FJELD OG VANN AS
PFAS in fish and zoobenthos from River Randselva and
N Lake Tyrifjorden H
Report R1-2019
Project Proposal
PFAS in fish and invertebrates from Randselva river and Lake Tyrifjorden Prepared for: Tom Tellefsen (Nature manager – leading consultant), Rambøll Norge AS Prepared by: Eirik Fjeld (Manager – environmental scientist), Fjeld og vann AS 2 May 2018
Proposal number: R1-2018
Prepared for Rambøll Norge AS and Huhtamäki Oyj April 2019
Org. nr. 820 371 542 Terrasseveien 31A Phone: +47 944 08 100 www.fjeldogvann.no 1363 Høvik [email protected]
Org. nr. 820 371 542 MVA Terrrasseveien 31 A Phone: +47 944 08 100 www.fjeldogvann.no 1363 Høvik Norway [email protected]
FJELD OG VANN AS
N H
Title PFAS in fish and zoobenthos from River Randselva and Lake Tyrifjorden Author Fjeld, Eirik Series Fjeld og Vann Report Volume R1-2019 Number of Pages 36 Date April 2019 ISBN 978-82-691555-0-1 Publisher Fjeld og Vann AS Format PDF Clients Rambøll Norge AS, Huhtamäki Oyj
Project ProposalAbstract We here report results on per- and polyfluorinated alkyl substances (PFAS) in fish and benthic invertebrates from Lake Tyrifjorden and River Randselva. Fish (perch – Perca fluviatilis) and zoobenthos (crustaceans, pond snails) were sampled during the late summer of 2018 in the PFAS in fish and invertebrateshydroelectric reservoir from Svarthølen Randselva in Randselva river close to and Huhtamäki Lake Oyj’s former Tyrifjorden industrial area at Viul; upstream of this at Bergertjern hydroelectric reservoir; and downstream in Tyrifjorden. Perch Prepared for: Tom Tellefsen (Naturefrom manager Svarthølen – hadleading significantly consultant), elevated Rambøll concentrations Norge of PFASAS compared to the two other Prepared by: Eirik Fjeld (Manager locations.– environmental The levels werescientist), also clearly Fjeld elevated og vann in Tyrifjorden, AS whereas they were rather low in Bergertjern. PFOS was the dominating compound at all sites, followed by PFCAs (perfluoroalkyl 2 May 2018 carboxylic acids). At Svarthølen, FTSs (fluorinated telomer sulfonates) were also present in Proposal number: R1-2018 elevated concentrations. The elevated concentrations of PFAS in fish from Svarthølen are most likely related to the use of fluorinated seizing agents in the production of molded paper pulp articles at Viul. Key Words Pollutants; Polyfluorinated Alkyl Substances; PFAS; Freshwater; Fish; Zoobenthos Frontside Photo Mosaic showing: field work at Tyrifjorden, Viul hydroelectric dam, and Viul main farm at Svarthølen. Photos: Eirik Fjeld
Org. nr. 820 371 542 Terrasseveien 31A Phone: +47 944 08 100 www.fjeldogvann.no 1363 Høvik [email protected]
Org. nr. 820 371 542 MVA Terrrasseveien 31 A Phone: +47 944 08 100 www.fjeldogvann.no 1363 Høvik Norway [email protected]
Fjeld og Vann Report: R1-2019
Table of Contents 1. Extended abstract ...... 4 2. Sammendrag – Norwegian abstract ...... 5 3. Introduction ...... 6 4. Materials and methods ...... 7 4.1 Description of sampling locations ...... 7 4.1.1 Svarthølen and Viul factory site ...... 7 4.1.2 Bergertjern ...... 7 4.1.3 Tyrifjorden ...... 8 4.2 Fish and zoobenthos sampling ...... 9 4.3 Analysis of PFAS ...... 11 4.4 Stable N- and C-isotopes, δ15N and δ13C ...... 14 5. Results and discussion ...... 15 5.1 Fish length and weight ...... 15 5.2 Food web analysis ...... 17 5.3 Quantifiable PFAS and general levels ...... 21 5.4 PFOS, PFCAs and precursors ...... 24 5.4.1 About precursors ...... 24 5.4.2 PFOS and precursors ...... 24 5.4.3 PFCAs and precursors ...... 25 5.5 Site specific chemical fingerprints ...... 26 5.6 Accumulation of PFOS in perch ...... 27 5.7 Source evaluations ...... 28 6. References ...... 29 7. Appendix: Analytical and morphological data ...... 31
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1. Extended abstract Title: PFAS in fish and zoobenthos from River Randselva and Lake Tyrifjorden, 2018 Year: 2019 Author: Fjeld, Eirik Source: Fjeld og Vann AS, Report R1-2019 ISBN: 978-82-691555-0-1
This study is a part of Huhtamäki Oyj’s environmental survey at its former industrial facilities at Viul, Ringerike municipality, Buskerud county. We here document and discuss results on per- and polyfluorinated alkyl substances (PFAS) in fish and benthic invertebrates from Lake Tyrifjorden and River Randselva. The rationale for the survey is the elevated concentrations of PFAS found in sediments from Randselva outside and downstream of the company’s premises, and in sediments and fish from Tyrifjorden further downstream Randselva. Between 1999 and 2013 Huhtamäki manufactured disposable water and oil repellent paper-based food contact materials at Viul. PFAS were used in their production and treated process water was discharged to the recipient Randselva.
Fish (perch – Perca fluviatilis) and zoobenthos (crustaceans, pond snails) were sampled during the late summer of 2018 in the hydroelectric reservoir Svarthølen in Randselva close to Huhtamäki’s industrial area at Viul; upstream of this (Bergertjern hydroelectric reservoir); and downstream in Lake Tyrifjorden. From each location 8 pooled samples of fish liver (each based on 5-10 individuals) and 4 samples of zoobenthos were prepared and analyzed for a broad selection of PFAS at the laboratory of Norwegian Institute for Water Research (NIVA). A subsample of each fish and zoobenthos sample were sent to Institute for Energy Technology (IFE) and analyzed for stable N- and C- isotopes to determine their trophic level (position in food web) and carbon sources, respectively.
Perch from Svarthølen, downstream Viul industrial site, had significantly elevated concentrations of PFAS compared to the two other locations. The levels were also clearly elevated in Tyrifjorden, whereas they were rather low in Bergertjern compared to the other sites. PFOS was the dominating compound at all sites, followed by PFCAs (perfluoroalkyl carboxylic acids). At Svarthølen, FTSs (fluorinated telomer sulfonates) were also present in elevated concentrations. The mean sum of 18 different PFAS substances were: Svarthølen: 1278 ng/g w.w.; Tyrifjorden: 266 ng/g w.w.; Bergertjern: 41.8 ng/g w.w. An estimate for EQS value (Environmental Quality Standard) for PFOS in perch liver is 166 ng/g w.w. This limit is exceeded with a factor of 6.2 in Svarthølen and 1.2 in Tyrifjorden.
The total PFAS levels in zoobenthos samples were significantly lower than in perch, with only modest differences between sites (13.6 – 45.7 ng/g w.w.). In Svarthølen samples, PFCAs (perfluorinated carboxylic acids) and FTSs were the dominating substances and occurred in about equal concentrations, while PFCAs dominated in Bergertjern and Tyrifjorden.
The complex patterns of PFAS in perch from different locations were summarized by a cluster analysis and a principal component analysis (PCA). The cluster analysis identified three clusters, and correctly grouped all fish samples to their locations. Both analyses showed the Svarthølen fish to be characterized by high concentrations of PFOS, FTSs, long-chained PFCA, FOSA and et-FOSAA. Tyrifjorden fish had in general elevated concentrations of the C6 – C9 PFCAs compared to the other locations, moderate concentrations of PFOS, and low concentrations of FTSs and FOSA. Bergertjern fish had in general low concentrations of all PFAS substances.
The concentration of PFOS in fish increased exponentially with fish length and trophic level. Adjusted mean concentrations of PFOS for each location were computed by correcting for differences in length and trophic level. This increased the difference between Svarthølen and the two other locations and reflects better their actual PFOS loadings.
The elevated concentrations of PFOS, FTSs, FOSA and et-FOSAA in fish from Svarthølen are most likely related to the use of PFAS based seizing agents in the production of molded paper pulp articles at Viul. Discharges from the production have undoubtedly contaminated the aquatic environment downstream the production premises. Huhtamäki has recognized using fluorchemistry in their production of food contact materials here between 1999 and 2013. However, we have no information as to whether the former industrial companies situated here, Keyes Norway AS (1963 – 1982) and Van Leer (1982 – 1999), also used PFAS in their production of molded paper pulp articles. To what extent the discharges at Viul has caused elevated PFAS concentrations in the aquatic food web in Tyrifjorden cannot be quantified without further targeted studies.
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2. Sammendrag – Norwegian abstract Tittel: PFAS in fish and zoobenthos from River Randselva and Lake Tyrifjorden, 2018 År: 2019 Forfatter: Fjeld, Eirik Kilde: Fjeld og Vann AS, Report R1-2019 ISBN: 978-82-691555-0-1
Denne studien er en del av Huhtamäkis miljøundersøkelse ved deres tidligere industrianlegg på Viul, Ringerike kommune, Buskerud fylke. Her dokumenterer og diskuterer vi resultatene av analyser av per- og polyfluorerte alkylstoffer (PFAS) hos fisk og bunndyr fra Tyrifjorden og Randselva. Bakgrunnen for undersøkelsen er de forhøyede konsentrasjonene av PFAS i sedimenter fra Randselva utenfor og nedstrøms for selskapets lokaler, og i sedimenter og fisk fra Tyrifjorden nedstrøms Randselva. Mellom 1999 og 2013 produserte Huhtamäki papirbasert vann- og fettavvisende engangsservise og matemballasje ved Viul. PFAS ble brukt i produksjonen og renset prosessvann ble sluppet til Randselva.
Fisk (abbor - Perca fluviatilis) og zoobenthos (krepsdyr, damsnegler) ble samlet inn i løpet av sensommeren 2018 i kraftverksdammen Svarthølen i Randselva nær Huhtamäkis industriområde ved Viul, høyere oppe i vassdraget ved kraftverksdammen Bergertjern, samt nedstrøms i Tyrifjorden. Fra hver lokalitet ble det laget 8 blandprøver av fiskelever (hver basert på 5-10 individer) og 4 prøver av zoobenthos. Disse ble analysert for et bredt utvalg av PFAS ved laboratoriet ved Norsk institutt for vannforskning (NIVA). Videre ble prøvene også analysert for stabile N- og C-isotoper ved Institutt for energiteknologi (IFE), slik at deres trofiske nivå (posisjon i næringsnettet) og karbonkilder kunne bestemmes.
Abbor fra Svarthølen, nedstrøms Viul industriområde, hadde signifikant forhøyede konsentrasjoner av PFAS sammenlignet med de to andre stedene. Nivåene var også tydelig forhøyet i Tyrifjorden, mens de var ganske lave i Bergertjern sammenlignet med de andre områdene. PFOS var den dominerende forbindelsen på alle steder, etterfulgt av PFCA (perfluoralkylkarboksylsyrer). I Svarthølen var FTS (fluorerte telomer sulfonater) også tilstede i forhøyede konsentrasjoner. Den gjennomsnittlige summen av 18 forskjellige PFAS-stoffer på våtvektsbasis var: Svarthølen: 1278 ng/g; Tyrifjorden: 266 ng/g; Bergertjern: 41,8 ng/g. Et estimat for EQS-verdi (Environmental Quality Standard) for PFOS i abborlever er 166 ng/g våtvekt. Denne grensen overskrides med en faktor på 6,2 i Svarthølen og 1,2 i Tyrifjorden.
De totale PFAS-nivåene i prøvene av zoobenthos var signifikant lavere enn i abbor, med bare beskjedne forskjeller mellom lokalitetene (13,6 - 45,7 ng / g w.w.). I prøvene fra Svarthølen var PFCA (perfluorerte karboksylsyrer) og FTS de dominerende forbindelsene og forekom i omtrent like store konsentrasjoner, mens PFCA dominerte i Bergertjern og Tyrifjorden.
De komplekse mønstrene av PFAS i abbor fra forskjellige lokaliteter ble beskrevet med en «clusteranalyse» (klyngeanalyse) og en «prinsipal komponentanalyse» (PCA, hovedkomponentanalyse). Clusteranalysen identifiserte tre hovedgrupper og grupperte alle prøvene korrekt i henhold til deres lokaliteter. Begge analysene viste at fisken fra Svarthølen var karakterisert ved høye konsentrasjoner av PFOS, FTS, langkjedet PFCA, FOSA og et-FOSAA. Tyrifjorden-fisk hadde generelt forhøyede konsentrasjoner av C6-C9 PFCA sammenlignet med de andre lokalitetene, men moderate konsentrasjoner av PFOS og lave konsentrasjoner av FTS og FOSA. Bergertjern-fisk hadde generelt lave konsentrasjoner av alle PFAS-stoffer.
Konsentrasjonen av PFOS i fisk økte eksponentielt med fiskelengde og trofisk nivå. Justerte gjennomsnittlige konsentrasjoner av PFOS for hver lokalitet ble beregnet ved å korrigere for forskjeller i fiskens lengde og trofisk nivå. Dette økte forskjellen mellom Svarthølen og de to andre lokalitetene, og reflekterer bedre deres faktiske PFOS- belastninger.
De forhøyede konsentrasjonene av PFOS, FTS, FOSA og et-FOSAA i fisk fra Svarthølen er mest sannsynlig knyttet til bruk av PFAS-baserte kjemikalier under produksjonen av papirbasert engangsservise og matemballasje ved Viul. Utslipp fra produksjonen har utvilsomt forurenset vannmiljøet nedstrøms produksjonslokalene. Huhtamäki har vedstått seg å ha brukt fluorbaserte kjemikalier ved produksjon av engangsservise og matemballasje her mellom 1999 og 2013. Vi har imidlertid ingen informasjon om hvor de tidligere industriselskapene lokalisert her, Keyes Norway AS (1963–1982) og Van Leer (1982–1999), også brukte PFAS i produksjonen av matvareemballasje og engangsartikler basert på papirmasse. I hvilken grad utslippene ved Viul har forårsaket de forhøyede PFAS- konsentrasjonene i Tyrifjordens akvatiske næringsnett kan ikke kvantifiseres uten videre målrettede studier.
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3. Introduction This study is a part of Huhtamäki Oyj’s environmental survey at its former industrial facilities at Viul, Nesmoveien 1, in Ringerike municipality, Buskerud county. Rambøll Norge AS, together with their sub-contractor Fjeld og Vann AS, have been assigned to carry out this survey. This part of the project is done by Fjeld og Vann AS, and documents the accumulation of PFAS in fish and aquatic invertebrates in the River Randselva and Lake Tyrifjorden and discuss possible sources for this. The other part of the survey is a sediment study, done by Rambøll Norway AS in collaboration with Fjeld og Vann AS (Helland and Tellefsen, 2019, in. prep.)
The rationale for the survey is the elevated concentrations of per- and polyfluorinated alkyl substances (PFAS1) found in sediments in River Randselva outside and downstream of the company’s premises at Viul, and in sediments and fish from Lake Tyrifjorden further downstream Randselva. Between 1999 and 2013 Huhtamäki manufactured disposable water and oil repellent food contact materials at Viul (molded fiber products; paper plates and bowls). PFAS were used in their production and the treated process water was discharged to the recipient Randselva.
The history behind the disclosure of elevated PFAS levels in Randselva was the findings of high levels of PFAS in fish from Tyrifjorden (Fjeld et al., 2015, 2016 and 2017). The concentrations of the substance PFOS in perch (Perca fluviatilis) from this lake, Norway’s fifth largest, were comparable to those of Lake Vansjø – a notably smaller lake polluted by runoff of PFAS from fire training fields at a neighboring military air base2. Apparently, Lake Tyrifjorden must have received considerably quantities of PFOS or its precursor substances.
To identify the sources of PFAS to the lake, the Norwegian Environment Agency conducted in 2017 a survey concluding that the largest active sources of PFAS contamination are residues of historical uses. The largest legacy sources identified were the sediments outside Huhtamäki’s closed-down industrial facilities at Viul and the training fields at the local fire station at Hønefoss town, close to Randselva (Aaasen Slinde and Høisæther, 2018). This survey included analyzes of perch liver from five locations in Tyrifjorden, but no fish samples from Randselva were analyzed. The authors reported significantly higher concentrations of PFOS in perch compared to the studies of Fjeld et al. (op. cit.), but the reason for these discrepancies remains unclear.
In a follow-up study in 2018, the Norwegian Environment Agency will try to document more thoroughly the levels and biomagnification of PFAS in the aquatic food web, reveal the pollution history of PFAS for lake Tyrifjorden, uncover other potential sources of PFAS, and sort out the relative importance of the identified sources. Irrespective of these studies, Huhtamäki has commenced an additional survey to provide a more complete picture of the impact and extent of PFAS contamination of the sediments and aquatic biota in the vicinity and downstream of the industrial premises at Viul.
The objective of this part of the survey is to provide an overview of the levels of different PFAS in fish and aquatic invertebrates from the River Randselva and Lake Tyrifjorden and identify possible sources for these compounds. The following goals were defined:
1. Analyze a broad selection of PFAS compounds, including structural isomers of PFOS, in liver samples of fish and in zoobenthos caught during the late summer of 2018 in the hydroelectric reservoir Svarthølen in Randselva close to Huhtamäki’s industrial area at Viul, upstream the factory site and downstream in Lake Tyrifjorden. 2. Compare the levels of different PFAS in fish between sites after adjusting for differences in trophic position (place in food web) by utilizing stable isotope analyses (δ15N). 3. Analyze the compositional data of different PFAS in fish and invertebrates to identify potential differences in profiles between sites and compare the profiles with sediment data in order to identify potential different sources. 4. Report the results in a scientific report written in English with a Norwegian extended abstract.
1 The release of PFAS compounds, such as PFOS, PFOA, and PFHxS, into the environment is an emerging concern globally, because these chemicals are highly persistent, bioaccumulate, can move long distances in the environment, and have adverse impacts on both humans and wildlife. The use of certain PFAS are now strictly regulated by both national and international legislations.
2 PFOS and its precursors were used in firefighting foam (AFFF: aqueous film-forming foam)
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4. Materials and methods
4.1 Description of sampling locations This survey were carried out at three different locations (i) in the hydroelectric reservoir Svarthølen in Randselva, close to Huhtamäki’s former industrial facilities at Viul; (ii) upstream Svarthølen, in the hydroelectric reservoir Bergertjern, close to the outlet of Lake Randsfjorden; and (iii) downstream in Lake Tyrifjorden, close to the Storøya island (Figure 1 and Figure 2).
Figure 1. View over Lake Tyrifjorden with Storøya island to the left. Photo: Stig Sunde. Wikimedia commons.
4.1.1 Svarthølen and Viul factory site The Svarthølen reservoir has a surface area of 0.45 km2, measured from the hydroelectric dam and to the bridge just downstream of the closed factory area. It is situated 92 m a.s.l., has a maximum depth of about 15 m, and an average waterflow of 59.2 m3/s. The sediments in the deeper areas have a high content of organic fiber, originating from the former wood processing industry at Viul. The hydroelectric dam at Svarthølen was built downstream of the waterfall Viulfoss, about 2.6 km from Huhtamäki’s former industrial facilities at Viul. The fish community here is dominated by perch (Perca fluviatilis) and minnows (Phoxinus phoxinus), but sparse populations of pike (Esox lucius) and large- bodied brown trout (Salmo trutta, lacustrine ecotype) are also known to inhabit the reservoir.
The Viul factory site, established downstream the waterfall Askerudfossen, has a long industrial history. The paper pulp mill Viul Tresliberi was established here in 1893 and modernized their production and increased their capacity up to the late 1950s. In 1961, the pulp mill was partially taken over by Follum Fabrikker, which needed pulp. Keyes Norway AS acquired the facilities in 1963 and started production of molded pulp products (paper bowls, plates etc.) in 1964. In 1982, the factory site was acquired by Van Leer from Netherland, continuing the same production as Keyes. In 1999, Huhtamäki acquired Van Leer, including their factory site at Viul, and started their production of disposable water and oil repellent paper-based food contact materials (FCMs).
Huhtamäki has recognized the use of PFAS based seizing agents in their production of FCMs, however emphasizing that the manufacturing process was closed. After the paper plates and bowls were formed in the mould drying machines in the end of the production line, the overflow fiber and water was recycled for re-usage in the production line and the treated process water was discharged to the recipient Randselva (at least from year 2000 and onwards, according to information given by Huhtamäki). One aim of the manufacturing process was to get as high as possible retention of fluorine-containing chemicals in their production. Unfortunately, we have no information as to whether Keyes Norway AS or Van Leer also have used PFAS containing chemicals in their production of molded paper pulp articles.
4.1.2 Bergertjern The Bergertjern reservoir is a containment of the outlet river Randselva from Lake Randsfjorden, Norway’s fourth largest lake. It is situated 135 m a.s.l., has surface area of 0.2 km2, and maximum depth is about 6 m. Average waterflow at the dam is 58.6 m3/s. Although Lake Randsfjorden is inhabited by 11 different fish species, only perch and minnows were observed in Bergertjern during the fish sampling. There are no known significant point sources
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4.1.3 Tyrifjorden Lake Tyrifjorden is Norway’s fifth largest lake with a surface area of 137 km2. It is situated 65 m a.s.l., has an average depth of 93 m, and a maximum depth of 288 m. The mean water residence time is about 2.7 years. The lake's primary inlet river is the Storelva river, which flows into Tyrifjorden downstream of Hønefoss town. At Hønefoss, the two rivers Randselva and Begna join and form Storelva. Tyrifjorden is a drinking water source but is also used as a recipient for several sewage treatment plants, scattered settlements, industry and agriculture.
Tyrifjorden has a rich fish community with 13 different species. Among these is the renowned large-bodied brown trout population, Tyrifjordørret, which spawns downstream the Viul dam (Museth et al., 2018). The lake is popular for recreation and outdoor activities, such as fishing, boating and bathing. Several nature reserves have been established around the lake, due to their unique flora and fauna – especially birds and waterfowls.
Figure 2. Map showing the different sampling locations for fish and zoobenthos collected in 2018.
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4.2 Fish and zoobenthos sampling Collection and sampling of biological material followed the guidelines of the Norwegian Environmental Specimen Bank procedures for freshwater fish (Miljøprøvebanken, 2013). This implies that personnel should avoid use of personal care product one day prior to sampling – or only use approved products. The organisms must not come into contact with potentially contaminating surfaces or substances during capture, later handling and sampling. Disposable gloves (nitrile rubber) should be used if possible. All tools used for dissection were in stainless steel and cleaned by machine washing, rinsed in Milli-Q water, acetone and methanol.
The fish and zoobenthos were caught in August and September (Table 1). From each location we collected eight samples of perch (Perca fluviatilis) from different length groups, and four samples of zoobenthos representing organisms at lower trophic levels (position in food web). The sampling areas at each location are given in Figure 3.
Table 1. Sampling dates for the different study locations, visited in 2018.
Location Organisms Activity Date
Tyrifjorden Fish Gill net fishing 6-7 Aug. Tyrifjorden Fish Gill net fishing 18-19 Aug. Tyrifjorden Zoobenthos Kick-net sampling 26 Aug. Bergertjern Fish Gill net fishing 9-10 Aug. Bergertjern Zoobenthos Kick-net sampling 25 and 27 Aug. Svarthølen Fish Gill net fishing 20-21 Aug. Svarthølen Zoobenthos Kick-net sampling 28 Aug. Svarthølen Zoobenthos Amphipod traps 1 Sept.
The fish were caught with gill-nets: 12 nets at each location, with mesh sizes varying from 8 mm to 45 mm. After collection, the fish were wrapped in clean aluminum foil, packed in clean polyethylene bags and kept cold (≈ 4 °C) or frozen (-20 °C) until dissection of samples.
Dissections of fish liver samples were done in the open to prevent contamination of PFAS from indoor sources. All surfaces that could come into contact with fish were covered by aluminum foil, rinsed with methanol and acetone (HPLC grade). Fish length, weight, sex and maturation stage were recorded. Liver tissue was dissected from each individual and transferred to polyethylene test tubes to be analyzed for PFAS. Each sample was a pooled sample (composite sample) made from several individuals from the same length group. This sampling strategy gives an average PFAS concentrations for a given length group, while reducing the influence of individual variation. As such it is a cost-efficient strategy while maintaining good statistical power (Bignert et al., 2014). The samples, approx. 2 g each, were stored frozen (-20 °C) until analysis.
Zoobenthos samples (crustaceans and snails) were mainly collected with macroinvertebrate sampling nets (kick-net sampling, mesh size 1.0 mm) in the littoral zone (near-shore, approx. 0-1 m depth). The one exception was a benthic sample of the crustacean Gammarus lacustris from Svarthølen, collected at 8–9 m depth with amphipod traps (cages filled with aquatic macrovegetation). The specimens were sorted out in the field and stored in clean polyethylene boxes and kept frozen (-20 °C) until transferred to the test tubes.
Initially, we wanted samples of the crustacean G. lacustris (common name: freshwater shrimp or scud) and the snail Radix peregra (wandering pond snail); two samples of each from every location. However, R. peregra was sparsely found at Tyrifjorden and Bergertjern, probably due to the low water level this summer season, and we had to substitute it with other organisms for these two locations. For Tyrifjorden we substituted it with two samples of the snail Lymnea stagnalis (great pond snail), whereas for Bergertjern we substituted it with one sample of L. stagnalis and one sample of the crustacean Asellus aquaticus (waterlouse).
The stored zoobenthos material were thawed and samples (approx. 2-3 g per sample) were transferred to the same type of test tubes as the fish samples and kept frozen until analysis (-20 °C). Whole bodies were used for the crustaceans, whereas soft body tissue was used for snails. We carefully crushed the snail shells and rinsed the soft body tissue for shell fragments before transferring the clean tissue to the sample tubes (3-5 individuals in each).
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Figure 3. Maps showing the sampling areas for fish and zoobenthos at the different locations, collected in 2018.
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4.3 Analysis of PFAS An aliquot of about 2 g of homogenized biota was spiked with 6 ng each of the mass-labeled internal standards. The sample was extracted with 7 mL of acetonitrile for 30 min. in an ultrasonic bath. Following centrifugation, the supernatant extract was removed, and the extraction was repeated with another 5 mL of acetonitrile. The combined acetonitrile extract underwent dispersive clean-up with graphitized carbon and acetic acid. A volume of 0.4 mL of the cleaned-up extract was added to 0.2 mL of aqueous ammonium acetate. The final extract was centrifuged before a clear supernatant was transferred to an autoinjector vial.
An Acquity Ultra Performance HPLC system (Waters) was used to inject aliquots of 7 µl extract onto a Waters Acquity BEH C18 reversed phase column (100 x 2.1 mm, 1,8 µm particles. The target compounds were separated at a flow rate of 0.5 ml/min using acetonitrile (A) and 5,2 mM NH4OAc in water (B). The following binary gradient was applied: 0-1.5 min, 12 % of A; 1.5-11 min, linear change to 99 % of A; 11-13 min, 99 % of A.
The Xevo G2-S Q-ToF-HRMS instrument (Waters) was employed in negative ion electrospray ionization (ESI(-)) mode. Mass spectra were registered in full scan mode (mass range m/z 150-1100). The following optimized parameters were applied: Capillary voltage, 0.7 kV; desolvation temperature, 500 °C; source temperature, 120 °C; nitrogen desolvation gas flow, 800 L/h. Quantitative analysis was performed employing extracted mass chromatograms from full scan recording using the m/z (typical mass tolerance of 0.03 u) for the different analytes.
A total of 89 different per- and polyfluoralkyl substances (PFAS) were analyzed (Table 2). Of these, standards were available for 46 substances. Hence, the results for the others must be considered as semi-quantitative. The level of quantifications (LODs) were in the range of 0.1–2 ng/g w.w.
Common classes and general formulas of different PFAS is given in Figure 4.
Table 2. Per- and polyfluoralkyl substances (PFAS) analyzed.
Name Acronym CAS No. Standard LOQ, ng/g perfluoro-n-butanoic acid PFBA 375-22-4 1.0 perfluoro-n-pentanoic acid PFPA 2706-90-3 1.0 perfluoro-n-hexanoic acid PFHxA 307-24-4 0.5 perfluoro-n-heptanoic acid PFHpA 375-85-9 0.5 perfluoro-n-octanoic acid PFOA 335-67-1 0.5 perfluoro-n-nonanoic acid PFNA 375-95-1 0.5 perfluoro-n-decanoic acid PFDA 335-76-2 0.5 perfluoro-n-undecanoic acid PFUnDA 2058-94-8 0.4 perfluoro-n-dodecanoic acid PFDoDA 307-55-1 0.4 perfluoro-n-tridecanoic acid PFTrDA 72629-94-8 0.4 perfluoro-n-tetradecanoic acid PFTeDA 376-06-7 0.4 perfluoro-n-pentadecanoic acid PFPeDA 1002-84-2 * 0.4 perfluoro-n-hexadecanoic acid PFHxDA 67905-19-5 0.4 perfluoro-n-heptadecanoic acid PFHpDA * 0.4 perfluoro-n-octadecanoic acid PFODA 0.4 perfluoro-1-propanesulfonate PFPrS 0.1 perfluoro-1-butanesulfonate PFBS 59933-66-3 0.1 perfluoro-1-pentanesulfonate PFPeS 22767-49-3 0.1 perfluoro-1-hexanesulfonate PFHxS 355-46-4 0.1 perfluoro-1-heptanesulfonate PFHpS 22767-50-6 0.1 perfluoro-1-octanesulfonate PFOS 754-91-6 0.1 perfluoro-1-nonanesulfonate PFNS 98789-57-2 0.1 perfluoro-1-decanesulfonate PFDS 335-77-3 0.1 perfluoro-1-undecansulfonate PFUdS * 0.3 perfluoro-1-dodecansulfonate PFDoS 79730-39-5 0.2 perfluoro-1-tridecansulfonate PFTrS * 0.3 perfluoro-1-tetradecansulfonate PFTeS * 0.3 perfluoro-7-methyloctanesulfonate ipPFNS 0.1 L-PFOSK with branched isomers br-PFOS * 0.1 8Cl-perfluoro-1-octanesulfonate 8Cl-PFOS 0.3 perfluoro-1-octanesulfonamide FOSA 754-91-6 0.1 N-methylperfluoro-1-octanesulfonamide me-FOSA 31506-32-8 0.3 N-ethylperfluoro-1-octanesulfonamide et-FOSA 4151-50-2 0.3
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Name Acronym CAS No. Standard LOQ, ng/g
2-(N-methylperfluoro-1-octanesulfonamido)-ethanol me-FOSE 24448-09-7 2.0 2-(N-ethylperfluoro-1-octanesulfonamido)-ethanol et-FOSE 1691-99-2 2.0 perfluoro-1-octansulfonamidoacetic acid FOSAA 2806-24-8 0.3 2-(N-methylperfluoro-1-octansulfonamido)acetic acid me-FOSAA 2355-31-9 0.3 2-(N-ethylperfluoro-1-octansulfonamido)acetic acid et-FOSAA 2991-50-6 0.3 1H,2H-perfluorohexan sulfonate (4:2) 4:2 FTS 757124-72-4 0.5 1H,2H-perfluorooctane sulfonate (6:2) 6:2 FTS 27619-97-2 0.3 1H,2H-perfluorodecane sulfonate (8:2) 8:2 FTS 39108-34-4 0.3 1H,2H-perfluorododecane sulfonate (10:2) 10:2 FTS 120226-60-0 0.3 1H,2H-perfluorotetradecane sulfonate (14:2) 12:2 FTS 149246-64-0 * 0.3 1H,2H-perfluorohexadecane sulfonate (14:2) 14:2 FTS * 0.3 Potassium 2-(6-chloro- 1,1,2,2,3,3,4,4,5,5,6,6- dodecafluorohexyloxy)- 1,1,2,2-tetrafluoroethane sulfonate, (C8 H F16 Cl O4 S) (6:2 chlorinated 6:2 F53B 73606-19-6 0.3 polyfluorinated ether sulfonate) C6 H F12 Cl O4 S (4:2 chlorinated polyfluorinated ether sulfonate) 4:2 F53B 0.3 Potassium 1,1,2,2- tetrafluoro-2- (perfluorohexyloxy) ethane sulfonate 53B 754925-54-7 * 0.5 3-perfluoroheptyl propanoic acid 7:3 FTAC 812-70-4 * 1.0 perfluoro butylphosphonic acid PFBPA 52299-24-8 * 0.5 perfluoro hexyl phosphonic acid PFHxPA 40143-76-8 * 0.5 perfluoro octyl phosphonic acid PFPOA 40143-78-0 * 0.5 perfluoro decyl phosphonic acid PFDPA 52299-26-0 * 0.5 1H,1H,2H,2H-perfluorooctyl phosphate 6:2 PAP 57678-01-0 * 0.5 1H,1H,2H,2H-perfluorodecyl phosphate 8:2 PAP 57678-03-2 * 0.5 bis (1H,1H,2H,2H-perfluorooctyl phosphate) 6:2 diPAP 57677-95-9 0.5 bis (1H,1H,2H,2H-perfluorodecyl phosphate) 8:2 diPAP 678-41-1 0.5 comb of 6:2 and 8:2 perfluoroalkyl phoshate 6:2/8:2 diPAP 943913-15-3 * 0.5 2-perfluorohexyl ethanol 6:2 FTOH 647-42-7 2.0 2-perfluorooctyl ethanol 8:2 FTOH 678-39-7 2.0 2-perfluorododecyl ethanol 10:2 FTOH 865-86-1 2.0 2-perfluorododecyl ethanol 12:2 FTOH 39239-77-5 * 2.0 2-perfluorohexyl ethanoic acid (6:2 FTA) 6:2 FTCA 53826-12-3 * 2.0 2-perfluorooctyl ethanoic acid (8:2 FTA) 8:2 FTCA 27854-31-5 * 2.0 2-perfluorodecyl ethanoic acid (10:2 FTA) 10:2 FTCA * 2.0 2H-perfluoro-2-octenoic acid (6:2 FTUA) 6:2 FTUCA * 2.0 2H-perfluoro-2-decenoic acid (8:2 FTUA) 8:2 FTUCA * 2.0 2H-perfluoro-2-dodecenoic acid (10:2 FTUA) 10:2 FTUCA * 2.0 perfluoro-1-butansulfonamide PFBSA 30334-69-1 * 0.5 perfluoro-1-pentansulfonamide PFPeSA 82765-76-2 * 0.5 perfluoro-1-hexansulfonamide PFHxSA 41997-13-1 * 0.5 perfluoro-1-heptansulfonamide PFHpSA 82765-77-3 * 0.5 N-methylperfluoro-1-butansulfonamide meFBSA 68298-12-4 * 0.5 N-methylperfluoro-1-pentansulfonamide meFPeSA 68298-13-5 * 0.5 N-methylperfluoro-1-hexansulfonamide meFHxSA 68259-15-4 * 0.5 N-methylperfluoro-1-heptansulfonamide meFHpSA 68259-14-3 * 0.5 N-ethylperfluoro-1-butansulfonamide etFBSA 40630-67-9 * 0.5 N-ethylperfluoro-1-pentansulfonamide etFPeSA 162682-16-8 * 0.5 N-ethylperfluoro-1-hexansulfonamide etFHxSA 87988-56-5 * 0.5 N-ethylperfluoro-1-heptansulfonamide etFHpSA 68957-62-0 * 0.5 2-(N-mehylperfluoro-1-butansulfonamido)-ethanol meFBSE 34454-97-2 * 2.0 2-(N-methylperfluoro-1-pentansulfonamido)-ethanol meFPeSE 68555-74-8 * 2.0 2-(N-methylperfluoro-1-hexansulfonamido)-ethanol meFHxSE 68555-75-9 * 2.0 2-(N-methylperfluoro-1-heptansulfonamido)-ethanol meFHpSE 68555-76-0 * 2.0 2-(N-ethylperfluoro-1-butansulfonamido)-ethanol etFBSE 34449-89-3 * 2.0 2-(N-ethylperfluoro-1-pentansulfonamido)-ethanol etFPeSE 68555-72-6 * 2.0 2-(N-ethylperfluoro-1-hexansulfonamido)-ethanol etFHxSE 34455-03-3 * 2.0 2-(N-ethylperfluoro-1-heptansulfonamido)-ethanol etFHpSE 68555-73-7 * 2.0 Ammonium 2-(heptafluoropropoxy)-2,3,3,3-tetrafluoropropanoate GEN X 62037-80-3 * 0.5 Sodium dodecafluoro-3H-4,8-dioxanonanoate ADONA 958445-44-8 * 0.5
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O O – – Perfluoroalkylsulfonates – – Perfluoroalkylcarboxylic Acids CF3(CF2)n–S–OH CF (CF ) –C–OH – – 3 2 n (PFSAs) O (PFCAs)
O
Telomer Alcohols – – X:2 Saturated Telomer Acids CF (CF ) CF CH CH OH CF (CF ) CF –CH –C–OH (FTOHs) 3 2 n 2 2 2 3 2 n 2 2 (X:2 FTCAs)
X:2 Telomer Sulfonates O O X:2 Unsaturated Telomer Acids – – – – (X:2 FTSs) CF3(CF2)n(CH2)2–S–OH CF3(CF2)nCF=CH–C–OH (X:2 FTUCAs) – – O
O
Perfluoroalkylsulfinates O – – X:3 Saturated Telomer Acids – – CF (CF ) CF –(CH ) –C–OH (PFASis) CF3(CF2)n–S–OH 3 2 n 2 2 2 (X:3 FTCAs)
O R O CH CH OH
– – 1
Perfluorooctanesulfonamides — – – 2 2 Perfluorooctanesulfonamido-
— — CF3(CF2)7–S–N CF (CF ) –S–N —
– – 3 2 7 (FOSAs) – – ethanols (FOSEs) O R2 O R
O
– – O Perfluorooctanesulfonamido- O CH C-OH – – Perfluoroalkylphosohonic Acids
– – 2
— — CF3(CF2)n–P–OH acetic Acids (FOSAAs) CF3(CF2)7–S–N – (PFAPAs) – – OH O R
O O – – Polyfluorinated Phosphate – – - CF (CF ) CH CH –O–P–OH CF3(CF2)2–O–CF–C-O GenX
3 2 n 2 2 – Esters (PAPs) – OH CF3
O Polyfluorinated Phosphate – – CF3(CF2)nCH2CH2–O–P–O–CH2CH2(CF2)nCF3 (diPAPs) di-Esters – OH
Figure 4. Common classes of different per- and polyfluoralkyl substances (PFAS).
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4.4 Stable N- and C-isotopes, δ15N and δ13C The ratio of the stable nitrogen isotopes 14N and 15N (δ15N) and the carbon isotopes 12C and 13C (δ13C) were determined by the Institute of Energy Technology (IFE, Kjeller). Briefly, the analytical method has the following steps: combustion in element analyzer, reduction of NOx in Cu oven, separation of N2 and CO2 on GC column and determination of δ13C and δ15N by a continuous flow isotope ratio mass spectrometer (IRMS).
All isotope values refer to primary standards. For C, the reference standard was marine carbonate, Pee Dee Belemnite, PDB (Craig 1953), while atmospheric N was the reference standard for N (Mariotti, 1983). The relationships between stable isotopes of C and N (δ 13C = 13C /12C and δ15N) = 15N /14N) are calculated as ‰ deviation from the reference standards and expressed by the following equation:
δR = [Rsample/Rstandard – 1] × 103
Here R represents the ratio between the heavy and light isotope (13C/12C or 15N/14N). The δ 13C values were not corrected for lipids due to the generally low C:N ratios, indicating negligible lipid content in samples.
As the δ15N values increase with 3.4 ‰ from primary consumer to secondary consumers etc., the relative trophic position to an organism can be defined as its δ15N value divided by 3.4 ‰ (∆15N, the enrichment factor per trophic level. Post, 2002). The slope (b) of the linear regression between log-transformed concentration of a compound [C] and stable N isotope δ15N values (log [C] = b·δ15N/∆15N + a), often termed the trophic magnification factor (TMF), has been routinely used as indicator of biomagnifying potential of contaminants in food webs since the early 1990s (Yoshinaga et al., 1992).
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5. Results and discussion
5.1 Fish length and weight The grand mean of length and weight for the perch used for the samples were in the range of 15.7 cm – 19.9 cm and 98 g – 114 g (Table 3). Although Tyrifjorden had the smallest average size, perch from this lake reached a larger individual size than perch from the other two locations. We also caught smaller size groups from Tyrifjorden than from the other locations. This can be seen in the weight – length scatterplots in Figure 5. The individual weight ranges (min – max.) were as follows — Bergertjern: 40 g – 247 g; Svarthølen: 9.2 g – 334 g; Tyrifjorden: 2.4 g – 526 g. The weight – length relationships (WLRs) given in these plots were not significantly different (analysis of covariance, ANCOVA), and the regression coefficients (b) were >3 demonstrating positive allometric growth (changes in proportion with increase in length).
Table 3. Data on the length and weight of the perch included in the samples. These are pooled samples, based on tissue from several individuals. The lower part of the table gives means of sample (grand means). Ni = number of individuals in pooled samples. Ns = number of samples. Mean: x̅. Standard deviation: ± SD. Individual samples Length, cm Weight, g
Location Sample ID Ni x̅ ± SD x̅ ± SD Tyrifjorden TA 1 4 11.2 1.1 17.4 6.1 Tyrifjorden TA 2 5 16.9 1.2 56.3 11.4 Tyrifjorden TA 3 4 18.6 0.5 82.5 9.9 Tyrifjorden TA 4 5 26.4 0.6 241.8 18.0 Tyrifjorden TA 5 5 30.5 2.7 374.4 116 Tyrifjorden TA 6 11 6.3 0.2 2.9 0.2 Tyrifjorden TA 7 7 7.9 0.2 5.7 0.3 Tyrifjorden TA 8 6 8.0 0.1 6.1 0.4 Bergertjern BA 1 5 25.5 1.0 230.8 9.7 Bergertjern BA 2 5 23.0 0.9 155.4 19.1 Bergertjern BA 3 5 21.5 0.6 129.2 9.2 Bergertjern BA 4 5 15.6 0.6 44.0 3.1 Bergertjern BA 5 5 23.5 1.1 154.4 24.7 Bergertjern BA 6 5 16.4 0.2 52.4 2.2 Bergertjern BA 7 5 16.6 0.3 51.8 3.0 Bergertjern BA 8 5 16.8 0.3 59.0 1.6 Svarthølen SA 1 5 27.4 1.5 272.0 39.8 Svarthølen SA 2 5 25.5 0.9 213.6 21.4 Svarthølen SA 3 5 10.7 0.5 13.6 2.0 Svarthølen SA 4 5 11.8 1.7 19.1 9.0 Svarthølen SA 5 5 15.4 0.4 44.8 2.0 Svarthølen SA 6 5 16.8 0.7 64.0 6.8 Svarthølen SA 7 5 19.7 0.7 91.8 8.9 Svarthølen SA 8 5 24.4 0.8 193.6 20.6
Sample means Length, cm Weight, g
Location Sample ID Ns x̅ ± SD x̅ ± SD Bergertjern BA 1-8 8 19.9 3.9 109.1 67.9 Svarthølen SA 1-8 8 19.0 6.3 114.1 98.6 Tyrifjorden TA 1-8 8 15.7 9.0 98.4 137.1
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Figure 5. Length — weight relationships for the perch included in the sample material. 95 % individual confidence bands are drawn around the regression lines. Determination coefficients: r2 = 0.99 for each location.
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5.2 Food web analysis The organisms’ trophic levels and carbon sources are important characteristics for the understanding of biomagnification of environmental pollutants in aquatic food webs. Fish size is also an important factor influencing their contaminant levels. Therefore, we present and discuss such information on the biological samples, before focusing on the PFAS levels.
The relationship between stable N- and C- isotopes indicate the organisms’ relative position in the food web (trophic level) and their dominate carbon sources (Vander Zanden and Rasmussen, 2001; Post, 2002), and in our study we found a significant variation δ15N and δ13C value in the samples, both between and within the different groups of organisms (Figure 6 A).
It is commonly assumed a δ15N enrichment of 3.4 ‰ per trophic level in aquatic food webs (Minagawa and Wada 1984; Post 2002), while the corresponding δ13C enrichment is rather low (0.4 ‰ per trophic level, Post 2002). The mechanism behind the δ15N enrichment is a mass dependent discrimination against the heavier 15N isotope during transamination/deamination of amino acids. Hence, heterotrophic organisms (consumers) in a food web will retain more of the heavy 15N, and preferentially excrete more of the light 14N. This results to an enrichment of 15N along a trophic gradient.
Figure 6. A: Scatterplot of δ15N vs. δ13C for fish and zoobenthos samples (N = 36). 90 % confidence ellipses are drawn for each location. B and C: Regressions of δ15N and δ13C on mean fish length for the perch samples. 95 % confidence bands drawn for each location. N = 8 per location.
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However, δ13C signatures may vary significantly, depending of the origin of organic carbon (Vander Zanden and Rasmussen 1999; Karlsson and Bystrom̈ 2005). In general, littoral and benthic food webs are believed to be more enriched in 13C compared with open-water or pelagic food webs (Hobson, 1999). Pelagic primary producers show δ13C values near -31 ‰, littoral-benthic primary producers show higher δ13C values near -17 ‰ and terrestrial primary producer δ13C values are near -27 ‰ (Borderelle et al., 2008). However, there is a significant variation around these figures.
The low δ13C values in pelagic food webs are attributed to the pool of respired inorganic carbon, depleted of 13C, utilized by phytoplankton during photosynthesis (Post 2002). Littoral or benthic producers (submerged macro vegetation and epilithic/periphytic algae mats) have a thicker boundary layer over their surfaces than free-floating phytoplankton. This may limit their CO2 supplies and reduce the mass dependent discrimination against the heavy 13C isotope. This boundary layer effect is reduced in running water, hence the primary producers in rivers and streams have a less depleted (more negative) δ13C signal than in the more protected littoral-benthic habitat (lotic versus lentic environment).
As the δ15N values increase with 3.4 ‰ from primary consumer to secondary consumers etc., the relative trophic position to an organism can be defined as its δ15N value divided by 3.4 (the enrichment factor per trophic level). At each of our study locations did pond snails have the lowest δ15N values and large sized perch the highest, reflecting their trophic levels. The maximum differences in trophic positions (range) were as follows — Bergertjern: 1.8; Svarthølen: 2.2; Tyrifjorden: 2.1.
Our intention by including pond snails in the survey was that they should represent primary consumers in the littoral-benthic food web, as they often feed on littoral-benthic algae mats growing on submerged macrovegetation and stones (periphytic and epilithic algae). As such they could be used to base-line adjust the δ15N values from each location, which permits a direct comparison of the trophic levels between locations. A baseline correction corrects for bias in δ15N values between locations due to differences in N-isotope ratios in the primary producers’ nitrate sources.
Pond snails from Bergertjern had noticeable lower δ15N values than snails from the two other locations (≈ 4 ‰ vs. 5-5.3 ‰), whereas the figures for Gammarus lacustris and Asellus aquaticus (crustaceans) from the three locations were quite similar (≈ 6.5 ‰). Initially, we had expected similar δ15N values for pond snails from Bergertjern and Svarthølen. Both locations are situated in the Randselva river and we have no large sources or sinks for nitrate between the locations that could affect the δ15N values at food web basis. However, the pond snails from Svarthølen were of a different species (Radix peregra) than those from Bergertjern and Tyrifjorden (Lymnea stagnalis). Both species may feed on algae mats, but while R. peregra has been characterized as a generalist herbivore (Brendelberger 1997), the larger L. stagnalis probably has a more diverse feeding strategy and may include animal matter in its diet (Nichols et al., 1971). We are therefore reluctant to use pond snails for δ15N baseline corrections.
However, the nearly identical δ15N signals in the crustaceans (G. lacustris and A. aquaticus) at all locations indicate that baseline corrections are unnecessary. These crustaceans have similar food preferences and are commonly considered as herbivorous scavengers and detritivores (Bagge, 1968; Moore, 1975 and 1977; Marcus et al., 1978).
The δ15N values in perch were positively correlated with fish length (Figure 6 B), reflecting their tendency for increased piscivory (fish-eating behavior) as they grow larger. The regression lines had about the same slopes, and at a given length the δ15N values for Svarthølen and Bergertjern were practically the same, whereas it was notably higher for Tyrifjorden. This was confirmed with a statistical analysis (ANCOVA): the length adjusted means (± SE) were 10.1 ± 0.2 ‰ for Svarthølen and Bergertjern compared to 11.3 ± 0.2 ‰ for Tyrifjorden (mean length: 18.2 cm). This suggests that perch from Tyrifjorden generally are at a higher trophic level than perch from the other two locations, which has implications for the later assessment of their PFAS levels.
It is noteworthy that also small individuals from Tyrifjorden have relatively elevated δ15N values compared to the smallest individuals from the other two locations. This suggests that even the juvenile and small size-classes of perch in Tyrifjorden may become piscivores, probably exploiting newly hatched fry of roach (Rutilus rutilus) – a recently introduced and abundant fish species in the littoral zone in Tyrifjorden. Although this is a rather uncommon or underreported foraging strategy for such small perch, similar behavior has been described by Beck et al. (2002) and Borcherding (2006).
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Analyses of δ13C in freshwater food webs can be used to distinguish between pelagic, littoral and terrestrial derived carbon sources. Pelagic primary producers and consumers have a more depleted δ13C signal (more negative) than organisms associated to littoral-benthic and terrestrial food webs. Both pond snails and Gammarus from Svarthølen had more negative δ13C values (-27.4 and -27.8 ‰) than those from Bergertjern (-23.6 and -19.3 ‰) and Tyrifjorden (-22.8 ‰ and -16.6 ‰. The reason for this is unsure, but it can reflect both an influence from terrestrial (allochthonous) organic matter in Svarthølen and a more riverine (lotic) δ13C signal in the primary producers here. The sediments in Svarthølen are rich in allochthonous organic matter due to the historical discharges of wood fiber originating from the paper pulp production at Viul. This may serve as an organic carbon source for scavengers as pond snails and Gammarus in the litoral-bentic habitat of Svarthølen.
The mean δ13C values in perch from Tyrifjorden were in general elevated compared to those from Svarthølen and Bergertjern (Figure 6 C). There was a tendency for increasing δ13C values with fish length at all locations, which indicates a gradually closer association to the littoral habitat as the fish grow larger.
As for δ15N, a statistical analysis (ANCOVA) could not prove any significant differences between the regression slopes (p = 0.5) but demonstrated differences between their adjusted means (p = 0.006). The length adjusted (18.2 cm) mean δ13C values (± SE) for Svarthølen and Bergertjern nearly identical (-26.2 ± 0.8 ‰ and -26.0 ± 0.8 ‰) but significantly higher for Tyrifjorden (-22.5 ± 0.8 ‰). These δ13C figures could indicate that perch from Tyrifjorden in general are closer connected to the littoral food web than perch from the other two locations. It is also reasonable to assume that the perch from Bergertjern with the most depleted δ13C signatures (≈ -28 ‰), belonging to the small size classes (mean length ≈16–17 cm), may have utilize drifting zooplankton produced in Lake Randsfjorden, as the outlet from this large lake is about 1 km upstream Bergertjern.
Table 4. Stable isotope ratios for samples analyzed for PFAS. These are pooled samples, based on tissue from several individuals. The lower part of the table gives sample means (grand means for fish length and weight). Sample matrix: fish, liver; crustaceans, whole body; snails, soft body. Number of individuals in pooled samples: Ni. Number of samples: Ns. Mean: x̅. Standard deviation: ± SD. Individual samples Stable isotopes 15 13 Location Organism Sample ID Ni δ N, ‰ δ C, Tyrifjorden Perch TA 1 4 10.40 -18.69 Perch TA 2 5 11.73 -20.54 Perch TA 3 4 11.20 -20.51 Perch TA 4 5 11.62 -22.47 Perch TA 5 5 12.22 -22.78 Perch TA 6 11 11.70 -26.59 Perch TA 7 7 10.04 -23.59 Perch TA 8 6 10.04 -27.49 Gammarus TGA 1 * 6.54 -15.66 Gammarus TGA 2 * 6.37 -17.49 Pond snail TDS 1 5 5.04 -22.11 Pond snail TDS 2 5 5.64 -23.47 Bergertjern Perch BA 1 5 11.26 -25.35 Perch BA 2 5 10.27 -25.62 Perch BA 3 5 10.30 -22.12 Perch BA 4 5 10.04 -25.14 Perch BA 5 5 10.23 -22.66 Perch BA 6 5 10.02 -27.93 Perch BA 7 5 10.12 -28.26 Perch BA 8 5 10.00 -27.95 Asellus BAS 1 * 6.23 -20.03 Gammarus BGA 1 * 6.27 -19.06 Gammarus BGA 2 * 6.78 -18.69 Pond snail BDS 1 3 4.98 -23.57 Svarthølen Perch SA 1 5 10.8 -25.21 Perch SA 2 5 10.95 -25.93 Perch SA 3 5 9.78 -27.34 Perch SA 4 5 9.36 -26.88
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Individual samples Stable isotopes 15 13 Location Organism Sample ID Ni δ N, ‰ δ C, Svarthølen Perch SA 5 5 9.67 -24.84 Perch SA 6 5 9.50 -25.98 Perch SA 7 5 10.24 -25.37 Perch SA 8 5 10.83 -25.50 Gammarus SGA 1 * 6.71 -27.52 Gammarus SGA 2 * 6.36 -28.09 Pond snail SDS 1 5 4.39 -27.33 Pond snail SDS 2 5 3.58 -27.47 Sample means δ15N, ‰ δ13C, ‰
Location Organism Sample ID Ns x̅ ± SD x̅ ± SD Bergertjern Perch BA 1-8 8 10.28 0.41 -25.63 2.36 Crustacean BGA 1-2, BAS 1 3 6.43 0.31 -19.26 0.69 Pond snail BDS 1 1 4.98 -23.57 Svarthølen Perch SA 1-8 8 10.14 0.65 -25.88 0.85 Crustacean SGA 1-2 2 6.54 0.25 -27.81 0.40 Pond snail SDS 1-2 2 3.99 0.57 -27.40 0.10 Tyrifjorden Perch TA 1-8 8 11.12 0.85 -22.83 3.03 Crustacean TGA 1-2 2 6.46 0.12 -16.58 1.29 Pond snail TDS 1-2 2 5.34 0.42 -22.79 0.96 * Not determined
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5.3 Quantifiable PFAS and general levels The samples were analyzed for 89 different PFAS, with levels of quantification (LOQ) in the range 0.1 – 2.0 ng/g w.w. Of these had 22 analytes quantifiable levels (Figure 7). However, four of these 22 compounds were found in very low concentrations (≤ 1.1 ng/g w.w.) and the percentage of samples with quantifiable results for these were also very low (≤ 11%). The data on these four compounds are therefore of limited value, only demonstrating that they occur in low concentrations. We have therefore excluded them from the main results, leaving us with 18 different PFAS for our further assessment (Table 5). In the following are all concentrations below LOQ substituted with LOQ/2.
Perch from Svarthølen had greatly elevated concentrations of PFAS compared to the two other locations (Table 5 and Figure 9). The mean sum (± SD) of the 18 different PFAS in fish samples were as follows — Svarthølen: 1278 ± 411 ng/g w.w.; Tyrifjorden: 266 ± 144 ng/g w.w.; Bergertjern: 41.8 ± 17.9 ng/g w.w. PFOS was the dominating compound at all locations, followed by PFCAs (perfluoroalkyl carboxylic acids). At Svarthølen, FTSs (fluorinated telomer sulfonates) were also present in significant concentrations.
There was a significant variation in concentrations of different PFAS within each location (Figure 9). Much of this variation can be attributed to differences in fish size or trophic level, as will be shown in the following subsections. The many samples from Bergertjern with concentrations < LOQ, especially for fluorinated telomer sulfonates (FTSs), FOSA and et-FOSAA, can also be observed in Figure 9.
The PFAS levels in zoobenthos samples were significantly lower than in perch, and the sum of the 18 compounds were in the range 13.6 – 45.7 ng/g w.w. (Table 5 and Figure 9). There were only modest differences in total PFAS for zoobenthos between sites. However, the presence of different substances varied between sites. In Svarthølen samples, PFCAs (perfluorinated carboxylic acids) and FTSs were the dominating substances and occurred in about equal concentrations, while PFCAs dominated in Bergertjern and Tyrifjorden.
Figure 7. Analyses of PFAS. A: Levels of quantification (LOQ) for the different analytes. B: Percentage of samples with quantified results (≥ LOQ) for the different analytes (green columns). PFAS with light green columns are omitted from the further assessment due to low concentrations and few quantifiable results. No. of samples = 36.
- 21 - Fjeld og Vann Report: R1-2019
Figure 8. Mean concentrations of main groups/substances of PFAS in biota samples. FTSs: Fluorinated telomer sulfonates; PFCAs: Perfluoroalkyl carboxylic acids. PFOS (l + br): linear and branched PFOS isomers. Sample matrix: fish, liver; crustaceans, whole body; snails, soft body. Number of samples per location: perch (8); crustaceans (2); pond snails (2) – except for Bergertjern, crustaceans (3) and snails (1).
Table 5. PFAS concentrations in biota samples (ng/g w.w.). Mean and standard deviation (x̅ ± SD) in for 18 individual compounds, the main groups and sum of PFAS (grey shaded rows). Concentrations Perch (liver) Crustaceans (whole body) Pond snails (soft body) Bergertjern Svarthølen Tyrifjorden Bergertjern Svarthølen Tyrifjorden Bergertjern Svarthølen Tyrifjorden PFAS n = 8 n = 8 n = 8 n = 3 n = 2 n = 2 n = 1 n = 2 n = 2 PFHxA 0.6 ± 0.5 0.5 ± 0.5 0.8 ± 0.5 1.4 ± 1.3 2.2 ± 0.4 0.5 ± 0.4 5.6 1.2 ± 0.1 2.3 ± 1.1 PFHpA 2.6 ± 0.7 3.6 ± 1.7 4.3 ± 2.5 5.1 ± 3.4 6.9 ± 1.4 3 ± 0.7 20 4.1 ± 0 3.3 ± 1.8 PFOA 1.9 ± 0.5 2.6 ± 1.5 3.6 ± 2.2 4.5 ± 2.9 7.5 ± 0.4 4.1 ± 0.8 16 3.4 ± 0.3 3 ± 1.8 PFNA 0.3 ± 0 0.5 ± 0.3 1.2 ± 0.5 0.5 ± 0.4 0.8 ± 0.1 0.9 ± 0.1 1.9 0.3 ± 0 0.3 ± 0 PFDA 1 ± 0.2 44.1 ± 31.7 10.1 ± 6.7 0.3 ± 0 0.3 ± 0 0.4 ± 0.2 0.3 0.3 ± 0 0.4 ± 0.2 PFUnDA 2.8 ± 1.1 23.5 ± 16.7 9.3 ± 5.6 0.2 ± 0 0.2 ± 0 2 ± 0.1 0.2 0.2 ± 0 1.7 ± 1.2 PFDoDA 1.8 ± 0.5 32.4 ± 7.2 14.4 ± 8.1 0.2 ± 0 2 ± 0.1 3.2 ± 0.8 0.2 0.2 ± 0 2.1 ± 1.1 PFTrDA 2.6 ± 0.6 8.3 ± 3 7.1 ± 4.1 0.2 ± 0 0.7 ± 0.1 2.3 ± 0.3 0.2 0.2 ± 0 0.7 ± 0.3 PFTeDA 0.9 ± 0.7 15.2 ± 4.7 5.2 ± 3.5 0.2 ± 0 1.1 ± 0.1 0.4 ± 0.3 0.2 0.2 ± 0 0.2 ± 0 PFPeDA 0.2 ± 0 0.9 ± 0.4 0.3 ± 0.3 0.2 ± 0 0.2 ± 0 0.2 ± 0 0.2 0.2 ± 0 0.2 ± 0 ∑PFCAs 14.6 ± 2.1 131.6 ± 53.9 56.3 ± 22 12.7 ± 7.9 21.8 ± 2.1 16.8 ± 3 44.8 10.2 ± 0.1 14 ± 1.9 PFOS 25.5 ± 16.8 836.5 ± 300.1 182.3 ± 98.2 0.1 ± 0 0.4 ± 0.1 0.8 ± 0.1 0.1 0.1 ± 0 0.1 ± 0 br-PFOS 1 ± 0.5 113.8 ± 52 6.5 ± 1.9 0.1 ± 0 0.1 ± 0 0.1 ± 0 0.1 0.1 ± 0 0.1 ± 0 ∑PFOS 26.5 ± 17.2 950.3 ± 340.4 188.7 ± 99.5 0.1 ± 0 0.4 ± 0.1 0.8 ± 0.1 0.1 0.1 ± 0 0.1 ± 0 FOSA 0.1 ± 0 27.8 ± 14.7 0.2 ± 0.3 0.1 ± 0 5 ± 1.7 0.2 ± 0.1 0.1 1 ± 0.1 0.5 ± 0.1 et-FOSAA 0.2 ± 0 44.6 ± 23.6 12.7 ± 32.1 0.2 ± 0 2.9 ± 0.8 0.2 ± 0 0.2 1.8 ± 0.2 0.7 ± 0.2 ∑FOSA(A) 0.2 ± 0 72.4 ± 35 13 ± 32.4 0.2 ± 0 7.9 ± 2.5 0.3 ± 0.1 0.2 2.8 ± 0.4 1.2 ± 0.4 8:2 FTS 0.2 ± 0 36 ± 21.3 1 ± 1.1 0.2 ± 0 0.2 ± 0 0.2 ± 0 0.2 0.2 ± 0 0.2 ± 0 10:2 FTS 0.2 ± 0 33 ± 16.4 0.4 ± 0.2 0.2 ± 0 1.2 ± 0.1 0.2 ± 0 0.2 3.7 ± 0.2 0.2 ± 0 12:2 FTS 0.2 ± 0 53.9 ± 16.6 6.9 ± 7.4 0.2 ± 0 10.2 ± 2.6 1 ± 0.2 0.2 10.5 ± 0.7 1.5 ± 0.4 14:2 FTS 0.2 ± 0 0.8 ± 0.3 0.3 ± 0.2 0.2 ± 0 0.9 ± 0.1 0.2 ± 0 0.2 0.5 ± 0.1 0.2 ± 0 ∑FTSs 0.6 ± 0 123.6 ± 39.9 8.5 ± 8.6 0.6 ± 0 12.3 ± 2.6 1.4 ± 0.2 0.6 14.8 ± 0.4 2 ± 0.4 ∑PFAS 41.8 ± 17.9 1278 ± 411 266 ± 144 13.6 ± 7.9 42.4 ± 3 19.3 ± 3.3 45.7 27.8 ± 0.9 17.2 ± 1.9 - 22 - Fjeld og Vann Report: R1-2019 z Figure 9. Concentrations of selected PFAS in perch (liver tissue) from the three locations. The box plots show median, interquartile range and max. and min. values. Observations below quantification limits (LOQ) are substituted with LOQ/2, and depicted with a triangle, ▽. No. of pooled samples per location = 8. - 23 - Fjeld og Vann Report: R1-2019 5.4 PFOS, PFCAs and precursors 5.4.1 About precursors PFOS, followed by PFCAs (perfluoroalkyl carboxylic acids), were the dominating substances or groups of PFAS in all the fish liver samples (Figure 10 A). These environmentally stable compounds can form as breakdown products from a variety of precursor molecules (Buck et al., 2011; Liu and Avendaño, 2013; Martin et al., 2010). PFOS is regarded as the terminal and environmental stable fluorinated end product of FOSA and et-FOSAA. These belong to a family compounds known as precursors to PFOS, chemically characterized by a PFOS moiety linked by the sulfonate to another molecular group. Among the precursors to PFCAs are FTSs (fluorinated telomer sulfonates). These share a polyfluorinated n:2 structure: n = number of perfluorinated carbon (C) bound to two C without fluorine (F). Figure 10. Mean concentrations of selected PFAS in perch (liver tissue) from the three locations. A: Total concentrations of different main groups of PFAS. B: Mean concentrations of PFOS, branched PFOS and their precursors. C: Mean concentrations of perfluorinated carboxylic acids (PFCAs) and precursors (Fluorinated Telomer Sulfonates, FTSs). “EQS liver” refers to an adjusted Environmental Quality Standard for liver, which for PFOS and PFOA are 166 ng/g w.w. and 1247 ng/g w.w., respectively. The average concentration of PFOS in Svarthølen and Tyrifjorden exceeded the “EQS liver” value, whereas the average PFOA concentrations were below this threshold. No. of pooled samples per location = 8. Observations below quantification limits (LOQ) are substituted with LOQ/2. 5.4.2 PFOS and precursors PFOS and its branched isomers (br-PFOS) were found with particularly high levels in Svarthølen compared to the other two locations (Figure 10 B). The mean concentrations were as follows — Svarthølen: 950 ± 340 ng/g w.w.; Tyrifjorden: 189 ± 100 ng/g w.w.; Bergertjern: 41.8 ± 17.9 ng/g w.w. As a percentage of total sum PFAS, this amounts to 76 %, 71 % and 63 %, respectively. Also, we found significantly elevated concentrations of FOSA and et-FOSAA in perch samples from Svarthølen (mean concentration: 72.5 ± 35 ng/g w.w.) compared to the two other locations (Tyrifjorden: 13.0 ± 32.4 ng/g w.w.; Bergertjern: < LOQ). The zoobenthos samples had in general very low concentrations of PFOS and its precursors. For PFOS (linear + branched) were the mean concentrations in the range 0.1 – 0.8 ng/g w.w. For the precursors FOSA and et-FOSAA, the levels in Svarthølen (2.8 – 7.9 ng/g w.w.) were elevated compared to Bergertjern and Tyrifjorden (0.2 – 1.2 ng/g w.w.). - 24 - Fjeld og Vann Report: R1-2019 Previous PFOS levels in liver of perch from Tyrifjorden (Storøya) for the years 2014 – 2016 have been reported to be in the range of 130 –190 ng/g w.w., based on pooled samples for fish with an average length and weight of 23 – 26 cm and 179 – 238 g (Fjeld et al., 2015 – 2017). This is comparable with the average results we report here (182 ng/g w.w.), but considerably lower than the levels of ≈ 850 – 1100 ng/g w.w. reported by Slinde and Høysether (2017) (fish size not known). The reason for the discrepancy between the results of Slinde and Høysether (op. cit.) and others is unknown. The ratio between linear and branched isomers of PFOS moieties in technical formulations may give insight in the manufacturing process employed, as electrochemical fluorination gives a yield of about 70 % linear and 30 % branched isomers — in contrast to exclusive linear PFOS for the more recent telomerization process (Benskin et al., 2010). Based on such information one could be tempted to conclude about different sources for PFOS in perch from Svarthølen and Tyrifjorden, as the percentage of branched isomers were about 7 % in Svarthølen compared to 2 % in Tyrifjorden and Bergertjern. However, isomer-specific bioaccumulation of PFOS and biotransformation of its precursors promote the accumulation of linear PFOS in wildlife and fish (for an overview, see Xing et al., 2018). An effect of such isomer-specific properties is that the biota in locations receiving fresh or little degraded discharges of PFAS have a higher ratio of branched isomers than locations with more aging sources. Thus, the isomer-profiles in aquatic biota cannot be used to support a theory predicting differences in the main sources of PFOS to Svarthølen and Tyrifjorden. The Environmental Quality Standard (EQS value) for PFOS and its derivatives (including precursors) in organisms is 9.1 ng/g w.w. (Norwegian Environmental Agency, 2016). A Swedish study has reported a ratio of 18.2:1 between PFOS concentrations in liver and muscle in perch (Faxneld et al., 2014). This is identical to the ratio for brown trout from Lake Mjøsa, Norway’s largest lake (Fjeld et al., 2017). If we accept this ratio as an adjustment factor, the corresponding EQS value for PFOS in liver will be 166 ng/g w.w. This EQS limit for liver is exceeded with a factor of 6.2 in Svarthølen and 1.2 in Tyrifjorden. 5.4.3 PFCAs and precursors PFCAs were clearly elevated in the perch samples from Svarthølen compared to the other two locations (Figure 10 C). The mean concentrations were as follows — Svarthølen: 131.6 ± 53.9 ng/g w.w.; Tyrifjorden: 56.3 ± 22.0 ng/g w.w.; Bergertjern: 14.6 ± 2-1 ng/g w.w. As percentage of total sum PFAS, this amounts to 10 %, 21 % and 30 %, respectively. In general, the more long-chained compounds (C10 – C14) were the dominating PFCAs in Svarthølen and Tyrifjorden. Deviating high concentrations of FTSs were also present in perch from Svarthølen. The levels here were comparable to those of PFCAs. In Bergertjern, all fish samples had concentrations below LOQ. In Tyrifjorden the levels were in general low, in average less than a tenth of those from Svarthølen. The mean concentrations were as follows — Svarthølen: 123.6 ± 39.9 ng/g w.w.; Tyrifjorden: 8.5 ± 8.6 ng/g w.w.; Bergertjern (substituted with LOQ/2): 0.6 ng/g w.w. As a percentage of total sum PFAS, this amounts to 10 %, 3 % and 1 %, respectively. The longer chained telomer 12:2 dominated among the FTSs in Svarthølen and Tyrifjorden. However, in Svarthølen the 8:2 and 10:2 telomers were also present in relatively high levels. Zoobenthos had in general low concentrations of PFCAs and FTSs, but the mean concentrations were clearly elevated compared to those of PFOS and its precursors. For crustaceans were the mean concentrations of PFCAs in the range 12.7 – 21.8 ng/g w.w., with the highest concentration in Svarthølen and lowest in Bergertjern. The mean levels in pond snails were in the range 10.2 – 44.8 ng/g w.w., with the highest concentration in Bergertjern. However, this was of a different species than those from Svarthølen and Tyrifjorden, making further comments on the differences difficult. In Svarthølen, the mean FTSs concentrations for crustaceans (12.3 ± 2.6 ng/g w.w.) and pond snails (14.8 ± 0.4 ng/g w.w.) were distinctly higher compared to the other two locations (0.6 – 2.0 ng/g w.w.). Again, as for perch, the 12:2 telomere was the dominating FTS in zoobenthos from Svarthølen. The national Environmental Quality Standard (EQS) value for PFOA in organisms is 91 ng/g w.w. (Norwegian Environmental Agency, 2016). If we apply a conversion factor of 13.7 based on the ratio between liver and muscle concentrations in perch (Faxneld et al., 2014), the corresponding EQS value for PFOA in liver will be 1247 ng/g w.w. The levels in the perch samples were far from exceeding this (mean values: 1.9 – 3.6 ng/g w.w.). - 25 - Fjeld og Vann Report: R1-2019 5.5 Site specific chemical fingerprints To summarize the complex patterns of PFAS in perch from the different locations, we used two different statistical techniques giving a simplified overview of their main chemical characteristics. We used a cluster analysis and a principal component analysis (PCA) to identify these “chemical fingerprints”. The analyses were based on log- transformed and standardized data (zero mean and unit variance for each variable) and were done by the statistical program JMP (SAS 2010). A cluster analysis tries to identify homogenous groups of objects or cases, with no prior information about the group membership for any of the objects. Our analysis (hierarchical clustering, Ward’s minimum variance method) identified three clusters, and correctly grouped all fish samples to their locations (Figure 11 A). A two-way clustering procedure revealed the influence different PFAS had on the solution. This is visualized with a “heat map” next to the dendrogram defining the clusters. The heat map shows the Svarthølen fish to be characterized by high concentrations of PFOS, FTSs, long-chained PFCAs (C10 – C14), FOSA and et-FOSAA. Tyrifjorden fish had in general elevated concentrations of the C6 – C9 PFCAs compared to the other locations, moderate concentrations of PFOS, and low concentrations of FTSs and FOSA. Bergertjern fish had in general low concentrations of all PFAS. In the principal component analysis (PCA) we have projected the data from its original multi-dimensional space to a two-dimensional subspace (the bi-plot in Figure 11 B). In a PCA are the original data transformed by a linear combination of the original variables to a new set of dimensions or principal components (PCs). This transformation is defined in such a way that the first PC has the largest possible variance, and each succeeding PC in turn has the highest variance possible under the constraint that it is orthogonal (not correlated) to the preceding PC. In this way most of the variance and the internal structure of the dataset can be expressed by a few PCs. In our case expressed the first and second PC 66.2 % and 14.6 % of the total variance in the dataset – or together 80.8 %. The first PC represents a dimension correlated with PFOS and most of the other PFAS, except for C6 – C9 PFCAs which were correlated to the second PC. The Svarthølen samples scored high on PC1, whereas Tyrifjorden and Bergertjern scored intermediate and low, respectively. For PC2, Tyrifjorden had in general the highest scores, whereas Bergertjern scored low. This pattern is in accordance with the results from the cluster analysis. Figure 11. Chemical fingerprinting of the perch samples based on log-transformed and standardized PFAS data (n = 8 for each location). A: Cluster analysis dendrogram and heat map showing the group affiliation and PFAS levels for each sample. B: Principal component analysis biplot showing the scores of fish (points) and variable loadings (arrows) on the principal components (PCs). 90 % confidence ellipses are drawn for the scores for each location. The percentage explained variance for each PC is written along the axes. Observations below quantification limits (LOQ) are substituted with LOQ/2. - 26 - Fjeld og Vann Report: R1-2019 5.6 Accumulation of PFOS in perch The concentration of PFOS in perch liver showed an exponentially increase or accumulation with both fish length and trophic level (expressed by their stable N-isotope ratio, δ15N) (Figure 12). Statistical models could not prove any significant differences between the regression coefficients for the different locations (ANCOVA, test for interaction), hence the regression lines for each location within a model should be considered as parallel. The regression plots (Figure 12) illustrate that all samples from Svarthølen and none from Bergertjern had concentrations exceeding the liver EQS limit for liver (166 ng/g w.w.). For Tyrifjorden did the concentration surpass the EQS limit at a length of 16.0 cm (95% confidence limits: 11.6 – 20.5 cm) and at δ15N) value of 11.2 ‰ (10.0 – 12.5 ‰). By correcting for the effects of length or δ15N, we could determine an adjusted mean concentration of ∑PFOS for each location. In this way we will have a better representation of the site specific PFOS levels than the raw arithmetic means. We found significantly differences between the adjusted means, after correcting for the effects of both length and δ15N (test for effect of “location” in the ANCOVA models) (Table 6). By length-adjusting, the main effects were a reduction in the means for both Svarthølen and Bergertjern compared to the unadjusted means. However, when adjusting for differences in trophic position (δ15N), the main effects were a marked increase in mean value for Svarthølen and a decrease for Tyrifjorden. We believe this δ15N adjustment gives a better impression of the PFOS loadings to the aquatic food web than the unadjusted figures. A) PFOS vs. Length B) PFOS vs. δ15N Bergertjern Svarthølen 1000 1000 Tyrifjorden EQS, liver 100 100 ∑PFOS, ng/g w.w. ∑PFOS, ng/g w.w. 10 10 5 10 15 20 25 30 9 9.5 10 10.5 11 11.5 12 12.5 Length, cm δ15N, ‰ ∑ ∑ 15 loge PFOS =3.834+0.064·Length loge PFOS =0.363+0.441·δ N -1.993;Bergertjern -1.783;Bergertjern + 1.739; Svarthølen + 1.953; Svarthølen 0.254;Tyrifjorden 0.170;Tyrifjorden 2 2 R =0.95,n =24,F3,20 =152.4 R =0.93,n =24,F3,20 =79.6 p <0.001 p <0.001 Figure 12. Relationship between PFOS concentration (linear and branched isomers) in perch liver and (A) mean fish length, and (B) stable isotope ratio (δ15N). Regression lines (with 95 % confidence bands) and equations are based on results from analyses of covariance (ANCOVAs). “EQS, liver” refers to an adjusted Environmental Quality Standard for PFOS concentration in liver (166 ng/g w.w.). The δ15N level is regarded to increase with 3.4 ‰ per trophic level. Table 6. Adjusted and unadjusted mean concentrations of ∑PFOS (linear and branched isomers, ng/g w.w.) in perch liver. Adjusted to average length and mean δ15N for all samples. L/U 95 % CI: Lower/Upper 95 % confidence intervals. Adjusted to mean length (16.0 Adjusted to mean δ15N (10.5 ‰) Unadjusted cm) Location Adj. mean L 95% CI U 95% CI Adj. mean L 95% CI U 95% CI Mean L 95% CI U 95% CI Bergertjern 20 15 26 25 17 36 26 12 41 Svarthølen 841 646 1095 1042 714 1521 950 666 1235 Tyrifjorden 191 146 250 125 83 188 189 106 272 - 27 - Fjeld og Vann Report: R1-2019 5.7 Source evaluations The PFOS precursors FOSA and et-FOSAA are both known as intermediate degradation products of parent compounds, such as et-FOSE based polyfluoroalkyl phosphate esters or acrylates, which have been incorporated into water and oil repellent paper-based food contact materials (FCM). Also, both PFCAs and their precursors FTSs are known to be applied in water and oil repellent formulations for FCM. These uses are well documented in scientific literature and registered patents (for an overview see Buck et al., 2011, Deisenroth et al., 1998; Jennings et al., 2002a and b; Schaider et al., 2017; Trier et al., 2018). Thus, the elevated concentrations of PFOS, FTSs, FOSA and et-FOSAA in fish from Svarthølen are most likely related to the use of PFAS based seizing agents in the production of molded paper pulp articles at Viul industrial site. Discharges from the production here have undoubtedly contaminated the aquatic environment downstream the factory premises. Huhtamäki has acknowledged the use of fluorchemistry in their production at Viul during their active period of 1999 – 2013. Unfortunately, we have no information as to whether the former industrial companies, Keyes Norway AS (1963 – 1982) and Van Leer (1982 – 1999), also used PFAS containing chemicals in their production of molded paper pulp articles. In the sediment study, done as a part of this survey for Huhtamäki and reported by Helland and Tellefsen (in prep.), significantly elevated PFAS concentration were found in the riverbed sediments outside the backfill of the closed down factory premises at Viul and further downstream in Svarthølen. The most abundant compounds were (i) the PFCA precursors 8:2, 10:2 and 12:2 FTS, and (ii) the PFOS precursors et-FOSE and et-FOSAA, whereas the PFOS concentration were relatively low. These two chemical groups probably reflect the use of different commercial, technical formulations of PFAS. The one with the PFOS moiety has supposedly been substituted with a FTS-based formulation after the regulation or voluntary phase-out of the production and use of PFOS and PFOS-related products (the use of commercial mixtures of PFAS substances containing PFOS or its precursors were strictly regulated in Norway in 2007). The presence of branched PFOS isomers in the samples indicates that the technical formulation containing this moiety have been produced by electrochemical fluorination. This production process was largely phased out in the western world in the early 2000s in favor of telomerization, which produces an isomerically pure product retaining the structure of the typically linear starting material (Benskin et al., 2010). The partitioning in the environment and accumulation in organisms of different PFAS substances are determined by their physiochemical properties – and can vary widely. Due to this, and the metabolic transformation of several PFAS species, concentrations of PFAS in the environmental compartments at one location do not necessarily reflect the biota concentrations (ITCR 2018). We can therefore expect to find quite different PFAS profiles between sediments and fish downstream the closed factory site at Viul. To what extent the PFAS contamination at Viul has affected the PFAS concentrations in the aquatic food web in Tyrifjorden is difficult to give any quantitative judgments of. The PFAS concentrations in fish here are clearly elevated compared to other large lakes in South-East Norway (Fjeld et al., 2015 – 2017). Hence, it is reasonable to assume we have one or several significant point sources of PFAS to this large lake. In the first assessment of PFAS sources to Lake Tyrifjorden (Aaasen Slinde and Høisæther, 2017), two major, historical sources were disclosed: (i) the industrial site at Viul, and (ii) the fire training facility at Hønefoss fire station further downstream Randselva. However, further targeted studies are needed to sort out their respective contributions to the pollution situation in Tyrifjorden. - 28 - Fjeld og Vann Report: R1-2019 6. References Aasen Slinde. G. and Høisether, Å. 2017. Source tracing of PFAS to Tyrifjorden. Norwegian Environmental Agency. 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The designing of a new grease repellent fluorochemical for the paper industry. Surface Coatings International 81(9): 440–447. Faxneld, S., Danielsson, S., and Nyberg, E. 2014. Distribution of PFAS in liver and muscle of herring, perch, cod, eelpout, arctic char, and pike from limnic and marine environments in Sweden. Swedish Museum of Natural History. Report No. 9:2014. Fjeld, E., Bæk, K., Rognerud, S., Rundberget, J.T., Schalbach, M. and Warner, N.A., 2015. Environmental pollutants in large Norwegian lakes, 2014. Norwegian Environmental Agency. Report M-349/2015. 101 pp. (In Norwegian) Fjeld, E., Bæk, K., Rognerud, S., Rundberget, J.T., Schalbach, M. and Warner, N.A., 2016. Environmental pollutants in large Norwegian lakes, 2015. Norwegian Environmental Agency. Report M-548/2016. 97 pp. (In Norwegian) Fjeld, E., Bæk, K., Rundberget, J.T., Schlabach, M. and Warner, N.A., 2017. Environmental pollutants in large Norwegian lakes, 2016. Norwegian Environmental Agency. 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Appendix: Analytical and morphological data - 31 - Fjeld og Vann Report: R1-2019 PFUnDA 5.8 17 18 8.9 11 3.9 5.1 4.8 3.2 5.1 3.5 2 2.4 2.3 2 1.8 41 42 6.4 8.8 13 11 20 46 0.8 2.5 2 1.9 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 0.4 PFUdA % recov 95 PFDA 4.5 13 13 18 20 3.7 4.3 4.4 1.2 1.5 1 0.8 1.1 1 0.9 0.7 78 59 11 22 25 16 44 98 <0,5 0.6 0.5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 PFDA % recov 96 PFNA 0.8 0.7 0.6 1.6 2 1.2 1.3 1 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 1.1 <0,5 0.7 <0,5 <0,5 <0,5 0.6 <0,5 <0,5 <0,5 0.9 0.8 1.9 0.9 <0,5 <0,5 <0,5 <0,5 0.9 0.7 0.5 PFNA % recov 99 PFOA 3.9 1.4 1.5 1.5 3 7.8 5.4 4.1 2.2 1.5 1.5 2.3 1.1 2.5 1.9 1.9 1.7 1.9 4.3 5.4 1.6 2.8 1.3 2.1 4.2 1.7 4.6 3.5 16 7.7 3.4 2.3 3.2 3.6 7.8 7.2 0.5 PFOA % recov 109 PFHpA 4.7 1.6 1.9 3.1 2.6 8.8 6.9 4.8 2.9 1.9 2 3 1.5 3.8 2.9 2.6 2.4 2.7 5.9 6.2 2.3 4.4 1.7 2.9 4.5 2 3.5 2.5 20 8.9 3.8 2.5 4.1 4.1 7.9 5.9 0.5 PFHpA % recov 105 PFHxA 0.96 <0,5 0.59 0.97 0.86 1.05 1.76 <0,5 1.05 <0,5 <0,5 1.2 <0,5 1.16 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 1.67 <0,5 0.81 3.1 1.5 0.8 <0,5 5.6 2.8 1.2 <0,5 1.3 1.1 2.4 1.9 0.5 PFHxA % recov 95 PFPA <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 1 PFPA % recov 101 PFBA <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 1 PFBA % recov 86 . . . . . 15 15 7.5 5.7 14.3 14.4 14.3 9.01 7.77 7.24 7.68 7.59 7.67 7.35 7.25 7.28 7.72 13.96 14.21 14.64 14.53 14.77 13.94 14.68 14.65 14.81 14.55 14.45 14.76 14.68 14.51 15.01 14.81 14.47 14.32 14.44 W%N . . . . . 47.1 45.8 46.4 41.8 41.3 46.41 46.41 47.15 47.33 47.91 47.39 46.06 47.57 46.58 47.12 47.23 45.51 45.79 47.77 47.28 47.24 47.95 46.85 46.23 45.16 47.88 42.36 38.91 40.02 34.27 36.24 39.15 46.37 47.91 38.67 38.27 W%C . . . . . -25.5 -18.69 -20.54 -20.51 -22.47 -22.78 -26.59 -23.59 -27.49 -25.35 -25.62 -22.12 -25.14 -22.66 -27.93 -28.26 -27.95 -25.21 -25.93 -27.34 -26.88 -24.84 -25.98 -25.37 -22.11 -23.47 -15.66 -17.49 -23.57 -20.03 -19.06 -18.69 -27.33 -27.47 -27.52 -28.09 13C, ‰ δ . . . . . 10 9.5 10.4 11.2 11.7 10.3 10.8 9.78 9.36 9.67 5.04 5.64 6.54 6.37 4.98 6.23 6.27 6.78 4.39 3.58 6.71 6.36 11.73 11.62 12.22 10.04 10.04 11.26 10.27 10.04 10.23 10.02 10.12 10.95 10.24 10.83 15N, ‰ δ ...... 3 2 9 2 18 6.1 9.9 0.2 0.3 0.4 9.7 9.2 3.1 2.2 1.6 6.8 8.9 116 11.4 19.1 24.7 39.8 21.4 20.6 SD Weight, g ...... 1 1.1 1.2 0.5 0.6 2.7 0.2 0.2 0.1 0.9 0.6 0.6 1.1 0.2 0.3 0.3 1.5 0.9 0.5 1.7 0.4 0.7 0.7 0.8 SD Length, cm ...... 44 59 64 2.9 5.7 6.1 272 17.4 56.3 82.5 52.4 51.8 13.6 19.1 44.8 91.8 241.8 374.4 230.8 155.4 129.2 150.4 213.6 193.6 Mean Weight, g ...... 8 23 6.3 7.9 11.2 16.9 18.6 26.4 30.5 25.5 21.5 15.6 23.5 16.4 16.6 16.8 27.4 25.5 10.7 11.8 15.4 16.8 19.7 24.4 Mean Length, cm ...... 4 5 4 5 5 7 6 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 11 N (fish) Sample ID TA 1 TA 2 TA 3 TA 4 TA 5 TA 6 TA 7 TA 8 BA 1 BA 2 BA 3 BA 4 BA 5 BA 6 BA 7 BA 8 SA 1 SA 2 SA 3 SA 4 SA 5 SA 5 SA 6 SA 7 TDS-1 TDS-2 TGA-1 TGA-2 BDS-1 BAS-1 BGA-1 BGA-2 SDS-1 SDS-2 SGA-1 SGA-2 Sample type Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Perch liver Pond snail Pond snail Gammarus Gammarus Pond snail Asellus Gammarus Gammarus Pond snail Pond snail Gammarus Gammarus Location Tyrifjorden Tyrifjorden Tyrifjorden Tyrifjorden Tyrifjorden Tyrifjorden Tyrifjorden Tyrifjorden Bergertjern Bergertjern Bergertjern Bergertjern Bergertjern Bergertjern Bergertjern Bergertjern Svarthhølen Svarthhølen Svarthhølen Svarthhølen Svarthhølen Svarthhølen Svarthhølen Svarthhølen Tyrifjorden Tyrifjorden Tyrifjorden Tyrifjorden Bergertjern Bergertjern Bergertjern Bergertjern Svarthhølen Svarthhølen Svarthhølen Svarthhølen LoQ spike 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Biota data, all conc. in ng·g-1 w.w. - 32 - Fjeld og Vann Report: R1-2019 me-FOSE <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2.0 me-PFOSE % recov 90 et-FOSA <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 et-PFOSA % recov 89 me-FOSA <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 me-PFOSA % recov 87 FOSA <0,1 <0,1 <0,1 1 0.45 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 16 24 52 13 12 31 45 29 0.4 0.6 0.2 0.1 <0,1 <0,1 <0,1 <0,1 0.9 1.1 3.8 6.2 0.1 PFOSA % recov 95 PFDoS <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 <0,2 0.2 PFDoS % recov 88 PFDS <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.2 0.1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.2 0.1 <0,1 <0,1 0.1 PFDS % recov 91 ipPFNS <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.1 ipPFNS % recov 92 PFNS <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.1 PFNS % recov 98 br-PFOS 4.0 8.0 5.0 10 7.3 5.3 5.8 6.3 1.2 1.5 0.8 0.5 1.7 1.3 0.5 0.8 150 137 112 35 75 60 175 166 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.10 PFOS 156 276 213 294 288 66 78 87 21 38 20 12 60 29 9.6 14 1782 1434 912 480 1071 915 1485 1860 <0,1 0.1 0.8 0.7 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.4 0.3 0.10 PFOS % recov 97 PFHpS <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.1 PFHpS % recov 92 PFHxS <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.1 PFHxS % recov 96 PFPeS <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.1 PFPeS % recov 98 PFBS <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.7 1.1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.2 0.1 <0,1 <0,1 0.1 PFBS % recov 91 PFPrS <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 <0,1 0.1 PFPrS % recov 86 PFODA <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 PFODA % recov 76 PFHpDA <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 PFHxDA <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 0.4 PFHxDA % recov 85 PFPeDA <0,4 <0,4 1.1 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 1.4 1.2 <0,4 <0,4 1 0.9 0.9 1.1 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 0.4 PFTeDA 2.6 7.3 8 9.8 8.2 0.9 2.5 2.2 1.1 1.2 0.5 <0,4 2.4 <0,4 1.1 0.6 19 17 8.8 8.1 14 18 21 16 <0,4 <0,4 <0,4 0.6 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 1 1.2 0.4 PFTeA % recov 91 PFTrDA 4.8 7.6 12 9.2 13 2.1 3.5 4.2 3.7 2.7 1.8 2.1 2.7 2.3 2.9 2.4 12 12 4.4 5.3 7.3 7.7 6.8 11 0.5 0.9 2.5 2.1 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 0.7 0.6 0.4 PFTrA % recov 88 PFDoDA 8.8 21 28 15 21 5.6 7.7 8.4 2.4 2.4 1.3 1.5 2.4 1.3 1.5 1.5 34 38 28 24 23 41 41 30 1.3 2.8 3.7 2.6 <0,4 <0,4 <0,4 <0,4 <0,4 <0,4 1.9 2.1 0.4 PFDoA % recov 98 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Biota data, all conc. in ng·g-1 w.w. - 33 - Fjeld og Vann Report: R1-2019 PFPeSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 etFBSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 meFBSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 PFBSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 7:3 FTAC <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 1 PFTeS <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 PFTrS <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 PFUdS <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 6:2 F53B <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 8:2 F53B % recov 91 4:2 F53B <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 4:2 F53B % recov 88 8Cl-PFOS <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 8Cl-PFOS % recov 97 14:2 FTS 0.5 0.4 <0,3 <0,3 0.5 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.5 0.6 1 1.4 0.5 0.6 0.8 0.6 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.5 0.4 0.9 0.8 0.3 12:2 FTS 1.4 5.6 7.5 24 8.4 1.9 3.5 2.5 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 48 37 49 59 50 52 92 44 1.8 1.2 1.1 0.8 <0,3 <0,3 <0,3 <0,3 10 11 8.3 12 0.3 10:2 FTS <0,3 0.6 0.6 0.5 0.7 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 26 21 22 25 22 69 42 37 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 3.8 3.5 1.1 1.2 0.3 10:2 FTS % recov 87 8:2 FTS <0,3 1.2 0.8 3.3 1.8 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 46 27 13 12 24 66 35 65 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 8:2 FTS % recov 105 6:2 FTS <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.9 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 6:2 FTS % recov 101 4:2 FTS <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 4:2 FTS % recov 95 et-FOSAA 0.7 4.4 1.2 92 1.1 0.5 1.1 0.9 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 55 29 53 9 35 38 90 48 0.5 0.8 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 1.6 1.9 2.3 3.5 0.3 et-FOSAA % recov 104 me-FOSAA <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 me-FOSAA % recov 109 FOSAA <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 <0,3 0.3 0.4 0.9 0.3 FOSAA % recov 68 et-FOSE <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2.0 et-PFOSE % recov 85 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Biota data, all conc. in ng·g-1 w.w. - 34 - Fjeld og Vann Report: R1-2019 6:2 PAP <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 diPAP 6:2/8:2 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 8:2 diPAP <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 8:2 diPAP % recov 90 6:2 diPAP <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 6:2 diPAP % recov 86 etFHpSE <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 meFHpSE <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 etFHxSE <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 meFHxSE <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 etFPeSE <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 meFPeSE <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 etFBSE <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 meFBSE <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 etFHpSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 meFHpSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 PFHpSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 etFHxSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 meFHxSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 PFHxSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 etFPeSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 meFPeSA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Biota data, all conc. in ng·g-1 w.w. - 35 - Fjeld og Vann Report: R1-2019 GEN X <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 ADONA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 53B <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 10:2 FTUCA <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 8:2 FTUCA <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 6:2 FTUCA <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 10:2 FTCA <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 8:2 FTCA <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 6:2 FTCA <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 12:2 FTOH <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 10:2 FTOH <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 10:2 FTOH % recov 73 8:2 FTOH <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 8:2 FTOH % recov 79 6:2 FTOH <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 6:2 FTOH % recov 75 PFDPA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 PFPOA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 PFHxPA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 PFBPA <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 8:2 PAP <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 <0,5 0.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Biota data, all conc. in ng·g-1 w.w. - 36 -