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REGIONAL COPPER-NICKEL STUDY:
FISH TISSUE ANALYSiS
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March 3, 1978
This document is made available electronically by the Minnesota Legislative Reference Library as part of an ongoing digital archiving project. http://www.leg.state.mn.us/lrl/lrl.asp
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REGIONAL COPPER-NICKEL STUDY:
FISH TISSUE ANALYSIS
MINNESOTA ENVIRONMENTAL QUALITY BOARDREGIONAL COPPER-NICKEL STUDY
Authors: Ron LawrenzDale Christensen
March 3, 1978
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TABLE OF CONTENTS
INTRODUCTI ON
QUALITY CONTROL .
TISSUE PREPARATIONFishMammals and Invertebrates
TISSUE DIGESTION . .General AnalysisMercury Analysis
ANALYSIS .
SPECIAL CONSIDERATIONS
PRESENTATION OF RESULTS
TISSUE ARCHIVE . .
LITERATURE CITED
APPENDIX
. .. . . . .
PAGE
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335
557
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INTRODUCTION
As part of a cooperative agreement between the Minnesota Department of
Natural Resources (MDNR) and the Minnesota Environmental Quality Board,
the MDNR analyzed fish tissues for body burdens of six metals as a functional
portion of the Regional Copper-Nickel Study. The primary assignment of this
study was the analysis of fish tissues from northeastern Minnesota. The
results of these analyses were to provide a regional characterization of
the body burdens of metals associated with copper-nickel mining and provide
a baseline for future comparison. In addition, tissues were to be archived
for future reference and analysis. The principal item of equipment, a
Perkin-Elmer Model 603 atomic absorption spectrophotometer equipped with a
graphite furance, was put on order in February, 1976, and delivered in
August, 1976. Plumbing, wiring, and general set-up were completed in
October, 1976, and preliminary analyses were run in November. The first set
of fi'sh tissue data was completed in December, and the total year's sample
assignment was completed in August, 1977. The following paper documents
these analyses.
QUALITY CONTROL
Since most of the data produced in this study depended on measurements near
the detection capabilities of the equipment, it was of primary importance
to exercise stringent quality control measures. The most probable vector
of contamination was the distilled water supply. Groundwater supplied to
the lab was initially treated in a water softener, distilled into a glass
holding tank and finally run through Barnstead cation removal and ultrapure
) deionizing columns, respectively. This system produced very pure water with
a specific conductance of less than 1.0 micro-ohms approaching ultrapure
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conditions. Blank samples of this water were continually tested and were
never found to contain measurable levels of any metal under consideration.
Standard stock solutions were prepared according to specifications outlined
in the Perkin-Elmer manual, Analytical Methods for Atomic Absorption
Spectropho~ometry, using either the pure metal (Ni, Cu, Zn, Cd) or a salt
of the metal [Pb (NOg)2HgC12]. Stock solutions were stored in specially
washed 1 liter polyethylene bottles at a pH of 2 or lower (Robertson, 1968;
Struempler, 1973).
Final standards were prepared fresh each day by serially diluting stock
solutions to desired concentrations. Standards were compared with the
Environmental Protection Agency in lab quality control samples. The results
of these analyses can be found in Table 1.
All glass and plasticware were carefully washed in Micro Detergent, rinsed
in deionized water, soaked in 20 percent HN03 usually overnight, and
rinsed several times with deionized distilled water. All acid used in the
preparation of blanks, stock, and standard solutions was Ultrex brand
(Baker) nitric acid from certified lots. All glassware was borosillicate
and plasticware, polyethylene. All liquid transfers in the preparation of
standards, digestions, and sample injections were done with various sized
Eppendorff micro pipets with disposable polyethylene tips. The tips were
rinsed_ three to four times before use in order to avoid contamination
(Benjamin and Jenne, 1976), and each tip was used only once. Efforts to
wash and reuse pipet tips proved to be unsuccessful. Polyethylene
(whirl-pak, 18 oz.) bags and polystyrene weighing boats were used for
tissue storage and transfer. To check if there might be a metal leaching
problem in each of these materials, a sample of each was filled with
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10 percent HN0 3 and allowed to sit overnight. The leachate was then tested
for the six metals under consideration and found to be free of contamination.
Extreme care was taken to eliminate possible sources of contamination during
dissection. All contact surfaces were limited to teflon, polyethylene, or
stainless steel. Stainless steel has been found to be safe for use in
tissue dissection (Christian and Feldman, 1970). Latex gloves were worn by
lab personnel handling fish, allowing for efficient clean-up between
dissections. To minimize metal contamination, analysis was performed in a
room separated from the main laboratory and supplied with a filtered air
source. Glassware used in the preparation of standards was designated
solely for that purpose and separated from. General Lab Glassware stores.
In general, the EPA handbook for Analytical Quality Control in Water and
Wastewater Laboratories (1972) was used as a guideline for quality control
measures.
In order to check analytical procedure; two samples of fish tissue (muscle
and liver), prepared by this lab, were distributed to four other labs for
analysis. These labs included the FDA Laboratory, Minneapolis, EPA Water
Quality Lab, Duluth; Agronomy Services Lab, St. Paul; and the Research Lab,
Department of Soil Sciences, University of Minnesota, St. Paul. The results
of these analyses can be found in Table VII. Generally, there appears to
be good comparison between the various labs using atomic absorption technique.
TISSUE PREPARATION
Fish
Fish were received from field crews wrapped in polyethylene bags and packed
on ice and/or dry ice. Subsamples of tissue were dissected as soon as
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possible in order to assure the best estimate of live weight. Muscle tissue
was taken from the area above the lateral line directly posterior to the
head of the fish. This allowed for a uniform sampling from an area of
maximum tissue bulk which was generally free of bones. Uniform sample
location appears to be important since recent studies indicate regionalized
concentrations of metals even in muscle tissue (Stiefel, 1976). Ideally,
a minimum of 10 gms of tissue were dissected out for analysis, duplicate,
analysis, possible sample splits, and tissue archives. In smaller fish
where 10 gms of tissue was not available, the entire fillet from one side
'was taken. In most cases, all of the liver tissue was extracted. The
exceptions were large fish (northern pike) with livers over 30 gms where
only part of the liver was taken. Fish which were very small and a few
suckers., which appear to decompose more rapidly, had too little liver tissue
to work with and were deleted from the collection. Dissected tissue was
placed in pre-tared and labeled polystyrene weighing boats and weighed to
the nearest one one-thousandth of a gram. Tissue and weighing boat were
then placed in 18 ounce polyethylene whirl-pak sample bags and frozen in
preparation for freeze-drying. The subsamples of tissue were transferred
to the Meat Science Laboratory, University of Minnesota, St. Paul, for
freeze-drying. Weighing boats and tissue were placed intact into the mass
freeze-dryer, thus minimizing sample handling. When the drying process was
complete, the boats were placed in new whirl-pak bags for storage. In
preparation for digestion, approximately one half of the dried muscle tissue,
and usually all of the liver tissue, was homogenized in an all glass mortar
and pestel. The resulting powder was stored in appropriately-labeled
whirl-pak bags pending digestion. Through the entire procedure, chain of
custody forms were maintained to ensure proper disposition of samples'.
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Mammals and Invertebrates
In addi';l to the primary assignment of fish tissue analysis, 16 small
mammal (Clethryonomys gapperi) and 38 invertebrate (composite and individual)
tissues were dissected and analyzed. These tissues were not freeze-dried,
but all other procedures applicable to fish tissues were applied to these
tissues. Mammal tissues included liver and muscle from the left hind leg.
Invertebrate tissues included clams foot muscle tissue, crayfish tail
muscle tissue, leech whole specimen, snail whole body free of shell, and
insect whole. Unfortunately, species designations were not afforded these
samples.
TISSUE DIGESTION
General Analysis
The assurance of a complete, homogenized digest became one of the most
critical points of the study. This was especially important in the case of
liver tissue which contains a higher percentage of oils which were difficult
to break down. A great deal of literature research and experimentation was
required to find a suitable technique. Anderson (1972) in a comparison of
wet and dry ashing technique recommends dry ashing over wet ashing of tissue.
In experiments conducted in this laboratory, both methods proved to be
unreproduceable. Dry ashing especially affected the more volatile metals,
while open wet ashing in HN0 3 failed to digest the samples fully. Both
methods required more steps than desired, thus increasing contamination
vectors. Fats and oils can be properly broken down in wet digestions by
using a combination of acids usually containing perchloric, sulfuric, and/or
nitric acids (Chernoff, 1975). However, these acid combinations can be
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dangerous and special precautions are required for perchloric acid fumes.
Since our lab is not equipped with a fume hood suitable for the use of
perchloric acid, this method was not considered. Base digestions can be
accomplished using a quaternary ammonium hydroxide such as the commercially
available Soluene-350 (Packard Instrument Company). Barlow and Khera (1975)
reported good solubilization of tissue using Soluene-350. However, pre
liminary experimentation in this study revealed precipitation problems and
inability to keep spiked samples and standards in solution. Recently,
pressure digestions, especially those involving teflon-lined digestion bombs
and nitric acid, have proved to be a very reproduceable analytical method
(Krinitz and Holak, 1974; Davies and Adrian, 1975; Holak, et al., 1972;
Pearce, et al., 1976; Sunderman and Wacinski, 1974; Holak, 1974; and
Bernas, 1967). Preliminary experiments confirmed these findings, and Parr
~igestion Bombs (Model 4547, 25 ml) were selected as ~he most feasible
method of tissue digestion for this project. Not only did this method pro
vide complete digestion, but it was rapid (2-3 hours), required small
volumes of expensive ultrex acid and it was applicable to small amounts
of tissue. In addition, it required few steps and was applicable to even
the most volatile metals, since it is a closed system. Recovery of spiked
samples digested in bombs proved to be well within limits of acceptability
(Table 2). Manufacturers' recommendations were followed in charging the
digestion bombs. Ideally, the conditions of digestion included 0.1 gm
dry tissue (~ 0.5 gm wet), and 3 ml ultrex HN0 3 at 1500 C for 2 hours. In
the case of mammal and some invertebrate samples, tissue was not freeze-dried,
and approximately 0.5 gm of wet tissue was used. Occasionally, less than
0.1 gm of dry tissue was available for analysis and conditions were changed
to accomodate the digestion.
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The digestion bomb produced a clear digestate which required no filtering.
The digestate was rinsed from the teflon cup into borosilicate glass
volumetric tubes and brought up to 10 ml with deionized distilled water.
The diluted digestate was then poured into specially washed and rinsed
(see Quality Control) 60 ml polyethylene screw cap bottles with a positive
seal (Nalgene 2114) and stored at 50 C pending analysis.
Mercury Analysis
Tissues for mercury analysis were not freeze-dried, since experimentation
indicates that some organic forms of mercury would be lost through vola
tilization. A separate digestion applicable to the technique was us~d for
the mercury analysis. Tissue was removed from the frozen specimen in the
same region used for the analysis of other metals by taking a plug with a
stainless steel corer~ This plug was trimmed at either end with a stainless
steel scalpel to approximately .6 gms, weighed, and placed in a graduated
50 ml (25 X 200 mm) pyrex folin digestion tube (Pyrex 7900). Five mls of
concentrated 4:1 sulfuric-nitric acid were added to each tube which was
in turn placed in a hot water bath (BOOC) for 2 hours. The digestate was
then cooled in an ice bath and 10 ml of saturated (6 percent) potassium
permagenate was added to each tube and mixed on a vortex mixer. These were
allowed to set in the ice bath until the reaction subsided. Then 5 ml of
20 percent hydroxalamine hydrochloride was added to clear the remaining
permangenate and the tubes were again mixed on the vortex mixer. The
digestate was brought up to 50 ml with deionized distilled water pending
analysis. Just prior to analysis, 5 ml of 20 percent stanous chloride was
added to each tube and immediately covered with a rubber septum. These were
shaken several .times to release the elemental mercury and analyzed. This
procedure is a modification of the cold vapor technique devised by Hatch
and Ott, 196B and expanded on by numerous other experimentors.
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ANALYSIS
Tissue digests were analyzed for Cu, Ni, Zn, Cd, Pb, and Hg by atomic
absorption spectropnotometry. The principal analytical instrument was a
Perkin-Elmer Model 603 atomic absorption spectrophotometer equipped with a
deuterium backround correction, HGA 2100 graphite furnace, PRS-IO printer
sequencer and a 056 recorder. The specific conditions of analysis for each
metal. are listed in Table 3. Generally, Cu and Zn were found to be in
concentrations applicable to the use of flame spectrophotometry while
flameless technique was required for Ni, Cd, Pb. Hg was analyzed by using
the standard cold vapor technique. For this purpose, the. graphite furnace
assembly was modified so that mercury vapor could be swept through the
graphite tube via the internal gas flow. Windows were removed from the
furnace to enhance the .signal. All heat modes on the furnace control box
were turned to zero. Drying and charring times were turned to zero while
atomizing time was set at the maximum of 30 seconds. In this way, the
spectrophotometer could be run just as if the full furnace capabilities were
being employed. With the push of one button, proper gas flow, chart recordings,
integration, micro-processing, and printout-sequencing we':~ fully coordinated
greatly simplifying the analysis. Mercury vapor was swepth through the
furnace by installing two 18 gauge hypodermic needles in the internal gas
flow line from the furnace control box. These were inserted through the
rubber septum covering the digestion tubes, thus blowing the head space and
mercury vapor in the tubes into the spectrophotometer light path. Tissue
values were compared to blanks and a series of standards prepared in the
same manner. Generally, the conditions of analysis for all metals follow
Perkin-Elmer recommendations found in the handbook of analysis. Graphite
furnace injections were performed using Eppendorff 10·and 20 ~£ pipets.
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Standards for all metals were prepared fresh each day by seria.lly diluting
stock solutions. Standards and blanks were acidified to match samples.
SPECIAL CONSIDERATIONS
Tissue digests frequently require the development of special technique in
order to separate the metal in question and the complex matrix. This
problem is compounded by the difficulties inherent to analysis at or near
detection capabilities. In the course of this study, these problems were
dealt with as they occurred. The solutions to these problems are basically
reflected in the final conditions of analysis in Table 3. However, an
expanded explanation of the measures employed to overcome certain diffi
culties could greatly facilitate future analysis. Additionally, different
techniques might be employed from the outset to compensate for problems and
save valuable time not only in analysis, but in impromptu experimentation
and analysis correction.
Complete digestion is of primary importance in overcoming matrix inter
ferences. If complex compounds can be disassociated, the likelihood of
interference is decreased. This is dealt with in the digestion section.
Bomb digestions have been shown to provide a complete hydrolysis of organic
constituents in rat liver (Sunderman and Wacinski, 1974). However, even
with this type of digestion, interfering compounds such as salts are
common problems.
The most troublesome metal in this study proved to be cadmium. There were
several reasons for this: 1) Cd was the least abundant of all metals
stUdied requiring work near detection levels; 2) Cd, outside of Hg, was the
most volatile of the metals studied; 3) Cd is one of the most sensitive
Page 10
metals in AA technique requiring standards of PPB accuracy andreproduceability;
and 4) the wavelength (228.8 nm) utilized in the analysis is also subject
to broadband absorption by sodium chloride (Pulido, et al., 1966; Schlesinger
and Potter, 1974).' These problems were overcome by experimenting with
charring times and temperatures. The working parameters finally utilized
managed to properly separate the cadmium from the matrix, but the time of
analysis was greatly lengthened. The use of the deuterium backround
corrector was imperative since backround absorption was evident. It is
recommended that future Cd analysis include the addition of P0 4, S04' or
ammonium salts, such as (NH4)2S04' to decrease the volatility of Cd so that
charring temperatures can be increased (Giesy and Wiener, 1977). This would
reduce the time required for analysis and facilitate Cd-matrix separation.
These matrix modifiers should be added to all standards and blanks for
proper comparisons.
Speci~l care should be taken in the analysis of nickel, since a narrow band
pass is required to eliminate a nearby nonabsorbing wavelength. Experience
has shown that any vibration could alter the monochrometer enough to
reduce sensitivity.
Previous mention has been made concerning pipet tips as possible vectors of
contamination. Another point of concern is variability in pipet delivery
and distribution of sample drops in the graphite tube. To eliminate pipet
delivery variation, each sample injection was followed by a rinse injection
of blank solution. This procedure helps to improve previously unexplained
variations in standard replications.
Finally, great care should be taken to ensure a laboratory air supply which
is free of water and oil. The system employed in this study included an
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oil-free compressor. A delteck oil filter, two cartridge air dryers, and
a pur-gas heatless air dryer. This system provided a more than adequate
clean air supply.
PRESENTATION OF RESULTS
The results of the tissue analysis are reported as ppm (mg/Kg) metal for
dry and wet weight. For reader convenience and reference, live fish length
and weight are reported to the left of the same table (Table 4). Results
for mammal and invertebrate samples are reported in a similar fashion in
Tables 5 and 6.
TISSUE ARCHIVE
Due to lack of funding, samples collected during the 1977 field season were
only processed, freeze-dried, and archived for future analysis. These
tiss~es, in addition to the remaining tissue from the 1976 collection, will
be stored at the Chemistry Laboratory, Carlos Avery Game Farm, Forest Lake.
The archive will be maintained with records until such time that it is
deemed feasible to analyze or destroy them. In case any question concerning
the analysis should arise, these records will remain open for review.
Page 12
LITERATURE CITED
AndersG .. , J. 1972. Wet digestion versus dry ashing for the analysis offish tissue for trace metals. Atomic Absorption Newsletter 11(4):88-89.
Barlow, P.J. and A.K. Khera. 1975. Sample preparation using tissuesolubilization by Soluene-350 for lead determinations by graphitefurnace atomic absorption spectrophotometry. Atomic AbsorptionNewsletter 14(6): 149-150.
Benjamin, M.M. and E.A. Jenne. 1976. Trace element contamination 1.Copper from plastic microlitre pipet tips. Atomic AbsorptionNewsletter 15(2): 53-54.
Bernas, B. 1968. A new method for decomposition and comprehensive analysisof silicates by atomic absorption spectrometry .. Analytical Chemistry40(11): 1682-1686. .
Chernoff, B. 1975.metal analysis.
A method for wet digestion of fish tissue for heavyTrans. Amer. Fish. Soc. 4: 803-804.
Giesy, J.P., Jr. and J.G. Wiener. 1977. Frequency distributions of tracemetal concentrations in five freshwater fishes. Trans. Am. Fish. Soc.106(4): 393-403.
Hatch, R. and W.L. Ott. 1968. Determination of submicrogram quantities ofmercury by A.A.S. Anal. Chern. 47: 150.
Holak, W. 1974. Atomic absorption in food analysis-special techniques.Progress in Analytical Chemistry, Vol. 7.
Holak, W., B. Krinitz, and J.C: Williams. 1972. Simple, rapid digestiontechnique for the determination of mercury in fish by flamelessatomic absorption. J. of the AOAC 55(4): 741-742.
Krinitz, B. and W. Holak. 1974. Simple, rapid digestion technique forthe determination of mercury in seafood by flameless atomic absorptionspectroscopy. J. of the AOAC 57(3): 568-569.
Pearce, 1.0., R.R. Brooks, and R.D. Reeves. 1976. Digestion of fishsamples for mercury determination by flameless atomic absorptionspectrophotometry. J. of the AOAC 59(3): 655-657.
Robertson, D.E. 1968. The adsorption of trace elements in seawater onvarious container surfaces. Anal. Chern. Acta. 42: 533-536.
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LITERATURE CITED (contd.)
Schlesinger, W.H. and G.L. Potter. 1974. Lead, copper, and cadmium concentrations in small mammals in the Hubbard Brook Experimental Forest.Oi kos 25: 148-152.
'Stiefel, R.C. 1976. Mercury in white bass and carp. Publication OhioDepar~ment of Natural Resources, Division of Wildlife.
Struempler, A.W. 1973. Adsorption characteristics of silver, lead, cadmium,zinc, and nickel on borosilicate glass, polyethylene, and polypropylenecontainer surfaces. Anal. Chern. 45(13): 2251-2254.
Sunde"rman, F.W. and E.T. Wacinski. 1974. Use of teflon digestion bombsfor tissue analysis: measurements of the effect of Estradiol-17Supon hepatic copper in rats. Annals. of Clinical and LaboratoryScience 4(4): 299-305.
Table 1. EPA quality control samples (trace metals),recovery of metal, pg/liter.
-' -~
METAL SAMPLE 1 SAMPLE 2 SAMPLE 3ANALYZED TRUE ANALYZED TRUE ANALYZED TRUE
VALUE VALUE-EPA VALUE VALUE-EPA VALUE VALUE-EPA
Cd 4.8 5.2 -- -- --- ---
Cu 19 16 76 72 104 102
Ni 27 26 47 45 147 152
Pb 25 22 -- -- --- ---
Zn 12 11 31 30 180 174
Table 2. Recovery of five metals in tissuedigests vs. expected concentrations.
CONCENTRATION (mq/lMETAL EXPECTED CONC. RECOVERED %RECOVERED
Cu .020 .020 100
Zn .200 .207 104
Cd .0050 .0049 98
Ni .100 .089 89
Pb .050 .045 90
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Table 3. Instrument settings.
Perkin-Elmer AAS, Model 603
Hollow Cathode LampCurrentWave LengthSlit OpeningSignalModeRecorderIntegration TimeExpansionBackground Correction
ZINC COPPER
zinc copper
15 rna 15 rna214 nm 325 nm.7 nm .7 nmConc. Conc.Hold HoldABS. ABS.5 sec. 5 sec.
1 1no no
CADMIUM LEAD NICKEL
cadmium lead ni ckel
4 rna 10 rna ·20 rna
228.8 nm 283.3 nm 232 nm
.7 nm .7 nm .2 nm
Conc. Conc. Conc.
PK HI. PK HT PK HTABS. ABS. ABS.
10 sec. 7 sec. 5 sec.
1 1 1yes yes yes
HGA-2100 Graphite Furnace &Controller
Graphite TubeDrying Temp, TimeCharring Temp, TimeAtomizing Temp, TimeParge GasLine Pressure, Flow RateGas ModeRecorderFlame &Burner Control Box
non-pyrolized pyrol ized pyrolyzednooc, 50 sec. 110°C, 50 sec. nooc, 60 sec.250oC, 60 sec. nooc, 40 sec. 10000C, 50 sec.21000C, 10 sec. 2300oC, 10 sec. 2700oC, 10 sec.Argon Argon Argon40psi, 20 div 40psi, 20 div 40psi, 20 divInterrupt Interrupt InterruptAuto Auto Auto
Flame Type C2H2/Air C2H2/Air ------ ------ ------Oxidant: Line Pressure, 30psi, 30psi, - ----- ------ ------
Flow 70 dive 70 diveFuel: Line Pressure, Flow 12psi, 8psi, ------ ------ ------
30 dive 30 diVe
KEY TO TABLE 4)
I"
Sample No. Length{cm) Height{gm) Cu Ni Zn Cd Pb Hg
Bob Bay-Birch Lake'FOOOl NP-M 35.5 190FOOOI NP-L
Keeley Creek Bay-Birch LakeF0019 BC-MF0019 BC-L
F0165 BB-M ---F0165 BB:..L
Key to Abbreviations
BB - BurbotBC Black CrappieBG - Bl uegi 11CC - CreekchubCM - Composite MinnowCS Common ShinerD - Dace~S - Golden ShinerHS - Hybrid SunfishLMB - Large Mouth BassME - MuskellungeNP - Northern PikeRB - Rock BassSHR - Short Head RedhorseTP - Trout PerchWE - WalleyeWS - White SuckerYP - Yellow PerchM - MuscleL - Liver
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Table 7. Quality assurance sample split. Comparisonof analytical results from five cooperatinglabs for two tissues.
LAB INSTRUMENT Cu Ni Zn Pb Cd
Muscle ug/g Dried Tissue
A A.A.S. 0.7 0.16 15.0 .016
B Induced plasma arc. 8.5 10 28.6 10 1
C Polarograph A.A.S. 16.5 0 0
D* A.A.S. 1.05 1. 30 18.4 .31 .15
E A.A.S. 1.04 17.9 .34 .14
Liver ug/g Dried Tissue
A 13.3 .35 85.9 .29
B 27.0 10 73.4 10 1
C 83.8 .40 .15
D 14.8 1. 68 63.9 .39 .32
E 13.12 --- 60.6 .34 .21
*Ecological Services Chemistry Lab
