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Prioritization of Contaminants of Emerging Concern in Wastewater Treatment Plant Discharges using Chemical:Gene Interactions in Caged Fish Edward J. Perkins, Tanwir Habib, Barbara L. Escalon, Jenna E. Cavallin, linnea Thomas, Matthew Weberg, Megan N. Hughes, Kathleen M. Jensen, Michael D. Kahl, Daniel L. Villeneuve, Gerald T. Ankley, and Natalia Garcia-Reyero Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01567 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 28, 2017
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Prioritization of Contaminants of Emerging Concern in Wastewater Treatment Plant
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Discharges using Chemical:Gene Interactions in Caged Fish.
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Edward J. Perkins †*, Tanwir Habib∥, Barbara L. Escalon†, Jenna E. Cavallin§, Linnea
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Thomas§, Matthew Weberg§, Megan N. Hughes§, Kathleen M. Jensen§, Michael D. Kahl§,
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Daniel L. Villeneuve§, Gerald T. Ankley§, Natàlia Garcia-Reyero †
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†
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Halls Ferry Road, Vicksburg, MS, USA
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§
U.S. Army Engineer Research and Development Center, Environmental Laboratory, 3909
U.S. EPA, National Health and Environmental Effects Research Laboratory, Duluth, MN,
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USA
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Badger Technical Services, 3909 Halls Ferry Road, Vicksburg, MS, USA
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KEYWORDS. effects based monitoring, fathead minnows, Great Lakes, microarrays
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AUTHOR INFORMATION
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* Corresponding author:
[email protected]; ERDC, 3909 Halls Ferry Rd,
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Vicksburg, MS 39180; phone: +1-601-634-2872.
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ABSTRACT. We examined whether contaminants present in surface waters could be
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prioritized for further assessment by linking the presence of specific chemicals to gene
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expression changes in exposed fish. Fathead minnows were deployed in cages for 2, 4, or
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8 days at three locations near two different wastewater treatment plant discharge sites in
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the Saint Louis Bay, Duluth, MN and one upstream reference site. The biological impact of
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51 chemicals detected in the surface water of 133 targeted chemicals was determined
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using biochemical endpoints, exposure activity ratios for biological and estrogenic
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responses, known chemical:gene interactions from biological pathways and knowledge
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bases, and analysis of the co-variance of ovary gene expression with surface water
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chemistry. Thirty-two chemicals were significantly linked by co-variance with expressed
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genes. No estrogenic impact on biochemical endpoints was observed in male or female
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minnows. However, bisphenol A (BPA) was identified by chemical:gene co-variation as the
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most impactful estrogenic chemical across all exposure sites. This was consistent with
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identification of estrogenic effects on gene expression, high BPA exposure activity ratios
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across all test sites, and historical analysis of the study area. Gene expression analysis
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also indicated the presence of nontargeted chemicals including chemotherapeutics
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consistent with a local hospital waste stream. Overall impacts on gene expression
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appeared to related to changes in treatment plant function during rain events. This
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approach appears useful in examining the impacts of complex mixtures on fish and offers a
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potential route in linking chemical exposure to adverse outcomes that may reduce
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population sustainability.
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Table of Contents (TOC)/Abstract Art
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INTRODUCTION A growing list of xenobiotics including legacy contaminants (such as metals and
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persistent organic pollutants) and chemicals of emerging concern (CEC) such as
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pharmaceuticals, personal care products, flame retardants, industrial chemicals, and
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pesticides are being released into the environment.1,2 Because the potential environmental
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hazards of a large number of CECs in surface waters have not been well characterized, a
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number of screening and prioritization approaches have been developed to assess their
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potential impacts.3-6 Approaches for assessing CECs impacts on fish health have included
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determining chemical concentrations in water relative to the amount established as causing
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a biological effect or Exposure Activity Relationships (EAR) 7,8, in vitro assays 5,6,9,10,
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laboratory exposures of fish to field-collected water samples 11 and direct exposure of fish
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to surface water via cages or flow through systems.3,12-19 Gene expression changes have
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been employed to assess effects of complex chemical mixtures on fish, such as the
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potential of wastewater treatment plant (WWTP) discharge to cause estrogenic effects 20,
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identify differences in exposure sites 12,13,15,16,19,21, and infer the potential of specific
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chemicals to cause effects through the use of prior knowledge of chemical:gene or
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chemical:protein interactions.19,22,23
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A complicating factor in applying existing knowledge to infer impacts of specific chemicals
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in complex mixtures is the limited availability of chemical:gene interaction data for many
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chemicals. Existing databases of chemical:gene interactions, such as the Comparative
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Toxicogenomics Database (CTD) 24, STITCH (Search Tool for Interacting Chemicals) 25 or
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Ingenuity Knowledge Base26, are biased towards well studied chemicals and relevant
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interactions are under-represented for less studied compounds. Since specific chemicals
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can induce expression of specific genes in most organisms 27,28, the lack of existing
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chemical:gene interaction data could potentially be overcome by identifying changes in
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changes in gene expression in exposed caged fish that co-vary with chemical
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concentrations in exposure water. Using similar logic, Davis et al3 used covariation of
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metabolomic data from livers of caged fathead minnow and surface water analytical
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chemistry data to prioritize chemicals for further assessment in surface waters from the
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Great Lakes. Davis et al3 found that 32% of the 86 detected chemicals could not be
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significantly linked to the biological effect of metabolite changes and used this to place
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these chemicals at a low priority for further assessments. Since changes in gene
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expression and metabolites generally occur earlier and at chemical lower concentrations
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than apical effects, these approaches could facilitate more rapid analysis of the interact of
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CECs with the Great lakes ecosystem.
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Since gene expression could be used to identify chemicals as well as possible
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mechanisms of toxicity, we examined whether existing knowledge-based approaches
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combined with analysis of gene expression covariance with surface water chemistry can be
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used to link specific chemicals to biological effects. We then examined if gene expression
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level impacts can be used to prioritize chemicals for further studies. We applied the gene
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regulatory network inference algorithm Context Likelihood of Relatedness (CLR) 29 to infer
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the ability of a chemical detected in surface water to influence the expression levels of
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specific genes. CLR has been applied to infer relationships among mixed data sets
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including gene or protein expression, hormones, and endogenous metabolite profiles.30,31
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For the current analysis, fathead minnow (Pimephales promelas) were deployed in cages
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for 2, 4 or 8 d near two WWTP discharge sites in the St Louis Bay and at an upstream
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reference site near Duluth, MN, USA. Rain events occurred during deployment decreasing
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chemical input from a small WWTP and increasing output from the second. Biological
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impacts of WWTP discharges were assessed using a combination of approaches including
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Exposure Activity Ratios, plasma steroid hormone and vitellogenin (VTG, egg yolk protein
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precursor) assays of both male and female minnows and changes in ovary gene
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expression. The analysis indicated that many, but not all, of the chemicals measured in
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surface waters could plausibly have impacted gene expression in caged fathead minnows
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based on co-variance or previously established relationships. Estrogenic effects on gene
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expression were detected and could be linked to the presence of bisphenol A (BPA). We
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expect that further development of these types of approaches will lead to improved
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assessment of CEC exposure and effects in aquatic systems and could be applied broadly
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to a diversity of environmental matrices.
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MATERIALS AND METHODS Deployment and Exposure of Fathead Minnows at WWTP sites. Fish
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used in the study were reproductively mature fathead minnows (5-6 months old) from the
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US EPA Mid-Continent Ecology Division (Duluth, MN). All procedures involving animals
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were reviewed and approved by the Animal Care and Use Committee in accordance with
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Animal Welfare Act and Interagency Research Animal Committee guidelines. Field
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exposures were performed in September, 2010. Fish were deployed at four sites in the St
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Louis River near Duluth, MN, USA (SI, Figure S1). One site upstream of the two WWTPs
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(Fond du Lac, FDL; N 46° 39.517, W 92° 16.951) was chosen as a ‘reference’ location as it
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is located near a large forest away from urban, industrial and agricultural input. Two sites
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were located near the discharge of the Western Lake Superior Sanitary District (WLSSD), a
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tertiary treatment plant. One (WLSSD-1; N 46° 45.460, W 92° 7.227) was located ≈10 m
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from the discharge, while the second (WLSSD-2; N 46° 45.347, W 92° 7.213) was located
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further in the main channel, ≈100 m from the discharge. A fourth site was located near the
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discharge of Superior Municipal Treatment Plant, a secondary treatment plant (SMTP; N
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46° 43.758, W 92° 4.125). A detailed description of the caging system and deployment can
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be found in Kahl et al.32 Briefly, three buoys were anchored to the bottom sediment at each
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site. Three cages of fish, each containing six male and six female adult fathead minnows
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were attached to a buoy and its anchor, suspending cages at a depth of 1-2 m. The well-
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mixed water and the open nature of the St Louis Bay made it difficult to obtain separate
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water samples to determine chemical exposure for each buoy. Therefore, a grab sample of
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water was obtained near the three buoys at a depth of 1-2 m on exposure days 0, 2, 4, and
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8 at a consistent time at each site for chemical exposure analysis. Chemicals were
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assumed to be well mixed in the surface water at the time of grab sampling. Grab samples
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were considered to be representative of the surface water at the time of sampling and were
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used as measure of chemical exposure for all caged fish exposed at that site and time.
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Each fish exposed at a site was considered an independent exposure replicate due to the
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well-mixed and open nature of the bay. After 2, 4, or 8 d of exposure, a total of 6 fish from
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two cages, each from a separate buoy, were transferred into separate buckets containing
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surface water collected at the appropriate site, and transported to the laboratory (transit
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time
