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Aroma See Sensory Evaluation: Sensory Characteristics of Human Foods; Food Acceptability and Sensory

Evaluation; Practical Considerations; Sensory Difference Testing; Sensory Rating and Scoring Methods;

Descriptive Analysis; Appearance; Texture; Aroma; Taste

Aroma Compounds See Flavor (Flavour) Compounds: Structures and Characteristics; Production

Methods

ARSENIC

Contents

Properties and Determination

Requirements and Toxicology

Properties and Determination

N Hata, I Kasahara, S Taguchi and K Goto, Toyama

University, Toyama, Japan

Background

0001 Arsenic is located in group 5A of the periodic table

and has an atomic number of 33 with an atomic

weight of 74.922. The electron configuration is

(1s)

(2s)

(2p)

(3s)

(3p)

(3d)

(4s)

(4p)

Chemical Properties

0002 The structures of some of several important arsenic

compounds and their acid dissociation constants

(pK

) are shown in Figure 1.

0003 Inorganic arsenic (As) occurs in four oxidation

states, As(V) as in arsenate (AsO

3

), As(III) as in

arsenite (AsO

3

), As(0), and As(-III) as in arsine

(AsH

). In aerated water, arsenate is a stable form.

Since As(III) is considerably more toxic than As(V),

the form in which arsenic exists, whether as arsenate

or arsenite, is important. Doses of 70–180 mg of

arsenic trioxide (As

) are fatal. Arsenic acid is a

fairly strong acid, while arsenous acid behaves in

water practically as a weak, monobasic acid (see

Figure 1).

0004Arsenic forms a variety of organoarsenic com-

pounds. Arsenic compounds of the types, R

As,

RAs(OH)

AsOH, RAsO(OH)

AsO(OH),

and R



are called arsines, arsenous acids, arsi-

nous acids, arsonic acids, arsinic acids, and arsonium

salts, respectively. It is believed that organoarsenic

compounds are much less toxic than the inorganic

arsenic compounds. Biochemical conversion of inor-

ganic arsenic compounds to organoarsenic com-

pounds has been known for more than 100 years.

Occurrence

0005Arsenic is distributed everywhere in the environment.

The use of arsenic is decreasing gradually but is still

important. Commercial elemental arsenic is mainly

employed as an additive for alloys. High-purity ar-

senic (at least 99.999%) is employed in electronics as

gallium arsenic (GaAs) or indium arsenic (InAs). Fur-

thermore, arsenic has been used in wood preserva-

tion, medical supplies (Salvarsan), pigment (Scheele’s

Green: CuHAsO

, Paris’s Green: 3Cu(A-

)

Cu(CH

COO)

), agricultural chemicals, food

additives (arsanilic acid, H

AsO(OH)

), and

poison gas (lewisite). British anti-lewisite (BAL),

which was developed as an antidote for lewisite, is

also one of the antidotes for heavy metal poisoning.

0006Arsenic occurs naturally in the earth’s crust (1.5–

2mgkg

1

). Hot-spring and well waters sometimes

contain appreciable quantities of arsenic, mostly in

304 ARSENIC/Properties and Determination

the form of arsenate. Arsenic is associated with sulfide

ores. Waste water discharged from sulfide mines

and ore-dressing plants often contains arsenic in

appreciable quantities. Arsenic contamination caused

by flue gases from lead and copper smelters has been

frequently reported. Most authorities adopt a value of

10 mgl

1

, measured as total arsenic, as the maximum

permissible concentration in drinking water.

0007The arsenic levels in ocean water are around

2 mgl

1

, and although arsenate is the stable form

in aerated waters, ocean water sometimes contains

appreciable proportions of arsenite. It is believed that

some bacteria are able to reduce arsenate to arsenite.

0008Because arsenic compounds have been used as

insecticides, herbicides, and animal-feed additives,

some soils, vegetation, swine, and poultry may be

contaminated with arsenic. The value of 1 mg kg

1

as As

has been the maximum allowable limit for

fruits and vegetables in Japan. Soil containing more

than 15 mg of arsenic per kilogram is considered

unsuitable for agriculture.

0009Edible marine organisms, such as lobsters, shrimps,

and brown kelps, contain considerable concentra-

tions of arsenic frequently in the mg kg

1

range, oc-

casionally at concentrations as high as several tens of

mg kg

1

on a wet-weight basis. Fortunately, however,

the arsenic is mostly in the form of organoarsenic

compounds, which are rapidly excreted and therefore

relatively nontoxic to humans. On average, about

80% of the arsenic in marine organisms is found in

the water-soluble fraction. Mostly, arsenobetaine

(AsB) is the predominant arsenic species in marine

animals, with arsenosugars in marine algae and

some marine bivalves. Concentrations in freshwater

plants and animals, and terrestrials are usually lower

than in marine species. Arsenic has been found in

mushrooms (Agaricus bisporus): As(III), 0.07

mg kg

1

; As(V), 0.14 mg kg

1

; monomethylarsonic

acid, 0.05 mg kg

1

; dimethylarsinic acid, 0.28 mg

1

; and AsB, 0.46 mg kg

1

0010In 1900, there was an outbreak of arsenic

poisoning among beer-drinkers in England. The

cause of poisoning was traced to the use of invert

sugar produced using arsenic-contaminated sulfuric

acid. In 1955, there was an outbreak of unusual

disease and fatalities in Okayama and Hiroshima

Prefectures in Japan among infants drinking arsenic-

contaminated milk. The arsenic content of the

powdered milk that caused this disaster was esti-

mated to be 15–24 mg kg

1

as As. The dose of arsenic

in the infants was estimated to be 1.3–3.6 mg day

1

and the source of arsenic was traced to arsenic-

contaminated sodium phosphate added as a stabilizer.

The sodium phosphate had been produced from

waste bauxite processing liquor and renamed by

resale, but it contained about 3% arsenic.

0011Recently, a large number of fatal arsenic intoxica-

tions were traced from drinking water contaminated

HO OH

COH

CCH

COOH

OCH

CH(OH)CH

(a) Arsenic acid

= 9.2

= 12.1

= 13.4

(b) Arsenous acid

= 2.3

= 6.8

= 11.6

MMAA (methanearsonic acid)

= 3.6

= 8.2

(d) Dimethylarsonic acid,

DMAA (cacodylic acid)

= 6.2

(g) Arsenobetaine, AsB

(h) Arsenocholine, AsC

( i ) Arsenosugars

= 2.2

(e) Trimethylarsine oxide,

TMAO

( f ) Tetramethylarsonium ion

= 3.6

(as (CH

)

OH)

fig0001 Figure 1 Structure of some important arsenic compounds

and their acid dissociation constants. (i1) R ¼ -SO

H, 2-hydroxy-

3-sulfopropyl-5-deoxy-5-(dimethylarsinoyl)-b-ribofranoside; (i2)

R ¼-OH, 2,3-dihydroxypropyl-5-deoxy-5-(dimethylarsinoyl)-b-

ribofranoside; (i3) R ¼-OPO

HCH

CH(OH)CH

OH, 3-‘glyceropho-

sphoryl’-2-hydroxy-1-[5-deoxy-5-(dimethylarsinoyl)-b-ribofrano-

syloxy] propane; (i4) R ¼-OSO

H, (2S)-3-[-5-deoxy-5-

(dimethylarsinoyl)-b-

D-ribofranosyloxy]-2-hydroxypropyl hydro-

gen sulfate.

ARSENIC/Properties and Determination 305

by inorganic arsenic species. The main sources of

arsenic contamination of drinking water are natu-

ral, and high concentrations of arsenic in drinking

water are found in various parts of the world. In

Taiwan, patients with blackfoot disease are found in

areas where drinking water contains arsenic. In Ban-

gladesh and West Bengal in India, arsenic poisoning

became evident in the 1980s, and tens of millions of

people are estimated to be at risk. The survey is not

complete because the survey area is so large. The

source of arsenic is geological.

Speciation of Arsenic in Food and Water

0012 Various arsenic species show large differences in

their toxicity. Their acute toxicity decreases in the

following order: As(-III) (AsH

) >> arsenite

{As(III)} > arsenate {As(V)} >> dimethylarsinate > mo-

nomethylarsonate >> arsenobetaine and arsenocho-

line (organoarsenic compounds in seafoods).

Therefore, speciation of arsenic is required for reli-

able toxicological estimation. Arsenic in seafoods has

been well studied and is mostly in the form of orga-

noarsenic compounds, including arsenobetaines and

arsenosugars.

0013 Extraction and purification are needed, especially

for speciation of arsenic. For the speciation of orga-

noarsenic compounds in seafoods, the samples are

usually extracted with methanol or a methanol–

water mixture. The samples are then purified with

cartridge, centrifugation, and/or filtration.

The purified sample is subjected to gel-permeation

chromatography, ion-pair chromatography, ion-

exchange chromatography, and preparative thin-layer

chromatography. Buffered ion-exchange chromatog-

raphy, close to neutrality, is necessary to prevent de-

composition of the compounds to dimethylarsinic

acid occurring at extremes of pH.

0014 Arsenic compounds extracted with methanol from

sea animals (cockle, trough shell, tuna, crab, sea cu-

cumber, squid, etc.) have been separated by liquid

chromatography. No single column has been found

to be satisfactory for separating all organoarsenic

compounds present in the seafoods. Four compounds

(Figure 1f, g, i2, i3 )havebeenfoundinanimals.

Arsenobetaine (Figure 1g) was found in all sea

animals tested. There is considerable evidence that

arsenocholine and arsenobetaine are rapidly excreted

and almost nontoxic to humans.

0015 Van Elteren and S

lejekovec investigated the stabil-

ity of several arsenic compounds in food treatment

procedures. Under usual food treatments (micro-

wave treatment of 300 W of power for 120 min

and boiling on a hot plate for 100 min), aqueous

solutions of monomethylarsonic acid (MMAA),

dimethylarsinic acid (DMAA), arsenobetaine (AsB),

and tetramethylarsonium ion remained stable. Using

g-irradiation treatment with a dose of 10 kGy, 5% of

AsB decomposed to trimethylarsineoxide (TMAO),

and 1.5% of MMAA and 1% of DMAA decom-

posed to inorganic arsenic. After dry heating for

30 min at 160



C, 10% of AsB decomposed to

TMAO, 9% of MMAA to As(III), 6% of DMAA

to MMAA, and tetramethylarsonium ions remained

stable. Under unusually harsh treatments, however,

decomposition of some compounds was found.

Sample Preparation

0016To determine total arsenic, the sample must be

brought into solution. Care must be taken during

the destruction of the sample to ensure that no arsenic

is lost by vaporization of trivalent arsenic halides.

Loss can usually be avoided by oxidizing the arsenic

at an earlier stage by boiling with nitric acid under

reflux.

0017In determining arsenic at trace levels in food, the

Association of Official Analytical Chemists (AOAC;

Article 9.1.01 D) recommends that a sample be

digested with HNO

(heated in an oven at 150



oven for 2 h) in a closed system. In the closed system,

a sample is placed in a digestion vessel (or cylinder),

usually constructed of a fluorinated polymer such as

polytetrafluoroethylene (PTFE) or perfluoroalkoxy

(PFA). After the addition of digestion reagents, the

cylinder is tightly sealed. The microwave-assisted

alkali digestion with tetramethylammonium hydrox-

ide ((CH

)

NOH, TMAH), which is used as a tissue

solubilizer, is useful for the pretreatment of various

biological samples prior to analysis. A mixture of

nitric, sulfuric, and perchloric acids is also used for

wet ashing of food samples. Monomethylarsonate,

dimethylarsinate, arsenobetaine, and phenylarsonate

are decomposed to inorganic arsenate. Dry ashing at

600



C with magnesium nitrate for the destruction of

meat and poultry is also used. The ash is dissolved in

dilute hydrochloric acid.

0018To examine arsenic contamination of soils in

Japan, arsenic is extracted by shaking the soil with

1 M hydrochloric acid (50 ml per 10 g of sample) for

30 min at 30



C. The mixture is filtered through dry

paper and subjected to hydride generation atomic

absorption spectrometry.

0019Low levels of arsenic in water can be enriched by

coprecipitation with ferric hydroxide after being

oxidized to the pentavalent state with potassium

permanganate. Triton X-100 has been used to aid

the formation of large precipitates. It is claimed that

both arsenate and arsenite are coprecipitated with

iron hydroxide.

306 ARSENIC/Properties and Determination

Methods of Analysis

0020 There are several methods available for the determin-

ation of low levels of arsenic, and various analytical

methods for determining arsenic in various concen-

trations are shown in Figure 2.

High-performance Liquid chromatography

0021 High-performance liquid chromatography (HPLC)

separation is most frequently employed for the speci-

ation of arsenic species. Gas chromatography (GC)

separation can also be used but requires compli-

cated analyte derivatization steps to form volatile

derivatives. Ion-pair chromatography in reverse

mode, ion-exchange chromatography, and gel-perme-

ation chromatography are often used. In reverse

mode, C

columns and polymer resin columns such

as Hamilton polymer reversed-phase (PRP) are com-

monly used. Ion-pair chromatography is based on the

distribution of ion-pairs between the aqueous mobile

phase and the hydrophobic stationary phase. A qua-

ternary ammonium ion such as tetrabutylammonium

ion is often adopted for the separation of the anionic

species. An alkyl sulfonate such as hexanesulfonate is

often adopted for the separation of cationic species.

Detection can be by UV absorption, conductivity, or

Species

(Liquid sample)

Flame AAS

XRF

GDS

Redox titration

Coulometric iodimetry

Magnesium ammonium

arsenate gravimetry

Molybdenum blue (Sp)

Iodimetry

TCD

HPLC/Flame-AAS HPLC/ICP-AES HPLC/ICP-MS

Cold trap/Fractional

volatilization/DC-AES

FPD

TC sensor

Controlled-potential electrolysis

Ozone chemiluminescence

HPLC/Conductivity detector HPLC/ICP-MS

HPLC/ICP-AES HPLC/ICP-MS

Ag-DDC (Sp)

(depth resolution: 0.01 µm) (space: 0.1 µm) (space: 0.1 µm, depth: 0.005 µm)

EPMA SIMS

GD-MS

Radio activation analysis

(sample several mg)

ICP-AES HG-AAS

GF-AAS

ICP-MS

Organoarsenic

compound

(Inorganic

arsenic

compound)

(Solid sample)

AsO

3−

AsO

3−

AsH

Analytical method (%)

−1

−2

−3

−4

−5

−6

−7

−8

−9

−1

−2

−3

−4

−5

−6

−7

−8

−9

fig0002 Figure 2 Analytical method for arsenic in various concentrations. From Mochizuki T (1993) Table of determination methods in

seventeen elements for various concentrations (in Japanese). Bunseki: 262 with permission. AAS, Atomic absorption spectrometry;

Ag-DDC, silver diethyldithiocarbamate; DC-AES, direct current ‘discharge’ atomic emission spectrometry; EPMA, electron probe

microanalysis; FPD, flame photometric detector; GD-MS, glow discharge mass spectrometry; GDS, glow discharge emission spec-

trometry; GF-AAS, graphite furnace atomic absorption spectrometry; HG-AAS, hydride generation atomic absorption spectrometry;

HPLC, high-performance liquid chromatography; ICP-AES, inductively coupled plasma atomic emission spectrometry; ICP-MS,

inductively coupled plasma mass spectrometry; SIMS, secondary-ion mass spectrometry; Sp, spectrophotometry; TCD, thermal

conductivity detector; XRF, X-ray fluorescence.

ARSENIC/Properties and Determination 307

hyphenated techniques (atomic absorption and mass

spectrometry).

Hydride Generation Method

0022 There are two approaches for hydride generation

(HG). The first is a selective HG for speciation

followed by detector and is limited to the determin-

ation of the hydride-forming arsenic species. Differ-

entiation of several arsenic species is possible in the

HG technique with sodium borohydride as a reduc-

tant. Inorganic arsenic, monomethylarsonic acid, and

dimethylarsinic acid are reduced, in acid solution, to

arsine, monomethylarsine, and dimethylarsine,

respectively, and collected in a cold trap. Differenti-

ation of these arsenic compounds is possible by frac-

tional volatilization of the arsines collected in the cold

trap, because there are great differences in boil-

ing point between arsine (AsH

, 55



C), mono-

methylarsine (CH

AsH



C) and dimethylarsine

((CH

)

AsH, 36



C). The choice of a proper packing

material in the cold trap is important for the mutual

separation of these arsines. A silanized diatomaceous

earth impregnated with phenylmethylsilicon (15%)

or hydrofluoric-acid-etched glass beads (0.3 mm) has

been used. Arsenite (As(III)) (pH 6) and arsenate

(As(V)) (pH 1) can be separated by pH-selective

HG. Hydride generation for differentiation of arsenic

species is becoming obsolete because very stable

organoarsenic compounds such as arsenobetaine

cannot form arsine.

0023 The second removes total arsenic from the matrix

and is generally combined with HPLC followed by

decomposition (microwave digestion or UV decom-

position) as a postcolumn prior to detector. The gen-

erated arsines are often concentrated by introduction

to a cold trap before detection. The HG technique is

used in combination with many different detection

systems, including atomic absorption spectrometry

(flame, quartz furnace, and graphite furnace), atomic

emission spectrometry, mass spectrometry, atomic

fluorescence spectrometry, spectrophotometry, and

colorimetry. The HG technique is incorporated into

the Gutzeit method and silver diethyldithiocarbamate

(AgDDC) method.

0024 Arsenic is reduced in acid solution by zinc or

sodium borohydride to form arsine (AsH

), which is

volatile and is stripped by the hydrogen generated

during the reduction; it can be collected by a trap

cooled in liquid nitrogen or by a dilute iodine or

bromine solution. With zinc as the reductant, prior

reduction of arsenic to arsenite is necessary for the

conversion of arsenate into arsine. A mixture of po-

tassium iodide and tin(II) chloride also helps the re-

duction of zinc. In a strongly acid solution, both

arsenate and arsenite are converted into arsine by

sodium borohydride.

Gutzeit Method (Colorimetry)

0025The Gutzeit method is becoming obsolete, although it

is highly sensitive and has been employed for many

years for the detection and semiquantitative deter-

mination of traces of arsenic in foods, soils, and fer-

tilizers. The sample is brought into solution by wet

ashing or by other means. Arsenic is reduced to

arsenite with potassium iodide and tin(II) chloride

and further to arsine with zinc in acid solution. The

arsine is stripped by the hydrogen formed during the

reduction with zinc, and passed through a tube con-

taining a strip of HgBr

-impregnated paper. The

paper darkens on reaction with arsine, and the length

of the darkened area is proportional to the amount of

arsenic.

0026In the field kit based on this method, the paper

turns yellow on reaction with arsine, the concentra-

tion of arsenic is determined by comparison on a

color chart, and its detection limit is sub-mg kg

1

levels.

Spectrophotometry

0027Spectrophotometric methods used for the determin-

ation of arsenic are based on either the formation of

heteropolymolybdate, e.g., the molybdenum blue

method, or the formation of arsenic diethydithiocar-

bamate. Only arsenic(V) reacts with molybdic acid to

form heteropolymolybdate. For the determination of

total inorganic arsenic, prior oxidation of arsenic(III)

to arsenic(V) is required.

0028In the molybdenum (Mo) blue method, arsenic(V)

reacts with molybdic acid in the presence of a

reducing agent to form heteropolymolybdenum

blue. Since this method is interfered with phosphate

and silicate, it is used after isolation of arsenic by the

hydride generation method. First, arsenic is converted

into arsine. The arsine is then passed through an

arsine trap solution containing sodium hypobromite.

An ammonium molybdate–sulfuric acid solution is

added to the arsine trap solution to react as follows:

AsO

3

þ 12MoO

2

þ 24H

! AsMo

3

þ 12H

After reduction with hydrazinium sulfate, absorbance

at 845 nm is measured. The color develops slowly

(* 75 min) at room temperature but is stable.

0029The silver diethyldithiocarbamate ((C

)

NCSSAg, AgDDC) method involves arsine gener-

ation and bubbling the arsine formed through a trap-

ping solution in pyridine. Figure 3 shows an example

of the apparatus used in this method. The glass wool

in the sulfide scrubber is moistened with lead acetate

308 ARSENIC/Properties and Determination

solution, and AgDDC–pyridine solution is placed in

the arsine trap.

0030 An appropriate volume of sample solution is placed

into an arsine generator flask, and hydrochloric acid,

potassium iodide, and tin(II) chloride are added to

the flask. The mixture is allowed to stand for more

than 15 min at room temperature. Zinc metal is

added, and the generator flask is immediately sealed

and the tube connected as illustrated. The solution

is then allowed to react for 30 min. The trap is dis-

connected, and the trapping solution is mixed by

gently drawing back and forth five times with

an aspirator assembly. The absorbance based on an

intense red color is measured at about 522 nm. The

wavelength for the absorption maximum changes

slightly with the composition of the trapping

solution. If AgDDC–brucine–chloroform solution is

used as a trapping solution, to prevent the production

of foul-smelling pyridine, an appropriate volume of

chloroform should be added to the trapping solution

at the end of the trapping operation to compensate for

the loss of chloroform evaporation.

Atomic Absorption Spectrometry

0031 A hollow cathode lamp or an electrodeless discharge

lamp can be employed as a light source. The primary

absorption line is 193.7 nm.

0032Direct nebulization of extracts into a flame atomic

absorption spectrometry is impractical at low concen-

trations because the flame strongly absorbs radiation

at the wavelengths of sensitive absorption lines.

Better sensitivity is obtained by the hydride gener-

ation technique, in which arsenic (as arsine vapor)

is introduced into a hydrogen–argon reducing flame

or into a heated tube for measurement of atomic

absorption.

0033Graphite furnace atomic absorption spectrometry

(GF-AAS) can also be employed. Arsenic sublimes at

613



C and arsenic trichloride (AsCl

) boils at 130



but nickel arsenide melts at 968



C. Successful results

are obtained when nickel nitrate is added as a modi-

fier to prevent loss of arsenic during the ashing pro-

cess and to reduce interference. Similarly, magnesium

nitrate, palladium and magnesium nitrates, and zir-

conyl nitrate can also be used as modifiers.

0034Instead of GF-AAS, quartz furnace atomic absorp-

tion spectrometry coupled with HG is also employed.

The temperature in the quartz furnace is elevated

to 900



Inductively Coupled Plasma Atomic Emission

Spectrometry

0035Inductively coupled plasma atomic emission spec-

trometry (ICP-AES) is a simultaneous multielement

analysis technique with a dynamic range. In ICP-

AES, arsenic can be measured simultaneously in vari-

ous emission lines (188.979, 180.042, 193.696,

197.192, or 228.812 nm) with different sensitivities.

Atomic emission at 193.7 nm is less subject to inter-

ference, giving a detection limit of c. 0.02 mg l

1

.At

228.8 nm, cadmium interferes. Atomic emission at

188.979 nm, which is recommended in recent years,

is giving a detection limit of mg1

1

levels.

0040At 228.812 nm cadmium interferes. Arsenic (V) is

converted into an arsenomolybdate, which is sorbed

by the filter as the ion-associate with tetrapentylam-

monium ion, and the filter and the arsenomolybdate

sorbed are dissolved in concentrated sulphuric acid,

diluted with water and nebulized into the plasma

torch. An emission line at 228.812 nm can be used.

Interference from cadmium is eliminated because

cadmium is not collected on the filter. Arsenic (III)

dose not form arsenomolybdate and is not deter-

mined.

0041The sensitivity of conventional direct nebulization

ICP-AES for arsenic is high but not sufficient to de-

termine traces of arsenic. The sensitivities of ultra-

sonic nebulization ICP-AES for arsenic are about ten

times better than that of conventional direct nebuli-

zation ICP-AES, but matrix (salt concentration) effect

becomes larger. The sensitivity of ICP-AES can be

improved by using HG techniques because of a

Sulfide scrubber

(glass wool wet with

lead acetate solution)

Generation flask

Arsine trap

fig0003 Figure 3 Apparatus for silver diethyldithiocarbamate method.

ARSENIC/Properties and Determination 309

more efficient sample introduction and matrix re-

moval. A continuous hydride generator is convenient.

The detection limits of HG-ICP-AES for arsenic are

about two orders in magnitude better than that of

conventional direct nebulization. ICP-AES is possible

to use a continuous monitoring. Do et al. investigated

the speciation of arsenic in urine by HPLC-HG-ICP-

AES. The limits of quantification were found to be

36 mgl

1

for AS(III), 9 mgl

1

for DMA, 57 mgl

1

for

MMA, and 101 mgl

1

for As(V), applying a 100 ml

sample injected for analysis.

Inductively Coupled Plasma Mass Spectrometry

0036 Inductively coupled plasma mass spectrometry (ICP-

MS) uses an inductively coupled plasma as an ion

source and is one of the most sensitive methods for

elemental determination. Arsenic is a monoisotopic

element, that is, 100% of arsenic is

As.

0037 The determination of arsenic using ICP-MS is inter-

fered with by any molecular ion having a mass/charge

ratio (m/z) equal to

As. Thus, the

ion is

interfered with by

formed by the com-

bination of plasma gas and chloride ions, and so

separation of the arsenic species from chloride ions

is often required. The removal or reduction of argon

from the excitation source by the addition of nitrogen

gas or helium gas also eliminates, or significantly

reduces, the formation of

. Before ICP-

MS, an HG method or a high performance liquid

chromatography (HPLC) coupled with ICP-MS is

required for the determination of arsenic species.

The concentration of organic solvents must be lower

because the introduction of organic solvents destabil-

izes the inductively coupled plasma.

Other Methods

0038 Atomic fluorescence spectrometry (AFS) combined

with HG is also available for trace arsenic determin-

ation. With LC (anion and cation exchange, liquid

chromatography)–UV (decomposition)–HG–AFS

system, six arsenic species (As(III), DMAA, MMAA,

As(V), AsB, tetramethyarsonium ion) can be separ-

ated, with detection limits of about 0.5 mg of As per

liter (100 ml injected). The limits of determination for

arsenic in AFS, neutron activation without chemical

separation, and pulse polarography are 0.8, 0.02, and

0.004 mg l

1

, respectively.

Certified Reference Materials

0039 For quality control, the adoption of certified refer-

ence materials (CRM) is the best choice. A number

of CRM for total arsenic are available, although only

a few are available for speciation. Several easily

available CRM for arsenic, relating to foods, are as

follows: NIES CRM 9 Sargasso (certified value:

115 + 9mgkg

1

), BCR CRM 422 cod muscle

(21.1 + 0.5 mg kg

1

), NIST SRM 1566 oyster tissue

(14.0 mg kg

1

), BCR CRM 279 Ulva lactuca (sea

lettuce) (3.09 + 0.20 mg kg

1

), NIST SRM 1548

total diet (0.20 + 0.020 mg kg

1

), NIST SRM 1570a

spinach leaves (0.068 mg kg

1

), BCR CRM 185

bovine liver (0.024 + 0.003 mg kg

1

), NIST SRM

8433 corn bran (0.002 mg kg

1

). CRM are produced

by several institutions:National Research Council of

Canada, Community Bureau of Reference EC, Brus-

sels, Belgium (Standards, Measurements and Testing

Programme), International Atomic Energy Agency,

Vienna, Austria, National Institute of Environmental

Studies, Tsukuba, Japan, and National Institute of

Standards and Technology, Gaithersburg, Maryland,

USA.

See also: Arsenic: Requirements and Toxicology;

Chromatography: High-performance Liquid

Chromatography; Mass Spectrometry: Principles and

Instrumentation; Applications; Spectroscopy: Atomic

Emission and Absorption; Visible Spectroscopy and

Colorimetry

Further Reading

Benramdane L, Bressolle F and Vallon JJ (1999) Arsenic

speciation in humans and food products: a review.

Journal of Chromatographic Science 37: 330.

Cullen WR and Reimer KJ (1989) Arsenic speciation in the

environment. Chemical Reviews 89: 713.

Do B, Alet P, Pradeau D, Poupon J, Gulley-Gaillot M and

Guyon F (2000) On-line reversed-phase liquid chroma-

tography hydride generation emission spectrometry:

speciation of arsenic in urine of patients intravenously

treated with As

. Journal of Chromatography B 740:

179.

Ferguson JF and Gavis J (1972) A review of the arsenic cycle

in natural waters. Water Research 6: 1259.

The Geological Society of Japan (1998) Environmental

Problems about Arsenic (in Japanese) Tokyo: Tokai

University Press.

Gerhartz W (ed.) (1985) Ullmann’s Encyclopedia of

Industrial Chemistry, 5th edn, vol. A3, pp. 113–141.

Weinheim: VCH.

Guerin T, Astruc A and Astruc M (1999) Speciation of

arsenic and selenium compounds by HPLC hyphenated

to specific detectors: a review of the main separation

techniques. Talanta 50: 1.

McIver KA (ed.) (1996) Official Methods of Analysis of the

Association of Official Analytical Chemists, 16th edn.

Arlington, VA: Association of Official Analytical

Chemists.

Mochizuki T (1993) Table of determination methods in

seventeen elements for various concentrations: Arsenic

(in Japanese). Bunseki: 262.

310 ARSENIC/Properties and Determination

Morita M and Shibata Y (1987) Speciation of arsenic com-

pounds in marine life by high performance liquid chro-

matography combined with inductively coupled argon

plasma atomic emission spectrometry. Analytical

Science 3: 575.

Nriagu JO (ed.) (1994) Arsenic in the environment, Part I:

Cycling and characterization In: Advance in Environ-

mental Science and Technology, vol. 26. New York:

John Wiley.

US National Research Council, Committee on Medical and

Biologic Effects of Environmental Pollutants (1977)

Arsenic. Washington, DC: National Academy of Science.

Van Elteren JT and S

lejekovec Z (1997) Ion-exchange sep-

aration of eight compounds by high-performance liquid

chromatography–UV decomposition–hydride generat-

ion–atomic fluorescence spectrometry and stability

tests for food treatment procedures. Journal of Chroma-

tography A 789: 339.

Van Elteren JT, S

lejekovec Z and Byrne AR (1998) A dual

arsenic speciation system combining liquid chromato-

graphic and purge and trap-gas chromatographic separ-

ation with atomic fluorescence spectrometric detection.

Analytica Chimica Acta 358: 51.

Requirements and Toxicology

G I Rehner, Institut fu

r Erna

hrungswissenchaft,

Giessen, Germany

Arsenic in the Environment

0001 Arsenic is widely distributed in the natural environ-

ment. Virtually all soils and waters contain a variety

of different arsenic compounds. As a byproduct of

melting copper, lead, and several other metals, as

well as from coal-fired power plants, high quantities

of this metalloid are emitted into the atmosphere.

Arsenic compounds are also used by the metallurgical

and chemical industry. Phosphate fertilizer, as well

as many herbicides, fungicides, wood preservatives,

insecticides, and rodenticides, contains arsenic. Thus

agriculture contributes significantly to arsenic pollu-

tion. Legislation in countries with developed conser-

vation systems attempts to minimize the quantity of

these pesticides as well as other sources of arsenic

pollution.

0002 Both the trivalent (arsenite) and the pentavalent

(arsenate) state of arsenic can be detected in bio-

logical material. The biochemically important forms

of arsenic are however methylated compounds:

dimethylarsenic acid, methylarsonic acid, trimethyl-

arsine oxide, trimethylarsonium ion, arsenocholine,

arsenobetaine, and unstable arsenyl esters.

0003Arsenic accumulated in the environment can be

subject to various biotransformations – reduction,

oxidation, and methylation – performed by micro-

organisms. Methylation of inorganic arsenic in

methanogenic bacteria under anaerobic conditions

may be a detoxification mechanism for arsenic. Also

marine algae transform arsenate into nonvolatile

methylated arsenic compounds (methanoarsenic

acid and dimethylarsenic acid) in seawater. These

less toxic compounds are concentrated in higher

marine organisms consuming the plankton. Several

fungi are able to transform aerobically inorganic

and organic arsenic compounds into very toxic vola-

tile methylarsines.

0004Owing to the ubiquity of arsenic in the environ-

ment, foodstuffs of plant and animal origin as well as

drinking water also contain arsenic. The maximally

permissible arsenic concentration in drinking water,

vegetables, fruits, and meat is regulated by law in

most industrially developed countries. The US drink-

ing water standard, for example, is fixed at 50 mgl

1

However, several springs and spa waters contain

0.5–1.3 mg of arsenic per liter.

0005The range of arsenic levels in foodstuffs in the

absence of significant pollution is as follows: cereals

0.05–0.4 mg kg

1

, fruits 0.03–1.0 mg kg

1

, vege-

tables 0.05–0.8 mg kg

1

; meat 0.05–1.4 mg kg

1

;

dairy products 0.01–0.23 mg kg

1

. Higher concentra-

tions – 1.5–15.3 mg kg

1

– are registered in seafoods

because marine organisms accumulate arsenic in the

form of nontoxic organic compounds. The average

daily intake of arsenic is estimated in the range of

12–40 mg. When diets contain substantial amounts

of seafood, as is usual in Japan, 70–170 mg could be

the realistic daily intake. (See Shellfish: Contamin-

ation and Spoilage of Molluscs and Crustaceans.)

Arsenic Metabolism

Intestinal Absorption

0006Arsenic is generally well absorbed by the gastrointest-

inal tract, although the solubility of the compounds

seems to correlate with the rate of absorption. Intes-

tinal absorption of most inorganic and organic com-

pounds of arsenic is a quick process. However, some

unusual compounds, such as Na-P-N-glycolylarseni-

late, given orally to humans, are poorly absorbed.

0007Some investigations in chicks, mice, rats, and

rabbits indicate that arsenate as well as arsenocholine

readily penetrates both the mucosal and the serosal

surfaces of the epithelial membrane and that arsenate

and phosphate do not share a common transport path-

way. Both arsenic compounds are apparently absorbed

by simple diffusion following a concentration

ARSENIC/Requirements and Toxicology 311

gradient. On the other hand, in vivo and in vitro

experiments in rats revealed a saturable transport

process for pentavalent arsenic which was signifi-

cantly reduced by phosphate. This result might indi-

cate that arsenic and phosphate share the same

secondary active carrier-mediated transport system.

Arsenic in Human Tissues

0008 Once absorbed, arsenic is quickly distributed to all

organs and tissues, probably as a complex with

a-globulins. Apart from inorganic arsenic, blood

also contains methylated metabolites formed in the

liver.

0009 If the uptake of arsenic is low, no significant accu-

mulation in the soft tissues occurs. However, in unex-

posed humans relatively high arsenic concentrations

are usually found in skin, nails, and hair. Because hair

levels correlate with arsenic in the air as well as with

renal arsenic excretion, hair concentration has often

been used as an index of contamination with toxic

amounts of arsenic. The median arsenic content of

human hair was found to be 0.51 mgg

1

. The hair of

workers at plants with arsenic-containing emissions

was found to be 10–31 mg of arsenic per g of hair.

Values greater than 3 mgg

1

indicate toxic levels of

uptake.

Arsenic Excretion

0010 In general, the biological half-life of arsenic is short:

the metalloid is excreted mainly in urine with a little

in feces. Some compounds, such as arsenobetaine,

which is the form found in marine animals, pass

through into the urine unchanged. For this reason,

despite the high arsenic content, intoxication by sea-

food is unlikely. Different methylated compounds are

the major urinary metabolites.

0011 Excretion via the bile is low after dietary intake of

arsenic, but can significantly increase when drugs

containing arsenic are administered intravenously.

The hepatobiliary transport of arsenic is gluta-

thione-dependent and associated with a profound

increase in biliary excretion of glutathione; thus, hep-

atic glutathione depletion and diminished glutathione

conjugation result.

Biotransformation of Arsenic

0012 Inorganic arsenic is methylated in the liver with S-

adenosylmethionine as methyl donator. As the first

step, arsenate is reduced to arsenite by using glu-

tathione. Arsenite is methylated to dimethylarsenic

acid with monomethylarsenic as a precursor. Investi-

gations with germfree animals have shown that

methylation is mainly performed by the liver cells,

with little contribution from the intestinal microflora.

0013The rate of methylation of inorganic arsenic varies

considerably between species and individuals. As

shown by studies in human volunteers exposed to

specific doses of inorganic arsenic, the rate of renal

excretion of arsenic increases with methylation effi-

ciency. It could be assumed that the variations in

interindividual susceptibility to arsenic in humans

could be due to differences in the capacity of arsenic

methylation.

0014Some organic compounds, especially arsenobe-

taine, are excreted unchanged. Arsenocholine can be

incorporated into phospholipids, replacing choline,

but the major part of this compound is excreted by

the kidney.

0015Some mold species are able to produce volatile

neurotoxic compounds by biotransforming arsenical

pigments contained especially in wallpapers. Some

lethal poisoning cases have been described resulting

from such toxic products of aerobic and nonaerobic

fungi as well as other primitive organisms.

Biochemical Function and Essentiality of

Arsenic

0016Although a specific function for arsenic on a molecu-

lar level is unknown, some evidence has been accu-

mulated indicating a distinct role for this element in

several metabolic processes.

0017This could be the case in CH

-group metabolism.

Almost all organic forms of arsenic produced in vivo

are methylated compounds. One of the trimethylated

ones is arsenocholine, which can be incorporated into

phospholipids, replacing choline. Choline is a methyl

donator for different methylation reactions in inter-

mediary metabolism. There is some evidence indicat-

ing that arsenocholine might have a similar function

in labile methyl metabolism. As some experiments

have shown that arsenic deprivation in the rat,

chick, and hamster affects labile methyl metabolism,

it could be speculated that the role of arsenocholine

cannot be fulfilled by choline.

0018Arsenic binds strongly to sulfydryl groups which

are known to participate in hundreds of enzymatic

reactions. In this context it behaves like heavy metals

that impair enzymatic catalysis. It is likely that the

biochemical basis is the inhibition of a wide range of

SH-enzymes by arsenic. But it cannot be ruled out

in advance that such an interaction is not only of

toxicological but also of biological significance.

0019Some investigators have reported that arsenic can

provide partial protection from chronic selenosis,

with the vitamin E level influencing the incorporation

of selenium into the tissues. It has been shown that

selenium toxicity for cultured mice fibroblasts can

also be counteracted by arsenic. Interactions between

312 ARSENIC/Requirements and Toxicology

arsenic and cadmium have also been shown. Con-

sidering the common mode of action that these elem-

ents have at the cellular level, especially through

interaction with SH-groups, these facts are not

surprising. (See Cadmium: Toxicology; Selenium:

Physiology.)

0020 Studies with chicks suggest that arsenic is closely

related to the metabolism of zinc. Depressed growth

caused by arsenic deprivation was alleviated when

dietary zinc was given in excess. It seems, however,

not only that interactions between arsenic and zinc

exist but also that the arginine status of the chicks

plays a distinct role in these complicated correlations

between the two trace elements and the amino acid.

There is support for the possibility that arsenic par-

ticipates – in a way that has yet to be clarified – in

utilizing amino acids for protein synthesis, or in pro-

tein degradation, as well as in uric acid metabolism.

(See Zinc: Physiology.)

0021 Effects of arsenic on carbohydrate metabolism

have been observed in experiments with guinea pigs.

The most prominent finding after repeated arsenic

(As

) administration was a marked decrease in

total carbohydrate content of the liver, mainly

owing to depletion of glycogen. (See Carbohydrates:

Digestion, Absorption, and Metabolism.)

0022 Some other biochemical facts in connection with

arsenic – used in physiological as well as toxicological

amounts – have been registered in the last few

years. We are far from being able to classify the

frequently conflicting facts and their significance for

metabolism.

0023 Since 1975, some conclusive evidence has been

published supporting the suggestion of nutritional

essentiality of arsenic. An element is considered es-

sential if a dietary deficiency consistently results in a

suboptimal biological function that is preventable or

reversible by intake of a physiological amount of that

element. Signs of arsenic deprivation were studied in

four animal species – chick, goat, miniature pig, and

rat. Safe and adequate intakes of arsenic for these

species are not precisely defined. However, a diet

containing less than 50 mg of arsenic per kg is denoted

as an arsenic-deficient ration in these animal experi-

ments, whereas 350–500 mgkg

1

is considered a

normal arsenic supply and 3.5–5mgkg

1

as a thera-

peutic dose.

0024 The essentiality of arsenic has been systematically

investigated in growing, pregnant, and lactating

goats, as well as in miniature pigs and their offspring.

Deficient rations contained less than 10 mg of arsenic

per kg of semisynthetic diet, and control rations

350 mgkg

1

0025 The following deficiency symptoms were de-

scribed: feed consumption was reduced and this was

correlated with diminished growth rates and milk

production; a high abortion rate for goats and

increased perinatal mortality for miniature pigs and

rats were also registered; intrauterine arsenic deple-

tion in goats, miniature pigs and rats resulted in sig-

nificantly reduced birth weight of the offspring. The

decreased milk-fat concentration could be a conse-

quence of lowered triglyceride concentration in the

blood serum of arsenic-deficient goats.

0026Histological changes, accompanied by ultrastruc-

tural alterations of the mitochondria, have been seen,

especially in skeletal muscles, the myocardium, and

liver tissue. Some modifications in the mineral com-

position of different organs with a remarkable reten-

tion of manganese were also connected to arsenic

deficiency.

0027Many recent findings from studies with sophisti-

cated new techniques have revealed possible sites of

essential action of arsenic. It seems to have an import-

ant role in the conversion of methionine to different

metabolites, e.g., S-adenosylmethionine, S-adenosyl-

homocysteine, and taurine. Additionally, arsenic

affects the formation of the polyamines spermine,

spermidine, and putrescine from arginine and the

metabolism of labile methyl groups.

0028As shown in recent studies, arsenic induces the

cellular synthesis of certain heat shock or stress pro-

teins. This event is controlled at the transcriptional

level and may involve changes in the methylation of

core histones. Thus, arsenic seems to regulate gene

expression. Arsenic also enhances DNA synthesis in

unsensitized and in stimulated human lymphocytes.

0029Beneficial effects of arsenic preparations have been

known – or at least believed – for centuries. Arsanilic

acid used to be incorporated into pig and poultry feed

in pharmacological doses as a growth-promoting

agent. Intake of relatively high amounts of inorganic

arsenic salts by humans and horses was reported from

several parts of Europe. ‘Arsenic eaters’ were con-

vinced that these preparations had protective effects

against diseases, and increased physical condition and

virility. Regular daily intakes of 0.5 g of arsenic have

been reported; this kind of dose can only be tolerated

once you are accustomed to arsenic for a long time.

(See Trace Elements.)

0030Nevertheless, there is as yet no convincing evidence

that arsenic is essential for humans, but the possibility

cannot be ruled out. For definitive judgment of essen-

tiality of arsenic, further critical investigations are

necessary.

0031Only data from animal studies are available for

estimating the approximate amount of arsenic re-

quired by humans, if it is essential for humans at all.

Cautious assessments support a daily requirement of

about 12–15 mg for persons consuming 8.4 MJ per

ARSENIC/Requirements and Toxicology 313