NT TR 499 - Laboratory and Reporting Instructions For The CEN-BT-TF 120 Oil Spill Identification - Nordtest Technical Report

SINTEF REPORT
TITLE
Laboratory and reporting instructions
for the CEN/BT/TF 120 Oil Spill Identification
Round Robin Test May, 2001
AUTHOR(S)
Per S. Daling and Liv-Guri Faksness
CLIENT(S)
SINTEF Applied Chemistry
Address: N-7465 Trondheim,
NORWAY
Location: S.P. Andersens vei 15A
Telephone: +47 73 59 20 80 / 12 12
Fax: +47 73 59 70 51
Enterprise No.: NO 948 007 029 MVA
Nordtest, attn.: Mads Schreiber
CEN/BT/Task Force 120
REPORT NO. CLASSIFICATION CLIENTS REF.
STF66 A02027 Unrestricted Nordtest Project no. 1520-00, Nordtest Technical Report no. 499
CLASS. THIS PAGE ISBN PROJECT NO. NO. OF PAGES/APPENDICES
Unrestricted 82-14-02677-6 661219.17 54
ELECTRONIC FILE CODE PROJECT MANAGER (NAME, SIGN.) CHECKED BY (NAME, SIGN.)
Report lab instructions.doc
Per S. Daling Alf G. Melbye
FILE CODE DATE APPROVED BY (NAME, POSITION, SIGN.)
2002-05-24 Tore Aunaas, Research Director
ABSTRACT
This report gives instructions for laboratory analysis / methodology and reporting procedures for the
CEN/BT/Task Force 120 Round Robin test performed in May 2001. The Norwegian General
Standardisation Body (NAS) and SINTEF co-ordinated the Round Robin test, and with financial
support from NORDTEST.
The instructions are based on the technical improvements and recommendations obtained through the
on-going NORDTEST project Revision of the NORDTEST- methodology- a collaboration project
between Forensic Oil Spill Identification laboratories in Denmark, Finland, Norway, Sweden, and the
USA.
The existing Nordtest methodology (1991) still forms the conceptual platform. The intention has been to
refine the existing Nordtest methodology into a technically more robust and legally defensible oil spill
identification methodology. The approach is to make more use of:
- (semi-) quantitative analysis of biomarkers and PAHs
- more systematic use of diagnostic ratios and data treatment tools.
This will make the methodology more measurable and objective, which means that the conclusions
will be less based on visual inspections of chromatograms and subjective interpretations of the results.
At the same time, the methodology should be operational and open for flexibility from case to case (i.e.
cost-effective).
KEYWORDS ENGLISH NORWEGIAN
GROUP 1 Chemistry Kjemi
GROUP 2 Oil spill Oljesl
SELECTED BY AUTHOR Round Robin Round Robin
Laboratory instructions Laboratorie instruksjoner
2
I:\CH661219 NORDTEST\Adm\Rapport\Report lab instructions.doc
TABLE OF CONTENTS
1 Background ........................................................................................................................... 5
1.1 Revision of the Nordtest Methodology for Oil Spill Identification.............................. 5
1.2 CEN/BT/Task Force 120: Harmonization of oil spill identification methodology.......... 5
1.3 Development of improved methodology.......................................................................... 6
2 Summary of chemical analytical methodology ................................................................... 7
3 Sample preparation ............................................................................................................... 9
3.1 Oil sample ........................................................................................................................ 9
3.2 Sample clean up................................................................................................................ 9
4 Gas chromatography ............................................................................................................. 9
4.1 Calibration standard ......................................................................................................... 9
4.2 GC conditions................................................................................................................. 10
4.3 Calibration...................................................................................................................... 10
4.4 Sample analysis .............................................................................................................. 11
4.5 Calculations.................................................................................................................... 11
5 Gas chromatography/mass spectrometry.......................................................................... 12
5.1 GC-MS conditions.......................................................................................................... 12
5.2 Preparation for full-scan analysis................................................................................... 13
5.3 Preparation for selected ion monitoring (SIM) analysis ................................................ 13
5.4 Procedures ...................................................................................................................... 14
5.4.1 Calibration....................................................................................................... 14
5.5 Sample analysis .............................................................................................................. 14
5.5.1 Relative retention time .................................................................................... 15
5.5.2 Sample spectrum............................................................................................. 15
5.5.3 Signal to noise ratio......................................................................................... 15
5.5.4 Peak symmetry................................................................................................ 15
5.5.5 Patterns............................................................................................................ 15
5.5.6 Minimum area ................................................................................................. 15
5.5.7 Alkyl homologues ........................................................................................... 15
5.5.8 Tentative identification of unknowns and alkyl homologues ......................... 15
5.5.9 Semi-quantification of PAH-analytes and alkyl homologues......................... 16
5.5.10 Semi-quantification of biomarkers.................................................................. 16
6 Calculations ......................................................................................................................... 17
6.1 Initial and continuing calibration ................................................................................... 17
6.2 Semi-quantification of PAHs ......................................................................................... 17
6.3 Weathering check........................................................................................................... 17
6.4 Diagnostic ratios of PAH ............................................................................................... 18
6.5 Diagnostic ratios of biomarkers ..................................................................................... 19
7 Quality control /Elimination of data .................................................................................. 22
8 Reporting ......................................................................................................................... 23
9 Attachments ......................................................................................................................... 23
10 References ......................................................................................................................... 24
3
Attachment 1: Internal standards (optional) and analytical standards................................. 25
Attachment 2: PAHs and biomarkers ....................................................................................... 29
Attachment 3: Quantification and calibration ......................................................................... 32
Attachment 4. Alkyl homologues patterns of PAHs............................................................ 35
Attachment 5. Peak identity of the biomarkers........................................................................ 45
Attachment 6. Examples, reporting........................................................................................... 48
4
Summary
This report gives instructions for laboratory analysis / methodology and reporting procedures for
the Round Robin test performed in May 2001. The Norwegian General Standardisation Body
(NAS) and SINTEF co-ordinated the Round Robin test, and with financial support from
NORDTEST.
The instructions are based on the technical improvements and recommendations obtained through
the on-going NORDTEST project Revision of the NORDTEST- methodology- a collaboration
project between Forensic Oil Spill Identification laboratories in Denmark, Finland, Norway,
Sweden, and USA.
The existing Nordtest methodology (1991) still forms the conceptual platform. The intention has
been to refine the existing Nordtest methodology into a technically more robust and legally
defensible oil spill identification methodology. The approach is to make more use of:
- (semi-) quantitative analysis of biomarkers and PAHs
- more systematic use of diagnostic ratios and data treatment tools.
This will make the methodology more measurable and objective, which means that the
conclusions will be less based on visual inspections of chromatograms and subjective
interpretations of the results. At the same time the methodology should be operational and open
for flexibility from case to case (i.e. cost-effective). This is illustrated in the schematic Decision /
Protocol Chart in fig. 2.1.
In these instructions for the Robin study, it is opened for a semi-quantitative approach of PAHs in
the oil. Hence, the peak areas of the PAHs could be internally normalised using C30-hopane, and
spiking of internal standards will not be necessary. This should ensure that calculated diagnostic
ratios would express statistical significance, and do the job in comparing two (or more) samples
analysed in the same lab, on the same instrument and at the same day. Optional: a full quantitative
characterisation of PAHs in the oil samples using internal standards. This quantitative option is
specified Attachment 1. A quantitative approach will ensure more comparable results of
diagnostic ratios between different laboratories. PAHs.
5
1 Background
1.1 Revision of the Nordtest Methodology for Oil Spill Identification.
Nordtest (Internet: http://www.vtt.fi/nordtest/) is an institution under the Nordic Council of
Ministers, and acts as a joint Nordic body in the field of conformity assessment. In April 2000,
Nordtest initiated the ongoing project called Revision of the Nordtest Methodology for Oil Spill
Identification. The project is a collaboration project between the National Oil Spill Identification
laboratories in Finland, Denmark, Norway, Sweden and Battelle Laboratories in USA. Senior
Scientist Per Daling at SINTEF in Norway is the leader for this project team.
The main objectives in this project are:
1. to refine the existing Nordtest methodology into a technically more robust and legally
defensible oil spill identification methodology.
2. to adjust the revised methodology into European standard guidelines for oil spill
identification.
During 2000, the following tasks / activities have been carried out:
1. Literature studies
2. Development of improved analytical techniques in the laboratory
3. Round Robin test (series A):
Samples taken at the Deep Spill field experiment (in the Norwegian Sea June 2000).
Samples were analysed according to the existing practice at the individual laboratories. All the
laboratories in the project team participated. This series of Round Robin test has been
reported to Nordtest. (Faksness et. al, 2001 and is available at:
http://www.vtt.fi/nordtest/finalrep/1520-1mic.pdf).
Conclusions:
This Round Robin Test series A demonstrates the potential in improving and harmonizing the
existing Nordtest methodology by more use of (semi-) quantitative approach for PAHs, and in
use of diagnostic ratios between selected PAHs and biomarker components as a data
treatment tool for documenting positive/non-positive match.
1.2 CEN/BT/Task Force 120: Harmonization of oil spill identification methodology
Parallel and in addition to this ongoing Nordtest project, the Technical Board (BT) of the
European Committee for Standardisation (CEN) in May last year established a Task Force
(working group) called: CEN/BT/Task Force 120 Oil Spill Identification. The Norwegian
Standardising Association (NSF, by Mr. Rolf Duus) has the secretariat and senior scientist Per S.
Daling, SINTEF, is convenor for the working group.
The 1
st
CEN- meeting of the CEN/BT/Task Force 120 Oil Spill Identification was held in
Helsinki on November 3
rd
2000. Participants from 10 European countries participated. Minutes
from the CEN- workshop are given in Attachment 2. A resume of this meeting is also given at:
http://www.vtt.fi/nordtest/info/info04-3.pdf.
In order to achieve a more complete standardization of guidelines / protocols within oil spill
identification, the CEN working group recommended strongly to extend the deliverables from the
ongoing Nordtest project from one protocol (only describing the methodology) to three protocols /
guidelines covering the following issues:
Issue 1: Guidelines on the methodology for identification of waterborne oils.
Issue 2: Guidelines for sampling of waterborne oils for oil spill identification.
6
Issue 3: Analytical and data processing specifications.
.
It is also the intention of the CEN working group that the establishment of these CEN guidelines
can form the norms for further international use (e.g. as ISO-standards).
A new series (Round Robin test series B) is planned to take place in April / May 2001.
This will be based on revised /improved methodology recommended through the findings in the
ongoing Nordtest project. As decided at the CEN-meeting in November, other European laboratories
within the CEN working Group are welcome to participate. An invitation was sent out to the
individual laboratories from CEN dated December 5, 2000.
The aim of this Series B Round Robin test is to ensure:
- that the recommended methodology is operational for the individual laboratory
- that the recommended methodology is a robust and a legally defensible identification
methodology that improve the probability for making specific conclusion in oil spill
identification
- that the results from the individual laboratories can form a basis for statistical evaluation of
the analytical precision and conclusion quantitative criteria
- that the results within the laboratories are reproducible and end up in consistent
conclusions (according to the terminologies sketched in fig. 2.1.)
1.3 Development of improved methodology
The analytical methodology suggested in this report for the coming Round Robin Testing is a result
of extensive literature studies (i.e. recent Oil Spill identification publications and learnings from
the geo-chemists), specific laboratory studies and consensus based on the experiences among the
project team.
We therefore hope that the participating laboratories find this recommended methodology useful and
can adapt it into the their laboratory routines before they receive the test sample in the beginning of
May.
7
2 Summary of chemical analytical methodology
In general, the petroleum-specific target analytes that may need to be chemically characterised for
oil source identification and environmental assessment include the following:
Individual saturated hydrocarbons including n-alkanes (C10-C40) and selected isoprenoids
pristane and phytane (in some cases, another three highly abundant isoprenoid compounds
farnesane (2,6,10-trimethyl dodecane), trimethyl-C13 (2,6,10-trimethyl tridecane) and
norpristane are also included).
The GC-FID fingerprints reveal the overall boiling (carbon) range of the oil, which is very
valuable especially when deciding which PAH and biomarker are most relevant for the spill.
Therefore, it may be prudent to conduct the GC-FID analysis prior to planning the GC-MS
analysis
The polycyclic aromatic hydrocarbons are dominated by the EPA priority parent PAHs and, in
particular, the petroleum specific alkylated (C1-C4) homologues of selected PAHs (that is,
alkylated naphthalene, phenanthrene, dibenzothiophene, fluorene, and chrysene series). These
alkylated PAH homologues (Table A2.1 (Attachment 2)) are the backbone of chemical
characterisation and identification of oil spill assessments.
Biomarker terpane and sterane compounds (Table A2.2 (Attachment 2)). Analysis of selected
ion peaks produced by these characteristics, environmentally-persistent compounds generates
information of great importance in determining source(s), weathered state and potential
treatability (Wang et al, 1999)
In addition to qualitative criteria, semi-quantitative criteria (optional: quantitative) was defined to
recognise sources of PAHs. Numerous quantitative diagnostic ratios have been used as indicators
for oil spill identification.
The results and the overall conclusions should be reported for the combined results of the test
methods used. For each standard method, results are specified as Positive match, Probable match,
Inconclusive and Non-Match. These categories represent standardised degrees of differences
between the analyses of two oils (criteria to be specified later).
A suggested protocol/decision chart is given in Figure 2.1
8
Weathering check
(semi-quantitative
distribution of PAH's)
Evaluate diagnostic
ratios / analytical precision /
correlation treatment
Positive match Conclusion
Level 3
Level 2
Level 1
Probable match Non-match Inconclusive
Diagnostic ratios
match?
Optional:
Ni/V-determination
GC - FID
GC - MS
Compare spill oils with suspected sources
Chromatograms and
n-alkane
distribution different?
Biomarker and PAH
pattern different?
Ground-truth any
positive correlations
using all available data
Differences possibly
caused by weathering
Differences possibly
caused by weathering /
sample inhomogeneity
Weathering check
(semi-quantitative
distribution of n-alkanes)
Yes
Yes
Yes
Yes
No
No
No ? Yes Partly
Physical and visual
characterization
Extraction /
eventual sample clean up
Spilled oil and suspected
source samples
Figure 2.1 Suggested protocol / decision chart for oil spill identification
9
3 Sample preparation
The same sample preparation procedure shall be followed for both GC-FID and GC-MS analysis
(the same extracts are analysed for both analyses).
If the oil sample can not be prepared immediately after arrival at the laboratory, it should be
stored in a refrigerator. Sample preparation should not, in any case involving emulsions or
samples containing visible amounts of water, be delayed more than three days.
3.1 Oil sample
Oil samples should be adjusted to room temperature and homogenized before sample preparation.
The oil (10-30 mg) is weighted into a tarred volumetric flask (5 mL) and diluted with
dichloromethane (DCM) to final volume. An aliquot (1 mL) of the oil/DCM extract is added
internal standards for PAH and TEOC, before analysis (optional).
3.2 Sample clean up
If sample clean up is necessary, pre-packed Bond Elute silica columns (500 mg silica, e.g. Varian,
Supelco or Isolute) are recommended. The solvent is exchanged from DCM to hexane by adding 1
mL of hexane and evaporate to 1 mL. This step is repeated three times. The sample is
fractionated on the pre-packed silica columns. Three bed volumes are used. The hexane extract is
concentrated to 1 mL, transferred to a GC-vial and added recovery internal standards, if required.
4 Gas chromatography
A GC with a non-discriminating injection system (e.g. splitless or on-column) equipped with a
capillary column and flame ionisation detector (FID) shall be used. The signal output must be
connected to a data acquisition system analyser for the chromatograms.
GC Column: Non-polar or slightly polar, 10 30 m length, 0.2-0.53 mm internal diameter, film
thickness 0.25-1.2 m. The column shall allow for the resolution of alkanes from n-C10 to n-C40
and C17/pristane and C18/phytane.
4.1 Calibration standard
Calibration standards are not necessary if qualitative analysis is performed, but shall be mandatory
as a QA/QC prerequisite for the instrument. A stock solution comprising of the alkanes from C
10
to C
40
(or C
36
), and pristane and phytane is recommended. The concentration is 100 g/mL (of
each analyte) in hexane (or DCM). Calibration standards shall be prepared to cover, at a
minimum, the concentration ranges from 1 to 100 g/mL. Internal standards shall be added at the
20 g/mL level to all calibration standards, if required. Preparations of calibration standards are
described in Attachment 1.
An external calibration curve can be established by using the source oil, e.g. from 0.1 mg/mL to
10 mg/mL oil (minimum 5 levels).
10
4.2 GC conditions
Suggested instrumental conditions:
Samples are analysed by capillary GC (equipped with Electron pressure control) using the
following GC conditions:
Inlet: Splitless
Detector: Flame ionisation
Column: 30-m long x 0.25 mm internal diameter, and 0.25 m film thickness, high resolution
capillary HP-5 column (5% diphenyl and 95% dimethylpolysiloxane stationary phase)
Gases: Carrier: Hydrogen 2 mL/min (or Helium)
Make up: Helium 25 mL/ min (can be ignored)
Detector: Air 360 mL/min
Hydrogen 30 mL/min
Temperatures:
Injection port: 275C
Detector: 325C
Oven Program:
40C for 5 min, then 6C/min to 310C, hold time 10 min.
The GC oven temperature program may be modified to improve resolution.
Daily calibration: Alkane standard mixture
Quantification: Internal standard/calibration standard
4.3 Calibration
A minimum of five concentration levels should be analysed. One of the concentration levels
should be at a concentration near, but above the method detection limit. The remaining
concentrations should correspond to the expected working range of the GC, or the range expected
in the samples. A range from 1 to 100 g/mL is recommended. The calibration factor shall be
linear within this range (average relative standard deviation for each analyte less than 15%). Once
linearity is established, an average response factor (RF) may be used for the analysis of each
analyte. The initial calibration shall be verified each working day by the measurement of one or
more calibration standards.
A mid-level standard is analysed immediately prior to conducting any analyses, and after each
group of 12 samples. The response factor (RF) must be within 15% of the average RF calculated
from the initial calibration for at least 90% of the analytes, and within 20% for remaining 10% of
the analytes.
Suggestions to improve the accuracy of the method
Mass discrimination shall be kept to a minimum by placing a small plug of silanized glass wool
one-cm from the base of the glass injection liner. The capillary column shall be placed just below
the glass wool. A full range alkane standard should be run to test the degree of mass
discrimination before performing any actual sample analyses. The area ratio of C32/C21 shall be
greater than 0.8. If less then 0.8, reposition the column in the glass liner until the mass
discrimination is minimised.
Target compounds, and surrogate and internal standards (optional) shall be resolved from one
another and from interfering compounds. Potential problems may arise from the lack of baseline
11
resolution of these compounds. Corrective action shall be taken to correct resolution problems, i.e.
rerun of samples with a different temperature program.
Samples should be pre-screened based on colour. Low level clear samples should be analysed
separately from high level, coloured samples in order to minimise base line drift and potential
carry over.
Holding the column temperature at 310C for 5 minutes after the GC run is useful to minimise
carry over. DCM runs shall be compared to monitor any baseline shift.
Column resolution
The resolution values for normal n-alkanes C17 and C18 from pristane and phytane, respectively,
are used in defining column performance.
The resolution for the peak pairs n-C17 and pristane and for n-C18 and phytane are determined
using the following formula:
Resolution (R
s
) = [(t
2
t
1
)/(0.5 x (w
1
+w
2
))]
Where
t
1
= time for C
17
(or C
18
) maximum
t
2
= time for pristane (or phytane) maximum
w
1
= peak width at baseline of pristane (or phytane)
w
2
= peak width at baseline for C
17
(or C
18
)
The resolution of components for a well performing column will give resolution values for
capillary columns of 0.80 or greater for the two peak pairs.
Columns should be replaced and operating conditions thoroughly checked should resolution
values become less than 0.50 for either peak pair.
4.4 Sample analysis
The samples will follow an analysis sequence initiated with a continuing calibration, followed by
ten samples, and ending with a continuous calibration. If the response factor (RF) for any analyte
in the continuing calibration fails to meet the criteria established in section 4.3, instrumental
maintenance shall be performed and the continuing calibration repeated. All samples that were
injected after the sample exceeding criteria must be reinjected.
Sample injections should preferably be made by an autosample device (or on-column) and consist
of 1 L of the sample extract.
4.5 Calculations
Comparing biodegradation indicators (n-C17/pristane and n-C18/phytane) for the spilled oil with
the source oil can be used as to monitor the effect of microbial degradation on the loss of
hydrocarbons on the spill site.
A weathering check is to integrate the n-alkanes in the GC-FID chromatogram and present the
sample comparison in bar-charts (e.g. by Excel) normalised to a non-weathered n-alkane (see
example in Figure 4.1). This will also reflect information on eventual wax/paraffin redistribution
as part of the weathering process (see figure 4.1).
12
Weathering check n-alkanes
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
C
1
0
C
1
1
C
1
2
C
1
3
C
1
4
C
1
5
C
1
6
C
1
7
p
r
i
s
t
a
n
e
C
1
8
p
h
y
t
a
n
e
C
1
9
C
2
0
C
2
1
C
2
2
C
2
3
C
2
4
C
2
5
C
2
6
C
2
7
C
2
8
C
2
9
C
3
0
C
3
1
C
3
2
C
3
3
C
3
4
C
3
5
C
3
6
N
o
r
m
a
l
i
z
e
d

c
o
n
c
e
n
t
r
a
t
i
o
n

(
r
e
l
a
t
i
v
e

t
o

C
2
5
)
Weathered oil Fresh oil
Figure 4.1 "Weathering check" of spilled sample versus a suspected non-weathered source,
normalised relative to n-C25.
5 Gas chromatography/mass spectrometry
Polycyclic aromatic hydrocarbons are separated via high-resolution capillary gas chromatography,
and identified and quantified using electron impact mass spectrometry. A data system interfaced
to the GC-MS is used to control acquisition and to store, retrieve, and manipulate mass spectral
data. This method provides procedures for the identification and measurement of the selected
PAH listed in Table A2.1 (Attachment 2) in selected ion-monitoring mode (SIM). Table A2.1
may be amended to meet project-specific protocols.
5.1 GC-MS conditions
Prior to the analysis of analytical standards and/or samples the mass spectrometer shall be tuned.
Perfluorotributlyamine (PFTBA) should be used to tune the mass spectrometer:
The mass spectrometer can be tuned according to the following specifications:
M/z 69 at 100% of base peak (isotope ratio ca 1)
M/z 219 at 60 to 140% of base peak (isotope ratio ca 4)
M/z 502 at 3 to 13% of base peak (isotope ratio ca 10)
The GC-MS shall be fitted with a non-discriminating injection system (e.g. splitless or on-
column) and a non-polar or slightly polar column, 30-60 m length, 0.20 - 0.32 mm internal
diameter, film thickness 0.25 1.2 m. A 60 m column gives better resolution and is therefore
recommended.
13
Suggested GC-MS conditions:
The gas chromatograph is fitted with a 60-m x 0.25-mm internal diameter, 0.25-m film
thickness, HP-5MS (5% diphenyl and 95% dimethylpolysiloxane stationary phase) fused silica
capillary column.
The GC oven temperature program:
Injection Port: 300C
Transfer Line: 280C
Initial Temp: 40C
Initial Hold: 1 minute
Ramp Rate: 6C/min
Final Temp: 300C
Final Hold: 20 minutes
Initial Pressure: 15 psi
Inlet: Pulsed splitless, initial temp 300C
Carrier gas: Helium, 1 mL/min (constant flow)
Ionisation: Electron Impact 70 eV
Ion source temp: 230C
5.2 Preparation for full-scan analysis
If a new method is established, or the instrumental conditions are changed, a full scan analysis is
recommended. A 1-L injection volume is analysed. The mass spectrometer shall be operated in
electron impact mode at a manifold pressure of less than 6 x 10
-5
torr (for a MS equipped with a
diffusion pump). The mass range scanned will be 50 to 450 atomic mass units (AMU) unless
specifically stated otherwise in the project protocols. The quantification and confirmation ions
used in the analysis of selected PAH are listed in Table A2.1 (Attachment 2).
5.3 Preparation for selected ion monitoring (SIM) analysis
The suggested oven temperature outlined in section 5.1 is used for SIM analysis. A 1 uL injection
volume will be analysed. The mass spectrometer should be operated in electron impact mode at a
manifold pressure of less than 6 x 10
-5
torr. The electron multiplier voltage shall be set a minimum
of 100 V above the tune value.
5.3.1 The quantification and confirmation ions used in the analysis of selected PAH are listed
in Table A2.1 (Attachment 2). If possible ion groups should be selected so that no more
than 20 ions are monitored in a single group (Newer instruments and software might
allow 30 ions in each group). It should be noted that as the number of ions scanned per
group increases and the individual dwell times decrease, sensitivity will also decrease.
Each ion in a group should have identical dwell times to ensure that correct ion ratios are
preserved. Total group dwell time should not exceed 500 ms, individual dwell times
should be a minimum of 20 ms.
5.3.2 If a new method is established or the instrumental conditions are changed, a 1 g/mL
analytical standard and reference sample (an oil sample) shall be analysed in full-scan
mode, prior to the first analysis of analytical standards and/or samples in SIM. The
full-scan total ion chromatograms (TIC's) from these analyses should be used to
determine the proper group start and stop times in the SIM acquisition method.
14
For this particular method, it is recommended to divide the SIM acquisition method into
8 groups to obtain as good detection as possible.
5.3.3 In the event that a section of the analytical column is removed, or the analytical column
is replaced, a 1 g/mL analytical standard and reference sample should be analysed in
SIM mode under the conditions presented in Section 5.2. The extracted ion profiles
(EIP's) from these analyses should be used to reassign the proper group start and stop
times of the SIM acquisition method
The polycyclic aromatic hydrocarbons are dominated by the EPA priority parent PAHs and, in
particular, the petroleum specific alkylated (C1-C4) homologues of selected PAHs (that is,
alkylated naphthalene, phenanthrene, dibenzothiophene, fluorene, and chrysene series). These
alkylated PAH homologues (Table A2.1 (Attachment 2)) are the backbone of chemical
characterisation and identification of oil spill assessments.
Biomarker terpane and sterane compounds are given in Table A2.2 (Attachment 2). Analysis of
selected ion peaks produced by these characteristics, environmentally-persistent compounds
generates information of great importance in determining source(s), weathered state and potential
treatability (Wang et al, 1999).
In addition to qualitative criteria, quantitative criteria should be defined to recognise sources of
PAHs. Numerous quantitative diagnostic ratios have been used as indicators for oil spill
identification, and the suggested ones are given in table 6.1.
The biomarkers are analysed in the same run as the PAHs, but no quantitative measurements are
performed (no internal standards for biomarker quantification are added). The biomarkers are
used for measurements of diagnostic ratios, using the peak heights. Table A2.2 (Attachment 2)
gives the biomarkers recommended for use when calculating the diagnostic ratios, which are
given in table 6.2.
5.4 Procedures
5.4.1 Calibration
Demonstration of a linear initial calibration is required prior to the analysis of samples. A
continuing calibration is required at the beginning and approximately every tenth injection during
which analyses are performed. Initial calibrations from previous sequences may be used if the
continuing calibrations still show acceptable linearity and no major instrument maintenance has
been performed (i.e. source cleaning, column change, etc.)
The laboratories are using their established procedures for initial calibration and continuing
calibration. Suggested calibration procedures are given in Attachment 3.
5.5 Sample analysis
Samples are analysed under the same analytical conditions used in the analysis of the analytical
standards. If required, a recovery internal standard (RIS) is added to the samples immediately
preceding analysis. The type and amount of RIS added is project specific, consult the project
protocols. The preparations of standards are described in Attachment 1.
The criteria presented in Sections 5.5.1 through 5.5.7 shall be satisfied to verify identification of
an analyte in a sample. Analyte peaks, which do not satisfy these criteria in the preliminary
quantification, are deleted from the final quantification reports/files.
15
5.5.1 Relative retention time
The relative retention time of the sample component (RRT) should be within 0.1 min (6 sec) of
the standard component. The standard must have been run within 12 hours of the sample.
5.5.2 Sample spectrum
For each analyte suspected to be present in the sample, the corresponding ions listed in Table
A2.1 (Attachment 2) must be present and the relative intensities must agree to within 20 percent
of the relative intensities found in spectra of the standard component. This criteria may be
modified for trace level analysis and is left to the project manager's/analyst's discretion.
Modifications to sample spectrum criteria will be documented. If the intensity of the
quantification ion in a sample spectrum is greater than the intensity of the same ion in the high
concentration analytical standard the sample should be diluted and reanalysed.
5.5.3 Signal to noise ratio
A quantifiable analyte peak should (but is not limited to) exhibit a signal:noise ratio of 3:1 or
greater unless specified otherwise in the project protocol.
5.5.4 Peak symmetry
Suspected peaks should be examined for proper shape and symmetry.
5.5.5 Patterns
Suspected sample components should display established patterns. See Attachment 4 (PAHs) and
Attachment 5 (biomarkers).
5.5.6 Minimum area
It is recommended that each of the sample components have a minimum area of 1/2 the
abundance count of the component in the low standard. Quantification of a component is not
limited to these criteria however. For instance, a component may have an area abundance slightly
less than 1/2 the low standard area abundance, but exhibit a signal to noise ratio of 5:1. This
component would be considered for quantification.
5.5.7 Alkyl homologues
Certain projects require identification and quantification of the alkyl homologues of selected
PAH. Alkyl homologues are identified by the procedures in Sections 5.5.8. Semi-quantification
of alkyl homologues is covered in Section 5.5.9, below.
5.5.8 Tentative identification of unknowns and alkyl homologues
For components not contained in the analytical standards, a library search may be performed on
the sample spectrum for the purpose of tentative identification (NIST Mass Spectral Search
Program) during a full scan run. Computer-generated search algorithms should not employ
unusual normalisation, tilting, or smoothing of the unknown spectrum.
5.5.8.1 Major ions (ions >10 percent of the primary ion) in the reference spectrum should
be present in the sample spectrum.
5.5.8.2 Relative intensities of the ions in the sample spectrum should agree with the
reference spectrum to within 20 percent.
16
5.5.9 Semi-quantification of PAH-analytes and alkyl homologues
Quantification of analytes identified in samples can be performed by the internal standard method,
using the average response factor from the initial calibration. See Attachment 3 for additional
information regarding calculations used for quantitative determination of target analyte
concentrations in samples.
For a semi-quantitative calculation approach, all analytes are internal normalized to the area of
C30-hopane (peak#10 in Attachment 5), if hopanes are present in the sample, or to fluoranthene if
not. Alkyl homologue groups are quantified by the internal normalisation method. The molecular
ion of the alkyl homologue should be extracted, and the areas of the individual isomers summed
by a straight baseline integration from the first to the last peak in each homologous series
(according to examples in Attachment 4).
5.5.10 Semi-quantification of biomarkers
The biomarkers given in Table A2.2 (Attachment 2) are analysed in the same run as the PAHs,
but no quantitative measurements are performed (no internal standards for biomarker
quantification are added). The biomarkers are used for measurements of diagnostic ratios of peaks
within the fragmentograms, using the peak heights. The identity and ion patterns for the
biomarkers are given in Attachment 5.
17
6 Calculations
6.1 Initial and continuing calibration
See Attachment 3 for details regarding initial and continuing calibration.
6.2 Semi-quantification of PAHs
The quantification of samples is based on internal normalization using C30-hopane (or
fluoranthene if hopane is not present in the oil sample).
(Normalized concentration)
a
= A
a
/A
i
A
a
= area of quantification ion for target analyte
A
i
= area of quantification ion for internal normalization (C30-hopane or fluoranthene)
6.3 Weathering check
The quantitative results from the PAH analysis are suggested used for weathering check. This can
be presented several ways, but it is suggested that all data is normalised relative to C30-hopane,
and presented graphically.
If hopane not are present in the sample, the data can be normalised relative to C2-chrysenes
(which are assumed to be resistant to weathering). A ratio between C2-chrysenes concentration in
an unweathered sample and C2-chrysene concentration in are measured, and all target
concentrations are multiplied with this ratio. The data are presented as shown in figure 6.1.
The major compositional changes of alkyl PAH compounds as the evaporative weathering
percentage increases can be summarised as follows:
There occurs a pronounced decrease in naphthalene and its alkyl homologues relative to other
PAH homologues series
Most homologous groups show the same evaporative loss trend: C0 >C1 >C2 >C3>C4
No loss of alkylchrysenes (4-rings) due to evaporation has been observed.
18
Relative distribution of PAHs (Aquila)
0.0
5.0
10.0
15.0
20.0
25.0
30.0
D
E
D
E
2
D
E
4
B
T
1
B
T
3
N
N
2
N
4
A
N
Y
D
B
F
F
1
F
3
A
P
2
P
4
D
1
D
3
F
L
F
L
1
F
L
3C
C
2
C
4
B
K
F
B
A
P
I
N
B
P
E
N
o
r
m
a
l
i
z
e
d

c
o
n
c
e
n
t
r
a
t
i
o
n

(
r
e
l
a
t
i
v
e

t
o

h
o
p
a
n
e
)
Fresh oil 250+
Figure 6.1 Relative distribution of PAH: Normalised concentration relative to C30-hopane.
(Aquila fresh crude and an Aquila 250C+ weathered residue)
6.4 Diagnostic ratios of PAH
Petroleum from different fields can have very different PAH distributions. Several diagnostic
ratios are used for oil identification.
When crude oil is distilled to manufacture diesel fuel, higher boiling fractions such as the
chrysene family and above are almost entirely removed.
An ideal source ratio would be unique to that particular source and the two analytes would
degrade at the same rate. One group of analytes that varies widely in different oils is the
dibenzothiophene family; their concentrations reflect the sulphur content of the oil, and this varies
widely in different oils. The ratios between C2-phenanthrenes and C2-dibenzothiophenes and
between C3-phenanthrenes and C3-dibenzothiophenes are good candidates for source ratios that
will remain reasonably constant as the oil is degraded. These compounds also have similar
chromatographic retention times and are self-normalising to changes in GC conditions (e.g. mass
discrimination, tune shifts, etc.) resulting in ratios with low analytical variance (Douglas et al.,
1996).
Methyl-phenanthrenes (C18-C19 boiling range, m/z 192) are sensitive to the geological thermal
maturity of parent oil, and are a subject to preferential biodegradation of 2-methylphenanthrene in
the reservoir (Wang and Fingas, 1995). The ratio 2-MP/1-MP is used as a diagnostic ratio. If there
are problems with the separation, the ratios between the double peaks can be used.
Methyl dibenzothiophenes (C18-C19 boiling range, m/z 198) are also sensitive to the geologically
thermal maturity of parent oil. Subject to preferential biodegradation of 2-/3- methyl
dibenzothiophene in the reservoir (Wang and Fingas, 1995). The ratio 4-MD/1-MD is used as a
diagnostic ratio.
19
33.0033.1033.2033.3033.4033.5033.6033.7033.8033.9034.0034.1034.2034.3034.40
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
Time-->
Abundance
Ion 192.00 (191.70 to 192.70): SV100103.D
2-
9-/4-
3-
1-
Methyl phenathrenes
Figure 6.2 Methyl phenanthrenes (m/z 192).
32.5032.6032.7032.8032.9033.0033.1033.2033.3033.4033.5033.6033.70
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
160000
170000
Time-->
Abundance
Ion 198.00 (197.70 to 198.70): SV100103.D
1-
3-
2-
4-
Methyl dibenzothiophenes
Figure 6.3 Methyl dibenzothiophenes (m/z 198)
The suggested diagnostic ratios for PAHs are given in table 6.1.
Table 6.1 Suggested diagnostic ratios for PAHs
Parameter name Definition
C2-dbt/C2-phe (%) 100 * [C2-dibenzothophene/ (C2-dibenzothophene + C2-phenanthrene)]
C3-dbt/C3-phe (%) 100 * [C3-dibenzothophene/ (C3-dibenzothophene + C3-phenanthrene)]
C3-dbt/C3-chr (%)
100 * [C3-dibenzothophene/ (C3-dibenzothophene + C3-chrysene)]
2-MP/1-MP (%) 100 * [2-methyl phenanthrene/ (2-methyl phenanthrene + 1-methyl phenanthrene)]
4-MD/1-MD (%) 100 * [4-methyl dibenzothiophene/(4-methyl dibenzothiophene + 1-methyl dibenzothiophene)]
6.5 Diagnostic ratios of biomarkers
Biomarkers are complex "molecular fossils" derived from once-living organisms. Biomarkers are
ubiquitous in sediments, rocks and crude oils and they provide information that reduces the risk
associated with finding petroleum accumulations. The most common biomarkers used by organic
geochemists include terpanes, steranes and mono and triaromatic steroids. These biomarkers are
20
useful because they are resistant to secondary processes, such as biodegradation, compared to n-
paraffins or acyclic isoprenoids (Peters and Moldowan, 1993, Stout et al., 2000).
Full compound names tend to be very long, therefore abbreviations are frequently used. Of the
many alternatives, the abbreviations (peak labels) defined in NIGOGA (The Norwegian Industry
Guide to Organic Geochemical Analysis) combine relevant chemical information with shortness
(Table A2.2 (Attachment 2)). These abbreviations are used in this procedure.
The peak ratios shall be based on quantified peak heights (steranes, terpanes). When diagnostic
peak ratios for biomarkers are reported, they should preferably be of the type 100(a/(a+b)) [%].
Reporting of decimals is unnecessary, as it would suggest a higher precision than the one that can
be achieved in reality.
Experience from petroleum geochemistry shows that it is absolutely necessary to define in detail
how the ratios should be named and calculated.
This would include
The mass fragmentograms to be used
Height should be used as the basis for quantification
Which precautions must be taken to guarantee that results from different laboratories, or from
the same laboratory, but obtained at different times, can be compared.
This section attempts to define and describe a "useful minimum selection" of saturated biomarker
parameters for oil identification and correlation. If an oil shows any additional characteristic
compositional features (such as "extra" peaks like gammacerane, oleanane, extended tricyclic
terpanes etc.), these should of course always be included in the characterisation and considered in
the identification and correlation. The suggested diagnostic ratios are given in table 6.2, and the
ion patterns are shown in Attachment 5.
Table 6.2 Diagnostic ratios for biomarkers in oil spill identification (The abbreviations are
described in Table A2.2 (Attachment 2), and the ion fragmentograms are given in
Attachment 5)
Terpane ratios (m/z 191)
%27Ts 100 * [27Ts(191)] ) / ( [27Ts(191)] + [27Tm(191)] )
%28ab 100 * [28ab(191)] / ( [28ab(191)] + [30ab(191)] )
%25nor30ab 100 * [25nor30ab(191)] / ( [25nor30ab(191)] + [30ab(191)] )
%30O 100 * [30O(191)] / ( [30O(191)] + [30ab(191)] )
%30G 100 * [30G(191)] / ( [30G(191)] + [30ab(191)] )
%29ab 100 * [29ab(191)] / ( [29ab(191)] + [30ab(191)] )
%30d 100 * [30d(191)] / ( [30d(191)] + [30ab(191)] )
%32abS 100 * [32abS(191)] / ( [32abS(191)] + [32abR(191)] )
%35ab 100 * ( [35abS(191)] + 35abR(191)] ) / ( [34abS(191)] + [34abR(191)] + [35abS(191)] + 35abR(191)] )
21
Sterane ratios (m/z 217 and 218)
%27dia 100 * ([27dbS(217)]+[27dbR(217)] ) / ( [27dbS (217)]+ [27dbR(217)] + [27bbR(217)] + [27bbS(217)] )
%29aaS 100 * [29aaS(217)] / ( [29aaS(217)]+ [29aaR(217)] )
%29bb 100 * ([29bbR(217)]+[29bbS(217)] ) / ( [29bbS(217)]+ [29bbR(217)] + [29aaS(217)] + [29aaR(217)] )
%27bbSTER 100 * [27bb(S+R)(218)] / ( [27bb(S+R)(218)]+[28bb(S+R)(218)]+[29bb(S+R)(218)] ) )
%28bbSTER 100 * [28bb(S+R)(218)] / ( [27bb(S+R)(218)]+[28bb(S+R)(218)]+[29bb(S+R)(218)] ) )
%29bbSTER 100 * [29bb(S+R)(218)] / ([27bb(S+R)(218)]+[28bb(S+R)(218)]+[29bb(S+R)(218)]) )
Triaromatic steroid hydrocarbon ratios (m/z 231)
%TA21 100 * [C21TA(231)] / ( [C21TA(231)]+ [RC28TA(231)] )
%TA26 100 * [SC26TA(231)] / ( [SC26TA(231)]+ [SC28TA(231)] )
%TA27 100 * [RC27TA(231)] / ( [RC27TA(231)]+ [RC28TA(231)] )
It is important to do a visual check of the ratio data (look at the chromatograms, and not only the
measured ratios) before conclusions are made.
22
7 Quality control /Elimination of data
Hardcopies of all initial and continuing calibrations are maintained with the project data. The
number of procedural blanks, spiked blanks, matrix spikes, and other types of quality control
samples shall be specified in individual project protocols. Acceptability criteria and reporting
limits will also be outlined in the project-specific protocols.
7.1 Each day that analysis is performed, the daily calibration standard shall be evaluated to
determine if the chromatographic system is operating properly.
Peak shape should be evaluated for proper peak shape and symmetry.
The instrumental response should be comparable to previous calibrations.
The system must be recalibrated if the analytical column is replaced.
7.2 There shall be an initial calibration of the instrument as specified in Section 5.4.1.
7.3 Sample analysis: All samples shall be analysed under the same instrumental conditions
(same sequence). There shall be at least one triplicate analysis conducted in the course of
the investigation. This triplicate analysis provides the only means to assess the precision of
the ratios measured. Without this measure of precision (e.g. % relative standard deviation)
there is no way to defensibly state that one sample is different from another. It is difficult to
quantify exactly what qualifies as a "good RSD" versus a "bad RSD", so this is up to the
individual laboratory to assess. Regardless, without some triplicate analysis, there is no
basis to determine if any given ratio is meaningful (e.g. 54 versus 58; are they really
different ?) when assessing the data collected. We therefore strongly encourage including at
least one triplicate analysis of the spilled oil.
23
8 Reporting
The results and the overall conclusions shall be reported for the combined results of the test
methods used. For each standard method, results are specified as Positive Match, Probably Match,
Inconclusive and Non-Match. These categories represent standardised degrees of differences
between the analyses of two oils. They are not intended, by themselves, to make a statement about
the origin of the oil. The analyst interprets the results of all the tests in the light of experience and
the existing body of knowledge about oil analysis, and draws conclusions about whether or not
certain samples came from the same source. The overall conclusion should be consistent with the
combined results. Supporting documentation shall be included in the full report, consisting of the
gas chromatograms, selected ion fragmentograms and histograms, a discussion of weathering
factors, and the measured diagnostic ratios.
Attachment 6 gives examples of tables and histograms of how the results shall be presented in
tables, chromatograms and bar charts.
9 Attachments
Attachment 1: Internal standards and analytical standards
Attachment 2: PAHs and biomarkers
Attachment 3: Quantification and calibration
Attachment 4: Alkyl homologue patterns
Attachment 5: Biomarker patterns
Attachment 6: Examples, reporting
24
10 References
Douglas, G.S., A.E. Bence, R.C. Prince, S.J. McMillen and E.L. Butler (1996). Environmental
Stability of Selected Petroleum Hydrocarbon Source and Weathering Ratios. Environ. Sci.
Technol. 1996, 30, 2332-2339.
EN ISO 3170: 1998 E. Petroleum liquids Manual sampling (ISO 3170:1988, including
Amendment 1:1998)
Nordtest Method NT CHEM 001 Oil spill identification (Edition 2, approved 1991)
Peters, K.E., and J.M. Moldowan (1993). The Biomarker Guide. Interpreting Molecular Fossils in
Petroleum and Ancient Sediments. (Prentice Halls Inc., New Jersey, USA).
Stout, S., W.P. Naples, A.D. Uhler, K.J. McCarthy, L.G. Roberts and R.M. Uhler (2000). Use of
Quantitative Biomarker Analysis in the Differentiation and Characterization of Spilled Oil. Paper
prepared for the SPE International Conference on Health, Safety, and the Environment, Stavanger,
Norway, 26-28 June 2000.
Wang, Z., M. Fingas and D.S. Page (1999). Oil spill identification. J. Chrom. A 843, 369-411.
Wang, Z. and M. Fingas, 1995: Use of Methyldibenzothiophenes as Markers for Differentiation
and Source Identification of Crude and Weathered Oils. Environmental Science and Technology,
vol. 29, no. 11, 1995. page 2842-2849.
25
Attachment 1: Internal standards (optional) and analytical standards
Recommended standards and concentrations
A1.1 Surrogate internal standard - SIS THC (o-terphenyl )
The concentration of the stock solution is 2 mg/ml (optional). 50 mg of the component is
weighted and transferred to a 25 mL volumetric flask using a small volume of hexane. When the
component is dissolved, bring the solution to volume with hexane and mix well.
The working standard solution is diluted in hexane, and an appropriate concentration is 200
g/mL.
Surrogate compound is added to each sample before sample preparation, and to obtain an
approximate final extract concentration of 20 g/mL
A1.2 Recovery internal standard RIS THC (5 -androstane)
The concentration of the stock solution is 2 mg/ml (optional). 50 mg of the component is
weighted and transferred to a 25 mL volumetric flask using a small volume of hexane. When the
component is dissolved, bring the solution to volume with hexane and mix well.
The working standard solution is diluted in hexane, and an appropriate concentration is 200
g/mL.
The recovery internal standard (RIS) should be added to each sample extract prior analysis in such
a manner as to obtain an approximate final extract concentration of 20 g/mL.
A1.3 Analytical standards for n-alkanes
The concentration of the stock solution is 100 g/ml. 5 mg of each component are weighted and
transferred to a 50 mL volumetric flask using a small volume of hexane. When all compounds are
dissolved, bring the solution to volume with hexane and mix well.
The n-alkane stock standard can consist of the following compounds:
nC
10
nC
20 nC
30
nC
12
nC
21 nC
32
nC
14
nC
22 nC
34
nC
15
nC
23 nC
36
nC
16
nC
24 nC
40
nC
17
nC
25
Pristane nC
26
nC
18
nC
28
nC
19
nC
29
26
Phytane
The compound is dissolved in isooctane and supplied in flame sealed 1mL vials (from Chiron).
The concentration is 1 mg/mL. Three vials are transferred to a 10 mL volumetric flask. Rinse the
vials carefully with hexane. Bring the solution to volume with hexane and mix well (0.3 mg/mL).
A1.4 Calibration standard THC (GC-FID)
A minimum of five concentration levels should be analyzed. A range of approximately 1 to 100
g/mL is recommended. The calibration factor must be linear within this range (average relative
standard deviation for each analyte less than 15%). Once linearity is established, an average
calibration factor (i.e., response factor, RF) may be used for the analysis of each analyte.
Recovery internal standard and surrogate compounds should be added at the 20 g/mL level to all
calibration standards.
The working standards concentrations are:
o-terphenyl (200 g/mL)
5-androstane (200 g/mL)
Calibstd THC (1g/mL)
Calibstd THC (5g/mL)
Calibstd THC (10 g/mL)
A1.5 Surrogate internal standard SIS PAH (optional)
The concentration of the stock solution is 500 g/mL. E.g. 12.5 mg of each component is
weighted and transferred to a 25 mL volumetric flask using a small volume of xylene. When all
compounds are dissolved in xylene, bring the solution to volume with hexane and mix well.
SIS-PAH
d
10
-naphthalene
d
10
-phenanthrene
d
12
-chrysene
The working standard solution is diluted in hexane, and an appropriate concentration is 10 g/mL.
Surrogate compounds are added to each sample before sample preparation. The approximate final
extract concentration is 1 g/mL.
A1.6 Recovery internal standard RIS-PAH (optional)
The concentration of the stock solution is 100 g/mL. E.g. 2.5 mg of each component is weighted
and transferred to a 25 mL volumetric flask using a small volume of DCM. When all compounds
are dissolved, bring the solution to volume with hexane and mix well.
RIS PAH
d
12
-perylene
d
10
-acenaphthene
d
10
-fluorene
27
The working standard solution is diluted in hexane, and an appropriate concentration is 10 g/mL.
The recovery internal standard (RIS) should be added to each sample extract prior to analysis in
such a manner as to obtain an approximate final extract concentration of 1 g/mL
Analytical standards for NPD and PAH
A1.7 16 PAH from the EPA-list
are dissolved, bring the solution to volume with DCM and mix well.
16 PAH
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo(a)anthracene
Chrysene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Indeno(1,2,3-c,d)pyrene
Dibenzo(a,h)anthracene
Benzo(g.h.i)perylene
A1.8 Other PAH-compounds, dibenzothiophenes, decalins etc.
are dissolved, bring the solution to volume with DCM and mix well.
PAH -others
1-methyl naphthalene
2,3-dimethyl naphthalene
biphenyl
1-methyl phenanthrene
3,6-dimethyl phenanthrene
1-methyl fluorene
9-ethyl fluorene
dibenzothiophene
2,8-dimethyl dibenzothiophene
perylene
dibenzofuran
benzo(b)thiophene
decahydronaphthalene (decalin)
28
A1.9 Calibration standard GC-MS
The standards used to establish the calibration curve are added 1 g/mL SIS PAH and 1 g/mL
RIS PAH (at all concentration levels). If all compounds in Table A2.1 (Attachment 2) are
analyzed, the calibration standard includes the standards "16 PAH" and "PAH others", for
example with the following concentrations:
0.01 g/mL
0.025 g/mL
0.05 g/mL
0.10 g/mL
0.50 g/mL
1.0 g/mL
5.0 g/mL
10.0 g/mL
The calibration standards are diluted in hexane.
A1.10 Storing of standard solutions
All standard solutions should be stored in a freezer (< -20C). Internal standards in daily use can
be stored in a refrigerator (5C), but only for a limited time (a few days). Solvent evaporation
rates increase with increasing temperature.
29
Attachment 2: PAHs and biomarkers
Table A2.1. Selected PAH quantification (Qion) and confirmation ions(Cion) for GC-MS full
scan and SIM analysis. The group# refers to the recommended grouping of ions. Cx
represents the retention intervals. The numbers refer to the retention window to the
n-alkanes with the carbon number indicated.
Abbrev # Rings Qion Cion Group# Cx interval
Fluorene-d
10
(RIS) 3 176 174 3 16-17
Naphtalene- d
8
(SIS) 2 136 134 2 11-12
Decalin DE 138 1 10-11
C1-decalins DE1 152 2 11-12
C2-decalins DE2 166 2 11-13
C3-decalins DE3 180 3+4 13-14
C4-decalins DE4 194 3+4 13-15
Benzo(b)thiophene BT 2 134 2 12
C1-benzo(b)thiophenes BT1 2 148 2 13-14
Naphthalene N 2 128 127 2 11-12
C1-naphthalenes N1 2 142 141 2 13-14
C2-naphthalenes N2 2 156 141 3 14-15
C3-naphthalenes N3 2 170 155 3 15-17
C4-naphthalenes N4 2 184 169,141 3+4 15-18
Biphenyl- d
10
(SIS) 2 164 162 3 14
Biphenyl B 2 154 152 3 14
Acenaphthylene ANY 3 152 153 3 14-15
Acenaphtene - d
10
(RIS) 3 164 162 3 15-16
Acenaphthene ANA 3 154 153 3 15-16
Dibenzofuran DBF 3 168 169 3 15-16
Fluorene F 3 166 165 3 16-17
C1-fluorenes F1 3 180 165 4 17-18
C2-fluorenes F2 3 194 179 4 18-19
C3-fluorenes F3 3 208 193 4+5 19-21
Phenanthrene - d
10
(SIS) 3 188 184 4 18-19
Phenanthrene P 3 178 176 4 18-19
Anthracene A 3 178 176 4 18-19
C1-phenanthrenes/anthracenes P1 3 192 191 4 19-20
C4-phenanthrenes/anthracenes P4 3 234 219,191 5+6 22-25
2-methyl phenanthrene 2-MP 3 192 191 4 18-19
1-methyl phenanthrene 1-MP 3 192 191 4 18-19
Dibenzothiophene D 3 184 152,139 4 17-18
C1-dibenzothiophenes D1 3 198 184,197 4 18-20
C2-dibenzothiophenes D2 3 212 197 5 19-21
C3-dibenzothiophenes D3 3 226 211 5 20-23
C4-dibenzothiophenes D4 3 240 5 21-24
4-methyl dibenzothiophene 4-MD 3 198 184,197 4 18-20
1-methyl dibenzothiophene 1-MD 3 198 184,197 4 18-20
30
Abbrev # Rings Qion Cion Group# Cx interval
Fluoranthene FL 4 202 101 5 21
Pyrene PY 4 202 101 5 21-22
C1-fluoranthrenes/pyrenes FL1 4 216 215 6 22-24
C2-fluoranthenes/pyrenes FL2 4 230 215 6 23-25
C3-fluoranthenes/pyrenes FL3 4 244 229,215 6 25-27
Chrysene - d
12
(SIS) 4 240 236 6 25-26
Benz(a)anthracene BA 4 228 226 6 25-26
Chrysene C 4 228 226 6 25-26
C1-chrysenes C1 4 242 241 6 26-27
C2-chrysenes C2 4 256 241 6+7 27-29
C3-chrysenes C3 4 270 255 7 28-30
C4-chrysenes C4 4 284 269,241 7 29-32
Benzo(b)fluoranthene BBF 5 252 253,125 7 28-29
Benzo(k)fluoranthene BKF 5 252 253,125 7 28-29
Benzo(e)pyrene BEP 5 252 253 7 29-30
Benzo(a)pyrene BAP 5 252 253,125 7 29-30
Perylene - d
12
(RIS) 5 264 260 7 29-31
Perylen PER 5 252 253 7 29-31
Indeno(1,2,3-c,d)pyrene IN 6 276 277,138 8 32-33
Dibenz(a,h)anthracene DBA 5 278 279,139 8 32-33
Benzo(g,h,i)perylene BPE 6 276 277,138 8 32-34
Triterpanes (Hopanes ) 5 191 412,177 7+8 29-36
Diasteranes and -Steranes 4 217 6+7 27-31
-Steranes 4 218 6+7 29-31
Triaromatic steroids 4 231 6+7 25-32
Surrogate correction (optional)
Fluorene-d
10
(or acenaphthene-d
10
) is used to quantify all target compounds and surrogate
compounds. Once RFs are generated for all the targets and surrogates versus fluorene-d
10
(or
acenaphthene-d
10
), the samples are quantified. These final results are considered "non-surrogate
corrected". In order to report surrogate corrected data, the non-SIS corrected data and "surrogate
correct" it using specific a SIS compound: Decalin and C
1
-decalins, benzothiophene, C
1
-
benzothiophenes, naphthalene and C
1
-naphthalenes are surrogate corrected versus naphthalene-d
8
,
target compounds benzo(a)anthracene through benzo(g,h,i)perylene are surrogate corrected versus
chrysene-d
12
. The remaining compounds listed in table A2.1 (except the biomarkers) are
surrogate corrected versus phenanthrene-d
10.
.
31
Table A2.2 Biomarkers recommended for measurements of diagnostic ratios. The peak# is
given in Attachment 5.
Peak # Abbreviation Name M/z Cx
1 27Ts 18(H)-22,29,30-trisnorhopane 191 29-30
2 27Tm 17(H)-22,29,30-trisnorhopane 191 29-30
3 28ab 17(H),21(H)-28,30-bisnorhopane 191 30-31
4 25nor30ab 17(H),21(H)-25-norhopane 191 30-31
5 29ab 17(H),21(H)-30-norhopane 191 31-32
6 29Ts 18(H)-30-norneohopane 191 31-32
7 30d 15-methyl-17(H)-27-norhopane (diahopane) 191 31-32
8 29ba 17(H)-21(H)-30-norhopane (normoretane) 191 32-33
9 30O 18(H)-oleanane 191 32-33
10 30ab 17(H),21(H)- hopane 191 32-33
11 30ba 17 (H)-21-(H)-hopane (moretane) 191 32-33
12 31abS 17(H),21(H), 22S-homohopane 191 33-34
13 31abR 17(H),21(H), 22R-homohopane 191 33-34
14 30G Gammacerane 191 33-34
15 32abS 17(H),21(H), 22S- bishomohopane 191 33-34
16 32abR 17(H),21(H), 22R-bishomohopane 191 34-35
17 33abS 17(H),21(H), 22S- trishomohopane 191 34-35
18 33abR 17(H),21(H), 22R-trishomohopane 191 34-35
19 34abS 17(H),21(H), 22S- tetrakishomohopane 191 35-36
20 34abR 17(H),21(H), 22R-tetrakishomohopane 191 35-36
21 27dbS 13 (H), 17(H), 20S - cholestane (diasterane ) 217 27-28
22 27dbS 13 (H), 17(H), 20R - cholestane (diasterane ) 217 27-28
27 28aaR 24-methyl-5(H), 14(H), 17, 20R- cholestane 217 30-31
28 29aaS 24-ethyl-5(H), 14(H), 17, 20S- cholestane 217 30-31
29 29bbR 24-ethyl-5(H), 14 (H), 17, 20R- cholestane 217 30-31
30 29bbS 24-ethyl-5(H), 14 (H), 17, 20S- cholestane 217 30-31
31 29aaR 24-ethyl-5(H), 14(H), 17, 20R- cholestane 217 30-31
23 27bbR 5 (H), 14(H), 17(H), 20R-cholestane 218 29-30
24 27bbS 5 (H), 14(H), 17(H), 20S-cholestane 218 29-30
25 28bbR 24-methyl-5(H), 14 (H), 17, 20R- cholestane 218 29-30
26 28bbS 24-methyl-5(H), 14 (H), 17, 20S- cholestane 218 29-30
29 29bbR 24-ethyl-5(H), 14 (H), 17, 20R- cholestane 218 30-31
30 29bbS 24-ethyl-5(H), 14 (H), 17, 20S- cholestane 218 30-31
32 C20TA C20-triaromatic steroid hydrocarbon 231 25-31
33 C21TA C21-triaromatic steroid hydrocarbon 231 26-27
34 SC26TA C26, 20S-triaromatic steroid hydrocarbon 231 29-30
35
RC26TA+SC27TA
C26, 20R- +C27, 20S-triaromatic steroid hydrocarbon 231 30-31
36 SC28TA C28, 20S-triaromatic steroid hydrocarbon 231 31-32
37 RC27TA C27, 20R-triaromatic steroid hydrocarbon 231 31-32
38 RC28TA 26 C28, 20R,triaromatic steroid hydrocarbon 231 32-33
32
Attachment 3: Quantification and calibration
Quantification
A3.1 Quantification of analytes
Quantification of analytes identified in samples will be performed by the internal standard
method, using the average response factor from the initial calibration, unless otherwise specified
in the project protocols. See Section A.3.8 for additional information regarding calculations used
for the determination of target analyte concentrations in samples.
A3.2 Quantification of alkyl homologues
Alkyl homologue groups are quantified by the internal standard method. The molecular ion of the
alkyl homologue should be extracted, and the areas of the individual isomers summed by a
straight baseline integration from the first to the last peak in each homologous series.
The response factor used to quantify a specific homologous series (e.g. C
1
,-naphthalenes) should
be the response factor of parent compound (naphthalene) unless specified otherwise in the project
protocols.
A3.3 Quantification of tentatively identified compounds
Tentatively identified compounds (see Section 5.5.8) should be quantified by the internal standard
method assigning a response factor of 1. The data report should indicate that these compounds are
uncorrected for response.
Calibration
Demonstration of a linear initial calibration is required prior to the analysis of samples. A
continuing calibration is required at the beginning and end of each 12 hour period during which
analyses are performed (approximately every tenth injections). Sample data may be used if
bracketed by a passing continuing calibration. Initial calibrations from previous sequences may be
used if the continuing calibrations still show acceptable linearity and no major instrument
maintenance has been performed (i.e. source cleaning, column change, etc.)
A3.4 Initial calibration
Analyse a minimum of a low, medium, and high concentration analytical standard which will
represent sample concentration range, but a 6 to 8 level calibration curve is recommended
(attachment A1.9).
The concentration of the low standard should be 2 to 3 times the detection limit. The medium
concentration standard should be near the concentration range expected in the samples. The
high concentration standard should be at least 1 order of magnitude more concentrated than
the low concentration standard.
For each level of calibration, calculate the response factors for each analyte of interest using
the software supplied with the GC-MS data system. For each analyte, calculate the average
response factor (RF) and the percent relative standard deviation (RSD). For a valid initial
calibration, the percent RSD for each analyte should be less than 25 percent, unless
specifically stated otherwise in project protocols (see Section A3.6 for calculation of RF and
RSD).
33
A3.5 Continuing calibration
A continuing calibration will be performed using the following procedure at the beginning and
end of each 12 hour period in which samples are analysed.
Analyse a medium concentration analytical standard under the same analytical conditions used
in the initial calibration. Calculate response factors for each analyte of interest and compare
them to the average response factor calculated in Section A3.4, above.
The percent difference between the average response factor and the continuing calibration
response factor is less than 25 percent, the initial calibration is still valid and sample analyses
may continue (see Section A3.7 for calculation of percent difference). If the percent difference
exceeds 25 percent, remedial action should be taken and the medium concentration standard
reanalysed. -If the continuing calibration falls again, then sample analyses should be
terminated, maintenance performed, and a new initial calibration analysed, unless specifically
stated otherwise in project protocols. In addition, analysts should be visually monitoring the
retention time (RT) deviation (in minutes) and percent area deviation recorded on the
continuing calibration reports. Ideally, these values should not exceed 0.5 minutes for RT, and
<50% or >200% area deviation.
A3.6 Initial calibration
The average response factors from the initial calibration are calculated using the following
equation:
RF = (A
a
/A
b
x (C
b
/C
a
)
where:
A
a
= Area of quantification ion for target analyte in the standard
A
b
= Area of quantification ion for internal standard in the standard
C
a
= Concentration of target analyte in the standard
C
b
= Concentration of internal standard in the standard
The percent relative standard deviation (RSD) is evaluated with following equation:
RSD = (SD/RF) x 100
where:
SD = standard deviation of the mean RF, calculated as n-l.
See Section A3.4 for details regarding initial calibration acceptability criteria.
34
A3.7 Continuing calibration
The response factors determined from the continuing calibration are checked against those
determined from the initial calibration. The percent difference is calculated using the following
Percent Difference = [(RF
i
- RF
c
)/RF
i
] x 100
where:
RF
i
= Average response factor from initial calibration
RF
c
= Response factor from continuing calibration
See Section A3.5 for continuing calibration acceptability criteria.
A3.7 Quantification of samples
Samples are quantified as detailed in Section 5.5. The concentration of target analytes is
determined using the following equation:
C
a
= (A
a
x Amt
i
x D) / (A
i
x RF
i
x V
a
)
where:
C
a
= Concentration target analyte
A
a
= Area quantification ion for target analyte
A
i
= Area quantification ion for internal standard
Amt
i
= Amount internal standard added to sample
RF
i
= Average RF for analyte determined from initial calibration
D = Dilution factor if applicable
V
a
= Sample size
Sample size may refer to sample volume or sample dry/wet weight. The project protocol will
specify reporting criteria.
35
Attachment 4. Alkyl homologues patterns of PAHs
36
37
38
39
40
41
42
43
44
45
Attachment 5. Peak identity of the biomarkers
Hopanes (m/z 191)
1 27Ts 18(H)-22,29,30-trisnorneohopane
2 27Tm 17(H)-22,29,30-trisnorhopane
3 28ab 17(H),21(H)-28,30-bisnorhopane
4 25nor30ab 17(H),21(H)-25-norhopane
5 29ab 17(H),21(H)-30-norhopane
6 29Ts 18(H)-30-norneohopane
7 30d 15-methyl-17(H)-27-norhopane (diahopane)
8 29ba 17(H)-21(H)-30-norhopane (normoretane)
9 30O 18(H)-oleanane (not present in North Sea oils)
10 30ab 17(H),21(H)- hopane
11 30ba 17(H)-21-(H)-hopane (moretane)
12 31abS 17(H),21(H), 22S- homohopane
13 31abR 17(H),21(H), 22R-homohopane
14 30G Gammacerane
15 32abS 17(H),21(H), 22S- bishomohopane
16 32abR 17(H),21(H), 22R-bishomohopane
17 33abS 17(H),21(H), 22S- trishomohopane
18 33abR 17(H),21(H), 22R-trishomohopane
19 34abS 17(H),21(H), 22S- tetrakishomohopane
20 34abR 17(H),21(H), 22R-tetrakishomohopane
Time-->
Abundance
Ion 191.00 (190.70 to 191.70): SV100102.D
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
20000
21000
22000
23000
24000
25000
26000
48.00 50.00 52.00 54.00 56.00 58.00 60.00 62.00 64.00
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
46
Steranes and diasteranes (m/z 217 and 218)
21 27dbS 13 (H), 17(H), 20S - cholestane (diasterane )
22 27dbR 13 (H), 17(H), 20R - cholestane (diasterane )
27 28aaR 24-methyl-5(H), 14(H), 17, 20R- cholestane
28 29aaS 24-ethyl-5(H), 14(H), 17, 20S- cholestane
29 29bbR 24-ethyl-5(H), 14 (H), 17, 20R- cholestane
30 29bbS 24-ethyl-5(H), 14 (H), 17, 20S- cholestane
31 29aaR 24-ethyl-5(H), 14(H), 17, 20R- cholestane
23 27bbR 5 (H), 14(H), 17(H), 20R-cholestane
24 27bbS 5 (H), 14(H), 17(H), 20S-cholestane
25 28bbR 24-methyl-5(H), 14 (H), 17, 20R- cholestane
26 28bbS 24-methyl-5(H), 14 (H), 17, 20S- cholestane
29 29bbR 24-ethyl-5(H), 14 (H), 17, 20R- cholestane
30 29bbS 24-ethyl-5(H), 14 (H), 17, 20S- cholestane
44.00 45.00 46.00 47.00 48.00 49.00 50.00 51.00 52.00
44.00 45.00 46.00 47.00 48.00 49.00 50.00 51.00 52.00
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
Ion 217.00 (216.70 to 217.70): SV100110.D
Time-->
Time-->
Abundance
Abundance
Ion 218.00 (217.70 to 218.70): SV100110.D
21
22
27
28 29
30
31
23
24
25
26 29
30
47
Triaromatic steroids (m/z 231)
32 C20TA C20-triaromatic steroid hydrocarbon
33 C21TA C21-triaromatic steroid hydrocarbon
34 SC26TA C26, 20S-triaromatic steroid hydrocarbon
35 RC26TA+SC27TA C26, 20R- +C27, 20S-triaromatic steroid hydrocarbon
36 SC28TA C28, 20S-triaromatic steroid hydrocarbon
37 RC27TA C27, 20R-triaromatic steroid hydrocarbon
38 RC28TA C28, 20R,triaromatic steroid hydrocarbon
41.00 42.00 43.00 44.00 45.00 46.00 47.00 48.00 49.00 50.00 51.00 52.00 53.00 54.00
0
2000
4000
6000
8000
10000
12000
14000
16000
Ion 231.00 (230.70 to 231.70): SV100110.D
Time-->
Abundance
32
33
34
35
36
37
38
48
Attachment 6. Examples, reporting
The following tables and chromatograms and bar charts (histograms) should be used by each
laboratory in the presentation of / reporting of the results from the Round Robin tests.
Table A6.1 Ratios from GC-FID analysis
Sample ID Spill sample Source A Source B Source C Source D
n-C17/pristane 4.58 1.18 5.26 1.62 0.14
n-C18/phytane 3.10 1.40 2.98 2.74 0.17
Pristane/phytane 0.65 1.22 0.65 1.58 1.47
min 0 10 20 30 40 50
counts
0
10000
20000
30000
40000
50000
FID1 A, (130299\035F1701.D)

o
-
t
e
r
p
h
e
n
y
l

5
a
-
a
n
d
r
o
s
t
a
n
e
GC-FI D chromatogram: Spill sample
min 0 10 20 30 40 50
counts
0
10000
20000
30000
40000
50000
FID1 A, (130299\034F1601.D)

o
-
t
e
r
p
h
e
n
y
l

5
a
-
a
n
d
r
o
s
t
a
n
e
GC/FI D chromatogram: Source sample B
49
Weathering check n-alkanes
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
C
1
0
C
1
1
C
1
2
C
1
3
C
1
4
C
1
5
C
1
6
C
1
7
p
r
i
s
t
a
n
e
C
1
8
p
h
y
t
a
n
e
C
1
9
C
2
0
C
2
1
C
2
2
C
2
3
C
2
4
C
2
5
C
2
6
C
2
7
C
2
8
C
2
9
C
3
0
C
3
1
C
3
2
C
3
3
C
3
4
C
3
5
C
3
6
N
o
r
m
a
l
i
z
e
d

c
o
n
c
e
n
t
r
a
t
i
o
n

(
r
e
l
a
t
i
v
e

t
o

C
2
5
)
Weathered oil Fresh oil
Weathering check of spilled sample versus non-weathered suspected source, normalised to
n-C25.
50
Table A6.2 Relative distribution of PAHs, normalized to hopane
The data are normalized to hopane Spill sample Source A Source B Source C Source D
Decalin DE 0.03 0.60 0.26 0.27 2.10
C1-decalins DE1 0.11 1.43 0.48 0.53 3.17
C2-decalins DE2 0.29 3.08 1.11 0.89 4.16
C3-decalins DE3 0.41 3.10 0.80 1.16 3.74
C4-decalins DE4 0.44 2.83 0.81 1.27 3.02
Benzo(b)thiophene BT 0.06 0.07 0.21 0.03 0.12
C1-benzo(b)thiophenes BT1 0.60 0.35 1.33 0.12 0.43
Naphthalene N 0.87 6.10 3.10 6.45 26.9
C1-naphthalenes N1 4.12 16.1 8.58 18.0 56.3
C2-naphthalenes N2 11.7 31.8 18.6 24.3 63.5
C3-naphthalenes N3 14.2 27.7 19.4 17.6 43.3
C4-naphthalenes N4 12.3 16.8 15.4 9.43 21.3
Biphenyl B 0.11 2.20 0.21 3.85 9.74
Acenaphthylene ANY 0.03 0.13 0.03 0.13 0.30
Acenaphthene ANA 0.09 0.15 0.09 0.07 0.14
Dibenzofuran DBF 0.09 0.44 0.12 0.97 1.86
Fluorene F 0.43 1.65 0.49 1.43 2.42
C1-fluorenes F1 1.69 4.55 1.99 2.64 5.39
C2-fluorenes F2 4.13 6.33 4.66 3.77 6.89
C3-fluorenes F3 5.45 6.08 5.40 2.91 5.42
Phenanthrene P 1.08 4.96 1.23 3.16 7.14
Anthracene A 0.03 0.21 0.07 0.12 0.10
C1-phenanthrenes/anthracenes P1 4.26 12.9 4.34 6.41 15.0
Dibenzothiophene D 3.74 1.21 4.32 0.75 0.76
C1-dibenzothiophenes D1 13.6 4.53 15.4 2.23 2.96
Fluoranthene FL 0.02 0.19 0.02 0.06 0.24
Pyrene PY 0.18 0.49 0.18 0.20 0.39
C1-fluoranthrenes/pyrenes FL1 0.63 2.95 0.53 1.16 2.61
C2-fluoranthenes/pyrenes FL2 1.46 4.03 1.45 1.31 3.33
C3-fluoranthenes/pyrenes FL3 2.53 5.15 2.65 1.46 3.78
Benz(a)anthracene BA 0.04 0.16 0.03 0.08 0.10
Chrysene C 0.23 0.90 0.24 0.31 0.70
C1-chrysenes C1 0.70 2.13 0.67 0.63 1.53
C2-chrysenes C2 1.42 2.96 1.41 0.77 1.99
C3-chrysenes C3 1.44 2.18 1.37 0.51 1.43
C4-chrysenes C4 0.64 1.55 0.67 0.33 1.38
Benzo(b)fluoranthene BBF 0.03 0.14 0.03 0.01 0.13
Benzo(k)fluoranthene BKF 0.01 0.04 0.01 0.04 0.03
Benzo(e)pyrene BEP 0.07 0.23 0.07 0.10 0.23
Benzo(a)pyrene BAP 0.01 0.05 0.03 0.09 0.03
Perylene PE 0.01 0.15 0.01 0.01 0.02
Indeno(1,2,3-c,d)pyrene IN ND 0.01 ND ND 0.01
Dibenz(a,h)anthracene DBA ND 0.03 ND 0.01 0.02
Benzo(g,h,i)perylene BPE 0.02 0.08 0.02 0.03 0.07
17B,21B-Hopane 1.00 1.00 1.00 1.00 1.00
3-methyl phenanthrene 0.79 0.88 0.00
2-methyl phenathrene 1.20 2.81 1.29 1.66 3.60
9-/4-methyl phenanthrene 1.10 1.18
1-methyl phenathrene 0.79 2.76 0.84 1.26 3.18
4-methyl dibenzothiophene 6.16 2.05 6.88 1.00 1.18
51
Relative distribution of PAHs in spill sample
0.0
5.0
10.0
15.0
20.0
25.0
30.0
D
E
D
E
2
D
E
4
B
T
1
B
T
3
N
N
2
N
4
A
N
Y
D
B
F
F
1
F
3
A
P
2
P
4
D
1
D
3
F
L
F
L
1
F
L
3
C
C
2
C
4
B
K
F
B
A
P
I
N
B
P
E
N
o
r
m
a
l
i
z
e
d

c
o
n
c
e
n
t
r
a
t
i
o
n

(
r
e
l
a
t
i
v
e

t
o

h
o
p
a
n
e
)
Relative distribution of PAHs in source sample B
0.0
5.0
10.0
15.0
20.0
25.0
30.0
D
E
D
E
2
D
E
4
B
T
1
B
T
3
N
N
2
N
4
A
N
Y
D
B
F
F
1
F
3
A
P
2
P
4
D
1
D
3
F
L
F
L
1
F
L
3
C
C
2
C
4
B
K
F
B
A
P
I
N
B
P
E
N
o
r
m
a
l
i
z
e
d

c
o
n
c
e
n
t
r
a
t
i
o
n

(
r
e
l
a
t
i
v
e

t
o

h
o
p
a
n
e
s
)
52
Relative distribution of PAHs in spill sample compared to source oil
0.0
5.0
10.0
15.0
20.0
25.0
30.0
D
E
D
E
2
D
E
4
B
T
1
B
T
3
N
N
2
N
4
A
N
Y
D
B
F
F
1
F
3
A
P
2
P
4
D
1
D
3
F
L
F
L
1
F
L
3C
C
2
C
4
B
K
F
B
A
P
I
N
B
P
E
N
o
r
m
a
l
i
z
e
d

c
o
n
c
e
n
t
r
a
t
i
o
n

(
r
e
l
a
t
i
v
e

t
o

h
o
p
a
n
e
)
Source B Spill sample
Table A6.3 Diagnostic ratios of PAH's
C2-dbt/C2-phe (%) 78 28 78 25 17
C3-dbt/C3-phe (%) 77 27 78 30 17
C3-dbt/C3-chr (%) 89 52 89 63 45
2-MP/1-MP (%) 60 50 60 57 53
4-MD/1-MD (%) 77 77 77 83 85
53
Table A6.4 Diagnostic ratios biomarkers
m/z 191
%27Ts 26 57 26 47 56
%28ab 3.9 19 4.0 9.1 12
%25nor30ab 1.1 15 1.2 5.2 1.1
%29ab 64 34 64 34 34
%C29Ts 12 22 12 14 17
%30d 1.0 17 1.0 10 13
%30O ND ND ND ND ND
%30G 17 ND 17 ND ND
%32abS 59 57 59 59 57
m/z 217
%29aaS 50 47 50 52 44
%29bb 54 53 54 58 52
m/z 218
%27bbSTER 36 39 36 34 36
%28bbSTER 22 28 22 26 30
%29bbSTER 42 33 42 39 35
m/z 231
%TA21 68 56 68 59 60
%TA26 15 44 15 29 32
%TA27 41 58 41 49 49
54
Ion chromatograms, hopanes (m/z191)
48.00 50.00 52.00 54.00 56.00 58.00 60.00 62.00
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
75000
80000
Time-->
Abundance
Ion 191.00 (190.70 to 191.70): SV100237.D
Spill sample
46.00 48.00 50.00 52.00 54.00 56.00 58.00 60.00 62.00
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
Time-->
Abundance
Ion 191.00 (190.70 to 191.70): SV131007.D
5
10
Source sample A
48.00 50.00 52.00 54.00 56.00 58.00 60.00 62.00
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
36000
38000
40000
42000
Time-->
Abundance
Ion 191.00 (190.70 to 191.70): SV100236.D
Source sample B
10
5

NT TR 499 - Laboratory and Reporting Instructions For The CEN-BT-TF 120 Oil Spill Identification - Nordtest Technical Report

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