United States
                                  Environmental Protection
                                  Agency
                                  Health Effects Research
                                  Laboratory
                                  Research Triangle Park NC 27711
xvEPA
                                  Research and Development
                                  EPA-600/S1-82-010  Nov  1982
Project Summary
                                  Speciation  of  Arsenic
                                  Compounds in Water  Supplies
                                  Kurt J. Irgolic
                                   The objectives of this project were
                                  to develop and testanalytical methods
                                  that would allow the chemical form
                                  (i.e.  valence  state or compound) of
                                  arsenic in drinking  waters to be
                                  determined, and to use the methods to
                                  analyze samples of drinking water
                                  from  sources where adverse health
                                  effects in consumers had been attri-
                                  buted to arsenic. Analytical techniques
                                  were developed for the determination
                                  of arsenate (differential pulse polaro-
                                  graphy),  for  inorganic and organic
                                  arsenic  compounds (high pressure
                                  liquid chromatography with graphite
                                  furnace atomic absorption spectro-
                                  metry as element-specific detector)
                                  and for the detection of arsenocho-
                                  line, arsenobetaine, and iodoarsines
                                  (mass spectrometry). These tech-
                                  niques, inductively coupled argon
                                  plasma emission spectrometry, and
                                  hydride generation/DC-helium arc
                                  emission were used for the character-
                                  ization of water samples from Utah,
                                  Alaska, Antofagasta, Taiwan and
                                  Nova Scotia.  The total arsenic con-
                                  centration ranged  from 18 ppb to 8
                                  ppm with arsenite/arsenate ratios
                                  between 0.007 and 3.4. No organic
                                  arsenic compounds were detected in
                                  any of the water samples.  The trace
                                  elements Al, B, Ba, Ca, Cu, Fe, Li, Mg,
                                  Mn, Na, P, S,  Si and Sr were present in
                                  most of the water samples. The results
                                  show that the various physiological
                                  effects observed in populations ex-
                                  posed to the arsenic-containing water
                                  supplies could  not be caused by
                                  arsenic compounds other than ar-
                                  senite  or arsenate. Other trace ele-
                                  ments acting in concert with arsenite
                                  and/or arsenate might produce these
                                  symptoms. However, sufficient data
                                  are not yet available to evaluate these
                                  hypotheses.
                                    This  Project Summary was devel-
                                  oped by EPA's  Health Effects Re-
                                  search Laboratory. Research Triangle
                                  Park. NC, to announce key findings of
                                  the  research project  that is fully
                                  documented in a separate report of the
                                  same title (see Project Report ordering
                                  information at back).


                                  Introduction
                                   Arsenic is an element possessing a
                                  rich  chemistry Inorganic and  organic
                                  arsenic compounds may contain tri-
                                  valent arsenic The trivalent arsenic
                                  compounds are generally more toxic
                                  than the pentavalent derivatives. Many
                                  inorganic and organic arsenic com-
                                  pounds  are linked in  a cycle with
                                  chemical and biologically  mediated
                                  reactions changing the compounds into
                                  each other The input of arsenic into this
                                  cycle is supplied by weathering of
                                  arsenic-containing rocks, human use,
                                  and  disposal of various arsenic com-
                                  pounds

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  Since arsenic is ubiquitous, man
consumes small amounts of  arsenic
compounds with the food he eats and
the water he drinks. Life developed in
the presence  of arsenic. Therefore,
organisms are expected to tolerate a
certain, not yet clearly defined, dosage
of arsenic.  Certain geographically
limited groups  oT  people  have taken
arsenic compounds into their systems
over extended  periods of time. These
arsenic compounds are present in their
drinking water supplies The  most
famous localities  where  arsemc-con-
tainmg waters have been consumed are
certain regions in Taiwan and the city of
Antofagasta  in  Chile Hyperpigmenta-
tion, skin cancer, vascular problems and
other  ailments have been attributed to
the arsenic present in the drinking
water Other groups, such as the people
in Fallen, Nevada, have been exposed to
similar arsenic  levels in their drinking
water without any  ill  effects  This
project was undertaken in order to
determine the  arsenic compounds and
other trace elements present in arsenic-
containing water supplies and to check
whether these drinking  water supplies
contain the same  or different arsenic
compounds.
  Thus far, arsenite, arsenate, methy-
larsmic  acid, dimethylarsinic acid,
methylarsines, arsenobetame, trimeth-
ylarsoniolactic acid and arsenic-contain-
ing lipids have  been  identified in
environmental samples.

Preservation of Arsenic
Compounds in  Aqueous
Solutions
  Water samples generally cannot be
analyzed for trace elements immediate-
ly after collection. Several hours or even
several days elapse between collection
and  analysis.  During  this time  the
chemical nature of a trace element,
such  as  arsenic,  can  change. Trace
elements  can  be  lost  by  volatization
and/or can be  adsorbed on container
walls. The absorption of many metal
ions and of phosphate  ions has been
studied, but arsenic was  rarely included.
  Disagreements exist in the literature
as to  the extent of  loss of arsenic  from
solutions stored in various containers.
Even  less certainty  exists about the
conditions under which  various arsenic
compounds can be preserved Experi-
ments have  shown that arsenite,
arsenate and dimethylarsinic acid are
not adsorbed from 1 ppm solution on the
walls of Cubitainers* (soft polyethylene
containers manufactured by Kimberly)
Walls of Pyrex  containers removed
approximately  one  percent  of the
arsenic  from  the solutions. Ascorbic
acid at a concentration of 1 mg/mL has
been found to prevent the oxidation of
arsenite to  arsenate in  distilled water
solutions at room temperature On the
basis of these  results most water
samples were collected and stored  in
Cubitainers and  some of the samples
were preserved by adding ascorbic acid

Development of Analytical
Techniques for  the
Determination of Arsenic
Compounds
  Whereas  adequate methods for the
determination of total arsenic concen-
trations  do  exist, the choice of tech-
niques for  the  estimation  of  arsenic
compounds is limited All the methods
available for the speciation of arsenic
compounds at the time this project was
initiated had  severe limitations. The
methods were applicable only to the
determination of arsenite,  arsenate,
methylarsonic acid, dimethylarsinic
acid,  trimethylarsine  oxide,  ethyl-,
propyl- and butylarsonic acid,  and the
arsmes  obtainable from these com-
pounds. Non-volatile  arsenic  com-
pounds  and arsenic compounds not
reducible to volatile  arsmes could not be
determined with  the existing methods.
Therefore, an analytical system with an
element-specific detector  had to be
developed that was capable of separat-
ing volatile and non-volatile  arsenic
compounds in complex matrices.
  The development efforts produced a
high pressure liquid chromatography-
Hitachi Zeeman  graphite furnace ato-
mic absorption  system, a  differential
pulse polarographic method  for the
determination of arsenite and arsenate
and the elucidation  of the mass spectral
behavior of organylarsenic  acids,  or-
ganyl lodoarsmes,  arsenocholine and
arsenobetaine.

Hitachi Zeeman Graphite
Furnace Atomic Absorption
Spectrometer as an Element-
Specific Detector for High
Pressure Liquid
Chroma tograph y
  Liquid chromatography and specially
high pressure liquid chromatography
(HPLC) with the great resolving power of
its microparticulate columns are poten-
'Mention  of tradenames or commercial products
 does not constitute  endorsement or recommen-
 dations for use
tially the best techniques for the
simultaneous detection and determina-
tion  of arsenic compounds  A water
sample may contain many substances
in  addition to arsenic compounds. The
common  detectors  will not respond
specifically to arsenic compounds. The
identification  of arsenic-containing
fractions  is, therefore,  difficult if not
impossible unless  element-specific
detectors with high sensitivity are
available  A graphite furnace atomic
absorption spectrometer (GFAA) com-
bines the advantage of element-speci-
ficity  with  high  sensitivity for many
elements  An  HPLC-GFAA analytical
system was developed employing a
Hitachi Zeeman GFAA with a sample
valve, an  injector, and associated
electronics to control  the analysis
sequence


  The HPLC-GFAA  system  has func-
tioned almost flawlessly during the past
three years  Aliquots of the column
effluent are automatically transferred
into the graphite cup of the GFAA for
analysis  The  time  interval between
consecutive analyses  can be made
within 30 seconds. The Hitachi Zeeman
GFAA Model 170-70 has a detection
limit for arsenic of 10 picograms. This
sensitivity is, of course, retained in the
HPLC-GFAA system  for each injection.
Upon migration through the chromato-
graphic column the arsenic compounds
are  separated and  spread out  into
bands. Aliquots of 40 fjL are withdrawn
from  the effluent. The 40 ^L aliquots
taken from the center of the band must
each  contain at least 10 picograms of
arsenic. The detection limit of the HPLC-
GFAA system is, therefore, strongly
dependent  on the  degree of band
spreading.


   Conditions have been  found which
allow the  separation  of  inorganic
arsenic (arsenite and arsenate), arsen-
ocholine and arsenobetaine employing
a  C-18 reverse phase column, organ-
ylsulfonates as countenons and mix-
tures of water/acetonitrile/acetic acid
as the mobile phase. Arsenite, arsenate,
methylarsonic acid and dimethylarsinic
acid  were  similarly separated using
water/methanol  mixtures saturated
with  tetraheptylammonium nitrate  as
the  mobile phase. The HPLC-GFAA
system, of  course, is neither limited to
the analyses of the arsenic compounds
listed above nor to compounds contain-
ing only arsenic.

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Differential Pulse
Polarographic (DPP)
Determination of Arsenate
and Arse nit e
  Arsenite is reducible at the dropping
mercury electrode and can be deter-
mined  polarographically at concentra-
tions as low as 0 3 ppb. Arsenate is
polarographically inactive  under these
conditions. Addition  of  polyhydroxy
compounds to an  acidic solution of
arsenate makes arsenate reducible
Among 11  polyhydroxy compounds, D-
mannitol at 0.5 M concentration in 2 0
M aqueous perchloric acid produced the
largest reduction peak for arsenate The
DPP curve of  arsenate under these
conditions is characterized by maxima
at -0 55 V and -075 V  Above an As
(arsenate)  concentration of 500 ppb a
current maximum appears at -0 59 V,
which increases  in  intensity with
increasing concentration  The peak at
-0 55 V merges into the current maxi-
mum  and  becomes  a  shoulder  at
As(arsenate) concentrations of approx-
imately 5 ppm The rather low intensity
peak between-0 Wand-0.8 V might be
obscured at low arsenate concentra-
tions  by  the  solvent  breakdown and
at high arsenate concentrations by the
current maximum The arsenite reduc-
tion wave  in 2 0 M  perchloric  acid
solution shifts from -0.425 V to -0 34V
upon addition of mannitol
  When  arsenite  and arsenate  are
present  in  solution,  the arsenate
reduction peak at -0 55 V can be  used
for the determination of arsenate with
some  confidence only when the  con-
centration of As(arsemte) is between
100 ppb and approximately 500 ppb, the
current is  not lower than 2 /uA and the
arsenate  concentration is equal to or
higher than the arsenite concentration
If these conditions are not fulfilled,
arsenite must be oxidized to arsenate by
cenumflV) ammonium nitrate.  Excess
cenum(IV) must  be  reduced with hy-
droxylamme hydrochlonde Arsenate is
then determined in  the  presence of
mannitol  using the peak at -0 55 V.
Arsenite  is determined in another
aliquot of the sample in the absence or
presence  of mannitol. The  arsenate
concentration in the sample is obtained
as  the difference between  the  total
arsenic concentration and the Asfarse-
nite) concentration The detection limits
for arsenate under these conditions are
6 ppb at the 95 percent confidence level
Mass Spectrometry of
Organylarsonic Acids,
Diorgan ylarsinic A cids,
Organyliodoarsmes,
Arsenocholine and
Arsenobetaine

  Organic arsenic compounds could
perhaps be determined by mass spectro-
metry in  the residues obtained by
evaporation  of the  water samples
Therefore, the mass spectral behavior of
several organic arsenic compounds was
studied
  At  probe  temperatures between
110°C and 250°C required to obtain
satisfactory mass spectra, organylar-
sonic acids, RAs03H2, and diorganylar-
smic  acids,  R2AsOOH, formed anhy-
drides and decomposed The products of
these thermal  reactions  were then
ionized  and fragmented yielding com-
plicated mass spectra with many peaks
at m/e values higher  than those
expected for the molecular  ions  A
detailed investigation of the spectra of
five arsonic acids and nine arsinic acids
indicated that mass spectrometry was
of little  value for  the  identification of
arsonic acids, but can  be  used to
establish the  presence of diorganylar-
smic acids in the  residues from water
samples Exact mass measurements by
high  resolution mass spectrometry
might be necessary to  distinguish
arsenic-containing from arsenic-free
ions
  Organyliodoarsmes, RnAsU-n (n=1,2),
are much more volatile than arsinic or
arsonic acids and are easily prepared by
treating these acids with hydnodic acid
All of the 14 organyliodoarsmes investi-
gated gave intense molecular ion peaks
Fragmentation  proceeded by loss of
alkyl  groups, iodine and hydrogen
abstraction  Organyliodoarsmes are
well  suited for the mass spectrometric
identification  of organic  arsenic com-
pounds  which  can be converted to
iodoarsmes
  Arsenocholine chloride, [(CH3)3AsCH2
CHgOHJCI, and arsenobetame chloride,
[(CH3)3AsCH2COOH] Cl,  produce rich
mass spectra  which do  not  contain
molecular ion peaks The highest mass
peaks correspond to (CH3);3AsCH2CH20
and  (CH3)2AsCH2COOH, which  were
formed by thermal cleavage of HCI and
CHsCI from the arsonmm salts In spite
of the absence of molecular ions, mass
spectrometry can provide an indication
of the  presence  of  arsenocholme
and/or arsenobetaine in a sample.
Analysis of Water Samples
  Samples of arsenic-containing drink-
ing water supplies selected by the EPA
project  officer were collected  and
shipped to College Station as quickly as
possible  Total arsenic concentrations
and the concentrations of arsenite and
arsenate  were determined by several
methods  Each  water  sample  was
checked for the presence of methylated
arsenic compounds and  other organic
arsenic  derivatives  Water  samples
from  Hmckley,  Utah,  Delta, Utah,
Barefoot Site, Alaska,  Mauer  Site,
Alaska, Antofagasta, Chile,  Yenshei,
Taiwan;  Hartlm Site, Nova Scotia; and
Sullivan Site, Nova Scotia were invest-
igated
  Graphite  furnace atomic absorption
spectrometry, differential pulse polaro-
graphy, high pressure liquid chromato-
graphy with a GFAA as an  element-
specific detector, the hydride genera-
tion technique with a DC-helium arc
detector, and inductively coupled argon
plasma emission spectrometry  were
employed for the determination of
concentrations of total  arsenic, and
trace  elements  The samples  were
collected  and stored in Cubitamers or
Pyrex glass containers  Unpreserved
samples and samples preserved  with
ascorbic  acid  or nitric acid were
analyzed  The analyses were carried out
as soon as possible after receipt of the
samples
  The water samples had total arsenic
concentrations in the range of 18 ppb to
8  ppm  The arsenite/arsenate ratios
were in the range of 0 007 to 3 4 (Table
1) No indications of the presence of
methylated arsenic compounds, which
are reducible to  methylarsme or di-
methylarsme, have been  found Exper-
iments with the  high pressure  liquid
chromatograph/graphite furnace atomic
absorption spectrometer system, which
would provide information about the
presence of organic arsenic compounds
not reducible to methylarsmes, detected
only arsenite and  arsenate Comparison
of total arsenic concentrations with the
sum of the arsenite and arsenate
concentrations placed an  upper limit on
the concentrations of any other arsenic
compounds  which might be present
These upper limits were  m most cases
in the low ppb range
  The other trace elements  found in
these water samples by ICP are also
listed  m Table 1. There were no signifi-
cant concentrations of these  elements

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Table 1.
Summary of Total Arsenic, Arsenite, Arsenate and Trace Element Concentrations in Drinking Water Samples^
               Hinckley  Delta
                     Barefoot]1^
        Antofagasta Antofagasta
/Wauertt  Untreated     Treated    Yenshei I  Yenshet II  Nova Scotia 1 Nova Scotia 2
Total As
Arsenite
Arsenate
Arsenite/
Arsenate Ratio
Al
B
Ba
Be
Ca
Cu
Fe
K
Li
Mg
Mn
Na
P
Pb
S
Si
Sr
Ti
V
Zn
0 18
0010
0 18
006
	
12
0026
—
324
008
—
—
—
7 83
—
233
—
—
*
734
0 10
—
—
—
002
0010
0010
-7 0
	
007
0045
—
154
026
0007
—
—
665
—
65
—
7 77
*
753
052
—
—
—
3 7
24
07
34
006-050
—
0 77
—
180-291
—
29-53
12
—
52-113
042
37-47
027
—
28
11 0
047
0008
0 15
0 14
45-60
0 35-4 6
01-43
0 06-0 54
—
023
—
200-309
—
25
13-118
0007
55-139
056-0 76
35-50
—
~
775
77 0
060
0009
0 77
030
0 75
0016
0 74
002
008
28
0008
74
200
0003
0 11
133
0 62
74
0002
102
034
—
«
38 7
028
—
—
0 70
047"
0003
047
0007
3 7
2,5
0008
—
203
0007
030
732
064
75
0006
703
026
—
•
363
029
—
—
0 JO
0 85
0023
084
003
	
—
—
—
43
—
046
7 0
0007
86
—
250
49
—
05
34
005
—
—
—
7 7
0024
1 08
002
	
057
—
—
775
—
0.50
736
007
23
—
796
23
—
75
34
020
—
—
—
SO
45
35
7 3
»
*
*
»
»
0008
<0. 1
»
*
»
24
60
*
*
*
*
*
<0 7
»
*
063
037
032
1 0
»
*
»
*
*
027
<0 7
»
*
*
<0 7
50
*
+
*
*
*
<09
*
*
 "Not determined
'**As in treated water is normally less than 100 ppb See text for discussion
 '[The concentrations are given in ppm
^Results from two different samples collected one year apart
in  these  water  supplies  with  the
exception  of  beryllium found  in  the
untreated Antofagasta sample.
  The various  physiological  effects
observed  in  populations exposed to
these arsenic-containing drinking water
supplies  (Table 2) could  have been
caused by  the presence  of varying
amounts of arsenite and arsenate  It is
also conceivable that one or more of the
trace elements  present  m  the  water
supplies acted in concert with arsenicto
cause the  observed  effects   More
samples  need to be analyzed and the
results of these  analyses  correlated
with  epidemiological  studies before  a
definite statement can be made about
the interactions of trace elements with
arsenite or arsenate
  The chromatographic  work  on  the
fluorescent compounds in  the Taiwan
well waters strongly suggests  the
presence  of alkaloids, such  as D-
lysergic acid, ergometrme and calciferol
Additional experiments (preparative
chromatography,  mass spectrometry)
could not  be carried out  because of
insufficient samples
                             Table 2.
                             Sampling
                             Location
        Arsenic-Containing Water Supplies and Their Physiological
        Manifestations in Man
                             Taiwan
                             (37 villages)
                             Chile
                             (Antofagasta)
                             Bskersfield, CA.
                             Fallon. NV
               Type of Water and Range of
               the Total Arsenic
               Concentration
Symptoms Observed in the Pop-
ulation
               Artesian well waters used
               for 45 years,  arsenic leached
               from geologic deposits, 0 017-
               1.097 ppm; median
               0 5 ppm
               Drinking water supply since
               1958, 0 8 ppm before water
               treatment, 0  1 after water
               treatment
               Community drinking water
               supply, 0.3-0 7 ppm
               Drinking water, 0.1 ppm.
Melanosis, keratosis, skin cancel
15% prevalence among males
over age 60, normal incidence
2-3%

Melanosis, hyperkeratosis,
vascular manifestations:
myocardialischemia, hemipleglia
with occlusion of the carotid
artery, mesenteric arterial
thrombosis, pneumonia
No adverse effects reported *

No known adverse physiological
effects. *
                              *(n a study of the arsenic exposure of populations in Bakersfield, Ca , and Fallon, Nv.,
                               by a questionnaire designed to elicit information about arsenic related symptoms
                               and diseases, very few symptoms were reported The incidence of these symptoms
                               was not significantly different from control populations not exposed to arsenic
                                   4

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Recommendations
  The  use of methods such as the
hydride generation technique; differential
pulse  polarography,  high  pressure
liquid chromatography with  sensitive,
element-specific detectors and colori-
metnc  methods for  determining con-
centrations of total arsenic and arsenic
compounds, and  the availability of
simultaneous, inductively coupled
argon plasma  emission spectrometers
for trace element determinations and of
ion chromatography for anion analyses
make the  thorough characterization of
water samples a relatively easy and not
too-time-consuming task. Additional
arsenic-containing water samples must
be analyzed in support of or in prepara-
tion for epidemiological studies. Exper-
ience has shown that analysis by one
method is not sufficient  to  produce
reliable results. At least two independent
techniques  should  be used  for the
determination of arsenic compounds.
Kurt J. Irgolic is with Texas A&M University, College Station, TX 77843.
Frederick C. Kopfler is the EPA Project Officer (see below).
The complete report, entitled "Speciation of Arsenic Compounds in  Water
  Supplies, "(Order No. PB 82 -257 817; Cost: $ 12.00, subject to change) will be
  available only from:
        National Technical Information Service
        5285 Port Royal Road
        Springfield, VA 22161
        Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
        Health Effects Research Laboratory
        U.S. Environmental Protection Agency
        Research Triangle Park, NC 27711
                                                                             •&U. S. GOVERNMENT PRINTING OFFICE: 1982/659-095/553

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