measuring sources of endocrine disrupting compounds in north carolina waterways: a gis-based sampling approach for studies on intersex fish Dana K. Sackett, Matthew Rubino, Crystal Lee-Pow, D. Derek Aday, W. Gregory Cope, Seth Kullman, James A. Rice, Thomas J. Kwak and Mac Law The presence of endocrine disrupting compounds (EDCs), particularly estrogens and estrogen mimics, has drawn public concern across the globe.

the endocrine system Chemical messages sent through the blood for cell to cell communication that activates physiological response Various glands located throughout the body which synthesize and secrete hormones to regulate an array of biological processes: Ductless, Rich Supply of Blood Endocrine disruption refers to the interference of endocrine system function by environmental chemicals. The endocrine system participates in virtually all important functions of an organism, such as sexual differentiation before birth, sexual maturition during puberty, reproduction in adulthood, growth, metabolism, digestion, cardiovascular function,and excretion.

Endocrine disruptors: “exogenous agents that interfere with the production, release, transport, binding, action or elimination of natural hormones in the body…” Mention here about lots of different chemicals with different affinities. In fact the “binding domain” of the ER is oriented in space as a pocket and the estradiol molecule fits into this pocket in a precise fashion. In addition to this fit, attractive forces due to chemical interactions between hormone and receptor contribute to the strength (or affinity) of this binding. Perhaps the most provocative mechanistic issue in the field pertains to the “promiscuity” of the estrogen receptor (ER) or why the ER is receptive to a large number of seemingly diverse chemicals displayed in Fig. 1. An explanation of this phenomenon requires an understanding of how estradiol binds to its’ receptor and of the chemical similarities that exist between this steroid and nonsteroidal ER ligands (ligand refers to a substance that binds). As mentioned previously, a hormonereceptor relationship is analogous to a key fitting into a lock. In fact the “binding domain” of the ER is oriented in space as a pocket and the estradiol molecule fits into this pocket in a precise fashion. In addition to this fit, attractive forces due to chemical interactions between hormone and receptor contribute to the strength (or affinity) of this binding

Estrogens and Estrogen Mimics Males producing vitellogenin Yolk precursor typically seen only in females Intersex Individuals with both male and female characteristics Feminized males Endocrine disrupting compounds have been associated with high incidence of fish intersex, defined as the presence of individuals with both male and female characteristics, and the expression of vitellogenin in male fish, a yolk precursor produced in the liver that is stimulated by estrogen (typically seen only in females). mortality is due to excessive production of vitellogenin, which causes kidney failure (other organs probably also suffer severe toxicity), followed by death(102), but it takes a few days for the blood vitellogenin concentration to reach a level high enough to severely disrupt kidney function.

Ronny Van Aerle et al. 2001: Gudgeon (Gobio gobio) in UK at locations that receive high-volume discharges of sewage treatment works [STW] effluent and that contain intersex roach, Intersex gonads were found at all sites. High vitellogenin found in fish as well Histological sections of (a) a normal male testis (3214), (b) a normal female ovary in vitellogenesis (354), (c) an intersex gonad containing primary oocytes only (category A, 3214)), and (d) a grossly intersex gonad containing both primary and secondary (vitellogenic) oocytes (category C, 354)). CA 5 cortical alveolus–stage oocyte, PO 5 primary oocyte, Sc 5 spermatocytes, Sg 5 spermatogonia, SO 5 secondary (vitellogenic) oocyte, Sz 5 spermatozoa, St 5 spermatids, Tt 5 testicular tissue, * 5 degenerative tissue

Study recently reported widespread intersex in the US with the highest incidence (67-91%) in the Pee Dee River in North Carolina and South Carolina Evidence links EDCs with fish reproductive impairment and may lead to fish population declines Kidd et al. 2007 Collapse of natural fathead minnow population with cascading affects on a population of lake trout

Another troubling aspect of EDCs: Currently lack a clear understanding of extent and distribution in surface waters and their relation to potential sources i.e. waste water treatment plants, concentrated animal feeding operations, land application of municipal waste especially given there potential impacts Large estrogen loads associated with animal feeding operations (AFOs) arise not only from steroidal treatments given to livestock to enhance growth and fecundity, but also from the current trend towards concentrating animals into small geographic areas, magnifying waste related issues. Manure disposal presents a potential source for surface and groundwater contamination, consequently threatening human health and imperiling wildlife communities.

Objectives: Identify locations of sources of EDCs (focusing on estrogens and estrogen mimics) Quantify the potential impact of these sources in North Carolina water bodies using GIS Sample water to validate GIS and examine extent of EDCs in surface waters This study provides information on the extent and distribution of EDCs in surface waters, an important first step towards developing a management strategy and determines the relative contribution of EDCs from different sources. Blazer et al. 2012 showed land-use (application of manure and pesticides influence seasonal observations in intersex and vitellogenin (smallmouth bass). Also saw severity of intersex affected by %ag, total # of CAFOS, and animal density Excrete hormones like people, steroid treatments given to livestock to enhance growth and fecundity, concentrating animals to small geographic areas magnifies waste related issues. Despite corresponding concerns over the presence of hormones originating from agricultural production in drinking water and aquatic systems, data collection on estrogen concentrations in livestock waste streams has only recently begun. Likewise, little information on the potential off-site transport of estrogens exists. Nevertheless, initial results find not only elevated estrogenic concentrations, but also evidence supporting migration to both surface and groundwater systems.

Data Sources: Point Sources: NPDES, EPA, NCDENR Wastewater treatment facilities Sewage and industrial Permitted discharge Non-point Sources: Concentrated animal feeding operations Permitted number of animals Agricultural applications of municipal waste Mean of CAFO Superfund sites Mean CAFO National Pollutant Discharge Elimination System, Environmental Protection Agency, and North Carolina Department of Environmental and Natural Resources

National Pollutant Discharge Elimination System, Environmental Protection Agency, and North Carolina Department of Environmental and Natural Resources

Point-source Non point-source Data Use: Used to create point and non-point source maps depicting the relative and cumulative contribution of potential sources of EDCs 12-digit HUCs Non point-source

Measured estrogen activity in water using a bio-assay (YES) - Doesn’t matter what chemicals there as long as they light up an ER - Can get an equivalence of 17β-estradiol

Extreme low dose effects Our range 0.00 – 34.5 ng/L No Risk < 1 ng/L High Risk > 10 ng/L Extreme low dose effects (4.28 ng/L next highest) Williams et al. 2009 30, 60, 120 ng/L in lab another study had 0 then 1ng/l, then higher 10 100 etc (ppt – ppb) Bisphenol A is a nonsteroidal xenoestrogen that exhibits approximately 10−4 the activity of estradiol. The activity of most xenoestrogens is weak (from about 1/1000th to 1/1,000,000th that of estradiol)

P = 0.02 B A A Blazer et al. 2012 showed land-use (application of manure and pesticides) influence seasonal observations in intersex and vitellogenin (smallmouth bass). Also saw severity of intersex affected by %ag, total # of CAFOS, and animal density

Model: Point P R2 N Model: Point P R2 N Model: Non-point P R2 N Ecoregion, Map Category 0.03 0.59 21 Map Category 0.06 0.35 Ecoregion 0.15 0.26 Model: Point P R2 N Ecoregion, Map Category 0.03 0.59 21 Map Category 0.06 0.35 Ecoregion 0.15 0.26 Model: Non-point P R2 N Ecoregion, Map Category 0.34 0.4 21 Map Category 0.12 0.35 Ecoregion 0.73 0.07

Actual log10 (std estradiol picomol/L) Predicted log10 (std estradiol picomol/L)

Actual log10 (std estradiol picomol/L) Predicted log10 (std estradiol picomol/L)

Point Source Ecoregion Chowdhury et al. 2011  water chemistry (No3 and Fe and humic acid) increase photodegradation, turbidity can reduce photodegradation of E2…other chemicals can also be affected differently by water chemistry…flushing another potential hypothesis Ecoregion

Overall low concentrations However, extreme low doses may cause negative impacts GIS approach useful for point sources Caution: unrecorded historical contamination Non-point contributions may only occur seasonally Intersex more reflective of map predictions Incorporate long term low dose exposures Our study examined the distribution of EDCs in surface waters and relative contribution from different sources Vandenberg et al. 2012 Blazer et al. 2012 showed land-use (application of manure and pesticides) influence seasonal observations in intersex and vitellogenin (smallmouth bass). Also saw severity of intersex affected by %ag, total # of CAFOS, and animal density Study by Iwanowicz et al. 2009 used passive sampling devices and were unable to detect natural or synthetic estrogens (<2.5ng/L) and still found a high incidence of intersex (82-100% of SMB) Similar low levels 90% < 0.1 ng/L, Quiros et al. 2005 (Portugese Rivers)

Thanks to… Cam Mcnutt Bryn Tracy Chuck Cranford Charles Bryan Endagered Species Branch, Fort Bragg Paul Super Great Smokey Mountains National Park

Questions adsorption on to granular activated carbon Estrogens display characteristics typical of organic compounds, including low solubility and high affinity to organic matter