Assessment and monitoring of persistent organic pollutants in lotic ecosystems methodological guide

Abstract

The Assessment and monitoring of persistent pollutants in lotic ecosystems methodological guide include the following main chapters: persistent organic pollutants (POP) definition and classification; POP assessment in lotic type ecosystems; POP sampling and sample preservation; and POP chemical analysis.

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ASSESSMENT AND MONITORING OF
PERSISTENT ORGANIC POLLUTANTS IN
LOTIC ECOSYSTEMS
METHODOLOGICAL GUIDE
Angela Curtean–Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
DDT

Angela Curtean–Bănăduc
Jan Ludvig Lyche
Vidar Berg
Alexandru Burcea
Doru Bănăduc
ASSESSMENT AND MONITORING OF
PERSISTENT ORGANIC POLLUTANTS IN
LOTIC ECOSYSTEMS
METHODOLOGICAL GUIDE
Publisher "Lucian Blaga" University of Sibiu
Sibiu, 2016

Designer: Maria Iasmina Moza
Cover: Maria Iasmina Moza, Alexandru Burcea
Descrierea CIP a Bibliotecii Naţionale a României
Assessment and monitoring of persistent organic pollutants in lotic ecosystems:
methodological guide / Curtean-Bănăduc Angela, Lyche Jan Ludvig, Berg Vidar, .... -
Sibiu : Editura Universităţii "Lucian Blaga" din Sibiu, 2016
Conţine bibliografie
ISBN 978-606-12-1414-3
I. Curtean-Bănăduc, Angela
II. Lyche, Jan Ludvig
III. Berg, Vidar
504.064:547
© All rights reserved. No part of this work can be copied without permission for
publishing from Editura Universității "Lucian Blaga" of Sibiu.
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Copyright ©2016
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The material was developed within the project „Support
instrument for decision making in management of
persistent organic pollutants. Case study: Mureș
Catchment Area", funded by a grant from Iceland,
Liechtenstein and Norway (EEA 2009-2014), in the frame
of RO04-Reduction of Hazardous Substances Program, the
total value of the project being 4,455,637.00 RON, out of
which 668,345.55 RON were co-financed from the
national budget through the Ministry of Environment.

Contents
CONTENTS ...................................................................................................... V
FIGURES LIST .............................................................................................. VII
TABLES LIST .................................................................................................. IX
ABBREVIATIONS ......................................................................................... XI
I. PERSISTENT ORGANIC POLLUTANS. DEFINITION,
CLASSIFICATION ........................................................................................ 15
II. POP ASSESSMENT IN LOTIC TYPE ECOSYSTEMS ................ 23
III. SAMPLING AND SAMPLE PRESERVATION .............................. 35
1. Sediment ............................................................................................ 37
2. Water ................................................................................................... 41
3. Biota..................................................................................................... 45
Fish ....................................................................................................... 45
Benthic macroinvertebrates ..................................................... 47
IV. CHEMICAL ANALYSIS ....................................................................... 51
1. Specific gas cromatograph coupled with mass
spectrometry methods ........................................................................ 55
2. Control parameters ....................................................................... 64
Criteria for data validation ........................................................ 65
Internal standard (IS) .................................................................. 66
The calibration curve ................................................................... 66
Recoveries and blind control .................................................... 67
3. Samples analysis ............................................................................ 68
Sediment ............................................................................................ 73
V

Contents
a) Method principle ................................................................ 73
b) Work protocol (LLE method) ........................................ 75
Water ................................................................................................... 77
a) Method principle ................................................................ 77
b) Work protocol (SPE method) ........................................ 81
Animal tissues ................................................................................. 82
a) Method principle ................................................................ 82
b) Rough guide of quantities and specific
consideration between matrices ......................................... 85
c) Work protocol (LLE - type extraction) ...................... 87
Explaining the injection sequence in GS-QqQ (sediment,
water, tissues) ................................................................................. 91
Data imterpretation and concentration calculus ............. 93
The toxic equivalence (TEQ) ..................................................... 96
Determination of parameters describing the
environmental faith of POPs ..................................................... 97
GLOSSARY ..................................................................................................... 98
REFERENCES ............................................................................................. 102
VI

Figures list
FIGURES LIST
Figure 1. Classification of persistent organic pollutants according to
origin.
17
Figure 2. Structure of pesticides classified as POPs.
18
Figure 3. Polychlorobiphenyl type organochlorinated compounds.
18
Figure 4. Perfluorinated compounds.
19
Figure 5. Brominated compounds.
19
Figure 6. Dibenzofurans and dibenzodioxin compounds.
20
Figure 7. Stages in POP assessment in lotic ecosystems.
31
Figure 8. POP monitoring in lotic ecosystems.
33
Figure 9. Sediment sampling for POP determination.
39
Figure 10. List of equipment and materials required for sediment
sampling.
40
Figure 11. List of required equipment and materials for water
sampling.
43
Figure 12. Water sampling for POP determination.
44
Figure 13. List of required equipment and materials for fish
sampling.
47
Figure 14. List of required equipment and materials for benthic
macroinvertebrates sampling.
49
Figure 15. POP analyses, associated to different matrix.
51
Figure 16. Gas chromatograph coupled with mass spectrometer.
56
Figure 17. MRM method chromatogram. Capillary 60 m db-5MS.
61
Figure 18. Calibration curve for α-HCH.
72
Figure 19. Equipment for POP extraction from sediment.
75
Figure 20. Lab equipment.
81
Figure 21. Concentration of the samples using the thermoblock.
91
VII

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
VIII

Tables list
TABLES LIST
Tabel 1. The estimated half-life – HL (days) of POPs in various
environments.
16
Table 2. List of POPs under the incidence of the Stockholm
Convention.
20
Table 3. Minimum and maximum concentrations of POPs in
continental aquatic ecosystems.
23
Table 4. POPs elution order and relative retention times.
57
Table 5. Transitions of ions used in MRM for POP quantification.
61
Table 6. Control parameters and their use.
64
Table 7. Criteria and limitations in data validation.
65
Table 8. Standards used for POP analysis.
68
Table 9. List of equipment, reagents and consumables required for
POP extraction from the sediment.
74
Table 10. List of equipment, reagents and consumables required for
POP extraction from water.
80
Table 11. List of equipment, reagents and consumables required for
POP extraction from tissues.
83
Table 12. Homogenisation methods for various matrices.
84
Table 13. Tissue quantities for various matrices.
85
IX

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
X

Abbreviations
ABBREVIATIONS
ATSDR – Agency for Toxic Substances and Disease Registry
APHA – American Public Health Association
bdl – below detection limit
CAS – Chemical Abstracts Service
Da – Dalton
DDT – Dichlorodipheniltrichloroethane
ECD – Electron Capture Detector
IS – Internal Standard
EPA – Environemental Protection Agency
EQS – Environmental quality standards
EU – European Union
GC – Gas Chromatograph
GPC – Gel Permeation Chromatography
HBCD – Hexabromocyclododecane
HCB – Hexachlorobenzene
HCH – Hexachlorocyclohexane
HES – High Efficiency Source
HL – Half Life
HPLC – High Performance Liquid Chromatography
ISO – International Organization for Standardization
LLE – Liquid Liquid Extraction
LOD – Limit of Detection
LOQ – Limit of Quantification
LSE – Liquid Solid Extraction
MAC – Maximum Allowable Concentrations
MASE – Membrane Assisted Solvent Extraction
XI

Abbreviations
MRM – Multiple Reaction Monitoring
Ms – Micropterus salmoides
MS – Mass spectrometry
MS/MS – Tandem mass spectrometer or triple quadrupole mass
spectrometry
NPDWRs – National Primary Drinking Water Regulations
OCP – Organochlorurate pesticides
Orbitrap – Ultra resolution mass spectrometer
PAH – Polycyclic aromatic hydrocarbons
PC – Control parameter
Pc – Procambarus clarkii
PCB – Polychlorinated biphenyls
PCDD – Polychlorodibenzo-p-dioxins
PCDF – Polychlorinated dibenzofurans
PCP – Pentachlorophenol
PCNs – Polychlor naphtalene
PLE – Pressurized Liquid Extraction
POP – Persistent Organinc Pollutant
ppb – parts per billion
ppt – parts per trillion
qMS – Quadrupole mass spectrometer
R2 – Regression coefficient
RF – Radio Frequency
RPSW – Protection of Surface Waters
SE – Solvent Extraction
SFE – Supercritical Fluid Extraction
SIFI – Simultan Full Scan and Selected Ion Monitoring
SIM – Selected Ion Monitoring
XII

Abbreviations
SPE – Solid Phase Extraction
SPMD – Semipermeable Membrane Device
Ss – Salmo salar
TCDD – 2,3,7,8-tetrachlorodibenzo-p-dioxin
TEF – Toxic equivalency factors
TEQ – Toxic equivalency
TIC – Total Ion Current
TOFMS – Time of flight mass spectrometry
USEPA – U.S. Environmental Protection Agency
WFD – Water Framework Directive
WHO – World Health Organization
γ-HCH – γ-hexachlorocyclohexane (lindane)
XIII

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
I. PERSISTENT ORGANIC POLLUTANS.
DEFINITION, CLASSIFICATION
Persistent organic pollutants (POPs) are organic compounds with
toxic properties, resistant to decay, which bioaccumulate in living
organisms and can be transported through air, water and migratory
species at great distances, over international borders and therefore
deposited away from the place of origin (Stockholm Convention 2001,
**http://chm.pops.int/, *European Commission Regulation (CE) no.
850/2004).
As lipophilic substances with high stability, POPs bioaccumulate
and concentrate in food chains (bioamplification), becoming toxic at
some threshold level (Van den Berg et al. 1998, El-Shahawi et al. 2010,
Polder et al. 2008, Zhou et al. 2007, Berg et al. 2013, Deribe et al. 2011,
Verhaert et al. 2013).
An organic compound may be classified as a POP if it meets the
following criteria:
- to be persistent, with long half-life in air, water, soil or sediment. A
persistent substance must have a half-life (HL) of more than 180
days for substances in soil and sediment, 60 days in the water and
more than 2 days for substances in the air (Ene 2014) (Tab. 1);
- transport capacity at great distance through air, water or migratory
species (Wania and MacKay 1996);
- bioaccumulation and bioamplification properties (Deribe et al. 2011,
Hong et al. 2009, Wania and MacKay 1996);
- toxic, with adverse effects for both human health and the
environment (Ashraf 2015).
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Because of to these features, POPs are among high-risk pollutants
to human health and the environment (Barni et al. 2014, Jacob 2013,
Dabrowski et al. 2004, Wilson et al. 2016b, Wilson et al. 2016a).
Table 1. The estimated half-life – HL (days) of POPs in various
environments (source: Gramatica and Papa 2007).
Number
POP
HL air
HL water
HL soil
HL sediment
1.
Aldrin
0.21
710
710
2300
2.
Chlordane
2.3
710
710
2300
3.
DDT
7.1
230
710
2300
4.
Dieldrin
2.3
710
710
2300
5.
HCB
710
2300
2300
2300
6.
PCB3
7.1
230
710
710
7.
PCB209
2300
2300
2300
2300
8.
TCDDa
7.1
23
710
2300
9.
OCDFb
23
230
2300
2300
10.
Toxaphene
7.1
2300
2300
2300
a - 2,3,7,8-tetrachlorodibenzo-p-dioxin; b - Octachlorodibenzofurane
POPs can be of natural origin - dioxins and dibenzofurans,
compounds from volcanic activity and vegetation fires (Jacob 2013, Jacob
and Cherian 2013) or of anthropogenic origin - chemicals used in
agriculture, such as pesticides (DDT, aldrin, dieldrin, endrin, chlordane,
heptachlor, toxaphene, mirex, hexachlorobenzene), in industry
(polychlorobiphenyls, hexachlorobenzene, brominated compounds,
perfluorinated compounds) or unintentionally produced, by-products of
industrial processes or resulting from the combustion of certain
materials (dioxins and furans, Fig. 6) (El-Shahawi et al. 2010, Ferreira
2013)(Fig. 1).
16

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Figure 1. Classification of persistent organic pollutants according to
origin.
Organochlorinated pesticides – OCP (Fig. 2) are characterized by
the presence of chlorine atoms in the structure which makes them toxic
but also resistant to degradation in nature, being hard to remove from
soil or water.
Organochlorinated compounds of the polychlorobiphenyl type -
PCBs (Fig. 3) are a group of chlorinated aromatic hydrocarbons with
biphenyl ring structure, with at least one hydrogen atom substituted by a
chlorine atom; there are 209 PCB isomers which have the same basic
structure, but a variable number of chlorine atoms in different positions.
Only about 70 of them having been identified in the various mixtures
available on the market (*Decree 1179/2010). Mixtures of various
polychlorobiphenyls are termed as aroclor.
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Figure 2. Structure of pesticides classified as POPs
(source: https://pubchem.ncbi.nlm.nih.gov).
Figure 3. Polychlorobiphenyl type organochlorinated compounds
(source : https://pubchem.ncbi.nlm.nih.gov).
Perfluorinated compounds – PFCs (Fig. 4) are used in flame
retardant foams or for materials treatment (textiles, packaging,
construction materials), due to their property to repeal water and oils.
18

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Figure 4. Perfluorinated compounds
(source : https://pubchem.ncbi.nlm.nih.gov).
Brominated compounds – BFRs (Fig. 5) present a structure with
aromatic rings as well as bromine, have flame retardant properties and
are used for fireproofing materials.
Figure 5. Brominated compounds
(source: https://pubchem.ncbi.nlm.nih.gov).
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Figure 6. Dibenzofurans and dibenzodioxin compounds
(source: https://pubchem.ncbi.nlm.nih.gov).
The pollution caused by POPs is a cross-border problem that
requires action at the international level. Thus, in 2001 the Stockholm
Convention was adopted, which aims to protect human health and the
environment from the adverse effect of POPs by ensuring safe disposal of
such substances and reducing production and their use
(**http://chm.pops.int/).
Currently covered by the Stockholm Convention are the
compounds and family of compounds presented in table 2, but this list
may be completed with new compounds whose presence in the
environment and whose eco-toxicological effects are supported by
scientific studies.
Table 2. List of POPs under the incidence of the Stockholm Convention
(source: *European Commission 2014, http://chm.pops.int/).
No.
POP
CAS
Annex
1. a
Aldrin
309-00-2
A.
2. a
Chlordane
57-74-9
A.
3. a
Dichloro-diphenyl-trichloroethane
(DDT)
50-29-3
B.
4. a
Dieldrin
60-57-1
A.
5. a
Endrin
72-20-8
A.
20

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
No.
POP
CAS
Annex
6. a
Heptachlor
76-44-8
A.
7. a,b,c
Hexachlorobenzene (HCB)
118-74-1
A. C.
8. a
Mirex
2385-85-5
A.
9. a
Polychlorobiphenyls (PCB)
Diverse
A. C.
10. c
Polychlorodibenzo-p-dioxins (PCDD)
Diverse
C.
11. c
Polychlorodibenzofurans (PCDF)
Diverse
C.
12. a
Toxafen
8001-35-2
A.
13. a
α- hexachlorocyclohexane (α-HCH)
319-84-6
A.
14. a
β- hexachlorocyclohexane (β-HCH)
319-85-7
A.
15. b
Perfluorooctane sulphonic acid (PFOS)
1763-23-1
B.
16. a
Chlordecone
143-50-0
A.
17. a
Endosulfan
115-29-7
A.
18. b
Ether hexabromobiphenyl
68631-49-2
A.
19. b
Ether heptabromobiphenyl
207122-16-5
A.
20. b
Ether tetrabromobiphenyl
5436-43-1
A.
21. b
Ether pentabromobiphenyl
60348-60-9
A.
22. b
Perfluorooctane sulphonic acid (PFOSF)
307-35-7
B.
23. b,c
Hexabromobiphenyl
36355-01-8
A.
24. b
Hexabromocyclododecane (HBCD)
25637-99-4
A.
25. a
Lindane (γ-HCH)
58-89-9
A.
26. a,b,c
Pentachlorobenzene
608-93-5
A. C.
27. a
Pentachlorophenol, its salts and esters (PCP)
87-86-5
A
28. b,c
Chlorinated naphthalene (PCNs)
70776-03-3
A. C.
29. a
Hexaclorobutadiene
87-68-3
A.
a – Substances used as pesticides
b – Substances resulting from chemical industry
c – Unintentional by-products
CAS registry number Chemical Abstracts Service
Annex A. - measures are necessary to eliminate the production and use of these POP
Annex B. - restriction measures are necessary in the production and use of these POP
Annex C - measures are needed to reduce the emission of such unintentional POP
21

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
22

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
II. POP ASSESSMENT AND MONITORING IN LOTIC TYPE
ECOSYSTEMS
The main classes of persistent organic pollutants (POPs) that have
been reported in continental aquatic ecosystems are: organochlorinated
pesticides (OCPs), polychlorobiphenyls (PCBs), aromatic hydrocarbons
(PAHs), brominated flame retardants (BFRs) and perfluorinated
compounds (PFCs) (Fernández et al. 2005, Muir et al. 1990, Sharma et al.
2009, Schmid et al. 2007, Yang et al. 2010, Cui et al. 2016, Kanzari et al.
2014, Quesada et al. 2014, Megson et al. 2016, Liu et al. 2016, Berg et al.
2013, Deribe et al. 2011, Barni et al. 2016, Smalling et al. 2016, Jacob and
Cherian 2013, Camino-Sánchez et al. 2012, Verhaert et al. 2013,
Blocksom et al. 2010, Eslami et al. 2016, Lorgeoux et al. 2016, Inostroza
et al. 2016, Kukučka et al. 2015, Pariatamby and Kee 2016, Seguí et al.
2013); (Tab. 3).
Table 3. Minimum and maximum concentrations of POPs in continental
aquatic ecosystems.
Matrix
POP
Min*
Max*
Gheographical area
Ref.
water
Σ DDT
0.011
0.014
Slovakia/
Lake Ladove
(Fernández et al. 2005)
Σ DDT
bdl
0.0096
Spain/Lake Redon
Σ DDT
bdl
0.014
Austria/Lake
Gossenkölle
Σ DDT
bdl
0.00059
Norway/Lake Øvre
Neådalsvatn
Σ DDT
0.004
1
0.0031
Spain/Lake Redon
HCB
0.001
0.01
Slovakia/ Ladove
23

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Matrix
POP
Min*
Max*
Gheographical area
Ref.
water
HCB
0.006
0.0084
Spain/Lake Redon
(Fernández et al. 2005)
HCB
bdl
0.004
Austria/
Lake Gossenkölle
HCB
bdl
0.0062
Norway/Lake Øvre
Neådalsvatn
HCB
bdl
0.0044
Mozambique/
Lake Malawi
HCB
bdl
0.021
Canada/Lake Bow
HCB
0.000
5
0.0024
Spain/Lake Redon
Σ PCB
0.05
0.083
Slovakia/
lake Ladove
Σ PCB
0.048
0.079
Spain/Lake Redon
Σ PCB
bdl
0.11
Austria/Lake
Gossenkölle
Σ PCB
bdl
0.026
Norway/lake Øvre
Neådalsvatn
Σ PCB
bdl
0.68
Anglia/lake
Easthwaite Water
Σ PCB
bdl
0.008
Sweden
Σ PCB
0.022
0.023
Spain/Lake Redon
α-HCH
0.054
0.11
Slovakia/
Lake Ladove
β-HCH
0.077
0.204
Slovakia/
Lake Ladove
α-HCH
0.313
0.41
Spain/Lake Redon
β-HCH
bdl
2.5
Spain/Lake Redon
α-HCH
bdl
0.064
Austria/
Lake Gossenkölle
24

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Matrix
POP
Min*
Max*
Gheographical area
Ref.
water
β-HCH
bdl
0.93
Austria/
Lake Gossenkölle
(Fernández et al. 2005)
α-HCH
bdl
0.11
Norway/Lake Øvre
Neådalsvatn
β-HCH
bdl
0.2
Norway/Lake Øvre
Neådalsvatn
α-HCH
bdl
0.0098
Mozambique/
Lake Malawi
β-HCH
bdl
0.014
Mozambique/
Lake Malawi
α-HCH
bdl
0.21
Canada/Lake Bow
β-HCH
bdl
0.13
Canada/Lake Bow
Σ PAH
bdl
0.0008
6
Austria/
Lake Gossenkölle
Σ PAH
bdl
0.0011
Norway/Lake Øvre
Neådalsvatn
Σ PCB
0.123
0.242
Bosnia and
Herzegovina/
River Bosna
(Harman
et al.
2013)
∑ PAH
4
36
France/River Sena
(Fernandes
et al.
1997)
river
particles
in
suspension
aldrin
bdl
0.222
Spain/River Ebro
(Quesada et al.
2014)
o,p′-DDT
bdl
4.885
p,p′-DDT
5.81
1467
HCB
0.817
435.98
α-HCH
bdl
bdl
β-HCH
0.178
2.182
lindane
0.937
5.203
25

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Matrix
POP
Min*
Max*
Gheographical area
Ref.
δ-HCH
0.024
0.218
Σ PAH
75.6
146.4
sediment
aldrin
0.01
1.3
Bosnia and
Herzegovina/
River Bosna
(Harman et al. 2013)
o,p′-DDE
bdl
0.04
p,p′-DDE
bdl
1.8
o,p′-DDD
bdl
0.52
p,p′-DDD
bdl
1
o,p′-DDT
bdl
0.14
p,p′-DDT
bdl
0.5
dieldrin
0.02
0.64
endrin
bdl
3.2
heptachlor
0.5
9.2
heptachlore1
bdl
0.27
heptaclore2
0.1
2.1
Σ PCB
0.78
16
trans-
chlordane
bdl
0.05
cis-chlordane
bdl
0.13
oxichlordane
bdl
0.33
α-HCH
bdl
0.01
β-HCH
bdl
0.01
lindane
bdl
0.03
muscleMs
aldrin
0.4
11.1
Africa
(Hicks 2012)
Σ DDT
4.6
345.5
o,p'-DDT
1.5
186.6
p,p'-DDT
2.6
100.7
p,p'-DDE
0.5
58.2
dieldrin
1.8
265.8
lindane
3.7
1595.9
26

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Matrix
POP
Min*
Max*
Gheographical area
Referen
ce
muscleSs
Σ DDT
5.2
249.5
Scotland, Ireland and
Norway
(Jacobs et
al. 2002)
Σ PCB
94
459.9
Σ HCH
9.8
23
musclePc
aldrin
0.8
70.7
Africa
(Hicks 2012)
Σ DDT
bdl
o,p'-DDT
0.5
11.9
p,p'-DDT
1.1
25
p,p'-DDE
0.3
4.2
dieldrin
0.4
11.1
lindane
0.2
10.4
fish oilSs
Σ DDT
2.8
20.5
Scotland, Ireland and
Norway
(Jacobs et
al. 2002)
Σ PCB
8.8
450.3
ΣHCH
10.9
218.3
meat
products
Ss
Σ DDT
35.1
51.7
Scotland, Ireland and
Norway
(Jacobs et
al. 2002)
Σ PCB
75.6
1153.2
Σ HCH
2.4
46.8
*bdl = below detection limit; **(ng/g)/(ng/l)
MsMicropterus salmoides; SsSalmo salar; PcProcambarus clarkii
e1exo-epoxid; e2endo-epoxid
POPs are hydrophobic substances with very low solubility in
water, being nonpolar organic compounds (Vallack et al. 1998), these
substances can be easily adsorbed on sediment particles or water
suspended particles, especially on organic particles (Kanzari et al. 2014),
they are lipophilic substances, a trait which causes accumulation in fatty
tissues of the organisms (La Merrill et al. 2013, Sharma et al.2009, Yu et
al. 2009, Tomy et al. 2004, Deribe et al. 2011). These properties
determine the distribution and concentration of POPs in different
compartments of the aquatic ecosystems (water, sediment, biota).
27

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Thus, for the assessment and monitoring of continental aquatic
ecosystems for POPs pollution it is necessary to determine their
concentration in the tissues of aquatic organisms (organisms belonging
to the groups that have a life span sufficiently long to accumulate these
compounds) and in the sediment where, due to the physical and
chemical properties of POPs and accumulation in time, concentrations
may be much higher than in water (Darko et al. 2008, Covaci et al. 2005,
Verhaert et al. 2013, Eljarrat et al. 2004); the presence of POPs in water
is linked to the presence of suspended particulate matter, especially of
suspended organic matter (Pozo et al. 2014) and for this reason the
analysis of the particles is also recommended.
The analysis of POPs concentration in aquatic organisms’ tissues
is highly relevant to the assessment of the water ecosystems pollution
with such chemicals. The species selected for analysis must fulfil the
following criteria:
- to accumulate POP in their tissues. Accumulation is conditioned by
the diet type – in most aquatic groups the main mechanism through
which POPs enter the organisms is feeding (Rignell-Hydbom et al.
2010, Gallo et al. 2015). Due to the biomagnification phenomena, the
species at the top of the food chain accumulate larger quantities of
pollutants. The accumulated pollutant quantity varies proportionally
to the total lipid quantity from the tissue, is directly influenced by
triacylglycerol (Sprague et al. 2012), and at the same time depends
on the types of lipids present in the tissue (the lipid chain length and
if it is saturated or unsaturated) and also on their classes (Elskus et
al. 2005);
- to have a life cycle in the aquatic environment that would be long
enough as to accumulate POPs;
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
- to be commonly present in the study area;
- to have a wide distribution and well represented populations in the
analysed watershed.
In the lotic ecosystems, these criteria are fulfilled by some benthic
invertebrate groups and by predatory or invertebrate eating fish
species.
Among fish species suitable for POP assessment in Romanian rivers
we mention: Perca fluviatilis Linnaeus, 1758 (lifespan - 22 years, feeds
on benthic invertebrates, fish and zooplankton; presents precocious
ichtyophagia), Squalius cephalus (Linnaeus, 1758) (lifespan – 22 years,
feeds on benthic invertebrates, fish sometime on batrachians), Barbus
barbus (Linnaeus, 1758) (lifespan - 15 years, feeds mainly on benthic
invertebrates, seldom on detritus and fish). It is recommended to collect
large individuals (2 kg minimum weight). The following categories of
tissue are analysed: liver (due to the high lipid content, the liver
accumulates the highest POP levels), muscle (analysed also due to the
fact that is the part of the fish most often consumed by man), kidneys
and gonads.
The use of benthic macroinvertebrates in the monitoring
programs has the advantage of the relatively easier sampling and of the
fact that the analysis reflects more accurately the pollution of the
sampled river sector, since these organisms are moving on much smaller
distances than fish, but usually the accumulated pollutants levels are
smaller than the levels found in fish. Among the groups of benthic
macroinvertebrates that can be analysed to this purpose, we mention
Amphipods, Ephemeropterans and Trichopterans (Gordon et al. 2004,
Simon 1998, Flotemersch et al. 2006).
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
In the environment, POPs are subjected to volatilisation processes
and to dispersion, biodegradation, bioaccumulation and
biotransformation, making difficult to establish their primary source.
The dispersion models and transfer through biogeochemical cycles are
too complex and difficult in this case, therefore hard to interpret.
Hence, in the case of rivers for which there are no data on POP
pollution it is highly recommended to implement an assessment
program for these chemicals concentration in sediment, water and biota
(Fig. 7). In order to determine the sampling stations, the following
options are to be considered:
- according to the presence of potential pollution sources –
downstream of these sources. Hotspots of pollution and diffuse
sources will be considered, and, if possible, historical pollution will
also be considered.
- at equal distances on the length of the river (distance among stations
is established according to geomorphologic and hydrologic
characteristics of the river).
After sample analysis, the results interpretation and the
identification of the polluted river sectors, of the present categories of
pollutants and their quantification in various matrices (sediment, water,
and biota), the persistent organic pollutants monitoring program will be
established for the analysed ecosystem (Fig. 8).
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
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Figure 7. Stages in POP assessment in lotic ecosystems.
31

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Due to the complexity of the POPs nature in the environment, it is
recommended to employ investigation methods that will allow detection
and quantification of the presence of a large number of contaminants
prior to using specific methods targeting only a limited number of
analytes (Megson et al. 2016).
The sample collection, preservation, preparation and analysis
methods must guarantee results with a level of accuracy adequate for
achieving the objectives of the evaluation.
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
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Figure 8. POP monitoring in lotic ecosystems.
33

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
34

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
III. SAMPLING AND SAMPLES PRESERVATION
Having representative samples is a basic requirement for having
relevant information on POP pollution in the analysed river sector.
In most cases, for sediment, benthos and water samples, it is
necessary to obtain a series of samples (3 – 5 samples taken in different
points in the cross section of the riverbed) from the same river section,
the sampling points being established according to the habitat specificity
(minor riverbed morphology, variation of water flow in the riverbed
cross section, etc.) (Wetzel et al. 1996, Curtean-Bănăduc 2001, Chapman
1996, Giller and Malmqvist 1998, Sawyer et al. 2010).
The protocol for sampling, sample labelling and sample
preservation must be established a priori, so it would be adequate for the
scope of the study, consistent for all studied river sectors (fact that
would allow comparison of results obtained for different river sectors)
and it must be strictly observed by the sampling operators.
The labelling of the obtained samples must be performed
distinctively and in such way as for the samples to remain identifiable
during transport and storage at the lab.
Samples must be preserved until testing to insure preservation of
the levels of pollutants to the values from sampling.
The methods of sampling and sample preservation are of the
utmost importance since errors due to incorrect sampling and/or
preservation cannot be corrected after the fact.
The samples will be accompanied by sampling records like:
- sampling location (GIS coordinates),
- sampling date and hour,
- sampling method,
35

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
- sample type,
- information included on sample label, including the coding
system, according to the case,
- meteorological conditions at the time of the sampling,
- name of the sampling operator.
Other information can also be included regarding:
- minor riverbed structure in the sampled sector: maximum,
minimum, and average width; maximum, minimum, and average depth;
presence of meanders, presence of potholes, inclination, type of flow
rate, type of bedrock – expressed in percentage; usually these
parameters are assessed for a 50 meters long river sector (Biedenharn et
al. 1997, Infante et al. 2009),
- presence and type of hydro-technical works,
- type of riparian vegetation,
- land use next to the river,
- presence and type of pollution sources.
Samples must be obtained, manipulated and processed in such
way as to maximise the possibility of detecting the targeted substances.
The methods used in sample manipulation must prevent contamination
or accidental loss of analytes (*Decree 2002).
Sampling procedures and equipment must be documented, and
field observations and measuring must be recorded adequately (*SR ISO
5667-6: 2009) in order to make sure that the information regarding
sampling is preserved for study replicability purposes.
The sampling operator is responsible for the security and
traceability of any sample and of samples records and documentation.
All obtained samples will be recorded in the sample register,
document offering a clear and strict evidence of the samples.
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
1. Sediment
During sediment sampling (Fig. 9), it is important to remember
that the obtained sample must be representative and must preserve the
qualitative characteristics of the sediment from the time and place of
sampling.
Sediment samples can be obtained with the aid of a bottom
sampler (Ekman–Birge or Pettersen); once the sediment is brought to
the surface, the sample will be taken from the middle of the sampler box
in order to avoid its contamination by contact with the metal of the
sampler. The sediment sampled in one point must have a weight of at
least 30 g, the minimum quantity allowing analysis with minimum three
technical replicas. A list of required equipment and materials is
presented in Figure 10.
It is recommended to sample a series of 3 – 5 samples from one
sampling station, from different points in the riverbed cross section.
If the sediment layer is deeper than one meter and we wish to
analyse separately the POP levels in various layers, samples can be
obtained with soil drill rigs; from each layer minimum 30 g of sediment
will be taken. This sampling technique allows the division of samples in
sub-samples in order to provide data on depth profiles.
If the minor riverbed in the analysed river sector is wider than 50
m, there is the chance for sediment deposits to have different ages and
structures, case for which it is recommended to sample in more than one
point in the same cross section of the riverbed.
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
The obtained samples may be stored in fluorocarbon polymer
cans or aluminium foil (tin foil) recipients, the latter being introduced in
inert (non-reactive) plastic bags, resistant to freezing (*APHA-AWWA-
WEF 1998). The recipient/bag containing the sample will be labelled
with a permanent marker in order to preserve the sample identity, the
recipient being introduced in a plastic bag together with a calking paper
label written in crayon. The label will bear the code of the station from
which the sample was taken, the sample code and the sampling date.
The sampling operator will wear adequate protection equipment
and gloves in order to avoid sample contamination.
During sampling, it is important to make sure that the redox state
of the sediment is preserved (oxic or anoxic), reason for which the
contact with air must be avoided; immediately after sampling, the
samples will be frozen to -20°C in order to preserve the characteristics of
the contaminants and of their association with sediment (*UNEP
Chemicals 2007).
The transport to the lab and the storage of the sediment samples
will be done at -20°C (*UNEP Chemicals 2007). The time frame
recommended for sample storage is of two weeks until extraction, 40
days until POP identification and quantification, several years for
archived samples – it is recommended to keep individual subsamples for
reanalysis purposes (*UNEP Chemicals 2007).
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Figure 9. Sediment sampling for POP determination.
39

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Figure 10. List of equipment and materials required for sediment
sampling.
Required equipment and materials:

dredge or sediment sampler/soil drill rig
 boat
 mobile freezer or liquid nitrogen recipient
 two decimals precision balance
 stainless steel trays
 stainless steel spatula
 fluorocarbon recipients
 tin foil (aluminium)
 polyethylene bags
 calking paper and crayon

protection equipment for the operator
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
2. Water
In order for the water samples to be representative, it is
recommended to take 3 – 5 samples from one sampling station, from
different points in the riverbed cross section, with the stream, from 20 –
30 cm depths (*SR ISO 5667-6: 2009). In case of the river sectors with
depths that do not reach 30 cm and have sediments deposits, the
sampling must be performed in such way as to avoid inclusion of the
sediment particles in the sample because they can (falsely) determine
the presence in the sample of large amounts of POPs, knowing the fact
that these chemicals are adsorbed on solid particles. Sampling is
performed directly in the sample recipient (in order to avoid sample
contamination by using repeatedly a sampling device), the recipient
being filled up to 80% – 90% of its volume; after the water intake, and
the recipient is sealed. The utilised recipients must be made of dark
brown glass with sanded glass corks or screwed tops with inner PTFE
lining (*SR EN ISO 6468). From each point, a volume of minimum 3 L of
water will be obtained, volume that allows analysis with minimum three
technical replicas (Fig. 12).
When the water flow in the sampled section is low, an
incremental sampling will be performed (*SR ISO 5667-6: 2009), taking
care that all increments are protected from contamination.
In case of running water, it is enough to take samples from a
depth of 20 – 30 cm, since the chemical characteristics of the water are
not vertically layered. Exceptionally, in the case of very deep rivers, with
very low flow rates and sediment deposits on the bottom, the POP levels
from the surface layer might differ from the bottom layer, case for which
water samples must be taken from the bottom layer as well. In order to
41

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
sample water at various depths devices that allow water intake are used
at the desired depth and that can be manipulated from the distance (i.e.
the Ruttner water sampler); these devices must be made of stainless
steel; in order to transfer the sample from the sampler to the sample
recipient no plastic tubes will be used, in order to avoid the loss of
analytes from the water sample or interference of other chemicals in the
sample. If necessary, control checking will be performed in order to
prove that no sample contaminations or adsorption losses occurred.
The sample recipients are labelled with waterproof labels, the
label containing at least the following information: code of the station
were the sample was taken from, sample code and sampling date.
The sampling operator will wear adequate protection equipment
and gloves in order to avoid samples contamination.
Samples will be transported and stored until analysis in dark
places and at temperatures of 1°C - 4°C. In order to avoid the
degradation of the chemicals of interest from the sample, it is
recommended to perform the extraction as soon as possible, preferably
within 24 h from sampling (*SR EN ISO 6468). A list of required sampling
equipment and material is presented in Figure 11.
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Figure 11. List of required equipment and materials for water sampling.
Required equipment and materials:

boat
 mobile freezer or liquid nitrogen recipient
 Ruttner water sampler (optional)
 dark brown glass bottles with sanded cork

waterproof labels

protection equipment for the operator
43

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Figure 12. Water sampling for POP determination.
44

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
3. Biota
Fish
In order for the analysis results to be relevant, it is necessary to
collect a number of 5 – 10 individuals of each species selected according
to the assessment/monitoring methodology. A list of required sampling
equipment and material is presented in Figure 13.
Fish fauna collection is performed by electro-narcosis or with the
aid of net fishing gear (Curtean-Bănăduc 2001). A 1 km long river sector
is browsed upstream.
The sampling operator will wear adequate protection equipment.
The captured individuals belonging to other species than the ones
of interest or the individuals of the species of interest smaller than the
protocol-established weight will be released immediately after capture
in the habitat from which they were collected. The sampling protocol
must explicitly specify this action.
After capture, the individuals belonging to the species of interest
and having the right size will be stunned, measured (the total body
length is measured, from the tip of the head to the tip of the longest lobe
of the caudal fin), weighed, killed (by decapitation) and then dissected
for tissue sampling.
Usually, samples of liver, muscle, gonads and kidneys are taken,
minimum 15 g of each type of tissue (an amount covering the three
technical replicas). The muscle tissue will be sampled from the caudal
region, between the dorsal fin insertion to the caudal. Stainless steel
tools are used, and the operator will wear gloves. Measures are to be
taken in order to avoid sample contamination.
45

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Each tissue sample will be packed in aluminium foil and then
introduced in an inert plastic bag, together with a calking paper label on
which the code of the sampling station is marked in crayon, the sample
code (indicating the species, the number of the individual from the batch
collected in that given sampling station, the type of tissue) and the
sampling date; in order to make sure the sample identity is preserved,
the label information will be written in permanent marker on the sample
bag. The label and the bag must be kept from contact with the tissue.
Samples will be transported and stored at temperatures between -20°C
and -80°C (*UNEP Chemicals 2007).
Besides the general data, the sampling record will also indicate:
length, weight and sex of each dissected individual.
The timeframe recommended for sample storage is of two weeks
before extraction, 40 days until POP identification and quantification,
several years for archived samples – it is recommended to keep
individual subsamples for reanalysis purposes (*UNEP Chemicals
2007).
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Figure 13. List of required equipment and materials for fish sampling.
Benthic macroinvertebrates
The sampling of benthic macroinvertebrate groups selected
according to the assessment/monitoring methodology must take into
account the fact that each sample must insure a minimum quantity of 15
g of tissue in order to allow the analysis of three technical replicas. The
sampling can be performed with various types of bentometers (in the
case of shallow, 10-70 cm deep river sectors with rocky bottom), by
Required equipment and materials:

electro-narcosis fishing gear/nets
 boat
 mobile freezer or liquid nitrogen recipient
 two decimals precision balance
 tape meter
 stainless steel trays
 stainless steel scalpel
 stainless steel dissecting scissors
 stainless steel clamps
 tin foil (aluminium)
 polyethylene bags
 calking paper and crayon

protection equipment for the operator
47

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
dredge or sediment sampler (in the case of deep river sectors with
sediment deposits on the bottom) (Curtean-Bănăduc 2001). From the
collected material individuals belonging to the taxonomic groups of
interest will be kept, in the case of the insect larvae it is recommended to
keep in the sample the larger development stages, fact that involves the
sorting of the collected material on the spot.
A list of the required sampling equipment and materials is
presented in Figure 14.
It is recommended to take 3 – 5 samples from one sampling
station, from different points in the riverbed cross section.
Each sample will be frozen wet, in fluorocarbon polymer
recipients, labelled with permanent marker; in order to secure the
sample identity preservation, the sample recipient will be introduced in
a plastic bag, together with a calking paper label written in crayon. The
label will mention the sampling station code, the sample code (indicating
also the collected taxonomic group) and the sampling date. Samples will
be transported and stored at temperatures between -20°C and -80°C
(*UNEP Chemicals 2007).
The timeframe recommended for sample storage is of two weeks
before extraction, 40 days until POP identification and quantification,
several years for archived samples – it is recommended to keep
individual subsamples for reanalysis purposes (*UNEP Chemicals 2007).
48

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Figure 14. List of required equipment and materials for benthic
macroinvertebrates sampling.
Required equipment and materials:

bentometer, dredge, silt sampler
 boat
 mobile freezer or liquid nitrogen recipient
 two decimals precision balance
 magnifying glass
 stainless steel trays
 glass Petri dishes
 stainless steel clamps
 fluorocarbon bottles
 polyethylene bags
 calking paper and crayon

protection equipment for the operator
49

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
50

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
IV. CHEMICAL ANALYSIS
There are numerous methods for POP extraction from various types
of matrices. Figure 1 presents some of those methods and the type of
matrix for which they are most suitable.
Figure 15. POP analyses, associated to different matrix
(source: El-Shahawi et al. 2010).
SE – (solvent extraction) is an analyte purification method using
two immiscible phases. The principle is based on the passage of
hydrophobic POPs from aqueous solutions to organic solvent (for water
and tissue).
LSE – (liquid solid extraction) is an analyte purification method
using a solvent for the extraction of a compound from a solid matrix
(sediment, soil).
LLE – (liquid liquid extraction) is an analyte purification method
using a solvent for the extraction of the solute from the solution. The
solvent and the solution must be immiscible (similar to SE).
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
SFE – (supercritical fluid extraction) is a process of separation of
the analyte from the matrix using supercritical fluids as solvents.
MASE – (membrane assisted solvent extraction) it is a method
related to the LLE, where a membrane is used for the separation of the
aqueous phase from the organic one.
SPE – (solid phase extraction) is a method for separating analytes
dissolved in the solvent by passing them through a solid phase (to which
the analytes having affinity).
PLE – (pressurized liquid extraction) is a technique of extraction of
analytes from a solid matrix using a pressurized fluid.
GPC – (gel permeation chromatography) is a method for separating
analytes from the solvent by passing them through a gel phase that
allows the size-based separation of the compounds.
SPMD – (semipermeable membrane device) is a method for
separating analytes based on semipermeable membranes which allow
passage to an organic phase (oil). Due to their hydrophobic character,
over time most of the POPs will be concentrated in the oil.
After extraction, the POP sample is passed to compounds
separation based on the molecular mass and characteristics; for this, the
literature mentions several methods such as liquid chromatography, gas
chromatography or multidimensional gas chromatography. Liquid
chromatography was used for compounds such as organochlorinated
pesticides (Alder et al. 2006, del Mar Gómez-Ramos et al. 2015), with the
advantages of high speed separation and large variety of compounds that
can be analysed (volatile, non-volatile, polar compounds, ionic
compounds) (Megson et al. 2016). A disadvantage of liquid
chromatography is its low resolution, especially when compared to gas
chromatography. The latter has been used in the study of many types of
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
POPs (Kim et al. 2008; Salem et al. 2016) and has the advantage of
accurate separation of the compounds, the disadvantage being the need
for the gas chromatography analyte to be volatilisable (Megson et al.
2016). The multidimensional gas chromatography method provides
excellent separation and has been used in studies to determine the
concentration of organochlorinated pesticides (Majola et al. 2011), PCB
(Megson et al. 2015), etc.
In addition to the extraction method and separation method, the
detector type is also important. There are many types of analyte
detectors such as:
1. ECD-Electron Capture Detector. It works by using a β particle
source to bomb the carrier gas producing low energy
electrons that form the background noise. When analytes elute
from the chromatographic column they form ions under the β
particles bombing resulting in a signal being recorded which is
directly proportional to the concentration of the analyte
(Walsh, 2001),
2. qMS quadrupole Mass Spectrometer. A mass spectrometer is
operated based on a source of electrons that is bombing
compounds resulting in a m/z ratio where z is the number of
unit charges per ion and m is the mass of the ion. A mass
spectrometer can run in SCAN mode obtaining a TIC
chromatogram where all analytes exiting the chromatographic
column are being ionized and subsequently detected, or in SIM
mode whereby only certain analytes (ions) are detected
(Tanaka et al. 2000). The SIM method is made possible
because the Mass Spectrometer operates as a quadrupole,
selecting and directing the ions to the detector,
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
3. TOFMS- time of flight Mass Spectrometer. In this type of
spectrometer m/z value is deduced from the flight time (time
of flight) of the ion under vacuum through a tube of known
length (Danielle and Aebersold 2006),
4. MS/MS- triple quadrupole mass spectrometer (tandem mass
spectrometry). Such a spectrometer consists of two
quadrupole mass spectrometers between which lies a
quadrupole RF (Radio Frequency) that allows the splitting of
ions by collision-induced dissociation. In the first mass
spectrometer, detection of ions takes place through SCAN or
SIM, in the collision cell, ions are split and in the second mass
spectrometer, detection is performed by SCAN or SIM
(Javahery and Thomson 1997). When both mass
spectrometers within the MS/MS are operated in the SIM
method, the method is called MRM, with increased selectivity
and sensitivity (Danielle and Aebersold 2006),
5. Orbitrap mass spectrometer with ultra-high resolution. It is a
complex type of mass spectrometer based on capture of ions
on an orbit specific to each ion (Perry et al. 2008).
The quantitative determination is considered to be the most
important application of mass spectrometry and the triple quadrupole
mass spectrometer is the most often used tool for this purpose
(Hopfgartner et al. 2004; Zubarev and Makarov 2013).
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
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1. Specific gas chromathograph coupled with mass
spectrometry methods
In the case of POPs such as PCBs, organochlorinated pesticides,
perfluorinated compounds and brominated flame retardants, one of the
methods of analysis is gas chromatography coupled with mass
spectrometry (GC/MS/MS) offering a good resolution in compound
identification (Megson et al. 2016). In figure 16 the components of a gas
chromatograph coupled with a triple quadrupole mass spectrometer of
Agilent technology are presented. Triple quadrupole-type mass
spectrometry provides a higher level of specificity compared to mass
spectrometry. In 2014, Kanzari et al. published a paper on using a gas
chromatograph coupled with a mass spectrometer to verify the
concentrations of some POPs, choosing to use a splitless type injection
and a 5MS type column. This method is widely used in the analysis of
pesticides, being accompanied by a standard temperature software for
gas chromatography as well as methods of analysis for the mass
spectrometer which vary between SIFI (Simultaneously Full Scan and
Selected Ion Monitoring) (Kanzari et al. 2014) and SIM (Selected Ion
Monitoring) (Deribe et al. 2011; Zheng et al. 2011). SIM method provides
more specific data in terms of quantity compared to SCAN, the first being
the most widely used method in quantitative analysis of POPs.
55

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Figure 16. Gas chromatograph coupled with mass spectrometer
(source: Wylie and Meng, 2009).
Legend: a – inlet, b – chromatographic column, c – transfer line, d –
electron source and mass detector.
For POPs such as OCPs and PCBs, a temperature software can be
used for gas chromatography modified according from Polder et al. 2008,
with the following steps:
1. 2 minutes at 90°C
2. 2 minutes at 180°C with a slope of 25°C/min
3. 2 minutes at 220°C with a slope of 1,5°C/min
4. 12 minutes at 275°C with a slope of 3°C/min
By using this protocol, various compounds can be separated in
the 60 meters long db-5MS column, which has a diameter of 250 µm with
a film thickness of 0.25 µm. For the ion source (HES – High Efficiency
Source) a temperature of 230°C was used. The temperature software
may be followed by a column cleaning programme at a temperature
between 300 and 320°C in order to remove possible contaminants.
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
In the qualitative analytes characterization, the triple quadrupole
mass spectrometer may be used in the SCAN mode. For this mode and by
separately running each standard, specific retention times can be
obtained for each analysed POP, the retention times being relatively
identical between various analysed samples and the elution order of
these compounds is presented in table 4 and is relatively constant in the
case of the used method, regardless of the analysed matrix.
Table 4. POPs elution order and relative retention times.
Elution
POP
Retention time
(time units)
Molecular mass (Da)
1
α-HCH
16.263
288
2
HCB
16.718
282
3
β-HCH
17.626
288
4
γ-HCH
18.066
288
5
δ-HCH
19.505
288
6
ε-HCH
20.229
288
7
PCB29
20.71
256
8
PCB31
21.609
256
9
PCB28
21.723
256
10
heptachlor
22.969
370
11
PCB52
24.293
290
12
PCB47
24.895
290
13
aldrin
25.679
362
14
PCB74
29.023
290
15
cis-heptachlor epoxid
29.03
386
16
oxichlordan
29.088
420
17
trans-heptachlor epoxid
29.414
386
18
PCB66
29.69
290
19
trans-chlordan
31.108
406
57

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Elution
POP
Retention time
(time units)
Molecular mass (Da)
20
PCB56
31.209
290
21
o,p'-DDE
31.648
316
22
PCB101
31.828
324
23
endosulfan I
32.289
404
24
PCB99
32.334
324
25
cis-chlordane
32.509
406
26
PCB112
33.146
324
27
PCB87
34.368
324
28
p,p'-DDE
34.654
316
29
dieldrin
34.733
378
30
PCB136
35.075
358
31
PCB110
35.347
324
32
o,p'-DDD
35.505
318
33
PCB151
36.624
358
34
endrin
36.94
378
35
PCB149
37.85
358
36
endosulfan II
37.896
404
37
PCB118
38.028
324
38
p,p'-DDD
38.933
318
39
PCB114
39.132
324
40
o,p'-DDT
39.249
352
41
PCB153
40.219
358
42
PCB105
40.59
324
43
PCB141
41.393
358
44
PCB137
42.021
358
45
p,p'-DDT
42.421
352
58

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Elution
POP
Retention time
(time units)
Molecular mass (Da)
46
PCB138
42.741
358
47
PCB187
44.185
392
48
PCB183
44.6
392
49
PCB128
45.045
358
50
PCB156
46.951
358
51
PCB157
47.329
358
52
metoxichlor
47.364
344
53
PCB180
48.252
392
54
PCB199
49.163
426
55
mirex
50.097
540
56
PCB170
50.42
392
57
PCB196
51.441
426
58
PCB189
52.423
392
59
PCB207
53.919
460
60
PCB194
54.788
426
61
PCB206
57.427
460
62
PCB209
59.832
494
Based on the retention time observed through the implemented
SCAN method, a MRM (Multiple Reaction Monitoring)-type quantitative
method may be created, focusing on the selection of specific ions and
their fragmenting using a characteristic collision energy. For the MRM
method a chromatographic time of 71.6 time units are used, with a delay
of 14 times units for the acquisition in order to eliminate the solvent
signals and the acquisition may be stopped 63 time units after the last
signal of interest. The method uses a 3 time unit period in which the
column is heated to 320°C in order to eliminate possible contaminants.
59

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
For the gas chromatograph, one can use a temperature
programme modified according to Polder et al. 2008, with the following
steps:
1. 2 minutes at 90°C
2. 2 minutes at 180°C with a slope of 25°C/min
3. 2 minutes at 220°C with a slope of 1.5°C/min
4. 12 minutes at 275°C with a slope of 3°C/min
The source of the mass spectrometer (for a HES-type source -
High Efficiency Source) is set at 230°C and the collision cell has set a flow
of 2.25 mL/min of helium while the collision gas (nitrogen) has a flow set
at 1.5 mL/min.
Table 5 presents the ion transitions (precursor ion → product
ion) specific to each compound of interest for the analysis, together with
the collision energies required to split the compound’s ions. For the
MRM analysis at least two transitions are required, one for quantification
and the other one for specificity. Figure 17 presents a MRM analysed
chromatogram, observing the elution order specific to the analysed
POPs. The MRM method is based on mass spectrometry studies such as
Wylie and Meng, 2009, Mariappan and Kumar, 2014, Xianyu and Zhai,
2015, and Ciscato et al. 2015 that are confirmed in the laboratory during
the performed analysis.
60

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Figure 17. MRM method chromatogram. Capillary 60 m db-5MS.
Table 5. Transitions of ions used in MRM for POP quantification.
POP
Transition Q
(m/z)
CID
(eV)
Transition q
(m/z)
CID
(eV)
α-HCH*
219.0 → 183.0
5
219.0 → 181.0
5
HCB
284.0 → 214.0
30
282.0 → 212.0
30
β-HCH
219.0 → 183.0
5
219.0 → 109.0
25
γ-HCH
219.0 → 183.0
5
181.0 → 145.0
15
δ-HCH
219.0 → 183.0
5
219.0 → 181.0
5
ε-HCH
219.0 → 181.0
5
219.0 → 109.0
25
PCB29
256.0 → 186.0
25
256.0 → 220.0
25
PCB31*
256.0 → 186.0
25
256.0 → 220.0
25
PCB28
256.0 → 186.0
25
256.0 → 220.0
25
heptachlor
272.0 → 237.0
15
274.0 → 239.0
15
PCB52
292.0 → 222.0
25
292.0 → 220.0
25
PCB47*
292.0 → 222.0
25
292.0 → 220.0
25
aldrin
263.0 → 193.0
35
263.0 → 191.0
35
61

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
POP
Transition Q
(m/z)
CID
(eV)
Transition q
(m/z)
CID
(eV)
cis-heptachlor epoxide
353.0 → 263.0
15
355.0 → 265.0
15
PCB74
292.0 → 222.0
25
292.0 → 220.0
25
oxychlordane
387.0 → 263.0
15
387.0 → 323.0
15
trans-heptachlor epoxide
183.0 → 119.0
25
289.0 → 219.0
30
PCB66
292.0 → 222.0
25
292.0 → 220.0
25
trans-chlordane
272.0 → 237.0
15
373.0 → 266.0
15
PCB56
292.0 → 222.0
25
292.0 → 220.0
25
o,p’-DDE
246.0 → 176.0
30
248.0 → 176.0
30
PCB101
326.0 → 256.0
25
326.0 → 254.0
25
endosulfan I
241.0 → 206.0
20
239.0 → 204.0
15
PCB99
326.0 → 256.0
25
326.0 → 254.0
25
cis-chlordane
373.0 → 266.0
20
372.0 → 237.0
15
PCB112
326.0 → 256.0
25
326.0 → 254.0
25
PCB87
326.0 → 256.0
25
326.0 → 254.0
25
p,p’-DDE
246.0 → 176.0
30
316.0 → 246.0
15
dieldrin*
263.0 → 193.0
35
263.0 → 191.0
35
PCB136
326.0 → 256.0
25
326.0 → 254.0
25
PCB110
326.0 → 256.0
25
326.0 → 254.0
25
o,p’-DDD
235.0 → 165.0
20
237.0 → 165.0
20
PCB151
360.0 → 290.0
25
360.0 → 288.0
25
endrin
263.0 → 193.0
35
263.0 → 228.0
20
endosulfan II
241.0 → 206.0
15
241.0 → 136.0
40
PCB149
360.0 → 290.0
25
360.0 → 288.0
25
PCB118
326.0 → 256.0
25
326.0 → 254.0
25
p,p’-DDD
235.0 → 165.0
20
237.0 → 165.0
20
PCB114
326.0 → 256.0
25
326.0 → 254.0
25
62

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
POP
Transition Q
(m/z)
CID
(eV)
Transition q
(m/z)
CID
(eV)
o,p’-DDT
235.0 → 165.0
20
237.0 → 165.0
20
PCB153
360.0 → 290.0
25
360.0 → 288.0
25
PCB105
326.0 → 256.0
25
326.0 → 254.0
25
PCB141
360.0 → 290.0
25
360.0 → 288.0
25
PCB137
360.0 → 290.0
25
360.0 → 288.0
25
p,p’-DDT*
235.0 → 165.0
20
237.0 → 165.0
20
PCB138
360.0 → 290.0
25
360.0 → 288.0
25
PCB187
396.0 → 326.0
25
396.0 → 324.0
25
PCB183
394.0 → 324.0
25
394.0 → 322.0
25
PCB128
360.0 → 290.0
25
360.0 → 288.0
25
PCB156
360.0 → 290.0
25
360.0 → 288.0
25
metoxichlor
227.0 → 141.0
40
227.0 → 169.0
25
PCB157
360.0 → 290.0
25
360.0 → 288.0
25
PCB180
394.0 → 324.0
25
394.0 → 326.0
25
PCB199
430.0 → 360.0
25
430.0 → 358.0
25
mirex*
272.0 → 237.0
15
274.0 → 239.0
15
PCB170
394.0 → 324.0
25
394.0 → 322.0
25
PCB196
430.0 → 360.0
25
430.0 → 358.0
25
PCB189
394.0 → 324.0
25
394.0 → 322.0
25
PCB207
392.0 → 322.0
25
392.0 → 357.0
25
PCB194
430.0 → 360.0
25
430.0 → 358.0
25
PCB206
392.0 → 322.0
25
392.0 → 357.0
25
PCB209
428.0 → 358.0
25
428.0 → 356.0
25
* compound with structure presented in Figure 3.
CID – collision energy
Q – quantification ion
q – identification ion
63

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
2. Control parameters
For each series of samples (one set of samples prepared and
analysed at the same time), different control parameters will be
prepared and analysed (PC). The usually observed parameters are
presented in Table 6. For each series at least three recoveries are
analysed, a control sample, a sample blank and a reagents blank. Besides,
a blind recovery control will be used if the content of the sample is
unknown. Besides these control parameters on the chromatograph drift
controls will also be analysed, these being useful in determining the
possible drift in the system.
Table 6. Control parameters and their use.
Control parameter
Utilization
Blank
Contamination control (air, solvents,
reagents, glassware).
Blind
If the matrix at hand contains analytes,
subtract their values before calculating the
recovery percentage.
Recoveries
Three or more if there is room in the series.
Calibration curve
At least five levels (including zero) with
description of the curve mathematical model.
Drift
A drift sample for each 10 samples.
Control sample
One for each series.
64

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
The most practical number of samples in a series is related to the
number of tubes in the centrifuge and evaporator, including the
necessary control parameters. If there is room, the controls number may
be increased as follows:
 more recoveries,
 more blanks,
 more sample replicas (if there is enough sample material).
Criteria for data validation
There are a series of criteria for data validation that must observe
the set standards; some of these criteria are presented in Table 7.
Table 7. Criteria and limitations in data validation.
Criterion
Limit
Standard
curve
R2 > 0,985
Recovery
percentage
Between 80 - 120% (or 70 - 130% or 60 - 140%).
For PCB 80 - 120% (for tetra- and penta-chlorinated
PCBs 60% recoveries are also accepted).
For PCDD/PCDF 50 - 130% (for hepta- and octa-
chlorinated PCDD/PCDF 40 -150% recoveries) are also
accepted (UNEP Chemicals, 2007)
Blind
If analytes are present, they will be subtracted from the
recovery.
Control
samples
± 2
σ
(standard deviation).
Blank
No specific peaks of the analytes.
65

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Internal standard (IS)
The possible losses of the analytes during the tests are
compensated by adding internal standards to all the samples, including
to the recoveries, blinds, control sample and blanks. It is important that
the IS peaks would not be too big or too small compared to the other
peaks of the chromatogram and that the signal: noise ratio would be of
minimum 20:1 (UNEP Chemicals, 2007), the internal standards
concentration must be close to the analytes expected concentration in
the sample. The analytes are quantified compared to the internal
standard with the most similar properties, from a physical and chemical
point of view as well as with the closest retention time. The internal
standards must be added in such a manner as for them to have the same
concentration in all samples in the series, a concentration that must be
found as well as in the calibration curve points.
The calibration curve
The quantitative analysis of the samples is performed by making
standardisation curves in at least five (including zero) points for the
specific standards mixes. The acceptable linear regression of the curve
(R2) is ≥0,985. The calibration curves must not be forced through the
origin point (0). The ideal calibration curve would include the values of
the analytes observed in the sample. If the origin point is used in the
calibration curve, it is included and not forced. For the accuracy of the
method on can also test a blank (solvents and internal standards only) as
part of the calibration curve and use it as 0-concentration point. For the
calibration curve the equation of the straight line y=ax+b usually is used
(Decision 2002).
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Recoveries and blind control
In order to continuously measure the way various analytes react
during preparation, one will always prepare an uncontaminated matrix
(or with low contamination levels) similar to the sample to be analysed
that will be separated in one blind to which no analytes are added
(except internal standards) and several recoveries to which a known
concentration of analytes is added.
The uncontaminated matrix must be as similar to the sample
matrix as possible, e.g. use already analysed sediment if you analyse the
sediment contamination, use distilled water if you analyse river water,
etc.; if the matrix contains analytes of any sort, they must be subtracted
(meaning the blind concentrations) before calculating the recovery
percentage. The analytes are added in the same concentrations as the
ones predicted in the sample or at 1, 1.5 and 2 times the required
minimum functioning limit or 0.5, 1 and 1.5 times the authorised limit
(Decision 2002). If the sample needs to be upgraded, then you must also
upgrade a low concentration recovery as well.
Acceptable recovery percentages (%) (all figures apply to
apparent recovery) for PCB and organochlorinated pesticides are falling
between 80-120% and, if low concentrations are added, between 60-
140%.
Actions to be taken in case of inacceptable recoveries:
1. One may consider rerunning the sample series in the gas
chromatograph, an important thing especially for easily
degradable analytes in general environmental conditions
(e.g. HBCD).
67

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
2. Double check the concentrations in the recovery standards,
the added quantities and the final volumes for accuracy,
also check if these values are correctly employed in the
applied mathematical formulas.
3. By comparing the height of the IS peak in the GC recovery
standards with the one in the relevant recoveries one may
identify a calculus error or a manipulation error.
4. In case of inacceptable recoveries is very important to
identify the reason for the deviation. When the exact
motive is found, it must be corrected before performing
the analysis of other similar samples.
3. Samples analysis
For the analysis of PCBs and organochlorinated pesticides Table 8
shows the standards used to create calibration curves, recovery
standards and internal standards.
Table 8. Standards used for POP analysis.
No.
POP analyte
1.
α-HCH 10 μg/mL in iso-octane
2.
β-HCH 10 μg/mL in iso-octane
3.
γ-HCH 10 μg/mL in iso-octane
4.
δ-HCH 10 μg/mL in iso-octane
5.
ε-HCH 10 μg/mL in iso-octane
6.
o,p’-DDE 10 μg/mL in iso-octane
7.
p,p’-DDE 10 μg/mL in iso-octane
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
No.
POP analyte
8.
p,p’-DDE 10 μg/mL in iso-octane
9.
o,p’-DDD 10 μg/mL in iso-octane
10.
p,p’-DDD 10 μg/mL in iso-octane
11.
o,p’-DDT 10 μg/mL in iso-octane
12.
p,p’-DDT 10 μg/mL in iso-octane
13.
p,p’-Methoxychlor 10 μg/mL in iso-octane
14.
Aldrin 10 μg/mL in iso-octane
15.
Dieldrin 10 μg/mL in iso-octane
16.
Endrin 10 μg/mL in iso-octane
17.
Heptachlor 10 μg/mL in iso-octane
18.
trans-Heptachlor epoxide 10 μg/mL in iso-octane
19.
cis-Heptachlor epoxide 10 μg/mL in iso-octane
20.
Endosulfan I 10 μg/mL in iso-octane
21.
Endosulfan II 10 μg/mL in iso-octane
22.
Oxychlordane (unmarked) 100 μg/mL in Nonane
23.
trans-Chlordane (unmarked) 100 μg/mL in Nonane
24.
cis-Chlordane (unmarked) 100 μg/mL in Nonane
25.
Mirex (unmarked) 100 μg/mL in Nonane
26.
Hexachlorobenzene (unmarked) 100 μg/mL in Nonane
27.
2,4,4'-Trichlorobiphenyl (PCB 28), 100 μg/mL in Hexane
28.
2,4,5-Trichlorobiphenyl (PCB 29), 100 μg/mL in Hexane – Internal
Standard
29.
2,4',5-Trichlorobiphenyl (PCB 31), 100 μg/mL in Hexane
30.
2,2',4,4'-Tetrachlorobiphenyl (PCB 47), 100 μg/mL in Hexane
31.
2,2',5,5'-Tetrachlorobiphenyl (PCB 52), 100 μg/mL in Hexane
32.
2,3,3',4'-Tetrachlorobiphenyl (PCB 56), 100 μg/mL in Hexane
33.
2,3',4,4'-Tetrachlorobiphenyll (PCB 66), 100 μg/mL in Hexane
69

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
No.
POP analyte
34.
2,4,4',5-Tetrachlorobiphenyl (PCB 74), 100 μg/mL in Hexane
35.
2,2',3,4,5'-Pentachlorobiphenyl (PCB 87), 100 μg/mL in
Hexane
36.
2,2',4,4',5-Pentachlorobiphenyll (PCB 99), 100 μg/mL in
Hexane
37.
2,2',4,5,5'-Pentachlorobiphenyl (PCB 101), 100 μg/mL in
Hexane
38.
2,3,3',4,4'-Pentachlorobiphenyl (PCB 105), 100 μg/mL in
Hexane
39.
2,3,3',4',6- Pentachlorobiphenyl (PCB 110), 100 μg/mL in
Hexane
40.
2,3,3’,5,6-Pentachlorobiphenyl (PCB 112), 100 μg/mL in
Hexane – Standard Intern
41.
2,3,4,4',5-Pentachlorobiphenyl (PCB 114), 100 μg/mL in
Hexane
42.
2,3',4,4',5-Pentachlorobiphenyl (PCB 118), 100 μg/mL in
Hexane
43.
2,2',3,3',4,4'-Hexachlorobiphenyl (PCB 128), 100 μg/mL in
Hexane
44.
2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136), 100 μg/mL in
Hexane
45.
2,2',3,4,4',5-Hexachlorobiphenyl (PCB 137), 100 μg/mL in
Hexane
46.
2,2',3,4,4',5'-Hexachlorobiphenyl (PCB 138), 100 μg/mL in
Hexane
47.
2,2',3,4,5,5'-Hexachlorobiphenyl (PCB 141), 100 μg/mL in
Hexane
48.
2,2',3,4',5',6-Hexachlorobiphenyl (PCB 149), 100 μg/mL in
Hexane
49.
2,2',3,5,5',6-Hexachlorobiphenyl (PCB 151), 100 μg/mL in
Hexane
50.
2,2',4,4',5,5'-Hexachlorobiphenyl (PCB 153), 100 μg/mL in
Hexane
51.
2,3,3',4,4',5-Hexachlorobiphenyl (PCB 156), 100 μg/mL in
Hexane
52.
2,3,3',4,4',5'-Hexachlorobiphenyl (PCB 157), 100 μg/mL in
Hexane
53.
2,2',3,3',4,4',5-Heptachlorobiphenyl (PCB 170), 100 μg/mL in
Hexane
70

Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Nr.
Analit POP
54.
2,2',3,3',4,4',5,5'-Octachlorobiphenyl (PCB 194), 100 μg/mL in
Hexane
55.
2,2',3,3',4,4',5,6'-Octachlorobiphenyl (PCB196), 100 μg/mL in
Hexane
56.
2,2',3,3',4,5,6,6'-Octachlorobiphenyl (PCB 199), 100 μg/mL in
Hexane
57.
2,2',3,3',4,4',5,5',6-Nonachlorobiphenyl (PCB 206), 100 μg/mL
in Hexane
58.
2,2',3,3',4,4',5,6,6'-Nonachlorobiphenyl (PCB 207), 100 μg/mL
in Hexane – Internal Standard
59.
Decachlorobiphenyl (PCB 209), 100μg/mL in Hexane
In order to create the calibration curve the solutions in Table 5
are used. They are diluted by preserving the concentration of the
internal standards (27, 39, and 61) constant for each point of the
calibration curve (50 ng/ml or 50 ppb). Keep in mind that 1 μg/mL =
1000 ng/mL. Solutions are obtained as follows:
a. Calibration curve point 0.1 ng/mL (100 ppt)
b. Calibration curve point 0.25 ng/mL (250 ppt)
c. Calibration curve point 0.5 ng/mL (500 ppt)
d. Calibration curve point 1 ng/mL (1 ppb)
e. Calibration curve point 2.5 ng/mL (2.5 ppb)
f. Calibration curve point 5 ng/mL (5 ppb)
g. Calibration curve point 10 ng/mL (10 ppb)
h. Calibration curve point 25 ng/mL (25 ppb)
i. Calibration curve point 50 ng/mL (50 ppb)
j. Calibration curve point 100 ng/mL (100 ppb)
71

Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
The solutions from the calibration curves may be used for
investigating the concentrations at various levels. For low
concentrations the points from a. to e. may be used, while for high
concentrations the points from f. to j. may be used. Figure 18 presents a
calibration curve for α-HCH.
Figure 18. Calibration curve for α-HCH.
For the internal standards 0.5 mL of each stock solution 27, 39, 61
(Table 5) is added resulting a volume of 1.5 mL to which are added 3.5
mL cyclohexane, obtaining a solution with the concentration of 10000
ppb or 10000 ng/mL stock solution of internal standards. This internal
standard stock solution must be used to spike samples, blinds, but also
the calibration curve points.
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
For 1 mL final volume eluted in the case of the water or sediment
or tissue workflow, the internal standards (IS) are added first (before
extraction or clean-up). Consider that 1 mL of the final volume must have
a concentration of internal standards of 50 ng/mL, so that from the IS
stock solution of 5 mL 10000 ng/mL 5 µL are added to the matrix (before
processing). For method accuracy purposes it is preferred that one
would not pipette volumes under 10 µL, thus from the IS stock solution
working dilutions can be made for more accurate tests.
In order to create recovery standards, the solutions from Table 5
are used except internal standards. The recovery standards making is
based on final concentrations of 500 ppt, 1 ppb, and 1.5 ppb.
In order to guarantee the proper preservation of the samples,
possible contamination checking, method standardisation, proper
calibration and maintenance of the equipment, several requirements
must be observed (UNEP Chemicals, 2007):
1. The workspace must be sufficient as to avoid
contamination. The samples of different origin must be
placed accordingly, and chemical niches must be in place.
Proper waste disposal procedures must be observed.
2. Samples must be stored in adequate conditions, in proper
temperature controlled containers according to the analyte
and matrix type.
3. The selection and validation of the method by
repeatability, selectivity, recovery, LOD, LOQ and accuracy.
The solvent quality must be adequate. Clean glassware
must be employed in order to avoid contamination. The
equipment must be periodically maintained and calibrated.
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Sediment
a) Method principle
For POP extraction from sediment samples, a LLE-type method
was used (liquid-liquid extraction) by which pollutants adsorbed on
sediment particles (Collings et al. 2006) go in to the organic phase
following homogenisation. After clean-up of the organic phase, removing
impurities with sulphuric acid, it is concentrated in order to allow
detection of very low POPs concentrations in the sample. The use of the
sulphuric acid is a destructive clean-up method degrading the aldrin,
heptachlor, endosulfan, dieldrin, endrin, and metoxychlor traces. The
concentration must take place in such way as not to lose the analyte
(nitrogen flow evaporation is recommended). A list of equipment,
reagents and consumables required for POP extraction from the
sediment is presented in table 9.
Table 9. List of equipment, reagents and consumables required for POP
extraction from the sediment.
Equipment
High precision scale
Pipettes
Glass flasks with volumetric
dispenser
Vortex
Sonicator
Centrifuge
Mortar and pestle
GC/MS/MS
Chemical niche
Thermoblock for evaporation
50 mL glass tubes
15 mL graded glass tubes
10 mL glass tubes with cap
Chart of POP extraction from
sediment:
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Reagents
NaCl
Ultra pure water
Acetone HPLC
Cyclohexane HPLC
Sulphuric acid 96%
Green malachite
Internal standards
Recovery standards
Consumables
Pipette tips with barrier
Glass pipettes
pH paper
GC tubes
b) Work protocol (LLE method):
Figure 19. Equipment for POP extraction from sediment.
Legend: a – sonicator, b – thermoblock.
1. The sediment is left to dry out overnight in the desiccator,
2. The sediment is grinded into a homogenous mass,
3. All test tubes are rinsed with a 1:1 acetone:cyclohexane
mix and left to dry,
4. 5 g of sediment are weighed in a 50 mL tube,
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
5. The internal standards are added (minimum 10 µL, 50
ng/mL = final concentration),
6. Recovery standards are added (minimum 10 µL),
7. Add 2 mL of NaCl 6% solution, 10 mL ultra-pure water, 15
mL acetone, 20 mL cyclohexane,
8. The solution is vortexed,
9. The sonicator is used for homogenisation (Figure 19.a),
10. The solution is centrifuged for 10 minutes at 2000 G,
11. The supernatant is transferred in a new 50 mL tube,
12. 5 mL acetone and 10 mL cyclohexane are added over the
sediment left in the old tube,
13. The solution is vortexed,
14. The sonicator is used for re-homogenisation,
15. The solution is centrifuged for 10 minutes at 2000 G,
16. The supernatant is transferred in the new 50 tube,
17. The solution is evaporated from the tube down to 2 mL,
18. The 2 mL solution is transferred in 10 mL capped tubes,
19. 6 mL of sulphuric acid 96% are added,
20. It is vortexed until cone formation to the lid in short,
strong pulse,
21. Mix by inversing one time,
22. Place in the dark at room temperature for one hour,
23. Green malachite solution is added to the blanks,
24. It is centrifuged for 10 minutes at 3000 G,
25. If the supernatant is gelatinous and if it is not clear, the
samples are frozen for 1 hour at -50°C or overnight at -
21°C and it is centrifuged again for 10 minutes at 3000 G
after defrosting during 1 hour,
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26. The supernatant is transferred into a 15 mL graded tube,
checking with a droplet on pH paper that acid is not
transferred as well,
27. In the 10 mL tube containing sulphuric acid 1 mL
cyclohexane is added, it is vortexed again and re-
centrifuged 10 minutes at 3000 G,
28. If the supernatant is gelatinous and if it is not clear, the
samples are frozen for 1 hour at -50°C or overnight at -
21°C and it is centrifuged again for 10 minutes at 3000 G
after defrosting during 1 hour,
29. The supernatant is transferred into the 15 mL graded tube,
checking with a droplet on pH paper that acid is not
transferred as well,
30. If sulphuric acid was transferred, the solution is
transferred back into the 10 mL tube, re-centrifuged 10
minutes at 3000 G and transferred into a new 15 mL
graded tube using a new pipette,
31. The solution is concentrated on the thermoblock down to a
final volume of 1 mL (Figure 19.b),
32. The solution is centrifuged for 10 minutes at 3000 G,
33. For 1 mL solution, 0.5 mL from the interface is transferred
into the GC tube and loaded on the GC/MS/MS.
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Water
a) Method principle
For POP extraction from water there is an ISO international
standard used for identification and quantification of some POPs such as
OCPs, PCBs and chlorobenzenes, the standard being EN ISO 6468:1996
(revised in 2014) „Water quality - Determination of certain
organochlorinated insecticides, polychlorinated biphenyls and
chlorobenzenes - Gas chromatographic method after liquid-liquid
extraction”. The standard mentions that 1 L volume of water is most
often used for POP extraction.
Under the EN ISO 6468:1996 (revised in 2014) the extraction can
be performed in two ways:
1. Extraction by agitating the recipient and use of a
separation funnel. In this case, solvent is added to the
sample and the solution is agitated followed by the
separation of the phases using the funnel. The extract is
dried with the aid of a desiccation column, using
anhydrous sodium sulphate and an evaporator, or by
freezing and introducing the sample into the evaporator.
After extraction a concentration follows using a rotating
evaporator and then the solvent is transferred to the GC
tube.
2. Extraction by magnetic agitator and separation by
microseparator. In this case, the sample over which solvent
is added is stirred with a magnetic agitator. The extract is
dried with the help of a desiccation column, using
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
anhydrous sodium sulphate and an evaporator, or by
freezing and introducing the sample into the evaporator.
After extraction a concentration with a rotating evaporator
follows and the solvent is transferred to the GC tube.
With the standard specified methods, without a precursory
treatment involving water filtering, POPs will be extracted from the
suspended particles from the water mass (Zhang et al. 2003). If filtered,
the particles may provide information on POPs concentration on the
particles suspended in the water column such as in the studies
performed by Quesada et al. 2014, Pohlert et al. 2011, Fernández et al.
2005, etc.
Besides the above-mentioned international standard, a SPE-type
method is also used (solid phase extraction) modified according to Sabin
et al. 2009, that uses Phenomenex Strata-X Polymeric SPE Reverse Phase
x33 µm 100 mg, 6 mL extraction cartridges. In the case of this method
the water may be filtered through nylon membrane of 0.45 µm mesh
considering that the determination is performed for dissolved
compounds (even in the case of low water solubility) and it is not
recommended for compounds that are adsorbed on the suspended
particles from the water column. The SPE method filters a certain water
quantity through the cartridge, a more rapid method using less reagents
than the standard mentioned methods. All SPE type methods have in
common the following steps: conditioning and equilibration, used for
removal of air from the cartridge, for solid phase conditioning and for
expanding the phase to its maximum size, in the following step the
sample is added and the analytes are retained by the sorbent, the
cartridge is rinsed in order to remove contaminants and then the
compounds from the solid phase are eluted (Sabin et al. 2009).
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Several of the advantages of using the SPE method for POP
extraction from water are mentioned: short extraction time, low
reagents consumption, large concentration factor, good recovery,
applicable to a wide range of compounds (Wells 2003; Fernandez-Alba
2005). The extraction using SPE cartridges is non-destructive.
Due to the hydrophobic nonpolar character of most of the POPs
like the PCB and OCP types, the extraction using SPE cartridges is
auspicious due to the reverse phase polymers to which nonpolar organic
compounds have affinity to (larger than in water or aqueous solutions).
In table 10 the equipment, reagents and consumables that are
necessary for the purification of POPs from water are presented.
Table 10. List of equipment, reagents and consumables required for POP
extraction from water.
Equipment
Glass recipients 1L
Filtering system
Vacuum pumps
Extraction system
Automated pipettes
Thermoblock
Vortex
Centrifuge
GC/MS/MS
Chemical niche
Graded glass tubes 15 mL
Chart of the POP extraction from water
:
Reagents
Ultra-pure water
Cyclohexane HPLC
Methanol HPLC
Internal standards
Recovery standards
Consumables
Filter membranes 0.45 µm
mesh; pipette tips with
barrier; glass pipettes
pH testing paper; GC tubes;
cartridges
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b) Work protocol (SPE method):
Figure 20. Lab equipment.
Legend: a – filtering system, b – extraction system.
1. The glass recipients of 1 L and the 15 mL tubes are rinsed with
cyclohexane,
2. 1L of water is filtered through the filtering system (Figure 20. a)
connected to a vacuum pump withnylon filtering membranes of
0.45 µm mesh,
3. The water is transferred in the 1 L glass recipient,
4. Internal standards are added to the filtered sample (minimum 10
µL, 50 ng/mL),
5. Recovery standards are added to the filtered water sample
considered as recovery (maximum cumulated of100 µL
cyclohexane to 1L water),
6. Vortex for 30 minutes,
7. Load the Phenomenex Strata-X Polymeric SPE Reverse Phase x33
µm 100 mg, 6 mL cartridge on the extraction system connected to
the vacuum (Figure 20.b),
8. Condition the cartridge with 10 mL methanol at a speed of 2
mL/minute,
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
9. Equilibrate the cartridge with 10 mL ultra-pure water at a speed of
2 mL/minute,
10. Run the 1 L filtered water sample at a speed of 10 mL/minute,
11. Rinse with 20 mL ultra-pure water at a speed of 2 mL/minute,
12. Dry the cartridge for 10 minutes under vacuum,
13. Load the 15 mL graded tubes into the extraction system,
14. Elute with 5 mL cyclohexane at a speed of 1 mL/minute,
15. Evaporate the solution down to a final volume of 1 mL,
16. Centrifuge the 15 mL graded tubes for 10 minutes at 3000 G,
17. For 1 mL solution, 0.5 mL of the interface is transferred to the GC
tube and loaded on the GC/MS/MS.
Animal tissues
a) Method principle:
The present method is based on the method developed and published
by Brevik, 1978. Initially the method was developed for human milk, but
it was later modified in order to include most of the biological matrices
and more analytes.
When a LLE type extraction method is employed, lipids from the
animal tissues (e.g.: liver, muscle, etc.) are also extracted, these being
removed by using concentrated sulphuric acid that degrades the
lipids by oxidative dehydration leaving behind the cyclohexane and
the PCB-like POPs as well as some pesticides (except: heptachlor,
endosulfan, dieldrin, endrin and methoxychlor) (Barcelo 2000). List of
equipment, reagents and consumables required for POP extraction
from tissues. In Table 11 the list of equipment, reagents and
consumables is presented.
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Assessment and monitoring of persistent organic pollutans in lotic ecosystems.
Methodological guide
Table 11. List of equipment, reagents and consumables required for POP
extraction from tissues.
Equipment
Ultra Turrax T25 homogeniser
Blender
Analytical balance
Volumetric dispenser flasks
Sonicator
Automated pipettes
Thermoblock for evaporation
Vortex
Centrifuge
GC/MS/MS
Chemical niche
Glass tubes 50 mL
Graded glass tubes 15 mL
Glass tubes 10 mL capped
Glass vials 30 mL
Chart for POP extraction
from tissues:
Reagents
Ultra-pure water
Sulphuric acid 96%
Cyclohexane HPLC
Acetone HPLC
NaCl
Ethanol 96%
Internal standards
Recovery standards
Consumables
Pipette tips with barrier
Glass pipettes
pH test paper
GC tubes
Cartridges
Homogenisation and pre-analysis preparation
Sample homogenisation is essential for obtaining representative
results for the tissue of origin or for the individuals mix (in the case of
insects). Considering the matrix used in the extraction keep in mind the
details of Table 12.
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Angela Curtean-Bănăduc, Jan Ludvig Lyche, Vidar Berg, Alexandru Burcea, Doru Bănăduc
Table 12. Homogenisation methods for various matrices.
Matrix
Homogenisation method*
Fat tissue and fatty substance
- small fragments
Ultra Turrax homogenisation.
Fat tissue and fatty substance
– big fragments
Blender homogenization followed by Ultra
Turrax homogenization.
Milk
Defreeze, warm for 1 hour at 37°C and vortex
20 seconds.
Eggs
Blender homogenization in 5 series of 5
seconds or more if the developmental stage is
advanced.
Liver
Ultra Turrax homogenization.