Supporting Information for 

Biodegradation and Product Identification of [14C]Hexabromocyclododecane in 

Wastewater Sludge and Freshwater Aquatic Sediment 

John W. Davis et al. 

 1
Supporting Information 

Experimental Section 

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

isocratic separation with 75/25 acetonitrile/water and 0.1% formic acid. The liquid 

stream was vaporized with a nebulizer pressure of 45 psig, nitrogen drying gas delivered 

at 7 L/min and 250 °C, and the vaporizer held at 300 °C. The capillary voltage was held

at 5000 V and the fragmentor was operated at 75 V or 145 V. Product ion scans were

obtained in negative ion mode over a scan range of 75 to 750 m/z with the electron 

multiplier operated at 2000 V. 

Confirmation of degradation products by GC/MS. The 1 μL injections were made

in the splitless mode, with a purge time of 60 s. The injection port was held at 285 °C.

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

mm, 0.50 μm 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. The MSD transfer line was held at 280 °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. 

 2
SI Figure 1. HPLC/RAM Chromatograms for analysis of HBCD diastereomers and products in extracts of aqua

 (Aerobically Prepared). A) Day 0; B) Day 56 

γ - HBCD

β - HBCD

α - HBCD

β - HBCD

α - HBCD

Product II

Product I

-

P

5 10 15 20

Time [min]

20406080

100

Re

sponse[

m

V]

A 

-

P

5 10 15 

Time [min]

B 

20406080

100120140160

Response [mV]
SI Figure 2. Concentration of the HBCD in the viable (solid diamonds []) and biologically-inhibited controls (

concentration in digester sludge (A) and freshwater aquatic sediments: Anaerobically prepared (B) and Aerobilines represent the predicted first order decay curve based upon the experimental data. The r2 represents co

each model prediction. 

C

t

/C

0

0.00000.30000.60000.90001.2000

0 20 40 60 80 100 120

B

0.00000.30000.60000.90001.2000

0 20 40 60 

C

t

/C

0

Day

C

t

/C

0

Day

r2viable 0.915controls 0.583

r2viable 0.718controls 0.123

0.00000.30000.60000.90001.2000

0 10 20 30

r2 Aviable 0.806controls 0.869

Day

C

t

/C

0

0.00000.30000.60000.90001.2000

0 20 40 60 80 100 120

B

0.00000.30000.60000.90001.2000

0 20 40 60 

C

t

/C

0

Day

C

t

/C

0

Day

r2viable 0.915controls 0.583

r2viable 0.718controls 0.123

0.00000.30000.60000.90001.2000

0 10 20 30

r2 Aviable 0.806controls 0.869

Day

0.00000.30000.60000.90001.2000

0 20 40 60 80 100 120

B

0.00000.30000.60000.90001.2000

0 20 40 60 

C

t

/C

0

Day

C

t

/C

0

Day

r2viable 0.915controls 0.583

r2viable 0.718controls 0.123

0.00000.30000.60000.90001.2000

0 20 40 60 80 100 120

B

0.00000.30000.60000.90001.2000

0 20 40 60 

C

t

/C

0

Day

C

t

/C

0

Day

r2viable 0.915controls 0.583

0.00000.30000.60000.90001.2000

0 20 40 60 80 100 120

B

0.00000.30000.60000.90001.2000

0 20 40 60 

C

t

/C

0

Day

C

t

/C

0

Day

0.00000.30000.60000.90001.2000

0 20 40 60 80 100 120

B

0.00000.30000.60000.90001.2000

0 20 40 60 

0.00000.30000.60000.90001.2000

0 20 40 60 80 100 120

B

0.00000.30000.60000.90001.2000

0 20 40 60 

C

t

/C

0

Day

C

t

/C

0

Day

r2viable 0.915controls 0.583

r2viable 0.718controls 0.123

0.00000.30000.60000.90001.2000

0 10 20 30

r2 Aviable 0.806controls 0.869

Day

0.00000.30000.60000.90001.2000

0 10 20 30

r2 Aviable 0.806controls 0.869

Day
SI Figure 3. Typical HPLC/-APPI/MS mass spectrum of an authentic tetrabromocyclododecene standard 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

800000

850000

Average of 15.801 to 16.303 m in.: 072604B.D (-)

125 (Average of 15.801 to 16.303 m in. m inus Average of 16.909 to 17.204 m in.)160

79 

561

m/z

Abundance
SI Table 1. Mass balance determinations for viable reaction mixtures containing [14C]HBCD: Digester Sludge 

Percent of Initial Radioactivitya

Day 0 5 7 14 21 

Total HBCD 114.2 ± 3.4 55.7 ± 6.5 30.1 ± 25.2 29.5 ± 16.6 27.9 ± 11.8 11

Product I nd b 2.7 ± 0.2 1.3 ± 1.8 <1 nd 

Product II nd 39.7 ± 0.1 50.1 ± 13.4 44.4 ± 10.6 38.0 ± 4.0 33

Product III nd 7.0 ± 1.3 9.3 ± 3.0 10.7 ± 3.0 11.5 ± 1.7 13

Other nd <1 1.7 nd 1.7 

% Recovery: 114 106 93 86 79 

a 100% equates to the nominal percentage of radioactivity at dosing on day 0. Most values represent duplicate samples ± b nd – not detected; approximate detection limit (3 x signal/noise) = 0.6% of initial radioactivity cThe first value represent the radioactivity recovered from the digester sludge while the second value includes both 14C 

butyl-rubber stoppers used to seal microcosms. 
SI Table 2. Mass balance determinations for viable reaction mixtures containing [14C]HBCD: Freshwater Sediment 

Percent of Initial Radioactivitya

Day: 0 5 7 14 21 28 56 

Total HBCD 95.6 ± 2.2 91.5 ± 1.3 88.0 ± 1.2 78.8 ± 1.8 65.9 ± 10.9 66.1 ± 3.2 48.2 ± 

Product I <1 4.6 ± 0.5 5.8 ± 0.5 12.3 ± 1.9 18.8 ± 2.0 19.5 ± 1.0 11.7 ± 

Product II <1 1.4 ± 2.0 <1 4.0 ± 1.5 10.4 ± 3.3 10.3 ± 1.8 30.5 ± 

Product III <1 1.0 ± 1.4 ndb <1 1.5 ± 2.1 1.4 ± 0.1 5.0 ± 

Trace Products <1 nd 1.1 <1 <1 <1 1.0 

Recovery: 98 98 95 99 97 98 97 

a 100% equates to the nominal percentage of radioactivity at dosing on day 0. Most values represent duplicate samples ± bnd – not detected; approximate detection limit (3 x signal/noise) = 0.8% of initial radioactivity 
SI Table 3. Mass balance determinations for viable rReaction mixtures containing [14C]HBCD: Freshwater Sediment (A

Percent of Initial Radioactivitya 

Day: 0 5 12 21 28 56 

Total HBCD 95.4 ± 4.6 90.2 ± 1.5 86.6 ± 1.4 71.2 ± 16.3 71.1 ± 0.5 49.8 ± 0.2 50

Product I ndb 4.8 ± 1.0 8.0 ± 0.3 12.1 ± 2.5 13.9 ± 0.1 12.5 ± 1.8 4

Product II nd 1.8 ± 0.2 3.6 ± 1.2 9.0 ± 2.9 6.6 ± 9.4 31.8 ± 0.3 30

Product III <1 nd nd 1.2 ± 1.6 2.2 ± 0.9 4.3 ± 0.6 6

Trace Products nd <1 <1 2.3 6.3 nd 

Total Recovery 96 97 99 96 100 98 

a 100% equates to the nominal percentage of radioactivity at dosing on day 0. Most values represent duplicate samples ± bnd – not detected; approximate detection limit (3 x signal/noise) = 0.8% of initial radioactivity 
SI Table 4: Ratio of the relative degradation rates of the individual HBCD diastereomer (i.e. α, β, and γ) in diges

sludge and aerobically or anaerobically prepared freshwater aquatic sediments.

α / β

(kα / kβ)* 

α / γ

(kα / kγ) 

β / γ

(kβ / kγ) 

Total HBCD 

Viable/Abiotic

(kViable / kAbitoic

Digester Sludge 0.87 

(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 in parentheses are the first-order degradation rate constant (k) for individual diastereomer or total HBCD.