1. 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

2. 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)

3. 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

4. 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

5. 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

6. What chemicals can we measure by immunoassay?

7. 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,

8. 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

9. 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

10. 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

11. (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]

12. 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

13. 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

14. 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

15. 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)

16. 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]

17. 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)

18. ENVIRONMENTAL MONITORING
Mokelumne River Sampling Sites For Estrogenic Activity Screening

19. 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 South Fork, RRF Road
3. Middle Fork, Taylor Bridge 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.

20. 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

21. 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

22. 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

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

24. 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

25. 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

26. 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

27. 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

28. 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!

29. 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 ( )
[Australia, USA, Malaysia] – Drinking water, water reuse, water treatment 29

30. 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