United States Environmental Protection Agency Office of Research and Development
National Exposure Research Laboratory
Research Abstract
Government Performance Results Act Goal: Clean and Safe Water
Significant Research Findings:
Methods for Detecting Arsenic Species in Food
Scientific Problem and The maximum contaminant level (MCL) for arsenic in drinking water
Policy Issues is currently being revised. The formulation of this MCL is influenced
by a variety of factors including best available treatment technology,
analytical monitoring capability, and risk assessments based on health
data and relative exposure source estimates. The relative exposure
source estimate is used to assess arsenic exposure from all possible
routes. For arsenic, the two major exposure routes are dietary and
drinking water ingestion. Arsenic dietary exposure data is needed to
improve cancer risk estimates. Food, unlike drinking water, can
contain numerous forms or species of arsenic which vary significantly
in relative toxicity. Many foods are comprised of mostly less toxic
forms of arsenic. However, one of the primary dietary sources of
arsenic is seafood, and there is great uncertainty whether or not
seafoods are a significant source of toxic forms of arsenic. In many
cases, the predominant "extractable" arsenical associated with seafood
is a low toxicity species, arsenobetaine. However, in certain seafood
matrices, less than 50% of the arsenic can be extracted and identified.
The remaining "unextractable" arsenic fraction in these foods can lead
to considerable uncertainty in dietary arsenic risk estimates because
the risk/toxicity of the unextractable arsenic is unknown. Other dietary
samples, such as carrots and rice, have similar issues regarding
species-specific determinations and extractability. Thus, the main
objective of dietary arsenic exposure research focuses on developing
analytical approaches that provide a complete or quantitative
extraction of the arsenic prior to speciation analysis. This research by
the U.S. EPA's National Exposure Research Laboratory (NERL) has
involved separate collaborative efforts with both the U.S. Food and
Drug Administration (FDA) and U.S. EPA Region 10.
Research Approach Research has focused on minimizing the unextractable arsenic in
dietary samples known to be high in arsenic. These samples were
selected based on total arsenic analyses conducted for the FDA's
market basket survey. Because most of the arsenic in a diet is
associated with only a few foods, it is possible to obtain a fairly robust
National Exposure Research Laboratory — October 2001
-------
estimate of dietary arsenic by analyzing a target set of foods. For
example, seafood represents greater than 60% of the total arsenic in the
diet for the general U.S. population, but has poor extraction
efficiencies. Rice is another food group which is known to be high in
arsenic and is problematic in extracting and speciating arsenic.
The analytical approach initially used to extract the solid dietary
samples was Accelerated Solvent Extraction (ASE). This extraction
technique has the advantage of being a semi-automated approach
which minimizes the overall cost of the analysis and provides
procedural repeatability. However, while the ASE was found to
produce quantitative extraction in certain seafoods, carrot and rice
samples, other seafood (oysters, clams) and rice (long grain, brown)
samples produced extraction efficiencies of less than 60%. The
extraction step was followed by a separation via Ion Chromatography
(IC). Arsenic species have either a net positive or negative charge
depending on the pH used in the chromatographic separation.
Positively charged arsenic are separated on a cation column, and the
negatively charged species are separated on a anion column. Because
seafoods contain a wide variety of arsenicals, both cation and anion
separations are required to properly quantitate each individual species.
Rice, carrots and apples contain fewer arsenicals, and for this reason an
anion separation was found to be adequate. In all cases, Inductively
Coupled Plasma Mass Spectrometry (ICP-MS) was used as the
detector for the IC separation. In some early development work, Ion
Chromatography Electrospray Ionization Mass Spectrometry/ Mass
Spectrometry (IC-ESI-MS/MS) was used to structurally identify
unknown arsenic compounds which eluted from the IC column.
Results and
Implications
This Annual Performance Measure (APM 65) supports FY01 Annual
Performance Goal 009 which states: "By 2001, reduce uncertainties
and improve methods associated with the assessment and control of
risks posed by exposure to arsenic in drinking water." Significant
findings were as follows:
1.) ASE was evaluated as an extraction procedure by both the EPA
and the FDA. ASE compared favorably to conventional extraction
techniques, but, in certain seafoods, both ASE and conventional
extraction failed to produce quantitative extractions (.JAAS, 1999, 14,
607). Subsequent analysis of seaweed products by Gallagher (FJAC,
2001, 369,71) indicated that extraction efficiencies in seaweed can be
as low as 25%. Similar results were obtained by the FDA for carrots
{Analyst, submitted 3/01 ), rice (JAAS, 2001, 16, 299) and apples
(Analyst, 2001, 126, 136). A 25% extraction efficiency implies that
75%) of the arsenic remains in the sample undetected. For example,
National Exposure Research Laboratory — October 2001
-------
seafood samples which can contain up to 10,000 ppb (|_ig/L) arsenic
would contain 7,500 ppb of undetected and potentially toxic arsenic.
This dietary source of arsenic may be significant when compared to the
old drinking water MCL of 50 ppb, and the relative significance would
increase with lower drinking water MCLs. Species-specific losses
associated with the ASE sample container and dispersion media (used
to improve contact time between the sample and extraction solvent)
was also investigated (Gallagher, submitted 9/01) and were reduced by
utilizing inert dispersion media. However, subsequent evaluations
indicated that the ASE could not quantitatively extract samples such as
oysters, clams and certain varieties of rice. These results clearly
indicated that the risk associated with certain exposures would be
underestimated using the ASE or conventional extractors.
2.) Arsenosugars, commonly associated with clams, oysters and
seaweed products, produced false positives due to chromatographic co-
elution with other toxic species present in the sample. Because
arsenosugar standards are not available commercially, they were
structurally identified and characterized by IC-ESI-MS/MS and IC-
Hydride ICP-MS (JAAS, 1999,14,607). This characterization allowed
for the development of a chromatographic separation which fully
separated the arsenosugars from other arsenic species. Because the
arsenosugars can be as high as 5,000 ppb in certain seafoods, the
misidentification of these as more toxic species can influence risk
assessments associated with seafood ingestion. Although there is
currently a large degree of uncertainty regarding the relative toxicity of
arsenosugars, the elimination of this false positive is likely significant
when viewed relative to a drinking water MCL of 50 ppb.
In summary, this research has resulted in a number of analytical
insights for speciating arsenic in dietary samples. The use of IC-ICP-
MS in combination with IC-ESI-MS/MS for the structural verification
of arsenosugars in natural product extracts indicates that these sugars
can produce false positives at concentration levels of 1,000 ppb (which
greatly exceeds the old drinking water MCL of 50 ppb) (Gallagher
JAAS, 1999,14,607). A second analytical discovery is that the use of
ASE as a semi-automated extraction device, as well as conventional
extraction techniques, produce matrix dependent extraction
efficiencies. Therefore, the risk associated with certain seafoods and
rice samples may be highly uncertain given the inherently poor
extraction efficiencies.
Research
Collaboration and
Publications
This research has been a collaborative effort between the U.S. EPA's
National Exposure Research Laboratory and the U.S. Food and Drug
Administration (FDA) and between NERL and U.S. EPA Region 10.
National Exposure Research Laboratory — October 2001
-------
U.S. EPA Publications and Presentations:
Gallagher, P.A., Creed, J.T., Wei, X. Murray, S., Brockhoff, C.A. "An Evaluation of
Sample Dispersion Medias with ASE for the Extraction and Recovery of
Arsenicals in LFB and DORM-2 with ICP-MS Detection." Analyst submitted
9/01.
Gallagher, P.A., Gamble, B.M., Heck, A.N., Freeman, D.M., Schwegel, C.A., Creed,
J.T. "Characterization of Arsenosugars and Associated Degradation Products
Following and Aggressive Acid / Base Extraction Procedure" Presented at the
International Symposium on Environmental Toxicology of Metals and
Metalloids - Environmental Chemistry, Toxicology and Health, Queensland,
Australia, July, 2001.
Gallagher, P.A., Creed, J.T., Wei, X., Shoemaker, J., Schwegel, C.A. "Extraction and
Detection of Arsenicals in Seaweed via ASE with IC-ICP-MS Detection."
Fresenius Journal of Anal. Chem. 369: 71-80, 2001.
Gamble, B.M., Gallagher, P.A., Heck, A.N., Schwegel, C.A., Creed, J.T. "An
Investigation of the Chemical Stability of Arsenosugars in Extraction Solvents
Utilized to Quantitatively Extract Arsenicals from Seafood Products using IC-
ICP-MS Detection" Presented at the European Winter Conference on Plasma
Spectrochemistry, Lillihamer, Norway, February, 2001.
Gallagher, P.A., Heck, A.N., Wei, X., Schwegel, C.A., Creed, J.T. "A Comparison
of Extraction Efficiencies in Seafood Matrices using a Synthetic Stomach and an
Accelerated Solvent Extraction approach with IC-ICP-MS Detection" Presented
at the European Winter Conference on Plasma Spectrochemistry, Lillihamer,
Norway, February, 2001.
Creed, J.T., Gallagher, P.A., Wei, X., Schwegel, C.A. "Extraction Techniques for the
Removal of Arsenicals from Seafood Exposure Matrices with ICP-MS
Detection" Presented at the Fourth International Symposium on Speciation of
Elements in Biological, Environmental and Toxicological Sciences, British
Columbia, Canada, July, 2000.
Creed, J.T., Gallagher, P.A., Wei, X., Schwegel, C.A., Larenzana, R., Chamberlain, I.
"Accelerated Solvent Extraction of Arsenicals from Seafood Matrices with Ion
Chromatography and ICP-MS Detection" Presented at the Fourth International
Conference on Arsenic Exposure and Health Effects, San Diego, CA, June,
2000.
Gallagher, P.A., Wei, X., McKiernan, J.W., Schwegel, C.A., Murray, S., Creed, J.T.
"Accelerated Solvent Extraction of Arsenicals from Environmental Matrices
with Ion Chromatography Separation and ICP-MS Detection" Winter
Conference on Plasma Spectrochemistry, Ft. Lauderdale, FL, January, 2000.
Wei, X., Shoemaker, J.A., Gallagher, P.A., Schwegel, C.A., Creed, J.T. "Extraction
and Identification of Arsenosugars in Commercially Available Seaweeds"
Presented at the International Ion Chromatogarphy Symposium, San Jose, CA,
September, 1999.
National Exposure Research Laboratory — October 2001
-------
Gallagher, P.A., Creed, J.T. Wei, X., Shoemaker, J., Brockhoff, C.A. "Detection of
Arsenosugars from Kelp Extracts via IC-ESI-MS/MS and IC Membrane Hydride
Generation ICP-MS." Journal of Analytical Atomic Spectrometry 14: 1829-
1834, 1999.
McKeirnan, J., Brockhoff, C.A., Creed, J.T., Caruso, J. "A Comparison of
Automated and Traditional Methods for the Extraction of Arsenicals from Fish."
Journal of Analytical Atomic Spectrometry 14: 607-613, 1999.
Gallagher, P.A., Creed, J.T., Brockhoff, C.A., Wei, X., McKiernan, J.W., Caruso, J.,
Shoemaker, J. "Accelerated Solvent Extraction of Arsenicals in Seaweed with
Ion Chromatographic Separation and ICP-MS Detection" Presented at the
American Chemical Society, Anaheim, CA, March, 1999.
Gallagher, P.A., Creed, J.T., Brockhoff, C.A., Wei, X., McKiernan, J.W. "The
Extraction and Detection of Arsenicals in Seaweed via Accelerated Solvent
Extraction with Ion Chromatography Separation and ICP-MS Detection"
Presented at the European Winter Conference on Plasma Spectrochemistry, Pau,
France, January, 1999.
McKiernan, J.W., Creed, J.T., Brockhoff, C.A., Chamberlain, I. "An Evaluation of
Accelerated Solvent Extraction as a Semi-Automated Means of Extracting
Arsenicals Prior to Speciation Analysis via IC-ICP-MS" Presented at the
European Winter Conference on Plasma Spectrochemistry, Pau, France, January,
1999.
U.S. FDA Publications and Presentations:
Vela, N.P., Heitkemper, D.T. "Arsenic Extraction and Speciation in Carrots using
Accelerated Solvent Extraction, Ion Chromatography and Plasma Mass
Spectrometry." Analyst 126: 1011-1017, 2001.
Heitkemper, D.T., Vela, N.P., Stewart, K.R., Westphal, C. "Determination of Total
and Speciated Arsenic in Rice by Ion Chromatography and Inductively Coupled
Plasma Mass Spectrometry." Journal of Analytical Atomic Spectrometry 16:
299- 306, 2001.
Heitkemper, D.T., B'hymer, C.B., Caruso, J.A. "Evaluation of Extraction
Techniques for Arsenic Species from Freeze Dried Apple Samples." Analyst
126: 136-140, 2001.
Heitkemper, D.T., Vela, N.P., Creed, J.T., Wei, X., Shoemaker, J., Gallagher, P.A.,
Schwegel, C.A. "Investigation of Methylated Arsenic Species in Carrots"
Presented at the Fourth International Conference on Arsenic Exposure and
Health Effects, San Diego, CA, June, 2000.
Future Research The poor extraction efficiencies mentioned above have led to the
development of a tetramethylammonium hydroxide (TMAOH)
extraction procedure for seafoods (generally high in protein) and a
trifluoroacetic acid (TFA) extraction procedure for rice (high in
National Exposure Research Laboratory — October 2001
-------
starch). TMAOH "softens" the protein in seafoods, and TFA "softens"
the starch in rice allowing more of the arsenicals to be released and
speciated. This release means that the extraction procedures have
removed almost all of the arsenic present in the sample. The extract is
then injected onto a chromatographic column. Future research will
address the inability to elute certain species from the chromatographic
column. The inability to elute all species from a column is correlated
to the presence of proteins in the extract. The unchromatographable
species may be bound to a protein or other substrate which may require
the addition of reagents which denature the protein and liberate the
bound arsenicals. The minimization of this unwanted binding to
substrates within the extract is essential to developing fully
quantitative arsenic speciation methodologies.
Questions and inquiries can be directed to:
John T. Creed, Ph.D.
US EPA, Office of Research and Development
National Exposure Research Laboratory
Cincinnati, OH 45268-1564
Contacts for
Additional
Information
Phone: 513/569-7833
E-mail: creed.jack@epa.gov
Federal funding for this research was administered through an
interagency agreement between the U.S. EPA and the U.S. FDA
(#DW75938526) and through a Regional Applied Research Effort with
U.S. EPA Region 10.
National Exposure Research Laboratory — October 2001
------- |