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Measuring Mercury Levels
in Fish
By: Hazel
Dickson From Technology and
Operations, Published:
12/3/2009
Mercury is a highly toxic element that can be
fatal to humans. It can occur naturally in the
environment as a metallic element, an inorganic
salt and/or an organic compound. However, human
activities produce most of the mercury found in
the environment. Coal-fired power plants, waste
incineration, metal processing and cement
production are the main sources of mercury air
pollution, producing approximately 75 percent
of the mercury released into the atmosphere
each year.1
Once in the atmosphere, mercury eventually
settles into rivers, lakes or oceans, where
certain microorganisms and abiotic reactions
convert it to methyl mercury. Through a process
called biomagnification, methyl mercury builds
up in predatory fish such as swordfish, tuna,
king mackerel and shark as well as in some
types of shellfish. Methyl mercury accounts for
more than 90 percent of the total mercury in
fish and seafood. In the
U.S., mercury has been estimated to have
polluted 30 percent of lakes, estuaries and
wetlands and 473,000 miles of streams, rivers
and coasts.2
Methyl mercury is acutely toxic to humans
because of its ability to pass through the
meninges into the brain. Similarly, in pregnant
women, methyl mercury can cross the placenta
and damage the developing nervous system of the
fetus.
In order to ensure maximum product safety and
protect the health of consumers, regulatory
bodies throughout the world have introduced
stringent legislation to monitor mercury and
methyl mercury levels in
seafood.
Regulatory
framework
According
to the action levels for poisonous or
deleterious substances in human food and animal
feed enforced by the
U.S.
Food and Drug Administration, the
maximum allowable concentration of methyl
mercury in seafood is 1
mg/kg.3
The
regulation is applicable to edible portions of
fresh, frozen or processed fish, shellfish,
crustaceans and other aquatic animals. Any
inspected products found to reach or exceed
this limit are withdrawn from the market, and
any further distribution, import or export is
prohibited unless otherwise implied by future
inspections.
The
U.S. Environmental Protection
Agency has introduced a methyl mercury
guideline that recommends a limit on mercury
consumption based on bodyweight, more
specifically, 0.1 mg/kg bodyweight per
day.4
The CODEX
alimentarius
193-19955
general
standard for contaminants and toxins in foods
specifies a maximum concentration of 0.5 mg/kg
wet weight of methyl mercury in fresh or
processed noncarnivorous fish and crustaceans
moving in international trade. The guideline
level for methyl mercury in carnivorous fish
such as shark, swordfish, tuna and pike is 1
mg/kg wet weight.
The
Zero
Mercury Working Group, a coalition
of different environmental organizations, has
recently published a report indicating that
fish tested in different locations around the
world show that internationally accepted
exposure levels for methyl mercury are
exceeded, often by wide margins. Based on the
fact that the consumption of fish is the major
source of ingestionrelated mercury exposure in
humans, the group claims that seafood products
should be labeled to ensure that consumers are
fully aware of the potential risks associated
with their
consumption.6
In order to ensure that concentrations of
mercury and methyl mercury in fish and fish
products are within the above specifications, a
powerful analytical method needs to be
implemented. Atomic absorption (AA)
spectrometry has emerged as a state-of-the-art
technique, offering precise, dependable
measurements of low levels of mercury in
seafood.
AA spectrometry advanced
capabilities
In cases where total mercury measurements are
required, AA spectrometry enables fast and
accurate analysis of samples with detection
limits below 0.07 ppb (μg/L) in solution, when
used in conjunction with a vapor-generation
accessory. This equates to 0.014 mg/ kg in the
original fish sample, based on a 0.5 g in 100
mL preparative method, which easily meets the
maximum levels set by food safety
regulations.
For the analysis of methyl mercury, AA
spectrometry provides a fast, cost-effective
and easy-to-use screening tool compared to more
complex and expensive techniques such as
HPLC-ICP-MS or
GC-ICP-MS.
Application
example
Analysis
was performed using a
Thermo
Scientific iCE 3500 AA spectrometer.
The spectrometer was combined with a Thermo
Scientific VP100 vapor-generation accessory,
which uses a continuous flow system to produce
a steady-state signal for excellent analytical
precision. The continuous flow of reagents
ensured that the system was self-cleaning,
reducing memory effects and increasing sample
throughput. The VP100 was controlled by the
Thermo Scientific SOLAAR software, simplifying
method setup and analysis. A mercury cell
provided as standard with the VP100 was also
used. This accessory offered an increased path
length compared to a normal vapor cell and
achieved exceptionally low detection
limits.
Sample preparation
Three different fish samples were chosen for
this application: fresh salmon purchased from a
supermarket; canned sardines also obtained from
a supermarket; and DORM-2 certified reference
material provided by the National Research
Council of Canada, Institute for National
Measurement Standards, Ottawa, Canada. Samples
were prepared following a four-step procedure
that included sample drying, sample
preparation, sample digestion and mercury
reduction
1. Sample drying phase is not necessary if the
final concentration of mercury is needed for a
wet-weight sample.
2. Refer to the manufacturer’s guidelines when
designing a digestion
program.
3. CARE: The reaction is exothermic and the
flask may become hot. Also, make sure to add
the hydroxylamine chloride slowly, otherwise
the solution may foam and eject some sample
from the flask.
Sample drying is necessary only if the final
mercury concentration needs to be measured as a
dry weight value. In that case, the fish
samples must be homogenized and dried in an
oven at 80°C until they reach a constant
weight. Fish tissue can be otherwise
freeze-dried and homogenized using a mortar and
pestle. After drying, portions of around 0.5 g
must be accurately weighed out for
digestion.
The FDA and CODEX alimentarius specify
concentrations of mercury in a wet-weight
sample, whereby fresh fish must be homogenized
in a food processor and a portion of
approximately 0.5 g must be precisely weighed
and placed in a microwave digestion vessel. In
that way, a representative fish sample is
produced.
For the
purposes of this experiment, 1 mL of 1,000 ppb
Hg standard solution was added to half of the
salmon and sardine samples. This spike gave a
concentration of 10 ppb Hg in the final 100 mL
sample. No mercury was added to the other half
of the samples to allow for the calculation of
spike recoveries. A set of microwave digestion
vessels containing the samples was placed in a
fume extraction hood prior to adding 10 mL of
concentrated
HNO.3
The
vessels were left for at least 30 minutes
without their lids on, to allow gases to
escape, and they were subsequently placed into
a microwave digestion system. A hot-block
digestion could also have been
used.
Upon completion of digestion, the samples were
transferred to a 100 mL graduated flask and 60
mL of 6 percent potassium permanganate solution
was added. The sample vessel was left for at
least two hours to ensure that all mercury in
the sample was reduced to Hg2+. It is very
important to ensure that the vessel is not
sealed at this stage, since any gases produced
can increase pressure. Following mercury
reduction, 15 mL of 20 percent hydroxylamine
chloride solution was added to the sample to
remove the excess potassium permanganate. Care
was taken during the addition of the
hydroxylamine chloride, as this produces an
exothermic reaction and the vessel may become
hot. The hydroxylamine chloride was added
slowly while the solution was gently mixed
during the addition. Without these precautions,
a violent reaction may occur that could eject
some sample from the flask, leading to
inaccurate results. The solution was then left
to cool, and deionized water was added to make
the volume up to 100
mL.
Standard
preparation
Standards were prepared from a 1,000 ppm (mg/L)
mercury standard solution. This standard was
first diluted to produce a 1,000 ppb (μg/L)
stock solution to allow simple preparation of a
range of standards. To demonstrate the linear
range of AA spectrometry, a wide range of
standards was used (1 to 100 ppb). The
standards were matrix-matched and prepared in
the same order as the
samples.
VP100 reagent
preparation
The vapor-generation accessory requires both a
reductant and an acid solution to perform the
reactions that form the gaseous mercury. For
this application, the reductant was a solution
of 7.5 percent stannous chloride (SnCl2)
stabilized in 10 percent HCl. The acid solution
was 50 percent HCl.
Instrument
conditions
The analysis was performed using the most
sensitive absorption wavelength for mercury,
253.7 nm. Five resamples were used, with each
resample taking four seconds, to thoroughly
assess the short-term stability of the
spectrometer. For normal use, three resamples
would be adequate. Deuterium background
correction was implemented throughout the
analysis.
Results
The calibration curve exhibited excellent
linearity up to 100 ppb (Figure 2), which is
equivalent to 20 mg/kg in a fish sample
(assuming a sample weight of 0.5 g) with an R2
value of 0.9989. This proves the superb
performance of AA spectrometry over a wide
concentration range. This calibration is
equivalent to concentrations of 0 to 20 mg/kg
mercury in the original fish samples, assuming
a sample mass of exactly 0.5 g. The percent
relative standard deviations (%RSDs) for each
of the standards were less than 2.5 percent.
This demonstrates the excellent stability of
both the spectrometer and the vaporgeneration
accessory.
The method detection limit (MDL) and
characteristic concentration were calculated
using the automated “Instrument Performance”
Wizard available in the SOLAAR software. This
user-friendly feature guides users through the
steps necessary to quantify the performance of
the method. It also automates all the data
processing, making the entire procedure quick
and easy. A detection limit of 0.068 ppb (μg/L)
in solution was identified. This equates to an
MDL of 0.014 mg/kg in the original fish sample
(assuming a sample mass of 0.5 g). The MDL
provides a measure of the noise and stability
of the system. A lower detection limit allows
for confident determination of lower
concentrations of mercury in samples. The
characteristic concentration, which is related
to the sensitivity of the method, was measured
at 0.724 ppb in solution. This is the
equivalent of 0.145 mg/kg in the initial fish
sample (assuming a sample weight of 0.5
g).
Salmon and sardine samples were spiked with 10
ppb mercury prior to digestion and compared
with unspiked samples to calculate recoveries.
These 10 ppb spikes would correspond to a
concentration of 2 mg/kg in normal fish samples
(assuming a sample weight of 0.5 g) and show
the accuracy of the analysis at levels
appropriate to current legislation. The spike
recoveries are shown in Tables 1 and 2. The
agreement with expected results was excellent,
with the recovered values all falling within 6
percent of the expected values. This
demonstrated the repeatability and precision of
both the sample digestion procedure and the
vapor analysis using AA
spectrometry.
Three separate samples of the DORM-2 standard
reference material were also analyzed to
guarantee the accuracy of the sample
preparation, digestion and analysis (Table 3).
The recoveries from these samples were
excellent, with an accuracy of ±2 percent or
better.
Conclusion
The recognition of the acute toxicity of methyl
mercury and the realization that fish is the
major source of human exposure has led to the
introduction of strict legislation in order to
protect consumers. A dependable analytical
method is required to ensure seafood product
safety and compliance with the regulations.
Vaporgeneration AA spectrometry has been
demonstrated to achieve precise, reliable
analysis of low levels of mercury in fish.
Offering excellent linear range, stability,
sensitivity and detection limits, the technique
easily meets the maximum concentration levels
set by regulatory bodies. The method is also
very fast, with an analytical cycle taking
approximately 90 seconds per
sample.
For more information on the iCE 3000 Series AA
spectrometers, please call +1 800-532-4752,
email analyze@thermofisher.com or,
alternatively, visit www.
thermo.com/ice.
References
1. United Nations Environment Programme (2002),
“Global Mercury Assessment,”
http://www.chem.unep.
ch/MERCURY/Report/GMA-report-TOC.htm
2. Natural Resources Defense Council Website,
“Mercury Contamination in Fish: A Guide to
Staying Healthy and Fighting Back,” “Global
Sources of Mercury,” http://
www.nrdc.org/health/effects/mercury/sources.asp
3. U.S. Food and Drug Administration, Industry
Activities Staff Booklet, August 2000, Action
Levels for Poisonous or Deleterious Substances
in Human Food and Animal Feed,
http://www.cfsan.fda.gov/~lrd/fdaact.
html#merc
4. Public Affairs Television, Science and
Health section, “The Mercury Story,”
http://www.pbs.org/now/science/
mercuryinfish.html
5. CODEX General Standards for Contaminants and
Toxins in Foods CODEX STAN 193-1995,
Rev.3-2007,
http://www.codexalimentarius.net/search/advancedsearch.
do?=&cat=3&doctext=&key=&qlang=&titletex
t=&type=5
6. Food Safety Information Website,
http://www.foodhaccp.
com/1news/021309k.html
Hazel Dickson, applications
chemist, Thermo Fisher Scientific, can be
reached at
hazel.dickson@thermofisher.com
or by phone at +44 (0) 1223 347
400
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