Bioassay and bioanalytical approaches to chemical detection in water samples Michael Denison, Thomas Young, Candace Bever Superfund Research Center UC Davis CLU-IN Seminar || June 27, 2016

Screening and Monitoring Approaches for Environmental Chemicals of Concern (Known and Unknown) SAMPLE EXTRACTION AND CLEAN-UP Instrumental Immunoassay Bioassay Issues to consider: 1. Chemicals to be measured (known and unknown) 2. Measurement and screening (speed, cost, accuracy, precision) 3. Biological/toxic potency estimates (TEQs, EEQs, BEQs, etc.) 4. Mixture Interactive Effects (inhibition, additivity, synergism)

Screening and Monitoring Approaches for Environmental Chemicals of Concern (Known and Unknown) SAMPLE EXTRACTION AND CLEAN-UP Instrumental Immunoassay Bioassay Issues to consider: 5. Integration between methods – Instrumental methods confirm and validate bioanalytical methods and bioassay methods can provide biological/toxicological relevance to instrumental analysis results

Screening and Monitoring Approaches for Environmental Chemicals of Concern (Known and Unknown) SAMPLE EXTRACTION AND CLEAN-UP Instrumental Immunoassay Bioassay Immunoassay: Bioanalytical method that takes advantage of the ability of antibodies to specifically and selectively bind a target chemical or structurally related chemicals – provides a quantitative chemical measurement

Detection by immunoassay antibody with label sample Qualitative or quantitative Can be made to a variety of targets (eg., chemicals, bacteria) High throughput Field portable antigen coated platform signal is inversely proportional to chemical concentration

What chemicals can we measure by immunoassay?

TCS detection by immunoassay Used pAb assay in a plate-based method for analysis of water and biosolids. Recently, I have been funded to adapt this assay for use in detecting TCS in breast milk. I am currently working on extraction procedures that generate a matrix that does not cause interferences. TCS in breast milk has been determined by instrumental techniques, but it requires large sample volumes because the levels are quite low and breast milk is not often produced in excess. Breast milk is also a food source for infants and as antimicrobial it could be potentially interfering with establishing beneficial gut microbes. Ahn et al., 2016, Environmental Science & Technology, 50, 3754-3761

Estuarine monitoring by immunoassay Years of creosote pollution/industrial activity Dredge up and remove PAH contaminated sediments Concerned about release of PAHs during dredging Standard instrumental methods would take days Immunoassay could be performed on site field site Used the PAH biosensor in the field. Evaluated effectiveness of dredge curtains and how far up and down stream the contamination went. photo courtesy of the ERP 8 8

Estuarine monitoring by immunoassay Low PAH concentrations (below environmental concern), but showed gradient Results reported in real-time to managers on shore Results were used to determine where samples should be taken dredge PAH concentration = 4.0 μg/L = 2.0 μg/L = <0.3 μg/L Spier et al., 2011, Env. Tox. and Chem. 9

Screening and Monitoring Approaches for Environmental Chemicals of Concern (Known and Unknown) SAMPLE EXTRACTION AND CLEAN-UP Instrumental Immunoassay Bioassay Bioassay: Bioanalytical method that takes advantage of the ability of a chemical(s) to affect a specific biological target/pathway/mechanism (e.g. receptor, enzyme, etc.) – can provide quantitative or qualitative measurements of activity, but not individual chemical identification

(Firefly Gene Activity – Light) Mechanism of Dioxin Action – CALUX Bioassay Protein Dioxin (TCDD) NUCLEUS Dioxin:AhR Message PROTEIN SYNTHESIS NEW DIOXIN-LIKE ACTIVITY Dioxin-Responsive Cell Dioxin-Like Chemical (Firefly Gene Activity – Light) Firefly Luciferase Gene Ah Receptor (AhR) O Cl PCDDs PCBs PCDFs CALUX: Chemically-Activated LUciferase eXpression [USEPA Method 4435]

CALUX Cell Bioassay Procedure CALUX Cells Plated Into 96-Well Microplates Chemicals or Extracts Added to Each Well and Incubated for 24 Hours Wells are Washed, Cells Lysed, and Luciferase Activity Measured in a Microplate Luminometer

Flow Diagram for CALUX Analysis of Unknown Chemicals & Extracts Sample Extraction/Clean-up AhR/ER-CALUX Bioassay Analysis No Compounds that Activate the AhR/ER and/or Contains Compounds that Block Activation (i.e. Antagonists) Positive Negative • Estimate of Relative Activity (BEQs) (concentration-response analysis) • Other Confirmatory Assays (for ER) • Instrumental Analysis to ID Chemicals False True BEQs – Bioanalytical equivalents

Bioassay-BEQ (ppt) GC/HRMS TEQs (ppt) Double-Blind CALUX Analysis of Biological and Environmental Matrices CALUX BEQ Activity in Environmental Samples is Typically Greater than TEQs Calculated from Instrumental Analysis (Additional AhR Active Chemicals) XDS - Hiyoshi Corporation

The AhR-CALUX Bioassay for Water Quality Monitoring: Stream Samples From Southern California (2015) Negative correlation between AhR CALUX bioassay results and California Stream Condition Index (CSCI) AhR active chemicals remain to be determined – GC:HRMS identified various flame retardants Collaborative Study with Southern California Coastal Water Research Project (SCCWRP)

Bioassay for Estrogenic/Antiestrogenic Chemicals Protein ESTROGEN NUCLEUS Estrogen Receptor Message PROTEIN SYNTHESIS NEW ESTROGENIC ACTIVITY Estrogen-Responsive Cell Estrogen Active Chemical o,p-DDT BPA TBBPA (Firefly Gene Activity – Light) Firefly Luciferase Gene OECD [Methods TG455/TG457] – USEPA [EDSP]

Chemical library: 176 chemicals ((2) 96-well plates, 10 mM test conc.) ER-CALUX High-Throughput Screening For Rapid Identification of Estrogenic Chemicals Plate 1 Plate 2 VM7Luc4E2 (ERa) VM7LucERbc9 (ERa/ERb) Chemical library: 176 chemicals ((2) 96-well plates, 10 mM test conc.) (Pesticides, Herbicides, Fungicides, Industrial Chemicals, Drugs, Detergents, etc)

ENVIRONMENTAL MONITORING Mokelumne River Sampling Sites For Estrogenic Activity Screening

Measurement of Estrogenic Activity of Water and Sediment Samples from Upper Mokelumne and Calaveras Rivers Samples: Extracts of 1 liter of water or 10 g of sediment 1. Bridge, Sheep Ranch 2. South Fork, RRF Road 3. Middle Fork, Taylor Bridge 4. North Fork, Hwy 26 Bridge * Significant levels of estrogenic activity in all Mokelumne River samples, but the sediment has significantly less activity. The responsible chemical(s) remain to be identified. Effects–directed analysis (EDA) - Combination of bioassays and chemical fractionation methods provides an avenue in which to identify the responsible bioactive chemical(s) in a complex mixture.

Screening and Monitoring Approaches for Environmental Chemicals of Concern (Known and Unknown) SAMPLE EXTRACTION AND CLEAN-UP Instrumental Immunoassay Bioassay Instrumental Analysis: Gas or liquid chromatography with high resolution mass spectrometry to identify target and non-target chemicals in a sample – quantify concentrations for target (and selected non-target) chemicals

Identifying Causes of Aquatic Toxicity Hyallela azteca Chemcatcher® Passive Sampler rain event Jan 2016 rain event March 2016 WWTP Vacaville UB C1 grab samples for pesticide analysis every day C2 Li C3 C4

Extraction, Concentration and Analysis Methods polar chemicals 27 targets LC-QTOF 21 targets GC-QTOF non-polar chemicals Filtration: separate analysis water and filter Filtration: only water analysis SPE: multilayer cartridge (Oasis, anion & cation exchanger) adapted from EAWAG, Switzerland Water: SPE Oasis Filter: sonication extraction adapted from USGS, CA Analysis: Agilent LC-QTOF-MS/MS ESI pos, ESI neg Analysis: Agilent GC-QTOF-MS NCI mode, EI mode Selected targets based on use data and structural diversity www.agilent.com

Suspect Screening Example: LC-TOF  Screen against exact mass library of 1600 pesticides and transformation products - Peak found for mass 330.1100 - Isotope pattern match C19H14F3NO (score 98)  1 database match: Fluridone

Suspect Confirmation Example: LC-QTOF  Library spectra  4 fragments confirmed Similar approach for GC-EI measurement. However, no molecular mass present. Used library containing retention times, we adapted our method to get the same RTs. Detected in all samples of March event, no samples in Jan event  Confirmed by reference standard

Pesticide Screening Results Overview Analytical Method Targets Detected Suspects Detected LC-QTOF-MS 21 57* GC-QTOF-MS 16 43 Total 37 90** 15-25 targets in every sample * 21 reference standards for LC suspects available, 18 confirmed ** 10 in both GC-MS and LC-MS, 25 not confirmed with MS/MS GC-QTOF-MS Targets 7 Pyrethroids, e.g. Cyhalothrin, Bifenthrin, Cypermethrin, Chlorpyrifos Fipronil and degradates LC-QTOF-MS Targets Insecticides: e.g. Methoxyfenozide, Imidacloprid, Dimethoate Fungicides: e.g. Azoxystrobin, Boscalid, Cyprodinil Herbicides: e.g. Diuron, 2,4-D, Hexazinone Biocides: e.g. Triclosan, DEET Propiconazole for example in LC and in GC. (used in almonds  700 pounds) Norflurazone, Triclopyr + Quanclorac rights of way Dacthal: vegetables Bromacil (herbi), low application rights of way BAM: TP from dichlobenil ( landscape, rights of way) Diazinone: apples, pear GC-QTOF-MS Suspects Dacthal, 2,6-Dichlorobenzamide (BAM), Bromacil, Oxadizone, Propiconazole, Kinoprene, Diazinone LC-QTOF-MS Suspects Propiconazole, Norflurazone, Triclopyr, Fluridone, Quinclorac, Diethofencarb

Finding Similar Features showing similarity to 2,4-D abundance > 0.75 indicator of diffuse source Normalized intensity 235 features showing similarity to sucralose abundance > 0.75 indicator of point source Sample Agilent MPP software

Acute Toxicity and Pyrethroid Concentration January Event March Event Cyfluthrin + Bifenthrin Bifenthrin, Cyhalothrin, Cypermethrin, Deltamethrin Of course, we finally want to know what happened with our Hyallela that were swimming in this cocktail of pesticides during the rain event. Pyrethroids most sensitive for Hyallela, so although no pyrethroids showed up in the Top12 deteted compounds, they might have a large influence on the toxicity. Plotted sum of pyrethroid concentration, as mode of action is the same for all pyrethroids. LC50 (96h) for Cyfluthrin is 3 ng/L, so we are in the range of concentrations which are critical for Hyallela. March event concentrations not as high as in Jan event, but over longer period of time. This could have been the cause of showing effects down to C3 location. Permethrin Cypermethrin Permethrin

Conclusion and Outlook Broad scope suspect/non-target screening finds many more compounds than those on a typical target list Significance of non-target analytes being confirmed by toxicity correlations and genomic profiling Statistical analysis can group molecular features to provide information regarding contaminant sources, similar fate processes With over 100 detected pesticides from varied classes, mixture toxicity likely important—bioassays can help!

CALUX Technology Transfer CALUX cell lines and reporter plasmids have been provided to more than 200 laboratories in 28 countries for research, screening and monitoring purposes • Regulatory Acceptance: AhR-CALUX - US EPA (Method 4435) for DLCs in environmental matrices (2007) ER-CALUX - Organization of Economic Cooperation and Development (2012) (OECD Test Method 455/457) - estrogenic/antiestrogenic chemicals - USEPA (endocrine disruptor Screening Program) • Commercialization: Xenobiotic Detection Systems (Durham, NC) – developing and using bioassays International commercial/governmental licensing agreements (US, Germany, Japan, Belgium, Poland, China, Chile) – monitoring applications • International Workshops on Bioassays for Monitoring Water (2015-2016) [Australia, USA, Malaysia] – Drinking water, water reuse, water treatment 29

Acknowledgments FUNDING: Bruce Hammock Shirley Gee Isaac Pessah Guochun He Jane Rogers Jennifer Brennan COMMUNITY PARTNERS • Elizabeth River Project • Southern California Coastal Water Research Project (SCCWRP) • Upper Mokelumne River Watershed Council and Central Sierra Resource Conservation & Development 30