 12 KAE2L- 13

preVg: PGCs: "spc-like"

Kaempferol - Month 2 - Concentration 0.5 mg/L

KAE2H-I KAEZH-2 KAEZH-3 KAE2H-4

KAE2H-6 KAEZH-7 preVg; PGCs KAE2H-8 preVg: PGCs: OL: "spc- KAE2H-9 TO KAEZH- I 0 SPg KAEZH-I 1 SP_r KAE2H- 12 preVg; atresia KAEZH- I3 preVg; atresia KAE2H- 14 SPg KAE2H- 1 5 preVg: PGCs

Kaempferol - Month 3 - Concentration 0.05 mg/L

KAE3L-l 10.5 0.006 0.52 F U U U preVg; atresia: "spg-like" KAE3L-2 13.0 0.015 0.68 M U F? F? SP~ KAE3L-3 15.0 0.022 0.65 M F F M spd KAE3L-4 15.5 0.026 0.70 M M M M spd; abnormal KAE3L-5 15.0 0.027 0.80 M M M M spd KAE3L-6 17.0 0.037 0.75 F F M M preVg; atresia: OL; "spc-like" KAE3L-7 13.5 0.016 0.65 F U U U preVg: PGCs; atresia Gonadal 2" Sex Characters Sample TL (mm)Wt (g) C.F. Sex UGP DF AF Observations

U U preVg; PGCs; "spc-like" F preVg; PGCs;atresia M spd U preVg: PGCs: atresia U spd U preVg; atresia u SPg

Kaempferol - Month 3 - Concentration 0.5 mg/L

all stages: abnormal preVg; PGCs: atresia preVg; PGCs; atresia spd: fibrosis SPcr preVg: PGCs: OL: "spc-like" spd preVg; PGCs; atresia all stages all stages SPg spd preVg; atresia: "spc-like" SPg preVg: atresia

Kaempferol - Month 4 - Concentration 0.05 mg/L

preVg; atresia; stroma all stages preVg: PGCs: atresia: OL TO preVg atresia SPg preVg: atresia SPg all stages all stages Gonadal 2" Sex Characters Sample TL (mm)Wt (g) C.F. Sex UGP DF AF Observations

KAEBL-I I F preVg; PGCs; atresia KAEJL-12 M all stages KAEJL- I3 M preVg; PGCs; atresia KAE4L- I4 U spd KAE4L- 15 F preVg; PGCs; atresia KAEJL- 16 F- M preVg; atresia KAE3L- t 7 F preVg; atresia KAEJL- 18 M all stages KAEBL- 19 F-M SPg KAEJL-20 F preVg; atresia; "spc-like"

Kaempferol - Month 4 - Concentration 0.5 mgL

KAESH-I preVg; atresia; OL KAEJH-2 all stages. advanced KAE4H-3 all stages KAE4H-4 all stages. advanced KAEJH-5 all stages KAEJH-6 preVg; PGCs; atresia: OL KAEJH-7 preVg; PGCs; atresia: OL KAEJH-8 preVg; PGCs; atresia; OL KAE4H-9 preVg; atresia; OL: "spc-like" KAEJH- I0 prevg; OL KAEJH-I 1 all stages

KAE4H- 13 all stages KAEJH-14 preVg; OL KAEJH-I 5 preVg; atresia: OL

-KAWH-17 prevg KAE4H- 18 preVg: atresia 4eHH-9 KAEJH-20

Kaempferol - Month 5 - Concentration 0.05 mg/L

KAESL-I 25.5 0.120 0.72 M M(E)? M MM advanced KAESL-2 19.0 0.021 0.77 M U U U spd GonadaI 2" Sex Characters Sample TL (mrn) Wt (a) C.F. Sex UGP DF AF Observations

KAESL-3 M all stages. advanced KAESL-4 F preVg; atresia: OL KAESL-5 FF early Vg; OL: atresia: PGCs KAE5 L-6 MM advanced KA E5 L-7 M preVg; atresia; OL KAE5L-8 F preVg; OL KAESL-9 M Vg:OL KAESL- I0 F Vg; OL; atresia KAESL- II M all stages. advanced KAESL- I? U all stages KAESL- 1 3 M preVg; PGCs: Ot KAESL-14 F ail stages KAE5 L- 15 M preVg; atresia KAESL-16 M a11 stages, advanced KAESL-17 MM all stages KAESL-18 M preVg; atresia KAESL- 1 9 M preVg: PGCs: atresia; OL KAESL-20 M preVg; 01. KAE5L-2 I F all stages KAESL-22 MM all stages, advanced KA E5 L-23 M all stages KA EjL-24 MM preVg; PGCs KAESL-25 M all stages KAESL-26 F Vg: OL KAESL-27 M all stages KAESL-28 MM all stages KA ES L-29 M all stages KAESL-30 M advanced KAE5L-3 1 U spd KAESL-32 F all stages KAESL-33 M all stages KAESL-34 M all stages

Kaempferol- Month 5 - Concentration 0.5 mg/L

KAESH-I 22.0 0.088 0.83 M M? MM all stages, advanced KAESH-2 19.0 0.060 0.87 M MM MM all stages, advanced. TO KAESH-3 25.0 0.129 0.83 M MM MM a11 stages. advanced KAESH-4 24.0 0.117 0.85 F F M Vg; atresia Gonadal T Sex Characters Sample TL (mm)Wt (g) C.F. Sex UGP DF AF Observations

(6tEStfJ KAESH-6 preVg; atresia; OL KAESH-7 early Vg; OL KAE5H-8 early Vg; OL; PGCs KAESH-9 early Vg: OL; atresia KAESH- I0 all stages. advanced KAESH-I I vg: OL KAESH- 12 preVg; OL KAE5H-13 Vg; OL; atresia KAE5H- 14 prdg KAESH-I 5 all stages, advanced KAE5 H- 16 preVg; OL: atresia KAE5H- I7 late Vg: atresia; stroma KAE5H-18 all stages. advanced

KAESH-20 preVg: PGCs: atresia KAESH-2 I preVg; atresia KAES H-22 early Vg: OL; atresia KAESH-23 early Vg; OL; atresia KAESH-24 all stages KAESH-25 preVg; atresia KAE5 H-26 all stages. advanced KAESH-27 advanced KAESH-28 Vg: atresia KAE5H-29 advanced KAES H-30 early Vg; OL Naringenin - Month 2 - Concentration 0.1 mg/L

prevg SPg prevg sPg preVg; PGCs; atresia: "spc-like"

prevg SPg TO preVg; PGCs; atresia: "spc- SPg Gonadal 2" Sex Characters Sample TL (mrn) Wt (g) C.F. Sex UGP DF AF Observations

preVg; PGCs: "spc-like" =Pg preVg; atresia

Naringenin - Month 2 - Concentration I mg/L

preVg; atresia SPg

SPC preVg; PGCs; atresia

preVg; PGCs; atresia SPg preVg; PGCs: "spg-like" preVg: PGCs: OL SPg preVg; PGCs: OL sPS preVg; PGCs preVg; PGCs; OL

Naringenin - Month 3 - Concentration 0.1 mg/L

preVg; atresia preVg: atresia preVg; atresia preVg: atresia preVg; PGCs: atresia preVg; PGCs: atresia SPg preVg; atresia preVg; PGCs; atresia preVg: atresia preVg: PGCs preVg: atresia preVg; atresia SP_g . . . - . . .- . Gonadal 2" Sex Characters Sample TL (mm) Wt (g;) C.F. Sex UGP DF AF Observations

NAWL-IS 13.0 0.024 1.09 F U U F preVg: atresia: "spc-like"

Naringenin - Month 3 - Concentration 1 mg/L

M? spg; abnormal U preVg; stroma; "spc-like" F TO F preVg; atresia U preVg: atresia M? TO M TO U preVg; atresia M F preVg: atresia: stroma M spd: abnormal U preVg; atresia U preVg: atresia M SPg M preVg; PGCs: atresia: "spc-like"

Naringenin - Month 4 - Concentration 0.1 mg/L

U F all stages. advanced F preVg; PGCs; atresia M all stages F? preVg; atresia: OL U preVg; OL: PGCs F-M preVg; PGCs: atresia U all stages M preVg; atresia M? all stages U preVg: atresia M early Vg; PGCs: atresia; OL u preVg: atresia; stroma MM all stages: abnormal, TO M preVg; atresia F spd F preVg; atresia Gonadal 2" Sex Characters Samp tr TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

MM all stages, abnormal F preVg: atresia F all stages. abnormal

Naringenin - Month 4 - Concentration 1 mg/L

MM M preVg; atresia; stroma MM MM all stages U U preVg: atresia F-M? M ? all stages. advanced F F preVg: atresia F M? all stages; abnormal M MM preVg: atresia; PGCs F F prevg F? F? prevg M M spd M U all stages u U preVg F F-M all stages M M preVg; atresia: OL M M preVg: atresia; OL M M all stages. advanced F F Vg: atresia; OL M F? all stages F M? preV_r; atresia: "spc-like" F F prevg

Naringenin - Month 5 - Concentration 0.1 rngL

NARSL-I M Vg NAFWL-2 M late Vg: atresia NARSL-3 M all stages NAR5L-J F-M Vg; atresia NARSL-5 MM all stages. advanced NARSL-6 F? preVg; PGCs NAR5L-7 M vg; OL NARSL-8 MM Vg; atresia: PGCs NARSL-9 MM ail stages NARSL- I0 F-M prevg NARSL-11 MM all stages. advanced Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

NARSL-lt M? prevg; OL NARSL- I3 M vg; OL NARSL- 14 MM all stages NARSL- I5 MM all stages. advanced NARSL-16 MM preVg; PGCs; atresia NAWL-I7 M all stages. advanced NARSL-18 U preVg; OL NARSL-19 M all stages N ARSL-20 MM all stages. advanced NARS L-2 I F-M all stages NARSL-22 M all stages. advanced NARS L-23 M preVg; PGCs: OL; stroma NARSL-24 MM advanced NARSL-25 M early Vg: OL; atresia: ectopic NAR5L-26 U prevg NAR5L-27 F- M preVg: atresia: OL NARSL-28 M all stages NARS L-29 MM all stages NARS L-30 M all stages NARSL-3 I F-M Vg: OL: atresia NARSL-32 MM MM.ppl all stages. advanced N ARSL-3 3 M M preVg: atresia

Naringenin - Month 5 - Concentration 1 mg/L

NAR5H-I MM Vg; atresia NAR5H-2 F-M early Vg: OL; atresia NARSH-3 MM all stages. advanced NAR5H-4 MM preVg: OL: atresia NARSH-5 M? all stages. advanced NARSH-6 MM all stages, abnormal NAR5H-7 MM all stages. advanced NARSH-8 MM Vg; 0L NARSH-9 MM all stages. advanced NARS H- I0 MM all stages. advanced NARSH-I I MM early Vg; atresia NARSH-12 MM all stages, advanced NARSH- 13 U spd NARSH-14 MM all stages. advanced Gonadal 2" Sex Characters Sample TL (mrn) Wt (g) C.F. Sex UGP DF AF Observations

MM MM all stages. advanced F-M F? early Vg: OL; stroma M MM early Vg; OL;"spg-like" MM MM all stages. advanced M F-M all stages, advanced MM MM early Vg; stroma F-M M ealy Vg: OL M M preVg: OL MM M preVg; OL: stroma M M allstages M M preVg; atresia: OL F-M F Vg:OL M F early Vg: OL: some atresia MM MM.ppl TO M MM advanced F-M MM advanced M? F? Vg; PGCs; atresia; 0L M MM all stages. advanced M MM all stages. advanced

Apigenin - Month 2 - Concentration 0.05 mg/L

SPC preVg; PGCs

preVg; PGCs

SP?2 Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

Apigenin - Month 2 - Concentration 0.5 mg/L

AP12H- I SPg AP12H-2 =Pg AP12H-3 SPg APEH-4 preVg; PGCs; atresia API2H-5 a Pg APEH-6 SPg API2H-7 =Pg API2H-8 TO API2H-9 preVg: atresia; "spc-like" API2H-10 SPg

APIZH- I2 SP9 APEH- I3 preVg; atresia APIZH- I4 preVg: atresia; "spc-like" API2H- IS preVg; aresia: PGCs

Apigenin - Month 3 - Concentration 0.05 mg/L

preVg; PGCs; "spg-like" spd preVg; atresia, PGCs =Pg TO preVg: OL; atresia. PGCs SPg

s PC preVg: stroma; atresia PGCs

preVg; atresia preVg: atresia spd; abnormal preVg; atresia

Apigenin - Month 3 - Concentration 0.5 mg/L Gonadal 1" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

spd TO spd TO preVg; atresia: "spc-like" preVg; atresia; PGCs TO all stages preVg; PGCs; atresia preVg; atresia; "spc-I i ke" all stages preVg TO preVg; PGCs; "spg-like" preVg: PGCs: "spg-like": TO? preVg; strorna: OL preVG: PGCs; OL: "spg-like" TO TO preVg; OL; atresia; "spg-like"

Apigenin - Month 4 - Concentration 0.05 mg/L

preVg all stages. abnormal all stazes preVg TO TO all stages; abnormal all stages preVg; atresia all stages preVg: PGCs:atresia:OL:"spc-like" preVg; PGCs;atresia;OL;"spc-like" Vg: PGCs: atresia preVg; PGCs:atresia;OL;"spc-like" preVg: PGCs: OL: atresia

Apigenin - Month 4 - Concentration 0.5 mglL Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

APIJH- I MM MM preVg; atresia: OL; "spg-like" AP14H-2 M M? all stages, advanced AP14H-3 MM MM all stages. advanced AP14H-4 F F preVg; atresia: OL: "spg-like" API4H-5 M U all stages. advanced APIJH-6 F F? all stages. advanced API4H-7 MM MM all stages, advanced API4H-8 M M preVg; atresia; OL; "spg-like" AP14H-9 U U preVg: OL APIJH- I0 M? M? preVG: OL: "spg-like" APfSH-I I U U preV2; atresia API4H- I2 M M all stages. advanced API4H-13 MM MM all stages, advanced APMH-15 M M all stages APIJH- I5 U U preVg: atresia; "spg-like"

Apigenin - Month 5 - Concentration 0.05 mg/L

APISL- I M M early Vg; OL: atresia API5L-2 MM MM APISL-3 MM MM Vg; atresia: OL: "spc-like" APISL-4 IMM MM TO APISL-5 F-M F-M preVg; OL APUL-6 M M Vg: OL APISL-7 MM MM all stages. advanced AP15L-8 MM M preVg; atresia; OL APISL-9 MM M Vg; OL; atresia APISL-I0 F? F vg; OL APISL-I 1 M MM.ppl advanced AP15L- 12 MM M Vg: atresia; OL: stroma APISL- I3 M M early Vg; atresia; stroma: OL APISL-14 MM M all stages. advanced APISL-I 5 F-M M Vg; OL; some atresia API5L- 16 MM MM all stages. advanced AP15L- 17 MM MM.ppl advanced APISL- I8 M MM,ppl advanced APISL-19 M? MM Vg; atresia: stroma; OL: "spc-like" AP15L-20 M M early Vg; atresia: OL APISL-2 I F-M F-M? late Vg: atresia: stroma AP15L-22 M MM a11 stages. advanced Gonadal 2" Sex Characters Sample TL (mrn) Wt (g) C.F. Sex UGP DF AF Observations

APISL-23 11 -5 0.072 0.79, F F F-M F? late Vg; atresia; stroma; OL APISL-24 21.5 0,074 0.74 M F(A) M M? all stages. advanced APISL-25 23.0 0. 108 0.89 M F MM MM advanced AP15L-26 24.5 0.1 I7 0.80 M F(A) MM MM,ppl advanced APISL-27 23.5 0.100 0.77 M M* MM MM.ppl advanced

Apigenin - Month 5 - Concentration 0.5 mg/L

APISH-1 MM all stages. advanced APISH-2 MM all stages APISH-3 MM Vg; OL;atresia: "spc-like" APISH4 MM Vg; OL; stroma APISH-5 M preVg; OL APISH-6 MM Vg: OL; stroma APISH-7 M early Vg; OL; atresia APISH-8 M late Vg; 01: PGCs APISH-9 M early Vg; atresia; "spg-like" APISH-I 0 MM advanced APISH-I 1 MM all stages. advanced APISH-12 MM all stages APISH-13 M early Vg; OL; atresia APISH-I4 MM all stages. advanced APISH- I5 MM all stages. advanced APISH- I6 MM TO APISH-1 7 MM TO APISH-18 M preVg; atresia APISH-19 MM.ppl advanced APISH-20 M early Vg; OL: atresia APISH-:! I M early Vg; OL; atresia APISH-22 MM all stages. advanced APISH-23 MM,ppl all stages. advanced APISH-24 MM at1 stages. abnormal APISH-25 MM ail stages. advanced

Galangin - Month 1 - Concentration 0.05 mg/L Gonadal 2" Sex Characters Sample TL(mm)Wt(g) C.F. Sex UGP DF AF Observations

Galangin - Month 2 - Concentration 0.5 mg/L

GAL2M-I preVg: PGCs; atresia GALZM-2 SPg GAL2M-3 SP_P GAL2M-I GAL2M-5 TO GALZM-6 preVg; PGCs: atresia: "spg-like" GAL2M-7 SPg GALZM-8 GALZM-9 GAL2M- I0 GALZM-I I GALZM- 12 GAL2M- I3 GAEM- I4 GAUM- I 5

Galangin - Month 3 - Concentration 0.05 mg/L

F sPg U preVg u SPg M M spd M preVg; PGCs; atresia M preVg; PGCs; OL F preVg; OL: "spc-like" - - Gonadal 3" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

GAL3L-9 12.5 0.012 0.6 I ? U GAL3L-10 14.0 0.020 0.73 F U preVg; atresia; PGCs: OL GAEL-I I 15.0 0.025 0.74 M M spd GAL3L-12 15.0 0.022 0.65 F M preVg; atresia: PGCs: OL GAL3L-I3 17.5 0.045 0.84 M M all stages: abnormal GAL3L-14 15.0 0.024 0.7 1 F M preVg: PGCs; atresia; "spc-like" GAL3L-I5 15.0 0.026 0.77 M F spd: abnormal

Galangin - Month 3 - Concentration 0.5 mg/L

prevg: atresia al I stages all stages SPg all stages. advanced preVg; PGCs: atresia preVg; PGCs: OL: atresia all stages SPg TO SPg preVg; OL: atresia: PGCs preVg: OL: atresia; PGCs SPg preVg; PGCs: "spc-like": atresia

Galangin - Month 4 - Concentration 0.05 mg/L

GALJL- 1 all stages. advanced GAL4 L-2 all stages, advanced GAL4L-3 all stages GAL4L-J preVg; PGCs; "spg-like" GAL4L-5 spd GAUL-6 all stages. advanced GAL4L-7 all stages. advanced GALJL-8 all stages GAL4L-9 all stages GALJL-10 early Vg: OL GAL4L-1 I early Vg: OL GAUL- I2 early Vg; PGCs: OL: atresia Gonadal 2" Sex Characters Sample TL (rnm) Wt (g) C.F. Sex UGP DF AF Observations

GAL4L-13 19.5 early Vg: OL GALJL- I4 19.5 all stages, advanced GAL4L-15 14.0 all stages GAL4L-16 15.0 prevg GAL4L-17 18.0 alI stages, advanced GAL4L-18 17.0 all stages GAL4L-19 19.5 early Vg; atresia GALJL-20 20.0 all stages, advanced

Galangin - Month 4 - Concentration 0.5 mg/L

GAL4M-I a1 Istages, advanced GALJM-2 all stages. advanced GALJM-3 all stages, advanced GALJM-4 preVg; atresia GALJM-5 early Vg; atresia: OL: stroma GAL4M-6 Vg: atresia: OL GAL4M-7 preVg: PGCs GALJM-8 preVg GALJM-9 preVg GALSM-10 preVg; PGCs: atresia GAL4M-I 1 preVg; some atresia GALJM- I2 all stages. advanced GAL4M- 13 all stages GAL-IM- I4 preVg: some atresia GAL4M-15 preVg: stroma GALJM- 16 prevg GAL4M-17 spd GAL4M-I 8 preVg; PGCs GALJM- 19 all stages GALJM-20 all stages

Galangin - Month 5 - Concentration 0.05 mg/L

GALSL-I 19.0 0.054 0.79 F M? bt preVg; OL: atresia: "spc-like" GALSL-2 19.0 0.045 0.66 M M MM all stages. advanced GALSL-3 17.0 0.035 0.71 M M M? all stages GALSL-4 22.5 0.088 0.77 M MM MM,ppl all stages, advanced GALSL-5 21.5 0.069 0.69 M MM MM ail stages. advanced GAL5L-6 11.5 0.074 0.74 M MM M all stages. advanced Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Obsewations

GALSL-7 M? all stages; reduced no. spz GALSL-8 MM advanced GALSL-9 F early Vg; OL GAUL-I0 U all stages GAUL-I I M advanced GALSL-I 2 MM all stages. advanced GALSL- 13 M preVg; OL; atresia; "spg-like" GALSL-I 4 M all stages GALSL-15 M? Vg; OL: atresia: "spc-like" GALSL- 16 MM all stages. advanced GALSL- I 7 MM advanced GALSL-18 M TO GALSL- I9 M al I stages GALSL-20 MM MM.ppl all stages. advanced GALSL-2 I M Vg; OL: stroma: atresia GALSL-22 MM advanced G ALSL-23 MM all stages. advanced GALSL-24 MM advanced GAL5L-25 M all stages GALSL-26 M? Vg: OL GAL5L-27 F-M Vg: OL; atresia GAL5L-28 M early Vg; atresia; "spc-like" GALS L-29 M TO GALSL-3 0 M early Vg; PGCs; "spc-like" GALSL-3 I F-M TO GALSL-32 F Vg; OL; atresia GALSL-33 MM all stages. advanced

Galangin - Month 5 - Concentration 0.5 rn@L

GALSM-I M all stages. advanced GAL5M-2 MM MM.ppl all stages, advanced GALSM-3 M M all stages. advanced GAL5M-J M? M? Vg: OL: atresia GALSM-5 F F Vg; OL: atresia GAL5M-6 MM MM all stages. advanced GALSM-7 MM MM.ppl all stages. advanced GAL5M-8 F F Vg; OL; atresia GALSM-9 M? MM all stages, advanced GALSM- I0 M MM all stages, advanced Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

GALSM- I1 early Vg; PGCs: OL; "spc-like" GALSM- I2 Vg; OL: PGCs: atresia GALSM-13 all stages. advanced GALSM- I4 all stages, advanced GALSM- 15 all stages. advanced GALSM- I6 early Vg; OL GALSM- I7 preVg; abnormal GAL5M- 18 preVg; PGCs GALSM-19 TO GALSM-to all stages, advanced GAL5M-2 I all stages GALSM-22 vg; OL GALSM-23 preVg; some atresia GALS M-24 preVg: OL; PGCs GALSM-25 all stages. advanced GALSM-26 preVg: OL; atresia GAL5M-27 all stages. advanced GALSM-28 advanced GALSM-29 early Vg: OL: atresia GAL5M-30 all stages. advanced GALSM-3 I late Vg GALSM-32 all stages. advanced

Catechin - Month 2 - Concentration 0.1 mg/L

preVg; few PGCs SPS preVg; PGCs SPS preVg: PGCs; atresia

SPe SP_e preVg: atresia; PGCs: "spc-like" TO preVg SPg TO preVg; PGCs; "spc-like" Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

Catechin - Month 1 -Concentration I mg/L

SPg SPg SPg SPg sPg SPg sl'g SPg preVg: PGCs; atresia preVg: PGCs SPg preVg; PGCs preVg: PGCs: atresia sPg SPg

Catechin - Month 3 - Concentration 0.1 mg/L

preVg; PGCs preVg: some atresia preVg: PGCs: fib: "spc-Ii kes" preVg: PGCs: fib: "spc-likes" preVg; PGCs; atresia preVg: some atresia preVg; some atresia all stages preVg: PGCs; "spc-like" all stages preVg; atresia preVg: PGCs: atresia SPC SPg preVg: atresia

Catechin - Month 3 - Concentration 1 mg/L Gonadal 2" Sex Characters Sampte TL (mrn) Wt fg) C.F. Sex UGP DF AF Observations

preVg; PGCs spd preVg SPg spd preVg: atresia preVg: atresia preVg; OL; atresia: PGCs spd preVg; atresia spd spd: abnormal preVg: atresia preVg: atresia preVg; atresia

Catechin - Month 4 - Concentration 0.1 mg/L

a1 l stages. advanced all stages. advanced all stages all stages. advanced preVg TO preVg; PGCs all stages. advanced all stages. advanced preVg: OL; atresia all stages. advanced preVg; OL: atresia prevg all stast. abnormal preVg: PGCs: OL all stages; reduced spz; abnormal all stages. advanced preVg: atresia; OL preVg: "spc-like" preVg; OL; atresia: PGCs

Catechin - Month 4 - Concentration I m@

260 Gonadal 3" Sex Characters Sample TL (mm)Wt (g) C.F. Sex UGP DF AF Observations

CAT4H-I CAT4H-2 prevg. CAT4H-3 preVg: OL: PGCs: "spc-like" CAT4H4 all stages. advanced CATJH-5 preVg; OL; atresia; PGCs; fib CATJH-6 ali stages. advanced CATJH-7 preVg: atresia CATJH-8 all stages CATJH-9 all stages. advanced CAT4H-! 0 preVg: OL; atresia; PGCs CATJH-I I early Vg; atresia; fib CATJH- 12 all stages CAT4H- 13 preVg: atresia CAT4H- 14 all stages CATJH- 1 5 a11 stages CAT4H- 16 all stages. advanced CATJH-I7 preVg: PGCs CATJH-18 all stages CATJH- 19 TO C ATJH-20 TO

Catechin - Month 5 - Concentration 0.1 mg/L

CATS L- I M preVg; OL; some atresia CATSL-2 F all stages. advanced CATSL-3 F-M Vg; OL: atresia: PGCs CATSL-4 M all stages C ATSL-5 M early Vg; OL: some atresia C ATSL-6 M preVG: OL; atresia CATSL-7 M al t stages. advanced CAT5 L-8 M all stages. advanced CATSL-9 MM all stages. advanced CATSL-10 MM all stages CATSL-I I F? all stages. advanced CAT5 L- 12 F prevg; OL CATSL-13 M early Vg; OL: tib: atresia CATSL- 14 M? all stages CATSL-i 5 MM spd CATSL- 16 M? early Vg CATSL- 1 7 F-M Vg; OL; atresia Gonadal 1" Sex Characters Sample TL (mrn) Wt (g) C.F. Sex UGP DF AF Observations

CATS L- 1 8 M MM preVg; OL; atresia CATSL- 1 9 MM MM.ppl all stages, advanced CATSL-20 M M all stages CATSL-2 1 MM MM early Vg; 01; atresia: PGCs CATSL-22 F-M F late Vg; stroma CATS L-23 M? F preVg CATS L-24 MM MM advanced CATS L-25 M M a11 stages CATSL-26 MM MM Vg: atresia: OL; PGCs CATSL-27 F F preVg CATSL-28 M? M spd CATS L-29 MM MM advanced CATS L-30 M M preVg: OL: atresia; fib: PGCs CATSL-3 1 F-M F early Vg: OL: atresia; PGCs CATS L-3 2 M? MM Vg; OL; atresia CATS L-3 3 M MM Vg: OL: atresia CATSL-34 F-M F Vg: OL; atresia CAT5L-35 M M,ppl all stages. advanced

Catechin - Month 5 - Concentration 1 mg/L

CATSH- 1 M M preVg; OL; "spg-like" CATSH-2 M M Vg; stroma CATSH-3 M M advanced CATSH-I F? F? preVg; OL; atresia; PGCs C ATSH-5 F-M F? early Vg; OL; atresia: PGCs CATS H-6 M M preVg CATSH-7 M M preVg; PGCs CATSH-8 F- M M Vg; OL; atresia: "spg-like" CATSH-9 M M all stages. advanced CATSH- 10 MM MM all stages. advanced CATSH-I 1 M MM ail stages, advanced CATSH- I2 MM MM,ppl all stages. advanced CATSH-13 M MM all stages. advanced CATSH- 14 M M preVg; OL; atresia; PGCs CATSH- 15 M M all stages. advanced CATSH- I6 M M all stages, advanced CATSH- 1 7 M M advanced CATSH-18 M M? Vg; OL: PGCs: atresia CAT5H- 19 M M preVg: OL; atresia; "spc-like" Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

CAT5H-20 MM early Vg; OL: atresia; "spc-like" CATSH-2 1 M all stages CATSH-22 M preVg: atresia: PGCs CATSH-23 MM.pp1 all stages, advanced C ATSH-24 MM all stages. advanced CATSH-25 MM CATSH-26 F-M Vg: some atresia CATS H-27 MM preVg; OL: atresia CATSH-28 MM all stages. advanced CATSH-29 M advanced CATSH-30 MM all stages, advanced CATSH-3 I MM all stages. advanced CAT2 MM Vg: OL; atresia; stroma t 7a-ethinyl estradiol (EEZ) - Month 2 - Concentration 1 ng/L

TO prevg preVg; many PGCs

TO prevg

advanced TO

1'la-ethinyl estradiol (EEZ) - Month 3 - Concentration 1 ng/L

E3 L- l 16.5 0.030 0.67 M F F F sPg E3 L-2 I40 0.024 0.87 F F M? F preVg and PGCs ES L-3 0 0.019 0.69 F F F F preVg and PGCs E3 L-4 17.0 0.040 0.81 F FF F F preVg: PGCs E3 L-5 15.5 0.032 0.86 F F U M preVg; PGCs in middle E3 L-6 16.5 0.032 0.71 F F F F preVg; PGCs

17~-ethinylestradiol (EEZ)- Month 4- Concentration I ng/L

EJL-I 14.5 0.020 0.66 M M M F SF% E4L-2 15.0 0.023 0.68 M F F F spd EJL-3 7 0.038 0.71 F F M-F M-F preVg: PGCs E4L-4 13.5 0.023 0.93 F F F F pteVg: PGCs; atresia E4L-5 19.0 0.059 0.86 F F F F early Vg E4 L-6 t9.0 0.024 0.87 F M-F M-F M-F preVg: auesia Gonadal 2" Sex Characters Sample TL (mm) Wt (g) C.F. Sex UGP DF AF Observations

E4L-7 16.5 0.033 0.73 M F F F spd E4L-8 19.0 0.053 0.77 M F F F all stages E4 L-9 16.0 0.033 0.81 M M(e) M F all stages; mostly spg EJL- 10 21.5 0.080 0.80 M M(e) M M all stages l7~~ethinylestradiol (EEZ) - Month 5- Concentration 1 ng/L

M M preVg MM MM,ppl advanced M? FF early Vg; atresia: PGCs M M ali stages M M all stages . advanced M F all stages . advanced F F preVg: PGCs: spg-like cells M M advanced M-F M all stages, advanced F? F? preVg; PGCs M-F M preVg: PGCs M M Vg stage F FF early Vg. one large post-Vg oocyte

17~-ethinylestradiol (EEZ)- Month 2 - Concentration 10 ng/L

preV~:PGCs TO advanced 5P_e ro. SP~ TO advanced

preVg; many PGCs (TO?) spg: increased IT preVg : PGGs spg; increased [T

17~-ethylestradiol (EE2) - Month 3 - Concentration 10 ng/L

E3H-1 6 0.032 0.71 M U M M spd E3 H-2 12.5 0.018 0.92 M F F F TO. spd E3 H-3 13.0 0.017 0.77 F FF F F preVg: PGCs E3 H-4 17.0 0.038 0.77 M IM M M spd E3 H-5 15.5 0.027 0.73 F M F F advanced.TO E3 H-6 18.0 0.042 0.72 M M M M a11 stages: abnormal Gonadal 2" Sex Characters Sample TL (mm)Wt (g) C.F. Sex UGP DF AF Observations

I'la-ethyl estradioi (EE2) - Month 4 - Concentration 10 ng/L

F U U U preVg; PGCs: atresia F U U U preVg: PGCs: atresia M F M-F F spd M F M-F F TO M F M-F F all stages M M(e) M M all stages F F F F preVg: PGCs; atresia F F M F early Vg: PGCs: atresia F M(e) M M-F preVg; TO F F F F preVg: PGCs; atresia

1 'la-ethinyl estradiol (EE2) - Month 5 - Concentration 10 ng/L

E5H-I M early Vg; atresia: PGCs E5H-2 M early Vg:PGCs E5H-3 M-F TO E5H-J M preVg; PGCs ESH-5 M early Vg: atresia ESH-6 F? all stages ESH-7 F all stages ESH-8 M all stages: advanced ES H-9 F preVg: PGCs (sex-reverse?) ESH- 10 M all stages E5H-I I F? preVg: PGCs: atresia E5H- t 2 M-F E5H-13 M Vg: many PGCs (sex-reverse?) E5H-I4 0.72 F M(e) F F Vg: many PGCs (sex-reverse?) Appendix 10: Determination of the gonadal phenotypic sex and expression of secondary sex characteristics in Japanese medaka (Oryzias latipes) exposed to various treatments of flavonoids from hatching to 2,3.4 and 5 months post-hatching (data used in Chapter 5). Total lenght (m),body weight (g) and condition factor from all individual medaka are also presented (Abbreviations in headings: TL = total length; Wt = wet weight; C.F. = condition factor; UGP = urogenital pore or urogenital papilla; DF = dorsalfin; AF = analfin. Abbreviations under "Gonadal Sex": M = male; F =/ernale. Abbreviations under "20 Sex Characters": U = unidentified; M = male; F =female; MM = definite mule: FF = definite female; ppl = papillary processes; M? or F? = ident8cution with nincertuinty; ..I = absent; D = deformed: F-A4 = femde probably chunging lo mule: M-F = male probably chunging to fern&: M(EI = esirogenized mule: F(A) = undrogenked jl.mu1. A bbrevirrtion rmd Terms under "Observutions ":spg = only spermutogoniu ure present, immuture; spc = spermatocytes present, immature; spd = spermatids, present, immture; all stages = spermatozoa present, immature; all stages, advanced = active spermatogenesis: advunced = advanced spermatogenesis; TO = testis-ova;fib = jibrosis; pre Vg = oocytes at the previtellogenic stage; early Fg = onset of vitellogenesis, oocytes in I I: and few in V stuges of' development: Vg = vitellogenic stage. oocytes at V and VI stuges uf'development: late Cg= oocytes at VII and up stage of development; PGCs = increased number of'primordiul germ cells; utresiu = increased number oj'utretic oocytes; OL = increuse in ovariun himen).

Specimen Wt (g) TL C.F. UGP DF AF Sex Observations (mm)

Control treatment

Gen C I a11 stages. immature Gen C 2 all stages (tissue not properly cut) Gen C 5 prevg Gen C 4 early Vg (few V) Gen C 5 spd Gen C 6 prevg Gen C 7 prevg Gen C 8 few spd Gen C 9 few spd Gen C I0 early Vg Gen C I I early Vg Gen C 12 prevg Gen C 13 SPC Gen C 14 all stages GenC 15 vg (W Gen C 16 vg (V) GenC 17 all stages. immature GenC 18 early Vg (few V) Gen C 19 vg (V) Specimen Wt (g) TL C.F. UCP DF AF Sex Observations (mm)

Gen C 20 all stages. immature Gen C 21 all stages Gen C 22 SPg Gen C 23 prevg Gen C 24 all stages, immature Gen C 25 prevg Gen C 16 P~W Gen C 27 all stages - advanced Gen C 28 preVg Gen C 29 early Vg (V) Gen C 30 prevg

all stages: immature advanced 'fg O/) all stages: advanced all stages v9 cw all stages all stages vg cw PW vg (W; 01. all stages; advanced all stages; advanced all stages; advanced all stages; advanced preVg; some atresra Vg (V); some atresra all stages vg (V1) advanced early Vg earty ~g vg cv, preVg - Vibrasis all stages vg (v, early Vg - some atresia early Vg all stages - advanced earty Vg v9 0 Specimen Wt (g) TL C.F. UGP Sex Observations (mm)

Genistein treatment ( 1 p&)

Gen 11 1 FF F spawning Gen I/ 2 MM M all stages - part A CT Gen ll3 F M all stages - vv#germ cells - ,'fib Gen 114 M Gen I/ 5 F( me F late Vg (VlIl) Gen 11 6 M M SPg Gen I/ 7 U F prevg Gen 1/ 8 FF F Vg(V) - some atresia Gen 1/ 9 FF F preVg: ""OL;"atresia Gen 11 10 M M spc - "CT Gen 11 I I M M spc - ^CT Gen 11 12 F F early Vg (few V): some atresia Gen 11 13 F F early Vg (few V) Gen 11 14 F F prevg Gen I/ 15 M M SPC Gen I1 16 MM M a11 stages Gen li I7 MM M all stages Gen 11' 18 M F pre''g Gen 11 19 U F prevg Gen 11 20 M M sP c Gen 1/21 FF M (not good for evaluation) Gen 1 / 22 F F prevg Gen 1/ 23 F F prevg Gen 1 / 24 F F preVg - some atresia Gen I/ 25 M M spd - TT Gen 11 26 U M spd - ^CT Gen !/ 27 UM M spd Gen I/ 28 F F early Vg (few V) - PGCs Gen 11 29 M M all stages. immature; "CT Gen I/ 30 F F preVg - many PGCs Gen 1/31 MM M all stages, immature: "CT Gen 11 32 U F preVg - PGCs Gen I/ 33 M M all stages. immature; .'CT Gen 11 34 M M all stages. immature; 'CT

I Gen 1 1 Gen 2 MF F Vg 0;some atresia; OL I Gen 3 MF F v9 0 1 Gen 4 MM M all stages; immature; some A in Cf 1 Gen 5 M M all stages; immature; some A In CT 1 Gen 6 FF F vg 0;WL t Gen 7 F F early Vg I Gen 8 MF F early Vg 1 Gen 9 MF M all stages; immature; some A ~nCT Specimen Wt (g) TL C.F. UGP DF AF Sex Observations (mm)

1 Gen I0 F? vs 0 I Gen I I M all stages - decreased spgnss - ACT 1 Gen 12 U.I. all stages; spgns inhibited: mod. A CT 1 Gen 13 F? vgo-OL 1 Gen I4 - SPg I Gen IS F '4 ('4 1 Gen 16 UM all stages, immature I Gen 17 F early Vg I Gen 18 M? early Vg (V); AOL; Aatresia 1 Gen 19 M all stages-advanced; ACTaround lobs I Gen 20 M vg cv, I Gen 21 U early Vg; some atres~a 1 Gen 22 M early Vg; some atresia 1 Gen 23 U early Vg 1 Gen 24 M? vs cv, I Gen 25 MM all stages-advanced - ACTpartial I Gen 26 M all stages; immature I Gen 27 M immature; few spz; ACT I Gen 28 M early Vg; some atresta I Gen 29 M all stages-advanced I Gen 30 ? preVg; OL; some atres~a I Gen 3 F vs (W 1 Gen 32 MM all stages - advanced

Genistein treatment ( 10 pg/L)

Gen 10/1 vg (V) - OL Gen 10 :'2 advanced; v;: spz; some ' IT space Gen 10 13 prevg Gen 10 /4 immature (not good for evaluation) Gen 10 /5 SPg Gen I0 /6 late Vg (VIII) Gen 10 17 preVg - some atresia Gen 10 /8 early Vg (V) Gen 10 19 all stages. immature - 'CTmod. Gen 10 :I0 all stages Gen 10 /I 1 preVg - PGCs - some atresia Gen 10 112 Gen 10 113 all stages; small testis; some "CT Gen 10114 spc (mostly spg) Gen 10 115 preVg - in parts "CT Gen 10 /I6 early Vg (V) - OL - some atresia Gen 10 117 pre''g Gen 10 /i8 prevg Gen 10 119 all stages - few spz Specimen Wt (g) TL C.F. UGP DF AF Sex Observations (mm)

Gen 10 /20 preVg - some PGCs Gen I0/21 preVg - some PGCs Gen 10 /22 prevg Gen 10 i23 Vg (V) -^^OL- '"atresia Gen 10 I24 preVg - some atresia - PGCs Gen 10 1'25 prevg Gen 10 126 preVg - some atresia Gen 10 127 preVg - many PGCs Gen 10 f28 prevg Gen 10 29 prevg Gen 10 /30 preVg Gen 10 !3l preVg Gen 10 /32 all stages - small testis: 'CT part. Gen 10 I33 all stages, immature

10 gen I F early Vg; some atresla 10 gen 2 F early Vg; some atres~a 10 gen 3 F "9 ('4 10 gen 4 F v9 ('4 10 gen 5 M all stages; moderate ACT I0 gen 6 F preVg; some atres~a I0 gen 7 10 gen 8 10 gen 9 10 gen 10 iO gen 11 10 gen 12 10 gen 13 10 gen I4 10 gen 15 10 gen 16 F early Vg 10 gen 17 all stages ; signA CT (posterior) I0 gen 18 10 gen 19 10 gen 20 10 gen 21 10 gen 22 10 gen 23 10 gen ZJ 10 gen 25 10 gen 26 10 gen 27 I0 gen 28 I0 gen 29 F PreVg 10 gen 30 M all stages - advanced 10gen 31 Specimen C.F. Sex Observations

10 gen 32 1-07 10 gen 33 0.63 F preVg; some atresia 10 gen 34 0.74 10 gen 35 0.56 10 gen 36 0.79 M all stages; immature I0 gen 37 0.62 I0 gen 38 0.77 10 gen 39 0.64 M all stages; v spgns; "spg lobules I0 gen 40 0.59 F preVg - some atresia I0 gen 41 0.6 1 F preVg - some atresia 10 gen 32 0.68 10 gen 43 0.59 F preVg; some atresla I0 gen 44 0.69 I0 gen 45 0.68 F preVg; some atresia I0 gen 46 0.67 I0 gen 47 0.71 M all stages; mostly spg I0 gen 48 0.73 M all stages; immature I0 gen 49 0.69 F preVg; atresla 10 gen 50 0.7 1 F early Vg (V), Aatresta 10 gen 5 1 0.7 1 10 gen 52 0.68 F prevs I0 sen 53 0.69 F early Vg 10 gen 54 0.7 1 F preVg; "atresia; some PGCs

Genistein treatment ( 100 pg/L)

Gen I00 /I F spawning Gen I00 12 F prevg Gen 100 13 M all stages: advanced: VVV3spz Gen 100 1'4 F early Vg Gen 100 iS F spawning Gen 100 I6 F preVg; some atresia Gen 100 17 F preVg (large IV) Gen 100 /8 F early Vg (few V): ""OL; ""atresia Gen 100 19 F preVg (large IV) Gen 100 /I0 M all stages. immature: ;'CT posterior Gen 100 I1 1 MM (d) MM, M all stages - advanced: pan. 'CT ppl (dl Gen 100 112 F FD F vg (V) Gen 100!13 D D M ail stages - some .'CT Gen 100 /I4 U F F vg (V) Gen 100 115 MM MD ppl M all stages: architecture:.' IT space - w#germ cells Gen 100 /I6 M all stages - v 8 germ cells in lobs - partial " CT Gen 100 I17 F Vg (V): PGCs: some atresia Specimen Wt (g) TL C.F. UGP DF AF Sex Observations (mm)

Gen 100/18 F VgW Gen 100 119 M all stages - part ,' CT Gen 100 /20 F early Vg - some ^OL Gen 100 12 1 F late Vg (VIII)

100 GEN 1 IM SPg 100 GEN 2 M all stages, immature - ^CT around lobs 100 GEN 3 I00 GEN 4 F early Vg; atresia I00 GEN 5 I00 GEN 6 I00 GEN 7 100 GEN 8 100 GEN 9 Vg (V); some atresia I00 GEN 10 I00 GEN 1 I all stages; immature; moderate 'CT 100 GEN I2 spd; 100 GEN 13 early Vg 100 GEN I4 all stages; spgns inhibited (?I; moderate A CT around lobules I00 GEN I5 all stages; ~mmature;few spz, mostly spg; ACT 100 GEN 16 SPC 100 GEN 17 preVg; some atresta 100 GEN 18 100 GEN 19 100 GEN 20 100 GEN 21 100 GEN 22 all stages; fewer lobules wtth spz; mostly spg; ^CT 100 GEN 23 I00 GEN 24 all stages; fewer lobules with spz: mostly spg; 'CT I00 GEN 25 LOO GEN 26 Prev9 100 GEN 27 all stages; either few spz or spg 100 GEN 28 early Vg; atresia 100 GEN 29 early Vg; some atresia 100 GEN 30 early Vg; atresia tOOGEN 31 Vg (V) - atresia I00 GEN 32 early Vg I00 GEN 33 preVg - atresia I00 GEN 34 all stages; immature; 100 GEN 35 early Vg t 00 GEN 36 preVg (large IV); atresia 100 GEN 37 SPC I00 GEN 38 early Vg; atresia 100 GEN 39 all stages-advanced - "CT partial Specimen Wt (g) TL C.F. UGP Sex Observations (mm)

100 GEN 40 0.037 17.5 0.69 F F Vg (V); atresia 100 GEN 41 0.016 16.0 0.63 M M SPg 100GEN42 0.046 19.0 0.67 F F preVg; atresia 100 GEN 43 0.048 19.0 0.70 M F preVg; AnOL;"atresia; PGCs 100 GEN SLF 0.076 23.0 0.62 M M all stages-advanced 100GEN45 0,041 17.5 0.77 F F early Vg 100 GEN 46 0.047 17.5 0.88 F F preVg; some atresia

Genistein treatment ( 1000 pg/L)

Gen 1000 / l FF F vg (VI) Gen 1000 12 FF F preVg; TO? - a Iobule with spg-like in the posterior end Gen 1000 i3 FF F vg (V) Gen 1000 /4 MM M sF'g Gen 1000 15 U M spd: immature; narrow testis: "CT Gen 1000 16 FF F preVg; ""OL; .'atresia Gen 1000 17 F F preVg: ^PGCs Gen 1000 /8 FF F vg (VII I) Gen I000 /9 M( E) F preVg: many PGCs Gen 1000!10 M M all stages Gen I000 /l l FF F preVg: 10 bules with spg-like (or PGCs) Gen 1000!12 F F early Vg: some 'OL; some atresia Gen 1000 :'I3 FF F Vg (V): 'i'OL; atresia: PGCs Gen I000 !14 FF F vg (V) Gen 1000 /15 FF F Vg (V): '*OL; atresia: '"PGCs Gen 1000 116 M( E) M all stages: immature: decrease spz: YT; - "TO-prone": thick lobules Gen 1000 / 17 FFF M spd: 'TC: thickening of lobules Gen 1000 118 M( E) M all stages; immature: architecture Gen 1000 119 FF F vg ('4 Gen I000 /20 M(E) M "TO-proneM-all stages: few spz: thick lobules; "CT Gen 1000 f21 F F preVg; many PGCs Gen 1000 I22 FF F early Vg """OL Gen 1000 I23 FF F Vg (V): atresia; 'OL

M M all stages; "CT; decrease spz In lobules 1000 GEN 2 F 1000 GEN 3 M M 1000 GEN 4 - 1000 GEN 5 F F early Vg; some atresia 1000 GEN 6 M 1000 GEN 7 - F

Specimen Wt (g) TL C.F. UCP DF AF Sex Observations (mm)

1000 GEN 52 I000 GEN 53 1000 GEN 54 1000 GEN 55 1000 GEN 56 F preVg; atres~a 1000 GEN 57 M sW 1000 GEN 58 l= F Vg (W; some atresia; OL I000 GEN 59 M F preVg; atresla 1000 GEN 60 M M immature 1000 GEN 6 t rM F preVg; atresia