Supporting Information for. Cats internal exposure to selected BFRs and organochlorines correlated to house dust and cat food

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1 Supporting Information for Cats internal exposure to selected BFRs and organochlorines correlated to house dust and cat food J. Norrgran Engdahl 1, A. Bignert 4, B. Jones 2, I. Athanassiadis 1, Å. Bergman 1,3, J.M. Weiss 1* 1 Department of Environmental Science and Analytical Chemistry, Stockholm University, SE Stockholm, Sweden 2 Department of Clinical Sciences, Swedish University of Agricultural Sciences, SE Uppsala, Sweden 3 Swedish Toxicology Sciences Research Center (Swetox), Forskargatan 20, SE Södertälje, Sweden 4 Swedish Museum of Natural History, Frescativägen 40, SE Stockholm, Sweden * Corresponding author: Jana.Weiss@aces.su.se 1

2 Table of Contents Materials and Methods... 3 Samples... 3 Table S Table S Analysis... 5 Clinical analysis... 5 Thyroid hormone levels... 5 Blood lipid determination... 5 Chemical analysis... 5 Cat serum... 5 House dust... 6 Cat food... 7 Instrumental analysis and quantification... 8 QA/QC and recovery... 9 Figure S Results Table S3A Table S3B Table S4A Table S4A (cont.) Table S4B Table S4B (cont.) Table S5A Table S5B Figure S Correlation between cat serum and external exposure Table S Figure S Figure S Figure S References

3 Materials and Methods Samples Table S1. Constellation of the household and description of the cats. Household Free-standing House 53% Detached house 18% Apartment 29% Number of rooms (excl. kitchen) 4.2 (2-6) Size of the living space (m 2 ) 116 (60-200) Number of family members 3.9 (2-6) Number of children 2.0 (1-4) Children age (range) 5.9 (1-13) Number of cats 1.6 (1-4) Pet cat(s) Domestic (shorthair) 57% Main coon 25% Burma 7% Persian 7% Bengal 4% Male/female 61%/36% Age (year) 7 (1-13) Weight (kg) 4.8 (3.5-14) Total T4 (nmol/l) 27 (9-43) TSH (ng/l) 80 (30-200) 3

4 Table S2. Brands of cat foods selected for chemical analysis of BFRs, other brominated compounds, and organochlorines. The cat food represents both dry (d) and wet food (w). Cat food Dry/wet Brand (n=28) (d/w) (Taste) F1 d HILLS SCIENCE PLAN (Feline Adult Optimal Care Chicken) F2 d ROYAL CANIN (Light Cat - Calorie Reduction) F3 w MJAU (Turkey) F4 w MJAU (Salmon) F5 w MJAU (Meat) F6 d ROYAL CANIN (Indoor Long hair) F7 d IAMS (Kitten& Junior) F8 w COOP (Shrimp, Salmon &Tuna, Cattle, Brain & Duck) F9 w WHISKAS (Ocean fish, Beef, Chicken) F10 d FOURFRIENDS (Sterilized) F11 d ROYAL CANIN (Obesity management) F12 d ROYAL CANIN (Main coon Adult) F13 d ROYAL CANIN Neutered young female ( Neutered Satiety Balance) F14 w FRISKIES (Tuna, Rabbit, Chicken, Turkey) F15 d FRISKIES (Salmon, Beef, Chicken) F16 d PUSSI (Meaty sensations) F17 w BILANX (Beef, Chicken, Lamb, Salmon) F18 d PURINA ProPlan Sterilized (Salmon, Turkey) F19 d BOZITA (Feline Outdoor and Active) F20 d MJAU (Fish, Meat, Chicken, Shrimp) F21 d MJAU (Fish, Meat, Chicken, Shrimp) F22 w WHISKAS (Ocean fish, Beef, Chicken) F23 w FRISKIES (Tuna, Rabbit, Chicken, Turkey) F24 d HILLS SCIENCE PLAN Adult (Lamb and Rabbit) + Kitten (Chicken and Tuna) F25 w PUSSI Trout & Salmon F26 w PUSSI Beef F27 w PUSSI Chicken F28 d HILLS SCIENCE PLAN Adult (Lamb and Rabbit) + Kitten (Chicken and Tuna) 4

5 Analysis Clinical analysis Thyroid hormone levels For the feline samples, the serum levels of total T 4 were determined using a solid-phase chemiluminiscent competitive immunoassay (Canine Total T 4 ) and the serum levels of total TSH were determined using a solid-phase enzyme labeled chemiluminiscent immunometric assay (Canine TSH). Both assays were performed on an Immulite 2000 (Siemens Healthcare Diagnostics Ltd., Llanberies, Gwynedd LL55 4EL, UK) according to the manufacture s instructions. Blood lipid determination The lipid determination in cat serum samples were calculated from enzymatically-based measurements of cholesterol (CHOL) and triglycerides (TG) concentrations in the blood. Total lipid was calculated using the Covaci equation: 1 TL=1.33*TG (mmol/l) *CHOL (mmol/l) in g/l Average molecular weight for TG and CHOL was estimated to be 807 and 571 (g/mol), respectively, assuming free and esterified cholesterol in a ratio of 1:2. TG and CHOL were measured at the laboratory of the university animal hospital at SLU. Blood serum cholesterol was determined by an enzymatic method 2 on an Architect c4000 (Abbott laboratories, Abbott Park, IL USA) using reagents from Abbott according to the manufacturer s instructions. Triglycerides were determined by an enzymatic method 3 using the same analyser and per the manufacturer s instructions. Lipid content of the dry and wet cat feed was determined gravimetrically after lipid extraction. 4 Chemical analysis Cat serum The chemical analysis of cat serum performed in this study was recently reported in detail in the supporting information for Norrgran et al. 5 The clean-up and extraction method used is described by Hovander et al. 6 with the modification that normal-hexane was exchanged to cyclo-hexane during sample preparation. In addition, the Hovander method was further validated for analysis of bromophenols, OH-PBDEs and Me-O- 5

6 PBDEs as described by Norrgran et al 5. In short, cat serum (1 ml) was weighed in a glass test tube and 1% potassium chloride (w/v) (4 ml) and surrogate standard (SS) (BDE-71, 13 C12-BDE- 209, CB-201, 2,3,4,6-tetrabromophenol, and 4 -OH-BDE121) was added. The serum samples were further denaturized with hydrochloric acid (6 M) and 2-propanol and extracted with cyclohexane (chx): methyl tert-butyl ether (MTBE) (1:1, v/v). Separation of neutral and phenolic compounds was achieved by partitioning with potassium hydroxide (0.5 M in 50% ethanol). The organic phase including the neutral compounds was transferred to a new test tube and the alkaline phase was acidified by adding hydrochloric acid (2 M) to re-protonate phenols. The phenolic compounds were then re-extracted with chx: MTBE (9:1, v/v). The phenolic compounds were further derivatized to their corresponding methylated analogues with diazomethane before lipid removal. Removal of blood lipids was conducted by adding concentrated sulfuric acid. As a last clean-up step, the samples were run through a silica gel column (0.1g active silica gel g sulfuric acid: active silica gel (1:2, w/w)). The phenolic compounds were eluted with dichloromethane (DCM) and the neutral compounds with chx:dcm (1:1, v/v). Last, the samples were concentrated; solvent was exchanged to n-hx and spiked with two volumetric standards (VS): CB-189 and BDE-139 for analysis of chlorinated and brominated compounds respectively. The final volume of sample before injection was 75 µl. All solvent and chemicals used were of the highest available quality. The internal (surrogate) and volumetric standards and the calibration standards for BFR analysis were purchased from Wellington Laboratories Inc. (Guelph, Ontario, 67 Canada) and organochlorines from AccuStandard, Inc. (New Haven, CT, USA), except CB-189, which was purchased from Larodan Fine Chemicals AB 61 (Malmö, Sweden). House dust Dust samples (50 mg) were weighed and surrogate standard (BDE-128) was added. Extraction and clean-up followed roughly the method described by Van den Eede.et al. 7 In short, extraction was carried out in iso-hexane:acetone (3:1, v/v) (2 ml). Samples were mixed in a vortex mixer for 1 min, then sonicated for 10 min, and finally centrifuged for 3 min. The supernatant was transferred to a new test tube and the procedure was repeated twice. The collected supernatants were evaporated to dryness under a nitrogen flow and re-dissolved with 300-µL iso-hexane. The extract was then quantitatively transferred to a prewashed (iso-hexane, 10 ml) Florisil SPE 6

7 cartridge (Thermo Scientific, HyperSep FL 500 mg/3 ml) and two fractions were collected. The first fraction, containing PBDEs, was eluted with iso-hexane (10 ml). The second fraction was eluted with ethyl acetate (10 ml) and stored for future analysis of organophosphate ester flame retardants (OPFRs). The PBDE fraction was evaporated to a volume of 250 µl and applied on dried silica i.e., a sulfuric acid (2:1, w/w) gel column (0.9 g). A fraction of iso-hexane (10 ml) was collected and the solvent was evaporated to near dryness and then reconstituted with n- hexane followed by addition of a volumetric standard (BDE-77). The extracts were transferred to a sample vial and the final volume was adjusted to 150 µl. Cat food The dry cat food pellet samples were weighed (15 g) and reconstituted in HPLC grade water (1:2, w/w) for min before homogenization. Each wet cat food sample was homogenized before a sub-sample (10 g) was taken out for extraction. Fat extraction was carried out in centrifuge tubes as described by Jensen et al. 4 The cat food samples were homogenized in a mixture of 2-propanol and iso-hexane/diethyl ether. The liquid phase was decanted into a separatory funnel after centrifugation (5 min at 900g) and the procedure was repeated. The combined extracts were washed twice by inverting the funnel with hydrochloric acid (0.2 M) in aqueous sodium chloride (0.9% w/v, ml). The aqueous phase was discarded, and the organic phase, decanted into a pre-weighed beaker (100 ml), was left overnight to evaporate in the fume hood. Any remaining solvent in the samples was evaporated with a gentle stream of nitrogen, and the flask was reweighed until constant weight. Extracted fat amounts ( mg) were transferred to a test tube, weighed, and fortified with SS. The following clean-up and separation of neutral and phenolic compounds continued as with the cat serum method and described by Hovander et al. 6 The phenolic compounds (i.e., OH-PBDE, PCP, 2,4,6-TBP) were analysed as their methylated analogues after methylation with diazomethane. 5 Work with diazomethane was approved by the Swedish work authority and in according with AFS 2005:17 (No IMS 2009/40104). In addition, all handling with diazomethane was carried out with great care and according to standard safety procedures. The naturally methylated PBDEs (MeO-PBDEs) associated to the neutral fraction were analysed separately from the derivatized halogenated phenols. 7

8 Instrumental analysis and quantification Analysis of organochlorines (except for PCP) was performed by a gas chromatography (GC) equipped with an electron capture detector (ECD) as described elsewhere. 5 The GC (Agilent Technologies 7890A) was fitted with a non-polar Varian CP-SIL 8CB capillary column (25m x 0.15mm, i.e., 0.12µm film thickness). Helium was used as a carrier gas at constant flow (1.25 ml/min). A multimode injector was set at 260 C and operated in splitless mode (1 min). Injection volume was 2 µl. The detector temperature was set at 325 C and nitrogen was used as make-up gas. The column oven temperature program used was 80 C (1 min), 40 C/min to 200 C/min (1 min), and 10 C/min to 310 C/min (8 min). Analysis of brominated compounds was performed by gas chromatography/mass spectrometry (GC/MS). Two instruments (Varian 450 GC connected to a Varian 320 MS and Varian GC PAL auto sampler, Bruker) were used in electron capture negative ionization (ECNI) mode with different analytical capillary columns. For the PBDE and PCP analysis in serum, house dust, and cat food, a Varian CP-SIL 5CB (10m x0.25mm DF=0.12µm) column was used. Oven temperature program for PBDE analysis was as follow: 60 C (2 min), 25 C/min to 200 C/min (0.4 min), and 10 C/min to 310 C/min (5 min). The carrier gas was helium at constant flow (1 ml/min). Injection volume was 1µL in splitless mode (2 min). Injector temperature was 260 C and ion source and transfer line temperatures were 250 C and 310 C, respectively. Methane (4.5 Torr) was used as reagent gas. For analyses of MeO-PBDEs and OH-PBDEs in serum and cat food, a J&W DB-5 HT (15m x 0.25mm DF=0.1 um) capillary column was used. Oven temperature program for phenolic compounds was 45 C (2 min), 5 C/min to 80 C/min (1 min), and 15 C/min to 320 C/min (5 min). The oven temperature program for MeO-PBDE compounds was 55 C (2 min), 15 C/min to 310 C/min (1 min), and 2 C/min to 320 C/min (5 min). The carrier gas was helium at constant flow (1.5 ml/min). Injection volume was 1µL in splitless mode (2 min). Injector temperature was 280 C and ion source and transfer line temperatures were 250 C and 310 C, respectively. Methane (4.5 Torr) was used as reagent gas. The analyses were performed using selected ion monitoring (SIM) scanning for bromine ions m/z: 79, 81 for all brominated compounds except for BDE-209 (m/z 484.5, 486.5) and 13C-BDE-209 (m/z 494.5, 496.5) and PCP (m/z 278, 280). In the indoor dust analysis, only the bromine ions m/z: 79, 81 were monitored for all brominated compounds. 8

9 The BFRs were identified by comparing retention times to authentic reference substances. The quantification was performed using surrogate standards, reference standards, and multi-point calibration curve. For the quantification of the dust samples, the volumetric standard was used as surrogate standard for calculation of sample amount against multi-point calibration curves. The amounts were then corrected against the recovery of the surrogate standard. QA/QC and recovery This study (n=5) Certified NIST values ng/g dust Figure S1. Average concentration (ng/g dust) and error bars (max and min) of PBDE congeners determined in NIST SRM dust 2585 sample measured in house (n=5) vs. NIST certified values. 9

10 Results Table S3A. Concentrations (pmol/g lipid weight) of brominated compounds analysed in cat serum. Cats Serum Lipid content BDE- BDE- BDE- BDE- BDE- BB- 2 -MeO- 6-MeO- 2,4,6-2 -OH- 6-OH- (n=19) (g) (mg) (%) BDE68 BDE47 TBP BDE68 BDE47 S1 p <LOQ <LOD <LOD <LOQ <LOQ 18 S4 p <LOQ <LOQ <LOD <LOD <LOD <LOQ <LOD ǂ S5 p <LOD <LOD 86 <LOQ ǂ S7 p <LOQ <LOQ <LOD <LOQ <LOD <LOD <LOQ <LOD ǂ S9 p <LOQ 9.1 <LOD <LOD <LOD <LOQ <LOQ 18 S <LOD <LOQ S12 p <LOQ <LOD ǂ S <LOQ <LOQ <LOD <LOQ <LOD <LOD <LOQ <LOD ǂ S <LOQ 5.4 <LOD <LOD <LOD 79 <LOD ǂ S16 p <LOQ 11 <LOD <LOD <LOD <LOQ <LOD 62 S <LOQ <LOD <LOD <LOQ <LOD ǂ S <LOD <LOD <LOQ S <LOQ <LOD <LOD S <LOQ <LOQ <LOQ <LOD <LOD <LOQ <LOD 39 S23 p <LOQ <LOQ <LOD <LOD <LOD <LOQ <LOD ǂ S <LOD <LOD 188 <LOD 58 S <LOQ <LOD <LOD S27 p <LOQ <LOD <LOD 120 <LOD ǂ S <LOQ <LOD <LOD <LOQ <LOD 18 detection >LOQ (%) LOQ+(pg) mean <LOD median <LOD <LOD 73 <LOD 58 min <LOD 2.3 <LOD <LOD <LOD 36 <LOD 18 max <LOD No IS in the sample ǂ Amount in sample >LOQ, but recovery <50%, sample deleted p Pooled samples + Highest LOQ for the analysed samples Table S3B. Concentrations (pmol/g lipid weight) of chlorinated compounds analysed in cat serum. 10

11 Cats Serum Lipid content CB-138 CB-153 4,4 -DDT 4,4 -DDE PCP (n=19) (g) (mg) (%) S1 p <LOD S4 p <LOD 20 <LOD S5 p S7 p <LOD <LOD <LOD <LOD 52 S9 p <LOD <LOD <LOD <LOQ 110 S S12 p S <LOQ 42 <LOQ S <LOD <LOQ S16 p <LOD <LOD <LOQ 25 <LOQ S <LOD <LOD <LOQ S S <LOQ S <LOD <LOD <LOQ 50 <LOQ S23 p S S <LOQ S27 p <LOD <LOD <LOQ S <LOQ detection >LOQ (%) LOQ+ (pg) mean median min <LOD <LOD <LOQ <LOD <LOQ max Highest LOQ for the analysed samples Pooled samples 11

12 Table S4A. Concentrations (pmol/g dry weight) of brominated compounds analysed in Swedish house dust. 1= Children s bedroom, 2 = Parents bedroom, and 3 = Living room House/apt. Dust BDE-28 BDE-47 BDE-66 BDE-99 BDE-100 BDE-153 BDE-154 BDE-183 BDE-194 BDE-196 BDE-197 (n=17) (mg) D <LOD D D D D D D D D <LOD D D D D D D <LOD D <LOD D <LOD D D <LOD D D D D D <LOD D <LOD D <LOD D D <LOD D D <LOD D <LOQ <LOD <LOD <LOD D <LOQ <LOD <LOD <LOD D <LOD D <LOD 1.0 <LOD D <LOQ 0.8 <LOD D <LOD D

13 Table S4A (cont.). Concentrations (pmol/g dry weight) of brominated compounds analysed in Swedish house dust. 1= Children s bedroom, 2 = Parents bedroom, and 3 = Living room House/apt. Dust BDE-28 BDE-47 BDE-66 BDE-99 BDE-100 BDE-153 BDE-154 BDE-183 BDE-194 BDE-196 BDE-197 (n=17) (mg) D D <LOD D D detection >LOQ (%) LOQ (Dx-1+2)(pg) LOQ (Dx-3)(pg) mean median min <LOD <LOD <LOD max

14 Table S4B. Concentrations (pmol/g dry weight) of brominated compounds analysed Swedish in house dust. 1= Children s bedroom, 2 = Parents bedroom, and 3 = Living room House/apt. Dust BDE-198 BDE-201 BDE-202 BDE-206 BDE-207 BDE-208 BDE-209 BB-209 HBB DBDPE (n=17) (mg) D <LOQ <LOD D <LOD D D <LOQ <LOD D <LOD D <LOD <LOD D <LOD D <LOD D <LOD D D <LOD D <LOD D D <LOD D D <LOD D <LOD <LOD D <LOD <LOD D <LOD D <LOD D <LOD D <LOD D <LOD D <LOD <LOD D <LOD D <LOD <LOD D D D D <LOD D <LOD <LOD <LOD D <LOD <LOD <LOD <LOD D <LOD <LOD <LOD D <LOD <LOD <LOD D <LOD <LOD D D <LOD

15 Table S4B (cont.). Concentrations (pmol/g dry weight) of brominated compounds analysed Swedish in house dust. 1= Children s bedroom, 2 = Parents bedroom, and 3 = Living room House/apt. Dust BDE-198 BDE-201 BDE-202 BDE-206 BDE-207 BDE-208 BDE-209 BB-209 HBB DBDPE (n=17) (mg) D <LOD <LOD D <LOD <LOD 93 D <LOD D <LOD detection >LOQ (%) LOQ (Dx-1+2)(pg) LOQ (Dx-3)(pg) mean median <LOD min <LOD <LOD <LOD <LOD <LOD 7.7 max

16 Table S5A. Concentrations (pmol/g lipid weight) of brominated compounds analysed in cat food. Cat food dry/wet Lipid BDE- BDE- BDE- BDE- BDE- BB- DBDPE 2 -MeO- 6-MeO- 2,4,6-2 -OH- 6-OH- (n=28) (d/w) (mg) BDE68 BDE47 TBP BDE68 BDE47 F1 d <LOQ <LOQ <LOD 0.02 F2 d <LOQ <LOD <LOQ <LOD <LOD F3 w <LOQ <LOD <LOQ <LOQ <LOD F4 w <LOQ <LOD <LOD <LOD <LOD <LOD 0.19 F5 w 235 <LOQ <LOQ <LOD <LOD <LOD <LOD <LOD 0.03 F6 d 268 <LOQ <LOQ <LOD <LOD <LOQ <LOD <LOQ <LOD <LOD F7 d <LOD <LOQ <LOD <LOD F8 w <LOD <LOQ <LOQ <LOD F9 w 260 <LOQ 0.12 <LOD <LOD <LOD <LOD F10 d 300 <LOQ 0.09 <LOD <LOD 1.7 <LOD 2.7 <LOD <LOD 1.2 <LOD <LOD F11 d 253 <LOQ 0.11 <LOD <LOD 0.04 F12 d <LOD <LOQ 2.3 <LOD <LOD 0.03 F13 d <LOD <LOD <LOD 4.3 <LOD 0.02 F14 w <LOD <LOQ <LOQ F15 d <LOQ <LOD <LOQ <LOD <LOD 1.8 <LOD <LOD F16 d 247 <LOQ 0.11 <LOD <LOQ 0.55 <LOD <LOD 3.3 <LOD <LOD F17 w <LOD <LOD <LOQ ǂ ǂ F18 d <LOD <LOQ <LOQ <LOD 0.03 F19 d <LOD <LOQ <LOQ <LOD 1.2 <LOD <LOD 0.02 F20 d <LOD <LOQ <LOQ <LOD <LOD 0.02 F21 d <LOD <LOQ <LOQ <LOD <LOD 0.02 F22 w 260 <LOQ 0.12 <LOD <LOD <LOD <LOD F23 w <LOD <LOQ <LOQ F24 d <LOD 0.05 <LOD <LOQ <LOQ <LOQ 0.02 F25 w <LOQ <LOD <LOD <LOD <LOD 0.21 F26 w 256 <LOQ <LOQ <LOD <LOD <LOQ <LOD ǂ ǂ F27 w <LOD <LOQ <LOD <LOD <LOD ǂ ǂ F28 d <LOD 0.05 <LOD <LOQ <LOQ <LOQ 0.02 detection >LOQ (%) LOQ (pg) mean median <LOD 0.04 <LOD <LOD 0.02 min <LOD <LOD <LOD <LOD 0.13 <LOD <LOD <LOD max ǂAmount in sample >LOQ, but recovery <50%, sample deleted 16

17 Table S5B. Concentrations (pmol/g lipid weight) of chlorinated compounds analysed in cat food. Cat food Dry/wet Lipid CB-138 CB-153 HCB 4,4 -DDT 4,4 -DDE PCP (n=28) (d/w) (mg) F1 d <LOQ <LOD F2 d 231 <LOD <LOD <LOD <LOD <LOD 1.5 F3 w <LOD <LOD F4 w <LOD F5 w <LOD F6 d <LOQ <LOD <LOQ F7 d <LOD <LOQ F8 w F9 w <LOD F10 d <LOD <LOD 6.2 <LOQ F11 d F12 d <LOD F13 d 254 ǂ ǂ ǂ ǂ ǂ 43 F14 w <LOD F15 d F16 d <LOD <LOD 9.9 <LOQ F17 w <LOD 8.5 <LOQ F18 d <LOD F19 d F20 d F21 d F22 w <LOD F23 w <LOD F24 d <LOD F25 w <LOD F26 w <LOD <LOD F27 w <LOD <LOD <LOD 5.3 <LOD F28 d <LOD detection >LOQ (%) LOQ (pg) mean median min <LOD <LOD <LOD <LOD <LOD <LOD max ǂAmount in sample >LOQ, but recovery <50%, sample excluded from the results 17

18 Figure S2. Principal component analysis (PCA) on the congener pattern (relative concentrations) of the four most abundant brominated congeners (BDE-47, -99, -209, and DBDPE) in dust collected from various rooms. The larger dots show the centre of gravity and the ellipses 95% confidence. 18

19 Correlation between cat serum and external exposure Table S6. Matched serum samples with dust and cat food samples. Cat serum Dust Cat food Cat food samples samples samples dry/wet (n=19) (n=17) (n=28) (d/w) S1 D1 F1 d S4 D2 F2 d S5 D3 F3 w S5 D3 F4 w S5 D3 F5 w S7 D4 F6 d S9 D5 F7 d S11 D6 * d/w S12 D7 F8 w S12 D7 F9 w S14 D8 F10 d S15 D9 F11 d S16 D9 F12 d S19 D10 F13 d S20 D11 F14 w S20 D11 F15 d S21 D12 F16 d S21 D12 F17 w S22 D13 F18 d S23 D14 F19 d S25 D15 F20 d S26 D16 F21 d S26 D16 F22 w S26 D16 F23 w S27 D17 F24 d S27 D17 F25 w S27 D17 F26 w S27 D17 F27 w S28 D17 F28 d *No specific brand. The cat was given assorted dry and wet food purchased from grocery stores and no correlation between serum and food for cat no. S11 was determined. 19

20 Figure S3. Concentrations of BDE-47 in cat serum and house dust (paired samples) from the living room, children s bedrooms, and parent s bedroom and all of the rooms combined. n = number of cats, r 2 = coefficient of determination, and τ = Kendall s tau. If p< 0.05 for both r 2 and τ, a significant correlation was considered. 20

21 Figure S4. Concentrations of BDE-153 in cat serum and house dust (paired samples) from the living room, children s bedroom, and parent s bedroom and all of the rooms combined. n = number of cats, r 2 = coefficient of determination, and τ = Kendall s tau. If p< 0.05 for both r 2 and τ, a significant correlation was considered. 21

22 Figure S5. Concentrations of BB-209 in cat serum and house dust (paired samples) from the living room, children s bedroom, and parent s bedroom and all of the rooms combined. n = number of cats, r 2 = Pearson s correlation coefficient, and τ = Kendall s tau. If p< 0.05 for both r 2 and τ, a significant correlation was considered. 22

23 References 1. Covaci, A.; Voorspoels, S.; Thomsen, C.; van Bavel, B.; Neels, H., Evaluation of total lipids using enzymatic methods for the normalization of persistent organic pollutant levels in serum. Sci. Total Environ 2006, 366 (1), Roeschlau, P.; Bernt, E.; Gruber, W., Enzymatic determination of total cholesterol in serum. Z Klin Chem Klin Biochem 1974, 12 (5), McGowan, M. W.; Artiss, J. D.; Strandbergh, D. R.; Zak, B., A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin. Chem. (Winston-Salem, N. C. ) 1983, 29 (3), Jensen, S.; Lindqvist, D.; Asplund, L., Lipid extraction and determination of halogenated phenols and alkylphenols as their pentafluorobenzoyl derivatives in marine organisms. J. Agric. Food Chem 2009, 57 (13), Norrgran, J.; Jones, B.; Bignert, A.; Athanassiadis, I.; Bergman, A., Higher PBDE Serum Concentrations May Be Associated with Feline Hyperthyroidism in Swedish Cats. Environ. Sci. Technol 2015, 49 (8), Hovander, L.; Athanasiadou, M.; Asplund, L.; Jensen, S.; Wehler, E. K., Extraction and cleanup methods for analysis of phenolic and neutral organohalogens in plasma. J. Anal. Toxicol 2000, 24 (8), Van den Eede, N.; Dirtu, A. C.; Ali, N.; Neels, H.; Covaci, A., Multi-residue method for the determination of brominated and organophosphate flame retardants in indoor dust. Talanta 2012, 89,