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Archives of Environmental Contamination and Toxicology (v.42, #1)
Interaction of 2,4,6-Trinitrotoluene (TNT) and 4-Amino-2,6-Dinitrotoluene with Humic Monomers in the Presence of Oxidative Enzymes by C.-J. Wang; S. Thiele; J.-M. Bollag (pp. 1-8).
Oxidative coupling of nitroaromatic compounds involving soil organic matter was examined as a means of soil remediation. Humic monomers, serving as model compounds for soil humic substances, were used as cosubstrates, applying phenoloxidases (laccase from Trametes villosa and peroxidase from horseradish) as oxidative biocatalysts. Without the addition of a cosubstrate, only 30% of 4-amino-2,6-dinitrotoluene (4ADNT) and no 2,4,6-trinitrotoluene (TNT) were transformed in the presence of the laccase. Adding various phenolic monomers produced differing effects on the enzyme-mediated transformation, which indicated that xenobiotics are preferentially bound to quinoid and phenolic moieties of soil humic substances. In the presence of the humic monomer catechol and the enzyme, up to 100% of 4ADNT and up to 80% of TNT were transformed. Enzymatic transformation of 4ADNT in the presence of catechol reached a maximum at pH 6.8. TNT transformation, however, further increased at pH values above 6.8, even in the absence of the enzyme, due to chemical polymerization of catechol. We postulate a two-step reaction mechanism. The humic monomer is initially oxidized to a semi-quinone radical by a phenoloxidase. Subsequent oxidative coupling involves reactions with additional humic monomers or anilinic products derived from TNT, forming an anilinoquinone via nucleophilic addition or a benzoquinone-imine through condensation.
Assessment of Toxicity Reduction in Wastewater Effluent Flowing Through a Treatment Wetland Using Pimephales promelas, Ceriodaphnia dubia, and Vibrio fischeri by J. M. Hemming; P. K. Turner; B. W. Brooks; W. T. Waller; T. W. La Point (pp. 9-16).
Effluent toxicity is regularly assessed with Ceriodaphnia dubia short-term chronic and Vibrio fischeri toxicity tests. Condition factor and hemotocrit of fish have recently been used to assess fish health following exposure to xenoestrogens in complex municipal effluents. To assess the ability of a treatment wetland to reduce or remove toxicity of a municipal effluent, we compared C. dubia and V. fischeri bioassays to Pimephales promelas responses in situ. Final whole effluent was diverted to a constructed wetland and effluent samples were taken daily from four sites, at incremental distances from the inflow, for a 3-week study. Overlapping 7-day C. dubia tests and V. fischeri assays were conducted with samples from each wetland site concurrent with a 3-week fish exposure. C. dubia survival and fecundity were significantly (< 0.0001) reduced at the inflow, but steadily improved with distance from the inflow. Fish condition and hematocrit were lower (α < 0.05) at wetland sites closer to the inflow than other wetland sites and laboratory controls. However, effluent toxicity was not detected by V. fischeri bioassays. Our findings indicate that 7-day C. dubia bioassays were most sensitive to effluent toxicity and suggest that other bioassays should be used concurrent with V. fischeri assays for municipal effluent toxicity testing.
Age- and Sex-Related Variation in Sensitivity to the Pyrethroid Cypermethrin in the Marine Copepod Acartia tonsa Dana by M. Medina; C. Barata; T. Telfer; D. J. Baird (pp. 17-22).
Acute effects of cypermethrin, a pesticide used to treat ectoparasite infestations of salmon, were assessed using the calanoid copepod Acartia tonsa. A. tonsa has been widely used for hazard assessment of chemicals in the marine environment using acute toxicity tests, but only with randomly selected adults, assuming a sex ratio of 1:1. The present study assesses the environmental hazard of cypermethrin by exposing nauplii and adult males and females, separately. Our results showed that the naupliar stages were 28 times more sensitive to cypermethrin than adults after 96 h of exposure, with LC50s of 0.005 μg L−1 and 0.142 μg L−1, respectively. Significant differences in sensitivity between sexes were only found during the first 24 h of exposure, with males being approximately twice as sensitive as females. The results of age-related variation in sensitivity are supported by studies with other species of copepods and toxicants, where the increased capacity of the adults for detoxification, the allometric differences in weight and size, and the molting process are given as explanations. Variation in sensitivity between sexes is discussed in terms of faster depuration rates in females through egg production and implications of feeding rate changes after 24 h of exposure. Our results suggested that standard toxicity test methods using A. tonsa are unsatisfactory because the most sensitive life stage is not included and sex-related differences in tolerance are not taken into account. We also found that cypermethrin caused significant mortality in Acartia at exposures concentrations from one to three orders of magnitudes lower than the recommended field treatment concentration for copepodic infestations.
Comparison of Methods to Obtain a Liquid Phase in Marine Sediment Toxicity Bioassays with Paracentrotus lividus Sea Urchin Embryos by R. Beiras (pp. 23-28).
Sediment toxicity bioassays using planktonic organisms are widespread tools in environmental quality assessment, but they have limitations because the method for extracting and diluting the liquid phase may affect the final toxicity. The present study compares the toxicity on sea urchin embryos and larvae of elutriates and pore water obtained from intertidal sediments by various methods. The effects of mixing method, dilution protocol, aeration, and pH adjustment of the pore water are discussed. For a quantitative assessment of toxicity, a posteriori dilution of the elutriate yielded results comparable to mixing the equivalent proportions of sediment and sea water. The highest sensitivity was found by using pore water. Rotator mixing in air-tight closed flasks was a second-best choice. In both cases the geochemical parameters of the liquid phases obtained were not suitable for the incubation of water-column organisms. To avoid the environmentally irrelevant toxicity caused by these natural conditions typical of highly reduced sediments, gentle aeration or dilution with O2-saturated control sea water is necessary prior to testing, despite the unavoidable changes in redox conditions. Measuring length of 48-h-old larvae improves the sensitivity and the discriminating power of the test.
Mechanism-Based Quantitative Structure-Phytotoxicity Relationships Comparative Inhibition of Substituted Phenols on Root Elongation of Cucumis sativus by X. Wang; Y. Wang; Y. Chunsheng; L. Wang; S. Han (pp. 29-35).
Phytotoxicity of selected substituted phenols to Cucumis sativus (log1/RC50, root elongation half inhibition concentration [mol/L] in logarithmic form) was determined by using a root elongation inhibition method and quantitative structure-activity relationships (QSARs) were developed. Hydrophobicity, described by 1-octanol/water partition coefficient in logarithmic form (log K ow) and electronic effect (characterized by the energy of the lowest unoccupied orbital, E lumo) proved to contribute mainly to the phytotoxicity of phenols in this study. Log K ow was used as a probe to classify the tested chemicals into subsets according to the modes of toxic action. The result indicated that the substituted phenols fell into two classes in general: narcotic phenols and bioreactive phenols. Most phenols elicited their toxic response via a polar narcosis mechanism and an excellent log K ow–dependent QSAR was developed (log1/RC50= 0.94 log K ow+ 2.04, n = 22, r 2 adj= 0.89). Selected phenols with bioreactive substructures exhibited elevated bioreactive toxicity and a strong E lumo-dependent correlation was achieved (log1/RC50=−0.91 E lumo+ 2.73, n = 6, r 2 adj= 0.81). This implied that their reactive toxicity involved mainly their in vivo electrophilic reactions. In an effort to model all chemicals without regard to their mechanisms, a highly predictive response-surface was developed with the phytotoxicity, hydrophobicity, and electrophilic reactivity (log1/RC50= 0.66 log K ow− 0.57 E lumo+ 2.31, n = 28, r 2 adj= 0.88).
Induction of Specific Isoforms of Metallothionein in Mussel Tissues After Exposure to Cadmium or Mercury by F. Geret; R. P. Cosson (pp. 36-42).
The synthesis of metallothioneins has been established for Mytilus edulis exposed to cadmium and mercury. We checked if this induction resulted in the synthesis of tissue- or metal-specific isoforms in the gills, the mantle, and the digestive gland that could be used as tool for the characterization of undefined metallic contamination of aquatic ecosystems. An accumulation of metals was observed in the selected organs after 21 days of exposure. The levels of metallothioneins measured by using the polarographic method were significantly increased by cadmium and mercury in the gills (21 days).Size exclusion chromatography showed the presence of a monomer and a dimer of metallothionein of respective apparent molecular weight about 12 kDa and 20 kDa in all samples. They were resolved into five components by anion exchange chromatography in the gills of control or Hg-treated mussels, whereas a sixth isoform was isolated in the gills of cadmium-exposed mussels. In the mantle of mussels exposed or not, five isoforms were separated, and in the digestive gland of mussels exposed or not, six isoforms were separated. The occurrence of a specific cadmium-binding isoform in the gills has to be confirmed in cadmium-contaminated specimens collected in situ before its detection may be used as biomarker of cadmium contamination.
Polycyclic Aromatic Hydrocarbon Metabolites in the Bile of a Territorial Benthic Fish, Oyster Toadfish (Opsanus tau) from the Elizabeth River, Virginia by A. D. Deshpande; R. J. Huggett; R. A. Halbrook (pp. 43-52).
Reverse-phase high-performance liquid chromatographic analyses of bile of a territorial benthic fish, oyster toadfish (Opsanus tau), indicated that fish from reference stations in the York and Elizabeth Rivers, Virginia, contained lower concentrations of polycyclic aromatic hydrocarbon (PAH) metabolites than fish from polluted stations. PAH metabolite levels in the bile of fish from mildly polluted stations were 7 to 10 times greater than those from reference stations. PAH metabolite levels in fish from a moderately polluted station and a highly polluted station were, respectively, 20 and 50 times greater than those from reference stations. Differential patterns of five major PAH metabolites in fish from the same station suggested individual variability in metabolic pathways possibly further convoluted by the differential inductions or suppressions of hepatic mixed-function oxygenase isozyme systems under various natural or anthropogenic habitat parameters. PAH metabolite levels in the bile of oyster toadfish correlated well with the gradient of PAH contamination in the Elizabeth River sediments. High levels of biliary PAH metabolites were not detected in muskrats (Ondatra zibethicus) collected along the polluted sections of the Elizabeth River, probably due to their primarily herbivorous nature. Assuming that the hepatobiliary system and the gastrointestinal tract are the major routes of biotransformation and excretion of PAHs in the muskrats, the contaminated diet appears to be a more important routes of exposure of muskrats (and possibly oyster toadfish) to PAHs than the transdermal transfer. Occurrence permitting, we propose oyster toadfish as a useful biological sentinel for early detection of estuarine PAH pollution.
Assessment of Exposure of Larval Razorback Sucker to Selenium in Natural Waters by D. W. Beyers; C. Sodergren (pp. 53-59).
This investigation evaluated effects of exposure of larval razorback sucker to waterborne and dietary selenium and other contaminants that occur in nursery habitats. Site waters were collected from three localities on the Colorado River near Grand Junction, CO; a total of five test waters (including control) were studied. Razorback sucker larvae were exposed to site-water contaminants via waterborne and dietary exposure using a laboratory food chain (algae, rotifer, razorback sucker). Fish were exposed for 28 days to site waters and food organisms cultured in site waters. Survival data were analyzed by inspection. Growth data were analyzed using analysis of variance to describe the response of fish in each site water and to describe the relative contribution of waterborne versus dietary exposure to constituents in site waters.Selenium concentrations in test-water treatments ranged from < 1 to 20.3 μg/L in water, < 0.702 to 21.8 μg/g in diet, and 2.34 to 42.0 μg/g in fish. Negative effects from dietary exposure to site-water constituents were detected, but the data suggest that they were caused by cocontaminants in the diet, not selenium exposure. Lack of detection of adverse effects from exposure does not imply that razorback sucker populations are not affected by increased environmental selenium concentrations. There are a variety of factors not included in this investigation that may influence sensitivity of razorback sucker populations to selenium.
Relating Body Condition to Inorganic Contaminant Concentrations of Diving Ducks Wintering in Coastal California by J. Y. Takekawa; S. E. Wainwright-De La Cruz; R. L. Hothem; J. Yee (pp. 60-70).
In wild waterfowl, poor winter body condition may negatively affect migration, survival, and reproduction. Environmental contaminants have been shown to adversely affect the body condition of captive birds, but few field studies have examined body condition and contaminants in wild birds during the winter. We assessed the body condition of carcasses from a collection of canvasbacks (Aythya valisineria) and lesser (A. affinis) and greater scaup (A. marila) wintering in coastal California. We used Akaike information criterion (AIC) to select the model with the best balance of parsimony and goodness of fit that related indices of body condition with concentrations of Cd, Cu, Hg, Se, and Zn. Total ash-free protein in canvasbacks decreased with increasing Se concentrations, and pancreas mass decreased with increasing Hg. We combined the closely related lesser and greater scaup in analyses and found that total carcass fat, pancreas mass, and carcass mass decreased with increasing Zn concentrations, and pancreas mass decreased with increasing Hg. Our AIC analysis indicated that some indices of body condition in diving ducks were inversely related to some environmental contaminants in this collection, but additional AIC analyses should be conducted across a wider range of contaminant concentrations to corroborate our findings.
Mercury and Selenium Concentrations in Livers and Eggs of Common Loons (Gavia immer) from Minnesota by K. Bischoff; J. Pichner; W. E. Braselton; C. Counard; D. C. Evers; W. C. Edwards (pp. 71-76).
Mercury and selenium act as mutual antagonists in mature birds, but their toxicity is additive or synergistic in avian embryos and immature birds. Twenty eggs and livers from 18 mature and 9 immature common loon carcasses found in Minnesota were collected. Livers and eggs were analyzed for mercury and selenium by atomic emission and atomic absorption spectroscopy. Liver mercury concentrations were significantly higher in mature loons compared to those of immature loons and eggs. Liver selenium concentrations were significantly higher in mature loons than those of immature loons, and selenium concentrations in immature loon livers were significantly higher than levels in eggs. There was a significant positive correlation between mercury and selenium in loon livers. There was a negative correlation between mercury and selenium in eggs.
Physiological, Growth, and Immune Responses of Japanese Quail Chicks to the Multiple Stressors of Immunological Challenge and Lead Shot by J. M. Fair; R. E. Ricklefs (pp. 77-87).
Exposure to the combination of a contaminant and an immunological challenge during development may greatly increase the impact of either or both of these stressors on an individual. This study investigated the interacting effects of a nonpathogenic immunological challenge and lead shot exposure early in the development of a precocial species. Seventy-one quail (Coturnix coturnix japonica) chicks orally received either one #9 lead shot (0.05 g), four lead shots (0.2 g), or no lead at the age of 8 days. A third of each of these groups of chicks were intraperitoneally injected with either 0.075 ml of 10% chukar (Alectoris graeca) red blood cells (CRBCs), Newcastle disease virus (NDV), or a placebo oil vaccine at 13 and 35 days of age. There was no difference in the survival between any of the lead or antigen treatment groups. Lead concentrations in blood were greater for the lead-dosed groups on day 49 but not on day 128. Growth curves were approximated by Weibull functions; growth parameters did not differ between lead-treated and antigen-treated birds. CRBC antigen increases fluctuating asymmetry (FA) for primary feather five, while NDV reduced FA. Lead did not affect antibody production or cell-mediated immune response. White blood cell numbers increased 7 days after antigen injection on days 27 and 49 of age. Granulocyte numbers were significantly higher for the lead-treated quail than the control quail, and both antigen-treated groups had lower granulocyte numbers than control quail. The treatment groups of combining NDV and lead shot had an average plasma protein 14% lower than other groups. Lead shot in the highest of these doses lowered asymptotic weights, increased hematocrits, lowered plasma protein, and increased granulocyte numbers of quail.
Tissue Levels of Lead in Experimentally Exposed Zebra Finches (Taeniopygia guttata) with Particular Attention on the Use of Feathers as Biomonitors by T. Dauwe; L. Bervoets; R. Blust; M. Eens (pp. 88-92).
We tested experimentally whether zebra finch feathers can be used as a biomonitor for lead pollution, and we examined whether lead exposure influences the accumulation of zinc into feathers. Two groups of eight adult male zebra finches were dosed with, respectively, 0 and 25 ppm lead as lead acetate in their drinking water. After 30 days, lead-treated zebra finches accumulated significantly higher lead concentrations in brain, fat, kidney, liver, muscle, testes, and regrown outer tail feathers than control individuals. Lead levels in regrown outer tail feathers were significantly higher than in original outer tail feathers in the exposed group. The concentration of lead in original (not regrown) fifth tail feathers at the end of the experiment was significantly higher than lead levels in the original outer tail feathers. Our results indicate that lead in regrown feathers originates both from internal deposition and external contamination through the excretion of the uropygial gland during preening. Lead levels in regrown feathers were significantly correlated with levels in liver, kidney, and muscle, suggesting that feathers can be used as a biomonitor for lead. We found that lead had an influence on the metabolism of zinc. Zinc concentrations in the regrown feathers were significantly lower in the lead-treated group although zinc levels in the liver did not differ significantly. Moreover, lead and zinc concentrations in the feathers were significantly negatively correlated.
Interactions in Developmental Toxicology: Effects of Concurrent Exposure to Lead, Organic Mercury, and Arsenic in Pregnant Mice by M. Bellés; M. L. Albina; D. J. Sánchez; J. Corbella; J. L. Domingo (pp. 93-98).
The development toxicity of lead nitrate (25 mg/kg, SC), methylmercury chloride (12.5 mg/kg, PO), and sodium arsenite (6 mg/kg, SC) was assessed in CD1 mice following administration on gestation day 10 of these chemicals separately or in their binary and ternary combinations. Cesarean sections were performed on day 18 of gestation, and fetuses were examined for malformations and variations. Three fetuses from each dam were used for whole-body analyses of Pb, Hg, and As. Maternal toxic effects were more remarkable in the group concurrently exposed to Pb, Hg, and As than in those given binary combinations of the elements. In turn, maternal toxicity was more notable in these groups than in those given separately the test compounds. With regard to developmental toxicity, the most relevant effects (decreased fetal weight, cleft palate) corresponded to the Hg-treated groups. It is in agreement with the finding that in all experimental groups the levels of Pb and As in whole fetuses were under their respective detection limits. In general terms, the present data suggests that at the current doses, the interactive effects of Pb and As on Hg-induced developmental toxicity were not greater than additive. In contrast, exposure of pregnant mice to Pb and As at doses that were practically nontoxic to dams, concurrently with organic Hg at a toxic dose, caused supra-additive interactions in maternal toxicity.
Assessing Regional Variation of Environmental Fluoride Concentrations in Western Germany by Analysis of Antler Fluoride Content in Roe Deer (Capreolus capreolus) by U. Kierdorf; H. Kierdorf (pp. 99-104).
To assess regional variation in ambient fluoride levels, we analyzed the fluoride content of 188 antlers of roe deer (Capreolus capreolus) killed between 1990 and 1999 in 14 areas of the federal state of North Rhine–Westphalia, Germany. Individual antler fluoride concentrations ranged between 113 and 11,995 mg F−/kg bone ash, and sample means differed significantly (p < 0.0001) among the study areas. Low average concentrations (geometric means of 262 and 277 mg F−/kg bone ash, respectively) were found in antler samples from two control areas, located quite remote to major fluoride emission sources. The highest geometric mean fluoride content (1,677 mg F−/kg bone ash) was recorded for an antler sample from a study area exposed to fluoride emissions from an aluminium smelter and two mineral coal-fired power stations. In eight antlers obtained from roe deer living in the direct vicinity of the aluminium smelter, fluoride values ranged between 2,067 and 11,995 mg F−/kg bone ash, thereby demonstrating the strong impact of this emission source on its surroundings. The study showed that by analyzing antler fluoride concentrations, large-scale surveys of environmental contamination by fluoride can be performed in a standardized and cost-effective way in areas inhabited by deer. Due to its rather narrow home range, abundance, and high adaptability, the roe deer is particularly suited for such studies.
Identification of Hydroxylated PCB Metabolites and Other Phenolic Halogenated Pollutants in Human Blood Plasma by L. Hovander; T. Malmberg; M. Athanasiadou; I. Athanassiadis; S. Rahm; . Bergman; E. Klasson Wehler (pp. 105-117).
A growing number of studies have reported phenolic halogenated compounds (PHCs) that are retained in the blood of humans and wildlife. These PHCs may be industrial chemicals; metabolites thereof, as in the case with polychlorobiphenylols (OH-PCBs); or of natural origin. The present study was aimed to identify hitherto unknown PHCs in human plasma with chemical structures that are consistent to PHCs known to possess endocrine-disrupting activity. For this purpose, samples of blood plasma from 10 randomly selected male blood donors from Sweden were pooled and analyzed by GC/ECD and GC/MS. Brominated, bromochlorinated, and chlorinated methyl derivatives of phenols and OH-PCBs were synthesized to be used as authentic reference standards. More than 100 PHCs were indicated in the plasma, and among those a total of 9 monocyclic brominated or chlorinated phenol-, guaiacol-, and/or catechol-type compounds were identified as their methylated derivatives. The two major compounds were 2,4,6-tribromophenol and pentachlorophenol. Thirty-eight OH-PCB congeners were structurally identified on two GC columns of different polarity. The origin of the OH-PCB metabolites in the context of their parent PCB congeners are suggested. Other PHCs identified in the male plasma were Triclosan (5-chloro-2-[2,4-dichlorophenoxy] phenol), a common bactericide; 4-hydroxy-heptachlorostyrene, a metabolite of octachlorostyrene; and 3,5-dibromo-2-(2,4-dibromophenoxy)phenol, a natural compound and a potential metabolite of polybrominated diphenyl ethers.
Persistent Organochlorine Contaminants and Enantiomeric Signatures of Chiral Pollutants in Ringed Seals (Phoca hispida) Collected on the East and West Side of the Northwater Polynya, Canadian Arctic by A. T. Fisk; M. Holst; K. A. Hobson; J. Duffe; J. Moisey; R. J. Norstrom (pp. 118-126).
To examine the influence of diet and age on organochlorine contaminant (OC) concentrations in two closely related ringed seal (Phoca hispida) populations enantiomeric fractions (EFs) of chiral contaminants and stable isotopes of nitrogen (δ15N) and carbon (δ13C) were measured along with OCs in ringed seals collected from the east and west side of the Northwater Polynya. Seals from these two locations were feeding at the same trophic level based on δ15N values in muscle but had slightly different sources of carbon based on δ13C measurements in muscle. After removing the influence of age, sex, and blubber thickness, OC concentrations did not vary between ringed seals from the east and west side of the polynya. ΣPCB, ΣDDT, and Σchlordane were found to increase with age for both male and female seals. The inclusion of older (>20 years) female seals, which may have a reduced reproductive effort, may influence the relationships in females. Stable isotopes failed to describe OC concentrations in ringed seals suggesting that diet was not a major factor in variation of OC concentrations within this ringed seal population. Cis- and trans-chlordane, oxychlordane, and heptachlor epoxide were all nonracemic in the ringed seal blubber but did not vary with age, sex, or collection site. α-HCH appeared racemic (enantiomeric fraction = 0.50 ± 0.01) in the seals, although this EF is different than those previously observed in their prey species, and was found to vary significantly with age. EF values in the ringed seals varied considerably from other Arctic marine mammals and seabirds, providing addition evidence that the type(s) and characteristic(s) of the enzymes involved in biotransformation of chiral OCs vary between these organisms.
Regional Assessment of Atrazine Exposure and Incidence of Breast and Ovarian Cancers in Kentucky by C. Hopenhayn-Rich; M. L. Stump; S. R. Browning (pp. 127-136).
The association between breast and ovarian cancers with endogenous estrogen or hormonally related events has led to the hypothesis that exposures to exogenous estrogenic compounds in the environment may increase the risk of these cancers. Atrazine, the most commonly used herbicide in the United States, belongs to this group of compounds and is widely used in corn production. This study is an expansion of a previous investigation conducted in Kentucky. Using secondary data, we derived several indices of environmental exposure to atrazine and examined the association between these measures and the incidence of breast and ovarian cancer in Kentucky over a 5-year period (1993–97). Exposure indices to atrazine were derived based on public water measurements, acres of corn planted, and pounds of atrazine sold. Data on breast and ovarian cancer incidence were obtained from the Kentucky Cancer Registry by county and by the 15 Area Development Districts (ADDs) in which the 120 counties are grouped. Poisson regression analyses adjusted for education and race were conducted separately for each index of exposure and for a combined total exposure score. All exposure measures were divided in quartiles for analysis. A null association was found for breast cancer across all exposure indices, both by county and by ADD. For ovarian cancer, the data suggest an inverse association, with increasing exposure linked to decreasing incidence rates, both at the county and ADD level. The following are the rate ratios (RR) and corresponding 95% confidence intervals, for the summary exposure scores in the three upper quartiles, using the lowest quartile as baseline (RR = 1.0), and the county as the unit of analysis: 1.01 (0.83–1.21), 0.77 (0.66–0.90), and 0.76 (0.65–0.88). Due to the ecologic nature of this study and inherent limitations, it is possible that other factors may be contributing to these findings. Studies using individual-level data are recommended to elucidate the relationships between estrogenic environmental exposures and female reproductive cancers.