Environmental Toxicology and Chemistry, Vol. 33, No. 3, pp. 553–559, 2014 # 2013 SETAC Printed in the USA

PHYTOESTROGENS IN THE ENVIRONMENT, I: OCCURRENCE AND EXPOSURE EFFECTS ON FATHEAD MINNOWS DANIEL C. REARICK,y NATHAN T. FLEISCHHACKER,z MEGAN M. KELLY,x WILLIAM A. ARNOLD,zx PAIGE J. NOVAK,zx and HEIKO L. SCHOENFUSS*y yAquatic Toxicology Laboratory, St. Cloud State University, St. Cloud, Minnesota, USA zDepartment of Civil Engineering, University of Minnesota, St. Paul, Minnesota, USA xWater Resources Science Graduate Program, University of Minnesota, St. Paul, Minnesota, USA

(Submitted 12 August 2013; Returned for Revision 23 September 2013; Accepted 8 November 2013) Abstract: Naturally occurring phytoestrogens may mimic biogenic estrogens and modulate endocrine action in vertebrates. Little is known, however, about their temporal and spatial variability in the environment and the biological effects associated with exposures. The present study assessed the environmental presence of phytoestrogens in human-impacted and relatively pristine areas. The response in larval and sexually mature fathead minnows to environmentally relevant concentrations of 3 common phytoestrogens (genistein, daidzein, and formononetin), both singly and in mixture, was also quantified. Phytoestrogens were only present in the human-impacted surface waters. When detected, mean concentrations were low (standard deviation) in an urban lake: 1.4  0.5 ng/L, 1.6  0.7 ng/L, and 1.1  0.2 ng/L for genistein, daidzein, and formononetin, respectively, and in treated wastewater effluent: 1.6  0.4 ng/L, 1.8  1.3 ng/L, and 2.0 ng/L. Biochanin A was detected twice, whereas zearalenone and coumestrol were never detected. No clear temporal trends of aqueous phytoestrogen concentration were evident. Larval survival was significantly reduced in genistein, formononetin, and mixture treatments, whereas adult male fish only exhibited subtle changes to their anatomy, physiology, and behavior. Daidzein-exposed adult females produced greater quantities of eggs. The present study indicates that genistein, daidzein, and formononetin are likely attenuated rapidly and are unlikely to cause widespread ecological harm in the absence of other stressors. Environ Toxicol Chem 2014;33:553–559. # 2013 SETAC Keywords: Endocrine-active compounds

Reproduction

Behavior

Pimephales promelas

responses following phytoestrogen exposures singly and in mixtures will provide information regarding the cumulative effect of natural phytoestrogen mixtures. Exposures across adult and larval life stages will help us assess windows of vulnerability, which may coincide with seasonal variations in environmental phytoestrogen concentrations. Despite possible effects on wild populations of fish, the presence of phytoestrogens in the environment, in both humanimpacted and relatively pristine surface waters, is still widely unknown. Several studies have assessed phytoestrogen occurrence downstream from pulp and paper mill discharges, finding elevated concentrations of isoflavones and phytosterols [4,18– 20]. Municipal and industrial effluents are also highly variable sources of phytoestrogens to surface water, containing concentrations ranging from 1 ng/L to 250 000 ng/L genistein plus daidzein [14,21–23]. Nonpoint agricultural runoff can also contribute phytoestrogens to surface water, although such runoff generally contains much lower concentrations of phytoestrogens (1 ng/L–10 ng/L) [24,25]. Combined or individually, these sources could convey significant quantities of phytoestrogens to aquatic ecosystems. It is not known, however, whether and at what concentrations phytoestrogens are present in most surface waters because dilution factors are likely to vary substantially between sources. Given the remaining uncertainty regarding the environmental presence and biological effects of phytoestrogen exposure on fish species such as the fathead minnow (Pimephales promelas), 2 major objectives were evaluated during the present study. First, the environmental presence of phytoestrogens in humanimpacted and relatively pristine areas was assessed. Second, organismal response (larval and sexually mature fish) was quantified following exposure to 3 common phytoestrogens

INTRODUCTION

Numerous studies have evaluated the sublethal consequences for androgenic, estrogenic, and antiandrogenic contaminants originating from agricultural runoff, municipal wastewater, and industrial effluent [1,2]. These effects range from reproductive impairments and skewed sex ratios in adult fish [3–6] to reduced locomotion and predator avoidance performance in larval fish [7–9]. Less attention, however, has been focused on naturally occurring phytoestrogens with similar endocrine modulating capabilities. Phytoestrogens are produced naturally by many wild and cultivated plants and are especially abundant in legumes, including soybeans, which are an abundant row crop in North America. The phytoestrogens genistein and daidzein and their methylated derivatives biochanin A and formononetin, respectively, are shown in Figure 1. These compounds enter surface waters through agricultural nonpoint sources as well as industrial and municipal wastewater effluents. Analogous to biogenic estrogen counterparts, phytoestrogens can influence development [10–14], alter behavior [11,15,16], and impair reproductive success [10–17] in individuals. Population significance is yet to be established. Although these studies suggest deleterious effects of phytoestrogen exposure, they were either conducted in mammalian models [10,13,17], used adult animals [10–12,17], did not include methylated derivatives of phytoestrogens [10–12,15,17], or did not include phytoestrogen mixtures [11,12]. Advancing our understanding of biological All Supplemental Data may be found in the online version of this article. * Address correspondence to [email protected]. Published online 19 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2461 553

554

Environ Toxicol Chem 33, 2014

HO

HO

O

OH

D.C. Rearick et al. O

O

O

OH

OH

Genistein HO

Daidzein

O

OH

HO

O

O

O

O

Biochanin A

O

Formononetin

Figure 1. Structures of the common phytoestrogens genistein, daidzein, biochanin A, and formononetin.

(genistein, daidzein, and formononetin), singly and in mixture at concentrations relevant to environmental exposures. Taken together, these objectives allow a much clearer assessment to emerge regarding the phytoestrogen risk to ecologically relevant fish species based on their environmental presence, apparent persistence, and biological effects. METHODS

Surface water samples Sample collection. To understand the magnitude and variability of environmental concentrations of phytoestrogens, surface water samples were taken at 5 sites in the Upper Midwest. At 3 sites, Lake Vadnais (Vadnais Heights, MN, USA), the Metropolitan Wastewater Treatment Plant (Metro Plant) effluent channel (St. Paul, MN, USA), and Straight Lake (Straight Lake State Park, WI, USA), sampling was performed to determine the temporal variability of phytoestrogens. Sampling occurred over 2 campaigns (5 February 2011–21 July 2011 and 5 May 2012–1 June 2012 for Lake Vadnais; 10 May 2011–19 September 2011 and 5 May 2012–1 June 2012 for the Metro Plant; and 25 April 2011–30 October 2011 and 5 May 2012–13 May 2012 for Straight Lake). Samples were taken in triplicate, every 2 weeks, at the same locations. The remaining 2 sites, Minnesota River (Mankato, MN, USA) and Okabena Creek (Brewster, MN, USA) were sampled to determine the spatial variation of phytoestrogen concentration at a single general location. To determine spatial variation, 7 or 8 sample locations, both upstream and downstream from suspected phytoestrogen point sources, were sampled on 3 dates (20 June 2011 and 8 November 2011 at the Minnesota River location and 6 June 2012 at the Okabena Creek location). Samples from the upstream locations, the suspected phytoestrogen discharges themselves, and 1 to 2 points downstream of the discharges were sampled in triplicate. Samples were collected from the top 0.5-meter of the water column in solvent-rinsed 1-L amber glass containers. After collection, all samples were packed on ice for transport to the laboratory, where they were refrigerated at 4 8C. Once at the laboratory, samples were filtered through glass wool, split into 0.6-L aliquots, and amended with chemical surrogates (d3genistein and d4-daidzein, Cambridge Isotopes). Samples were then concentrated via solid phase extraction (SPE), cleaned with silica gel, and analyzed via liquid chromatography–tandem mass spectrometry (LC–MS/MS; described in LC-MS/MS analysis of phytoestrogens). Sample preparation for phytoestrogen analysis. Solid phase extraction cartridges (6 mL, 200 mg HLB Oasis cartridges) were conditioned with 2 column volumes of acetone followed by 2

column volumes of ultrapure (Milli-Q, Millipore) water. Samples were loaded onto the SPE cartridge at a flow rate

Phytoestrogens in the environment, I: occurrence and exposure effects on fathead minnows.

Naturally occurring phytoestrogens may mimic biogenic estrogens and modulate endocrine action in vertebrates. Little is known, however, about their te...
194KB Sizes 0 Downloads 0 Views