UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
• SUBJF.CT: tissue Analysis for use in FY78 Regional
Toxic Data Collection
FROM: Robert Crim, chief
Monitoring Branch (WH-553)
TO Surveillance and Analysis
.Division Directors
DATE: 2. 5
The methods included in the enclosed "Sampling and Analysis
Procedures for Screening of Fisli for Priority Pollutants" should
pe used for tissue analyses in your FY78 Toxic Data Collection
Activity. This program is discussed in Eckardt C. Beck's
memorandum of June 9, 1977, subject: FY78 Regional Toxic Data
Collection.
Technical questions or comments on these methods should be
addressed to the Cincinnati Laboratory.
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI. OHIO 40268
ENVIRONMENTAL MONITORING AND
SUPPORT LABORATORY - CINCINNATI
SUBJECT: Fish Tissue Analysis DATE: August 23, 1977
FROM : Dwight G. Ballinger,
Environmental Monitoring and Support
Laboratory - Cincinnati
TO : Surveillance and Analysis Division Directors
Attached is a copy of "Sampling and Analysis Procedures
for Screening of Fish for Priority Pollutants" for your use
in collecting and analyzing fish samples in response to the
State/Regional monitoring operating guidance. As with the
recent document sent to you on sediment analysis (see memo
of August 12, 1977), this document should also be considered
as interim guidance. While we consider it the Agency's best
source for the analysis of fish samples for priority pollutants,
we recognize that changes may be warranted as a result of your
regional experiences on a variety of collected fish samples.
As noted in the foreword, we encourage your careful review of
this protocol and your identification of any problems that may
arise.
•Attachment
As stated above
cc: Charles Brunot (w/attachment)
-------
SAMPLING Ai-iD ANALYSIS rRCClilHJRES FOR'
SCREENING OF FISH FOR PRIORITY POLLUTANTS
U. S. ENVIRONMENTAL PROTECTION AGENCY
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45263
August 23, 1977
-------
Foreword
These guidelines for sample preparation and analysis of fish
have been prepared by the staff of the Environmental Monitoring and
Sqpport Laboratory - Cincinnati, at the request of the Monitoring and
Data Support Division, Office of Water and Hazardous Wastes, with the
cooperation of many EPA Regional Laboratories, the Food and Drug Admin-
istration, the Southeast Water Research Laboratory, the Environmental
Research Laboratory - Duluth, and the National Institute for Occupational
Safety and Health.
The procedures represent the current state-of-the-art, but improve-
me,its are anticipated as more experience is obtained. Users of these
methods are encouraged to identify problems to assist in updating the
test procedures by contacting the Environmental Monitoring and Support
Laboratory, EPA, Cincinnati, Ohio 45268.
-------
CONTENTS
SAMPLE HANDLING .,
ANALYSIS OF FISH FOR CHLORINATED PESTICIDES AND POI.YCHLORI.NATED
BfPHENYLS 3
ANALYSIS OF FISH FOR GENERAL ORGANICS BY SOLVENT EXTRACTION ...... 8
ANALYSIS OF FISH FOR VOLATILE ORGANICS BY HEAD SPACE ANALYSES .... 14
ANALYSIS OF FISH FOR CYANIDE Vl9
ANALYSIS OF FISH FOR PHENOL 22
ANALYSIS OF FISH FOR MERCURY . 26
t
ANALYSIS OF FISH FOR METALS 30
•
REFERENCES 40
-------
LIST OF TABLES
Table I. Priority Tollutants Analyzed by Pesticide and
PCB Procedures 7
Table II. Base - Neutral F.xtractables 11
Table IJI. Acid Extractables 13
Taole IV. Characteristic Ions of Volatile Organics 17
Table V. Method References for Metals 36
-------
Sampling and Analysis Procedures for
Screening of. Fish for Priority Pollutants
Sample Handling
1. Collection
Separate analyses for all priority pollutants are done '
on the same sample of fish. A minimum of 2SO grams is required
for the total protocol. Small fish must be combined by species
to obtain this minimum weight.
2. Preservation
Field sampling requires an ice chest packed with dry ice.
Collected samples are wrapped in aluminum foil, labeled with freezer
tape, and transported in the chest. The dry ice must be replaced as
needed until subsamples are obtained for purgeoble organics.
3. Processing
To prepare samples for analytical pretrea'cments, wrap and
weigh each fish. Combine small fish by site and species until a
minimum combined weight of 250 grains is obtained. Chop the sample
into one-inch chunks using a sharp knife and mallet. Grind the sample
witfr a large commercial meat grinder that has been precooled by grinding
dry ice. Thoroughly mix the ground material. Regrind and mix material
*
two additional times. Clean out any material remaining in the grinder;
add this to the sample and mix well. Weigh five 10.0 gram portions
*
•
of the sample into separate 125 vials. Using a crimper, quickly
U. S. Environmental Protection Agency
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45263
-------
- "2 - .
and tightly secure a septum to each bottle with a seal. Store these
sample aliquots in a freezer until ready for Volatile Organics analyses.
Transfer remaining fish samples to a glass container and store in a
freezer for later subsampling and analysis.
4, - Special Equipment and Materials
4.-1 Knife, heavy blade (or meat cleaver)
4.2 Mallet, plastic faces, 2-3 pounds.
4.3 Electric meat grinder, 1/2 HP.
4.4 Ice chest.
4.5 Dry ice.
.4.6 Aluminum foil.
4.7 Freezer tape, for labels.
4.8 Freezer
4.9 Vials, 125 ml "Hypo-Vials" (Pierce Chemical Co., #12995,) or
equivalent.
4.JO Septa, "Tuf-Bond" (Pierce #12720,) or equivalent.
4.^1 Seals, aluminum, (Pierce #13214,) or equivalent.
4.\2 Crimper, hand, (Pierce #13212,) or equivalent.
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- 3 -
Analysis of Fish for Chlorinated Pesticides and Polychlorinntcd Biphenyls
1. Scope
1.1 The chlorinated pesticides and polychlorinatcd biphcnyls (PCBs)
listed in Table I are extracted from fish with either of the two
procedures described below. Method A employs a blender, while a
Tissumizer or equivalent is required for Method B. Both proce-
dures result in an extract that can be incorporated directly into
1 2
current EPA test procedures for pesticides or PCBs as cited
in the Federal Register .
2. Special Apparatus and Materials
. 2.1 Method A only
2.1.1 Blender, high speed - Waring Blender, Gourdes, Oir.ni-Mixer,
or equivalent. Explosion proof model recommended. Quart
container is suitable size for routine use.
2.1.2 Buchner funnel - Porcelain, 12 cm.
2.1.3 Filter paper - 110 mm "Sharkskin" circles.
2.1.4 Flask, vacuum filtration - 500 ml.
2.2 Method B only
2.2.1 Tissumizer SDT-182EN (available from Tckmar Company,
P. 0. Box 37202, Cincinnati, Ohio 45222) or equivalent.
2.2.2 Centrifuge - capable of handling 100 ml centrifuge tubes.
2.3 Method A ^ ^
2.3.1 Kuderna - Danish concentrator - 500 ml, with 10 ml
-------
-4-
graduatcd receiver and 3-ball Snyder column.
2.3.2 Chromatographic column - pyrcx, 20 mm ID x approximately
400 mm long, with coarse fritted plate on bottom.
3. Procedures
3.1 METHOD A: Weigh a 25-50.g portion of frozen, ground fish and add
to a high speed blender. Add 100 g anhydrous Na^SO. to combine with
the water present and .to disintegrate the sample. Alternately,
blend and mix with a spatula until the sample and sodium sulfate
are well mixed . Scrape down the sides of the blender jar and break
up the caked material with the spatula. Add 150 ml of hexane and
blend at high speed for 2 min.
H
3.1.1 Decant the hexane supernatant through a 12 cm Buchner
filter with two shark-skin papers, into a 500 ml suction
flask. Scrape down the sides of the blender jar and break
up the caked material with the spatula. Re-extract the
residue in the blender jar with two 100 -J. portions of
hexane, blending 3 min. each time. (After one min.
blending, stop the blender, scrape the material from the
sides of the blender jar and break up the caked material
with the spatula; continue blending for one min.) Scrape
down the sides of the blender jar and break up the caked
material between extractions.
tt
3.1.2 Decant the hexane supernatants through the Buchncr and
combine with the first extract. After the last blending,
I!
transfer the residue from the blender jar to the Buchner,
ti
rinsing the blender jar and material in t^ie Buchner with
-------
- 5 -
three 25-50 ml portions of hcxanc. Immediately after
ii
the last rinse, prcvss the residue in the Buchncr with
the bottom of a clean beaker to force out the remaining
hcxane.
3.1.3 Pour the combined extracts and rinses through a column of
anhydrous Na-,SO., 20 mm x 100 mm long, and collect the
eluate in a 500 ml Kuderna-Danish concentrator. Wash- the
flask and then the column with small portions of hexane
and concentrate the extract below 10 ml.
3.2 METHOD B: Weigh 20.0 grams of frozen, ground fish in a 100 ml
centrifuge tube. Add 20 ml of hexane and insert the Tissumizer
into the sample. Turn on the Tissumizer and disperse the fish in
the solvent for 1 minute. Centrifuge and decant the solvent
through a column of anhydrous Na_SO., 20 mm x 100 mm long, and
collect the eluate in a 500.ml Kuderna-Danish concentrator. Repeat
the dispersion twice more using a 20 ml aliquot each time, com-
bining all dried portions of solvent in the concentrator. Rinse
the Tissumizer and the column with small portions of hexane and
concentrate the extract below 10 ml.
3.3 Cleanup and Analysis: Unless prior experience would indicate the
fish species fat content is low (less than 3g/cxtract), the hexane-
acetonitrile clean-up procedures described in the reference methods
should be followed. In all cases, Florisil column chromatography
should be used to clean up the extracts before gas chromatography. ''
£n electron capture detector is used for final measurement, and
results are calculated in micrograms per kilogram. Identifications
-------
- 6 -
may be confirmed by GC/MS techniques as described in the analytical
(41
protocol for wastewaters .
3.4 Quality Control: Standard quality assurance protocols should be
employed, including blanks, duplicates, and dosed samples as described
in the "Analytical Quality Control Handbook"^ . Dose fish
sample aliquots by injecting minimum amounts (<20 yl total)
of concentrated pesticide or PCB solutions into the solid sub-
sample 10-15 minutes before extraction.
4. Reporting of Data
4.1 Report results in ug/kg on a wet tissue basis. Report all quality
control (QC) data along with the analytical results for the samples.
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- .7 -
TABLE 1 PRIORITY POLLUTANTS ANALYZED BY
PESTICIDE AND PCS PROCEDURES
Pesticides
Aldrin
a-BHC
b-BHC
d-BHC
g-BHC
Chlordane
ODD
DDE
DDT
Dieldrin
a-Endosulfai
b-Endosulfan
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Toxaphene
PCBs
Aroclor 1016
Aroclor 1221
Aroclor 1232
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
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- 8 -
Analysis of Fish for General Organic? by Solvent Extraction
1. Scope
1.1 Thi"s method is designed to determine those "unambiguous priority
f4)
pollutants" associated with the Consent Decree that are solvent
extractable and amenable to gas chromatography. These compounds
are listed in Tables II and III of this section. It is a gas chroma-
tographic-mass spectrometric method intended for qualitative and
semi-quantitative determination of these compounds. While this
approach has not been sufficiently tested through extensive experi-
mentation, it is based on laboratory experience and is presently
our best analytical approach for these organic materials in fish.
2. Special Apparatus and Materials
\
2.1 Tissumizer SDT-182EN (available from Tekmar Company, P. 0. Box
37202, Cincinnati, Ohio 45222) or equivalent.
2.2 Centrifuge - capable of handling 100 ml centrifuge tubes.
2.3 Separatory Funnels - 2 liter with Teflon stopcock.
2.4 Organic Free Water - prepared by passing distilled water through an
activated carbon column.
3. •Procedure
3.1 Weigh 20.0 grains of ground, homogeneous fish in a 100 ml
centrifuge tube. Add 20 ml acetonitrile and insert the Tissumizer
into the sample. Turn on Tissumizer and disperse the fish into
the solvent for 1 minute. Centrifuge and decant the solvent into
-------
-9-
a 2 liter separator/ funnel which contains 1300 ml of a 2
percent aqueous solution of sodium sulfate. Repeat the dis-
persion twice using a 20 ml aliquot each time and combine the
acetpnitrile in the separator/ funnel.
CAUTION^ The dispersion should be carried out in a fume hood to
avoid exposure to acetonitrile.
3.2 Adjust the pH of the sodium sulfate acetonitrile solution with 6N NaOi-
to pH 11 or greater. Use multirange pH paper for the measurement. E>
tract the aqueous acetonitrile solution with 60 ml hexane. Shake the
separator/ funnel for two minutes. Drain the aqueous layer into a 2
liter Erlenmeyer flask and pour the hexane extract through a short
column of prerinsed anhydrous sodium sulfate. Collect the dried
extract in a 500 ml Kuderna-Danish (K-D) flask fitted with a 10 ml
ampul. Repeat the extraction and drying steps twice combining the ex
tracts. Evaporate the extract to 5-10 ml in a 500 ml K-D apparatus
fitted with a 3-ball Snyder column and a 10 ml calibrated receiver
tube. Allow the K-D to cool to room temperature. Remove the receive
and adjust the volume to 10 ml. Analyze by GC/MS. If additional
•sensitivity is required, add fresh boiling chips, attach a two-ball
micro-Snyder column, and carefully evaporate to 1.0 ml or when
active distillation ceases.
3.3 Return the aqueous acetonitrile solution to the separatory funnel
and adjust the pH with 6N HC1 to pH 2 or less. Extract three
times with 60 ml hexane each time. Dry and concentrate as described
above. Analyze by GC/MS.
-------
- -10 --
NOTE: Should the partition used in this procedure not sufficiently
remove the lipid material, gel permeation may be employed. However,
special expensive equipment is necessary for this procedure .
3.4 Quality Control: Standard quality assurance protocols should be
employed, including blanks, duplicates and dosed samples as described
in the "Analytical Quality Control Handbook" . Dosing can be ac-
complished by injecting 1-20 ul of a standard solution, into the
homogenized tissue contained in a centrifuge tube.
5. Reporting of Data
4.1 Report results in yg/kg on a wet tissue basis. Report all quality
control (QC) data along with the analytical results for the samples.
-------
.J.J.
Ccrr.pc-jr.c Narr.s
'—chloropher.ol
: hsr.ol
;, •-.-Cic'.-.lorophor.ol
I-r.i~rccr.c-r.ol
i-chioro-rn-c-Gsol
1,4, 6-trichlcrcphenol
2.. -;-di-otr.ylphenol
(2-nitrooher.ol)
eut-sratcd anthracene (dlO)
0
0
0
1
1
1
• 1
1
1
1
1
0) 1
.63
.66
.96
.00
.05
.14
.32
.34
.42
.43
.64
.68
Limit of
Detection
fng)
100 •
100
100
100
100
100
100
2 pg
2 yg
100
100 .
40
Characteristic
El ions (Rel. Int.)
128 (100) , .64 (54), 130(31)
94 (100) , 65(17) , 66(19)
162 (100) , 164(58) , 98(61)
133 (ICO)', 65 (35) , 109 (8)
142(100) , 107(80)', 144 (32)
196(100), 193(92), 200(26)
122 (100) , 107(90) , ,121 (55)
184 (100) , 63 (59) , 154 (53)
198 (ICO) , 182(35) , 77 (28)
65(100) , 139 (45) , 109 (72)
266(100), 264(62), 268(63)
188(100), 94(19-)', 80(13)
CI ions
(Methane)
129
95,
163
140
143
197
123
135
199
140
257
189
/ 1-31,
123,
,. 1G5,
/ 168,
, 171,
, 199,
, 131,
, 213,
, 227 ,
/ ies,
, 255,
, 217
1
12
^,
J_
7
.1
_
s
^_
_
__
;olu.T.n: 6' clciss, 2 ITCT i.d.
Tenax GC
°
- 60/80 ^.
- 300° @ 3°/nin.
180
He •? 3C nil/min
-------
Comound NTar.e
(hexachloro-
benzene)
V
/ — '
• •
- ;.;
"V
is
.-.
?:•:
r •"
.' ^
— ' "'I
- •«-
" r
- -
zc
-*• w
T *""*
— V-
t '^
•^
t •'-
t '-
-«
zx
— ^J
-t
-« * . .
Z- o
J.U
"r
i-
Z:*1
-cich lorcbenzene
-dichlorobenzene
achloroethane
-r.icrlorcber.zene
( 2-chlorciscpropyl)
-'- * N O v-
c.r!-.lor obutadiene
, -, - tr icnlcrobenzene
( "'-cu "; o "oethvl ) ether
r. ch lor ccycl open tadiene
"DLc". ro.r.e
hlcrcr. cphthalene
:-.^"'hthylene
. . -^. ^- * . w • ^ ^ : . i^.
::I-.cron5
crc-r.s
-dinitro toluene
-ci phony Ihydrazine
_ ^ ^' T-» ^ 4- .- A^ J- — . T * T ^ *-v p
_•.—-. .^ U — O *-,wiL*U-i*O
ic.voscdiphenylanune
achlorcbensene
rc.-cphenyl phenyl ether'-
_,_.,i.;-,v-£rj-%
>..vftcer.e
•3 1 h y 1 ~ h t ha 1 a ta
thy ip^. thai axe
ci'cir.tier.e
cnr:
n-butylphthalate
2 id ins
' 0.35
0.36
0.38
0.39
0.47
.0.55
Or- ^
. 55
0.57
0 . 61
0.64
0.64
n £fl
VJ . O O
0.76 '
0.83
0.86
0.07
0.91
0.93
0.96
0.93
0.99
1.00
1.01
1 .09
1.09 •
1.10
1.15
1.23
1.3Q
1.31
1.38
bir.sylphthalate
1.46
Limit of
Detection
(ng)
40.
40
40
. 40
40
40
40
40
40
40
40
40
40
40
40
40
. 40
40
40*
40
40*
40
40 '
40
40
40
40
40
40.
40
40*
40
Characteristic
El ions (Rel. Int.)
146(100), 148(64), 113(12)
146(100),- 148 (64) , 113(11)
117(100), 199(61), 201(99)
146(100), 140(64), 113(11)
45(100) ,
225 (100,
74 (100),
128 (100)
93 (100) ,
237(100)
77 (100),
93(100) ,
162(ICO)
152(100)
154 (100)
82(100) ,
166 (ICO)
165(100)
77(100) ,
165(100)
169(100)
284 (100)
248 (100)
178 (100)
170 (100)
163(100)
149(100)
202(100)
202(100)
149(100)
184 (100).
149(100)
77(19), 79(12)
223(63), 227(65)
109 (CO) , 145 (52)
, 127(10), 129(11)
63(99), 95(31)
, 235(63), 272(12)
123(50), 65(15)
95(32), 123(21)
, 164 (32) , 127(31)
, 153(16), 151(17)
, 153(95), 152(53)
95(14) , 138 (10)
, 165(80), 167(14)
, 63(72) , 121(23)
93(58), 105(28)
, 63(72), 121(23)
168(71),
142(30),
250 (99) ,
179(16)-,
179(16),
164(10) ,
178(25) ,
101(23) ,
101(26),
150(27) -,
167(50)
249 (24)
141(45)
176(15)
176(15)
194(11)
150(10)
100(14)
.100(17)
104(10)
(
146,
146,
199,
146,
77,
223,
181,
129,
63,
235,
124,
65,
163,
152,
154,
139,
166,
183,
185,
103,
169,
284,
249,
178,
17S,
1C 1
~J JL ,
177,
203,
203,
149,
CI lone '
Methane)
143,
•14-C,
201 ,
I/O
. - '» -j i
135,
225,
133,
157,
107,
237,
152,
107,
191,
153,
155,
167,
1G7,
211,
213,
211,
170,
236,
251,
179,
179,
163,
223,
231,
231,
I- Ub~,
15
15
2C
1!
137
2 '
2C
1C
10?
^ -
^_ 'w
137
2C
1 1
^ \
i >
^. ^.
22
21
2 I
1 c
2 :;
27
2C
2T
1:
25
24
2--
Z/
,92(24) ,
,,91)50)
185(13)
135, 213, 22
149, 2S9, 32
^ * t.
-------
Table II Base-neutral Extractables (Cor.t'd.)
(hexachlcro-
benzene)
~ •.• •; .-. •p /a
!2-cthylhexyl)phthalate
:o (a)anthracene
:oi b)fluoranthene
:o (k)fluoranthene
:c (a)pyre.n.e
• •"•^. ,'T 9 "5 — Tl\*rvfPv^P
-•»^- \_/*./^/ \^w.y^/
-------
Table II
- 11 -
Base-neutral Extractables
:hlorcbenzene
:r:lorober.zer.G
.oroethar.e
:hlorcber.zcne
:hloroiscpropyl)
Lorobutadiene
-richlcrober.zene
:. lone
::*.loroethyl) ether
Lorccyclopentadiene
RRT1
(hexachloro-
benzene)
" 0.35
0.36
0.33
0.39
~ '•.'. Icroethcxy) methar.e
nitre toluene
pi-.or.yihydrazine
.-.itrc toluene
oscdiher.lanu.ne
r.yl phenyl ether'
ylphthalate
Iph thai ate
r.thene
utylphthalate
i:ie
bansylphthalate
0
0
0
0
0
0
0,
0
0
0
47
55
55
57
61
64
68
76
83
0.06
0.07
0.91
0.93
0.96
93
99
00
01
09
0
0
1
I
1
1.09
1.10
1,
1,
1,
1,
T_ _
1.
15
,23
3Q
31
38
46
Limit of
Detection
(ng)
40. . '
40
40
. 40
40
40
40
40
40
40
40
40
40
40
40
40
. 40
40
40*
40
40*
• 40
40
40
. 40
40
40
40
4Q
40
40*
40
Characteristic
El ions (Rel. Int.)
•146(100), 148(64), 113(12)
146(100),- 148 (64) , 113(11)
117(100), 199(61), 201(99)
146(100) , 140 (64), 113(11)
45 (100),
225 (100,
74 (100) ,
12S (100) ,
93(100) ,
237(100) ,
77(100) ,
93 (100) ,
162 (100) ,
152(100),
154(100) ,
82 (100) ,
166 (100) ,
165(100) ,
77(100) ,
165(100) ,
169(100),
284 (100) ,
248(100),
178 (100) ,
178 (100),
163(100) ,
149(100) ,
202(100),
202(100),
77(19), 79(12)
223(63), 227(65)
109 (CO) , 145 (52)
127(10) , 129 (11)
63(99), 95(31)
235(63) , 272 (12)
123(50) , 65(15)
95(32), 123(21)
164(32), 127(31)
153(16), 151(17)
153(95), 152(53)
95(14) , 138(18)
165(80) , 167 (14)
63 (72) , 121(23)
93(58), 105(23)
63(72), 121(23)
167(50)
168(71),
142(30),
250(99),
179(16),
179(16),
164(10),
178(25),
101(23),
101(26),
150(27),
149(100) ,
184 (100)-,92(24)',
149(100), 91)50)
249(24)
141(45)
176(15)
176(15)
194(11)
150(10)
100(14)
100(17)
104(10)
CI iono
(Methane)'
146, 143, -150
146, 14C, 150
199, 201, 203
146, 143, 150
77,
223,
151,
129,
63,
235,
124,
65,
163,
152,
154,
139,
166,
183,
185,
103,
169,
284,
249,
178,
17S,
151,
177,
203,
203,
149,
135,
225
133
157
107,
237
152
107,
191
153
155
167
167
211
213
211
170
236
251
179
179
153
223
231
231,
137
i 227
, 2C9
, .169
109
, 239
, 164
137
203
101
1C3
173
195
223
225
223
190
230
277
207
207
164
251
243
243
279
185(13)
135, 213, 225
149, 299, 327
-------
Table II
- 12 -'
Base-neutral Extractables (Cor.t'd.)
:our.d Xarr.e
(he.xachloro-
benzene)
-r.thyihexyl ) phthalate
\'a) anthracene
ib) f luorantheno
•.';•:) fluoranthene
.'.•' ) ^v"-^ne
,».•/ ,^ V ^ +^ + , \^f
: a",2,3-cd)?yrer.e
.-.D (2, h) anthracene
(c h i)pcrylene
1.46
1 . 50
1.54
66
66
173
J* * I fj
2.07
2.12
2.18
1
Limit of
Detection
(ng)
40
40
40
40
40
40
100
100
100
Characteristic
El ions (Rcl. Int.)
m
228 (100) , 229(19) , 226(23)
149(100) , 167(31) , 279(26)
228/100) , 229(19) , 226 (19)
252 (100) , 253(23) , 125(15)
252(100) , 253(23) , 125(1G)
252(100) , 253(23) , 125(21)
' 276 (100) , 138 (28) , 277(27)
278 (100) , 139 (24) , 279 (24)
276 (100) , 138 (37) , 277 (25)
CI iqr.s
(Methane)
223, 229,
149
223, 229,
252, 253,
252, 253,
252,
276,
253,
277/
278, 279,
276, 277,
237
281
231
231
30:
3C7
305
-csodirr.ethylanir.e
„• rsccli-n-prcpy la^.ine
:rc-T:her.yl phenyl ether
.-. r.idchyrle
lichlcrobcr.sidinc
,G-tetrachlcrocibenzo-
L o *•'. in '
.-;.! oror.ethyl) ether
42(100), 74(88), 44(21)
130 (22), 42(64) , 101(12)
204 (100) , 206 (34), 141(29)
252(100), 254 (66), 126(16)
322(100), 320(90), 59(95)
45(100) , 49(14) , 51(5)
£?-2250 on 100/120 rr.esh Supelcoport in s. 6' x 2 mm id glass column;. He @ 30 ml/min;
;-rar.: 50° for 4 min', then 8 /min to 260 and hold for 15 min.
nditicr.ing of column \vith base is required.
-------
. - 13 -
Table III Acid Extractables .
ur
(2-nitrooher.ol)
ropr.-2r.ol
j'.-.lorophcr.ol
cpr.C'r.oi
ro-m-c;resol
t rich lor onhcnol
r.troper.o
.-.itrc-o-cresol
•'r.Ioi-cpher.ol
ated anthracene (dlO)
0
0
0
1
1
1
• 1
. 1
1
1
1
0) 1
.53
.66
.96 ' .
.00
.05
.14
.32
.34
.42
.43
.64
.63
Limit of .
Detection
(ng)
100
100
100
100
100
100
100
2 yg
2 yg '
100 •'
100 .
40
Characteristic
El ions (Rel. Int.)
128 (100) , .64 (54) , 130(31)
94 (100) , 65(17) , 66(19)
1G2 (100) , 164 (58) , 93 (61)
133(100)', 65(35) , 109(3),
142(100) , 107 (80), 144 (32)
19G (100) , 193 (92) , 200(26)
122 (100) , 107 (90) , 121(55)
184 (100) , 63 (59) , 154 (53)
198 (ICO) , 132(35) , 77(28)
65(100) , 139(45) , 109 (72)
265 (100) , 264 (62) , 268 (63)
188(100), 94(19)', 80(13)
CI ions•
(Methane)
129, 131
95, 123,
163, 1G5
140,
143,
197,
123,
185,
199,
1-10,
267,
189,
168
171
1?9
151
213
227
1GS
255
217
, 157
135
, 167
, 122
, 133
, 201
, 153
, 225
, 23 v
, 122
, 26S
.unui: 6' glass, 2 irm i.d.
GC - 60/80 r.esh
300° @ S°/nin.
100° -
He -S 3C nl/.v.in
-------
-14-
Analysis of Fish for Volatile Orgarucs by Head Space Analyses
1. Scope
1.1 This method is designed to determine those "unambiguous priority
f4")
pollutants" associated with the Consent Decree that are
amenable to head space analyses. These compounds are listed in
Table IV of this section. It is a gas chromatographic-mass
spectromctric (GC-MS) method intended for qualitative and semi-
quantitative determination of these compounds.
The head space analyses and the liquid-liquid extraction methods
are complementary to one another. There is an area of overlap
between the two and some compounds may be recovered by either method.
The efficiency of recovery depends on the vapor pressure
and water solubility of the compounds involved. Generally, the area
of overlap may be identified by compounds boiling between 130°C and
150°C with a water solubility of approximately two percent. When
compounds are efficiently recovered by both methods, the chromato-
graphy determines the method of choice. The gas chromatographic
conditions selected for the head space method are, generally, not •
i
• suitable for the determination of compounds eluting later than
chlorobenzene.
1.2 While the above approach has not been sufficiently tested
through extensive experimentation, it is based on laboratory ex-
perience and is presently our best analytical approach for volatile
organic materials in fish.
2. Special Apparatus and Materials
2.1 Sonificr Cell Disrupter W-350 with microprobc (manufactured by
-------
- 15 -
Brawson Sonic Power Co., Manbury, Connecticut) or equivalent.
2.2 Gas-tight syringe - Sec.
2.3 Organic Free Water - Prepared by passing distilled water through
an activated carbon column.
. 2.4 Head Space Standard Solutions - Prepare three standard methanol
solutions of the compounds listed in Table IV at the following
concentrations: 50 ng/yg, 150 ng/yl and 300 ng/yl. The standard
solutions should be stored in the freezer at less than 0°C. Solutions
should be allowed to warm to room temperature before dosing. Fresh
standards should be prepared weekly. Procedures for preparing stan-
dards are outlined in the purge and trap section of ref. 4.
3. Procedure
3.1 Remove four of the sample vials containing 10.0 grams of homo-
genized fish from the freezer. Open the vials and add 10 ml of
organic free water to each while the fish is still frozen. Sonify
the fish for 30 seconds at maximum probe pov.-er. Immediately rcseal
the vials!!
3.2 Dose one sample vial, through the septum, below the water level with
10 yl of the 50 ng/yl standard solution. Dose a second vial, with
10 yl of the 150 ng/yl standard and a third vial with 10 yl of the
*
300 ng/yl standard.
3.3 Place all four sample Vials into a 90°C water bath for 1 hour.
3.4 K'hile maintaining the sample at 90°C, withdraw 2.0 ml of the head
gas with a gas tight syringe and analyze by injecting into a GC,
operating under the conditions recommended in ref. 4.
-------
- 16 -
NOTE: Specific GC detectors may be substituted for the MS.
3.5 Analyze the undosed sample first, followed by the 50 ng/yl
dosed sample.- If no compounds of interest are found in the
undosed sample and the dosed sample produces peaks to indicate
recovery of the protocol compounds, do not analyze the remaining
samples. Calculate lower limits of detection from the response
obtained from the dosed sample. If compounds are observed
in the undosed sample, analyze the two remaining dosed • samples
in exactly the same manner. Subtract the peak areas of compounds
found in the undosed sample from the corresponding compounds con-
tained in the dosed data; quantify the unknown. (NOTE: If the
calculated sample concentration is greater than the concentration
of the dosed standard used in the dosing step, it is necessary
to prepare additional standards in order to "bracket" the unknown.
Utilize the remaining sample in the freezer for this purpose.)
3.6 Quality Control: Standard quality assurance protocols should be
employed, including blanks, duplicates, and dosed samples as described
in the "Analytical Quality Control Handbook"^ .
4. Reporting of Data
4,1 Report all results in ug/kg on a wet tissue basis. Report all
i
quality control (QC) data along with the analytical results for
the samples.
-------
- 17 -
Table iv
Characteristic lens of Volatile Organics
Compound
chloromathane
dichlcrodifluororne thane
bromomethane
vinyl chloride
chloroethane
methylepe chloride
trichlcrcfluoromethane
1,1-dicJiloroethylene
broiriochlorome thane (IS)
1,1-dichlo^oethane
trans-1,2-dichloroethylene
chloroform
1,2-dichloroethane
1,1,1-trichloroethane
carbon tetrachloride
bromod;Lchlorome thane
bis-ch|croniethyl ether
•1,2-dichloropropane
trans-1,3-dichloropropene
trichloroethylene
dibromoch lor orr.e thane
\
cis-1, ij-dichloropropene
El Ions (Relative
intensity)
50(100); 52(33)
85(100)
101(13)
87(33);
103(9)
94(100); 96(94)
62(100); 64(33)
64(100); 66(33)
49(100);51(33);
84(86).; 86(55)
101(100); 103(66)
61(100); 96(80); 98(53)
49(100); 130(88);
128(70); 51(33)
63(100); 65(33); 83(13)
85(8); 98(7); 100(4)
61(100); 96(90); 98(57)
83(100); 85(66)
62(100); 64(33);
98(23); 100(15)
98(100); 99(66);
117(17); 119(16)
j.on usod to
quantify ;
50
101
. 94
62
64
84
101
96
128
63
• 96
83
98
117(100); 119(96); 121(30)
83(100)
127(13)
85 (66) ;
129(17)
79(100); 81(33)
63(100); 65(33);
112(4); 114(3)
75(100): 77(33)
95(100)
130(90)
97(66) ;
132(85)
129(100); 127(78)
208(13); 206UO)
75(100); 77(33)
97
117
127
79
112
75
130
127
75
-------
TABLE IV Continued
CcrTpound
1,1,2-hrichioroethane
benzene
2~chloro2thylvinyl ether
2-bronio-l-chloroprcpane (IS)
broTr.oform
1,1,2,2-tetrachlorocthene
1,1,2,2-tetrachloroethane
1,4-dichlorobutane(IS)
toluene
chlorobenzene
ethylbenzene
. acrolein
acrylonitrile
El Ions (Holci
^ intensity)
Ion uncd to
quantify
83(95) ; 35 (GC) ; 97(100) ;
. 99(63) ; 132(9) ; 134(8)
73(100)
63(95) ; 65(32) ; 105(18)
77(100) ; 79(33) ;156(5)
171(50) ;173 (100) ; 175(50)
250(4) ; 252(11) ; 254(11) ;
25G(4)
129(64)
164(78)
131(62);
166(100)
83(100); 85(66); 131(7)
133(7); 166(5); 168(6)
90(30); 92(10)
92 (78)
55(100);
91(100);
112(100); 114(33)
91(100); 106(33)
26(49); 27(100);
55(64); 56(83)
26(100); 51(32);
52(75); 53(99)
97
78
106
77
173
164
JL68
55
92
112
106
56
' 53
-------
- -19 -
Analysis of Fish for Cyanide
1. Scope and Application
1.1 This method is to be used for the determination of cyanide in
fish. All samples must be distilled prior to the analytical deter-
mination. For cyanide levels exceeding 0.2 rr.g/200 ml of absorbing
liquid, the silver nitrate titrimctric method is to be used. For cyan-
ide levels below this value, the colorimetric procedure is to be used.
4
2. Samp1c Preparation
2.1 A 5-gram portion of the frozen ground fish as described under
"Sample Handling" is used for the analysis. The sample should be
thawed before the analysis begins.
3. Preparation of_ Calibration Curve
3.1 The calibration curve 13 prepared by using portions of "spiked" fish
tissue, distilled in the same manner as the tissue sample being
analyzed. For preparation of the calibration standards, choose a 50 g
portion of fish, weigh and blend in a Waring blender (or equivalent) •
Vfith 10 ml of 10% NaOH and sufficient deionized distilled water so
that the volume of the mixture will be 500 ml.
f
3.2 Using a volumetric pipet whicli lias had the tip removed, withdraw
«
eight 50 ml portions, and place in a series of 1 liter boiling flasks.
Seven of the flasks should be spiked with increasing volumes of the
cyanide standard as given in 3.8 (Ref. 7). Adjust the final volume
of each flask to 500 ml with deionized distilled water.
3,3 Add 50 ml of a 5% NaOH'solution to the absorbing tube and dilute,
if necessary, with deionizc-d distilled water to obtain an adequate
-------
- 20 •
depth of liquid in the absorber. .Connect the boiling flask,
condenser, absorber and trap in the train as shown in Fig. 1 (Ref. 7).
3.4 Continue with step 8.2 through 8.7 (Ref. 7).
3.5 The calibration curve is prepared by plotting the absorbance versus
the cyanide concentration. The blank absorbance value must be sub-
tracted from each value before plotting the curve.
4. Sample Procedure
4.1 Place a weighed portion of the ground fish (approximately 5 g) in
a blender along with 100 ml of deionized distilled water and 1 ml
of a 5% NaOH solution.
4.2 Blend until a homogeneous mixture is obtained and transfer to a
1 liter boiling flask. Rinse the blender with several portions of
deionized distilled water totaling 400 ml ond add to the boiling flask.
4.3 Add 50 ml of a 5% NaOH solution to the absorbing tube and dilute
if necessary with deionized distilled water to obtain an adequate
depth of liquid in the absorber. Connect the boiling flask, condenser,
absorber and trap in the train and continue with step 8.2 through
8.7 (Ref. 7).
4.4 Read the absorbance and determine the cyanide concentration from
the calibration curve.
5. Quality Assurance
•5.1 Initially, demonstrate quantitative recovery with each distillation
digestion apparatus by comparing distilled aqueous standards
to non-distilled aqueous standards. Each day, distill at least
one standard to confirm distillation efficiency and purity of reagents.
-------
- 21 -
5.2 At least 15% of the cyanide analysis should consist of duplicate
and spiked samples. Quality control Jimits should be established
and confirmed as described in Chapter 6 of the "Analytical Quality
Control Handbook" (Ref. 6).
6. Reporting o_f_ Data
6.J Report cyanide concentrations as follows: less than 1.0 mg/kg,
nearest 0.01 mg; 1.0 mg/kg and above, two significant figures.
6.2 Report all quality control (QC) data along with the analytical
results for the samples.
-------
Analysis of Fish for Phenol
1. Scope and Application
1.1 This me_thod is to be used for the determination of phenol in fish.
Phenols are defined as hydroxy derivatives of benzene and its
condensed nuclei. The 4-amino-ant.ipyrinc colorimctric method given
determines phenol, the ortho- and msta-substituted phenols, and
under proper pH conditions, those para-substituted phenols in which
the substitution is a carboxyl, halogen, methoxyl or sulfonic
acid group. Presumably, the 4-amino-antipyrine method does not
determine those para-substituted phenols in which the substitution
is, an alkyl, afyl, nitro, benzoyl, nitroso, or aldehyde group.
1.2 All samples must be distilled prior to the determination of phenols
using the procedure given on page 576 (Ref. 8). Use method 510 B
for samples that contain less than 1 mg/kg and method 510 C for
samples that contain more than 1 mg/kg.
2. Sample Preparation
.; 2.1 A 5-gram portion of the frozen, ground fish as described under
"Sample Handling" is used for the analysis. The sample should be
thawed before the analysis begins.
< •
3. Preparation p_f Calibration Curve
3.1 The calibration curve is prepared by using portions of "spiked"
«
fish tissue, distilled in the same manner as the tissue samples
being analyzed. For preparation of the calibration standards,
choose a 50 g portion of fish, weigh, and blend in a Waring blender
-------
- 23 -
(or equivalent) with sufficient cleionizcd distilled water so that
the total volume of the mixture will be SOU ml.
3.2 Transfer a 50 ml portion of mixture to a beaker using a volumetric
pipet which has had the tip removed and determine the volume
of (1 + 9) H PO required to lower the pll to 4.0 using either
methyl orange indicator or a pll meter. This volume of (1 + 9)
H,PO. is to be added to each 50 ml portion of fish mixture prior
to the distillation step which follows.
3.3 Transfer 50 ml portions of the blended fish mixture to the dis-
tillation apparatus as shown in Fig. 318:1, p. 241 (Ref. 3), adding
the volume of H..PO. (determined above) to lower the pll to 4.0.
34
Add 5 ml of a 10% CuSO. solution to each distillation flask along
with appropriate volumes of the standard phenol solution (Ref. 8,
p. 579, 3C). A blank and 7 standards should be distilled for
preparation of the calibration curve. [NOTE: The ir.inimujn
detectable quantity is 1 pg/1 phenol in a 500 ml distillate.]
Adjust the volume in the distillation flask to 500 ml. Use a
500 ml graduated cylinder as a receiver.
3.4 Begin the distillation and continue until a distillate volume
i
of 450 ml is obtained. Stop the distillation and add 50 ml
deionized distilled water to 'the distillation flask after
boiling has ceased. Continue the distillation until a total of
500 ml has been collected. If the distillate is turbid, acidify
with (1 + 9) H,PO. and repeat the distillation as described.
-------
- 24 •-
3.5 Continue with the procedure, as j'.ivcn in the chloroform extraction
method 510 B, p. 577 (Rcf. 8). Rcr.d the absorbancc of the standards
against a reagent blank at a wavelength of 460 nm. Plot absorbancc
against micrograms of phenol for the calibration curve.
3.6 Alternatively, follow the direct photometric method (510 C, p. 580,
Ref. 8), for those samples in which the phenol concentration exceeds
1 nig/kg.
4. Sample Procedure
4,.! Place a weighed portion of the ground fish (approximately 5 g)
in a blender along with 100 ml of dcionized distilled water. Blend
un,til a homogeneous mixture is obtained and transfer to a 1 liter
boiling flask.
4..2 Rinse the blender with several portions of dcionized distilled
water and add to the distilling flask. Add a volume of (1 + 9)
H PO. to bring the pH of the mixture to 4.0 (the same volume as
O ^T
determined for preparation of the calibration standards may be
used). 'Add 5 ml of a 10% CuSO solution and adjust the total
volume to approximately 500 ml. Use a 500 ml graduated cylinder
as a receiver.
4,.3 Begin the distillation and continue as described in 3.4 - 3.6
above'. Read the absorbance and determine the ug of phenol from
the calibration curve.
5. -Cuality Assurance
1 ~
£.1 Demonstrate quantitative recovery with eacli distillation apparatus
by comparing aqueous distilled standards to non-distilled standards.
Each day, distill at least one standard to confirm the distillation
efficiency and purity of reagents.
-------
5.2 At least 15* of the phenol analyses should consist of duplicate
and spiked samples. Quality control limits should be established
*
and confirmed as described in Ref. 6.
6. Reporting p_f_ Data
6.1 Report phenol concentrations as follows:
Method 510 B to the nearest pg/kg
' Method 510 C - when less than 1.0 wg/kg to the nearest
0.01 yg; 1.0 nig/kg and above'to two significant figures.
6.2 Report all quality control data when reporting results of sample
analysis.
-------
Analysis of Fish for Mercury
Scope and Application
J.I This Method is. to be used for the determination of total mercury
(organic and inorganic) in fish. A weighed portion of the sample
is digested with sulfuric and nitric acid at 58°C followed by
ovornight oxidation with potassium permanganate at room temperature.
Mercury is subsequently measured by the conventional cold vapor
technique (Ref. 7, page 118).
1.2 The range of the method is 0.2 to 5 iig/g but may be extended
above or below the normal range by increasing or decreasing sample
size or through instrument and recorder control.
Sample Preparation
', \
2.1 The sample may be prepared as described under "Sample Handling"
or the special metal procedure may be used. A 0.2-0.3 g portion
should be taken for each analysis. The sample should not be allowed
to thaw before weighing.
Freparation of_ Calibration Curve
3.1 The calibration curve is prepared by using portions of "spiked"
fish tissue, treated in the same manner as the tissue samples
being analyzed. For preparation of the calibration standards
choose a 5 g portion of fish, and blend in a Waring blender.
3.2 Remove six equal and accurately weighed portions (0.2 g) with a
spatula and transfer to each of six dry BOD bottles. "Add 4 ml
, K*e
of cone. H,,SQ. and 1 ml of cone. HNQ_ and place in a water bath^ o
at f>8°C until the tissue is completely dissolved (30-60 minutes).
-------
3.3 Cool, and transfer 0, 0.5, 1.0, 2.0, 5.0 and 10.0 nil aliquots
of the working mercury solution containing 0 to 1.0 pg of. mercury
to, the BOD bottles. Cool to 4°C in an ice bath and cautiously
ad.d IS ml of potassium permanganate solution. Allow to stand
overnight at room temperature under oxidizing conditions.
3.4 Add enough distilled water so that the total volume is approximately
125 ml. Add 6 ml of sodium chloride-hydroxylaminc sulfate solution
to reduce the excess permanganate.
3,.5 Wait -it least 30 seconds after the addition of the hydroxylamine.
Treating each bottle individually, add 5 ml of the stannous sulfate
solution and immediately attach the bottle to the aeration apparatus.
5.6 Continue with the procedure as given on page 121 (Ref. 7). The
calibration curve is prepared by plotting the peak height versus
the mercury concentration. The peak height of the blank is
subtracted from each of the other values.
4. Sampl_c_ Procedure
4..1 Weigh 0.2-0.3 portions of the sample and place in the bottom
of a dry BOD bottle. Care must be taken that none of the sample
adheres to the side of the bottle. Add 4 ml of cone. H?SO and
1' nil of cone. UNO and place in water bath at 58°C until the tissue
is completely dissolved (30-60 minutes).
•4.2 Cool, to 4°C in an ice bath and cautiously add 5 ml of potassium
permanganate solution in I ml increments. Add 10 ml additional
permanganate, or more if necessary to maintain oxidizing conditions.
Allow to stand overnight at room temperature (sec NOTE 1). Continue
-------
- 28 -
.as described under (2.4J. NOTE 1: As an alternate to the overnight
digestion, the solubilization of the tissue may be carried out in a
water bath at 80°C for 30 minutes. The sample is then cooled and
15 »nl of potassium permanganate solution added cautiously. At this
point the sample is returned to the water bath and digested for an
additional 90 minutes at 80°C (Ref. 0). If this method is
followed, the calibration standards must also be treated in this
manner. Continue as described under (3.4).
5. Calculation
5.1 Measure the peak height of the unknown from the chart and read the
mercury value from the standard curve.
5.2 Calculate the mercury concentration in the sample by the formula
.. , yg Hg in aliquot
Vg He/gram = —*—^—n '—-
6 b 6 wt. of aliquot in gms
5,.3 Report mercury concentrations as follows:
Below 0.1 yg/gm, <0.1: between 0.1 and 1 yg/gm, to nearest
0.01 yg; between 1 and 10 yg/gm, to nearest 0.1 yg; above
10 yg/gm, to nearest yg.
6. Quality Assurance
§..l Standard quality assurance protocols should be employed, including
blanks, duplicates and spiked samples as described in the "Analytical
Quality Control Handbook" (Ref. 6).
•6.2 Report all quality control data when reporting results of sample analyses.
7. Precision and Accuracy
7.1 The following standard deviations on replicate fish samples were re-
corded at the indicated levels: 0.19 yg/gm ±0.02, 0.74 yg/gm ±0.05
and 2.1 yg/gm ±0.06. The coefficients of variation at these levels
-------
- 29 -
at these levels, added as methyl mercurjc chloride, were 112,
93 and 86%, respectively.
-------
- 30 -
Analysis of Fish for Metals .
1. Scope
1.1 This method is designed to determine in whole fish those priority
pollutants listed in the Consent Decree that are classified as
heavy metals or considered toxic as they exist in their elemental
form and associated compounds. Those pollutants are the following:
antimony, arsenic, beryllium, cadmium, chromium, copper, lead,
nickel, selenium, silver, thallium, and zinc.
2. Summary p_f Method
T..\ The fish is prepared for analysis by first being chopped into
small pieces, homogenized in a blender with dry ice, and then
solubilized by either dissolution after dry ashing or a wet
oxidation digestion. After sample preparation, atomic absorption,
either direct aspiration, gaseous hydride, or a flameless technique,
is used to measure the concentration of the pollutant in the fish.
3. Preservation and Handling
£.} While an aliquot of the ground fish as prepared under "Sample
f
Handling" may be taken for the metals determination, it may be
more desirable to prepare an individual fish to avoid possible
i
metal contamination from the grinder. Dust in the laboratory
environment, impurities in reagents and impurities on laboratory
apparatus which the sample contacts are all sources of potential
contamination. All glassware should be thoroughly washed with
i
detergent and tap water, rinsed with 1:1 nitric acid, tap water,
and finally deionizcd distilled water in that order.
-------
- .31 -
NQTli: Cliromic acid may be useful to remove organic deposits
from glassware; however, the analyst should be cautioned that
the glassware must be thoroughly rinsed with water to remove
the last trace of chromium. This is especially important if
chromium is to be included in the analytical scheme. A com-
mercial product--NOCHUOMlX--available from Codax Laboratories,
6 Yarick Street, New York, NY 10013, may be used in place of
chromic acid.
4. Sample Homogenization
4.1 If a separate fish sample, other than that prepared under "Sample
Handling," is to be used for metals analyses, unwrap and weigh the
frozen fish at the time of processing. Select a fisli that weighs
between 50 and 300 grams. If an analysis is required on a larger
(>300 grams) a 50 gram representative portion must be taken from
the sample after it has been pretrcatcd as described, in "Sample
Handling" on page 1 of this document and proceed to step 4.3.
^.2 After weighing, the fish should be chopped into approximately
one-inch chunks or smaller with a meat cleaver or a knife.and
mallet (2-3 pounds). Smaller pieces ensure efficient grinding.
4.3 Place crushed or pelleted dry ice into the blender container. The
weight of dry ice should be approximately equal to, or greater
« *
th.an, the weight of the fish.
4,4 Turn on the blender for 10 seconds to pulverize the ice and
chill the blender.
-------
- 32 -
1.5 Add the pieces of fish and blend at high speed until the
mixture is homogeneous. This usually requires 2-5 minutes.
Add more dry ice.if needed.
4.6 Pour the homogcnate into a plastic bag and close the bag with a
rubber band. Do not seal the bag tightly so that CCL may escape.
^.7 Place the bag in the freezer (-12°C for at least 16 hours) until
ready to proceed with the digestion step.
NOTE: If desired, the blender blades can be modified in order to
have the leading edge of the blades (the sharpened edge) turned down
so that, as it rotates, the blade will throw the material upwards.
Stainless steel blades may be a possible source of nickel and
chromium contamination and should be noted if detected. If a
•f \
^tantalum blade is available, it should be substituted for the
)
stainless steel.
The hole in the blender lid should bo enlarged sufficiently
to allow the evolved gas to escape (1/2 inch - quartsizc, 1 inch -
gallonsize). Hold a cloth or labwipe over this hole when blending
to prevent loss of the sample material. A glove should be worn
to prevent possible freezing of the skin by escaping gas.
5. Reagents
5.1 Lfeionizcd distilled water: Prepare by passing distilled water
through a mixed bed of cation and anion exchange resins. Use
deionized distilled water for the preparation of-all rcngents,
calibration standards, and as dilution water.
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-.35 -
5.2 Nitric acid (cone.)'- If metaJ impurities arc found to be present,
distill reagent grade nitric ncid in a borosilicatc glass.dis-
tillation apparatus.
5.3 SuIfuric acid, ACS grade (95.5 - 36.5%).
5.4 Sulfuric acid - 20% v/v solution. Carefully add 200 ml concentrated
H-,SO. to 500 ml water. Cool and dilute to 1 liter with water.
2 4
5.5 Hydrochloric acid, ACS grade (37-38%).
5.6 Hydrogen Peroxide, 50% stabilized ACS grade.
5.7 Dry ice (frozen carbon dioxide), pellet form preferred.
6. Apparatus
(,.} Blender, Waring, two-speed, stainless steel blade or tantalum blade
if available, glass container capacity 1000 ml, or equivalent
equipment.
(.2 Drying oven - Controllable with the range of 100"C to 150°C witli
less than ±5°C variation. Check calibration of oven temperature
control to ensure accurate ashing temperatures.J Furnace must be
' " • vi' operated in suitable fume hood.
-
(..3 Hot plate, controllable within the range of 80°C to 400°C.
Hot plate must be operated in fume hood.
7. lirocedure
Except for mercury which employs a cold vapor technique, the other
pollutants can be divided into two groups for continued processing.
GROUP I: Be, Cd, Cr, Cu, Pb, Ni, Ag, Tl, and Zn
GROUP II: As and Se
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Group I is digested by a dry ashing process (10) with the use of an ashing
aid, whi}e Group II is prepared utilizing a wet ashing procedure.
7. \ Group I_ - Metals
7.1,1 Remove the homogenized sample from the freezer and weigh
approximately 10 grams into a tared 100 ml tail form Pyrcx
beaker. Subtract the beaker weight from the total and
record the wet sample weight.
7.1,2 Add 25 ml of 20% sulfuric acid. Mix each sample thoroughly
with a glass stirring rod ensuring all sample material is
wetted by the acid. Rinse the stirring rod with water into
the ashing vessel and cover the sample with a ribbed
watch glass.
7.1.3 Dry the samples in an oven or furnace at 110+ 5°C until
a charred viscous sulfuric acid/sample residue remains.
Usually 12 to 16 hours (overnight) is sufficient. Transfer
the ashing vessels containing the dried samples to a cold,
clean muffle furnace which is provided with good external
ventilation (fume hood), ensuring that the sample remain
covered during the transfer. Initially set the furnace at
125°C and increase the temperature approximately every hour
in 50°C increments up to 275°C. Mold the temperature at
275°C for 3 hours. Finally, increase the temperature
to 450°C (at 50°C per hour) and hold for 12 to 16 hours
(overnight). Remove the covered ashing vessels from the
furnace and allow to cool to room temperature in a clean,
draft-free area.
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7.1.4 After initial overnight ashing, some residual carbon nwy
remain in the samples. Treat each sample asli with 0.5 ml
of water and 1 ml of concentrated nitric acid (whether or
not they are already white). Evaporate carefully just to
dryncss on a warm hotplate (in a fume hood). Place the
ashing vessels (covered with watch glasses) in a cool muffle
furnace and raise the temperature to 300°C and hold for
exactly 30 minutes. Remove eacli covered sample ash from
the furnace and allow to cool as before. If residual
carbon remains, repeat the nitric acid treatment until a
carbon-free white ash is obtained. The covered ashing
vessels containing the ash may be stored in a dessicator
or in a laminar flow clean hood. NOTE: Copious carbon
residues (i.e., black ashes) after overnight ashing may
indicate inefficient or uneven heating within the furnace.
Routine calibration of the furnace is advised.
7.1.5 Add 0.5 ml of nitric acid and 10 ml uf water to each cool
ashing vessel, then warm gently on a hotplate at 80-90°C
for 5 to 10 minutes to effect dissolution of the ash.
A small amount of insoluble white siliceous-like residue
may remain undissolved; do not filter the residue because
of the possibility of contamination. Quantitatively
transfer the contents of each ashing vessel into a 100 ml
volumetric flask, dilute to volume with water, and shake
thoroughly. Allow any residue to settle to the bottom
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- 30 -
of the flask (about 2 hrs.). Do not shake the sample
further before taking yn aliquot for analysis. The sample
is now ready for analysis. Note: The presence of a
precipitate other than the insoluble siliccous-like material
may result in low or eratic results for ?b. Precipitate
formation can result from heating the samples too long or
at too high a temperature after nitric acid treatment of
the ash. Precipitate formation must be avoided by main-
tenance of appropriate ashing temperatures.
7.1,6 The prepared sample should be analyzed by atomic absorption
using either direct aspiration or furnace techniques. For
a discussion of basic principles, the method of standard
addition, the chelation/solvent extraction procedures,
general instrumental operating parameters, and preparation
of standards and calibration see the section on "Atomic
Absorption Methods," pages 78-91 (Kef. 7) and the individual
analyses sheets (page numbers listed in Table V).
Table V_
Element Methods for Chemical Analysis
AC
Be
Cd
Cr
Cu
Ni
I'b
Sb
Tl
Zn
of Water
P-
P-
P-
P-
P-
P-
P-
P-
P-
P-
and Wastes, 1974
146
99
101
105
108
141
112
94
149
155
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- 37 -
7.1.7 Because of the adequate sensitivity by conventional flame
AA and the expected concentration levels of cadmium, copper
and zinc in the sample, these three elements should be
analyzed by direct aspiration. The furnace technique is
preferred for the analysis of the other Group I metals because
of their expected low concentration. When using the furnace
technique, the operating parameters and instructions as-speci-
fied by the particular instrument manufacturer should be
followed. If the detected concentration by the furnace pro-
cedure is beyond the working range of the standard curve, the
sample should either be diluted and reanalyzed or analyzed by
direct aspiration. The method of standard additions should be
employed when needed. If the sample matrix is so complex
that sample dilution followed by furnace analysis cannot be
used, or if the use of chclation/solvent extraction technique
for concentration Ag, Ni, Pb and Tl is preferred, the pro-
cedure as described starting on page S9 (Rcf. 7). should
be utilized.
7.2 Group II - Metals
7.2.1 Remove the homogenized sample from the freezer and weigh approx-
imately 5 grams into a tared 125 ml conical beaker. Subtract
the beaker weight from the total and record the wet sample weight.
7.2.2 Add 5 ml of cone. HNOj. Then slowly add 6 ml cone. H2S04
and cover with a watch glass.
7.2.3 Place beaker on hot plate and warm slightly. (NOTE: Remove
beaker If foaming becomes excessive.) Continue heating
until t IT* m i Y 111 t'f bprntn<--c; d n rk o v n no<; ^ i h I r Tfr\\ \c i n o
-------
condition is evident. Po not allow the mixture to char.
Remove beaker from hotplate and allow to cool.
7.2.4 Add an additional 5 ml of cone. HNO_, cover witli a watch
glass, and return beaker to hot plate. Repeat step 7.2.3.
7.2,5 When mixture again turns brown, cool, and slowly add 5 ml
of 50% hydrogen peroxide. Cover with watch glass and
heat gent.ly until the initial reaction has ceased. If
the solution becomes dark, repeat the peroxide addition,
several times if necessary, and heat to S0_ fumes. If
charring occurs, add further 1 ml portions of hydrogen
peroxide until the fuming sulfuric acid remains colorless
or very light yellow. (If at any stage it seems that the
sulfuric acid may approach dryness, cool, add 2 to 3
ml of sulfuric acid, and continue.)
7,2.6 Cool, add 40 ml of cone. IIC1 and dilute to 100 ml with
deionized distilled water. The sample is now ready for
analysis.
7,2.7 The Group II metals should be analyzed by atomic absorption
using the gaseous hydride technique. The apparatus setup,
standard preparation and calibration, and analysis procedure
that is to be followed is given starting on page 159 (Uef. 8)
From the prepared sample a 25 ml aliquot should be withdrawn
and the analysis continued as described in section 3.d,
page 162 (Rcf. 8).
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- 39 -
8. Calculation
£.1 From the values read off the appropriate calibration curve
calculate the concentration of each metal pollutant in the fish
as follows:
If the concentration of standards in the calibration curve is
plotted as mg/1,
mg/1 of constituent volume of prepared
in prepared sample X sample in ml
yg/gram = weight of wet sample in g
If the concentration of standards in the calibration curve is
plotted as pg/1,
pg/1 of constituent
in prepared sample X volume of prepared
1000 sample in ml
pg/gram = weight of wet sample in g
9. Quality Assurance
,-i
?.l Standard quality assurance protocols should be employed, including
blanks, duplicates and dosed samples as described in the
"Analytical Quality Control Handbook" (6).
S.2 Report all quality control data when reporting results of
sample analyses.
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- 40
REFERENCES
1. "Method for Orcjanochlorir.c Pectisidar, in Industrial Efflucntr.,"
U. S. Environmental Protection Agency, Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio, 45268 1973.
2. "Method for Poll/chlorinated Biphenyls in Industrial Effluents, "
Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio 45268 1973.
3. Federal Register, Volume 41, number 232, p. 52780, Wednesday,
December 1, 1976.
^
4. "Sampling and Analysis Procedures for Screening of Industrial
Effluents for Priority Pollutants, " Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, rev.
April, 1977.
5. Stalling, D. t..; Tindle, R. C.; Johnson, J. L. ; (1972) JAOAC.
55, 32-38. "Cleanup of Pesticide and Polychlorinated Bip};enyl
Residues in Fish Extracts by Gel Peiineation Chromatography. "
6. Handbook for Analytical Quality Control in l-'ater and Xasteioater
Laboratories (1972). U. S. Environmental Protection Agency,
Technology Transfer.
7. "Methods for Chemical Analysis of Water and Wastes (1974), U. S.
Environmental Protection Agency, Technology Transfer.
8. "Standard Methods for the Examination of Waster and Wastewater,
14th edition (1975).
9. Bishop, J. N., "Mercury in Fish," Ontario Water Resources Connn.,
Toronto, Ontario, Canada, 1971.
10. Jones, J. W. ; Gajan, R. J.; Buyer, X. W.; Fiorino, J. A.; (1977)
JAOAC_, 60, 826. "Dry Ash - Volttmnetric Determination of Cadmium,
Copper, L'ead, and Zinc in Foods."
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