# Supporting Information for

Biodegradation and Product Identification of [ 14 

# C]Hexabr omocyclododecane in

Wastewater Sludge and Freshw ater Aquatic Sediment

John W. Davis et al

.



# 1

# Supporting Information

Experimental Section

Product Identification : Separations for the LC/ M S/APPI was achieved using an

isocratic se paration with 75/25 acetonitrile/wa t er and 0.1% formic a c id. The liquid

stream was vaporized with a nebulizer pressure of 45 psig, n i trogen drying gas delive r ed

at 7 L/min a nd 250 °C, and the vap o rizer held a t 300 °C. T he capillary voltage was held

at 5000 V a nd the fragmentor was operated at 75 V or 14 5 V. Produc t ion scans were

obtained in negative ion mode o v er a scan range of 75 to 750 m/z wi th the electr on

multiplier op erated at 20 00 V.

Confirmation of degrad ation products by GC/MS. The 1 μL inject ions w e re made

in the split le ss mode, with a purge time of 60 s. The injectio n port was held at 285 °C.

Separations were achieved on a Phenomonex ZB5-MS capillary colu mn (30 m x 0.25

mm, 0.50 μ m film thickness) using a helium carrier gas pressure of 15 psi and an oven

temperature program of 70°C for 1 min, ramp to 350°C at 15°C/min with a final

temperature hold for 2.3 minutes. T he MSD tra n sfer line was held at 2 80 °C and the ion

source was held at 250 °C. Ions were produced via electron impact ionization at 70 eV.

Product ion scans were obtained over the range of 50 to 750 amu.



# 2SI Figure 1. HPLC/RAM Chromatograms for analysis of HBCD diastereomers and products inextracts of aquatic sediment(Aerobically Prepared).A) Day 0;B) Day 56

γ - HBCD

β - HBC D

α - HBCD β - HBC D γ - HBCD

α - HBCD Product II Product III

Product I-P5101520Time[min]20406080100Response[mV]A-P2015510Time[min]B20406080100120140160

R e spon se [m V ]SI Figure 2.Concentration of the HBCD intheviable (soliddiamonds []) and biologically-inhibited controls (stars [*])normalized to initialconcentrationin digestersludge (A) and freshwater aquatic sediments: Anaerobicallyprepared (B) and Aerobically prepared (C).The solidlines represent the predicted first order decaycurve basedupon the experimental data. The r2represents coefficientof determination foreach model prediction.4

C t /C 00.00000.30000.60000.90001.2000020406080100120B0.00000.30000.60000.90001.2000020406080100120C

C t /C 0Day C t /C 0Dayr2viable0.915controls0.583r2viable0.718controls0.1230.00000.30000.60000.90001.20000 102030Ar2viable0.806controls0.869Day

C t /C 00.00000.30000.60000.90001.2000020406080100120B0.00000.30000.60000.90001.2000020406080100120C

C t /C 0Day C t /C 0Dayr2viable0.915controls0.583r2viable0.718controls0.1230.00000.30000.60000.90001.20000 102030Ar2viable0.806controls0.869Day0.00000.30000.60000.90001.2000020406080100120B0.00000.30000.60000.90001.2000020406080100120C

C t /C 0Day C t /C 0Dayr2viable0.915controls0.583r2viable0.718controls0.1230.00000.30000.60000.90001.2000020406080100120B0.00000.30000.60000.90001.2000020406080100120C

C t /C 0Day C t /C 0Dayr2viable0.915controls0.5830.00000.30000.60000.90001.2000020406080100120B0.00000.30000.60000.90001.2000020406080100120C

C t /C 0Day C t /C 0Day0.00000.30000.60000.90001.2000020406080100120B0.00000.30000.60000.90001.2000020406080100120C0.00000.30000.60000.90001.2000020406080100120B0.00000.30000.60000.90001.2000020406080100120C

C t /C 0Day C t /C 0Dayr2viable0.915controls0.583r2viable0.718controls0.1230.00000.30000.60000.90001.20000 102030Ar2viable0.806controls0.869Day0.00000.30000.60000.90001.20000 102030Ar2viable0.806controls0.869DaySI Figure 3. Typical HPLC/-APPI/MS mass spectrum of an authentic tetrabromocyclododecenestandard56080100120140160180200220240260280300320340360380400420440460480500520540560580050000100000150000200000250000300000350000400000450000500000550000600000650000700000750000800000850000Average of15.801to16.303 min.:072604B.D(-)(Averageof15.801 to16.303min.minusAverageof16.909 to17.204 min.)12516079561m/z

Abu ndanc eSI Table 1. Mass balance determinations for viable reactionmixtures containing [14C]HBCD: Digester SludgePercent of Initial RadioactivityaDay05714212860Total HBCD114.2±3.455.7±6.530.1±25.229.5±16.627.9±11.811.2±2.3-Product Indb2.7±0.21.3±1.8<1nd<1-Product IInd39.7±0.150.1±13.444.4±10.638.0±4.033.4±1.0-Product IIInd7.0±1.39.3±3.010.7±3.011.5±1.713.2±1.0-Othernd<11.7nd1.75.3-%Recovery:1141069386796353.9/85ca100% equates to the nominal percentage of radioactivity atdosingon day 0. Most valuesrepresent duplicate samples±1 standarddeviationbnd – not detected; approximate detection limit (3x signal/noise) =0.6% of initial radioactivitycThe first value representthe radioactivity recoveredfrom thedigester sludgewhile thesecond value includes both14C activity from thesludgeand thebutyl-rubber stoppers usedto seal microcosms.6SI Table 2.Mass balance determinations for viable reaction mixtures containing [14C]HBCD: Freshwater Sediment (Anaerobically Prepared)Percent of Initial RadioactivityaDay:0571421285684113Total HBCD95.6±2.291.5±1.388.0±1.278.8±1.865.9±10.966.1±3.248.2±0.637.5±3.437.0±6.9Product I<14.6±0.55.8±0.512.3±1.918.8±2.019.5±1.011.7±3.67.2±4.65.5±2.2Product II<11.4±2.0<14.0±1.510.4±3.310.3±1.830.5±2.143.0±3.944.9±7.4Product III<11.0±1.4ndb<11.5±2.11.4±0.15.0±0.77.1±2.19.5±1.2TraceProducts<1nd1.1<1<1<11.01.6ndRecovery:989895999798979797a100% equates to the nominal percentage of radioactivity atdosingon day 0. Most valuesrepresent duplicate samples±1 standarddeviationbnd – not detected; approximate detection limit (3x signal/noise) =0.8% of initial radioactivity7SI Table 3. Mass balance determinations for viable rReaction mixtures containing [14C]HBCD: Freshwater Sediment (Aerobically Prepared)Percent of Initial RadioactivityaDay:051221285684112Total HBCD95.4±4.690.2±1.586.6±1.471.2±16.371.1±0.549.8±0.250.4±7.053.3±9.7Product Indb4.8±1.08.0±0.312.1±2.513.9±0.112.5±1.84.5±0.34.5±0.9Product IInd1.8±0.23.6±1.29.0±2.96.6±9.431.8±0.330.6±4.231.0±7.1Product III<1ndnd1.2±1.62.2±0.94.3±0.66.5±0.78.2±2.6Trace Productsnd<1<12.36.3nd<1<1Total Recovery96979996100989298a100% equates to the nominal percentage of radioactivity atdosingon day 0. Most valuesrepresent duplicate samples±1 standarddeviationbnd – not detected; approximate detection limit (3 xsignal/noise) =0.8% ofinitial radioactivity8SI Table 4:Ratio of the relative degradation rates of the individual HBCD diastereomer (i.e.α,β, andγ) in digestersludge andaerobicallyor anaerobically prepared freshwater aquatic sediments.α/β(kα/ kβ)*α/γ(kα/ kγ)β/γ(kβ/ kγ)Total HBCDViable/Abiotic(kViable/ kAbitoic)Digester Sludge0.87(0.1327/0.1522)1.06(0.1327/0.1248)1.22(0.1522/0.1248)2.7(0.1278/0.0470)Freshwater Sediment(Anaerobically Prepared)0.43(0.0060/0.0140)0.53(0.0060/0.0113)1.24(0.0140/0.0113)3.2(0.0109/ 0.0034)Freshwater Sediment(Aerobically Prepared)0.56(0.0054/0.0097)0.72(0.0054/0.0075)1.29(0.0097/0.0075)10.9(0.0074/0.0007)*Values inparentheses are the first-order degradation rateconstant (k) for individual diastereomer or total HBCD.9