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Perfluoroalkyl Substances (PFASs) in Marine Mammals from the South China Sea and Their Temporal Changes 2002 – 2014: Concern for Alternatives of PFOS? James C.W. Lam, Jinling Lyu, KAREN YING KWOK, and Paul Kwan-Sing Lam Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b06076 • Publication Date (Web): 18 Feb 2016 Downloaded from http://pubs.acs.org on February 19, 2016
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Environmental Science & Technology
Submitted for publication in Environmental Science & Technology
Perfluoroalkyl Substances (PFASs) in Marine Mammals from the South China Sea and Their Temporal Changes 2002 – 2014: Concern for Alternatives of PFOS?
James C.W. Lam, ,*, Jinling Lyu, , Karen Y. Kwok, , Paul K.S. Lam, ,*
State Key Laboratory in Marine Pollution (SKLMP), Department of Biology and Chemistry,
City University of Hong Kong, Kowloon, Hong Kong SAR, PR China
Research Centre for the Oceans and Human Health, Shenzhen Key Laboratory for Sustainable
Use of Marine Biodiversity, City University of Hong Kong, Shenzhen Research Institute Building, Shenzhen 518057, China
Corresponding authors: *James C. W. Lam
*Paul K. S. Lam
State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City City University of Hong Kong,
University of Hong Kong,
Tat Chee Avenue, Kowloon, Hong Kong Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China
SAR, PR China.
Tel: +852-3442-4126
Tel: +852-3442-6828
Fax: +852-3442-0524
Fax: +852-3442-0303
E-mail:
[email protected] E-mail:
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Environmental Science & Technology
Abstract
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Perfluorinated sulfonic acids (PFSAs) and perfluorinated carboxylic acids (PFCAs), as well as
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the replacement for the phase-out C8 PFSAs were determined in the liver samples of Indo-
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Pacific humpback dolphins (Sousa chinensis) and finless porpoises (Neophocaena phocaenoides)
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from the South China Sea between 2002 and 2014. Levels of total perfluoroalkyl substances
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(PFASs) in samples ranged from 136 – 15,300 and 30.5 – 2,720 ng/g dw for dolphin and
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porpoise respectively. Significant increasing trends of several individual PFCAs and
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perfluorobutane sulfonate (PFBS) were found in cetacean samples from 2002 to 2014, whereas
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no significant temporal trends of ΣPFASs appeared over the sampling period. This pattern may
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be attributed to the increasing usage of PFCAs and C4-based PFSAs following the
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restriction/voluntary withdrawal of the production and use of perfluorooctane sulfonate (PFOS)
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related products. In addition, significantly increasing temporal shifting trends of PFOS to PFBS
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were observed in the dolphin liver samples. This pattern may be attributed to the substitution of
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PFOS by its alternative – PFBS. The highest levels of PFOS were observed in the liver samples
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of dolphin as compared with other marine mammal studies published since 2006, indicating high
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contamination of PFAS in the South China region. An assessment of relatively high
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concentrations of C8-based PFASs in the liver samples of cetaceans predicted that concentrations
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of PFOS would be expected to affect some proportion of the cetacean populations studied, based
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on the toxicity thresholds derived.
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INTRODUCTION
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Perfluoroalkyl substances (PFASs) are a large class of highly fluorinated organic chemicals
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which are used as surfactants and surface protectors in numerous industrial and commercial
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applications.1-2 In view of their recalcitrant nature, strong potential to accumulate in biota and
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toxic effects on humans and animals, the phase-out of perfluorooctane sulfonate (PFOS) related
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products was started by the manufacturer 3M in 2001.3 Later on, these chemicals were added to
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the list of the Stockholm Convention for global elimination in 2009.4 These compounds, thus,
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should no longer be used in China. However, it has been reported that the industrial and
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manufacturing sources of PFASs has been shifted geographically from the US, Europe and Japan
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to emerging Asian countries, especially China.5 Over the past ten years, the production of PFASs
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in China has increased dramatically due to the high domestic demand and wide range of
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applications in consumer products for export, as well as caused by the restriction of PFOS
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production in other countries.6 In addition, the phasing out of PFOS requires that other chemicals
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with similar physiochemical properties be synthesized and used to meet the large demand for
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manufactured products. The production and use of shorter-chain perfluoroalkyl substances, such
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as perfluorobutane sulfonate (PFBS) as replacements for PFOS in the products has been
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reported.7 Hence, the production and use of both PFOS and its replacements is anticipated to
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increase rapidly in China in order to cope with an increasing market demand. To date, most of
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the studies have elucidated the changes of levels of PFOS and PFOA only and the trend analyses
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were only conducted up to 2008. In addition, there is scarcity of information on the
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environmental fate of PFOS-replacements (C4-based compounds) in the region. Although these
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chemicals were detected in some of the environmental matrices such as sediment, no clear
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temporal changes of these PFOS-replacements have been reported so far. In addition, there is no
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control on the production and use of PFBS anywhere in the world and there is a lack of
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information on the current status and trends of both banned PFASs and their replacements in the
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coastal environment of China. Therefore, the results of the temporal changes of shorter and
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longer chains of PFASs over the past decade will fill an important knowledge gap in the region.
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Archive environmental specimens are important resources for elucidating geographic and
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temporal changes of conventional and emerging contaminants in the environment.8,9 In addition,
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trend analysis of contaminants in archive samples is valuable for better understanding variations
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in emission, trends in exposure levels, assessing the effectiveness of restrictions and regulations
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on their production and use, and for evaluating environmental clearance rates of pollutants.1,10 Of
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the specimens, marine mammals are the target for long-term systematic storage for retrospective
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analysis of different environmental contaminants.9 Cetaceans have frequently been used as
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sentinels for determining status, trends, and spatial and temporal differences in concentrations of
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persistent contaminants and as species of concern in ecological risk assessments.9,11-13
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Indo-Pacific humpback dolphin (Sousa chinensis) is the most conspicuous cetacean species in
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the coastal environment of South China. Another resident species in the region is finless porpoise
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(Neophocaena phocaenoides). These two species of marine mammals are not only aquatic
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symbols for the coastal environment, but also have a high conservation value in the local marine
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ecosystem. Dolphins and porpoises are the top predators of the marine food chain; thus they can
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accumulate relatively high concentrations of persistent organic pollutants (POPs), making them
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useful in studies of organic pollutants and possible effects of POPs on wildlife in aquatic
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environments. In recent years, studies have reported the increasing anthropogenic stress on these
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vulnerable species resulting from different impacts including contamination.14,15 However,
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information on the toxic effects of PFASs on wildlife is very limited. There are some recent
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studies suggesting that PFASs may cause adverse health effects to marine mammals such as
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disruption of the hepatic PPARα-CYP4A signalling pathway of marine mammals by exposure to
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PFASs,16 and a significant association between infectious diseases and elevated concentrations of
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PFASs in the livers of sea otters from California.17 Nonetheless, information on the occurrence
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and distribution of these toxic chemicals in the wildlife from China is very limited.
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As the Pearl River Delta (PRD) region of South China is one of the most heavily
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industrialized and urbanized regions in China,12,18,19 it is conceivable that this region is
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contaminated by PFASs. Moreover, the PRD including Guangdong province has been reported
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to be one of the industrial hotspots with PFAS-related production facilities in China.6 Our recent
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monitoring study carried out in the South China Sea has revealed that the highest total PFAS
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concentrations in seawater were observed at the mouth of the PRD.18 In addition, the
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concentrations in this study were found to be two times higher when compared to the PFOS
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concentrations measured nine years ago at the same sampling locations. PFASs have emerged as
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global environmental contaminants; however, the information on PFASs, particularly the
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changes in levels and patterns of the possible replacements for PFOSs, is still very limited in the
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region. It is hypothesized that replacement of C8-based PFSA by C4-based compounds may
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increase the concentrations of PFBS in the marine environment of South China. This study
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therefore aims to examine temporal trends in PFAS concentrations in two species of marine
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mammals, the Indo-Pacific humpback dolphin and finless porpoise, in the PRD region of China.
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The changes in levels and composition profiles over time may be able to reflect the current
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national manufacturing practices and usage patterns of PFASs. In addition, a preliminary risk
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assessment on this ecologically important marine species due to exposure to this group of
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persistent and toxic substances was conducted in this study.
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MATERALS AND METHODS Details of the chemicals and reagents used in the present study are given in the Supporting
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Information (SI).
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Sample Collection
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Indo-Pacific humpback dolphins (n = 17) and finless porpoises (n = 50) were found stranded
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in Hong Kong, China, between 2002 and 2014 and between 2004 and 2014, respectively (Table
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S1). All the samples were stored at -20°C.
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Extraction and cleanup
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Liver samples were extracted with 10 mM NaOH in acetonitrile, followed by purification
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with an ENVI-Carb and OASIS WAX-SPE method. The procedures are a slight modification of
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previously described methods.19-21 0.3 g of liver tissue was transferred to a 50-mL PP centrifuge
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tube, and surrogates (i.e.
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PFDA, 13C2-PFUnDA and 13C2-PFDoDA), and 5 mL of 10 mM NaOH/ACN were added to the
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15 mL PP tube and sonicated for 1 hour. The mixture was separated by centrifugation at 3000
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rpm at room temperature (~25oC) for 15 min. 4 mL of extract was removed and transferred to a
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second 15 mL PP tube. The extraction was repeated twice as described above, except that 5 mL
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of extract was removed each time, instead of 4 mL. All three extracts were combined in the
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C4-PFOS,
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C4-PFBA,
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C2-PFHxA, 13C4-PFOA,
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C5-PFNA, 13C2-
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second 15 mL PP tube. 0.25 mL of 2M HCl was added to the final extract which was then
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concentrated to 1 mL under high-purity nitrogen for further ENVI-Carb and SPE cleanup.
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The Supelco ENVI-Carb cartridges were preconditioned by passing through 2 mL of
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methanol three times. All the sample extracts were loaded onto the preconditioned cartridges and
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collected. The samples were further eluted by passing through 1 mL of methanol three times.
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After ENVI-Carb cleanup, all the extracts were diluted in 125 mL of Milli-Q water and subjected
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to Oasis WAX cartridges for further SPE cleanup.
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The Oasis WAX cartridges were preconditioned by passing through 4 mL of 0.1% ammonium
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hydroxide in methanol (0.1% NH4OH/MeOH), followed by 4 mL of methanol, and 4 mL of
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Milli-Q water at a rate of 1 drop per second. 125 mL of samples were then passed through the
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pre-conditioned cartridges at a rate of 1 drop per second. After loading the samples, the
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cartridges were washed with 4 mL of 25 mM ammonium acetate buffer at pH 4. Water
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remaining in the cartridges was removed completely by centrifugation at 3000 rpm for 2 min.
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The target analytes were then eluted into two fractions, first with 4 mL of methanol, followed by
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4 mL of 0.1% NH4OH/MeOH at a rate of 1 drop per second separately. The eluates of fractions 1
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and 2 were then concentrated to 1 mL under a gentle stream of high-purity (
