**2. Materials and methods**

**1. Introduction**

the Canadian Arctic and Greenland [8–12].

54 Emerging Pollutants in the Environment - Current and Further Implications

adverse impacts of PFCs on local and global scales [8].

Past research shows that legacy persistent organic pollutants (POPs) such as dichlorodiphe‐ nyltrichloroethane (DDT), polychlorinated biphenyls (PCBs), and hexachlorocyclohexanes (HCHs) pose substantial problems related to environmental and ecosystem health on a global scale [1–5]. POPs can be transported over very long distances, biomagnify in food webs, and cause adverse health effects in high trophic level species such as birds and marine mammals. Cold regions that are typically isolated from anthropogenic activity, such as the Arctic and the Antarctic, are particularly vulnerable to POPs because of the global distillation phenomenon, which causes many pollutants to concentrate in these regions [6, 7]. The Arctic Monitoring Assessment Program (AMAP), in association with the United Nations Environment Pro‐ gramme (UNEP) Stockholm Convention on Persistent Organic Pollutants, has played a key role in documenting the fate, transport, and effects of these pollutants in the Arctic, and has promoted global initiatives to monitor, manage, and control these substances [6, 8]. Despite enhanced understanding of POP contamination in the Arctic, limited information exists on the state of pollution in Antarctic food webs. Researchers have identified a lack of comparative data between the polar regions of the world, where many efforts have been directed toward understanding POP contamination in the high latitudes of the Northern Hemisphere such as

Ongoing research has identified emerging contaminants of concern, including perfluorinated contaminants (PFCs), which are expected to pose significant risks to the environment and wildlife, particularly in the Arctic and the Antarctic [13–15]. Although PFCs have been detected in some Antarctic ecosystems and biota, the environmental transport and bioaccumulation patterns of PFCs, mainly perfluoroalkyl acids (PFAAs) such as perfluorinated carboxylates (PFCAs) and perfluorinated sulfonates (PFSAs), remain relatively unexplored within Antarc‐ tica. PFCs are highly fluorinated anthropogenic compounds, often utilized as repelling agents, with applications including coatings for paper or food packaging and textiles, industrial surfactants, insecticides, and historically, aqueous film-forming foams [16,17]. Due to their widespread use, PFCs are now considered environmentally ubiquitous substances, found in all areas around the world. In response, numerous measures have been taken to reduce the

PFCs are extremely persistent, can travel long distances (predominantly via ocean currents), bioaccumulate in food webs, and achieve highest concentrations in marine mammals and birds. PFCs are of particular ecological and toxicological concern due to their tendency to biomagnify in food webs and cause adverse health effects, including reproductive damage, immunotoxicity, and hepatotoxicity [18]. Of further interest is the unique physicochemical nature of PFCs. Whereas many legacy POPs are lipophilic and therefore accumulate in fatty tissues, PFCs tend to accumulate primarily in protein-rich tissues, such as the liver. Two PFAAs, perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA), represent the most commonly investigated PFCs of significant risk to wildlife and humans due to their ubiquitous nature, global fate and transport, high biomagnification potential, and toxicity risks, especially in aquatic and marine food webs of the northern hemisphere and Arctic [18–21]. Phase out

### **2.1. Study Area and sampling**

The Ecuadorian Research Station "Pedro Vicente Maldonado" (Maldonado Station, hereafter) is located at Fort William Point, Greenwich Island (62°31'S; 59°46'W; Figure 1). The study area encompassed the Barrientos Island (62°24'01"S; 59°43' 52"W), Dee Island (62º25'48.5" S; 59º47'69.6" W), Punta Ambato (62º26'33" S; 59º47'28.8" W), and the surroundings of the Maldonado Station (62º27'59''S; 59º43'32.5''W), as illustrated in Figure 1. Sampling was conducted using three tracks established by the Maldonado Station to access the coastline of Fort Williams, which enclose two sampling zones: Ensenada Guayaquil and Bahia Chile. These sectors are only used by technical and military personnel that work at the Station and visiting scientists that come to the island for research purposes. Barrientos Island is used principally as a tourist stopover for cruise ships where tourists land and walk around the island for birdwatching. In Dee Island and Punta Ambato, sampling was deployed around the coastline. All sampling was done during the Austral summer and seabird breeding seasons of 2009. The collection of abiotic and biotic samples is described as follows.

#### *2.1.1. Sediments*

Sediment samples were collected from three locations in the Antarctic Peninsula including Dee Island (*n* = 1 site), Maldonado Station (*n* = 2 sites), and Punta Ambato (*n* = 2 sites) (Figure 1). Sediment samples were directly collected using 100 mL centrifugation tubes, stored at < 4°C until transportation to the laboratory in Canada.

#### *2.1.2. Seabirds*

Gentoo penguins (*Pygoscelis papua*) and southern giant petrels (*Macronectes giganteus*), two species of seabirds that inhabit the Antarctic Peninsula, were identified as potential bioindicators of PFCs contamination. The main reason for selecting seabirds is based on studies showing that bird populations are most affected by contaminants, specifically POPs, among wildlife species (see [26] for a review). Bird species have the greatest capacity to biomagnify chemicals because of their highly energy-efficient metabolic system and also because of their high trophic position within the food web. Bird populations are therefore often at high risk from bioaccumulative substances, and can act as the "canary in the coal mine" for the larger Antarctic ecosystem.

In this context, we conducted a noninvasive sampling technique to minimize or completely avoid the impacts of lethal or invasive sampling on the local bird populations. Sampling focuses on the collection of shed/molted feathers and excreted fecal matter from nesting sites. Because of the very high affinity of PFCs for protein, feathers are good noninvasive sampling media for PFCS, as they consist mainly of protein matter (i.e., keratin, a high molecular weight protein). Feathers have also been used to successfully monitor mercury in seabird populations such as brown skuas*, Catharacta lonnbergi*, chinstrap penguins, *Pygoscelis antarctica*, and gentoo penguins, *P. papua*), in our study area [27], as well as PFCs in the feathers of aquatic and marine birds, including grey heron (*Ardea cinerea*) and herring gull*,* (*Larus argentatus*) from the Northern Hemisphere [28]. Fecal matter is known to contain some of the highest concentration of contaminants due to the gastrointestinal magnification that occurs in the intestinal tract of consumer organisms. In addition, the contaminant concentrations in fecal matter are related to compounds absorbed by the organism, such that they can provide a measure of accumulated concentrations. The low capacity to migrate to the gaseous phase (i.e., air) and high octanolair partition coefficient (*KOA*) of the analytes (Table 1) cause minimal losses of the contaminants from feces or feathers to the air after feathers or fecal matter have been dropped. This means that the concentrations of the chosen analytes can remain a measure of bird exposure levels long after the feces have been excreted or feathers have been shed.

Molted feathers were collected randomly in and around nests and colonies of petrels sur‐ rounding the Maldonado's Station and stored in ziploc-type plastic bags (*n* = 5). Only one bag of feather samples for gentoo penguin was collected from the Maldonado's Station. Fecal matter samples from gentoo penguins were collected from nesting sites and colonies around the Maldonado's Station (*n* = 9), Barriento Island (*n* = 7), and Dee Island (*n* = 3). All feces samples were placed into 20 mL glass vials. Both feather and fecal samples were stored in coolers and transported by airplane with dry ice (–20°C) until transportation to the lab in Canada.

#### *2.1.3. Lichens*

Fort Williams, which enclose two sampling zones: Ensenada Guayaquil and Bahia Chile. These sectors are only used by technical and military personnel that work at the Station and visiting scientists that come to the island for research purposes. Barrientos Island is used principally as a tourist stopover for cruise ships where tourists land and walk around the island for birdwatching. In Dee Island and Punta Ambato, sampling was deployed around the coastline. All sampling was done during the Austral summer and seabird breeding seasons of 2009. The

Sediment samples were collected from three locations in the Antarctic Peninsula including Dee Island (*n* = 1 site), Maldonado Station (*n* = 2 sites), and Punta Ambato (*n* = 2 sites) (Figure 1). Sediment samples were directly collected using 100 mL centrifugation tubes, stored at < 4°C

Gentoo penguins (*Pygoscelis papua*) and southern giant petrels (*Macronectes giganteus*), two species of seabirds that inhabit the Antarctic Peninsula, were identified as potential bioindicators of PFCs contamination. The main reason for selecting seabirds is based on studies showing that bird populations are most affected by contaminants, specifically POPs, among wildlife species (see [26] for a review). Bird species have the greatest capacity to biomagnify chemicals because of their highly energy-efficient metabolic system and also because of their high trophic position within the food web. Bird populations are therefore often at high risk from bioaccumulative substances, and can act as the "canary in the coal mine" for the larger

In this context, we conducted a noninvasive sampling technique to minimize or completely avoid the impacts of lethal or invasive sampling on the local bird populations. Sampling focuses on the collection of shed/molted feathers and excreted fecal matter from nesting sites. Because of the very high affinity of PFCs for protein, feathers are good noninvasive sampling media for PFCS, as they consist mainly of protein matter (i.e., keratin, a high molecular weight protein). Feathers have also been used to successfully monitor mercury in seabird populations such as brown skuas*, Catharacta lonnbergi*, chinstrap penguins, *Pygoscelis antarctica*, and gentoo penguins, *P. papua*), in our study area [27], as well as PFCs in the feathers of aquatic and marine birds, including grey heron (*Ardea cinerea*) and herring gull*,* (*Larus argentatus*) from the Northern Hemisphere [28]. Fecal matter is known to contain some of the highest concentration of contaminants due to the gastrointestinal magnification that occurs in the intestinal tract of consumer organisms. In addition, the contaminant concentrations in fecal matter are related to compounds absorbed by the organism, such that they can provide a measure of accumulated concentrations. The low capacity to migrate to the gaseous phase (i.e., air) and high octanolair partition coefficient (*KOA*) of the analytes (Table 1) cause minimal losses of the contaminants from feces or feathers to the air after feathers or fecal matter have been dropped. This means that the concentrations of the chosen analytes can remain a measure of bird exposure levels

long after the feces have been excreted or feathers have been shed.

collection of abiotic and biotic samples is described as follows.

56 Emerging Pollutants in the Environment - Current and Further Implications

until transportation to the laboratory in Canada.

*2.1.1. Sediments*

*2.1.2. Seabirds*

Antarctic ecosystem.

Lichen (*Usnea aurantiaco-atra*) samples (*n* = 5) were collected from rocky areas around the surroundings of the Maldonado's Station and wrapped with clean, sterile aluminum foil and stored in ziploc plastic bags until further transportation to the lab. The rationale to select lichens is based on the premise that this biological matrix can be used as a potential monitor and indicator of global atmospheric transport of some PFCs to the Antarctic Peninsula.

**Figure 1.** Geographical location of the study area and sampling sites in the Antarctic Peninsula

#### **2.2. PFC physical – Chemical properties**

Table 1 summarizes the compiled physical–chemical properties for the various PFCs studied, including molecular weights (MW), log octanol–water partition coefficients (log *KOW*), log octanol–air partition coefficients (log *KOA*), and log *D* values. Because the physicochemical properties of PFCs are considerably different from that of many other legacy POPs (i.e., they can be ionized at environmentally relevant pH), it is important to recognize that relationships applicable to other POPs may be less relevant when applied to PFCs and other ionizable compounds. For instance, many organic compounds of concern, including numerous agricultural and pharmaceutical compounds, are lipophilic in nature, and will tend to accumulate in fatty tissues [3]. The octanol–water partition coefficient (*KOW*) has become a common property used to describe the tendency of a substance to partition into lipid, as the behavior of octanol and lipid are quite similar. Octanol thus serves as a suitable surrogate for lipid, particularly within predictive bioaccumulation models [29]. However, *KOW* describes the lipophilicity of neutral compounds, and is not necessarily applicable to ionizable organic compounds (IOCs) such as PFCs, where the measure of lipophilicity is pH-dependent [30]. Many PFCs are almost completely ionized at environ‐ mentally relevant pH [31]. A more applicable indicator for predicting the lipophilicity of ionizable substances is log *D*, where both the neutral and the ionic species of the com‐ pound are accounted for [30].

#### **2.3. PFC analysis: Extraction and quantification**

Sediment and biological samples were extracted and analyzed at the Institute of Ocean Sciences (IOS), Fisheries and Oceans Canada (DFO), Sidney, British Columbia, Canada. PFC concentrations were analyzed by liquid chromatography tandem mass spectrometry with double mass detectors (LC/MS/MS), as described elsewhere [19]. Analyte concentrations were determined with respect to the mass labeled quantification and internal standards using isotope dilution method. Fifteen PFCs were examined in this study (Table 1). High purity (>95%) analytical standards, including perfluorobutane sulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), PFOS, perfluorobutanoic acid (PFBA), perfluoropen‐ tanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecano‐ ic acid (PFUnA), perfluorododecanoic acid (PFDoA), perfluorotetradecanoic acid (PFTA), and perfluorooctanesulfoamide (PFOSA), were used. Mass-labeled internal standards included six PFCs (13C2 PFOA, 13C2 PFDA, 13C2 PFDoA, and 13C4 PFOS, 13C4-PFOA). Calibration curves were constructed from the analysis of calibration standard solutions (range 0.08–5.0 ng/mL).

Various calibration standards and standard additions were prepared and used as quality assurance/quality control (QA/QC). QA/QC measures included initial method validation work, consisting of analyte recovery experiments of native PFCs in clean sediments and biota. The method of detection limit (MDL) was set equal to the concentration of the method's level of quantification (MLOQ) for samples and subtracted from quantified concentrations of each analyte (Table 2). Only corrected data above the MLOQ are reported in this work. Concentrations of PFCs were expressed on a wet weight basis (ng/g ww). Extraction methods are briefly described as follows.

#### *2.3.1. Sediment*

**2.2. PFC physical – Chemical properties**

58 Emerging Pollutants in the Environment - Current and Further Implications

pound are accounted for [30].

(range 0.08–5.0 ng/mL).

**2.3. PFC analysis: Extraction and quantification**

Table 1 summarizes the compiled physical–chemical properties for the various PFCs studied, including molecular weights (MW), log octanol–water partition coefficients (log *KOW*), log octanol–air partition coefficients (log *KOA*), and log *D* values. Because the physicochemical properties of PFCs are considerably different from that of many other legacy POPs (i.e., they can be ionized at environmentally relevant pH), it is important to recognize that relationships applicable to other POPs may be less relevant when applied to PFCs and other ionizable compounds. For instance, many organic compounds of concern, including numerous agricultural and pharmaceutical compounds, are lipophilic in nature, and will tend to accumulate in fatty tissues [3]. The octanol–water partition coefficient (*KOW*) has become a common property used to describe the tendency of a substance to partition into lipid, as the behavior of octanol and lipid are quite similar. Octanol thus serves as a suitable surrogate for lipid, particularly within predictive bioaccumulation models [29]. However, *KOW* describes the lipophilicity of neutral compounds, and is not necessarily applicable to ionizable organic compounds (IOCs) such as PFCs, where the measure of lipophilicity is pH-dependent [30]. Many PFCs are almost completely ionized at environ‐ mentally relevant pH [31]. A more applicable indicator for predicting the lipophilicity of ionizable substances is log *D*, where both the neutral and the ionic species of the com‐

Sediment and biological samples were extracted and analyzed at the Institute of Ocean Sciences (IOS), Fisheries and Oceans Canada (DFO), Sidney, British Columbia, Canada. PFC concentrations were analyzed by liquid chromatography tandem mass spectrometry with double mass detectors (LC/MS/MS), as described elsewhere [19]. Analyte concentrations were determined with respect to the mass labeled quantification and internal standards using isotope dilution method. Fifteen PFCs were examined in this study (Table 1). High purity (>95%) analytical standards, including perfluorobutane sulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), PFOS, perfluorobutanoic acid (PFBA), perfluoropen‐ tanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecano‐ ic acid (PFUnA), perfluorododecanoic acid (PFDoA), perfluorotetradecanoic acid (PFTA), and perfluorooctanesulfoamide (PFOSA), were used. Mass-labeled internal standards included six PFCs (13C2 PFOA, 13C2 PFDA, 13C2 PFDoA, and 13C4 PFOS, 13C4-PFOA). Calibration curves were constructed from the analysis of calibration standard solutions

Various calibration standards and standard additions were prepared and used as quality assurance/quality control (QA/QC). QA/QC measures included initial method validation work, consisting of analyte recovery experiments of native PFCs in clean sediments and biota. The method of detection limit (MDL) was set equal to the concentration of the method's level of quantification (MLOQ) for samples and subtracted from quantified concentrations of each analyte (Table 2). Only corrected data above the MLOQ are reported

Sediment samples (≈ 10 g wet weight) were added to 50 mL polypropylene centrifuge tubes and spiked with internal surrogate spiking solution (360 ng of 13C2 PFOA, 120 ng of 13C2 PFDA, 120 ng of 13C2 PFDoA, and 120 ng of 13C4 PFOS; Table 1). After 20 min, 10 mL of 0.1% acetic acid in MeOH was added, and samples were extracted on a shaker table for 16 h. After extraction and centrifugation, 1 mL was pipetted into 1.5 mL Ependorf vial containing 25 mg of activated carbon. Then the vial was subject to centrifugation for 30 min at 14,000 rpm; 300 *μ*L of supernatant was taken and combined with 300 *μ*L of water and 50 *μ*L of 20 ppb of recovery standard and centrifuged again for 15 min at 14,000 rpm. Then, 300 *μ*L of supernatant was used for LC/MS/MS analysis (i.e., injection volume=100 *μ*L for LC/MS/MS).

#### *2.3.2. Feathers*

Approximately 0.74 g of feather was weighed, and then homogenized by adding first HNO3 (e.g., 4 × 0.9 mL, 2 × 0.9 mL) with a series of vortexing steps until the whole particulates completely disappeared within 3 h. Samples were set up for digestion at room tempera‐ ture (RT) for 12 h. Afterwards, 15 mL of 5 M NaOH prepared in water was added to samples and shaken on a shaker table for 5 min. The pH was measured to ensure the sample was acidic enough (i.e., pH~3–4) prior to direct injection in LC/MS/MS (large volume injection). After neutralization and extraction with 2.5 mL MeOH for a total volume 27.5 mL, ion suppression was found from recoveries; therefore, additional dilution (10×) was done until ion suppression was reduced (i.e., injection volume = 200 *μ*L).

#### *2.3.3. Feces*

Penguin fecal matter (~0.65 g of feces) was weighed and homogenized with HNO3 (e.g., 4 × 0.9 mL, 2 × 0.9 mL and vortexing). Samples were set at RT for digestion during 12 h. After digestion, samples were neutralized to pH equal to 3.2–4.2, brought up to 50 mL and centrifuged at 6000 rpm for 25 min; 100 *μ*L of recovery standard (13C4-PFOA) was added to an aliquot of 400 *μ*L and injected into LC/MS/MS (i.e., injection volume= 200 *μ*L).

#### *2.3.4. Lichen*

Lichen (2 g) was extracted based on the methodology described in reference [32]. After extraction, 4 mL of solution was blown down to 2 mL, followed by collecting 1 mL aliquot and added into 1.5 mL Ependorf vial containing 25 mg of activated carbon. The vial was subject to centrifugation for 30 min at 14,000 rpm and 300 *μ*L of supernatant was ob‐ tained and combined with 300 *μ*L water and 50 *μ*L of 20 ppb recovery standard and centrifuged for 15 min at 14,000 rpm. Then, 300 *μ*L of supernatant was used for LC/MS/MS analysis (i.e., injection volume=100 *μ*L).


MW: molecular weight

\*Log *K*OW and *K*OA values of individual PFCs were compiled from published values calculated using SPARC general partitioning model [33].

\*\*Log *D* values were calculated at pH = 7.5 and T = 21°C using SPARC.

*a* used to quantify PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA

*b* used to quantify PFDA and PFUnA

*c* used to quantify PFDoA and PFTA

*d* used to quantify PFBS, PFHxS PFOS, PFDS, PFOSA

*e* used to quantify recovery of mass labeled surrogates

**Table 1.** List of target perfluoroalkyl chemicals (PFCs) and radiolabeled surrogates monitored using LC/MS/MS.

Perfluorinated Chemicals Assessment in Sediments, Lichens, and Seabirds from the Antarctic Peninsula… http://dx.doi.org/10.5772/60205 61


**Table 2.** Method's limit of quantification (MLOQ) for PFC analytes measured by LC/MS/MS.
