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Application of an antibody-based biosensor for rapid assessment of PAH fate and toxicity at contaminated sediment sites SRP Progress in Research Biogeochemical Interactions Affecting Bioavailability for in situ Remediation May 13, 1-3 pm EDT


  1. Application of an antibody-based biosensor for rapid assessment of PAH fate and toxicity at contaminated sediment sites SRP Progress in Research Biogeochemical Interactions Affecting Bioavailability for in situ Remediation May 13, 1-3 pm EDT Michael Unger Professor Aquatic Animal Health VIMS munger@vims.edu 804-684-7187

  2. Application of an antibody-based biosensor for rapid assessment of PAH fate and toxicity at contaminated sediment sites • PAH and their importance as environmental contaminants • Sources & concerns • PAH biosensor, what is it and how do we make it? • Biosensor applications to PAH fate and transport • Elizabeth River, VA: Evaluating PAH transport • Oil spill detection: ExxonMobil and Ohmsett • Biosensor applications to PAH bioavailability/toxicity • Factors affecting bioavailability in sediments • Baltimore Harbor, MD: Toxicity of contaminated sediments • Current and future work • Kristen Prossner’s SRP Research-Bioaccumulation in oysters • Krisa Camargo SRP TAMU Research- Soil screening • Continued Technology Development-Sapidyne and VIMS • Fate and Toxicity Assessment

  3. PAH: Bioavailability is governed by partitioning Polycyclic Aromatic Hydrocarbons (PAH) Potentially toxic and carcinogenic Common target of Superfund cleanup (historical/legacy contaminants) Oysters are potential vector for human exposure Sources include: combustion products, creosote, oil Superfund driven by reducing Human Risk Limited water solubility “hydrophobic” very low concentrations in water Under “equilibrium” conditions High affinity for lipid material “Lipophilic” organic carbon rich sediments and biota (bivalves) are a “sink” or reservoir NIEHS-SRP Research Focus Can we predict how PAH fate will affect bioaccumulation from contaminated sediments?

  4. Available Analytical Methods for FTS Dura Dry Bulk Freeze Dryer 48 hours or until dry, aliquots removed for % solids, Organics can be Slow and Expensive grain size, and organic carbon 2 days How slow? Spike with surrogate standards PCB 30, PCB 65, PCB 204, 1,1’binaphthyl, BDE-77, perinaphthenone, d-10 Environmental samples are acenaphthene, d-12 chrysene, d-8 naphthalene, d-12 perylene, d-10 phenanthrene, and 1,4-dichlorobenzene extremely complex: 100,000’s of 1-2 days compounds Dionex ASE 300 extracted 100% methylene chloride at 100 ° C and 1500psi Multiple steps to separate, isolate 1 days and concentrate the target Copper Column to remove sulfur molecules- 1 day Instrument and time intensive HPLC-SEC Days- Weeks up to $1000/sample Waters HPLC with a Phenomenex Envirosep (data point) ABC GPC column in methylene chloride Evaluate QA/QC 1 day 1-2 days Varian Saturn Silica gel to remove polar compounds 1 day GCMS-SIMS Spike with Internal Standards 1-2 days pentachlorobenzene, p-terphenyl, decachlorodiphenyl ether(DCDE), & BDE-166

  5. Near real-time PAH analysis: VIMS Biosensor Our Approach Bio Sensor Monoclonal Antibodies Electronic detection of against Contaminants mAb Binding Boise, Idaho

  6. How to make new antibodies to PAH and other small targets? Hapten- Target surrogate with linking arm H 2 ↑ not immunogenic H protein H molecule H H 2 immunogenic →

  7. How to make antibodies to pollutants? Hapten Y Pollutant Y Y protein Pollutant Monitor sera for titer Y Immunize Y Y Pollutant Y Y Hybridoma-antibody producing cells Screening of Hybidomas an important step Several month process from immunization to mAb Provides an endless (Li et al 2016, Immunoassay and Immunochemistry) supply of antibodies in cell culture

  8. Goal: Quantification of mAb binding Inline Sensor (Biosensor) features: 1. Automated sample handling 2. Precise fluidics for analyzing small quantities accurately 3. Fluorescence emission/detection for heightened sensitivity Boise, Idaho

  9. samples reagents Sample with • Beads NO PAH antigen Y Y Sample with Y Y Y • AF647 Y PAH Y Y labeled mAb Fluorescent source • high signal Flow cell •

  10. samples reagents Sample with • Beads NO PAH antigen Y Sample with Y Y Y • AF647 Y Y PAH Y Y labeled mAb sample with NO PAH = high signal sample with high PAH = low signal Fluorescent source • low signal Flow cell •

  11. VIMS Antibody Biosensor: new technology for contaminant analysis allows quantification at low concentrations at new spatial and temporal scales PAH in Pore Water Biosensor vs. GC-MS Total PAH (GC-MS μg/L) 200 20 2 y = 0.56x R² = 0.99 0.2 0.02 0.0 0.2 2.0 20.0 200.0 Total PAH (Biosensor μg/L) 2G8 Affinity for a wide Good correlation to GC-MS range of PAH (3-5 ring) SMALL volume samples (1-5 ml) FAST analysis (8 m) near real-time LOW concentrations (0.1 ppb total PAH) Environmental Fate Studies: spatial and temporal resolution to identify sources and transport mechanisms Toxicity Evaluation: spatial and temporal resolution to understand what is driving bioavailability and toxicity PAH selective antibodies (Spier et al., 2009, Anal. Biochem., Spier et al., 2011, Environ. Chem. Tox.; Xin et al., 2016, J. Immunoassay and Immunochemistry, Xin et al. 2016, Sensing and Bio-sensing Research

  12. Study Site Money Point: Contaminated with PAH and DNAPL from Historical Creosote Facilities in the Southern Branch of the Elizabeth River, VA Elizabeth River Chesapeake Bay Atlantic Wood Industries Superfund Site Contact: Randy Sturgeon, EPA Money Point ERP Sediment Remediation Site Contact: Joe Rieger, ERP Methods are needed to better understand and •Sites contain a wide range of PAH contamination and various stages of ongoing predict PAH transport at sediment remediation sediment remediation •Surface sediments meet criteria for PAH remediation, biota with reduced effects sites to assure long-term success •Some areas contain DNAPL on surface p ost-remediation dred in ( g g & ca pp g) in

  13. Methods: Porewater sampling surface sediments • Real-time analysis can be used to map [PAH] in water/sediment porewater in the field • Dissolved phase (0.47 μm) porewater samples are collected and analyzed on board and up to 30 stations can be surveyed in 1 day • Small volume samples analyzed on board by biosensor PAH in Pore Water and larger volume samples can be brought back to the lab Total PAH (GC-MS μg/L) Biosensor vs. GC-MS 200 for GC-MS 20 2 • Good correlation between biosensor & GC-MS in complex environmental samples 0.2 0.02 0.0 0.2 2.0 20.0 200.0 Total PAH (Biosensor μg/L)

  14. Results: Money Point, Phase 2 Mapping water/porewater in a day Southern branch Money Point Phase 2 (MP) Site Survey 08-09-12 Id Conc(ug/L) Station 1 0.08 MP-5 Bot Surface water <1μg/L-3μg/L 2 0.12 MP-5 Surf Porewater 50μg/L – 450 μg/L 3 0.25 MP-4 Bot 4 0.2 MP-4 Surf Phase 2 remediation area 5 0.11 MP-1 Bot 6 0.19 MP-1 Surf 7 0.3 MP-7 Bot 8 0.13 MP-7 Surf Mapping of site 9 0.1 MP-2 Bot porewater and 10 0.15 MP-2 Surf surface water and 11 0.1 MP-8 Bot 12 0.07 MP-8 Surf bottom water in 13 0.07 MP-6 Bot one day 14 0.09 MP-6 Surf 15 3 MP-9 Bot 27 samples 16 0.1 MP-9 Surf 17 0.13 MP-3 Bot 18 0.08 MP-3 Surf 19 190 MP-3 PW 20 120 MP-9 PW 21 400 MP-6 PW 22 450 MP-7 PW 23 230 MP-8 PW 24 130 MP-2 PW 25 220 MP-1 PW 26 50 MP-5 PW 27 50 MP-4 PW

  15. PAH Transport within sediment : Methods In-situ porewater measurements Small volume (mls) sample 0.45 μm filtered Drive-point Piezometer Salinity by refractometer Total PAH by biosensor Sampling at various depths within the sediment

  16. Porewater PAH Concentration Profiles within the Sediment at Money Point PAH Concentration (μg/L) PAH Concentration (μg/L) 0 200 400 600 0 200 400 600 Porewater sampling stations-Money Point 0 0 Depth in sediment (cm) MPF-2 Depth in sediment (cm) MPF-6 50 50 MPF-1 MPF-6 100 MPF-5 100 150 150 MPF-4 200 PAH Concentration (μg/L) PAH Concentration (μg/L) MPF-3 0 200 400 600 0 200 400 600 0 0 MPF-3 MPF-4 Depth in sediment (cm) Depth in sediment (cm) 50 50 MPF-2 100 100 150 150 600 PAH Concentration (μg/L) PAH Concentration (ug/L) y = -22.2x + 458 0 200 400 600 500 R² = 0.47 0 400 MPF-5 20 Depth in sediment (cm) MPF-1 300 40 200 60 100 80 100 0 0 10 20 30 120 Salinity (ppt) 140 Saline surface water is mixing with more contaminated fresh pore water at depth in the sediment

  17. PAH Flux Transport to the water column: Seepage meter/Biosensor data Porewater sampling stations-Money Point Seepage Meters Direct hourly flow measurements MPF-6 PAH concentrations by biosensor MPF-5 Short-term concentration/flux measurement MPF-4 MPF-3 35 160 MPF-2 Porewater Flow 140 30 Ebb Flow Rate Flood Flow Rate PAH Flux 120 MPF-2 25 25 20 Water height (cm) 18 100 PAH Flux (μg/m 2 /hour) Flow Rate (mLs/min) 20 20 16 Salinity (ppt) 80 Dredged 14 15 15 12 and 60 10 capped 10 10 8 40 6 5 5 4 Salinity Seepage 20 Meter 2 MPF-1 0 0 0 0 '08/18/ 2017… '08/18/ 2017… '08/19/ 2017… '08/19/ 2017… '08/19/ 2017… '08/20/ 2017… '08/20/ 2017… '08/20/ 2017… '08/21/ 2017… 0 0.2 0.4 0.6 0.8 1 1.2 Avg Water Height (m) Date/Time Highest flux at remediated sites with CTD data logger provides evidence of coarse sediment cap and low total tidal driven advection PAH Data from the Biosensor is now helping to guide future remediation plans to limit flux to the water column. Revisit problem sites and engineered caps in new areas

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