-------
PRIORITY POLLUTANTS: Base/Neutral Extractables
Page 2
Green List
Nuraber
Compound Name
54
Isophorone
80
Fluorene
35
2,5-0-initrotolue.ie
37 / \
l,2-0iphenyl-hydr22in9
3S
2,4-Oinitrotoluer:s
62
N-Nitrosodiphenyi jST-e
9
Hexachlorobenzene
41
4-3romo?h«nyl phervl
ether
31
Phen»nthrene
78
Anthracene
71
Oimetnylphthalate
70
Olethylphthalate
39
Fluoranthene
STORET
Number
34408
34381
34626
34346
34611
34433
39700
34636
34461
34220
34341
34336
34376
S4
Sample Number
—
£
/ s" W
i ijij
0,o
,tr
j [g (f
ni
»7J>
193
.«
IS'J
/u
234,
2Vt
.„
^
Q.S2
Q.9C
(0.9 s-;
a...
a.n
J.OG
/.oo
,.o
,.-
O.SS
Q.fO
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=55-
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Wl
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l^i Q
m
?2^
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lol
,c
S7
w
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9^
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"
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no
,
-
Pyrene
34469
;^^fisas»ae5g*
>.'&«&nftJu£riCAr>
-------
PRIORITY POLLUTANTS: Sase/Seutral Extractable*
Green List
Number
Compound Name
63
Oi-n-butyl-phthalsts
5
Senzidine
57
Suty 1-benzy 1 -phtfta : its
76
Chrysene
65
Bis(2-ethylhexyl)
phthaJate
72
Benzo(a)Anthrscars
74
8enzo(b)Fluoranth«re
75
8enzo(k)Fluoranthana
73
Senzo(a)Pyrene
33
Indeno( 1 ,2 ,3-cd ) -pyrene
82
Oibenzo( a, h) -Anthracene
79
3enzo(g,h,i) perylene
fil ,
H-N i trosod i methyl ami ne
STORET
Number
39110
39120
34292
34320
39100
34525
34230
34242
34247
34403
34556
34521
34438
Sample Number
S3- n*.
a/4
253
^1
CS7
W
a?7
3C.7
ij \t *
fi Q ^
"T
W
«r
...
MS
I.BO-
|.?8
i.^
,../
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/ 70
u?
Q'^
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!
1ZS
7.^
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7.23
lit
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1 ZS
as-3
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1 2s'
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-U
135
JU
IcS"
"~*
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63
22
/C7
/of
1 «•»
1 ^-'
131
111
42
4V
-
'
i
K -(H trosod i-n-
3^423
lol
-70
133
-------
PRIORITY PCLLb'TA?!TS: Base/Neutral Extrsctables
Green List
Number
Compound Haas
40
4-Chloro-phenyl p^eryl
ether
28
3 ^'-Oictilorobenz-iiirie
69 .
V
Oi-n-octyl pntalcta
77
Acenaphthylene
°*££££,,f
STORET
Number
34641
34631
34596
34JOO
, »• » -i
Sample f.'umber
,,,
ISM
,«s
^
1 30
,«
•
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,.«
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-
i
—
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' 4
f
•sf^.-.f ' •••£•;
134
I -.— '.^^T:
» ^,
1
-------
PRIORITY POLLUTANTS: Volatile Organic*
Green List
Number
Confound Nasie
2
Acrolein
3
Acrylonitrile
45
Chlorome thane
50
Qichlorodifluoromethane
45
Bromonethane
38
Vinyl Chloride
16
Chloroethane
44
Methylene Chloride
49
Trichlorofluoromat.line
23
1 , 1 -0 i cii 1 oroethy 1 ene
30
Trans-1 ,2-Oichloro-
ethylene
23
Chloroform
10
1, 2-0 1 Chloroethane
STORET
Number
34210
342TS
34418
32105
34413
39175
34311
344Z3
34438
34501
34546
32106
34531
11 1
1,1,1-Tnchloroethane 34505
V^JUIJJLU.I, '
Sample Number 's C1-
SS^TJN-A
(VUlx^etV
—
—
—
—
1
IS"
11
OS"
2C
33"
S3
74
S3
=H
^
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jaj
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SI.
">«
01
<•
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61
t,t
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— ., .. _..-_,*»- .^.^^. . . >i'_«.a»sS8SS
H
;
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'- --X-v' V;*V < >7l-:y^v;-^illf
?i^;'£j^'i>af^&>V<<-''^^=^
-------
PRIORITY POLLUTANTS: Volatile Orgamcs
f
2.:
I-.- .
• *»-*s--
Green List
Compound Name
6
Carbon tetrachlorida
48
Bromodichlorone thane
17
Sis-chloromethy] ethsr
32
1,2-Oiciiloropropane
33 A
Trans-1 ,3-Oichlor3-
propene
51
D i bromoch 1 ortj-st -i-s
33 3
C-is-1,3-Oicnloroprojene
14
1 ,1 ,2-TMch Joroetnane
4
Benzene
19
2-Chloroethylvinyl ether
47
Sronwfortn
15
1 ,1,2,2-Tetrachloroethan
35
1,1,2,2-Tetrachloroether
STORET
Number
32102
32101
34268
34541
34561
34306
34561
345 n
34030
3457S
32104
34475
34516
13 !
Sample Number
O ^
l Q
in
,*.
133
isa
'**
* r
5=1
1 3^
,5,
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205
. a n
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atlM
Q.U1
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1.0-1
1-14
i ii"?
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•
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~^
.37,
-------
PRIORITY POLLUTANTS: Volatile Orgamcs
Page 3
Green List
Number
Compound Name
Z7
Trichloroethylene
86
Toluene
7
Chlorobenzene
38
Ethyl benzene
<&&Qf>vi ^W(.C JIG
P-|
i"3
QQ7
•
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0.73
\.\^
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1.34
o.a^
1.13
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97
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130
l 13
s-s-
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93.
•
j
• i
-------
PRIORITY POLLUTANTS: Pesticides
Green List
Number
Compound Naine
96
a-endosulfan
'02
o-SHC
!04
Y-8HC
(03
3-3HC
39
Aldrin
iCO
Hsptachlor
'01
Heptachlor epoxiss
95
a-sndosulfan
93
Oieldn'n
93
4, 4' -ODE
94
4, 4 '-000
92
4,4'-OOT
93
EndHn
STORET
Number
34356
39337
34264
39333
39330
39410
39420
34361
393SO
39320
39310
39300
39390
97 1
San"p'e Number
ql^SG-iQ
(Son-
TO
,.r.
*.«
*n
C.fl
l.oo
o.rz
1.37
,.-»
2,'t
1.1*
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-TO
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138
-------
Environmental Research Laboratory
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
„„_,„ Athens, Georgia 30605
DATE: January 18, 1978
SUBJECT;Action Concerning Region VII Comments on "Sampling and
Analysis Procedure for Screening of Industrial Effluents for
Priority Pollutants"
F«OM:W. M. Shackelford
Analytical Chemistry Branch
TO: William A. Telliard
Environmental Protection Agency
Effluent Guidelines Division
WH-552, 401 M Street, SW
Washington, DC 20460
In a memo dated 2 November chemists at the Region VII
Surveillance and Analysis Laboratory commented on strengths
and weaknesses of the analysis protocol for the consent
decree analysis program. Several of the "deficiencies and
errors" mentioned in the Region VII memo refer to typographical
errors, while others are the result of differences in judgment.
This memo will deal with each comment from the consecutive
sections "Base/Neutral Compounds"—"Radian Consent Decree
Standards" in the Region VII memo.
Base/Neutral Compounds
A) The protocol should be corrected such that 177 is one
of the characteristic masses for diethylphthalate
instead of 178.
B) After reflecting on the ions for 2,6-dinitrotoluene and
2,4-dinitrotoluene, it appears that corrections should
be made such that:
2,4-dinitrotoluene—165(100) , 63(31), 182(11)
Apparently, the ions for 2,6-dinitrotoluene were put in
the space for 2,4-dinitrotoluene by mistake.
C) It is true that bis(2-chloroethyl ether) elutes before
bis(2-chloroisopropyl ether) on 3% OV-17 but this order
is reversed on 1% SP-2250. We observed this in our lab
as well.
D) Published values for the 42 ion for N-nitroso-di-n-
propylamine all indicate a significant intensity. Mass
42 is also characteristic of all alkyl nitrosamines.
E) The diphenylhydrazine problems have been commented upon
in a memo to you from Wayne Garrison and Fred Haeberer.
EPA FORM 1320-6 (REV. 3-7S)
-------
-2-
F) 3% OV-17 was tried in this lab and found to give bleed
problems. The 1% SP-22SO is equivalent to 1% OV-17.
Our work showed essentially equivalent chromatography
with the two packings (3% OV-17 and 1% SP-2250) except
for some changes in retention times .
G) Chromatography of benzidine is a necessary evil, but
the amount used could be increased to 100 ng.
H) We are aware of several PAH isomers that cannot be
separated on the recommended column. They cannot be
separated easily on capillary columns either. We will
have to live with reporting them as the sum of the two
isomers .
Extraction Recoveries
*.
A) A provision for the determination of extraction recovery
efficiencies should definitely be made for the verifica-
tion stage. Other labs have not experienced troubles
extracting hexachlorocyclopentadiene. This probably
needs study .
Phenols
A)
Relative retention ti-T.es fcr phenols in the protocol
should be corrected as listed below:
RHT
Phenol 0.79
2-chlorophenol 0.34
2-nitropher.oi 1.00
2, 4-dime-chylphencl 1.02
2, 4-dichlorophenoi 1.06
p-chloro-ni-crasol 1.27
2 , 4 , S--crich.lcrophe.rioi 1.34
2,4-ci2itrcphenci 1.53
. 4-nitrophenol 1.73
4, 6-dinicro-o-cresol 1.32
pentachlorophenol 2.01
These agree reasonably well with other data. The
original data was taken before Tenax GC had been fully
evaluated in this lab.
3) One help for t£e gaps in Tenax is to condition a new
column at <_225 C, pack together when spaces develop,
then use normally. This can minimize the gap forma-
tion.
140
-------
-3-
C) You have commented previously on the use of the 4AAP
method for phenols.
Data Storage
A) It has been stressed that each VOA, B/N, and Acid
extract must be run in the GC/MS and the data saved.
, In following up this point with Dr. Kleopfer, he
assured me that he is running all the samples on the
GC/MS and not just screening with GC to avoid GC/MS
samples with no flame detected peaks.
Radian Consent Decree Standards
A) Radian inadvertantly left ethylbenzene out of the VOA
mix. Vinyl chloride and bis(chloromethyl ether) are
present in the mix. Dr. Tom Bellar has commented that
the vials must be opened at 20 C to keep from losing
these two. The 2-chloroethyl vinyl ether was put in
the B/N vial. New standards have been promised by Dr.
Larry Keith of Radian. He has been made aware of the
shortcomings of the first set. The new sets will have
more divisions for more ease of identification of
individual components. Concentrated samples of the
d, ,,-anthracene have been on order for several months.
Dr. Keith said that contractual problems in HQ were the
hold up.
3) Corrections to the standards identification sheets have
been made to reflect the proper concentrations.
141
-------
THIS PAGE LEFT BLANK
INTENTIONALLY
142
-------
THIS PAGE LEFT BLANK
INTENTIONALLY
143
-------
UNCHLORINATED BASE/NEUTRAL PRIORITY POLLUTANTS
ORDER OF ELUTION
Protocol (EPA)
Identification by
Gas Chromatography
(Radian)
Compound RRT
naphthalene 0.57
acenaphthylene 0.83
acenaphthene 0.86
isophorone 0.87
fluorene 0.91
phenanthrene 1.09
anthracene 1.09
dimethyl phthalate 1.10
diethyl phthalate 1.15
fluoranthene 1.23
pyrene 1.30
di-n-butyl phthalate 1.31
butyl benzyl phthalate 1.46
chrysene 1.46
bis(2-ethylhexyl) phthalate 1.50
benzo(a)anthracene 1.54
benzo(b)fluoranthene 1.66
benzo(k)fluoranthene 1.66
benzo(a)pyrene 1.73
indeno(l,2,3-c,d)pyrerre 2.07
dibenzo(a,h)anthracene 2.12
benzo(ghi)perylene 2.12
Compound RRT
isophorone 0.46
naphthalene 0.51
acenaphthalene 0.81
acenaphthene 0.83
dimethyl phthalate 0.88
fluorene 0.92
diethyl phthalate 0.98
phenanthrene 1.10
anthracene 1.10
dibutyl phthalate 1.23
fluoranthene 1.30
pyrene 1.34
butyl benzyl phthalate 1.51
benzo(a)anthracene 1.57
bis(2-ethylhexyl) phthalate 1.57
chrysene 1.57
benzo(b)fluoranthene 1.74
benzo(k)fluoranthene 1.77
benzo(a)pyrene 1.80
indeno(l,2,3-c,d)pyrene 2.07
dibenzo(a,h)anthracene 2.13
benzo(ghi)perylene 2.13
144
-------
Calspah
19 December 1977
PMT:hf-67
Mr. William Telliard
Chief, Energy and Mining Branch
Effluent Guidelines Division (WH-5S2)
USEPA
Washington, DC 20460
Dear Mr. Telliard:
This letter is for the purpose of updating you on the situation
regarding the transport of hazardous materials which was mentioned at the
Denver analytical seminar. As you are probably aware, the Department of
Transportation has a regulation (49CFR172-101 Hazardous Materials Table)
forbidding the transport of nitric acid aboard passenger aircraft. In
keeping with the requirements of the regulation and in view of the fact
that the EPA Standard Method for metal analyses calls for acid stabiliza-
tion of samples to pH <2, Calspan filed for an exemption to this regulation
so that field sampling for LOE Task 11 would proceed uninterrupted.
On December 15, 1977, Calspan received a reply from the DOT
denying our request to transport nitric acid (MOO ml) in a specially
prepared field sampling kit. (Enclosed is a copy of Calspan's request
for exemption describing the conditions under which the acid shipment
would take place and also a copy of the denial.)
Since the most recent revision of the sampling protocol specifies
field stabilization of metal samples (verbally given by you at the Denver
seminar) and in view of the recent ruling by DOT on our request, we feel
that this situation should be brought to the attention of all contractors
involved in the field sampling phase of the Effluent Guidelines Program.
This regulation by the DOT may seriously jeopardize the ability of con-
tractors to provide accurate metal analyses on unstabilized wastewater
samples. We would appreciate any assistance on your behalf to resolve
this situation with DOT and kindly request you inform us of any change
which may be affected by your action. Thank you.
Sincerely,
P. Michel Terlecky, Jr. Ph.D. ' (
Head, Environmental Sciences Section
Environmental § Energy Systems Dept.
Enclosures
-------
Caispan
PC. Box23i
Buffalo, New York14 221
Tel. 1716j 632-7500
28 September 1977
PMT:pL-37
Office of Hazardous Material Operations
U.S. Department of Transportation
Washington, D.C. 20590
Attn: Exemptions Branch
Gentlemen:
Item 1.
In accordance with subpart B, Section 107.103, Caispan Corporation
seeks exemption of the requirements of 49CFR 172.101 Hazardous Material
Table (HNO, forbidden aboard passenger aircraft) and seeks to determine
what may be carried in "Chemical reagent kits" as described in Section
173.286. According to Section 107,103,b(l), three copies of this request
are submitted herein for your review and approval.
It em 2.
Specifically, we seek to carry aboard passenger aircraft chemical
reagent test kits during sampling expeditions in support of requirements of
the U.S. Environmental Protection Agency (USEPA Contract 68-01-3281) to set
national effluent standards for various point source categories pursuant to
P.L. 92-500 (Federal Water Pollution Control Act Amendments - 1972) and
various state, local, and regional agencies and industrial customers. The
chemical reagent test kits are necessary in order to properly preserve
wastewater samples for subsequent analysis in our laboratories in Buffalo,
New York. Without certain stabilizing agents, these samples degrade
resulting in a loss of value of the sample for analytical and regulatory
purposes.
Item 3.
The applicant for this exemption is:
Caispan Corporation
A, tn: Environmental and Energy Systems Department
P.O. Box 235
Buffalo, New York 14221
716-632-7500
146
-------
Item 4.
In accordance with DOT 15(A) spec. 173.263(a); d(l) and i(l), the
proposed -nejthod of shipping is as follows: a small plastic container (bottle)
with a threaded acid-resistant cap cushioned by absorbent packing material
is enclosed in a glass bottle with threaded acid-resistant plastic cap. This
glass container is then enclosed in an individual, tightly sealed metal can
and surrounded by vermiculite (mineral matter) packing inside. The metal
cans are then placed in wooden boxes, surrounded by cushioning material
(vermiculite). The wooden boxes are then secured with lids, screwed into
place and properly labeled as to the items contained therein.
The wooden boxes mentioned above are constructed of white pine
stock with 3/4" walls and have reinforced ends with a total thickness of
1 1/2". The lid is also constructed of 3/4" stock and when secured in place
with 1 1/4" screws affords an effective seal capable of withstanding trans-
portation handling. Photographs of this proposed method of shipping are
attached. Construction blueprints were not utilized in the assembly of this
item and hence are not included in this report.
Drop tests have been performed on the proposed transport containers
(wooden boxes) in accordance with DOT 15(A) spec. 178.168-6: Gluing Efficiency
Wood Drop Test. The specification states that for containers with a gross
weight less than one hundred and fifty (150) pounds, the container, when filled
to capacity with sand and/or sawdust, shall be capable of withstanding eight (S)
drops from 1 foot (12 inches) onto solid concrete, 1 on each corner, without
exposure of contents. Such performance has been attained (and exceeded) on
the proposed containers and it is expected that these containers will withstand
the type of handling normally associated with transportation of materials on
commercial carriers.
Item S.
See attached table.
Item 6.
We believe that the level of safety achieved will meet or exceed
that required by the regulations and will ensure that no additional risks
to life or property will occur as a result of the granting of this exemp-
tion. Our record of shipment of explosives and other materials over the
past 30 years is exemplary with four full time personnel at Calspan devoted
to packaging.- shipping and receiving activities alone. Because of our
experience, and because the small amounts of reagents needed present no
additional risk, we respectfully request approval of our exemption at the
earliest possible time.
147
-------
Item 7.
The proposed mode of transportation for the chemical kits is
by commercial aircraft. Separate shipment of chemical reagent kits by
other carriers or by cargo aircraft will seriously affect our ability
to support EPA requirements in rural areas of the U.S. and would adversely
affect our acquisition of some $1 million worth of environmental sampling
and analytical business annually. Receipt by our engineers and technicians
of these kits simultaneously with receipt of sampling equipment and con-
tainers (shipped as baggage) is essential to the timely and economical
conduct of our work for which the Federal government is the main supporter,
Due to the small amounts of material being shipped (3 containers
of 100 ml (3 oz.) each) and considering the extraordinary care under which
these kits are prepared and packaged, it is felt that there will be np_
increased risks associated with the shipment of these materials.
Item 8.
As previously mentioned in Item 2, these kits are required for
the proper execution of the water sampling phase of a variety of EPA-
sponsored programs directed toward the establishment of national effluent
guideline regulations for numerous point source categories. This work as
described by P.L. 92-500 is a continuous effort and is due to be continued
for an indefinite period of time. Each sampling trip arranged for data
collection in conjunction with the above-mentioned programs lasts an
average of 4 days. During this time the chemical kits are transported to
the sampling site, used as required and when empty, returned to Calspan.
Item 9.
The small amounts of materials due to be transported do not
constitute any increased safety hazard when packaged and shipped as
proposed above. It is strongly felt that the time and effort invested
in the execution of precautionary measures for shipment of these hazardous
substances is consistent with the public interest and will adequately
protect against the risks of life ana property which are generally asso-
ciated with the transportation of hazardous materials.
Item 10.
It is not necessary to process this application on a priority
basis. We do ^espectfully request, however, that the handling of this
resubnitted application be given all due consideration with regard to
expeditious review. This is necessary in order that our company may not
experi -,ze any interruption in the acquisition of the aforementioned
govern, .;nt contracts which represent a significant financial investment.
148
-------
If there are any technical questions related to the materials
to be carried or the kits themselves, contact our Dr. P. Michael Ter.1 eoky
at (716) 632-75CC, x538.
Sincerely,
/
Poland J. Pilie, Head
Environmental § Energy Systems Department
149
-------
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A
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151
-------
\ /'' i'\ \ DEPARTMENT Or TRANSPORTATION "^P
I f,' / ' -, •]: A\ATER1AL5 TRANsPOkTAl'ON BUREAU
^V- " ' ""V^ WASHINGTON, O.C. IOS9O
i
| . DEC S 1377
I iSr . Xoland 3 , Pilie
1 Environmental and Energy
) Systems Department
.CaJLspan Corporation
•j P.O. Sax. 235
•I Boffalo, ItewTork 14221
j
1 Jleax Mr, Pilie:
i
| This is in response to your application dated September 2B. 1977
| (7844-N), iiled in accordance with Section 107. 103 of Title 49, Code of
i .Federal Regulations, (49 CPU), for -permission to snip cnemical kits
containing 70 percent .nitric acid, .sulfuric acid, -pnosphorii; acid ana
..solid .sodium iivdroxide pellets by passenger, carrying aircraft.
i
^ -3si accordance -with the Code of Jedsral- Regulations, Title 49, Section
\ . 107^ 109(0, .the request -is denied.
^ . The TEason/T-or denial is failure of -tne application TO saxisxy
\ requirements of Section 107.103 as follows:
] 1. In accordance -with 49 CFR 107. 103 Cb) (9) (±) you have made general
3 .statements as to why you believe that your, proposal to include 7H^
I ; nitric acid in 'the chemical kit -will scnxevt a level oi satet? at. iaa-_-
equivalent to that specified in the regulation from which the exempt ici
* is sought, .^owevex, it .is .obvious -that ,-n.o. ;yractT cal pactogiog ^tor &&•••
4 hazardous material will result in the same level of safety as -will be
| achieved by -precluding that material from transport.
j . 2~ Jlso. 49 CTS 173.286(b) and (n) (1) limits the contents of chemical
j .kits to 'corrosive liquids for vhicb B3^eption5 are provided in 49 CFR
i 172,101. Therrexore, nitric acid 01 caacentTanon of 4ui. OT less is n. ^
\ anthorized to be iticluded in a chemical kit. It. 'would, theraf ore, bvj
' more hazardous to permit nitric acid of concentration exceeding % ro
4 ie .included in a. chemical . kit .thereby further reducinp. the specif ieil
1 level of safety.
152
-------
idition as -noted below., -same of your requests axe unnecessary in
that the regulations already .authorize .shipment by passenger carrying
il-craft.
s tion T73 286 (b) provides ior shipioent cf sulfuric acid and -phosphoric
-• ' in .chemical .kit?. under conditions, that jaake your request for exemption
f<-r" these commodities unnecessary.
-tion 173 744 provides ior -shipment of sodium hydroxide solid as a
Intc-d quantity"such that no exemption is necessary for the package you
, -..rribi-a in your application .
Sincerely ,
Alan I. Roberts
Director
Office of Hazardous -Materials
Operations
153
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL RESEARCH LABORATORY
ATHENS. GEORGIA 30601
December 22, 1977
Mr. J. B. Anderson, Editor
Analytical Quality Control Newsletter
EMSL
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
Dear Mr. Anderson:
Enclosed is an article describing preliminary results of
investigations by Dr. Fred Haeberer of the Analytical
Chemistry Branch into the stability of two of the Consent
Decree Pollutants. This should be of interest to the many
of your readers who are involved in analysis of these
pollutants, and we request that you publish it in your
Newsletter. We are also planning to publish these results
in the Athens ERL quarterly report, which will probably be
published in 2 or 3 months.
Sincerely yours,
Arthur W. Garrison
Analytical Chemistry Branch
cc: Dr. Jim Lichtenberg, EMSL, Cincinnati
Dr. William Telliard, Effluent Guidelines
Division, EPA v-
Dr. Walter Shackelford, ACB
Dr. Ron Webb, ACB
154
-------
Analysis of Consent Decree Pollutants
Various researchers and contractors involved in the analysis
of the base-neutral extractable priority pollutants have
noted that both the GC retention time and the mass spectral
fragmentation pattern of N-nitrosodiphenylamine (one of the
priority pollutants) and diphenylamine axe apparently
identical. Our studies on this problem have shown that as
N-nitrosodiphenylamine (mp 67 C) is heated it begins to
decompose as soon as it is in the liquid state. Above 145 C
the decomposition proceeds very quickly yielding diphenyla-
mine and tetraphenylhydrazine. The identities of these two
compounds were established by infrared and mass spectral
data.
When N-nitrosodiphenylamine is subjected to gas chromatography
under the conditions imposed by the Consent Decree Protocol,
i.e., inlet temperature 275 C, decomposition occurs in the
inlet, resulting in a single sharp symetrical peak that has
been identified as diphenylamine (elution temperature 161°C,
RRT 0.97). This compound is formed in 40 to 80% yield.
Formation of tetraphenylhydrazine in the GC inlet may also
occur, but has not been established since this compound does
not elute under the protocol conditions. No GC peak that
could be identified as N-nitrosodiphenylamine has to date
been observed.
Identification of this nitrosamine via the regimen of the
protocol is inconclusive and it is therefore suggested that
the apparent presence of this compound be currently reported
as "N-nitrosodiphenylamine and/or diphenylamine" until a
valid analytical method can be developed. We are investigating
liquid chromatography as a separation tool for nitrosamines,
including N-nitrosodiphenylamine.
Preliminary work with 1,2-diphenylhydrazine (hydrazobenzene—
another priority pollutant) indicates that it also degrades,
perhaps not in the GC inlet, but definitely in solution,
forming azobenzene as the major product, along with aniline
and an unknown of mw 184. Current data have eliminated
benzidine as the unknown's identity and indicate that it
might be a N-phenylphenylenediamine. This decomposition
occurs in methanol and methylene chloride (the solvent for
the protocol standards), as well as in water. Additional
work is needed on this problem before any concrete recommenda-
tions can be made. (Alfred F. Haeberer, 404-546-3187, FTS-
250-3187).
155
-------
Index of References
1. letter: Examples of trap packing deterioration. NUS,
Miss C. Ellen Gonter, November 15, 1977.
2. A Brief Evaluation of Phenol Extraction Procedures. Environ-
mental Science and Engineering, Inc., November 9, 1977.
3. Analytical Problems in Effluent Analysis, Dow Chemical Company,
R.O. Kagel.
4. Draft - Priority Pollutant Validation Protocol, Dow Chemical Co.
R.O. Kagel and R. H. Stehl.
5. Memo, dated November 23, 1977, Subject: Metals Analysis-
Chicago Regional Laboratory.
6. Preliminary Interim Procedures for Fibrous Asbestos, Charles H.
Anderson and J. MacArthur Long, U.S. E.P.A., Environmental
Research Laboratory, Athens, Georgia.
7. Analytical Methodology for the Determination of Asbestos by
Transmission Election Microscopy. Walter C. McCrone Assoc.,
Inc.
8. Preservation of Phenolic Compounds in Wastewaters, M.J. Carter
and M.T. Huston, E.P.A., Central Regional Laboratory.
9. Diagram of Liquid-Liauid extractor, MIDWEST Research Institute,
C.L. Haile.
156
-------
IMUS
CORPORATION
CYRUS WM. RICE DIVISION
November 15, 1977
ANALYTICAL SERVICES LABORATORY
15 NOBLE AVENUE • PITTSBURGH. PA. 15205
Mr. William A. Telliard
Chief, Electric Utilities &
Mining Branch
Effluent Guidelines Division
U.S. Environmental Protection Agency
Waterside Mall
401 M Street, S.W.
Washington, D.C. 20460
Dear Bill:
Enclosed are examples of what is believed to be trap packing deterioration.
1. February 2, 1977, 5.0 ml of sample was purged according to the Bellar-
Lichtenberg procedure (EPA-670/4-74-009). The trap was sealed with
stainless steel caps and frozen. July 14, 1977, the trap was allowed
to warm to room temperature and run on GC/MS. The trap was sealed
with Teflon caps, and stored in a drawer at ambient temperature. July
21, 1977, the trap was desorbed onto the GC column, and the resultant
curve 1 obtained.
2. July 21, 1977, 5.0 ml of the retain sample was purged, trapped, desorbed,
etc. according to the Bellar-Lichtenberg procedure, (March 1977 trap
packing) and the resultant curve 2 obtained.
This was not an isolated case.
The peaks at 5.3 and 13.5 have appeared in most of the traps that were frozen.
The peak at 15.5 "grows" larger with time in an eight-hour period, even
though the trap is heated at 180°C with nitrogen at 40 ml/min flowing through
it for 20 to 30 minutes between runs..
At present we are using one trap per day, following the procedure as written
(some samples are diluted before purging), and not using the freezing technique.
Sincerely,
(Miss) C. Ellen Gonter, Manager
Water Laboratories Department
Enclosure
157
-------
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environ mental srivni'p and
P.O. BOX 13454 9 GAINESVILLE, FLORIDA 32604
szc.
904/372-3318
75-054-104
A BRIEF EVALUATION OF
PHENOL EXTRACTION PROCEDURES
Prepared by:
ENVIRONMENTAL SCIENCE AND ENGINEERING, INC.
P. 0. BOX 13454, UNIVERSITY STATION
GAINESVILLE, FLORIDA 32604
NOVEMBER 9, 1977
For:
EFFLUENT GUIDELINES DIVISION
ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
ATLANTA , GEORGIA
404 / 688-502S
JACKSONVILLE , FLORIDA
904 / 398-8303
ST. LOUIS , MISSOURI
314 / 567-4600
TAMPA , FLORIDA
313 / 886-6672
160
-------
Introduction
This report is the end result of a very brief study on the effectiveness
of the current protocol method for extraction and analysis of the acidic
(phenolic) fraction of the semi-volatile extractables. Alternative methods were
also investigated and the results are discussed here.
It should be noted that this study, which was designed and executed
in about 150 man hours, is in no way conclusive. The presentation of the
data in this report is for discussion purposes and to help in the solution
of a very complex problem.
161
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Procedures for Phenol Analysis
I Base/Neutral and Acidic Extraction (Similar to EPA Protocol)
1) The sample should be preserved with CuSO and phosphoric acid
to pH=4 as in Standard Methods, 14th Edition, p. 576.
2) Measure 100 ml of the sample into a 250 ml separatory funnel.
3) Adjust the pH of the sample to pH=12 with 6N NaOH solution.
4) Extract the sample with 50 ml of methylene chloride. Shake
for 2 min. and let emulsion break. Repeat the extraction with
25 ml and 25 ml of methylene chloride. Save the methylene
chloride layers for base-neutral analysis.
5) Adjust the pH of the sample to pH=2 with concentrated HC1 solution.
6) Extract with 50 ml, 25 ml, 25 ml of methylene chloride. Transfer
the methylene chloride layers through a 75 mm diameter glass funnel
containing a glass wool plug and a 2 cm layer of anhydrous sodium
sulfate to a 500 ml Kuderna-Danish apparatus with a 10 ml receiver.
7) Evaporate the extract down to 1.0 ml.
8) Add 10 ul of a 2 ug/ul d-^-anthracene internal standard solution to
the 1.0 ml of the extract.
9) Inject 2 ul of the extract on the gas chromatograph/mass spectrometer
using the GC conditions given in part II.
10) Calculate the amount of phenols in the sample using relative response
factors with respect to the dj_Q-anthracene internal standard obtained
from standard solutions.
162
-------
II Steam Distillation and Extraction Method
1) The sample should be preserved with CuSO and phosphoric acid
to pH=4 as in Standard Methods, 14th Edition, p. 576.
2) Measure 500 ml of the sample into the 1 liter boiling flask
of the distillation apparatus. Add several glass boiling beads.
3) Distill 450 ml of the sample, stop the distillation and when
boiling ceases add 50 ml phenol-free distilled water to the
distilling flask. Continue distillation until a total of 500 ml has
been collected.
4) Transfer the distillate to a 1 liter separatory funnel washing
the distillate container with several rinses of distilled water.
Combine the washings into the separatory funnel.
5) Adjust the pH of the distillate to pH=12 with 6N NaOH solution.
Check the pH with pH paper. Extract with 250 ml, 100 ml, 100 ml
of methylene chloride. Each time the separatory funnel should be
shaken for 2 minutes. Let the layers separate and draw off and
discard the methylene chloride layer.
6) Adjust the pH of the remaining aqueous distillate in the separatory
funnel to pH=2 with concentrated HC1. Check pH with pH paper.
7) Extract with 200 ml, 100 ml, 100 ml of methylene chloride. The
funnel should be shaken for at least 2 minutes each time.
8) Draw off the methylene chloride layers and pass through a glass
funnel containing a small amount of anhydrous sodium sulfate into
a 500 ml Kuderna-Danish apparatus with a 10 ml receiver.
9) Add a glass boiling bead and concentrate the combined methylene
chloride extracts to 1.0 ml on a boiling water bath.
10) Add 10 ul of a 20 ug/ul solution of d-, Q-anthracene to the 1.0 ml extract.
163
-------
II Continued
11) Inject 2 ul of the sample onto a gas chromatographic column using
the conditions given below.
MS/GC conditions:
Column: 6 ft x 2 mm i.d. glass
Packing: Tenax GC, 60/80 mesh
Flow: 30 tnl/min, Helium
Column Temperature: 180°C to 300°C at 8°C/min
Injector Temp.: 250°C
Jet Temp: 290°C
Transfer Line: 290°C
12) Calculate the amount of phenols in the sample using mass
spectrometer detection based upon relative response factors with
respect to d,Q-anthracene obtained from standard solutions.
164
-------
TABLE I
Distilled Water Spiking Experiments
Compound
Acidic Extraction
Only
92.5
89.1
1 88.2
88.4
.enol 89.6
57.7
sol 97.5
87.5
%RSD
11.4
5.6
5.1
0.8
1.5
0.6
3.7
1.7
II
1) Base Neutral Extraction
2) Acidic Extraction
III
1) Steam Distillation
2) Acidic Extraction
Phenol
0-Chlorophenol
0-Nitrophenol
2,4-Dichlorophenol
4-Chloro-m-cresol
2,4,6-Trichloroph
2,4-Dinitrophenol
p-Nitrophenol
4,6-Dinitro-o-cresol
Pentachlorophenol
I and II} 1 liter of water spiked with 100 ug of each phenol
III } 500 ml of water spiked with 100 ug of each phenol
Phenol and 2,4-Dinitrophenol were not included in standard solution
Creosote Waste Spiking Experiments
% Recovery
88.9
90.4
91.2
87.9
94.9
56.5
95.4
89.5
%RSD
8.0
6.1
7.0
4.4
5.7
5.1
8.4
5.7
75.7
73.5
78.2
85.9
78.1
3.1
67.8
80.7
%RSD
5.4
6.7
6.2
4.9
8.4
0
16.8
5.7
IV
1) Base-Neutral Extraction
2) Acidic Extraction
V
90.7
88
85
77
88
76
47
76
67
93
% Recovery
%RSD
9.2
17.
4.2
4.7
22.
11.7
16.5
10.7
3.
12.
1) Steam Distillation
2) Base-Neutral 3) Acidic Extract
Phenol
0-Chlorophenol
0-Nitrophenol
2,4-Dichlorophenol
4-Chloro-m-cresol
2,4,6-Trichlorophenol
2,4-Dinitrophenol
p-Nitrophenol
4,6-Dinitro-o-cresol
Pentachlorophenol
IV } 1 liter of water spiked with 1 ing of each phenol
V } 500 ml of water spiked with 1
-------
PROPOSED ALTERNATE METHODS
III Liquid-Liquid Extraction Using Labeled Internal Standard
1) The sample should be preserved with CuSO, and phosphoric acid
to pH=4 as in Standard Methods, 14th Edition, p. 576.
2) Measure 500 ml of the sample into a 1 liter separatory funnel.
3) Prepare a stock solution in acetone of the labeled internal
standard (e.g. phenol-dg) containing 100 ug/ml of the labeled compound,
4) Spike the sample with an aliquot of the labeled internal standard
solution. (e.g. 10 ml x 100 ug/ml=1000ug)
5) Adjust the pH of the sample to pH=12 with 6N NaOH solution.
Add 250 ml of methylene chloride to the sample and shake very
gently for about 5 minutes. A gentle rolling action is used to
prevent emulsion formation. Draw off the methylene chloride
layer and repeat the extraction with 100 ml and 100 ml of methylene
chloride. Discard the methylene chloride layers.
6) To the remaining aqueous sample in the separatory funnel, add
concentrated hydrochloric acid to bring the pH to 2.
7) Extract the sample with 200 ml of methylene chloride using a
gentle rolling motion as before. Repeat the extraction with 100
ml and 100 ml more of methylene chloride. Transfer the extracts
through a glass funnel containing a small amount of Na^SO,
(anhydrous) into a 500 ml Kuderna-Danish apparatus which includes
a 10 ml receiver.
8) Concent-rate the methylene chloride extracts down to 1.0 ml on
a boiling water bath.
166
-------
Ill Continued
9) Inject 2 ul of the concentrate onto a gas chromatograph/mass
spectrometer using the conditions given in Procedure II.
10) Calculate the extraction efficiency of the labeled internal
standard based upon response factors determined from
standard runs. Correct the response for the other phenols
assuming the same extraction efficiency as the internal
standard.
11) a) From Standard Solution,
= Areao
™
FIS
W
IS
= response factor for internal standard
Area = observed MS response for internal standard
JL o
W = amount of internal standard injected (ng).
J- O
\ = response factor for phenolic component A,B,,
,...;
W, . = amount of component A,B,... injected (ng)
(,A,D ,. . .)
b) From Sample Extract Injection,
Area
Extraction Efficiency (EEf ) = IS_
for internal standard R . WTr.
FIS IS
Area = observed MS Area for internal standard in sample
injection
167
-------
(b) Continued
W = Amount of internal standard expected in sample
injection (ng)
(ug/ml) ppm, , = Area(A,B,...) x WIS x 500 x
(A'B"-0 AreaIS vT
WHERE,
ppm. = concentration of phenolic component
U,B,...) ln sample in (Ug/mi)
Area,, ^ = MS area of phenolic component in sample
\" »•£*)•••/ . . «
injection
Area = MS area of internal standard in sample injection
(.LS)
W = amount of internal standard expected in sample
injection (ng)
VI = volume of injection (ul)
500 = dilution factor
168
-------
ANALYTICAL PROBLEMS IN EFFLUENT ANALYSIS
R. 0. KAGEL
ENVIRONMENTAL SERVICES
DOW CHEMICAL COMPANY
628 BUILDING
MIDLAND, MICHIGAN
169
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ANALYTICAL PROBLEMS IN EFFLUENT ANALYSIS
The most frustrating aspect of analyzing a complex effluent stream
for specific organic compounds is the almost total lack of validated
analytical method for those compounds, in that media, at low
concentrations. Specifically, these analyses usually involve the
effluent stream at the point where it interfaces with public waters.
Here, the concentration of any specific organic compound is most
certainly well below the part per million level. A few comparative
definitions of parts per million (ppra), parts per billion (ppb), and
parts per trillion (ppt) are given in Figure 1 in order to put the
magnitude of the analytical problem into proper perspective.
At these levels it is extremely difficult, very tedious and usually
costly - but not impossible - to obtain statistically meaningful
analytical data. A statistically meaningful result can be obtained
only by using validated analytical methods. Analytical procedures
as such are not validated. Validation involves the statistical
treatment of the data to determine the accuracy, precision, sensitivity
and reproducibility of an analytical procedure from laboratory to
laboratory or even from analyst to analyst within a laboratory. In
other words, validation provides a common denominator for agreement
on what an analytical result really means.
During the last five years, many industrial and government laboratories
have been busy developing analytical procedures for determining trace
levels of specific organic compounds in aqueous media. The increased
activity in this direction is the result of two things. First (Figure 2),
is the stagnation of analytical technology associated with those
methods that represent the shot gun approach to effluent analysis -
the BOD's, TOD's, TOC's etc. These methods provide gross parameters
that characterize the quality of the effluent and are used as control
parameters in most vasts treatment plants. The state of the art of
170
-------
this methodology has not changed appreciably over the past 10-15 years
ana for all practical purposes, this area of analytical technology has
become stagnant.
During the same time frame, significant state of the art developments
did occur in separations and detection technology. Gas chromatography-
inass spectrometry (GC-MS) is rapidly becoming a common place tool in
most analytical laboratories. Applied to effluent analyses, the
GC-MS represents the high powered rifle with a telescopic sight for
it allows the analytical chemist to zero-in on some specific compounds.
The combined use of separations technology, extraction of organic
components from a. waste water using an organic solvent such as hexane
or ether, followed by preconcentration and then detection by GC-MS
appears to be the universal approach to trace component analysis.
Figure 3 shows a typical example of this type of approach. Three
liters of a synthetic mixture of several compounds were extracted
with diethyl ether, preconcentrated by a factor of 3000 and analyzed
by GC-MS. All of these components are present at the ppb level.
Once the identity of each peak in the chromatogram has been established
by GC-MS, then subsequent analysis (Figure 4) are performed - in this
case - by election capture gas chromatography. This is simply to
avoid tying up a GC-MS which can range in price from $40K to $350K
with routine analysis which could easily be done on a $5K to $6K gas
chromatograph. The latter are readily available in most laboratories,
can easily be set up to do the analysis, and can be readily interfaced
with a computer to massage the data. These analytical procedures
for identifying and quantitating most of the components in an effluent
stream tend to be exceedingly tedious, time consuming and hence
quite costly. A good chrornotographer working in concert with a good
mass spectroscopist, given enough time,, the proper instrumentation,
a wide choice of column packings, will eventually develop an analytical
procedure for analyzing just about any system.
171
-------
A good example of Che kind of data generated by this approach is the
Environmental Protection Agency (EPA) study of the New Orleans area
water supply. Some 66 organic compounds, 10 of which are shown in
Figure 5, were reported to be present many at concentrations at or
less than 1 ppb. This study was one of the sources used, by EPA, to
develop the list of 65. The EPA research people who did this study
were very careful to emphasize that the values reported represent
highest concentration values rather than absolute values. This is
because when a component was determined by different methods, the
reported concentrations differed to some extent. Also, efficiency
values (recovery) for each stage of the analytical procedure were
not determined, i.e., the efficiency of carbon absorption of the
compound from water, losses incurred in drying the carbon, the efficency
of desorption, and losses incurred in concentrating the solvent to
low volumes. Without knowledge of these factors, one is hard pressed
to judge how good the results are because each step in the analytical
procedure introduces some error into the determination. The results
are probably good to ±50% at best and perhaps as much as ±100%, or
even more. The difference between 1 ppb and 2 ppb is probably not
significant in terms of the over all goals of the New Orleans study.
The New Orleans study was an exceptionally fine piece of work but
unfortunately it was not carried to completion - the procedures were
not validated. Hence it would be difficult for any two analysts to
produce numbers that would satisfactorily agree. It is obvious
that for the purpose of establishing effluent guidelines and monitoring
effluents one needs to establish better control and better technical
criteria on the analytical data. In general, analytical chemist
whether in industry or government are as genuinely concerned about
the accuracy, precision, sensitivity, and reproducibility of their
numbers as they are about developing the analytical procedures which
generate the numbers.
For 3. number of years, the residue analytical chemists were faced with
a similar analytical problem that involved the generation of meaningful
analytical data for pesticide residues in animal tissue, plants, soil
and water. Working together with USDA and FDA, they developed a mutually
172
-------
acceptable technical protocol for validating their analytical methods.
This protocol, the 10-10-10 principle, could easily be extended to
the case of effluent analysis, which in the broadest sense is a form
of residue analysis.
The mechanics of the 10-10-10 principle are shown in Figure 6. Ten
determinations are made on a control sample to determine interferences.
In residue studies the control is an untreated crop, soil, animal, etc.
A suitable control for effluent analysis is a synthetic sample of
plant effluent spiked with all compounds known to be present except
the one being analyzed. In this way the level of interference is
determined.
The fortified samples are used to determine recoveries. Samples of
control are usually spiked with the compound of interest at various
concentration and then spike is run through the entire analytical
procedure. This will show losses due to absorption on glassware,
charcoal absorption and desorption efficiencies, extraction efficiencies,
and the like. Generally, recoveries better than 85% are acceptable.
Realistically recoveries may range from 50% to 85%. The lower values
are acceptable if consistent results are obtained with replicate
samples.
Finally, 10 determinations are run on different aliquots of the same
samples to determine the precision of the procedure. The statistics
of the analytical procedure are usually verified by two independent
laboratories.
The 10-10-10 principle first surfaced in the 1950's. It was later
advocated by Harris and Cummings , USDA, (in 1964) as the absolute
minimum data requirement necessary to support the registration of a
pesticide use. In recent years the 10-10-10 principle has become
accepted protocol for validating analytical procedures in most
pesticide studies. It would be ironic if any data less than this
would be deemed adequate for drawing conclusions about effluent
studies.
173
-------
An example of the application of the 10-10-10 principle to a residual
herbicide metabolite in soil is shown in Figure 7. The chromatograms
represent a 5 ppb standard of the material, a soil control and the
control samples spiked at 5, 10, 100 and 500 ppb. The control is a
soil sample which has not been exposed to the herbicide.
Figure 8 shows the results of 10 determinations on 3 different
control soils. An interference is noted at the 0.4 ppb level. The
percent error at 2a (2 standard deviations) or the 95% confidence
level is ±50%. The blank is normally subtracted from recovery and
precision data. In this case, the blank is negligible.
The recovery data from spiked controls is shown in Figure 9. The
spike normally extends to 1/10 of the value of interest. The average
recovery shown here is 90% with an error of ±3% at the 2a level.
The small error is due to the large number of determinations, 25.
The error would have been larger if only 10 determinations had been
run.
As shown in Figure 10, the precision of the analytical procedure
based on 10 determinations of different aliquots of a sample each
containing about 500 ppb, is ±13%.
These statistics apply to any subsequent single operator determination
as long as there is no deviation from the method. For example, as
shown in Figure 11, a value of 484 ppb corrected for recovery and
blank translates into 537± 63 ppb.
This is of course an idealized example. Normally blanks are not
negligible, the recoveries are not 90% and the error may easily
range up to ±50%. However, once the procedure has been validated
any competent analytical chemist should be able to generate numbers
which are within the range of the stated errors.
174
-------
This type of validation scheme was recently used by Synons, et al ,
EPA Cincinnatti Labs, to determine sinsla operator precision and
accuracy for the determination of organohalides in chlorinated drinking
waters. The single operator precision for two replicate determinations,
shown in Figure 12, varies between 5 and 20% at the Ic? level. The
accuracy determined by two different laboratories on solutions of known
concentration, Figure 13, shows recoveries ranging, for example,
between 64 and 94% for a given compound. At the present, no known
data of this nature appears in the open literature for specific
organic compound in an effluent stream. There is a rather significant
difference between analyzing drinking water and an effluent stream.
The latter is a more complicated system and the procedure and method
that apply to drinking water are probably not transferable to effluent
analysis. The EPA is aware of this and is presently developing appropriate
analytical protocol for effluent analysis. Industry will be a contributing
party to the development of this protocol.
In conclusion, by applying validated analytical methods, statistically
meaningful values for the concentration of an organic compound in our
effluent can be obtained and, at least these numbers, mutually agreed
upon. Once the precision, accuracy, sensitivity, and reliability
of the methods has been established, it is then possible to establish
effluent guidelines, to monitor, B.A.T., and to affect protection of
the environment with a reasonable cost/benefit ratio.
References
1. Environmental News, Nov. 8, 1974; Draft Analytical Report; New
Orleans area water supply study. EPA, Lower Mississippi River
Facility, Region VI, S&A.
2. T. H. Harris & J. G. Cumniings, Residue Rev. _6, 104 (1964).
3. J. M. Symons et al, J. Am. Water Works Assoc. 67, 634 (1975).
175
-------
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FIGURE 2
ANALYSIS OF WASTE STREAMS
-STATE OF THE A R T -
GROSS PARAMETERS
BIOCHEMICAL OXYGEN DEMAND (BOD)
TOTAL OXYGEN DEMAND (TOD)
TOTAL ORGANIC CARBON (TOO
TOTAL DISSOLVED SOLIDS (TDS)
INDIVIDUAL COMPONENTS
SEPARATION TECHNOLOGY
LIQUID CHROMATOGRAPH (LC)
GAS CHROMATOGRAPH (GO
GAS CHROMATOGRAPH-MASS SPECTROMETRY (GC-MS)
177
-------
178
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FIGURE 4
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179
-------
FIGURE 5
COMPOUND
ORGANIC COMPOUND IDENTIFICATION
1
NEW ORLEANS AREA WATER SUPPLY STUDY
HIGHEST MEASURED CONCENTRATION yg/1 (ppb)
CARROLLTON JEFFERSON SI JEFFERSON =2
WATER PLANT WATER PLANT WATER PLANT
1 Acetaldehyde
D-VOA
NE
NE
Acetone
D-VOA
NE
3 AlkyIbenzene-C2 isoroer
4 Alkylbenzene-C2 i
5 AlkyIbenzene-C2 isomer
0.05
0.33
0.11
ND
ND
0.03
ND
ND
ND
6 ALkylbenzene-C3 isoner
0.01
ND
ND
7 AUcylbenzene-C3 isorner
0.04
0.05
0.02
isoner
0.02
ND
ND
Atrazine *
(2-chloro-4-ethylamino-
6-isopropylarnino-
s-triazine)
5.0
4.7
5.1
10 Deethylatrazine
(2- ch lore- 4-amino-
6-isopropylamino-
s-triazine)
0.51
0.27
0.27
180
-------
FIGURE 6
METHODS VALIDATION
THE "10-10-10" PRINCIPLE
10 DETERMINATIONS OF A CONTROL TO DETERMINE INTERFERENCES
10 DETERMINATIONS OF A FORTIFIED SAMPLE TO DETERMINE
RECOVERY VALUES
10 DETERMINATIONS OF ACTUAL SAMPLE TO DETERMINE PRECISION
OF THE METHOD
181
-------
FIGURE 7
J-
Q.
ro a.
o
^ §
-------
FIGURE 8
PRECISION OF BLANK
Concentration ppb
X.
1 0.5
2 0.3
3 0.4
4 0.2
5 0.3
6 0.4
7 0.4
8 0.3
9 0.4
10 0.5
X .4
E(Xj_ - X)2 = .1
a = ,/ KX, - X) 2 = .1
n - 1
Relative standard deviation at 951 confidence level
2 100% = 50%
+ 2£ 100%) = .4 + ,2ppb
X
183
-------
FIGURE 9
AGR
Number
113549
121954
122195
118092
127406
114598
121955
112239
117038
119526
114597
121954
122194
113424
132581
114596
113548
121921
130768
128288
130510
112240
122982
130767
128286
Location
Corvallis
Davis
Davis
Fargo
Fargo
Bozeman
Davis
Pendleton
Pendleton
Corvallis
Bozeman
Davis
Davis
Fargo
Bozeman
Bozeman
Corvallis
Davis
Bozeman
Pendleton
Fargo
Pendleton
Bozeman
Bozeman
Pendleton
ppb
Added Found
5 4.8
4.3
4.5
3.9
4.8
4.3
10 9.2
8.3
9.6
9.7
8.6
50 43.6
45.1
44.3
43.7
44.7
49.7
100 86.7
99.5
99.8
500 405
410
484
1000 888
973
Recovery
96
86
90
78
96
86
92
83
96
97
86
87
90
89
87
89
99
87
100
100
81
82
97
89
97
90 + 3*
*95% confidence limits for the mean.
AVERAGE PERCENTAGE RECOVERY
Rn = Rn = 90% = .90
n
Relative standard deviation at 95% confidence level of R
n
R
nfn"
R = 90+3%
n —
184
-------
FIGURE 10
PRECISION OF AN ANALYSIS
n Concentration ppb
Xi
1 436
2 451
3 410
4 484
5 447
6 451
7 443
8 ' 437
9 433
10 492
X = 448
Z(Xi - X)2 = 5210
a = V to ^xi "' = 24
n - 1
Relative standard deviation at 951 confidence level
2_£ 100% = 11%
X
X(1± X2g 100%) = 484 +53ppb
X
185
-------
FIGURE 11
ACTUAL CONCENTRATE
11%) = 537(1+ 11%) = 537 ± 63PPB
,90(1+ 3%)
CONC FOUND BLANK I RECOVERY CONCENTRATE AFTER
PPB PPB DETERMINED CORRECTION
484 ± 53 0,4+2, 90 + 3% 537 ± 63PPB
186
-------
FIGURE 12
DETERMINATION OF PRECISION3
Compound
LOW CONCENTRATION
Spiked Relative
Cone, yg/l a percent
HIGH CONCENTRATION
Spiked Relative
Cone, yg/l a percent
Chloroform
18
1,2-dichlor-
oethane
Carbon tet-
rachloride
14
Bromo-dichloro-
methane
20
Dibromo-chloro
methane
10
30
13
Bromoform
20
30
12
*Not determined at high concentration
187
-------
FIGURE 13
DETERMINATION OF ACCURACY5
(CONCENTRATION - yg/£)
Chloroform
1,2-Dichloro-ethane
Carbon Tetrachloride
Calculated
Lab A
Lab B
75 60_(+6-7%)
63(84) 46(77)
65(87) 46(77)
61(81) 54(90)
76(101)69(98)
10
5(+5%)
10
6(+14%)
9(90)
10(100)
10(100)
10(100)
6(120)
5(100)
5(100)
4(80)
9(90)
8(80)
S(80)
6(60)
5(83)
6(100)
6(100
4(67)
Calculated
Bromo-dichloro
methane
40
24(+5-7%)
Dibrcrro-chloro-rne thane
24
Bromoform
19(+10-13%) 40
23(+12-20%)
Lab A
Lab B
39(98) 22(92)
40(100)23(96)
35(88) 21(88)
38(95) 19(75)
23(96)
23(96)
17(71)
15(63)
14(74)
18(94)
13(68)
12(63)
40(100)
38(95)
48(120)
45(13)
18(78)
24(108)
24(104)
29(126)
188
-------
DRAFT
PRIORITY POLLUTANT VALIDATION PROTOCOL
R. 0. Kagel & H. H. Stehl
The Dow Chemical Company
Midland, Michigan 48640
1. ANALYTICAL METHODOLOGY
The methods for the priority pollutants are those listed
in "Analytical Methods for the Verification Phase of the Bat
Review" issued by Effluent Guidelines Division, Office of Water
and Hazardous Materials, U.S. Environmental Protection Agency.
Alternate Analytical methods will be considered if they are
prcperly substantiated in accordance with the following validation
Protocol. Methods must be described in sufficient detail in a
step-wise fashion that a competent analyst, unfamiliar with the specific
procedure can apply the method. Modifications of published methods
must be described fully. One method may suffice for simultaneous
analysis of several components.
2. VALIDATION PROTOCOL
The validation protocol is a modification of the EPA-EMSL
Analytical Quality Control Program1 and the Winter2 (EPA-EMSL)
interlaboratory validation study program. Each analytical
procedure must be validated by an adequate number of control
values and recovery values to establish the precision and
accuracy. Validation is necessary for an analytical procedure
to become an analytical method. The validation should be
repeated by at least three independent laboratories. Participating
laboratories must conform to the requirements specified by
Winter, in accordance with EPA-EMSL analytical quality control
programs, seven determinations for control and recovery values are
a minimal data requirement.
A. Best Achievable Limit of Detection (LOD)
The best achievable LOD'is obtained from the analyses of
seven samples of organic free water carried through the
entire analytical procedure. The observed peakrto-peak
noise, 0B, and the average peak-to-peak noise, 0B,
are determined. The best achievable limit of detection,
LOD-Q is defined as:
LODB = 2.5 6B
189
-------
Page 2
B. Control Values
Control values are th 2.5 DB, then LOD,
b. If 61 2.5 !o?
2.5 ff-I
0
then LOD-j. = 2.5 0J
Recovery Values
A single sample of plant effluent is analyzed to obtain
the observed sample 4oncentrati°n' °S- The amount found, C-=,
Cf = 0s - 61
Recovery values are
-------
Page 3
D. Precision Values
The precision of a single determination, 0s, at the 95% confidence
level is calculated from the recovery data as:
= 2 E (R? -
n - 1
E. Calculation of Data
The actual concentration, Ca, and the standard deviation,
tfr is: Ca + 231 = 0s (1 +_ 2 CTRs . 100%) - O1 (1 + 2 OlT . 1005
^-a ~ Ca -- ~
O
O1
5s (1 + 2 0Rs . 100%;
tt — ^f
Reporting of Data
a. Any result where 0s < LOD, is reported as "N.D. (LODj)
meaning, not detected, with the detection limit given
in parenthesis.
b. If LCD-,- ^ 0s < 4 LODj the result is reported as a
qualitative result. A second determination must be
run. The two separate results and an average will
be reported as quantitative results.
c. For 0s >, 4 LODj a single determination is reported
as a quantitative result.
REFERENCES
1. Handbook for Analytical Quality Control in Water and Wastewat
Laboratories EPA-EMSL 1976.
2. J. A. Winter, "Validation of Environmental Measurement
Methodology" Int. Conf. On Environmental Sensing and
Assessment, September 14-19, 1975.
191
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^v
INITED STATES ENVIRONMENTAL PROTECTION AGENCY
tfyi -
DATE:
SUBJECT: Metals Analysis - Chicago Regional Laboratory
William A. Tell lard, Chief
Energy and Mining Branch
Branch Chiefs, EGD
Project Officer, EGD
The following memo addresses a recent meeting that was held
between representatives of this office and several staff members
from the Region V Laboratory, with regards to the negotiation for
additional analytical support. Arrangements have been made for
an additional 1,000 samples to be analyzed by the Chicago
Regional Laboratory.
The following information pertains to the labeling, sampling and
type of containers to be utilized in the forth coming program for
metal analysis.
During the previous period of time, a number of points have been
raised regarding the use of uniformity in both the container and
sample size. The following notes should be made available to
contract personnel as well as the Surveillance and Analysis
Divisions:
1. Labeling Codes - Attachment A of this memo contains a
list of codes numbers to be used on labels which will be supplied
to you for those samples to be analyzed for metal parameters. A
code number shall be a six digit code, the first two digits
indicate the industrial category, the second two digits refer to
the contractor or sampling group and the third set is the sample
number. These codes and the labeling information are contained
in Appendix A. The regional lab will utilize this coding system
for their computer which handles the data output.
192
EPA FORM 1320-S (REV. 3-76) iz"~
-------
Example:
Be ~Mg Sn
TI
jCa. Hy V
11 22 57
Sampl e UoY
This code number means that this particular sample contains coal mining
water (11) taken by Versar (22) and that this is the 57th bottle
or sample taken.
2. Samples shall not be preserved with acid as it is
written in the Screening Protocol. This procedure in the only
way to comply with the Department of Transportation regulations
against shipping corrosive materials. Samples shall be prepared
for analysis of total metals by a hard digestion at the Chicago
Regional Labs. This means that a combination of nitric acid and
hydrochloric acid shall be added for samples analyzed by the
plasma unit.
3. Data Turnaround Time - Twenty-two elements can presently
be determined with the plasma unit. A number of parameters must
still be done by either flame!ess AA or flame AA for the purpose
of identification. To enable a better utilization of time, it is
recommended that the primary contractor (most of which have
atomic absorption capabilities) run the following parameters;
selenium, arsenic, antimony thallium and silver. Twenty-two
additional parameters can be supplied by the Central Regional Lab
with the plasma unit. This will cut down on the time delays due
to the limited instrumentation available in the laboratory.
4. Sample Type - As has previous been the case, samples
from the screening portion of the program shall be taken from the
composite sample (either influent or effluent or both) well mixed
and then put into a properly labeled container.
Additional sample capabilities will hopefully be made available,
some time after the first of the year. Until then, we will be
193
-------
limited to the 1,000 samples that have been negotiated. The need
for metals analysis for screening samples by all project officers
should be made known to myself or Gail Goldberg, as soon as
possible, so that scheduling can be afforded.
5. Quality Control - The Central Regional Laboratory will
continue to maintain a quality control file for all samples run
for EGD. This quality control file will be periodically supplied
to the Division and as needed can be incorporated into any court
record. The quality control file is probably the most complete
effort that the Division has been able to obtain. It specifies
the recoveries, performance of the instrument, and the individual
sample variability on a day-by-day basis. This information will
be made available through the Energy and Mining Branch to the
project officers and their contractors, as the need arises. The
quality control program at Central Regional Lab is far superior
to any program that has previously existed in the Division. It
can insure you that the metals analysis data are properly framed
and within the confines in definition of the performance
standards specified under 304(g). As each project comes to a
conclusion this data will be made available to the individual
project officers and Branches for inclusion in their record. A
period of a two weeks notice will be greatly appreciated, this
notification again, should be made in writing to Gail S. Goldberg
so that we may solicit the computer output for the quality
control data for those samples.
6. Samples, are collected as before meaning that there
exists only one sample for metal analysis per sampling site. The
possibility of collecting duplicates was considered. This idea
had to be rejected in view of time limits and financial
constraints.
7. The use of old labels for metals analysis, like the one
shown in the screening protocol should be discontinued. New
labels as shown in point 1. of this memo shall be distributed to
you. Only these new labels are compatible with the Chicago Lab
computer.
194
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Sampling Contractor Code Number
01. EPA Region I
02. EPA Region II
03. EPA Region III
04. EPA Region IV
05. EPA Region V
06. EPA Region VI
07. EPA Region VII
08. EPA Region VIII
09. EPA Region IX
10. EPA Region X
11. National Enforcement Investigations Center
12.
13. Hamilton Standard
14. Colin A. Houston & Associates
15. Environmental Science & Engineering , Inc.
16. Ryckman, Edgerley, Tomlinson and Associates, Inc.
17. E.H. Richardson Associates
18. Mid-West Research Instutute
19. NUS - Cyrus Rice Division
20. Burns & Roe, Inc.
21. Calspan Corporation
22. Versar Incorporated
23. Jacobs Engineering Company
24. E.G. Jordan Co., Inc.
25. Sverdrup 4 Parcel and Associates, Inc.
195
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26. Carborundum Corporation
27. TRW
28. Industrial Environmental Research Lab, Cincinnati
196
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Industrial Code Numbers
01 - Timber Products
02 - Steam Electric
03 - Leather Tanning
04 - Iron & Steel mfg.
05 - Petroleum Refining
06 - Nonferrous Metals
07 - Paving & Roofing
08 - Paint & Ink
09 - Printing & Publishing
10 - Ore Mining
11 - Coal Mining
12 - Organic Chemicals
13 - Inorganic Chemicals
14 - Textile Mills
15 - Plastics & Synthetics
16 - Pulp & Paper
17 - Rubber Processing
18 - Soaps & Detergents
19 - Auto & other Laundries
20 - Pesticides mfg.
21 - Photographic Industries
22 - Gum & Wood Industries
23 - Pharmaceuticals
24 - Explosives
25 - Adhesive & Sealants
26 - Battery mfg.
27 - Plastics mfg.
28 - Foundries
29 - Coil Coating
30 - Porcelain/Enameling
31 - Aluminum
32 - Copper
33 - Electronics
34 - Shipbuilding
35 - Electroplating
36 - Oil and Gas Extraction
197
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
SUBJECT: Sample Codes
FROM: William Telliard, Chief
Energy and Mining Branch
TO.- All EGO Project Officers
You will inform your sampling contractor to use the same digit coding
system for metals and organic samples. This six digit code system is
explained in the attached memo. Keeping the same code number for all
portions of samples greatly reduces the amount of data processing and
any changes for error in correlating samples.
198
EPA FORM 1320-6 (REV. 3-76)
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
DATE= NOV231977
SUBJECT: Chicago Lab - New address
FROM: William Telliard, Chief
Energy and Mining Branch
TO: All EGD Project Officers
The Chicago Lab has moved. The new address is:
U.S. Environmental Protection Agency
Region V, Central Regional Laboratory
536 South Clark
Chicago, Illinois 60605
Please send screening samples for metals analysis only, to the above
address.
EPA FORM 1320-6 (REV. 3-76)
200
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PRELIMINARY INTERIM PROCEDURE
FOR
FIBROUS ASBESTOS
by
Charles H. Anderson and J. MacArthur Long
Analytical Chemistry Branch
U.S. Environmental Protection Agency
Environmental Research Laboratory
College Station Road
Athens, Georgia 30601
-------
FIBROUS ASBESTOS
(Preliminary Interim Procedure)
(Transmission Electron Microscopy Method)
1. Scope and Application
1.1 This method is applicable to drinking water and water
supplies.
1.2 The method determines the number of asbestos
fibers/liter, their size (length and width), the size
distribution, and total mass. The method
distinguishes chrysotile from amphibole asbestos. The
detection limits are variable and depend upon the
amount of total extraneous particulate matter in the
sample as well as the contamination level in the
laboratory environment. Under favorable circumstances
0.1 MFL (million fibers per liter) can be detected.
The detection limit for total mass of asbestos fibers
is also variable and depends upon the fiber size and
size distribution in addition to the factors affecting
the total fiber count. The detection limit under
favorable conditions is in the order of 0.1 ng/1.
1.3 The method is not intended to furnish a complete
characterization of all the fibers in water.
1.4 It is beyond the scope of this method to furnish
detailed instruction in electron microscopy, electron
diffraction or crystallography. It is assumed that
those using this method will be sufficiently
knowledgeable in these fields to understand the
methodology involved.
1.5 The method outlined below is based upon what is
considered to be state-of-the art practice but it is
emphasized that at present no single analytical
procedure for asbestos is universally accepted. As a
result no inter-laboratory comparisons are presented
and the procedure should not be considered as a
standard method.
2. Summary of Method
2.1 A variable, known volume of water sample is filtered
through a membrane filter of sufficiently small pore
size to trap asbestos fibers. A small portion of the
filter with deposited fibers is placed on an electron
microscope grid and the filter material removed by
gentle solution in organic solvent. The material
remaining on the electron microscope grid is examined
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in a transmission microscope at high magnification.
The asbestos fibers are identified by their morphology
and electron diffraction pattern and their length and
width are measured. The total area examined in the
electron microscope is determined and the number of
asbestos fibers in this area is counted. The
concentration in MFL (millions of fibers/liter) is
calculated from the number of fibers counted, the
amount of water filtered, and the ratio of the total
filtered area/sampled filter area. The mass/liter is
calculated from the assumed density and the volume of
the fibers.
3. Definitions
Asbestos - A generic term applied to a variety of
commercially useful silicate minerals that may have a
fibrous structure.
Fiber - Any particle that has parallel sides and a
length/width ratio greater than or equal to 3:1.
Aspect Ratio - The ratio of length to width.
Chrysotile - A nearly pure hydrated magnesium silicate, the
fibrous form of the mineral serpentine, possessing a
unique layered structure in which the layers are
wrapped in a helical cylindrical manner about the
fiber axis.
Amphibole - A silicate mineral whose basic structural unit
is a double silica chain (Si^Ojj), but with a variable
composition and a layered structure that is easily
cleaved to form a fiber.
Detection Limit - The calculated concentration in MFL,
equivalent to one fiber above the background or blank
count.
Statistically Significant - Any concentration based upon a
total fiber count of five or more in 20 grid squares.
H. Sample Handling and Preservation
4.A Sampling
It is beyond the scope of this procedure to furnish detailed
instructions for field sampling; the general principles of
sampling waters are applicable. There are some
considerations that apply to asbestos fibers, a special type
of particulate matter. These fibers are small, and in water
range in length from .1 ym to 20 ym or more. Because of the
range of size there may be a vertical distribution of
particle sizes. This distribution will vary with depth
2
203
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depending upon the vertical distribution of temperature as
well as the local meteorological conditions. Sampling
should take place according to the objective of the
analysis. If a representative sample of a water supply is
required a carefully designed set of samples should be taken
representing the vertical as well as the horizontal
distribution and these samples composited for analysis.
U.1 Containment Vessel
The sampling container shall be a clean polyethylene,
screw-capped bottle capable of holding at least one
liter. The bottle should be rinsed at least two times
with the water that is being sampled prior to
sampling.
NOTE: Glass vessels are not suitable as sampling
containers.
ft. 2 Quantity of Sample
A minimum of approximately one liter of water is
required and the sampling container should not be
filled. It is desirable to obtain two samples from
one location.
4.3 Sample Preservation
No preservatives should be added during sampling and
the addition of acids should be particularly avoided.
If the sample cannot be filtered in the laboratory
within US hours of its arrival, sufficient amounts (1
ml/1 of sample) of a 2.71X solution of mercuric
chloride to give a final concentration of 20 ppm of Hg
may be added to prevent bacterial growth.
5. Interferences
5.1 Misidentification
The guidelines set forth in this method for counting
fibrous asbestos require a positive identification by
both morphology and crystal structure as shown by an
electron diffraction pattern. Chrysotile asbestos has
a unique tubular structure, usually showing the
presence of a central canal, and exhibits a unique
characteristic electron diffraction pattern. Although
halloysite fibers may show a similar streaking to
chrysotile they do not exhibit its characteristic
triple set of double spots or 5.3A layer line. It is
highly improbable that a non-asbestiform fiber would
exhibit the distinguishing chrysotile features.
Although amphibole fibers exhibit characteristic
morphology and electron diffraction patterns, they do
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not have the unique properties exhibited by
chrysotile. It is therefore possible though not
probable for misidentification to take place.
Hornblende is an amphibole and, in a fibrous form,
will be mistakenly identified as amphibole asbestos.
It is important to recognize that a significant
variable fraction of both chrysotile and amphibole
asbestos fibers do not exhibit the required
confirmatory electron diffraction pattern. This
absence of diffraction is attributable to unfavorable
fiber orientation and fiber sizes. The results
reported will therefore be low as compared to the
absolute number of asbestos fibers that are present.
5.2 Obscuration
If there are large amounts of organic or amorphous
inorganic materials present, some small asbestos
fibers may not be observed because of physical
overlapping or complete obscuration. This will result
in low values for the reported asbestos content.
5.3 Contamination
Although contamination is not strictly considered an
interference, it is an important source of erroneous
results, particularly for chrysotile. The possibility
of contamination should therefore always be a
consideration.
5.4 Freezing
The effect of freezing on asbestos fibers is not known
but there is reason to suspect that fiber break down
could occur and result in a higher fiber content than
was present in the original sample. Therefore the
sample should be transported to the laboratory under
conditions that would avoid freezing.
6. Equipment and Apparatus
6.1 Specimen Preparation Laboratory
The ubiquitous nature of asbestos, especially
chrysotile, demands that all sample preparation steps
be carried out to prevent the contamination of the
sample by air-borne or other source of asbestos. The
prime requirement of the sample preparation laboratory
is that it be sufficiently free from asbestos
contamination that a specimen blank determination
using 200 ml of asbestos-free water yields no more
than 2 fibers in twenty grid squares of a conventional
200 mesh electron microscope grid.
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In order to achieve this low level of contamination,
the sample preparation area should be a separate
conventional clean room facility. The room should be
operated under positive pressure and have incorporated
electrostatic precipitators in the air supply to the
room, or alternatively absolute (HEPA) filters. There
should be no asbestos floor or ceiling tiles, transite
heat-resistant boards/ nor asbestos insulation. Work
surfaces should be stainless steel or Formica or
equivalent. A laminar flow hood should be provided
for sample manipulation. Disposable plastic lab coats
and disposable overshoes are recommended.
Alternatively new shoes for all operators should be
provided and retained for clean room use only. A mat
(Tacky Mat, Liberty Industries, 589 Deming Rd.,
Berlin, Connecticut 06037, or equivalent) should be
placed inside the entrance to the room to trap any
gross contamination inadvertently brought into the
room from contaminated shoes. Normal electrical and
water services, including a distilled water supply
should be provided. In addition a source of ultra-
pure water from a still or filtration-ion exchange
system is desirable.
6.2 Instrumentation
6.2.1 Transmission Electron Microscope. A
transmission electron microscope that operates
at a minimum of 80 KV, has a resolution of 1.0
nm and a magnification range of 300 to
100,000. If the upper limit is not attainable
directly it may be attained through the use of
auxiliary optical viewing. It is mandatory
that the instrument be capable of carrying out
selected area electron diffraction (SAED) on
an area of 300 nm. The viewing screen shall
have either a millimeter scale, concentric
circles of known radii, or other devices to
measure the length and width of the fiber.
Most modern transmission microscopes meet the
requirements for magnification and resolution.
An energy-dispersive X-ray spectrometer is
useful for the identification of suspected
asbestiform minerals; this accessory to the
microscope, however, is not mandatory.
6.2.2 Data Processor. The large number of
repetitive calculations make it convenient to
use computer facilities together with
relatively simple computer programs.
206 5
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6.2.3 Vacuum Evaporator. For depositing a layer of
carbon on the Nuclepore filter, and for
preparing carbon coated grids.
6.2.4 Low Temperature Plasma Asher. To be used for
the removal of organic material (including the
filter) from samples containing so much
organic matter that asbestos fibers are
obscured. The sample chamber should be at
least 10-cm diameter.
6.3 Apparatus* Supplies and Reagents
6.3.1 Jaffe Wick Washer. For dissolving Nuclepore
filter (if Nuclepore is used in sample
preparation). Assemble as in 8.2A.1. It is
illustrated in Figure 1.
6.3.2 Condensation Washer. For use in dissolving
the Millipore filter when using the Millipore
sample preparation method. A system with
controlled heating, controlled refluxing, and
a cold finger for holding the electron
microscope (EM) sample grids. At least two
systems are commercially available. Figure 2
is an illustration of one design that has
proven satisfactory.
6.3.3 Filtering Apparatus. 47-mm funnel (Cat. No.
XX1504700, Millipore Corporation, Order
Service Dept., Bedford, MA 01730). Used to
filter water samples. 25-mm funnel (Millipore
Cat. No. XX1002500). Used to filter dispersed
ash samples.
6.3.1 Vacuum Pump. For use in sample filtration.
Should provide vacuum up to 20 inches of
mercury.
6.3.5 EM Grids. 200-mesh copper or nickel grids,
covered with carbon-coated collodion for use
with the Millipore-condensation washing
technique. Formvar-backed grids, without a
carbon coating are used in the Nuclepore-Jaffe
sample preparation method. These grids may be
purchased from manufacturers of electron
microscopic supplies or prepared by standard
electron microscopic grid preparation
procedures. Finder grids may be substituted
and are useful if the re-examination of a
specific grid opening is desired.
207
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Screen Support
with Grid
Ridge
Petri Dish
Glass Slides
A.
Layer of Filter Papers
Nuclepore Filter
Carbon
Chloroform
Formvar
Grid
Carbon
Grid
B.
Figure 1. Modified Jaffe Wick Method
A. Washing Apparatus
B. Washing Process
208
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1.70 mm
\
3.30 mm
3.14 mm
Condenser
RJ
25 min
View A
Water Source
X
Brass Holder
(See View A)
Cold Finger
Adapter
Heating Mantle
Powerstat
_2 mm
Water Drain
•Figure 2. Condensation Washer
209
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6.3.6 Membrane Filters.
47-mm diameter Millipore membrane filter, type
HA; 0.45 urn pore size. For filtration of
water sample.
47-mm diameter Nuclepore membrane filter; 0.1
ym pore size. (Nuclepore Corp, 7035 Commerce
Circle, Pleasanton, CA 94566) For filtration
of water sample.
47-mm diameter Millipore membrane filter type
BS; 2 ym pore size. Used as a Nuclepore filter
support on top of the glass frit.
25-mm diameter Millipore membrane filter, type
HA; 0.45 ym pore size. To filter dispersed
ashed Millipore filter.
25-mm diameter Nuclepore membrane filter; 0.1
ym pore size. To filter dispersed ashed
Millipore filter.
25-mm diameter Millipore membrane filter, type
BS; 2.0 ym pore size. To be used as a
Nuclepore filter support on top of the glass
frit.
6.3.7 Glass Vials. 30-mm diameter x 80-rran long.
For holding filter during ashing.
6.3.8 Glass Slides. 5.1-cm x 7.5-cm. For support
of Nuclepore filter during carbon evaporation.
6.3.9 Scalpels. With disposable blades and
scissors.
6.3.10 Tweezers. Several pairs for the many handling
operations.
6.3.11 "Scotch" Doublestick tape. To hold filter
section flat on glass slide while carbon
coating.
6.3.12 Disposable Petri dishes, 50-mm diameter, for
storing membrane filters.
6.3.13 Static Eliminator, 500 microcuries Po-210.
(Nuclepore Cat. No. V090POL00101) or
equivalent. To eliminate static charges from
membrane filters.
6.3.14 Carbon rods, spectrochemically pure, 1/8" dia.,
3.6 mm x 1.0 mm neck. For carbon coating.
9
210
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6.3.15 Carbon rod sharpener. (Cat. No. 1204, Ernest
F. Fullam, Inc., P. O. Box 444, Schenectady,
NY 12301) For sharpening carbon rods to a
neck of specified length and diameter.
6.3.16 Ultrasonic Bath. (50 watts, 55 KHz). For
dispersing ashed sample and for general
cleaning.
6.3.17 Graduated Cylinder, 500 ml.
6.3.18 Spot plate.
6.3.19 10 yl Microsyringe. For administering drop of
solvent to filter section during sample
preparation.
6.3.20 Carbon grating replica, 2160 lines/mm. For
calibration of EM magnification.
6.3.21 Cork borer (1/8 inch diameter). For sampling
prepared Millipore filters.
6.3.22 Filter paper. S & S #589 Black Ribbon or
equivalent (9-cm circles). For preparing
Jaffe Wick Washer.
6.3.23 Screen supports (copper or stainless steel) 12
mm x 12 mm, 200 mesh. To support specimen
grid in Jaffe wick Washer.
6.3.24 Brass holder. For holding specimen in
condensation washer. See Figure 2, View A.
6.3.25 Chloroform, spectro grade, doubly distilled.
For dissolving Nuclepore filters.
6.3.26 Acetone, reagent grade or better. For
dissolving Millipore filters,
6.3.27 Asbestos. Chrysotile (Canadian), Crocidolite,
Amosite. DICC (Onion Internationale Centre le
Cancer) Standards. Available from Duke
Standards Company, 445 Sherman Avenue, Palo
Alto, CA 94306.
6.3.28 Petri dish, glass (100 mm diameter x 15 mm
high). For modified Jaffe Wick Washer.
6.3.29 Alconox. (Alconox, Inc., New York, NY 10003)
For cleaning glassware. Add 7.5 g Alconox to
a liter of distilled water.
10
211
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6.3.30 Aerosol OT, 0.1X solution (Cat. No. So-A-292,
Fisher Scientific Company, 711 Forbes Avenue,
Pittsburgh, PA 15219) Used as dispersion
medium for ashed Millipore filter. Prepare a
0.1% solution by diluting 1 ml of the 10$
solution to 100 ml with distilled water.
Filter through 0.1-ym Nuclepore filter paper
before using.
6.3.31 Parafilm. (American Can Company, Neenah, WI)
Used as protective covering for clean
glassware.
6.3.32 Pipets, disposable, 5 ml and 50 ml.
6.3.33 Distilled or deionized water. Filter through
0.1-ym Nuclepore filter for making up all
reagents and for final rinsing of glassware,
and for preparing blanks.
6.3.3* Mercuric chloride, 2.71* solution w/v. Used
as sample preservative. See 4.3. Add 5.42 g
of reagent grade mercuric chloride (HgCl2) to
100 ml distilled water and dissolve by
shaking. Dilute to 200 ml with additional
water. Filter through 0.1-pm Nuclepore filter
paper before using.
7. Preparation of Standards
Reference standard samples of asbestos that can be used for
quality control for a quantitative analytical method are not
available. It is, however, necessary for each laboratory to
prepare at least two suspensions; one of chrysotile and
another of a representative amphibole. These suspensions
can then be used for intra-laboratory control and furnish
standard morphology photographs and diffraction patterns.
7.1 Chrysotile Stock Solution.
Grind about 0.1 g of UICC chrysotile in an agate
mortar for several minutes, or until it appears to be
a powder. Weigh out 10 mg and transfer to a clean 1
liter volumetric flask, add several hundred ml of
millipore filtered distilled water containing 0.1
percent Aerosol OT and one ml of a 20,000 ppm solution
of mercury and then make up to 1 liter with the 0.1
percent Aerosol filtered distilled water. To prepare
a working solution, transfer 10 ml of the above
suspension to another 1-liter flask, add 1 ml of a
20,000 ppm solution of mercury and make up to 1 liter
with the same 0.1 percent aerosol OT solution. This
suspension contains 100 yg per liter. Finally
transfer 1 ml of this suspension to a 1-liter flask,
11
212
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add 1 ml of a 20,000 ppm solution of mercury and make
up to volume with the 0.1 percent aerosol OT solution.
The final suspension will contain 5-10 MFL and is
suitable for laboratory testing.
7.2 Amphibole Stock Dispersion.
Prepare amphibole suspensions from UICC amphibole
samples as in Section 7.1.
7.3 Identification Standards
Prepare electron microscopic grids containing the UICC
asbestos fibers according to 8, Procedure, and obtain
representative photographs of each fiber type and its
diffraction pattern for future reference.
8. Procedure
8.1 Filtration.
The separation of the insoluble material, including
asbestiform minerals, through filtration and
subsequent deposition on a membrane filter is a very
critical step in the procedure. The objective of the
filtration is not only to separate, but also to
distribute uniformly the particulate matter such that
discreet particles are deposited with a minimum of
overlap.
The volume filtered will range from 50-500 ml. In an
unknown sample the volume can not be specified in
advance because of the presence of variable amounts of
particulate matter. In general sufficient sample is
filtered such that a very faint stain can be observed
on the filter medium. The maximum loading that can be
tolerated is 20 yg/cm2, or about 200 yg on a 47-mm
diameter filter; 5 yg/cm2 is near optimum. If the
total solids content is known, an estimate of the
maximum volume tolerable can be obtained. In a sample
of high solids content, where less than 50 ml is
required, the sample should be diluted with filtered
distilled water so that a minimum total of 50 ml of
water is filtered. This step is necessary to allow
the insoluble material to deposit uniformly on the
filter. The filtration funnel assembly must be
scrupulously clean and cleaned before each filtration.
The filtration should be carried out in a laminar flow
hood.
NOTE 1: The following cleaning procedure has been
found to be satisfactory:
12
213
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Wash each piece of glassware three times with
distilled water. Following manufacturer's
recommendations use the ultrasonic bath with an
Alconox-water solution to clean all glassware. After
the ultrasonic cleaning rinse each piece of glassware
three times with distilled water. Then rinse each
piece three times with deionized water which has been
filtered through 0.1-ym Nuclepore filter. Dry in an
asbestos-free oven. After the glassware is dry, seal
openings with parafilm.
8.1.1 Filtration
a. Assemble the vacuum filtration apparatus
incorporating either the . 1-um Nuclepore
backed with 2-^m Millipore, or the . 45-ym
Millipore filter. See 8.2A. 2 or 8.2B.2.
b. Vigorously agitate the water sample in its
container.
c. If the required filtration volume can be
estimated, either from turbidity estimates of
suspended solids or previous experience,
immediately withdraw the proper volume from
the container and add the entire volume to the
47-mm diameter funnel. Apply vacuum
sufficient for filtration but gentle enough to
avoid the formation of a vortex. If a
completely unknown sample is being analyzed, a
slightly modified procedure must be followed.
Pour 500 ml of a well-mixed sample into a 500
ml graduated cylinder and immediately transfer
the entire contents to the prepared vacuum
filtration apparatus. Apply vacuum gently and
continue suction until all of the water has
passed through the filter. If the resulting
filter appears obviously coated or discolored,
it is recommended that another filter be
prepared in the same manner, but this time
using only 200 or 100 ml of sample.
NOTE 1: Do not add more water after
filtration has started and do not rinse the
sides of the funnel.
d. Disassemble the funnel, remove the filter
and dry in a covered petri dish.
8.2 Preparation of Electron Microscope Grids.
The preparation of the grid for examination in the
microscope is a critical step in the analytical
procedure. The objective is to remove the organic
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filter material from the asbestos fibers with a
minimum loss and movement and with a minimum breakage
of the grid support film. Two alternative procedures
are acceptable:
A. Nuclepore Filter, Modified Jaffe Wick
B. Millipore Filter, Condensation Washer
If the sample contains organic matter in such amounts
that interfere with fiber counting and identification
a preliminary ashing step is required. See 8.5.
NOTE 1: Two alternatives for grid preparation are
suggested because the superiority of one technique
over the other has not been substantiated by
sufficient experimental evidence. The differences
between the two techniques of sample preparation lie
in the filtering medium (Nuclepore vs. Millipore) ,
whether the filter is carbon coated, and in the method
of dissolving the filter material. There is evidence
that the condensation washing procedure can lose
amphibole fibers and that amphiboles are more
susceptible to loss than chrysotile.
8.2A Nuclepore Filter, Modified Jaffe Wick Technique.
8.2A.1 Preparation of Modified Jaffe Washer
Place three glass microscope slides (75 mm x
25 mm) one on top of the other in a petri dish
(100 mm x 15 mm) along a diameter. Place 14 S
& S #589 Black Ribbon filter papers (9-cm
circles) in the petri dish over the stack of
microscope slides. Place three mesh copper
screen supports (12 mm x 12 mm) along the
ridge formed by the stack of slides underneath
the layer of filter papers. Place an EM
specimen grid on each of the screen supports.
See Fig. 1.
8.2A.2 Vacuum Filtration Unit
Assemble the vacuum filtration unit. Place a
2-ym Millipore filter type BS on the glass
frit and then position a 0.1-ym Nuclepore
filter, shiny side up, on top of the Millipore
filter. Apply suction to center the filters
flat on the frit. Attach the filter funnel
and shut off the suction.
8.2A.3 Sample Filtration
See 8.1.1.
14
215
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8.2A.U Sample Drying
Remove the filter funnel and place the
Nuclepore filter in a loosely covered petri
dish to dry. The petri dish containing the
filter may be placed in an asbestos-free oven
at 45° C for 30 minutes to shorten the drying
time.
8.2A.5 Selection of section for carbon coating
Using a small pair of scissors or sharp
scalpel cut out a retangular section of the
Nuclepore filter. The minimum approximate
dimensions should be 15 mm long and 3 mm wide.
Avoid selection near the perimeter of the
filtration area.
8.2A.6 Carbon Coating the Filter
Tape the two ends of the selected filter
section to a glass slide using "Scotch" tape.
Take care not to stretch the filter section.
Identify the filter section using a china
marker on the slide. Place the glass slide
with the filter section into the vacuum
evaporator. Insert the necked carbon rod and,
following manufacturer's instructions, obtain
high vacuum. Evaporate the neck, with the
filter section rotating, at a distance of
approximately 7.5 cm from the filter section
to obtain a 30-50 nm layer of carbon on the
filter paper. Evaporate the carbon in several
short bursts rather than continuously to
prevent overheating the surface of the
Nuclepore filter.
NOTE 1: Overheating the surface tends to
crosslink the plastic, rendering the filter
dissolution in chloroform difficult.
NOTE 2: The thickness of the carbon film can
be monitored by placing a drop of oil on a
porcelain chip that is placed at the same
distance from the carbon electrodes as the
specimen. Carbon is not visible in the region
of the oil drop thereby enabling a visual
estimate of the deposit thickness by the
contrast differential.
8.2A.7 Grid Transfer
Remove the filter from the vacuum evaporator
and cut out three sections somewhat less than
15
216
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3 mm x 3 mm and such that the square of
Nuclepore fits within the circumference of the
grid. Pass each of the filter sections over a
static eliminator and then place each of the
three sections carbon-side down on separate
specimen grids previously placed in the
modified Jaffe Washer. Using a microsyringe,
place a 10-yl drop of chloroform on each
filter section resting on a grid and then
saturate the filter pad until pooling of the
solvent occurs below the ridge formed by the
glass slides inserted under the layer of
filter papers. Place the cover on the petri
dish and allow the grids to remain in the
washer for approximately 24 hours. Do not
allow the chloroform to completely evaporate
before the grids are removed. To remove the
grids from the washer lift the screen support
with the grid resting upon it and set this in
a spot plate depression to allow evaporation
of any solvent adhering to the grid. The grid
is now ready for analysis or storage.
8.2B Millipore - Condensation Washer Technique
8.2B.1 Operation of Condensation Washer
Fill the extractor flask to UQ% capacity with
acetone. Filter the acetone through 0.1-ym
Nuclepore filter paper before using. Adjust
the tap water flow rate to 10 ml/sec by
allowing the water exiting from the cold
finger to run into a graduated cylinder for 30
seconds. Set the variable transformer,
regulating the heater power-input, to
approximately 45 volts. Sufficient heat
should be applied to generate acetone vapors
at the required condensation or reflux level
without boiling or simmering. The reflux
level should be even with the top of the cold
finger and just below the stainless steel
grid. (Important: See Note 1). After the
system has been running for twenty minutes,
check the reflux level of the acetone. Place
a heavily lined index card behind the adaptor
so that when viewed from the front of the
adaptor the cold finger is parallel to the
heavy lines on this card. Locate the reflux
level by noting the illusionary wavy motion of
the heavy lines on the index card. If the
reflux level is too high, increase, or if too
low, decrease the tap water flow rate. Do not
allow pooling of the solvent to occur on the
grids. At very low flow rates keep a careful
217"
-------
check to ensure that the water valve does not
shut itself off. To account for changes in
the tap water temperature, establish the *
correct flow rate daily.
NOTE 1: The relative position of the acetone
condensation level to the grid level is
critical to the successful operation of the
condensation washer. If the condensation
level is too low, the Millipore filter will
not be sufficiently removed within a
reasonable period of time and the asbestos
fibers cannot be successfully counted; if the
level is too high, excessive washing occurs
with a resulting loss of fibers and rupture of
the carbon film. As each extractor has
different characteristics, several test runs
should be made on blank Millipore-loaded grids
to determine the optimum operating conditions.
NOTE 2: It has been suggested that the rate
of acetone condensation, observed as drops
from the end of the cold finger, should be 10
drops per 30-45 seconds.
NOTE 3: A constant pressure regulator may be
required in the water line if a constant flow
cannot be otherwise attained.
8.2B.2 Vacuum Filtration Unit
Assemble the vacuum filtration unit. Place a
O.U5-ym Millipore filter on the glass frit.
Turn on the suction and center the filter on
the frit. Attach the filter funnel and turn
off suction.
8.2B.3 Sample Filtration
See 8.1.1.
8.2B.4 Sample Drying
Remove the filter funnel and place the
Millipore filter in a petri dish in an
asbestos-free oven at 45° C for at least two
hours to dry.
8.2B.5 Sampling of Filter
Using a well sharpened (1/8 inch diameter)
cork borer, cut three circular sections from
the filter. Keep one-half of the filter
undisturbed for future reference or if an
17
218
-------
ashing step is required (See 8.5). Avoid
sampling near the perimeter of the filtration
area.
8.2B.6 Grid Transfer
Pass each filter section over a static
eliminator and then place each section
particulate side down on carbon coated
specimen grids previously placed in the brass
holder. Add a 10-yl drop of acetone to each
of the grids using a micro syringe. Place the
brass holder on the cold finger of the
condensation washer which has been charged
with acetone. After the correct reflux level
has been established (8.2B.1) insert the brass
block holding the grids and check a few
minutes later to make certain that the acetone
reflux is near but below grid level. Allow
the acetone to reflux for 7-8 hours to
dissolve away the filter and leave the residue
deposited on the carbon substrate of the grid.
Turn off the heating mantle. Remove the brass
block holding the grids when no drops of
acetone can be seen falling from the cold
finger. The grids are now ready for analysis
or storage.
NOTE 1: The addition of 10 yl of acetone
directly to the filter, while recommended, may,
in the opinion of some investigators, increase
the risk of removing particulates from the
filter. There are no data available to show
it has a deleterious effect.
8.4 Electron Microscopic Examination
8.1.1 Microscope Alignment and Magnification
Calibration
Following the manufacturer's recommendations
carry out the necessary alignment procedures
for optimum specimen examination in the
electron microscope. Calibrate the routinely
used magnifications using a carbon grating
replica.
NOTE 1: Screen magnification is not
necessarily equivalent to plate magnification.
8.4.2 Grid Preparation Acceptability
After inserting the specimen into the
microscope adjust the magnification low enough
18
219
-------
(300X - 1000X) to permit viewing complete grid
squares. Inspect at least 10 grid squares for
fiber loading and distribution, debris
contamination, and carbon film continuity.
Reject the grid for counting if:
1) The grid is too heavily loaded with fibers
to perform accurate counting and diffraction
operations, A new sample preparation either
from a smaller volume of water or from a
dilution with filtered distilled water must
then be prepared.
2) The fiber distribution is noticeably
uneven. A new sample preparation is required.
3) The debris contamination is too severe to
perform accurate counting and diffraction
operations. If the debris is largely organic
the filter must be ashed and redispersed (see
8.5). If inorganic the sample must be diluted
and again prepared.
4) The majority of grid squares examined have
broken carbon films. A different grid
preparation from the same initial filtration
must be substituted.
8.4.3 Procedure for Fiber Counting
There are two methods commonly used for fiber
counting. In one method (A) 100 fibers,
contained in randomly selected fields of view,
are counted. The number of fields plus the
area of a field of view must be known when
using this method. In the other method (B),
all fibers (at least 100) in several grid
squares or 20 grid squares are counted. The
number of grid squares counted and the average
area of one grid square must be known when
using this method.
NOTE 1: The method to use is dependent upon
the fiber loading on the grid and it is left
to the judgement of the analyst to select the
optimum method. The following guidelines can
be used: If it is estimated that a grid square
(80 ym x 80 ym) contains 50-100 fibers at a
screen magnification of 20000X it is
convenient to use the field-of-view counting
method. If the estimate is less than 50, the
grid square method of counting should be
chosen. On the other hand, if the fiber count
19
220
-------
is estimated to be over 300 fibers per grid
square, a new grid containing less fibers must
be prepared (through dilution or filtration
of a smaller volume of water).
8.4.3A Field-of-View Method
After determining that a fiber count can be
obtained using this method adjust the screen
magnification to 10-20000X. Select a number
of grid squares which would be as
representative as possible of the entire
analyzable grid surface. From each of these
squares select a sufficient number of fields
of view for fiber counting. The number of
fields of view per grid square is dependent
upon the fiber loading. If more than one
field of view per grid square is selected,
scan the grid opening orthogonally in an
arbitrary pattern which prevents overlapping
of fields of view. Carry out the analysis by
counting, measuring and identifying (see
8.4.4) approximately 50 fibers on each of two
grids.
The following rules should be followed when
using the field of view method of fiber
counting. Although these rules were derived
for a circular field of view they can be
modified to apply to square or rectangular
designs.
1) count all fibers contained within the
counting area and not touching the
circumference of the circle.
2) Designate the upper right-hand quadrant as
I and number in clockwise order. Count all
fibers touching or intersecting the arc of
quadrants I or IV. Do not count fibers
touching or intersecting the arc of quadrants
II or III.
3) If a fiber intersects the arc of both
quadrants III and IV or I and II count it only
if the greater length was outside the arc of
quadrants IV and I, respectively.
U) Count fibers intersecting the arc of both
quadrants I and III but not those intersecting
the arc of both II and IV.
These rules are illustrated in Fig. 3.
20
221
-------
I
r
L 1
i
I
»
\
V
/
/
\
\
-\
III
\
Counted
Not Counted
Figure 3. Illustration of Counting Rules
for Field-of-View Method
222
21
-------
8.4.3B Grid Square Method
After determining that a fiber count can be
obtained using this method adjust the screen
magnification to 10-20000X. Position the grid
square so that scanning can be started at the
left upper corner of the grid square. While
carefully examining the grid, scan left to
right, parallel to the upper grid bar. When
the perimeter of the grid square is reached
adjust the field of view up one field width
and scan in the opposite direction. The
tilting section of the fluorescent screen may
be used conveniently as the field of view.
Examine the square until all the area has been
covered. The analysis should be carried out
by counting, measuring and identifying (see
8.4.4) approximately 50 fibers on each of two
grids or until 10 grid squares on each of two
grids have been counted. Do not count fibers
intersecting a grid bar.
8.4.4 Measurement and Identification
Measure and record the length and width of
each fiber having an aspect ratio greater than
or equal to three. Disregard obvious
biological, bacteriological fibers and diatom
fragments. Examine the morphology of each
fiber using optical viewing if necessary.
Tentatively identify, by reference to the UICC
standards, chrysotile or possible amphibole
asbestos. Attempt to obtain a diffraction
pattern of each fiber. Move the suspected
fiber image to the center of the screen and
insert a suitable selected area aperture into
the electron beam so that the fiber image, or
a portion of it, is in the illuminated area.
The size of the aperture and the portion of
the fiber should be such that particles other
than the one to be examined are excluded from
the selected area. If an incomplete
diffraction pattern is obtained move the
particle image around in the selected area to
get a clearer diffraction pattern or to
eliminate possible interferences from
neighboring particles.
Determine whether or not the fiber is
chrysotile or an amphibole by comparing the
diffraction pattern obtained to the
diffraction patterns of known standard
asbestos fibers. Confirm the tentative
identification of chrysotile and amphibole
22
223
-------
asbestos from their electron diffraction
patterns. Classify each fiber as chrysotile,
amphibole, non-asbestos, no diffraction and
ambiguous.
NOTE 1: It is convenient to use a tape
recorder during the examination of the fibers
to record all pertinent data. This
information can then be summarized on data
sheets or punched cards for subsequent
automatic data processing.
NOTE 2: Chrysotile fibers occur as single
fibrils, or in bundles. The fibrils generally
show a tubular structure with a hollow canal,
although the absence of the canal does not
rule out its identification. Amphibole
asbestos fibers usually exhibit a lath-like
structure with irregular ends, but
occasionally will resemble chrysotile in
appearance.
NOTE 3i The positive identification of
asbestos by electron diffraction requires some
judgement on the part of the analyst because
some fibers give only partial patterns.
Chrysotile shows unique prominent streaks on
the layer lines nearest the central one and a
triple set of double spots on the second layer
line. The streaks and the set of double spots
are the distinguishing characteristics of
chrysotile required for identification.
Amphibole asbestos requires a more complete
diffraction pattern to be positively
identified. As a quantitative guideline,
layer lines for amphibole, without the unique
streaks (some streaking may be present), of
chrysotile, should be present and the
arrangement of diffraction spots along the
layer lines should be consistent with the
amphibole pattern. The pattern should be
distinct enough to establish these criteria.
NOTE <*: Chrysotile and thin amphibole fibers
may undergo degradation in an electron beam;
this is particularly noticeable in small
fibers. It may exhibit a pattern for a 1-2
seconds and disappear and the analyst must be
alert to note the characteristic features.
NOTE 5: An ambiguous fiber is a fiber that
gives a partial electron diffraction pattern
resembling asbestos, but insufficient to
provide positive identification.
23
224
-------
8.ft.5 Determination of Grid Square Area
Measure the dimensions of several
representative grid squares from each batch of
grids with an optical microscope. Calculate
the average area of a grid square. This
should be done to compensate for variability
in grid square dimensions.
8.5 Ashing
Some samples contain sufficiently high levels of
organic material that an ashing step is required
before fiber identification and counting can be
carried out. If a Nuclepore filter was used for the
original preparation and if the preliminary
examination of the initial preparation shows that this
condition exists, carry out the filtration step on a
new water sample using a . 45-ym Millipore filter. If
a Millipore filter was used initially the unused half
from 8.26.5 can be ashed.
NOTE 1: A Millipore filter is specified because it is
more readily oxidized under the specified ashing
conditions.
Place the dried Millipore filter paper containing the
collected sediment into a glass vial (28 mm diameter x
80 mm high). Position the filter such that the
filtration side touches the glass wall. Place the
vial in an upright position in the low temperature
asher. Operate the asher at 50 watts (13.56 MHz)
power and 2 psi oxygen pressure. Ash the filter until
a thin film of white ash remains. The time required
is generally 6 to 8 hours. Allow the ashing chamber
to slowly reach atmospheric pressure and remove the
vial. Add 10 ml of filtered distilled water
containing 0.1 percent filtered Aerosol OT to the
vial. Place the vial in an ultrasonic bath for 1/2
hour to disperse the ash. Dilute the sample if
required.
Assemble the 25 mm diameter filtering apparatus. (See
Note 1) Center a 25 mm diameter O.U5-ym Millipore or
.1-ym Nuclepore filter (with the 2-pm Millipore
backing) on the glass frit. Apply suction and
recenter the filter if necessary. Attach the filter
funnel and turn off the suction. Add the water
containing the dispersed ash from the vial to the
filter funnel. Apply suction and filter the sample.
After drying this filter it is ready to be used in
preparing sample grids as in 8.2A or 8.2B.
24
225
-------
NOTE 2; In specifying a 25-mm diameter filter it is
assumed that the ashing step is necessary mainly
because of the presence of organic material and that
the smaller filtering area is desirable from the point
of view of concentrating the fibers. If the sample
contains mostly inorganic debris such that the smaller
filtering area will result in over-loading the filter,
the 47-mm diameter filter should be used.
NOTE 3: It will be noted that a 10-ml volume is
filtered in this case instead of the minimum 50-ml
volume specified in 8.1.1. These volumes are
consistent when it is considered that there is
approximately a 5-fold difference in effective
filtration area between the 25-mm diameter and 47-mm
diameter filters.
8.6 Determination of Blank Level
Carry out a blank determination with each batch of
samples prepared, but a minimum of one per week.
Filter a fresh supply (500 ml) of distilled, deionized
water through a clean ,1-ym membrane filter. Using
the selected filter type, filter 200 ml of this water,
prepare the electron microscope grid, and count
exactly as in the procedures 8.1 - 8.U. Examine 20
grid squares and record this number of fibers. A
maximum of two fibers in 20 grid squares is acceptable
for the blank sample.
NOTE 1: The monitoring of the background level of
asbestos is an integral part of the procedure. Upon
initiating asbestos analytical work, blank samples
must be run to establish the initial suitability of
the laboratory environment, cleaning procedures, and
reagents for carrying out asbestos analyses.
Analytical determinations of asbestos can be carried
out only after an acceptably low level of
contamination has been established.
9. Calculations
9.1 Fiber Concentrations
Grid Square Counting Method - If the Grid Square
Method of counting is employed, use the following
formula to calculate the total asbestos fiber
concentration in MFL.
C = (F x Af) / (G x A x VQ x 1000)
If ashing is involved use the same formula but
substituting the effective filtration area of the 25-
mm diameter filter for A^ instead of that for the 47-
25
226
-------
mm diameter filter. If one-half the filter is ashed,
multiple C by two.
C = Fiber concentration (MFL)
F * number of fibers identified in "G" grid
squares
Af * effective filtration area of filter paper
(mm*) used in grid preparation used for fiber
counting
Ag » Average area of one grid square (mm2)
G = number of grid squares analyzed
Vo = original volume of sample filtered (ml)
Field-of-View Counting Method - If the Field-of-View
Method of counting is employed use the following
formula to calculate the total asbestos fiber
concentrations (MFL)
C = (F x Af x 1000) / (Ay x Z x VQ)
If ashing is involved use the same formula but
substituting the effective filtration area of the 25-
mm diameter filter for Af instead of that for the 47-
mm diameter filter.
C = fiber concentration (MFL)
F = number of fibers identified in area
examined (Ay x Z)
Af = effective filtration area of filter paper
(mm2) used in grid preparation for fiber
counting
Ay = area of one field of view (
Z = number of fields of view examined
VQ = original volume of sample filtered (ml)
9.2 Estimated Mass Concentration
Calculate the mass ftig) of each fiber counted using
the following formula:
M=LxW«xDx 10-*
If the fiber content is predominantly chrysotile, the
following formula may be used:
26
227
-------
M = xLxW«xDx 10-*
where M * mass (yg)
L * length (urn)
W » width (urn)
D » density of fibers (g/cm3)
Then calculate the mass concentration (yg/1) employing
the following formula.
Mc * C X Mf x 10*
where Mc = mass concentration (yg/1)
C = fiber concentration (MFL)
M.f = mean mass per fiber (yg)
To calculate Mf use the following formula:
n
fff - 2lMi/n
i - 1
where M = mass of each fiber, respectively
n * number of fibers counted
NOTE 1: Because many of the amphibole fibers are lath
shaped rather than square in cross section the
computed mass will tend to be high since laths will in
general tend to lie flat rather than on edge.
NOTE 2: Assume the following densities: Chrysotile
2.5, Amphibole 3.25
9.3 Aspect Patio
The aspect ratio for each fiber is calculated ty
dividing the length by the width,
10. Reporting
10,1 Report the following concentration as MFL
a. Total fibers
b. Chrysotile
c. Amphibole
27
228
-------
10.2 Use two significant figures for concentrations greater
than 1 MFL, and one significant figure for
concentrations less than 1 MPL.
10.3 Tabulate the size distribution, length and width.
10.4 Tabulate the aspect ratio distribution.
10.5 Report the calculated mass as yg/1.
10,6 Indicate the detection limit in MFL.
10.7 Indicate if less than five fibers were counted.
10.8 Include remarks concerning pertinent observations,
(clumping, amount of organic matter, debris) amount of
suspected though not identifiable as asbestos
(ambiguous) .
11. Precision
11.1 Intra Laboratory
The precision that is obtained within an individual
laboratory is dependent upon the number of fibers
counted. If 100 fibers are counted and the loading is
at least 3.5 fibers/grid square, computer modeling of
the counting procedure shows a relative standard
deviation of about 10% can be expected.
In actual practice some degradation from this
precision will be observed but should not exceed ± 15%
if several grids are prepared from the same filtered
sample. The relative standard deviation of analyses
of the same water sample in the same laboratory will
increase due to sample preparation errors and a
relative standard deviation of about ± 25 - 30% will
occur. As the number of fibers counted decreases, the
precision will also decrease approximately
proportional to /N where N is the number of fibers
counted.
11.2 Inter Laboratory
While there have been numerous inter laboratory
testing programs, there have been few carried out
using the same procedure. Those that have been done
indicate that agreement within a factor of two is
achieved if 100 fibers can be counted.
229
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12, Accuracy
12.1 Fiber concentrations
As no standard reference materials are available, only
approximate estimates of the accuracy of the procedure
can be made. At 1 MFL, it is estimated that the
results should be within a factor of 10 of the actual
asbestos fiber content.
This method requires the positive identification of a
fiber to be asbestos as a means for its quantitative
determination. As the state-of-the art precludes the
positive identification of all of the asbestos fibers
present, the results by this method, as expressed as
MFL, will be biased on the low side and assuming no
fiber loss represent .2 - .6 of the total asbestos
fibers present.
12.2 Mass concentrations
As in the case of the fiber concentrations, no
standard samples of the size distribution found in
water are available. The accuracy of the mass
determination should be somewhat better than the fiber
determination because a larger fraction of the large
fibers, which contribute the major portion of the
mass, are identifiable. This will reduce the bias of
low results due to difficulties in identification. At
the same time, the assumption that the thickness of
the fiber equals the width will result in a positive
error in determining the volume of the fiber and thus
give high results for the mass.
SELECTED BIBLIOGRAPHY
Seaman, D. R. and D. M. File. Quantitative Determination of
Asbestos Fiber Concentrations. Anal. Chem. 48(1): 101-110, 1976.
Lishka, R. J., J. R. Millette, and E. F. McFarren. Asbestos
Analysis by Electron Microscope. Proc. AWWA Water Quality Tech.
Conf. American Water Works Assoc., Denver, Colorado XIV - 1 - XIV
- 12, 1975.
Millette, J.R. and E. F. McFarren. EDS of Waterborne Asbestos
Fibers in TEM, SEM and STEM. Scanning Electron Microscopy/1976
(Part III) 451-460, 1976.
Cook, P. M., I. B. Rubin, C. J. Maggiore, and W. J. Nicholson.
X-Ray Diffraction and Electron Beam Analysis of Asbestiform
Minerals in Lake Superior Waters. Proc. Inter. Conf. on Environ.
Sensing and Assessment 34(2): 1-9, 1976.
McCrone, W. C. and I. M. Stewart, Asbestos. Amer. Lab. 6(4):10-
18, 1974.
230 "
-------
Mueller, P. K., A. E» Alcocer, R. L. Stanley, and G. R. Smith,
Asbestos Fiber Atlas. U.S. Environmental Protection Agency
Technology Series, EPA 650/2-75-036, 1975.
Glass, R. w., improved Methodology for Determination of Asbestos
as a Water Pollutant. Ontario Research Foundation Report, April
30, 1976. Mississauga, Ontario, Canada.
30
231
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Analytical Methodology for the Determination of Asbestos
by Transmission Electron Microscopy
The analytical procedure used by Walter C. McCrone Associates,
Inc., for the determination of asbestos in environmental samples is substan-
tially similar to that given in the U.S. EPA "Preliminary Interim Procedure
for Determining Fibrous Asbestos". * Although this procedure was written
for water samples, the techniques for preparation of the filter for examination
and the criteria for the identification of the asbestiform minerals are equally
applicable to air samples. Details of the procedure follow.
Working in a laminar flow clean bench (see attached laboratory
description), discs approximately 3 mm in diameter are punched out of the
filter. These discs are then placed face-down on previously carbon-coated
electron microscope support grids either of copper, if only chrysotile is
expected, or nylon. Nylon is used for samples in which there is a reason-
able likelihood of amphibole fibers in order that chemical analyses may be
performed on the fibers, by either the X-ray energy or wavelength dispersive
system fitted to the microscope. The use of nylon minimizes extraneous
X-ray signals from the support grid which would otherwise saturate the
detector system. Such an analysis is essential in order to classify the amphi-
bole type present. The grids are then transferred to a cold finger in a Soxhiet
extraction apparatus in which the membrane filter is dissolved using acetone
for Millipore Type MF and for Gelman GN-6 Metricel filters or chloroform
for Nuclepore filters. A "wicking" method may also be used for Nuclepore
filters but is unsuitable for the Millipore or Gelman types. Previous work
has shown us that there is very little risk of contamination in transferring
the filter on the electron microscope grid to the Soxhiet extractor. Further-
*Available from U.S. EPA Environmental Research Laboratory, Athens,
GA 30601.
232
waiter c. me crone associates, inc.
-------
more, by dissolving the filter in situ on the grid ("direct transfer"), the
risk of losing portions of the sample is minimal., Techniques involving
transfer of a liquid suspension directly to the electron microscope grid
are more subject to error since there is frequently a size separation as
the meniscus of the drying drop recedes. Procedures involving "rub-out"
techniques, though of some value in obtaining mass concentration data are
not applicable to fiber number or size distribution determinations as they
intentionally degrade the fibers to unit fibrils thus altering their size and
simultaneously increasing their numbers.
The sample grids are examined on the electron microscope
1 2
(JEM 200 * or EMMA 4* ) using a magnification such that the intermediate
lens aperture is in focus in the specimen plane. It is thus possible, by
inserting the aperture and switching to the diffraction position, to obtain a
selected area electron diffraction (SAED) pattern of the fiber with no other
adjustments to the microscope. In this way it is possible to spot check the
diffraction pattern, of individual fibers very rapidly. The JEM 200 is used on
those samples in which only chrysotile is of interest. EMMA 4, with the
capability for X-ray fluorescence analysis of individual fibers, is used where
the identification of amphibole types present is required. Both instruments
have a selected area electron diffraction capability.
Prior to commencing measurement the electron microscope
grid is scanned at a low magnification, approximately 2000-4000X to ensure
uniformity of dispersion on the filter. In the case of non-uniform deposition,
which may occur for example with cemented or aggregated fibers, several
grids may be examined from the same filter. This prior examination indicates
to the analyst which areas should be examined to obtain a truly representative
analysis of the sample. Magnifications in excess of 10,000 X are required for
*1 JEM 200. 200 Kv transmission electron microscope manufactured by Japan
Electron Optics Laboratories (JEOL)
*2 EMMA 4. Combined 100 Kv transmission Electron Microscope-Micro-
probe Analyzer manufactured by Associated Electrical Industries (AJEI)
233 waiter c. me crone associates, inc,
-------
the observation of the smallest chrysotile fibrils present.
The magnification of examination used in the JEM 200 is
14, 600 on the viewing screen; that used in EMMA 4 is 24,800. As stated
above, these magnifications are based on user convenience in switching from
viewing to diffraction.
The length and width of each asbestos fiber is recorded. Only
fibers which are positively identified as asbestos are measured. Interpolation
from intervals scribed on the viewing screen allows an accuracy of measure-
ment on the screen of approximately 0. 05 cm. This corresponds to an
accuracy in size measurement of about 0.02-0.04 pm. Measurements of the
individual fibers are computer processed to give listings of the length and
width of the fibers, together with a computed mass of each fiber computed on
7
the basis of density, D, and dimensions, L and W (D x L x W ). A value of
3.3 is taken as the mean density of amphibole fibers: a density of 2.3 is used
for chrysotile. Because many of the amphiboles are lath-shaped rather than
square in cross section, this figure may well be slightly high, since the laths
will, in general, tend to lie flat rather than on edge. There is, however, a
finite possibility that some laths will be on edge and, due to the very small
size of many of the fibers of interest, the approximation to a square fiber will
not give more than a slightly high bias to the mass readings. The program
automatically assigns the longest dimension to the fiber length and excludes all
particles with an aspect ratio below three.
Also presented in the computer printout are the calculated num-
ber of fibers per unit volume, the calculated mass of fiber per unit volume,
the size distribution of the fibers based on length and width, and the distribu-
tion of fibers by aspect ratio together with the relevant statistical information
on these parameters. A physical description of the sample accompanies the
measurements and is considered an integral and essential part of the analysis.
A sample of a complete analysis, description and computer printout is attached.
234
waiter c. me crone associates, inc.
-------
Sample
Much chunky and chiplike material ranging to quite large sizes
and showing a wide range of composition — Mg, Si and some Fe, mainly Si,
mainly Ca or Fe-Si types — although the Mg-Si type predominates. Additionally,
fine agglomerate material, organic matter, some spherical inorganic particles
and chrysotile are found,in this sample. The spheroidal particles are found
normally, to be Al-Si-Fe composition but wide ranging variation does occur.
Antigorite laths (e.g. fibers) noted. Occasional";massive" fibers are present or
fibers which are lathlike. These are found to contain Mg-Si-Ca-Fe and some S,
consistently.
• -..'iSiap^g^fwi^spc^sa'Js
Probe of large chunky material
Probe of large chunky material
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waiter c. me crone associates, inc.
235
-------
Sample
Probe of chrysotile
Probe of typical spherical particle
Probe of chrysotile bundle
^^^^^^^al^^^^S^&S^S^^S^sS^S
Probe of antigorite lath
Probe of small lathlike fiber
welter c, me crone associates, inc.
236
-------
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t '
' * . - -6
PRESERVATION OF PHE1IOLIC COMPOUNDS
IN WASTEWATERS
Mark J. Carter * and Madeliene T. Huston
Central Regional Laboratory
Environmental Protection Agency
1819 W, Pershing Road
Chicago, Illinois 60609
240
-------
BRIEF
The combination of strong acid or base with sample storage at 4°C stabilized
phenolic compounds in wastewaters for at least 3-4 weeks. There is a positive
relationship between microbiological activity and chemical stability of the
samples studied.
ABSTRACT
Copper sulfate and phosphoric acid with sample-storage at 4°C is a common
preservative technique used for phenolic compounds in wastewaters. However,
there are no data showing its effectiveness. A study was conducted to compare
the preservation method with the addition of strong base or acid and sample
storage at 25° and 4°C. The addition of 1 ml cone H.SO./l with sample storage
at 4°C was most consistently effective in preserving stability for 3-4 weeks.
However, the other chemical preservatives were found to be effective for at
least 8 days. Substantial loss of phenolic compounds rapidly occured in all
samples unless the chemical preservative was added immediately after sample
collection. A positive correlation was found between loss of phenolic compounds
and microbiological activity suggesting the latter was the dominant factor in
determining sample stability.
INTRODUCTION
The 1972 Amendments to the Clean Water Act have resulted in limitations on
the concentration and loading of pollutants that can be discharged by industries
and municipalities (1). The need to monitor these discharges has substantially
increased the number of environmental samples requiring analysis, especially for
toxic substances such as phenolic compounds.
*
Xelly has reviewed the literature for methods of analyses of phenolic compounds
in wastewaters (2). The most common methods are the 4-aminoantipyrine (4-AAP)
(3-6) and 3-methyl-2-faenzothiazolinone hydrazone (MBTH) (7,8) colorimetric
procedures and the ultraviolet bathochromic shift method (9). The difficulty and
equipment requirements for these methods often results in samples being shipped
241
-------
Chemical Analysis of Samples. The first analysis of each sample was completed
within two hours of sample collection. All samples, standards and blanks were
distilled from acidic solution, to separate phenolic compounds from potential
interferences (13). The distillates were analyzed by an automated version of the
4-aminoantipyrine method shown in figure 1.
The buffered potassium ferricyanide reagent was prepared by adding 2.0 g
potassium ferricyanide, 2.1 g boric acid, 3.75 g potassium chloride, 44 ml of
1 S sodium hydroxide and 0.5 ml Brij-35 (Technicon Corp. No. T21-011Q) to a
volumetric flask and diluting to 1 1. The 4-aminoantipyrine reagent was preparad
by diluting 0.65 g of the chemical to 1 1. Both reagents were filtered through
a 0.45 im membrane filter before use.
Two control standards were prepared and preserved with copper sulfate and
phosphoric acid by an independent analyst at the beginning of each study to
checJc on the consistency of the day-to-day standard preparation and instrument
calibration.
Standard Plate Count. Plate count agar was prepared fresh just before use, added
to petri dishes and 1, 0.1 and 0.01 ml of each sample was plated in triplicate
(13). All samples were incubated at 35°C for 24 hrs. Only those plates having
30-300 colonies were considered valid and the values reported in Table I are an
average of the three replicate dilutions.
RESULTS AND DISCUSSION
The stability of phenolic compounds in ncn-wastewaters aqueous solutions has
been studied by several investigators. Phenolic compounds are good preservatives
at high concentrations (>0.5%) but are readily biodegraded at lower concentrations
(14-17). Chambers and Kabler found no detectable nonbiological degradation (15).
Extremes in pH (18-22), temperature (23-26) and the use of toxic chemicals (27-29)
have been used to reduce microbiological activity in aqueous solution. Strong base
(4,30,31), acid (4) and copper sulfate-phosphoric acid (11,12) in combination
with temperature control have been used to stabilize phenolic compounds in surface
waters.
- 3 -
243
-------
to a large centralized laboratory for analysis. The shipping process can lead
to substantial delays between sample collection and analysis. As a result, the
effectiveness of the sample preservation technique will substantially affect the
accuracy of the data. It is also necessary to consider sample stability during
the collection process, especially if the 24-hour composite method is used (10) .
Very little data exist in. the literature to give guidance about the best method
to stabilize phenolic compounds in wastewaters. The current recommended technique
(11) is a modification of work performed over thirty years ago (12) . Ettinger,
et al., found that copper sulfate effectively preserved river water and river
water seeded with sewage for two to four days when the samples were stored at
25°C. Subsequent to the work of Sttinger, phosphoric acid was added to the copper
sulfate preservative to keep the metal ions in solution when added to alkaline
samples (11). In addition, it was recommended that all samples be stored at
4°C until analysis.
No data was presented until 1974 about the effectiveness of the combined
phosphoric acid, copper sulfate, 4CC storage technique for preserving phenolic
compounds in water samples. Afghan, et al., showed that either strong acid
or base has more effective in retarding bacterial activity and stabilizing phenol
in Great Lakes' waters than the combined copper sulfate preservative (4).
•
The observation of Afghan raises doubt that the copper sulfate - phosphoric acid
preservation method is best for stabilizing phenolic compounds in wastewaters.
Therefore, a study was undertaken to determine the most effective and practical
preservation method and maximum allowable holding time for phenolic compounds in
wastewaters.
METHODS
Preparation of Samples. Fresh samples were collected in 5 gal high density
polyethylene jugs and immediately brought to the laboratory. The water samples
were homogenized with a Tekmar Super. Dispax system, preserved and split into 250 ml
high density polyethylene bottles. Some samples were spiked with phenol to raise
the starting concentration to a level that could be accurately measured. The
samples were preserved and stored as described in figures 2-5.
- •> 242 e
• .. \ *
-------
Chemical Analysis of Samples. The first analysis of each sample was completed
within two hours of sample collection. All samples, standards and blanks were
distilled from acidic solution, to separate phenolic compounds from potential
interferences (13). The distillates were analyzed by an automated version of the
4-aminoantipyrine method shown in figure 1.
The buffered potassium ferricyanide reagent was prepared by adding 2.0 g
potassium ferricyanide, 2.1 g boric acid, 3.75 g potassium chloride, 44 ml of
1 N sodium hydroxide and 0.5 ml Brij-35 (Technicon Corp. No. T21-0110) to a
volumetric flask and diluting to 1 1. The 4-aminoantipyrine reagent was preparad
by diluting 0.65 g of the chemical to 1 1. Both reagents were filtered through
a 0.45 yjn membrane filter before use.
Two control standards were prepared and preserved with copper sulfate and
phosphoric acid by an independent analyst at the beginning of each study to
check on the consistency of the day-to-day standard preparation and instrument
calibration.
Standard Plate Count. Plate count agar was prepared fresh just before use, added
to petri dishes and 1, 0.1 and 0.01 ml of each sample was plated in triplicate
(13). All samples were incubated at 354C for 24 hrs. Only those plates having
30-300 colonies were considered valid and the values reported in Table I are an
average of the three replicate dilutions.
RESULTS AND DISCUSSION
The stability of phenolic compounds in ncn-wastewaters aqueous solutions has
been studied by several investigators. Phenolic compounds are good preservatives
at high concentrations (>0.5%) but are readily biodegraded at lower concentrations
(14-17). Chambers and Kabler found no detectable nonbiological degredation (15).
Extremes in pH (18-22), temperature (23-26) and the use of toxic chemicals (27-29)
have been used to reduce microbiological activity in aqueous solution. Strong base
(4,30,31), acid (4) and copper sulfate-phosphoric acid (11,12) in combination
with temperature control have been used to stabilize phenolic compounds in surface
waters.
- 3 -
243
-------
The stability of phenolics in three different wastewaters preserved with copper
sulfate - phosphoric acid and stored at 4°C was studied first. The results arc
shown in Figure 2. The raw sewage was fairly weak with a biochemical oxygen
demand (BOO) of only 95 mg/1 and the treated sewage sample was collected after
secondary biological treatment but before chlorination. The industrial wastewater
was collected from the Grand Calumet River which is essentially a composite from
the South Chicago, industrial area.
Since the samples chosen for study often had low background concentrations of
phenolics, each was spiked with phenol as needed so that changes in phenolic
concentration wovld be easier to determine. Phenol was chosen for the spike
because Kaplin, et al., found phenol to be the least stable of all the phenolic
compounds in natural waters (32-35).
The most important result of study 1 was the rapid loss of phenolics from the
samples at 4°C with no addition of any chemical preservative. The percentage
loss of phenolics within 24 hrs. for the industrial waste, raw and treated
sewage saoples was 35, 80 and 40%, respectively. Ettinger, et al., reported that
an unpreserved river water sample stored at 2°C lost only 15% of the original
phenolic content after 4 days (12). However/ the same sample stored at 25°C lost
all of its phenolic compounds within 2 days. The results from these two studies
show that the loss of phenolics from unpreserved samples is variable depending
upon sample type but significant in all cases. The expected precision of sample
analysis was determined from daily analysis of the control A and 3 samples to be
± 12 vg/1 (20). There was no statistically significant change in phenolic
concentration over the 22 day study for the samples preserved with copper sulfara-
phosphoric acid and stored at 4°C. However, there were large day-to-day changes
in the concentration of phenolics measured. This poor precision was determined
to be caused by the problem in taking a representative sample for analysis due
to the presence of particulate matter. The problem was solved in later studies
by homogenizing all samples before analysis.
In the second study (figure 3), the effectiveness of the combined copper sulfate-
phosphoric acid preservative was sutdied vs. sample type. Activated sludge was
added to raw sewage to create a sample that was organically rich and biologically
active. This sample was stable for 12 days, but degraded to 85% of the original
244
— 4 -
-------
phenol concentration after 33 days. The other samples were stable for the duration
of the study. The dip in all values on day 1 of the study was attributed to
improper calibration.
Baylis reported the use of 1.2 ml 1 M NaOH/1 of sample to preserve phenolic
compounds in potable water samples(30). However, Ettinger, et al., found Baylis1
procedure to be ineffective for sewage seeded stream samples (12). Kaplin and
Frenko found that a hundred-fold increase in base concentration was effective for
preserving stream waters (31). Afghan, et al., verified the effectiveness of the
higher concentration of base for preserving lake waters (4). Afghan
also showed that 0.1 M HCl was an effective preservative.
The effectiveness of strong base or acid in preserving phenolic compounds was
compared with copper sulfate in the third study (figure 4). The concentration
of phenolic compounds was stable in the raw sewage sample studied when stored at
4°C regardless of the preservative used. However, the sulfuric acid and copper
sulfate preserved samples deteriorated rapidly after eight and two days, respective
when stored at 25°C.
Doetsch and Cook reported that a common feature of acidophilic bacteria was a
resistance to copper ions (23). Growth of acidophilic bacteria occurs at pH 2-5
which is the pH range for the copper sulfate preservative. These facts makes the
use of copper sulfate at pH 4 suspect as a good preservative, especially if the
samples are not stored at 4aC. The same sample with 2 ml cone H.SO./l - which
produces a pH cf about 1.5 - at 2S°C was stable for eight days. Kushner has
reported far fewer microorganisms can tolerate pH 1.5 than 4 (22). It is
interesting to note that even at pH 1.5 but at 25°C that the phenolic concentration
decreased substantially. This observation indicates that, while neither acidifi-
cation or cold storage stabilizes phenolic compounds in a wastewater, the
combination does.
In order to evaluate the biological induced degredation of phenolic compounds,
microbiological activity was measured on a raw and secondary treated sewage.
Samples were preserved as indicated in Table I and total plate counts taken after
1 hr. (day 0), 8 and 20 days. The only secondary sewage aliguot that showed any
significant activity was the chemically unpreserved sample stored at 4°C. The
-5-245
-------
microbiological activity noted corresponds very closely with the chemical stability
of phenolics in treated sewage found in studies 1 and 2.
i
The unpreserved raw sewage sample stored at 4°C showed very great microbiological
activity which corresponds to the phenolic instability noted in Study 1. The
addition of 2 ml cone H.SO /I initially reduced the microbiological activity
significantly. "However, by day eight, the activity increased five-fold and then
decreased slightly again by day twenty. This trend corresponds closely with the
rapid loss of phenolics in Study 3, after day eight and then a moderate but
continued loss thereafter. The same sample stored at 4°C with 2 ml cone H.SO /I
showed at least a ten-fold lower microbiological activity and a corresponding
increase in chemical stability shown in Study 3.
The raw sewage sample with copper sulfate-phosphoric acid and stored at 4°C
exhibited greater microbiological activity than the aliquot with sulfuric acid.
This observation corresponds to the moderate effectiveness of this preservative -
stability in studies 1 and 3 and instability of the raw sewage in Study 2. Addition
of strong caustic also lowered the microbiological activity of the raw sewage
sample. However, the higher concentration of caustic, 10 ml 10 N NaOH/1, was
required for a quick initial kill. The high initial microbiological activity
of the 2 ml 10 N NaOH/1 aliquot did not affect the chemical stability found in
Study 3.
•
Increasing the concentration of H _SO . two-fold with storage at 2S°C, reduced the
microbiological activity to the same level as the aliquot stored at 4°C with
2 ml cone H.SO./l. A fourth study was conducted to detemine if a greater acid .
concentration could preserve phenolic stability without cold storage. The results
in Figure 5, show good stability for the aliguots preserved with 2 ml H SO /I at
4°C and 4 ml H2SO4/1 at 25°C. The aliquot with 2 ml H SO4/1 at 2S°C showed a
substantial loss of phenolic compounds after the eighth day.
The enhanced stability of the samples preserved with the higher acid concentration
is- excellent evidence that the greatest cause of sample instability is caused by
microbiological and not chemical acitivity. Gordon claimed that phenolic compounds
in many refinery effluent waters can be oxidized in acid solution (35). However,
246
-------
he did not state the temperature conditions for storage or provide any data to
support the claim. Emerson noted that phenol was less reactive under oxidizing
acidic than basic conditions (37). Stewart'(38) and Waters (39) also noted that
phenolic compounds were more reactive in basic solution. However, in practice
basic preservation does not cause instability of phenolic compounds especially
if stored at 48C (4,30,31,36).
CONCLUSIONS AND RECOMMENDATIONS
All samples quickly lost phenolic compounds in the absence of a chemical
preservative, even if stored at 4°C. Therefore, all samples must be chemically
preserved at the'time of collection. The chemical preservative must be added to
the first aliquot of a composite sample.
The desired time period for holding samples determines the choice of chemical
preservatives. All preservatives studied, NaOH, 3,30 and CuSO. - H PO , were
effective - no more than 5% phenolic compound loss - for at least 12 days whan
the samples were stored at 4°C. Strong base or acid were effective for 26 and
28 days, respectively, when the samples were stored at 4aC.
The use of acid or base preservation has the advantage of eliminating the use of
one separately preserved bottle specifically for phenolics analysis (11,13). The
choice of acid or base preservation will depend on whether cyanide (base preserved)
or nutrient (acid preserved) analyses will be performed. The advantage of acid
preservation is that sulfides, a common interference in the colorinetrie methods,
will be driven out of the sample (35). The basic preservation will be advantageous
if any organic extraction is required in the analysis method to remove organic
interferences (13).
*
Use of 2 ml cone H.SO./l with sample storage at 4°C is recommended over the use
Of 4 ml cone H_SO /I at 25°C. The former conditions combine the preservative
Dualities of low temperature and pH and are milder conditions chemically which
should reduce the possibility of undesirable chemical reactions.
- 7 -'
247
-------
ACKNOWLEDGEMENT
The authors would like to thank M. Anderson, C. Steiner and 0. Grothe, EPA,
Chicago for .their assistance with the microbiological work and data interpretation.
Mention of trade names or commercial products does not- imply endorsement by
the Environmental Protection Agency or the Central Regional Laboratory.
8 -
248
-------
LITERATURE CITED
I
1. Amendment to the Federal Water Pollution Control Act, Public Law 92-500,
October 18, 1972.
2. Kelly, J.A., "Determination of Phenolic - Type Compounds in Water and
Industrial Wastewaters," Oklahoma State Univ., Stillwater, Okla., NTIS
* ORO-4254-11, 1972.
3. Mohler, E.F., Jr. and Jacob, L.N., Anal. Chem., 29, 1369 (1957).
4. Afghan, B.K., Belliveau, P.E., Larose, R.H. and Ryan, J.F., Anal. Chim. Acta,
JU, 355 (1974) .
5. Ettinger, M.S., Ruchhoft, C.C. and Lishka, R.J., Anal.Chem., 23_, 1783 (1951).
6. Gales, M.E., Jr. and Booth, R.I., Journal AHWA, 63, 540 (1976).
7. Friestad, J.O., Ott, D.E. and Gunther, F.A., Anal.Chem., 41, 1750 (1969).
3. Goulden, P.O., Brooksbank, P. and Day, M.B., Anal.Chem., 45, 2430 (1973).
9. Fountaine, J.E., Joshipura, P.B., Keliher, P.N. and Johnson, J.D., Anal.Chan.,
46_, .62 (1974).
10. "Handbook for Monitoring Industrial Wastewater," U.S. Environmental Protection
Agency, Technology Transfer, Cincinnati, CH, 1973.
11. "Manual of Methods for Chemical Analysis of Water and Wastes," U.S. Environments
Protection Agency, Technology Transfer, Cincinnati, OH, 1974.
12. Ettinger, M.S., Schott, S. and Ruchhoft, C.C., Journal AWWA, 35, 299 (1943).
13. "Standard Methods for the Examination of Water and Wastewater," 14th ed.,
American Public Health Association, Washington, D.C., 1976.
14. Harlow, I.P., Ind. Sng. Chem., 3_1_, 1346 (1939).
15. Chambers', C.W. and Kobler, P.W., in "Developments in Industrial Microbiology,"
Vol. 5, American Institute of Biological Sciences, Washington, D.C., 1964.
16. Erikson, D., Jour. Bact., 41, 277 (1941).
17. ZoBell, C.E. and Brown, B.F., J. Mar. Res., 5_, 178 (1944).
18. Ruchhoft, C.C., Ettinger/ M.B. and Walker, W.W., Ind. Eng. Chem., 32, 1394 (1949)
19. Hewitt, L.F., in "Microbial Ecology," University Press, Cambridge, England, 1957.
- 9 -
249
-------
20. Wood, E.J. Ferguson, "Microbiology of Oceans and Estuaries," Elsevier
Publishing Co., New York, N.Y., 1967.
21. Weiss, R.L., Liranol. and Oceanogr., 18, 877 (1973).
22. Kashner, D.J., in "Inhibition and Destruction of the Microbial Cell,"
W.B. Hugo, ed., Academic Press, London, 1971.
23. Waksman, S.A. and Carey, C.L., Jour. Bact., 29, 531 (1935).
24. ibid, p. 545.
25. Butterfield, C.T., Sew. Works Jour. > 5_, 600 (1933).
26. Olsen, R.H. and Metcalf, E.S., Science, 162., 288 (1968).
27. Ruchhoft, C.C. and Placak, Q.S., Sew. Works Jour., 14, 638 (1942).
28. Doetsch, R.N. and Cook, T.M., "Introduction to Bacteria and their Ecobiology,"
Oniversity Park Press, Baltimore, Md., 1973.
29. Pelczar, M.J., Jr. and Reid, R.D,, "Microbiology," 2nd ed., McGraw-Hill- Sook Co.
New York, N.Y., 1965.
30. Baylis, J.R., Water Works and Sewerage, 79, 341 (1932).
31. Kaplin, V.T. and Frenko, N.G., Gig. Sanit., 26, 68 (1961).
32. Kaplin, V.T., Fesenko, H.G., Babeshkina, 2.M. and Simirenko, V.I.,
Gidrokhim. Materialy, 37_, 158 (1964). Chea.Abs. , 62_, 14332.
33. Kaplin, V.T., Panchenko, S.S. and Fesenko, N.G., Gidrokhim.Materialy, 40,
134 (1965). Chem.Abs., 64, 13906.
34, Kaplin, 7.T., Semenchenko, L.V. and Ivanov, E.G., Gidrokhin,Materialy, 46,
199 (1968). Chem.Abs. 69, 69563.
35. Kaplin, V.T., Panchenko, S.E. and Fesenko, N.G. Gidrokhim.Matarialy, 42,
262 (1966). Chem.Abs., 67, 57105.
36. Gordon, G.E., Anal.Chem. , 3_2_, 1325 (1960).
37. Emerson, E., Jour. Orgr. Cham. , 8_, 417 (1943) .
38. Stewar-t, R., "Oxidation Mechanisms, Applications to Organic Chemistry,"
W.A. Benjamin, Inc., N.Y., N.Y., 1964.
39. Waters, W.A., "Mechanisms of Oxidation of Organic Compounds," John Wiley & Sons,
Inc., N.Y., N.Y., 1964.
10 -
250
-------
Table I. Effectiveness of Preservatives in Sterilizing Sewage as
Indicated by Total Plate Counts
Preservation Method
Raw Sewage
4°C
2 ml cone H,SO., 2S°C
2 4
2 ml cone H_SO., 4°C
2 4
4 ml cone H-SO,, 25eC
2 4
CttSO., H.PO., 4°C
4 34
2 ml ION NaOH, 4°C
10 ml ION NaOH, 4°C
Total Plate County, Colonies/ml
Day 0
Day 8
Day 20
>»30,000
730
560
6,300
28,000
230
>»30,000
3,500
70
40
800
110
90
>»30,000
2,200
200
b
600
270
100
Secondary Treated Sewage Before Chlorination
4°C . 23,000
<30
2 ml cone H_SO., 25°C
2 4
2 ml cone H.SO., 4°C
2 4
4 ml cone H.SO., 2S"c
2 4
CaS04/ H3P04, 4°C
2 ml ION NaOH, 4aC
10 ml ION NaOH, 4°C
<30
<30
40
<30
20,000 5,400
<30 <30
<30 <30
<30 <30
<30 <30
<30 <30
<30 <30
Volume of acid or base added per liter of sample. Copper sulfate, phosphoric
acid preservative prepared as described in ref. 13. Temperatures refer to storage
conditions. .
b
Confluent colonies
Elated within 1 hr. of preservation
- 11 -
251
-------
Figure 1. Automated phenol manifold diagram. Numbers in parentheses
correspond to the flow rate of the pumptubes in ml/min. Numbers
adjacent to glass coils and fittings are Technicon Corp. part numbers
Figure 2. Plot of stability of phenolic compounds in several wastcwaters with
time; Study 1. All samples with points plotted as "B" were preserved
with l.Og CuSO. • 5 H 0/1, the pH brought to 4.0 with phosphoric acid
and then stored at 4°C. Samples plotted as "A" were stored at 4°C
with no chemical preservatives. Both industrial waste, raw and
treated sewage samples were spiked with phenol to bring their initial
concentrations to 50, 100 and 60 wg/l» respectively.
Figure 3. Plot of stability of phenolic compounds in several wastewaters with
time; Study 2. 'All samples were stored at 4°C. The industrial
waste, raw and treated sewage samples were spiked with phenol to
bring their initial concentrations to 110, 165 and 110 yg/1^ respecti
Figure 4. Plot of stability of phenolic compounds in a raw sewage sample presex
with several chemicals; Study 3, Aliquots 1 and 2 were preserved wit
copper sulfate and phosphoric acid and stored at 25 and 4°C, respecti
Aliquots 3 and 4 were preserved with 2ml cone H.SO./l and stored at
25 and 4°C, respectively. Aliquot 5 was preserved with 2 ml 10 N
NaOH/1 and stored at 4°C, Aliquots 1-4 were spiked with phenol to
bring their initial concentrations to 125 ug/1. Aliquot 5 was spikec
with phenol to bring its initial concentration to 130 ug/1.
Figure 5. Plot of stability of phenolic compounds in a raw sewage sample
preserved with several concentrations of sulfuric acid. Aliquots
3 and 5 were preserved with 2 ml cone H-SO./l and stored at 25 and
4°C, respectively. Aliquot 4 was preserved with 4 ml cone H_SC./1
and stored at 25°C. All samples were spiked with phenol to cring
their initial concentration to 130 yg/1.
- 12 -
252
-------
flj
a.
CO
«N
s
I
a
in
.a
I
x
O
UL
il
253
-------
Figure 2.
100
50-
50-
\.
8-
Treated Sewage
a— „ ,a
Treated Sewage
»8 Industrial Waste
.A Industrial Waste
'10 r15
DAYS
T20
25
254
-------
Rgure 3.
150-
<
o.
100-
\/\
V
V
70- *
»^ Raw Sewage
Treated Sewage
-2,
Control A
Industrial Waste
3—
Control 8
A
10 ' '20
DAYS
V
33
255
-------
180-
ngure
100-
Control B
a
J
O
100-
50-
0
-rt
lU
1 '20
DAYS
30
256
-------
figure
150-
1
Control A ,,
100-
o
z
50-.
ControJ B
10
DAYS
257
'20
'30
-------
MIDWEST RESEARCH INST1T1
425VolkerBoule
Kansas City, Missouri 6
Telephone (816) 753-;
February 1, 1978
Dr. W. A. Telyard, Chief
Energy and Mining Branch
Effluent Guidelines Division
WH-552
Environmental Protection Agency
401 M Street, S.W.
Washington, D.C. 20460
Dear Bill:
Enclosed is a drawing of the liquid-liquid extractor we used for extracting
tannery wastewaters for base/neutral and acidic priority pollutants. The
design was patterned after the Hershberg-Wolf extractor sold by Ace Glass.
The precision bore leveling device was eliminated to facilitate getting them
easily fabricated locally.
This simplified model is not totally automated. The stopcock requires periodic
adjustment to maintain the appropriate solvent level in the sample chamber.
As I told Gail, we have designed a simple modification of the solvent return
to eliminate periodic readjustments but have not had time to check it out to
our satisfaction. We'll pass along this modification as soon as we can.
Please let me know if we can help further.
Best regards,
CSa
Clarence L. Haile
Senior Chemist
Program Manager,
Mass Spec Center
Enclosure
CLH:1m
259
-------
2.5cm l.D.
250ml
5 24/4°
24/40 ^. t—i -1—
45/50
TFE Sfopcock
1 cm l.D
?fiO
-------
Attendees
Seminar on Analytical Methods
Environmental Protection Agency
November 9 & 10, 1977
Charles E. Stephan, Chemist
U.S. EPA
6201 Cangdon Blvd.
Duluth, Minnesota 55804
218-727-6692 X510/ FTS: 783-9510
Phil Cook
EPA Duluth
6201 Congdon Blvd.
Duluth, Mn 55804
Ian M. Stewart, Manager
Electron Optics Group
Walter C. McCrone Assoc. Inc.
2820 S. Michigan Ave.,
Chicago, Illinois 60616
(312) 342-7100
John D. Hallett, Staff Engineer
Shell Oil Co.
P.O. Box 2463
Houston, Texas 77068
(713) 241-5778
Gary Seidel, Chemist
Bunker Hill
1508 Northwest Blvd.
Coeur d'Alene, Idaho 83814
(208) 667-6797
Stephen Wright, Lab Manager
Edward C. Jordan, Co., Inc.
Portland, Maine
(207) 775-5401
Joe C. Watt
Environmental Development Coordinator
Catalytic Inc.
1500 Market St.,
Philadelphia, Pennsylvania 19101
(215) 864-8109
261
-------
Bruce W. Long
Associates
Ryckman, Edgerley, Tomlinson & Asso., Inc.
12161 Lackland Road
St. Louis, Mo. 63141
(314) 434-6960
Carol A. Hammer, Associate
RETA/Envirodyne Engineers
12161 Lackland Road,
St. Louis Mo. 63141
(314) 434-6960
Robert A. Fluegge, Program Manager
Carborundum
Niagara Falls, New York
(716) 278-2992
Bernard S. MacCabe
Business Development Manager
Carburundum Co,
P.O. Box 1054
Niagara Falls, New York 14302
(716) 278-6347
E. Ellen Gonter, Manager
Water Laboratories Department
Cyrus Wm. Rice Division, NUS Corp.
15 Noble Avenue,
Pittsburgh, Pennsylvania 15205
(412) 343-9200
Liz Privitera
Environmental Scientist
Calspan Corp.
4455 Geneva Street,
P.O. Box 235
Buffalo, New York 14221
(716) 632-7500
Barry Langer
Chemical Engineer
Burns and Roe
P.O. Box 663,
Paramus, New Jersey
(201) 265-8710
Dr. Joseph N. Blazevich, Chemist
EPA, Region X, Lab
1555 Alaskan Way So.
Seattle, Washington 98134
442-5840/ FTS 8-399-5840
-------
David C. Hemphill, Chemist
U.S. EPA
EMSL/Las Vegas
P.O. Box 15027
Las Vegas, Nevada 89114
(702) 736-29697 FTS: 595-2969
Walter Shackelford, Research Chemist
U.S. EPA - Athens ERL
Athens, Georgia
(404) 546-3186
E. William Loy, Jr., Chemist
U.S. EPA, S & A Division, Region X
College Station Rd.
Athens, Georgia 30605
FTS: 250-3165/ Commercial (404)546-3165
Edward Taylor, Chief
Chemistry Section
Region I EPA - New England Regional Lab
60 Westview
Lexington, Ma
(617) 861-6700
Larry A. Parker, Chief
Laboratory Section
U.S. EPA, Region III, Wheeling, WV
303 Methodist 81dg.,
Wheeling, West Virginia 26003
(304) 233-127V FTS: 923-1049
Walter E. Andrews, Chief
Rochester Program Support Branch
U.S. EPA, Region II
Rochester, New York
(716) 473-3166
Francis T. Brezenskis, Laboratory Director
EPA, Region II
Hilton Inn
Fred Haeberer, Research Chemist
EPA - Athens, Georgia
College Station Rd.,
Athens, Georgia 30605
(404) 546-3781
Bill Donaldson, Chief
Analytical Chemistry Branch
U.S. EPA (Athens Environmental Research Lab,
-------
Dr. Larry D. Johnson, Research Chemist
U.S. EPA, Industrial Environmental Research Lab., R.T.P,
Research Triangle Park,
NC 27711
FTS: 629-2557
Commercial: (919) 541-2557
Dr. T.O. Munson, Chief
Organics Analysis Unit
U.S. EPA Annapolis Field Office
Annapolis Science Center
Annapolis, Maryland 21401
(301) 224-2740/ FTS: 922-3753
Thomas Bellar, Research Chemist
EPA - EMSL
Cincinnati, Ohio 45226
(513) 684-7311
Kathleen A. Carl berg, Chemist
EPA - Nat1! Enforcement Investigations Center
Bldg. 53, Denver Federal Center,
Denver, Colorado 80225
(303) 234-4661
Bob Claeys, NCASI
Engineering Experiment Station, O.S.U.
Corvallis, Oregon 97331
(503) 754-2015
O.J. Loaspon II, Chemist
U.S. EPA, NEIC
Box 25227, Bldg. 53 DFC
Denver, Colorado 80225
(303) 234-4661
Billy Fair!ess, Deputy Director
EPA
1819 W. Pershing
Chicago, Illinois
(312) 353-8370
Mark J. Carter, Deputy Chief
Chemistry Branch
EPA - NEIC
P.O. Box 25227, Bldg. 53
Federal Center
Denver, Colorado 80225
(303) 234-4661
-------
Gerard F. McKenna
Reg. Q.A. Coordinator
EPA - Region II
Edison, New Jersey
FTS: 340-6645/ (201) 321-6645
Richard D. Spear, Chief
Surv. & Monitor Branch
EPA - Region II,
Edison, New Jersey
8-340-6685/6 - 321-6685/6
James J. lichtenberg, Chief
ORganic Analyses Section
U.S. EPA
EMSL - Ci
(513) 684-7308
P. Michael Terlecky, Head
Environmental Science Section
Calspan Corporation
P.O. Box 235
Buffalo, New York 14221
(716) 632-7500
Martha Bronstein, Chemist
Calspan Corporation
Box 235
Buf-alo, New York
(716) 632-7500
Larry Wapensky, Organic Chemist
U.S. EPA - Region VIII
Box 25366 DFC
Denver, Colorado 80225
(303) 985-7725
C. H. Anderson, Research Chemist
U.S. EPA
Athens, Georgia
(404) 546-3452
Leon Myers, Sup. Research Chemist
U.S. EPA, RSKERL
Box 1198,
Ada, Ok
(405) 332-8800 Ext. 202
William B. Prescott, Manager
Research Services
American Cyanamid Company
Bound Brook, New Jersey 08805
(201) 356-2000 X2167
-------
Richard A. Javick, Senior Res. Chemist
FMC Corporation
Box 8
Princeton, New Jersey 08540
(609) 452-2300 X328
Robert T. Rosen, Research Chemist
Mass Spectroscopist
FMC Corporation
P.O. Box 8
Princeton, New Jersey 08540
(609) 452-2300
Dr. S. T. Mayre, Staff Chemist
Duke Power Company
422 South Church St.
Charlotte, North Carolina 28242
(704) 373-8283
Dr. S. C. Blum, Research Associate
Exxon Research and Engineering Co.
P.O. Box 121,
Linden, New Jersey 07036
(201) 474-3303
Frank Hochgesang
Environmental Analytical Coordinator
Mobil Research S Development Corp.
Bill ingsport Rd.
Paulsboro, New Jersey 08066
(609) 423-1040 X2479
Robert F. Sabcock, Research Chemist
Standard Oil Co. (Ind.)
P.O. Box 400
Naperville, Illinois 60540
(312) 420-5229
R. 0. Kagel, Senior Research Specialist
Dow Chemical Co.
574 Bldg.
Midland, Michigan 48640
(517) 636-2953
R. M. Dille, Supervisor
Texaco Inc.
P.O. 1108
Port Arthur, Tx
(713) 982-5711
Dr. R. F. Stubbeman, Section Leader
Celanese Chemical
P.O. Box 9077
Corpus Christi, Texas 78408
(512) 241-2343
-------
P.A. Wadsworth, Staff Research Physicist
Shell Development Co.
P.O. Box 1380
Houston, Texas 77001
(713) 493-7723
James E. Norn's, Group Leader
Analytical Environmental Technology Dept.
CIBA - Geigy Corp.
P.O. Box 113,
Mclntosh, AL 36553
(205) 944-2201
Judith Thatcher, Sr. Environmental Assoc.
American Petroleum Institute
2101 L St. N.W.
(202) 457-7079
Max Lazar, Manager
Quality Control
Hoffman - LaRoche (Representing PMA)
P.O. Box 238
Belvidere, New Jersey
(201) 475-5381
Gary D. Raw!ings, Sr. Research Engineer
Monsanto Research Corp.
1515 Nicholas Rd.
Dayton, Ohio
(513) 268-3411
William G. Krochta, Sr. Supervisor Analytical
PPG Industries
Box 31
Barberton, OH 44203
753-4561
Will M. Oil 1 son, Staff Chemist
API (2101 L St., N.W.)
Washington, D.C. 20037
(202) 457-73757 333-7711
George Stanko, Sr. Research Chemist
Shell Development Co. (Also MCA & API Rep.)
Box 1380
Houston, Texas 77001
(713) 493-7702
Charles P. Hensley, Chemist
EPA Region VII
25 Funston Rd.
Kansas City, Kansas 66115
(816) 374-42857 FTS: 758-4285
-------
Will 1am F. lully, Project Scientist
U.C.C.
So. Charleston, West Virginia
(304) 747-4755
Bob Fisher, Research Chemist
National Council Paper Industry
3434
Gainesville, Florida 32608
John W. Way, Research Supervisor
E. I. Dupont DeNemours & Co.
Industrial Chemistry Oept.
Experimental Station
81dg. 336
Wilmington, Delaware 19895
(302) 772-4376
Roger 0. Holm, Group Leader
Waste Water Effluents
Monsanto Research
1515 Nicholas Rd.
(513) 268-3411 X354, 385
Paul X. Riccobono, Manager
Materials Evaluation
J.P. Stevens
Garfield, New Jersey
Janine Neils, Manager
Laboratory Service
MRI/Northside Div.
10701 Red Circle Drive
Minnetou
(612) 933-7880
Clarence L. Haile, Program Manager
for Mass Spec
Midwest Research Institute
425 Volke Blvd.
Kansas City, Mo 64110
(816) 753-7600
M.L. (Bud) Moberg, President
Analytical Research Labs Inc.
160 Taylor St.
Monrovia, California 91016
(213) 357-3247
Robert Z. Muggli, Sr. Research Chemist
W.C. McCrone Associates
2820 S. Michigan Avenue
Chicago, Illinois 60616
(312) 842-7100
-------
Roderick A. Carr, Sr. Project Manager
Versar, Inc.
6621 Electronic Drive
Springfield, Va. 22151
(703) 750-3000
Richard Kearns, Manager
Field Sampling Operations
Hamilton Standard
Airport Rd.
Windsor Locks, Ct. 06096
(203) 623-1621 ext. 8868
H. V. Myers, Consulting Engineer
NUS Corooration
Manor Oak Two,
1910 Cochran Rd.,
Pittsburgh, Pennsylvania 15220
(412) 343-9200
Jack R. Hall, Manager
Analytical Services
Hydroscience
9041 Executive Park Drive
Knoxville, Tenn. 37919
(615) 690-3211
Linda B. Kay
Environmental Scientist
Versar, Inc.
6621 Electronic Dr.
Springfield, Va.
(703) 750-3000
Jay L. Crane, Project Manager
Jacobs Engineering
251 So. Lake Ave.,
Pasadena, California 91101
(213) 449-2171
Bonnie Parrott, Environmental Engineer
Jacobs Engineering Co.
251 So. Lake
Pasadena, California 91101
(213) 449-2171
H. Dwight Fisher
V.P. Technical Director
West Coast technical Service, Inc.
17605 Fabrica Way
Cerritos, Ca. 90701
Jack Northington, Asst. Technical Director -> / &
West Coast Technical Director
17605 Fabrica Wav. Suit.p n
-------
10
Jim Spigarelli, Associate Director
for Analytical Chemistry
Midwest Research Institute
425 Volker Blvd.
Kansas City, Mo 64110
(816) 753-7600
Authur J. Condren, Manager
Analytical Services
E. C. Jordan Co.
PP.0. Box 7050,
Downtown Station,
Portland, Maine 04112
(207) 775-5401
Donald M. Shilesky, Project Manager
SCS Engineer
11800 Sunrise Valley Dr., Suite 432
(703) 620-3677
John H. Taylor, Laboratory Director
Jacobs Engineering Co.
660 S. Fair Oaks,
Pasadena, California 91105
(213) 795-7553
Charlie Westerman, Sr. Chemist
Environmental Science & Engineering, Inc.
P.O. Box 13454
Gainesville, Florida 32604
(904) 372-3318
Paul A. Taylor, President
California Analytical Lab., Inc.
401 N. 16th St.
Sacramento, California
(916) 444-9602
David D. Conway, Supervisor
Conservation Section
Marathon Oil Co.
P.O. Box 269
Littleton, Colorado
(303) 794-2601
Robert D. Kleopfer, Chief
EPA, Region VII
25 Funston Rd.,
Kansas City, Kansas 66115
(816) 374-428S/ FTS: 758-4285
-------
11
Charles W. Amelotti
Sverdrup & Parcel and Associates
800 N. 12th Blvd
St. Louis, Mo 63101
(314) 436-7600
Stuart A. Whitlock, Manager
Organic Chemistry Group
Environmental Science & Eng. Inc.
P.O. 13454
University Station
Gainesville, Florida
((904) 372-3318
Kendall B. Randolph, Chemical Engineer
Versar, Inc.
6621 Electronic Dr.,
Springfield, Va.
750-3000
D.R. Rushneck
PJ8 Labs
Pasadena, California 91101
Rick Johnston, AA Specialist
Edward H. Richardson Associates
P.O. Box 935
Dover, Delaware 19901
(302) 697-2183
Donald R. Wilkinson, Ph.D.
Director of Organic Analyses
Edward H. Richardson Associates
Dover, Delaware 19901
(302) 697-2183
Ronald G. Oldhan, Sr. Staff Scientist
Radian Corp.
P.O. Box 9948
Austin, Texas 78756
(512) 454-4797
Larry Keith, Head
Organic Chemistry Department
Radian Corporation
P.O. Box 9948
Austin, Texas 78766
(512) 45404797
James K. Rice, Consulting Engineer
Utility Water Act Group
17415 Batchellors Forest Rd.
Olney, Maryland 20832 ^ n O
(301) 774-2210 * '
-------
James Ryan, Manager
Gulf South Research Institute
P.O. Box 70186
(504) 283-4223
7
-------
r-
U.S. Environmenta! Protection Agency
Region V, Library
230 South Dearborn Street
Chicago. Illinois 60604
-------