SETAC North America Focused Topic Meeting Risk Assesment of Chemical Mixtures: From Scientific Evidence to Environmental Regulation Meeting Program

Wednesday, 6 September Problem Formulation

Time Title Speakers 8:00 a.m.–8:30 a.m. Opening Remarks Eric Van Genderen and Mary Ann Ottinger, Program Committee Co-Chairs 8:30 a.m.–9:00 a.m. 1 Toxicological and Environmental Risk Assessment of Chemical Mixtures: A Thomas Backhaus Critical Overview of Concepts, Methods and Approaches University of Gothenburg, Sweden 9:00 a.m.–9:30 a.m. 2 Mixture Toxicology in the 21st Century: Pathway-Based Concepts Gerald Ankley and Tools USEPA, USA 9:30 a.m.–10:00 a.m. 3 A Brief Overview of Regulatory Frameworks and Approaches to the Karen Eisenreich Ecological Assessment of Mixtures USEPA, USA 10:00 a.m.–10:30 a.m. Break 10:30 a.m.–11:00 a.m. 4 Troubling Mismatch Between Research on Compounds of Emerging Concern Gunilla Öberg (CECs) and Societal Needs University of British Columbia, Canada

Exposure Assessment

Time Title Speakers 11:00 a.m.–11:30 a.m. 5 Identifying Complex Mixtures in the Environment With Cheminformatics and Emma Schymanski Non-targeted High-Resolution Mass Spectrometry EAWAG, Switzerland 11:30 a.m.–12:00 p.m. 6 The Use of Non-Targeted Analysis (NTA) and Suspect Screening Analysis Mark Strynar (SSA) for Human Exposure Assessment at the USEPA: Successes and USEPA, USA Challenges 12:00 p.m.–1:30 p.m. Break 1:30 p.m.–2:00 p.m. 7 Extensive Target-Chemical Analysis and Biological Assays Illustrate Mixed- Paul Bradley Organic-Contaminant Exposure and Effects in USA Streams USGS, USA 2:00 p.m.–2:30 p.m. 8 Combined Exposure Assessment of 37 Substances From Mixed Land Use Christopher Holmes Catchments to Assess Prospective Aquatic Risk Waterborne Environmental, USA 2:30 p.m.–3:00 p.m. 9 Assessing Chemical Mixture Exposures Using spatial Principle Components Kristin Eccles Analysis (sPCA) and Geospatial Methods University of Ottawa, Canada 3:00 p.m.–3:30 p.m. Break 3:30 p.m.–4:00 p.m. 10 Species Sensitivity May Influence the Determination of Additivity in Metal– Elizabeth Traudt Mixture Toxicity Colorado School of Mines, USA 4:00 p.m.–4:30 p.m. Facilitated Discussion Kevin Brix and Ed Furlong, Program Committee 4:30 p.m.–5:30 p.m. Poster Social Thursday, 7 September Effects Assessment

Time Title Speakers 8:00 a.m.–8:30 a.m. 11 The ADME of Toxicological Interactions and Mixtures Risk Assessment Moiz Mumtaz Agency for Toxic Substances and Disease Registry, USA 8:30 a.m.–9:00 a.m. 12 Embracing Biological and Chemical Complexity Improves the Ecological Travis Schmidt Relevance of Effect Assessment U.S. Geological Survey, USA 9:00 a.m.–9:30 a.m. 13 Mesocosm Experiments Conducted With Metal Mixtures Often Reveal William Clements Ecological Surprises Colorado State University, USA 9:30 a.m.–10:00 a.m. 14 Integration of Chemical, Organismal, and Transcriptomic Data Reveals David Bertolatus Landscape-specific Exposure Effects to Complex Chemical Mixtures University of Colorado 10:00 a.m.–10:30 a.m. Break 10:30 a.m.–11:00 a.m. 15 Mixture Toxicity Prediction via Quantitative Toxicogenomics-based Na Gou Approach Northeastern University, USA 11:00 a.m.–11:30 a.m. 16 Toxicity Modeling With Parallel Factor (PARAFAC) Decomposition From the Sheikh Rahman Perspective of Mixture Toxicity Prediction With Time Series Toxicogenomic Northeastern University, USA Data 11:30 a.m.–12:00 p.m. Facilitated Discussion Thomas Backhaus and Mark Johnson, Program Committee 12:00 p.m.–1:30 p.m. Break

Risk Assessment and Management

Time Title Speakers 1:30 p.m.–2:00 p.m. 17 An Evidence-based Tiered Risk Assessment Scheme for Metal Mixtures in Karel A.C. De Schamphelaere Freshwater Ecosystems Ghent University, Belgium 2:00 p.m.–2:30 p.m. 18 Use of the Basic Hazard Index Mixtures Screening Method for Rapid Valerie Adams Assessment U.S. Army Public Health Center, USA 2:30 p.m.–3:00 p.m. 19 Risk Assessment and Regulation of Chemical Mixtures – Plant Protection Mick Hamer Products Syngenta, UK 3:00 p.m.–3:30 p.m. Break 3:30 p.m.–4:00 p.m. 20 Developing Management Strategies for Mixtures: A Path Forward Based on Nathalie Vallotton a Review of Cumulative Risk Assessment Dow Europe GmbH, Switzerland 4:00 p.m.–4:30 p.m. 21 Assessing the Effects of Chemical Mixtures Using a Bayesian Network Valerie Chu Relative Risk Model (BNRRM) Integrating Adverse Outcome Pathways (AOPs) Western Washington University, USA 4:30 p.m.–5:00 p.m. 22 Application of the MIXTOX Model in the Characterization of Neonicotinoid Erin Maloney Mixture Toxicity to Chironomus dilutus University of Saskatchewan, Canada 5:00 p.m.–5:30 p.m. Facilitated Discussion Amy Beasley and Kevin Brix, Program Committee 5:30 p.m.–6:30 p.m. Poster Social

Friday, 8 September Exposure Assessment

Time Title Speakers 8:30 a.m.–9:00 a.m. 23 Combining Chemical Analysis, Bioanalysis and Risk Assessment to Prioritize Juliane Hollender Risk Driving Substances in Wastewater-Impacted Streams EAWAG, Switzerland 9:00 a.m.–9:30 a.m. 24 A Multipronged, Effects-Driven Exposure and Impact Assessment of Edward Perkins Chemicals of Emerging Concern in Rivers of the Great Lakes US Army Engineer Research and Development Center, USA 9:30 a.m.–10:00 a.m. 25 Important Considerations for the Environmental Risk Assessment of Katherine Coady Mixtures of Alkylphenol Ethoxylates and Their Degradation Products The Dow Chemical Company, USA 10:00 a.m.–10:30 a.m. Break 10:30 a.m.–11:00 a.m. 27 Mixtures Risk Assessment: Finding Opportunity Amidst Chaos Eric Van Genderen International Zinc Association, USA 11:00 a.m.–12:00 p.m. Closing Discussion Eric Van Genderen, Mary Ann Ottinger and Thomas Backhaus, Program Committee abstract book

SETAC North America Focused Topic Meeting Risk Assessment of Chemical Mixtures: From Scientific Evidence to Environmental Regulation

6–8 SEPTEMBER 2017 | DENVER, COLORADO, USA This book comprises the abstracts of the presentations for the platform and poster presentations of the SETAC North America Focused Topic Meeting on “Risk Assessment of Chemical Mixtures: From Scientific Evidence to Environmental Regulation,” which was conducted from 6–8 September 2017 at the The Curtis Hotel in Denver, Colorado. The abstracts are reproduced as accepted by the Scientific Program Committee and appear in numerical order. In each abstract, the presenting author’s name is underlined.

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WWW.SETAC.ORG ENVIRONMENTAL QUALITY THROUGH SCIENCE® PLATFORM ABSTRACTS Problem Formulation 3 A Brief Overview of Regulatory Frameworks and Approaches to the Ecological Assessment of Mixtures 1 Toxicological and Environmental Risk Assessment of Chemical K.M. Eisenreich, USEPA / Office of Pollution Prevention and Toxics Mixtures: A Critical Overview of Concepts, Methods and Approaches Regulatory frameworks from various jurisdictions have highlighted the T. Backhaus, University of Gothenburg / Department of Biological and complexities of characterizing fate, exposure, hazard and risk of complex Environmental Sciences mixtures of substances (e.g., multi-constituent substances (MCS) and The realization that drug combinations are frequently more potent than unknown, variable composition and biological substances (UVCBs). their individual components and might have a different spectrum of activ- This presentation will cover approaches for registering or assessing ity has led early on to the development of concepts that can serve as null mixtures (at varying levels of complexity) within the regulatory context hypotheses for expected mixture effects. First landmark works include the of REACH, the Canadian Environmental Protection Act (CEPA 1999) publications by the German pharmacologist Loewe from 1926 onwards, and various USEPA programs in the Office of Pollution Prevention and and the papers written by the US biometrician Bliss from 1930 onwards. Toxics (OPPT), Office of Pesticide Programs (OPP), Office of Water (OW) The resulting concepts, Loewe Additivity and Bliss Independence, were and Office of Land and Emergency Management (OLEM). Case studies put to the test during the last almost hundred years, resulting in a plethora will be used to illustrate how different types of mixtures have been of documented experimental studies with a broad range of mixtures assessed in the New Chemicals Program under the Toxics Substances and bioassays. Neither humans nor other organisms in the environment Control Act (TSCA). The case studies will focus on how fate and hazard are ever exposed to single, pure chemicals. In fact, chemical monitor- determinations are made (e.g., persistence, bioaccumulation and toxicity) ing studies routinely find complex mixtures comprising dozens or even for mixtures and how data gaps may be filled through modeling or testing. hundreds of chemicals in virtually every environmental and biological The views expressed in this abstract are those of the authors and do not sample. It was therefore only logical to explore whether the classical represent Agency policy or endorsement. mixture concepts can also be applied for toxicological and environmen- tal risk assessments of chemical mixtures. Current empirical evidence 4 Troubling Mismatch Between Research on Compounds of strongly suggests that this is indeed the case, although the specifics of Emerging Concern (CECs) and Societal Needs environmentally realistic exposure scenarios pose new challenges. This G. Oberg, UBC / IRES presentation will first provide a brief overview of the conceptual founda- The scientific uncertainties that surround compounds of emerging con- tions of the classic mixture toxicity concepts in their various incarnations. cern (CECs) pose several challenges for policy makers. The present study It will then explore their applicability and limitations for toxicological is a critical review of peer-reviewed research on CECs with a focus on and environmental risk assessments, which usually involves exposure what researchers are concerned about, how risks, uncertainties and igno- scenarios that are fundamentally different from the situation found dur- rance are portrayed, whether policy-relevance is used as a justification ing drug development. Toxicologists and ecotoxicologists alike are often and whether or not findings are related to challenges facing regulators. challenged by multi-component mixtures whose composition might be Relevant publications were identified from 1950 to present through partially unknown and comprise only low concentrations of individual advanced searches in online databases using a variety of search terms. A compounds for which only fragmentary (eco)toxicological knowledge is content analysis was carried out, iteratively creating thematic categories at hand. In this context, I will in particular discuss (i) reasonable defini- and noting new topics as they emerged. The review shows that the focus tions of “synergisms” and “antagonisms” and the expectable quantitative of CEC-studies has changed over time: for example from drinking water consequences of such interactions, (ii) how component-based and whole quality in the 1990s to more attention to wastewater and antibiotics, fol- mixture approaches can mutually support mixture evaluations, and, lowed by an increasing number of studies on land-application of sludge finally, (iii) how the conceptual and empirical understanding of mixture on arable land. During the past decade, potential challenges related to (eco)toxicology can be used to guide the evaluation of complex exposure indirect potable use are added to the growing list, as are illicit drugs. Over scenarios, and to develop tiered approaches that are suitable for the regu- time, an increasing number of studies make references to policy-relevance latory risk assessment of mixtures in toxicology and ecotoxicology. to justify the work. Very few studies do, however, clarify how their find- ings or associated uncertainties relate to challenges facing policy-makers 2 Mixture Toxicology in the 21st Century: Pathway-Based Concepts or regulatory agencies. A handful authors discuss strengths and weak- and Tools nesses with different policy mechanisms and regulatory tools, heavily G.T. Ankley, USEPA / National Health and Environmental Effects criticizing the present system as being obsolete because the methods used Research Laboratory to assess risk are antiquated. This is held to be so because the methods are The past decade has witnessed a notable evolution of approaches focused inevitably outpaced by the production, use and release of new chemicals on predicting chemical hazards and risks in the absence of empirical as well as by the developments in analytical chemistry that allows detec- data from resource-intensive in vivo toxicity tests. In silico models, in tion at lower levels. Also, methods do not take into account the toxicity vitro high-throughput toxicity assays, and short-term in vivo tests with of chemical mixtures in the environment and they are also held to be too pathway-specific molecular and biochemical endpoints provide the expensive. Calls are made for alternative approaches, but resources are mechanistic data to support these predictive approaches. And, the adverse mainly directed to ‘more of the same’. Our review suggests a mismatch outcome pathway (AOP) framework provides a conceptual basis for between the majority of conducted research and the type of knowledge translation of the resultant mechanistic knowledge into the types of apical that is needed to handle the problem, which is troubling. It appears as if (whole organism) responses needed for risk assessment (e.g., changes in the scientific uncertainties that surround CESs pose problems for policy survival, growth and reproduction). To date, these predictive tools and makes not only because such compounds are surrounded by “known concepts have largely been applied only to prospective assessments of unknowns” (uncertainty) and “unknown unknowns” (ignorance) but single chemicals. However, the various approaches can be just as useful also “unknown knowns” (overlooked/excluded). We discuss and analyze for assessing effects of contaminant mixtures in both prospective and proposals to solutions made in the literature. diagnostic assessments. This presentation will describe the conceptual and technical underpinnings of various tools and approaches used in predictive toxicology, and provide “real world” examples of their applica- tion to ecological assessments of the occurrence and effects of chemical mixtures. The views expressed in this presentation are those of the author and do not represent the views or policies of the USEPA.

4 | Risk Assessment of Chemical Mixtures PLATFORM ABSTRACTS Exposure Assessment molecular formula or chemical assignment. Such databases of highly curated chemicals, (e.g.,. DSSTox), make chemical information avail- 5 Identifying Complex Mixtures in the Environment With able for usage in these types of studies. The ultimate goal is the marriage Cheminformatics and Non-targeted High Resolution Mass between HRMS and HTT assessments to prioritize chemical mixtures Spectrometry detected against a chemicals potential for adverse health effects. Non- E. Schymanski, Eawag Swiss Federal Institute of Aquatic Science and targeted analysis (NTA) becomes necessary when analytes are detected in Technology / Environmental Chemistry; A. Williams, USEPA / National a sample that are not in a database being used for SSA. In these instances, Center for Computational Toxicology; J. Hollender, Eawag, Swiss Federal additional analyses are needed to elucidate the structure of unknowns. Institute of Aquatic Science and Technology / Environmental Chemistry Once confirmed, a chemical then needs to be added to databases for future SSA and included in planned bioactivity screening. With this Non-targeted high resolution tandem mass spectrometry (HR-MS/ dynamic feedback between approaches real and continued progress is MS) techniques are ideal for exploring complex environmental samples made. This presentation will focus on the progress made at the USEPA beyond target and suspect screening for “knowns.” Non-target HR-MS/ RTP toward this goal. The workflows, data analysis and output on a select MS, combined with advanced cheminformatics and data processing, set of samples will demonstrate the state of the science with this approach. offers huge potential for exploring complex mixtures, yet faces many Through in-house application examples of these techniques to chemical challenges. Peak inventories of several non-target studies reveal that mixtures in multimedia samples (e.g.,. BRITA filters, house dust, surface only a few percent of detected masses are “known” compounds, while water, wastewater) successes and challenges will be demonstrated. The several thousand peaks remain unidentified. Mass-based homologue pros and cons of this novel approach like how chemical are prioritized, screening shows that many of these peaks are interconnected through databases used for matching, threshold cutoffs and viable instrumentation a web of related, discrete masses. Homologue screening in wastewater to use will be presented. studies confirms this for several surfactant series. A better understanding of all “unknowns” in samples, including homologues, will be essential 7 Extensive Target-Chemical Analysis and Biological Assays to unravel the role that complex mixtures play in environmental Illustrate Mixed-Organic-Contaminant Exposure and Effects in observations. Improved chemical identification is hampered by the USA Streams difficulty in exchanging information on substances, yet incorporating P.M. Bradley, USGS / South Atlantic Water Science Center; D.W. Kolpin, chemical knowledge and fragment information is vital to unravelling USGS / Iowa Water Science Center; D.L. Villeneuve, USEPA / National the identities of these “unknowns” and assessing the environmental and Health and Environmental Effects Research Laboratory toxicological relevance of mixtures. The collaborative efforts between the USEPA’s CompTox Chemistry Dashboard (https://comptox.epa. Assessing human and ecological health risks associated with exposure gov/) and the NORMAN Network’s Suspect Exchange (http://www. to complex chemical mixtures is acknowledged as a significant norman-network.com/?q=node/236) and MassBank (www.massbank. environmental health challenge. The current shortage of information on eu) initiatives showcase how improved information exchange can help the composition and variability of environmental mixtures combined address this challenge. Connecting these initiatives into non-target with limited understanding of their combined effects are fundamental workflows and in silico structure elucidation approaches such as obstacles to management of adverse human and ecological effects of MetFrag (https://msbi.ipb-halle.de/MetFragBeta/) allows rapid tentative chemical mixtures. This presentation describes a USGS/USEPA national identification of potential candidates amongst thousands of non-target field-based reconnaissance exploring chemical mixtures and biological masses. These efforts will be showcased on European studies, showing activity associated with stream waters affected by a wide range of how non-target HR-MS helps turn the “unknowns” into “knowns” for contaminant sources. Stream sites were selected from watersheds that improved assessment of complex mixtures in the environment. Note: This are vulnerable to multiple contaminant sources, including industrial and abstract does not reflect USEPA policy. municipal wastewater discharges, crop and animal agriculture, land- applied manures and biosolids, urban runoff, and other point and nonpoint 6 The Use of Non-Targeted Analysis (NTA) and Suspect Screening contaminant sources. The study combined comprehensive target-chemical Analysis (SSA) for Human Exposure Assessment at the USEPA: characterization of organic contaminants in water and a battery of Successes and Challenges bioassays to evaluate biological activity and toxicity. Surface water from M.J. Strynar, USEPA / National Exposure Research Laboratory; J. 38 streams nationwide was assessed using 14 target-organic methods (719 McCord, ORISE / NERL; J. Sobus, USEPA / National Exposure Research compounds). Designed-bioactive anthropogenic contaminants (biocides, Laboratory; E.M. Ulrich, USEPA / Office of Research and Development pharmaceuticals) comprised 57% of 406 organics detected at least once. National Exposure Research Laboratory; S. Newton, USEPA / Cumulative detections and concentrations ranged 4−161 compounds -1 Department of Applied Environmental Science ITM; A.D. McEachran, (median 70) and 8.5−102 847 ng L , respectively, and correlated USEPA / ORISE National Risk Management; A. Williams, USEPA / significantly with wastewater discharge, watershed development, ORDNCCT and toxic release inventory metrics. Log10 concentrations of widely monitored HHCB, triclosan, and carbamazepine explained 71−82% Human exposure assessment has classically revolved around a targeted of the variability in the total number of compounds detected (linear analyte list in select media sometimes coupled with biomonitoring regression; p-values: < 0.001−0.012), providing a statistical inference information. Historically the drive behind such studies has been the tool for unmonitored contaminants. Due to multiple modes of action, discovery of an analyte either in environmental media or biomonitoring high bioactivity, biorecalcitrance, and direct environment application samples and the relationship between toxicology and exposure. Recently (pesticides), designed-bioactive organics (median 41 per site at μg L-1 there has been a revolution in the approaches available to answer both cumulative concentrations) in developed watersheds present aquatic exposure and risk assessment questions. The advent of high-throughput health concerns, given their acknowledged potential for sublethal effects toxicology (HTT) (e.g.,. Toxcast, Tox21) has allowed for the screening of to sensitive species and lifecycle stages at low ng L-1. Estrogen, androgen, thousands of chemicals for bioactivity as an indicator of health effects. and glucocorticoid receptor binding activities verified varying biological Formerly these studies required dosed animal approaches, and though activity in collected water samples. Additional possible effects of the they were very thorough, they are costlier and can only be done with one complex chemical exposures observed in this study were computationally or very few analytes at a time. Parallel to HTT has been the introduction predicted using knowledgebase-leveraging tools. of high resolution mass spectrometry (HRMS) which has revolution- ized the analytical abilities available for human exposure assessments. Through the use of HRMS, one can screen for analytes against large databases of known analytes (SSA) with a high degree of confidence in

SETAC North America Focused Topic Meeting | 5 PLATFORM ABSTRACTS

8 Combined Exposure Assessment of 37 Substances From Mixed principal component (PC), to create an index of metal exposure for each Land Use Catchments to Assess Prospective Aquatic Risk sample site. The PCs, as well as the developed index values, were mapped C.M. Holmes, Waterborne Environmental, Inc.; C. Brown, University of in ArcGIS 10.3. Further methods of global clustering (Moran’s I) and York / Environment Department; D. De Zwart, Mermayde / Centre for local clustering (Getis and Ord’s G*) were used to assess spatial patterns Sustainability Environment and Health; J.M. Diamond, Tetra Tech, Inc.; of the principal components. Results allow us to identify hotspots of either S.D. Dyer, The Procter & Gamble Company / Environmental Stewardship single metal or metal mixture in the study area. These results show that and Sustainability Organization; S. Marshall, Independent Consultant; L. sPCA is a useful method for assessing sources and drivers of chemical Posthuma, RIVM / Centre for Sustainability, Environment and Health mixture across a landscape. ® In 2015, a SETAC Pellston workshop was held to help inform decision 10 Species Sensitivity May Influence the Determination of Additivity making around aquatic mixture risk assessments of chemicals using in Metal–Mixture Toxicity exposure scenarios for workgroups focused on agricultural, domestic, and E.M. Traudt, Colorado School of Mines / Department of Chemistry; J.F. urban scenarios. Each workgroup produced chemical mixture “signa- Ranville, Colorado School of Mines / Chemistry and Geochemistry; J.S. tures,” and combined with effects thresholds, mixture risk profiles. Based Meyer, Applied Limnology Professionals LLC on these workgroup outputs, prospective emissions of 37 chemicals were combined into daily mixture profiles over a 10-year period assessed at Surface waters may contain metal mixtures that are potentially toxic the bottom of a hypothetical catchment containing a changeable configu- to aquatic life, yet mixture toxicity is currently difficult to predict due ration of sub-catchments defined by three land use types (agricultural, to apparent non-additive interactions among metals. For example, we city [domestic + urban], natural). Ultimately the mixture risk assessment conducted binary- and ternary-metal mixture acute toxicity tests with looked at daily individual substance risk quotients (RQs) and multiple Daphnia magna exposed to Cd, Cu, and Ni in USEPA moderately hard substance ∑RQ (assuming concentration addition). A catchment-scale water with 3 mg/L dissolved organic carbon (DOC) and observed non- exposure assessment was implemented to integrate daily loadings of additive toxicity in all mixture combinations (Cd-Cu, Cd-Ni, Cu-Ni, individual chemicals from each sub-catchment to produce a time series of Cd-Cu-Ni). We hypothesized that part of this effect may be related to mixture profiles at the catchment outlet. Catchment hydrology was based metal-metal competition for binding to DOC in the exposure water on 10 years of rainfall data on different land use surfaces, coupled with because in the presence of Ni, Cu ions (which are more toxic than Ni) a simplified hydrologic model within the catchment. Catchment con- should displaced from DOC, resulting in greater-than-predicted toxicity. figuration can be changed by varying the placement, type and number of To test this hypothesis, we used geochemical-speciation software sub-catchments with resulting changes in exposure automatically cascad- WHAMVII to predict changes in bioavailable Cu speciation as a function ing though to the risk summary output of the model. For sub-catchments of Ni and DOC concentrations by considering metal-metal competition with municipal discharge, constant daily emissions of household chemicals for binding to DOC. We analyzed our mixture-toxicity results in a produced variable river concentrations because of changing river flow due two-way matrix: assuming either a concentration-addition (CA) or to rainfall. Urban discharge via storm water runoff was episodic, triggered independent-action (IA) mixture-toxicity model, and using either the if daily rainfall exceeded a threshold value (the 95th percentile daily inten- measured metal concentrations or the calculated metal-ion activities sity), with resulting chemical “signatures” in the runoff water. Agricultural to predict toxicity. In binary Cu-Ni mixtures, the toxicity was always sub-catchment exposures were also episodic, being driven by rainfall more-than-additive; however, in ternary Cd-Cu-Ni mixtures, the toxicity events, but also pesticide applications within the sub-catchment (based on was less-than-additive, additive, or more-than-additive, depending on actual application regimes for a UK wheat field). The outcome of the over- the metal concentrations and the assumed additivity model (i.e., CA all Pellston study demonstrated that a scenario-based approach can be used predicted the toxicity more accurately than IA at intermediate to high to determine whether mixtures of chemicals pose risks over and above any concentrations of the varied metal). At concentrations causing 100% identified using existing approaches for single chemicals, how often and to mortality to D. magna, Cu ion activities increased considerably in the what magnitude, and ultimately which mixtures (and dominant chemicals) presence of Ni, consistent with our prediction. However, at intermediate- cause greatest concern. mortality concentrations, the computed ion activities did not increase enough to account for the observed toxicity. Thus, using ion activities 9 Assessing Chemical Mixture Exposures Using Spatial Principle did not reconcile the apparently more-than-additive toxicity, suggesting Components Analysis (sPCA) and Geospatial Methods an alternative mechanism may also be important. Therefore, the toxicity K. Eccles, University of Ottawa / Department of Biology; P. J Thomas, conclusion for a less-sensitive species exposed to higher Ni concentrations B. Pauli, Environment and Climate Change Canada / National Wildlife might differ from the conclusion for the more-sensitive D. magna exposed Research Centre; L. HM Chan, University of Ottawa / Biology to lower Ni concentrations. These results demonstrate that conclusions about additivity of metal mixtures can depend not only on complex Modelling impacts of chemical mixture exposures across a landscape interactions of toxicity-controlling geochemical mechanisms, but also on is challenging due to the spatial heterogeneity and complexity of the the sensitivity of the test organism to the metals. sources, pathways, and fate of the multiple chemicals. This is especially true for wildlife living in areas of intense anthropogenic disturbance such as the Athabasca Oil Sands. Biomonitoring is used to assess the contami- Effects Assessment nant exposure in wildlife but the significance of the chemical mixture is often unknown. Principal Component Analysis (PCA) is a common 11 The ADME of Toxicological Interactions and Mixtures Risk method to reduce the data complexity of highly dimensional multivari- Assessment ate contaminant data. Moreover, spatially mapping PCA outputs can be M. Mumtaz, CDC/ATSDR / Division of Toxicology and Human Health used to assess spatial patterns of the biomonitoring data. In this study, we Sciences; K. Krishnan, H. Pohl, CDC/ATSDR used biomonitoring data collected under the Joint Oil Sands Monitoring (JOSM) to demonstrate the application of sPCA for risk assessment. Risk assessment landscape is always in a continuous flux presenting new Concentrations of arsenic, lead, cadmium and mercury in adult and real life exposure scenarios to mixtures of environmental pollutants, recently metamorphosed wood frog (Lithobates sylvaticus) collected chemical and non-chemical agents. Their environmental or public health from Northern Alberta, Canada were measured. Data was collected for assessment consists of identifying specific agent(s), studying their adverse 83 wood frogs across 23 sampling sites. First a PCA was completed on effects, and using this information to improve public health. As dynamic z-score standardized data in R 3.4.0. Then we used the linear combina- changes take place in the environment, the introduction of new chemicals tion of the PCA scores, which are comprised of the PCA eigenvalues and associated emerging issues are challenges the risk assessment commu- and eigenvectors, weighted by the amount of variance explained by each nity addresses on an ongoing basis. Following sequential or simultaneous

6 | Risk Assessment of Chemical Mixtures PLATFORM ABSTRACTS exposures, biological systems have capabilities to process chemicals and these mixtures often include chemicals that cause both toxicological their mixtures to limit their impacts on their normal functioning. These (e.g., Cd, Cu and Zn) as well as physical (e.g., Fe hydroxide deposition) capabilities, absorption, distribution, metabolism and excretion (ADME) effects, mesocosm experiments are necessary to quantify responses. form a network of pathways that can be perturbed and can be modified by Mesocosm experiments conducted with mixtures Cu and Zn showed interactions of multiple chemicals in a system. The resulting changes in that Cu alone was significantly more toxic to aquatic insect communities the physiological or physical changes (breathing rates, flow rates, protein than the combination of these two metals. In contrast, macroinvertebrate binding, metabolic rates or rate constants) can lead to temporal concentra- community responses to a mixture of Cd+Cu+Zn were greater than either tion changes and inhibition or enhancement of net toxicological impact Zn alone or Cd+Zn. A second set of field and mesocosm experiments was across the adverse outcome pathways involved. Every federal agency designed to separate direct effects of aqueous metals from the physical develops a strategic plan to meet its mandate. ATSDR’s mixtures program, effects of Fe hydroxide deposition on stream benthic communities. to improve currently available methods and models, consists of identifica- Although previous investigators have reported that ferric Fe has a tion of chemical mixtures found at hazardous waste sites, identification of protective effect on the toxicity of other metals, we observed only weak data gaps related to these mixtures, and targeted research. The goal of such evidence for antagonistic interactions between aqueous metals and programs is to formulate generalizable rules for assessment of mixtures ferric Fe. Results showed that direct exposure to metals was highly that can be integrated into guidelines for risk assessors. Examples of toxic to some taxa; however, reduced colonization of Fe-contaminated pharmacokinetic model simulations of change in internal dose resulting substrate was primarily a result of behavioral avoidance of metal-oxides. from chemical interactions and their use in cancer and non-cancer risk Understanding the effects and potential interactions among individual assessment of mixtures of environmental pollutants will be presented. constituents in chemical mixtures is critical for assessing effectiveness of restoration programs. Although the focus of most restoration efforts in 12 Embracing Biological and Chemical Complexity Improves the mining-contaminated streams is on improving water quality, removing Ecological Relevance of Effect Assessment this single stressor may not be sufficient to restore structural and T.S. Schmidt, USGS / Colorado Water Science Center functional integrity of these systems. Because of a limited number of Regulations set to protect aquatic life consider a single chemical at a experimental units in most mesocosm studies, assessing responses to time and are developed using organisms like crustaceans and amphipods mixtures containing numerous chemicals will remain challenging. An that are easily cultured in aquaria. However, aquatic ecosystems are approach that integrates mesocosm experiments, field experiments and dominated aquatic insects with complex life histories that are difficult to standard laboratory toxicity tests could increase the reliability of water culture in the lab. As a result, effect assessment science based on beaker- quality criteria and help explain the discrepancies between laboratory and scale toxicological evaluations and standard toxicity testing organisms field responses to contaminants reported in the literature. are easily criticized for their lack of ecological relevance. Notably, field 14 Integration of Chemical, Organismal, and Transcriptomic Data observations are infrequently used in regulation development because Reveals Landscape-specific Exposure Effects to Complex Chemical of the difficulty of attributing any single ecological response to an Mixtures individual chemical compound found in the complex mixtures of natural ecosystems. We have a vast knowledge of the effects of water quality on David Bertolatus, University of Colorado Denver / Integrative Biology; the bioavailability of metals, mostly derived from single-metal, short- Larry Barber, USGS / National Research Program; Christopher term, standard toxicity tests. Aqueous solution based predictive models Martyniuk, University of Florida / Physiological Sciences; Alan Vajda, that account for the effect of water quality on metal speciation have University of Colorado Denver / Integrative Biology become popular tools of decision makers, but they too are criticized Aquatic habitats are often contaminated with complex mixtures of pol- for over simplifying ecosystems and ironically for their complexity. lutants. Interpreting biological effects caused by mixture exposures is Despite the latter, my lab to field investigations fill these knowledge and an ongoing challenge in ecotoxicology and is expected to improve with credibility gaps and aim to understand the complexity of ecosystems. chemical and geographical data. Here, we employed integrated chemi- These investigations focused on multiple levels of biological organization cal and biological analyses to determine how environmental mixtures from cells to ecosystems and have shown that metals interact in mixture, affected biological responses in watersheds with different landuse. A thereby altering their own effects on natural ecological communities. better understanding of the relationships between landuse, contaminant These effects are not due to direct exposure to aqueous metals alone, occurrence, and exposure effects will increase predictive power for regu- but are rather the culmination of direct exposure to metals and metal lators and managers. Adult male fathead minnows (Pimephales promelas) altered inter-species interactions. I will show that focusing on a single life were exposed to water from different locations within the Shenandoah stage of organisms with complex life histories can result in seemingly River watershed (VA, USA) in 2014 and 2015. The exposure locations contradictory findings. Finally, while models that account for both were chosen to capture unique landuse in surrounding watersheds, includ- the biological and chemical complexity of ecosystems have improved ing agricultural, municipal, mixed-use, and forested sites. Endpoints prediction of the effects of metal mixtures on these systems, these models from multiple levels of biological organization were measured, including do not account for the total effect of solution chemistry on organisms nor condition factor, gonadosomatic index (GSI), number of nuptial tubercles, how organisms regulate metals within their bodies. and hepatic gene expression profiles. Water samples were taken 4 times during the fish exposure and analyzed for over 460 chemical constituents. 13 Mesocosm Experiments Conducted With Metal Mixtures Often Each location had a unique chemical profile that was generally consistent Reveal Ecological Surprises with landuse. Whole-organism and molecular responses also differed W.H. Clements, B. Wolff, C. Kotalik, Colorado State University / Fish, between the locations. In 2014, fish exposed at agricultural and WWTP Wildlife, and Conservation Biology; P. Cadmus, Colorado Parks and impacted sites showed signs of endocrine disruption including a reduced Wildlife number of nuptial tubercles and decreased GSI. However, transcript bio- Predicting fate and effects of chemical mixtures on aquatic organisms markers of estrogen exposure, including er1, er2, vtg1, and vtg3 showed is complicated by our relatively poor understanding of contaminant little to no differential expression, suggesting that these fish were not interactions and our inability to quantify effects on ecologically affected in a significant way by estrogenic compounds in the mixture. meaningful endpoints. Mesocosm experiments using natural communities Pathway analysis of transcriptomic data revealed that lipid related pro- provide a unique opportunity to assess the direct and indirect effects cesses were affected by exposure to agricultural, WWTP impacted and of chemical mixtures. Over the past 25 years we have focused much of mixed-use watersheds. Immune-related processes were down-regulated at our research on assessing responses of stream benthic communities to the agricultural site and up-regulated at the mixed-use site. Hierarchical mixtures of metals associated with historical mining activities. Because clustering of total transcriptome profiles showed individuals generally

SETAC North America Focused Topic Meeting | 7 PLATFORM ABSTRACTS clustered by exposure location, demonstrating that exposure to water from binary mixture of two metals (As and Cr) from their time-series transcrip- sites with different landuse results in unique and site specific responses tomic responses of over 100 genes of all known stress pathways generated at the transcript level. These data generate new hypotheses regarding the by GFP-fused E. coli K12, MG1655. PARAFAC model decomposed 3D effects of exposure to different types of complex mixtures and demon- toxicogenomic data (gene x time x concentration) of the mixture and strate the value of our complex mixture/landscape research approach. its constituent chemicals into multiple components/factors and revealed the relative importance of the genes, associated temporal profiles, and 15 Mixture Toxicity Prediction via Quantitative Toxicogenomics- concentration-specific responses. Subsequent gene set enrichment analy- based Approach ses, conducted on the decomposed gene profiles, revealed the dominant N. Gou, A. Gu, Northeastern University / Department of Civil and molecular toxicity mechanism of the decomposed factors — represent- Environmental Engineering ing conserved fundamental biological/molecular features. The intrinsic Mixture assessments from toxicogenomic studies is as yet mainly temporal profiles and concentration-specific responses differed across observational and quantitative prediction of the combined effects chemicals and decomposed factors, representing varying biological is lacking. In this study, we demonstrated the application of a responses of the conserved sub-modules. In addition, concentration- toxicogenomic-based approach for investigating the combined effects dependent responses of the sub-modules showed typical dose-response of various binary mixtures with a range from environmental relevant behavior. We further demonstrated the efficacy of the conserved profiles concentration to the benchmark level. A high-throughput mechanistic of the individual chemicals as prediction parameters to predict intrinsic toxicogenomics assay was employed to measure and quantify (as mixture toxicity profiles via multiple linear regression. Highly signifi- quantitative omic-index TELI) the time series altered gene expression cant associations (p< 0.001) were observed between mixture PARAFAC level in over 100 genes that cover all known stress response pathways in5 model and the individual chemical models at all three modes (gene, time, binary mixtures. Gene Set Enrichment Analysis (GSEA) and Principal and concentration), indicating the regression models’ ability to predict Component Analysis (PCA) were performed. Two additive models, the conserved mixture toxicity profile. The proposed novel application of concentration addition (CA) and independent action (IA), were explored PARAFAC analysis can provide significant insights into toxicogenomics for mixture toxicity predication and comparison. Distinct gene expression based mixture toxicity characterization and prediction at molecular level. profiles were obtained for the 5 mixture combinations as well as their single components. The toxicity profile were both chemical-specific and concentration-dependent. The toxicity profiles indicate both conservative Risk Assessment and Management and distinct responses among mixtures and their single components. The 17 An Evidence-based Tiered Risk Assessment Scheme for Metal GSEA also show similar and dissimilar overrepresented stress categories Mixtures in Freshwater Ecosystems between mixtures and their single components. PCA analysis of stress response profiles among the chemicals and their mixtures show that some K. De Schamphelaere, Ghent University / Applied Ecology and mixtures are clearly separated from with their single components, while Environmental Biology; C. Nys, University of Ghent / Laboratory others are mixed and shared some profile spaces with their components. for Environmental Toxicology and Aquatic Ecology GhEnToxLab Cellular level responses (indicated by our quantitative omic-index TELI) unit; T. Van Regenmortel, Ghent University / GhEnToxLab; K. Oorts, evoked by most of the binary mixtures exposure exhibited clear dose- Arche Consulting; E.E. Smolders, KULeuven / Department of Earth response patterns as single chemicals did. The modified CA and IA model Environmental Sciences predicts the mixture effects well for some mixtures. And the negative Although metal mixture toxicity has been studied relatively intensely, r2 values for some mixture combination with the model prediction there is no general concensus yet on how to incorporate metal mixture indicate that the mixture toxicity do not follow the trend of certain model toxicity into aquatic risk assessment. Here, we combined existing data prediction, even worse than the null hypothesis — a horizontal line. Our on chronic metal mixture toxicity at the species level with species- results for the first time demonstrated that the concept of combined effects sensitivity-distribution-based in-silico metal mixture risk predictions are presented at molecular level and they seem to comply with current at the community-level, in order to develop a tiered risk assessment mixture toxicity model for some chemicals binaries. The possibility to scheme for metal mixtures for freshwater. Generally, independent action explicitly predict the combined effects at the molecular level has been (IA) predicts chronic metal mixture toxicity at the species level most showed. Although most of the mixture exhibited additive effect in our accurately, while concentration addition (CA) is the most conservative results, further investigation is required for better understanding of the model. At low effect sizes (around 10% effect), CA overestimates metal factors that influence the combined effects. mixture toxicity at the species-level on average by 1.5-fold. To extrapolate from species-level to community-level, bioavailability-normalization 16 Toxicity Modeling with Parallel Factor (PARAFAC) procedures were combined with applying CA or IA to species-sensitivity Decomposition From the Perspective of Mixture Toxicity Prediction distributions (SSD) for predicting metal mixture risk, totaling 4 different With Time Series Toxicogenomic Data methods. We found that the method in which CA was directly applied to S. Rahman, Northeastern University / Civil & Environmental the SSD, i.e. CASSD, the most simple method, was the most conservative Engineering; C. Gao, Northeastern University / Bioengineering; N. Gou, method. For binary metal mixtures CASSD has a margin of safety of about A. Gu, Northeastern University / Department of Civil and Environmental 1.5-fold on average relative to the theoretically more consistent method of Engineering applying CA directly to the DRC (i.e., CADRC) of each species individu- Prediction and modeling of mixture toxicity are very challenging due ally prior to estimating the fraction of affected species. This Margin of to complex interactive modes of action that may include concentra- Safety increases with an increasing number of metals. When applying our tion addition, independent action, synergistic, or antagonistic effects. methods to European monitoring datasets, we found that CASSD yielded a Toxicogenomic technologies in combination with bioinformatics show considerable number of mixture risk predictions, even when metals were promises to transcend the limitation of conventional approaches by at background concentrations. In contrast, metal mixture risks predicted quantifying and predicting mixture toxicity. However, several limitations with the theoretically more consistent methods (e.g., CADRC) occured — including lack of quantitative dose-dependent toxicogenomic data, significanly less frequently (and were in fact very limited under natural data complexities due to small sample size coupled with large no. of vari- background metal concentrations). Based on the combined evidence of ables, scarcity of established theoretical and modeling frameworks, and chronic mixture toxicity predictions at the species-level and of in silico lack of consensus in computational methodologies— hinder their wider risk predictions at the community-level, a tiered risk assessment scheme applicability. Here, we explored the application of a tensor decomposition for evaluating metal mixture risks is presented, with CASSD functioning method, Parallel Factor (PARAFAC), to model the molecular toxicity of a as a first, simple conservative tier. The more complex, but theoretically

8 | Risk Assessment of Chemical Mixtures PLATFORM ABSTRACTS more consistent methods, like CADRC, can be used in higher tiers assess- for risk management. If it is identified that an registration of a new ments. The proposed tiered risk assessment scheme will be illustrated via chemical will result in a risk threshold being breached, what will be the application to an actual monitoring dataset. consequences and options for risk management ?

18 Use of the Basic Hazard Index Mixtures Screening Method for 20 Developing Management Strategies for Mixtures: A Path Rapid Assessment Forward Based on a Review of Cumulative Risk Assessment V. Adams, C.L. Procell, T.P. Simon, U.S. Army Public Health Center N. Vallotton, Dow Europe GmbH / Toxicology Environmental Research Environmental health risk assessments often involve assessing the and Consulting; A. Beasley, The Dow Chemical Company / TERC; H.M. potential health effects of exposure to mixtures (i.e., multiple concurrent Hollnagel, Dow Europe GmbH / Toxicology and Environmental Research chemical exposures). Because the number of possible chemicals is very and Consulting large, few controlled in vivo toxicological studies with chemical mixtures Current analytical methods support the detection of a large number of have been performed. When mixture-specific toxicity data are lacking substances present in samples collected during monitoring surveys. As and the cumulative HI score exceeds unity, the segregated hazard index a result, several monitoring datasets have been analysed to evaluate the (HI) approach have been used to determine if adverse effects from potential risks to humans or the environment from the co-exposure to exposure to multiple chemicals warrants further investigation. The multiple substances. While the reviews indicate that co-exposures to segregated HI approach assumes that risk is a function of toxicity from humans are of low concern, assessments focusing on ecological receptors exposures to multiple chemicals that all act through additive processes such as surface water indicate that a subset of co-exposures may lead to affecting the same target site of toxicity. Mixtures of concern (MOC) potential cumulative effects to aquatic species. As a first tier approach, were identified using a subset of substances identified within the national the studies typically assume additivity and estimate cumulative risks by priorities list. A comprehensive and standardized list of toxicity target selecting reference values such as regulatory thresholds to characterise organs and systems (TTOS), where each chemical was assigned to one the effect of each substance, while Tier 2 assessment may discuss cumula- or more target organs based on critical effect, was developed and used. tive risk at the trophic level. In studies where cumulative risk is suspected, Target organ system and subsequently target organ-specific HIs for these details key to a conclusion are rarely discussed: for example, determin- mixtures were gengerate by summing the individual hazard quotients and ing whether the risk is driven by a single component of the mixture, or the segregated HI using TTOS were calculated. The basic hazard index whether other variables may contribute to the appearance of cumulative mixtures screening (BHIMS) approach standardized the evaluation of risk. Tools such as the maximum cumulative ratio (MCR) have been the MOC and permitted the rapid identification of HI above unity. The shown to support not only the identification of mixtures of potential con- BHIMS strategy is a screeing step in a tiered approach for risk assessment cern that may require further consideration, but also highlight that single that identifies MOC that may need further and more sophisticated substances are, in the majority of cases, drivers of a cumulative risk. assessment. Further, exposures characterised as complex mixture driven hazard based on the number of detects were shown to involve toxicity limited to just a 19 Risk Assessment and Regulation of Chemical Mixtures – Plant few chemicals in the mixture. The aim of this paper is to present a review Protection Products of the findings of recently published cumulative risk assessments with M. Hamer, Syngenta / Environmental Safety regards to the types of exposures encountered in priority mixtures and The perception is that chemicals are regulated as single substances and suggest how current cumulative risk assessment approaches may inform that there is an issue with the potential for exposure to and effects from the risk management of co-exposures. chemical mixtures. Indeed, across most chemical sectors, the majority 21 Assessing the Effects of Chemical Mixtures Using a Bayesian of environmental risk assessment and any risk mitigation as part of risk Network-Relative Risk Model (BN-RRM) Integrating Adverse management is largely done on a single chemical basis. Nevertheless, Outcome Pathways (AOPs) the success of regulation in different chemical sectors in reducing the overall environmental risk from exposure to chemicals should not be V. Chu, Western Washington University / Environmental Science; M.J. underestimated. However, there is still a need for methodology to risk Harris, Whatcom Conservation District; C. Mitchell, Washington State assess the reality of environmental exposure to multiple chemicals and to University / School of the Environment; J.D. Stark, Washington State answer the question whether existing risk assessment methods cover the University Puyallup / Dept of Entomology; K. von Stackelberg, Harvard potential effects of mixtures or whether additional protection measures School of Public Health / Environmental Health; W.G. Landis, Western are required. Under Regulation (EC) No 1107/2009 for Plant Protection Washington University / Institute of Environmental Toxicology Products in EU, in a prospective assessment, the risk from single active There are long-standing questions about toxicity of chemical mixtures substances is assessed at an EU level, whereas formulated products, to populations. Laboratory toxicity tests have confirmed synergistic and chemical mixtures which may contain several active substances, are antagonistic effects to individuals, but not to populations. We have con- assessed at Member State level. Thus mixture assessment is an integral ducted a regional-scale ecological risk assessment by evaluating the risk part of the regulatory process and EFSA Guidance is available on how chemical mixtures to populations with a Bayesian Network- Relative Risk to conduct these assessments for terrestrial and aquatic organisms. Risk Model (BN-RRM) incorporating an Adverse Outcome Pathway (AOP). is currently assessed in-field or edge-of field where exposure is likely to We applied this BN-RRM framework in a case study with organophos- be highest, using standard exposure models. In addition, some member phate pesticide (OP) mixtures (diazinon, chlorpyrifos, and malathion) in states are starting to address the risk from tank mixes. There have also four watersheds (Nooksack, Lower Skagit, Cedar and Lower Yakima) in been attempts to address the risk from seasonal spray programs, with the state of Washington (USA). Acetylcholinesterase (AChE) inhibition both experimental and modelling based approaches and these can give an is the molecular initiating event and the Puget Sound Chinook salmon indication of the level of protection of the existing procedures. Although (Oncorhynchus tshawytscha) Evolutionary Significant Units (ESU) relatively simplistic, scenario based approaches, these methods have were chosen as population endpoints. Laetz et al. (2009,2013) found that shown the importance of identifying “risk drivers” and the importance of organophosphate pesticide mixtures act synergistically and impair AChE pragmatic approaches to the effects assessment, to provide an estimate of activity which leads to a change in swimming behavior and mortality, combined risk. Challenges remain - moving away from in-field and edge which then leads to changes in population productivity. Exposure- of field will be required to further address exposure to multiple chemicals. response equations have been generated for single chemicals, binary and This is likely to require the further development of both exposure and ternary mixtures and integrated into the BN-RRM. Dissolved oxygen and effects modelling at a landscape scale and possibly new scenario based temperature data from the four watersheds were also included in our risk approaches. These prospective risk assessments will bring challenges analysis. Spatially explicit matrix metapopulation models were generated

SETAC North America Focused Topic Meeting | 9 PLATFORM ABSTRACTS from data rich information on Puget Sound. Overall risk results indicated samples were taken of WWTPs effluent, as well as upstream, downstream that single chemical toxicological effects were as important as ecological during eight time points and analyzed for almost 400 substances. Besides stressors. Synergism between the binary and ternary mixtures changed pharmaceuticals and household chemicals, also many pesticides were risk. Synergism indicated more risk due to binary mixtures of malathion included as we wanted to investigate whether the higher loaded phar- (malathion and diazinon, malathion and chlorpyrifos) and less risk due to maceuticals or the episodically discharged pesticides – during low flow the binary mixture of diazinon and chlorpyrifos. Ternary mixtures indi- conditions – contribute most to the risk towards aquatic organisms. Acute cated about the same risk as binary mixtures containing malathion. This risk was predicted using the multi-substance potentially affected frac- research demonstrated a probabilistic and a multiple-stressor approach tion approach and compared with biomonitoring data using the SPEAR to estimate the risk of mixtures to populations of Chinook salmon can be index indicative of pesticide sensitivity. Macroinvertebrate data was accomplished. collected at the sites during two time points in spring. In a parallel study 13 ecotoxicological bioassays were conducted at three selected sites and 22 Application of the MIXTOX Model in the Characterization of mixture toxicity modeling was performed to assess the contribution of Neonicotinoid Mixture Toxicity to Chironomus dilutus the detected chemicals to the observed effects. A multitude of micropol- E. Maloney, University of Saskatchewan / Toxicology; C.A. Morrissey, lutants was regularly detected and the concentrations were mostly higher University of Saskatchewan / Biology; J. Headley, University of Regina; downstream than upstream. Further, a positive correlation of pesticides K.M. Peru, Environment and Climate Change Canada / Watershed upstream and arable land use could be observed. While pharmaceuticals Hydrology and Ecology Research Division; K. Liber, University of and other household chemicals were regularly detected in the effluents, Saskatchewan / Toxicology Centre many pesticides were detected only during episodic events and are thus Neonicotinoids are a popular class of systemic insecticides that are, in underrepresented with grab samples. Nevertheless, occasional pesticides Canada, commonly applied as seed treatments to protect young crops concentration peaks in the stream as well as in the effluents drove the against biting-sucking insects. Extensive use of these insecticides in acute toxic pressure, but the lack of effect data for pharmaceuticals limits Canadian Prairie agricultural regions has resulted in the detection of interpretation. Overall, rather low acute risk was predicted of affected neonicotinoid mixtures in ecologically important wetlands in these areas. species (up to 2.1%) over all sites and time points with only a few sub- Yet the impact of these mixtures on local insect communities is poorly stances – mainly pesticides and diclofenac – explaining the total risk. The understood. This research aimed to address that knowledge gap through mixture toxicity modelling conducted at three sites underlined the rel- characterization of the cumulative toxicity of binary and ternary mixtures evance of single substances as toxicity drivers. However, the lack of effect of the most common neonicotinoids (imidacloprid (IMI), clothianidin data limited the bioassay evaluation, in many cases only a small fraction (CLO), and thiamethoxam (TMX)) to larvae of the sensitive aquatic of the effect was explained by the detected chemicals indicating a joint insect Chironomus dilutus under acute (96 h) and chronic (28 d) exposure effect of many unknown substances. Overall, despite the low predicted scenarios. Mixture toxicity was modeled using MIXTOX, a descriptive, risk a significant correlation with the SPEAR index was observed, high- regression-based approach to modeling the cumulative toxicity of lighting the importance of pesticides in wastewater-impacted streams. chemical mixtures. Briefly, preliminary assessments of single-compound This relevance also during low flow conditions seems to be typical for catchments where urban and agricultural land use co-occur. toxicity (LC50, EC50) were completed and used to develop parametric models describing potential cumulative effects. These parametric models 24 A Multipronged Effects-Driven Exposure and Impact Assessment were then statistically compared to data obtained from similar toxicity of Chemicals of Emerging Concern in Rivers of the Great Lakes studies with binary and ternary neonicotinoid mixtures, to determine the model of best-fit. Interestingly, even though all neonicotinoids are E.J. Perkins, L. Burgoon, US Army Engineer Research and Development assumed to have a similar mechanism of action, for many neonicotinoid Center / Environmental Laboratory; S.R. Corsi, USGS / Wisconsin mixtures tested the model of best fit significantly deviated from Water Science Center; D. Ekman, USEPA / National Exposure Research concentration addition. These results indicate that, under both acute and Laboratory; B.R. Blackwell, ORISE / National Health and Environmental chronic exposure scenarios, the cumulative toxicity of neonicotinoid Effects Research Laboratory; G.T. Ankley, USEPA / National Health and mixtures cannot be adequately predicted by concentration addition, Environmental Effects Research Laboratory; N. Vinas, Mississippi State suggesting that a different approach will have to be implemented to more University / Environmental Laboratory accurately predict effects and protect aquatic insect in contaminated A wide variety of chemicals are discharged into the Great Lakes region wetlands. Ongoing limnocorral (in situ enclosure) experiments will allow of North America including industrial, agricultural, pharmaceutical, for further assessment of whether neonicotinoid mixtures display similar personal care products, dietary, and legacy contaminants such as polyaro- deviations from concentration addition under more environmentally matic hydrocarbons or organochlorines. Many of these compounds have realistic exposure paradigms, and whether that poses a risk to natural little information available on their potential to effect fish and wildlife aquatic insect communities. health relative to legacy contaminants. As a result, the potential impact of introducing large numbers of chemicals into the environment through land run off or effluent discharge is unknown. In order to better under- Case Studies stand the impact of these chemical mixtures, we have developed an effects-driven multipronged approach to assess what chemicals fish in 23 Combining Chemical Analysis, Bioanalysis and Risk Assessment the environment are exposed to and to assess potential health impacts of to Prioritize Risk-Driving Substances in Wastewater-Impacted these mixtures. The multipronged approach is composed of several novel Streams approaches including: Determining the potential of contaminants in water J. Hollender, N. Munz, Eawag, Swiss Federal Institute of Aquatic Science to cause effects based on Exposure Activity Ratios determined using high and Technology / Environmental Chemistry; F. Burdon, Eawag, Swiss throughput in vitro data; In vitro assay assessment of water extracts; In Federal Institute of Aquatic Science and Technology / Aquatic Ecology; situ effects assessment using caged fathead minnows; and Toxicogenomics D. De Zwart, Mermayde / Centre for Sustainability Environment and and metabolomics analysis of caged fish to identify specific chemical and Health; B. Escher, Helmholtz Centre for Environmental Research GmbH health impacts. We applied this approach to two case studies to under- - UFZ / Helmholtz Institute Ulm; P.A. Neale, University of Queensland / stand the impact of chemical mixtures. The first examined the impact of ENTOX; H. Singer, C. Stamm, Eawag, Swiss Federal Institute of Aquatic effluent on fish in St. Louis Bay, MN. Here, 32 of 51 chemicals detected Science and Technology / Environmental Chemistry in the surface water were linked to specific changes in gene expression In this study, we investigated concentration patterns of micropollutants in and metabolome profiles indicating that these chemicals could potentially 24 Swiss wastewater-impacted streams during low flow conditions. Grab impact the fish. BPA in the water was linked to estrogenic effects on gene

10 | Risk Assessment of Chemical Mixtures PLATFORM ABSTRACTS expression, high Exposure Activity Ratios, and upstream regulator predic- such statute where mixtures of pesticides have been evaluated in respect tions suggesting that BPA is a chemical of concern in this system. The to their toxicity to threatened and endangered species. We conducted second case study examined impacts of agricultural areas versus urban multiple assessments to address potential mixture effects from registra- areas with effluent discharges in the Maumee River and the Detroit River. tion of current use pesticides. Here we describe a process for assessing Here, the impact of 82 different chemicals were linked to gene expression toxicity of three categories of pesticide mixtures; formulated products changes, however gene expression analysis was consistent with effects (one product containing multiple active ingredients), tank mixes (multiple related to PAHs suggesting that PAHS maybe chemicals of concern in this pesticides applied simultaneously), and environmental mixtures (resulting area. When combined these novel approaches have proven useful in iden- from unrelated pesticide use over the landscape). Mixtures were assumed tifying exposure effects and potential health impacts of complex mixtures to be either dose-additive or response-additive, depending on the modes including chemicals of emerging concern on fish in the Great Lakes. of action of the individual pesticide components. Estimates of additive toxicity utilized twoWITHDRAWN main pieces of information - exposure concentrations 25 Important Considerations for the Environmental Risk and taxa-specific toxicity values. Exposure concentrations were generated Assessment of Mixtures of Alkylphenol Ethoxylates and Their using USEPA’s Pesticide Water Calculator (PWC), which incorporates Degradation Products chemical and application-specific parameters to calculate anticipated K.K. Coady, The Dow Chemical Company / Toxicology Environmental water concentrations over different durations. Standard measures of toxic- Research and Consulting; C.A. Staples, Assessment Technologies Inc; B. ity (typically the LC50, or the concentration that is lethal to 50% of the Losey, RegNet Environmental Services test organisms) were gathered from various sources and used to represent The exposure, hazard and risk of C8 and C9 alkylphenol ethoxylates taxa groups with different sensitivities to a given pesticide. Reflecting (APnEO, where n=4-40) and their degradation products can be assessed the three mixture categories, we predict toxicity for formulated products using a mixtures-based approach. Based on the knowledge of use containing organophosphates, co-applications reported in California’s patterns, releases to the environment, biodegradation pathways, and envi- Pesticide Use Reporting System, and ambient water quality monitoring ronmental monitoring efforts, mixtures of APnEO and their degradation data from Washington State. Results show that estimating mixture toxic- products have been well characterized in the environment. Both in vitro ity is possible with currently available information, and these predictions and in vivo toxicity studies on commercial APnEOs and various degrada- can be used in regulatory situations such as risk assessments under the tion products, including both C8 and C9 alkylphenols (AP), have been Endangered Species Act. Importantly, failing to consider mixtures may conducted such that relative toxicity of the components in the mixture can underestimate pesticide risk to such an extent as to lead to erroneous risk be characterized. These toxicity studies include endpoints that are rel- conclusions and ineffective protections to threatened and endangered evant to estrogenic activity as well as endpoints that integrate effects from species. multiple toxicity pathways. Mixture toxicity studies with these chemicals 27 Mixtures Risk Assessment: Finding Opportunity Amidst Chaos indicate that taking an additive approach to the assessment is appropri- ate for this class of chemicals. However, additivity methods such as toxic E.J. Van Genderen, International Zinc Association / Environment & equivalency factors, are not perfect and it is necessary to be aware of, and Sustainability; M. Ottinger, University of Houston / Department of account for uncertainties in additivity (e.g., differences across cell types, Animal and Avian Sciences tissues, species and environmental matrices) even when a common mode The state of the science for evaluating risks to multiple substances has of action exists. Several environmental risk assessments for APnEOs and/ matured, now incorporating novel analytical techniques for characterizing or APs have been conducted by regulatory authorities or in response to exposures and effects to human and ecological receptors. In addition, the product-stewardship needs. Some of these risk assessment approaches practical experiences of risk assessors and risk managers acquired from consider the mixture of APnEOs and their degradation products, using field investigations have contributed to new assessment frameworks and slightly different methodologies, while other risk assessment approaches identified remaining research needs. However, several challenges remain focus on single chemicals of the mixture that are deemed most critical. that will continue to constrain the development of comprehensive risk Several examples of these risk assessments will be presented with consid- assessment approaches for mixtures across chemical classes and levels erations for the appropriate scope of these risk assessments, potential data of biological organization. In particular, the absolute number of chemi- gaps, and recommendations for future efforts to refine environmental risk cal combinations and exposures limits our ability to adequately assess assessment of C8 and C9 APnEOs and their degradation products. even a small fraction of relevant scenarios. It is believed that attempts to meaningfully characterize risks to human and ecological receptors must 26 Evaluating Toxicity to Endangered Species From Three implement multi-disciplinary approaches. To that end, mixtures risk Categories of Pesticide Mixtures assessment could foreseeably become the nexus between novel analyti- D.H. Baldwin, NOAA Fisheries / Northwest Fisheries Science Center; cal tools (chemical fingerprinting, omics, etc.), screening procedures C.A. Laetz, NOAA NMFS / Northwest Fisheries Science Center; S.A. (micro-arrays, high-throughput techniques, etc.), statistical interpretations Hecht, T. Hawkes, NOAA / National Marine Fisheries Service (species/genus extrapolation, etc.), and computational frameworks (AOP). Pesticides are widely used throughout the United States and are fre- This presentation will wrap up the FTM with perspectives from the past, quently detected as complex mixtures in aquatic habitats of threatened present, and future of risk assessment for complex mixtures. and endangered species. Therefore, pesticide mixture toxicity is an important component of risk assessments performed within different statutory and regulatory contexts. The Endangered Species Act is one

SETAC North America Focused Topic Meeting | 11 POSTER ABSTRACTS Problem Formulation Exposure Assessment

WP001 GCxGC-based Property Estimation for Risk Assessment WP003 Chemical and Biological Assessment of Endocrine of Complex Petrochemical Mixture Including the Generated Disrupting Chemicals in Wastewater and Coastal Waters in Kuwait Compounds in the Engine Oils N.J. Al-Jandal, Kuwait Institute for Scientific Research / Environmental Y. Zushi, National Institute of Advanced Industrial Science and Management Program; T. Saeed, I. Azad, KISR Technology / Research Institute of Science for Safety and Sustainability; The present study aimed to investigate the status and sources of endo- J. Nataga, Shimadzu Japan; D. Nabi, Bigelow Laboratory for Ocean crine disrupting chemicals (EDCs) and their effect on local fish in Sciences Kuwait’s cpatal areas, from the chemicals and biological perspectves. The comprehensive two-dimensional gas chromatography (GC×GC) is These cghemicals find their way to the marine environment via the now a well-known technique for its separation power in greatly resolving sewage treatments plants (STPs). Therefore, assessment of three STps complex chemical mixtures. Recently, a new potential of GC×GC is indicated the existance of significant levels of phthalates (19 and 31 ug/l), revealed, whereby diverse environmental partitioning properties can alkylphenols (85 and 159 ng/l), and estrogens (30 and 368 ng/l) in both reliably be estimated for the nonpolar analytes detected in complex inflow and outflow samples. The analysis of samples from field expo- mixtures. Using first- and second-dimension retention information of sure sites revealedWITHDRAWN significant levels of ECDs in seawater (phthalates: nonpolar analytes, 42 properties relevant to bioaccumulation and baseline 2.1 to 4.6 ug/l; alkylphenols: 1.2 to 16.4 ng/l; estrogens: 0 to 36.2 ng/l) toxicities were reliably predicted. In this study, we further developed and sediment (phthalates: 2.1 to 15.7 mg/kg dry wt; alkylphenols: 2.5 to this technique for the risk assessment of original and newly generated 15.1 ug/kg dry wt; estrogens: 4.1 to 214.2 ug/kg dry wt.). The biological products in complex mixtures. We analyzed continually sampled engine perspective investigated through the exposure of fish to sewage outlets at oil from a vehicle in use and estimated properties of detected compounds five sites proved that the exposure resulted in elevated levels of vitel- including the generated compounds during burning process. The GC×GC logenin (Vtg) and Vtg gene expression. The hepatosomatic index (HSI) detected around a hundred of the generated compounds in addition to revealed a higher level in winter samples (0.48 to 0.79%) than in sum- originally presented compounds in the engine oils. The GC×GC-based mer samples (1 to 1.5%). Histological and immunolocalization evidences technique provided us valuable information, which is vital for risk showed the effect of EDCs on Vtgdeposition in the liver tissue. The Vtg assessment of chemicals. This approach will be useful for risk assessment gene of seabream (Acanthopagrus latus) yielding 275 base pair amplicon of complex mixtures. was similar to that of the European seabream (Sparus aurata). Vtg gene expression was stronger in summer than in winter with relative expression WP002 In the Mix – the Pitfalls of Assessing Mixtures for levels ranging from 0.05 to 37. Chemicals and biological inb=vestigations Regulatory Purposes indicated the presence of significant levels of phthalates, alkylphenols and C. Phillips, Cefas Lowestoft Laboratory / Science Directorate – estrogens in the Kuwait Bay, and these levels were enough to initiate Vtg Advice and Assessment; R. Suhring, Cefas Lowestoft Laboratory; H. gene expression and Vtg deposition in the liver tissue of fish exposed to Walton, Cefas Lowestoft Laboratory / Ecotoxicology; A. Smith, Cefas the sewage outlets in Kuwait for two weeks / Ecotoxicology and Molecular Ecology; B. Rowles, Cefas Lowestoft Laboratory; M. Shields, Department of Business, Energy and Industrial WP005 Challanges of Testing Chemical Mixtures in Aquatic Strategy Toxicology Studies The European REACH and OSPAR regulatory frameworks are based H.O. Krueger, Wildlife International / Aquatic, Plant and Insect on the assessment of substances, however multi-constituent substances Toxicology are effectively mixtures, which can incorporate both hazardous and A key component of any aquatic toxicology test is the delivery of the non-hazardous compounds. For regulatory risk assessments, this means test substance in the test system. This is difficult with a single chemical that individual substances in a formulation might have different physico- and is even more complicated with mixtures. The more chemicals in the chemical properties but are marketed as a single substance reaction mixture the more difficult it becomes to design a test that maximizes mixture. This characterisation as a single substance can go as far as exposure for each chemical. But before one even begins testing, it is to include additives like water, alcohols and surfactants with a range important to know 1) how the toxicity of the mixture is to be expressed, of properties. There is a concern that the presence of non-hazardous 2) physical and chemical properties of the chemical(s), 3) the suitability components can mask the true environmental risk of more problematic of a particular test type, and 4) proper analytical methodology. While it is substances. To investigate this concern, Cefas tested two marketed appealing to test at nominal concentrations and not measure each of the forms of a dye, Rhodamine B, comprising of the isolated solid and an components in a mixture, it is also desirable to know which component(s) aqueous solution of Rhodamine B and acetic acid using standard OECD are causing toxicity or if there is an interaction between chemicals. It is test guidelines for aquatic toxicity and biodegradation. Test results uncertain as to whether the toxicity should be expressed based on the were compared with those presented by a chemical supplier as part of toxicity of the entire mixture, key components, or surrogate measures registration for offshore use in the UK. Toxicity results for the isolated such as TOC. Most regulatory tests require maintaining concentrations in solid were in good agreement with those provided by the chemical water between 80 and 120% of nominal for the duration of the test. What supplier. The data for Rhodamine B mixture showed lower toxicity than does that mean for mixtures? Do all chemicals in the mixture need to be the single substance, but when the water content of the mixture was maintained in that range or only the ones suspected to be toxic at con- taken into account, the toxicity was similar to that of the single substance centrations of concern? For a single chemical or mixture it is important despite the presence of acetic acid. Similarly, the biodegradation test to understand the physical and chemical properties of the chemical(s) in results for the single substance were in good agreement with the supplied order to design a proper test. Prior to testing it is desirable to know if the data – indicating high persistence. However, when carried out on the chemical is volatile, stable in water, its water solubility, whether it adsorbs reaction mixture the biodegradation was over 20%, which is considered to components of the test system, or reacts to light. That will determine if moderately persistent. Adjusting the test concentrations for the correct the test can be run as a static, static-renewal, or flow-through test. Closed addition rate of the dye led to biodegradation results < 20% – again systems can be used for volatile materials, water accommodated frac- indicating high persistence. Our results showed a that the biodegradation tions were developed for petroleum products and are also useful complex potential of mixtures can be masked by the more readily biodegradable materials with low solubility, saturator columns or passive dosing systems compounds in the mixture. Therefore, a test as a single substance can are newer techniques that also are useful. There are other options for skew the results, making the product look more biodegradable than it is. bioaccumulation tests in which the test material can be delivered via diet instead of a water exposure or sediment tests in which test materials are

12 | Risk Assessment of Chemical Mixtures POSTER ABSTRACTS added to the sediment for testing. The more chemicals in the mixture the Pseudokirchneriella subcapitata was used due to its easy manipulation more difficult it becomes to design a test that maximizes exposure for under laboratory conditions and the higher sensitivity to chemicals each chemical. Choices and trade-offs based on regulatory concerns will compared to other species such as fish. The testing solution consisted be necessary in designing aquatic toxicology studies for mixtures. in Milli-Q water buffered with 3-(N-Morpholino) propanesulfonic acid (MOPS) at pH 6.5. The target metal (Cu, Pb or Zn with a concentration WP006 Assessing Exposure of Bald Eagle Nestlings to from 0 to 300µg/L) was included individually to the testing solution for Organochlorine Compounds in Michigan (2015–2017) and evaluation. The microalgae was exposed to the metal for 6 hours using Voyageur’s National Park, Minnesota (2011–2017) a transparent microplate. The algae growth was obtained measuring R. Eberius, University of Maryland at College Park / Environmental the fluorescence (435/685 nm wavelength of excitation/emission) with Science and Technology; M.E. Barton, University of Maryland / Animal a microplate reader every 1 hour. Under this conditions the EC50 at 6h Sciences; W.W. Bowerman, University of Maryland / Environmental for Cu, Pb and Zn were 150, 189 and 88 µg/L, respectively. We believe Science & Technology; T. Pittman, FL Fish and Wildlife Commission / that this method may be an alternative base to evaluate the future mixed Fish and Wildlife Research Institute; K.L. Simon, University of Maryland, effects of metal avoiding non target metal effect College Park / Environmental Science and Technology; L. Fuentes, University of Maryland / Department of Environmental Science and WP008 Occupational Exposure of Butchers to Toxic Mixtures in Technology Fumes From Burning Tyres Employed in De-furring of Animals in Nigerian Abattoirs Historic organochlorine contamination has been a concern in the Great Lakes region since the middle of the twentieth century. The bald F.O. Okonkwo, A.E. Ogbu, University of Nigeria Nsukka / Biochemistry; eagle (Haliaeetus leucocephalus) population decline in this region, I.N. Onwurah, University of Nigeria / Biochemistry and elsewhere, was largely attributed to contamination by P,p’-DDE, Fumes from the burning tyres used in de-furring animals meant for a metabolite of widely used organochlorine pesticide DDT. However, consumption in Nigerian abattoirs are known to contain myriad of toxic the effects of P,p’-DDE and other organochlrorine contaminants, such mixtures especially particulate matter (PM), volatile organic compounds as PCBs, can be difficult to isolate due to the co-occurrence of these (VOCs), hazardous air pollutants (HAPs), and metals some of which are toxicants in the environment. Bald eagles have been utilized as a known human carcinogens. This study determined the effect of exposure bioindicator of persistent organic pollutants in Michigan since the late to these toxicants on abattoir workers. Determination of urinary hydroxy- 1980s and Voyageur’s National Park, Minnesota has been used as a PAH, urinary phenolic compounds levels and urinary heavy metal reference site for this ongoing study since 1989. Recent contaminant concentration, coupled with lung function measured using digital spirom- trends indicate that the concentrations of many organochlorine pollutants etry were extrapolated to adverse toxic injury. The results showed that the have decreased. Additionally, the bald eagle population has largely concentrations of PM1, PM2.5, PM7, PM10, and TSP were generally low recovered, with 776 fledged young in Michigan in 2016. However, this at 7 am, but increased with time until 8.30 am, after which they dropped population recovery is not uniform across the region. Concentrations of at 9 am, irrespective of the point of sampling. Statistically significant twenty two PCBs and twenty four organochlorine pesticide compounds variations in the concentrations of the studied particulate matter (except were extracted from bald eagle nestling blood plasma. Compounds were PM1) with time were found only at the de-furring point (Point A). Their separated from plasma using solid phase micro extraction and quantified concentrations at the wash area (Point B) and the administrative housing using gas chromatography. Contaminant mixtures were sub-divided into (Point C) were statistically similar at the time intervals and points studied. three groups: (1) DDTs (2) PCBs (3) non-DDT organochlorine pesticides. Other than indeno(1,2,3-cd)pyrene, (0.0350 ± 0.0032; p-value = 0.511) Here we report preliminary results from plasma collected in Michigan and benzo(b)fluoranthene, (0.0495 ± 0.0039; p-value = 0.252) at Point B and Voyageur’s National Park from 2015-2017 and 2011-2017 respectively. relative to Point A), the other studied PAHs had concentrations in samples These preliminary results indicate that concentrations are greater in some from Point A that were significantly higher P( < 0.05) than concentra- watersheds Michigan than others, and nesting territories on the Great tions found in Points B and C. It is therefore obvious that the de-furring Lakes have higher contaminant loads than inland territories. Additionally, point significantly contained more naphthalene, anthracene, fluoranthene, concentrations seem to vary annually across Voyageur’s National Park pyrene, and benzo-a-pyrene compared to the wash area and the adminis- and spatially between the park’s major impoundments. It has yet to be trative building. The concentration of 1-Hydroxypyrene (1-OHPyr) (µg/ establish what, if any, environmental factors initiate these fluctuations. molCret), a PAH metabolite, in the post-shift urine samples of the butch- The source of these fluctuations and their impact on population recovery ers was significantly higher, (P < 0.05) with mean concentration ±SD will be the subject of further analysis. value of 0.52±0.13µg/molCret in the butchers relative to the control with 0.20±0.07µg/molCret value. Similarly the concentrations of the heavy WP007 New Approach for Toxicity Assessment Avoiding the metals zinc and nickel were significantly higher in the butchers compared Chemical Reaction Between Target Metals and Culture Medium to the control (0.91±0.19 vs 0.31±0.28 mg/l and 0.11±0.06 vs 0.06±0.02 Contents mg/l, respectively). The result of the spirometry showed significant G. Pascual, Tohoku University / Civil and Environmental Engineering; I. deterioration of lung function in the workers at Point A when compared to Garcia, N. Tanaka, Tohoku University; F. Takeda, Public Works Research the administrative staff. It is the opinion of the authors that interventions Institute of Japan; O. Nishimura, Tohoku University / Architecture Civil in the form of personal protective equipment are put in place to reduce and Environmental Engineering exposure through inhalation and the skin. Water pollution is a global problem, and metals as pollutants have a WP009 Trends of Known and New Contaminants of Emerging special importance because they cannot be degraded in biological Concern Measured in a Wastewater-Influenced Stream Using metabolism processes. Copper (Cu) and Zinc (Zn) are essential Passive Samplers micronutrients for living organisms but they are toxic at high concentrations. On the other hand, Lead (Pb) is considered toxic and have E.T. Furlong, L.K. Kanagy, W.T. Foreman, USGS / National Water not been reported yet any biological function. For these three metals, it Quality Laboratory; D.A. Alvarez, USGS-CERC / Environmental has been reported a wide range of medium effective concentration (EC50) Chemistry in different species including microalgae; this variation in EC50 may be Integrating passive samplers, such as the polar organic compound integra- due to the testing medium composition which causes toxicity inhibition tive sampler (POCIS), provide the means to collect an integrated profile of the target metal. For this reason, our approach is to use a simple of many polar organic contaminants present in surface water whose testing solution to evaluate the metals effect on pre-cultured microalgae, presence may be continuous or episodic, reflecting changes in hydrologic avoiding the interference of non-target metal. Green microalgae or source input. POCIS permits contaminant trend assessment by (1)

SETAC North America Focused Topic Meeting | 13 POSTER ABSTRACTS identification and quantitative concentration using methods for targeted Risk Assessment and Risk Management contaminants, such as analgesics, antidepressants, stimulants, and their degradates, as well as (2) identification and semiquantitative estimation WP011 Risk Assessment Methods That Simplify Chemical Mixtures of nontargeted contaminants using a comprehensive screening strategy. Associated With Treated Domestic Wastewater Discharges We present results from structured taget and nontarget analysis of POCIS J.M. Diamond, Tetra Tech, Inc.; S.D. Dyer, The Procter & Gamble extracts deployed over a year-long period under differing flow conditions. Company / Environmental Stewardship and Sustainability Organization; The extracts were analysed using a Quadrupole Time-of-Flight Mass M. Focazio, USGS / Environmental Health; K.A. Kidd, McMaster Spectrometer (QToF MS) operated in the positive electrospray ionization University; M.R. Servos, University of Waterloo / Department of Biology; mode and coupled to an ultra performance liquid chromatograph, using J. Snape, AstraZeneca UK Ltd. / AstraZeneca Global Environment; J. a C-18 reversed phase column for separation of analytes. The QToF MS Tolls, Henkel AG & Co. KGaA was operated in a scan mode combining spectra with alternating low and Municipal waste water treatment plants are a source of “down the drain” high collision cell energies. The resulting data were then aligned and chemicals that are assessed for their potential to cause environmental analysed instrument software and an extended pesticide and toxicology effects both independently and as mixtures. A framework is presented library to identify known and suspected contaminants. Results from the that facilitates the evaluation of potential aquatic ecological risks result- QToF analysis were compared to targeted analysis using high-perfor- ing from discharges of down the drain chemical mixtures. A scenario is mance liquid chromatography/tandem quadrupole mass spectrometry presented using representatives of many types of chemicals that are used (LC/MS/MS). Methyl-1H-benzotriazole, carbamazepine, citalopram, domestically and that have a wide range of physical/chemical properties desvenlafaxine, fexofenadine, and tramadol were among the compounds and modes of action. Predicted treated effluent chemical concentrations in POCIS extracts detected by LC/MS/MS and confirmed by QToF MS. are based on reported loading rates and removal rates for three types of Lamotrigrine and DEET, not determined by LC/MS/MS but anticipated wastewater treatment: trickling filter (TF), activated sludge secondary to likely be present, were identified by QtoF MS from elemental composi- treatment (AS), and AS plus advanced oxidation treatment (ASO). In Tier tions of the parent pseudomolecular ions and the associated fragments I of the framework, predicted effluent concentrations were compared with from the linked high-energy collisional dissociation spectra. The hydroxyl the lowest predicted no effect concentration (PNEC) obtained from the degradation products of carbamazepine and lamotrigrine were similarly literature for algae, daphnia, and fish. A cumulative risk characteriza- identified in these extracts, as were ketamine, which was not initially tion ratio (cumRCR) was computed for the mixture. A cumRCR is < 1.0 expected to be present. Trends in the relative abundances of suspected and indicates risk is unlikely and no further action is needed. If the cumRCR newly identified contaminants were consistent with those observed for > 1.0, then a Tier 2 assessment is recommended, in which a more refined targeted compounds by LC/MS/MS. The complementarity of targeted and examination of effects information is used based on trophic level. If the nontargeted analysis is also well demonstrated by the trends in relative Tier 2 assessment indicates a possible risk, then a targeted retrospec- abundance under differing flow conditions and in different sections of the tive assessment is recommended. In Tier 1 of our scenario, the cumRCR stream reach. was > 1.0 for all three treatment types in our scenario, even though no WP010 Unpredictable Effects on Target Gene Expression Evaluated chemical exceeded a hazard quotient of 1.0 in AS or ASO. In Tier 2, AS in Zebrafish Exposed to Pharmaceutical Mixtures yielded a lower cumRCR than TF due to higher removal rates and the cumRCR in ASO was 1.0, particularly as more chemicals are considered. G.V. Aguirre-Martinez, Arturo Prat University / Health Science Faculty; A more refined Tier 2 mixture risk approach is useful, as well as receiv- H. Reinardy, The University Centre in Svalbard / Arctic Technology; L. ing waterbody assessments. We present a process in which a retrospective Martín-Díaz, University of Cadiz / Physical Chemistry; T. Henry, Heriot- assessment can test whether certain chemicals hypothesized as potential Watt University / School of Life Sciences drivers in the prospective assessment could have, or are having, deleteri- Although present at low concentrations at the ng L-1 to μg L-1 range, ous effects on aquatic life. pharmaceutical substances are found simultaneously as complex mixtures that have unknown and difficult to evaluate effects on aquatic biota. It WP012 A Novel Methodology for Screening Hazard and Risk From is possible that individual substances can act in a synergistic or additive Environmental Exposures to Mixtures manner, which suggests management for environmental protection should H. Plugge, 3E Company take into consideration mixtures of substances rather than models based Increasingly regulatory methodology incorporates a hazard/risk screening on individual effects. The aim of this study was to investigate whether step prior to proceeding with an in-depth assessment phase. Heretofore effects on target gene expression and enzymatic activity of individual such a screening step has only been performed on single chemicals. Even substances tested were additive when mixed. To reach this aim, zebraf- comparison of single chemicals encounters the problem of comparing ish (Danio rerio) larvae (72-96 hour post fertilization) was exposed to apples and oranges: most of these screening systems employ buckets, caffeine-CAF, ibuprofen-IBU, and carbamazepine-CBZ (0.05 and 5 μM), low, medium, high without allowing for summation of effects. Given tamoxifen-TMX (0.003 and 0.3 μM), and to pharmaceutical mixtures (low the absence of unified effect data for even single chemicals, addressing mix: 0.05 μM of CAF, IBU, CBZ and 0.003 μM of TMX, and high mix: 5 hazards/risks of chemical mixtures is impossible. What is thus needed is μM of CAF, IBU, CBZ and 0.3 μM of TMX). After exposure period (96 a screening methodology that allows for unified expression of both envi- h) expression of zebrafish cytochrome P4501A cyp1a( ) and vitellogenin ronmental and health effects. In response, we developed Green Score, a (vtg) genes as well as activity of ethoxyresorufin-O-deethylase (EROD) screening method that expresses hazard in unitless numerical scores, thus were analyzed. Results indicated that pharmaceuticals tested individu- allowing easy comparison of single chemicals and chemical mixtures. ally down regulated studied genes. Single pharmaceuticals did not induce Green Score begins with a data identification phase, where (raw) scientific EROD activity and exposure to low mix did not alter gene expression; data is combed from both the literature as well as (regulatory) databases. however, the high mix up regulated significantly cyp1a gene expression This function can be performed both automatically and manually. (The (p< 0.05). Up regulation of cyp1a was consistent with induction of EROD manual method allows for the immediate filling of identified datagaps activity in larvae exposed to high mix. Complex mixtures of pharma- using Read-Across or other methodologies.). The raw data are then trans- ceutical substances can have unpredictable effects on toxicity endpoints formed using logarithmic algorythms into normalized scores on a scale than those observed by the individual substances. Results don’t indicate of 1-1000, with 1 being the worst, “ungreenest” score. Given that the data additive toxicity, nevertheless, these results highlight the need to evaluate are most often not normally distributed, geometric means provide the best mixtures when performing an environmental risk assessment of drugs, available estimate of an average. Presently 18 health effects endpoints are rather than studies based on individual effects, since in the environment scored, while 6(8) environmental endpoints are scored. Additional end- pharmaceutical active compounds are not found isolated and the effects of points are under development. Given that scores for individual endpoints their mixtures is poorly understood and might be underestimated. 14 | Risk Assessment of Chemical Mixtures POSTER ABSTRACTS are not normalized, intermediate and final scores are derived using bioassays protocols as the environmental risk assessment followed the equally weighted geometric means of individual endpoint scores. Here USEPA guidelines and recommendations and other authors. Mainly geometric means beside accounting for outliers, tend to weight the lower ecotoxicological results with barite and soluble forms, showed a negative score, biasing the final score(s) toward the lower number, i.e. the worse/ effect of barite on cell growth of epicontinental microalgae Chlorella sp. ungreenest score. Mixtures are assessed using a weight fraction weighted (96 h), which recorded an IC50 of 0.1 g/l and an unobservable effective partial contribution algorythm, where the overall score is the average concentration (NOEC) of 0.02 g/l. Likewise, a negative effect of barium of the weighted geometric means. For screening purposes relative risk on leaf growth of monocot terrestrial plant Z. mays (10 d) which recorded calculations can be made using exposure surrogates where risk= hazard an EC50 of 0.0011 g/l and NOEC of 0.0002 g/l. Finally, it concludes that * exposure. Various exposure surrogates have been used imcluding Csat there is high environmental risk (RQ) for barium sulfate (RQ = 1.224) and in occupational settings as well as emission/inventory mass balances, in soluble forms (RQ = 37 500) using the ecotoxicological response of twelve order to calculate screening risk estimates. non-target organisms.

WP013 A Web-based Integrated Platform for Mixture Risk WP015 Human Dietary Intake and Hazard Characterization for the Assessment Tools Residues of Neonicotinoides and Organophosphorus Pesticides in J. Kim, H. Jeong, K. Akhlaq, K. Kume, Y. Kim, KIST Europe / Egyptian Honey Environmental Safety Group; S. Kim, KIST Europe / Chemical Y. Alnaggar, Saskatchewan University / Department of Zoology; G. Management Lab Codling, University of Saskatchewan / Toxicology; J. Giesy, University of The paradigm shift of chemical risk assessment is in progress from Saskatchewan / Department of Veterinary and Biomedical Sciences and “chemical safety focusing on single chemicals” to “product safety Toxicology Centre extending to mixtures and articles” since mixture toxicity can be caused Abstract Two studies published recently looked into the health risks of among chemicals even at levels below no observed effect concentrations. organophospate and neonicotinoid plant protection products (PPP), upon However, requiring to conduct toxicity tests for all possible mixture the European honey bee (Apis mellifera L.) in Egypt. Using the concentra- products is unfeasible due to the extremely large number of combinatorial tions reported in those studies a hazard assessment for consumption of complexity. Thus, developing computational toxicology methods as Egyptian honey from the Nile delta was performed for both spring and cost savings, fast and reliable approaches to realize the new paradigm summer pesticide exposure to humans. A total of 28 compounds includ- needs. In this study, we designed and developed a web-based integrated ing metabolites were assessed for adult Egyptians based on a best and platform for mixture risk assessment tools which includes three different worst case scenario. Even for the worst case scenario, exposure to these open access tools: CLP Mix-Tool for estimating the hazard classification two classes of pesticides from honey was 15-fold less than hazard index of mixture products; Eco-PDS for eco-friendly product design support (HI) value of effect and people exposed are unlikely to suffer adverse tool; and MixExpo for the risk assessment for occupational exposure health outcomes. to mixtures. Chemical industries in the EU have to classify and label mixtures, according to the European regulation on classification, labelling WP016 Incorporating the Joint Toxicity of Co-applied Pesticides and packaging of substances and mixtures (CLP). CLP Mix-Tool based on Into the Ecological Risk Assessment Process the technical guidance for the CLP regulation supports the component- J.B. Belden, Oklahoma State University / Integrative Biology; R. Brain, based estimation approaches appear to be practically employed for Syngenta Crop Protection, Inc. / Department of Environmental Risk the CLP estimation, due to the lack of toxicity test data on mixtures Characterization and chemical combinations. Eco-PDS provides the chemical industry Pesticides are frequently formulated as mixtures of active ingredients. with a possible solution to calculate the mixture toxicity of chemical Although traditionally ecological risk assessments (ERA) have focused products before conducting experimental toxicity tests. In the tool, two on individual active ingredients, there is an ongoing effort in many juris- conventional models, i.e., first tier models, and two advanced models, dictions to more formally include assessment of mixtures. The overall i.e., higher tier models, were combined to predict the mixture toxicity goal of this project was to describe an approach for conducting ERA of in different models. A prototype of MixExpo (ver. 0.3) supports a lead jointly applied pesticides. We suggest that standard testing of formulation component identification (LCID) method suggested by the European mixtures is not warranted due to the low probability of synergy occurring chemical industry council (Cefic) and German Chemicals Industry at a high enough magnitude to be measurable above experimental vari- Association (VCI) for mixture risk assessment. This study highlights ability. Thus, empirical testing should focus on formulations where there that the integrated platform for mixture risk assessment tools can be is a greater likelihood of synergy due to known toxicological interactions useful to screen the potential toxicity of mixture product; and to check of the pesticide class or a priori knowledge of synergy, such as intellectual which components are critical, and to what extent they can be modified or property claims. Additionally, empirical testing should focus on species substituted to be safer products in time- and cost-saving methods. that are above levels of concern and limit testing on species where it is unlikely that synergy would significantly change the outcome of the ERA. WP014 Ecological Risk Assessment of Barite (BaSO ) Using 4 If empirical testing is warranted, we suggest that results be compared Ecotoxicological Tests With Twelve Non-Target Organisms to the concentration addition model (CA). If the empirical data deviates C. Paredes, Instituto del Mar del Perú / Área Funcional de from the model by a factor of greater than five, then synergy is considered Investigaciones Marino Costeras likely and the ERA is based on the empirical data. Otherwise, the ERA The effect of barite in aquatic ecosystems related to where hydrocarbon may use CA to calculate risk quotients or be based on the most toxic activities are performed nationally is unknown. Therefore, the active ingredient. To evaluate the approach, we reviewed formulation environmental risk of barite was undertaken using the ecotoxicological mixtures where data were available. Only 3 of 24 mixture studies were response of twelve non-target organisms in order to know the effects found to deviate from CA by more than 5. The majority of the studies had that this compound may be cause to organisms related to marine and a single component that dominated toxicity suggesting that the ERA for continental ecosystems where hydrocarbon activities are developed. these formulations will not be meaningfully different if based on the most Ecotoxicological tests included the microalgae Isochrysis galbana, toxic active ingredient. Overall, this approach balances risk assessment and Chlorella sp., terrestrial plants Medicago sativa and Zea mays, conservatism and reduces testing that would likely not result in improve- crustaceans Daphnia magna, Emerita analoga, and Apohyale sp., ment of the ERA. the echinoderm Tetrapygus niger, the aquatic insect Chironomus calligraphus, and fishes Odontesthes regia regia, Poecilia reticulata, and Paracheirodon innesi. Measurements of parameters and

SETAC North America Focused Topic Meeting | 15 POSTER ABSTRACTS

WP017 Selenoenzymes as Molecular “Targets” of Irreversible increase Mg toxicity. Further work at lower Mg:Ca ratios will determine Inhibition by Mercury, Cadmium, and Similarly Electrophilic the COPC, or combination thereof, responsible for toxicity. Mixture Inorganic and Organic Neurotoxicants toxicity is also being assessed through Direct Toxicity Assessments N. Ralston, University of North Dakota / Earth Systems Science and of several water types from the mine, including tailings and process Policy; L. Raymond, Translational Medicine Independent Research water, which vary in COPC concentration and composition (especially Consultants Mg:Ca ratios and pH). The tailings water of pH 3, with the greatest concentrations of COPCs, was the most toxic. At 0.06% strength (99.94% High methylmercury (MeHg) exposures irreversibly inhibit selenium (Se) dilution, corresponding to Mn 1550 µg/L, Mg 4.9 mg/L, U 24 µg/L and dependent enzymes that are required to prevent and reverse oxidative TAN 0.4 mg/L), a 50% reduction in population growth and growth rate damage in the brain. Now that the selenoenzyme inhibition mechanisms occurred for the hydra and alga respectively, and an ~80% reduction in Hg toxicity and their sequelae have been defined, many previously inex- reproduction for the gastropod and cladoceran. The latter two species plicable aspects of the seafood mercury issue are simple to understand were also the most sensitive to process water (pH 6, at 1% strength) with and entirely consistent with biochemical principles. For example, the an 80% and 100% effect for the gastropod and cladoceran, respectively latency effect, tissue specificity, and seemingly variable nature of effects (at Mn 9920 µg/L, Mg 28 mg/L, U 44 µg/L and TAN 5.4 mg/L). A 10% of high MeHg exposures were all challenging before the biochemical effect occurred for the cladoceran in process water at 0.06% strength, mechanisms were recognized. For example, since maternal consumption equating to Mn 596 µg/L, Mg 1.7 mg/L, U 2.6 µg/L and TAN 0.3 mg/L. of seafoods with high MeHg contents such as pilot whale and shark meats By analysing the responses of these organisms to different mine waters was associated with adverse effects on child neurodevelopment and it had a site-specific model will be built to predict toxic effects based on been assumed that low level MeHg exposures from ocean fish might also concentrations of COPCs and the local conditions. be associated with adverse outcomes. However, epidemiological stud- ies have instead shown that increasing maternal consumption of typical WP019 Assessing the Impacts of Toxic Mixtures Over a Broad varieties of ocean fish improves child neurodevelopment rather than Geographic Scale: Challenges and First Steps degrades it. Reanalysis of the findings of the Faroes Study, the popula- D.H. Baldwin, J.A. Spromberg, NOAA Fisheries / Northwest Fisheries tion that current seafood safety advisories were based on, indicate that Science Center; J. Lundin, NOAA NMFS; C.A. Laetz, NOAA NMFS / the more ocean fish mother’s ate during pregnancy, the better protected Northwest Fisheries Science Center; N. Scholz, NOAA Fisheries their children were from the adverse effects of Hg exposures from eating pilot whale meat. The reason why MeHg exposures from shark and pilot Assessing the risks posed by chemical mixtures is a complex process. whale meat are harmful while eating ocean fish is beneficial appears to Ideally, details are available on exposure (e.g., which chemicals and what be related to differences in their Se contents. To provide consumers with concentrations) and effects (e.g., mechanisms of action and toxicity data). a reliably accurate and more easily understood index of seafood safety, Even for a single location and time such as a lab or field site (a small scale), we developed the Se-Health Benefit Value (HBV). Seafoods that contain this can be challenging. Unfortunately, risk assessments often need to more MeHg than Se have a negative HBV, while those that are rich in cover much larger scales such as an entire watershed or a wide-ranging Se relative to MeHg have a positive HBV. The HBV of shark (-110) and species. This increase is scale substantially increases the complexity whale (-80) meats that were associated with adverse effects stand in stark of a risk assessment. Thousands of chemicals are in use throughout the contrast to those of commonly consumed varieties of ocean fish which are United States leading to potential environmental mixture exposures. uniformly positive (generally ranging between +10 and +30). While MeHg Examples include pesticide runoff from urban, agricultural, and forested effects on brain selenoenzymes has been increasingly well recognized, areas and wastewater discharges from municipal treatment sites, etc. At the effects of other neurotoxic soft electrophiles such as cadmium and the landscape scale, therefore, the nature of chemical mixtures will vary various other inorganic and organic toxicants need to be assessed so their across space and time. At this increased complexity, available monitor- additive or synergistic effects can be assessed and applied in public health ing data are inadequateWITHDRAWN for describing realistic exposure scenarios and risk assessments. For certain cases, where the HBV may be affected by effects on aquatic species. Therefore, creative solutions are required to these other electrophiles, their relative effects will need to be quantified. utilize sources of data that are available to identify where and when risk is the greatest. Sources of data are available for beginning to develop a WP018 Toxicity of Uranium Mining Contaminant Mixtures to less-detailed, but still useful, landscape scale risk assessment for mixtures. Tropical Freshwater Species These include data on potential use (e.g., pesticide labels) or release (e.g., M. Trenfield, Environmental Research Institute of the Supervising mapping of NPDES permits) sites. For example, the use of crop designa- Scientist; A.J. Harford, C.J. Pease, C.L. Humphrey, R.A. van Dam, tions to represent allowable use of specific pesticides can be a surrogate of Environmental Research Institute of the Supervising Scientist / actual use to establish when exposure has the potential to be the highest. Department of the Environment and Energy This landscape scale risk assessment for mixtures will establish priority watersheds for monitoring and further study that pose the greatest relative Magnesium (Mg), uranium (U), manganese (Mn) and ammonia risk of exposure. Further efforts to describe aquatic species exposure to (NH3) are key Contaminants Of Potential Concern (COPCs) from complex mixtures discharged in wastewater will be related to urban land the Ranger uranium mine, Northern Territory. Site-specific water uses and permit distributions. We will discuss considerations for migratory quality guideline values (GVs; 99% species protection), have been species, such as salmon, that traverse hundreds of miles of waterways bor- derived using local species and are currently in place for Mg (3 mg/L), dering multiple states. Additionally, effluent mixtures are a point source of Mn (75 µg/L), U (2.8 µg/L) and NH3 (interim GV of 0.7 mg/L TAN exposures to unregulated compounds such as pharmaceutical and personal adjusted for local conditions). A study commenced in April 2017 to care products with emerging data on measurable concentrations in aquatic gain understanding of interactions amongst COPCs and ensure that species. The goal is to develop a process to prioritize the relative risks and operational limits and rehabilitation standards are sufficiently protective identify important data needs necessary for more detailed mixture analy- where these contaminants occur as mixtures. Exposure of six tropical ses in the context of a landscape scale risk assessment. freshwater species to a mixture of the COPCs near their site-specific GV concentrations resulted in toxicity to five species. Based on growth or reproduction compared to a control, there was a 100% effect for the duckweed, Lemna aequinoctialis; a 15-30% effect range for green hydra, Hydra viridissima; green alga, Chlorella sp.; fry, Mogurnda mogurnda; cladoceran, Moinodaphnia macleayi and no effect for the gastropod, Amerianna cumingi. The unexpected sensitivity of the duckweed may have been due to a high Mg:Ca ratio, which is known to

16 | Risk Assessment of Chemical Mixtures POSTER ABSTRACTS Effects Assessment In addition to enhanced oxidation rates, the preliminary data indicate that within the urban transport timescale, malathion is transitioning from RP001 Integration of Chemical, Organismal, and Transcriptomic the coarse aerosol of the initial spray application (i.e., 5 to 30 µm) to fine Data Reveals Landscape-Specific Exposure Effects to Complex aerosol (i.e., < 2.5 µm) and that malaoxon is present predominantly in the Chemical Mixtures fine aerosol. The efficacy of permethrin, which is applied with a syner- D. Bertolatus, University of Colorado Denver / Integrative Biology; L.B. gist compounds and a carrier (mineral oil) may also be impacted by the Barber, USGS / National Research Program; C.J. Martyniuk, University presence of atmospheric oxidants. Data derived from filter-based samples of Florida / Physiological Sciences; A.M. Vajda, University of Colorado of particulate matter and real-time measurements of NOx, O3, and VOCs, Denver / Integrative Biology will be overlayed to develop adulticide plume models that will predict malathion oxidation, and will lead to better preventative measures of the Aquatic habitats are often contaminated with complex mixtures of pol- spread of mosquito-borne diseases. lutants. Interpreting biological effects caused by mixture exposures is an ongoing challenge in ecotoxicology and is expected to improve with RP003 Combination of Linear Process Yields Non-linear Toxic chemical and geographical data. Here, we employed integrated chemical Effect in Chemical Mixtures and biological analyses to determine how environmental mixtures affected M. Kamo, Advanced Industrial Science and Technology; H. Yokomizo, biological responses in watersheds with different landuse. A better Center for Health and Environmental Risk Research understanding of the relationships between landuse, contaminant occur- rence, and exposure effects will increase predictive power for regulators The effects of drug and chemical mixture has been studied long time. and managers. Adult male fathead minnows (Pimephales promelas) were Loewe’s concentration addition (CA) and Bliss’s independent action (IA) are exposed to water from different locations within the Shenandoah River the two majors. These models are linear models which predicts toxicities of watershed (VA, USA) in 2014 and 2015. The exposure locations were chemical mixture by a summation of concentration in CA and by a summa- chosen to capture unique landuse in surrounding watersheds, including tion of response in IA when concentrations of chemicals are low. Testing CA agricultural, municipal, mixed-use, and forested sites. Endpoints from is one of big challenges in empirical studies, and many toxicity tests showed multiple levels of biological organization were measured, including condi- significant deviations from predictions by CA. When such non-linear toxici- tion factor, gonadosomatic index (GSI), number of nuptial tubercles, and ties are observed, it tends to be thought that toxic mechanism should be hepatic gene expression profiles. Water samples were taken 4 times during complicated. However, the complexities are in a continuous spectrum and in the fish exposure and analyzed for over 460 chemical constituents. Each some cases the toxic mechanisms are very complicated and they are not in location had a unique chemical profile that was generally consistent with other cases. In the present study, we make mathematical models to argue the landuse. Whole-organism and molecular responses also differed between condition leading to non-linear effect and to argue how complicate it should the locations. In 2014, fish exposed at agricultural and WWTP impacted be to cause non-linear toxic effects in chemical mixture. We considered two sites showed signs of endocrine disruption including a reduced number types of models. One is an enzyme-substrate reaction model (ES-model) of nuptial tubercles and decreased GSI. However, transcript biomarkers in which there are at least two ES-reactions in a chain reaction. The other of estrogen exposure, including er1, er2, vtg1, and vtg3 showed little to no model is a biotic ligand model (BLM) which is now widely used to predict differential expression, suggesting that these fish were not affected in a toxicities of metals. In the ES models, we assumed product (P) produced significant way by estrogenic compounds in the mixture. Pathway analysis by ES reaction is essential substance to living organisms. Chemicals cause of transcriptomic data revealed that lipid related processes were affected toxicity by disturbing ES reaction and by reducing P. In BLM, processes are by exposure to agricultural, WWTP impacted and mixed-use watersheds. involved. One is a process for the metal speciation, and the other is that for Immune-related processes were down-regulated at the agricultural site metals binding to biotic ligand. These two processes are treated separately and up-regulated at the mixed-use site. Hierarchical clustering of total in most BLM studies. We coupled these processes in a group of differential transcriptome profiles showed individuals generally clustered by exposure equations, and investigated how non-linear effects appear. Mathematical location, demonstrating that exposure to water from sites with different analysis revealed that in the ES-model, a linear (or additive) toxicity is only landuse results in unique and site specific responses at the transcript level. possible when two chemicals disturb exactly the same ES reaction. When These data generate new hypotheses regarding the effects of exposure they disturb different part of ES reaction, then toxicities are non-linear. In to different types of complex mixtures and demonstrate the value of our BLM, mathematical analysis revealed that even if toxicities are linear as a complex mixture/landscape research approach. bioavailable (free ion) concentrations, but are non-linear as total concentra- tions. Our mathematical analysis shows that even if each process is simple RP002 Atmospheric Aging of Pesticides Mixtures Used to Control and linear, toxicity can be non-linear when such simple processes are Mosquito Populations coupled. The result implies that there is a possibility that non-linear effect S. Usenko, Baylor University / Department of Environmental Science; is not a product of complicated toxic mechanisms, and the world may be R.J. Sheesley, Baylor University; C.W. Matson, S. Guberman, Baylor simpler than we think. University / Environmental Science RP004 Gene Expression Profile in the Diatom T. pseudonana Urban areas are literal hot spots of mosquito-borne disease transmission Upon Exposure to the Chemical Mixtures at Environmental and air pollution during the summer months. Public health authorities Concentrations. What Did We Learn? release pesticides targeting adult mosquitoes (“adulticides”) directly in to D. Napierska, R. Carvalho, S. Tasselli, I. Sanseverino, European the atmosphere as aerosols to control mosquito populations and reduce the Commission Joint Research Centre; M. Potalivo, S. Balzamo, ISPRA threat of diseases, such as Zika. The pesticides permethrin and malathion Institute for Environmental Protection and Research; R. Loos, European are the primary weapons for controlling adult mosquito populations in Commission Joint Research Centre; T. Lettieri, European Commission Houston, TX and are typically sprayed at night. Houston, TX has a his- Joint Research Centre / Directorate D Sustainable Resources Water and tory of NO radical and O pollution, supported by NO emissions: these 3 3 x Marine Resources Unit two nighttime oxidants are known to oxidize both malathion and VOCs. Periods of high NOx and O3 atmospheric concentrations in Houston coin- Thousands different chemicals are discharged into the environment cides with the mosquito season, and therefore peak of adulticide usage. from agriculture, industry, medical facilities, house-holds. Currently, Preliminary atmospheric data has been collected for malathion and per- there is an increasing concern for the environmental impact of mixture methrin post-application in Houston. For malathion, diurnal atmospheric of compounds since the additive and eventual synergistic effects are samples (nighttime and daytime) indicate that it is oxidizing to malaoxon unknown and could produce serious adverse effects. To address this issue, at a much more rapid rate in the urban atmosphere of Houston than would a joint-effort of 16 European and associated research groups participated be predicted using oxidation rates based on historical agricultural studies. to an exercise to test a 14-substance synthetic reference mixture at safety

SETAC North America Focused Topic Meeting | 17 POSTER ABSTRACTS environmental concentration under the Water Framework Directive RP006 Transcriptomics Provides Mechanistic Indicators of Mixture (Environmental Quality Standard, EQS). The mixture, was tested on Toxicology for IMX-101 and IMX-104 Formulations in Fathead the own routine bioassays to investigate the chemical mixtures effects Minnows (Carvalho et al., 2014). The bioassays covered the entire ecosystem from K.A. Gust, G.R. Lotufo, US Army Engineer Research and Development bacteria to fish as well in vitro assays providing an unique scenario from Center / Environmental Laboratory; J.K. Stanley, Stanley Environmental ecological risk assessment perspective. The results showed that effects Consulting / Environmental Laboratory; M.S. Wilbanks, US Army were observed at very low concentration on algal-bacteria composition in Engineer Research and Development Center / Environmental Lab; P. a marine microcosm, immobilization in crustacean, fish embryo toxic- Chappell, SOL Engineering; N. Barker, Bennett Aerospace ity and frog embryo development. Transcriptomics was performed for The mixture effects and toxicology of the two predominant insensitive the marine diatom Thalassosiria pseudonana exposed either to single munitions (IM) formulations IMX-101 and IMX-104 were characterized compound or the mixture to investigate whether the single exposure and in acute (96h) larval fathead minnow (Pimephales promelas) exposures. multiple exposure would show different gene expression profile pattern. IMX-101 consists of 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one The results show that the mixture induces a pattern similar to the ones (NTO), and nitroguanidine (NQ) while IMX-104 is composed of DNAN, induced by the single herbicides Diuron and Isoproturon. Signatures NTO, and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). DNAN was induced by the Cadmium or Nickel partially overlapped with the mixture the most potent constituent in IMX-101 eliciting an LC50 of 36.1 mg/L, signature. The exposure to the other compounds did in general not induce whereas NTO and NQ did not elicit significant mortality in exposures up relevant signatures, although a weak overall consistent signature is pres- to 1,040 and 2,640 mg/L, respectively. IMX-101 elicited toxicity repre- ent for some of them. In conclusion the effects of the mixtures could be sentative of the component concentration of DNAN within the mixture explained mainly by the exposure to the two herbicides. where toxic-unit (TU) calculations indicated non-interactive toxicity RP005 Eco/Genotoxicity Assessment of Sediment Samples Under the among constituents. Transcriptomic responses for individual IMs indicated Influence of a Submarine Outfall in Brazil unique functional responses characteristic of: oxidative stress, impaired energy metabolism, tissue damage, and inflammatory responses in DNAN M. Artal, University of São Paulo / Toxicology and Toxicology Analysis; exposures; impaired steroid biosynthesis and developmental cell-signaling F.I. Vacchi, A. dos Santos, University of Campinas / LEAL Laboratory of in NQ exposures; and altered mitogen-activated protein kinase signaling in Ecotoxicology and Environmental Microbiology School of Technology; L. NTO exposures. Transcriptional responses to the IMX-101 mixture were Ferreira, University of Campinas / LEAL Laboratory of Ecotoxicology driven by the fractional equivalent of DNAN. Given that the functional and Environmental Microbiology; K. Pereira, São Paulo State University responses among the individual constituents of IMX-101 were unique, and UNESP; A. Luchessi, Campinas State University; T. Henry, Heriot- NTO and NQ did not interact with DNAN within the IMX-101 mixture Watt University / The School of Energy, Geoscience, Infrastructure and exposure, the overall toxicity and toxicogenomic responses indicated that Society; G. Umbuzeiro, School of Technology – UNICAMP / LEAL the mixture toxicity of affective exposures to the IMX-101 formulation Submarine outfall is an alternative manner to discharge of sewage in were “independent.” Alternatively, the toxic response to the IMX-104 expo- many countries and presents unique challenges for ecotoxicology risk sure indicated at least slight potential for toxicological synergism where the assessments. Prior to discharge, chlorination of sewage effluents may toxicity was 18% greater than expected compared to the ΣTU for DNAN, occur and can generate numerous chlorinated decomposition products RDX and NTO. Functional transcriptomic responses to DNAN were that are both persistent and of toxicological concern. In Santos city, São conserved in the IMX-104 exposures, however with less fidelity compared Paulo, Brazil, discharge of chlorinated urban effluent through a subma- to IMX-101. Based on previous transcriptomics responses to acute RDX rine outfall have led to accumulation of toxic substances in the sediments exposures in fathead minnow larvae, we hypothesize that the potentially and a risk of toxicity to aquatic organisms. The objective of this study synergistic responses within the IMX-104 mixture are related to interactive was to investigate toxicity of these sediments in the amphipod Parhyale effects of each DNAN and RDX on systemic energy metabolism. hawaiensis, an ecologically relevant species developed for ecotoxicol- ogy. Collection of sediment samples impacted by the Santos submarine RP007 Predicting Sediment Metal Toxicity to Hyalella azteca Using outfall occurred in October 2016 and February 2017. Tests investigated a Metals Mixtures Version of the Biotic Ligand Model acute toxicity of sediment elutriate, pore water, and organic extracts R.C. Santore, A.C. Ryan, K.E. Croteau, Windward Environmental, LLC (prepared with dichloromethane and methanol) obtained from natural The Biotic Ligand Model (BLM) is a computational framework used and dried sediments. The Salmonella/microsome test, with and without for predicting the effects of water chemistry on metal bioavailability. exogenous metabolic activation, were also performed to assess mutagen- In this presentation we describe the development of an extension of the icity in organic extracts. There were no differences in mortality between BLM framework that can be applied to mixtures of multiple metals (the P. hawaiensis neonates exposed to elutriate samples and the negative mBLM). The mBLM was used to predict toxicity in chronic expo- control, but exposure to organic extracts and pore water resulted in mor- sures with Hyalella azteca. Calibration of the mBLM to H. azteca was tality of 50 to 95% and 22 to 72%, respectively. Most (86%) of the dried accomplished by comparison with water-only toxicity tests. Equilibrium sediment samples were toxic with mortality ranging from 17 to 100%. partitioning theory predicts that the toxic effect that may result from The natural sediment samples caused 25 to 50% mortality in three of exposure to sediment pore-water is similar to effects resulting from expo- the four samples tested. For all samples tested, there was no detection of sure to the water column. the calibrated model was then used to predict mutagenicity based on strains YG1041, YG5185 and TA100. Investigations whether field sediments from several sites characterized by elevated of DNA damage (Comet assay) and dysregulation of gene expression metal concentrations would be toxic to H. azteca. Predicted toxicity was (q-PCR of target gene transcripts) after adult P. hawaiensis exposure to compared with measured toxicity in chronic tests with H. azteca from contaminated sediments are underway. Toxicity appears related to the several field sites and high predictability was observed. The mBLM organic compounds adsorbed within the sediment matrix. Fractionation analysis allows sediments to be ranked to identify which samples would and chemical analysis of the organic extracts are underway to determine be most likely to cause toxicity. The mBLM can also identify which classes of compounds responsible for toxicity. Acknowledgements: The metals in the mixture of metals are most likely to be responsible for any authors thank CNPq PVE (400362/2014-7), FAPESP (2014/08829-7, observed toxicity. The overall good performance of the mBLM suggests 15/24758-5), CAPES, Solutions Project and CETESB. that it is a powerful predictive tool for use in assessing risk from metal concentrations in field sediments and could be used as part of a general methodology in the development of sediment guidelines.

18 | Risk Assessment of Chemical Mixtures POSTER ABSTRACTS

RP008 Effects of the Discharge of Coalbed Methane Produced effluent). The chemicals evaluated in phase 1 include: estrone, metformin, Water on Macroinvertebrate Stream Communities in the Bosque del triclosan, triclocarban, chlorpyrifos, 4-nonylphenol, bisphenol-A, diel- Oso State Wildlife Area, Colorado drin, and atrazine. Phase 2 morphological studies are underway. K. Kringel, L. Martinez, Colorado State University-Pueblo / Aquatic RP010 Microbial Community Responses to Individual Metals and Ecology Biology Metal Mixtures In the arid West, water is a scarce and precious resource. At the same B. Wolff, W. Clements, Colorado State University / Fish, Wildlife and time, energy from fossil fuel extraction has played an important role in Conservation Biology; E. Hall, Colorado State University / Ecosystem the economy. Coalbed methane (CBM) production lies at the nexus of Science and Sustainability; T.S. Schmidt, USGS / Colorado Water Science these resources as it both releases copious amounts of natural gas and Center entrains large quantities of deep, briny water, bringing it to the surface. The quality of this “produced water” varies and options for its manage- There is currently very limited knowledge regarding the effects of met- ment include deep well injection, treatment, and surface discharge into als and metal mixtures on natural microbial communities. In this study, streams. Although laboratory studies have indicated that CBM produced we used controlled mescosm experiments to evaluate the responses of water can be toxic to some aquatic organisms, field studies assessing the benthic stream microbial communities to various metals mixtures (e.g., impact of discharge into stream ecosystems are few, and no investigations Cd, Cu, and Zn) along ecologically relevant concentration gradients. have been made in the Raton Basin of southern Colorado where surface Microbial communities were examined by sequencing the 16S rRNA gene discharge is the primary method of disposal. We examined the effects of using high-throughput sequencing following a 40-day exposure period. the discharge of CBM produced water on stream health in a 29,000 acre Using this approach, we could separate the effects of individual metals State Wildlife Area in the Purgatoire River watershed of Las Animas and their combinations on certain operational taxonomic units (OTUs), County, CO. Seven contaminated streams (below discharge points), and to entire microbial community assemblages. This information is along with four comparable reference streams (having no discharge) in an important step needed determine if metals and their mixtures have the study area, were sampled and analyzed for differences in macroin- predictable effects of microbial communities and further refine the useful- vertebrate community structure, metrics, and water quality parameters. ness of microbial communities as indicators of ecosystem health. Conductivity, alkalinity, dissolved oxygen, chloride, fluoride, sodium, barium, boron, as well as metrics such as the Colorado multi-metric index RP011 SynToxTool: Prediction of Synergistic Toxicity of Mixtures (MMI) differed between the two groups. Non-metric multidimensional by Using Chemical–Protein And Protein–Protein Networks scaling was used to visualize community differences between sites. Our J. Kim, KIST Europe / Environmental Safety Group; M. Fischer, KIST study results may inform future produced water management require- Europe; V. Helms, University of Saarland / Center for Bioinformatics ments, especially where new coalbed methane development is to occur. Mixture toxicity studies are continuously increasing in ecotoxicology since the mixture toxicity can be caused by joint effects of substances RP009 Evaluating Mixture Toxicity Using Two Aquatic Model even at their no-observed-effect concentrations. The joint effects can be in Organisms and a Multi-Tiered Approach general divided into additivity, synergism, antagonism, and potentiation. D. Pitts, Wayne State University / Pharmaceutical Sciences; N. Reddy, Conventionally, concentration (CA) and independent action (IA) models Wayne State University; M. Monshi, K. Alame, E. Crofts, Wayne have been frequently used to predict the additive toxicity. Integrated CA State University; S. McElmurry, Wayne State University / Civil & and IA models have been also developed to overcome the limitations of the Environmental Engineering; D. Kashian, Wayne State University / conventional models. However, there is still a lack of synergism models Biological Sciences; T. Baker, Wayne State University / Institute of to predict the synergistic toxicity of mixtures. The objectives of this study Environmental Health Sciences were to develop an approach to predict the synergistic toxicity of mixtures The progress is described on the development of a multi-tiered approach by using protein-chemical and protein-protein interaction (PPI) networks, to the evaluation of the complex mixtures of chemical contaminants that and to apply it to Vibrio fischeri, a luminescent bacteria. This study are often found in surface and ground water. Contaminants of emerg- highlights that the PPI network based approach showed a potential to be ing concern (CECs) that are not routinely monitored or regulated often used for predicting the synergistic toxicity. Further studies are required to include chemicals that are known or suspected endocrine disrupting evaluate this method with different organisms and target mixtures. chemicals (EDCs). The EDCs have the potential to have adverse effects on environmental health and human health at very low concentrations. RP012 The Synergistic Potential of Azole Fungicides in the Aquatic Given the diversity of CECs that may affect endocrine function at very Invertebrate Gammarus pulex low concentrations, there is an urgent need to develop more efficient A. Rösch, Eawag, Swiss Federal Institute of Aquatic Science and biologically-based methods of evaluation that can complement sophisti- Technology / Environmental Chemistry; C. Vignet, Eawag, Swiss Federal cated analytical chemistry. We have implemented a multi-tiered biological Institute of Aquatic Science and Technology / Environmental Toxicology; approach to evaluate the endocrine disrupting potential of water samples N. Cedergreen, University of Copenhagen / Department of Plant and using two aquatic organisms, Danio rerio (zebrafish) and Daphnia Environmental Sciences; J. Hollender, Eawag, Swiss Federal Institute of pulex (waterflea). The initial focus is on developing a model to predict Aquatic Science and Technology / Environmental Chemistry the estrogenicity or anti-androgenic quality of water by evaluating a Azole fungicides are known inhibitors of the important enzyme class series of selected individual compounds with the aim to create predictive cytochrome P450 monooxygenases (CYPs), thereby influencing the mathematical model. The first tier is behavioral where the concentration detoxification of co-occurring substances via biotransformation. This range for sub-lethal chemical effects on swimming behavior is identi- synergism in mixtures containing an azole has mostly been studied by fied. The second tier employs morphological analysis to identify effects effect measurements, while the underlying mechanism has been less well on development (e.g., change in sex-ratio, feminized male fish, etc.) that investigated. In this study, six azole fungicides (cyproconazole, epoxi- likely represent EDC-like responses. The third tier examines the effects conazole, ketoconazole, prochloraz, propiconazole and tebuconazole) of single chemicals on gene expression, and utilizes the information from were selected to investigate their synergistic potential and their CYP the concentration-dependent biological responses observed in the first two inhibition strength in the aquatic invertebrate Gammarus pulex. As co- approaches to determine specific concentrations of interest. A mathemati- occurring substrate, the strobilurin fungicide azoxystrobin was chosen. It cal model will be developed that combines findings in order to evaluate is biotransformed by various reactions and 18 biotransformation products the estrogenicity or anti-androgenic quality of water samples with (BTPs) were identified. By measuring internal concentrations of azoxys- known or unknown mixtures of chemical contaminants (e.g., wastewater trobin and its BTPs with high resolution tandem mass spectrometry in the presence and absence of azole fungicides and by toxicokinetic modelling, SETAC North America Focused Topic Meeting | 19 POSTER ABSTRACTS we showed that the observed synergism is indeed due to inhibited In the lower tiers of the Framework, without enough knowledge on all biotransformation of CYP-catalysed reactions. However, synergism was of the candidate chemicals for CAGs, so many chemicals were included only observed for prochloraz at environmentally realistic concentrations. into the respective CAGs. In the course of the tiered assessment, some Increased uptake rate constants, an increase in the total internal concen- of the chemicals were eliminated under semi-quantitative or qualitative tration of azoxystrobin and its BTPs, in vivo assays for measuring CYP judgment so as to conduct reasonable assessment. In the higher tiers activities, and G. pulex video-tracking suggested that the twofold increase quantitative detailed assessment was applicable to limited numbers of in bioaccumulation and thereby raised toxicity of azoxystrobin in the chemicals for both exposure and hazard, and thus reasonable “extrapola- presence of prochloraz is not only caused by inhibited biotransformation tion” to other chemicals should be considered. In addition to the case but also due to increased azoxystrobin uptake induced by hyperactivity. studies, we are identifying which tier in the Framework should be applied to any particular part of the existing regulatory environmental RA prac- Case Studies tices. Detailed situation of the case studies and potential application of the Framework into RA practices will be presented at the meeting. RP013 Comparison of Macroinvertebrate Communities at Organic- Polluted River Sites With Different Zinc Concentrations: Is RP015 MixToxDB Ver. 1.0: An Online Database on the Toxicity of Single-Chemical Regulation Worth It? Binary Chemical Mixtures to Environment-Living Organisms Y. Iwasaki, National Institute of Advanced Industrial Science and J. Kim, K. Kume, H. Jeong, KIST Europe / Environmental Safety Group Technology Japan / Research Institute of Science for Safety and Since the number of conceivable mixture combinations is extremely large, Sustainability; T. Kagaya, The University of Tokyo; H. Matsuda, conducting toxicity tests for all mixtures seems to be unfeasible or non- Yokohama National University sense. In company with toxicity testing, predictive models for assessing and More than half of river sites that had total zinc concentrations higher screening the toxicity of mixtures are also essentially needed to facilitate than the environmental water quality standard (EQS; 30 μg/L) were the development of reliable mixture risk assessment. Various mixture contaminated with organic matter (biological oxygen demand (BOD) of toxicity datasets can contribute not only to provide information of potential > 3 mg/L) in Japan. However, field surveys investigating the impacts of hazard of mixtures, but also to be employed in developing prediction mod- metals have been usually conducted in mountainous or upland streams, els for estimating mixture toxicity. However, there is a lack of systematic which are rarely contaminated with organic matter. It is thus uncertain databases on the ecotoxicity of mixtures. Therefore, in this study, an online whether those previous results can simply be applied to zinc-contaminated database on the toxicity of binary chemical mixtures to environment-living lowland rivers that have higher values of BOD. For example, because some organisms (MixToxDB Version 1.0, www.mixtox.de), was developed as an metal-sensitive macroinvertebrates such as heptageniid mayflies are well open access resource, which effectively provides available information on known to be intolerant to organic pollution, metals may have minimal mainly binary combinations of chemical substances, toxicity effects, target impact on species-poor communities in organic-polluted rivers. Here, organisms, toxicity end-points, model deviation ratios, and citations. based on results from (1) a regional-scale macroinvertebrate survey and (2) RP016 Retrospective Application of “Concentration Addition” to smaller-scale field surveys, we compared macroinvertebrate communities Ni Toxicity Thresholds Determined for Field Studies in Soils With at sites with different zinc concentrations in lowland rivers contaminated Elevated Co and Cu with organic matter. We observed significant reductions in many richness metrics in organic-contaminated sites compared to uncomtaminated sites B.A. Hale, University of Guelph / School of Environmental Sciences; S. in the regional-scale survey. Both the regional- and smaller-scale surveys Siciliano, University of Saskatchewan / Department of Soil Science showed that total zinc concentrations of approximately twice the Japanese Emissions to terrestrial ecosystems from metals mining, smelting and EQS did not lead to remarkable reductions in richness or population-level refining are typically mixtures, as many metals geologically co-occur in abundance of macroinvertebrates in rivers contaminated with organic ores. Thus, while toxicity observed in the field is usually attributed to the matter, as previously shown in upland rivers. Our findings suggest that metal to which organisms are most sensitive, or to the metal of highest reduction in zinc concentration is not a first choice for the recovery of concentration, the observed toxicity could be a combination of toxicities, macroinvertebrates in organic-contaminated rivers. depending on the concentrations of the co-occurring metals. To evaluate whether the combined toxicity is additive or not, there needs to be knowl- RP014 How Is the Tiered Framework of Mixtures Assessment edge of the expected toxic unit (TU) leading to a specified toxic effect, Applicable to Regulatory Environmental Risk Assessment? An for example, reduction in yield of 50%, or EC50, for each of the mixture Attempt Through Case Studies metals in single exposure. For risk assessment of field-contaminated sites, K. Yamazaki, Ministry of the Environment / Environmental Health this can be determined in reference soils amended with individual metals, The goal of our study on mixtures assessment is to develop the way to or the metals mixture, using standardized ecotoxicity threshold testing. introduce “assessment of combined exposure to multiple chemicals” into Using reference soils with similar properties as the contaminated site is environmental risk assessment practices for regulatory objectives, such as important, due to the effect of those properties on metals’ bioavailability. selecting high-priority chemicals, setting criteria/standards, or identifying Collecting sufficient reference soils to conduct the required studies can chemicals to be regulated. The WHO/IPCS Framework on risk assess- be limited by the effect of the contaminants on the properties of concern, ment (RA) of combined exposure to multiple chemicals would be useful and also by the availability of soils proximal to these sites. As an alterna- for regulatory approach, while it focuses on RA of human health, not of tive, the PNEC calculator tool which arose from the REACH activities in the environment. We have been conducting case studies on environmental the EU can be used to calculate site-specific, bioavailability adjusted TU RA to see how we should adapt the framework to our RA practices, from values for NOEC/EC10, for Co, Cu, Ni, Pb and Zn. These values are the screening-level to comprehensive ones. In our chemical-by-chemical median HC5 for the SSD of terrestrial organism endpoints, thus are con- environmental RA, target chemicals have been selected based on antici- servative. Dose response relationships between soil Ni and crop yield and pated or potential environmental risks. Screening-level RA of combined woodlot community structure/function (field studies), as well as for native exposure from multiple chemicals would be initiated similarly. Once grass and soil invertebrate responses (pot studies) to field collected soils, one or a few target chemicals have been selected, cumulative assessment were further characterized for the additional contributions of soil Co and group (CAG) of chemicals should be identified in a reasonably conserva- Cu to Ni toxicity. For all studies, soil Co and Cu were elevated along with tive way within limited information. How many chemicals with similar Ni, as the soils received emissions from Ni mining, smelting and refining. structure would be enough to be included into the CAG? Can we be Bioavailability-adjusted Cu toxicity as predicted by the PNEC calcula- confident enough to exclude any of the chemicals of similar structure at tor for these soils was able to explain some but not all of the discrepancy the problem formulation stage? We have conducted case studies on tiered between predicted and observed Ni toxicity, following the “concentration approach to environmental RA of acrylates and phthalates respectively. addition” approach.

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