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Effects of nonylphenol on stress response in Rainbow Trout, Oncorhynchus mykiss Suto, Akiko 2000

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EFFECTS OF NONYLPHENOL ON STRESS RESPONSE FN RALNBOW TROUT, Oncorhynchus mykiss. by AKIKO SUTO B.Sc. Waseda University, Tokyo. 1997. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Resource Management and Environmental Studies) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 2000 © Akiko Suto, 2000 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Resource Management and Environmental Studies The University of British Columbia Vancouver, Canada June 25, 2000 A B S T R A C T During the last few decades, there has been a great concern about the impact of endocrine disrupting chemicals (EDCs) on the health of wildlife and human populations. Nonylphenol, a degraded product of a widely used surfactant, has recently been identified as an estrogenic EDC. A set of experiments were conducted to examine how exposure to three levels (4, 20, and lOOppb) of nonylphenol for certain periods (1-3 weeks) affected physiological function in rainbow trout over time, in terms of their response to an acute stress of air exposure. It was shown that a high level (lOOppb) of nonylphenol suppresses the elevation of plasma Cortisol level in rainbow trout in response to the acute stress of air exposure. In addition, the fish treated with nonylphenol were incapable of reducing their Cortisol levels back to pre-stress levels after 24 hours. The normal response of plasma glucose levels to the stress was observed. Plasma levels of thyroid hormones decreased significantly (p<0.0\) after exposure to the high level of nonylphenol for 3 weeks. Nitrite, a toxic chemical naturally present in the aquatic environment, was used in the experiment for comparison and had no effect on the normal stress response of Cortisol. The results from these experiments suggest that nonylphenol may have negative impacts on fish, such as a lower chance of survival and vulnerability to diseases, by interfering with normal function of the endocrine system. T A B L E O F C O N T E N T S ABSTRACT ii TABLE OF CONTENTS • • • ill LIST OF FIGURES V LIST OF TABLES ix LIST OF ABBREVIATIONS X PREFACE xi ACKNOWLEDGEMENTS xiii CHAPTER 1. INTRODUCTION 1 1.1. NONYLPHENOL 1 1.1.1. Production and use of nonylphenol 1 1.1.2. Nonylphenol in aquatic environment 2 1.1.3. Toxicity of nonylphenol 3 1.2. NITRITE 10 1.3. DLMETHYLSULFOXIDE (DMSO) 10 CHAPTER 2. STRESS IN FISH 12 2.1. STRESS RESPONSE IN FISH 12 2.2. REGULATION OF CORTISOL AND THYROID HORMONES 12 2.2.1. The hypothalamo-pituitary-interrenal (HPI) axis 12 2.2.2. Functions of C o r t i s o l 13 2.2.3. The hypothalamo-pituitarty-thyroid (HPT) axis 14 2.2.4. Functions of thyroid hormones 14 2.3. RESPONSE OF FISH TO SECONDARY STRESS 15 iii CHAPTER 3. MATERIALS AND METHODS 19 3.1. OBJECTIVE 19 3.2. EXPERIMENTAL PROTOCOL 20 3.2.1. Experiment I: Short term exposure to nonylphenol 3.2.2. Experiment H : Time course of stress response in fish exposed to nonylphenol 3.2.3. Experiment HI: Short term exposure to nitrite 20 23 24 3.3. FISH 25 3.4. STRESS APPLICATION AND SAMPLING 25 3.5. BLOOD COLLECTION AND PLASMA SEPARATION 26 3.6. MEASUREMENT OF PHYSIOLOGICAL PARAMETERS 27 3.7. BLOOD ANALYSIS 27 CHAPTER 4. RESULTS 29 4.1. EXPERIMENT I: SHORT TERM EXPOSURE TO NONYLPHENOL 29 4.2. EXPERIMENT H : T I M E COURSE OF STRESS RESPONSE IN FISH EXPOSED TO NONYLPHENOL 30 4.2.1. Physiological status of the fish throughout the experimental period 30 4.2.2. Stress experiment 31 4.3. EXPERIMENT D T : SHORT TERM EXPOSURE TO NITRITE 32 CHAPTER 5. DISCUSSION 58 CHAPTER 6. CONCLUSIONS AND COMMENTS 62 REFERENCES 66 iv L I S T O F F I G U R E S page Figure 1.1. Nonylphenol. 1 Figure 1.2. Degradation of nonylphenol polyethoxylate (NPnEO). 8 Figure 1.3. Chemical structures of Cotiosol and 17fj-estradiol. 9 Figure 2.1. Schematic representation of hypothalamo-pituitary-interrenal (HPI) axis. 17 Figure 2.2. Schematic representation of hypothalamo-pituitary-thyroid (HPT) axis. 18 Figure 4.1. Experiment I. Effects of stress (air exposure for 5min) on hematocrit in rainbow trout treated with nonylphenol (lOOppb) for 14 days. 34 Figure 4.2. Experiment I. Effects of stress (air exposure for 5min) on total hemoglobinin rainbow trout treated with nonylphenol (lOOppb) for 14 days. 34 Figure 4.3. Experiment I. Effects of stress (air exposure for 5min) on plasma Cortisol in rainbow trout treated with nonylphenol (lOOppb) for 14 days. 35 Figure 4.4. Experiment I. Effects of stress (air exposure for 5min) on plasma glucose in rainbow trout treated with nonylphenol (lOOppb) for 14 days. 36 Figure 4.5. Experiment I. Effects of stress (air exposure for 5min) on plasma free T3 in rainbow trout treated with nonylphenol (lOOppb) for 14 days. 36 Figure 4.6. Experiment I. Effects of stress (air exposure for 5min) on plasma total T3 in rainbow trout treated with nonylphenol (lOOppb) for 14 days. 37 Figure 4.7. Experiment I. Effects of stress (air exposure for 5min) on plasma free T 4 in rainbow trout treated with nonylphenol (lOOppb) for 14 days. 37 Figure 4.8. Experiment I. Effects of stress (air exposure for 5min) on plasma total T 4 in rainbow trout treated with nonylphenol (lOOppb) for 14 days. 38 page Figure 4.9. Experiment I. Effects of stress (air exposure for 5min) on plasma 38 lactate in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Figure 4.10. Experiment I. Effects of stress (air exposure for 5min) on plasma 39 ammonia in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Figure 4.11. Experiment I. Effects of stress (air exposure for 5min) on plasma 39 total protein in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Figure 4.12. Experiment II. Total hemoglobin in rainbow tout treated with 40 DMSO and nonylphenol (4-100 ppb). Figure 4.13. Experiment II. Plasma Cortisol in rainbow tout treated with DMSO 41 and nonylphenol (4-100 ppb). Figure 4.14. Experiment II. Plasma glucose in rainbow tout treated with DMSO 42 and nonylphenol (4-100 ppb). Figure 4.15. Experiment II. Plasma free T3 in rainbow tout treated with DMSO 43 and nonylphenol (4-100 ppb). Figure 4.16. Experiment II. Plasma free T 4 in rainbow tout treated with DMSO 44 and nonylphenol (4-100 ppb). Figure 4.17a. Experiment II. Effects of stress (air exposure for 3 min) on total 45 hemoglobin in rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 7 days. Figure 4.17b. Experiment II. Effects of stress (air exposure for 3 min) on total 46 hemoglobin in rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 21 days. Figure 4.18a. Experiment II. Effects of stress (air exposure for 3 min) on plasma 47 C o r t i s o l in rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 7 days. Figure 4.18b. Experiment II. Effects of stress (air exposure for 3 min) on plasma 48 Cortisol in rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 21 days. vi page Figure 4.19. Experiment II. Effects of exposure to high level (lOOppb) of 49 nonylphenol on stress (air exposure for 3 min) response of plasma Cortisol in rainbow trout. Figure 4.20a. Experiment II. Effects of stress (air exposure for 3 min) on plasma 50 glucose in rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 7 days. Figure 4.20b. Experiment II. Effects of stress (air exposure for 3 min) on plasma 51 glucose in rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 21 days. Figure 4.21. Experiment III. Effects of stress (air exposure for 5min) on 52 hematocrit in rainbow trout treated with nitrite (0.2mgN-NO2_/L) for 14 days. Figure 4.22. Experiment III. Effects of stress (air exposure for 5min) on total 52 hemoglobin in rainbow trout treated with nitrite (0.2mgN-NO27L) for 14 days. Figure 4.23. Experiment III. Effects of stress (air exposure for 5min) on plasma 53 Cortisol in rainbow trout treated with nitrite (0.2mgN-NO2_/L) for 14 days. Figure 4.24. Experiment III. Effects of stress (air exposure for 5min) on plasma 53 glucose in rainbow trout treated with nitrite (0.2mgN-NO2"/L) for 14 days. Figure 4.25. Experiment III. Effects of stress (air exposure for 5min) on plasma 54 free T3 in rainbow trout treated with nitrite (0.2mgN-NO2_/L) for 14 days. Figure 4.26. Experiment III. Effects of stress (air exposure for 5min) on plasma 54 total T 3 in rainbow trout treated with nitrite (0.2mgN-NO27L) for 14 days. Figure 4.27. Experiment III. Effects of stress (air exposure for 5min) on plasma 55 free T 4 in rainbow trout treated with nitrite (0.2mgN-NO2_/L) for 14 days. Figure 4.28. Experiment III. Effects of stress (air exposure for 5min) on plasma 55 total T4 in rainbow trout treated with nitrite (0.2mgN-NO27L) for 14 days. vii page Figure 4.29. Experiment III. Effects of stress (air exposure for 5min) on plasma 56 lactate in rainbow trout treated with nitrite (0.2mgN-NO2"/L) for 14 days. Figure 4.30. Experiment III. Effects of stress (air exposure for 5min) on plasma 56 ammonia in rainbow trout treated with nitrite (0.2mgN-NO2"/L) for 14 days. Figure 4.31. Experiment III. Effects of stress (air exposure for 5min) on plasma 57 total protein in rainbow trout treated with nitrite (0.2mgN-NO27L) for 14 days. viii L I S T O F T A B L E S page Table 3.1. Nonylphenol concentration in the water, determined by 22 capillary electrophoresis. The chemical exposure of fish was started on December 2nd. Table 3.2. Quality of the fish tank water in Experiment I, II and III. 22 Table 3.3. Concentration of ammonia in water to which stock solution of 24 known concentration (0.9mg/L=100%) of ammonia chloride was administered by peristatic pump (3mL/min). ix L I S T O F A B B R E V I A T I O N S ACTH adrenocorticotropic hormone APnEO alkylphenol ethoxylate BAF bioaccumulation factor CRH corticotropin-releasing hormone DMSO dimethylsulfoxide EDC endocrine disrupting chemical ELISA enzyme-linked immunosorbent assay EO ethoxylate GBP glococorticosteroid binding protein HPI hypothalamo-pituitary-interrenal HPT hypothalamo-pituitary-thyroid Kow octanol-water partition coefficient LC50 50% lethal concentration NPnEO nonylphenol ethoxylate PAH polycyclic aromatic hydrocarbon PCB polychlorinated biphenyl SD standard deviation T 3 triiodothyronine T 4 thyroxine TRH thyrotropin releasing hormone TSH thyrois stimulating hormone P R E F A C E Human ingenuity has given us a vast variety of synthetic chemicals. In the past few decades, there has been increasing concern about anthropogenic chemicals (xenobiotics) that are capable of impairing the delicately-tuned endocrine functions. The chemicals are named endocrine disrupting chemicals (EDCs). EDCs and their effects on organism are of particularly great concern because they are potent, persistent, and their effects can be irreversible. One of the initial indications of actions by EDCs was seen in cases of reproductive failures in wild animals such as lake trout, river otters, bald eagles and other fish-eating birds (Carson 1962). Field workers noticed that these animals suffered from a variety of deficiencies, including precocious sexual development, eggshell thinning, and impaired incubation. A series of studies suggested that the exposure to polychlorinated biphenyls (PCBs) and agricultural chemicals to be responsible for the adverse responses of these animals (Spitsbergen et al. 1991; Wren 1991; Giesy et al. 1994), even though many of the alleged chemicals had already been eliminated from use. Nonylphenol is a degraded product of nonylphenol ethoxylates (NpnEO) that are extensively used as surfactant. It is much more persistent than its mother product and is lipophilic, as a result nonylphenol tends to bioconcentrate. A series of toxicology studies on the endocrine disrupting actions of nonylphenol have been conducted since Soto et al. (1991) accidentally found that nonylphenol mimics estrogen. At present, many reports have investigated the estrogenic action of nonylphenol, although other xi possible effects are less studied. The focus of this work is to study the effects of nonylphenol on one of the fundamental endocrine functions of animals and humans; the stress response. ACKNOWLEDGEMENT I would like to show my gratitude to two of the greatest scientists on this planet; Dr. Dave Randall and Dr. Izuru Kakuta for their guidance and inspiring discussion through my research. I appreciate the patient support from Dr. Les Lavkulich who greatly helped me fulfill the master's degree. Gratefulness is also given to Dr. David Chen, Dr. Philip Britz-McKibbin and Junji Kobayashi, who kindly performed measurement of nonylphenol in water by capillary electrophoresis. I thank Dr. George Iwama and his lab members for generously allowing me to use their microplate reader for analyzing Cortisol and thyroid hormones. Thank you to Bev Wicks who put up with sharing the lab with me for this past year. In addition to a number of rainbows who have given their lives to this research, I would like to finally thank my friends and family for being so understanding, supportive and encouraging. Thank you all very much!! xiii CHAPTER 1. INTRODUCTION 1.1. Nonylphenol 1.1.1. Production and use of nonylphenol Alkylphenol polyethoxylates (APnEOs) are non-ionic surfactants that are widely used in industry and households. The APnEOs were first introduced during the 1940s. Nonylphenol ethoxylates comprise a significant portion of the surfactant chemical market, accounting for 73.6% of the production in 1980 (Cahn and Lynn 1983). Nonylphenol ethoxylates (NPnEOs) are used for a variety of domestic and industrial purposes. Five sectors of industry are believed to be responsible for the discharge of these chemicals; pulp and paper manufacturing, petroleum production, C 9 H 1 9 household/industrial/institutional cleaning, textile manufacturing and leather manufacturing (Reeviewed by Maguire 1999). They are eventually degraded into the F i &> T e 1 • 1 • Nonylphenol more stable and toxic nonylphenol once released in the aquatic environment. Nonylphenol (Figure 1.1) itself has been used in aminocarb (4-dimethylamino-3-methylphenyl-N-methylcarbamate) insecticide sprays against the 1 spruce budworm in Canada. Nonylphenol has also been reported to leach from polyvinyl chloride tubing for milk processing (Junk et al. 1974) and plastics used in food packaging (Gilbert etal. 1992). The total supply of nonylphenol in Canada in 1993 was estimated as approximately 7.0 kilotones (Shang et al. 1999). 1.1.2. Nonylphenol in the aquatic environment NPnEOs with more than 8 ethoxylate (EO) units (the most common commercial products) are readily degraded anaerobically in the treatment systems, usually with >92% efficiency (Figure 1.2; Reviewed by Servos 1999). The primary products that eventually end up in the aquatic environment are NP1EO and NP2EO, and they are significantly less biodegradable than their mother compounds. These metabolites are partially removed from the water body by adsorption onto sludge. They are then further degraded to nonylphenol, which accumulates in the digested sludge (Reviewed by Maguire 1999). It is shown that nonylphenol is a predominant species in the sediment (Ahel et al. 1994). Relatively high levels of persistent metabolites of NPnEOs were detected in the natural water environment which receives effluents from sewage plants. The typical concentration of nonylphenol found in field studies ranges from less than 1 ppb in pristine environments up to 100 ppb in sites adjacent to sewage treatment plants (Ahel et al. 1994). In a particular study, however, 330 ppb of nonylphenol was found in sewage discharge into the river Aire in England, which reseives high inputs of 2 surfactants from the textiles industry (Blackburn and Waldock 1995). Up to 180 ppb of nonylphenol was also measured at 6 sites downstream from near the source to the river mouth. Once diffusing into the fish body, nonylphenol is stored mainly in the liver as well as the kidney, and excreted through the bile (Coldham et al. 1998; Lewis and Lech 1996). The value of log Kow for nonylphenol is determined as 4.2 (McLeese et al. 1981). A study about the bioaccumulation factor (BAF) of nonylphenol has shown that the chemical has a significant potential to bioaccumulate in freshwater organisms and birds (Ahel et al. 1993). 1.1.3. Toxicity of nonylphenol Acute toxicity According to Servos (1999), the toxicity of NPnEOs to most organisms generally increased as the length of the EO chain decreases. Acute toxicity test shows that 96-h LC50 of nonylphenol for rainbow trout is in a range of 221 to 250 ppb, while the values of NP8EO is as much as 4700 ppb. Estrogenic actions It was realized that nonylphenol was estrogenic by researchers (Soto et al. 1991) who observed proliferation of breast cancer cells in plastics tubes. They later discovered that growth of the cancer cells was caused by nonylphenol which was used 3 as a stabilizer in the plastic. Vitellogenin is the protein responsible for making egg yolk in oviparous female fish, and the biosynthesis of vitellogenin is stimulated by 1713-estradiol. Vitellogenin is produced in the liver, released into blood, and then modified and deposited as yolk in oocytes. Normally, little or no vitellogenin is found in the blood of male fish (Sampter and Jobling 1991). Male fish have a gene which can produce vitellogenin, but male fish ordinarily lack sufficient 1713-estradiol to trigger the vitellogenin-making gene. Since disorders in the reproductive system have become a major concern in both humans and wildlife, it has been suggested to use in vivo induction of vitellogenin synthesis as a biomarker for oviparous animals (Christiansen et al. 1998). The expression of vitellogenin driven by 1713-estradiol has been used for studying estrogenic effects in aquatic species. Induction of vitellogenin synthesis by nonylphenol has been observed in male fish (Ren et al. 1996; Schwaiger and Negele 1998; Korsgaad and Pedersen 1998; Miles-Richardson et al. 1999). Studies show that nonylphenol produces estrogenic effects in rainbow trout in vivo at concentrations well below the LC50 (Lech et al. 1996). It is reported that nonylphenol induce plasma vitellogenin in rainbow trout in vivo is approximately 150 ppb with 9 days exposure in water (Pedersen et al. 1999). Evidence of estrogenicity of nonylphenol can be explained by its chemical structure and properties. Computer-graphic overlays of nonylphenol and 1713-estradiol 4 reveal their structure resemblance (Leadley et al. 1998). RMS7 (root mean square) deviation for the two compouds is 0.36 A, while the values lower than 1.0 A are considered to indicate close structural similarity in three-dimentional structure between any two compounds compared (Wiseman et al. 1997). In fact, nonylphenol is reported to compete with 1713-estradiol for binding to the rainbow trout estrogen receptors (White et al. 1994). White et al. (1994) reported that nonylphenol has intrinsic estrogenic activity, and is 103 -104 less potent than 1713-estradiol. Effects on Cortisol stress responce Although nonylphenol has been demonstrated to be estrogenic, there is little known about the impact of this chemical on other endocrine functions. Since natural estrogen, 1713-estradiol, plays many different roles in fish, it is quite reasonable that the environmental xenoestrogen, nonylphenol, influences other important physiological processes besides reproduction. Cortisol is one of the glucocorticoid hormones and has very similar structure to that of estradiol (Figure 1.3). Being synthesized from cholesterol, they are both steroid hormones and possess a lipophilic nature. Increases of plasma Cortisol level are commonly observed in fish exposed to a variety of stresses. Hontela et al. (1992) studied fish in several sites, that were chronically polluted by high levels of chemicals such as polycyclic aromatic ' The RMS deviation is determined by measuring each atom's distance from the position in the other structure, squaring this distance and averaging these results, and calicurating the square-root of this average. It is widely used for comparison of complicated molecule structures. 5 hydrocarbons (PAHs), PCBs, and mercury2. They found that the contaminated fish were unable to increase their serum Cortisol in response to the acute stress of capture, compared to fish from reference sites. This means that contaminated fish will display either retarded or reduced response to acute stress. Effects on thyroidfunctions Little is known about the effects of nonylphenol on Thyroid hormones ( T 3 and T 4 ) . It is said that 1713-estradiol suppresses hepatic synthesis of T 3 (Leatherland 1994) and has no effect on the plasma T 4 level (Cyr and Eales 1990). Nonylphenol has been shown to have effects on the growth of fish. Ashfield, et al (1998) have reported that juvenile rainbow trout exposed to nonylphenol (10 ppb for 35 days) showed significant decreases in body weight and folk length 55 days after exposure was terminated. Rainbow trout exposed to 1713-estradiol also have reduced weight and length (Johnstone et al. 1978). In addition, a field study by Leatherland and Sonstegard (1981) found thyroid hyperplasia and goitre in fish that were chronically polluted by industrial effluents. These observations suggest that it is possible that specific kinds of xenobiotics impair thyroid gland function, including production of thyroid hormones. Fairchild et al. (1999) reported the coincidence of a decline in a salmon run and application of the insecticide Matacil 1.8D, that occurred in 1997. Matacil 1.8D consists of aminocarb as the carbamate insecticide and nonylphenol as the primary 2 These compounds are suggested to be endocrine disrupting chemicals O^ ingerman et al. 1996). 6 solvent. They studied each tributary of the river with this salmon run and found a significant relationship between the catch for each tributary and how much area was sprayed within that drainage basin. They also point out that there was an unusualy heavy salmon smolt mortality in 1997. It has been suggested that estrogenic compounds, including nonylphenol, may negatively influence smoltification (Madsen et al. 1997). Therefore, it is highly probable that the presence of nonylphenol in the environment was responsible for the poor survival rate in salmon smolt and the subsequent migration stock of salmon. 7 \ / -0-(CH 2CH 20) n-H NPnEO - 0 - ( C H 2 C H 2 0 ) n . i - H NP(n-l)EO Anaerobic R \ / -0-(CH 2CH 20) 2-H NP2EO Anaerobic \ / -0 -CH 2 CH 2 OH NP1EO Anaerobic \ / -OH Nonylphenol Figure 1.2. Degradation of nonylphenol polyethoxylate (NPnEO). -R=-C 9Hi 9. 1.2. Nitrite Nitrite (NOV) is a naturally occurring anion in fresh and saline waters. Nitrite is intermediate in oxidation of ammonia to nitrate, and its concentration in natural environment is typically less than 0.005mg/L (Lewis and Morris 1986). However, today its concentration is increasing due to the extensive use of fertilizers in agriculture. Nitrite changes hemoglobin to methemoglobin which does not carry oxygen, potentially causing anoxia in fish and other organisms. Although nitrite is toxic to fishes at relatively low concentration, the actual effects of nitrite is dependent on the water conditions such as pH and concentration of chloride and calcium. In aqueous solution, nitrite exists in the following equilibrium. N02" + H + HN02(aq) (1) Since this equilibrium is dependent on pH, so is the toxicity of nitrite. It has been show that the acute (96-h LC505) toxicity of NCV decreases, as pH increases (Russo et al. 1981). Chloride in the external environment offsets the toxicity of nitrite by competing with nitrite for uptake through the chloride cells of the gills (Russo and Thurston 1977). 1.3. Dimethylsulfoxide (DMSO) Due to low solubility of nonylphenol in water'' (MacKay et al. 1992), 3 Ranges from 0.5 mgN-N027L (when [Cl"]=1.4 mg/L, [Ca2+]=8mg/L, and pH=6.2) to 12.6 mg N-N02" IL (when [Cl']=40.8mg/L, [Ca2+]=50mg/L, and pH=7.7). 4 Approximately 5 ppm. 10 dimethylsulfoxide (DMSO) was used as organic solvent in these experiments. DMSO is a chemical that has been used primarily as an industrial solvent. In addition to ethanol and acetone, it is used as an organic solvent for water-insoluble chemicals in aquatic bioassays. Physicians have also been using DMSO as a vehicle to help absorb other therapeutic agents through the skin, although the clinical effectiveness of DMSO is controversial (Herschier and Jacob 1980). A toxicity test reports the 4-d and 12-d LC50 of DMSO for the grass shrimp are 22.57 and 12.33 g/L. respectively (Rayburn and Fisher 1997), and DMSO was shown to be less toxic in terms of LC50, than both ethanol and acetone. 11 C H A P T E R 2. S T R E S S IN F I S H 2.1. Stress response in fish Fish have a series of physiological responses to physical stress. Firstly, upon perception of the stress by the nervous system, catecholamines (adrenaline and noradrenaline) are released into the bloodstream by the chromaffin cells. The secretion of these hormones initiates an alarm reaction, enabling the fish to deal with threatening situations quickly. Cortisol, as well as corticosterone that exists in relatively low concentrations, also have an important role in the physiological response to stress for all vertebrates. Following to the actions of these hormones, blood glucose levels, red blood cell counts, heart and ventilation rates all increase. Cortisol provides an important backup for catecolamine, ensuring glucose supplies when the body is under stress and in need of extra energy (Solomon et al. 1993). 2.2. Regulation of Cortisol and thyroid hormones 2.2.1. Thehypothalamo-pituitary-interrenal (HPT) axis Cortisol is synthesized and secreted by steroidogenic cells located in the interrenal tissue of fish. They use cholesterol as a substrate for the synthesis of steroid hormones, including Cortisol and estradiol. The primary stimulant of the steroidogenic cells is adrenocorticotropic hormone (ACTH) released from the pituitary gland. Other 12 factors that possess corticotropic activity in fish include thyroxine (T4) and growth hormone (Schreck et al. 1989). The activity of the cells that synthesize ACTH, pituitary corticotropes, is regulated by the corticotropin releasing hormone (CRH) and other hypophyseal peptides (Fryer 1989). Production of ACTH and CRH are also regulated by a negative feedback exerted by C o r t i s o l (Figure 2.1). 2.2.2. Functions of Cortisol. Cortisol results in the production of glucose from short-chain precursors by hepatic gluconeogenesis. Gluconeogenesis is also stimulated by adlenaline, thyroxine, glucagon and growth hormone. Cortisol is also known to have effects on reproduction through inhibition of reproductive hormones (Carragher and Sumpter 1990). Studies show that starvation has little or no effect on Cortisol and glucose level in fish plasma (White and Fletcher 1986; Sumpter etal. 1991). Physical stresses, such as handling, chasing, and air exposure, typically increase fish plasma Cortisol levels. Glucose and lactate concentrations in plasma also increase, but that is known to be more gradual; reaching their peaks, in case of handling stress, about 2 and 4 hours later than Cortisol, respectively (Vijayan et al. 1994). It has been shown that exposure to chemical pollutants such as heavy metals, pesticides and acid water also increase blood Cortisol levels (Webb and Wood 1998; Bennett and Wolke 1987; Brown etal. 1989). Studies of the effects of cadmium5 observed return of plasma Cortisol levels in 3 Cadmium is suggested to be endocrine disrupting (Ricard et al. 1998; Pundir and Saxena 1992). 13 fish to normal levels following initial rise, after several weeks exposure to the metal (Reviewed by Hontela 1997). It is suggested that this pattern of Cortisol response to the chemical stress may reflect an exhaustion stage of the HPI axis by a continuous application of stress, following an alarm stage and a resistance stage (Selye 1973). 2.2.3. The hypothalamo-pituitary-thyroid (HPT) axis Thyroxine (T4) is secreted by the thyroid gland, and a part of the production is converted to triiodothyronine (T3) in plasma and liver. The production of T 4 is regulated by thyroid stimulating hormone (TSH) or thyrotropin, whose synthesis and secretion in pituitary gland are regulated by thyrotropin releasing hormone (TRH). Food intake also stimulates the production of T3 and T 4. Elevated levels of thyroid hormones suppress the production of TSH in the pituitary and TRH in hypothalamus (negative feedback; Figure 2.2). T 4 is produced in greater quantities than T3, but is approximately a quarter as potent (McNabb and King 1993). 2.2.4. Functions of thyroid hormones Thyroid hormones are important in tissue growth and development, and also play an important role in salmonid smolting, as well as in amphibian metamorphosis. Increased thyroid hormone production is observed consistently in smolting fish (Dickhoff 1993). The hormones act synergistically with other hormones to influence the rate of smolt development, rather than triggering smolting (Dickhoff and Sullivan 1987). 14 Thyroid hormones have been much less extensively monitored than plasma Cortisol in fish in response to stress. Plasma T3 and T4 levels are reported to decrease in fish exposed to heavy metals and pesticides (Ricard et al. 1998; Yadav and Singh 1986). There are some suggestions that circulating T3 and T 4 may be affected by various forms of stress although this response appears to be dependent on the particular type of imposed stress (Brown et al. 1989). 2.3. Response of fish to secondary stress A series of studies were conducted to examine effects of xenobiotic exposure on fish response to additional stresses. Gill et al. (1993) exposed eel to cadmium and then applied them a controlled stressor (1 min exposure to CO2 bubbles). They detected a significant rise in plasma Cortisol in fish exposed to the heavy metal and they still had an ability to further raise the plasma C o r t i s o l , although the change was small. Impaired ability to elevate plasma Cortisol in response to acute sampling stress was observed in fish exposed PCBs (Quabius et al. 1997). Hontela et al. (1992) argued that the observation of impaired stress response in polluted fish indicates that under chronic exposure to the chemicals including PAHs and PCBs , the normal responsiveness of the HPI axis to acute stress is impaired, possibly as a result of prolonged hyperactivity of the axis. To test which stage of HPI axis is impaired, Brodeur et al. (1997) assessed the response of the interrenal tissue to an ACTH pulse in vitro. They found an impaired response to ACTH, and diminished capacity to synthesize Cortisol in yellow perch 15 sampled at sites contaminated by heavy metals in Northern Quebec and mixtures of organic contaminants and heavy metals in the St. Lawrence River (Hontela et al. 1997). Quabius et al. (1997) exposed fish dietary to PCB and found direct toxic effects of the xenoestrogen on the interrenal cells in fish. These results suggest that the functional impairment of HPI axis by exposure to the pollutants is attributed to the impairment of the interrenal tissues. 16 Stress i ACTH t Negative feedback I (Inhibition) Target cells Figure 2.1. Schematic representation of hypothalamo-pituitary-interrenal (HPI) axis. 7f Pituitary Negative feedback (Inhibition) TSH T • I T 4 t Target cells Figure 2.2. Schematic representation of hypohtalamo-pituitary-thyroid (HPT) axis. CHAPTER 3. MATERIALS AND METHODS 3.1. Objective More than three decades ago Carson (1962) pointed out the potential relationship between observed reproductive failures in wildlife and use of agricultural chemicals. Since that time, researchers have investigated incidents in wildlife such as decreases in clutch size (Woodward et al. 1993), egg shell thinning (Hunt and Hunt 1977), and defective sex organs (Colborn et al. 1996), through extensive field and experimental studies. Evidence has been gathered to suggest that the failures were caused by exposure to synthetic chemicals. The chemicals interfere with proper function of the endocrine system, and they are termed endocrine disrupting chemicals (EDCs). Consequently, extensive studies were conducted to reveal EDCs' impacts on reproductive systems. Just as hormones that operate in very low concentrations in a body, EDCs are shown to affect the endocrine system when in very minute amount, although in many cases manifestation of EDCs require a very long time (Colborn et al. 1996). Since discovered as being estrogenic, nonylphenol has been studied looking of its effects on reproductive functions, especially in relation to the induction of vitellogenin in male fish. However, there have been relatively fewer studies on its impact on the other functions of the endocrine system, including stress mediation and thyroid hormone regulation. The objective of this study is to contribute to further understanding of the 19 effects of nonylphenol, focusing on its impact on the endocrine function of stress response. Cortisol is synthesized through similar pathways and thus has a resemble structure as 17 B-estradiol. Therefore, it is likely that estrogenic nonylphenol affects regulation of Cortisol. The impact of nonylphenol exposure on thyroid hormones is also studied, since previous studies (Fairchild et al. 1999; Aahfield et al. 1998; Letherland and Sonstregard 1981) have suggested that thyroid functions may be impaired by exposure to nonylphenol. 3.2. Experimental protocol 3.2.1. Experiment I: Short term exposure to nonylphenol Chemicals. A group of 8 rainbow trout (average weight ± SD: 243 ± 44.6) was divided into two subgroups (n=4) and were exposed to nonylphenol (technical mixture. Aldrich, Milwaukee, WI, USA) for 14 days in water contained in glass tanks covered with styrene foam sheets over black vinyl sheets. A control group (n=8) was also divided into subgroups (n=4) and received no chemical. Each subgroup was put in a tank individually and the volume of each tank was approximately 143 L. The nonylphenol was dissolved in dimethylsulfoxide (DMSO. Aldrich, Milwaukee, WI, USA) because of the hydrophobic nature of nonylphenol. Adequate amounts of the solution were dispensed into the water containing the fish so that the concentration of nonylphenol and DMSO would be lOOppb and 22.4ppm, respectively. Maintenance of the water quality. Throughout the experiment, filtering 20 systems with zeolite stones plus glass filters were used to prevent ammonia accumulation in water. In addition, the water in the tanks was exchanged by half the volume every three days to maintain the level of nonylphenol in the water. The concentrations of nonylphenol in the water were determined by means of capillary electrophoresis (Table 3.1), through the courtesy of Dr. David Chen and his group (Department of Chemistry, UBC). The concentration of ammonia in the water was measured according to the salicylate-hypochlorite assay for N H 4 + (Verdouw et al. 1978). Concentrations of other ions (Na+ and Ca2+) in water were determined using an atomic absorption spectrophotometer, and pH was measured by a pH meter (PHM 64, Radiometer-Copenhagen; Table 3.2). 21 Table 3.1. Nonylphenol concentration in the water, determined by capillary electrophoresis. The chemical exposure of fish was started on December 2nd. Sampling date Concentration (ppb) SD (ppb) Dec. 2, 1999 l ) 143 6.65 Dec. 5, 1999 2 ) 81.0 5.79 Dec. 5, 1999 ! ) 147 8.21 Dec. 8, 1999 2 ) 112 4.53 ' the water sample was taken 1 hour after the chemical was added. ' the water sample was taken 3 days after the addition of the chemical and immediately before the water exchange. Table 3.2. Quality of the fish tank water in Experiment I, II and III. Level in test water Experiment I and III Experiment II Na+ *> 4 mg/L 4mg/L Ca 2 + ] ) 1 mg/L lmg/L N H 4 + l ) 1.2 mg/L <62ug/L Temperature 2 ) 7.6 °C 6.5 °C pH 1 } 6.9 6.7 Values are average and , '^ =2,^ =10. 3.2.2. Experiment II: Time course of stress response in fish exposed to nonylphenol Fish (average weight ± SD: 44.3 ± 10.lg) were exposed to 3 concentrations; 4, 20, and 100 ppb of nonylphenol in separated tanks with a flow-through system. Each tank had a capacity of approximately 670 L. The two lower concentrations represent environmentally relevant concentrations. Stock solutions were prepared in bottles made of polycarbonate (18 L), by dissolving nonylphenol in DMSO and then in dechlorinated water. Two control groups were used; one received DMSO and another did not. The concentrations of DMSO were 20 ppm in each test water that the fish were exposed to. The onset of exposure was carried out instantly by adding the chemicals such that the concentration of the test water will be immediately obtained. Then the stock solutions were delivered continuously to the fish tanks to maintain the concentration, at the rate of 3 mL/min via a peristatic pump (Gilson, France). Water flow to each tank was at the rate of 4 L/min. These flow rates were checked everyday throughout the experimental period, to ensure that the intended concentrations of the test chemicals were maintained in each test water. The test water from the fish tanks was discharged through charcoal filters. The concentration of nonylphenol in water was not determined for this experiment. However, results of a set of tests using eosin Y (Fisher, Fair Lawn, NJ, USA) and known concentrations of ammonia chloride in place of nonylphenol (Sigma, St. Louis, MO, USA) indicate that the water successfully contained the intended concentrations of nonylphenol throughout the experiment (Table 3.3). 23 Table 3.3. Concentration of ammonia in water to which stock solution of known concentration (0.9mg/L=100%) of ammonia chloride was administered by peristatic pump (3mL/min). The time 0 designates when addition of the chemical by pump was started with instant administration of ammonia solution into the water. The expected values from calculated ammonia concentrations are 100% at any time. Time NFL,"1" concentration (%) 5 min 84 2h 91 6h 95 24 h 96 3.2.3. Experiment III: Short term exposure to nitrite Chemicals. A groups of 8 rainbow trout (average weight ± SD: 223 ± 42.3g) was divided into 2 subgroups and exposed to sodium nitrite (Sigma, St. Louis, MO, USA) in water contained in glass tanks covered with styrene foam sheets over black vinyl sheets. A control group (n=8) were also divided into subgroups (n=4) and received no chemical. Each subgroup was put in a tank individually and the volume of each tank was approximately 143 L. Adequate amount of stock solution was dispensed into the water so that the concentration of nitrite would be 0.2 mg N-NO2VL. Maintenance of the water quality was the same as in Experiment I (See 3.1.1). The concentration of nitrite in water was not monitored. 24 3.3. Fish Experiment I and III. Rainbow trout (Oncorhynchus mykiss) were obtained from Spring Valley Trout Hatchery (Langley, B.C., Canada) and held prior to experimentation in flowing, aerated, dechlorinated tapwater. They were fed a ration of 1% body weight of commercial trout pellets (Moore Clark, Vancouver, B.C., Canada) daily. All fish were starved from the onset to the end of experimentation. No mortality was observed throughout the experiment. Experiment II. Rainbow trout (Oncorhymchus mykiss) were obtained from Spring Valley Trout Hatchery (Langley, B.C., Canada) and held for 2 months prior to experimentation in flowing, aerated, dechlorinated tapwater. They were fed a ration of 1% body weight of commercial trout pellets (Moore Clark, Vancouver, B.C., Canada) daily. All fish were starved from 7 days prior to and until the end of experimentation. No mortality was observed throughout the experiment. 3.4. Stress application and sampling Experiment I and III. The fish were captured and exposed to the air for 5 min and then put back into a tank with water without nonylphenol or nitrite. Immediately before the stressor was given, the fish from the test tanks were sampled and designated as the pre-stress groups. The fish, to which the stress was applied, were captured again 2 hours after they were released into the water. The fish were euthenized by a sharp blow on the head, and body length and weight were measured. 25 Experiment II. Fish sampling (n=6-10) was conducted 2 hours, 3days, 7days, 14days and 21 days after the onset of chemical exposure. The stress test was conducted on day7 and dayl4. When sampled, the fish were euthenized by a sharp blow on the head and blood sample was taken and body length and weight were measured for each fish. To minimize the potential disturbing stress to the other fish, the fish that are used for the stress experiment were placed in cages in water beforehand. Groups of fish (n=6) were put in cages (24.7 L) with water flowing through, more than 48 hours prior to the application of the stress. The stress of air exposure was applied to fish by simply pulling up the cage into the air for 3 minutes. Fish were then put back into the tank again until they were sampled 0, 0.75, 2, 4 and 24 hours after the stress. The group of fish (n=6-10) that was not in a cage was sampled on the next day of stress experiment, due to potential impact of the sampling stress. They were designated control fish. 3.5. Blood collection and plasma separation The blood was drawn from the caudal vasculature into a 1 to 3 ml heparinized syringe. The blood was collected for the measurements of total hemoglobin and hematocrit, and then centrifuged at 5,000 x g for 5 min, and the plasma was collected (see below) and stored at -80°C until when they are analyzed as described below. 26 3.6. Measurement ofphysiological parameters Experiment I and III. The following physiological parameters were measured for each sample; hematocrit and total hemoglobin in blood, and Cortisol, glucose, free T3, total T3, freeT4, total T4, lactate, ammonia and protein in plasma. Experiment II. Total hemoglobin, plasma C o r t i s o l , and plasma glucose were measured in all the samples. Free T3, free T4, and protein were measured in all the samples except for the ones that were placed in cages and used for the stress experiment. 3.7. Blood analysis Physiological parameters. Blood was collected in heparinized micro-hematocrit capillary tubes and centrifuged at 13,460 x g for 5 min, and the hematocrit values were measured using a standard hematocrit reader. Total hemoglobin in blood was measured Total Hemoglobin Kit (Sigma Diagnostics, St. Louis, MO, USA). Plasma Cortisol was measured in duplicate for each sample using a Neogen Cortisol ELISA Kit (Medicorp Inc., Montreal, Quebec, Canada). Plasma glucose, lactate and ammonia were measured using Glucose, L[+]Lactate and Ammonia Kit (Sigma Diagnostics, St. Louis, MO, USA), respectively. Plasma free T3, total T3, free T4 and total T4 were measured mostly in duplicate for each sample using ELISA kits for free T 3 , total T 3 , free T 4 and total T 4 (Monobind, Costa Mesa, CA, USA). Plasma total protein was measured by colorimetric determination using Total Protein Regent (Sigma 27 Diagnostics, St. Louis, MO, USA) and bovine albumin (Sigma, St. Louis, MO, USA) as a standard. Statistical Analysis. The concentration of plasma Cortisol, thyroid hormones, glucose, lactate, ammonia and total protein were compared for significant differences using two-way student t-test. Statistical differences were taken at levels of /?=0.05 (represented by *or+) and/?=0.01 (represented by **or++). 28 CHAPTER 4. RESULTS 4.1. Experiment I: Short term exposure to nonylphenol Hematocrit and Total hemoglobin. Following the application of the acute stress, hematocrit and total hemoglobin both increased significantly in fish blood (Figure 4.1 and Figure 4.2). Cortisol. Both control and nonylphenol groups elevated their C o r t i s o l levels significantly in response to the acute stress (Figure 4.3). The fish exposed to nonylphenol showed a considerably altered response to the stress, producing significantly less Cortisol 2 hours after the application of the stress. Glucose. The pre-stress level of plasma glucose of the fish exposed to nonylphenol was significantly less than the control level (Figure 4.4). Both groups showed significant increase of plasma glucose levels following the stress. Thyroid hormones. Exposure to nonylphenol increased the initial level of the all thyroid hormones (Figures 4.5-4.8). There was no significant difference in levels of the hormones between pre-stress and post-stress, for any of the hormones. Lactate. Increased plasma lactate levels were observed following the stress with a significant difference of p<0.01 (Figure 4.9). There were no differences observed between the nonylphenol group and the corresponding control group, for either pre or post stress level. Ammonia. Both groups significantly increased their plasma ammonia levels following the stress (Figure 4.10). 29 Protein. The plasma protein levels did not change significantly following the stress (Figure 4.11). Nonylphenol exposure also had no effect. 4.2. Experiment II: Time course of stress response in fish exposed to nonylphenol Comparison between the control group and the DMSO group showed that there was no detectable effect of the low concentration (20 ppm) of DMSO on fish (Figures 4.12-4.20b). 4.2.1. Physiological status of the fish throughout the experimental period Total hemoglobin. Hemoglobin levels increased significantly (p<0.05) in nonylphenol (20 ppb) treated fish 2 hours after the instant onset of the chemicals exposure (Figure 4.12). Control and DMSO groups increased their hemoglobin concentrations through the experiment period. The effects of nonylphenol were not significant, although the highest nonylphenol concentration seemed to suppress hemoglobin levels after 3 weeks. Cortisol. The significant effect of the instant onset of nonylphenol exposure on plasma Cortisol levels was observed (Figure 4.13). Control group, including DMSO exposed group, both increased their Cortisol levels in plasma after 7 days. However, in general, the other three groups that were exposed to nonylphenol had no significant changes in Cortisol levels, keeping their levels as low as during the first period of the 30 experiment. The Fish exposed to 100 ppb of nonylphenol had, in fact, decreased Cortisol levels after 21 days. Glucose. Plasma glucose level increased significantly (p<0.01) in the fish 2 hours after being exposed to lOOppb of nonylphenol instantly (Figure 4.14). The same group had decreased their glucose levels considerably (p<0.01) after 2weeks and 3 weeks of exposure. Thyroid hormones. No significant changes were observed in plasma free T 3 level in the fish 2 hours after the instant exposure onset (Figure 4.15). The high level (100 ppb) of nonylphenol exposure decreased free T 4 levels significantly by 2 hours (Figure 4.16). Fish exposed to 100 ppb of nonylphenol significantly decreased their plasma levels of both free T 3 and free T 4 21 days after initiation of the chemical exposure. On the other hand, lower concentrations of nonylphenol had a relatively minor effect on the plasma levels of the thyroid hormones. There was an overall increase in free T 3 in fish on day 14. 4.2.2. Stress experiment. Total hemoglobin. On day 7, no systematic change was observed in total hemoglobin concentrations (Figure 4.17a, b). On day 21, significant increase of the levels were observed in 2 hours in nonylphenol treated fish, and in 0.75 hour in control and DMSO groups. Cortisol. Elevation of plasma Cortisol was observed in all groups of fish, 31 following application of the acute stress. On day 7 (Figure 4.18a), no significant difference was observed between the five groups, in terms of the plasma Cortisol stress response. A significant increase was observed 0.75 hour after the stress for each group of fish, and the levels of Cortisol then gradually decreased, reaching the pre-stress level after 24 hours. On the other hand, a somewhat different observation was made for the day 21 measurement. The high (100 ppb) nonylphenol group fish were, in fact, not only unable to increase their Cortisol levels but they did not decrease the levels to the normal state even after 24 hours (Figure 4.18b). This is clearly demonstrated in Figure 4.19, in a comparison between the control and 100 ppb nonylphenol group. Glucose. A slow and prolonged increase was observed in glucose levels in response to the acute stress. On day 7, the control group increased plasma glucose concentration significantly in 2 hours (Figure 4.20a). The nonylphenol group (100 ppb), on the other hand, increased the level in 4 hours. On day 21, the plasma glucose levels in fish exposed to 100 ppb levels of nonylphenol were significantly lower overall than the control levels (Figure 4.20b). Elevation of plasma glucose concentration in any group of fish was not observed to be as significant as in the results from the day 7 experiment. 4.3. Experiment III: Short term exposure to nitrite Hematocrit and Total hemoglobin. The nitrite exposure did not affect the 32 hematocrit and total hemoglobin concentration in blood (Figure 4.21 and Figure 4.22). They increased following application of the stress, in both control and nitrite treated fish. Cortisol. The pre-stress levels of plasma Cortisol were not significantly different in control and nitrite treated fish (Figure 4.23). Nitrite exposure did not affect the normal Cortisol increase following the stress. Glucose. Nitrite exposure did not affect the pre-stress levels of plasma glucose (Figure 4.24). Nitrite did not affect the elevation of the glucose level following the stress. Thyroid hormones. Exposure to nitrite increased the initial level of the all thyroid hormones (figure 4.25-4.28). There was no significant difference in plasma levels of the hormones between pre-stress and post-stress. Lactate. The lactate pre-stress level was significantly (p<0.05) lower in nitrite treated group, than the level of control group (Figure 4.24). The increase of plasma lactate was not significant in nitrite group. Ammonia. Nitrite exposure did not affect the pre-stress levels of plasma ammonia. Both groups significantly increased their plasma ammonia levels following the stress (Figure 4.30). Exposure to nitrite resulted in as longer increase in plasma ammonia than control group levels. Protein. No significant change was observed for plasma protein (Figure 4.31). 33 Control Nonylphenol Figure 4.1. Experiment I. Effects of stress (air exposure for 5min) on hematocrit in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ±SD. Values with superscript are significantly different than either pre-stress level (*p<0.05, **p<0.0\) or corresponding control level (+/?<0.05). 12 -r 10 5 "Si 2 8 .a o o E 4 C o n t r o l N o n y l p h e n o l Figure 4.2. Experiment I. Effects of stress (air exposure for 5min) on total hemoglobin in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average +SD. Values with superscript are significantly different than pre-stress level (*/K0.05). 34 1200 1000 800 f 600 tn t o O 400 200 Control Nonylphenol Figure 4.3. Experiment I. Effects of stress (air exposure for 5min) on plasma Cortisol in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than either pre-stress level (**/?<0.01) or corresponding control level (++/?<0.01). 35 200 — 150 5 E 0) (A O U 3 • pre-stress • post-stress 100 Control Nonylphenol Figure 4.4. Experiment I. Effects of stress (air exposure for 5min) on plasma glucose in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than pre-stress level (*/?<0.05, **p<0.0\). Q. CO a> 9> 15 12.5 10 7.5 5 2.5 • pre-stress m post-stress Control Nonylphenol Figure 4.5. Experiment I. Effects of stress (air exposure for 5min) on plasma free T3 in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than corresponding control level (+p<0.05). 36 400 • pre-stress H post-stress 300 200 100 Control Nonylphenol Figure 4.6. Experiment I. Effects of stress (air exposure for 5min) on plasma total T 3 in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average + SD. Values with superscript are significantly different than corresponding control level (+p<0.05). 10 8 4 •S, 6 "3) c 0) • pre-stress • post-stress Control Nonylphenol Figure 4.7. Experiment I. Effects of stress (air exposure for 5min) on plasma free T 4 in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than corresponding control level (+/K0.05). 37 12 • pre-stress 1 post-stress Control Nonylphenol Figure 4.8. Experiment I. Effects of stress (air exposure for 5min) on plasma total T 4 in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than corresponding control level (+/K0.05). Figure 4.9. Experiment I. Effects of stress (air exposure for 5min) on plasma lactate in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than pre-stress level (**/?<0.01). 38 20 n pre-stress B post-stress Control Nonylphenol Figure 4.10. Experiment I. Effects of stress (air exposure for 5min) on plasma ammonia in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than pre-stress level (*/K0.05, **/K0.01). 10 • pre-stress post-stress Control Nonylphenol Figure 4.11. Experiment I. Effects of stress (air exposure for 5min) on plasma total protein in rainbow trout treated with nonylphenol (lOOppb) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. 14 12 10 6 t 2hours 3days 7days 14days Time after exposure onset 21days Figure 4.12. Experiment II. Total hemoglobin in rainbow tout treated with DMSO and nonylphenol (4-100 ppb). Each value (n=8-10) is the average ± SD. Values with superscript are significantly different than either control level (*/?<0.05) or initial control level (t=2h, +/?<0.05, ++/K0.01). 40 •Control DMSO 1 Low a Mid "High 2hours 3days 7days 14days Time after exposure onset 21days Figure 4.13. Experiment II. Plasma Cortisol in rainbow tout treated with DMSO and nonylphenol (4-100 ppb). Each value (n=8-10) is the average ± SD. Values with superscript are significantly different than either control level (*/?<0.05, **p<0.01) or initial control level (t=2h, +/X0.05, ++/K0.01). 41 2hours 3days 7days 14days 21 days Time after exposure onset Figure 4.14. Experiment II. Plasma glucose in rainbow tout treated with DMSO and nonylphenol (4-100 ppb). Each value (n=8-10) is the average ± SD. Values with superscript are significantly different than either control level (*/?<0.05, **/?<0.01) or initial control level (t=2h, +p<0.05, ++/K0.01). 42 2hours 3days 7days 14days Time after exposure onset 21days Figure 4.15. Experiment II. Plasma free T 3 in rainbow tout treated with DMSO and nonylphenol (4-100 ppb). Each value (n=8-10) is the average ± SD. Values with superscript are significantly different than either control level (**/?<0.01) or initial control (t=2h, +/K0.05, ++p<0.01). 43 Time after exposure onset Figure 4.16. Experiment II. Plasma free T 4 in rainbow tout treated with DMSO and nonylphenol (4-100 ppb). Each value (n=8-10) is the average ± SD. Values with superscript are significantly different than either control level (*/?<0.05, **p<0.0\) or initial control level (t=2h, +/K0.05,). 44 • Control • DMSO 0 0.75 2 4 24 Time after stress onset Figure 4.17a. Experiment II. Effects of stress (air exposure for 3 min) on total hemoglobin in rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 7 days. Each value (n=6) is the average ± SD. Values with superscript are significantly different than either control level (*/?<0.05, **/?<0.01) or corresponding pre-stress level (t=0h, +p<0.05). 45 0 0.75 2 4 24 Time after stress onset Figure 4.17b. Experiment II. Effects of stress (air exposure for 3 min) on total hemoglobin in rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 21 days. Each value (n=6) is the average ± SD. Values with superscript are significantly different than either control level (*p<0.05, **p<0.0\) or corresponding pre-stress level (t=0h, +/?<0.05, ++p<0.0\). 46 Time after srtess onset Figure 4.18a. Experiment II. Effects of stress (air exposure for 3 min) on plasma Cortisol rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 7 days. Each value (n=6) is the average ± SD. Values with superscript are significantly different than either control level (*/?<0.05) or corresponding pre-stress level (t=0h, +/?<0.05, ++/K0.01). 47 0 0.75 2 4 24 Time after srtess onset Figure 4.18b. Experiment II. Effects of stress (air exposure for 3 min) on plasma Cortisol rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 21 days. Each value (n-6) is the average ± SD. Values with superscript are significantly different than either control level (*/?<0.05, **/?<0.01) or corresponding pre-stress level (t=0h, +/?<0.05, ++/7<0.01). 48 450 0 -I 1 1 1 1 1 1 1 0 4 8 12 16 20 24 28 Time after stress onset (h) Figure 4.19. Experiment II. Effects of exposure to high level (lOOppb) of nonylphenol on stress (air exposure for 3 min) response of plasma Cortisol in rainbow trout. Values are the average and selected from Figure 4.18b. 49 350 + • Control [ I DMSO • Low I Mid • h i g h 0.75 2 4 Time after srtess onset 24 Figure 4.20a. Experiment II. Effects of stress (air exposure for 3 min) on plasma glucose in rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 7 days. Each value (n=6) is the average ± SD. Values with superscript are significantly different than either control level (*/?<0.05, **/?<0.01) or corresponding pre-stress level (t=0h, +/?<0.05, ++p<0.01). 50 • Control I DMSO 0 0.75 2 4 24 Time after srtess onset Figure 4.20b. Experiment II. Effects of stress (air exposure for 3 min) on plasma glucose in rainbow tout treated with DMSO and nonylphenol (4-100 ppb) for 21 days. Each value (n=6) is the average + SD. Values with superscript are significantly different than either control level (*/?<0.05, **/?<0.01) or corresponding pre-stress level (t=0h, +/K0.05, ++p<0.01). 51 70 60 _ 50 ¥ 40 o | 30 X 20 10 0 n pre-stress 8 8 post -s t ress Cont ro l Nitrite Figure 4.21. Experiment III. Effects of stress (air exposure for 5min) on hematocrit in rainbow trout treated with nitrite (0.2mgN-NO2_/L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value is (n=4) the average ± SD. Values with superscript are significantly different than pre-stress level (V<0.05). 12 10 2 8 6 4-Control • p r e - s t r e s s • p o s t - s t r e s s N i t r i t e Figure 4.22. Experiment III. Effects of stress (air exposure for 5min) on total hemoglobin in rainbow trout treated with nitrite (0.2mgN-NO2"/L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average + SD. Values with superscript are significantly different than pre-stress level (*/?<0.05). 52 Figure 4.23. Experiment III. Effects of stress (air exposure for 5min) on plasma Cortisol in rainbow trout treated with nitrite (0.2mgN-NO2"/L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than pre-stress level (**p<0.01). 200 150 • pre-stress i l post-stress 5 E "ST 100 </> o o 3 o 50 Control Nitrite Figure 4.24. Experiment III. Effects of stress (air exposure for 5min) on plasma glucose in rainbow trout treated with nitrite (0.2mgN-NO2"/L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value is the average ± SD. Values with superscript are significantly different than pre-stress level (•/K0.05). 53 15 i £ 5 1 2.5 4 • pre-stress II post-stress Control Nitrite Figure 4.25. Experiment III. Effects of stress (air exposure for 5min) on plasma free T 3 in rainbow trout treated with nitrite (0.2mgN-NO2"/L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than corresponding control level (+p<0.05). 400 • pre-stress H post-stress 300 c CO H 200 4 ra o 100 0 -I 1 1 l , ' 1 1 i Control Nitrite Figure 4.26. Experiment III. Effects of stress (air exposure for 5min) on plasma total T 3 in rainbow trout treated with nitrite (0.2mgN-NO2_/L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than corresponding control level (+p<0.05). 54 10 • pre-stress I I post-stress •5, 6 Control Nitrite Figure 4.27. Experiment III. Effects of stress (air exposure for 5min) on plasma free T 4 in rainbow trout treated with nitrite (0.2mgN-NO2*/L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than corresponding control level (+p<0.05). 12 • pre-stress • post-stress 3 "3) * 6 4-(0 o Control Nitrite Figure 4.28. Experiment III. Effects of stress (air exposure for 5min) on plasma total T 4 in rainbow trout treated with nitrite (0.2mgN-NO2_/L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than corresponding control level (+/K0.05). 55 250 200 |> 150 S 100 o RJ - J 50 • pre-stress 1 post-stress Control Nitrite Figure 4.29. Experiment III. Effects of stress (air exposure for 5min) on plasma lactate in rainbow trout treated with nitrite (0.2mgN-NC«27L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. Values with superscript are significantly different than pre-stress level (**/?<0.01) or corresponding pre-stress level (+/?<0.05). 20 3 15 .« 10 4 o E E < • pre-stress H post-stress + ** * i Control Nitrite Figure 4.30. Experiment III. Effects of stress (air exposure for 5min) on plasma ammonia in rainbow trout treated with nitrite (0.2mgN-NO2"/L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average + SD. Values with superscript are significantly different than pre-stress level (**/?<0.01) or corresponding pre-stress level (+/?<0.05). 56 Control Nitrite Figure 4.30. Experiment III. Effects of stress (air exposure for 5min) on plasma total protein in rainbow trout treated with nitrite (0.2mgN-NO2"/L) for 14 days. Post-stress levels were measured 2 hours after the stress application. Each value (n=4) is the average ± SD. 57 CHAPTER 5. DISCUSSION Experiment II suggests that the acute effects of nonylphenol are not significant. It has been found, however, that 14 days long (Experiment I) and 21 days (Experiment II) long exposure to lOOppb level of nonylphenol impair the normal increase of Cortisol plasma Cortisol following the air exposure stress. Similar response was observed in both field (Hontela et al. 1992; Jardine et al. 1996; Norris et al. 1999; Lappivaara and Oikari 1999) and experiment (Quabius et al. 1997) in fish that were chronically exposed to such chemicals as PCBs, polycyclic aromatic hydrocarbons (PAHs), mercury and cadmium. There are three possible explanations for the impaired Cortisol response to stress: i) the function of interrenal tissue to produce Cortisol is inhibited; ii) the activation of the interrenal tissue by ACTH is inhibited; iii) the rate of circulating Cortisol breakdown is enhanced. It is not clear if nonylphenol affects all or any of these functions. The mechanisms of the abnormal Cortisol response reported here should be further elucidated. It would be useful, for example, to quantify the functional integrity of the interrenal tissue by a standardized functional test such as in vitro ACTH challenge test. It is also possible that xenobiotics such as nonylphenol can affect plasma Cortisol levels by altering the availability and affinity or metabolism of the hormones' carrier proteins, such as plasma glucocorticosteroid binding protein (GBP). Cortisol, as well as other steroid hormones, is not stored within the cells in which it is synthesized, but rather it diffuses across the cell membranes as it is synthesized. Therefore, secretion 58 of the hormone into plasma is dependent on the rate of its biosynthesis (Atterwill and Flack 1992). Once being released into plasma, Cortisol either circulates free or bound to the carrier protein. The free hormone only can interact with the receptors of target cells, and the bound form is metabolized at a slower speed than the free hormone. Thus, an alteration in the availability and affinity of the carrier proteins potentially affect the plasma concentrations of the hormone together with its physiological actions. Therefore, measurement of GBP concentration in plasma would be worthwhile in order to know whether or not this is the case. In Experiment I, plasma glucose levels of the fish treated with nonylphenol showed a normal increase similar to that observed in control fish. Cortisol secretion stimulates gluconeogenesis and eventually contributes to glucose level increase in blood. Adrenaline and glucagon are also responsible for gluconeogenesis. Therefore, it is suggested that nonylphenol exposure did not affect the function of chromaffin cells by which adrenaline, a stress response indicator, is produced. On the other hand, in Experiment II, The high level of nonylphenol that had a significant impact on Cortisol level in plasma influenced as well the subsequent increase of glucose. This may be due to the long period (28 days) of starvation on the fish of small size. The fish exposed to nonylphenol showed a relatively slower and prolonged increase of plasma glucose following the stress. This tendency was especially distinct for the group of fish that was exposed to the highest level of nonylphenol. Experiment III suggested that exposure to nitrite does not affect the normal elevation of plasma Cortisol in fish in response to the acute stress under the 59 experimental conditions as shown in Table 4.2. Impairment of HPT axis by nitrite was not observed. Nonylphenol is shown not only to suppresses the plasma level of Cortisol, but also to prevent recovery to the normal state (Figure 4.19). This observation is the first to be made of this type and is important, when applied to the natural environment. There is no aquatic natural environment where fish can live without any "stress" that includes escaping from predators, territorial competition between individuals and temperature changes in water. This means that the fish polluted by nonylphenol may keep abnormally high levels of Cortisol, without being able to reduce the level back to the normal state each time they get stressed. Chronically excess level of Cortisol can affect fish adversely, leading to muscle wasting, reduced growth, and disturbances of the reproductive axis (Freeman and Idler 1973; Bilard et al. 1981). The inhibited growth of salmonids exposed to nonylphenol (Fairchild et al. 1999) may be attributed to this hypersecretion of Cortisol. In Experiment I and III, air exposure resulted in an increase in anaerobic metabolism as indicated by the elevation in plasma lactate. The most noteworthy result from thyroid hormone measurements is seen on day 21, that is a significant decrease in T 3 and T 4 in fish plasma in response to 100 ppb level of nonylphenol exposure. Letherland and Sonstegard (1980) observed thyroid hyperplasa in fish exposed to industrial effluent. This may be caused by suppressed thyroid hormone levels and their the negative feedback, that eventually hyperactivated the thyroid gland. The decrease in both free T 3 and free T 4 in Experiment II can partly 60 be caused by starvation (Holloway et al. 1994). In addition, both values are significantly different also from control levels. This finding can be explained by the possible explanation for the observation reported by may have been caused by hyperactvation of thyroid gland due to the negative feedback from suppressed thyroid hormone production. 61 CHAPTER 6. CONCLUSION AND COMMENTS The major conclusion of the experimental portion of the research is that these experiments demonstrated that nonylphenol modifies the stress response of rainbow trout in terms of Cortisol level increase. Glucose response to stress was not significantly impaired, suggesting that nonylphenol solely affects the HPI axis. Nonylphenol also inhibited the return of plasma Cortisol levels 24 hours after application of stress. Thyroid hormone levels were low in the fish exposed to 100 ppb nonylphenol for 21 days. How nonylphenol instigates these changes is unknown. Cortisol, along with adrenaline, plays a crucial role in metabolism enabling fish to cope with stress as well as their ability to respond to a challenging environment. Being unable to secrete normal levels of Cortisol could lead to disastrous consequences such as a lower chance of survival and vulnerability to diseases. Insufficient thyroid hormone may spur this impact, by causing failures in proper growth and development in the fish. To address some contextual issues the following section provides personal comments on the research. Ever since the industrial revolution, the productivity of industrial activities has steadily increased. Advanced technology has made it possible for humans to develop and produce chemicals with extremely complicated structures in massive amount6. 6In 1997, there were approximately 70,000 different synthetic chemicals on the global market. Their total number would be even higher if their by-products of production and incineration were taken into account (Mitchel 1997). 62 Most notably in the latter part of this century and in less than a lifetime, the production of synthetic xenobiotics (e.g. dyes, plastics and solvents) has increased more than a thousand-fold in the United States alone. Many of these anthropogenic compounds were not originally present in the environment. Thus it is not surprising that they can cause significant, either positive or negative, effects on exposed organisms. A variety of xenobiotics are used for industrial and pharmaceutical purposes which has greatly changed humans' lifestyle through improving conditions of material development, comfort, convenience, and overall physical well-being. However, proper control, storage and disposal are critical for these chemicals to prevent unwanted contamination of the environment. The results of this study highlight an example of the case in which a widely marketed chemical turns out to potentially endanger ecosystem health in an unexpected manner. The observation made in the Cortisol response to stress is highly relevant to the situation in the natural environment where fish from highly polluted areas must manage the pollutant and also react aptly to predators and various environmental stressors to maintain homeostasis. The ability to activate the normal endocrine functions and elevate plasma Cortisol level in response to stress is vital for the fish to cope with the stress. The disruptions of the process, consequently, may reduce the survivorship of the fish. Carson (1962) noted in her famous book that synthetic substances were being released into the environment "with little or no advance investigation of their effects on 63 soil, water, wildlife, and man himself." Almost four decades later, this situation does not seem to have changed. The present world view of most industrialized countries are "economy-centric," as opposed to "ecology-centric." That is they tend to put priority on economical advantages over speculated concern for damaging ecosystem integrity, which is usually not easy to perceive as a result of the long period of time required to manifest environmental effects, as well as lack of inclusive scientific data. Newly synthesized chemicals, once recognized as effective in certain aspects, have a tendency to be marketed widely without conscientious elucidation about their possible side effects. The outcome of this kind of action could be very dangerous and is hardly reversible, as seen in countless cases of environmental pollution. It is not possible to fully ensure the safety of xenobiotics before they are produced commercially. However, once a harmful property is discovered, actions should be made immediately for prevention of the chemical's further release into the environment. Persistent chemicals do not disappear from the environment, even if their release is terminated. PCBs, for example, are still found ubiquitously in water, sediment, and in organisms including humans, even though they have been banned for more than 20 years. As a result of long term appreciation of reductionism and Newtonian mechanism in science, it had become challenging for humans to see the significance of comprehensive discussion involving potentiality and ethical decision-makings. As discussed earlier, the impacts of EDCs are in most cases very subtle and can require very long term (may be a few generations) to manifest. In addition, the mechanisms 64 that explain how EDCs operate in animals are still far from complete comprehension. However, whether these factors are an excuse for responsible regulative actions to decide on continuing the use of these potentially dangerous chemicals is certainly worthy of debate. 65 REFERENCES Ahel, M., McEvoy, J. and Giger, W. 1993. Bioaccumulation of the lipophilic metabolites of nonionic surfantants in freshwater organisms. Environmental Pollution. 79: 243-248. Ahel, M., Giger, W. and Schaffner, C. 1994. Behaviour of Alkylphenol Polyethoxylate Surfactants in the Aquatic Environment-II. Occurrence and Transformation in Rivers. Water Research. 28: 1143-1152. Ashfield, L. A., Potting, T. G. and Sumpter, J. P. 1998. Exposure of female juvenile rainbow trout to alkylphenolic compounds results in modifications to growth and ovosomatic index. Environmental toxicology. 17: 679-686. Atterwill, C. K. and Flack, J. D. 1992. Endocrine Toxicology. Cambridge University Press, New York. Bennett, R. O. and Wo Ike, R. E. 1987. The effects of sublethal endrin exposure on rainbow trout, Salmo gairdneri Richardson. 1. Evaluation of serum Cortisol concentration and immune responsiveness, J. Fish Biol. 31: 375-385. Billard, B., Bry, C. and Gillet, C. 1981. Stress, environment and reproduction in teleost fish [In: Pickering, A. D. (Ed.) Stress in Fish]. Academic Press, London. Blackburn, M. A. and Waldock, M. J. 1995. Concentration of alkylphenol in rivers and estuaries in England and Wales. Water Research. 29: 1623-1629. Brodeur, J. C , Girard, C , Fortin, R. and Hontela, A. 1997. Use of perifusion to assess in vitro the functional integrity of interrenal tissue in feral fish from polluted sites. Environ. Toxicol. Chem. 16: 2171-2178. Brown. J. A., Edwards, D. and Whitehead, C. 1989. Cortisol and thyroid hormones responses to acid stress in the brown trout, Salmo trutta L. J. Fish. Biol.-35: 73-84. Cahn, A and Lynn J. L. Jr. 1983. Surfactants and detersive systems [Irv.Kirk-Othmer Encyclopedia of Chemical Technology, 3rd ed., vol. 22]. John Wiley and Sons, Inc., New York, Inc., New York, ISBN 0-471-02075-3. Carragher, J. F. and Sumpter, J. P. 1990. The effect of Cortisol on the secretion of secretion of sex steroids from cultured ovarian follicles of rainbow trout. Gen. Comp. Endocrinol. 77: 403-407. Carson, R. 1962. Silent Spring. Houghton Mifflin, Boston. 6 6 Christiansen, L. B., Pedersen, K. L., Korsgaard, B. and Bjerregaard, P. 1998. Estrogenicity of Xenobiotics in Rainbow Trout (Oncorhynchus nykiss) using in vivo Synthesis of Vitellogenin as a Biomarker. Marine Environmental Research. 46: 137-140. Colborn, T., Dumanoski, D. and Myers, J. P. 1996. Our Stolen Future: are we threatening our fertility, intelligence, and survival-a scientific detective story. A Plume Book, New York. Colby, H. D. and Longhurst, P. A. 1992. Toxicology of the adrenal gland [In: Endocrine Toxicology]. Cambridge Press, 243-281. Coldham, N. G., Sivapathasundaram, S., Dave, M., Ashfield, L. A., Pottinger, T. G. Goodall, C. and Sauer, M. J. 1998. Biotransformation, tissue distribution, and persistence of 4-nonylphenol residues in juvenile rainbow trout (Oncorhynchus Mykiss). Drug Metaboliam and Disruption. 26: 347-354. Cyr, D. G. and Eales, J. G. 1990. Influence of short-term oestradiol treatment on plasma thyroid hormone kinetics in rainbow trout, Salmo gairdneri. J. Fish. Biol. 36: 391-400. Dickhoff, W. W. 1993. Hormones, Metamorphosis, and Smolting [In: Schreibman, M. P., Scanes, C. G., and Pang P. K. T. (Eds.) The endocrinology of growth, development, and metabolism in vertebrates]. Academic Press, San Diego. Dickhoff, W. W. and Sullivan, C. V. 1987. Common Strategies of Anadromous and Catadromous Fishes. American Fisheries Society, Bethesda, Maryland. Fairchild, W. L., Swansburg, E. O., Arsenault, J. T. and Brown, S. B. 1999. Does an Association between Pesticide Use and Subsequent Decline in Catch of Atlantic Salmon (Salmo salar) Represent a Case of Endocrine Disruption? Environmental Health Perspectives. 107: 349-357. Fingerman M., Manjula, D., Reddy Palla, S., R. and Rajesh, K. 1996. Impact of heavy metal exposure on the nervous system and endocrine-mediated processes in crustaceans. Zoological Studies. 35: 1-8. Freeman H. C. and Idler, D. R. 1973. Effects of corticosteroids on liver transaminases in two salmonids. the rainbow trout (Salmo gaidneri) and the brook trout (Salvelinus fontinails). Gen. Comp. Endocrinol. 20: 69-76. Fryer, J. N. 1989. Neuropeptides regulating the activity of goldfish corticotropes and melanotropes. Fish Physiol. Biochem. 7: 21-27. Giesy, J. P., Ludwig, J. P. and Tillitt. D. E. 1994. Deformities in birds of the Great Lakes Region. Environ. Sci. Technol. 2: 101-110. 67 Gilbert, M. A., Shepherd, M. K., Startin, J. R. and Wallwork, M. A. 1992. Identification by gas chromatography-mass spectrometry of vinylchloride oligomers and other low molecular weight components of poly(vinylchloride) resins for food package applications. J. Chromatogr. 237:249-261. Gill, T. S., Leitner, G., Porta, S. and Epple, A. 1993. Response of plasma Cortisol to environmental cadmium in eel, Anguilla rostrata Lesueur. Comp. Biochem. Physiol. 3: 489-495. Herschier, R. and Jacob, S. W. 1980. The case of dimethylsulfoxide [In: Lasagna, L. (Ed.) Controversies in Therapeutics]. Saunders, Philadelphia. Holloway, A. C , Reddy, P. K., Sheridan, M. A. and Letherland, J. F. 1994. Diurnal Rhythms of Plasma Growth Hormone, Somatostatin, Thyroid Hormones, Cortisol and Glucose Concentrations in Rainbow Trout, Oncurhynhus mykiss, during Progresive Food Deprivation. Biological Rhythm Research. 25: 415-432. Hontela, A. 1997. Endocrine and physiological response of fish to xenobioties: Role of glucocorticoid hormones. Reviews in Toxicology. 1: 1-46. Hontela, A., Rasmussen, J. B., Audet, C. and Chevalier, G. 1992. Impaired Cortisol Response in Fish from Environment Polluted by PAHs, PCBs, and Mercury. Arch. Environ. Contam. Toxicol. 22: 278-283. Hunt, G. and Hunt, M. 1977. Female-Female Pairing in Western Gulls (Larus occidentalis) in Southern California. Science. 196: 1466-1467. Jardine, J. J., Van Der Kraak, G. J. and Munkittrick, K. R. 1996. Capture and Confinement Stress in White Sucher Exposed to Pleached Kraft Pulp Mill Effluent. Ecotoxicology and Environmental Safety. 33: 287-298. Jobling, S., Sheahan, D., Osborne, J. A., Matthiessen, P. and Sumpter, J. P. 1996. Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenol chemicals. Environ. Toxicol. Chem. 15: 194-202. Johnstone, R., Simpsojn, T H. and Youngson, A. F. 1978. Sex reversal in salmonid culture. Aquaculture. 13: 115-134. Junk, G. A., Svec, H. J., Vick, R. D. and Avery, M. J. 1974. Contamination of water by synthetic polymer tubes. Environ. Sci. Technol. 8: 1100-1106. Korsgaard, B. and Pedersen, K. L. 1998. Vitellogenin in Zoarces viviparus: Purification, quantification by ELISA and induction by estradiol-1713 and 4-nonylphenol. Comp. Biochem. Physiol. 120C: 159-166. 68 Lappivaara, J. and Oikari, A. 1999. Altered Challenge Response in Whitefish Subchronically Exposed in Areas Polluted by Bleached Kraft Mill Effluents. Ecotoxicology and Environmental Safety. 43: 212-222. Leadley, J., Lewis, D. F. V., Wiseman, H., Goldfarb, P. S. and Wiseman, A. 1998. Environmental Oestrogen-Mimics Display Liposomal Membrane-Antioxidant Ability: Importance of Molecular Modelling Predictions. J. Chem. Technol. Biotechnol. 73: 131-136. Leattherland, J. F. 1994. Reflections on the thyroidology of fishes: From molecules to humankind. Guelph Ichthyol. Rev. 2:1-67. Leatherland, J. F. and Sonstegard, R. A. 1981. Thyroid function, pituitary structure and serum lipids in Great Lakes coho salmon, Oncorhynchus kisutch Walbaum, 'jacks'compared with sexually immature spring salmon. Journal of Fish Biology. 18: 643-653. Lech, J. J., Lewis, S. K. and Ren, L. 1995. In Vivo Estrogenic Activity of Nonylphenol in Rainbow Trout. Fundamental and Applied Toxicology. 30: 229-232. Lewis, S. K. and Lech, J. J. 1996. Uptake, disposition, and persistence of nonylphenol from water in rainbow trout (Oncorhynchus mykiss). Xenobiotica. 26: 813-819. Lewis, W. M. and Morris, D. P. 1986. Toxicity of Nitrite to Fish: A Review. Transactions of the American Fisheries Society. 115: 183-195. MacKay, D., Ying, S. W. and Ching, M. K. 1992. Illustrated handbook of physical-chemical properties and environmental fate for organic chemicals. Lewis Publishers, Boca Raton. Madsen, S. S., Mathiesen, A. B. and Korsgaard, B. 1997. Effects of 17p-estradiol and 4-nonylphenol on smoltification and vitellogenesis in Atlantic salmon (Salmo salar). Fish Physiology and Biochemistry. 17: 303-312. Maguire, R. J. 1999. Review of the Persistence of Nonylphenol and Nonylphenol Ethoxylates in Aquatic Environments. Water Qual. Res. J. Canada. 34: 37-38. Manson, J. M. and Wise, D. L. 1991. Teratogens [In: Amdur, M. O., Doull, J. and Klaassen, C. D. (Eds.) Casarett and Doull's Toxicology: The Basic Science of Poisons]. Elsevier, New York. McNabb, F. M. A. and King, D. B. 1993. Thyroid hormone Effects on Growth, Development, and Metabolism [In: Schreibman, M. P., Scanes, C. G., and Pang P. K. T. (Eds.) The endocrinology of growth, development, and metabolism in vertebrates]. Academic Press, San Diego. 69 Miles-Richardson, S. R., Pierens, S. L., Nichols, K. M., Kramer, V. L., Snyder, E. M., Snyder, S. A., Render, J. A., Fitzgerald, S. D. and Giesy, J. P. 1999. Effects of Waterborne Exposure to 4-Nonylphenol and Nonylphenol Ethoxylate on Secondary Sex Characteristics and Gonads of Fathead Minnows (Pimephales promelas). Environmental Research. 80A: S122-S137. Mitchell, J. D. 1997. Nowhere to Hide: The Global Spread of High-Risk Synthstic Chemicals. World Watch. March/April. Norris, D. O., Donahue, S., Dores, R. M., Lee, J. K., Maldonedo, T. A., Ruth, T. and Woodling, J. D. 1999. Impaired Adrenocortical Response to Stress by Brown Trout, Salmo trutta, Living in Metal-Contaminated Waiters of the Eagle River, Colorado. General and Comparative Endocrinology. 113: 1-8. Pedersen, S. N., Christinansen, L. B., Pedersen, K. L. and Korsgaard, B. 1999. In vivo estrogenic activity of branched and linear alkylphenols in rainbow trout (Oncurhynchus mykiss). Science of the Total Environment. 233: 89-96. Pundir, R. and Saxena, A. B. 1992. Chronic toxic exposure of cadmium on the pituitary gland of fish Puntius ticto and pattern of recoupment. Journal of Environmental Biology. 13: 69-74. Quabius, E. S., Balm, P. H. M. and Bonga, S. E. W. 1997. Interrenal Stress Responsiveness of Tilapia (Oreochromis mossambicus) Is Impaired by Dietary Exposure to PCB 126. General and Comparative Endocrinology. 108: 472-482. Rayburn, J. R. and Fisher, W. S. 1997. Developmental Toxicity of Three Carrier Solvents Using Embyos of the Grass Shrimp, Palaemonetes pugio. Arch. Environ. Contam. Toxicol. 33: 217-221. Ren, L., Lewis, S. K. and Lech, J. J. 1996. Effects of estrogen and nonylphenol on the post-transcriptional regulation of vitellogenin gene expression. Chemico-Biological Interactions. 100: 67-76. Ricard, A. C , Daniel, C , Anderson, P. and Hontela, A. 1998. Effects of Subchronic Exposure to Cadmium Chloride on Endocrine and Metabolic Functions in Rainbow Trout Oncorhynchus mykss. 34: 377-381. Russo, R. C. and Thurston, R. V. 1977. The acute toxicity of nitrite to fishes [In: Tubb, R. A. (Ed.) Recent advances in fish toxicology]. U. S. Environmental Protection Agency, Ecological Research Series EPA-600/3-77-085. Corvallis, Oregon. Russo, R. C , Thurston, R. V. and Emerson, K. 1981. Acute Toxicity of Nitrite to Rainbow Trout (Salmo gairdneri): Effects of pH, Nitrite Species, and Anion Species. Can. J. Fish. Aquat. Sci. 38: 387-393. 70 Schreck, C. B., Bradford, C , Fitzpatrick, M. S. and Patino, R. 1989. Regulation of the interrenal of fishes: non-classical control mechanisms. Fish Physiol. Biochem. 7: 259-265. Schwaiger, J. and Negele, R. D. 1998. Plasma Vitellogenin-a Blood Parameter to Evapolate Exposure of Fish to Xenoestrogens. Acta vet. Brno. 67: 257-264. Selye, H. 1973. The Evolution of the Stress Concept. American Scientist. 61: 692-699. Servos, M. R. 1999. Review of the Aquatic Toxicity, Estrogenic Responses and Bioaccumulation of Alkylphenols and Alkylphenol Poly ethoxylates. Water Qual. Res. J. Canada. 34: 123-177. Shang, D. Y., MacDonald, R. W. and Ikonomou, M. G. 1999. Persistence of Nonylphenol Ethoxylate Surfactants and Their Primary Degradation Products in Sediments from near a Municipal Outfall in the Strait of Georgia, British Columbia, Canada. Environ. Sci. Technol. 33: 1366-1372. Solomon, E. P., Berg, L. R., Martin, D. W. and Villee, C. 1993. Biology. Saunders College Publishers, Toronto. Soto, A. M., Justica, H, Wray, J. W. and Sonnenschein, C. 1991. P-Nonyl-phenol: an estrogenic xenobiotic xenobiotic released from "modified" polystylene. Environ. Health. Persp. 92: 167-173. Spitsbergen, J. M., Walker, J. R. Olson, J. R. and Peterson, R. E. 1991. Pathologic alterations in early life stages of lake trout, Salvelinus namaycush, exposed to 2, 3, 7, 8-tetrachlorodibenzo-jp-dioxin as fertilized eggs. Aquatic Toxicology. 19: 41-72. Sumpter, J. P. and Jobling, S. 1995. Vitellogenesis as a Biomarker for Estrogenic Contamination of the Aquatic Environment. Environ. Health. Persp. 103: Suppl. 7. 173-177. Sumpter, J. P., Le Bail, P. Y., Pickering, A. D., Pottinger, T. G. and Garragher, J. F. 1991. The effect of starvation on growth and plasma growth hormone concentrations of rainbow trout, Oncorhyncuss mykiss. General & Comparative Endocrinology. 83: 94-102. Toppari, J., Larsen, J. C , Christiansen, P., Giwercman, A., Grandjean, P., Guillette, L. J., Jegou, B., Jensen, T. K., Jouannet, P., Keiding, N., Leffers, H., McLachlan, J. A., Meyer, O., Muller, J., Rajpert-De Meyts, E., Scheike, T., Sharpe, R, Sumpter, J and Skakkebeak, N. E. 1996. Male reproductive health and environmental xenoestrogens. Environ. Health. Persp. 104: Suppl. 4. 741-803. 71 Verdouw, et al. 1078. Ammonia determination based on indophenol formation with sodium salicylate. Water Res. 12: 399-402. Vijayan, M. M., Pereira, C. and Moon, T. W. 1994. Hormonal stimulation of hepatocyte metabolism in rainbow trout following an acute handling stress. Comp. Biochem. Pysiol. 108C: 321-329 Webb, N. A. and Wood, C. M. 1998. Physiological Analysis of the Stress Response Associated with Acute Silver Nitrate Exposure in Freshwater Rainbow Trout (Oncorhynchus mykiss). Environmental Toxicology and Chemistry. 17: 579-588. White, A. and Fletcher, T. C. 1986. Serum Cortisol, glucose and lipids in plaice (Pleuronectes platessa L.) exposed to starvation and aquarium stress. Comp. Biochem. Physiol. 84A: 649-653. White, R., Jobling, S., Hoare, S. A., Sumpter, J. P. and Parker, M. G. 1994. Environmentally Persistent Alkylphenolic Compounds Are Estrogenic. Endocrinology. 135: 175-182. Willers, W. B. 1981. Trout biology: an angler's guide. University of Wisconsin Press, Madison, Wisconsin. Wiseman, H., Cannon, M., Arnstein, H. R. V. and Barlow, D. J. 1992. The structural mimicry of membrane sterols by tamoxifen: evidence from its cholesterol coefficients and molecular-modelling for its action as a membrane anti-oxidant and an anticancer agent. Biochim. Biophys. Acta. 1138: 197-202. Woodward, A., Percival, H., Jennings, M. and Moore, C. 1993. Low Clutch Viability of American Alligators on Lake Apopka. Florida Science. 56: 52-63. Wren, C D . 1991. Cause-effect linkage between chemicals and populations of mink (Mustela vison) and otter (Lutra canadensis) in the Great Lakes basin. Journal of Toxicology and Environmental Health. 33: 549-585. Yadav, A. K. and Singh, T. P. Effect of pesticide on circulating thyroid hormone levels in the freshwater catfish, Heteropneustes fossilis (Bloch). Environ. Res. 39: 136-142. 72 REFERENCES Ahel, M., McEvoy, J. and Giger, W. 1993. Bioaccumulation of the lipophilic metabolites of nonionic surfantants in freshwater organisms. Environmental Pollution. 79: 243-248. Ahel, M., Giger, W. and Schaffner, C. 1994. Behaviour of Alkylphenol Polyethoxylate Surfactants in the Aquatic Environment-II. Occurrence and Transformation in Rivers. Water Research. 28: 1143-1152. Ashfield, L. A., Potting, T. G. and Sumpter, J. P. 1998. Exposure of female juvenile rainbow trout to alkylphenolic compounds results in modifications to growth and ovosomatic index. Environmental toxicology. 17: 679-686. Atterwill, C. K. and Flack, J. D. 1992. Endocrine Toxicology. Cambridge University Press, New York. Bennett, R. O. and Wo Ike, R. E. 1987. The effects of sublethal endrin exposure on rainbow trout, Salmo gairdneri Richardson. 1. Evaluation of serum Cortisol concentration and immune responsiveness, J. Fish Biol. 31: 375-385. Billard, B., Bry, C. and Gillet, C. 1981. Stress, environment and reproduction in teleost fish [In: Pickering, A. D. (Ed.) Stress in Fish]. Academic Press, London. Blackburn, M. A. and Waldock, M. J. 1995. Concentration of alkylphenol in rivers and estuaries in England and Wales. Water Research. 29: 1623-1629. Brodeur, J. C , Girard, C , Fortin, R. and Hontela, A. 1997. Use of perifusion to assess in vitro the functional integrity of interrenal tissue in feral fish from polluted sites. Environ. Toxicol. Chem. 16:2171-2178. Brown. J. A., Edwards, D. and Whitehead, C. 1989. Cortisol and thyroid hormones responses to acid stress in the brown trout, Salmo trutta L. J. Fish. Biol. 35: 73-84. Cahn, A and Lynn J. L. Jr. 1983. Surfactants and detersive systems \\w.Kirk-Othmer Encyclopedia of Chemical Technology, 3rd ed., vol. 22]. John Wiley and Sons, Inc., New York, Inc., New York, ISBN 0-471-02075-3. Carragher, J. F. and Sumpter, J. P. 1990. The effect of Cortisol on the secretion of secretion of sex steroids from cultured ovarian follicles of rainbow trout. Gen. Comp. Endocrinol. 77: 403-407. Carson, R. 1962. Silent Spring. Houghton Mifflin, Boston. 66 Christiansen, L. B., Pedersen, K. L., Korsgaard, B. and Bjerregaard, P. 1998. Estrogenicity of Xenobiotics in Rainbow Trout (Oncorhynchus nykiss) using in vivo Synthesis of Vitellogenin as a Biomarker. Marine Environmental Research. 46: 137-140. Colborn, T., Dumanoski, D. and Myers, J. P. 1996. Our Stolen Future: are we threatening our fertility, intelligence, and survival-a scientific detective story. A Plume Book, New York. Colby, H. D. and Longhurst, P. A. 1992. Toxicology of the adrenal gland [In: Endocrine Toxicology]. Cambridge Press, 243-281. Coldham, N. G., Sivapathasundaram, S., Dave, M., Ashfield, L. A., Pottinger, T. G. Goodall, C. and Sauer, M. J. 1998. Biotransformation, tissue distribution, and persistence of 4-nonylphenol residues in juvenile rainbow trout (Oncorhynchus Mykiss). Drug Metaboliam and Disruption. 26: 347-354. Cyr, D. G. and Eales, J. G. 1990. Influence of short-term oestradiol treatment on plasma thyroid hormone kinetics in rainbow trout, Salmo gairdneri. J. Fish. Biol. 36: 391-400. Dickhoff, W. W. 1993. Hormones, Metamorphosis, and Smolting [In: Schreibman, M. P., Scanes, C. G., and Pang P. K. T. (Eds.) The endocrinology of growth, development, and metabolism in vertebrates]. Academic Press, San Diego. Dickhoff, W. W. and Sullivan, C. V. 1987. Common Strategies of Anadromous and Catadromous Fishes. American Fisheries Society, Bethesda, Maryland. Fairchild, W. L., Swansburg, E. O., Arsenault, J. T. and Brown, S. B. 1999. Does an Association between Pesticide Use and Subsequent Decline in Catch of Atlantic Salmon (Salmo salar) Represent a Case of Endocrine Disruption? Environmental Health Perspectives. 107: 349-357. Fingerman M., Manjula, D., Reddy Palla, S., R. and Rajesh, K. 1996. Impact of heavy metal exposure on the nervous system and endocrine-mediated processes in crustaceans. Zoological Studies. 35: 1-8. Freeman H. C. and Idler, D. R. 1973. Effects of corticosteroids on liver transaminases in two salmonids. the rainbow trout (Salmo gaidneri) and the brook trout (Salvelinus fontinails). Gen. Comp. Endocrinol. 20: 69-76. Fryer, J. N. 1989. Neuropeptides regulating the activity of goldfish corticotropes and melanotropes. Fish Physiol. Biochem. 7: 21-27. Giesy, J. P., Ludwig, J. P. and Tillitt. D. E. 1994. Deformities in birds of the Great Lakes Region. Environ. Sci. Technol. 2: 101-110. 67 Gilbert, M. A., Shepherd, M. K., Startin, J. R. and Wallwork, M. A. 1992. Identification by gas chromatography-mass spectrometry of vinylchloride oligomers and other low molecular weight components of poly(vinylchloride) resins for food package applications. J. Chromatogr. 237:249-261. Gill, T. S., Leitner, G., Porta, S. and Epple, A. 1993. Response of plasma C o r t i s o l to environmental cadmium in eel, Anguilla rostrata Lesueur. Comp. Biochem. Physiol. 3: 489-495. Herschier, R. and Jacob, S. W. 1980. The case of dimethylsulfoxide [In: Lasagna, L. (Ed.) Controversies in Therapeutics]. Saunders, Philadelphia. Holloway, A. C , Reddy, P. K., Sheridan, M. A. and Letherland, J. F. 1994. Diurnal Rhythms of Plasma Growth Hormone, Somatostatin, Thyroid Hormones, Cortisol and Glucose Concentrations in Rainbow Trout, Oncurhynhus mykiss, during Progresive Food Deprivation. Biological Rhythm Research. 25: 415-432. Hontela, A. 1997. Endocrine and physiological response of fish to xenobiotics: Role of glucocorticoid hormones. Reviews in Toxicology. 1: 1-46. Hontela, A., Rasmussen, J. B., Audet, C. and Chevalier, G. 1992. Impaired Cortisol Response in Fish from Environment Polluted by PAHs, PCBs, and Mercury. Arch. Environ. Contam. Toxicol. 22: 278-283. Hunt, G. and Hunt, M. 1977. Female-Female Pairing in Western Gulls [Larus occidentalis) in Southern California. Science. 196: 1466-1467. Jardine, J. J., Van Der Kraak, G. J. and Munkittrick, K. R. 1996. Capture and Confinement Stress in White Sucher Exposed to Pleached Kraft Pulp Mill Effluent. Ecotoxicology and Environmental Safety. 33: 287-298. Jobling, S., Sheahan, D., Osborne, J. A., Matthiessen, P. and Sumpter, J. P. 1996. Inhibition of testicular growth in rainbow trout {Oncorhynchus mykiss) exposed to estrogenic alkylphenol chemicals. Environ. Toxicol. Chem. 15: 194-202. Johnstone, R., Simpsojn, T H. and Youngson, A. F. 1978. Sex reversal in salmonid culture. Aquaculture. 13: 115-134. Junk, G. A., Svec, H. J., Vick, R. D. and Avery, M. J. 1974. Contamination of water by synthetic polymer tubes. Environ. Sci. Technol. 8: 1100-1106. Korsgaard, B. and Pedersen, K. L. 1998. Vitellogenin in Zoarces viviparus: Purification, quantification by ELISA and induction by estradiol-1713 and 4-nonylphenol. Comp. Biochem. Physiol. 120C: 159-166. 68 Lappivaara, J. and Oikari, A. 1999. Altered Challenge Response in Whitefish Subchronically Exposed in Areas Polluted by Bleached Kraft Mill Effluents. Ecotoxicology and Environmental Safety. 43: 212-222. Leadley, J., Lewis, D. F. V., Wiseman, H., Goldfarb, P. S. and Wiseman, A. 1998. Environmental Oestrogen-Mimics Display Liposomal Membrane-Antioxidant Ability: Importance of Molecular Modelling Predictions. J. Chem. Technol. Biotechnol. 73: 131-136. Leattherland, J. F. 1994. Reflections on the thyroidology of fishes: From molecules to humankind. Guelph Ichthyol. Rev. 2:1-67. Leatherland, J. F. and Sonstegard, R. A. 1981. Thyroid function, pituitary structure and serum lipids in Great Lakes coho salmon, Oncorhynchus kisutch Walbaum, 'jacks'compared with sexually immature spring salmon. Journal of Fish Biology. 18: 643-653. Lech, J. J., Lewis, S. K. and Ren, L. 1995. In Vivo Estrogenic Activity of Nonylphenol in Rainbow Trout. Fundamental and Applied Toxicology. 30: 229-232. Lewis, S. K. and Lech, J. J. 1996. Uptake, disposition, and persistence of nonylphenol from water in rainbow trout (Oncorhynchus mykiss). Xenobiotica. 26: 813-819. Lewis, W. M. and Morris, D. P. 1986. Toxicity of Nitrite to Fish: A Review. Transactions of the American Fisheries Society. 115: 183-195. MacKay, D., Ying, S. W. and Ching, M. K. 1992. Illustrated handbook of physical-chemical properties and environmental fate for organic chemicals. Lewis Publishers, Boca Raton. Madsen, S. S., Mathiesen, A. B. and Korsgaard, B. 1997. Effects of 17p-estradiol and 4-nonylphenol on smoltification and vitellogenesis in Atlantic salmon (Salmo salar). Fish Physiology and Biochemistry. 17: 303-312. Maguire, R. J. 1999. Review of the Persistence of Nonylphenol and Nonylphenol Ethoxylates in Aquatic Environments. Water Qual. Res. J. Canada. 34: 37-38. Manson, J. M. and Wise, D. L. 1991. Teratogens [In: Amdur, M. O., Doull, J. and Klaassen, C. D. (Eds.) Casarett andDoull's Toxicology: The Basic Science of Poisons]. Elsevier, New York. McNabb, F. M. A. and King, D. B. 1993. Thyroid hormone Effects on Growth, Development, and Metabolism [In: Schreibman, M. P., Scanes, C. G., and Pang P. K. T. (Eds.) The endocrinology of growth, development, and metabolism in vertebrates]. Academic Press, San Diego. 69 Miles-Richardson, S. R., Pierens, S. L., Nichols, K. M., Kramer, V. L., Snyder, E. M., Snyder, S. A., Render, J. A., Fitzgerald, S. D. and Giesy, J. P. 1999. Effects of Waterborne Exposure to 4-Nonylphenol and Nonylphenol Ethoxylate on Secondary Sex Characteristics and Gonads of Fathead Minnows (Pimephales promelas). Environmental Research. 80A: S122-S137. Mitchell, J. D. 1997. Nowhere to Hide: The Global Spread of High-Risk Synthstic Chemicals. World Watch. March/April. Norris, D. O., Donahue, S., Dores, R. M., Lee, J. K., Maldonedo, T. A., Ruth, T. and Woodling, J. D. 1999. Impaired Adrenocortical Response to Stress by Brown Trout, Salmo trutta, Living in Metal-Contaminated Waiters of the Eagle River, Colorado. General and Comparative Endocrinology. 113: 1-8. Pedersen, S. N., Christinansen, L. B., Pedersen, K. L. and Korsgaard, B. 1999. In vivo estrogenic activity of branched and linear alkylphenols in rainbow trout (Oncurhynchus mykiss). Science of the Total Environment. 233: 89-96. Pundir, R. and Saxena, A. B. 1992. Chronic toxic exposure of cadmium on the pituitary gland of fish Puntius ticto and pattern of recoupment. Journal of Environmental Biology.13: 69-1 A. Quabius, E. S., Balm, P. H. M. and Bonga, S. E. W. 1997. Interrenal Stress Responsiveness of Tilapia (Oreochromis mossambicus) Is Impaired by Dietary Exposure to PCB 126. General and Comparative Endocrinology. 108: 472-482. Rayburn, J. R. and Fisher, W. S. 1997. Developmental Toxicity of Three Carrier Solvents Using Embyos of the Grass Shrimp, Palaemonetes pugio. Arch. Environ. Contam. Toxicol. 33: 217-221. Ren, L., Lewis, S. K. and Lech, J. J. 1996. Effects of estrogen and nonylphenol on the post-transcriptional regulation of vitellogenin gene expression. Chemico-Biological Interactions. 100: 67-76. Ricard, A. C , Daniel, C , Anderson, P. and Hontela, A. 1998. Effects of Subchronic Exposure to Cadmium Chloride on Endocrine and Metabolic Functions in Rainbow Trout Oncorhynchus mykss. 34: 377-381. Russo, R. C. and Thurston, R. V. 1977. The acute toxicity of nitrite to fishes [In: Tubb, R. A. (Ed.) Recent advances in fish toxicology]. U. S. Environmental Protection Agency, Ecological Research Series EPA-600/3-77-085. Corvallis, Oregon. Russo, R. C , Thurston, R. V. and Emerson, K. 1981. Acute Toxicity of Nitrite to Rainbow Trout {Salmo gairdneri): Effects of pH, Nitrite Species, and Anion Species. Can. J. Fish. Aquat. Sci. 38: 387-393. 70 Schreck, C. B., Bradford, C , Fitzpatrick, M. S. and Patino, R. 1989. Regulation of the interrenal of fishes: non-classical control mechanisms. Fish Physiol. Biochem. 7: 259-265. Schwaiger, J. and Negele, R. D. 1998. Plasma Vitellogenin-a Blood Parameter to Evapolate Exposure of Fish to Xenoestrogens. Acta vet. Brno. 67: 257-264. Selye, H. 1973. The Evolution of the Stress Concept. American Scientist. 61: 692-699. Servos, M. R. 1999. Review of the Aquatic Toxicity, Estrogenic Responses and Bioaccumulation of Alkylphenols and Alkylphenol Poly ethoxylates. Water Qual. Res. J. Canada. 34: 123-177. Shang, D. Y., MacDonald, R. W. and Ikonomou, M. G. 1999. Persistence of Nonylphenol Ethoxylate Surfactants and Their Primary Degradation Products in Sediments from near a Municipal Outfall in the Strait of Georgia, British Columbia, Canada. Environ. Sci. Technol. 33: 1366-1372. Solomon, E. P., Berg, L. R., Martin, D. W. and Villee, C. 1993. Biology. Saunders College Publishers, Toronto. Soto, A. M., Justica, H, Wray, J. W. and Sonnenschein, C. 1991. P-Nonyl-phenol: an estrogenic xenobiotic xenobiotic released from "modified" polystylene. Environ. Health. Persp. 92: 167-173. Spitsbergen, J. M., Walker, J. R. Olson, J. R. and Peterson, R. E. 1991. Pathologic alterations in early life stages of lake trout, Salvelinus namaycush, exposed to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin as fertilized eggs. Aquatic Toxicology. 19: 41-72. Sumpter, J. P. and Jobling, S. 1995. Vitellogenesis as a Biomarker for Estrogenic Contamination of the Aquatic Environment. Environ. Health. Persp. 103: Suppl. 7. 173-177. Sumpter, J. P., Le Bail, P. Y., Pickering, A. D., Pottinger, T. G. and Garragher, J. F. 1991. The effect of starvation on growth and plasma growth hormone concentrations of rainbow trout, Oncorhyncuss mykiss. General & Comparative Endocrinology. 83: 94-102. Toppari, J., Larsen, J. C , Christiansen, P., Giwercman, A., Grandjean, P., Guillette, L. J., Jegou, B., Jensen, T. K., Jouannet, P., Keiding, N., Leffers, PL, McLachlan, J. A., Meyer, O., Muller, J., Rajpert-De Meyts, E., Scheike, T., Sharpe, R., Sumpter, J and Skakkebeak, N. E. 1996. Male reproductive health and environmental xenoestrogens. Environ. Health. Persp. 104: Suppl. 4. 741-803. 71 Verdouw, et al. 1078. Ammonia determination based on indophenol formation with sodium salicylate. Water Res. 12: 399-402. Vijayan, M. M., Pereira, C. and Moon, T. W. 1994. Hormonal stimulation of hepatocyte metabolism in rainbow trout following an acute handling stress. Comp. Biochem. Pysiol. 108C: 321-329 Webb, N. A. and Wood, C. M. 1998. Physiological Analysis of the Stress Response Associated with Acute Silver Nitrate Exposure in Freshwater Rainbow Trout {Oncorhynchus mykiss). Environmental Toxicology and Chemistry. 17: 579-588. White, A. and Fletcher, T. C. 1986. Serum Cortisol, glucose and lipids in plaice (Pleuronectes platessa L.) exposed to starvation and aquarium stress. Comp. Biochem. Physiol. 84A: 649-653. White, R., Jobling, S., Hoare, S. A., Sumpter, J. P. and Parker, M. G. 1994. Environmentally Persistent Alkylphenolic Compounds Are Estrogenic. Endocrinology. 135: 175-182. Willers, W. B. 1981. Trout biology: an angler's guide. University of Wisconsin Press, Madison, Wisconsin. Wiseman, H., Cannon, M., Arnstein, H. R. V. and Barlow, D. J. 1992. The structural mimicry of membrane sterols by tamoxifen: evidence from its cholesterol coefficients and molecular-modelling for its action as a membrane anti-oxidant and an anticancer agent. Biochim. Biophys. Acta. 1138: 197-202. Woodward, A., Percival, H., Jennings, M. and Moore, C. 1993. Low Clutch Viability of American Alligators on Lake Apopka. Florida Science. 56: 52-63. Wren, C D . 1991. Cause-effect linkage between chemicals and populations of mink {Mustela vison) and otter {Lutra canadensis) in the Great Lakes basin. Journal of Toxicology and Environmental Health. 33: 549-585. Yadav, A. K. and Singh, T. P. Effect of pesticide on circulating thyroid hormone levels in the freshwater catfish, Heteropneustes fossilis (Bloch). Environ. Res. 39: 136-142. 72 

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