Hydroxylated polychlorinated biphenyls (OH-PCBs) in the blood of mammals and birds from Japan: Lower chlorinated OH-PCBs and profiles

Chemosphere 74 (2009) 950–961

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Hydroxylated polychlorinated biphenyls (OH-PCBs) in the blood of mammals and birds from Japan: Lower chlorinated OH-PCBs and profiles Tatsuya Kunisue 1, Shinsuke Tanabe * Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan

a r t i c l e

i n f o

Article history: Received 2 July 2008 Received in revised form 8 September 2008 Accepted 5 October 2008 Available online 2 December 2008 Keywords: OH-PCBs Whole blood Mammal Bird Japan

a b s t r a c t An analytical method was developed to measure tri- to octa-chlorinated OH-PCBs and pentachlorophenol (PCP) in the whole blood. Further, baseline data on the levels and profiles of these phenolic compounds in Japanese mammals (human, cat, dog, raccoon dog, and northern fur seal) and birds (black-tailed gull, common cormorant, and jungle crow) were obtained. Eighteen identifiable and fifty unknown peaks of OH-PCBs were detected and the major congeners identified were 40 OH-CB101/120, 4OH-CB107/40 OHCB108, 4OH-CB146, 4OH-CB178, 4OH-CB187, 40 OH-CB172, 4OH-CB202, and 40 OH-CB199. Relatively higher concentrations of OH-PCBs were found in animal species than humans; OH-PCB levels in dog, raccoon dog, black-tailed gull, and common cormorant blood were one order of magnitude higher than in humans. Penta- to hepta-chlorinated OH-PCB congeners were predominant in human blood, but profiles of OH-PCBs in other animals widely varied by species. Elevated composition of tri- and tetra-chlorinated OH-PCBs in cat blood and octa-chlorinated OH-PCBs in dog and raccoon dog blood were observed. In cat blood, elevated PCP concentration was also found. When concentration ratios of OH-PCBs to PCBs were calculated in all the animal blood, the ratios in dog, raccoon dog, and cat were notably higher than in other species. These results indicate that animals other than humans, especially cat and canine species such as dog and raccoon dog, might be at risk from OH-PCBs. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Hydroxylated polychlorinated biphenyls (OH-PCBs) are formed by oxidative metabolism of PCBs by cytochrome P450 monooxygenases and can disturb thyroid hormone homeostasis (Morse et al., 1995, 1996; Darnerud et al., 1996; Sinjari and Darnerud, 1998; Meerts et al., 2002). The competition of OH-PCBs and thyroxine (T4) to transthyretin (TTR), a carrier protein of T4, in the blood is the mechanism involved in the disturbance of TH homeostasis. In fact, some competitive binding assays demonstrated that the binding of para-substituted high chlorinated OH-PCB congeners with chlorine atoms on each of adjacent meta-positions to TTR was distinctly high and the binding affinity of several OH-PCB isomers was stronger than that of T4 (Lans et al., 1993; Cheek et al., 1999; Meerts et al., 2002). Such para-substituted OH-PCBs have high retention in the blood and a few OH-PCB isomers have longer half-life than parent compounds (Sinjari et al., 1998; Öberg et al., 2002). Studies of OH-PCBs in human blood have shown the prevalence of these metabolites (Sandau et al., 2000; Sjödin et al., 2000;

* Corresponding author. Tel./fax: +81 89 927 8171. E-mail address: [email protected] (S. Tanabe). 1 Present address: Wadsworth Center, New York State Department of Health, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, USA. 0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.10.038

Fängström et al., 2002; Hovander et al., 2002; Sandanger et al., 2004). OH-PCBs have also been detected in umbilical cord blood, suggesting transfer of these metabolites across the placenta to the fetus (Sandau et al., 2002; Soechitram et al., 2004). Sandau et al. (2002) found a significant negative correlation between concentrations of free T4 and the sum of OH-PCBs and pentachlorophenol (PCP) in umbilical cord blood and suggested that these chlorinated phenolic compounds are possibly altering TH status in newborns. It has been shown from a competitive binding assay that PCP also bind to human TTR and the binding affinity was about twice that of T4 (Van den Berg, 1990). Detailed information regarding OH-PCBs in animals except humans is still scarce, although OH-PCBs have been detected in the blood of several wildlife species such as marine mammals and birds (Klasson-Wehler et al., 1998; Olsson et al., 2000; Hoekstra et al., 2003; Sandala et al., 2004; Houde et al., 2006; Verreault et al., 2008). Nevertheless, only a small number of OH-PCB congeners were investigated earlier or no data of individual congener is available (only total OH-PCB concentration was shown) for these wildlife studies. In addition, penta- (OH-P5CBs), hexa- (OHH6CBs), and hepta- (OH-H7CBs) chlorinated OH-PCBs were quantified in most studies including humans. Recently, species-specific accumulation of octa-chlorinated OH-PCBs (OH-O8CBs) in polar bear (Ursus maritimus) plasma (Verreault et al., 2008) and relatively high accumulation of lower chlorinated OH-PCBs such as

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Table 1 Concentrations (pg/g wet wt.) of PCP, OH-PCBs, and PCBs and concentration ratios of OH-PCBs to PCBs (OH-PCBs/PCBs) in the blood of Japanese mammalian and avian species. Mammalian species

Avian species

Species Sex Sampling site Collected year

Human Male Ehime 1998

Cat M(1) F(4)a Kochi 2001

Dog Male Osaka 2001

Raccoon dog Male Osaka 2004

Northern fur seal Male Off Sanriku 1998

Black-tailed gull Male (2)a Hokkaido 2000

Common cormorant Female Shiga 2007

Jungle crow Male Osaka 2004

PCP OH-PCBs Unknown OH-T3CBb 40 OH-CB79 Unknown OH-T4CBc Total OH-P4CB 40 OH-CB101/120 30 OH-CB118 4OH-CB107/40 OH-CB108 Unknown OH-P5CBd Total OH-P5CB 4OH-CB146 30 OH-CB138 40 OH-CB130 40 OH-CB159 Unknown OH-H6CBe Total OH-H6CB 4OH-CB178 30 OH-CB182/183 4OH-CB187 4OH-CB177 30 OH-CB180 40 OH-CB172 Unknown OH-H7CBf Total OH-H7CB 4OH-CB202 4OH-CB201 40 OH-CB198/30 OH-CB203 40 OH-CB199 Unknown OH-O8CBg Total OH-O8CB R OH-PCBs R PCBsh OH-PCBs/PCBs

740

2200

150

1100

110

36

730

130

<0.5 3.7 7.3 11 10 2.1 35 23 69 26 5.4 <0.5 <0.5 29 61 2.2 1.3 59 0.99 1.2 7.0 2.7 74 2.4 <0.5 <0.5 5.8 <0.5 8.2 220 590 0.37

140 <0.5 320 320 120 10 100 50 280 6.9 <0.5 <0.5 <0.5 72 79 <0.5 <0.5 8.8 <0.5 <0.5 1.6 1.5 12 1.2 <0.5 <0.5 1.9 <0.5 3.1 840 160 5.3

<0.5 <0.5 <0.5 <0.5 2.4 <0.5 15 11 28 89 <0.5 <0.5 25 280 390 85 <0.5 310 4.9 8.9 140 350 890 280 <0.5 9.4 730 <0.5 1000 2300 79 29

16 5.2 45 50 8.7 1.6 20 35 66 160 7.8 <0.5 2.8 210 380 67 <0.5 740 21 <0.5 70 240 1100 1000 22 37 1200 99 2400 4000 920 4.3

15 4.1 93 97 32 2.6 46 100 180 4.8 <0.5 <0.5 <0.5 150 150 2.2 1.6 4.8 <0.5 0.73 1.9 46 57 1.7 1.0 <0.5 0.97 1.2 4.9 510 7800 0.065

52 4.9 390 390 12 <0.5 17 42 70 67 6.9 2.9 <0.5 50 130 23 8.8 690 4.8 3.1 6.4 15 750 100 39 2.0 61 <0.5 200 1600 21 000 0.076

<0.5 <0.5 190 190 26 67 140 280 510 270 35 43 <0.5 630 970 46 5.0 470 49 20 30 42 660 51 12 4.4 26 4.8 98 2400 17 000 0.14

<0.5 <0.5 <0.5 <0.5 0.72 <0.5 3.7 3.6 8.1 4.6 1.5 <0.5 1.8 17 23 4.3 <0.5 40 1.2 1.6 1.4 5.0 53 5.1 2.5 <0.5 30 <0.5 38 120 11 000 0.011

a

The number of samples in parentheses were pooled. Four (raccoon dog), 7 (cat), 3 (northern fur seal), and 6 (black-tailed gull) isomers were detected. c Four (raccoon dog), 8 (cat), 2 (human), 9 (northern fur seal), 10 (cormorant), and 5 (black-tailed gull) isomers were detected. d Seven (raccoon dog), 7 (cat), 3 (dog), 4 (human), 11 (northern fur seal), 8 (cormorant), 8 (black-tailed gull), and 2 (jungle crow) isomers were detected. e Six (raccoon dog), 7 (cat), 5 (dog), 8 (human), 15 (northern fur seal), 10 (cormorant), 12 (black-tailed gull), and 6 (jungle crow) isomers were detected. f Four (raccoon dog), 1 (cat), 4 (dog), 2 (human), 6 (northern fur seal), 2 (cormorant), 2 (black-tailed gull), and 3 (jungle crow) isomers were detected. g One (raccoon dog), 1 (northern fur seal), and 1 (cormorant) isomers were detected. h Sum of CB28, 52, 74, 95, 101, 99, 119, 87, 110, 118, 114, 105, 151, 149, 153, 138, 158, 128, 167, 156, 157, 178, 187, 183, 177, 171, 180, 170, 189, 202, 201, 199, 194, 205, 208, 206, 209. b

tri- (OH-T3CBs) and tetra- (OH-T4CBs) congeners in bottlenose dolphin (Tursiops truncates) plasma (Houde et al., 2006) has been found. The studies on polar bear and bottlenose dolphin suggested that the levels and profiles of OH-PCBs in the blood vary by animal species and several animals may be at higher risk from these metabolites including congeners which are not detected in the human blood. The present study aimed at developing an analytical method for OH-T3CBs to OH-O8CBs and PCP in the whole blood. Data for OHPCB levels and patterns in Japanese mammals and birds are determined. 2. Materials and methods 2.1. Samples In this study, we used the whole blood samples (10 g of each species) of human (a 22 year-old student), pet cat, pet dog, raccoon dog (Nyctereutes procyonoides), northern fur seal (Callorhinus ursinus), black-tailed gull (Larus crassirostris), common cormorant

(Phalacrocorax carbo), and jungle crow (Corvus macrorhynchos) stored in the Environmental Specimen Bank (es-BANK) of Ehime University at 25 °C (Tanabe, 2006). The samples of pet cat (n = 5; each 2 g) and black-tailed gull (n = 2; each 5 g) were pooled. The information on sex, sampling location, and sampling year are shown in Table 1. 2.2. Chemical analysis In previous OH-PCB studies, blood plasma or serum samples have been used and hence cleanup process before derivatization was simple. Nevertheless, in the case of whole blood, a cleanup process is indispensable before derivatization, because many interferences in the extract can reduce derivatization efficiency. In addition, sulfuric acid cannot be used for cleanup, because lower chlorinated OH-PCB congeners with only OH-group (no chlorine atom) on the phenyl ring such as 13C12-labeled 40 OH-T3CB29 and 40 OH-T4CB61, which are used as internal standards in this study, are unstable. Thus we adopted an inactive silica-gel cleanup procedure before derivatization. Other analytical procedures were

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Human Cat Dog Raccoon dog

Northern fur seal

Black-tailed gull Common cormorant Jungle crow 0

25

50

75

100

Contribution to total OH-PCBs (%) OH-T3-T4CBs

OH-P5-H7CBs

OH-O8CBs

Fig. 1. Contribution of OH-T3-T4CBs, OH-P5-H7CBs, and OH-O8CBs to total OH-PCBs in the blood of Japanese mammalian and avian species.

60

Contribution to total OH-PCBs (%)

Identified

Unknown

40

20

OH-T3CBs OH-T4CBs OH-P5CBs OH-H6CBs OH-H7CBs OH-O8CBs

OH-T3CBs OH-T4CBs OH-P5CBs OH-H6CBs OH-H7CBs OH-O8CBs

OH-T3CBs OH-T4CBs OH-P5CBs OH-H6CBs OH-H7CBs OH-O8CBs

OH-T3CBs OH-T4CBs OH-P5CBs OH-H6CBs OH-H7CBs OH-O8CBs

OH-T3CBs OH-T4CBs OH-P5CBs OH-H6CBs OH-H7CBs OH-O8CBs

OH-T3CBs OH-T4CBs OH-P5CBs OH-H6CBs OH-H7CBs OH-O8CBs

OH-T3CBs OH-T4CBs OH-P5CBs OH-H6CBs OH-H7CBs OH-O8CBs

OH-T3CBs OH-T4CBs OH-P5CBs OH-H6CBs OH-H7CBs OH-O8CBs

0

Human

Cat

Dog

Raccoon dog

Northern fur seal

Black-tailed gull

Common cormorant

Jungle crow

Fig. 2. Homolog composition of OH-PCBs in the blood of Japanese mammalian and avian species.

performed following our previous report (Kunisue et al., 2007a), with slight modification. Whole blood sample (10 g) was transferred to a centrifuge tube and denatured with HCl (6 M). 13C12-labeled PCP, OH-PCBs (40 OHT3CB29, 40 OH-T4CB61, 40 OH-P5CB120, 40 OH-H6CB159, 40 OHH7CB172, 4OH-H7CB187) and PCBs (CB28, 52, 95, 101, 105, 118, 138, 153, 156, 167, 170, 178, 180, 189, 194, 202, 206, 208, 209) (Wellington Laboratories, Canada) were spiked as internal standards. 2-Propanol and then 50% methyl t-butyl ether (MTBE)/hexane were added and mixed by a homogenizer. After centrifugation, the organic phase was transferred to a separatory funnel and the aqueous phase including precipitate was extracted two more times

with 50% MTBE/hexane. The organic phases were combined and washed with 5% NaCl in hexane-washed water. The organic phase was evaporated by a rotary evaporator and dissolved in hexane. 1 M KOH in 50% ethanol/hexane-washed water was added and shaken. The KOH solution phase was transferred to another separatory funnel. The partition process was repeated with fresh 1 M KOH and the alkaline phases were combined. The remaining organic phase was concentrated by a rotary evaporator. Lipid in organic phase was removed by gel permeation chromatography (GPC) with Bio-Bead S-X 3(Bio-Rad Laboratories, Hercules CA). Fifty percent dichloromethane (DCM)/hexane was used as mobile phase at a flow rate of 5 ml/min. First fraction

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T. Kunisue, S. Tanabe / Chemosphere 74 (2009) 950–961

Cat

Raccoon dog

OH-T3CB [M]+

300000

[M]+

[M+2]+

250000

[M+2]+

Intensity

Intensity

200000 160000 120000

*

80000

* * 16

18

150000

20

22

24

26

28

16

30

18

[M-COCH3]+

240000

[M-COCH3]+

[M+2-COCH3]+

200000

[M+2-COCH3]+

200000 150000

50000

40000

20

22

24

26

28

30

18

22

24

26

28

Retention Time (min)

28

24

30

26

28

30

28

30

[M-COCH3]+ [M+2-COCH3]+

16000 12000

16

18

20

22

24

26

Retention Time (min)

[M-CH3Cl]+ 7000

Intensity

[M+2-CH3Cl]+

8000 6000

[M+2-CH3Cl]+

6000 5000 4000 3000 2000

2000

6000 26

20000

30

4000

24

22

[M-CH3Cl]+ Intensity

Intensity

20

8000

22

20

Retention Time (min)

10000

20

18

4000 16

10000

12000

18

*

Retention Time (min)

[M+2-CH3Cl]+

16

IF

16

30

24000

[M-CH3Cl]+

14000

*

8000

Retention Time (min)

16000

28

120000 80000

18

26

160000

100000

16

24

4’OH-CB79 20000 10000

Intensity

Intensity

Intensity

250000

22

30000

Retention Time (min)

Retention Time (min)

300000

20

[M+2]+

*

* IF * * * * *

50000

**

40000

200000

100000

*

40000

*

[M]+

* * Intensity

*

240000

OH-T4CB

OH-T4CB

16

18

20

22

24

26

Retention Time (min)

28

30

16

18

20

22

24

26

28

30

Retention Time (min)

Fig. 3. SIM chromatograms monitored parent and distinctive daughter (for OH-PCB congeners with OH-group substituted on meta- or para-position [MCOCH3]+ and on ortho-position [MCH3Cl]+) ions of OH-T3CB and OH-T4CBs in the blood of cat and raccoon dog. *Unknown peak.

containing lipid was discarded, and the following fraction containing PCBs was collected, concentrated and passed through activated silica-gel (Wako-gel S-1: Wako Pure Chemical Industries Ltd., Japan) packed in a glass column. PCBs were eluted with hexane for GC–MS analysis. This fraction was concentrated and 13C12-labeled hexabrominated diphenyl ether (BDE139) was added as a syringe spike. The combined alkaline phase was acidified with sulfuric acid (pH 2). Then 50% MTBE/hexane was added and the separatory funnel was shaken. The phases were allowed to separate and the organic phase containing OH-PCBs and PCP was transferred to a pyriform flask. The aqueous phase was extracted one more time with 50% MTBE/hexane. The organic phases were combined and evaporated by a rotary evaporator. The solvent-evaporated residue was dissolved in hexane and passed through 1.2 g inactive silicagel (Wako-gel DX: Wako Pure Chemical Industries Ltd., Japan) packed in a glass column. OH-PCBs and PCP were eluted with 100 ml of 50% DCM/hexane, concentrated, and dissolved in 1 ml of hexane. Then OH-PCBs and PCP were methylated by reaction with trimethylsilyldiazomethane overnight. The derivatized solution was treated with sulfuric acid and then washed with hexane-washed water. After concentration, the solution containing CH3O-PCBs and -PCP was passed through activated silica-gel (Wako-gel S-1) packed in a glass column. CH3O-PCBs and -PCP were eluted with 10% dichloromethane/hexane and this fraction

was concentrated nearly to dryness. 13C12-labeled CB77 and CB157 were then added as syringe spikes. Identification and quantification were performed using a gas chromatograph (GC: Agilent 6890 series) with an auto-injection system and a high-resolution mass spectrometer (HRMS: JEOL JMS-800D) with a resolving power of more than 10 000. CH3O-PCBs and -PCP were monitored by selective ion monitoring (SIM) mode with a lock mass system at two most intensive ions ([M]+, [M+2]+, and [M+4]+) of the molecular ion cluster. Details of analytical condition for GC–HRMS were described in our previous study (Kunisue et al., 2007a). Identifiable native OH-PCB isomers were 40 OHT4CB79, 20 OH-P5CB114, 3OH-P5CB118, 40 OH-P5CB97, 40 OHP5CB101, 40 OH-P5CB104, 4OH-P5CB107, 40 OH-P5CB108, 40 OH-P5 CB120, 40 OH-P5CB127, 30 OH-H6CB138, 40 OH-H6CB130, 4OHH6CB134, 4OH-H6CB146, 40 OH-H6CB159, 4OH-H6CB162, 4OH-H6 CB163, 30 OH-H7CB180, 30 OH-H7CB182, 30 OH-H7CB183, 30 OHH7CB184, 40 OH-H7CB172, 4OH-H7CB177, 4OH-H7CB178, 4OH-H7 CB187, 4OH-H7CB193, 30 OH-O8CB203, 40 OH-O8CB198, 40 OH-O8 CB199, 40 OH-O8CB200, 40 OH-O8CB201, and 4OH-O8CB202 (Wellington Laboratories, Canada). In addition, the peaks, which were within 15% of the theoretical ratio of two monitor ions and were more than 10 times of noise (S/N > 10), were also quantified as unknown OH-PCB isomers. Unknown isomers were quantified using mean values of relative response factors (RRFs) determined from all identifiable 12C12-homologues and the corresponding 13C12-iso-

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Present study Jungle crow Northern fur seal Black-tailed gull Common cormorant Human Raccoon dog Cat Dog

References Human-Inuit

1)

-Quebec

1)

Laysan albatross

2)

Black-footed albatross

2)

Polar bear- female

3)

-male

3)

0.001

0.01

0.1

1.0

10

100

OH-PCBs/PCBs ratio Fig. 4. Concentration ratios of OH-PCBs to PCBs in the blood of Japanese fauna and in human and wildlife blood reported previously. (1) Sandau et al. (2000), (2) KlassonWehler et al. (1998), (3) Sandala et al. (2004).

mer in standard solution. OH-O8CB isomers were quantified using 13 C12-40 OH-H7CB172 because no 13C12-labeled OH-O8CB isomer was present in internal standards. Identification and quantification of native 62 PCB isomers (BPMS; Wellington Laboratories Inc., Canada) were performed by a GC (Agilent 6890)–MS (Agilent 5973N) using EI-SIM mode at two most intensive ions ([M]+, [M+2]+, and [M+4]+) of the molecular ion cluster. 2.3. Quality assurance and quality control OH-PCBs, PCP, and PCBs were quantified using isotope dilution method to the corresponding 13C12-internal standards according to the formula described previously (Kunisue et al., 2007a), when the peaks met the following criteria: (1) the retention time matches that of the standard compound within ±0.1 min, (2) the signalto-noise ratio (S/N) is higher than 10, (3) the deviation of ion intensity ratio is within 15% of that of the standard compound. Procedural blanks were analyzed simultaneously with every batch of four samples to check for interferences or contamination from solvent and glassware. Low peak of PCP was found in two blank (these peak areas were almost same), and hence PCP concentration in samples were corrected by subtracting the blank value. The limit of quantification (LOQ) was defined as the amount of target compound that resulted in a S/N of 10:1. Recoveries for the 13C12labeled internal standard in this analytical procedure were within 50–95% for OH-PCBs and PCP, and 85–105% for PCBs. When OH-PCBs (CH3O-PCBs after methylation) were analyzed using scan mode by GC–MS, OH-PCB congeners with OH-group substituted on meta- or para-position and ortho-position show distinctive fragment daughter ion of [MCOCH3]+ and [MCH3Cl]+, respectively. We re-analyzed OH-PCBs in all the animal blood samples by monitoring two intensive ions of [MCOCH3]+, [M+2COCH3]+, and [M+4COCH3]+ and of [MCH3Cl]+, [M+2CH3Cl]+, and [M+4CH3Cl]+ by SIM mode of GC–HRMS, to confirm the reliability of OH-PCB peaks.

3. Results and discussion 3.1. Residue levels Eighteen identifiable and fifty unknown OH-PCB peaks (CH3OPCBs after methylation) were detected in the blood of Japanese mammals and birds analyzed in this study (Appendix A). Total concentrations (on a wet weight basis) of OH-PCBs including unknown isomers were as follows; raccoon dog – 4000 pg/g, common cormorant – 2400 pg/g, dog – 2300 pg/g, black-tailed gull – 1600 pg/g, cat – 840 pg/g, northern fur seal – 510 pg/g, human – 220 pg/g, and jungle crow – 120 pg/g (Table 1). Thus higher OH-PCB concentrations in animals (except jungle crow) than in humans were found. It is noteworthy that the highest concentration of OH-PCBs was detected in raccoon dog blood, because the level of total PCBs in this species was the lowest compared with other wildlife such as black-tailed gull, common cormorant, jungle crow, and northern fur seal (Table 1). Total PCB concentrations (on a wet weight basis) were in the decreasing order of black-tailed gull (21 000 pg/g), common cormorant (17 000 pg/g), jungle crow (11 000 pg/g), northern fur seal (7800 pg/g), raccoon dog (920 pg/g), humans (590 pg/g), cat (160 pg/g), and dog (79 pg/g) (Table 1). Elevated PCB concentrations in Japanese bird tissues, especially piscivorous species, were also observed in our previous studies (Kunisue et al., 2003, 2008a). It is likely that PCBs pollution is still great in Japanese fish and many fish-eating birds have been exposed to relatively high levels of PCBs. Interestingly, the highest concentration of PCP was found in cat blood (2200 pg/g), followed by raccoon dog (1100 pg/g), humans (740 pg/g), common cormorant (730 pg/g), dog (150 pg/g), jungle crow (130 pg/g), northern fur seal (110 pg/g), and black-tailed gull (36 pg/g) (Table 1). It has been recently reported that elevated concentrations of PCP were detected in Japanese house and office dust (Suzuki et al., 2008). Also in a recent investigation in the USA, PCP was detected in indoor air and dust samples collected at houses

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4OH-CB107+108/CB105+118 101

4'OH-CB101+120/CB99+101+118 101

7.5

5.9

4.4 1.7

100

100

0.8

0.24

0.2

10-1

0.09

10-1 0.041

0.04

0.031 0.02

10-2

10-2

0.0063

0.0062 0.0035 0.0015

10-3

10-3

4OH-CB146/CB138+153 101

5.2

100 0.42

10-1

0.09

0.076 0.043

10-2

0.0071

0.0015 0.00085

Concentration ratio

10-3

10-4

4OH-CB187/CB183+187

4'OH-CB172/CB170+180 101

103

2.4 160

102

100

76

101

10-1

0.58

0.056

0.069 0.024

1.8

100

0.8

10-2

0.72 0.4

0.0027 0.087

10-1

0.0024

10-3

0.00049

0.025

10-2

10-4

4'OH-CB199/CB199

4OH-CB202/CB202 103

103

1000

730

280

102

92

102 11

101

5.1

101

3.1

2.4

1.9 1.2

100

100

10-1

0.53

0.45

0.34

0.26

10 -1

0.05

0.015

Jungle crow

Common cormorant

Black-tailed gull

Northern fur seal

Raccoon dog

Dog

Cat

Human

Jungle crow

Common cormorant

Black-tailed gull

Northern fur seal

Raccoon dog

Dog

Cat

10-2

Human

10-2

Fig. 5. Concentration ratios of OH-PCB metabolites to their possible parent PCB congeners in the blood of Japanese mammalian and avian species. PCB congeners with the detection limit value (i.e. <2.0 pg/g) was treated as the half concentration, 1.0.

and daycare centers and the children’s potential exposures to PCP were predominantly through inhalation but not through dietary ingestion (Wilson et al., 2007). Relatively high concentration of

PCP in human blood analyzed in this study may support this observation. Pet cats may ingest PCP-laden dust that deposit on their fur, because of their grooming behavior, as pointed out in a pet cat

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study on polybrominated diphenyl ethers (PBDEs) (Dye et al., 2007). 3.2. Profile variation by species A lot of OH-PCB peaks were detected in animal blood, but their chromatogram profiles widely varied depending on the species (Appendix A). OH-P5-H7CB congeners were predominant in human blood (Fig. 1). Some previous reports analyzed only OH-P5-H7CB congeners (Sandau et al., 2000; Sjödin et al., 2000; Fängström et al., 2002; Sandanger et al., 2004; Soechitram et al., 2004) almost reflect total OH-PCBs in human blood. A study that analyzed OHO8CBs in umbilical cord blood also showed notably lower levels of OH-O8CBs than OH-P5-H7CBs (Sandau et al., 2002). Although the OH-PCB homolog pattern in common cormorant was similar to that in humans, relatively higher proportions of OH-T3-T4CBs in cat, black-tailed gull, and northern fur seal and OH-O8CBs in raccoon dog, dog, and jungle crow were observed; especially the contributions of OH-T3-T4CBs in cat and OH-O8CBs in raccoon dog to total OH-PCBs were above 50% (Fig. 1). Relatively high accumulation of OH-T3-T4CBs and OH-O8CBs has been recently reported in bottlenose dolphin (Houde et al., 2006) and polar bear plasma (Verreault et al., 2008), respectively. These variations could be due to species-specific metabolic capacity by phase I CYP and/or phase II conjugation enzymes, binding affinity to TTR, and exposure profiles of parent PCBs. The major congeners identified in animal blood analyzed in this study were 40 OH-CB101/120, 4OH-CB107/40 OH-CB108, 4OHCB146, 4OH-CB178, 4OH-CB187, 40 OH-CB172, 4OH-CB202, and 40 OH-CB199 (Table 1). These congeners with OH-group substituted on para-position have been also detected as predominant congeners in humans and other wildlife blood samples. This could be due to the fact that these 4OH-PCB congeners have strong binding affinity to TTR in the blood because of the structural similarity with T4 (Lans et al., 1993; Brouwer et al., 1998; Cheek et al., 1999; Purkey et al., 2004). Among OH-T3CB to –H6CB homologues, however, some predominant unknown congeners existed in almost all species analyzed (Appendix A). The presence of unknown congeners in the blood has been also reported in previous human and wildlife studies (Klasson-Wehler et al., 1998; Olsson et al., 2000; Sandau et al., 2000; Hovander et al., 2002; Houde et al., 2006). Higher proportions of unknown OH-T4CB congeners in cat and black-tailed gull and unknown OH-H6CB congeners in northern fur seal and common cormorant were observed (Fig. 2). Additionally, cat blood showed the highest proportion (Fig. 2) and concentration (Table 1) of unknown OH-T3CB congeners. When OH-PCBs (CH3O-PCBs after methylation) were analyzed using scan mode by GC–MS, OH-PCB congeners with OH-group substituted on meta- or para-position and ortho-position show distinctive fragment daughter ion of [MCOCH3]+ and [MCH3Cl]+, respectively. For example, 40 OHCB79, which was the only identifiable OH-T4CB isomer in this study, detected in raccoon dog blood is detectable by monitoring [MCOCH3]+ and [M+2COCH3]+, but not by [MCH3Cl]+ and [M+2CH3Cl]+ (Fig. 3). When we re-analyzed OH-T3-T4CBs in cat blood by monitoring these distinctive fragment daughter ions, predominant unknown OH-T3CB and –T4CB congeners observed in cat blood were substituted at meta- or para-position (Fig. 3). It is necessary to further address the identification of these predominant unknown OH-PCBs, because these congeners might have high binding affinity to TTR in the blood. 3.3. OH-PCBs/PCBs concentration ratio When concentration ratios of OH-PCBs to PCBs (OH-PCBs/PCBs ratios) were calculated, the highest value was found in dog blood, followed by cat, raccoon dog, humans, common cormorant, black-

tailed gull, northern fur seal, and jungle crow (Table 1 and Fig. 4). The OH-PCBs/PCBs ratio in human blood analyzed in this study was similar to that reported for the Canadian blood (Quebec) (Sandau et al., 2000) and the ratios observed in avian and northern fur seal blood were comparable to or lower than those for albatrosses (Klasson-Wehler et al., 1998) (Fig. 4). By contrast, the OH-PCBs/ PCBs ratios in dog, cat, and raccoon dog blood were 1–3 orders of magnitude higher than those in humans and avian species and the values were comparable to those in polar bear blood (Sandala et al., 2004) (Fig. 4). This could be mainly due to higher metabolic capacity of these mammals, although it is also speculated that the binding affinity of OH-PCBs to TTR may be higher in these species. We previously suggested that pet dogs have higher metabolic capacity to PCBs than pet cats based on PCB profiles in tissues and diet samples (Kunisue et al., 2005). It has been reported that beagle dogs quickly form metabolites of PCBs compared to cynomolgus monkeys (Sipes et al., 1982a,b). In addition, it is highly possible that raccoon dogs induce drug-metabolizing enzymes such as CYP families depending on the hepatic levels of contaminants and metabolize organohalogen compounds including PCBs, as indicated in our previous studies (Kunisue et al., 2006, 2007b, 2008b). Cats may preferentially metabolize lower chlorinated OH-PCBs and retain these metabolites in the blood, although no experimental evidence is available. When concentration ratios of OH-PCB metabolites to their possible parent PCB congeners were calculated, the highest 40 OHCB101 + 120/CB99 + 101 + 118 and second highest 4OH-CB107 + 108/CB105 + 118 ratios were observed in cat blood (Fig. 5), supporting the above explanation. On the other hand, 4OHCB146/CB138 + 153, 4OH-CB187/CB183 + 187, 40 OH-CB172/ CB170 + 180, 4OH-CB202/CB202, and 40 OH-CB199/CB199 ratios were extremely higher in canine blood than other animal species (Fig. 5). Elevated 4OH-CB187/CB183 + 187 and 40 OH-CB199/ CB199 ratios were recently reported in polar bear blood and it was suggested that these metabolites might be formed by catalysis of CYP2B and/or CYP2D (Verreault et al., 2008). It has been demonstrated that phenobarbital (PB)-induced isozymes, CYP2B and/or CYP2C subfamilies, play a primary role in the metabolism of CB153 in beagle dogs (Duignan et al., 1987; Ariyoshi et al., 1992). Based on the above observations, it is likely that CYP2B-like enzymes are responsible for the metabolism of higher chlorinated OH-PCBs in raccoon dog. Considering especially higher ratios of 4OH-CB187/CB183 + 187, 4OH-CB202/CB202, and 40 OH-CB199/ CB199, it is speculated that these metabolites could be retained in the blood for a long time compared with lower chlorinated OH-PCB congeners. In an experimental study using Sprague–Dawley rats, it has been shown that the half-life of 4OH-CB187 in the blood was about four times longer than that of 4OH-CB107 (Malmberg et al., 2004). Thus, species-specific formation and retention of OH-PCBs is evident and OH-PCBs might pose risk to cat and canine species such as dog and raccoon dog.

4. Conclusions This study presents a valuable baseline data on OH-PCBs in the whole blood of Japanese mammalian and avian species. Sixty eight OH-PCB congeners including unknown peaks were detected. Higher OH-PCB concentrations were found in animal species than in humans. Concentrations of OH-T3CB and –T4CB congeners in pet cat blood and OH-H7CB and –O8CB congeners in pet dog and raccoon dog blood were notably high. Elevated OH-PCBs/PCBs ratios were also observed in these terrestrial mammals compared with humans and birds, indicating cat and canine species might be at higher risk from OH-PCBs. Considering the existence of unknown OH-PCB congeners and variation in OH-PCB profiles by species,

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further studies on the identification of unknown congeners, accumulation features, and the relationship with TH levels are needed to assess the toxicological risk posed by these metabolites.

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tific Research (S) (No. 20221003) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) and Japan Society for the Promotion of Science (JSPS).

Acknowledgments Appendix A We are grateful to the following scientists for their help in sample collection; Prof. Haruo Ogi (Hokkaido University), Dr. Susumu Nakatsu (Nakatsu Veterinary Surgery Hospital), Dr. Norihisa Baba (National Research Institute of Fisheries Science), Dr. Akihiko Sano (Sano Veterinary Surgery Hospital), Dr. Akiko Sudo (Eaglet Office Inc.), and Ms. Kumiko Yoneda (Japan Wildlife Research Center). The authors thank Dr. K. Kannan (Wadsworth Center, New York State Department of Health and State University of New York at Albany) for the critical reading of this manuscript. This study was supported by ‘‘Global COE Program” and Grants-in-Aid for Scien-

SIM chromatograms of OH-PCBs (CH3O-PCBs after methylation) in the blood of Japanese mammalian and avian species. (A) 40 OHCB79 (B) 40 OH-CB101/120 (C) 3OH-CB118 (D) 4OH-CB107/40 OHCB108 (E) 4OH-CB146 (F) 30 OH-CB138 (G) 40 OH-CB130 (H) 40 OHCB159 (I) 4OH-CB178 (J) 30 OH-CB182/183 (K) 4OH-CB187 (L) 4OH-CB177 (M) 30 OH-CB180 (N) 40 OH-CB172 (O) 4OH-CB202 (P) 40 OH-CB201 (Q) 40 OH-CB198/30 OH-CB203 (R) 40 OH-CB199, *Unknown peak, IF = interfere (peaks with more than 50% of the theoretical ratio of two monitor ions).

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