-------
s r
- 9'? - &-H
3
.
B-157
-------
L,
*
s0
f
--75-00
fi-158
-------
PRESURVEY DATA SHEETS
I. NAME OF COMPANY,
ADDRESS
NAME OF CONTACTS
DATE OF
SUMMARY
PHONE
MRC PERSONNEL
EPA PERSONNEL
STATE PERSONNEL
INDUSTRY TYPE
PHONE
PHONE
PHONE
PHONE
PHONE
PHONE
l
PORTION OF PROCESS TO BE SAMPLED
OCn
v /
II. PROCESS DESCRIPTION
B-159 ..
-------
II. Con't.
Raw materials and amount* _
Fuels C^Qfll Atlh Of I -FnjZ
Products and amounts
Operating Cycle:
Check: Batch y Continuous \J Cyclic
* ,_ v , /n£TAt£ T&*rw*J*r <3*S?*f is
Timing of batch or cycle /ggfggTb *&**
Best time to sample
Length of Operating day g^T hit
Length of operating week / #n
Scheduled shutdowns
Other
III. WASTEWATER TREATMENT PLANT DESCRIPTION:
A
Chemicals added and amounts
Handles rainfall runoff?
To Cfrffatf £>fp
Includes sanitary waste, flow //Q
Source of plant intake water O/Q/T\f S
Hydraulic retention tiroe:^ Thru plant
Thru treatment
unit operations _____
Recent treatment plant performance T^ffy "/T
B-160
-------
III. Con't.
MPDES permit parameters and*limits
Final effluent flow rate
vfe
List of potential pollutants
(Lappe* ."T>Q*/
/) rj ' ' r\ .
/ Aff/Miitm , i,hygTn>/
Recent analyses available?
Sampling point description
Qo
' Use automatic sampler?
? >
Electricity available
Extension cord and type of outlet?
IV. Safety Checklist
A. Personnel Protection Equipment (check If required)
Item
Safety glasses
Goggles
Side shields
Face shields
Hard hats
Ear plugs
Safety shoes
Life belt
Ladder climbing
device
Plant
MRC
Item
Dust masks
Vapor masks
Air purifying
Air supply
Air packs
Chem. res't clothes
Heat res't clothes
Chem. res't gloves
Heat res't gloves
First aid
Plant
MRC
B-161
-------
B. SAMPLE SITE
.1. Smoking restrictions
2. Vehicle traffic rules
- Possible set-up/clean-up facilities?
. -Evacuation procedures
5. Alarms
6. -Hospital location
7. Hospital Phone
Emergency Numbers
V.. Plant Entry
-£.£>.
A. Plant Requirements
Special time constraints:
B. MRC Agreement
C. Potential Problems
B-162
-------
VI. SAMPLING HANDLING
A. Ice availability
B. Sample splitting requested
Describe
C. Nearest airport;
D. Chemical available: H2S04
HN03 _
NaOH
B-163
-------
VIL, Field Test Schedule
Time
Day
AM
PM
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
B-164
-------
PRESURVEY DATA SHEETS
I. NAME OF COMPANY
ADDRESS
NAME OF CONTACTS
DATE'OF
SUMMARY
PHONE
MRC PERSONNEL
EPA PERSONNEL
STATE PERSONNEL
INDUSTRY TYPE
PHONE
PHONE
PHONE
PHONE
PHONE
PHONE
PORTION OF PROCESS TO BE SAMPLED
\\ 001
II. PROCESS DESCRIPTION
B-165
-------
II. Con
Raw materials and amounts
Fuels
Products 'and amount£
Operating Cycle:
Check: Batch
Continuous
Timing of batch or cycle
Best time to sample oS°o
Length of Operating day
Length of operating week
Scheduled shutdowns
Other
17
W
Cyclic
III. WASTEWATER TREATMENT PLANT DESCRIPTION:
TO
Chemicals added and amounts
Handles rainfall runoff?"
Includes sanitary waste, flow
Source of plant intake water
Hydraulic retention time:. Thru plant N6
Thru treatment
unit operations
Recent treatment plant performance
B-166
-------
III. Con't.
NPDES permit parameters and" limits Qj'
Final effluent -flow rate
List of potential pollutants
Recent analyses available?
Sampling point description • 3)\SCLW\g££.
' Use automatic sampler?
&Rftn
Electricity available
i £
Extension cord and type of outlet?
) 00"fr.
IV. Safety Checklist
A. Personnel Protection Equipment (check if required)
Item
Safety glasses
Goggles
Side shields
Face shields
Hard hats
Ear plugs
Safety shoes
Life belt
Ladder climbing
device
Plant
MRC
Item
Dust masks
Vapor masks
Air purifying
Air supply
Air packs
Chem. res't clothes
Heat res't clothes
Chem. res't gloves
Heat res't gloves
First aid
Plant
MRC
B-167
-------
B. SAMPLE SITE
1. Smoking restrictions
2. Vehicle traffic rules
Possible set-up/clean-up facilities?_
Evacuation procedures
5. Alarms
6. Hospital location
7. Hospital Phone
Emergency Numbers
V.. Plant Entry
A. Plant Requirements X.^X C.Atj)
Special time constraints:
B. MRC Agreement
C. Potential Problems
B-168
-------
VI. SAMPLING HANDLING
A. Ice availability
B. Sample splitting requested
Describe
C. Nearest airport;
D. Chemical available: HjSO
HN03
NaOH
B-169
-------
VIL. Field Test Schedule
Time
Day
AM
PM
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
B-170
-------
PRESURVEY DATA SHEETS
I. NAME OF COMPANY_
ADDRESS
NAME OF CONTACTS
DATE OF
DATE OF y /
SUMMARY S/12/gf
PHONE
MRC PERSONNEL
EPA PERSONNEL
STATE PERSONNEL
INDUSTRY TYPE
PHONE
PHONE
PHONE
PHONE
PHONE
PHONE
PORTION OF PROCESS TO BE SAMPLED
00 \ 5loRlt\
II. PROCESS DESCRIPTION
^, S.lAPKv.pic. ftc
U
To
J.^ Ac
B-171
-------
II. Con't.
Raw materials and amounts K//^ • ____; • _/")
Fuels
Products and amounts M/fi O
Operating Cycle:
Check: Batch Continuous Cyclic_
Timing of batch or cycle .''"- '"' f ^ • ' - - • -
Best time to sample
Length of Operating day Q^QO fT
Length of operating week > J> fi ^
Scheduled shutdowns
Other
III. WASTEWATER TREATMENT PLANT DESCRIPTION; jj Qfj — CONT7\Cl^
Chemicals added and amounts
Handles rainfall runoff?
Includes sanitary waste, flow
Source of plant intake water
Hydraulic retention time:. Thru plant (LQI^Tl>JVvQV.5
Thru treatment
unit operations
Recent treatment plant performance
B-172
-------
III. Con't.
NPDES permit parameters and' limits
Final effluent flow rate
List of potential pollutants
OK;!
Recent analyses available?
Sampling point description
1 Use automatic sampler?
Electricity available
Extension cord and type of outlet?
IV. Safety Checklist
A. Personnel Protection Equipment (check if required)
Item
Safety glasses
Goggles
Side shields
Face shields
Hard hats
Ear plugs
Safety shoes
Life belt
Ladder climbing
device
Plant
MBC
/
^ /
Item
Dust masks
Vapor masks
Air purifying
Air supply
Air packs
Chem. res't clothe:
Heat res't clothes
Chem. res't gloves
Heat res't gloves
First aid
Plant
MRC
B-173
-------
B. SAMPLE SITE
1. Smoking restrictions
2. Vehicle traffic rules
3- Possible set-up/clean-up facilities?^
4. Evacuation procedures
5. Alarms
6. Hospital location
7. Hospital Phone
ljf/in\ploy.
Emergency Numbers
yVbV"H>iK QT°
V.; Plant Entry
A. P-lant Requirements
Special time constraints: "^/7|e V"VW~T
•
v£{- tVoc^Ss./.e,
j
B. MRC Agreement
C. Potential Problems f^/ONH
4
B-174
-------
VI. SAMPLING HANDLING
A. Ice availability
B. Sample splitting requested
Describe
C. Nearest airport:
D. Chemical available: H-SO.
HN03
NaOH
B-175
-------
VIL, Field Test Schedule
\Tlme
Day ^v
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
AM
-
PM
X
-
B-176
-------
PRESURVEY DATA SHEETS
I. NAME OF COMPANY_ * _ SUMMARY
ADDRESS PHONE
NAME OF CONTACTS
MRC PERSONNEL PHONE
PHONE
EPA PERSONNEL PHONE_
PHONE_
STATE PERSONNEL PHONE_
PHONE
INDUSTRY TYPE ( ^(\\^t\Ot\) ^PiJAfrrg. \t^
PORTION OF PROCESS TO BE SAMPLED EffJUfvy niLTTftH 00 \
II. PROCESS DESCRIPTION
B-177
-------
XX. Conft.
Raw materials and amounts
Fuels
Products and amounts ty / ^
/
Operating Cycle:
Y
Check: Batch _ Continuous Y _ Cyclic_
Timing of batch or cycle
Best time to sample O&&O~fe /7
-------
III. Con't.
NPDES permit parameters and limits
Final effluent flow rate 10
List of potential pollutants
.O
Recent analyses available?^
Jp
Sampling point description
' Use automatic sampler?
110
Electricity available
Extension cord and type of outlet?^
IV. Safety Checklist
A. Personnel Protection Equipment (check If required)
Item
Safety glasses
Goggles
Side shields
Face shields
Hard hats
Ear plugs
Safety shoes
Life belt
Ladder climbing
device
Plant
MRC
Item
Dust masks
Vapor masks
Air purifying
Air supply
Air packs
Chem. res't clothes
Heat res't clothes
Chem. res't gloves
Heat res't gloves
First aid
Plant
MRC
B-179
-------
B. SAMPLE SITE
1. Smoking restrictions
2. Vehicle traffic rules
3- Possible set-up/clean-up facilities?
4. Evacuation procedures
5. Alarms
6. Hospital location /7lf&/ fA / LOfff°^ O-f
-^
7. Hospital Phone
Emergency Numbers
V.. Plant Entry
A. Plant Requirements
Special time constraints;
B. MRC Agreement
C. Potential Problems
B-180
-------
VI. SAMPLING HANDLING
A. Ice availability
B. Sample splitting requested
Describe
C. Nearest airport;
jfS
D. Chemical available: H-SO,
HN03
NaOH
B-181
-------
VIL, Field Test Schedule
X^Tiroe
Day^s^
Sunday
Monday
Tuesday
•
Wednesday
Thursday
Friday
Saturday
AM
PM
-
B-182
-------
PRESURVEY DATA SHEETS
I. NAME OF COMPANY
ADDRESS
NAME OF CONTACTS
DATE OF
SUMMARY
PHONE
MRC PERSONNEL
EPA PERSONNEL
STATE PERSONNEL
PHONE
PHONE
PHONE
PHONE
PHONE
PHONE
PORTION OF PROCESS TO BE SAMPLED \J\
00 (
II. PROCESS DESCRIPTION
B-183
-------
XI. Con't.
Raw materials and amounts ¥£00
/
Fuels
Z/JM&ftftfi
Products and amounts
Operating Cycle:
Check: Batch
G? R)$l? ffattlgft / (, /
Continuous
- /
y
Cyclic '
Timing of batch or cycle
Best time to sample
Length of Operating day
Length of operating week
Scheduled shutdowns
Other
ZZZ. WASTEWATER TREATMENT PLANT DESCRIPTION
ksft
Chemicals added and amounts (ln/4 Yififc,
Handles rainfall runoff?
75%
Includes sanitary waste, flow
Source of plant intake water
Hydraulic retention time: Thru plant
Thru treatment
unit operations
~Tb
Recent treatment plant performance
B-184
-------
III. Con't.
NPDES permit parameters and limits'
i ^ *
Final effluent flow rate
List of potential pollutants
Recent analyses available?
Sampling point description
Use automatic sampler?
GRAB
Electricity available
YES
Extension cord and type of outlet?
//0 V /vt?
IV. Safety Checklist
A. Personnel Protection Equipment (check if required)
Item
Safety glasses
Goggles
Side shields
Face shields
Hard hats
Ear plugs
Safety shoes
Life belt
Ladder climbing
device
Plant
KRC
Item
Dust masks
Vapor masks
Air purifying
Air supply
Air packs
Chem. res't clothe:
Heat res't clothes
Chem. res't gloves
Heat res't gloves
First aid
Plant
MRC
B-185
-------
B. SAMPLE SITE
1. Smoking restrictions
2. Vehicle traffic rules
3- Possible set-up/clean-up facilities?
4. Evacuation procedures
5. Alarms
6. Hospital location
7. Hospital Phone
Emergency Numbers
V.; Plant Entry
A. Plant Requirements
Special time constraints;
B. MRC Agreement
C. Potential Problems /vo>/t
B-186
-------
VI. SAMPLING HANDLING
A. Ice availability
B. Sample splitting requested
Describe
C. Nearest airport:
D. Chemical available: HjSO
HN03
NaOH
B-187
-------
VIL, Field Test Schedule
\Time
Day^v
Sunday
Monday
Tuesday
•
Wednesday
Thursday
Friday
Saturday
AN
PM
B-188
-------
PRESURVEY DATA SHEETS
I. NAME OF COMPANY_
ADDRESS
NAME OF CONTACTS
DATE OF
SUMMARY
PHONE
MRC PERSONNEL
EPA PERSONNEL
STATE PERSONNEL
INDUSTRY TYPE
T"KW\
0
PHONE
PHONE
PHONE
PHONE
PHONE
PHONE
\
PORTION OF PROCESS TO BE SAMPLED
00 \
II. PROCESS DESCRIPTION
B-189
-------
II. Con't.
III.
Raw materials and amounts
Fuels
Products and amounts
Operating Cycle:
Check: Batch
ItyfflLfflL
Timing of batch or cycle
Continuous
NA
Cyclic
Best time to sample OYOO — 1
Length of Operating day
Length of operating week
Scheduled shutdowns
Other
r ft
>S
WASTEWATER TREATMENT PLANT DESCRIPTION
: Coo] IN
a
*
vr.,
Chemicals added and amounts
Handles rainfall runoff?
Includes sanitary waste, flow
Source of plant intake water
Hydraulic retention time:. Thru plant
Thru treatment
unit operations
Recent treatment 'plant performance
B-190
-------
III. Con't.
NPDES permit par
'o
Final effluent flow rate
List of potential pollutants
ters and' limits
Recent analyses available?
Sampling point description
' Use automatic sampler?
Electricity available
Ye*
Extension cord and type of outlet?
IV. Safety Checklist
A. Personnel Protection Equipment (check If required)
Item
Safety glasses
Goggles
Side shields
Face shields
Hard hats
Ear plugs
Safety shoes
Life belt
Ladder climbing
device
Plant
MRC
Item
Dust masks
Vapor masks
Air purifying
Air supply
Air packs
Chem. res't clothes
Heat res't clothes
Chem. res't gloves
Heat res't gloves
First aid
Plant
MRC
B-191
-------
B. SAMPLE SITE
1. Smoking restrictions ^
2. Vehicle traffic rules /Wv kAty." 7£> 77
Possible set-up/clean-up facilities? N
Evacuation procedures
5. Alarms
6. Hospital location
7. Hospital Phone
Emergency Numbers
V., Plant Entry
A. Plant Requirements J» \J
Special time constraints;
B. MRC Agreement^
C. Potential Problems
B-192
-------
VI. SAMPLING HANDLING
A. Ice availability
B. Sample splitting requested
Describe
C. Nearest airport;
D. Chemical available: H-SO,
HN03 _
NaOH
B-193
^
-------
VII. Field Test Schedule
Time
Day
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
PM
B-194
-------
PRESURVEY DATA SHEETS
I. NAME OP COMPANY
ADDRESS
DATE OF
SUMMARY
PHONE
NAME OF CONTACTS
MRC PERSONNEL
EPA PERSONNEL
STATE PERSONNEL
INDUSTRY TYPE
PORTION OF PROCESS TO BE SAMPLED
r r
II. PROCESS DESCRIPTION
PHONE
PHONE
PHONE
PHONE
PHONE
J£
V
^^'i I C/t^
J
B-195
-------
II. Con't.
Raw materials and amounts CsJ Ac^^ <2~i& ^zg^L &J*Z 5"«//, S
-------
III. Con't.
NPDES permit parameters and limits
Final effluent flow rate
List of potential pollutants -j2^e
Recent analyses available?
Sampling point description
n^.S2£ £
' Use automatic sampler?
Electricity available
f //.y 1/J
Extension cord and type of outlet?
IV. Safety Checklist
A. Personnel Protection Equipment (check if required)
Item
Safety glasses
Goggles
Side shields
Face shields
Hard hats
Ear plugs
Safety shoes
Life belt
Ladder climbing
device
Plant
^*
/*
MRC
/
y
/
Item
Dust masks
Vapor masks
Air purifying
Air supply
Air packs
Chem. res't clothes
Heat res't clothes
Chem. resrt gloves
Heat res't gloves
First aid
Plant
MRC
B-197
-------
B. SAMPLE SITE
1. Smoking restrictions
3* Possible set-up/clean-up facilities?
4. Evacuation procedures (Zr-**^ st*r££f £e
5. Alarms
6. Hospital location
7. Hospital Phone
Emergency Numbers
V.. Plant Entry
A. Plant Requirements t^Z-g-^ &£* <
Special time constraints;
B. MRC Agreement
C. Potential Problems
B-198
-------
VI. SAMPLING HANDLING
A. Ice availability
B. Sample splitting requested
yi
Describe
C. Nearest airportt
D. Chemical available:
HN03
NaOH
B-199
-------
fit Field Te
\Time
Day X^
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
st Schedule c^^^"^^^-^
\
AM
^
PM
B-200
-------
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B-202
-------
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B-203
-------
B-204
-------
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B-205
-------
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B-206
-------
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B-207
-------
f
U.S
-------
PR£SURVEY DATA SHEETS
X. NAME OF COMPANY^
ADDRESS
„ ,_
/5/T 3
DATE OF
SUMMARY
PHONE
NAME OF CONTACTS
MRC PERSONNEL
EPA PERSONNEL
STATE PERSONNEL
INDUSTRY TYPE T,
PORTION OF PROCESS TO BE SAMPLED
PHONE
PHONE
PHONE
PHONE
PHONE
PHONE
QQb/O
II. PROCESS DESCRIPTION
B-209
-------
CONFIDENTIAL
CONFIDENTIA!
I
Raw materials and amount* Vie (jron>p)f »/? fo,/ 4x?
Fuels
Product* and amount* T.t>i(
3oe>oo T/*/^ C i
^ ^ r ,
Operating Cycle:
Check: Batch
COMPANY CONFIDENTIAL
Continuous C I
Cyclic
Timing of batch or cycle
Best time to sample
Length of Operating day
Length of operating week
Scheduled shutdowns _£/
—I
Other
III. WASTEWATER TREATMENT PLANT DESCRIPTION;
Chemical* added and amount*
Handle* rainfall runoff?
Includes sanitary waste, flow
Source of plant intake water
Hydraulic retention time: Thru plant
Thru treatment
unit operation* C/
Recent treatment plant performance
B-210
-------
III. Con't.
NPDES permit parameters and limits TS*fV33o */*&..
Final effluent flow rate J"" r xfr
List of potential pollutants
Recent analyses available?
Sampling point description
_/x
/
£L
A/
Use automatic sampler?
Electricity available
Extension cord and type of outlet?
IV. Safety Checklist
A. Personnel Protection Equipment (check If required)
Item
Safety glasses
Goggles
Side shields
Pace shields
Hard hats
Ear plugs
Safety shoes
Life belt
Ladder climbing
device
Plant
MRC
/
Item
Dust masks
Vapor masks*
Air purifying
Air supply
Air packs
Chem. res't clothes
Heat res't clothes
Chem. res't gloves
Heat res't gloves
First aid
Plant
MRC
IX
B-211
-------
B. SAMPLE SITE
1. Smoking restrictions
2. Vehicle traffic rules
3- Possible set-up/clean-up facilities?
4. Evacuation procedures
5. Alarms
6. Hospital location Q
7. Hospital Phone
Emergency Numbers
-------
VI. SAMPLING HANDLING
A. Ice availability
B. Sample splitting requested
Describe
r
C. Nearest airportt
D. Chemical available: H.SOj
BN03 m
NaOH
B-213
-------
VIL Field Test Schedule
Time
Day
AN
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
B-214
-------
7",
-------
/ /
t*
7^7^
fl* A^&ty&a J €+<*£- jt*ud*K&fT* ****£*& e*
4*^lu*&J jCtMfre£&«*0 ttrit^f C+~CJ? &&
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B-216
-------
B-217
-------
B-218
-------
T
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B-219
-------
U.S.
' 4* rfSSeSS''**'* 1* £>f +/*. ~/9ff,iY/9?+ x» 7* ^^^^7^0 S
B-220
-------
u
f
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C^.
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B-221
-------
C^€^t
-------
I.
SHSURVEY 9ATA SHEETS
NAME .;;' C
MRC PERSONNEL
EPA PERSONNEL
STATE PERSONNEL
INDUSTRY TYPE
•*£&*£
s
DATE
SUMMARY
PHONE
JPHONE_
_PHONE_
_PHONE_
_PHONE_
_PHONE_
PHONE
( S I
PORTION OF PROCESS TO BE SAMPLED
£>O /-
XI. PROCESS DESCRIPTION
B-223
-------
II. Con't.
Raw materials *ina tut.ounc*
Fuels
Products and aipounfr.s
• •:> : i \ :
Operating Cycle": jA:t*iir5>
5V ••* / -
Check: Batch • Continuous tX" Cyclic
Timing of batch or cycle
Best time to sample
/
Length of Operating day ^^/^^^n^i^j
Length of operating week
J
Scheduled shutdowns £>/'?'?— *~Z//*/
Other
III. WASTEWATER TREATMENT PLANT DESCRIPTION;
sLs> Jrs/
Chemicals added and amounts
Handles rainfall runoff?
/^
Includes sanitary waste, flow
Source of plant intake water
Hydraulic retention time: Thru plant
Thru treatment .
unit operations //. A.
Recent treatment plant performance y(/ /^
B-224
-------
III. Con't.
NPDES permit
Final effluent fie?
List of potential
'-' * . ^:-.,-jf. -f Sfi£.\.C'.%£&&
Recent analyses aval lab le?_
Sampling point description
' Use automatic sampler?
Electricity available
Extension cord and type of outlet?
IV. Safety Checklist
A. Personnel Protection Equipment (check If required)
Item
Safety glasses
Goggles
Side shields
Face shields
Hard hats
Ear plugs
Safety shoes
Life belt
Ladder climbing
device
Plant
MRC
Item
Dust masks
Vapor masks
Air purifying
Air supply
Air packs
Chem. res't clothes
Heat res't clothes
Chem. res't gloves
Heat res't gloves
First aid
Plant
MRC
B-225
-------
B. SAMPLE SITE
1.^, Smoking restrictions _ '"'
2. Vehicle traffic rules
Possible set-up/clean-up facilities?
Evacuation procedures
5. Alarms
6. Hospital location
7. Hospital Phone
Emergency Numbers ILe*.
V.. Plant Entry
A. Plant Requirements
Special time constraints
B. MRC Agreement
C. Potential Problems
B-226
-------
VI. SAMPLING HANDLING
A. Ice availability
B. Sample splitting requested
Describe X e^.jf ^T
C. Nearest airport:
D. Chemical available:
HN03
NaOH
B-227
-------
VIL Field Test Schedule
m.
^v Time
Day >^
Sunday
Monday
Tuesday
•
Wednesday
Thursday
Friday
Saturday
AH
•V
PM
B-228
-------
*d&&>C'£**~
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B-230
-------
B-231
-------
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B-232
-------
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B-233
-------
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B-234
-------
«' C
of
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B-235
-------
APPENDIX C
PHASE III SAMPLING AND ANALYTICAL METHODS
C.I INTRODUCTION
The chemical analysis scheme implemented in Phase III was designed
to collect sufficient data to screen 28 effluent samples and 22
sediment samples for the presence of chemical species known or
suspected to be present, and to identify as many of the other
compounds as possible within the time and economic constraints.
Samples were collected in Maryland and Virginia; in Maryland 8
effluent and 5 sediment samples were taken, and in Virginia 20
effluent and 17 sediment samples were taken.
The objectives of the Phase III chemical and physical analysis
scheme were as follows:
1. Quantitative analysis of NPDES parameters, anions and metals;
2. Semiquantitative analysis of organic compounds known or
suspected to be present in the samples (based on an engi-
neering evaluation of the plant production processes),
identified as being potentially toxic;
3. Qualitative analysis of other organic compounds suspected to
be present in the sample, but not particularly toxic;
4. Qualitative analysis of other unknown organic compounds ,
detected in the sample by gas chromatography/mass spec-
trometry (GC/MS);
C.l-1
-------
5. Determination of the potential for organic compounds in the
samples to accumulate in the food chain; and
6. Presentation of the data in a format consistent with-other
Bay Program studies.
This appendix contains a detailed description of the methods
employed and the purpose and goal of each test. Examples are in-
cluded to brief the reader on the thought process involved in
analyzing the data.
C.l-2
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C.2 FIELD SAMPLING METHODLOGY
Sampling was designed to collect sufficient water to determine if
the data generated by the chemical and physical analysis protocol
were sufficient to measure the type and amount of pollutants being
discharged at the site. Sampling was conducted in Maryland by MRC
personnel with the help of Maryland Department of Health person-
nel. Typically, sampling was performed by a two-man crew, start-
ing at mid-morning and lasting about three hours. In Virginia,
effluents and sediments were sampled by State Water Control Board
personnel.
Table C.2-1 presents a field sampling logistics checklist that
was used by the sampling crews. Listings include analyses to be
conducted, volume required, type of container, preservative used,
and analysis laboratory. This chicklist (filled out prior to the
site visit) was used to organize the crew during sample splitting
and packing and details the final destination of each bottle of
effluent.
C.2.1 Maryland Sites
In order to minimize sample contamination, grab samples were col-
lected in Teflon®-lined buckets and transferred to one of two
compositing containers. One sample was collected and placed in a
190-liter (50-gallon) plastic container. Aliguots were removed
from this container for analysis of NPDES parameters and inorganic
species. The second sample was collected and placed in a 19-liter
(5-gallon) glass container. Aliguots were removed from this con-
tainer for analyses of all organic compounds. By segregating the
samples in different types of containers, contamination from the
collection vessels, such as plasticizers or leached metals, was
avoided. The glass vessel was packed in ice during the sampling
period to reduce the possibility of loss of volatile compounds
and biodegradation.
C.2-1
-------
TABLE C.2-1. FIELD SAMPLING LOGISTICS CHECKLIST FOR PHASE III PLANTS
PtJ\NT CODE
SAMPLING TRAM
Sampling
required
at site
Analysis
PH
Flow
Filtration
Volume required
100 ml,
ISO ml
Container
Beaker
Filter apparatus
Preservative
None
Ship to
Analyze on site
Determine at site
Perform on site
o
•
10
to
Plant spill potential
Fish/Daphnia
Algae (freshwater)
Sheepshead/mysid/oyster larvae
Algae (marine)
HERL/RTP
Battelle, Columbus
Battelle, Duxbury
Annapolis - AFO will supply
prepreserved bottles for
NPDES and anion analysis
Filtered ICAP metals
Unfiltered ICAP metals
Filtered Hg analysis
Unfiltered Hg analysis
Volatile organics
Nonvolatile organics
Extra sanple/bioaccumulation
Bioassay (Ames/CHO)
TOC
Special analysis
Direct water injectables No
Aldehyde analysis
Nitrogen-phosphorus
detector FID/GC No
Derivatization NO
Sulfur analysis
Inorganics
25 gal
5 gal
25 gal
S gal
1 gal
15 gal
S gal
50 mL
50 mL
100 mL
100 mL
80 mL
3 gal
1 gal
3 gal
500 mL
separate sample required
1 gal
separate sample required
separate sample required
2 SO mL
1 L
5 5-qal c'ubitainers
1 5-gal cubitainers
5 5-gal cubitainers
1 5-gal cubitainers
1 1-gal glass
3 5-gal cubitainers
1 5-gal cubitainer
Plastic
Plastic
Plastic
Plastic
2 40-mL glass vials
3 1-gal glass
1 1-gal glass
3 1-gal glass
Glass
1 1-gal glass
Plastic
Plastic
4*C
4«C
4«C
4«C
4"C
4'C
4*C
4'C, S mL UNO,
4*C, S mL HNOj
4°C, S mL HNOj
4°C, 5 mL HNOj
4«C
4«C
4«C
4«C
HaSO«, pH<2
4»C
Determine at aite
EG&G - Hareham
EGSO - Pensacola
EG&G - Pensacola
EG&G - Pensacola
Shabeg Sandhu-EPA-HERL/RTP
Columbus, Ohio
Duxbury, Mass.
Annapolis Field Office
Annapolis Field Office
Annapolis Field Office
Annapolis Field Office
Annapolis Field Office
Monsanto Research Corporation
Monsanto Research Corporation
Monsanto Research Corporation
Monsanto Research Corporation
Monsanto Research Corporation
4°C, 1% sodium bisulfite Monsanto Research Corporation
4«C
4«C
Monsanto Research Corporation
Monsanto Research Corporation
-------
Grab samples for purgeable organics analysis were taken by col-
lecting a sample in a Teflon®-lined bucket and then filling the
40-mL vial by completely immersing it in the bucket. These sam-
ples were collected at the beginning and ending of the sampling
period. Samples were hermetically sealed immediately after sam-
pling, then labeled and stored at 4°C until shipment. The vials
were shipped in ice to maintain this temperature.
After the sampling period was completed, the crew thoroughly
mixed the sample in the composite vessels with a Teflon®-coated
rod and then divided the effluent into appropriate bottles. Pre-
servatives were added to the bottles when needed to maintain sam-
ple integrity. Samples were carefully labeled with the type of
analysis to be run, the plant code, and the name of the analytical
laboratory that was to perform the analysis. Samples were then
packed in ice for shipment. Once packing was complete, samples
were shipped that day by air freight to the appropriate labora-
tory and were normally delivered in less than 24 hours.
In addition to the samples taken, other pertinent information was
collected. Discussions with plant representatives gave the team
leader an indication of the treatment operation for the day
(i.e., upset or normal operation). Temperature and other weather
factors that may have affected the samples or sampling procedures
were also noted. Flow measurements were requested from the plant
in order to determine the total discharge into the Chesapeake Bay
basin. All significant sampling procedure deviations were also
noted.
C.2.2 Virginia Sites
During the month of April 1981 effluent from 20 plants discharg-
ing into the James and Elizabeth Rivers were sampled by Virginia
State Water Control Board (SWCB) personnel. At each plant a
carefully cleaned 110 gallon linear polyethylene tank was rinsed
C.2-3
-------
with the effluent and then filled using a submersible pump with
nonreactive fittings. Aliquots of effluent samples used for
chemical analyses were taken from the tank in the field. The
remaining effluent was transported back to the SWCB facility for
fish bioassays and Microtox® tests. When no fish bioassays were
performed, a 13-gallon linear polyethylene container was filled
with effluent and samples were taken from this container. SWCB
personnel took dissolved oxygen, pH, temperature, and conductivity/
salinity readings at each plant, and the instantaneous flow rate
was obtained from the plant recorder. Plant operators were also
queried as to any current treatment problems at their facility.
All clean sample containers were rinsed three times with efflu-
ent prior to filling, with the exception of the filtered metals
and oil and grease containers. The samples taken at each plant
were as follows:
1. Four 1-gallon amber glass bottles for nonvolatile
organics.
2. Two 40-mL glass vials with Teflon septa for volatile
organics—these vials were filled while submerged in
effluent in a stainless steel bucket to prevent air
bubbles.
3. One 500-mL amber glass bottle for TOC—fixed with
H2SO4 such that the pH was less than 2.
4. Two 125-mL wide-mouth plastic bottles, one for ion
chromatographic analysis and one for total sulfur.
5. One 1-gallon cubitainer for BOD, TSS, NO2", N03",
ortho-phosphate, and color.
6. Four 1-quart cubitainers, one each for:
C.2-4
-------
(a) Total Kjeldahl nitrogen, total phosphorus, NH3, fixed
with H2SO4 such that the pH was less than 2.
(b) Total metals, fixed with HNO2 such that the pH
less than 2.
(c) CN~, fixed with NaOH such that the pH was greater
than 12.
(d) Phenol, fixed with H2SO4 such that the pH was less
than 2, then 5 mL CuS04 added.
To sample for filtered metals, a Biichner funnel apparatus and
portable electric pump were used. Initially, the funnel, flask,
and quart cubitainer were rinsed with deionized water. Then
150 mL of effluent were measured in a graduated cylinder; and
vacuum-filtered through a pre-weighed 0.45 pm paper filter on the
Buchner apparatus. The filter was removed from the Biichner fun-
nel, sealed in a plastic Petri dish for shipment to MRC, and
analyzed for metals associated with solids in the sample. The
filtrate was poured into the quart cubitainer and acidified with
HN03 to a pH less than 2.
At selected plants, several additional samples were also collected:
1. One 1-quart cubitainer for COD, fixed with H2SO4 such
that the pH was less than 2.
2. One 1-quart cubitainer for sulfite.
3. One 1-quart glass jar for oil and grease.
4. One 5-gallon cubitainer of effluent, sent to the
E.G.&G. Bionomics laboratory in Pensacola, Florida
for mysid shrimp bioassay.
C.2-5
-------
All samples were hermetically sealed with stretch tape, wrapped
in bubble packing, placed on ice in sealed coolers, and mailed to
the appropriate locations for analysis.
For the sediment sampling, SWCB personnel collected sediments in
the vicinity of eleven of twenty outfalls. A joint Virginia
Institute of Marine Science—Maryland Geological Survey crew col-
lected sediments at another six outfalls. No sediments were col-
lected for the three remaining outfalls due to substrate limita-
tions at each site.
The object of the sediment sampling program was to perform the
same set of chemical tests on fine-grained sediments near each
outfall which were performed on the effluent itself. If persis-
tent toxic substances found in the analysis of the effluent were
also found in the sediment, a possible link could be formed between
the discharge of this chemical and its appearance in the
environment.
Sediment sampling goals were straightforward: obtain a fine-
grained sediment sample as closely as possible to each outfall.
Equipment used in the sampling were either the 6 in. x 6 in. or
9 in. x 9 in. Ponar sediment grab sampler. The undisturbed top
3 cm of each sediment sample were removed with a stainless steel
scoop and placed in specially cleaned glass one-liter jars sup-
plied by the Virginia Institute of Marine Science. Jar lids were
lined with a sheet of Teflon plastic. After labeling, the samples
were frozen by placing them in coolers with dry ice. The samples
were shipped on dry ice via air freight to Monsanto Research
Corporation's lab in Dayton, Ohio.
C.2-6
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C.3 NPDES PARAMATERS
In Phase III, the Central Regional Laboratory of EPA analyzed
grab samples for NPDES parameters. The test, method, and quality
control performance are given in Table C.3-1.
TABLE C.3-1. NPDES PARAMETERS ANALYZED BY EPA IN PHASE III
Test
Method
Average QC
accuracy, %
BOD
COD
Turbidity
Nitrate
Total dissolved
phosphorus
Total phosphorus
Ortho-phosphate
Ammonia
Phenol
Cyanide
Chromium VI
Dissolved mercury
Total mercury
TKN
Color
TSS
Fluoride
Winkler/Probe [1] 101.0
Standard Methods [1] 104.9.
Standard Methods [1] -c
Automated Cadmium Reduction [2] . 104.4
Automated Colorimetric Ascorbic
Acid Reduction [2] 100.8
Automated Colorimetric Ascorbic
Acid Reduction [2] 104.3
Automated Colorimetric Ascorbic
Acid Reduction [2] 100.8
Automated Phenate Colorimetric [2] 109.2
Distillation/Colorimetry [1] 98.0
Distillation/Pyridine
Colorimetric [1] 99.0.
Colorimetric, Diphenylcarbazide [1] -c
Cold Vapor [2] 98.2
Cold Vapor [2] 101.5
Automated Phenate Method [2] 94.1
Colorimetric, Platinum Color t
Units [1]
Standard Methods [1] 103.2
Electrode [2] 100.0
Blanks indicate no QC data reported.
5Not applicable.
[1] APHA, AWWA, WPCF, Standard Methods for the Examination of
Water and Wastewater (14th Edition). American Public Health
Association, Washington, D.C., 1977.
[2] U.S. EPA, Methods for Chemical Analysis of Water and Wastes.
EPA-625/6-76-003a, National Environmental Research Center,
Cincinnati, Ohio, 1976.
C.3-1
-------
C.4 ION CHROMATOGRAPHY FOR ANALYSIS OF ANIONS
C.4.1 Method
Ion chromatography (1C) was used to measure the anions F~, Cl~,
SO3~2, and SO4~2 in the plant effluent samples. Ion chromatog-
raphy is a highly selective instrumental technique for rapidly
detemining ionic species. The technique is based on well-
established ion-exchange principles used in a novel way that
allow electrical conductance to be used to detect and quantitate
ions which are selectively eluted from a chromatographic column.
This novel adaptation of ion exchange principles involves the
use of a background ion suppressor column to eliminate or mini-
mize the ionic character of the mobile phase. In the case of
anion analyses, sodium carbonate and/or bicarbonate in the mobile
phase is converted to the weakly conductive carbonic acid, while
the anions to be measured are converted to strongly conducting
acid forms. As the anions elute from the chromatographic column,
the change in electrical conductance of the mobile phase is
measured and recorded as a function of time on a strip chart
recorder or data system. For relatively clean waters, detection
limits of low ppm or ppb levels can be attained.
Both qualitative and quantitative data were generated in the
analysis. The retention times (time of elution from the column
after sample introduction) are correlated with individual anions
and can be used to identify the anion. However, it is important
to note that very high anion concentrations can result in signi-
ficant shifting of retention times for the anions. Suitable
quality control/quality assurance standards must be analyzed along
with the unknowns to validate the qualitative identifications.
To protect the separator column from the effects of overloading
and contamination, a guard column was used which contained the
C.4-1
-------
same ion exchange resin as the separator column. The guard
column is replaced or regenerated as necessary and insures con-
sistent response from the separator column.
Because of the diverse nature of the plant effluents in this
program and the complexity of the sample matrix, the analytical
procedures were optimized to yield maximum sensitivity for all
sample types. Samples were run in several dilutions to obtain
an acceptable response for each anion of interest. Samples were
analyzed for the anions F~, Cl~, SO3""2, and SO4~2 using a Model
10 Dionex ion chromatograph which utilized a 3 mm x 150 mm pre-
column, a 3 mm x 500 mm anion separator column as the analytical
column, a 6 mm x 250 mm anion supressor column and 0.003M NaHCO3/
0.0024M Na2CO3 in deionized water as the eluent, with an operat-
ing pressure of 240 psi to 360 psi. Sample volumes of 0.1 mL
were injected into the chromatograph.
The only sample preparation involved filtration through a 0.45-
micron nitrocellulose filter. In the cases where the chloride
and sulfate concentrations exceeded the working range of the
instrument, samples were diluted with deionized water.
Calibration curves were generated for each anion by plotting peak
height of the anion versus concentration of that anion in stand-
ard anion solutions. Four different concentrations of each ion
were plotted and response factors calculated using linear regres-
sion analysis. Peak heights obtained from samples were converted
to concentration units using these response factors. Spiked
samples were run to verify peak identification and recovery. To
assure proper quantitative measurements, replicate analyses and
measurement of a sufficient number of quality control/quality
assurance standards were also performed.
C.4-2
-------
The analysis was performed without further sample preparation.
Typical calibration curves for the anions detected are given in
Figures c.4-1 through C.4-4.
The analytical method was the basic Dionex procedure for anions.
Quantitative measurements were made by comparing the chromato-
graphic response for the unknown with the response for known con-
centrations of anions in deionized water. Initially, after the
filtration step, single analyses were performed on the samples
as received. Because of the high concentrations of chloride and
sulfate ions, additional analyses were performed on samples
diluted in deionized water.
Figure C.4-5 shows a representtive ion chromatographic pattern
for a typical sample. Particularly notable are the peak shapes
of the two major components: chloride (major early eluting com-
ponent) and sulfate (major later eluting component). The greater
broadening of the peak characteristic of sulfate anion than the
peak characteristic of chloride anion is due partially to dif-
ferences in anion size and charge. Ion size and charge determine
the ion interaction with the resin of the chromatographic column.
Ions that have a charge that can be polarized toward the func-
tional group of the resin will have a slower rate of exchange
between the resin and mobile phase. Therefore, ions such as
sulfate will elute later and the chromatographic peaks will be
broader than those for chloride or fluoride ions. Additional
peak broadening and tailing can result from overloading the active
sites of the resin. An additional result of resin overload is a
decrease in retention time of the eluting components as samples
are processed in sequence.
C.4.2 QA/QC for Anion Analysis
To assure proper performance of the ion chromatograph during the
sequence of analyses, the electrical conductivity response of the
C.4-3
-------
o
•
•*»
i
120
no
100
90
80
E 70
5E
a w
50
40
30
20
10
0
CONCENTRATION vs. RESPONSE
Cl"
CORRELATION COEFFICIENT: 9991
4567
Cl" CONCENTRATION, ppm
10
120
110
100
90
70
•
o
3
Q.
50
40
30
20 \-
10
CONCENTRATION vs. RESPONSE
r
CORRELATION COEFFICIENT: 9996
0.5 1 2 3
F" CONCENTRATION, ppm
figure C.4-1. Peak height vs. chloride
ion concentration.
Figure C.4-2. Peak height vs. fluoride
ion concentration.
-------
Ol
70
60
50
E
40
2
a.
30
20
10
CORRELATION vs. RESPONSE
so,-'
CORRELATION COEFFICIENT: 9954
5 10 15 20 25 30 35 40
S03"2 CONCENTRATION, ppm
Figure C.4-3. Peak height vs. sulfite
ion concentration.
80
70
60
50
30
20
10
CONCENTRATION vs. RESPONSE
CORRELATION COEFFICIENT: 9939
5 10 15 20 25 30 35 40
S04" 2 CONCENTRATION, ppm
Figure C.4-4. Peak height vs. sulfate
ion concentration.
-------
INTENSITY
INJECTION
1.23
1:1 DILUTION
113mm
5.20min Cl
86mm
17.47minS04~
Figure C.4-5. Ion chromatographic pattern for typical sample,
C.4-6
-------
detector was determined using standard conductance solution and
the column quality was verified. The system was found to be with-
in the Dionex instrument specifications for the Model 10.
Analyses of deionized water blanks were performed with each set
of samples. The calibration curves were prepared prior to the
analysis of each set of samples and a representative standard
solution of anions was checked after the analysis of each set of
samples.
C.4-7
-------
C.5 ICAP SPECTROSCOPY FOR METALS ANALYSIS OF BAY SEDIMENT
C.5.1 Introduction
Inductively Coupled Argon Plasma (ICAP) was used to measure the
concentration of 24 metals in the Chesapeake Bay sediment sam-
ples. The ICAP technique is a very powerful method for the
determination of metals in solution. The ICAP has several ad-
vantages over conventional flame atomic absorption, such as simul-
taneous, multi-element determinations, high temperature with low
background, and linearity often extending over 5 orders of mag-
nitude in concentration. In routine analyses, the ICAP has
detection limits similar to flame AA for most transition metals
and alkaline earths and far superior detection limits for re-
fractories and nonmetals.
C.5.2 Sample Prep
Approximately 0.5 g (weighed to 0.1 mg) of the freeze dried sedi-
ment sample was prepared for analysis by the Parr Teflon Bomb
method using 12 mL of concentrated Ultrex HNOa as the digestion
media. After digestion, the sample was filtered to remove any
insoluble particulates (probably silicates) and diluted to a
known volume. At this point, the sample was analyzed with no
further preparation.
C.5.3 Analysis
ICAP operations followed the manufacturers instructions. In
addition, the guidelines given in EPA interim ICAP method 200.7
were followed. The Parr Bomb blank was subtracted from the sam-
ple values. Background correction was used to compensate for a
shift caused by high concentrations of sodium and aluminum. Also,
interelement correction factors were used to correct for the
spectral overlap of several metals caused by high levels of iron.
C.5-1
-------
For equipment used, operating conditions and elemental wavelengths,
see Tables C.5-1, C.5-2, and C.5-3, respectively.
TABLE C.5-1. EQUIPMENT USED
Spectrometer
Computer
Terminal
Nebulizer
RF generator
Pump
JY48P 1 meter focal length
Digital Equipment Corporation PDP-1103 16K memory
LA120 DECWRITER
Meinhard glass concentric
Plasma-Therm Model 2500D
Gilson, Model HP-4
TABLE C.5-2. SYSTEM OPERATING CONDITIONS
Incident RF power
Reflected RF power
Coolant Ar flow rate
Auxiliary air flow rate
Nebulizer pressure
Sample uptake rate
Observation height
Number of integrations
Integration time
Background correction
Sample flush time
1.4 kW
S5 W
16 L/min
0.6 L/min
18 psi
~0.75 mL/min
14 mm above load coil
3
10 s o
+0.5 A
60 s
TABLE C.5-3. WAVELENGTHS
Element
o
\, A
Element
o
X, A
Element
X,
o
A
Ag
Al
B
Ba
Be
Ca
Cd
Co
3,280.68
3,082.15
2,089.59
2,335.27
3,130.42
3,158.87
2,265.02
2,286.16
Cr
Cu
Fe
Mg
Mn
Mo
Na
Ni
2,677.16
3,247.54
2,382.04
2,795.53
2,576.10
2,020.30
3,302.37
2,316.04
P
Pb
Sb
Si
Sr
Ti
V
Zn
2,149.14
2,203.53
2,068.33
2,516.11
4,077.71
3,349.41
3,102.30
2,138.56
C.5-2
-------
C.5.4 QA/QC for ICAP Analysis
To insure that the instrument was operating properly, reference
standards were analyzed at a frequency of 10% (one reference stand-
ard with every 10 samples). Five samples were digested in dupli-
cate. Duplicate samples showed high relative percent differences
for a number of elements. This is indicative of problems with
sample homogeneity. Na, Si, and Zn had consistently high relative
percent differences. The high differences in Zn values are prob-
ably due in part to contamination from filters used in digestion
and from contact with polyethylene tubing and bottles. Two samples
were to be spiked at the time of digestion. However, from the low
recoveries obtained for sample B143S, it appears that the spike was
inadvertently omitted.
Sample C158D had acceptable recoveries where the unspiked sample
value was less than 10 times the concentration of the spike. It
should be noted that variations in sample homogeneity will have
a large bearing on percent recoveries.
C.5-3
-------
C.6 ANALYSIS OF PURGEABLE ORGANICS
C.6.1 Bellar Purge and Trap Technique For Purgeable Organics
The 40-mL vials were analyzed for volatile organics by the purge
and trap method using a standard packed-column GC/MS [3]. This
method was designed for trace-level volatile organics contained in
a wide variety of water sources. For quantitative determinations
the method is limited to organic compounds that are less than 2%
soluble in water and that boil below 200°C. Most compounds boil-
ing above 200°C would be found in the methylene chloride extracts
of the water, the analysis of which is described later.
This method of volatiles analysis is useful at levels from 1 pg/L
to 2,500 mg/L. At concentrations exceeding 2,500 mg/L, flooding of
the chromatographic column and nonlinear detector responses gen-
erally occur. It typically works well except on those samples
where foaming is a problem. Water entering the trap causes non-
quantitative trapping and severe gas chromatographic interferences.
C.6.2 GC/MS Analysis of Purgeables
The two vials in which the effluent samples were collected were
stored at 4°C. Before analysis the contents of the vials were
composited in an ice bath and returned to the original vials,
again with no headspace. The samples were first allowed to warm
to room temperature to prepare them for analysis. Next, the
plunger from a 5-mL syringe equipped with a valve was removed.
The sample to be analyzed was poured into the syringe body, with
the valve closed, until the sample overflowed. The syringe plunger
was then replaced and the residual air and excess volume of sample
[3] Sampling and Analysis Procedures for Screening of Industrial
Effluents for Priority Pollutants. Final Draft Report, U.S.
Environmental Protection Agency, Cincinnati, Ohio, April 1977.
C.6-1
-------
was removed. Internal standards (1,4-dichlorobutane and bromo-
chloromethane) were added to the water sample in the syringe by
means of a 10 pL syringe inserted through the valve at the de-
livery end of the 5 mL syringe. The 5 mL of sample was introduced
into the purging device and sparged with high purity helium at a
rate of 40 mL/min at room temperature for 10 minutes. The purged
organics were sorbed onto a 2.7 mm x 15.2 cm (1/8 in. x 6 in.)
stainless steel tube packed with 6.4 cm (4 in.) of Tenax GC
(60/80 mesh) and 3.2 cm (2 in.) of type 15 silica gel (35/60 mesh).
The tube was then desorbed by backflushing at 180°C for 10 min
onto the head of the chromatographic column which was maintained
at -40°C. The sequence used consisted of analyzing a particular
tube from an organic-free water sparge, and then using the same
tube for the sparging of an effluent sample, so that the immediate
history of the tube was known.
Samples were analyzed using a modified Hewlett-Packard 5983 GC/MS,
operated in the positive ion, electron impact mode, with a 5934A
data system. The following parameters describe the system.
- 6 ft x 2.7 mm stainless steel column
- 0.2% Carbowax 150 on 80/100 mesh Carbopak C
- Flow rate, 30 mL/min helium
- Initial temperature, -40°C
- Time at initial temperature, 0 min
- Heating rate, 8°C/min
- Time at final temperature, 5 min
- Glass jet separator and glass-lined transfer lines, 260°C
- Electron energy, 70eV
- Emision current, 300 pa
- Source temperature, 200°C
- A/D rate, 5 measurements/0.1 amu; scan rate 41.6 amu/s
- Mass spectrometer scan delay, 2 min
C.6-2
-------
The GC/MS data were examined for priority pollutant compounds and
for other substances present in identifiable amounts. The EPA
volatile priority pollutants (Consent Decree compounds) are listed
in Table C.6-1, along with their typical retention times, major
masses, and associated intensities. To indicate the presence of a
priority pollutant compound by GC/MS, three conditions must be met.
First, the characteristic ions for the compound (see Table C.6-1)
must maximize in the same spectrum. Second, the time at which the
peak occurs must be within a window of ±1 min for the retention
time of the compound. Finally, the ratios of the ion intensities
must agree with the relative intensities given in Table C.6-1
within ±20%.
Substances not identified in the specific search for priority
pollutants were sought in a "wide scan" mode. In this procedure,
mass spectra are obtained for peaks not accounted for in the pre-
vious search, and the ions observed are compared with those listed
in the Eight Peak Index to produce a tentative identification [4].
The amounts of these substances are estimated by comparing their
total ion areas with those of similar compounds for which standards
are available, or of the internal standards, assuming a similar
ionization cross section (hence semiquantitation).
C.6.3 Example of Quantitation Performed in Purgeables Analysis
From the mass spectral analysis of a standard mixture one obtains
the response of the major ions of each species relative to those
of the internal standards present in the misture. For example:
[4] Eight Peak Index of Mass Spectra, Vol. Ill, 2nd Ed., Mass
Spectrometry Data Center, AWRE, Aldermaston, Reading,
United Kingdom, 1974.
C.6-3
-------
TABLE C.6-1.
TYPICAL RETENTION TIMES AND CHARACTERISTIC IONS OF VOLATILE CONSENT
DECREE COMPOUNDS MEASURED IN THE PURGE AND TRAP TECHNIQUE
Retention
time, min
Compound
El ions (relative intensity)
Ion used
to quantify
O
I
if*
6.2
6.4
6.1
6.9
8.
10.
12.
13.
13.
14.2
15.2
15.1
16.2
17.0
17.6
17.9
19.
19.
20.
20.
20.
20.7
21.3
22.8
25.9
24.9
25.4
27.3
28.2
30.0
Chloromethane
Dichlorodifluoromethane
Bromomethane
Vinyl chloride
Chloroethane
Methylene chloride
Trichlorofluoromethane
1,1-Dichloroethylene
Bromochloromethane (IS)
1,1-Dichloroethane
trans-l-2,-Dichloroethylene
Chloroform
1,2-Dichloroethane
1,1,1-Trichloroethane
Carbon tetrachloride
Bromodichloromethane
bis-Chloromethyl ether
1,2-Dichloropropane
trans-l,3-Dichloropropene
Trichloroethylene
Dib romochloromethane
ois-l,3-Dichloropropene
1,1,2-Trichloroethane
Benzene
2-Chloroethylvinyl ether
Bromoform
1,1,2,2-Tetrachloroethene
1,1,2,2-Tetrachloroethane
1,4-Dichlorobutane (IS)
Toluene
Chlorobenzene
Ethylbenzene
Acroleiii
Acrylonitrile
50(100); 52(33)
85(100); 87(33); 101(13); 103(9)
94(100); 96(94)
62(100); 64(33)
64(100); 66(33)
49(100); 51(33); 84(86); 86(55)
101(100); 103(66)
61(100); 96(80); 98(53)
49(100) 130(88); 128(70); 51(33)
63(100); 65(33); 83(13); 85(8);
98(7); 100(4)
61(100); 96(90); 98(57)
83(100); 85(66)
62(100); 64(33); 98(23); 100(15)
98(100); 99(66); 117(17); 119(16)
117(100); 119(96); 121(30)
83(100); 85(66); 127(13); 129(17)
79(100); 81(33)
63(100); 65(33); 112(4); 114(3)
75(100); 77(33)
95(100); 97(66); 130(90); 132(85)
129(100); 127(78); 208(13); 206(10)
75(100); 77(33)
83(95); 85(60); 97(100); 99(63);
132(9); 134(8)
78(100)
63(95); 65(32); 106(18)
171(50); 173(100); 175(50); 250(4);
252(11); 254(11); 256(4)
129(64); 131(62); 164(78); 166(100)
83(100); 85(66); 131(7); 133(7);
166(5); 168(6)
55(100) 90(30); 92(10)
91(100); 92(78)
112(100); 114(33)
91(100); 106(33)
26(49); 27(100); 55(64); 56(83)
26(100); 51(32); 52(75); 53(99)
50
85
96
62
64
84
101
61
130
63
61
83
62
97
117
83
79
63
75
130
129
75
83
78
106
173
166
83
55
91
112
91
56
53
-------
Compound Identified
(Int. Std.) Bromochloromethane
(Int. Std.) 1, 4-dichlorobutane
Vinyl chloride
Chloroform
Benzene
Major
ion
130
55
62
83
78
Peak
area
9,763
14,271
43,315
39,313
69,903
Cone . ,
pg/L
400
400
800
400
400
Rvalj
_
-
2.22
4.03
7.16
Rval2
_
-
1.52
2.75
4.90
The R values are calculated as follows, using chloroform for
example:
_ Area chloroform, mass 83 _ Cone . bromochloromethane
1 ~ Area bromochloromethane, mass 130 Cone, chloroform
= 39.313 400
9,763 X 400
Area chloroform, mass 83 Cone . 1 , 4-dichlorobutane
2 Area 1 ,4-dichlorobutane, mass 55 Cone, chloroform
400
14,271 400 -
The R values, obtained as above, are then employed to guantitate
compounds identified in actual water samples, for example:
Major Peak Cone.,
Compound Identified ion area J'1 * Xz pg/L
(Int. Std.) Bromochloromethane 130 9,741 - - 400
(Int. Std.) 1, 4-dichlorobutane 55 12,245 - - 400
Chloroform 83 1,343 4.03 2.75
Benzene 91 2,681 7.16 4.90
The concentrations are calculated in the following manner, again
using chloroform for an example:
, . - _ Area chloroform, mass 83 _ Cone. Bromochloromethane
cnxorororm cone.! - Arga bromochloromethane/ mass 130 x tfy^ chloroform
C.6-5
-------
Area chloroform, mass 83 Cone . 1 ,4-dichlorobutane
cruorororm conc.2 - Afea ^4-aichiorobutane, mass 55 * Rval2 chloroform
Average = 15 pg/L
C.6.4 QA/QC for Purge able Organics
The concentrations reported were measured relative to Supelco
Standards A, B, and C, calculated as explained in the previous
section. The absorption tubes used for trapping the volatile
species were conditioned prior to use under vigorous conditions
to eliminate carry-over from one sample to another. This was done
by sparging organic-free water into a tube for 20 minutes, and
then heating the tube for 1 hour at 200 °C with helium flowing
through it at 40 mL/min. In addition, each sample was analyzed
using a tube that had been previously used for organic-free water,
as mentioned in the section on analysis. The multiple blanks
were averaged and used for background subtraction in calculating
the results reported. The duplicate analyses of three samples
that were performed gave an average percent deviation of ±21%.
C.6-6
-------
C.7 EXTRACTABLE ORGANICS FROM EFFLUENTS
Figures C.7-1 through C.7-4 show the detailed scheme employed in
work-up and analysis of the extractable organics fractions of the
Phase III effluent samples.
C.7.1 Extraction Procedure
Figure C.7-5 is a facsimile of MRC's sample extraction QA/QC flow
sheet used by the analyst to ensure that every element of the
extraction has been performed. The circles were checked as each
step of the procedure was performed.
For ease of handling, each 10-L sample was divided into four ali-
quots of 2.5 L, which were then spiked with deuterated recovery
standards (listed in Table C.7-1). After allowing the spiked
samples to equilibrate 0.5 hr, the pH of each aliquot was ad-
justed to £12 using 6% NaOH; each aliquot was then extracted with
100 mL of methylene chloride (CH2C12) followed by two additional
75 mL portions. For each extraction, the separatory funnels are
manually shaken for 2 min (by the clock), and then allowed to
TABLE C.7-1. DEUTERATED RECOVERY STANDARDS ADDED
TO 10-LITER EFFLUENT SAMPLE
Concentration,
Compound
Phenol-d6a 112
l,2-Dichlorobenzene-d4 118
Biphenyl-d10 102
Pyrene-dj 0 99
Chrysene-d! 2 83
Perylene-D12 85
aRecovered as Phenol-d5, due to exchange
of the labile deuterium with protons
from the water.
C.7-1
-------
o
I
SAW
FORTIFY Wlft
RECOVERY !
ORG.
1
B/N FRACTION
GRAV ^ 1 DRY & CONCENTRATE
^^^5 I
ImL
GC/FID (TCO) -*— B/N CONCENTRATE
GC/EC^ 1 5 mL
IPLE
\ OEUTERATED
STANDARDS
L pH>!2
f ~ AQ.
ORG.
I
A FRACTION
PH>2 ^
\
REMAINDER
1 DRY & CONCENTRATE 1
A CONCENTRATE
STORE
GC/MS
LC
SEE FIG. C.7-3
4rnL
TCO GRAV
GC/FID
GRAV GRAV GRAV
GC/FID (TCO) TCO TCO
GC/MS GC/MS GC/MS
CONCENTRATE
/ImL
GC/EC
Figure C.7-1. Overall Phase III effluents analytical scheme.
-------
MJOUOTt
me. c.7-51
HMAIMIU
Figure C.I-2. Extraction procedure for Phase III effluent samples,
C.7-3
-------
GRAV
TCO
( COMBINE N
All HI'S IN
ly^LtRLENMEYEiy
\
PASS THROUGH Na2S04
COLUMN INTO K- D RECEIVER
\
CONC. TO 10 mL
*
^\ REMOVE 4 mL
\ REMAIN
1 mL I
•»--| REMOVE ImL
DER (6 nt)
1 REMAINDER (5 nt)
GC/MS
•i^ REMOVE 1 nt
4 REMAINDER (4 mU
/PUREN
(PURE N.
WJREN
\^ J HHJREN
DILUTE TO ImL
\
GC/EC
PLACE IN 40°C BLOCK
I
jV—1 EVAP. TO 1 mL
*
ADD 2 mL HEXANE
*
,)-»j EVAP. TO 1 mL
\
ADD 1 mL HEXANE
*
.W EVAP. TO 1 mL
\
ADD 2 nt HEXANE
\
jV— EVAP. TO 1 nt
I
— 1 REMOVE 50 ML
{ REMAINDER
STORE
SOLVENT
> EXCHANGE
FOR GC/EC
Figure C.7-3. Concentration and analysis scheme
for base/neutral fraction.
C.7-4
-------
I
C COMBINE ^1
ALL[0'slN
12L ERLENMEYERJ
PASS THROUGH Na2S04
COLUMN INTO K-D RECEIVER
CONC. TO 10 ml
REMOVE 4 ml
REMAINDER (6 ml)
REMOVE 1 ml
REMAINDER (5 mU
REMOVE 1 nt
REMAINDER 14 mU
PLACE IN 40°C BLOCK
EVAP. TO 1 mL
ADD 2 mL HEXANE
GC/EC
EVAP. TO 1 mL
S. /
SOLVENT
EXCHANGE
FORCC/EC
S
EVAP. TO 1 mL
ADD 2 mL HEXANE
ADD 2 mL HEXANE
EVAP. TO 1 mL
Figure C.7-4. Concentration and analysis
scheme for acid fraction.
C.7-5
-------
n
•
•sj
I
Figure C.7-5. Phase III extraction flow sheet.
-------
stand a-minimum of 10 min to complete separation of the liquid
phases. The larger volume used in the initial extraction is to
allow for the solubility of the solvent in the sample. The ex-
tracts were combined, dried over sodium sulfate, and concentrated
to 10 mL using Kuderna-Danish evaporative concentrators, giving a
1,000-fold concentration of extractable base/neutral organics,
assuming 100% extraction efficiency. Four mL of this 10 mL con-
centrate was removed for GRAV analysis and 1 mL each for TCO and
GC/MS was stored in screw-cap vials with Teflon closures. The
remaining 4 mL of concentrate was solvent exchanged with hexane
and further concentrted to 1 mL for GC/EC analyis. The check
sheets for these procedures are given in Figures C.7-6 through
C.7-8.
Each supernatant aqueous portion from the base/neutral CH2C12
extractions was acidified to pH £2 using 6N H2S04, and extracted
with CH2C12 (3 x 75 mL), as before. The combined acidic extracts
were concentrated to 10 mL, giving an extractable acidic organics
fraction enriched 1,000-fold from the original effluent, again
assuming 100% extraction efficiency. Four mL of this 10 mL con-
centratate was taken for GRAV analysis while 1 mL each for TCO
and GC/MS was stored, as above, for TCO and GC/MS analyses. The
remaining concentrate was solvent exchanged with hexane and con-
centrated to 1 mL for GC/EC. The check sheets for these pro-
cedures are given in Figures C.7-6 through C.7-8.
C.7.2 Liquid Chromatographic (LC) Fractionation
None of the effluent samples that were part of the Phase III pro-
gram required liquid Chromatographic (LC) fractionation. However,
in those cases where fractionation should be required based on TCO/
TCG, the following procedures (graphically shown in Figure C.7-9)
would be followed to improve identification of individual species.
C.7-7
-------
Sample No.:
Color of extract:
Date:
Analyst:
10 mL concentrate
Grav
GRAV
Hood/24 hr
Date/Time In:
Date/Time Out:
TCO
TCO
Store/Ref.
5 mL to vial
Label BNP-1
Store/Ref.
go to Sheet 2
Date/Time In:
Date/Time Out:
Desiccator/24 hr
Pan and Sample:
Pan Tare:
Residue:
Figure C.7-6. Base/neutral extract flow sheet 1.
€.7-8
-------
SIMPLE TCO
Sample No.:
(should contain -BNP-1)
Hexane Lot No.:
Date:
Analyst:
5 mL
GC/MS
1 mL to vial
GC/MS
QStore/Ref.
Label Solvent extraction/GC-EC
-BNPD-4 O Cone, to 1 mL (N2/40°)
+ 2 mL hexane
Cone, to 1 mL
+ 2 mL hexane
Cone, to 1 mL
+ 2 mL hexane
Cone, to 1 mL
iDilute to 1 mL
(hexane)
Label
BNPS-5
GC/EC
O Store/Ref.
Remove 50 |jL to vial
Label Residue
BNPS
Residue
Store/Ref.
Figure C.7-7. Base/neutral extract flow sheet 2
C.7-9
-------
Sample No.:
Color of Extract:
Hexane Lot No.:
Date:
Analyst:
10 mL concentrate
Grav
TCO/GC-MS
Q4 mL to pan
-------
PLACE SAMPLE IN
GRAD. RECEIVER w
200 mg Na.SO.
1
PLACE IN 40°C BLOCK
»-
»-
k»
*-
1
EVAP. TO 1 mL
1
ADD 2 mL HEXANE
1
EVAP. TO 1 mL
1
ADD 2 mL HEXANE
*
EVAP. TOltnL
*
ADD 2 mL HEXANE
1
EVAP. TO 1 mL
*
CENTRIFUGE
»
REMOVE 50 ML
s
50 Ht
) REMAINDER
RE-SUSPEND Na2S04
SOLVENT
^EXCHANGE FOR
GC/EC AND LC
(HEXANEJ
i
- nil HIT in i mi
UILU 1C IU 1 MIL
i
GC/EC
CONTINUED
Figure C.7-9. Liquid chromatographic fractionation.
C.7-11
-------
TRANSFER LI QUID AND
Na2S04 TO LC COLUMN
niiTTiuTimi urvAhir ^*_.
(» mL
15 mL
GRAV -*— REMOVE 15 mL
^-^ 4 REMA 1 NDER (20 mU
(PURE N2)— *• EVAP. TO 2 mL
1 mL f ^"^ 1 mL
TCO GC/MS
-
*
prunv/r m mi — ^^» />D*V/
1
EVAP. TO 2 mL ---/PURE N?)
if \*^_^s
I mL f J 1 mL
TCO GC/MS
ELUTE w 20 mL
50/50 CH^yCHjOH
*
10 mL
ElOmL
^-^ | REMAINDER (10 mL)
(PURE N^— »• EVAP. TO 2 mL
x^^x j
1 mL r — ^^^1 mL
TCO GC/MS
> LC SEPARATION
Figure C.7-9 (continued)
C.7-12
-------
A 5-mL portion of the B/N concentrate remaining after the GRAV
and TCO/TCG analyses is solvent-exchanged into hexane in the
presence of sodium sulfate and concentrated to 1 mL. After re-
moving 50 pL for GC/EC analysis, the hexane concentrate was trans-
ferred as a slurry tp a prepared silica gel column. Details of
the column preparation are as follows:
Column: 200 mm x 10.5 nun ID, glass with Teflon stopcock,
equipped with cooling water jacket.
Adsorbent: Davison, Silica Gel, 60-200 mesh, Grade 950 (avail-
able from Fisher Scientific Company) should be used. This mate-
rial is cleaned prior to use by sequential Soxhlet extraction
with methanol, methylene chloride, and hexane. The adsorbent
is then activated at 110°C for at least 2 hr just prior to
use, and cooled in a desiccator.
Drying Agent: Sodium sulfate (anhydrous, reagent grade).
Cleaned by sequential Soxhlet extraction for 24 hr each with
methanol, methylene chloride, and hexane. The cleaned sulfate
is dried for at least 2 hr at 110°C and cooled in a desiccator,
just prior to use.
Procedure for Column Preparation: The chromatographic column,
plugged at one end with a small portion of glass wool, is
slurry-packed with 6.0 g of freshly activated silica gel in
hexane. The total height of the silica bed, in this packed
column, is about 10 cm. After packing the silica gel column,
3 g ± 0.2 g of clean sodium sulfate is added to the top of the
column and vibrated for 1 min to compact the column. The
sodium sulfate is used to remove any remaining traces of water
from the organic extract and/or the solvents used. Once the
column is fully prepared, the pentane level in the column is
dropped to the top of the sulfate so that the sample can be
loaded for subsequent chromatographic elution.
C.7-13
-------
The elation sequence consists of 35 mL of hexane (Fraction 1),
30 mL of CH2C12 (Fraction 2), and 20 mL of 50:50 CH2C12zmethanol
(Fraction 3). This sequence should yield primarily aliphatics
and lower molecular weight aromatics and PNA's in Fraction 1;
higher molecular weight aromatics and PNA's, halogenated compounds,
and moderately polar substances in Fraction 2; and very polar
substances in Fraction 3. In order to insure adequate resolution
and reproducibility, the column elution rate should be maintained
at 1 mL/min. The cooling water should be adjusted so as to main-
tain a constant temperature throughout the column.
The volume of solvents mentioned above represents volume added to
the column for that fraction. If the volume of solvent collected
is less than the volume actually added, due to evaporation, the
fraction volume is restored to the proper level with fresh solvent.
In all cases, the solvent level in the column is maintained at or
above the top of the gel/sulfate bed; i.e., the sample-containing
zone.
Each new solvent should be slowly added to the column to minimize
disturbing the gel/sulfate bed and to eliminate trapped air bubbles,
particularly in the zone of the sample-containing sodium sulfate.
From each fraction collected, nearly half is removed for a frac-
tional GRAV analysis. The remainder is evaporated to 2 mL to be
divided between TCO and GC/MS analyses. Figure C.7-10 is a
facsimile of the LC fractionation QA/QC flow sheet used for each
sample.
C.7-14
-------
(lli» Otl Lot No.i
Heaana lot to. i
Date:
OltCIt Lot No.:
MOM Lot Ho.:
o
•
^1
I
M
tn
11 ai./»iei
)UlMl
Mem 10 •*.
. (cant, tube)
Cone, to 2 el.
) Label
-•PC?
GC/N5
) Store/let.
) Store/Kef.
) Label
) ltood/24 hr
leaove II at
(cent, tube)
Cone, to 2 el.
I I Bt/, .
Tarai
Datt/TiM In,
Date/TiM Outt
Deilcc
-------
C.8 EXTRACTABLE ORGANICS FROM SEDIMENTS
C.8.1 Extraction Procedure
The frozen sediment samples were fractured into suitably sized
pieces for the freeze-drying containers, and were freeze dried
in a Virtis freeze dryer at 0.01 mm Hg pressure. In certain sam-
ples, this process required in excess of 96 hours to complete, as
the percentages of water in the samples were as high as 78.5%.
The freeze-dried samples were homogenized and aliquots removed
(coning/quartering method) for extraction. Another aliquot was
removed for metals determination. The dried samples (approximately
30 g) were weighed into glass Soxhlet thimbles, equipped with
sintered glass frits, and were each spiked with 1 mL CH2C12 solu-
tion containing 100.6 jjg d10-biphenyl; 101.0 pg dt2-chrysene;
107.6 pg d4-l,2-dichlorobenzene; 106.8 pg d6-phenol; 102.0 pg
d12-perylene; and 102.8 pg d10-pyrene. These spiked samples
were allowed to stand 0.5 hour to equilibrate. Each spiked sedi-
ment was extracted with 500 mL of methylene chloride for 24 hours.
The organic extract was removed from the Soxhlet apparatus and
was concentrated to -^5 mL in a rotary evaporator under water aspir-
ation with a water bath temperature of •v35°C. The concentrated
extracts were quantitatively transferred to a screw-cap vial with
Teflon closure and the volume adjusted to ^10 mL. These concen-
trates were processed through a gel permeation chromatography
(GPC) separation procedure.
The procedure used in processing sediments is diagrammed in
Figure C.8-1.
C.8.2 GPC Cleanup Procedure
Methylene chloride extracts of sediment samples were fractionated
to eliminate various compounds known to interfere with subsequent
analytical measurements; i.e., fatty acids and molecular sulfur (S8)
C.8-1
-------
(FROZEN SEDIMENT^
I
FRACTION I
(HOLD)
BIO-8EAOS SX-8
SEPARATION
FRACTION III
(HOLD)
FRAaiON
IIJ
CONCENTRATE
TOlOmL
BIOACCUMULATION
ImL
jml
1ml ^
5 ml
[GC/MS
DILUTE TO
ImL
4.95mL
•SAME PROCEDURE AS
FOR EFFLUENTS
GRAVh*-
TCOJ-»-
GRAVh*-
GC/MS
GRAVh*"
GC/MSI
Figure C.8-1. Diagram of sediment processing and analysis scheme,
C.8-2
-------
In Phase II, the chromatographic method employed to process the
sediment extracts was based upon that developed by Stalling and
co-workers [5]. The chromatographic support employed was Bio-
Beads SX-3, manufactured and distributed by BioRad Laboratories,
Richmond, California. When an eluent of 1:1 (V/V) cyclohexane/
roethylene chloride is employed, chlorinated pesticides, PCB's,
dioxins, dibenzofurans, and all organic priority pollutants should
elute over a reproducible volume. Fish oils, triglycerides, fatty
acids, stearates, phthalates, and sulfur, which are commonly found
in sediment extracts, should elute in other fractions (see
Table C.8-1).
In Phase III, Bio-Beads SX-8® was employed as the chromatographic
support due to its increased separation capabilities for the
polynuclear aromatic compounds (fraction two) and sulfur. 5.0 mL
of a 10.0 mL extract was injected onto a 50 cm x 2.2 cm stainless
steel column packed with 200/400 mesh Bio-Beads SX-8® and eluted
with 30% acetonitrile in methylene chloride (V/V) at 4.0 mL/min.
The first fraction, 0-88 mL, and the third fraction, 256-^370 mL,
were collected and archived. Fraction two, 88-256 mL, was col-
lected, evaporated in a Kudema-Danish concentrative evaporator
to approximately 10-15 mL, and subsequently blown down with
purified N2 to less than 2-3 mL. At this point, some extracts
precipitated in what appeared to be almost entirely acetonitrile.
The fractions were easily redissolved with addition of methylene
chloride to the original extract volume injected, 5.0 mL.
Figure C.8-2 is the UV chromatogram obtained at 254 nm when the
standard spiking solution is fractionated according to the
[5] Stalling, D. L., L. M. Smith, and J. D. Petty. Measurement
of Organic Pollutants in Water and Wastewater. C. E. VanHall,
ed., American Society for Testing and Materials, Philadelphia,
Pennsylvania, 1979. pp. 302-323.
C.8-3
-------
TABLE C.8-1.
ELUTION VOLUMES OF VARIOUS COMPOUNDS
ON BIO-BEADS SX-3 2.0 cm X 100 cm WITH
1:1 (V/V) CYCLOHEXANE/METHYLENE CHLORIDE
Fish lipids
Stearic Acid & p-Carotene
DEHP
DBF
C12-C24 Aliphatics
Cholesterol
Butylbenzene
Chlorinated Pesticides
Pentachloroanisole
PCB's
PCDF's
PCDD's
Hydroxy PCB's
Toluene
Biphenyl
Acenaphthene
Alkyl- and Chlorophenols
Hexabromobiphenyl
Anthracene
Phenanthrene
2,3-Benzofluorene
Cnloronaphthalenes
Naphthalene and Fluorene
2,4-D
Pyrene
Fluoranthene
Benzo(a)pyrene
Coronene
4-Nitrophenol
Sulfur
53 - 207 mL
> 207 - 373 mL
390 - 420 mL
C.8-4
-------
previously described protocol. This chromatogram shows the ex-
cellent separation of the PNA's and sulfur. Figure C.8-3 shows
a UV chromatogram at 254 nm obtained from a typical sediment
extract. The value of this cleanup procedure is readily evident.
C.8-5
-------
o
•
00
I
22 mm I. D. X 500 mm
BIO-BEADS SX-8
30% CH3CN/CH2CI2
4.0mL/min
MONITORING 254 nm
AMBIENT TEMP.
•FRACTION 1
PHTHALATES
0 25 50 75 100 125 150 175 200 225 250 275 300 325
VOLUME, ml
Figure C.8-2. Chromatogram of spiking standard.
-------
o
•
00
I
-J
22 mm I. D. X 500 mm
BIO-BEADS SX-8
30% CH3CN/CH2CI2
4.0 ml/ml n
MONITORING 254 nm
AMBIENT TEMP.
32
64
96 128 160 192
VOLUME, ml
224 256
288 320
Figure C.8-3. Chromatogram of sample 8400
-------
C.9 ORGANIC CARBON TRACKING SYSTEM
C.9.1 Total Organic Carbon Procedure
The effluent samples were analyzed for total organic carbon fol-
lowing Procedure 505 given in Standard Methods [6]. The samples
were acidified and sparged with CO2-free nitrogen to remove inor-
ganic carbon prior to analysis. The analysis was conducted via
catalytic combustion to convert the organic to CO2 followed by
detection of the C02 with an infrared detector.
C.9.2 Total Chromatographable Organics (TCO) and Total
Chromatographable Gravimetrics (TCG)
C.9.2.1 TCO/TCG Method—
An automated capillary GC/FID analysis was used to provide four
types of information on the Phase III samples: total Chromato-
graphable organics (TCOs), total Chromatographable gravimetrics
(TCGs), relative retention indices, and recoveries of deuterated
spike compounds.
The TCO analysis is a gas chromatographic procedure for quanti-
tating materials boiling in the 100°C to 300°C range. The area
of all peaks eluting between compounds having boiling points of
98°C and 302°C is integrated, and by comparison with standards,
is reported as the TCO value in mg/L of original effluent water
[7], or mg/g of sediment.
[6] Standard Methods for the Examination of Water and Wastewater,
APHA, 14th Ed., Method 505, p. 532 (1975).
[7] U.S. Environmental Protection Agency, IERL-RTP Procedures
Manual: Level I Environmental Assessment, 2nd ed., EPA-600/
7-78-201, October 1978.
C.9-1
-------
The end-points of the boiling range are defined by the retention
times of n-heptane (C7, b.p. 98°C) and n-heptadecane (C17, b.p.
302°C). Quantitation is based on standard solutions of known
amounts of normal C8, C12, and C16 hydrocarbons, with corrections
made for solvent blanks and spiked deuterated compounds.
The TCG analysis was included in the Phase III protocol to deter-
mine what fraction of the GRAV organics can actually be chroraato-
graphed by capillary GC. For TCG, the areas of all peaks eluting
between the retention times of n-heptadecane and the end of the FID
chromatogram (51.00 min) are integrated. The boiling point range
is from 300°C to about 500°C. Quantitation is based on a standard
composed of n-C2o and n-C24 alkanes, with corrections made for sol-
vent blanks and spiked deuterated compounds. The results are
reported in mg/L or original effluent water, or mg/g of sediment.
Figure C.9-1 depicts the division of a sample FID chromatogram
into TCO and TCG regions.
The samples were analyzed using a Hewlett-Packard 5880 capillary
column gas chromatograph with the following instrument conditions:
- 30 m fused silica column with SE-54 stationary phase
- linear flow velocity - 30 cm/s
- septum purge on at 0.5 min, at 100 mL/min
- initial temperature, 40°C
held at initial temperature, 4 min
- then heated at a rate of 8°C/min
- with a final temperature of 280°C
- total analyis time, 51 min
- injector, splitless mode, temperature, 250°C
- FID temperature - 250°C
- Hewlett-Packard Model 3356 Laboratory Data System
- A/D acquisition rate, 8 samples/s
- injection volume, 1 \iL
C.9-2
-------
10-
o
•
vO
(A)
o
5 5
I—I '
n
ii
*
i
i
1-7
i
r-
i
. L
T(
CO
\
.1 ...
C17
I
i
* — i —
IVAJ
>v
L
10
20
TIME, min
40
Figure C.9-1. Typical TCO/TCG trace on FID/GC.
-------
Four types of standards were analyzed during the GC/FID determina-
tion of TCO's, TCG's, relative retention indices, and spike re-
coveries. Hydrocarbon standards containing C8, C12, C16, C20, and
C24 at 20, 10O, or 250 vg/mL were analyzed to obtain FID response
factors in the TCO and TCG ranges. A standard of C7 and C17 was
used to determine the beginning (C7) and end (C17) of the TCO
range of the chromatogram. A standard of six PNA retention time
marker compounds was analyzed to calibrate the relative reten-
tion index segment of the analysis (see Section C.10), and stand-
ards of deuterated spike compounds were used to determine recover-
ies of spike compounds (see Section C.9.2.3). The numbers of each
type of standard analyzed with each sample set are presented in
Table C.9-1. The numbers of standards were adjusted for each set
to be proportional to the number of samples. Retention time
standards and hydrocarbon standards were run at the beginning
and end of each analysis set to check for any variations in
instrument parameters.
TABLE C.9-1. NUMBERS OF STANDARDS AND BLANKS ANALYZED WITH
EACH GC/FID ANALYSIS SET FOR PHASE III
C7 & C17PNA
Spike retention retention Methylene
recovery Hydrocarbon time time chloride
Analysis set standards standards standards standards blanks
Effluents
Effluent duplicates
Sediments
Sediment duplicates
5
2
6
3
13
4
6
4
7
2
3
2
8
2
4
2
10
3
6
3
C.9.2.2 Example TCO Calculation—
The following describes the calculation of a TCO value for the
sediment sample, C169S: First, a raw TCO, in mg/mL, is obtained
by computer data reduction of an FID run by summing all of the
area integrated between the C7 and C17 retention time limits.
C.9-4
-------
From this raw TCO is subtracted the average raw TCO for the sol-
vent blank, as well as the concentrations of the spiking compounds
eluting in the TCO range, as determined by GC/FID. (GC/MS concen-
trations are used when interferences prevent accurate GC/FID
guantitation.) Finally, the appropriate factors are used to con-
vert this corrected TCO value for the extract back to the TCO
for the original sample.
For example, a raw TCO of 1.369 mg/mL was obtained for the sedi-
ment extract. From this was subtracted the background blank TCO
of 0.002 mg/mL and the spike concentration of 0.133 mg/mL, to
give a corrected TCO of 1.234 mg/mL. The corrected TCO was then
converted from extract concentration to sample concentration as
follows: Sample TCO (mg/g) = Corrected TCO (jj-2) x final extract
mij
volume (mL)
x dilution factor at the instrument x 10-° mL initial extract
5.0 mL of initial extract
fractionated by Bio-Beads
sample wt (g)
- i o-ayi 513 ~ c n »T ~ 10.0 mL
= 1.234 —r- X 5.0 mL X c A -
mL 5.0 mL
A 30.0 g
=0.41 pg/g of sediment
C.9.2.3 QA/QC Results—
C.9.2.3.1 Reproducibility of TCO/TCG analyses—Duplicate analyses
were performed for acid and base/neutral extracts of five plant
effluent samples and for extracts of eleven sediment samples. The
presented in Table C.9-2, while those of the sediments are pre-
sented in Table C.9-3. The duplicates were taken from separate
C.9-5
-------
TABLE C.9.-2.
TCO AND TCG RESULTS FROM DUPLICATE ANALYSES OF ACID AND
BASE NEUTRAL FRACTIONS OF FIVE PLANT EFFLUENT SAMPLES
O
•
vO
I
Plant
No.
B112D
B149S
C150D
C156D
C161D
TCO-Acid fraction, TCO-B/N fraction,
Plant mg/L mg/L
No. Run #1 Run #2 Avg Run tt
8219 0.
8193 0.
8237 0.
8238 1 .
99 0.
23 0.
61 0.
04 0.
8239 0.89 0.
93 0
26 0
.96 2.59
.24 13.38
69 0.65 0.45
95 1
66 0
.00 0.54
.78 1.57
1 Run #2
2.41
TCG-Acid fraction,
mg/L
Avg Run #1 Run HI Avg
2.50
12.49 12.94
0.35
0.35
1.03
0.40
0.45
1.30
0.88
0.22
0.48
0.32
1.29
1.04 0.
0.27 0.
0.61 0.
0.36 0.
1.07 1.
96
25
54
34
18
TCG-B/N fraction,
mg/L
Run HI Run H2
1.68 1.77
7.79 11.91
0.55 0.49
0.29 0.33
2.26 2.16
Avg
1.72
9.85
0.52
0.31
2.21
TABLE
C.9-3.
TCO
AND TCG
OF EXTRACTS
Plant
No.
C161D
C154D
C153D
B126S
B141S
B142S
B143S
C150D
C169S
B124D
A101
Plant
No.
8377
8381
8382
8384
8385
8386
8387
8393
8396
8402
8406
RESULTS FROM DUPLICATE
OF ELEVEN
PLANT
TCO, mg/g
Run HI
0.01
0.26
0.04
0.22
0.21
0.13
1.75
0.04
0.41.
ND°
ND
Run #2 Run #3 Avga
0.01
0.22
0.04
0.16
0.18
0.11
1.73
0.03
0.40
ND
ND
0.01
0.21 0.23
0.04
0.17 0.18
0.20
0.12
1.74
0.04
0.42 0.41
ND ND
ND ND
OR TRIPLICATE ANALYSES
SEDIMENT SAMPLES
TCG,
mg/g
Run HI Run H2 Run H3
0.24
2.08
0.31
3.29
1.26
1.25
3.25
0.38
3.56
0.08
0.11
0.30
1.86
0.32
2.72
1.10
1.14
4.09
0.32
3.65
0.15
0.24
1.70
2.78
3.27
0.20
0.23
Avg
0
1
0
2
1
1
3
0
3
0
0
.27
.88
.32
.93
.18
.20
.67
.35
.49
.14
.19
Avg = average.
ND = less than 0.03 mg/g.
-------
sample vials provided for TCO and GC/MS analyses, and were fre-
quently of different dilutions. In order to isolate instrument
variability from variations due to sample storage and dilution
factors, several sediment samples were analyzed in triplicate.
Referring to Table C.9-3, runs #2 and #3 were taken from the same
vial, were at the same dilution, and were analyzed within four
days of each other, while run #1 was analyzed at an earlier date,
from a separate vial, and usually with less dilution. As expected,
runs #2 and #3 generally provided better agreement.
C.9.2.3.2 Accuracy of TCO/TCG analyses—Measurement of recoveries
of standards spiked into a sample and then carried through the
workup scheme is a very useful tool for gauging the accuracy of
an analysis in terms of the actual percentage of sample that is
quantified at the instrument. Since all samples were not ana-
lyzed by GC/MS for Phase III, the decision was made to measure
recoveries of six deuterated spikes by GC/FID. While there are
some problems with interferences when measuring spike concentra-
tions using a. nonspecific detector such as an FID, this measure-
ment provides a feel for the quantity of material making it
through the sample work-up procedure, and provides a quite accu-
rate measurement when samples are relatively clean. The good
agreement between the GC/MS and GC/FID recovery data attests to
the value of the FID.
Recoveries of the acid spike (phenol-ds) in the acid extract of
the plant effluents can be found in Table C.9-4, while recoveries
of base/neutral spikes (l,2-dichlorobenzene-d4, biphenyl-d10,
pyrene-d12, chrysene-d!2, and perylene-di2) in the base/neutral
extract can be found in Table C.9-5. Recoveries of all deuter-
ated spikes in the sediment extracts are presented in Table C.9-6.
Where samples were analyzed in duplicate or triplicate, spike
recoveries were calculated in duplicate or triplicate, also.
C.9-7
-------
TABLE C.9-4.
RECOVERIES OF DEUTERATED SPIKE COMPOUND (PHENOL-D5) IN ACID
FRACTIONS OF PLANT EFFLUENT SAMPLES,AS DETERMINED BY GC/FID
O
•
to
i
00
Effluent No.
Phenol-D5
Cone, spiked, ug/L
Cone, found, M9/L
% Recovery
Phenol-D5
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Phenol-D5
Cone, spiked, (Jg/L
Cone, found, \iq/L
% Recovery
8182
Method
Blank
223.0
55.0
24.7
8204
B113D
45.8
20.6
8218
C160D
54.1
24.3
8183
B133S
55.7
25.0
8205
B119D
_c
_c
8219
B112D
59.4b
26. 6b
8184
B142S
ND9
0
8206
B124D
53.9
24.2
8225
C153D
59.9
26.9
8185
B141S
54.9
24.6
8207
B111D
54.4
24.4
8230
C155D
66.6
30.0
8186
Reagent
Blank
130
58.2
8208
C169D
50.0
22.4
8233
A109
46.6
20.9
/Plant No.
8188
B143S
82.5
37.0
8209
C151D
446. Od
57.8
13.0
8237
C150D
200.6.
42'8b
21. 3b
8191
B126S
48.7
21.8
8210
C158D
223.0
37.3
16.7
8238
C156D
69. 7b
34. 7b
8192
B147S
48.8
21.9
8211
C157D
223.0
51.9
23.3
8239
C161D
72. 2b
36. Ob
8193
B149S
42. 2b
18. 9b
8212
C164D
59.0
26.4
8240
C154D
59.4
26.6
8194
C169S
68.2
30.6
8217
A101C
55.1
24.7
8245
C159D
54.8
27.3
Not detected.
Average of duplicate analyses.
cCound not be quantitated by FID because co-eluting interference was present.
Spike concentration double because sample volume was only 5 L.
-------
TABLE C.9-5. RECOVERIES OF DEUTERATED SPIKE COMPOUNDS IN BASE/NEUTRAL
FRACTIONS OF PLANT EFFLUENT SAMPLES AS DETERMINED BY GC/FID
O
•
\D
Effluent No. /Plant No.
Ph«nol-Ds
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
1 ,2-Dichlorobenrene-D-4
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Biphenyl-D|0
Cone, spiked, pg/L
Cone, found, \>g/L
% Recovery
Pyrene-D1B
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Chrysene-Du
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Perylene-D|2
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
8182
Method
blank
223.0
ND*
0
118.1
92.1
80.0
101.6
89.3
87.9
99.3
97.1
97.8
83.0
74.2
89.4
85.0
37.3
43.9
8183
8133S
12.1
5.4
76.4
64.7
87.4
86.1
85.4
86.0
8.5
9.0
106
100.0
81.5
81.5
8184
B142S
18.9
8.5
75.1
63.6
76.8
75.5
68.0
68.5
6.5
77
56.9
56.9
6185
B141S
ND
0
130
110
105
103
c
6.1
72
45.2
45.2
8186
Reagent
blank
25.4
11.3
108
91.8
105
104
106
107
10.4
123
88.0
88.0
8188
B143S
ND
0
78.5
66.5
90.6
89.1
61.9
62.3
5.6
65
80.6
80.6
8191
B126S
ND
0
87.9
74.4
87.4
86.0
74.8
75.4
83.0
68.0
81.9
85.0
62.6
73.7
8192
' B147S
ND
0
88.3
74.8
92.4
90.9
63.6
64.1
81.1
97.8
111
131
8193
B149S
B"
0D
w
81 4b
68. 9b
t>
85 -9H
84. 6b
h
so.ej
50.9°
h
46.8°
56. 4b
25.9'
28.6
8194
C169S
5.3
2.4
64.5
54.6
59.3
58.3
43.0
43.3
24.7
29.8
18.2
21.5
8204
B113D
ND
0
80.6
68.2
88.8
87.4
69.9
70.4
77.1
92.9
89.1
105
8205
B119D
ND
0
58.6
49.6
67.4
66.4
59.4
59.8
64.3
77.5
62.3
73.3
8206
B124D
NO'
0
65.6
55.5
72.0
70.8
54.8
55.2
60.6
73.0
60.5
71.2
8207
B111D
ND
0
53.5
45.3
62.3
61.3
49.9
50.2
52.9
63.7
49.5
58.2
8208
C169D
ND
0
81.1
68.7
88.8
87.4
93.1
59.4
63.8
78.8
60.4
72.7
54.7
64.3
(continued)
-------
TABLE C.9-5 (continued)
O
•
vO
I
Effluent No. /Plant No. ' .
Phenol-D8
Cone, spiked, \ig/L
Cone, found, pg/L
% Recovery
1 . 2-Dichlorobenzene-D4
Cone, spiked, pg/L
Cone, found. pg/L
% Recovery
Biphenyl-D|0
Cone, spiked, pg/L
Cone, found. pg/L
% Recovery
Pyrene-D|2
Cone, spiked, pg/L
Cone, found. pg/L
% Recovery
Chrysene-D12
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Perylene-Dj2
Cone, spiked, pg/L
Cone, found/ pg/L
% Recovery
8209
C151D
ri
446. Od
ND
0
A
236.2°
172.8
73.2
d
203.2°
162.4
80. 0
f|
198.6°
162.8
82.0
A
171. 6d
162.0
94.4
A
181.2°
150.0
82.8
8210
C1S80
223.0
NO
0
118.1
63.6
53.9
101.6
63.4
62.4
99.3
64.5
65.0
85.8
75.6
88.1
90.6
88.4
97.6
8211
C157D
223.0
ND
0
118.1
80.1
67.8
101.6
78.4
77.1
99.3
74.2
74.7
65.8
69.1
80.5
90.6
73.1
80.7
8212
C164D
ND
0
68.1
57.7
66.2
65.2
56.3
56.7
62.8
73.2
82.0
90.6
8217
A101
ND
0
79.0
66.9
80.4
79.2
74.4
74.9
70.1
81.7
75.7
83.5
8218
C160D
ND
0
76.1
64.5
77.3
76.1
73.8
74.4
53.8
62.7
54.2
59.9
8219
B112D
NB
0D
K
79.6J
67.4
K
l°7b
106b
W
49. 6b
50. Ob
L
53 -8h
62. 6b
fc-
46. Ob
50. 8b
8225
C153D
ND
0
74.9
63.4
73.9
72.8
72.7
73.2
46.3
53.9
25.8
28.5
8230
C155D
5.7
2.5
86.8
73.5
84.3
82.9
47.8
48.1
49.1
57.2
75.5
83.3
8233
A109
ND
0
59.5
50.4
65.1
64.1
39.3
39.6
33.8
39.5
38.6
42.6
8237
C150D
200.6
BD
0D
K
69 -S
58.8°
h
52. 4b
51. 5b
K
35. 3b
35.6b
1%
31. 4b
36. 6b
L
35- IK
38. 8b
8238
C156D
b
2'°b
1.0°
h
106 b
89.7°
h
38.0b
37. 4b
K
26. 4b
26. 5b
W
22. 4b
26. 2b
»_
24. Ob
26. 5b
8239
C161D
b
8'2b
4.0°
h
148b
126°
w
102b
101b
h
56. 2b
56. 6b
w
56. 5b
65. 8b
44. 5b
49. lb
8240
C1S4D
ND
0
83.5
70.7
78.9
77.6
82.4
83.0
55.3
64.5
39.5
43.6
8245
C159D
ND
0
82.7
70.0
77.0
75.8
69.8
70.3
66.5
77.5
72.5
80.0
*Not detected.
b
Average of duplicate analyses.
Large interfering peak prevented determination by GC/FID.
Spike concentration double because sample volume was only 5 L.
Values for perylene were obtained from a third analysis of this sample, in which no dilution was made. Poor peak shape for perylene
in this extremely dirty sample prohibited accurate quantitation in the first two analyses, in which the sample was diluted 1:10.
-------
TABLE C.9-6.
O
•
u>
I
RECOVERIES OF DEUTERATED SPIKE COMPOUNDS IN ANALYSES OF
EXTRACTS OF SEDIMENT SAMPLES, AS DETERMINED BY GC/FID
Sediment No. /Plant Ho.
Phenol-D5
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
1 ,2-Dichlorobenzene-D4
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Biphenyl-D|0
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Pyrene-D12
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Chrysene-D,2
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Perylene-D12
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
8377
C161D
35.6
"•9a
33.4*
35.9
9.2
25.6*
33.5
14 -5a
43.3*
20 !l*
58.6
33.7
25.0*
74.0*
34.0
27.7*
81.5*
8379 8380
C156D B112D
24.0 53.5
67.4 150
16.8 25.0
46.8 70.0
24.6 -C
73.4 -
34.6 -;
101 -C
f»
42.6 -;
126 -C
52.0 -C
153 -
83B1
C154D
»_
9'°b
25. 4b
1_
V
102B
Jk
24.2*
72. 2b
24.2*
70.7*
jk
32 '4a
96.1*
JK
28.0*
82.5*
8382
C153D
16l
4.4*
20.6*
57.4*
•
26.4*
78.8*
27 •-»:
80.0*
n
36.8-
110*
jk
30.6*
89.8*
8383
C159D
30.8
86.5
21.1
58.8
34.0
101
33.1
96.5
38.6
115
32.9
96.8
B384
B126S
19. 9b
53. 2b
15.3?
45. 3b
h
22 -7h
67. Bb
22. 5b
65. 7b
h
29. 4b
87. 2b
>>
29 -4K
86. 6b
B385
B141S
5'9a
16.6*
18.1-
50.4
m
28.8-
86.0a
22.1*
64.4
m
32 -4a
95.4*
JB
3B.2*
112*
8386
B142S
20.8*
58.3*
15'4«
42.7*
Jk
23.0*
68.7*
24.0*
70.1
104*
j|
33.8*
99.4*
8387
B143S
24.7*
69.4*
19.7*
54.9*
• ,
28.7*
85.7*
26.0*
75.7*
Jk
341*
101*
jk
32.8
96.3*
8393
C150D
12.0*
33.6*
4.0*
11.2*
23 -4a
69.9*
21 •>:
62.1*
Jk
25 -8a
76.6
Jk
28.0*
82.6*
8394
C158D
25.0
70.2
18.4
51.3
26.9
80.3
25.6
74.6
28.3
84.0
30.4
89.4
(continued)
-------
TABLE C.9-6 (continued)
n
•
vO
I
M
to
Sediment No. /Plant
Phenol-Ds
Cone, spiked.
Cone, found.
% Recovery
pg/g
pg/g
8395
C151D
17.5
49.2
8396 8400
C169S A109
h
7.5£ 24.1
21.0° 67.7
8401
B113D
35.6
25.8
72.5
8402
B124D
h
25.5b
71. 7D
8403
C157D
22.7
63.8
8404
B119D
25.0
70.2
No.
8405
C160D
22.1
62.1
8406
A101
h
24. 6b
69.2
8407
C164D
28.6
80.3
8420
Spiked
water
m>
0
1 , 2-Dichlorobenzene-D4
Cone, spiked.
Cone, found.
% Recovery
Biphenyl-Dto
Cone, spiked.
Cone, found.
% Recovery
Pyrene-D12
Cone, spiked.
Cone, found,
% Recovery
Chrysene-D12
Cone, spiked.
Cone, found.
% Recovery
Perylene-Du
Cone, spiked.
Cone, found.
% Recovery
pg/g
pg/g
pg/g
pg/g
pg/g
pg/g
pg/g
pg/g
pg/g
pg/g
10.0
27.9
24.6
73.4
25.1
73.2
27.8
82.6
26.0
76.5
k
3.9? 17.2
10.8 47.9
k
24. 5b 25.4
73.1D 75.8
K
22.2° 20.0
54.7° 58.3
32.3? 21.2
95.9 62.9
k
27.2° 23.2
79.9 68.2
35.9
17.0
47.4
33.5
28.8
86.0
34.3
24.9
72.6
33.7
26.6
78.9
34.0
32.2
94.7
h
17. Ob
47. 3b
h
27.1?
80. 9b
26. 4b
76. 5b
U
31. 4b
73. 2D
k
31.3?
92. 2b
17.1
47.6
24.9
74.3
22.8
66.5
f.
2*.4
78.3
31.6
92.9
19.7
54.9
29.1
86.9
25.9
75.5
29.6
87.8
32.5
95.6
15.5
43.2
25.4
75.8
22.3
65.0
25.1
74.5
30.6
90.0
K
14.6?
40. 8b
k
27.4°
81.9
.
24.9?
72.6b
29.5J
87.7°
h
29. 5b
86. 9b
19.7
54.9
30.0
89.6
27.0
78.7
31.4
93.2
36.7
108
Nt>
0
NO
0
3.7
10.8
31.1
92.3
35.3
104
Average of duplicate analyses.
Average of triplicate analyses.
cCould not be quantitated by GC/FID because interference(s) present.
-------
Tables C.9-7, C.9-8, and C.9-9 provide results of duplicate (or
triplicate) recovery determinations for acid extracts of efflu-
ents, base/neutral extracts of effluents, and sediment extracts,
respectively.
In general, the recovery tables indicate that there is some sample-
to-sample variability in the extraction efficiency of organic com-
pounds, probably due primarily to matrix effects. None of the
GC/FID or GC/MS data have been corrected for recovery, but the
reader should be aware that the actual levels of organics in the
samples are probably higher than those stated in the tables, espe-
cially for the sediment samples. The sediment spiking (Table C.9-6)
was performed after freeze-drying, a process in which large quan-
tities of semivolatile compounds may be lost. The recoveries of
deuterated spikes from a water sample spiked prior to freeze-
drying (sample 8420-spiked water) demonstrate this phenomenon.
C.9.3 GRAV Analysis
Gravimetric analyses were used for quantitative determination of
the mass of organics with boiling points in excess of 300°C. In
the case of the effluents, 4 mL aliquots of the concentrated
extracts were evaporated to dryness in a hood and then desiccated
over Drierite® for 24 hours and weighed to constant weight
(±0.1 mg). For sediments, 3 mL of the GPC fraction II concentrate
was used for the GRAV determination. The evaporated residue
weights were then used to calculate the GRAV results in terms of
mg/L of original effluent sample and pg/g lyophillized sediment.
C.9-13
-------
TABLE C.9-7.
DEUTERATED SPIKE RECOVERIES RESULTING FROM DUPLICATE GC/FID
ANALYSES OF ACID FRACTIONS OF FIVE PLANT EFFLUENT
O
Effluent No. /Plant No.
Phenol-D5
Cone, spiked, (jg/L
Cone, found, pg/L
% Recovery
Phenol-D5
Cone, spiked, |Jg/L
Cone, found, \ig/L
% Recovery
8193
B149S
223.0
35.0
15.7
8238
C156D
69.7
34.7
8193 Dup
B149S
49.4
22.1
8238 Dup
C156D
82.7
41.3
6193 Avg
B149S
42.2
18.9
8238 Avg
C156D
76.2
38.0
8219
B112D
53.4
23.9
8239
C161D
72.2
36.0
8219 Dup
B112D
65.4
29.3
8239 Dup
C161D
66.5
33.2
8219 Avg 8237
B112D C150D
200.6
59.4 33.5
26.6 16.7
8239 Avg
C161D
69.4
34.6
8237 Dup 8237 Avg
C150D C150D
52.0 42.8
25.9 21.3
-------
TABLE C.9-8. DEUTERATED SPIKE RECOVERIES RESULTING FROM DUPLICATE GC/FID
ANALYSES OF BASE/NEUTRAL FRACTIONS OF FIVE PLANT EFFLUENT SAMPLES
I
M
in
Effluent
Phenol-Dg
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
1 ,2-Dichlorobenzene-D«
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Biphenyl-D|0
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Pyrene-Dijj
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Chrysene-Dt2
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
Perylene-Dj2
Cone, spiked, pg/L
Cone, found, pg/L
% Recovery
"Not detected.
8193
8193 Dup
B149S B149S
223.0
ND ND
0 0
118.1
77.3 85.5
65.4 72.4
101.6
79.9 91.9
.78.7 90.4
99.3
46.9 54.3
47.2 54.6
83.0
46.3 47.4
55.8 57.1
85.0
.ND .ND
ob ob
t^J*..^* _ ft_I_a_ 1
8193
Avg 8219
B149S B112D
ND ND
0 0
81.4 64.3
68.9 54.5
85.9 87.7
84.6 86.4
50.6 49.6
50.9 50.0
85.8
46.8 43.5
56.4 50.6
90.6
.ND 41.8
0 46.1
B219
Dup
B112D
ND
0
94. 8
80.2
127
125
49.7
50.0
64.1
74.7
50.3
55.6
__ A A. ,
B219
Avg
B112D
ND
0
79.6
67.4
107
106
49.6
50.0
53.8
62.6
46.0
50.8
.._. ^J 1
8237
C150D
200.6
ND
0
61.9
52.4
50.1
49.3
34.5
34.8
31.6
36.9
37.2
41.1
..a._.J • _
No. /Plant No.
8237
Dup
C150D
ND
0
76.9
65.1
54.6
53.7
36.1
36.4
31.2
36.4
33.0
36.4
« A £__
B237
Avg
C150D
ND
0
69.4
58.8
52.4
51.5
35.3
35.6
31.4
36.6
35.1
38.8
»U_. -*- —
8238
C156D
ND
0
94.8
60.3
37.2
36.6
24.6
24.7
21.4
24.9
24.5
27.1
1 1 __*._.
8238
Dup
C156D
4.0
2.0
117
99.1
38.7
38.1
2B.1
28.3
23.5
24.7
23.5
25.9
8238
Avg
C156D
2.0
1.0
106
89.7
38.0
37.4
26.4
26.5
22.4
26.2
24.0
26.5
~ uk 4 _l
8239
C161D
6.7
3.3
142
120
99.2
97.6
51.9
52.2
53.2
62.0
42.8
47.2
i
8239
Dup
C161D
96
4.8
155
131
106
105
60.6
61.0
59.8
69.7
46.2
51.0
t_*« 4M <
8239
Avg
C161D
8.2
4.0
148
126
102
101
56.2
56.6
56.5
65.8
44.5
49.1
tt..
perylene-d,2 spike being below the detection limit. The sample was analyzed a third time without dilution, and perylene-d(2
was found at 25.9 pg/L, or 28.6% recovery.
-------
TABLE C.9-9.
O
DEUTERATED SPIKE RECOVERIES RESULTING FROM DUPLICATE OR TRIPLICATE
GC/FID ANALYSES OF EXTRACTS OF ELEVEN PLANT SEDIMENT SAMPLES
Sediment 8377
Plant C161D
Phenol-D5
Cone, spiked, pg/g
Cone, found. \tg/g
% Recovery
1 , 2-Dichlorobenrene-D4
Cone, spiked, M9/9
Cone, found, \tg/g
% Recovery
Biphenyl-Dio
Cone, spiked, UQ/9
Cone, found, M9/9
% Recovery
Pyrene-D12
Cone, spiked, M9/9
Cone, found, (19/9
% Recovery
Chrysene-Djj
Cone, spiked, pg/g
Cone, found, M9/9
% Recovery
Perylene-DiS
Cone, spiked, M9/9
Cone, found, (Jg/g
% Recovery
Run il
35.6
12.0
33.7
35.9
9.3
25.9
33.5
14.7
43.9
34.3
19.6
57.7
33.7
22.7
67.4
34.0
30.9
90.9
Run #2
11.8
33.1
9.1
25.3
14.3
42.7
20.4
59.5
27.2
80.7
24.5
72.1
Avqa Run il
11.9 10.6
33.4 29.8
9.2 3.9
25.6 10.9
14.5 26.6
43.3 79.4
h
20.1 -?
58.6 -b
W
25.0 -I
74.0 -b
w
27.7 -I
81.5 -b
Sediment 8381
Plant C154D
Run 12
8.7
24.4
3.6
10.0
23.8
71.0
25.8
75.2
33.9
100.6
29.4
85.6
Run «3
7.8
21.9
3.5
9.7
22.2
66.3
22.7
66.2
30.9
91.7
26.7
78.5
Avg
9.0
25.4
3.7
10.2
24.2
72.2
24.2
70.7
32.4
96.1
28.0
82.5
Sediment 8382
Plant C153D
Run ftl
1.5
4.2
20.6
57.4
26.2
78.2
23.9
69.7
29.2
86.6
31.9
93.8
Run «2
1.6
4.5
20.7
57.7
26.5
79.1
31.0
90.4
44.3
131
29.2
85.8
Avg
1.6
4.4
20.6
57.4
26.4
78.8
27.4
80.0
36.8
110
30.6
89.8
Run «1
22.7
63.8
16.8
46.8
25.6
76.4
26.4
77.0
37.4
111
41.9
123
Sediment 8384
Plant B126S
Run *2
18.0
50.6
16.8
46.8
22.4
66.9
20.8
60.6
26.2
77.7
24.3
71.5
Run «3
16.1
45.2
15.2
42.3
20.1
60.0
20.4
59.5
24.6
73.0
22.1
65.0
Avg
19.9
53.2
15.3
45.3
22.7
67.8
22.5
65.7
29.4
87.2
29.4
86.6
(continued)
-------
TABLE C.9-9 (continued)
o
I
»-•
•J
Sediment 8385
Plant B141S
Phenol-Ds
Cone, spiked, pg/g
Cone, found, pg/g
% Recovery
1 , 2-Dichlorobenzene-D4
Cone, spiked, pg/g
Cone, found, pg/g
% Recovery
Biphenyl-Dio
Cone, spiked, pg/g
Cone, found, pg/g
% Recovery
Pyrene-Dla
Cone, spiked, pg/g
Cone, found, pg/g
% Recovery
Chrysene-Di2
Cone, spiked, pg/g
Cone, found, pg/g
% Recovery
Perylene-D18
Cone, spiked, pg/g
Cone, found, pg/g
% Recovery
Run «1
35.6
S.8
16.3
35.9
17.3
48.2
33.5
28.5
85.1
34.3
21.2
61.8
33.7
30.8
91.4
34.0
42.3
124
Run »2
6.0
16.9
18.9
52.6
29.1
86.9
23.1
67.3
34.1
101
34.2
101
Avg
5.9
16.6
18.1
50.4
28.8
86.0
22.1
64.4
32.4
95.4
38.2
112
Sediment 8386
Plant B142S
Run *1
23.0
64.6
15.7
43.7
25.8
77.0
24.5
71.4
34.0
101
41.8
123
Run 02
18.5
52.0
15.0
41.8
20.2
60.3
23.6
68.8
36.1
107
25.8
75.9
Avg
20.8
58.3
15.4
42.7
23.0
68.7
24,0
70.1
35.0
104
33.8
99.4
Sediment 8387
Plant B143S
Run «1
27.9
78.4
19.4
54.0
31.3
93.4
22.3
65.0
35.1
104
43.4
128
Run *2
21.5
60.4
20.0
55.7
26.1
77.9
29.6
86.3
33.1
98.2
22.1
65.0
Avg
24.7
69.4
19.7
54.9
28.7
85.7
26.0
75.7
34.1
101
32.8
96.3
Sediment 8393
Plant C150D
Run tl
12.0
33.7
4.0
11.1
23.7
70.7
21.5
62.7
25.9
76.9
30.2
88.8
Run 12
11.9
33.4
4.1
11.4
23.2
69.3
21.1
61.5
25.7
76.3
25.7
75.6
Avg
12.0
33.6
4.0
11.2
23.4
69.9
21.3
62.1
25.8
76.6
28.0
82.2
(continued)
-------
TABLE C.9-9 (continued)
o
O>
Sediment 8396
Plant C169S .
Phenol-Ds
Cone, spiked, M9/9
Cone, found, |ig/g
% Recovery
1 ,2-Dichlorobenzene-D4
Cone, spiked, pg/g
Cone, found, M9/9
% Recovery
Biphenyl-D10
Cone, spiked, M9/9
Cone, found, pg/g
% Recovery
Perylene-Di2
Cone, spiked, M9/9
Cone, found, M9/9
% Recovery
Chrysene-D12
Cone, spiked. M9/9
Cone, found, M9/9
% Recovery
Perylene-D12
Cone, spiked, (19/9
Cone, found, M9/9
% Recovery
Run ftl
35.6
10.2
28.7
35.9
4.6
12.8
33.5
29.5
88.1
34.3
25.0
72.9
33.7
37.9
112
34.0
25.5
75.0
Run «2
6.3
17.7
3.6
10.0
22.1
66.0
20.7
60.3
32.7
97.0
28.9
85.0
Run #3
5.9
16.6
3.5
9.7
21.8
65.1
20.9
60.9
26.4
78.3
27.1
79.7
Avg
7.5
21.0
3.9
10.8
24.5
73.1
22.2
64.7
32.3
95.9
27.2
79.9
Run 01
25.4
71.3
16.7
46.5
28.4
84.8
24.6
70.0
28.2
83.7
35.0
103
Sediment 8402
Plant B124D
Run «2
26.1
73.3
17.4
48.5
26.9
80.3
27.6
80.5
33.1
98.2
30.0
88.2
Run #3
25.1
70.5
16.8
46.8
26.0
77.6
27.1
79.0
32.9
97.6
29.0
85.3
Avg
25.5
71.7
17.0
47.3
27.1
80.9
26.4
76.5
31.4
93.2
31.3
92.2
Run 81
25.7
72.2
14.9
41.5
29.8
89.0
25.1
73.2
28.6
84.9
35.0
103
Sediment 8406
Plant A101
Run t»2
25. 0
70.2
14.9
41.5
27.4
81.8
25.7
74.9
31.2
92.6
28.5
83.8
Run t»3
23.2
65.2
14.1
39.3
25.1
74.9
23.9
69.7
28.8
85.5
25.1
73.8
Avg
24.6
69.2
14.6
40.8
27.4
81.9
24.9
72.6
29.5
87.7
29.5
86.9
Average.
Could not be quantitated when undiluted sample was analyzed because column overloading resulted in peaks for
spike compounds being poorly resolved from interferences.
-------
C.10 RELATIVE RETENTION INDICES
Relative retention indices for peaks eluting within the GC/FID
and GC/MS chromatograms were determined in order to compare the
data obtained during the TCO/TCG analyses with GC/MS data and
chroma tographic data generated at the Virginia Institute of Marine
Sciences Laboratories by R. J. Huggett. This approach shows the
feasibility of a preliminary identification of components present
in a given extract, based upon this index. A standard mixture of
PNAs was prepared and analyzed several times over the course of
each sample set. Retention times of all sample peaks falling in
the range of the PNA marker compounds
RRIphenanthrene = 200' ""pyrene = 300' ^'chrysene = 400'
RRIperyiene = 500' ^benzoCghiJperylene = 60°3 were automatic-
ally calculated using the HP 3356 Laboratory Automation System.
When long series of runs were scheduled, as was the case with the
56 effluent extracts, PNA marker standards were analyzed at fre-
quent intervals to monitor any retention time shifts. When slight
fluctuations did occur, the relative retention time data were
corrected for these shifts.
The method adopted by MRC uses the RRIs based on a group of PNA
marker compounds used by the VIMS group. This approach is very
similar to the Kovats indices developed in 1965 by E. Kovats [8].
A similar approach is also discussed by L. S. Ettre [9-11].
[8] E. Kovats, Advances in Chromatography, Vol. 1, J. C. Gidding
and R. A. Keller, eds. Marcel Dekker, Inc., New York, New
York, 1965. pp. 229.
[9] L. S. Ettre, Chromatographia, 6:489, 1973.
[10] L. S. Ettre, Chromatographia, 7:39, 1974.
[11] L. S. Ettre, Chromatographia, 7:261, 1974.
C.10-1
-------
The strength of the present approach lies in the large amount of
data presently available at VIMS on Chesapeake Bay sediment and
fish tissue extract analyses. The data presented in the present
section show the strength of this approach when using state-of-
the-art GC/FID and GC/MS instrumentation. However, other capil-
lary chromatographic/specific detector instrumentation (i.e.,
GC/EC, GC/Hall, GC/PID, etc.) may also be used for screening
large amounts of sample extracts, and these data can also be eas-
ily correlated with capillary GC/MS data through the use of RRIs
generated using the MRC protocol. The major weakness, however,
of this approach is the choice of the PNA marker compounds. A
much more versatile index such as the Kovats Retention Index (KRI),
or that developed by M. Lee [12], eliminates the problem of com-
ponents eluting before biphenyl or after benzo(ghi)perylene. In
the present study, if our approach had been the use of Kovats
Retention Indices, all data obtained could be assigned KRIs, since
the latter index is limited only by the high molecular weight
limit of the chromatographic analysis system. However, components
eluting in the first half of the TCO region could not be assigned
RRIs, since they eluted before the elution time of biphenyl. The
following sections show how the RRIs generated within the present
study could be used for the screening of sample extracts of interest
to the Chesapeake Bay Program, and address several problems in the
implementation of this approach.
C.10.1 RRIs of Deuterated Spiking Compounds
In order to evaluate how reproducible the present RRI approach is
in the analysis of real sample extracts, RRIs of the deuterated
surrogate spiking compounds present in each extract were compared
for all effluent and sediment extracts. Tables C.10-1 through
C.10-4 show the RRIs calculated for pyrene-d10, chrysene-dj2/ and
[12] Lee, M. L., D. L. Vossilaros, C. M. White, and M. Novotny,
Anal. Chem., 51:766, 1979.
C.10-2
-------
TABLE C.I 0-1. RELATIVE RETENTION INDICES FOR THREE SURROGATE
SPIKING COMPOUNDS IN PHASE III EFFLUENTS
, _ _ HRC Effluent workup number _ . _ .
Spike compound 8182 8183 8184 B185 8186 '8188 8191 8192 8193 8194 8204 8205 8206 6207 6208 8209 8Z10
Pyrene-d,0 299.12 299.28 299.46 Ia 300.04 298.22 298.36 298.21 297.42 297.99 298.24 298.01 298.11 298.66 297.84 298.18 298.42
Chrysene-d,2 398.02 397.71 397.98 398.24 398.50 397.23 397.37 397.42 396.69 397.11 397.37 397.22 397.30 397.42 396.83 397.44 397.53
Perylene-d,, 497.54 497.94 498.40 HPb 499.13 497.40 497.36 497.48 HP HP 497.14 497.22 497.36 497.26 496.44 497.44 497.14
, _ . _ MRC Effluent workup number _ . _ __ Standard
8211 8212 8217 8218 8219 8225 6230 6233 8237 8238 8239 8240 8245 Average Range deviation
Pyrene-dio 298.41 29S.9B 298.23 298.36 298.33 298.34 298.25 298.51 298.25 298.09 298.72 298.64 298.46 298.43 ±1.61 0.53
Chrysene-d,2 397.41 398.29 396.86 397.13 397.07 397.16 397.19 397.28 397.42 397.07 397.50 397.31 397.39 397.39 ±1.11 0.41
Perylene-dij 497.24 497.98 496.90 497.21 497.01 496.99 497.23 497.27 497.04 497.05 497.26 497.22 497.43 497.34 ±1.79 0.51
O 'interference present.
l-i No peak, or peak belo
I Range = ±( (Value showing greatest deviation from average) - (average)].
No peak, or peak below detection limit.
TABLE C.10-2. RELATIVE RETENTION INDICES FOR THREE SURROGATE SPIKING
COMPOUNDS IN PHASE III EFFLUENT SPIKING STANDARD
MRC Raw file no. for each analysis Standard
Spike compound BAY6 BAY30 BAY58 BAY80 BAY105 Average Range3 deviation
Pyrene-d10 298.54 297.96 299.96 298.56 298.80 298.76 ±1.20
Chrysene-d12 397.41 396.75 398.97 397.40 397.66 397.64 ±1.33
Perylene-d12 497.48 496.52 498.67 497.06 497.70 497.47 ±1.20
0.74
0.82
0.80
Range = ±[(Value showing greatest deviation from average) - (average)].
-------
TABLE C.10-3. RELATIVE RETENTION INDICES FOR THREE SURROGATE
SPIKING COMPOUNDS IN PHASE III SEDIMENTS
___^ HRC Sediment workup number
Spike compound 8377
8379
8380
8381
8382
8383
8384
8385
8386
8387
8393
8394
8395
Pyrene-d10 298.62 299.40 Ic
Chrysene-d,2 397.68 398.64 I
Perylene-d12 498.07 498.94 I
299.65 299.09 298.82 298.99 298.67 298.74 298.86 298.45 298.58 298.45
399.34 398.38 397.98 398.46 397.71 397.91 397.83 397.50 397.55 397.27
498.99 498.73 497.97 498.79 498.12 498.29 498.17 497.84 497.63 497.35
O
I
*>
8396
8400
8401
MRC Sediment workup number
8402
8403
8404
8405
8406
. Standard
8407 Average Range deviation
Pyrene-dto 298.83 298.67 298.70 298.68 298.62 298.66 298.66 298.67 298.70 298.79 ±0.86 0.29
Chrysene-d12 398.25 397.75 397.69 397.75 397.68 397.76 397.77 397.79 397.84 397.93 ±1.41 0.46
Perylene-d12 497.98 497.85 497.88 497.77 497.79 497.76 497.86 497.78 497.83 498.07 ±0.92 0.44
"interference present.
Range - ±[(Value showing greatest deviation from average) - (average)].
TABLE C.10-4. RELATIVE RETENTION INDICES FOR THREE SURROGATE SPIKING
COMPOUNDS IN PHASE III SEDIMENT SPIKING STANDARD
MRC Raw file no.
Spike compound
Pyrene-d10
Chrysene-d12
Perylene-d12
CBS6
298
397
497
.25
.07
.05
CBS 16
298.90
397.69
498.10
CBS32
298.64
397.54
497.68
for each
CBS35
298.50
397.56
497.68
analysis
CBS46
298.52
397.66
497.75
CBS23
298.51
397.44
497 . 50
Average
298
397
497
.55
.49
.63
Range
±0.35
±0.42
±0.58
Standard
deviation
0.21
0.23
0.34
Range = ±[(Value showing greatest deviation from average) - (average)].
-------
perylene-d12 in 30 effluent sample extracts, 5 effluent spiking
standards, 22 sediment extracts, and 6 sediment spiking stand-
ards analyzed by the GC/FID method. The standard deviations of
all of these values range from 0.21 to 0.82 RRI units and appear
to be independent of sample matrix effects. For example, the
average RRIs calculated for the three deuterated compounds are
independent of whether the matrix is a clean spiking standard or
a highly contaminated extract of an effluent or sediment sample.
Similar data are shown for RRIs of pyrene-d10/ chrysene-da2,
perylene-d12, and anthracene-d10 calculated from GC/MS analyses
of effluent and sediment extracts shown in Tables C.10-5 and
C.10-6. In fact, the absolute values of the RRIs calculated for
a given deuterated surrogate spiking compound are within one unit,
whether measured from GC/FID or GC/MS data. It should be empha-
sized that the RRIs were calculated using external PNA marker com-
pound standards, and not by spiking the matrix with the marker
compounds as is the standard protocol followed by the VIMS group.
This further shows that the routine analysis of extracts using
the instrumentation described within the present report, is highly
reproducible (at least for PNAs) and thus allows very accurate
determinations of RRIs without contaminating sample extracts with
native compounds.
C.10.2 RRIs of Native PNAs
A number of sample extracts were identified, from GC/MS data to
contain fluoranthene, phenanthrene, and pyrene. Table C.10-7
summarizes the RRIs calculated for these compounds from GC/MS
data (the GC/FID chromatograms were too complex to identify these
compounds unambiguously). As can be seen, excellent agreement is
observed for the RRIs for fluoranthene and phenanthrene.
C.10-5
-------
TABLE C.10-5. GC/MS RELATIVE RETENTION INDICES FOR FOUR SURROGATE
SPIKING COMPOUNDS IN PHASE III EFFLUENTS
Plant number
Spike compound C150D C169D B133S B142S B141S B119D C157D C164D B112D
Pyrene-d10 298.36 298.36 298.36 298.36 297.89 298.36 298.36 298.36 298.83
Chrysene-d12 397.91 397.91 397.91 397.39 398.70 397.91 397.91 397.91 398.17
Perylene-d12 498.02 497.52 498.02 498.02 497.52 498.02 498.02 497.27 498.51
Anthracene-djo 202.34 201.87 201.87 201.87 201.87 201.87 201.87 202.34 202.81
O
•
g Plant number _ Standard
Pyrene-d10
Chrysene-di 2
Perylene-dj 2
Anthracene-dj 0
C155D
298.83
398.17
498.51
202.34
A109
298.36
397.71
499.26
202.34
C156D
298.83
398.17
498.51
202.34
C161D
299.06
398.17
498.51
202.34
B149S
298.59
397.66
498.75
201.88
Average
298
397
498
202
.53
.99
.01
.14
Range
±0
±0
±1
±0
.64
.71
.26
.67
deviation
0
0
0
0
.40
.30
.71
.30
Range = ±[(Value showing greatest deviation from the average) - (average)].
-------
TABLE C.10-6. GC/MS RELATIVE RETENTION INDICES FOR FOUR SURROGATE
SPIKING COMPOUNDS IN PHASE III SEDIMENTS
a Plant number ,
Spike compound C161D C156D C159D B112D C154D C153D A109 B113D B124D C157D B119D C160D A101
Pyrene-d10 299.77 299.77 298.83 299.76 299.30 299.30 298.82 298.82 298.82 298.82 298.82 298.82 298.82
Chrysene-d12 399.22 399.22 398.44 397.14 397.92 398.70 397.66 397.66 397.66 397.66 397.66 397.66 397.66
Perylene-d,2 499.50 499.50 498.76 497.51 498.76 499.50 498.50 498.50 498.50 498.50 498.50 498.50 498.00
Anthracene-d10 201.88 202.35 202.35 202.35 202.35 201.88 201.88 202.35 201.88 201.88 201.88 201.88 201.88
n
•
!-•
o
I Plant number Standard
Pyrene-dlo
Chrysene-d12
Perylene-di2
Anthracene-d10
C164D
299.06
397.66
498.00
201.88
B126S
298.35
397.40
498.50
201.88
B141S
298.82
397.66
498.75
201.88
B142S
298.82
398.18
498.75
201.88
B143S
298.35
396.88
497 . 51
202.35
C150D
298.82
397.66
498.50
201.88
C158D
298.82
397.40
498.00
201.41
C151D
298.82
397.66
498.00
201.41
C169S
298.58
397.66
498.24
202.84
Average
298.95
397.84
498.49
202.00
Range
±0.82
±1.38
±1.01
±0.84
deviation
0.40
0.59
0.55
0.33
8Range = ±[(Value showing greatest deviation from the average) - (average)].
-------
TABLE C.10-7.
RELATIVE RETENTION INDICES FROM GC/MS DATA
FOR SOME PEAKS IDENTIFIED AS PNAs
Sediment
Compound identified Plant workup GC/MS
by GC/MS no. no. peak no.
Relative
retention
index
Fluoranthene
Phenanthrene
Pyrene
A109 8400 21 284.24
B112D 8380 10 284.74
C153D 8382 14 284.74
C154D 8381 13 284.74
C159D 8383 11 284.74
B126S 8384 10 284.25
Average 284.58
Rangea ±0.34
Standard dev. 0.26
B112D 8380 4 199.35
C153D 8382 9 199.68
C154D 8381 8 199.68
C159D 8383 6 199.68
B126S 8384 7 199.35
Average 199.55
Range 0.20
Standard dev. 0.18
B112D 8380 11 298.59
a
Range = ±[(value showing greatest deviation from average) -
(average)].
Standard deviation.
C.10-8
-------
C.10.3 RRIs Measured in Two Sediment Extracts
Two extracts from plants B143S and B141S contained large amounts
of aromatic compounds substituted with various lengths of long
chain hydrocarbons. Figure C.10-1 shows a comparison of the GC/
FID and GC/MS chromatograms obtained from analysis of the sediment
from plant B143S. Above each major peak are the RRIs calculated
from the GC/FID or GC/MS data shown. Values of RRIs calculated
from the GC/FID and GC/MS data from plant B141S are shown in paren-
theses. The good agreement between RRIs generated from GC/MS data
and the fair agreement for RRIs from GC/FID data can be seen in
this figure. This example comparison of sediment extract data
shows at least one problem encountered with the MRC protocol for
effluent analyses and also demonstrates the complexity of the
general problem of assimilating such large volumes of information
into an understandable format which can be used by environmental
scientists to study contamination of the Chesapeake Bay Basin.
The major problem encountered with the data shown in Figure C.10-1
is that the RRIs generated from GC/FID data deviate from RRIs
generated from GC/MS data by approximately 7 units in the worst
case shown in the figure. This is a much larger deviation than
was observed for the deuterated surrogate spiking compounds and
the native PNAs shown in Tables C.10-1 through C.10-7. The source
of this deviation is probably due to column overloading of the
sample analyzed by GC/FID which does not occur in the GC/MS anal-
ysis. The reason for this difference is that the GC/FID data were
generated from the injection of nondiluted extracts while the GC/MS
data were generated after first diluting the extracts by a factor
of 10. This can be seen from the increased tc "ng at the onset
of the major peaks in the GC/FID chromatogram. However, this is
not due to the classical sample overloading phenomenon, since the
RRIs calculated from GC/FID data are earlier than those calculated
from GC/MS data. Normal peak overloading causes retention time to
C.10-9
-------
I
I
i
.M.M M.M «.«• M.M M.M M.M
I tit Ml? IMCUt It 4lMltl W I
••MMl MtM Iwi CMM Pr«l
F.**
tf.M
M.M M
"
«.«• W.
II .M II .M
IMI
O
•
H
O
I
TS
*• i ^1-
a • *. *. »» «• * <•
S ** a «g
<^ fl M M O M
* *.: § s f r
« S * i "
»4 "5 °» *** •* •
-------
be shi f ted to longer retention times [13]. The phenomenon shown
in Figure C.10-1 is due to large amounts of organic material being
deposited on the chromatographic column which changes the retention
characteristics from that of an SE-54 liquid phase to that of the
phase containing the organic matrix of the sample. This change
is not apparent when examining the RRIs of the nonpolar deuterated
PNAs, within the sample, but is apparent when analyzing components
which have a large amount of hydrocarbon properties (such as long
chain alkyl benzenes) shown in Figure C.10-1. This example shows
the necessity to reduce the amount of column overloading by using
wide bore capillary columns and also taking care to dilute samples
which are known to contain large amounts of chromatographable
components.
This example comparison of the GC/FID and GC/MS RRIs for these
two plant sediment samples also shows the complexity of the data
that are generated from capillary GC/FID and GC/MS analysis of
extracts. Even with the highly computerized data acquisition and
analysis of samples presently employed in the current study, no
effort has been made to easily treat the data once it is acquired
and analyzed.
In fact, the time required to display the data shown in Figure
C.10-1, transfer RRIs to the figure, and evaluate the data required
from 8 to 16 hours of an experienced scientist's time. These data
only include 9 minutes of a total of 30 minutes of data collected
for the two sediment samples compared. In order to evaluate the
results of this total sediment extract analysis in a similar manner
would require from 3 to 6 days, or about 1 week (if there were
data in this total analysis time). Considering there are approxi-
mately 50 effluent extracts (base/neutral and acid) and 22 sediment
extracts, a similar comparison of the one sediment extract shown
[13] Fales, H. M. J. Chrom. Sci., 19:26, 1981,
C.10-11
-------
in Figure C.10-1 with these approximately 70 analyses would require
more than one-year's time of an experienced scientist. However,
to compare all extracts with the 70 analyses would require approx-
imately 70 years to evaluate in a similar manner.
The above calculation is obviously of a worst case and certain
assumptions can be made to reduce the numbers of comparisons re-
quired. However, it does demonstrate why any detailed analysis of
the capillary data generated is virtually impossible unless
selected analytes are chosen (such as the PNAs as chosen by the
VIMS group), or unless sophisticated pattern recognition approaches
are employed which reduce the time required for a given comparison.
An interim solution to data interpretation is presently being
employed by VIMS (Hugett, et al.) where they search effluent arid
sediment RRI data for components which they have compiled in a
file of known RRIs. However, it should be recognized that this
approach still limits the comparisons to those components which
are within the file of known RRIs. This obviously reduces the
data analysis time, but at the cost of limiting the analysis to
a finite number of components.
A more complete analysis approach for data interpretation by the
Chesapeake Bay Office would be to develop pattern recognition
approaches for the treatment of chromatographic data, similar to
that developed by Sweeley [14] and Bieman [15] for biological
profiling of body fluids. The general subject of pattern recog-
nition approaches to a wide variety of chromatographic analyses is
not new and is also the subject of a recent symposium given by the
Ohio Valley Chromatography Symposium (June 1982). Not until these
[14] Sweeley, C. C., N. D. Young, J. F. Holland, and S. C. Gates,
J. Chrom., 99:507, 1974.
[15] Nau, H., and K. Bieman. Anal. Chem., 46:426, 1974.
C.10-12
-------
methods can be used for data screening (which can also be applied
to all types of analytical and toxicological data) of very complex
environmental samples, will real progress be made in understanding
the sources and potential threats of a wide variety of industrial
discharges to the Chesapeake Bay Basin.
C.10-13
-------
C.ll GC/MS ANALYSES OF EXTRACTABLE ORGANICS
C.ll.l Introduction
All GC/MS analyses were performed by automated capillary column
chromatography using a Hewlett-Packard 5985-A GC/MS with a 5934
data system, which used Revision C Software and included a 7920
multi-drive disc unit. Since the peaks obtained in capillary
column GC/MS analysis are only a few seconds wide (much narrower
than packed-column peaks), it is necessary to scan the mass
spectrometer at its maximum rate to generate reliable mass spec-
tra — approximately 4500 spectra for a 40-minute run, depending
on the initial MS scan delay, as compared to perhaps 700 for a
40-minute, packed-column run. A low detection threshold must
also be used to capture the information contained in small peaks,
which combines with the large number of spectra to consume enor-
mous amounts of computer disc space in a given analysis. In
some cases several GC/MS analyses could fill a data disc cartridge.
Thus, to perform a series of 10 to 20 analyses, using the automatic
injector system, requires the use of several discs for data acqui-
sition. This is only possible in an automated mode on the Hewlett-
Packard system by using the multi-disc drive in which the equivalent
of up to 19 discs could be accessed simultaneously.
C.ll.2 Method
The relevant GC/MS parameters used for the Phase III analyses are
summarized below:
- 30 m fused silica SE-54 WCOT column
- Column head pressure, 7-9 psi_
- MS operating pressure, 1 x 10~s to 4 x 10~6 torr
- Septem purge on at 0.5 min
- Initial temperature, 50°C
- Initial temperature held for 4 minutes
- Heating rate, 8°C/min
- Final temperature, 280°C
C.ll-1
-------
- Total analysis time/ 40 min
- Injector, splitless mode, temperature, 250°C
- Transfer line temperature, 250°C (fused silica column
through this zone, directly to the MS source)
- Electron energy, 70eV
- Source temperature, 200°C
- A/D rate, 1 measurement/0.125 amu;
scan rate, approximately 0.6 s/scan
- Mass spectrometer scan delay, 3.0 min
The routine procedure used for GC/MS analysis was to transfer
150 pL of the sample extract into 1.5 mL sample vials along with
150 pL of a 100 pg/rnL anthracene-d10 in methylene chloride inter-
nal standard. The vial was capped and placed in the autosampler,
which injected approximately 1 pL into the GC, operating in the
splitless mode.
C.11.3 Interpretation of Mass Spectra
The following protocol was followed in interpreting the mass
spectral data obtained from the Phase III extracts:
1) Spectra of compounds detected as peaks in the Hewlett-
Packard BATCH program with a 1% to 5% threshold (depending
upon the complexity of the sample) were automatically com-
pared with standard spectra in the computerized EPA/NIH
mass spectral data base using the Probability Based Search
(PBS) software supplied by Hewlett-Packard. A goodness of
fit parameter was generated (1.0 being a perfect match)
for the ten matches closest to 1.0.
2) The results from these searches were reviewed by an exper-
ienced mass spectroscopist using the Hewlett-Packard sup-
plied spectral comparison software, SPDIF, to compare the
sample spectra with the standard spectra identified by the
SEARCH program.
C.ll-2
-------
3) In cases where the spectral agreement was judged to be ade-
quate, compound identities were assigned. . When compound
isomers produced similar spectra, no specific isomer could
be identified based solely upon mass spectral data.
4) In cases where the agreement was not adequate, identifica-
tions were made on the basis of manual interpretation (when
obvious), or compounds were listed as unknown with their
major masses given in parentheses. If a high even mass
was present with odd fragment masses, the even mass was
underlined to indicate a possible molecular ion.
The approach outlined above was adopted to minimize the cost and
time required for the analyses of large amounts of capillary chro-
matographic mass spectra by maximizing the use of computerized
data interpretation techniques.
To illustrate the use of this protocol, a section of a total ion
chromatogram is reproduced in Figure C.ll-1. The mass spectrum
from the chromatographic peak designated by the arrow was auto-
matically compared with the spectra in the EPA/NIH mass spectral
data base using available Hewlett-Packard software options. The
ouput from this search is illustrated in Table C.ll-1. The loca-
tion of the unknown spectrum which was searched is shown in the
table as spectrum number 12 in File Reference Number (FRN)
23121. This latter data file was generated from the Hewlett-
Packard BATCH Quantification program which measured the areas
of peaks present in the total ion chromatogram of the original
capillary GC/MS data located in FRN 13121.
Library 3000 listed in Table C.ll-1 refers to the EPA/NIH Mass
Spectral Data Base (NSRDS-NBS 63) mass spectral library of over
30,000 compounds. The PBS SEARCH output shown in the table
indicates that 28,767 spectra were searched and seven probable
identifications were found. The seven "hits" are listed in
C.ll-3
-------
** SPECTRUM DISPLAY/EDIT *X
1 UL 125X125 Die + 8184-BNP3 CB142S) 8/13X81 BflH
BTL*8 D13121
FRN 13121
1ST SCXPQ. 463
» .59 V- 1.00
Figure C.11-1.
Portion of total ion chromatogram obtained from the analysis
of the base/neutral fraction from the effluent of Plant B142S,
-------
TABLE C.ll-1.
EXAMPLE OF THE OUTPUT OF THE HEWLETT-PACKARD
PBS SEARCH PROGRAM USED TO IDENTIFY COMPONETS
IN EFFLUENT AND SEDIMENT EXTRACTS
REF. SPECT #= 12 LSN- 12. MW= 0 FRN=23121 RET. TIME= 11.0
92 PEflKSi 24 SIGNIFICANT MflX K 23.8
LIBRflRY 3600 28767 SPECTRfl SERRCHED»
7 HITCS)
.9779 + Benzenet 1>2»3-trichloro- (8CI9CI)
SPEC- 4698 LSN- 10457. MW= 180 C6H3C13
FRN = 3005 CUBS 10458.] CRS « 0006987616 EPfl ft 0000827788
MflTCHING PERKS CONTRMINRTED MISSING PERKS QURL INDEX- 629
20.0 9 54-; .c 0 0* .e e e* MULTIPLIER= .74
.9778 + Benzene* 1»2»4-trichloro- (8CI9CI)
SPEC- 4700 LSN- 10459. MU= 180 C6H3C13
FRN = 3005 CNBS 10460.] CRS « 6000126821 EPfl # 6000027871
MRTCHING PERKS CONTRMINRTED MISSING PERKS QURL INDEX- 728
20.0 9 51* .0 0 0* .0 0 0* MULTIPLIER- .92
.9777 + Benzenei 1 >3»5-trichloro- (8CI9CI)
SPEC- 4699 LSN= 10453. MU= 180 C6H3C13
FRN = 3005 CNBS 10459.] CflS « 0000108703 EPfl ft 6000022208
MRTCHINC PERKS CONTRMINflTED MISSING PERKS QURL INDEX- 692
19.8 9 50* .0 00* .00 0* MULTIPLIER- .83
. CC'1! * ?-:nz-:;-.*• l-brohO-3»5-dichloro- (3CI9CI)
SPEC- 4882 LSN» 15755. MN= 224 C6H3BrC12
FRN = 3008 CNBS 15757.] CflS ft 6019752557 EPfl ft 6000010686
MflTCHING PEflKS CONTflMINflTED MISSING PERKS QUflL INDEX- 699
15.2 7 25* .0 0 0* 3.1 I 16* MULTIPLIER= .57
.6274 * Benzene» 2-brofio-l > 4-dichloro- (3CI9CI)
SPEC- 4880 LSN« 15753. MW= 224 C6H3BrC12
FRN = 3008 CNBS 15755.] CflS ft 0001435563 EPfl ft 0000010687
MflTCHING PEflKS CONTftMINflTED MISSING PEflKS QUflL 'INDEX* 655
15.2 7 24* .0 0 0* 8.1 3 33* MULTIPLIER- 1.04
.6182 * Benzoldeh>de» 3»4-dichloro- (8CI9CI)
SPEC- 3872 LSN- 9631. MW= 174 C7H4C120
FRN = 3005 CNBS
MflTCHING PEflKS
15.0 7 25*
9632.] CflS ft 6606287383 EPfl ft 6660007643
CONTflMINRTED MISSING PEflKS QUflL INDEX- 649
.0 0 O* 8.3 3 34* MULTIPLIER- .71
.6138 # Benzaldehydei 2»4-dichloro- (8CI9CI)
SPEC= 3871 LSN- 9630. MW= 174 C7H4C120
FRN = 3005 CNBS 9631.] CflS ft 6666874426 EPft ft 0600067042
MflTCHING PEflKS CONTflMINflTED HISSING PEflKS QUflL INDEX= 655
15.6 7 23* .0 0 0* 8.3 3 45* MULTIPLIER= .95
C.ll-5
-------
decreasing match order. For each identified compound, the good-
ness of fit value (with 1.0 being a perfect match), is followed
by the compound name and additional information relating to the
location of the standard spectrum and additional parameters cal-
culated as a part of the PBS SEARCH program.
Spectral comparison plots were generated from those "best fit"
candiate compounds identified in the computerized search using
the Hewlett-Packard SPDIF program. Three examples of compari-
sons of standard library spectra of three of the four most likely
candidate compounds with the actual spectrum obtained in the anal*
ysis of the extract are shown in Figures C.ll-2, C.ll-3, and
C.ll-4. Figures C.ll-2 and C.ll-3 show comparisons of two dif-
ferent trichlorobenzene isomers with the unknown spectrum. From
the difference of each of the two spectra plotted in these fig-
ures, it can be seen that less than 30% deviations with the two
standard spectra were measured. Since the two standard spectra
of trichlorobenzene isomers are very similar, the identification
of the peak at 11.0 minutes is listed in the effluent table for
this plant as "Benzene,trichloro-(isomer)." Figure C.ll-4 gives
an example of the comparison of a compound listed as a possible
identification, with the spectrum of the compound eluting at
11.0 minutes. The fact that the difference spectrum shows 100%
deviations, indicates that the particular computer identification
given is incorrect.
These comparative mass spectral plots were reviewed by an expert
mass spectroscopist who, based on experience in mass spectrometry
and mass spectral interpretation, accepted or rejected the
computer-generated identification. In the example, trichloro-
benzene (isomer) was determined to be the proper identification
for the compound in question. It should be emphasized that this
identification is considered to be tentative until confirmed by
the measurement of its relative retention time of an authentic
standard of isomers of the identified compound using similar
C.ll-6
-------
o
FRN 3C«4 SPECTRUM -4698 HU* 180 C6H3C13
B«n*«n«/ 1,2.3-trlchloro- (8CI9CI )
100.0*
60
80
100
120
"iir "|iril> — "'IT —
** * * II •**" * *
180
ll'l*'*
200
FRN 23121 SPECTRUM 12 RET. TIME- n.o
1 UL 125 /1 35 D1C «• 8184-BNP3 (B142S) 8X13x81 BMH DRNt 13121
27.9*
2O
40
,..«,»M,.t.,..-.,
60 80 10O 120 14
140
160
L
.100. OK
180
Figure C.ll-2.
Comparison of the mass spectrum of the highest matching
compound shown in Table C.ll-1 with that of the unknown
compound eluting at 11.0 minutes and shown in Figure C.ll-1
-------
o
I
00
FRN 3OO4 SPECTRUM 4699 MU- ISO C6H3C13
B«ns«n«/ 1,3,6-lrlchlore- (8CI9CI)
ll -n tl rlri r Jl flJIt J|f V T
J.
2O 40 6O 80 1OO 130 14O 160 ISO
30(
100.ON
FRN 33131 SPECTRUM 13 RET. TIHE- ll.O
1 UL 125/125 D10 + 8184-BNP3 (B143S) 8X13x81 BMH DRN* 13121
3O
40
6O
i A | N- r . | i l|' i | i i i |
8O
1OO
13O
14O
160
L
.100. OK
180
eo«
Figure C.ll-3. Comparison of the mass spectrum of the third highest matching
compound shown in Table C.ll-1 with that of the unknown compound
eluting at 11.0 minutes and shown in Figure C.ll-1.
-------
o
i
vO
FRN 3007 SPECTRUM 4882 MU- 224 C6H3BrCVS
B«ns«n«. l~bromo-3,6-dichloro- (8CIOCI)
I - i - | *t ' | - i - |
Y ' | ' I
20 40 60 80 100 120 140 160 180 200 220
100.OX
FRN 23121 SPECTRUfl 12 RET. TIME- 11.0
1 UL 125X125 D1O + 8184-BNP3 (B142S) 8x13x81 BP1H DRNt 13121
ioo.o*
4 t, V 'i
L
• i • i • i • i • !'•• i^ i r • i • T • 'i • i • i n • i • i • • i • i •
20 40 60 80 100 12O 14O 16O 180 200
i - i * i
'820
100.OH
Figure C.ll-4.
Comparison of the mass spectrum of the fourth hightest matching
compound shown in Table C.ll-1 with that of the unknown compound
eluting at 11.0 minutes and shown in Figure C.ll-1.
-------
analytical conditions as those used for the original sample
extract analysis.
Some considerations that go into making decisions on the identity
of components in effluent and sediment extracts include:
1) Unknown spectrum contains all peaks with intensities
greater than 10% of the base peak in the standard spectrum.
2) All mass intensities of multiplets present in the standard
spectrum must also be present in the unknown spectrum with
similar relative intensities.
3) Since the DFTPP spectrum in the low mass region is lower
than that given in the EPA criterion for this compound (see
Section C.11.5), quantitative agreement of ion intensities
was not required for masses below m/e 60. For example,
Figure C.ll-5 compares the spectrum for pentadecane found in
plant B124D sediment extract with that published in the EPA/
NIH mass spectral library. As can be seen, while the mul-
tiplets at m/e 43, 57, and 71 are present in both spectra,
the relative intensities for the lower masses in the spec-
trum generated during Phase III are approximately 50% of those
shown in the standard mass spectral library. However, this
does not prevent the correct identification of this compound.
4) If peaks are present additional to those in the standard spec-
trum of a compound, then it is assumed that the additional peaks
are due to the presence of an unknown co-eluting compound.
In obvious cases of mixed spectra, the PBS SEARCH program
identified the presence of a mixture of spectra with a "+" in
the column following the match factor. Where mixed spectra
are confirmed using the spectral comparison program, the major
identified compound was listed along with an estimate of the
percent of the unknown compound present in the mixed spectrum.
C.11-10
-------
n
FRN 3007
SPECTRUM 3683 flU-
(8CI9CI)
C16H32
J
100.0X
20 40
T[ T
?N 23454
60
r T ^ r • r ^ i •
80 100 12O
160 ISO 200 289
• «•*•".. ••••••^•|N>- , v ««lnM «^M» «*^^ •
FRN 234S4 SPECTRUn 5 RET. TIME- 16.7
1 UL 150/150 D10 + 8402-SED 9X16x81 BHH
69. 9*
DRNS 13450
d
,,
I il
100.0H
20
40 60 80 100 120 140 160 180 200 880
Figure C.ll-5. Comparison of the EPA/NIH Library Spectrum of pentadecane
with the compound eluting at 16.7 minutes in Plant B124D
sediment extract.
-------
Several mass spectra from Phases II and III are shown here to
illustrate those obtained. Figures C.ll-6 (Acenaphthylene) and
C.ll-7 (G!-naphthalene) show the quality of spectra obtained for
most of the compounds identified using the PBS SEARCH program.
Figures C.ll-8 and C.ll-9 show the spectra of substances which
could not be identified. Due to the large amounts of time gen-
erally required to identify unknown compounds which are not in
the EPA/NIH mass spectral library, no attempt was made to man-
ually determine the structures of compounds not identified using
computer search techniques. However, attempts were made to give
additional information such as the major masses found in the un-
known, the molecular weight if the spectrum appears to contain a
molecular ion, and the presence of nonhydrocarbon atoms. However,
the identification of the presence of chlorine, sulfur, or sili-
con in unknown compounds is based solely upon the occurrance of
multiplet ion patterns which are consistent with the naturally-
occurring abundances of isotopes for these species, and is used
only to give more information about the unknown compound's
spectrum.
The good chromatography generally observed in most water efflu-
ent analyses resulted in relatively pure mass spectra being ob-
tained for most compounds detected in water. However, many
sediment extracts contained a very broadly eluting component or
components which resulted in spectra containing ions from the
broadly eluting components and the narrowly eluting compounds.
An example of this is shown in Figures C.11-10 and C.11-11. The
mass spectrum obtained at the retention time marked by the arrow
in Figure C.11-10 is shown in C.11-11. As can be seen from the
mass spectrum, masses 83, 97, and 109 are major ions in the spec-
trum. Figure C.11-10 also shows the response of these three
masses as a function of chromatographic elution time. This dis-
play shows that the major ion chromatograms of the unknown component
or components, eluting as a very broad hump, have similar shapes
as does the broad hump in the total ion chromatogram. Therefore,
C.11-12
-------
o
U)
FRN 8001 SPECTRUn 13 RETENTION TIP1E 13.9
LARGST 41 153.3,100.0 151.3, 34.3 150.3, 14.7 153.1, 11.4
LAST 4J 163.3, 3.3 164.3, 3.0 178.3, 1.7 308.3, 1.7
1 UL GGH-53-13 (7464 B) 1x39x81 BMH 35m FS SP-31 DRN« 19731 PAGE 1
L00.
80
60
40
20
<
e
100
80.
60
40.
30
e
152
ACENAPHTHYLENE
20 40 60 80 100 120 140
16^
1S0 200 220 240 260 280 300 33ft
Figure C.ll-6.
Mass spectrum of acenaphthylene identified
by comparison with an authentic standard.
-------
o
UOF
Lft.^
. v
^ I
80
60
40
20
i*
100
60.
I
i
20
0
1
«C AREA SPECTRUM FRN 19719 PAQE 1 V • l.OO
r-SST 4t 142.1,10O.O 141.1, 89.7 115.8, 83.1 143.1, 10. O
>T 41 143.1, 10. O 158.0, 2.3 807.1, 1.4 815.3, 8.5
•88 •»• 789 -784 -783
Ci- NAPHTHALENE 141
mi iniltiil'lil liiiiii iiilillli iiiiilili'lliinlli'iiiliiill iktiilii'll linn utl hiilim ll'iiiiiim iiuiiih mli
142
80 40 60 80 100 120 14O 160
MS OF PEAK AT 10.88 WIN
1 r«3i - ""•""^fl- -'"'""g^ '""eiS; '""ae^ r"'aao' '""aoS '""" ^.
Figure C.ll-7. Mass spectrum of Cj-naphthalene, tentatively identified
by comparison with a reference spectrum.
-------
o
•
(-•
M
I
H
Ui
r
100
80
60
40
80
0
100
80
60
40
80.
«
0
~9N 13132 SPECTRUM 629
^ST 4» 86.2,100.0 72.2, 24.2
?T 4t 173.2, 1.4 174.3, .1
1 1
SO 40 6O 80
180 ' 200 ' 22O ' 240
RETENTION TIME 9.1
101.2, 22.4 57.3, 19.7
354.2, .0 446.3, .0
PAGE 1 V • 1.00
' ' l
i
100 180 140 160
1 26O ' 283 ' 3O 1 B 320
Figure C.ll-8. Mass spectrum of unknown compound eluting at 9.1 minutes
in plant A109 effluent base/neutral extract.
-------
RETENTION TIME 24.9
L*«
L*.
100
80
60
40
SO
0
100
80
60
40
20
*
0.
=>G9T 4i 216.1. 1C0. 0 71.2* 56.7 85.2, 56.3 57.2, 45.2
rT 4t 212.0, 31.8 216.1,100.0 217.0, 23.4 218.1, 14.6 :
PAGE IV- l.OO
i
, |J
.... ......... ......... ......... ....r... ......... .
20 40
In!
lj|| Ill Ill |l 4 JJ,L
....... . . ... ....|.. . ....| ... . ..r... ... | ... ......... ....|.... . .., ... ....| ... ....,
6O 80 10O 120 140 160
1
I
ISO 200 220 24O 26O 28O 3c D 320
Figure C.ll-9. Mass spectrum of unknown compound eluting at
24.9 minutes in Plant A109 sediment extract.
-------
o
SPECTRUM DISPLAYxEDIT **
1 UL 150X150 Die + 8384-SED (DIL 1*2) 9/17/81 BC1H
50(4)-S8e(89 BTLS4 D13464
FRN 13464
1ST SCxPQ'2444
X- .25 V- 1
83 0
Tt
.^Uw**-**'^^
.
,**»**>**^^
B
as
Figure C.11-10. Total ion chromatogram and selected ion chromatograms obtained
from the analysis of the sediment extract from Plant B126S.
-------
0
•
H .
M
1
00
FRN 13464
LARGST 4>
LAST 4-
100
80
60
40
20
0
100
80
60
40
20
0
SPECTRUM 2857 RETENTION TINE 31.3
97.2,100.0 95.2. 82.2 81.1. 76.2 83.2, 70.6
260.3, 6.0 261.2. 6.0 281.1. 6.5 354.1. 4.3
PAGE 1 V - 1.00
II
20 ' 40 '
1
||
Hill III
••I"" ••••
ia<
.
h'lllllll'l iill'llli '
.
60
llllllU.J
Kl. il.lfllll .li.|IHfiltf|l... ....,
0 200 220
LrJ,
1
'
ii
111 llJiliiJllliULi
80 100 120 140
tUrU
.....U.I..1 l.llll- . — I., ,
lliin.1,
160
|MH ni ,.... ......... ....,..,. „,.,.... ....,..„ .„.!„„ ......... ..„,.... ....,
240 260 280 3JBA 3->«
Figure C.11-11. Mass spectrum obtained at the retention time
shown by the arrow in Figure C.11-10.
-------
spectra taken of compounds eluting as discrete peaks on the
broadly eluting peak will also contain spectra similar to that
shown in Figure C.ll-1. Figure C.ll-9 is an example of this
phenomenon. Table D.l=5 in Appendix D gives results for the
base/neutral extract of the Plant A109 effluent. This unknown
compound gives mass 216 as a possible molecular ion and also lists
the other major ions present in the spectrum. However, most of
the ions listed are due to the broadly eluting hump rather than
the compound of interest. Due to the lack of time and funds for
a more complete analysis, no attempt was made to determine which
of the major ions present in the spectrum of coeluting components
were due to the discrete component measured by the BATCH Quanti-
fying program.
Figure C.11-10 also demonstrates a major difference in the method
of quantification of capillary gas chromatographic/mass spectrom-
etric data and capillary gas chromatographic/flame ionization
detector data. The quantification of mass spectral data given in
tables of Appendix D and E are the results of the Hewlett-Packard
BATCH program. This program attempts to determine peak areas of
discretely-chromatographable components such as those shown elut-
ing at 29.0 and 33.0 minutes in Figure C.11-10. The areas meas-
ured automatically are those shown in the figure. However, the
shaded areas below each of the two peak are not measured in the
BATCH program and should not be measured if the area of the dis-
cretely eluting compound is of interest. However, the values of
TCO and TCG, measured with capillary chromatography/flame ioni-
zation detection, measures the total area of eluting components
which cause a response above a baseline value. For the times
corresponding to the elution of the components at 29.0 and 33.0
minutes, the total areas measured in the TCO and TCG analysis
include A29 0 and Asa o plus B29 0 and Bss o- In addition, the
• * • • '
total area of the broadly eluting hump is also included in the
TCO and TCG analyis, but is not included in any of the areas of
discretely-chromatographable component areas measured from mass
C.11-19
-------
spectral data. This very important difference in quantitation ex-
plains major differences which occur between the TCO and TCG values
determined with flame ionization detection and the sum of compo-
nents measured from capillary chromatographic/mass spectrometric
data. Although a greater level of confidence can be placed on the
accuracy of some "tentative" identifications than of others, there
is no way to quantify this; consequently, many unknowns for which
no standards have been analyzed are given tentative identifications.
C.11.4 Quantitation of GC/MS Data
The calculations used for determining the concentration of or-
ganics in sample extracts were performed in two ways. All levels
of deuterated spiking compounds were determined using the response
of the molecular ion of each of the compounds, relative to the
anthracene-dj0 molecular ion. Therefore, all recovery data were
calculated using authentic standards.
The amounts of all other compounds were determined assuming the
total ion response of each compound to be equal to the response
of the anthracene-d!o internal standard. This latter calculation
was made using the following relationship:
. . . _ „ *.*..!» *v j Total ion area X in unknown
Concentration of X - concentration Anthracene-d10 Total ion area anthracene-d10
x (sample concentration factor)
The sample concentration factor takes into account the extraction
and concentration of the 10-liter sample to a 10-mL extract or
the extraction of 30 g sediment and final volume of 5 or 10 mL in
the sediment extract. None of these data were corrected for recov-
ery of the deuterated spike compounds.
C.ll-5. QA/QC for Extractable Organics
In Phase II, MRC developed a routine procedure for assessing the
condition of the mass spectrometer and the column in the gas
C.11-20
-------
chromatograph. The mass spectrometer was tuned approximately
once a week using perfluorotributylamine (PFTBA) which is the
basis for the Hewlett-Packard AUTOTUNE program. The output from
an average tune is shown in Figure C.11-12.
Samples were run in the automated BATCH AQUIRE mode overnight,
and with each batch of samples a system performance standard was
analyzed. This included the compound decafluorotriphenylphos-
phine (DFTPP) which was analyzed by the Hewlett-Packard program:
DFTPP EPA Criterion Verifier. The output from a typical analysis
by this program is shown in Figure C.11-13. If masses other than
51 and 127 fell outside the ranges for the DFTPP spectrum, the
tuning of the mass spectrometer was re-examined.
Typically, the m/e 51 and 127 on our instrument fell somewhat
below the normal range specified by the program. This is because
the DFTPP criteria were designed for and tested on Finnigan mass
spectrometers, which use DFTPP as their tuning compound. The
Finnigan mass spectrometers are typically tuned to optimize sen-
sitivity in the lower mass region. The Hewlett-Packard autotune
program, wich tunes to PFTBA, optimizes sensitivity for higher
mass ions (m/e 219 and 502). This deviation from the DFTPP
criteria poses very little problem in terms of data interpreta-
tion, once one is aware that intensities of the lower masses should
not be expected to be as high as in the library spectra. The
higher abundances of high mass ions often facilitates interpreta-
tion since the lower masses are common to a wider variety of
compounds.
The substances present in the system performace standard were:
(1) 2,6-dimethylphenol, (2) 2,6-dimethylaniline, (3) decanol,
(4) pentachlorophenol, (5) anthracene-dj0 added prior to analy-
sis, (6) octadecene, (7) octadecane, (8) DFTPP, (9) eicosane,
(10) heneicosane, (11) pyrene, (12) methyl stearate, and (13) chry-
sene. Figure C.11-14 shows a typical chromatogram obtained from
C.11-21
-------
-l0.84
133
ION FOCUS (V)-3S
ENT LENS C M V/ AMU >
X-RAV(V>«154
Ef1ISSION-70
LOG AMP OFFSET- 1
EH VOLTAGE(U)-gS00
Afiu GAIN* 115
AHU OFFSET-113
flASS AXIS GAIN-1.M1S7
HASS AXIS OFFSET— .190491
IONSI POSITIVE
ACTUAL SOURCE TEMP
O
I
K>
65 LIN-1349O
73
ACTUAL ABS REL
HASS ABUND ABUND
68.99 12489 190
218.97 8638 69.16
501.98 888 8.85
OPTION?
X16
A
815 LIN-8639
883 498 LIN-283
506
.54
.59
.58
ACTUAL
ISO
70
819.
503
ISO
ABUND
143
433
86
ACTUAL
ISO RATIO
1.14
5.91
9.81
EFF
WIDTH
.589
.58
.507
Figure C.11-12.
A typical result from tuning the Hewlett-Packard
5985 mass spectrometer with PFTBA.
-------
DFTPP EPA CRITERION UERIFIE*
DRNt 22512
SPECTRUM! 2011
i
to
O)
Mass
*X 51
68
69
70
XX 127
197
198
199
875
365
•441
442
443
R«l. Abund*
9.95296
0
29.7239
0
33.4849
0
100
6.69613
82.6732
1.70276
14.7049
73.8387
16.0357
NOTES 'XX' indicate* out of rang*I
Criterion
30-60)1 MASS 198
< 2* HASS 69
< 2* MASS 69
40-60* MASS 198
< 1* MASS 198
BASE PEAK
6-9)1 MASS 198
10-30* MASS 198
> 1* MASS 198
< MASS 443
> 40* MASS 198
17-23* MASS 448
PRESS TO RERUN...
Figure C.11-13. Computer analysis of mass spectrum of DFTPP,
-------
** S-KCTrtUll OISPLAVXE31T ** FRN 38513
1 UL 39/80 D10 + COL.PERF.STD. 4/10x81 BMH 30m FS 8E-541ST SCxPQt 61O
BTL*13 D33518 X- .25 V- l.OO
o
to
188. C
Figure C.11-14.
Chromatogram of the column performance standard
with the GC column in average condition. The
numbered peaks are identified in the text.
-------
1 UL
** SPECTRUn DISPLAY/EDIT ** FRN 2E51S
D10 •* COL.PERF.STD. 4/10/81 BHH 30m FS SE-541ST SC/PGH535
) BTL»12 DSS518 X- .25 V- 1.00
O
•
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Ui
188.0
I TI
8
11
12
Figure C.I1-14 (continued)
-------
x* spECTaun D:SPLAV/EDIT ** FRN aasia
i uu ase Die + COL.PERF.STD. -ixicxsi BHH 30m FS se-S4isT
Daasia x- .as v- i.oo
o
i
to
iss.e
TI
13
Figure C.11-14 (continued)
-------
the standard. By examining the resolution of the peaks ^20.7 min
and ^24.7 min, and by looking at the tailing of certain peaks
such as decanol and pentachlorophenol, we visually determined the
condition of the column. When the resolution and tailing were
substantially worse than in a new column, the injection port in-
sert was replaced and the front end of the column was broken off
or the column replaced.
In Phase II, to evalute the reproducibility of guantitation using
the BATCH mode for determining peak areas on an actual sample, the
unfractionated acid extract from B142S was analyzed 3 times (by
placing portions of the sample plus the anthracene-d10internal
standard in 3 vials in the autosampler). Taking the mean and the
percent deviation from the mean of the areas of 30 peaks identi-
fied by the BATCH ROUTINE in all 3 samples, it was found that the
average percent deviation was ±21%. It was also observed that
the percent deviations from the mean for 8 of these peaks were
substantially above 21%.
For these peaks the chromatograms were re-examind manually, and
it was apparent that due to the complexity of the sample, i.e.,
overlapping peaks, the computer routine had not correctly identi-
fied the areas to be measured. When they were measured manually,
the average percent deviation from the mean for these 8 peaks
amounted to 10%. Thus, the deviation one should expect on area
replications by automated peak guantitation routines is approxi-
mately 20%, with greater reproducibility possible if manual analy-
sis is warranted. During Phse III, the output of the BATCH peak
detection program was visually compared with the total ion chrb-
matogram and where relative peak areas did not agree with relative
peak heights, the areas of the peaks in question were measured
manually.
To evaluate the linearity of instrument response, a sample con-
taining indene was serially diluted, each time, by a factor of 2
C.11-27
-------
from 292 mg/L to 0.142 mg/L, and the samples were analyzed in the
standard fashion using BATCH mode. A least-squares line was cal-
culated for the actual concentration vs. the total ion area of
the indene peak divided by the m/e 168 area from anthracene-di 0
internal standard (the method always used to compensate for
instrument variations). This gave a slope of 0.0245, an inter-
cept of 0.0721, and a correlation coefficient of 0.998.
The difference between the true concentration at each measurement
and the concentration calculated using the measured area and the
least-squares line was obtained. The mean difference was 4.5 mg/L,
and the mean percent difference was 58%.
This large percent difference reflects the increase in inaccuracy,
when expressed on a percentage basis, at the low concentration
end of the analysis. If one considers only the points with con-
centrations from 2 mg/L to 292 mg/L, the mean percent difference
is 28%.
In order to assess the accuracy of GC/MS quantitation, two stand-
ards were compared with each other. A Supelco phenols standard
was analyzed on 8 February 1981, and a freshly prepared MRC
standard containing many of the same phenols was analyzed on
4 April 1981. In both standards the concentrations of the com-
ponents were close to 100 mg/L. Taking the concentrations in
the MRC standard to be correct, the concentrations of the com-
ponents in the Supelco standard were calculated and compared
with their stated values. The substances analyzed included
2-nitrophenol, 2,4-dinitrophenol, 2,4-dichlorophenol, 4-chloro-
a»-cresol, 2,4,6-trichlorophenol, 2,4-dinitrophenol, 4-nitrophenol,
4,6-dinitro-o-cresol, and pentachlorophenol.
Based on MRC standards the average percent deviation of the values
found from the stated values was 28%, excluding 4-nitrophenol,
where 7 mg/L was found vs. the stated 100 mg/L.
C.11-28
-------
The quantitation of 4-nitrophenol has been a source of difficulty
previously, as it appears to diminish in concenetration with stor-
age time of the standard. The indication from the comparison of
standards is that except for a particularly troublesome compound
such as the 4-nitrophenol, the average uncertainty in the quan-
titation of clean samples in which the unknown component is of
comparable concentration to the standard, is approximately ±30%.
Finally, to show what one may expect for retention times of hydro-
carbon, a standard consisting of normal alkanes from C8 to C34
was analyzed following the usual procedures. The results are
plotted in Figure C.11-15, where it is seen that the first normal
alkane that can be identified by the MRC GC/MS analysis procedure
is C9, b.p. 151°C, and the last one is C34, b.p. ^483°C. The
linearity of elution time vs. boiling point is excellent up to
C30, at which time the oven temperature reaches its maximum,
causing C32 and C34 to depart from the linear relationship.
C.11.6 Analysis of Standards
Three standard solutions were routinely analyzed along with
effluent or sediment extracts in order to verify that good qual-
ity capillary chromatography and mass spectrometric analyses
were being performed on sample extracts. In addition, capillary
chromatographic/flame ionization detector system performance was
also verified using the same set of standards analysis. Fig-
ure C.11-16 shows the total ion chroma*-gram obtained from the
analysis of a benzene standard containing all acid priority
pollutants; Figure C.11-17 shows the analysis of a benzene stand-
ard containing over 80% of the pesticide priority pollutants
(excluding the Aroclor and toxaphene multicomponent priority
pollutants). Figures C.11-19 through C.11-22 show capillary
GC/FID chromatograms obtained from the analysis of the system
performance standard, shown in Figure C.11-14, and the three
C.11-29
-------
u
o
10
20 30
RUN TIME, min
50
Figure C.11-15
Elution times for normal alkanes, C9-C34 in
the GC/MS system. Upper curve: Boiling points
of the alkanes indicated vs. retention time.
Lower curve: GC oven temperature profile.
C.11-30
-------
1 UL
** SPECTRUM DISPLAV/EDIT **
D10 ••• ACID PP STD. 9/83/81 Bf1H
BTL*3 D13488
FPN 13488
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Figure C.11-16.
Total ion chromatogram obtained from the analysis of a
200-pg/mL component acid priority pollutant standard.
-------
** SPECTRUM DISPUW/EDIT XX
UL 150/150 D1O •»• ACID PP STD. 9/23/81 Bf1H
BTL*3 D13483
FRN 13483
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Figure C.11-16 (continued)
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** SPECTRUM DISPLAV.-EDIT **
1 'JL 15OX150 D10 «• ACID PP STD. 9x23x81 BHH
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Fiure C.I1-16 (continued)
-------
** SPECTRUM DISPUW/EDIT ** FRN 13481
1 UL 1?0,'150 D1O •»• HRC B/N tl.8,3,4,BENZENE 9x33x81 BPtHlST SC/PGt 1
Se".4)-2Se BTLtS D13481 X" .25 V- 1.00
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Figure C.11-17,
Total ion chromatogram obtained from the analysis of a
100-pg/mL component base/neutral priority pollutant standard.
-------
** SPECTRUP1 DISPLAY/EDIT ** FRN 13481
I UL 150/150 DIO •*• nRC B/N *1,2,3,4,BENZENE 9x33^81 BMH1ST SC/PG: 929
S0'4>-280(8) BTLtS D13481 X- .85 V- 1.O0
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Figure C.11-17 (continued)
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** SPECTRUM DISPLrtVxEDIT XX FRN 13481
150x150 D10 ••• (IRC BXN *1, 2, 3, 4, BENZENE 9x23x81 BPIH1ST SCxPG*1857
)-380(8) BTLta D13481 *• .25 V- 1.00
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** SPECTRUM DISPLttWEDIT *X FRN 13481
1 UL 15O/15O D10 * MRC B/N *1,2,3,4,BENZENE 9x33/81 BMH1ST SCxPGt2785
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35
Figure C.11-17 (continued)
-------
priority pollutant standards, shown in Figures C.11-16 through
C.11-18. The identification of selected components is given in
each of the above figures. As can be seen, excellent agreement
was observed between the total ion chromatograms obtained from
capillary GC/MS analyses and chromatograms obtained from capil-
lary GC/FID analyses of these standards. Therefore, the correla-
tion of data obtained from these two analytical techniques should
exclude any differences between chromatography from these two
analytical systems.
C.11-38
-------
** SPECTRUM DISPLASVELIT **
UL 150X150 D10 •»• PEST. PP STD. 9x23x81 BflH
!) BTL*4 D13483
FPN 13483
1ST SCxPG:l857
- .35 V- 1
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Figure C.11-18,
Total ion chromatogram obtained from the analysis of a
100-pg/mL component pesticide priority pollutant standard.
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SRMPLE: D10 + SYS INJECTED OT Ili27:53 ON SEP 29, 1981
Method: BOY32 Raw: CBSD35 Proc : *PRC32
Figure C.11-19. FID chromatogram obtained from the capillary GC analysis of the
system performance standard, shown analyzed in Figure C.11-14.
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SRMPLE: D10 + SYS INJECTED PT 11:27:53 OH SEP 29, 1981
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Figure C.11-19 (continued)
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Figure C.11-19 (continued)
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Kl in IDinUi-S COMDI r. nt a a. fir- 1 n i u icr-rrn nr o.rD -50 1001
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acid priority pollutant standard, shown analyzed in Figure C.11-16.
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SRMPLE: D10 + RCID INJECTED RT 9:18:44 ON SEP 29, 1981
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Figure C.11-20 (continued)
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SPMPLE: D10 + B'H
INJECTED PT 7:09:22 ON SEP 29, 1981
Method: BRY32 Raw: CBSD31 Proc : *PRC32
Figure C.11-21.
FID chromatogram obtained from the capillary GC analysis of the base/
neutral priority pollutant standard shown analyzed in Figure C.11-17.
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Figure C.11-21 (continued)
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SPMPLE: Hie + PEST INJECTED OT 10:23:11 OH SEP 29, 1981
Method: BRY32 Raw: CESD34 Proc : *PRC32
Figure C.11-22. FID chromatogram obtained from the capillary GC analysis of the
pesticide priority pollutant standard shown analyzed in Figure C.11-18,
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Adjusted Peak Height =
E x H
where V_ = volume (pL) of methylene chloride extract after volume reduction
£
V. = volume (pL) injected (normally 10 pL)
H = peak height (cm)
If the volume of water extracted does not equal X, the adjusted peak height can be
determined by:
V. „
Adjusted Peak Height = ::= x £- x H
VI VW
where V_ = volume (pL) of methylene chloride extract after volume reduction
V_ = volume (pL) injected (normally 10 pL)
H = peak height (cm)
X = experimentally determined from instrument sensitivity (L)
volume of water extracted (L)
FSD
adjusted cm peak height
FSD (in cm)
X 100
Estimated concentration, ppb «
This calculation is based on the experimentally determined instrument sensitivity
tivity and may be in error by 2 orders of magnitude or more due to the varying UV
response of different compounds at 254 nm.
A positive response is defined as an instrumental response greater than or equal
to 25% full-scale deflection in the region of log P greater than or equal to 3.5.
Figure C.12-2 (continued)
C.12-6
-------
C.12.3 Sample Calculation for Peak 23 (44.08 min) of a Typical
Sample
C.12.3.1 Instrumental Response—
Adjusted Peak height ~ r== x ^- x H
VI VW
VE = 1,000
Vj = 10
H = 2.4 cm
X = 0.911 L
VW=1.0L
Adjusted Peak height = 1^P°° fL x °'g^L.L x 2.4 cm
J.U \JLi J. . U Li
Adjusted peak height = 220 cm
% Full-scale deflection = 'd1"s*|g **** * 100
• TTS «
% Full-scale deflection = 1,500
Note that this calculation results in %FSD >100%.
C.12.3. 2 Determination of Log P—
= 44.08; Log t = 1.6442
Using the linear regression equation Log P = 4.7565 Log
-1.0109, the value for log P is 6.81.
Therefore, Peak 23 gives a positive response since the instru-
mental response is greater than or equal to 25% of the full-scale
deflection and the value of log P is greater than or equal to 3.5.
C.12-7
-------
C.12.4 Data Correlation
An attempt was made to determine the chemical identity of the
positive bioaccumulation responses found in the samples. The
chemical structure of the presurvey compounds and GC/MS identi-
fied compounds listed with their literature value log P [18-26]
[18] Gould, R. F., editor. Biological Correlations - The Hansch
Approach. Adv. Chem. Ser. #114. American Chemical Society,
Washington, D.C., 1972.
[19] Veith, G. D., and D. E. Konasewich. Structure-Activity
Correlations in Studies of Toxicity and Bioconcentration
with Aquatic Organisms. International Joint Commission Pub-
lication, Windsor, Ontario, 1975. 347 pp.
[20] Carlson, R. M., H. L. Kopperman, and R. E. Carlson. Struc-
ture Activity Relationships Applied.
[21] Neeley, W. G., D. R. Branson, and G. E. Blau. The Use of the
Partition Coefficient to Measure the Bioaccumulation Potential
of Organic Chemicals in Fish. Environ. Sci. Technol. 8:1113-
1115, 1974.
[22] Chiou, C. T., V. H. Freed, D. W. Schmedding, and R. L. Kohnert.
Partition Coefficient and Bioaccumultion of Selected Organic
Chemicals. Environ. Science and Technol. 11(5):475-478, 1977.
[23] Vieth, G. D., and N. Austin. Detection and Isolation of Bio-
accumulable Chemicals in Complex Effluents. In: Identifica-
tion and Analysis of Organic Pollutants in Water, L. H. Keith
ed. Ann Arbor Science Publishers, Inc., Ann Arbor, Michigan,
1976. pp. 297-302.
[24] Hansch, C., and T. Fujita. A Method for the Correlation of
Biological Activity and Chemical Structure. J. Am. Chem. Soc.,
86:1616-1626, 1964.
[25] Leo, A., C. Hansch, and D. Elkins. Partition Coefficients
and Their Uses. Chem. Rev., 71:525-616, 1976.
[26] Hansch, C. Computerized Printout of Log P Values by Increas-
ing Log P and Increasing Molecular Carbon Content. Pomona
College, Claremont, California.
C.12-8
-------
The actual log P values found in the samples were then compared
with this information, and any potential correlations were noted.
Experiments conducted at MRC indicated that the HPLC technique
for log P and the published literature values agreed ±0.05 for
polar compounds which are typially more bioaccumulative than
nonpolar ones. This number was used as the criteria for deter-
mining any correlations.
In addition, log P values found in the samples which had no poten-
tial presurvey or GC/MS compound correlation were compared to the
log P literature to attempt to tentatively identify the species,
using the ±0.05 criteria while looking in the literature for com-
pounds of a similar nature to those identified by GC/MS (e.g.,
compounds that could be a wastewater treatment or extraction
artifact of a previously identified compound).
C.12.5 QC/QA For Bioaccumulation Analysis
The following QC/QA procedures were practiced when performing
Phase III bioaccumulation potential analyses:
(1) The instrument sensitivity (pg/25% full-scale deflection)
was determined daily by duplicate injection of a standard
solution containing 215 ug/mL benzene, 510 pg/mL bromo-
benzene, 10 pg/mL biphenyl, 510 ug/mL bibenzyl, 33 ug/mL
p,p'-DDE, and 93 pg/mL PCB.
(2) The linear regression equation was determined daily from
the values obtained from the log of the retention times
and the Federal Register log P values for the calibration
standards.
(3) A minimum of 10% deionized water blanks were run with each
set of analyses.
C.12-9
-------
(4) A minimum of 10% deionized water spikes (Federal Register
calibration standards) were run with each set of analyses.
(5) A minimum of 10% duplicate analyses were run with each set
of analyses.
C.12-10
-------
C.13 BI©ACCUMULATION FRACTIONATION
In order to identify the potentially bioaccumulative compounds
present in the effluent samples, the methylene chloride extracts
were separated and fractionated by high performance liquid chroma-
tography (HPLC). Each fraction was extracted and the extracts
were analyzed by GC/MS.
C.13.1 Method Development
A mixture of six compounds used to calibrate the HPLC response
used in the bioaccumulation studies, was used to develop an ex-
traction method for the identification of major components in
HPLC-fractionated samples. The standard solution contained ben-
zene at 215 ppm, bromobenzene at 510 ppm, biphenyl at 10 ppm,
bibenzyl at 510 ppm, p,p'-DDE at 33 ppm, and 2,2',4,5,5'-penta-
chlorobiphenyl (PCB) at 93 ppm in hexane. One hundred micro-
liters of this standard solution was added to 25 mL of a methanol/
water (85/15) solvent system identical to that used for HPLC sep-
aration and fractionation. The spiked methanoI/water solution was
concentrated to approximately 8 mL using a Kuderna-Danish appa-
ratus. Three, 3.5-mL portions of methylene chloride were used to
extract the concentrated methanol/water solution. After extrac-
tion, the methylene chloride extract was dried and concentrated
using either a stream of dry nitrogen or a Kuderna-Danish evap-
orator. Table C.13-1 summarizes removeries measured using these
two concentration steps. Generally better recoveries were ob-
served using Kuderna-Danish evaporation which was therefore used
for the concentration of the methylene chloride extracts of frac-
tions from Plants B112D, B149S, B141S, C161D, C150D, and B119D.
C.13.2 HPLC Fractionation
The methylene chloride extracts from selected plant effluents
were separated by HPLC and automatically fractionated using a
C.13-1
-------
TABLE C.13-1. EXTRACTION RECOVERIES
Compound
Bromobenzene
Biphenyl
Bibenzyl
p , p ' -DDE
2,2'/4,5,5'-PCB
Percent
Nitrogen
evaporation
0
59.2
80.0
66.9
71.7
recovery
Kuderna-Dani sh
evaporator
0
81.9
113
87.7
97.8
Hewlett-Packard 1084B liquid chromatograph and a fraction collec-
tion accessory. Ten, 10-pL injections were made, and two to five
fractions per extract were collected. The decision as to which
fractions to separate was effected by the amount of the compound
present and the calculated Log P values. The HPLC fractions were
extracted according to the procedure described in C.13.1 and ana-
lyzed by GC/MS.
C.13.3 Results
The extracts of the HPLC fractions were analyzed by capillary GC/MS.
The method blank, instrument blank, and fraction extracts contained
large amounts of well resolved peaks which did not interfere with
the determination of the major components of the plant fractions.
C.13.3.1 Plant B149S—
Figure C.13-1 shows the HPLC chromatogram of the methylene chlo-
ride extract of Plant B149S effluent with the four fractions :
indicated. The approximate Log P range of the components in the
fractions were:
Fraction Approximate log P range
1 3.6 to 3.8
2 4.2 to 4.5
3 4.9 to 5.0
4 5.7 to 5.8
C.13-2
-------
RREft
74620
4B67B
33B6B
3795B
27 IBB
1B41BB
13B6BB
4336B
1759B
4B67B
5869B
4B24B
1179BB
2183BB
3523B
316600
1542BB
2933BB
3255B
2944BB
594BBB
6998B
148600
9298B
757BBB
4229BB
1352BB
1619BB
31BBBB
4933BB
->
Z
Figure C.13-1.
HPLC chromatogram of extract
of effluent B149S.
C.13-3
-------
Figure C.13-2 compares the total ion chromatograms obtained .from
the analysis of the method blank and four fraction extracts of
Plant B149S. The major components of fraction 1 are 1- and 2-
methyl naphthalene which elute between 13 and 14 minutes. The
small component eluting at approximately 14.8 minutes is biphenyl.
The literature Log P value of biphenyl is 3.76 which agrees with
the 3.6 to 3.8 Log P range of fraction 1. The major components of
fraction 2 which elute in the 15 to 16 minute range, are dimethyl
naphthalene isomers. The group of peaks which elute between 9
and 11 minutes are tetramethyl benzene isomers.
Fraction 3 of the Plant B149S extract contains a large number of
low-level components. The compounds which elute between 17 and
18 minutes are trimethyl naphthalene isomers. Those which elute
between 10 and 14 minutes have not been completely identified.
However, dimethyl tetrahydronaphthalene appears to be present.
Fraction 4 of the Plant B149S extract also contains a large num-
ber of components. The major component which elutes at 11.4 min-
utes is n-dodecane (C12 normal hydrocarbon). The other compounds
which elute between 12 an 15 minutes also appear to be hydro-
carbons, however the levels are too low to attempt identification.
We were not requested to analyze completely the acid and base/
neutral fractions from Plant B149S. However, capillary GC/MS
data were acquired for these extracts, due to the very high TCO
values obtained. Figure C.13-3 shows the molecular ion chromato-
grams for methyl napthalene and dimethyl naphthalene isomers
obtained from the analysis of capillary GC/MS data of the diluted
base/neutral extract from Plant B149S. As can be seen, large
amounts of these two compounds are present in the base/neutral
extract. Note that the relative intensities of the m/e 142 mass
chromatogram are very similar to the total ion chromatogram ob-
tained from analysis of fraction 1 and that the relative intensi-
ties of the peaks in the m/e 156 mass chromatogram are very
C.13-4
-------
FPflf
13833
10-
o
•
M
CO
I
Ul
13888
10-
500
13381
10-
13380
10-
500
13884
600
TIME
.25
149-F4
149-F3
149-F8
149-F1
METHOD BLANK
10
11
18
13
14
15
16
Figure C.13-2. Total ion chromatograms obtained from capillary GC/MS analysis of the
method blank and four HPLC fractions of a methylene chloride extract
of effluent B149S (no significant response occurred after 25 min).
-------
o
•
M
U>
I
13383
id-
see
13882
10-
500
13881
10-
500
13880
500
10-
see
Tine
X
.25
Jx
L
149-F4
149-F3
149-F8
149-F1
METHOD BLANK
18
19
80
81
88
83
i
84
85
Figure C.13-2 (continued)
-------
o
W
I
-J
156. C
** S^ECTRUil DISPLrtV'EDIT **
I UL 12?.'i2S 013 > S193-BNP3 tDIL 1 * 10 > 8x14x81 BUM
^:K4v-2S*<8> BTL*8 D13143
«*EPPOR!
FRN 13143
1ST SCxPGi 919
X- .£S V- l.OO
142.fi
Mi,
TI
DinCTHVL NAPHTHALENE
XSOnERS
I1ETHVL NAPHTHALENE
XSOHERS
JL
AA
AAju. ftnftftfA f>A
i
Figure C.13-3. Molecular ion chromatograms of methyl and dimethyl naphthalene
isomers present in the base/neutral extract of effluent B149S.
-------
similar to the major components in fraction 2 (exclusive of the
large impurity peaks).
C.13.3.2 Plant B112D—
Figure C.13-4 shows the HPLC chromatogram of the methylene chlo-
ride extract of Plant B112D effluent with the four fractions indi-
cated. The approximate Log P ranges of the components in the
fractions were:
Fraction Approximate log P range
1 4.1 to 4.5
2 4.9 to 5.1
3 5.3 to 5.6
4 5.8 to 5.9
Figure C.13-5 compares the total ion chromatograms obtained from
the analysis of the method blank and four fraction extracts of
Plant B112D. Only the first fraction of the Plant B112D effluent
contained significant amounts of chromatographable compounds in
excess of those found in the method blank. The compounds eluting
between 15 and 16 minutes are dimethyl naphthalene isomers. The
prominent peak at 16.5 minutes is acenaphthene. The peak at
18.1 minutes is fluorene and the peak at 20.9 minutes is phenan-
threne. Experimentally determined Log P values for acenaphthene,
fluorene, and phenanthrene are 4.15, 4.11, and 4.30, respectively.
These values agree with the approximate Log P range of 4.1 to 4.5
for fraction 1. The concentration of acenaphthene in the original
water sample, based upon the analysis of fraction 1, is approxi-
mately 450 pg/L. This is in good agreement with a value of
302 pg/L measured for this compound in the base/neutral extract
of this plant's effluent and further demonstrates the feasi-
bility of this approach in the identification of high levels of
possible bioaccumulating compounds.
C.13-8
-------
IX
i
H
to
RRER
49170
6920B
11B1BB
6251B
1164BBB
12S1BBB
5B41BB
5496BB
6916BB
2537BB
3B49BB
1287BB
1497BB
17B3BB
4425BB
9826BB
3165BB
3619BB
262900
9616B
3943B
112900
3592BB
9396
Figure C.13-4.
HPLC chromatograms of extract
of effluent B112D.
C.13-9
-------
o
•
H
to
I
(-•
O
FPHl
13288
10-
500
13287
10-
500
13886
10-
5*0
13285
10-
500
13284
10-
500
TlrtE
.2?
8.00
A
112-F4
112-F3
112-F2
112-F1
METHOD 1LANK
8
i
10
i
11
Figure C.13-5. Total ion chrotnatograms obtained from Capillary GC/MS
analysis of the method blank and four HPLC fractions
of a methylene chloride extract of effluent B112D.
-------
o
•
M
U)
13888
1O-
500
13287
500
13286
10-
500
13285
10-
500
13284
10-
600
TIME
X •
113-F4
.25
V
3.00
112-F3
ne-Fi
METHOD BLANK
I 1 7 r-
21 22 23 24
i i i
26 27 28
Figure C.13-5 (continued)
-------
o
•
H*
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I
M
ro
FPNi
13388
10-
509
13887
10-
500
13886
10-
500
13385
10-
500
X •
.25
112-F8
na-Fi
13884
METHOD BLANK
600
TIME
13
14
IS
16
17
18
i
19
80
Figure C.13-5 (continued)
-------
C.13.3.3 Plant B119D—
Figure C.13-6 shows the HPLC chromatogram of the methylene chlo-
ride extract of Plant B119D effluent with the two fractions indi-
cated. The approximate Log P range of the components in the
fractions were:
Fraction Approximate log P range
1 3.5 to 3.6
2 3.9 to 4.0
Figure C.13-7 compares the total ion chromatograms obtained from
two fractions from the extract of Plant B119D effluent with an
HPLC method blank. The blank gave compound peaks at 14.8 min,
16.4 min, 17.9 min, 19.4 min, and 20.7 min attributed to C14-r
C15-, C16-, C17-, and C18-alkanes, respectively. The compound
eluting at 20.6 min is the anthracene-dj0 internal standard which
is added prior to GC/MS analysis.. No measurable components in
Plant B119C were detected in excess of the hydrocarbon contamina-
tion present in the blank.
C.13.3.4 Plant B141S—
Figure C.13-8 shows the HPLC chromatogram of the methylene chlo-
ride extract of Plant B141S effluent with the five fractions indi-
cated. The approximate Log P range of the components in the
fractions were:
Fraction Approximate log P range
1 3.2 to 3.4
2 3.4 to 4.5
3 4.5 to 5.5
4 5.7 to 5.8
5 6.8 to 6.9
Figure C.13-9 compares the total ion chromatograms obtained from
the analysis of the five fractions of the Plant B141S extract.
Only fractions 1 and 2 contained measurable components in excess
of the impurities present in these extracts.
C.13-13
-------
IT uo: —
RREfi
169900
145S00
20140B
395500
29560
92540
26050
104400
5964
vO
•
«•
ro
Figure C.13-6.
HPLC chromatogram of extract
of effluent B119D.
C.13-14
-------
o
u>
I
H
Ul
* . • . •
1
13491
10-
500
13490
10-
500
13489
10-
500
TIME
x • .13 v • i.ae
119-FB
i L
119-Fl
15 16 17 1
. • )
i .
HPLC BLANK
1 1,
8 19 80 21 22 83 84 85 86 87 88 !!9 30
Figure C.13-7. Comparison of the total ion chromatograms obtained from two
fractions of the bioaccumulation extract of effluent B119C
with an HPLC method blank.
-------
RRER
22
65560
256BB
31320
4877B
153000
3427B
16930
53300
28460
108000
12140
1787B
21B5B
2374B
9256
2668
2394BB
VD
•
M
to
Figure C.13-8. HPLC chromatogram of extract
of effluent B141S.
C.13-16
-------
o
•
H
U)
I
FPm
13-498
l
j 10-
! 500
13497
10-
500
13496
10-
500
13495
10-
500
13494
10-
500
TIME
X • .13 V • 1.00
1
1
1 .
I
_u
B141S-F5
1 , 1
IB1416-F4
, L
• B1418-F3
1 1 1.
B141S-F8
ill. . J
B141S-F 1
1,1 i ,
15 16 17 18 19 80 81 88 83 84 85 86 87 88 i 3 30
Figure C.13-9. Comparison of total ion chromatograms obtained from five
fractions of the bioaccumulation extract of effluent B141S
-------
Figures C. 13-10 to C.13-13 compare mass spectra of the four com-
ponents from Plant 8141S extracts with compounds producing similar
mass spectra in the EPA/NIH mass spectral library. Only the com-
ponent eluting at 22.9 minutes in the first fraction produced a
mass spectrum (Figure C.13-10) which agreed well with a library
spectrum. This component was identified as hexadecanoic acid.
The spectra shown in Figures C.13-11 and C.13-12 appear to be of
alcohols and the spectrum shown in Figure C.13-13 appears to be
similar to that identified as hexadecanoic acid. Therefore, it
is probably also a carboxylic acid.
C.13.3.5 Plant B150D—
Figure C.13-14 shows the HPLC chromatogram of the methylene chlo-
ride extract of Plant B150D effluent with the two fractions indi-
cated. The approximate Log P range of the components in the
fractions were:
Fraction Approximate log P range
1 3.5 to 3.6
2 4.2 to 4.3
Figure C.13-15 compares the total ion chromatograms from two
fractions of the bioaccumulation extract of Plant C150D with an
HPLC method blank. The mass spectra of the components in Plants
B141S and C150D fractions were the same for both sets of extracts.
C.13.3.6 Plant C161D—
Figure C.13-16 shows the HPLC chromatogram of the methylene chlo-
ride extract of Plant C161D effluent with the two fractions indir
cated. The approximate Log P values of the components in the
fractions were:
Fraction Approximate log P range
1 3.4 to 3.6
2 6.7 to 6.8
C.13-18
-------
F«N 3010 SPECTRUM 8930
Hvxadecvnolc acid COCI)
256 C16H32O8
20
60
try
w
i
H
vD
FRN 13495 SPECTRUM 2191 RET. TIME* 22.9
1 UL 150X150 D10 + B141S-A-F2 9x23x81 BflH
^
4-H-
i
80 100 120 140 160 180
100.0*
I • Y * ^ i ' i * i it } ' i i * i • i w i i
80 100 120 140 160 180 200 220 240 260
88.8)1
i
.100.0*
I I I I T ^ I
200 220 840 260
Figure C.13-10. Comparison of the mass spectrum of the compound eluting at 22.9
minutes, present in the first fraction of the bioaccumulation
extract of effluent B141S, with that of hexadecanoic acid.
-------
o
•
H*
U>
I
O
FRN 3O1O SPCCTRUn 2944
l-H«ptad«canol (8CIOCI)
256 C17H36O
100.OH
150
200
260
^ -prr- /. -- y
-prr
FRN 13495 SPCCTRUn 2428 RET. TIDE- 25.0
1 UL 150x150 D1O + B141S-A-F2 9/23x81 BflH
66.2H
Figure C.13-11.
100.OH
Comparison of the mass spectrum of the compound eluting at 25.0
minutes, present in the first fraction of the bioaccumulation
extract of effluent B141S, with that of 1-heptadecanol.
-------
FRN 39Id SPECTRUM 2944
1-H«ptad«e»noI < 8CIOCI>
PIU- 256 C17H360
tee.ex
o
w
I
N>
FRN 13495 SPECTRUfl 8438 RET. TIME- 85.8
1 UL ISC/ISC 019 •*• B141S-A-F3 9X83X81 BMH
66.8)1
ice.
Figure C.13-12,
Comparison of the mass spectrum of the compound eluting at 25.2
minutes, present in the second fraction of the bioaccumulation
extract of effluent B141S, with that of 1-heptadecanol.
-------
FRN 3019 SPECTRUM 1314
1-Dotriacontanol (8CI9CI)
MU- 466 C32H66O
5(
»
||
MM 1 * A f j
•i'i"i~iwi"iwi~i"i*i*i*i*i'i*i~i'i*i*i'i*i"i"i"i~iwiwi"i"iwi~i~*"i"i~i'
100 150 200 250 300 350 400
450
100. 0*
i
K>
FRN 13495 SPECTRUM 8468 RET. TIME' 25.4
1 UL 150X150 D10 + B141S-A-F2 9x23/81 BflH
98.9*
,
.pjT
5<
•
i
I,W,
^ "IT1 T*J™
100
art ' / 1 v / 1 i 1
150 200 250 300 350 400 450
Figure C. 13-13
Comparison of the mass spectrum of the compound eluting at 25.4
minutes, present in the second fraction of the bioaccumulation
extract of effluent B141S, with that of 1-dotriacontanol.
-------
IO
•
v£>
ro
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49190
1675B
40760
62570
114BBB
3362B
6778
6B59B
6522B
3B13
1845BBB
-)
z
Figure C.13-14.
HPLC chromatogram of extract
of effluent C150D.
C.13-23
-------
01
Is)
FRN:
i
1
i
13485
10-
500
13484
1O-
500
13489
10-
500
Tine
X • .13 V • 1.00
C1500-F8
1
C150D-F1
15 16 17 1
1 . i i.
HPLC BLANK
. 1
1 t
B 19 80 81 88 83 84 85 86 87 88 89 30
Figure C.13-15. Comparison of the total ion chromatograms from two
fractions of the bioaccumulation extract of effluent
C150D with an HPLC method blank.
-------
VO
fO
vT
B
•
f)
RT
1.59
2.42
2.88
3.B6
3.87
5.B1
6.BB
6.45
7.33
8.63
IB.22
36. BB
flREfl
3263B
8B14
2337B
4135B
112BB
472
1939
2651
475BB
911
7762
99B4BB
n
Figure C.13-16.
HPLC chromatogram of extract
of effluent C161D.
C.13-25
-------
Figure C.13-17 compares the total ion chromatograms obtained from
two fractions of the bioaccumulation extract of Plant C161D. The
mass spectrum (Figure C.13-18) of the compound eluting at 25.3 min-
utes present in the first fraction agrees with hexadecanoic acid
(Figure C.13-10).
Fraction 2 showed no compounds present other than those found in
the method blank. This could be explained by a) the components
have a very large UV absorption at 254 nm giving an inaccurate
indication of the actual amount of material present, or b) the
component is not gas chromatographable.
C.13.4 Conclusion
From the capillary GC/MS analyses of base/neutral and acid ex-
tracts shown in Appendix D, it is concluded that long chain fatty
acids or high molecular weight alcohols, are probably not the com-
ponents which are being measured in the bioaccumulation fractions
from Plants C150D, C161D, and B141S. Since the levels of total
organics present in these three plant extracts, as measured using
capillary GC/MS analysis techniques, are approximately one order
of magnitude less than those levels measured for the extracts of
Plants B112D and B149S, and these latter plants showed only barely
detectable components in excess of the large hydrocarbon inter-
ferences present in the various extracts, there appears to be
inadequate levels of detection for these lower concentrations of
components ,in plant extracts.
The results, however, for Plant B149S show the feasibility of
identifying major components which may be detected in the bioaccu-
mulation analysis of water extracts. However, the sensitivity
for this overall procedure is not very high. Both the results
from the direct capillary GC/MS analysis of the diluted plant
extract and of the fractionated plant extracts indicate that
there is approximately 400 pg/L methyl naphthalene isomers and
C.13-26
-------
o
•
H
W
I
K>
Fpm
1
13500
10-
500
13499
10-
600
TIME
1? 16
17 1
X •
1
I
B 19 80
.13 V • 1.00
C161D-F8
1 .
C161D-F1
1
81 88 83 84 85 86 87 88 8* 30
Figure C.13-17. Comparison of total ion chromatograms obtained from two fractions
of the bioaccumulation extract of effluent C161D.
-------
o
U)
I
K)
00
1
LA«
LH!
100
80
60
40
80
0
100
80
60
40
80.
0.
-RN 13499 SPECTRUM 2461 RETENTION TIME 25.3
?G3T 4t 73.3,100.0 60.3* 51.1 57.3, 42.5 129.3, 38.1
ST 4s 227.5, 2.0 241.5, 7*1 284.7, 11.0 285.7, 1.7
P*GE IV- 1.00
• •
I
""""• "Si ' '&
. ', ,
1B0 20a
Mmmrfi
-_U
60
IT. ....1.... ....,..., ....,
220
III 1
1 .lllllL Jill. ..l.,ll .. • .1
80 100 180 140 160
1 >,
240 260 2B0 30ii 320
Figure C.13-18. Mass spectrum of compound eluting at 25.3 minutes
present in the first fraction of the bioaccumulation
extract of effluent C161D.
-------
600 |jg/L dimethyl naphthalene isomers. The limit of detection
for components in the fractionated sample is approximately one
order of magnitude below this level. However, the large impuri-
ties in the solvents, and the limited amount of sample which can
be fractionated in a reasonable period of time, result in this
analytical technique only being applicable to gross components
of a water extract. If the capillary GC/MS analysis of the ori-
ginal plant effluent extract were conducted for a defined group
of suspected bioaccumulating compounds, levels of detection for
compounds such as PAHs and PCBs would be on the order of 1 pg/L.
Thus, this latter approach is more applicable to bioaccumulating
compounds which need to be detected in the low- or even sub-ppb
range.
C. 13-29
-------
C.14 AQUATIC TOXICITY ANALYSES OF EFFLUENTS
C.14.1 Mysid Shrimp Assay
Marine toxicity tests were conducted at E.G.&G. Bionomics Marine
Research Laboratory (BMRL), Pensacola, Florida, to determine the
acute effect on mysid shrimp Hyszdopis bahia of effluent samples
collected in the Chesapeake Bay area. Eight samples were col-
lected from the State of Maryland and 10 samples from the State
of Virginia. Of the 10 samples collected in the State of Virginia
the toxicity of those from Plants A109, B119D, and C161D was also
evaluated using fathead minnows as described in Section C.14.2.
C.14.1.1 Sample Collection and Shipping—
At each site a 10-gallon (38-liter) sample was collected in
5-gallon polypropylene cubitainers. Following collection, sam-
ples were packed in ice and shipped to BMRL via air freight.
Upon receipt at the BMRL facility, samples were stored at 4°C
until the bioassay testing was started.
C.14.1.2 Experimental Methods—
Methods for th 96-hour static tests were based on those given in
"IERL-RTP Procedures Manual: Level 1 Environmental Assessment
(Second Edition)1' [27]. The criterion for toxic effect was death
of the shrimp, and test results are expressed as 24-, 48-, 72-,
and 96-hour LC50 effluent concentration (the concentration of
sample estimated to be lethal to 50% of the test organisms at the
specified exposure duration).
[27] Lentzen, D., D. Wagoner, E. Estes, and W. Gutknecht. IERL-
RTP Procedures Manual: Level 1 Assessment (Second Edition).
EPA-600/7-78-201, U.S. Environmental Protection Agency,
Research Triangle Park, North Carolina, 1978.
C.14-1
-------
Juvenile mysids were isolated from adults and were acclimated in
flowing, natural seawater until testing was initiated. The ani-
mals were estimated to be six to nine days old at test initiation
and appeared to be in excellent condition.
Twenty-four hours prior to testing, the salinity of each effluent
was adjusted with Rila Marine Mix to a salinity that was approxi-
mately that in which test animals were being cultured and main-
tained. Artificial seawater used in control tanks and for dilu-
tion was deionized water containing an amount of Rila Marine Mix
equivalent to the amount that had been added to the effluent
sample. It was prepared 24 hours prior to test initiation.
Shrimp were definitively tested at nominal effluent concentra-
tions of 3% to 100% (expressed a percentage of effluent in arti-
ficial seawater on a volume/volume basis). Test concentrations
were prepared by adding appropriate volumes of effluent to each
test container and diluting to the appropriate concentration with
artificial seawater. A Rila Marine Mix control was also prepared
by adding an equivalent amount of salts to deionized water as had
been added to each effluent sample for purposes of salinity
adjustment.
Toxicity tests were conducted in one-liter covered glass beakers,
each of which contained one liter of test solution or control
(artificial) seawater. Test solution temperature was maintained
at 22 ±1°C, and aeration was not provided. Five animals were
tested per jar.
C.14.1.3 Quality Control/Quality Assurance Aspects—
For purposes of evaluating effluent toxicity data quality, all
tests were run in triplicate. In addition, control beakers were
used, each containing 100% artificial seawater.
C.14-2
-------
C.14.1.4 LCsn Calculation—
When data were amenable, LCSO values and their 95% confidence
limits were calculated by digital computer. The computer program
estimated LC50 values by one of three statistical techniques in
the following order: moving average angle analysis, probit anal-
ysis, or binomial probability. The method selected was determined
by the characteristics of the data, with the presence or absence of
0% and 100% shrimp mortality and the number of concentrations in
which mortality between 0% and 100% occurred serving as criterion
for selection [28]. The computer scanned the data, identified the
most suitable method, and performed the required calculations.
C.14.2 Fish Assay (Fathead and Sheepshead Minnows)
Effluent samples from 13 industrial operations or sewage treat-
ment plants in the Chesapeake Bay area of Virginia were collected
and transported to the State Water Control Board's (SWCB) bioassay
facility in Richmond. Tests were conducted on 12 of the effluents
to estimate their acute toxicity to fathead minnows, while the
toxicity of the remaining effluent stream (Plant C153D) was esti-
mated using sheepshead minnows as the test species. In addition,
the toxicity of three of these effluents (Plants A109, B119D, and
C161D) was also evaluated using the mysid shrimp assay procedure
described previously in Section C.14.1.
C.14.2.1 Sample Collection and Shipping—
At each site a 110-gallon (416-liter) sample was collected and
stored in upright 55-gallon (208-liter) tanks constructed of
linear polyethylene. After collection and during storage, these
tanks were sealed to prevent loss of volatile components from the
[28] Stephan, C. E. Methods for Calculating an LC50/ ASTM,
Aquatic Toxicology and Hazard Evaluation. ASTM STP 634,
F. L. Mayer and J. L. Hamclink, eds., 1977.
C.14-3
-------
samples. A submersible pump was used to fill each sample con-
tainer, and all connective tubing was polyethylene-lined plastic
(VEV-A-LINE V-HT®). Due to relatively short transport times and
potential experimental problems associated with rewarming the
effluent sample to the test fish acclimation temperature, samples
were not refrigerated during transport to Richmond or subsequent
storage.
The duration between sample -collection and start of bioassay test-
ing was usually less than three hours. To eliminate potential
toxic effects due to the presence of residual chlorine, chlori-
nated samples were aerated for 24 to 48 hours, until total chlo-
rine residual was 0.1 mg/L or less. One chlorinated sewage
effluent sample was tested immediately upon receipt at the SWCB
facility.
C.14.2.2 Experimental Methods—
The detailed basis for the fish toxicity testing methods can be
found in "Methods for Acute Toxicity Tests with Fish, Macroin-
vertebrates and Amphibians" [29] and "Methods for Measuring the
Aute Toxicity of Effluents to Aquatic Organisms" [30]. A 96-hour
static test was employed with the following modification: after
48 hours of test species exposure, the test solutions were renewed
using an aliquot of the initial 110-gallon sample.
The test fish species for all effluents except Plant C153D was the
fathead minnow, Pimpehales promelas. Because of the salinity of
sample C153D, a brackish water species, the sheepshead minnow,
Cyprinodon variegatus, was used. Fathead minnows were obtained
[29] Stephen, C. E. Methods for Acute Toxicity Tests with Fish,
Macroinvertebrates and Amphibians. EPA-600/3-75-009, U.S.
Environmental Protection Agency, 1975.
[30] Peltier, W. Methods of Measuring the Acute Toxicity of Efflu-
ents to Aquatic Organisms. EPA-600/4-78-Dl2, U.S. Environ-
mental Protection Agency, 1978.
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from Kurtz's Fish Hatchery, Elverson, Pennsylvania, and acclimated
in the test dilution water at the SWCB bioassay facility. The
sheepshead minnows were captured, wild stock, from a tidal creek on
Virginia's Eastern Shore (Accomack County). Batches of fish were
treated for specific pathogens only when necessary; no general
prophylaxis was administered. All fish appeared healthy, and pre-
vious observed mortality rates were well within tolerances when
the tests were begun.
Fathead minnows were held in continuous-flow 135-gallon (500-liter
fiberglass raceways prior to the tests. The water supply to these
raceways and also that used to make all test dilutions was tap
water obtained from the City of Richmond Public Utilities. Prior
to use in the holding tanks, this water was treated with carbon
filtration, ultraviolet light, and diffused air.
Sheepshead minnows were held in a recirculating, salt-water hold-
ing tank system having a salinity of 14,000 mg/L. This salinity
was subsequently lowered to 11,000 mg/L shortly before the efflu-
ent test to more closely match the salinity of effluent sample
C153D. Holding tank water was also used to make test dilutions.
Test vessels were all-glass, 10-gallon (36-liter) commercially
available aquaria. The following cleaning procedure was used on
these aquaria before the initial test and between each subsequent
test:
1. Submerge and scrub aquarium in a 2% solution of Micro-
wash®, a commercially available liquid detergent.
2. Allow aquarium to soak overnight in Microwash® solution.
3. Rinse aquarium thoroughly for several minutes with
running dilution water.
4. Rinse aquarium thoroughly, for several minutes, with
running laboratory deionized water and repeat.
5. Invert to drain and dry.
C.14-5
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A similar procedure was used to clean all glassware, tubing,
pumps, and other tanks used throughout the test series.
No fish screening tests were performed with the test species. In-
stead, 96-hour static effluent tests were immediately initiated up-
on receipt of the 110-gallon effluent sample from the field. Each
tank was loaded with ten randomly-selected fish. Five effluent
sample concentrations (10, 18, 32, 56, and 100%) were tested.
Dissolved oxygen, pH, and temperature in each holding tank were
measured initially and at 24-hour intervals through completion of
the testing. A YSI Model 57 D.O. meter was used to measure dis-
solved oxygen and temperature; this meter was calibrated daily
for dissolved oxygen versus the Winkler method (azide modifica-
tion). The meter's temperature function was compared with a
mercury-filled laboratory thermometer.
pH measurements were made with a Corning Model 610A pH meter,
which was calibrated daily using two buffers, one having a pH
of 4.0 and one of 7.0.
C.14.2.3 Quality Control/Quality Assurance Aspects—
As a means of evaluating the quality of the effluent toxicity
data, all tests were run in duplicate. In addition, two control
tanks were used, each containing 100% dilution water. Thus, a
total of 12 tanks and 120 fish were used for each sample tested.
C.14.2.4 96-Hour LCSO Concentration—
Dead fish were counted and removed from each holding tank at
24-hour intervals during the test duration. Data analysis was
accomplished using a log-probability graphical technique. The
fraction of test fish which had died after 96 hours of exposure .
was plotted on the probability scale versus the effluent sample
concentration (expressed as percent by volume) on the logarithmic
scale. These points were connected by straight lines and the -
C.14-6
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effluent concentration which would kill 50% of the test organisms
was read from the graph. This value is defined as the 96-hour
LC50 (lethal concentration).
C.14-7
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C.I5 REFERENCES
1. APHA, AWWA, WPCF, Standard Methods for the Examination of
Water and Wastewater (14th Edition). American Public Health
Association, Washington, D.C., 1977.
2. U.S. EPA, Methods for Chemical Analysis of Water and Wastes.
EPA-625/6-76-003a, National Environmental Research Center,
Cincinnati, Ohio, 1976.
3. Sampling and Analysis Procedures for Screening of Industrial
Effluents for Priority Pollutants. U.S. Environmental Protec-
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of Organic Pollutants in Water and Wastewater. C. E. VanHall,
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6. Standard Methods for the Examination of Water and Wastewater,
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7. IERL-RTP Procedures Manual: Level I Environmental Assessment,
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16. Bieri, R. H., M. K. Cueman, R. J. Huggett, W. Maclntyre,
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Partition Coefficient to Measure the Bioaccumulation Potential
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28. Stephan, C. E. Methods for Calculating and LC50/ ASTM,
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