Content uploaded by Lemnuel Aragones
Author content
All content in this area was uploaded by Lemnuel Aragones on Jul 10, 2021
Content may be subject to copyright.
1093
Development, Validation, and Application
of a Three-way Cleanup Method for GC/MS Measurement
of PCBs in Stranded Cetaceans in Philippine Waters
Jonah L. Bondoc1,2*, Lemnuel V. Aragones2, and Charita S. Kwan1,2
1Research and Analytical Services Laboratory (RASL)
Natural Sciences Research Institute (NSRI)
University of the Philippines, Diliman 1101 Quezon City, Philippines
2Institute of Environmental Science and Meteorology
University of the Philippines, Diliman 1101 Quezon City, Philippines
Polychlorinated biphenyls (PCBs) are a group of synthetic organic chemicals that enrich the
food chain via biomagnification where the highest levels have been detected in cetaceans. While
several studies have reported cetaceans as sentinels of environmental pollution, information
on the contamination status of PCBs in cetaceans found stranded along Philippine coasts has
been very limited over the past decades. An important contributory factor to this paucity is the
challenging analytical method for the analysis of PCBs in lipid-rich biological tissues due to
lipid interferences that can affect gas chromatographic systems especially column efficiency and
lifetime. Thus, a modified method consisting of a series of three cleanup steps – involving isolation
column chromatography, gel permeation chromatography (GPC), and solid-phase extraction
(SPE) using silica gel following Soxhlet extraction, plus macro- and micro-concentration prior
to gas chromatographic/mass spectrometric analyses – was used. Recovery of added analytes
corresponding to appropriate concentration ranges and repeatability of data values were
excellent as values obtained were within the established performance criteria of 40–120%
recovery, including a 106% mean recovery for the QC material (FAPAS T05100QC – Oily
Fish) and < 30% RSD for an analyte concentration of 1 µg kg–1. Thus, the modified cleanup
technique in conjunction with GC/MS detection is proven suitable for its intended use. Thirty-
eight (38) congeners (PCBs-8, -12, -19, -18, -33, -38, -35, -37, -52, -44, -57, -74, -79, -78, -81, -77,
-104, -99, -123, -118, -114, -126, -155, -153, -162, -167, -156, -157, -169, -188, -189, -202, -195,
-194, -205, -208, -206, and -209) were detected in cetacean blubber (n = 15) ranging from 21.7
ng g–1 lipid weight (in an adult female dwarf sperm whale (Kogia sima) found in Camarines
Sur) to 1460 ng g–1 lipid weight (in an adult male rough-toothed dolphin (Steno bredanensis)
found in Zambales.
Keywords: cleanup techniques, Philippines, polychlorinated biphenyls, stranded cetacean
*Corresponding Author: jlbondoc@up.edu.ph
Philippine Journal of Science
150 (5): 1093-1108, October 2021
ISSN 0031 - 7683
Date Received: 21 Jan 2021
1094
INTRODUCTION
PCBs are man-made chemicals that were extensively
manufactured in the late 1920s (Jonathan et al. 2018).
This class of organic compounds – with theoretically
209 possible congeners – was regarded as a perfect
industrial chemical being highly resistant to electricity,
unreactive, non-flammable, and stable (Faroon and Ruiz
2016). However, their production and use were banned in
Sweden (1970), Japan (1972), the US (1976), and in most
countries by the 1980s due to their reported persistence
in the environment (Borja et al. 2005; Luby-Secretan
et al. 2013), bioaccumulative properties (Walczak and
Reichert 2016), and potential toxic effects to animal and
human health (Faroon and Ruiz 2016). As such, PCBs
have been identified by the Stockholm Convention as one
of the original ubiquitous Dirty Dozen persistent organic
pollutants. Consequently, several studies have been
conducted to monitor and quantify their concentration
levels in different environmental matrices, e.g. in the air
(Dai et al. 2016), sediments (Kwan et al. 2014), and water
(Santiago and Kwan 2015). Previous work reported that
the marine environment is their final sink (Daewel et al.
2020) where high PCB residue levels have been detected,
e.g. in fatty fish such as herring and salmon (Webster et al.
2013). However, it is in the fatty tissues of top-level marine
predators (e.g. marine mammals) that these semi-volatile
organic compounds preferentially accumulate by virtue
of their lipophilicity and high octanol-water partitioning
coefficients (Kow). According to Zhang et al. (2013), the
observed log Kow values range from 4.10 for biphenyl to
9.60 for decachlorobiphenyl, with a striking correlation
between the degree of chlorination and lipophilicity.
These characteristics, including their capacities to undergo
atmospheric deposition and biochemical metabolism
(Birgul and Tasdemir 2011), have been identified to be the
key parameters in their pathways and fate in the marine
environment, particularly in the fatty tissues of marine
mammals. However, the measurement of the levels of
PCBs in these lipid-rich tissues can be challenging.
Isolation of PCBs from the complexity of lipid-rich
matrix requires exhaustive analytical processes. The
high-fat content can pose interferences in the accurate
identification and quantification of specific PCB
congeners due to the resulting high baseline signal
(Quehenberger et al. 2011). Thus, matrix effects need to
be minimized, if not totally eliminated. Conduct of one
cleanup procedure such as the standard GPC may not be
sufficient in eliminating or reducing the lipid matrix effects
in cetacean tissues. Additional cleanup techniques that are
efficient and simple can be combined with GPC to obtain
better chromatograms. Thus, the study was conducted to
develop a method that is easy to follow for the analysis of
PCBs in lipid-rich animal tissues by examining feasible
and efficient cleanup techniques and to demonstrate its
suitability by establishing the performance characteristics
of the method. Another important aim was to devise an
alternative analytical method that would be both cost-
efficient and environmentally sound in evaluating the
extent of PCB contamination in coastal environments
through measurements of the levels of PCBs in stranded
cetaceans, particularly those found in Philippine waters.
MATERIALS AND METHODS
Chemicals and Reagents
Fully-resolved native PCB mixture from di- to deca-CBs
in isooctane (98%, Cat. No. EC 5434) consisting of 42
congeners (PCBs-8, -9, -10, -12, -15, -18, -19, -33, -35,
-37, -38, -44, -52, -54, -57, -74, -77, -78, -79, -81, -99,
-104, -114, -118, -123, -126, -153, -155, -156, -157, -162,
-167, -169, -188, -189, -194, -195, -202, -205, -206, -208,
and -209 at concentration levels of 2000 ng mL–1) for the
di-CBs and 1000 ng mL–1 for the tri- to deca-CBs plus
13C12-labeled mono- to deca-CBs in nonane (99%, Cat. No.
EC 4189-A) consisting of 10 congeners (PCBs- 3, -15, -28,
-52, -118, -153, -180, -194, -208, and -209 at 1 µg mL–1)
were purchased from Cambridge Isotope Laboratories
(CIL) Inc., Massachusetts. For the evaluation of accuracy
and precision, the following mixtures and standards were
used: 1) CEN PCB Congener Mix 1 in heptane (Cat. No.
47927) comprising six congeners (PCBs-18, -44, -52, -118,
-153, and -194 at 10 µg mL–1 each component) purchased
from Supelco, USA; 2) Perylene-d12 (98%; Cat. No. DLM-
366-1.2) purchased from CIL; 3) FAPAS Oily Fish Quality
Control (QC) Test Material (Cat. No. T05100QC) from Fera
Science Ltd., Delaware; and 4) Phenanthrene-d10 (98%;
Cat. No. DLM-371-S), pyrene-d10 (98%, DLM-155-S), and
chrysene-d12 (98%; Cat. No. DLM-261-S) purchased from
CIL and used as internal standards. Pre-distilled solvents
such as acetone, dichloromethane (DCM) and hexane were
all analytical grade and purchased from JT Baker or Merck.
Glass fiber thimbles for Soxhlet extraction, glasswool, and
silica gel (powder, 60–200 mesh, JT Baker) for isolation
column chromatography and SPE were pre-baked at 400 °C.
Sodium sulfate (anhydrous, powder, JT Baker and Merck)
and GC/MS vials were baked at 550 °C prior to use.
Sample Extraction
Approximately 10 g of homogenized cetacean kidney
sample was mixed with 30 g pre-baked sodium sulfate in
a beaker. The mixture was quantitatively transferred into
the pre-baked glass fiber thimble which was then placed
into the main chamber of the Soxhlet extractor. The beaker
was rinsed thrice with 10-mL portions of hexane: acetone
(1:1, v v–1) with the rinses poured through the thimbles.
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1095
The sample was added with 25 µL of 1 µg mL–1 CEN
PCB Congener Mix 1, 50 µL of 2 µg mL–1 perylene-d12,
and 10 µL of 1 µg mL–1 13C12-labeled surrogate mono- to
deca-CBs. Prior to extraction, the thimble was covered
with a thin layer of glass wool. Flat-bottomed flasks
with glass beads and containing approximately 170 mL
hexane:acetone (1:1, v v–1) were used during extraction
for 12 h, maintaining at least 8 cycles/h. The hexane:
acetone extract was quantitatively transferred into the
rotary vapor flask and was concentrated to approximately
5 mL at 45 °C with rotation at 30–35 rpm and using
approximately 5 °C circulating water in the condenser.
The extract was solvent-exchanged with 5 mL DCM and
concentrated to approximately 1 mL.
Cleanup of Extracts
To eliminate potential interferences of the fat content
in the complex sample matrix (cetacean tissues), three
different cleanup steps – in series – were utilized for
the measurement of PCBs. Isolation column, GPC, and
SPE using silica gel were employed as cleanup methods
following homogenization, Soxhlet extraction, and macro-
concentration of the samples, where readily available and
reusable glassware were used.
First Cleanup: Isolation Column Chromatography
The initial cleanup procedure used a column (500 mm
x 5 mm) that was packed from bottom to top with the
following: a thin layer of glass wool, pre-baked silica gel
(2 g), and sodium sulfate (2 g). The sample extract and
the DCM rinses were loaded onto the column where the
PCBs were eluted with a total of 150 mL of DCM. The
extracts were concentrated to approximately 5 mL by
rotary evaporation with the temperature of the water bath
set at 30 °C. The extracts were quantitatively transferred
into a 10-mL volumetric flask, diluted to mark with DCM,
and vortexed to mix.
Second Cleanup: GPC
The DCM extract was further cleaned up by GPC, wherein
it was filtered through a 0.45-µm, 25-mm polyvinylidene
difluoride syringe filter into a scintillation vial before
injecting into the Agilent GPC system using the following
conditions: flow rate at 5 mL min–1 and absorbance set at
250 nm. Pre-rinsing of the GPC system was done prior
to repeated eight-time injections of the 1-mL volume of
the DCM extract. Fractions of the extract were collected
between 9.5–20.0 min. The pool of collected fractions
together with the DCM rinses were concentrated to ~ 5 mL
using a rotary evaporator utilizing the previous conditions.
The extract was solvent-exchanged with 5 mL hexane and
concentrated to a final volume of 1 mL.
Third and Final Cleanup: Solid Phase Extraction
Using Silica Gel
The GPC extract was further cleaned up by SPE using
pre-baked silica gel in hexane and packed in a glass SPE
column (6 mL tube volume, 1 g bed weight) using hexane.
The silica SPE column was conditioned by passing one-
column volume of DCM and two-column volumes of
hexane. One milliliter (1 mL) of GPC cleaned-up extract
was transferred quantitatively into the silica SPE column.
PCBs were eluted with a total of 20 mL of 25% DCM in
hexane. The extract was evaporated to near dryness using
a gentle stream of nitrogen gas. One hundred microliters
(100 µL) of 50 µg L–1 mixed internal standards were
added to the concentrate and transferred quantitatively
using a microsyringe into a glass insert inside a vial,
capped, and mixed in a vortex mixer prior to analysis
by GC/MS.
GC/MS Instrumentation
The PCBs in the extracts were analyzed by GC/MS using
the Shimadzu GC/MS QP 2010 (electron impact ionization
equipped with auto-injector AOC-20i) and a capillary
column (SPB-5 ms, 30 m long, 0.25 µm thickness, 0.25
mm diameter). The injector port and interface were set at
280 °C while the ion source was kept at 200 °C. Helium
was used as carrier gas at a constant linear velocity of 57.8
cm s–1. Forty-two (42) native PCB congeners from mono-
to deca-CB and one 13C12-labeled surrogate standard
representing each for mono- to deca-CB were analyzed
using the column temperature program of 55 °C held for
2 min, followed by 20 °C/min to 130 °C, 5 °C/min to 240
°C for 5 min, and 10 °C/min to 300 °C for 15 min. The
MS was set in the selected ion monitoring mode using the
target and reference mass ion charge ratios defined for
each native PCB, surrogate PCB, and internal standards
(Appendix Table I).
Validation of the Analytical Method: Evaluation of
Performance Parameters
The method performance parameters such as linearity
of the calibration curves, detection limits, accuracy, and
precision were established according to Eurachem Guide
(2014). A set of six-level calibration standards (0, 5, 10, 20,
50, 100 ng mL-1 mono- to deca- native PCBs in hexane)
were prepared for evaluation of linearity of the calibration
curve, where 0 represents the blank.
For the determination of instrument detection limits
(IDLs), low-level concentrations (1 and 5 ng mL–1) of
the native PCB congeners were injected into the GC/
MS, and the IDL for each PCB congener was computed
from results of either 1 or 5 ng mL–1 and calculated from
the calibration curves (0–100 ng mL–1). All IDLs were
determined by multiplying the standard deviation (s) of
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1096
the resulting concentrations by 3, i.e. IDL = 3s (Eurachem
Guide 2014; US EPA 2016). From the computed IDLs,
estimated method detection limits (EMDLs) were
computed by multiplying the IDLs with the factor and
the final sample volume of 100 µL divided by the sample
weight (2 g for liver and 0.5 g for blubber). The differences
in sample weights for the three types of tissues were based
on published literature (Aguilar and Borrell 1994; O’Hara
et al. 2004) on the % fat content of each tissue type, i.e.,
a 10-g kidney sample with a reported average % fat of
5% would constitute 0.5 g of fat. Likewise, a 2-g liver
sample with a reported average % fat of 20% will only
have 0.4 g of fat, and a 0.5-g blubber sample with 75%
fat will only have 0.4 g of fat also. Thus, the weight of
the kidney sample has provided a good representation in
terms of % fat and possible matrix interference.
An optimum weight of 10-g cetacean kidney sample
spiked with native PCB congeners at relatively low
concentrations of 2.5 ng g–1 was used. This spiking level
was based on US EPA (2016) stating that spiking levels
2–33 times the EMDL can be used. Accordingly, MDLs
were computed by multiplying the standard deviation
by 3 or t(n-1, 1- α = 0.99) (S), reliable detection limits
(RDLs) by multiplying the corresponding MDLs by
2, and LOQs equal to MDL times 3.33 or 10 times the
standard deviation of the resulting concentrations based
on Eurachem Guide (2014).
For the accuracy measurements of the analytical method,
four approaches were used: 1) the percent recoveries of the
42 native PCBs in the spiked cetacean kidney samples (n =
8, mass of 10 g and spiked level of 2.5 ng g–1), 2) recovery
of PCB-118 contained in the QC material (FAPAS QC
Material T05100QC – Oily Fish; n = 2, mass of 2.0 g,
with 95.8 µg kg–1 PCB 118), 3) the percent recoveries
of 1 ng g–1 surrogate 13C12-PCBs (IUPAC Nos. 3, 15,
28, 52, 118, 153, 180, 194, 208, and 209) and 10 ng g–1
perylene-d12 spiked in the cetacean kidney samples (n =
8, mass of 10 g), and 4) the percent recoveries of 2.5 ng
g–1 CEN PCB Congener Mix 1 in heptane (IUPAC Nos.
18, 44, 52, 118, 153, and 194) spiked in cetacean kidney
samples (n = 3, mass of 10 g).
For the evaluation of precision, three sets of spiked
cetacean kidney samples (mass of 10 g) were utilized, i.e.
1) n = 8 spiked native PCBs, 2) n = 8 spiked surrogate
13C12-PCBs and perylene-d12, and 3) n = 3 spiked CEN
PCB Congener Mix 1 in heptane. A method blank sample
was included for every batch not greater than five samples
analyzed. All PCB concentrations in the spiked and QC
samples were corrected with the PCB concentrations
found in the corresponding blank samples.
Applicability of the Method for the Determination of
PCBs in Blubber Tissues of Selected Cetacean Samples
The GC/MS method for the determination of PCBs in
lipid-rich animal tissues in combination with the three-
way cleanup techniques was applied to n = 15 blubber
samples from 15 stranded cetaceans of seven species
found in various locations in the Philippines during
2013–2015 (Table 1). Blubber tissues were sampled only
from cetaceans with carcass condition Code 2 (freshly
dead), following the standard protocols of Geraci and
Lounsbury (2005).
Concentrations of PCBs in Cetaceans
Measurement of the concentrations of 42 PCB congeners
in 15 cetacean blubber samples was done.
RESULTS AND DISCUSSION
Performance Parameters of the Analytical Method
of PCBs in Lipid-rich Animal (Cetacean) Tissues
Recognized by the AOAC (1998) as one type of method
validation procedure, single-laboratory (or within-
laboratory) method validation was carried out to evaluate
the different method performance characteristics of the
modified method. These include the linearity of the
calibration curves of the PCB congeners, detection limits
[both instrument and method detection limits (MDLs)
of each PCB congener], and the accuracy and precision
of the GC/MS method for the determination of PCBs in
lipid-rich animal tissues.
Linearity of the Calibration Curves of the PCB
Congeners
Linearity of the calibration curves of the PCB congeners
was established from the coefficient of correlation (r),
which ranged from 0.9960 (PCB-123) to 0.9999 (PCB-
126, -153) (Appendix Table II: pre-PCB analysis of
cetacean samples) and from 0.9961 (PCB-54) to 0.9999
(PCB-126) (Appendix Table III: during PCB analysis of
cetacean samples), providing evidence that the calibration
curve had remained linear – a positive indication of the
method performance in the validated analytical range.
Detection Limits of the PCB Congeners
From the results of the 1 or 5 ng mL–1 Mix Native
PCB congeners (n = 8 injections), the IDLs of 42 PCB
congeners were obtained at 2 ng mL–1 at a 95% confidence
interval, which allowed the calculation of the EMDLs
of the PCB congeners in the blubber and liver samples.
Obtaining IDLs that were relatively high than the usual
values, e.g. in instrument brochures, has been expected as
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1097
IDLs in the aforesaid literature are adjusted for dilution
to obtain far smaller detection limits inappropriate for
method validation (Thompson et al. 2002). In addition,
a wide statistical spread has been obtained resulting in
IDLs exceeding the value of the standard used, where
zero values were included in the calculations. From the
values of the IDLs, the EMDLs of the PCB congeners in
the blubber and liver ranged from 0.4–4 ng g–1 and 0.2–1
ng g–1, respectively (Table 2).
Initial experiments on establishing MDLs ensured
representative replication by calculating the standard
deviation of measured concentration values (Eurachem
Guide 2014) for samples (n = 8) processed through the
entire modified PCB method. Ideally, it is known that
better determination of MDL values can be obtained from
tissue-specific analysis; however, no stringent protocols
have been published requiring laboratories or scientific
studies to use matrix-specific MDLs (Ripp 1996). In fact,
more recent studies have utilized non-specific matrices
for their methods validation. For instance, cetacean blow
and blubber for determining EDCs in blood were used
by de Mello and de Oliviera (2016), whereas and spiked
silt, clay, and coarse sand were used by Coppock et al.
(2017) for extracting microplastics in marine sediments.
In reference to these and with the limitations of this study,
specifically on the amounts of PCB standards for two
cetacean tissues (i.e. blubber and liver), and with the fact
that endogenous substance (e.g. fats) in the sample tissues
will interfere in the analysis and will greatly contribute
to matrix effects (Zhang et al. 2013), cetacean kidney
samples – with the least reported average % fat of 11%
compared to blubber (75%) and liver (20%) (Kawai et al.
1988) – were used in the initial validation experiments.
From the results of the fat content determination, a 10-g
kidney sample was estimated at 1.1 g compared to the 0.4
g in a 2-g liver sample and 0.5-g blubber sample. Hence,
the EMDLs for each PCB congener in the blubber and
liver samples were referenced to the MDLs obtained from
the analysis of spiked cetacean kidney samples.
Table 1. Stranded cetaceans sampled from January 2013–August 2015, from which kidney and blubber samples were obtained.
Species Sample code Stranding location Sampling
year
Total PCBs
(ng g–1 lipid weight)gReference
Spinner dolphin
Sl15R7050513 Cordova, Cebu 2013 345 This study
Sl09R1140214 Magsingal, Ilocos Sur 2014 1190 This study
Sl04R4A100714 Calatagan, Batangas 2014 670 This study
–Mindanao 1996 250 (240–260) Minh et al. (2000)
–India 1997–1999 328 Karuppiah et al. (2005)
Fraser's dolphin
Lh05R1051114 Bangui, Ilocos Norte 2014 829 This study
Lh06R1260115 Aringay, La Union 2015 429 This study
Lh18R1270115 Alaminos, Pangasinan 2015 788 This study
–Mindanao 1996 620 (380–860) Minh et al. (2000)
Rough-toothed
dolphin
Sb04R3140113 Candelaria, Zambales 2013 1460 This study
Sb12R1310815 Sto Domingo, Ilocos Sur 2015 785 This study
–Brazil 2003–2012 7120 ± 7600 Lavandier et al. (2015)
Cuvier’s beaked
whale
Zc02R12141014 Maitum, Saranggani 2014 75.6 This study
Zc03R12141014 Maitum, Saranggani 2014 138 This study
–Pacic Islands
(Molokai, Saipan) 2008/2011 4250 (3130–5360) Bachman et al. (2014)
Risso's dolphin
Gg03R2071014 Sanchez Mira, Cagayan 2014 816 This study
Gg08R1010615 Dagupan, Pangasinan 2015 662 This study
–Italy 1992 593 Corsolini et al. (1995)
Dwarf sperm
whale
Ks02R5100413 Ragay, Camarines Sur 2013 21.7 This study
Ks01R11230714 Talomo, Davao 2014 292 This study
–Taiwan 2000–2001 211 Chou et al. (2004)
Blainville's beaked
whale
Md02R11150414 Bucana, Davao 2014 1300 This study
–Pacic Island (Maui) 2010 1450 Bachman et al. (2014)
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1098
The MDLs of the PCB congeners determined using the
spiked cetacean kidney samples ranged from 1 ng g–1
(PCBs -157 and -202) to 20 ng g–1 (PCBs -12, -35, -52,
and -194) (Table 2). Most of the EMDLs obtained for the
PCBs (38 out of the 42 congeners) were relatively low,
ranging from 1–10 ng g–1. The 20 ng g-1 level may be
higher than the usual MDL values obtained; for example,
in fish tissues (3 ng g–1; Batt et al. 2017). However, values
were relevant as they were obtained from spiked cetacean
kidney samples (n = 8), which passed through the entire
modified procedure, with three cleanup methods that
produced excellent accuracy and precision values. The
calculated MDLs, specifically those ranging from 1–2
ng g–1, are comparable with the MDL results of other
biological studies (Table 3).
Succeeding validation experiments that would have
established lower MDLs in the liver and blubber at sample
weights of 2 and 0.5 g, respectively using the spike level
of 0.025 µg (25 µL of 1 ppm) could no longer be done
due to limitations on the amount of the PCB standards.
Thus, the concept of EMDL in reporting the results in
the samples was used. While it can be noted that EMDLs
of the PCB congeners based on the MDLs determined
using the kidney samples were generally higher than
the EMDLs calculated from IDLs, EMDLs from the
kidney samples were more realistic as these MDLs were
established from samples that have passed through the
entire procedure. While different approaches can be
used for LOD estimation, confidence in the obtained
detection limits can only be established through repeated
Table 2. EMDLs of PCB congeners in lipid-rich animal (cetacean) tissues by GC/MS in combination with the three-way cleanup techniques.
PCBs
s
n = 8
EMDLs MDLs
PCB congener-specic
MDL literature values,
ng g-1 (from other
cetacean studies)
Congener No.
References
Based on
IDLsaKidney:
MDLd,
ng g-1
n = 8
Based on
MDLkidney
Homolog Congener
no.
Blubber:
EMDLb,
ng g–1
n = 8
Liver:
EMDLc, ng
g-1
n = 8
Homolog
Penta-CB –114 0.46 4 1 2 2 – –
–126 1.49 4 1 5 5 – –
Hexa-CB –155 1.17 4 1 4 4 – –
–153 0.63 4 1 2 2 2.2;
20.3
Binnington et
al. (2017)e
Lundin et al.
(2016)f
–162 2.48 4 1 8 8 – –
–167 2.06 4 1 7 7 – –
–156 0.50 4 1 2 2 – –
–157 0.24 4 1 1 1 – –
–169 0.50 2 0.4 2 2 – –
Hepta-CB –188 0.45 1 0.4 2 2 – –
–189 0.64 1 0.2 2 2 – –
Octa-CB –202 0.32 4 1 1 1 – –
–195 0.56 4 1 2 2 – –
–194 4.01 2 0.4 20 20 – –
–205 2.89 2 0.4 9 9 – –
Nona-CB –208 2.25 4 1 7 7 – –
–206 0.42 1 0.4 2 2 – –
Deca-CB –209 0.68 4 1 2 2 – –
aIDLs – 2 ng mL–1 at 95% confidence interval
bEMDLs – determined using the formula: [(final injection volume, Vf /sample weight)*IDL], where Vf = 100 µL and sample weight for blubber = 0.5 g
cEMDLs – determined using the formula: [(final injection volume, Vf /sample weight)*IDL], where Vf = 100 µL and sample weight for liver = 2 g
dEstablished, experimental MDL = 3S (using kidney with sample weight = 10 g)
Dash means no published PCB congener-specific MDL value
eUsed beluga whale skin and blubber samples
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1099
measurements showing high accuracy and precision
(Eurachem Guide 2014).
To the authors’ knowledge, this is the first Philippine study
reporting MDL values from a lipid-rich cetacean tissue,
providing initial and conservative PCB values in stranded
cetaceans. As for the RDLs, values ranged from 1–30 ng
g–1 and LOQs from 3–40 ng g–1. The MDLs, RDLs, and
LOQs of the other PCB congeners were in between those
reported ranges (Appendix Table IV).
Accuracy and Precision
The mean percent recoveries (n = 8) of the elucidated
42 native PCBs in the spiked cetacean kidney samples
ranged from 59.6% (PCB-78) to 120% (PCB-15). PCB-
118 extracted from the FAPAS QC Material showed an
average percent recovery of 106% (n = 2). Moreover, in the
spiked cetacean kidney samples, the percent recoveries of
the surrogate PCBs (labeled mono- to deca-CBs in nonane)
ranged from 64.6% (13C12-PCB-3) to 114% (13C12-
PCB-118) and 96% for perylene-d12. The good recoveries
obtained indicated that the desired extraction and cleanup
performance was achieved. Similarly, good recoveries were
obtained for the cetacean kidney samples spiked with CEN
PCB Congener Mix 1, i.e., ranging from 85.2% (PCB-52) to
113% (PCB-18). For precision, the following % RSD values
were obtained in the spiked cetacean kidney samples: 9%
(PCB-57) to 29% (PCB-33, -99, -155, -206) for the native
PCBs, 19% (13C12-PCB-28) to 28% (13C12-PCB-153) for
the surrogate 13C12-PCBs, and 15% for perylene-d12, and
5.6% (PCB-52) to 17.2% (PCB-194) for the CEN PCB
Congener Mix. Comparing the results obtained from the
accuracy and precision tests with the estimated recovery
and precision data as a function of analyte concentration by
the AOAC (1998), the % recoveries and % RSD obtained
were within the criteria of 40–120% and < 30% RSD,
respectively, for an analyte concentration of 1 µg kg–1
(Figures 1a and b).
Figure 1a. Accuracy (% mean recovery) and precision (% RSD) data for native PCBs in spiked cetaceankidney
samples.
Table 3. Reported MDLs for PCBs in cetacean tissues and other biological samples, ng g–1.
Reported MDL values or range of values for PCBs in
cetacean tissues and other biological samples ng/gTissue samples used References
1–20 Cetacean: kidney This study
1 Orca: blubber Jepson et al. (2016)
2 Rhesus monkey: brain, kidney, liver Mes et al. (1995)
20 Rhesus monkey: serum and adipose samples Barsotti and van Miller (1984)
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1100
The results indicated that the modified method has
substantiated the AOAC (1998) requirement for accuracy,
as an excellent recovery of added analytes corresponding
to appropriate concentration ranges has been obtained and
precision as good repeatability of data values was observed.
Detected PCB Concentrations in the Cetacean
Blubber Tissues
Out of the 42 possible PCB congeners that can be measured
reliably using the validated method, 38 congeners (PCBs-
8, -12, -19, -18, -33, -38, -35, -37, -52, -44, -57, -74, -79,
-78, -81, -77, -104, -99, -123, -118, -114, -126, -155, -153,
-162, -167, -156, -157, -169, -188, -189, -202, -195, -194,
-205, -208, -206, and -209) were detected in 15 blubber
samples of stranded cetaceans found in various locations
in the Philippines (Table 1). Other studies have also
reported a similar number of PCB congeners in cetacean
blubber samples, e.g. 32 and 47 congeners as reported by
Méndez-Fernandez et al. (2017) and Bachman et al. (2014),
respectively. In terms of the total PCB concentrations
in each blubber sample, the detected levels ranged from
21.7–1,460 ng g–1 lipid weight. These concentrations are
generally comparable to what has been reported in the
blubber tissues sampled from stranded cetaceans found
elsewhere (Table 4). Supported with good recoveries and
repeatability data for the QC and spiked cetacean kidney
samples, detected PCB levels in actual lipid-rich blubber
tissues are, therefore, accurate test results.
CONCLUSION AND
RECOMMENDATION
In-house single method validation of an analytical method
intended for routine PCB residue analyses in cetacean
tissues was carried out. The study was able to develop
an easy-to-follow, cost-effective, and reliable analytical
method with efficient cleanup techniques for lipid-rich
sample matrices, e.g. animal tissues such as the kidney.
The suitability of the method for its intended use was
also demonstrated. With the use of external QC material
and spiked samples, acceptable accuracy and precision
data were obtained, providing confidence in the modified
method. The GC/MS method in combination with three-
way cleanup techniques was successfully applied to
lipid-rich cetacean blubber samples to elucidate residual
PCBs at low detection limits. This method can be used for
routine detection of selected PCBs in fatty animal tissues.
ACKNOWLEDGMENTS
The authors are thankful to the University of the
Philippines Natural Sciences Research Institute’s
Research and Analytical Services Laboratory (UP-
NSRI-RASL) and its staff, Dr. Evangeline C. Santiago,
Philippine Marine Mammal Stranding Network, the
United Nations University, and Shimadzu Corporation
for the GC/MS QP-2010 donated to the UP-NSRI-RASL.
Figure 1b. Accuracy (% mean recovery) and precision (% RSD) data for surrogate PCBs, perylene-d12, CEN PCB
congener mix 1, and PCB-118 in FAPAS oily fish in spiked cetacean kidney samples.
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1101
Table 4. Comparison of total PCB concentrations in blubber of cetacean species from other countries.
Species Sample code Stranding location Sampling year
Total PCBs
(ng g–1 lipid weight)
g
Reference
Spinner dolphin
Sl15R7050513 Cordova, Cebu 2013 345 This study
Sl09R1140214 Magsingal, Ilocos Sur 2014 1190 This study
Sl04R4A100714 Calatagan, Batangas 2014 670 This study
–Mindanao 1996 250 (240–260) Minh et al. (2000)
–India 1997–1999 328 Karuppiah et al. (2005)
Fraser's dolphin
Lh05R1051114 Bangui, Ilocos Norte 2014 829 This study
Lh06R1260115 Aringay, La Union 2015 429 This study
Lh18R1270115 Alaminos, Pangasinan 2015 788 This study
–Mindanao 1996 620 (380–860) Minh et al. (2000)
Rough-toothed
dolphin
Sb04R3140113 Candelaria, Zambales 2013 1460 This study
Sb12R1310815 Sto Domingo, Ilocos Sur 2015 785 This study
–Brazil 2003–2012 7120 ± 7600 Lavandier et al. (2015)
Cuvier’s beaked
whale
Zc02R12141014 Maitum, Saranggani 2014 75.6 This study
Zc03R12141014 Maitum, Saranggani 2014 138 This study
–Pacic Islands
(Molokai, Saipan) 2008/2011 4250 (3130–5360) Bachman et al. (2014)
Risso's dolphin
Gg03R2071014 Sanchez Mira, Cagayan 2014 816 This study
Gg08R1010615 Dagupan, Pangasinan 2015 662 This study
–Italy 1992 593 Corsolini et al. (1995)
Dwarf sperm whale
Ks02R5100413 Ragay, Camarines Sur 2013 21.7 This study
Ks01R11230714 Talomo, Davao 2014 292 This study
–Taiwan 2000–2001 211 Chou et al. (2004)
Blainville's beaked
whale
Md02R11150414 Bucana, Davao 2014 1300 This study
–Pacic Island (Maui) 2010 1450 Bachman et al. (2014)
gSum of elucidated concentration levels of PCB congener
This study was supported by the Office of the Vice
Chancellor for Research and Development of UP Diliman
(Project No. 161611SOS), the Philippine Council for
Health Research and Development of the Department of
Science and Technology, and the UP-NSRI (Project No.
ESM-15-1-02).
NOTES ON APPENDICES
The complete appendices section of the study is accessible
at http://philjournsci.dost.gov.ph
REFERENCES
[AOAC] Association of Official Analytical Chemists.
1998. AOAC Peer-verified Methods Program, Manual
on Policies and Procedures. Gaithersburg, MD: AOAC
International.
AGUILAR A, BORRELL A. 1994. Assessment of
organochlorine pollutants in cetaceans by means of
skin and hypodermic biopsies. In: Nondestructive
biomarkers in vertebrates, 11th ed. Fossi MC, Leonzio
C eds. Boca Raton, FL: Lewis Publishers. p. 245–267.
BACHMAN M, KELLER J, WEST K, JENSEN BA.
2014. Persistent organic pollutant concentrations in
blubber of 16 species of cetaceans stranded in the
Pacific Islands from 1997 through 2011. Science of
the Total Environment 488: 115–123.
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1102
BARSOTTI D, VAN MILLER J. 1984. Accumulation
of a commercial polychlorinated biphenyl mixture
(Aroclor 1016) in adult rhesus monkeys and their
nursing infants. Toxicology 30(1): 31–44.
BATT AL, WATHEN JB, LAZORCHAK JM, OLSEN AR,
KINCAID TM. 2017. Statistical Survey of Persistent
Organic Pollutants: Risk Estimations to Humans and
Wildlife through Consumption of Fish from U.S.
Rivers. U.S. Environmental Protection Agency Papers
No. 268. Retrieved from https://digitalcommons.unl.
edu/usepapapers/268
BINNINGTON M, LEI Y, POKIAK L, POKIAK J,
OSTERTAG SK, LOSETO L, WANIA F. 2017. Effects of
preparation on nutrient and environmental contaminant
levels in Arctic beluga whale (Delphinapterus leucas)
traditional foods. Environmental Science: Processes &
Impacts 19(8): 1000–1015.
BIRGUL A, TASDEMIR Y. 2011. Seasonal atmospheric
deposition variations of polychlorinated biphenyls
(PCBs) and comparison of some deposition sampling
techniques. Environmental Science and Pollution
Research 18(3): 396–406.
BORJA J, TALEON D, AURESENIA J, GALLARDO
S. 2005. Polychlorinated biphenyls and their
biodegradation. Process Biochemistry 40(6): 1999–
2013.
CHOU C, CHEN Y, LI C. 2004. Congener-specific
polychlorinated biphenyls in cetaceans from Taiwan
waters. Archives of Environmental Contamination and
Toxicology 47: 551–560.
COPPOCK R, COLE M, LINDEQUE P, QUEIROS A,
GALLOWAY T. 2017. A small-scale, portable method
for extracting microplastics from marine sediments.
Environmental Pollution 230: 829–837.
CORSOLINI S, FOCARDI S, KANNAN K, TANABE
S, BORRELL A, TATSUKAWA R. 1995. Congener
profile and toxicity assessment of polychlorinated
biphenyls in dolphins, sharks and tuna collected from
Italian coastal waters. Marine Environmental Research.
40(1): 33–53.
DAEWEL U, YAKUSHEV V, SCHRUM C, NIZZETTO
L, MIKHEEVA E. 2020. Understanding the Role
of Organic Matter Cycling for the Spatio-Temporal
Structure of PCBs in the North Sea. Water 12(3): 817.
DAI Q, MIN X, WENG M. 2016. A review of
polychlorinated biphenyls (PCBs) pollution in
indoor air environment. Journal of the Air & Waste
Management Association 66: 941–950.
DE MELLO D, DE OLIVEIRA C. 2016. Biological
matrices for sampling free‐ranging cetaceans and the
implications of their use for reproductive endocrine
monitoring. Mammal Review 46(2): 77–91.
EURACHEM. 2014. The Fitness for Purpose of Analytical
Methods – A Laboratory Guide to Method Validation
and Related Topics, 2nd ed. Teddington, UK.
FAROON O, RUIZ P. 2016. Polychlorinated biphenyls:
new evidence from the last decade. Toxicology and
Industrial Health 32: 1825–1847.
GERACI J, LOUNSBURY V. 2005. Marine mammals
ashore: a field guide for strandings. National Aquarium
in Baltimore.
JEPSON P, DEAVILLE R, BARBER J, AGUILAR À,
BORRELL A, MURPHY S, BARRY J, BROWNLOW
A, BARNETT J, BERROW S, CUNNINGHAM A.
2016. PCB pollution continues to impact populations of
orcas and other dolphins in European waters. Scientific
Reports 6: 18573.
JONATHAN J, WOANANU-AGGOR S, ADATARA
P, PHIL M, ADJORLOLO-GASOKPO A. 2018.
Variations of polychlorinated biphenyls (PCBs)
levels in the bivalve Galatea paradoxa from Ada, in
Ghana, during the dry and wet seasons. Journal of
Environmental Chemistry and Toxicology 2: 48–56.
KARUPPIAH S, SUBRAMANIAN A, OBBARD JP.
2005. Organochlorine residues in odontocete species
from the southeast coast of India. Chemosphere, 60,
891-897.
KAWAI S, FUKUSHIMA M, MIZAYAKI N, TATSUKAWA
R. 1988. Relationship between lipid composition and
organochlorine levels in the tissues of striped dolphin.
Marine Pollution Bulletin 19(3): 129–133.
KWAN C, TAKADA H, BOONYATUMANOND R,
KATO Y, MIZUKAWA K, ITO M, ZAKARIA M,
SANTIAGO E. 2014. Historical occurrences of
polybrominated diphenyl ethers and polychlorinated
biphenyls in Manila Bay, Philippines, and in the upper
Gulf of Thailand. Science of the Total Environment 470:
427–437.
LAUBY-SECRETAN B, LOOMIS D, GROSSE Y,
EL GHISSASSI F, BOUVARD V, BENBRAMIN-
TALLAA L, IARC WORKING GROUP. 2013.
Carcinogenicity of polychlorinated biphenyls and
polybrominated biphenyls. Lancet Oncology, 14(4),
287-288.
LAVANDIER R, AREAS J, DIAS P, TANIGUCHI
S, MONTONE R, DE MOURA J, QUINETE N,
SICILIANO S, MOREIRA I. 2015. An assessment of
PCB and PBDE contamination in two tropical dolphin
species from the Southeastern Brazilian coast. Marine
Pollution Bulletin 101: 947–953.
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1103
LUNDIN J, YLITALO BR, ANULACION B,
HEMPELMANN J, PARSONS K, WASSER S.
2016. Modulation in persistent organic pollutant
concentration and profile by prey availability and
reproductive status in Southern Resident killer whale
scat samples. Environmental Science & Technology
50(12): 6506–6516.
MÉNDEZ-FERNANDEZ P, SIMON-BUOHET B,
BUSTAMANTE P, CHOUVELON T, FERREIRA
M, LOPEZ A, MOFFAT C, PIERRE G, RUSSELL M,
SANTOS M, SPITZ J. 2017. Inter-species differences
in polychlorinated biphenyls patterns from five
sympatric species of odontocetes: Can PCBs be used
as tracers of feeding ecology? Ecological Indicators
74: 98–108.
MES J, ARNOLD D, BRYCE F. 1995. Postmortem
tissue levels of polychlorinated biphenyls in female
rhesus monkeys after more than six years of daily
dosing with Aroclor® 1254 and in their non-dosed
offspring. Archives of Environmental Contamination
and Toxicology 29(1): 69–76.
MINH TB, NAKATA H, WATANABE M, TANABE
S, MIYAZAKI N, JEFFERSON, TA, PRUDENTE
M, SUBRAMANIAN A. 2000. Isomer-specific
accumulation and toxic assessment of polychlorinated
biphenyls, including coplanar congeners, in cetaceans
from the North Pacific and Asian coastal waters.
Archives of Environmental Contamination and
Toxicology 39: 398–410.
O’HARA T, HOEKSTRA P, HANNS C, MUIR D,
WETZEL D, REYNOLDS J. 2004. A preliminary
assessment of the nutritive value of select tissues from
the bowhead whale based on suggested nutrient daily
intakes. International Whaling Commission Scientific
Committee Report SC/56/E2. p. 1–19.
QUEHENBERGER O, ARMANDO A, DENNIS
E. 2011. High sensitivity quantitative lipidomics
analysis of fatty acids in biological samples by gas
chromatography–mass spectrometry. Biochimica et
Biophysica Acta (BBA)–Molecular and Cell Biology
of Lipids 1811: 648–656.
RIPP J. 1996. Analytical detection limit guidance &
laboratory guide for determining method detection
limits. Wisconsin Department of Natural Resources,
Laboratory Certification Program.
SANTIAGO E, KWAN C. 2015. POPs in selected
rivers and bays in the Philippines. In: Monitoring and
Governance of Persistent Organic Pollutants in Asia.
Ito O et al. eds. United Nations, New York. p. 169–193.
THOMPSON M, ELLISON S, WOOD R. 2002.
Harmonized guidelines for single-laboratory validation
of methods of analysis (IUPAC Technical Report). Pure
and Applied Chemistry 74(5): 835–855.
[US EPA] United States Environmental Protection
Agency Method 1668C. 2016. EPA-820-R-10-005
– Chlorinated Biphenyl Congeners in Water, Soil,
Sediment, Biosolids and Tissue by HRGC/HRMS.
Washington, DC.
WALCZAK M, REICHERT M. 2016. Characteristics of
selected bioaccumulative substances and their impact
on fish health. Journal of Veterinary Research 60:
473–480.
WEBSTER L, ROOSE P, BERSUDER B, KOTTERMAN
M, HAARICH M, VORKAMP K. 2013. Determination
of polychlorinated biphenyls (PCBs) in sediment and
biota. ICES Techniques in Marine Environmental
Sciences No. 53. 18p.
ZHANG F, YANG X, XUE X, TAO X, LU G, DANG
Z. 2013. Estimation of n- octanol/water partition
coefficients (log) of polychlorinated biphenyls by
using quantum chemical descriptors and partial
least squares. Journal of Chemistry http://dx.doi.
org/10.1155/2013/7405
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1104
APPENDIX
Table I. Retention time, target, and reference ions of each PCB (native and surrogate) and internal
standards analyzed by the modified clean-up/GC/MS method.
Native PCBs Retention time
(min)
Target ions
(m z–1)
Reference ions
(m z–1)
Homolog Congener no.
Native
Di-CB –10 11.197 221.90 152.05
–9 11.436 221.95 152.05
–8 12.291 152.05 221.90
–12 12.848 221.95 152.05
–15 14.236 221.95 152.00
Tri-CB –19 13.366 185.95 255.95
–18 13.467 185.95 255.90
–33 14.286 257.90 255.95
–38 16w.331 186.00 257.90
–35 17.507 255.90 186.00
–37 17.921 255.90 186.00
Tetra-CB –54 15.308 291.90 289.90
–52 17.187 291.90 289.90
–44 17.878 219.90 291.90
–57 18.744 291.90 289.90
–74 19.272 291.90 219.90
–79 20.706 291.90 219.90
–78 21.044 291.90 289.95
–81 21.411 291.90 289.95
–77 21.799 291.90 219.90
Penta-CB –104 17.625 325.85 323.85
–99 20.493 325.85 323.85
–123 22.520 327.85 325.85
–118 22.600 325.85 323.85
–114 22.600 325.85 323.85
–126 25.520 327.85 325.85
Hexa-CB –155 19.901 359.85 361.80
–153 23.271 359.80 361.80
–162 25.101 359.80 357.80
–167 25.265 359.85 361.80
–156 26.040 359.85 357.80
–157 26.168 359.85 359.80
–169 26.244 359.85 361.80
Hepta-CB –188 22.967 393.80 395.80
–189 28.748 393.80 395.75
Octa-CB –202 29.147 429.75 427.70
–195 27.743 429.75 325.85
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1105
Native PCBs Retention time
(min)
Target ions
(m z–1)
Reference ions
(m z–1)
Homolog Congener no.
–194 30.279 429.75 431.75
–205 30.399 429.75 431.75
Nona-CB –208 30.175 439.80 441.75
–206 31.961 463.70 461.70
Deca-CB –209 33.663 497.65 499.60
Surrogates
13C12 Mono-CB –3 10.860 152.05 200.00
13C12 Di-CB –15 13.954 234.00 219.90
13C12 Tri-CB –28 16.092 268.00 270.00
13C12 Tetra-CB –52 17.187 291.90 289.90
13C12 Penta-CB –118 23.218 369.80 371.85
13C12 Hexa-CB –153 26.512 405.85 407.80
13C12 Hepta-CB –180 29.175 477.70 202.80
13C12 Octa-CB –194 30.259 475.70 477.70
13C12 Nona-CB –208 30.175 439.80 441.75
13C12 Deca-CB –209 33.580 439.75 437.75
Perylene-d12 – 34.125 264.20 260.20
Internal standards
Phenanthrene-d10 14.434 188.00 184.00
Pyrene-d10 20.698 212.00 210.00
Chrysene-d12 24.928 240.00 236.00
Table II. Linearity coefficients of the calibration curves, IDLs for mono- to deca-native PCBs in hexane and EMDLs (calculated from IDLs)
(pre-PCB analysis on cetacean samples).
PCBsLinearitya r
n = 8
Mean concentration
ng/mL
n = 8
s
n = 8
IDLb
ng/mL
n = 8
EMDLc, ng/g
n = 8
Homolog Congener No. BlubberdLivere
Di-CB –10 0.9993 2.05f2.52 8 2 0.4
–9 0.9990 2.44f2.39 8 2 0.4
–8 0.9993 4.93g2.85 9 2 0.4
–12 0.9995 4.07g5.63 20 4 1
–15 0.9990 1.11f0.423 2 0.4 0.1
Tri-CB –19 0.9993 8.28g6.25 20 4 1
–18 0.9996 9.08g4.87 20 4 1
–33 0.9990 7.72g2.65 8 2 0.4
–38 0.9993 6.44g2.22 7 1 0.4
–35 0.9981 12.9g5.65 20 4 1
–37 0.9992 5.53g0.826 3 1 0.2
Tetra-CB –54 0.9961 8.28g4.50 20 4 1
–52 0.9998 8.86g4.34 20 4 1
–44 0.9996 4.01g5.10 20 4 1
–57 0.9992 3.60f0.488 2 2 0.4
–74 0.9993 8.75g0.514 2 2 0.4
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1106
PCBsLinearitya r
n = 8
Mean concentration
ng/mL
n = 8
s
n = 8
IDLb
ng/mL
n = 8
EMDLc, ng/g
n = 8
Homolog Congener No. BlubberdLivere
–79 0.9967 5.43g0.502 2 2 0.4
–78 0.9983 6.13g0.445 2 2 0.4
–81 0.9988 6.39g0.568 2 2 0.4
–77 0.9998 4.03g0.699 3 1 0.2
Penta-CB –104 0.9996 7.23g4.99 20 4 1
–99 0.9983 6.07g4.45 20 4 1
–123 0.9960 6.10g0.430 2 2 0.4
–118 0.9987 6.35g0.470 2 2 0.4
–114 0.9995 7.05g5.41 20 4 1
–126 0.9999 2.98f5.37 20 4 1
Hexa-CB –155 0.9995 4.86g5.47 20 4 1
–153 0.9999 1.14f4.08 20 4 1
–162 0.9995 8.20g3.79 20 4 1
–167 0.9990 3.53f3.71 20 4 1
–156 0.9996 2.56f5.59 20 4 1
–157 0.9996 4.25g3.99 20 4 1
–169 0.9995 1.51f0.410 2 2 0.4
Hepta-CB –188 0.9997 4.65g2.31 7 1 0.4
–189 0.9995 1.28f1.21 4 1 0.2
Octa-CB –202 0.9995 2.52f4.74 20 4 1
–195 0.9992 3.3g4.56 20 4 1
–194 0.9987 5.64g0.485 2 2 0.4
–205 0.9993 0.55f0.590 2 2 0.4
Nona-CB –208 0.9996 2.88f4.96 20 4 1
–206 0.9992 6.02g2.21 7 1 0.4
Deca-CB –209 0.9993 3.31f3.87 20 4 1
aLinearity – determined using six-level calibration solutions: 0, 5, 10, 20, 50 and 100 ng mL–1 native PCB congeners
bIDLs – determined using 1 or 5 ng mL–1 native PCB congeners; IDL = 3s (US EPA 2016; Eurachem Guide 2014)
cEMDLs = [(final injection volume, Vf /sample weight)*IDL], where Vf = 100 µL (Tiryaki 2016).
dSample weight for blubber = 0.5 g
eSample weight for liver = 2 g
fNative PCB concentration = 1 ng mL–1
gNative PCB concentration = 5 ng mL–1
PCBs
Linearityh, r
PCBs
Linearity, r
Homolog Congener No. Homolog Congener No.
Di-CB –10 0.9970 Penta-CB –99 0.9982
–9 0.9981 –123 0.9961
–8 0.9971 –118 0.9992
–12 0.9994 –114 0.9984
–15 0.9995 –126 0.9999
Tri-CB –19 0.9968 Hexa-CB –155 0.9995
Table III. Linearity coefficients of the calibration curves for mono- to deca-native PCBs in hexane (conducted during the PCB analysis on
cetacean samples).
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1107
PCBs
Linearityh, r
PCBs
Linearity, r
Homolog Congener No. Homolog Congener No.
–18 0.9997 –153 0.9969
–33 0.9976 –162 0.9974
–38 0.9975 –167 0.9981
–35 0.9974 –156 0.9969
–37 0.9965 –157 0.9962
Tetra-CB –54 0.9961 –169 0.9974
–52 0.9982 Hepta-CB –188 0.9964
–44 0.9991 –189 0.9970
–57 0.9979 Octa-CB –202 0.9977
–74 0.9984 –195 0.9995
–79 0.9969 –194 0.9977
–78 0.9994 –205 0.9965
–81 0.9979 Nona-CB –208 0.9988
–77 0.9977 –206 0.9992
Penta-CB –104 0.9991 Deca-CB –209 0.9996
Table IV. Performance parameters of the modified method for extracting PCBs from cetacean kidney.
PCB s
n = 8
MDLi
ng g–1
n = 8
RDLi
ng g–1
n = 8
LOQi
ng g–1
n = 8
Homolog Congener No.
Di-CB –10 2.36 7 20 30
–9 0.82 3 5 9
–8 1.30 4 8 20
–12 3.73 20 30 40
–15 2.07 7 20 20
Tri-CB –19 2.25 7 20 30
–18 2.27 7 20 30
–33 0.88 3 6 9
–38 2.91 9 20 30
–35 3.67 20 30 40
–37 1.83 6 20 20
Tetra-CB –54 0.98 3 6 10
–52 3.71 20 30 40
–44 3.35 10 20 40
–57 1.39 5 9 20
–74 1.13 4 7 20
–79 0.59 2 4 6
–78 1.00 3 6 10
–81 2.25 7 20 30
–77 1.36 4 8 20
Penta-CB –104 1.07 4 7 20
–99 0.95 3 6 10
–123 2.65 8 20 30
–118 0.48 2 3 5
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
1108
–114 0.46 2 3 5
–126 1.49 5 9 20
Hexa-CB –155 1.17 4 7 20
–153 0.63 2 4 7
–162 2.48 8 20 30
–167 2.06 7 20 30
–156 0.50 2 3 5
–157 0.24 1 2 3
–169 0.50 2 3 5
Hepta-CB –188 0.45 2 3 5
–189 0.64 2 4 7
Octa-CB –202 0.32 1 2 4
–195 0.56 2 4 6
–194 4.01 20 30 40
–205 2.89 9 20 30
Nona-CB –208 2.25 7 20 30
–206 0.42 2 3 5
Deca-CB –209 0.68 2 4 7
iMDLs, RDLs, LOQs – determined using cetacean kidney samples spiked with 2.5 ng g–1 native PCB congeners.
Philippine Journal of Science
Vol. 150 No. 5, October 2021
Bondoc et al.: PCBs in Stranded Cetaceans
