perﬂuoroalkyl substances in the blood of wild rats and ... · perﬂuoroalkyl substances in the...

Perfluoroalkyl Substances in the Blood of Wild Rats and Micefrom 47 Prefectures in Japan: Use of Samples from NationwideSpecimen Bank

Sachi Taniyasu • Kurunthachalam Senthilkumar • Eriko Yamazaki •

Leo W. Y. Yeung • Keerthi S. Guruge • Kurunthachalam Kannan •

Nobuyoshi Yamashita

Received: 20 September 2012 / Accepted: 29 January 2013 / Published online: 14 March 2013

� Springer Science+Business Media New York 2013

Abstract Numerous studies have reported on the global

distribution, persistence, fate, and toxicity of perfluoroalkyl

and polyfluoroalkyl substances (PFASs). However, studies

on PFASs in terrestrial mammals are scarce. Rats can be

good sentinels of human exposure to toxicants because of

their habitat, which is in close proximity to humans. Fur-

thermore, exposure data measured for rats can be directly

applied for risk assessment because many toxicological

studies use rodent models. In this study, a nationwide survey

of PFASs in the blood of wild rats as well as surface water

samples collected from rats’ habitats from 47 prefectures in

Japan was conducted. In addition to known PFASs, com-

bustion ion chromatography technique was used for analysis

of total fluorine concentrations in the blood of rats. In total,

216 blood samples representing three species of wild rats

(house rat, Norway rats, and field mice) were analyzed

for 23 PFASs. Perfluorooctanesulfonate (PFOS; concentra-

tion range \0.05-148 ng/mL), perfluorooctane sulfonamide

(PFOSA; \0.1–157), perfluorododecanoate (\0.05–5.8),

perfluoroundecanoate (PFUnDA; \0.05–51), perfluorode-

canoate (PFDA; \0.05–9.7), perfluorononanoate (PFNA;

\0.05–249), and perfluorooctanoate (PFOA) (\0.05–60)

were detected[80 % of the blood samples. Concentrations

of several PFASs in rat blood were similar to those reported

for humans. PFSAs (mainly PFOS) accounted for 45 % of

total PFASs, whereas perfluoroalkyl carboxylates (PFCAs),

especially PFUnDA and PFNA, accounted for 20 and 10 %

of total PFASs, respectively. In water samples, PFCAs were

the predominant compounds with PFOA and PFNA found in

[90 % of the samples. There were strong correlations

(p \ 0.001 to p \ 0.05) between human population density

and levels of PFOS, PFNA, PFOA, and PFOSA in wild rat

blood.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are

synthetic organic compounds used in a variety of industrial

and commercial applications, as surfactants in pesticides,

and as surface protectors in textiles, furnishings, and food

packaging (Giesy and Kannan 2001). PFASs were first

reported in wildlife in 2001 (Giesy and Kannan 2001);

since then, PFASs have been widely studied in surface

waters (Yamashita et al. 2005; Nakata et al. 2006; Senthil

Kumar et al. 2007; So et al. 2007; Raj et al. 2011), sedi-

ments (Higgins et al. 2005; Nakata et al. 2006; Ahrens

et al. 2010), biota (Houde et al. 2006; Senthil Kumar et al.

2007; Yeung et al. 2009a; Yamashita et al. 2012), and

humans (Kannan et al. 2004; Kannan 2011).

Perfluorooctanesulfonate (PFOS) and perfluorooctano-

ate (PFOA) have been the most studied chemicals due to

S. Taniyasu � E. Yamazaki � N. Yamashita (&)

National Institute of Advanced Industrial Science and

Technology (AIST), Ibaraki 305-8569, Japan

e-mail: [email protected]

K. Senthilkumar (&)

Department of Natural Sciences, Savannah State University,

Savannah, GA 31404, USA

e-mail: [email protected]

L. W. Y. Yeung

Department of Chemistry, University of Toronto, Toronto, ON

M5S 3H6, Canada

K. S. Guruge

Safety Research Team, National Institute of Animal Health,

Tsukuba, Ibaraki 305-0856, Japan

K. Kannan

Wadsworth Center, New York State Department of Health and

Department of Environmental Health Sciences, State University

of New York, Albany, NY 12201-0509, USA

123

Arch Environ Contam Toxicol (2013) 65:149–170

DOI 10.1007/s00244-013-9878-4

their widespread occurrence and potential toxic effects

(Seacat et al. 2002; Oakes et al. 2005; Senthil Kumar 2005;

Liu et al. 2012). Both of these chemicals have been shown

to have various effects on developmental, reproduction,

and immune functions in laboratory animals (Abbott et al.

2007; Fairley et al. 2007; Lau et al. 2007; Keil et al. 2008;

Liu et al. 2012). Concerns about the toxicity led to the

phasing out of PFOS production in the United States and

the European Union by some manufacturers in 2002, but

the phasing out of PFOA will not be completed until 2015

(http://www2.dupont.com/PFOA/en_US/). Therefore, PFOA

may still be produced in other parts of the world, including

China (UNEP United Nations Environment Programme

[UNEP] 2008). PFOS and its precursor, perfluorooctane

sulfonyl fluoride (PFOSF), were listed as restricted use

chemicals (Annex B) under the Stockholm Convention in

May 2009 (Wang et al. 2009), and this restriction was entered

into force in August 2010. In Japan, according to the Japa-

nese Chemical Substance Control Law (http://www.safe.

nite.go.jp/english/), PFOS and PFOSF were listed as Class I

Specified Chemical Substances in April 2010.

The first report on PFASs in Japanese environment was

published in 2003 (Taniyasu et al. 2003), and subsequent

monitoring reports investigated PFASs in different envi-

ronmental matrices (Kannan et al. 2002; Nakata et al. 2006;

Senthil Kumar et al. 2007; Harada et al. 2007; Guruge et al.

2008; Murakami et al. 2008; Murakami and Takada 2008;

Taniyasu et al. 2008; Harada and Koizumi 2009; Mak et al.

2009; Zushi et al. 2010). However, most wildlife bio-

monitoring data on PFASs in the world focused mainly on

aquatic biota, such as fish (Kannan et al. 2005), water birds

(Wang et al. 2008), and marine mammals (Yeung et al.

2009a). Very few studies have reported PFASs in terrestrial

wildlife (Dai et al. 2006; Senthil Kumar et al. 2007; Guruge

et al. 2008; Li et al. 2008).

Wild rat (Rattus spp. and Apodemus spp.) is a good

indicator of human exposure to environmental chemicals

because they live in proximity to humans (Takasuga et al.

2004; Senthil Kumar et al. 2005a). They feed on human

refuse from the garbage and drink water from drains and

water bodies. Rats have a life span of 1–2 years and have

short home ranges (approximately 100 m2) suggesting

that they could be used as ‘‘sentinels’’ for the detection of

local pollution in terrestrial environments (Scott 1966;

Ceruti et al. 2002; Takasuga et al. 2004; Senthil Kumar

et al. 2005a; Ishizuka et al. 2005). In this study, we report

geographical distribution of PFASs and total fluorine (TF)

concentrations in wild rat blood samples collected in 47

prefectures in Japan. Second, the relationship between

PFAS concentrations in wild rats with those in water

collected near their habitat was examined. To our

knowledge, this is the first report to document the use of

wild rats as sentinels of water contamination by PFASs.

Furthermore, we document the use of samples from a

wild rat specimen bank for the analysis of PFASs and TF

in Japan.

Materials and Methods

Sample Collection

A nationwide collection of wild rats in all 47 prefectures in

Japan started in June 2004 and was completed in December

2009. For this study, three species of wild rats (Norway rat

[Rattus norvegicus], House rat [R. rattus], and Japanese

field mouse [Apodemus specious]) were collected. Samples

were collected from two distinct geographical locations,

one in a densely populated area (i.e., industrial, commercial,

residential areas) and another in a remote area (i.e., country

side or nature reserve), in each prefecture. Approximately

five specimens were collected from each location, and the

number of specimens collected for each prefecture was

between 15 and 20. Overall, 700 specimens from all 47

prefectures in Japan were collected during a 6-year period.

The specimens were archived at Advanced Industrial Sci-

ence and Technology (Tsukuba, Japan) to evaluate potential

risks of hazardous chemicals to humans and wildlife.

The rats and mice were collected using box traps and

then transported to the laboratory alive. In the laboratory,

using a PFAS-free syringe, blood was drawn from the tail

vein and fresh livers removed immediately after cerebral

dislocation. After biometric measurements, whole body,

blood, and liver were frozen at -20 �C until analysis.

PFAS-free polypropylene (PP) tubes and containers were

used for the storage of samples. From the specimen bank,

216 specimens were selected for PFAS analysis. The

details of the samples are listed in Table 1. Water samples

(500 mL in PP bottles) were collected simultaneously from

62 locations near the nesting places of rats. All the water

samples were stored at –20 �C until analysis. The details of

water sampling are also listed in Table 1.

Chemicals and Reagents

Purities of all of analytical standards used in this study

were C 95 %. Potassium salts of perfluorodecane sulfonate

(PFDS), PFOS, perfluorohexane sulfonate (PFHxS), perfluo-

rooctane sulfonamide (PFOSA), perfluorononanoate (PFNA),

PFOA 13C4-PFOS 13C2-perfluorodecanoate (PFDA), perflu-

orodecanesulfinic acid (PFDSi), perfluorooctanesulfinic acid

(PFOSi), perfluorohexanesulfinic acid (PFHxSi) 13C5-PFNA,13C4-PFOA, and 13C4-perfluorobutanoate (PFBA) were pur-

chased from Wellington Laboratories (Guelph, Ontario,

Canada). N-ethyl perfluorooctanesulfonamide (N-EtFOSA)

and N-ethyl perfluorooctanesulfonamidacetate (N-EtFOSAA)

150 Arch Environ Contam Toxicol (2013) 65:149–170

123

http://www2.dupont.com/PFOA/en_US/

http://www.safe.nite.go.jp/english/

http://www.safe.nite.go.jp/english/

Table 1 Details of wild rat and water samples analyzed in this study

Region Prefecture City/town Sample code Date Description of sampling location Possible water sources to wild rat

Hokkaido Hokkaido Tomakomai HK1 Oct 05 River basin River

Sapporo HK2 Sep 06 River basin River

Yoichi HK3 May 06 River basin River

Tohoku Aomori Aomori AM1 Sep 06 Port Sump

Hachinohe AM2 Nov 05 Port Tap water

Iwate Kitakami IT2 Oct 06 Industrial area Channel

Shizukuishi IT3 Oct 06 River basin Channel

Akita Akita AT1 Nov 06 Forest Agricultural channel

Yamagata Yamagata YG2 Nov 06 River basin River

Miyagi Ishinomaki MG1 Jun 06 Port Channel

Fukushima Iwaki FS1 Mar 06 Port No corresponding water sample

Kanto Gunma Shibukawa GM1 May 07 River basin River

Tochigi Utsunomiya TG1 May 06 River basin River

Ibaraki Kashima IK1 Oct 04 Port Sump

Hitachi IK5 Sep 06 Port Channel

Chiba Choshi CB4 Jun 04 Port Sump

Chiba CB5 Oct 05 Residential area Sump

Saitama Saitama ST1 Nov 07 River basin River

Tokyo Hachioji TK4 Oct 04 Market place Sump

Kanagawa Kawasaki KG1 Jul 04 Park Sump

Chubu Niigata Niigata NG1 Jul 06 River basin Sump

Jyoetsu NG3 Jul 05 River basin Sump

Yamanashi Kofu YN1 May 05 Market place Channel

Fuefuki YN2 Jul 07 Forest Channel

Shizuoka Yaizu SO1 May 05 Port Sump

Nagano Suwa NN1 Sep 05 Lake basin Agricultural channel

Aichi Nagoya AC1 Nov 07 Port Channel

Toyama Uozu TY1 Jul 07 Port Sump

Toyama TY3 Jul 07 River area River

Ishikawa Kaga IK1 Jun 07 Clean center Agricultural channel

Fukui Fukui FK1 Oct 07 Forest Agricultural channel

Gifu Seki GF3 Jul 07 Agricultural area Agricultural channel

Kinki Mie Suzuka ME1 Jul 07 Port Channel

Shiga Nagahama SG3 Jul 07 Agricultural area Agricultural channel

Nara Nara NR2 Nov 07 Agricultural area Agricultural channel

Wakayama Arida WY1 Jan 07 River basin River

Kyoto Kyotanba KT1 Apr 07 Industrial area Channel

Osaka Izumisano OS1 Apr 07 Forest Channel

Hyogo Sanda HG3 May 07 Industrial area Agricultural channel

Shikoku Kagawa Higashikagawa KG2 Apr 07 Port Pond

Takuma KG3 Apr 07 Port Sump

Tokushima Shishikui TS2 Nov 07 Port Sump

Ehime Imabari EH2 Apr 07 Port Sump

Kochi Toyo KC3 Nov 07 Agricultural area Agricultural channel

Arch Environ Contam Toxicol (2013) 65:149–170 151

123

were gift from the 3M Company (St. Paul, MN). PFBS

was a gift from Chiron AS (Trondheim, Norway). Per-

fluoropropanesulfonate (PFPrS) was a gift from JEMCO

(Akita, Japan). Perfluorooctadecanoate (PFOcDA), perflu-

orohexadecanoate (PFHxDA), perfluorotetradecanoate

(PFTeDA), and 7:3 fluorotelmer carboxylate (FTCA) were a

gift from SynQuest Lab (Alachua, FL). Perfluoropentanoate

(PFPeA), perfluoroheptanoate (PFHpA), PFDA, perfluo-

roundecanoate (PFUnDA), and perfluorododecanoate

(PFDoDA) were purchased from Fluorochem Ltd (Derby-

shire, UK). PFHxA was purchased from Wako Pure Chemical

(Osaka, Japan). PFBA was purchased from Avocado

Research Chemicals, Ltd (Lancashire, UK). Saturated flu-

orotelomer carboxylate (8:2 FTCA) and unsaturated fluoro-

telomer carboxylate (8:2 FTUCA) were purchased from Asahi

Glass (Tokyo, Japan). Oasis weak anion exchange (WAX;

6 cc 150 mg, 30 lm) solid phase extraction (SPE) cartridges

were purchased from Waters (Milford, MA). Milli-Q water

was used throughout the experiment. Methanol and acetoni-

trile (residual pesticide and polychlorinated biphenyls [PCB]

analytical grade), ammonium acetate (97 %), ammonium

solution (25 %), and acetic acid (99.9 %) were purchased

from Wako.

PFAS Extraction

Water samples were extracted using Oasis-WAX SPE

cartridges according to methods published elsewhere

(Taniyasu et al. 2005, 2008; ISO25101 2009). The cartridges

were preconditioned by passing 4 mL of 0.1 % NH4OH in

methanol followed by 4 mL of methanol and 4 mL of Milli-

Q water. Water samples, 300 mL, were passed through the

preconditioned cartridges at a rate of 1 drop/s. The cartridges

were then pre-eluted with 4 mL of 25 mM ammonium ace-

tate buffer at pH 4 and the target analytes eluted with 4 mL of

methanol followed by 4 mL of 0.1 % NH4OH in methanol.

The eluates were then concentrated to 1 mL under a gentle

stream of nitrogen for instrumental analysis.

Whole blood samples were extracted by acetonitrile

(ACN) followed by SPE clean-up as described earlier

(Yeung et al. 2009b). In brief, 0.5 mL of whole blood sample

was mixed with 5 mL of ACN using a vortex mixer. The

mixture was shaken for 20 min at 250 rpm. The organic

phase was separated from the aqueous mixture by centrifu-

gation for 15 min at 3000 rpm. The supernatant, 5 mL, was

transferred to another PP tube. Five mL of ACN was added to

the sample again, and the extraction procedure was repeated.

Then 10 mL of combined extract was concentrated to

0.5 mL under a gentle stream of nitrogen. Milli-Q water,

10 mL, was added to the extract and then subjected to the

SPE clean-up step as described for water samples.

Instrumental Analysis

Separation of the analytes was performed using an Agilent

HP1100 liquid chromatograph (Agilent, Palo Alto, CA)

Table 1 continued

Region Prefecture City/town Sample code Date Description of sampling location Possible water sources to wild rat

Chugoku Okayama Wake OY3 Aug 07 Agricultural area Agricultural channel

Hiroshima Fukuyama HS1 Oct 07 Residential area Channel

Higashihiroshima HS3 Jun 07 Residential area Channel

Tottori Yonago TT1 Aug 07 Port Channel

Tottori TT2 Jun 07 Port Channel

Shimane Hamada SN2 Aug 07 Port Channel

Ooda SN4 Oct 07 Port Channel

Yamaguchi Shimonoseki YG1 Jun 07 Port Sump

Nagato YG3 Aug 07 Port Channel

Kyushu Fukuoka Kitakyusyu FO2 Mar 08 Port Channel

Nijyo FO3 Mar 08 Port Channel

Saga Taku SA3 Nov 07 Industrial area Agricultural channel

Oita Nakatsu OI1 Jun 07 Industrial area Agricultural channel

Nagasaki Sasebo NS4 Oct 07 River basin River

Miyazaki Kawaminami MZ1 Oct 06 Port Sump

Kumamoto Kamiamakusa KM1 Jun 07 Port Channel

Kagoshima Makurazaki KS2 Jul 05 Animal center Tap water

Okinawa Okinawa Ginowan ON3 Mar 07 Residential area No corresponding water sample

There were no corresponding water sources for wild rat collected from FS1 and ON3


123

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123

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0.2

90

.15

0.2

00

.13

0.3

4

SD

0.3

60

.22

0.0

20

.09

0.0

60

.09

0.1

10

.06

Min

imu

m0

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20

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0.2

30

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0.0

50

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Max

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0.2

00

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00

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LO

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1.5

0.0

81

.22

.01

.31

.00

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1.9

SD

0.1

51

.80

.04

0.6

00

.38

0.5

30

.17

0.4

31

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Min

imu

m1

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.41

0.0

50

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1.6

0.6

90

.92

0.5

00

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Max

imu

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40

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0.0

20

.07

0.1

20

.11

0.0

50

.21

Min

imu

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0.1

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0.0

30

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Max

imu

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10

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0.2

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40

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70

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0.6

11

.41

.00

.43

0.0

40

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Min

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Max

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ano

(rat

n=

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1M

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11

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2.7

5.6

3.9

4.8

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2.3

SD

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1.2

1.9

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Min

imu

m7

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Min

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1.6

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0.6

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4.5

0.2

90

.65

0.0

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3.0

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4.3

4.2

0.2

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0.0

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Min

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80

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Min

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0.1

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0.0

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0.2

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Min

imu

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0.1

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0.0

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0.0

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0.1

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Min

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.30

.59

n[

LO

Q0

30

31

03

33

33

02


123

interfaced with a Micromass Quattro Ultima Pt Mass

Spectrometer (Waters, Milford, MA) operated in the elec-

trospray negative ionization mode. A 10-lL aliquot of the

extract was injected onto two different analytical columns.

One of the columns was a Keystone Betasil C18-column

(2.1 mm i.d. 9 50 mm length, 5-lm 100 A pore size, end-

capped) with 2 mM ammonium acetate and methanol as

mobile phase for the quantification of C6 to C18 PFASs.

Another column was ion exchange column RSpak JJ-50 2D

(2.0 mm i.d. 9 150-mm length; Shodex, Showa Denko

K.K., Kawasaki, Japan) with 50 mM ammonium acetate

and methanol as mobile phase and was employed for the

quantification of C3 to C5 PFASs. The PFAS concentra-

tions (C6–C18) determined by these two stationary phases

were checked against each other for confirmation. The

variations in PFAS concentrations determined between

these two columns were \10 %. The desolation gas flow

and temperature were kept at 610 L/h and 450 �C,

respectively. The collision energies, cone voltages, and

double mass spectrometry (MS/MS) parameters for the

instrument were optimized for individual analytes and were

similar to those reported elsewhere (Taniyasu et al. 2005,

2008).

TF and Organic Fluorine Analysis

An aliquot of blood was subjected to fractionation pro-

cedure for the analysis of TF and extractable organic

fluorine (EOF) according to the extraction methods

described in Yeung et al. (2009a, c). TF was determined by

taking 0.1 mL of wild rat blood on a silica boat and placing

it directly into the combustion ion chromatograph (CIC).

EOF from fraction 1 (the extract of Methyl tertiary butyl

ether [MTBE] fraction of ion-pairing) and EOF from

fraction 2 (hexane extraction of residue after ion-pairing

extraction) were quantified using CIC. The method

involves modifications to traditional CIC by the combina-

tion of an automated combustion unit (AQF-100 type

AIST; Dia Instruments) and an ion chromatography system

(ICS-3000 type AIST; Dionex, Sunnyvale, CA). The cus-

tomized instrument, combustion ion chromatograph for

fluorine (CIC-F), has been described in detail elsewhere

(Yeung et al. 2009a, c). The sample extract was set on a

silica boat and placed into a furnace at 900–1000 �C.

Combustion of the sample in the furnace converted organic

fluorine and inorganic fluoride into hydrogen fluoride (HF).

The HF was absorbed into sodium hydroxide solution

(0.2 mmol/L). The concentration of F- in the solution was

analyzed using ion chromatography. Sodium fluoride

(99 % purity; Wako) was used as a standard for quantifi-

cation. Five calibration standards were prepared routinely

at 0.2, 1, 5, 25, and 100 mg/L and injected at 1.5 mL to

check for linearity of the instrument. Quantification wasTa

ble

2co

nti

nu

ed

Co

un

try

PF

DS

PF

OS

PF

Hx

SP

FO

SA

N-E

tFO

SA

AP

FT

eDA

PF

Do

DA

PF

Un

DA

PF

DA

PF

NA

PF

OA

PF

BA

7:3

FT

CA

Kag

osh

ima

Mak

ura

zak

i(r

atn

=3

)K

S2

Mea

n1

.00

.03

0.1

70

.81

2.7

1.3

1.8

0.2

30

.44

SD

0.6

80

.06

0.0

90

.56

2.0

1.2

1.7

0.1

10

.11

Min

imu

m0

.38

\0

.10

.06

0.2

20

.77

0.3

80

.51

0.1

70

.34

Max

imu

m1

.70

.10

0.2

31

.34

.82

.73

.70

.36

0.5

6

n[

LO

Q0

30

10

33

33

33

30

Ok

inaw

aG

ino

wan

(rat

n=

5)

ON

3M

ean

16

0.3

83

.00

.14

4.6

0.9

42

13

.20

.15

SD

21

0.2

36

.30

.06

8.1

0.5

84

23

.90

.24

Min

imu

m1

.80

.14

\0

.05

0.0

60

.25

0.2

70

.14

0.2

0\

0.0

5

Max

imu

m5

30

.67

14

0.2

01

91

.79

79

.70

.57

n[

LO

Q0

55

02

05

55

55

03

Ari

thm

etic

mea

nis

giv

enfo

rth

e2

16

wh

ole

blo

od

sam

ple

s,th

esa

mp

les

low

erth

anL

OQ

sis

con

sid

ered

asa

zero


123

based on the response of the external standards that brack-

eted the concentrations found in the samples. The analytical

parameters and conditions of the ion chromatography were

given in Yeung et al. (2009a, c). All solutions were prepared

in Milli-Q water, and the fluoride concentration in the Milli-

Q water was \0.025 mg/L. Furthermore, fluoride concen-

tration in the methanol was lower than the limit of quantifi-

cation (LOQ).

Quality Assurance/Quality Control

For individual PFASs, LOQs were defined as the lowest

mass of the compound injected that resulted in a reproduc-

ible measurement of the peak areas within ± 20 % of the

duplicate injection. The LOQ for each compound typically

ranged from 0.05–0.1 ng/mL for the blood samples and

0.02–0.50 ng/L for the water samples. LOQs were evaluated

based on several criteria, including (1) the smallest con-

centration of standard on the calibration curve that could be

accurately measured within ± 20 % of its theoretical value;

(2) a signal-to-noise ratio C 10; (3) the concentration factor;

and (4) the sample volume. PFAS concentrations were

quantified using external calibration curves consisting of a

concentration series of 2, 10, 50, 200, 1000, 5000, and

20,000 pg/mL. The standard calibration curve showed high

linearity (correlation coefficients [ 0.99). The linearity and

repeatability of these calibration curves were confirmed

before each batch of sample analysis. If the measured con-

centrations exceeded the range of the calibration curve,

the samples were diluted and reinjected into the liquid

chromatography-tandem mass spectrometer. A custom-

made standard solution (1 ng/mL) of target analytes,

including mass-labeled standards, was injected with every

10 sample injections to check for any shift and intensity

changes of peaks from the original retention time.

Procedural blanks were analyzed with every batch of

(n = 10) samples, and procedural recoveries were con-

ducted to check the accuracy of the methods used. Matrix

recovery tests (i.e., whole blood and water samples) were

also performed. Matrix recoveries were calculated by

IK1FI1 GM1 TG1

YN1 YN2

SO1AC1ME1

WY1

TS2

KG2 KG3EH2

KC3

ON3

TT1TT2

SN2 SN4YG1 YG3

OY3HS3 HS1

HK2HK3 HK1

IK1IK5 CB4

GF3

ST1TK4 CB5KG1

FS1

MG1

AM1

IT2

IT3YG2 AT1

AM2

NS4

KM1

MZ1

KS2

FO2FO3SA3

OI1OS1 NR2

HG3 SG3KT1

NG1NG3TY1TY3NN1

67.8

66.8

117

Low

High

High

High

High

Hokkaido

Tohoku

Kanto

Chubu

Kinki

Shikoku

Chugoku

Kyushu

Okinawa

Nine region andtheir boundary

PF

OS

PF

OSA

PF

OA

PF

NAC

once

ntra

tion

s(n

g/m

L) 20

10

0

Fig. 1 Concentrations of PFOS, PFOA, PFNA and PFOSA in the

blood of 216 wild rats from 47 prefectures in Japan (No bar indicates

sample below LOQ (0.05 ng/mL); blue, red and black colors circles

on the map indicates the rat species Rattus rattus, Apodemus specious

and Rattus norvegicus, respectively) (Color figure online)


123

comparing the relative response factors of the spiked

samples with those of the nonspiked ones. All procedural

blanks were lower than the corresponding LOQs for the

blood and water samples. The recoveries ranged from 70 to

120 % for blood samples and 87 to 105 % for water

samples, whereas matrix spike recoveries for blood sam-

ples ranged from 58 to 122 % and for water samples ran-

ged from 83 to 117 %. All of the samples were extracted in

duplicate, and the relative standard deviations for duplicate

analysis were \10 %. Carbon-13 (13C)–labeled standards

were spiked into blood and water samples to evaluate

overall recoveries. Recoveries for the 13C-standards spiked

in blood and water samples were between 76 and 110 %.

PFAS concentrations in samples were not corrected for the

internal standard recoveries.

For TF and EOF analysis, because contaminants could

be present in the gases used for CIC, high-purity gases

(argon = 99.9999 %, oxygen = 99.9995 %) were used

(Yeung et al. 2009a, c). Ion chromatograph tubing, gas

lines, valves, and regulators, which contained materials or

parts made of polytetrafluoroethylene, were replaced with

stainless steel, polyetheretherketone, or polyethylene tub-

ing. Furthermore, a gas purifier containing activated carbon

was placed in the gas line to remove trace levels of fluo-

rine from the gases. Analyses of TF were conducted in

duplicate. In-house reference material (pig blood) was

injected before and after 10 CIC injections, and one of the

liver homogenates was injected before and after of daily

injection to check for the overall efficiency of the com-

bustion process.

Statistical Analysis

Pearson correlation analysis was used to evaluate the

relationships between PFASs in water and rat blood and

population density. The significant level was set at

a = 0.05. Because the data were not normally distributed,

all data, including PFAS concentrations measured in blood

and water as well as the population density, were log-

transformed for statistical analysis.

Rat

blo

od c

once

ntra

tion

(n

g/m

L)

Location (Geography)

Hok

kaid

o

Toh

oku

Kan

to

Chu

bu

Kin

ki

Shik

oku

Chu

goku

Kyu

shu

Oki

naw

a

PFDS

PFOS

PFHxS

PFOSA

N-EtFOSAA

PFTeDA

PFDoDA

PFUnDA

PFDA

PFNA

PFOA

PFHpA

PFHxA

PFPeA

PFBAPFPrA

0

20

40

60

80

100

120

140

HK

1H

K2

HK

3A

M1

AM

2IT

2IT

3A

T1

YG

2M

G1

FS1

GM

1T

G1

IR1

IR5

CB

4C

B5

ST1

TK

4K

G1

NG

1N

G3

YN

1Y

N2

SO1

NN

1A

C1

TY

1T

Y3

IR1

FK

1G

F3

ME

1SG

3N

R2

WY

1K

T1

OS1

HG

3K

G2

KG

3T

S2E

H2

KC

3O

Y3

HS1

HS3

TT

1T

T2

SN2

SN4

YG

1Y

G3

FO

2F

O3

SA3

OI1

NS4

MZ

1K

M1

KS2

ON

3

143 301

PFASs

Fig. 2 Individual PFAS concentrations (ng/mL) in wild rat blood samples from 47 prefectures in Japan


123

Results and Discussion

Among 23 PFASs analyzed, 13 PFASs (% occurrence)

were detected in whole blood of wild rats: PFDS (detection

rate 28 %), PFOS (100 %), PFHxS (31 %), PFOSA (83 %),

N-EtFOSAA (53 %), PFTeDA (41 %), PFDoDA (95 %),

PFUnDA (100 %), PFDA (100 %), PFNA (100 %), PFOA

(95 %), PFBA (36 %), and 7:3 FTCA (55 %). The sum-

mary of minimum, maximum, arithmetic mean, and median

PFAS concentrations, as well as their frequency of detec-

tion, are listed in Table 2. PFDSi (\0.05 ng/mL), PFOSi

(\0.05 ng/mL), and PFHxSi (\0.05 ng/mL) were not

detected in any of the wild rat blood samples. PFHpA

(\0.05–2.02 ng/mL), PFHxA (\0.05–9.93 ng/mL), PFPeA

(0.05–0.263 ng/mL), and PFPrA (\0.2–0.276 ng/mL) were

detected in 10 % of the samples. PFBA and PFHxS were

detected in \40 % of the samples. PFSAs (mainly PFOS)

accounted for 45 % of total PFASs, whereas PFUnDA and

PFNA accounted for 20 and 10 % of total PFASs, respec-

tively. Median PFOS concentration was 5.7 ng/mL

followed by PFOSA at 0.48 ng/mL. Among PFCAs, odd-

number carbon-fluorine chain (i.e., PFNA [median 6.0 ng/

mL] and PFUnDA [median 2.5 ng/mL]) were greater than

those of even carbon-fluorine chain (PFDoDA [median

0.51 ng/mL], PFDA [median 1.3 ng/mL], and PFOA

[median 0.58 ng/mL]).

Japan has four major islands (i.e., Hokkaido, Honshu,

Shikoku, and Kyushu), which can be further divided into

nine regions (i.e., Hokkaido, Tohoku, Kanto, Kinki, Chubu,

Chugoku, Shikoku, Kyushu, and Okinawa). The geo-

graphical locations are characterized by different levels of

development and industrial activities. Average PFOS,

PFOSA, PFNA, and PFOA concentrations in wild rat blood

from 47 prefectures are shown in Figs. 1 and 2. A clear

geographical difference in PFAS distribution was observed

in Japan. Relatively lower PFAS concentrations were

found in Kyushu and Shikoku islands. The Kanto region in

Honshu island encompasses the largest industrial zone with

a wide range of commerce and heavy industries, such as

electronic production and semi-conductor industries, which

PFDS

PFOS

PFHxS

PFOSA

N-EtFOSAA

PFTeDA

PFDoDA

PFUnDA

PFDA

PFNA

PFOA

PFHpA

PFHxA

PFPeA

PFBAPFPrA

0

10

20

30

40

50

60

70

80

HK

1H

K2

HK

3A

M1

AM

2IT

2IT

3A

T1

YG

2M

G1

FS2

GM

1T

G1

IR1

IR5

CB

4C

B5

ST1

TK

4K

G1

NG

1N

G3

YN

1Y

N2

SO1

NN

1A

C1

TY

1T

Y3

IK1

FK

1G

F3

ME

1SG

3N

R2

WY

1K

T1

OS1

HG

3K

G2

KG

3T

S2

EH

2

KC

3

OY

3

HS1

HS3

TT

1

TT

2

SN2

SN4

YG

1

YG

3

FO

2

FO

3

SA3

OI1

NS4

MZ

1

KM

1

KS2

ON

2

Wat

er c

once

ntra

tion

s (

ng/L

)

Location (Geography)

Hok

kaid

o

Toh

oku

Kan

to

Chu

bu

Kin

ki

Shik

oku

Chu

goku

Kyu

shu

Oki

naw

a

180

PFASs

Fig. 3 Individual PFAS concentrations (ng/L) in water samples collected near the habitat of wild rats (n = 62) from 47 prefectures in Japan


123

use PFOS in their manufacturing processes (Hori et al.

2006; Tang et al. 2006). The Kinki region in Honshu Island

is the second most industrialized and commercial center in

Japan. Increased PFAS concentrations were found in rat

blood samples collected from the Kanto and Kinki regions

(Fig. 1). A gradual increase of PFAS concentration was

observed in the direction from Chubu to Tohoku and

Hokkaido (Table 2; Fig. 1). In addition, the composition of

PFASs varied among the sampling locations, even within

the same prefecture.

Studies have shown that bioaccumulation of PFASs is

related to the length of the fluorocarbon chain (Kudo et al.

2006; Liu et al. 2011). Another study showed that PFCAs

with fewer then seven fluorinated carbons have less bio-

magnification potential in wildlife (Conder et al. 2008),

which is similar to the results found in the present study.

Long-chain PFCAs are bioaccumulative in rats and (i.e.,

C8–C12) are readily enriched onto protein-rich tissues,

such as blood and liver (Senthil Kumar et al. 2005b). Faster

elimination (i.e. short half-lives) of short-chain PFASs in

rats (Chang et al. 2008) is a possible explanation for the

low accumulation of short-chain PFASs in wild rat blood.

Sixty-two water samples collected near the habitat of the

wild rats were also analyzed for the 23 PFASs. Similar to rat

blood, PFDSi, PFOSi, PFHxSi, PFDS, and PFHxS were not

detected in the water samples (Fig. 3). In contrast, PFCAs,

such as PFOA and PFNA, were detected frequently in water

samples (Figs. 2, 3). Comparatively higher concentrations of

PFHpA, PFBA, PFPrA, PFPeA, and PFHxA were observed

in rats from the Kinki, Kanto, and Chugoku regions (Fig. 3).

Among PFSAs, PFOS and N-EtFOSAA were detected at

considerable levels in water samples collected from the

Kinki (except for N-EtFOSAA), Kanto, Chubu, and Chug-

oku regions. Long-chain PFASs, including PFOS and C8 to

C12 PFCAs, were frequently detected in wild rat blood

samples (Fig. 2). In contrast, short-chain PFASs (i.e., C3–C7)

were detected more frequently in water samples (Fig. 3). In

addition, PFOS predominated in wild rat blood samples,

whereas PFOA predominated in water samples. These

findings agree well with those of many other environmental

monitoring studies that showed the predominance of PFOS

in biological samples but predominance of PFOA in water

(Moody et al. 2002; Taniyasu et al. 2003, 2005; Kannan et al.

2005; Nakata et al. 2006; Senthil Kumar et al. 2007, 2009;

Houde et al. 2008; Yeung et al. 2009a, b; Naile et al. 2010;

Wang et al. 2012). These results indicate that wild rats are

good indicators for the accumulation of long-chain PFASs.

Measured concentrations of PFOS, PFOSA, PFDoDA,

PFUnDA, PFDA, PFNA, PFOA, PFBA, and PFTeDA in

rat blood samples were plotted against the human popula-

tion density in the sampling area (Fig. 4a–i). Significant

positive correlations (p \ 0.001) were found between cer-

tain PFASs (i.e., PFOS, PFOSA, PFNA, and PFOA) and

human population density. However, no clear relationship

was observed for longer-chain PFASs (PFUnDA,

PFDoDA, and PFDA) as well as PFBA (Fig. 4b, c, e, h).

Domestic usage and release of PFAS-containing materials

by local populations is a source of contamination by PFASs

(Sinclair and Kannan 2006). A recent study suggested that

PFAS pollution in river water increased in areas of high

traffic (Zushi and Masunaga 2009) as well as urban

activities and population density (Murakami et al. 2008).

Lack of such a relationship for PFBA and long-chain

PFCAs is due to their formation from precursors, such as

fluorotelomer alcohols (FTOHs), which can be transported

to remote areas by atmospheric transport.

The measured concentrations of PFCAs were signifi-

cantly related to each other in the blood of wild rats except

for PFBA and PFTeDA (Table 3). PFBA was found to be

readily removed from the bodies of exposed animals

(Chang et al. 2008). Studies have shown that degradation

of FTOHs is one of the sources of PFCAs (Ellis et al.

2004). For example, degradation of 8:2 FTOH has been

shown to yield similar quantities of PFOA and PFNA (Ellis

et al. 2004), and longer-chain PFCAs have been shown to

be more bioaccumulative (i.e., PFNA [ PFOA [Conder

et al. 2008]). In this study, among many PFASs analyzed, a

7:3 ratio FTCA was found in 55 % of the samples,

although at low levels (Table 2) suggesting exposure of

Table 3 Pearson correlation analysis of PFASs in blood of wild rats

from Japan

PFOSA PFDoDA PFUnDA PFDA PFNA PFOA

PFOS 0.651 0.419 0.220 0.493 0.694 0.272

p ** ** ** ** ** **

n 45 51 58 56 57 47

PFOSA 0.275 0.368 0.370 0.460 0.256

p ** ** ** ** **

n 43 44 44 45 35

PFDoDA 0.711 0.746 0.303 0.289

p ** ** ** **

n 51 51 51 43

PFUnDA 0.806 0.672 0.342

p ** ** **

n 59 58 50

PFDA 0.661 0.318

p ** **

n 57 48

PFNA 0.346

p **

n 48

All data were log10-tranformed for the analysis. No significant cor-

relations were found for PFBA/PFTeDA. Other PFASs showed

positive correlation at the * p \ 0.05 and ** p \ 0.01 levels


123

Human population density (person/km2)

bloo

odco

ncen

trat

ion

( ng/

mL

)bl

ood

conc

entr

atio

n (n

g /m

L)

0.1

1

10

100

10 100 1000 10000

PFOS

R2 = 0.116**n = 58

0.01

0.1

1

10

10 100 1000 10000

PFDoDA

R2 = 0.011n = 51

R2 = 0.104*n = 58

0.01

0.1

1

10

10 100 1000 10000

PFDA

R2 = 0.150**n = 44

0.01

0.1

1

10

100

1000

10 100 1000 10000

PFOSA

R2 = 0.100*n = 60

0.1

1

10

100

10 100 1000 10000

PFUnDA

R2 = 0.172**n = 58

0.01

0.1

1

10

100

10 100 1000 10000

PFNA


a

ed

cb

f

0.01

0.1

1

10

10 100 1000 10000

PFBA


bloo

d co

ncen

trat

ion

(ng/

mL

) R2 = 0.147**n = 50

0.01

0.1

1

10

100

10 100 1000 10000

PFOA

R2 = 0.005n = 26

0.01

0.1

1

10 100 1000 10000

PFTeDA

R2 = 0.2138n = 33

agricultural area

animal center

clean center

forest

industrial area

lake basin

market place

park

parking

port

power plant

residential area

river basin

g ih

Fig. 4 The relationship between concentration of individual PFASs

in wild rat blood and population density. Strong correlations (Pearson

correlation significant at \0.05*, \0.001**), with human population

density were found for PFOS, PFOSA, PFNA and PFOA and no clear

relationship was found for both long chain (PFUnDA, PFDoDA,

PFDA) and short chain (PFBA) compounds


123

rats to FTOHs; FTCA is a partial metabolic transformation

product of FTOHs.

Drinking water is a source of human exposure to

PFASs (Loos et al. 2007; Ericson et al. 2008; Mak et al.

2009; Quinones and Snyder 2009). Exposure of certain

populations to high PFOA concentrations has been

reported (Emmett et al. 2006; Skutlarek et al. 2006;

Paustenbach et al. 2007; Ericson et al. 2008). In the

present study, correlation was performed between PFASs

detected in wild rat blood and concentrations found in

water from rats’ habitats. Only significant correlation

(p \ 0.05) was observed for PFNA (R2 = 0.273) and

Organic fluorine

Total fluorine

Fig. 5 Contributions of known PFASs, unknown organic fluorine and inorganic fluorine to TF concentrations in wild rat blood from Japan

0.01

0.1

1

10

100

1000

0% 20% 40% 60%

PF

ASs

con

cent

rati

ons

[ng/

mL

]

known PFASs / TF

0.01

0.1

1

10

100

1000

0% 20% 40% 60% 80%

PF

C c

once

ntra

tion

s [n

g/m

L]

EOF/ TF

PFOS

PFHxS

PFOSA

PFDoDA

PFUnDA

PFDA

PFNA

PFOA

a b

Fig. 6 Ratios of individual PFASs concentrations in wild rat blood to TF (known PFASs/TF) or EOF/TF) as indicators sources of PFASs in rats.

Dotted line indicates general (non-specific) sources of pollution and solid line indicates specific source of pollution


123

PFOA (R2 = 0.093). Several factors affect accumulation

of PFASs in rats, and dietary sources appear to be

important sources of PFASs in rats. Serum PFOS and PFOA

concentrations for the Japanese populations were in the

range of several tens of parts per billion (ppb) (Taniyasu

et al. 2003; Harada et al. 2004, 2007). Interestingly, con-

centrations of PFOS and PFOA in rat blood were similar to

those found in human serum. Furthermore, geographic

differences in PFAS concentrations were similar between

wild rats and humans. These results suggest that wild rats

can be good indicators of PFAS exposure in humans.

Inorganic fluorine is a naturally occurring element

(Zhang et al. 2010). Concentrations of known organic

fluorine (i.e., PFASs), EOF and inorganic fluorine were

analyzed in wild rat blood (Fig. 5). The proportion of

known organic fluorine, such as PFASs, was relatively

higher in the Shizuoka and Hokkaido regions than in other

regions in Japan. The proportion of EOF was relatively

greater in the Osaka region, and inorganic fluorine was

relatively greater in the Fukuoka, Hokkaido, and Okinawa

regions (Fig. 5). TF concentration in wild rat was variable,

and no significant relationship was found with the concen-

tration of individual PFASs. Fluorine readily forms com-

pounds with other elements, such as calcium, which can

affect bioavailability. In our previous study, we reported

that EOF concentrations (Fr1; MTBE extraction) in wild rat

blood ranged 61–134 ng-F/mL, whereas those in fraction 2

(Fr2; hexane) were lower than the LOQ (32 ng-F/mL); TF

concentrations in the blood of wild rats ranged from 60 to

192 ng-F/mL. The contribution of known PFASs in EOF-

Fr1 (MTBE) varied from 9 to 89 % (average 56 %), and

known PFAS concentrations in TF content were \25 %

(Yeung et al. 2009c). Miyake et al. (2007) reported that

nonextractable organofluorine concentrations in the blood

of the general human population in Japan were [70 % of

the TF. These results found for humans were similar to that

found for rat blood, which showed 45 to[95 % of nonex-

tractable organofluorines in rat blood.

The ratios of concentrations of individual PFAS with TF

and EOF were examined in rat blood as indicators of sources

of exposures (i.e., known PFASs/TF and EOF/TF). This

approach was useful to identify general (nonspecific sour-

ces) and specific sources of pollution by PFASs on a

regional scale (Fig. 6a, b). As shown in Fig. 6, the cluster

that indicated nonspecific sources of pollution was from

those samples that had PFASs/TF and EOF/TF ratios\40 %

(blue circle). The cluster that showed specific sources of

pollution was based on those samples with PFASs/TF ratios

of 60 % and EOF/TF ratios of 60 to 80 %. The general

nonspecific sources of PFASs pollution can be related to

environmental releases from human activities (e.g., use of

products such as wax, detergents, garbage, food, waste

water from residents), whereas specific sources of PFASs

pollution are related to industrial sources (e.g., metal and

fluoropolymer manufactures, industry wastewater).

In summary, a nationwide survey of a suite of PFASs,

including short- and long-chain compounds in wild rat

blood and water sources close to their habitat, was con-

ducted in Japan. Contamination profiles of PFASs in wild

rats showed high accumulation in the Kanto and Chiba

regions. There were strong correlations between human

population density and levels of PFOS, PFNA, PFOA and

PFOSA in wild rats. However, long- and short-chain

compounds showed no relationships with population den-

sity. Concentrations of PFASs in rat blood were compa-

rable with those found in human blood from Japan.

Therefore, we propose that wild rats are good indicators of

human exposure to PFASs.

Acknowledgments We thank to Paul Lam and staff in City Uni-

versity of Hong Kong for their support.

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