A SURVEY OF THE METHODS FOR ANALYSIS OF
METHYLMERCURY IN FISH AND PLANT MATERIAL
by
Jerrold R. Darm
Abstract
Five columns, two extraction procedures, and
three cleanup methods were tested for efficiency in
the detection and isolation of methylmercury in both
fish and plant material. A 1/8" x 6' stainless steel
column packed with chromasorb W (80-100 mesh) on carbo¬
wax 20 M was used in the final measurements. Between
85 and 100% of the mercury in Bonita samples was me¬
thylated. No methylmercury was found in macrocystis.
105
Introduction:
Considering the growing concern over mercury
levels in the marine environment, it is important to be
able to readily analyze for mercury compounds in marine
organisms. Several methods have been reported for the
extraction and detection of methylmercury; the most pre¬
valent and toxic mercury compound found in fish. This
study was initiated to compare two extraction technigues
and other related procedures for the removal and detection
of methylmercury in marine organisms.
Methods and Materials:
Standard Solutions:
Methylmercury chloride was obtained from MC/B
Manufacturing Chemists of Norwood, Ohio, and serially
diluted in pesticide quality benzene. No deterioration
of standards was detected over the five week period but
it is suggested that the most dilute solution be remade
from more concentrated solutions periodically. Standard
solutions of 500, 5, and 0.5 ng/ul (ug/ml) were used in
these experiments.
Standard curves:
Using injections of from 1 to 7 ul of standard so¬
lutions peak heights or peak weights were plotted against
nanograms injected to obtain standard curves (see Fig. 1).
Unknown samples were then weighed or measured to determine
ng/ul in the unknown (see Fig. 1).
10
e
S
2
N-
-
—
en

—.
s
a





2

—

O


—

—
10
Columns:
All the columns were tested on the Beckman GCA gas
liguid chromatograph. The first column to be tested was
a pesticide column containing a 1:1 mixed bed of 5% DC200
and 6% Ofl on chromosorb W (80-100 mesh) packed in a 6
x 1/4" pyrex tube. Operating at 200°C this column gave
insufficient separation of peaks as shown in Figure 2.
A more polar coating of 5% 1,4 Butanediol succinate on
chromasorb W (80-100 mesh) packed in a 6' x 1/8" stainless
steel column was used in the next trial. However, even at
column temperatures of as low as 140°C severe "tailing"
and incomplete separations of the benzene and methylmercury
peaks were observed.
The final three columns were packed with 5% carbowax
20M on chromasorb W (80-100 mesh) since this compound was
the most polar coating available to us. The first column
was a 6' x 1/4" glass tube. At a column temperature of
140°C this column gave good separation but poor sensitivity.
If the temperature had been increased both the peak shape
and sensitivity might have been improved but this was not
tested. The next column tested was 4' x 1/8" and was packed
with the same material. It yielded excellent peak shapes
and fair sensitivity at elevated temperatures (i.e. 175°c).
The final column was of stainless steel 6' x 1/8" and when
operated at a column temperature of 200°C it yielded good
separation and excellent sensitivity. The limit of detec¬
tion was approximately 0.5 ng, while the limit of detection
10
COMPARISONS
COLUMN
20 N6
nonpolar
10 NG

pesticide
column
Butanediol succinate
1,4
BASE LINE
(FIG. 2)
X 4
X
X 6
5
3NG
2 0 N6
10 N6
Carbowax 20 M on Chromosorb W
0
of the preceding column was 2.0 ng. On this column it
was found that peak area for methylmercury does not vary
with temperature. This would suggest that there is no
breakdown at high temperatures.
In conclusion, it was found that a polar coating
(carbowax 20M) in narrow columns at high temperatures
give the most desirable results.
Extractions:
Two procedures were used to extract methylmercury
from fish muscle.
Lafavor Extraction: The first procedure developed
by Dr. Herb Lafavor of the Berkeley Food and Drug Laboratory
(1), utilized sample weights which yielded 0.2 to 3.0 ug
depending upon column sensitivity. Samples were homogenized
with 20-30 ml of acetone, placed in a stoppered 50 ml centri¬
fuge tube and run at 2000 rpm for 15 minutes. The acetone
was then decanted and the sample shaken with another 30 ml
portion of acetone after breaking the cake with a spatula,
The sample was then centrifuged at 2000 ppm for 15 minutes
and the process repeated one more time. Although "nanograde"
acetone is suggested, reagent grade acetone was used without
pparent effects. After decanting the third time, the sample
was dried by blowing a filtered airstream (run through GLC
gas filter) across the broken up cake in the centrifuge
tube. Five ml of pesticide grade benzene and 10-20 ml of
3N HCI which had been washed in benzene were then added,
The benzene should be added first to prevent bubbling.
The solution was shaken vigorously for 3-5 minutes and
centrifuged at 2000 rpm for 30 minutes. The benzene
layer was then pipetted off and two more 5 ml aliguots
were added and extracted using the same procedure. All
three aliquots were placed in the same vial and submitted
to gas chromatography.
Westoo Extraction (4): Westöö's latest extraction
procedure was used (1968). The glassware was washed with
IN ammonium hydroxide, distilled water, and finally with
ethyl alcohol. Samples were homogenized with 55 ml of
distilled water and poured into a 250 ml centrifuge flask,
Fourteen ml of Hel and 70 ml of pesticide grade benzene
were added. The mixture was stoppered, shaken vigorously
for 5 minutes and centrifuged at 1500 rpm for 30 minutes.
Fifty ml of the benzene layer were pipetted off the top
and placed in a separatory funnel. The methylmercury was
then extracted with two 5 ml aliquots of 1% cysteine acetate
solution. This solution is made by dissolving 1.00 gram
of cysteine hydrochloride, 0.775 gram of sodium acetate
and 12.5 grams of anhydrous sodium sulphate in water and
made up to 100 ml (3). Finally, the 10 ml of cysteine
solution were acidified with 5.5 ml of 6N HCl and extracted
with 10 ml of benzene. This is dried with anhydrous sodium
sulphate using a micropipette stoppered with silvanized
galvanized wool and submitted to liquid gas chromatography.
Cleanup Procedures:
Several cleanup procedures were tested including
elution through silica gel and florisil columns packed in
micropipettes. The former method was both unsatisfactory
for removing plant pigments and for allowing passage of
methylmercury. The florisil column however removed plant
pigments while allowing two-thirds of the methylmercury in
a 10 ml sample of 0.5 ug/ul sample to pass through. A
third method which proved most satisfactory for recovery
was to extract the solution twice with 1% cysteine acetate
solution. Ten ml of this solution were sufficient to
transfer 100% of the methylmercury from 10 ml of a 2.0
ug/ml standard solution into a "clean" benzene solution.
These cleanup procedures were necessary for preparing ex¬
tractions of mixed plant and animal tissue.
Concentration Procedures:
In all cases, the concentration of standard solutions
of methylmercury in benzene yielded loss of a certain amount
of methylmercury. However, some were more efficient than
others. Aspiration and heating while exposed to a nitrogen
airstream were the least efficient procedures since approxi¬
mately one-half of the methylmercury was lost in 20-fold
concentration tests. Only one-third of the methylmercury
was lost using a reflux device on a steambath and the same
amount was vaporized in a 30-fold concentration test using
59
only a nitrogen airstream to evaporate the benzene. This
latter method then would be easiest and most efficient if
the column sensitivity is insufficient. However, further
tests would be need to determine percentage loss versus
original concentration.
Discussion:
Both the Westoo and Lafavor extraction procedures
were employed to test Bonita muscle samples with known
mercury concentrations (0.33 to 0.37 ppm) (5). Using the
latter method, 100% of the mercury in a 10 gram sample
was found to be in the form of methylmercury. Using the
former, two samples were found to contain 85 and 91%
methylmercury concentrations. Since the Lafavor extrac¬
tion is shorter, less complicated, and possibly more ef¬
ficient than the Westöö method it is recommended for
analysis of fish samples.
A 40 gram (dry weight) sample of macrocystis was
also tested. The Lafavor extraction was inadequate for
removing pigments before the benzene extraction. Thus
the cysteine method was used but had to be used twice for
sufficient cleanup. Of the 2 ug of total mercury present
(6) in the macrocystis sample none was shown to be in the
form of methylmercury.
6.
In another experiment ten grams of Perch intestinal
content were measured for methylmercury content and none
was found. Then to test for the possible methylation of
inorganic mercury (7) 50 parts per million of HgCl, were
added to 10 grams of gut content and incubated for two
weeks. However no methylmercury was found in the incubated
sample.
In conclusion then the Lafavor extraction seems to
be superior for fish samples yet inadequate when plant
pigments are present while the Westöö procedure is suffi¬
cient for the latter case.
14
gement
t
950 of the
am of the N
nder

1T
luate resear


——

articipation

Ackno


.

.


.

1

la
Bibliography:
(1) Lafavor, H. 1971. Tentative method for methyl
mercury measurement by gas chromatography
after release from animal tissue samples.
(personal communication).
G. 1966. Determination of Methylmercury
(2) Westöö,
Compounds in Foodstuffs. 1. Methylmercury
compounds in fish, identification and de¬
termination. Acta Chem. Scand. 20: 2131-2137.
G. 1967. Determination of Methylmercury
(3) Westöö,
Compounds in Foodstuffs. II. Acta Chem.
Scand. 21: 1798-1800.
G. 1968. Determination of Methylmercury
(4) Westöö,
Salts. Acta Chem. Scand. 22: 2279-2280.
(5) Wyland, J. 1971. Samples of Bonita muscle obtained
from Jacki Wyland, Hopkins Marine Station.
(6) Knauer,
G. 1971. Amount of Mercury in Macrocystis.
(personal communication); Hopkins Marine
Station.
(7) Jensen, S. and Al Jernelöv. 1969. Biological Methyla¬
tion of Mercury in Aquatic Organisms. Nature,
Vol. 223. p. 753-754.
11