Development and validation of a method for the
determination of polyfluorinated organic substances in
sea water, sediments and biota Occurrence of these Compounds in the North and Baltic
Seas
Norbert Theobald 1, Wolfgang Gerwinski1, Christina Caliebe 1 and Michael Haarich 2
1
Federal Maritime and Hydrographic Agency
Bernhard-Nocht-Str. 78, D-20359 Hamburg
2
Federal Research Centre for Fisheries
Marckmannstr. 129b, 20539 Hamburg
Project 202 22 213 of the Federal Environmental Agency, Germany
Executive Summary
Sensitive analytical procedures for the determination of perfluorinated carboxylic and
sulfonic acids (PFCs) in marine environmental matrices (sea water, sediment, fish
tissue) were developed and validated in the course of the research project. The
methods were used to analyse representative environmental samples from the North
and Baltic Seas in order to document the occurrence and distribution of these
compounds in the marine environment.
Background
In the past five years, there has been growing interest in the ecological aspects of
perfluorinated organic carbon and sulfonic acids. Perfluorinated organic acids are
used in a large number of industrial and consumer applications, e.g. in surface
treatment of textiles, carpets, and paper, and in lubricating oils, pigments, polishes,
food packaging and fire fighting foams. Perfluorooctane sulfonic acid (PFOS) is both
an important perfluorinated surfactant (PFT) and a precursor to other perfluorinated
compounds.
Besides
PFOS,
also
perfluorooctane
sulfonamide
(PFOSA),
perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNoA) and perfluorohexane
sulfonic acid (PFHxS) are used as precursors in the synthesis of perfluorinated
compounds or directly in numerous applications. Owing to their unique properties,
large amounts of PFCs have been synthesised and used since the 1950s.
1
F
F
F
F F F F
F
F
F
C C C
C
C C OH
F
F
F
F
F
O
F
PFHpA Perfluoroheptanoic acid
F
F
F
F
F
F
F
F
F
F
F
O
F
F
F
F
F
F
F
O
C C C C C C
S
F
O
F
F
F
F
F
OH
PFHxS Perfluorohexane sulfonic acid
C C C C C
F
O
PFBS Perfluorobutane sulfonic acid
......
C C C
S OH
F F F F
PFHxA Perfluorohexanoic acid
F
O
F C C C C
OH
F
F
PFOA Perfluorooctanoic acid
F
......
F
F
F
F
F
F
C C
C
C C
C
C C
S
F
F
F
F
F
O
F
F
F
F
O
OH
PFOS Perfluorooctane sulfonic acid
PFNoA Perfluorononanoic acid
F
F
F
F
F
F
F
F
F
F
O
F
F
C
F
C
F
C
C
F
F
C
C
C
C
C
F
F
F
F
F
C
OH
F
F
F
F
F
F
F
O
C C C C C C C C
S
F
O
F
F
F
F
F
F
F
NH2
PFOSA Perfluorooctane sulfonamide
PFDeA Perfluorodecanoic acid
Figure 1. Structural formulas of the PFCs investigated
Owing to their very strong and stable carbon-fluorine bonds, these compounds are
chemically nearly inert and are highly persistent in the environment. These acids
have surfactant properties resulting from the presence of both an ionic group and a
lipophilic end. PFOS has a certain bioaccumulative potential and toxic properties. In
rats and rabbits, not only alterations in liver enzyme values were found but also
developmental toxicities. Besides, a statistically significant association has been
found between PFOS exposure of industrial workers and bladder cancer. For these
reasons, OSPAR and OECD classified PFOS as a PBT compound.
Analytical problems and method development
Although PFCs have been produced for over 50 years now, their importance as
environmental contaminants did not become apparent until methods for their routine
determination became available. Because of their polar characteristics, they escaped
detection by the usual gaschromatographic methods and did not "become visible"
until the introduction of routine HPLC-MS-MS analyses. However, in 2003, when the
research project was started, routine methods sensitive enough for the determination
of ultratrace levels of PFC in marine environmental samples did not yet exist.
Therefore, in order to get a comprehensive overview of the presence and possible
distribution processes of PFCs, highly sensitive methods were developed for the
environmental compartments of water, sediment, and biota (fish liver and muscle
tissue). Eight perfluorinated carboxylic and sulfonic acids with chain lengths from C4
to C10, and perfluorooctyl sulfonamide (PFOSA) as derivative, were analysed (Fig. 1).
The main difficulties in the process development were the very low PFC
2
concentrations in the marine samples and the avoidance of artefacts due to blank
values. Extraction and enrichment of PFCs from sea water was performed by solid
phase extraction using a polymer resin. Samples of sediment and fish tissue were
extracted by shaking three times with methanol. The determination for all matrices
was carried out by HPLC-MS-MS analysis with an ESI (neg.) source (Figure 2).
Water Samples
Sediment Samples
Sampling with 10-L glass bowl
sampler (5 m water depth)
Addition of IS
Solid-Phase-Extraction (SPE) on
HR-P adsorbent (APOS-Workstation)
Washing and drying of
adsorbent
Sampling with box-corer
Sampling with net
Drying, grinding
Liver-, muscle dissection
Addition of IS
Extraction with methanol
(3 x Agitating at room temperature)
SPE-Clean-up on H-RP
(Removal of water and salts)
Elution with methanol,
Concentration of extracts (250 µl)
Fish Samples
Optional: Clean-up on Envicarb
(Removal of matrix components)
HPLC-MS-MS
Grinding of tissue,
Addition of IS, extraction with methanol
Concentration,
Sequential freezing of lipids
SPE-clean-up on HR-P
(Removal of water and salts)
HPLC-MS-MS
HPLC-MS-MS
Figure 2. Analytical procedures for the determination of PFCs in water, sediment, and fish
tissue samples
The limits of determination (LODs) in water samples ranged from 2 pg/L (PFOSA) to
30 pg/L (PFOA, PFNoA), in sediment from 0.02 to 0.13 µg/kg dw, and in fish tissue
from 0.1 to 1.1 µg/kg ww (Table 1). The developed procedures proved to be reliable
in routine analysis and were sensitive enough to detect and determine the PFCs in all
three matrices in the marine environment.
Table 1. Analytical characteristics (summary)
PFBS
PFHxA PFHxS
PFHpA PFOA
PFOS
PFNoA PFDeA PFOSA
10 - 24
0.01
0.2
6 -19
0.02
0.7
12 -39
0.03
0.5
21 - 30
0.13
0.5
10 - 21
0.05
0.6
21 - 30
0.03
0.2
17 -20
0.02
0.3
2-3
3-9
0.001
0.015
0-2
0
0.045
1-2
0.002
0.039
6 - 19
0.043
0.199
7 -11
0.003
0.041
10 -20
0
0.006
0-3
0
0.003
1 -3
0.001
0
Limits of determination
Water [pg/L]*
Sediment [µg/kg]
Biota [µg/kg]
15 - 41
0.03
1.1
0.1
Blank values (Total BV)
Water [pg/L]*
Sediment [µg/kg]
Biota [µg/kg]
0
0.001
0.062
* depending on sea area
3
PFCs in Sea Water, Sediments, and Biota in the North and Baltic Seas and in
the northern North Atlantic Ocean
Between 2003 and 2005, about 90 water samples taken at 55 representative stations
in the North and Baltic Seas were analysed for PFCs. Additional sampling was
carried out at 20 stations in the Greenland Sea during a research cruise in 2004. All
nine PFCs investigated were found to be present in the waters of the German Bight
and western Baltic Sea. In the North Sea, concentrations of PFOA and PFOS ranged
from 30 pg/L to 6 ng/L, with strong gradients from the coasts to the open sea (Fi.g 3
and Fig. 4). The rivers Elbe and Rhine/Schelde were identified as significant input
sources for the southern North Sea. In the Elbe, PFOA and PFOS concentrations
ranged from 8 to 30 ng/L. As a regional phenomenon, PFOS levels were found to
decrease faster than PFOA levels from the river estuaries toward the open sea.
Another peculiarity was the relatively high PFBS concentrations (3.9 ng/L) off the
Rhein/Schelde estuary.
3
4
5
8
7
6
9
56
56
ENTE 3
Ga 419 Mai 2004
DTENT
8
7
55
NSB 2
NSGR 2
SYLT 2
55
SYLT 1 LTIEF
6
ng/L
AMRU 2
5
4
EIDER
54
54
3
MEDEM
BRIFF
1
53
Stade
PFHxA
PFHxS
PFHpA
PFOA
PFOS
PFNoA
PFDeA
PFOSA
2
53
0
File: db2004.dsf
3
4
5
6
7
8
9
Figure 3. PFC concentrations [ng/L] in surface water (5 m) of the German Bight in May 2004
4
Gauss 446
53
52
51
46
45
43
40
7
6
36
5
39
29
33
26
n g /L
4
20
3
17
2
8
Cux
Stade
1
11
0
14
12
902
911
904
909
906
907
Figure 4. PFC concentrations [ng/L] in surface water (5 m) of the North Sea in August 2005
Although the distribution pattern was found to be much more even in the western part
of the Baltic Sea than in the North Sea (PFOA: 0.47 - 0.9 ng/L; PFOS: 0.33 - 0.58
ng/L), slightly elevated levels nevertheless occurred off the Odra estuary (PFOA: 1.1
ng/L; PFOS: 0.9 ng/L) (Fig. 5).
PFCs were found even in remote areas like the Greenland Sea, where PFOS and
PFOA concentrations reached up to 67 pg/L.
5
9
11
10
12
15
14
13
16
17
57
57
Gauss 442
June 2005
56
56
55
55
ARKO2
KALKG
ARKO3
SCHLEI
DARSS2
ECKFBU
FBELT2
KFOTN6
1,2
MEBU2
1,0
USEDOM
54
NEUBU
54
ODER
0,8
0,6
PFOSA
PFDeA
PFNoA
PFOS
PFOA
PFHpA
PFHxS
PFHxA
PFBS
ng/L
0,4
0,2
0
Filename:Ga442
53
9
10
11
12
13
14
15
16
53
17
Figure 5. PFC concentrations [ng/L] in surface water (5 m) of the western Baltic Sea in June
2005
In all investigations, PFOA and PFOS were found to be the substances with the
highest concentrations, and all other PFCs had lower levels. PFOA and PFOS
concentrations in sea water were comparable to the levels of many classical
pollutants (HCH, PAH, herbicides). In comparison with the lipophilic chlorinated
hydrocarbons (HCB, DDT, PCB), concentrations were considerably higher.
In 2004 and 2005, surface sediment was sampled at 15 stations in the German Bight
and western Baltic Sea. PFCs were detected in all of the 18 samples taken. At most
stations, PFOS had the highest concentrations of all PFCs analysed (max. 2.45
µg/kg dw). The highest dry weight related values were found at silty stations in the
area influenced by the Elbe plume (German Bight) and in the western Baltic, which
suggests a strong influence of sediment properties such as TOC content or the
proportion of fine-grained material (Fig. 6). After normalisation to TOC, sediments
from the German Bight were found to have clearly higher PFC levels than Baltic
sediments.
6
4
5
6
9
8
7
10
12
11
13
14
15
56
56
2005
UE 70
UE 67
55
55
WB 5
1,5
SSL
54
Arko1
Echernf.
bight
1,0
BL 4
710
BL 2
718
Ruden
0,5
ES 1
0,0
53
4
5
54
Oder
KS 11
PFHxA
PFHxS
PFHpA
PFOA
PFOS
PFNoA
PFDeA
PFOSA
µg/kg dw
WB 1
53
6
7
8
9
10
11
12
13
14
15
Figure 6. PFC concentrations [µg/kg dry weight] in surface sediments (0-2 cm)
Concentrations of PFOS in sediments were on the order of 0.02 - 2.4 µg/kg dw, those
of PFOA 0.06 - 1.57 µg/kg dw. They were comparable to levels of the classical
lipophilic pollutants DDD, PCB, and HCB. Most other PFCs had markedly lower
levels and were on the order of medium polar compounds such as HCH isomers.
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PFOS has a higher affinity to, and consequently higher enrichment in, sediment than
most other PFCs.
Because of the paucity of data available (9 fishes from 6 stations), the results of biota
analysis can only be used for general orientation. Two fish species from the North
Sea and Baltic Sea were sampled (dab and cod), and both muscle tissue and liver
samples were analysed. In all samples, PFOS was the compound with the highest
concentration. PFOS levels of 2.4 - 25.6 µg/kg ww were found in liver samples, and
0.65 - 4.2 µg/kg ww in muscle tissue. The values were of an order of magnitude
comparable to that of other persistent, bioaccumulating compounds such as DDE,
PCB, and HCB. Levels of the other PFCs were clearly lower, and some were even
below the limits of determination. PFC levels in fish had a tendency to be higher in
samples taken close to the coast than in offshore samples (Fig. 7).
10
9 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 9
60
60
59
59
58
58
L3
L9
57
57
56
56
N06
L2
L5
L7
µg/kg wet weight
30,0
54
53
52
51
25,0
L 10
54
N04
N01
20,0
53
15,0
52
10,0
51
PFOA
PFOS
PFNoA
5,0
50
55
N11
0,0
PFDeA
PFOSA
55
50
49
49
10
9 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 9
Figure 7. PFC concentrations [µg/kg wet weight] in liver samples of dab (single samples), 2003
8
K L8
K L4
10
11
12
13
14
15
D LB
D LA
55
55
B11
BMP
5,0
PFOA
PFOS
PFNoA
PFDeA
PFOSA
54
µg/kg
B 01
0,0
10
11
12
13
14
54
15
Figure 8. PFC concentrations [µg/kg wet weight] in liver samples (B01: dab; BMP: cod (single
samples), 2003
Recommendations
Because of the widespread distribution and relatively high levels of PFOA and PFOS
that have been found in the North and Baltic Seas (ng/L range in water, µg/kg range
in sediment and fish tissue), it is recommended to monitor these compounds routinely
within the framework of the national BLMP and the international CEMP and
COMBINE monitoring programmes. The compounds should be determined in the
matrices water, sediment, and fish liver. Other PFCs presently have a lower priority
but should also be monitored - though at a lower frequency – in order to identify
possible changes in production processes or product ranges.
9