Exposure to Perchlorate Induces the Formation of Macrophage Aggregates in the Trunk Kidney of Zebrafish and Mosquitofish Tim Capps Texas Tech University

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2004 Exposure to Perchlorate Induces the Formation of Macrophage Aggregates in the Trunk Kidney of Zebrafish and Mosquitofish Tim Capps Texas Tech University

Sandeep Mukhi Texas Tech University

Jacques Rinchard The College at Brockport, [email protected]

Chris W. Theodorakis Texas Tech University

Vicki S. Blazer U.S. Geological Survey

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Repository Citation Capps, Tim; Mukhi, Sandeep; Rinchard, Jacques; Theodorakis, Chris W.; Blazer, Vicki S.; and Patiño, Reynaldo, "Exposure to Perchlorate Induces the Formation of Macrophage Aggregates in the Trunk Kidney of Zebrafish and Mosquitofish" (2004). Environmental Science and Ecology Faculty Publications. 5. https://digitalcommons.brockport.edu/env_facpub/5 Citation/Publisher Attribution: Capps, T., Mukhi, S., Rinchard, J., Theodorakis, C.W., Blazer, V.S. and Patino, R., 2004. "Exposure to Perchlorate Induces the Formation of Macrophage Aggregates in the Trunk Kidney of Zebrafish and Mosquitofish." Journal of Aquatic Animal Health, 16(3): 145-151. This Article is brought to you for free and open access by the Environmental Science and Ecology at Digital Commons @Brockport. It has been accepted for inclusion in Environmental Science and Ecology Faculty Publications by an authorized administrator of Digital Commons @Brockport. For more information, please contact [email protected]. Authors Tim Capps, Sandeep Mukhi, Jacques Rinchard, Chris W. Theodorakis, Vicki S. Blazer, and Reynaldo Patiño

This article is available at Digital Commons @Brockport: https://digitalcommons.brockport.edu/env_facpub/5 Journal of Aquatic Animal Health 16:145±151, 2004 ᭧ Copyright by the American Fisheries Society 2004

Exposure to Perchlorate Induces the Formation of Macrophage Aggregates in the Trunk Kidney of Zebra®sh and Mosquito®sh

TIM CAPPS,SANDEEP MUKHI,JACQUES J. RINCHARD,1 AND CHRIS W. T HEODORAKIS Texas Cooperative Fish and Wildlife Research Unit and Department of Environmental Toxicology, Texas Tech University, Lubbock, Texas 79409-2120, USA

VICKI S. BLAZER U.S. Geological Survey, National Fish Health Laboratory, Leetown Science Center, 1700 Leetown Road, Kearneysville, West Virginia 25430, USA

REYNALDO PATINÄ O* U.S. Geological Survey, Texas Cooperative Fish and Wildlife Research Unit and Departments of Range Wildlife and Fisheries Management and of Biological Sciences, Texas Tech University, Lubbock, Texas 79409-2120, USA

Abstract.ÐEnvironmental contamination of ground and surface waters by perchlorate, derived from ammonium perchlorate (AP) and other perchlorate salts, is of increasing concern. Exposure to perchlorate can impair the thyroid endocrine system, which is thought to modulate renal and immune function in vertebrates. This study with zebra®sh Danio rerio and eastern mosquito®sh Gambusia holbrooki examined the histological effects of perchlorate on the trunk kidney, which in teleosts serves excretory and hemopoietic functions and therefore may be a target of perchlorate effects. Adult zebra®sh of both sexes were exposed in the laboratory to waterborne, AP-derived perchlorate at measured concentrations of 18 mg/L for 8 weeks. Adult male mosquito®sh were exposed to waterborne sodium perchlorate at measured perchlorate concentrations of 1±92 mg/L for 8 weeks. Control ®sh were kept in untreated water. The region of the body cavity containing the trunk kidney was processed from each ®sh for histological analysis. Macrophage aggregates (MAs), possible markers of contaminant exposure or immunotoxic effect, were present in the hemopoietic region of the kidney in both species exposed to perchlorate. The estimated percent area of kidney sections occupied by MAs was greater in zebra®sh exposed to perchlorate at 18 mg/L (P Ͻ 0.05) than in controls. In male mosquito®sh, the incidence of renal MAs increased proportionally with sodium perchlorate concentration and was signi®cantly different from that of controls at 92 mg/L (P Ͻ 0.05). These observations con®rm that in ®sh the kidney is affected by exposure to perchlorate. The concentrations of perchlorate at which the effects were noted are relatively high but within the range reported in some contaminated habitats.

Downloaded by [SUNY Brockport] at 12:03 19 December 2011 Ammonium perchlorate (AP) is a strong oxidant The best known biological impact of perchlorate used primarily as a rocket propellant in military in animals is the impairment of thyroid gland func- and aerospace industries and operations (Fisher et tion. Perchlorate alters thyroid function by com- al. 2000). After the dissociation of AP into am- petitively blocking iodide in¯ux at the sodium io- monium and perchlorate ions in water, the result- dide symporters that regulate the amount of iodide ing perchlorate anion persists in the environment transported into thyroid follicles. Because iodide for many years (Fisher et al. 2000; Urbansky is necessary for thyroid hormone production, this 2002). Environmental contamination of ground blocking reduces the levels of thyroid hormones and surface waters by perchlorate derived from AP in blood (Wolff 1998). As a result, the pituitary and other perchlorate salts is of increasing concern gland increases its production of thyroid-stimu- (Urbansky 2002). lating hormone (TSH) in an attempt to restore thyroid hormone levels. Prolonged exposure to per- * Corresponding author: [email protected] chlorate causes hyperstimulation of the thyroid 1 Present address: School of Natural Resources, Ohio gland (Wolff 1998; Soldin et al. 2001). For ex- State University, Columbus, Ohio 43210, USA. ample, histopathological studies with several ver- Received April 18, 2004; accepted June 6, 2004 tebrate species have revealed that exposure to AP-

145 146 CAPPS ET AL.

derived perchlorate leads to thyroid follicle alter- previous study of AP effects on ®sh reproduction ations such as hypertrophy, hyperplasia, angio- and thyroid function (PatinÄo et al. 2003b). Brie¯y, genesis, and colloid depletion (Fernandez adult zebra®sh were exposed to AP by using a Rodriguez et al. 1991; Siglin et al. 2000; York et static-renewal procedure at measured perchlorate al. 2001a; Goleman et al. 2002a; PatinÄo et al. concentrations of 6 ␮g/L (untreated control) and 2003b). Other studies have indicated that exposure 18 mg/L in 38-L glass aquaria. Four to ®ve rep- to environmentally relevant concentrations of per- licated tanks with 8 female ®sh each (female tanks) chlorate can affect circulating levels of thyroid and two replicated tanks with 12 male ®sh each hormones or TSH (Brechner et al. 2000; York et (male tanks) were used per concentration for a total al. 2001a; Goleman et al. 2002a). of six or seven replicates per treatment. The ex- Impairments in thyroid function caused by per- posures lasted 8 weeks. For the present study, 3± chlorate are likely to affect thyroid-dependent pro- 4 ®sh per aquarium were prepared for histological cesses. Indeed, exposure to perchlorate causes de- analysis of the kidney, giving a total of 22±26 ®sh velopmental abnormalities in African clawed frogs per treatment. Xenopus laevis (Goleman et al. 2002a, 2002b), Adult eastern mosquito®sh samples for this deer mice Peromyscus maniculatus (Thuett et al. study were generated in a separate study of the 2002), and zebra®sh Danio rerio (Brown 1997). effects of sodium perchlorate (EM Science, Gibbs- Conversely, perchlorate did not impair reproduc- town, New Jersey) on reproduction, in the presence tive function in adult zebra®sh (PatinÄo et al. of measured concentrations (Anderson and Wu 2003b), rats Rattus norvegicus, and New Zealand 2002) of 4 ␮g/L (untreated control) and 1, 8, and white rabbits Oryctolagus cuniculus (York et al. 92 mg/L, respectively, for 8 weeks with a static- 2001a, 2001b). Little attention has been paid to renewal process in 38-L glass aquaria (J. W. Park the effects of perchlorate on other physiological et al., Texas Tech University, unpublished). Five functions. replicate tanks per treatment were prepared. One In amphibians, perchlorate-induced thyroid dis- or two male ®sh per aquarium were collected and ruption has been implicated in immune dysfunc- analyzed for the present study, giving a total of tion (Rollins-Smith et al. 1993), and in mammals, six to eight ®sh per treatment. Four female ®sh thyroid hormones are known to in¯uence renal ex- were also housed in the same aquaria as the males, cretory functions (Katz et al. 1975; Capasso et al. but they were part of a separate study of repro- 1999; Prasad et al. 1999). Also, receptor activity ductive effects (J. W. Park et al., unpublished) and or receptor transcripts for thyroid hormones were not used for the present analysis. (MacLatchy and Eales 1992) and TSH (Dutton et For all experiments, aquaria were ®tted withbiof- al. 1997; Sellitti et al. 2000; Vischer and Bogerd iltration units to maintain water quality; ammonia, 2003) have been reported in the vertebrate kidney temperature, pH, and dissolved oxygen were mon- (including the teleost kidney); and perchlorate- itored regularly (PatinÄo et al. 2003b; J. W. Park et sensitive sodium iodide symporters, similar to al., unpublished). Animal use protocols were re- those found in the thyroid gland, are found in the viewed and approved by the Animal Care and Use mammalian kidney (Spitzweg et al. 2001). In most Committee of Texas Tech University. Downloaded by [SUNY Brockport] at 12:03 19 December 2011 teleost ®shes, the head and trunk regions of the Histological preparations.ÐAfter euthanasia by kidney serve hemopoietic functions; the trunk kid- immersion in1gofMS-222 per liter, the abdom- ney also produces the urine (Willett et al. 1999; inal cavity of the ®sh was exposed by incision; Powell 2000). Thus, the possibility exists that the full specimens were ®xed whole in Bouin's solu- teleost kidney is affected by perchlorate exposure. tion for 48 h at 4ЊC, rinsed in water for several The objective of this study was to examine the hours, and stored in 70% ethanol. The trunk region histological effects of perchlorate on the trunk kid- of the ®sh was embedded in paraf®n and serial ney of zebra®sh and eastern mosquito®sh Gam- sections of the trunk kidney (5±6 ␮m thick) were busia holbrooki. The information obtained is ex- obtained with a microtome. Slides were stained pected to contribute to a better understanding of with Harris's hematoxylin stain and counterstained the ecotoxicology of perchlorate in aquatic habi- with eosin (Sigma Diagnostics, St. Louis, Missou- tats. ri). For zebra®sh, select slides were also stained with periodic acid Shiff's (PAS) reagent and Methods Wright's iron stain (both from Sigma Diagnostics). Fish and perchlorate exposures.ÐZebra®sh Histological observations.ÐFor each sample, samples for this study were generated during a the tissue section for histological observation was PERCHLORATE AND KIDNEY MACROPHAGE AGGREGATES 147

chosen according to its histological integrity and the presence of granulomas with acid-fast bacteria quality, determined when viewing the ®rst row of (not shown). These two aquaria were not used in sections on the slide from left to right. A prelim- the subsequent analysis, leading to a ®nal number inary analysis of renal sections indicated that treat- of replicates of ®ve and six for the control and 18- ment with perchlorate induced a higher incidence mg/L treatments, respectively (the total number of of macrophage aggregates (MAs). No other ab- ®sh analyzed per treatment was consequently also normalities were apparent at the gross level in ei- reduced to 18 and 21 ®sh for the control and 18- ther the excretory or hemopoietic tissue compart- mg/L treatment, respectively). The omission of ments, but a detailed examination of excretory tis- these two male tanks left only one male tank each sues was not performed. To quantify the incidence for the control and 18-mg/L groups. Given the low of MAs, a 1-mm ϫ 1-mm ocular grid containing sample size of male tanks in this study, a statistical 121 crosshairs was positioned over the right kid- analysis based on sex was not feasible. ney lobe, covering as much kidney tissue area as In sections of zebra®sh kidney stained with he- possible, at a total magni®cation of 100ϫ for ze- matoxylin and eosin, MAs were recognized as bra®sh and 200ϫ for mosquito®sh. Crosshairs of clear or light-brown macrophage clusters of var- the grid that fell on MAs were counted. Crosshairs ious sizes (Figure 1A). Some of the cells in the that fell outside the kidney or on large blood ves- aggregates had inclusions that reacted with sels were regarded as misses and subtracted from Wright's iron stain (Figure 1B), and most of the the total crosshair count. The relative (percent) cells were positive for PAS (Figure 1C), indicating area of kidney sections covered by MAs was es- the presence of iron and polysaccharides, respec- timated by dividing the number of crosshairs fall- tively. Sections of mosquito®sh MAs stained with ing on MAs by the corrected total crosshair count. hematoxylin-eosin had a considerable amount of This quanti®cation strategy does not discriminate melanin and showed a dark-brown appearance between differences in area due to differences in (Figure 1D). In both species, renal MAs were the number of MAs or in the size of individual found within the hemopoietic tissue compartment. MAs. The same observer performed these mea- Zebra®sh from tanks containing 18 mg/L AP surements three separate times on each sample, had signi®cantly greater amounts of renal MAs using the same tissue section. than the ®sh in the control tanks (Figure 2; P ϭ Statistical analysis.ÐThe average of the three 0.015639). The same results were obtained when measurements per ®sh was designated as the ®sh the male tanks were excluded from the analysis value for MA incidence. All ®sh values within (data not shown). In male mosquito®sh, the in- each aquarium were then averaged to estimate the crease in the incidence of renal MAs was propor- respective tank value. Tank values were subjected tional to the concentration of sodium perchlorate to square root transformation, as suggested for the (Figure 3). The incidence of renal MAs in per- analysis of low-value percentage data (Steel and chlorate-exposed (92 mg/L) mosquito®sh was sig- Torrie 1980). Analyses were conducted with the ni®cantly different from that of the control ®sh STATISTICA for Windows 1998 software package (one-way ANOVA, P ϭ 0.0133; Duncan's multiple (StatSoft, Tulsa, Oklahoma). The data for zebra®sh range tests, P Ͻ 0.05). Downloaded by [SUNY Brockport] at 12:03 19 December 2011 were analyzed with a two-tailed Student's t-test, whereas the data for mosquito®sh were analyzed Discussion with one-way analysis of variance (ANOVA), fol- The results of the present study indicate that lowed by Duncan's multiple range tests. Homo- exposure to perchlorate increases the incidence of geneity of variances was assessed using the Coch- MAs in the hemopoietic tissue compartment of the ran C statistic and the Bartlett chi-square test. Sta- trunk kidney of zebra®sh and eastern mosquito®sh. tistical differences were considered signi®cant at Ammonia (un-ionized) is toxic to ®shes at rela- overall ␣ of 0.05. tively high concentrations (Wedemeyer 1996); ac- cordingly, a role for ammonia in the process of Results renal MA formation cannot be clearly ruled out There were six and seven aquarium replicates for zebra®sh because ammonium perchlorate was for the zebra®sh control and 18-mg/L treatments, used as source of perchlorate in these experiments. respectively (PatinÄo et al. 2003b). Preliminary his- However, based on water pH and temperature (Pa- tological analysis showed that ®sh from one con- tinÄo et al. 2003b) and measured total ammonia trol male tank and one AP-treated male tank had levels, the estimated value for un-ionized ammonia signs of kidney mycobacterial infection, based on in the AP-treated aquaria (approximately 0.03 mg/ 148 CAPPS ET AL.

FIGURE 1.ÐPhotomicrographs of macrophage aggregates (MAs; arrowheads) in trunk kidney of (A±C) zebra®sh and (D) mosquito®sh. Panel (A) shows a macrophage aggregate with light brown pigmentation (hematoxylin and eosin [H&E] stain), panel (B) an MA with hemosiderin (blue) deposits (iron stain), panel (C) an MA with polysaccharide (red) deposits (periodic acidϪSchiff stain), and panel D an MA with dark melanin deposits (H&E stain). Bars are as follows: (A±C), 50 ␮m; (D), 100 ␮m.

L; R. PatinÄo, unpublished) only slightly exceeds al. 1997; Sellitti et al. 2000; Vischer and Bogerd the range generally recommended for ®shes (Wed- 2003) have been reported in the vertebrate kidney, emeyer 1996). Further, the AP-treated ®sh ap- including the teleost kidney. Also, the hemopoietic peared healthy and showed normal feeding and compartment of the teleost trunk kidney contains reproductive behavior as well as normal spawning lymphocytes (Rijkers et al. 1980; Danilova and success (PatinÄo et al. 2003b). More importantly, Steiner 2002), and thyroid hormones are believed sodium perchlorate also increased the incidence of to in¯uence the process of lymphopoiesis in var- Downloaded by [SUNY Brockport] at 12:03 19 December 2011 renal MAs in mosquito®sh (present study). These ious vertebrate taxa (Rollins-Smith et al. 1993; observations suggest that increased renal MA for- Hastings et al. 1997; Dorshkind and Horseman mation in both species was a speci®c response to 2000). To examine the hypothesis that the thyroid perchlorate exposure. system is involved in perchlorate-dependent renal Histology of mosquito®sh thyroid follicles was MA formation, however, researchers will need to not performed, but histological examination of the localize thyroid hormone and TSH receptors to thyroid follicles of zebra®sh indicated that expo- speci®c tissue compartments (excretory or he- sure to perchlorate caused severe angiogenesis, hy- mopoietic) and cells within the trunk kidney. pertrophy, hyperplasia, and colloid depletion (Pa- Direct effects of perchlorate exposure on renal tinÄo et al. 2003b). Thus, the effect of perchlorate excretory function are also possible, given that on renal MA formation may have been indirect, perchlorate-sensitive sodium iodide symporters acting through alterations in brain±pituitary±thy- have been reported in mammalian kidney tubules roid activity and in thyroid hormone or TSH levels. (e.g., Spitzweg et al. 2001). At high doses that Consistent with this hypothesis, receptor activity severely affected several organ systems, AP was or receptor transcripts for thyroid hormones toxic to the mammalian kidney (Sellivanova and (MacLatchy and Eales 1992) and TSH (Dutton et Vorobieva 1968). However, even if sodium iodide PERCHLORATE AND KIDNEY MACROPHAGE AGGREGATES 149

FIGURE 3.ÐEstimated percent area (ϩSE) of mos- FIGURE 2.ÐEstimated percent area (ϩSE) of zebra®sh quito®sh kidney sections occupied by macrophage ag- kidney sections occupied by macrophage aggregates gregates (MAs). Male ®sh were exposed to control water (MAs). Male and female ®sh were exposed to control or water containing various concentrations of sodium water or water containing ammonium perchlorate for 8 perchlorate for 8 weeks. The incidence of renal MAs weeks. The incidence of renal MAs was signi®cantly was signi®cantly higher in mosquito®sh exposed to a higher in zebra®sh exposed to a measured perchlorate measured concentration of perchlorate of 92 mg/L, concentration of 18 mg/L (asterisk; Student's t-test on whereas the incidences of renal MAs at 1 and 8 mg/L square-root-transformed data, P Ͻ 0.05; n ϭ 5±6 aquaria were intermediate between those of the control and 92- per treatment; the untransformed data are shown on the mg/L treatments (bars with common letters are not sig- graph). ni®cantly different; one-way ANOVA and Duncan's multiple-range test on square-root-transformed data, P Ͻ 0.05; n ϭ 5 aquaria per treatment; the untransformed symporters were found in ®sh kidney tubules, it data are shown on the graph). is unclear how such occurrence could be tied to the perchlorate-dependent MA response of the hemopoietic tissue compartment observed in this trunk kidney is affected by perchlorate exposure. study. PAS-positive reactions indicate the presence of The role of macrophage aggregates or centers is polysaccharide material, a common feature of ®sh generally considered to be the recycling and re- tissue MAs (Herraez and Zapata 1986; PatinÄo et al. moval of cellular debris; the sequestering, destruc- 2003a). Renal MAs of various ®sh species typically tion, and removal of cellular toxicants; antigen trap- contain melanin (Tsujii and Seno 1990; Herraez and ping and presentation to lymphocytes; and other Zapata 1991; Weeks et al. 1992; Fournie et al. 2001; functions (Tsujii and Seno 1990; Wolke 1992; Agius and Roberts 2003). The signi®cance of mel- Agius and Roberts 2003). In ®shes, exposure to anin in MAs is unclear, but one possible function environmental contaminants is known to increase is to protect the cells against injury from oxidizing Downloaded by [SUNY Brockport] at 12:03 19 December 2011 the incidence of MAs in some tissues, a response radicals (Henninger and Beresford 1990; Roza- that may indicate the occurrence of tissue injury or nowska et al. 1999; Jacobson 2000). The results of immunotoxic response (Weeks et al. 1992; Wolke the present study con®rmed the presence of signif- 1992; Fournie et al. 2001; Agius and Roberts 2003). icant melanin deposits in renal MAs from mosqui- Because the MAs of zebra®sh and mosquito®sh kid- to®sh but not in zebra®sh. We do not know whether neys were associated with the hemopoietic tissue the difference in melanin content of renal MAs be- compartment, it is possible that the blood cell± tween the two species re¯ects species-dependent forming or immune function of this tissue was af- differences in their function or differences in the fected by perchlorate exposure. Zebra®sh MAs con- perchlorate exposure regimens used for each spe- tained hemosiderin and PAS-positive components. cies. The presence of hemosiderin, a protein-bound iron In conclusion, the results of the present study pigment, indicates the breakdown and storage of suggest that exposure to perchlorate can affect iron-containing cellular products such as hemoglo- physiological functions other than thyroid activity bin (Wolke 1992). Therefore, an increase in the and general development in ®shes. The concen- amount of hemosiderin-containing MAs supports trations at which perchlorate had clear effects on the notion that the erythropoietic function of the the incidence of renal MAs (18 mg/L in zebra®sh 150 CAPPS ET AL.

and 92 mg/L in mosquito®sh) are at the high end the National Academy of Sciences of the USA 94: of perchlorate concentrations previously reported 13011±13016. in the environmentÐperchlorate concentrations as Capasso G., G. De Tommaso, A. Pica, P. Anastasio, J. Capasso, R. Kinne, and N. G. De Santo. 1999. Ef- great as 33 mg/L have been reported in some con- fects of thyroid hormones on heart and kidney func- taminated aquatic habitats (Smith et al. 2001). tions. Mineral and Electrolyte Metabolism 25:56± Thus, the environmental relevance of the present 64. ®ndings is uncertain. However, it has been reported Danilova, N., and L. A. Steiner. 2002. B cells develop that the magnitude of the thyroidal effects of per- in the zebra®sh pancreas. Proceedings of the Na- chlorate are a function of not only exposure con- tional Academy of Sciences of the USA 99:13711± 13716. centration but also exposure duration (Fernandez Dorshkind, K., and N. D. Horseman. 2000. The roles Rodriguez et al. 1991; S. Mukhi et al., Texas Tech of prolactin, growth hormone, insulin-like growth University, unpublished). Thus, if the thyroid system factor-I, and thyroid hormones in lymphocyte de- were involved in the mechanism of perchlorate- velopment and function: insights from genetic mod- induced kidney MAs, exposures to perchlorate at els of hormone and hormone receptor de®ciency. lower concentrations but for longer periods of time Endocrine Reviews 21:292±312. than those used in the present study might also Dutton, C. M., W. Joba, C. Spitzweg, A. E. Heufelder, and R. S. Bahn. 1997. Thyrotropin receptor ex- affect the structure and function of the ®sh kidney. pression in adrenal, kidney, and thymus. Thyroid 7: 879±884. Acknowledgments Fernandez Rodriguez, A., H. Galera Davidson, M. Sal- guero Villadiego, A. Moreno Fernandez, I. Martin This study was funded by the U.S. Department Lacave, and J. Fernandez Sanz. 1991. Induction of of Defense through the Strategic Environmental thyroid proliferative changes in rats treated with Research and Development Program under a co- antithyroid compound. Anatomic Histological Em- operative agreement with the U.S. Air Force, In- bryology 20:289±298. stitute for Environmental Safety and Occupational Fisher, J., P. Todd, D. Mattie, D. Godfey, L. Narayanan, Health, Brooks Air Force Base, Texas; and by the and K. Yu. 2000. Preliminary development of a U.S. Geological Survey through the Texas Coop- physiological model for perchlorate in the adult male rat: a framework for further studies. Drug and erative Fish and Wildlife Research Unit. Com- Chemical Toxicology 23:243±258. ments on a draft of this manuscript were provided Fournie, J. W., J. K. Summers, L. A. Courtney, V. D. by Christine Densmore and Luke Iwanowicz. The Engle, and V. S. Blazer. 2001. Utility of macro- U.S. Geological Survey, Texas Tech University, phage aggregates as an indicator of ®sh exposure Texas Parks and Wildlife Department, and The to degraded environments. Journal of Aquatic An- Wildlife Management Institute jointly sponsor the imal Health 13:105±116. Texas Cooperative Fish and Wildlife Research Goleman, W. L., J. A. Carr, and T. A. Anderson. 2002a. Environmentally relevant concentrations of am- Unit. The views and conclusions contained herein monium perchlorate inhibit thyroid function and al- are those of the authors and should not be inter- ter sex ratios in developing Xenopus laevis. Envi- preted as necessarily representing the of®cial pol- ronmental Toxicology and Chemistry 21:590±597. icies or endorsements, either expressed or implied, Goleman, W. L., L. J. Urquidi, T. A. Anderson, E. E.

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