Certification

of

butyltins

and

phenyltins

in

marine

sediment

certified

reference

material

by

species-specific

isotope-dilution

mass

spectrometric

analysis

using

synthesized

organotin

compounds

A

new

marine

sediment

certified

reference

material

NMIJ

CRM

for

butyltin

and

phenyltin

analysis

has

been

prepared

and

certified

by

the

National

Metrological

Institute

of

Japan

at

the

National

Institute

of

Advanced

Industrial

Science

and

Technology

-LRB-

NMIJ/AIST

-RRB-

Candidate

sediment

material

was

collected

at

a

bay

near

industrial

activity

in

Japan

After

air-drying

sieving

and

mixing

the

material

was

sterilized

with

γ-ray

irradiation

The

material

was

re-mixed

and

packaged

into

glass

bottles

-LRB-

g

each

-RRB-

and

these

were

stored

in

a

freezer

at

°C

Certification

was

performed

by

use

of

three

different

types

of

species-specific

isotope-dilution

mass

spectrometry

-LRB-

SSID

MS

-RRB-

SSID

GC

ICP

MS

SSID

GC

MS

and

SSID

LC

ICP

MS

with

organotin

compounds

synthesized

from

metal

used

as

a

spike

The

mono-butyltin

-LRB-

MBT

-RRB-

dibutyltin

-LRB-

DBT

-RRB-

and

tributyltin

-LRB-

TBT

-RRB-

were

synthesized

as

a

mixture

whereas

the

di-phenyltin

-LRB-

DPhT

-RRB-

and

triphenyltin

-LRB-

TPhT

-RRB-

were

synthesized

individually

Four

different

extraction

methods

mechanical

shaking

ultrasonic

microwave-assisted

and

pressurized

liquid

extraction

were

adopted

to

avoid

possible

analytical

bias

caused

by

non-quantitative

extraction

and

degradation

or

inter-conversion

of

analytes

in

sample

preparations

Tropolone

was

used

as

chelating

agent

in

all

the

extraction

methods

Certified

values

are

given

for

TBT

μg

kg

as

Sn

DBT

μg

kg

as

Sn

MBT

μg

kg

as

Sn

TPhT

μg

kg

as

Sn

and

DPhT

μg

kg

as

Sn

These

levels

are

lower

than

in

other

sediment

CRMs

currently

available

for

analysis

of

organotin

compounds

Introduction

Organotin

compounds

have

wide

ranging

chemical

and

toxicological

properties

They

are

widely

applied

as

stabilizers

of

plastics

as

fungicides

and

pesticides

and

as

marine

antifoulants

-LSB-

-RSB-

Use

of

tributyltin

-LRB-

TBT

-RRB-

and

triphenyltin

-LRB-

TPhT

-RRB-

as

marine

antifouling

agents

has

led

to

their

almost

global

dispersal

Although

use

of

TBT

and

TPhT

has

been

controlled

in

Japan

since

the

compounds

are

still

prominent

in

the

coastal

sea

waters

of

Japan

Thus

these

organotins

and

their

degradation

products

di

and

mono-organotins

in

sediment

are

frequently

monitored

to

evaluate

organotin

pollution

in

water

environment

Quantification

of

organotin

species

in

environmental

samples

is

very

difficult

because

of

their

instability

and

the

low

concentrations

present

Because

long-term

and

worldwide

monitoring

is

necessary

for

these

compounds

comparability

of

the

analytical

results

is

required

-LSB-

-RSB-

The

National

Metrology

Institute

of

Japan

-LRB-

NMIJ

-RRB-

has

already

developed

in

a

marine

sediment

certified

reference

material

-LRB-

NMIJ

CRM

-RRB-

for

analysis

of

mono

di

and

tributyltins

-LRB-

MBT

DBT

and

TBT

-RRB-

-LSB-

-RSB-

Certification

was

performed

using

two

different

types

of

species-specific

isotope-dilution

mass

spectrometric

-LRB-

SSID

MS

-RRB-

method

gas

chromatography

coupled

with

inductively

coupled

plasma

mass

spectrometry

-LRB-

SSID

GC

ICP

MS

-RRB-

-LSB-

-RSB-

and

SSID

GC

MS

combined

with

a

microwave-assisted

extraction

in

which

a

mixture

of

butyltins

synthesized

in

our

laboratory

was

used

as

a

spike

In

we

developed

a

new

marine

sediment

certified

reference

material

NMIJ

CRM

certified

for

di

and

triphenyltins

-LRB-

DPhT

and

TPhT

-RRB-

and

the

butyltins

by

using

three

different

types

of

SSID

MS

method

In

the

SSID

methods

TPhT

and

DPhT

newly

synthesized

from

metal

and

a

mixture

of

butyltins

previously

synthesized

from

metal

-LSB-

-RSB-

were

used

as

spikes

The

certification

strategy

for

NMIJ

CRM

was

almost

the

same

as

that

for

NMIJ

-LSB-

-RSB-

but

with

two

improvements

to

ensure

the

reliability

of

the

CRM

One

was

the

sterilization

of

the

material

with

γ-ray

irradiation

NMIJ

was

not

irradiation-sterilized

because

of

possible

degradation

of

the

organotin

compounds

Yang

et

al.

reported

that

significant

losses

of

butyltins

in

methanol

occurs

during

γ-ray

irradiation

whereas

such

degradation

is

minimal

in

a

sediment

matrix

-LSB-

-RSB-

In

our

preliminary

test

no

substantial

loss

of

organotins

was

observed

on

γ-ray

irradiation

and

thus

irradiation-sterilization

was

performed

for

to

ensure

long

term

stability

The

other

improvement

was

that

a

total

of

six

combinations

of

four

extraction

methods

and

three

SSID

MS

methods

were

used

for

analysis

of

the

organotin

compounds

to

ensure

the

reliability

of

the

certified

values

The

overview

of

the

analysis

for

certification

is

shown

in

Fig.

Although

SSID

MS

methodology

can

correct

most

of

the

systematic

errors

that

occur

in

the

analysis

it

can

not

compensate

for

non-quantitative

extraction

from

the

sample

or

for

species

re-conformation

in

the

sample

preparation

process

-LSB-

-RSB-

Indeed

quantitative

extraction

of

MBT

is

not

an

easy

task

because

MBT

is

strongly

adsorbed

by

sediment

matrices

because

of

its

polarity

-LSB-

-RSB-

It

has

been

reported

that

DBT

is

degraded

during

microwave-assisted

extraction

-LSB-

-RSB-

and

pressurized

liquid

extraction

-LSB-

-RSB-

when

high

microwave

energy

or

high

extraction

temperatures

are

used

although

species

interconversion

can

be

taken

into

account

in

SSID

MS

techniques

that

furnish

data

for

more

than

one

enriched

isotope

as

has

been

described

by

Encinar

and

co-workers

-LSB-

-RSB-

In

this

certification

therefore

the

four

different

types

of

extraction

method

mechanical

shaking

ultrasonic

microwave-assisted

and

pressurized-liquid

extraction

were

adopted

to

avoid

possible

analytical

biases

that

caused

by

non-quantitative

extraction

and

degradation

or

interconversion

of

analytes

SSID

liquid

chromatography

-LRB-

LC

-RRB-

ICP

MS

was

adopted

as

an

SSID

MS

method

in

addition

to

SSID

GC

ICP

MS

and

SSID

GC

MS

because

organotin

separation

by

LC

does

not

need

a

derivatization

step

Thus

use

of

SSID

LC

ICP

MS

can

avoid

potential

degradation

or

interconversion

of

analytes

in

the

derivatization

step

This

paper

mainly

describes

SSID

MS

analysis

for

certification

of

NMIJ

CRM

Marine

Sediment

for

organotin

compounds

Fig.

of

analysis

for

certification

Experimental

Preparation

of

sediment

material

The

starting

material

for

the

CRM

was

collected

in

a

bay

near

industrial

activity

in

the

Kyushu

area

Japan

Approximately

kg

surface

sediment

was

sampled

and

the

water

was

removed

by

filtration

The

sediment

material

was

air-dried

for

weeks

at

room

temperature

-LRB-

°C

-RRB-

After

removal

of

visible

external

materials

-LRB-

rock

shell

etc.

-RRB-

the

sediment

material

was

ground

in

a

high-purity

alumina

ball-mill

Sediment

powder

that

passed

through

a

sieve

was

homogenized

using

a

pan-type

mixer

The

sediment

powder

was

then

bottled

in

glass

bottles

-LRB-

g

each

-RRB-

and

irradiation

sterilized

-LRB-

kGy

-RRB-

The

irradiated

powder

was

re-mixed

and

homogenized

by

use

of

a

rocking

mixer

-LRB-

Aichi

Electric

Japan

-RRB-

as

a

precaution

against

an

inhomogeneous

degradation

of

organotin

compounds

during

sterilization

Finally

the

powder

was

placed

in

amber

glass

bottles

-LRB-

g

each

-RRB-

and

stored

at

°C

Conversion

to

dry

mass

basis

The

concentrations

of

the

constituents

of

this

CRM

are

given

on

a

dry-mass

basis

A

dry

mass

correction

factor

for

sample

humidity

was

determined

by

drying

the

sample

at

°C

After

h

the

sediment

sample

reached

constant

weight

so

it

was

decided

the

drying

time

would

be

h

in

this

experiment

The

dry

mass

correction

factor

at

the

time

of

certification

was

-LRB-

average

standard

deviation

for

ten

different

bottles

-RRB-

Chemicals

Tributyltin

-LRB-

TBT

-RRB-

chloride

dibutyltin

-LRB-

DBT

-RRB-

chloride

and

triphenyltin

chloride

-LRB-

TPhT

-RRB-

were

purchased

from

Wako

Pure

Chemicals

-LRB-

Osaka

Japan

-RRB-

Monobutyltin

-LRB-

MBT

-RRB-

chloride

and

diphenyltin

chloride

-LRB-

DPhT

-RRB-

were

purchased

from

Aldrich

-LRB-

Milwaukee

WI

USA

-RRB-

Tripropyltin

-LRB-

TPrT

-RRB-

chloride

was

purchased

from

Merck

-LRB-

Darmstadt

Germany

-RRB-

Standard

solutions

of

each

organotin

compound

except

DPhT

were

separately

prepared

by

dissolving

each

compound

in

methanol

-LRB-

pesticide

analysis

grade

Wako

-RRB-

to

avoid

any

disproportionation

reactions

with

other

organotins

The

standard

solution

of

DPhT

was

prepared

by

dissolving

in

mol

L

HCl

in

methanol

to

prevent

self-disproportionation

All

standard

solutions

were

stored

at

°C

and

diluted

working

solutions

were

prepared

daily

before

the

analysis

Sodium

tetraethylborate

-LRB-

-RRB-

was

purchased

from

Wako

Pure

Chemicals

A

m/v

solution

of

was

prepared

in

a

glove

box

that

was

purged

with

gas

Other

chemicals

used

were

of

analytical

reagent

grade

Pure

water

prepared

by

use

of

a

Milli-Q

water-purification

system

-LRB-

resistivity

MΩ

cm

Nihon

Millipore

Kogyo

Tokyo

Japan

-RRB-

was

used

throughout

the

experiments

Synthesis

of

the

organotin

compounds

The

mixture

of

butyltin

compounds

used

as

the

spike

for

the

ID

methods

were

synthesized

from

tin

metal

-LRB-

enriched

-RRB-

purchased

from

Trace

Science

International

-LRB-

Ontario

Canada

-RRB-

The

synthetic

procedure

has

been

described

previously

-LSB-

-RSB-

DPhT

and

TPhT

were

individually

synthesized

from

tin

metal

-LRB-

enriched

-RRB-

purchased

from

Nippon

Sanso

-LRB-

Tokyo

Japan

-RRB-

The

synthetic

procedures

were

almost

the

same

as

those

described

elsewhere

-LSB-

-RSB-

A

mixture

of

ca.

g

metal

and

ca.

g

iodine

was

introduced

into

a

round-bottomed

flask

containing

acetic

acid

-LRB-

mL

-RRB-

and

acetic

anhydride

-LRB-

mL

-RRB-

A

small

crystal

of

potassium

iodide

was

added

as

catalyst

and

the

mixture

was

gently

heated

to

reflux

After

cooling

in

an

ice

bath

orange

crystals

of

-LRB-

g

-RRB-

were

collected

was

placed

in

a

flask

containing

mL

diethyl

ether

and

mol

L

phenyl

magnesium

bromide

in

THF

was

added

dropwise

After

heating

to

reflux

for

h

the

white-pink

precipitate

was

collected

by

filtration

after

the

Grignard

reagent

had

been

hydrolyzed

with

water

The

solid

was

dissolved

in

dichloromethane

and

the

solution

was

filtered

to

remove

insoluble

impurities

The

dichloromethane

was

removed

to

leave

-LRB-

TePhT

-RRB-

as

a

white

solid

this

was

washed

with

ethanol

-LRB-

g

-RRB-

and

-LRB-

g

-RRB-

were

mixed

in

a

glass

tube

and

heated

to

°C

The

reaction

products

were

dissolved

in

ethanol

and

the

insoluble

products

were

removed

by

filtration

By

the

addition

of

potassium

fluoride

solution

fluoride

was

obtained

as

an

insoluble

salt

The

fluoride

was

isolated

by

filtration

washed

with

a

minimum

amount

of

ethanol

and

then

treated

with

conc

hydrochloric

acid

and

extracted

with

pentane

The

extracts

were

dried

with

and

the

pentane

was

removed

to

yield

TPhT

chloride

as

a

white

powder

DPhT

dichloride

was

prepared

by

treating

TePhT

with

HCl

The

product

was

extracted

with

pentane

The

extracts

were

dried

with

and

the

pentane

was

removed

to

yield

DPhT

dichloride

Extraction

procedure

Ultrasonic

extraction

The

ultrasonic

extraction

procedure

for

GC

ICP

MS

and

GC

MS

was

as

follows

The

sediment

sample

-LRB-

ca.

g

-RRB-

was

placed

in

a

PFA

centrifuge

tube

and

spiked

with

an

appropriate

amount

of

the

spikes

Then

g

NaCl

mL

toluene

containing

tropolone

and

mL

acetic

acid

methanol

-LRB-

-RRB-

were

added

to

the

tubes

The

resulting

mixtures

were

extracted

in

an

ultrasonic

bath

for

min

at

°C

After

addition

of

mL

water

the

tubes

were

again

shaken

for

good

phase

separation

and

then

centrifuged

at

rpm

for

min

Finally

the

upper

toluene

layer

was

collected

as

the

extract

For

LC

ICP

MS

the

extraction

solvent

was

replaced

with

mL

acetic

acid

methanol

-LRB-

-RRB-

containing

tropolone

and

the

same

extraction

procedure

was

performed

Mechanical

shaking

extraction

The

sediment

sample

-LRB-

ca.

g

-RRB-

was

placed

in

a

PFA

centrifuge

tube

and

spiked

with

an

appropriate

amount

of

the

spikes

Then

g

NaCl

mL

toluene

containing

tropolone

and

mL

mol

L

HCl

in

methanol

were

added

to

the

tubes

and

the

resulting

mixtures

were

mechanically

shaken

for

min

After

addition

of

mL

water

the

tubes

were

again

shaken

for

good

phase

separation

and

then

were

centrifuged

at

rpm

for

min

Finally

the

upper

toluene

layer

was

collected

as

the

extract

Microwave-assisted

extraction

Closed-vessel

microwave-assisted

extraction

-LRB-

MAE

-RRB-

was

performed

The

microwave

system

used

was

Mars

X

-LRB-

CEM

USA

-RRB-

The

sediment

sample

-LRB-

ca.

g

-RRB-

was

placed

in

a

PFA

vessel

and

spiked

with

an

appropriate

amount

of

the

spikes

Then

g

NaCl

mL

toluene

containing

tropolone

and

mL

mol

L

acetic

acid

in

methanol

were

added

to

the

vessel

The

microwave

irradiation

program

was

ramp

to

°C

in

min

then

hold

for

min

The

mixture

was

transferred

to

a

PFA

tube

containing

mL

water

and

the

tube

was

shaken

for

good

phase

separation

and

then

centrifuged

at

rpm

for

min

Finally

the

upper

toluene

layer

was

collected

as

the

extract

Pressurized

liquid

extraction

Pressurized

liquid

extraction

-LRB-

PLE

-RRB-

was

performed

with

an

ASE

instrument

-LRB-

Dionex

USA

-RRB-

The

sediment

sample

-LRB-

ca.

g

-RRB-

was

placed

in

an

stainless

extraction

cell

containing

a

glass

filter

and

quartz

sand

and

was

spiked

with

an

appropriate

amount

of

the

spikes

After

filling

the

extraction

vessel

with

quartz

sand

the

cell

was

placed

in

the

PLE

system

The

extraction

solvent

used

was

mol

L

acetic

acid

in

methanol

containing

tropolone

The

extraction

conditions

were

oven

temp

°C

pressure

MPa

static

time

and

cycle

was

min

times

The

extract

obtained

was

transferred

to

a

PFA

tube

containing

mL

water

and

mL

toluene

and

the

tube

was

shaken

for

good

phase

separation

and

then

centrifuged

at

rpm

for

min

Finally

the

upper

toluene

layer

was

collected

as

the

extract

Derivatization

procedure

for

GC

ICP

MS

and

GC

MS

The

extracted

solutions

were

transferred

to

PFA

centrifuge

tubes

and

mL

ammonium-acetate

buffer

-LRB-

mol

L

pH

-RRB-

and

mL

solution

were

added

The

tubes

were

mechanically

shaken

for

min

for

ethylation

and

extraction

and

then

centrifuged

to

achieve

phase

separation

The

toluene

layers

were

also

transferred

to

the

tubes

and

mixed

with

g

anhydrous

sodium

sulfate

to

remove

the

water

Clean-up

procedure

Clean-up

on

a

Presep-C

Florisil

cartridge

-LRB-

Wako

Japan

-RRB-

was

performed

after

the

derivatization

except

for

LC

ICP

MS

measurement

The

toluene

layer

was

evaporated

to

ca.

mL

under

a

stream

of

gas

and

loaded

on

to

a

cartridge

previously

conditioned

with

hexane

The

eluent

from

the

cartridge

was

collected

in

a

glass

centrifuge

tube

Hexane

-LRB-

mL

-RRB-

containing

-LRB-

v/v

-RRB-

diethyl

ether

was

then

also

loaded

on

the

cartidge

and

the

eluent

was

collected

in

the

glass

tube

Finally

the

collected

eluent

was

evaporated

to

mL

under

a

stream

of

gas

and

used

as

the

measurement

sample

solution

For

the

LC

ICP

MS

measurement

a

clean-up

procedure

using

cartridge

-LRB-

Varian

Australia

-RRB-

was

performed

The

extract

was

loaded

on

to

a

cartridge

previously

conditioned

with

acetone

The

eluent

from

the

cartridge

was

collected

in

a

glass

centrifuge

tube

evaporated

to

mL

under

a

stream

of

gas

and

used

as

the

measurement

sample

solution

Determination

of

organotin

compounds

by

ID

GC

ICP

MS

The

GC

ICP

MS

used

was

that

the

GC

-LRB-

Agilent

-RRB-

was

coupled

with

an

ICP

MS

-LRB-

Yokogawa

Analytical

Systems

Tokyo

Japan

-RRB-

by

means

of

a

manufactured

transfer-line

-LRB-

Yokogawa

Analytical

Systems

-RRB-

The

GC

column

was

ms

-LRB-

m

mm

i.d.

μm

film

thickness

-RRB-

The

measured

masses

were

m/z

and

The

operating

conditions

and

procedures

for

GC

ICP

MS

were

similar

to

those

described

elsewhere

-LSB-

-RSB-

The

concentrations

of

organotin

compounds

were

calculated

by

inserting

each

value

into

the

ID

equation

Eq

-LRB-

-RRB-

based

on

a

double-ID

method

-LSB-

-RSB-

where

Cx

is

the

analyte

concentration

in

the

sample

-LRB-

mol

g

-RRB-

mx

the

mass

of

sample

-LRB-

g

-RRB-

used

for

the

sample-spike

mixture

my

the

mass

of

spike

solution

-LRB-

g

-RRB-

used

for

the

sample-spike

mixture

the

mass

of

spike

solution

-LRB-

g

-RRB-

used

for

the

standard-spike

mixture

mz

the

mass

of

standard

solution

-LRB-

g

-RRB-

used

for

standard-spike

mixture

R

is

the

ratio

in

the

sample

spike

mixed

solution

R

is

the

ratio

in

the

standard

spike

mixed

solution

Rx

is

the

ratio

in

the

sample

solution

Ry

is

the

ratio

from

the

spike

solution

Rz

is

the

ratio

in

the

standard

solution

w

is

the

correction

factor

for

dry

mass

n

is

the

number

of

replicate

measurements

and

k

Ky

and

K

are

the

mass

discrimination

correction

factors

for

each

isotope

ratio

which

were

calculated

from

the

area

ratio

of

for

TPrT

in

each

chromatographic

run

-LSB-

-RSB-

P

is

the

purity

of

each

organotin

chloride

reagent

used

for

preparing

the

standard

solutions

D

is

the

dilution

factor

for

each

organotin

chloride

in

the

gravimetric

dilution

of

the

standard

solutions

Mw

the

molecular

weight

of

each

organotin

chloride

B

the

procedure

blank

and

E

the

variation

introduced

by

extraction

to

the

analytical

results

Determination

of

organotins

by

ID

GC

MS

The

GC

MS

used

was

Agilent

model

-LRB-

Agilent

Technologies

Wilmington

DE

USA

-RRB-

with

an

ms

column

-LRB-

m

mm

i.d.

μm

film

thickness

-RRB-

The

measured

masses

were

m/z

and

for

MBT

m/z

and

for

DBT

and

TBT

m/z

and

for

DPhT

and

m/z

and

for

TPhT

The

other

operating

conditions

and

procedures

for

GC

ICP

MS

analysis

were

similar

to

those

described

previously

-LSB-

-RSB-

The

concentrations

of

organotins

were

calculated

by

inserting

each

value

into

Eq

-LRB-

-RRB-

below

in

which

it

was

assumed

that

the

relationship

between

mass

ratio

and

peak

ratio

was

linear

where

Cx

is

the

analyte

concentration

in

the

sample

-LRB-

mol

g

-RRB-

mx

the

mass

of

sample

-LRB-

g

-RRB-

used

for

the

sample-spike

mixture

my

the

mass

of

spike

solution

-LRB-

g

-RRB-

used

for

the

sample

spike

mixture

RWL

the

ratio

of

the

mass

in

the

spike

solution

-LRB-

g

-RRB-

to

the

mass

in

the

standard

solution

-LRB-

lower

level

-RRB-

RWH

the

ratio

of

mass

in

the

spike

solution

-LRB-

g

-RRB-

to

the

mass

in

the

standard

solution

-LRB-

higher

level

-RRB-

R

the

measured

abundance

ratio

in

the

sample

spike

mixed

solution

RL

the

measured

abundance

ratio

in

the

standard

spike

mixed

solution

-LRB-

lower

level

-RRB-

RH

the

measured

abundance

ratio

in

the

standard

spike

mixed

solution

-LRB-

higher

level

-RRB-

w

the

correction

factor

for

dry

mass

P

the

purity

of

each

organotin

chloride

reagent

used

for

the

preparing

the

standard

solutions

D

the

dilution

factor

for

each

organotin

chloride

in

gravimetric

dilution

of

the

standard

solutions

Mw

the

molecular

weight

of

each

organotin

chloride

B

the

procedure

blank

and

E

the

variation

introduced

by

the

extraction

to

the

analytical

results

The

mass

bias

correction

was

not

performed

for

GC

MS

measurements

In

this

experiment

we

prepared

several

sample

spike

mixed

solutions

of

different

mass

ratios

and

chose

the

solutions

having

values

of

RL

and

RH

slightly

lower

and

higher

than

the

R

value

for

calculation

Determination

of

organotin

compounds

by

ID

LC

ICP

MS

The

LC

ICP

MS

system

used

consisted

of

a

HPLC

pump

-LRB-

Inert

model

GL

Science

Tokyo

Japan

-RRB-

a

Nanospace

auto-injection

sampler

-LRB-

Siseido

Tokyo

Japan

-RRB-

and

an

Agilent

ICP

MS

-LRB-

Yokogawa

Analytical

Systems

Japan

-RRB-

The

LC

column

used

was

a

Mitysil

GP

-LRB-

mm

mm

i.d.

μm

-RRB-

All

tubing

used

was

in

-LRB-

mm

i.d.

-RRB-

PEEK

The

mobile

phase

was

acetonitrile

water

acetic

acid

tetraethylammonium

chloride

tropolone

-LRB-

v/v

-RRB-

The

mobile

phase

flow

rate

was

μL

min

and

the

sample

volume

injected

was

μL

The

sample-introduction

device

for

ICP

MS

was

modified

from

the

default

settings

as

follows

a

PFA

μflow

of

μL

min

was

used

as

a

nebulizer

the

double-path

Scott

type

spray

chamber

was

cooled

to

°C

a

mm

dimmer

injector

torch

was

used

and

additional

-LRB-

L

min

-RRB-

gas

was

mixed

with

the

argon

axial

gas

flow

via

a

T-adaptor

The

LC

column

was

directly

connected

via

a

PEEK

adaptor

The

concentrations

of

organotins

were

calculated

by

use

of

Eq

-LRB-

-RRB-

Homogeneity

study

The

between-bottle

homogeneity

of

the

CRM

was

determined

by

analyzing

sub-samples

taken

from

ten

bottles

selected

from

the

lot

of

bottles

All

the

organotin

compounds

were

determined

by

ID

GC

ICP

MS

after

ultrasonic

extraction

Analysis

of

variance

-LRB-

ANOVA

-RRB-

of

the

data

was

performed

and

mean

squares

within

group

-LRB-

MSwithin

-RRB-

and

among

group

-LRB-

MSamong

-RRB-

were

calculated

Then

standard

deviations

between

bottles

-LRB-

sbb

-RRB-

were

calculated

by

use

of

Eq

-LRB-

-RRB-

If

the

measurement

method

was

insufficient

repeatable

the

effect

of

analytical

variation

on

the

standard

deviation

between

units

ubb

was

calculated

and

used

to

estimate

the

inhomogeneity

-LSB-

-RSB-

The

ubb

was

calculated

by

use

of

Eq

-LRB-

-RRB-

where

denotes

the

degrees

of

freedom

of

MSwithin

Results

and

discussion

Homogeneity

study

In

the

homogeneity

study

between-bottle

inhomogeneity

-LRB-

sbb

-RRB-

was

only

observed

for

TPhT

so

ubb

was

used

as

uncertainty-derived

inhomogeneity

for

TPhT

A

relatively

large

sbb

value

-LRB-

-RRB-

was

obtained

for

DPhT

although

this

value

was

not

very

large

compared

with

uncertainties

from

other

sources

for

example

the

uncertainty

of

analytical

results

and

between-method

variance

as

is

shown

later

These

results

indicate

that

this

material

may

be

considered

homogeneous

for

butyltin

and

phenyltin

analysis

Stability

of

organotins

in

this

material

We

have

been

monitoring

the

stability

of

the

butyltin

compounds

in

NMIJ

since

All

three

butyltins

were

sufficiently

stable

during

storage

for

five

years

at

°C

even

though

was

not

sterilized

The

new

CRM

contains

almost

the

same

butyltin

concentrations

as

its

sample

composition

was

also

similar

and

it

was

sterilized

with

γ-ray-irradiation

Thus

the

butyltins

in

CRM

will

also

be

stable

for

at

least

five

years

Both

the

phenyltin

and

butyltin

compounds

in

BCR

which

was

pasteurized

at

°C

have

been

shown

to

be

stable

when

the

material

is

stored

at

below

°C

-LSB-

-RSB-

Therefore

we

decided

to

store

at

°C

and

to

assess

the

stability

of

the

material

by

measuring

each

organotin

by

ID

GC

ICP

MS

over

a

period

of

year

and

that

for

Characterization

of

the

synthesized

organotins

Characterization

of

the

synthesized

organotins

was

performed

by

GC

ICP

MS

after

mixing

of

each

dilution

with

methanol

and

ethylation

with

Figure

shows

the

GC

ICP

MS

chromatograms

obtained

at

m/z

and

for

the

synthesized

organotins

mixture

The

DPhT

and

TPhT

could

be

synthesized

individually

The

molar

ratio

of

the

and

in

the

synthesized

mixture

measured

by

GC

ICP

MS

was

approximately

Fig.

ICP

MS

chromatograms

of

and

obtained

for

the

mixture

of

organotin

compounds

The

chromatogram

of

was

shifted

s

and

cps

for

clarity

Ten

replicate

measurements

of

the

ethylated

organotin

mixture

by

GC

ICP

MS

were

performed

to

obtain

the

ratio

for

each

compound

The

mass

bias

was

corrected

with

the

in-run

correction

method

with

TPrT

-LSB-

-RSB-

The

isotope

ratios

for

were

-LRB-

mean

standard

uncertainty

n

-RRB-

with

no

significant

differences

The

isotope

ratios

for

DPhT

and

TPhT

were

and

respectively

Assay

for

the

standard

solution

of

the

organotins

To

obtain

a

standard

solution

with

a

well-defined

concentration

an

assay

was

performed

to

determine

the

purity

of

the

natural

abundance

organotin

chloride

reagents

used

to

prepare

the

standard

solutions

Details

of

the

assay

have

been

described

elsewhere

-LSB-

-RSB-

In

brief

the

amounts

of

organic

and

inorganic

tin

species

impurities

in

the

organotin

chloride

reagents

were

estimated

by

GC

ICP

MS.

The

inorganic

impurities

except

for

Sn

were

checked

by

ICP

MS.

The

non-organotin

organic

impurities

were

also

checked

by

GC

FID

The

water

content

of

the

organotin

chloride

reagents

were

evaluated

with

a

Karl

Fisher

coulometric

titrator

-LRB-

Model

Mitsubishi

Japan

-RRB-

The

main

impurities

in

the

organotin

chloride

reagents

used

are

water

and

tin

species

Small

amounts

of

inorganic

and

organotin

impurities

were

observed

in

both

the

ethylated

MBT

and

ethylated

DBT

solutions

and

significant

amounts

of

several

organotin

impurities

were

observed

in

the

ethylated

TBT

ethylated

TPhT

and

ethylated

DPhT

solutions

Among

the

organotin

impurities

observed

in

each

ethylated

organotin

solution

inorganic

tin

MBT

DBT

and

tetrabutyltin

-LRB-

TeBT

-RRB-

MPhT

and

DPhT

were

identified

from

their

retention

times

in

GC

ICP

MS

measurement

and

isomers

of

TBT

-LRB-

iso-TBT

-RRB-

and

dioctyldibutyltin

-LRB-

DOcDBT

-RRB-

were

identified

from

the

fragment-ion

patterns

obtained

by

GC

MS.

Two

organotin

impurities

that

could

not

be

identified

from

their

retention

time

were

also

observed

in

the

ethylated-MBT

solution

but

the

amounts

of

those

were

small

-LRB-

-RRB-

The

purities

of

the

organotin

chloride

reagent

obtained

were

for

MBT

for

DBT

for

TBT

for

DPhT

and

for

TPhT

The

stability

of

the

standard

solution

during

certification

is

also

important

Van

et

al.

reported

that

DPhT

in

methanol

was

not

stable

because

of

its

disproportionation

reaction

although

solutions

of

the

butyltins

and

TPhT

were

found

to

be

stable

-LSB-

-RSB-

DPhT

also

reacted

with

MBT

in

mixed

solutions

in

methanol

In

contrast

Arnold

et

al.

reported

that

DPhT

in

methanol

containing

mol

L

HCl

was

stable

for

six

months

-LSB-

-RSB-

Hence

the

stability

test

for

DPhT

in

methanol

with

and

without

mol

L

HCl

was

performed

by

GC

ICP

MS

after

storage

at

°C

for

one

month

Redistribution

of

DPhT

to

MPhT

and

TPhT

was

observed

for

the

methanol

solution

as

has

been

described

elsewhere

-LSB-

-RSB-

In

contrast

no

degradation

or

redistribution

of

DPhT

was

observed

for

the

methanol

solution

containing

mol

L

HCl

Plazzogna

et

al.

reported

that

the

redistribution

reaction

between

monomethyltrialkyltin

-LRB-

-RRB-

and

dimethyltin

dichloride

-LRB-

-RRB-

in

methanol

is

occurred

with

dissociation

of

-LSB-

-RSB-

They

also

reported

that

the

redistribution

reaction

can

be

prevented

by

addition

of

chloride

ion

as

NaCl

which

inhibits

the

dissociation

of

in

methanol

For

DPhT

in

methanol

the

disproportionation

reaction

occurred

with

dissociation

of

and

was

prevented

by

addition

of

HCl

which

inhibited

the

dissociation

Consequently

the

standard

solution

of

DPhT

was

prepared

by

dissolving

in

methanol

containing

mol

L

HCl

to

prevent

the

self-disproportionation

reaction

Evaluation

of

the

degradation

of

DBT

and

TPhT

during

extraction

Because

SSID

MS

methodology

can

not

compensate

for

non-quantitative

extraction

from

the

sample

aggressive

conditions

are

required

for

solid

liquid

extraction

of

strongly

adsorbed

species

for

example

MBT

Degradation

or

interconversion

of

species

can

occur

under

the

aggressive

extraction

conditions

-LSB-

-RSB-

Indeed

it

has

been

reported

that

degradation

of

DBT

occurs

during

microwave

assisted

extraction

-LSB-

-RSB-

and

pressurized

liquid

extraction

-LSB-

-RSB-

when

high-energy

microwaves

or

high

extraction

temperatures

are

used

Degradation

of

TPhT

during

sample

pretreatment

has

also

been

reported

-LSB-

-RSB-

In

this

certification

therefore

degradation

of

DBT

and

TPhT

during

extraction

was

checked

using

an

DBT

solution

obtained

from

the

LGC

UK

-LSB-

-RSB-

and

the

single

solution

of

the

synthesized

TPhT

Both

DBT

and

TPhT

solutions

were

used

to

spike

the

sediment

sample

and

then

the

four

extraction

methods

were

performed

After

derivatization

and

clean-up

the

extract

was

analyzed

by

GC

ICP

MS.

Because

the

TPhT

solution

does

not

contain

any

phenyltins

degradation

of

TPhT

in

each

extraction

was

evaluated

from

the

difference

between

the

natural

abundance

isotope

ratio

and

the

measured

ratios

of

for

DPhT

On

the

other

hand

the

DBT

solution

contains

small

amounts

-LRB-

ca.

-RRB-

of

MBT

as

an

impurity

Therefore

the

degradation

of

DBT

in

each

extraction

was

evaluated

from

the

difference

between

the

natural

abundance

isotope

ratios

and

the

ratios

of

for

MBT

corrected

for

MBT

In

this

experiment

the

measured

ratios

for

DPhT

obtained

for

all

four

extraction

methods

closely

matched

the

natural

abundance

and

the

results

suggest

that

no

significant

degradation

of

TPhT

to

DPhT

occurred

during

extraction

The

degradation

of

DBT

to

MBT

was

also

not

observed

in

ultrasonic

extraction

and

mechanical

shaking

extraction

Slight

degradation

of

DBT

to

MBT

was

observed

in

microwave-assisted

extraction

MAE

-LRB-

-RRB-

and

in

pressurized

liquid

extraction

PLE

-LRB-

-RRB-

these

degradation

levels

were

lower

than

those

reported

by

Encinar

-LSB-

-RSB-

-LRB-

in

both

extractions

under

their

optimized

conditions

-RRB-

These

differences

could

be

because

of

different

extraction

conditions

The

sample

size

used

in

our

experiments

was

two

to

four

times

that

used

by

Encinar

and

the

concentrations

of

acetic

acid

we

used

were

lower

In

addition

tropolone

was

used

as

a

chelating

reagent

in

all

the

four

extraction

methods

Formation

of

the

tropolone-chelating

complex

might

prevent

degradation

The

slight

degradation

of

DBT

observed

would

not

significantly

affect

the

analytical

results

obtained

by

use

of

MAE

and

PLE

because

the

typical

uncertainty

of

isotope-ratio

measurement

by

GC

ICP

MS

in

this

study

was

in

the

range

to

and

the

relative

standard

deviations

of

the

analytical

results

for

DBT

obtained

by

use

of

MAE

and

PLE

were

approximately

As

is

shown

later

the

analytical

results

obtained

by

use

of

the

four

different

extraction

methods

were

in

good

agreement

within

the

range

of

uncertainties

These

results

indicate

that

analytical

biases

caused

by

degradation

or

inter-conversion

of

analytes

in

the

extraction

would

be

negligible

in

this

certification

Analytical

results

obtained

by

each

method

Determination

of

the

organotin

compounds

in

the

candidate

material

by

the

six

analytical

methods

was

performed

for

the

certification

Figures

to

show

the

GC

ICP

MS

GC

MS

and

LC

ICP

MS

chromatograms

obtained

for

the

sample

extract

spiked

with

the

organotin

compounds

It

is

apparent

from

Figs.

and

that

adequate

sensitivity

for

all

the

organotins

was

obtained

in

GC

ICP

MS

and

GC

MS

measurements

Adequate

sensitivity

for

the

organotin

compounds

except

for

DPhT

was

also

obtained

in

LC

ICP

MS

the

sensitivity

for

DPhT

was

insufficient

to

obtain

a

reliable

isotope-ratio

measurement

The

relative

standard

deviation

for

DPhT

in

LC

ICP

MS

measurement

was

approximately

in

triplicate

measurement

and

thus

variation

of

the

analytical

results

obtained

was

quite

large

Thus

the

analytical

results

for

DPhT

obtained

by

ID

LC

ICP

MS

were

not

adopted

for

the

certification

The

analytical

results

with

their

uncertainties

obtained

by

both

analytical

techniques

are

summarized

in

Table

The

values

were

calculated

as

mass

fractions

-LRB-

based

on

dry

mass

-RRB-

The

combined

standard

uncertainties

for

the

analytical

results

obtained

by

each

method

were

calculated

by

use

of

Eqs

-LRB-

-RRB-

or

-LRB-

-RRB-

The

uncertainties

related

to

the

standard

solutions

-LRB-

uncertainty

of

P

and

D

in

Eqs

-LRB-

-RRB-

and

-LRB-

-RRB-

-RRB-

were

not

combined

into

uc

because

the

same

reagents

were

used

for

both

analyses

They

were

combined

later

when

the

uncertainties

in

the

certified

values

were

calculated

Fig.

ICP

MS

chromatograms

of

and

obtained

for

the

sample

extract

spiked

with

butyltin

and

phenyltin

compounds

The

chromatogram

of

was

shifted

s

and

cps

for

clarityFig

MS

chromatograms

of

each

measured

mass

obtained

for

the

sample

extract

spiked

with

butyltin

and

phenyltin

compounds

The

chromatogram

for

was

shifted

s

and

cps

for

clarityFig

ICP

MS

chromatograms

of

and

obtained

for

the

sample

extract

spiked

with

butyltin

and

phenyltin

compounds

The

chromatogram

of

was

shifted

s

and

cps

for

clarityTable

results

obtained

by

use

of

six

combinations

of

extraction

and

measurement

methods

Result

combined

standard

ua

-LRB-

μg

kg

as

Sn

-RRB-

ExtractionbMeasurementTBTDBTMBTTPhTDPhTUSEGC

ICP

USEGC

USELC

ICP

cMAEGC

ICP

PFEGC

ICP

MWEGC

ICP

aCalculated

by

use

of

Eqs

-LRB-

-RRB-

or

-LRB-

-RRB-

bUSE

ultrasonic

extraction

MSE

mechanical

shaking

extraction

PFE

pressurized

fluid

extraction

MAE

microwave

assisted

extractioncTechnically

invalid

The

analytical

results

obtained

by

use

of

the

six

methods

were

in

good

agreement

within

the

range

of

their

uncertainties

this

agreement

may

indicate

there

were

no

significant

analytical

biases

between

measurement

and

extraction

techniques

for

all

the

analytes

Therefore

all

the

analytical

results

obtained

were

treated

equally

for

calculation

and

evaluation

of

the

certified

values

and

their

uncertainties

Establishing

certified

values

The

analytical

results

in

Table

were

combined

to

provide

the

certified

values

listed

in

Table

Certified

values

are

the

weighted

means

of

these

results

from

the

six

methods

where

-LRB-

ui

is

the

uncertainty

of

each

result

-RRB-

was

used

as

a

weight

Certified

values

are

available

for

the

concentrations

of

MBT

DBT

TBT

DPhT

and

TPhT

as

tin

Table

values

and

their

uncertainties

for

mass

fractions

of

organotin

compounds

in

NMIJ

CRM

TBTDBTMBTTPhTDPhTCertified

value

-LRB-

mass

fraction

μg

kg

as

Sn

-RRB-

Relative

standard

uncertainty

-LRB-

-RRB-

Calibration

solution

Analytical

results

Between

method

In-homogeneity

sbb

or

Combined

uncertainty

Relative

-LRB-

-RRB-

Absolute

-LRB-

μg

kg

as

Sn

-RRB-

Expanded

uncertainty

U

-LRB-

k

-RRB-

-LRB-

mass

fraction

μg

kg

Sn

-RRB-

Uncertainty

of

the

certified

values

The

uncertainties

of

the

certified

values

included

the

combined

effects

of

method

imprecision

possible

bias

effects

among

methods

material

inhomogeneity

and

stability

Components

of

the

uncertainty

of

each

certified

value

are

listed

in

Table

Because

the

same

reagents

for

calibration

solutions

were

used

for

all

the

measurements

uncertainty

of

the

calibration

solutions

-LRB-

ucal

-RRB-

was

combined

with

the

uncertainty

of

the

certified

values

separately

from

the

uncertainty

of

the

analytical

results

as

is

described

above

The

property

value

was

the

weighted

mean

of

the

two

results

and

so

the

combined

uncertainty

of

each

analytical

result

-LRB-

uanal

-RRB-

was

given

by

Eq

-LRB-

-RRB-

where

xi

are

the

results

obtained

by

use

of

the

six

methods

and

wi

are

their

weights

The

between-method

variance

-LRB-

umethod

-RRB-

was

calculated

from

ANOVA

on

the

data

from

the

three

techniques

The

uncertainties

derived

from

material

inhomogeneity

-LRB-

ubb

-RRB-

were

the

estimates

in

the

homogeneity

study

As

is

described

above

the

butyltin

and

phenyltin

compounds

would

be

stable

at

lest

five

years

at

our

storage

temperature

-LRB-

°C

-RRB-

we

did

not

include

the

uncertainty

from

stability

The

expanded

uncertainty

in

each

certified

value

is

equal

to

U

kuc

where

uc

is

the

combined

standard

uncertainty

with

coverage

factor

k

corresponding

to

confidence

intervals

Comparison

with

other

CRMs

The

certified

values

for

and

the

are

summarized

in

Table

In

comparison

with

the

the

uncertainties

of

the

certified

values

for

DBT

and

MBT

in

are

significantly

smaller

despite

the

similar

concentrations

of

the

compounds

The

main

contributors

to

the

uncertainty

in

the

certified

values

for

DBT

and

MBT

in

were

ubb

and

uanal

-LSB-

-RSB-

The

homogeneity

of

CRM

was

improved

by

remixing

after

X-ray

sterilization

and

the

reliability

of

the

analytical

methods

used

for

certification

was

also

better

than

those

used

for

Thus

these

improvements

may

lead

to

the

small

uncertainties

Table

values

for

butyltin

and

phenyltin

in

and

other

CRMs

certified

by

use

of

SSIDMS

-LRB-

unit

µg

as

Sn

-RRB-

Most

recently

several

sediment

reference

materials

for

butyltin

analysis

certified

by

use

of

SSID

MS

have

been

issued

by

the

National

Research

Council

Canada

-LRB-

NRCC

Canada

-RRB-

The

certified

values

of

the

CRMs

are

also

summarized

in

Table

and

were

certified

and

was

recertified

by

NRCC

Their

certified

and

re-certified

values

were

based

on

results

from

the

Comite

Consultatif

pour

la

Quantite

de

Matiere

-LRB-

CCQM

-RRB-

comparisons

-LSB-

-RSB-

in

which

we

participated

These

CRMs

have

certified

values

for

DBT

and

TBT

only

and

their

concentrations

are

higher

than

those

in

and

The

BCR

freshwater

sediment

for

butyltins

and

phenyltins

are

also

available

from

the

European

Commission

Joint

Research

Centre

-LRB-

IRMM

Belgium

-RRB-

although

SSID

MS

was

not

used

for

the

certification

The

certified

concentrations

for

BCR

are

higher

than

those

for

In

Japan

typical

butyltin

and

phenyltin

concentrations

observed

in

environmental

monitoring

are

not

as

high

as

they

used

to

be

Reported

TBT

concentrations

in

sediment

were

below

μg

kg

as

Sn

in

environmental

monitoring

by

the

Ministry

of

the

Environment

Japan

in

-LSB-

-RSB-

By

using

these

CRMs

properly

a

wide

concentration

range

can

be

covered

in

routine

laboratories

B O O O O O O B B I I O O O O O O O O O B I O O O B B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O B I O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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