1. Analysis and Relative Distribution of OPFR Compounds in Wastewater Treatment Million B. Woudneh, Coreen Hamilton, Guanghui Wang, Richard Grace, John R. Cosgrove Sidney B.C., Canada, V8L 5X2

2.  OPFRs are commercial additives widely used to inhibit, suppress or delay the production of flames. Trialkyl phosphatesHalogenated phosphatesTriaryl phosphates Restrictions on polybrominated diphenyl ethers (PBDEs) has increased demand for OPFRs. Since they are not chemically bonded agents, OPFRs use as additives results in easy release to the environment. Importance of Organophosphate Flame Retardants (OPFRs)

3. Alkyl phosphates Short - hand Halogenated alkyl phosphates Short - hand Triethyl phosphate TEPTris(2-chloroethyl) phosphateTCEP Tripropyl phosphateTPrPTris(2-chloroisopropyl) phosphateTCPP Tributyl phosphateTBPTris(1,3-dichloro-2-propyl) phosphateTDCPP Tris(2-ethylhexyl) phosphateTEHPTris(2,3-dibromopropyl) phosphateTDBPP Aryl phosphatesOther phosphates Triphenyl phosphateTPPTris(2-butoxyethyl) phosphateTBEP Tricresyl phosphateTCrP Tetrakis(2-chlorethyl) dichloroisopentyldiphosphate V6 2-Ethylhexyl-diphenyl phosphateEHDPP Target compounds selected based on: CMP = Chemical management plan TSCA = EPA's Toxic Substances Control Act SFEI = San Francisco Estuary Institute CECBP = California Environmental Contaminant biomonitoring program list Leading researchers in the field Target Analytes of Interest to Regulators & Scientists

4. Matrices: Influent, Effluent, Soil, Sediment, Biosolid Solids + surrogates Extraction: Soxhlet, DCM:EtAOc Cleanup: -NH2 SPE, Silica Solvent exchange to MeOH Analysis by LC-MS/MS Aqueous + surrogates Extraction: L-L with DCM Cleanup: -NH2 SPE Surrogates 2 H 15 -TEP 2 H 12 -TCEP 2 H 21 -TPrP 2 H 18 -TCPP 2 H 15 -TDCPP 13 C 15 -TPP 2 H 27 -TBP Recovery 2 H 15 -TPP Initial and ongoing QCs: 5 replicates MS/MSD QC samples: 1 Lab blank 1 Spiked sample 1 Duplicate Isotope labeled surrogates: 7 Surrogates 1 Recovery Method Makes Use of Eight Isotope- Labeled Standards

5. min 6.0 10.014.018.022.0 327> 152 TPP min 431> 99 TDCPP min 583 >361 V6 min 329> 99 TCPP min 225> 99 TPrP min 285 > 223 TCEP min 183 > 99 TEP 8.59 9.69 13.14 13.31 14.24 16.08 16.67 Relative response min 17.021.025.029.0 435 >99 TEHP 27.07 min 363 > 251 EHDPP 22.01 min 369> 165 TCrP min 399 > 299 TBEP 18.85 min 267 > 99 TBP 17.98 min 716 >99 TDBPP 17.25 20.89 Relative response Column: - Waters C18MS Mobile phase: - 0.1% HCOONH4 + 0.1% HCOOH MeOH Two MRMs per compound All Analytes Separated by Mass and Time

6. Reporting limits: - 0.1 to 5 ng/L for a 0.5 L sample - 0.01 to 0.5 ng/g for a 5 g wet sample Quantitative measurement of OPFRs from aqueous samples, sediment and biosolids

7. P RIMARY E FFLUENT Surface Waters Primary Clarifier Aeration Basin Secondary Clarifier Disinfection Bio-GasDigestion DewateringLand Application P RIMARY S LUDGE W ASTE A CTIVATED S LUDGE T REATED B IOSOLIDS R AW I NFLUENT S ECONDARY E FFLUENT Sampling Throughout the Process = Sample points at the study site

8. 3292 3807 ND 1069 OPFRs survive secondary wastewater treatment Distribution of OPFRs

9. BDE influent and effluent data from Kim, et al., Water Research 47, no. 7: 2213–21. BDE influent and effluent data from Kim, et al., Water Research 47, no. 17: 6496–6505. BTBPE and DBDPE data from Grace et al. BFR 2011, Boston. Final effluent and biosolid from secondary wastewater treatment are significant sources of OPFRs. Relative to brominated diphenyl ethers, OPFRs have a much higher affinity for the aqueous phase. OPFR Concentrations Significantly Higher than Other FRs

10. AXYS OPFR Method Summary and Conclusions  A reliable method for quantitative measurement of OPFRs from aqueous, sediment and biosolid samples.  The method makes use of eight isotope-labeled surrogates.  OPFRs survive secondary wastewater treatment.  Final effluent and biosolids from secondary wastewater treatment are significant sources of OPFRs.  OPFRs have much more affinity for the aqueous phase relative to BDEs  OPFRs are present at significantly higher concentrations than BDEs and other FRs. For Follow-up: mwoudneh@axys.com

11. Acknowledgements  Dr. Shirley Anne Smyth and her research group at Environment Canada are thanked for their assistance in sample collection and water quality data analysis.  Dr. Heather M. Stapleton is thanked for providing ‘V6’ technical standard for the initial method development.

12. Supplemental Information

13. NH2 1 g SPE - Condition: - with 2 x col. vol of HXN and 2 x col. Vol of DCM - Load: 1 mL extract in Hexane - Wash: 5 mL 20:80 DCM:Hexane (DISCARD) - Elute: 12 mL of 1:9 EtOAc:DCM (COLLECT) Silica, 8 g, 5% deactivated - Pack column in hexane - Load: 1 mL DCM extract - Wash: 20 mL 15: 85 DCM:Hexane (DISCARD) - Elute : 100 mL of 1:2 DCM:ETOAc (COLLECT) Before cleanup After cleanup Clean extracts were achieved with NH2 and silica columns

14. * * Samples sizes: 0.25 L for influent 0.50 L for effluent and 2.5 g wet for biosolid 10 g for biosolid These samples sizes were demonstrated to have no matrix effects. No significant matrix effects Reagent

15. OPFR Standard Stability in Methanol

16. OPFR Extract Stability

17. MRM transitions Target Analyte Typical Retention Time (minutes) Parent Ion Mass 1 Daughter Ion Mass 1 Quantified Against TEP8.6818399.0 (127)d15-TEP TCEP9.79284.9222.9 (63.1)d12-TCEP TPrP13.32225.199.0 (140.9)d21-TPrP TCPP13.50329.1 (327.1)99.0 (99.0)d18-TCPP V614.38582.8360.8 (99.0)d15-TDCPP TDCPP16.28430.999.0 (208.9)d15-TDCPP TPP16.86327.1152.0 (77.1) 13 C 18 -TPP TDBPP17.44715.5 (698.6)99.0 (99.0)d15-TDCPP TBP18.17267.199.0 (155.0)d27-TBP TBEP19.09399.1299.0 (199.0)d27-TBP TCrP21.08369.1165.0 (91.0) 13 C 18 -TPP EHDPP22.20363.1250.9 (77.1) 13 C 18 -TPP TEHP27.21435.399.0 (211.0)d27-TBP Surrogate Standard d15-TEP 8.55198.0102 (134)d15-TPP d12-TCEP 9.70297.0231.9 (67.1)d15-TPP d21-TPrP 13.10246.2102 (150.1)d15-TPP d18-TCPP 13.41347.0 (345)102d15-TPP d15-TDCPP 16.18 446.0102 (216)d15-TPP 13 C 18 -TPP 16.86345.0164.1 (83.1)d15-TPP d27-TBP 17.93294.2102 (166.1)d15-TPP Recovery Standard d15-TPP16.67342.1160.1 (82.1)d15-TPP

18. LC Gradient Time (min)Flow mixture 1 LC Flow Rate Program Gradient Curve 0.0 15% solvent A 85% solvent B 0.2 mL/min1 3.0 50% solvent A 50% solvent B 0.2 mL/min6 10 70% solvent A 30% solvent B 0.2 mL/min6 18 85% solvent A 15% solvent B 0.2 mL/min6 21 95% solvent A 5% solvent B 0.2 mL/min4 30 95% solvent A 5% solvent B 0.25 mL/min6 31 15% solvent A 85% solvent B 0.2 mL/min1 35 15% solvent A 85% solvent B 0.2 mL/min1

19. Calculated Log KD vs Log Kow for OPFRs